EPA/600/R-17/356 I September2017
www.epa.qov/homeland-securitv-research
United States
Environmental Protection
Agency
Selected Analytical Methods
for Environmental
Remediation and Recovery
(SAM) 2017
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-17/358 | September 2017
Selected Analytical Methods for
Environmental Remediation and
Recovery (SAM) 2017
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Cincinnati, OH 45268
Office of Research and Development
Homeland Security Research Program
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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-15-012 to CSRA Inc. This
document is undergoing review and has not been approved for publication. The contents 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:
Romy Campisano
National Homeland Security Research Center
Office of Research and Development (NG16)
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, OH 45268
(513) 569-7016
campisano.romy@epa.gov
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.kathy@epa.gov
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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) Homeland Security Research Program (HSRP) to provide
selected analytical methods to laboratories tasked with analyzing environmental samples in support of
EPA remediation and recovery efforts following an intentional or accidental homeland security-related
contamination incident. The information 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
Selected Analytical Methods for Environmental Remediation and Recovery (SAM) website
(https://www.epa.gov/homeland-securitv-research/sam'). which provides searchable links to supporting
information regarding selected methods.
At this time, not all of the methods that have been selected have been validated for the listed analyte and
sample type. These methods, however, are considered to contain the most appropriate currently available
techniques, based on expert judgment. Method usability tiers are assigned to provide an indication of the
applicability of the methods that have been selected to address the chemistry, pathogen and biotoxin
analytes (i.e., the extent to which the methods have been tested and applied for analysis of the specific
analyte and sample type for which they have been selected). Unless a method 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 methods and 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, HSRP will continue to provide technical support regarding appropriate actions. HSRP has also
compiled information and published documents regarding sample collection, rapid screening/ preliminary
identification equipment, and disposal of samples corresponding to the analytes and sample types
addressed in this document. This information is available at https://www.epa.gov/homeland-securitv-
research/sam-companion-documents-and-sample-collection-procedures.
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Abbreviations and Acronyms
Abbreviations and Acronyms
ACS
American Chemical Society
AOAC
AOAC International (formerly the Association of Official Analytical Chemists)
APCI
Atmospheric Pressure Chemical Ionization
APHA
American Public Health Association
APHL
Association of Public Health Laboratories
ASM
American Society for Microbiology
ASR
Analytical Service Requests
ASTM
ASTM International (formerly the American Society for Testing and Materials)
ATP
Alternate test procedure
ATSDR
Agency of Toxic Substances & Disease Registry
AWWA
American Water Works Association
BA
Bacillus anthracis
BAM
Bacteriological Analytical Manual
BCYE
Buffered charcoal yeast extract
BCYE GPCV
Buffered charcoal yeast extract with glycine, polymyxin B, cycloheximide and
vancomycin
BCYE PCV
Buffered charcoal yeast extract with polymyxin B, cycloheximide and vancomycin
BEH
Ethylene-bridged hybrid
BGMK
Buffalo green monkey kidney
BHT
Butylated hydroxytoluene
BMBL
Biosafety in Microbiological and Biomedical Laboratories
BoNT
Botulinum neurotoxin
BSL
Biosafety level
BTX
Brevetoxin
BZ
Quinuclidinyl benzilate
ฐC
Degree Celsius
CAS RNฎ
Chemical Abstracts Service Registry Number
CBR
Chemical, biological and/or radiological
CCD
Charge-coupled device
CCID
Coordinating Center for Infectious Diseases
CDC
Centers for Disease Control and Prevention
CFR
Code of Federal Regulations
CFSAN
Center for Food Safety and Applied Nutrition (U.S. Food and Drug Administration)
CHCA
a-cyano-4-hydroxycinnamic acid
CLLE
Continuous liquid-liquid extraction
CLP
Contract Laboratory Program
CPE
Cytopathic effect
cps
Counts per second
Cx
Cycle threshold
CVAA
2-Chlorovinylarsonous acid
CVAFS
Cold vapor atomic fluorescence spectrometry
CVAOA
2-Chlorovinylarsonic acid
CWA
Chemical Warfare Agent
2,4-D
2,4-Dichlorophenoxyacetic acid
DA
Domoic acid
DAI
Direct aqueous injection
DAPI
4',6-Diamidino-2-phenylindole
DAS
Diacetoxyscirpenol
DAS-HG-HSA
Diacetoxyscirpenol hemiglutarate human serum albumin
DAS-HS-HRP
Diacetoxyscirpenol hemisuccinate horseradish peroxidase conjugate
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Abbreviations and Acronyms
DB-1 100% Dimethylpolysiloxane
DBPR Division of Bioterrorism Preparedness and Response
dcNEOSTX Decarbamoylneosaxitoxin
dcSTX Decarbamoylsaxitoxin
DHHS U.S. Department of Health and Human Services
DHS U.S. Department of Homeland Security
DIC Differential interference contrast
DIMP Diisopropyl methylphosphonate
DL Detection limit
DNA Deoxyribonucleic acid
2,4-DNPH 2,4-Dinitrophenylhydrazine
DOC U.S. Department of Commerce
DoD U.S. Department of Defense
DOE U.S. Department of Energy
DOT U.S. Department of Transportation
DPD N,N-Diethyl-p-phenylenediamine
DQO Data quality objective
DTPA Diethylenetriamine -pentaacetate
DVL Detection verification level
EA2192 S-2-(Diisopropylamino)ethyl methylphosphonothioic acid
EC Escherichia coli
ECD 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
ELFA Enzyme-linked fluorescent 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
ESI-MS-MS Electrospray ionization - tandem mass spectrometry
ETV Environmental Technology Verification
FA immunofluorescence 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
FID Flame ionization detector
FL Fluorescence detector
FPD Flame photometric detector
FRET Forster resonance energy transfer
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Abbreviations and Acronyms
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-MS-TOF
Gas chromatography-mass spectrometry-time of flight
GC-NPD
Gas chromatography-nitrogen-phosphorus detector
GD
Soman
GE
1-Methylethyl ester ethylphosphonofluoridic acid
Ge
Germanium
Ge(Li)
Germanium (Lithium)
GF
Cyclohexyl sarin
GFAA
Graphite furnace atomic absorption spectrophotometer or Graphite furnace atomic
absorption spectrophotometry
GTX
Gonyautoxins
HASL
Health and Safety Laboratory, currently known as National Urban Security
Technology Laboratory (NUSTL)
HAV
Hepatitis A virus
HCoV
Human coronavirus
HD
Sulfur mustard / mustard gas; bis(2-chloroethyl) sulfide
HEPA
High-efficiency particulate air
HEV
Hepatitis E virus
HFBA
Heptafluorobutyric anhydride
HFBI
Heptafluorobutyrylimidazole
HHS
U.S. Health and Human Services
HILIC
Hydrophilic interaction liquid chromatography
HILIC-MS-MS
Hydrophilic interaction liquid chromatography-tandem mass spectrometry
HLB
Hydrophilic-lipophilic-balanced
HMTD
Hexamethylenetriperoxidediamine
HMX
Octahy dro -1,3,5,7 -tetranitro -1,3,5,7 -tetrazocine
HN-1
Nitrogen mustard 1; bis(2-chloroethyl)ethylamine
HN-2
Nitrogen mustard 2; 2,2'-dichloro-N-methyldiethylamine N,N-bis(2-
chloroethyl)methylamine
HN-3
Nitrogen mustard 3; tris(2-chloroethyl)amine
HPGe
High purity germanium
HPLC
High performance liquid chromatography
HPLC-FL
High performance liquid chromatography-fluorescence
HPLC-MS
High performance liquid chromatography-mass spectrometry
HPLC-MS-MS
High performance liquid chromatography tandem mass spectrometry
HPLC-UV
High performance liquid chromatography-ultraviolet
HPLC-vis
High performance liquid chromatography-visible
HRP
Horseradish peroxidase
HSRP
Homeland Security Research Program
HTO
Tritiated water
HV
High volume
IC
Ion chromatograph or Ion chromatography
ICLN
Integrated Consortium of Laboratory Networks
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Abbreviations and Acronyms
ICP
Intestinal contents preparation (pathogens); Inductively coupled plasma (chemistry)
ICP-AES
Inductively coupled plasma - atomic emission spectrometry
ICP-MS
Inductively coupled plasma - mass spectrometry
ID.
Inner diameter
IDL
Instrument detection limit
IMPA
Isopropyl methylphosphonic acid
IMS
Immunomagnetic separation
10
Inorganic
IRIS
Integrated Risk Information System (EPA)
ISE
Ion specific electrode
ISG
Impregnated silica gel
ISM02.3
Inorganic Superfund Methods Multi-Media, Multi-Concentration ISM02.3
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
LC-ESI-MS-MS
Liquid chromatography-electrospray ionization-tandem mass spectrometry
LCMRL
Lowest common minimum reporting level
LC-MS
Liquid chromatography-mass spectrometry
LC-MS-MS
Liquid chromatography tandem mass spectrometry
LC-PIM-MS
Liquid chromatography-product ion monitoring-mass spectrometry
LC-UV
Liquid chromatography-ultraviolet
LD50
Median lethal dose
LFA
Lateral flow immunoassay
LFD
Lateral flow device
LLD
Lower limit of detection
LLOQ
Lower limit of quantitation
LOD
Limit of detection
LOQ
Limit of quantitation
LRN
Laboratory Response Network
LSC
Liquid scintillation counter
LSE
Liquid-solid extraction
M
Molar
mAbs
Monoclonal antibodies
MAE
Microwave-assisted extraction
MALDI
Matrix-assisted laser-desorption ionization
MALDI-TOF-MS
Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry
MARLAP
Multi-Agency Radiological Laboratory Analytical Protocols
MC
Microcystin
MDC
Minimum detectable concentration
MDCK
Madin-Darby canine kidney cells
MDEA
N-Methyldiethanolamine
MDL
Method detection limit
MFA
Monofluoroacetate
MIC
Methyl isocyanate
mLDso
Mouse lethal dose
MPA
Methylphosphonic acid
MRL
Minimum reporting level
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Abbreviations and Acronyms
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 tert-butyl ether
MW
Molecular weight
MWCO
Molecular weight cut-off
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
NCPDCID
National Center for the Prevention, Detection, and Control of Infectious Diseases
NCRP
National Council on Radiation Protection and Measurements
NEMI
National Environmental Methods Index
NEO
Neosaxitoxins
NERL
National Exposure Research Laboratory (EPA)
NHSRC
National Homeland Security Research Center (EPA)
NIOSH
National Institute for Occupational Safety and Health
NIST
National Institute of Standards and Technology
nM
Nanomolar
NMAM
NIOSH Manual of Analytical Methods
NNSA
National Nuclear Security Administration
NOD
Nodularins
NPD
Nitrogen-phosphorus detector
NRC
U.S. Nuclear Regulatory Commission
NRMRL
National Risk Management Research Laboratory (EPA)
nS
Nano Siemens
NTIS
National Technical Information Service
NTU
Nephelometric turbidity units
OAQPS
Office of Air Quality Planning and Standards (EPA)
OAR
Office of Air and Radiation (EPA)
OGWDW
Office of Water, Office of Ground Water and Drinking Water (EPA)
OLEM
Office of Land and Emergency Management (EPA)
ORAU
Oak Ridge Associated Universities
ORD
Office of Research and Development (EPA)
ORIA
Office of Radiation and Indoor Air (EPA)
ORISE
Oak Ridge Institute for Science and Education
OSHA
Occupational Safety and Health Administration
OVS
OSHA versatile sampler
OW
Office of Water (EPA)
PCDDs
Polychlorinated dibenzo-p-dioxins
PCDFs
Polychlorinated dibenzofurans
PCR
Polymerase chain reaction
PEL
Permissible exposure limit
PETN
Pentaerythritol tetranitrate
PFBHA
0-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine
PFE
Pressurized fluid extraction
PHILIS
Portable High Throughput Integrated Laboratory Identification Systems
PIM
Product ion monitoring
PLOS
Public Library of Science
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Abbreviations and Acronyms
PLRP-S
Polymeric reversed phase
PMPA
Pinacolyl methyl phosphonic acid
1,2-PP
1 -(2-Pyridyl)piperazine
ppbv
Parts per billion by volume
pptv
Parts per trillion by volume
PST
Paralytic shellfish toxin
PTFE
Polytetrafluoroethylene
PubMED
U.S. National Librarv of Medicine (http://www.pubmed.eov)
PUF
Polyurethane foam
PVC
Polyvinyl chloride
PVDF
Polyvinylidene fluoride
QA
Quality assurance
QAP
Quality assessment program
QC
Quality control
QL
Quantitation limit
qPCR
Quantitative polymerase chain reaction
ฎ
Registered trademark
R 33
Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] 0-2-methylpropyl ester (VR)
RBA
Receptor binding assay
RCRA
Resource Conservation and Recovery Act
RDX
Hexahydro-1,3,5 -trinitro-1,3,5 -triazine
RESL
Radiological and Environmental Sciences Laboratory
RFV
Relative fluorescence value
RLAB
Regional laboratory method
RNA
Ribonucleic acid
RNAse
Ribonuclease
rRNA
Ribosomal ribonucleic acid
RTECS
Registry of Toxic Effects of Chemical Substances
RTG
Radioisotope thermoelectric generator
RT-PCR
Reverse transcription polymerase chain reaction
RT-qPCR
Quantitative reverse transcription polymerase chain reaction
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
SEB
Staphylococcal enterotoxin type B
SEC
Staphylococcal enterotoxin type C
SED
Staphylococcal enterotoxin type D
SEE
Staphylococcal enterotoxin type E
SET
Staphylococcal enterotoxin
SIM
Selective ion monitoring
SIS
Selected ion storage
SM
Standard Methods for the Examination of Water and Wastewater
SOP
Standard operating procedure
SPE
Solid-phase extraction
SPR
Solid-phase receptacle
SRC
Syracuse Research Corporation
SRM
Single reaction monitoring
SRS
Savannah River National Laboratory, Savannah River Site
STEC
Shiga-toxigenic E. coli
STEL
Short term exposure limit
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Abbreviations and Acronyms
STS
Sample test source
STX
Saxitoxin
Stx
Shiga toxin
Stx-1
Shiga toxin Type 1
Stx-2
Shiga toxin Type 2
sw
Solid waste
To
Time zero
t2o
Tritium oxide
TBD
To be determined
TCLP
Toxicity Characteristic Leaching Procedure
TDG
Thiodiglycol
TEA
T riethanolamine
TEM
Transmission electron microscope or Transmission electron microscopy
TEPP
Tetraethyl pyrophosphate
TETS
Tetramethylenedisulfotetramine or tetramine
Tf
Time final
THF
T etrahydrofuran
TIC
Total ion chromatogram
TIOA
Tri-isooctylamine
TM
Unregistered trademark
1,3,5-TNB
1,3,5 -T rinitrobenzene
2,4,6-TNT
2,4,6-Trinitrotoluene
TO
Toxic Organic
TOF
Time-of-flight
TOF-MS
Time-of-flight mass spectrometry
TOPO
Trioctylphosphine oxide
TOXNET
Toxicology Data Network
TRU
Transuranic
TTX
Tetrodotoxin
UF
Ultrafiltration
U.S.
United States
USD A
U.S. Department of Agriculture
USGS
U.S. Geological Survey
uv
Ultraviolet
VBNC
Viable but non-culturable
VC
Vibrio cholerae
VCSB
Voluntary Consensus Standard Body
VE
Phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester
VG
Phosphonothioic acid, S-(2-(diethylamino)ethyl) 0,0-diethyl ester
vis
Visible detector
VM
Phosphonothioic acid, methyl-,S-(2-(diethylamino)ethyl) O-ethyl ester
VOA
Volatile organic analysis
VOC
Volatile organic compound
VR
Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (R
33)
VX
0-Ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate
WCIT
Water Contaminant Information Tool
WEF
Water Environment Federation
WHO
World Health Organization
WLA
Water Laboratory Alliance
WSD
Water Security Division (EPA, Office of Water)
YP
Yersinia pestis
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Acknowledgments
Dedication
This publication is dedicated to the memory of our friend and colleague, Terry Smith. Terry provided
critical support both in initiating the efforts resulting in this document, and in putting in countless hours
providing chemistry expertise beginning in 2004. Throughout the many updates to this document and its
information, Terry provided valuable input as a work group member, as well as an alternate lead during
the third revision. Terry was a friend and mentor to many chemists at the U.S. Environmental Protection
Agency (EPA). His contributions to the EPA Homeland Security Research Program's Sampling and
Analysis Methods program were always provided with devotion and camaraderie, all while keeping up
with his duties within the Office of Land and Emergency Management and with the Environmental
Response Laboratory Network. He left many friends throughout EPA and the chemistry community, and
will be greatly missed.
Acknowledgments
Contributions of the following individuals and organizations to the development of the information
provided in this document are acknowledged.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
(NHSRC)
Romy Campisano
Sandip Chattopadhyay
Kathy Hall - 2017 Radiochemistry Work Group Alternate Lead
Matthew Magnuson - 2017 Biotoxins Work Group Lead
Tonya Nichols
Emily Parry
Eric Rhodes - 2017 Pathogens Work Group Lead
Sanjiv Shah
Erin Silvestri
Sarah Taft
Stuart Willison - 2017 Chemistry Work Group Alternate Lead
Office of Air and Radiation, Office of Radiation and Indoor Air (ORIA)
James Burn
Malek Chatila
ORIA, National Analytical Radiation Environmental Laboratory (NAREL)
John Griggs - 2017 Radiochemistry Work Group Lead
Alison Tamasi
Office of Land and Emergency Management (OLEM)
Scott Hudson (Office of Emergency Management)
Lawrence Kaelin (Office of Emergency Management)
Terry Smith (Office of Emergency Management)
Shen-Yi Yang
Office of Water, Office of Ground Water and Drinking Water (OGWDW)
Jennifer Best (Standards and Risk Management Division)
Lesley D'Anglada (Water Security Division)
George Gardenier (Water Security Division)
James Sinclair (Technical Support Center)
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Acknowledgments
Office of Research and Development, National Exposure Research Laboratory (NERL)
Thomas Behymer
Jennifer Cashdollar
Ann Grimm
Jeffrey Morgan
Jody Shoemaker
Gerard Stelma
Jeanette Van Emon
Office of Research and Development, National Risk Management Research Laboratory
(NRMRL)
Laura Boczek - 2017 Pathogens Work Group Alternate Lead
Alan Lindquist
Heath Mash - 2017 Biotoxins Work Group Alternate Lead
Office of Enforcement and Compliance Assurance (OECA)
Jeff Cahill (National Enforcement Investigations Center)
John Reschl (National Enforcement Investigations Center)
EPA Regions
Jack Berges (Region 9)
Diane Gregg (Region 6)
Stephanie Harris (Region 10)
Michelle Kerr (Region 5)
Rick McMillin (Region 6)
Steve Reimer (Region 10) - 2017 Chemistry Work Group Lead
Troy Strock (Region 5)
Sue Warner (Region 3)
Larry Zintek (Region 5)
United States Department of Agriculture (USDA)
Luisa Chang (Foodborne Toxin Detection and Prevention Research Unit)
Xiaohua He (Foodborne Toxin Detection and Prevention Research Unit)
Marcus Head (Office of Public Health Science)
Larry Stanker (Foodborne Toxin Detection and Prevention Research Unit)
United States Department of Commerce (DOC)
Peter Moeller (National Oceanic & Atmospheric Administration)
United States Department of Defense (DoD)
Linda Beck (U.S. Navy)
David Burns (U.S. Air Force Technical Applications Center)
Gretchen Farming (U.S. Air Force)
Marty Johnson (U.S. Army, Radiation Standards Laboratory)
Daniel Mackney (U.S. Air Force)
Ty Richards (U.S. Air Force)
Aurelie Soreefan (U.S. Air Force)
United States Department of Energy (DOE)
Sherrod Maxwell (Savannah River Site)
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Acknowledgments
United States Department of Health and Human Services (DHHS)
Centers for Disease Control and Prevention (CDC)
Nicole Abbot (National Center for Environmental Health ORISE)
Thomas Blake (National Center for Environmental Health)
Jasmine Chaitram (National Center for Emerging and Zoonotic Infectious Diseases)
Rebecca Coleman (National Center for Environmental Health)
Jay Gee (National Center for Emerging and Zoonotic Infectious Diseases)
Vincent Hill (National Center for Emerging and Zoonotic Infectious Diseases)
Marcie Jaffee (National Center for Environmental Health)
Rudy Johnson (National Center for Environmental Health)
Suzanne Kalb (National Center for Environmental Health)
Susan Maslanka (National Center for Emerging and Zoonotic Infectious Diseases)
Stephen Morse (Office of Public Health Preparedness and Response)
Geovannie Ojeda-Torres (National Center for Environmental Health)
Dongxia Wang (National Center for Environmental Health)
Richard Wang (National Center for Environmental Health)
United States Food and Drug Administration (FDA)
Eric Garber
Sherwood Hall
Sara McGrath
Shashi Sharma
Sandra Tallent
United States Geological Survey (USGS)
Rebecca Bushon
Dale Griffin
Keith Loftin
State Agencies
Sanjib Bhattacharya (City of Milwaukee Health Department)
Sanwat Chaudhuri (Unified Public Health Laboratory)
Marinea Mehrhoff (State Hygienic Laboratory at the University of Iowa, retired)
Bahman Parsa (New Jersey Department of Health)
Bob Read (Tennessee Department of Health, Environmental Laboratory)
Christopher Retarides (Virginia Division of Consolidated Laboratories)
Municipalities
Earl Peterkin (Philadelphia Water Department)
Anthony Rattonetti (San Francisco Public Utility Commission)
National Laboratories
Donivan Porterfield (Los Alamos National Laboratory)
Sonoya Shanks (Sandia National Laboratories)
NorthStar Medical Radioisotopes, LLC
James Harvey
CSRA Inc.
Eric Boring
Yildiz Chambers
John Chandler
Joan Cuddeback
Emily King
Robert Rosson
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Table of Contents
Selected Analytical Methods for
Environmental Remediation and Recovery (SAM) 2017
Contents
Disclaimer ii
Use of This Document iii
Abbreviations and Acronyms iv
Dedication xi
Acknowledgments xi
Section 1.0: Introduction 1
Section 2.0: Background 4
Section 3.0: Scope and Application 7
Section 4.0: Points of Contact 10
Section 5.0: Selected Chemical Methods 11
5.1 General Guidelines 12
5.1.1 Standard Operating Procedures for Identifying Chemical Methods 12
5.1.2 General QC Guidelines for Chemical Methods 27
5.1.3 Safety and Waste Management 29
5.2 Method Summaries 30
5.2.1 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and
Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry 30
5.2.2 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry 31
5.2.3 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry 32
5.2.4 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking
Water by Ion Chromatography 33
5.2.5 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry 33
5.2.6 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate) 34
5.2.7 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by
Capillary Column Gas Chromatography / Mass Spectrometry 35
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 36
5.2.9 EPA Method 525.3: Determination of Semivolatile Organic Chemicals in Drinking
Water by Solid Phase Extraction and Capillary Column Gas Chromatography/ Mass
Spectrometry (GC/MS) 37
5.2.10 EPA Method 531.2: Measurement ofN-Methylcarbamoyloximes and N-
Methylcarbamates in Water by Direct Aqueous Injection HPLC With Postcolumn
Derivatization 38
5.2.11 EPA Method 538: Determination of Selected Organic Contaminants in Drinking
Water by Direct Aqueous Injection-Liquid Chromatography/Tandem Mass
Spectrometry (DAI-LC/MS/MS) 38
5.2.12 EPA Method 540: Determination of Selected Organic Chemicals in Drinking Water
by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS) 39
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5.2.13 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by
Liquid-Solid Extraction and High Performance Liquid Chromatography With
Ultraviolet Detection 40
5.2.14 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts,
Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by
Liquid-Liquid Extraction and Gas Chromatography With Electron-Capture Detection 40
5.2.15 EPA Method 556.1: Determination of Carbonyl Compounds in Drinking Water by
Fast Gas Chromatography 41
5.2.16 EPA Method 3015A (SW-846): Microwave Assisted Acid Digestion of Aqueous
Samples and Extracts 42
5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils 43
5.2.18 EPA Method 3051A (SW-846): Microwave Assisted Acid Digestion of Sediments,
Sludges, and Oils 44
5.2.19 EPA Method 3511 (SW-846): Organic Compounds in Water by Microextraction 45
5.2.20 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction 46
5.2.21 EPA Method 3535A (SW-846): Solid-Phase Extraction 46
5.2.22 EPA Method 3541 (SW-846): Automated Soxhlet Extraction 48
5.2.23 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE) 49
5.2.24 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSE) 50
5.2.25 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples 51
5.2.26 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples 52
5.2.27 EPA Method 6010D (SW-846): Inductively Coupled Plasma - Optical Emission
Spectrometry 53
5.2.28 EPA Method 6020B (SW-846): Inductively Coupled Plasma - Mass Spectrometry 54
5.2.29 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor
Technique) 55
5.2.30 EPA Method 747IB (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-
Vapor Technique) 56
5.2.31 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry 56
5.2.32 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography 57
5.2.33 EPA Method 8015D (SW-846): Nonhalogenated Organics Using GC/FID 58
5.2.34 EPA Method 8260D (SW-846): Volatile Organic Compounds by Gas
Chromatography-Mass Spectrometry (GC/MS) 59
5.2.35 EPA Method 8270E (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS) 60
5.2.36 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed Within the Laboratory 61
5.2.37 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC) 62
5.2.38 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC) 63
5.2.39 EPA Method 8318A (SW-846): 7V-Methylcarbamates by High Performance Liquid
Chromatography (HPLC) 63
5.2.40 EPA Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by
High Performance Liquid Chromatography (HPLC) 64
5.2.41 EPA ISM02.3 Cyanide: Analytical Methods for Total Cyanide Analysis 65
5.2.42 EPA Method 3135.21: Cyanide, Total and Amenable in Aqueous and Solid Samples
Automated Colorimetric With Manual Digestion 65
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5.2.43 EPA 10 [Inorganic] Compendium Method 10-3.1: Selection, Preparation, and
Extraction of Filter Material 66
5.2.44 EPA 10 [Inorganic] Compendium Method 10-3.4: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy 67
5.2.45 EPA 10 [Inorganic] Compendium Method 10-3.5: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry
(ICP-MS) 68
5.2.46 EPA 10 [Inorganic] Compendium Method 10-5: Sampling and Analysis for Vapor
and Particle Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic
Fluorescence Spectrometry (CVAFS) 69
5.2.47 EPA Air Method, Toxic Organics - 10A (TO-IO A): 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) 70
5.2.48 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) 71
5.2.49 EPA Air Method, Toxic Organics - 17 (TO-17): Determination of Volatile Organic
Compounds in Ambient Air Using Active Sampling Onto Sorbent Tubes 72
5.2.50 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) 73
5.2.51 EPA/600/R-12/653: Verification of Methods for Selected Chemical Warfare Agents
(CWAs) 74
5.2.52 EPA/600/R-13/224: Surface Analysis of Nerve Agent Degradation Products by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 75
5.2.53 EPA/600/R-15/097: Adaptation of the Conditions of U.S. EPA Method 538 for the
Analysis of a Toxic Degradation Product of Nerve Agent VX (EA2192) in Water by
Direct Aqueous Injection- Liquid Chromatography/Tandem Mass Spectrometry 76
5.2.54 EPA/600/R-15/258: Extraction and Analysis of Lewisite 1, by its Degradation
Products, Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) 77
5.2.55 EPA/600/R-16/114: Analytical Protocol for Measurement of Extractable Semivolatile
Organic Compounds Using Gas Chromatography/Mass Spectrometry 78
5.2.56 EPA/600/R-16/115: Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and
Sulfur Mustard Using Gas Chromatography/Mass Spectrometry 80
5.2.57 EPA/600/R-16/116: Analytical Protocol for VX Using Gas Chromatography/Mass
Spectrometry (GC/MS) 80
5.2.58 EPA-821-B-01-009: Method Kelada-01: Kelada Automated Test Methods for Total
Cyanide, Acid Dissociable Cyanide, and Thiocyanate 81
5.2.59 NIOSH Method 1612: Propylene Oxide 82
5.2.60 NIOSH Method 2016: Formaldehyde 82
5.2.61 NIOSH Method 2513: Ethylene Chlorohydrin 83
5.2.62 NIOSH Method 3509: Aminoethanol Compounds II 83
5.2.63 NIOSH Method 3510: Monomethylhydrazine 84
5.2.64 NIOSH Method 5600: Organophosphorus Pesticides 84
5.2.65 NIOSH Method 5601: Organonitrogen Pesticides 85
5.2.66 NIOSH Method 6001: Arsine 86
5.2.67 NIOSH Method 6002: Phosphine 86
5.2.68 NIOSH Method 6010: Hydrogen Cyanide 87
5.2.69 NIOSH Method 6013: Hydrogen Sulfide 88
5.2.70 NIOSH Method 6015: Ammonia 88
5.2.71 NIOSH Method 6402: Phosphorus Trichloride 88
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5.2.72 NIOSH Method 7905: Phosphorus 89
5.2.73 NIOSH Method 7906: Particulate Fluorides and Hydrofluoric Acid by Ion
Chromatography 90
5.2.74 NIOSH Method 7907: Volatile Acids by Ion Chromatography (Hydrogen Chloride,
Hydrogen Bromide, Nitric Acid) 90
5.2.75 NIOSH Method 9102: Elements on Wipes 91
5.2.76 NIOSH Method 9106: Methamphetamine and Illicit Drugs, Precursors and
Adulterants on Wipes by Liquid-Liquid Extraction 92
5.2.77 NIOSH Method 9109: Methamphetamine and Illicit Drugs, Precursors, and
Adulterants on Wipes by Solid Phase Extraction 93
5.2.78 NIOSH Method S301-1: Fluoroacetate Anion 93
5.2.79 OSHA Method 40: Methylamine 94
5.2.80 OSHA Method 54: Methyl Isocyanate (MIC) 95
5.2.81 OSHA Method 61: Phosgene 95
5.2.82 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres 96
5.2.83 OSHA Method ID216SG: Boron Trifluoride (BF3) 96
5.2.84 OSHA Method PV2004: Acrylamide 97
5.2.85 OSHA Method PV2103: Chloropicrin 97
5.2.86 ASTM Method D5755-09: Standard Test Method for Microvacuum Sampling and
Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos
Structure Number Surface Loading 98
5.2.87 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 98
5.2.88 ASTM Method D7597-16: 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 99
5.2.89 ASTM Method D7598-16: Standard Test Method for Determination of Thiodiglycol
in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass
Spectrometry 100
5.2.90 ASTM Method D7599-16: Standard Test Method for Determination of
Diethanolamine, Triethanolamine, 7V-Methyldiethanolamine and N-
Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 100
5.2.91 ASTM Method D7644-16: Standard Test Method for Determination of Bromadiolone,
Brodifacoum, Diphacinone and Warfarin in Water by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 101
5.2.92 ASTM Method D7645-16: 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) 102
5.2.93 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) 102
5.2.94 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) 103
5.2.95 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
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Pressurized Fluid Extraction and Analyzed by Liquid Chromatography/Tandem Mass
Spectrometry 104
5.2.96 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-
Transfer Transmission Electron Microscopy Method 105
5.2.97 Standard Method 4500-CN G: Cyanides Amenable to Chlorination after Distillation 105
5.2.98 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step 106
5.2.99 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method 107
5.2.100 Standard Method 4500-C1 G: Chlorine (Residual) DPD Colorimetric Method 107
5.2.101 Literature Reference for Chlorine in Air (Analyst, 1999. 124(12): 1853-1857) 108
5.2.102 Literature Reference for Hexamethylenetriperoxidediamine (HMTD) (Analyst, 2001.
126:1689-1693) 108
5.2.103 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-237) 109
5.2.104 Literature Reference for Cyanogen Chloride (Encyclopedia of Anal. Chem. 2006 DOI:
10.1002/9780470027318.a0809) 109
5.2.105 Literature Reference for 3-Chloro-l,2-propanediol (Eur. J. Lipid Sci. Technol. 2011.
113: 345-355) 110
5.2.106 Literature Reference for Methyl Hydrazine (Journal of Chromatography. 1993.
617(1): 157-162) Ill
5.2.107 Literature Reference for 3-Chloro-l,2-propanediol (Journal of Chromatography A.
2000. 866(1): 65-77) 112
5.2.108 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl Fluoroacetate
(Journal of Chromatography A. 2007. 1139: 271-278) 112
5.2.109 Literature Reference for Methamidophos (Journal of Chromatography A. 2007. 1154:
3-25) 113
5.2.110 Literature Reference for Paraquat (Journal of Chromatography A. 2008, 1196-1197,
110-116) 114
5.2.111 Literature Reference for Fentanyl (Journal of Chromatography A. 2011. 1218: 1620-
1649) 114
5.2.112 Literature Reference for BZ (Journal of Chromatography B. 2008. 874: 42-50 115
5.2.113 Literature Reference for Fluoroacetamide (Journal of Chromatography B. 2008.
876(1): 103-108) 116
5.2.114 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl Fluoroacetate
(Journal of Chromatography B. 2010. 878: 1045-1050) 117
5.2.115 Literature Reference for Carfentanil and 3-Methyl Fentanyl (J. Chromatogr. B. 2014.
962: 52-58 117
5.2.116 Literature Reference for Sodium Azide (Journal of Forensic Sciences. 1998. 43(1):
200-202) 118
Section 6.0: Selected Radiochemical Methods 120
6.1 General Guidelines 121
6.1.1 Standard Operating Procedures for Identifying Radiochemical Methods 122
6.1.2 General QC Guidelines for Radiochemical Methods 128
6.1.3 Safety and Waste Management 129
6.2 Method Summaries (Environmental Samples) 131
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary Sources... 131
6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water 131
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water 132
6.2.4 EPA Method 903.1: Radium-226 in Drinking Water - Radon Emanation Technique 134
6.2.5 EPA Method 905.0: Radioactive Strontium in Drinking Water 134
6.2.6 EPA Method 906.0: Tritium in Drinking Water 135
6.2.7 EPA Method 907.0: Actinide Elements in Drinking Water - Thorium, Uranium,
Neptunium, Plutonium, Americium and Curium 136
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6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium
in Water, Soil, Air, and Biological Tissue 137
6.2.9 EPA Method Rapid Radiochemical Method for Phosphorus-32 in Water for
Environmental Restoration Following Homeland Security Events 138
6.2.10 EPA Method R4-73-014: Radioactive Phosphorus 138
6.2.11 EPA Method: Determination of Radiostrontium in Food and Bioenvironmental
Samples 139
6.2.12 EPA Method: Rapid Radiochemical Method for Americium-241 in Water for
Environmental Restoration Following Homeland Security Events 140
6.2.13 EPA Method: Rapid Radiochemical Method for Plutonium-238 and Plutonium-
239/240 in Water for Environmental Restoration Following Homeland Security
Events 141
6.2.14 EPA Method: Rapid Radiochemical Method for Radium-226 in Water for
Environmental Restoration Following Homeland Security Events 141
6.2.15 EPA Method: Rapid Radiochemical Method for Radiostrontium in Water for
Environmental Restoration Following Homeland Security Events 142
6.2.16 EPA Method: Rapid Radiochemical Method for Isotopic Uranium in Water for
Environmental Restoration Following Homeland Security Events 143
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 144
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 146
6.2.19 Rapid Method for Radium in Soil Incorporating the Fusion of Soil and Soil-Related
Matrices with the Radioanalytical Counting Method for Environmental Remediation
Following Radiological Incidents 147
6.2.20 Rapid Method for Fusion of Soil and Soil-Related Matrices Prior to Americium,
Plutonium, and Uranium Analyses for Environmental Remediation Following
Radiological Incidents 148
6.2.21 Rapid Method for Sodium Carbonate Fusion of Soil and Soil-Related Matrices Prior
to Strontium-90 Analyses for Environmental Remediation Following Radiological
Incidents 149
6.2.22 Rapid Method for Sodium Hydroxide/Sodium Peroxide Fusion of Radioisotope
Thermoelectric Generator Materials in Water and Air Filter Matrices Prior to
Plutonium Analyses for Environmental Remediation Following Radiological Incidents... 150
6.2.23 EPA Method: Rapid Radiochemical Method for Californium-252 in Water, Air
Particulate Filters, Swipes and Soil for Environmental Restoration Following
Homeland Security Events 151
6.2.24 EPA Method: Rapid Radiochemical Method for Curium-244 in Water Samples for
Environmental Remediation Following Radiological Incidents 153
6.2.25 EPA Method: Rapid Radiochemical Method for Curium-244 in Air Particulate Filters,
Swipes and Soil 154
6.2.26 Rapid Radiochemical Method for Radium-226 in Building Materials for
Environmental Remediation Following Radiological Incidents 156
6.2.27 EPA Method: NAREL Standard Operating Procedure for Actinides in Environmental
Matrices by Extraction Chromatography 157
6.2.28 EML HASL-300 Method Am-01-RC: Americium in Soil 159
6.2.29 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters -
Eichrom's TRU Resin 159
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6.2.30 EML HASL-300 Method Am-06-RC: Americium and/or Plutonium in Vegetation 160
6.2.31 EML HASL-300 Method Ga-01-R: Gamma Radioassay 161
6.2.32 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air
Filters 162
6.2.33 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or
Sediments 162
6.2.34 EML HASL-300 Method Ra-03-RC: Radium-226 in Soil, Vegetable Ash, and Ion
Exchange Resin 163
6.2.35 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples 164
6.2.36 EML HASL-300 Method Tc-01-RC: Technetium-99 in Water and Vegetation 164
6.2.37 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water - TEVAฎ Resin 165
6.2.38 EML HASL-300 Method U-02-RC: Isotopic Uranium in Biological and
Environmental Materials 165
6.2.39 DOE FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air 166
6.2.40 DOE RESL Method P-2: P-32 Fish, Vegetation, Dry Ash, Ion Exchange 167
6.2.41 DOE SRS Actinides and Sr-89/90 in Soil Samples 168
6.2.42 DOE SRS Actinides and Sr-89/90 in Vegetation: Fusion Method 168
6.2.43 ORISE Method API: Gross Alpha and Beta for Various Matrices 169
6.2.44 ORISE Method AP2: Determination of Tritium 170
6.2.45 ORISE Method AP5: Determination of Technetium-99 171
6.2.46 ORISE Method AP7: Determination of Radium-226 in Water and Soil Samples Using
Alpha Spectroscopy 171
6.2.47 ORISE Method API 1: Sequential Determination of the Actinides in Environmental
Samples Using Total Sample Dissolution and Extraction Chromatography 172
6.2.48 ORISE Method Procedure #9: Determination of 1-125 in Environmental Samples 173
6.2.49 ASTM Method D3084-05 (2012): Standard Practice for Alpha Spectrometry in Water.... 174
6.2.50 ASTM Method D3972-09 (2015): Standard Test Method for Isotopic Uranium in
Water by Radiochemistry 175
6.2.51 ASTM Method D5811-08 (2013): Standard Test Method for Strontium-90 in Water 175
6.2.52 ASTM Method D7168-16: Standard Test Method for Technetium-99 in Water by
Solid Phase Extraction Disk 176
6.2.53 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total,
Suspended, and Dissolved) 177
6.2.54 Standard Method 7120: Gamma-Emitting Radionuclides 178
6.2.55 Standard Method 7500-Ra B: Radium: Precipitation Method 179
6.2.56 Standard Method 7500-Ra C: Radium: Emanation Method 179
6.2.57 Standard Method 7500-U B: Uranium: Radiochemical Method 180
6.2.58 Standard Method 7500-U C: Uranium: Isotopic Method 181
6.2.59 Y-12 (DOE) Preparation of Samples for Total Activity Screening 182
6.3 Method Summaries (Outdoor Infrastructure and Building Material Samples) 183
6.3.1 Rapid Radiochemical Method for Total Radiostrontium (Sr-90) In Building Materials
for Environmental Remediation Following Radiological Incidents 183
6.3.2 Rapid Radiochemical Method for Radium-226 in Building Materials for
Environmental Remediation Following Radiological Incidents 184
6.3.3 Rapid Method for Sodium Hydroxide Fusion of Concrete and Brick Matrices Prior to
Americium, Plutonium, Strontium, Radium, and Uranium Analyses for Environmental
Remediation Following Radiological Incidents 185
6.3.4 Rapid Radiochemical Method for Isotopic Uranium in Building Materials for
Environmental Remediation Following Radiological Incidents 187
6.3.5 Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Building
Materials for Environmental Remediation Following Radiological Incidents 188
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6.3.6 Rapid Radiochemical Method for Americium-241 in Building Materials for
Environmental Remediation Following Radiological Incidents 189
6.3.7 Rapid Method for Sodium Hydroxide Fusion of Asphalt Matrices Prior to Americium,
Plutonium, Strontium, Radium, and Uranium Analyses 190
6.3.8 Rapid Method for Sodium Hydroxide Fusion of Asphalt Roofing Material Matrices
Prior to Americium, Plutonium, Strontium, Radium, and Uranium Analyses 191
Section 7.0: Selected Pathogen Methods 194
7.1 General Guidelines 198
7.1.1 Standard Operating Procedures for Identifying Pathogen Methods 198
7.1.2 General QC Guidelines for Pathogen Methods 201
7.1.3 Safety and Waste Management 202
7.1.4 Laboratory Response Network (LRN) 203
7.2 Method Summaries for Bacteria 205
7.2.1 Bacillus anthracis [Anthrax] - BSL-3 205
7.2.2 Brucella spp. [Brucellosis] - BSL-3 208
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderiapseudomallei [Melioidosis]
-BSL-3 211
7.2.4 Campylobacter jejuni [Campylobacteriosis] - BSL-2 213
7.2.5 Chlamydophilapsittaci [Psittacosis] (formerly known as Chlamydiapsittaci) - BSL-2;
BSL-3 for Aerosols and Large Volumes 216
7.2.6 Coxiella burnetii [Q-fever] - BSL-3 218
7.2.7 Escherichia coli 0157:H7 - BSL-2 221
7.2.8 Francisella tularensis [Tularemia] - BSL-3 223
7.2.9 Legionella pneumophila - BSL-2 226
7.2.10 Leptospira interrogans [Leptospirosis] - BSL-2 228
7.2.11 Listeria monocytogenes [Listeriosis] - BSL-2 230
7.2.12 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2 233
7.2.13 Salmonella enterica serovar Typhi (S. Typhi) [Typhoid fever] - BSL-2; BSL-3 for
Aerosol Release 235
7.2.14 Shigella spp. [Shigellosis] - BSL-2 238
7.2.15 Staphylococcus aureus - BSL-2 240
7.2.16 Vibrio cholerae [Cholera] - BSL-2 242
7.2.17 Yersiniapestis [Plague] - BSL-3 245
7.3 Method Summaries for Viruses 248
7.3.1 Adenoviruses: Enteric and Non-enteric (A-F) - BSL-2 248
7.3.2 Astroviruses - BSL not specified 251
7.3.3 Caliciviruses: Noroviruses - BSL-2 254
7.3.4 Caliciviruses: Sapovirus - BSL-2 255
7.3.5 Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human
Coronavirus (HCoV) - BSL-2; BSL-3 for Propagation 258
7.3.6 Hepatitis E Virus (HEV) - BSL-2 261
7.3.7 Influenza H5N1 virus - BSL-3 264
7.3.8 Picornaviruses: Enteroviruses - BSL-2 267
7.3.9 Picornaviruses: Hepatitis A Virus (HAV) - BSL-2 270
7.3.10 Reoviruses: Rotavirus (Group A) - BSL-2 272
7.4 Method Summaries for Protozoa 276
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2 276
7.4.2 Entamoeba histolytica - BSL-2 282
7.4.3 Giardia spp. [Giardiasis] - BSL-2 284
7.4.4 Naegleria fowleri [Naegleriasis] - BSL-2 288
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7.4.5 Toxoplasma gondii [Toxoplasmosis] - BSL-2 291
7.5 Method Summaries for Helminths 294
7.5.1 Baylisascarisprocyonis [Raccoon roundworm fever] - BSL-2 294
Section 8.0: Selected Biotoxin Methods 298
8.1 General Guidelines 300
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods 300
8.1.2 General QC Guidelines for Biotoxin Methods 303
8.1.3 Safety and Waste Management 304
8.2 Method Summaries 305
8.2.1 Abrin/Abrine 305
8.2.2 Aflatoxin 308
8.2.3 a-Amanitin 309
8.2.4 Anatoxin-a 311
8.2.5 Botulinum Neurotoxins (BoNTs) 312
8.2.6 Brevetoxins (BTX) 317
8.2.7 Cylindrospermopsin 318
8.2.8 Diacetoxyscirpenol (DAS) 320
8.2.9 Domoic Acid (DA) 321
8.2.10 Microcystins 323
8.2.11 Picrotoxin 325
8.2.12 Ricin (Ricinine) 326
8.2.13 Saxitoxins 330
8.2.14 Shiga and Shiga-like Toxins (Stx) 333
8.2.15 Staphylococcal Enterotoxins (SETs) 335
8.2.16 T-2 Mycotoxin 337
8.2.17 Tetrodotoxin (TTX) 339
Section 9.0: Conclusions 341
Figures
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland Security Incidents 3
Figure 2-1. Method Selection Process 6
Figure 7-1. Sample Analysis During Site Characterization and Post Decontamination Phases
Following a Biological Contamination Event 195
Tables
Table 5-1. Chemical Methods and Corresponding Section Numbers 13
Table 5-2. Sources of Chemical Methods 26
Table 6-1. Radiochemical Methods and Corresponding Section Numbers 122
Table 6-2. Sources of Radiochemical Methods 127
Table 7-1. Sources of Pathogen Methods 199
Table 8-1. Sources of Biotoxin Methods 301
Appendices
Appendix A: Selected Chemical Methods A-l
Appendix Bl: Selected Radiochemical Methods for Environmental Samples B-l
Appendix B2: Selected Radiochemical Methods for Outdoor Building and Infrastructure Materials B-2
Appendix C: Selected Pathogen Methods C-l
Appendix D: Selected Biotoxin Methods D-l
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Table of Contents
Attachments
Attachment 1: Supporting Documents 1-1
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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-related incidents resulting in intentional or accidental
contamination. The need to improve the nation's laboratory capacity and capability to analyze
environmental samples following such incidents (i.e., chemical, biological and/or radiological [CBR]
contamination) was one of the most important areas identified. This need has proven to be an on-going
concern as the nation faces continued contamination incidents, such as the environmental contamination
introduced by the 2014 microcystins contamination of drinking water in Toledo, Ohio; the 2013 ricin
letter incident in Washington, D.C.; and the 2011 Fukushima power plant accident which threatened
contamination of the western coast of the United States.
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 Land and Emergency Management (OLEM, formerly
Office of Solid Waste and Emergency Management), Office of Environmental Information, Office of
Chemical Safety and Pollution Prevention, and several EPA regional offices.
A critical area identified by the Homeland Security Laboratory Capacity Work Group was the need for a
list of selected analytical methods to be used by all laboratories when analyzing contamination incident
samples and, in particular, when analysis of many samples is required over a short period of time. EPA's
Environmental Response Laboratory Network (ERLN) is a national network of laboratories that can be
accessed as needed to support responses to large-scale environmental contamination incidents by
providing analytical capabilities and data of consistent quality in a systematic, coordinated response. This
network includes the Water Laboratory Alliance (WLA), which can be accessed specifically for responses
pertaining to the nation's water sector.1 Using the same selected methods should 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 follow-up activities, including 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 preparedness and
response: (1) ongoing surveillance and monitoring; (2) response and rapid screening for determining
whether an incident 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)2 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
1 Information regarding EPA's Environmental Response Laboratory Network is available at:
https://www.epa.gov/emergencv-response/environmental-response-laboratorv-network.
Information regarding the Water Laboratory Alliance (WLA) is available at:
https://www.epa.gov/waterlabnetwork/learn-about-water-laboratorv-alliance
2 Formerly EPA's Standardized Analytical Methods for Environmental Restoration Following Homeland Security
Events. SAM and its methods are available at: https://www.epa.gov/homeland-securitv-research/sam
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Section 1 - Introduction
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.
In support of this document, EPA periodically assembles methods experts from within EPA, as well as
other federal, state and local agencies; and public utilities, to review and update SAM. Workgroup
members revise the methods listed or select new methods as appropriate, determine method tier
classifications, provide input into special considerations, and add analytes of interest as needed. 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, and toxicity). The sample types
listed are specific to each technical section and have been determined by technical work groups to be a
concern during site remediation. Work groups identify a single method or method group per
analyte/sample type to ensure a consistent analytical approach across multiple laboratories when
analyzing environmental samples.
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Section 1 - Introduction
Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland
Security Incidents
Surveillance and Monitoring
(if appropriate)
Immediate Response/
Credibility Determination
Preliminary Site
Characterization
Site Remediation
SAM
(Selected Analytical Methods for
Environmental Remediation and Recovery)
Assessment
Cleanup
Clearance
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Section 2 - Background
Section 2.0: Background
Method selection is based on consideration of specific criteria that emphasize method performance and
include existing laboratory capabilities, laboratory capacity, method applicability to multiple sample
types, and method applicability to multiple 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 work groups under the direction of EPA's Homeland Security
Research Program (HSRP). For analytes where limited laboratory capability exists, methods are selected
based on their applicability to similar analytes. In these cases, laboratory studies to evaluate the ability of
the selected method to measure the target analyte(s) are either underway or needed. Method usability tiers
are assigned to provide an indication of the applicability of the methods that have been selected to address
the chemical, pathogen and biotoxin analytes (i.e., the extent to which the methods have been tested and
applied for analysis of the specific analyte and sample type(s) for which they have been selected).
Figure 2-1 summarizes steps and provides the criteria used during the 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 when selecting methods.
Since 2004, the National Homeland Security Research Center (NHSRC) has brought together experts
from across EPA and other interested 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-related contamination incidents. Participants have included
representatives from EPA program offices, regions, and laboratories; U.S. 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 Energy (DOE),
Department of Agriculture (USDA), Geological Survey (USGS) and Department of Commerce (DOC);
and numerous state agencies, municipalities and universities. Methods and protocols are considered for
chemical, radiochemical, biological and biotoxin agents of concern in the types of environmental samples
that would be anticipated, including outdoor building and infrastructure materials containing
radiochemical contamination. Work groups have also begun considering methods that might be needed to
address waste generated during site decontamination and the analytical impacts of decontamination
agents.
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
Standard Methods for the Examination of Water and Wastewater (SM)
Scientific Literature
The primary objective of this effort is to support EPA's ERLN and WLA by identifying appropriate
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Section 2 - Background
methods that represent a balance between providing existing, documented techniques and providing
consistent and analytical results of known quality.
Since publication of Revision 1.0 of this document in September 2004, NHSRC has continued to convene
technical work groups to evaluate and update the analytes and methods that are listed. Details regarding
changes that have been incorporated into each revision are provided in Attachment 1. The 2012 revision
also reflects a title change agreed to by stakeholders (i.e., EPA's HSRP; Office of Groundwater and
Drinking Water [OGWDW], 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 Summit, to better reflect its focus on providing selected analytical methods for use across multiple
laboratories during environmental remediation and recovery. The current revision (2017) includes the
addition of outdoor building and infrastructure materials as sample types in the radiochemistry sections;
the addition of soil as a sample type in the pathogens sections; the assignment of method applicability
tiers for methods selected for pathogen and biotoxin analytes; several new methods added or replaced for
chemical, radiochemical, pathogen and biotoxin analytes; the addition of several new analytes to each of
these technical sections (chemistry, radiochemistry, pathogens and biotoxins); and considerations
regarding the potential impacts of decontamination agents on the analytical performance of selected
radiochemical methods.
In addition to updating analytes and methods, work groups identified areas for development of companion
documents to provide information regarding sample collection, rapid screening and preliminary analysis
equipment, and sample disposal to supplement the selected analytical methods. The information listed in
the companion documents generally corresponds to the analytes and methods in this document, and are
updated as needed and as resources allow. Currently available companion documents are listed below
and, with content descriptions, in Attachment 1.
Sample Collection Information Document for Chemicals, Radiochemicals and Biotoxins -
Companion to SAM 2017
Sample Collection Information Document for Pathogens - Companion to SAM 2017
Rapid Screening and Preliminary Identification Techniques and Methods - Companion to SAM
Revision 5.0
Laboratory Environmental Sample Disposal Information Document - Companion to SAM Revision
5.0
Guide for Development of Sample Collection Plans for Radiochemical Analytes in Environmental
Matrices Following Homeland Security Events
Sample Collection Procedures for Radiochemistry Analytes in Environmental Matrices.
Sample Collection Procedures for Radiochemistry Analytes in Outdoor Building and Infrastructure
Materials
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Section 2 - Background
Figure 2-1. Method Selection Process
Step 1
Is there an EPA
published method for
measurement of the
analyte in the sample
type of interest?
YES-
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
interest?
Is there an EPA
federal, or VCSB method
that has been developed for
measurement of the analyte
in another environmental
sample type?
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?
NO
Repeat Steps 1 - 4 to identify methods that measure
analytes similar to the analyte of concern
Evaluate method against
selection criteria
Use the following criteria as guidelines to assess which method is most
appropriate for inclusion:
Has the method been issued by a government agency or consensus body
(e.g., ASTM, Standard Methods)?
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 significantly higher than for other comparable
methods?
Has the method already been selected for other analytes?
Are modifications needed to accommodate the analytes or sample types?
Select method
If no methods are available, prioritize
for further research
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Section 3 - Scope and Application
Section 3.0: Scope and Application
The premise and purpose of this document is to identify the analytical methods that will be used in cases
when multiple laboratories are called on to analyze environmental samples following a homeland
security-related intentional or accidental contamination incident. 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 and outdoor building material samples containing radiochemical
contamination following such incidents. 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 and in outdoor building
materials containing radiochemical contamination. The majority of methods include detailed laboratory
procedures for confirming the identification of analytes and determining their concentrations in samples
and, 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.
Instead, NHSRC has developed companion documents that are intended to provide information regarding
sample collection, laboratory rapid screening/preliminary identification equipment, and sample disposal.
Currently available 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 and 6.3 (radiochemical
methods), 7.2 (pathogen methods) and 8.2 (biotoxin methods).
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 and outdoor building material samples in support of
EPA remediation and recovery efforts following a homeland security-related contamination incident. The
information 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
(https://www.epa.gov/homeland-securitv-research/sam). which provides searchable links to supporting
information based on the analytes and analytical methods listed.
At this time, only some of the methods selected have been validated for the analyte and sample type.
However, the methods are considered to contain the most appropriate currently available techniques based
on expert judgment. Method usability tiers are assigned to provide an indication of the applicability of the
methods that have been selected to address the chemical, pathogen and biotoxin analytes (i.e., the extent
to which the methods have been tested and applied for analysis of the specific analyte and sample type for
which they have been selected). Unless a published method 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 to the procedures described in the method may be required to accurately account
for variations in analyte/sample type characteristics, environmental samples, analytical interferences, and
data quality objectives (DQOs). Where further development and testing are necessary, EPA is continuing
to develop and evaluate analytical techniques based on the methods and protocols that are listed in this
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Section 3 - Scope and Application
document and based on current EPA policies for validating analytical methods. Once validation is
complete, data regarding the resulting method performance and DQOs will be available.
EPA recognizes that selection of a single method might limit laboratory capability and affect laboratory
capacity when techniques different from those provided in the methods are required for the analysis of
difficult samples. 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 (see
list of contacts in Section 4.0). Additional information is also provided in the Agency Policy Directive
Number FEM-2010-01.3 NHSRC has also compiled information and published documents regarding field
screening equipment, sample collection materials, rapid screening/preliminary identification equipment,
and disposal of samples. These documents are available at https://www.epa.gov/homeland-securitv-
research/sam-companion-documents-and-sample-collection-procedures.
EPA plans to continue to update the 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 periodically provides addenda to provide updates regarding methods,
information and issues that are not addressed by the most current versions of this document. Information
regarding the use of deviations from the selected methods is provided in Section 4.0.
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 contamination incident. EPA
recognizes that this advanced selection of such methods may pose potential risks, including the following:
Selecting technologies that may not be the most cost-effective technologies currently available for
addressing the particular situation at hand;
Selecting methodologies that may not be appropriate for use in responding to a particular incident
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 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 and protocols, 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 site-
3 U.S. EPA, Forum on 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. https ://www .epa. gov/sites/production/files/2015-
01/documents/emergencv response validity policv.pdf
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Section 3 - Scope and Application
specific DQOs for each response activity.4 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 intentional or
unintentional contamination incidents, 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:
https://www.epa.gov/sites/production/files/documents/erln lab requirements.pdf; project-specific
requirements also are included in individual Analytical Service Requests (ASRs).
4 Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/OUALITY/daos.html.
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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. As previously indicated, any deviations from the recommended method(s) should be reported
immediately to ensure data comparability is maintained when responding to intentional or unintentional
contamination incidents. In cases where laboratories are specifically tasked by EPA to use these methods
following an incident, 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 (https://www.epa.gov/homeland-securitv-research/sam').
General
Romy Campisano - Primary
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513) 569-7016 campisano.romvfSlepa.aov
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.kathvฎ,epa.aov
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(2),epa.aov
Stuart Willison - Alternate
National Homeland Security Research Center
U.S. EPA ORD (NG16)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513) 569-7253 willison.stuartฎ,epa.aov
Radiochemical Methods
John Griggs - Primary
U.S. EPA National Analytical Radiation
Environmental Laboratory, ORIA
540 South Morris Avenue
Montgomery, AL 36115-2601
(334) 270-3401 ariaas.iohnฎ,epa.aov
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.kathvฎ, epa.aov
Pathogen Methods
Laura Boczek - Primary
National Risk Management Research Laboratory
U.S. EPA ORD (681)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513) 569-7282 boczek.lauraiฎ,epa.aov
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.erinฎ,epa.aov
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 maanuson.matthew(ฎepa.aov
Heath Mash - Alternate
National Risk Management Research Laboratory
U.S. EPA ORD (681)
26 West Martin Luther King Jr. Drive
Cincinnati, OH 45268
(513) 569-7713 mash.heath(5),epa.aov
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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 contamination incident. 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. In some cases, procedures from peer-reviewed
journal articles or provisional methods are listed for those analyte-sample type combinations where
validated methods are unavailable. In these instances, the best available procedure was selected based on
its environmental application and on data quality objectives (DQOs). Appendix A includes method
usability tiers that have been assigned to each method to indicate its applicability to the specific analyte-
sample type combination(s) for which it has been selected. These tiers are described in Section 5.1.1
below, and are defined on the first page of Appendix A. As appropriate, when fully validated methods
become available, the literature references and alternative methods 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 4.0.
Appendix A is sorted alphabetically by analyte and includes the following information:
Analyte(s). The component, contaminant or constituent of interest.
Chemical Abstracts Service Registry Number (CAS RNฎ [Chemical Abstracts Service, Columbus,
OH]). A unique identifier for chemical substances that provides an unambiguous way to identify a
chemical or molecular structure when there are many possible systematic, generic or trivial names.
Determinative technique. An analytical instrument or technique used to determine the quantity and
identification of compounds or components in a sample.
Method type. Two method types (sample preparation and determinative) are used to complete
sample analysis. In some cases, a single method contains information for both sample preparation and
determinative procedures. In most instances, however, two separate methods may need to be used in
conjunction.
Solid samples. The recommended method / procedure to identify and measure the analyte of interest
in solid phase samples.
Non-drinking water samples. The recommended method / procedure to identify and measure the
analyte of interest in aqueous liquid phase samples other than drinking water.
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 an environmental contamination incident, 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 incident-specific goals, investigation of
background levels using methods listed in Appendix A is recommended.
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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://www.srcinc.com/what-we-
do/environmental/scientific-databases.html') contains information pertaining to chemical structures,
names, physical properties and persistence. PHYSPROP is sponsored by the U.S. Environmental
Protection Agency (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) (https://www.epa.gov/waterdata/water-
contarninant-information-tool-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 (https://ec.europa.eu/i rc/cn)
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.
Chemical Safety Data Sheets (http://www.ilpi.com/msds/).
Additional research on chemical contaminants is ongoing within EPA. Databases to manage this
information are currently under development and will be updated when additional information becomes
available.
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, 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 DQOs for a particular environmental
remediation activity. These selected chemical methods have been assigned tiers (below) to indicate a level
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Section 5.0 - Selected Chemical Methods
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 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 (e.g., due to potential interferences, alternate matrices, the need to address different
DQOs).
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 5.2.116 below provide summaries of the sample preparation and determinative
methods listed in Appendix A.
Table 5-1. Chemical Methods and Corresponding Section Numbers
Analyte
CAS RN
Method
Section
Ace p hate
30560-19-1
538 (EPA OW)
5.2.11
Chromatographia (2006) 63(5/6): 233-237
5.2.103
J. Chromatogr. A (2007) 1154(1): 3-25
5.2.109
Acrylamide
79-06-1
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8316 (EPA SW-846)
5.2.38
PV2004 (OSHA)
5.2.84
Acrylonitrile
107-13-1
524.2 (EPA OW)
5.2.7
3570 (EPA SW-846)
5.2.24
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
8290A Appendix A (EPA SW-846)
5.2.36
PV2004 (OSHA)
5.2.84
Aldicarb (Temik)
Aldicarb sulfone
Aldicarb sulfoxide
116-06-3
1646-88-4
1646-87-3
531.2 (EPA OW)
5.2.10
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8318A (EPA SW-846)
5.2.39
5601 (NIOSH)
5.2.65
D7645-16 (ASTM)
5.2.92
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
5030C (EPA SW-846)
5.2.25
Allyl alcohol
107-18-6
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
TO-15 (EPA ORD)
5.2.48
3535A (EPA SW-846)
5.2.21
4-Aminopyridine
504-24-5
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
350.1 (EPAOW)
5.2.6
Ammonia
7664-41-7
6015 (NIOSH)
5.2.70
4500-NH3 B (SM)
5.2.98
4500-NH3 G (SM)
5.2.99
200.7 (EPAOW)
5.2.1
200.8 (EPAOW)
5.2.2
3015A (EPA SW-846)
5.2.16
Ammonium metavanadate (analyze
as total vanadium)
7803-55-6
3050B (EPA SW-846)
5.2.17
3051A (EPA SW-846)
5.2.18
Arsenic, Total
7440-38-2
6010D (EPA SW-846)
5.2.27
Arsenic trioxide (analyze as total
arsenic)
1327-53-3
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
200.7 (EPAOW)
5.2.1
200.8 (EPAOW)
5.2.2
3015A (EPA SW-846)
5.2.16
Arsine (analyze as total arsenic in
non-air samples)
3050B (EPA SW-846)
5.2.17
7784-42-1
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
6001 (NIOSH)
5.2.66
9102 (NIOSH)
5.2.75
D5755-09 (ASTM)
5.2.86
Asbestos
1332-21-4
D6480-05 (ASTM)
5.2.87
10312:1995 (ISO)
5.2.96
Boron trifluoride
7637-07-2
ID216SG (OSHA)
5.2.83
3541 (EPA SW-846)
5.2.22
Brodifacoum
56073-10-0
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
Bromadiolone
28772-56-7
8290A Appendix A (EPA SW-846)
5.2.36
D7644-16 (ASTM)
5.2.91
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
BZ [Quinuclidinyl benzilate]
6581-06-2
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
TO-10A (EPA ORD)
5.2.47
J. Chromatogr. B (2008) 874: 42-50
5.2.112
200.7 (EPA OW)
5.2.1
200.8 (EPA OW)
5.2.2
3015A (EPA SW-846)
5.2.16
3050B (EPA SW-846)
5.2.17
Calcium arsenate (analyze as total
arsenic)
3051A (EPA SW-846)
5.2.18
7778-44-1
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
531.2 (EPA OW)
5.2.10
3570 (EPA SW-846)
5.2.24
Carbofuran (Furadan)
1563-66-2
8290A Appendix A (EPA SW-846)
5.2.36
8318A (EPA SW-846)
5.2.39
5601 (NIOSH)
5.2.65
D7645-16 (ASTM)
5.2.92
524.2 (EPA OW)
5.2.7
5030C (EPA SW-846)
5.2.25
Carbon disulfide
75-15-0
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
TO-15 (EPA ORD)
5.2.48
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
Carfentanil
59708-52-0
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
J. Chromatogr. B (2014) 962: 52-58
5.2.115
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
Chlorfenvinphos
470-90-6
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Chlorine
7782-50-5
4500-CI G (SM)
5.2.100
Analyst (1999) 124(12): 1853-1857
5.2.101
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
5030C (EPA SW-846)
5.2.25
2-Chloroethanol
107-07-3
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
2513 (NIOSH)
5.2.61
TO-10A (EPA ORD)
5.2.47
3-Chloro-1,2-propanediol
96-24-2
Eur. J. Lipid Sci. Technol. (2011) 113: 345-
355
5.2.105
J. Chromatogr. A (2000) 866: 65-77
5.2.107
551.1 (EPAOW)
5.2.14
Chloropicrin
76-06-2
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
PV2103 (OSHA)
5.2.85
Chlorosarin
1445-76-7
TO-17 (EPA ORD)
5.2.49
Chlorosoman
7040-57-5
EPA/600/R-16/115
5.2.56
Analyze as CVAA and CVAOA
EPA/600/R-15/258 (EPA NHSRC)
5.2.54
Analyze as total arsenic
200.7 (EPAOW)
5.2.1
200.8 (EPAOW)
5.2.2
3015A (EPA SW-846)
5.2.16
2-Chlorovinylarsonic acid (CVAOA)
64038-44-4
3050B (EPA SW-846)
5.2.17
2-Chlorovinylarsonous acid (CVAA)
85090-33-1
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
525.2 (EPA OW)
5.2.8
Chlorpyrifos
2921-88-2
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
540 (EPA OW)
5.2.12
Chlorpyrifos oxon
5598-15-2
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Crimidine
535-89-7
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Cyanide, Amenable to chlorination
NA
RLAB Method 3135.21 (EPA Region 7)
5.2.42
4500-CN G (SM)
5.2.97
335.4 (EPA OW)
5.2.5
Cyanide, Total
57-12-5
ISM02.3 CN (EPA CLP)
5.2.41
6010 (NIOSH)
5.2.68
TO-15 (EPA ORD)
5.2.48
Cyanogen chloride
506-77-4
Encyclopedia of Anal. Chem. (2006)
DOM 0.1002/9780470027318.a0809
5.2.104
Cyclohexyl sarin (GF)
329-99-7
TO-17 (EPA ORD)
5.2.49
EPA/600/R-16/115 (EPA NHSRC)
5.2.56
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
1,2-Dichloroethane
107-06-2
524.2 (EPA OW)
5.2.7
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
TO-15 (EPA ORD)
5.2.48
Dichlorvos
62-73-7
525.2 (EPA OW)
5.2.8
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Dicrotophos
141-66-2
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Diesel range organics
NA
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8015D (EPA SW-846)
5.2.33
8290A Appendix A (EPA SW-846)
5.2.36
Diisopropyl methylphosphonate
(DIMP)
1445-75-6
538 (EPA OW)
5.2.11
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Dimethylphosphite
868-85-9
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
TO-10A (EPA ORD)
5.2.47
Dimethylphosphoramidic acid
33876-51-6
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Diphacinone
82-66-6
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
D7644-16 (ASTM)
5.2.91
Disulfoton
Disulfoton sulfone oxon
Disulfoton sulfoxide
Disulfoton sulfoxide oxon
298-04-4
2496-91-5
2497-07-6
2496-92-6
525.2 (EPA OW)
5.2.8
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
5600 (NIOSH)
5.2.64
1,4-Dithiane
505-29-3
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
EA2192 [S-2-(diisopropylamino)ethyl
methylphosphonothioic acid]
73207-98-4
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
TO-10A (EPA ORD)
5.2.47
EPA/600/R-15/097 (EPA NHSRC)
5.2.53
Ethyl methylphosphonic acid (EMPA)
1832-53-7
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Ethyldichloroarsine (ED)
598-14-1
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
8270E (EPA SW-846)
5.2.35
TO-15 (EPA ORD)
5.2.48
9102 (NIOSH)
5.2.75
N-Ethyldiethanolamine (EDEA)
139-87-7
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
EPA/600/R-11/143 (EPA NHSRC / CDC)
5.2.50
3509 (NIOSH)
5.2.62
D7599-16 (ASTM)
5.2.90
Ethylene oxide
75-21-8
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
TO-15 (EPA ORD)
5.2.48
Fenamiphos
22224-92-6
525.2 (EPA OW)
5.2.8
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Fentanyl
437-38-7
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
J. Chromatogr. A (2011) 1218: 1620-1649
5.2.111
Fluoride
16984-48-8
300.1, Rev 1.0 (EPAOW)
5.2.4
Fluoroacetamide
640-19-7
J. Chromatogr. B (2008) 876(1): 103-108
5.2.113
Fluoroacetic acid and fluoroacetate
salts
NA
S301-1 (NIOSH)
5.2.78
J. Chromatogr. A (2007) 1139: 271-278
5.2.108
J. Chromatogr. B. (2010) 878: 1045-1050
5.2.114
2-Fluoroethanol
371-62-0
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
2513 (NIOSH)
5.2.61
Fluorosilicic acid (analyze as fluoride)
16961-83-4
300.1, Rev 1.0 (EPAOW)
5.2.4
SAM 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
Formaldehyde
50-00-0
556.1 (EPAOW)
5.2.15
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8315A (EPA SW-846)
5.2.37
2016 (NIOSH)
5.2.60
Gasoline range organics
NA
3570 (EPA SW-846)
5.2.24
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8015D (EPA SW-846)
5.2.33
8290A Appendix A (EPA SW-846)
5.2.36
H exa hy d ro-1,3,5-t ri n itro-1,3,5-tri azi ne
(RDX)
121-82-4
3535A (EPA SW-846)
5.2.21
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
Hexamethylenetriperoxidediamine
(HMTD)
283-66-9
3535A (EPA SW-846)
5.2.21
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
Analyst (2001) 126: 1689-1693
5.2.102
Hydrogen bromide
Hydrogen chloride
10035-10-6
7647-01-0
7907 (NIOSH)
5.2.74
Hydrogen cyanide
74-90-8
6010 (NIOSH)
5.2.68
Hydrogen fluoride
7664-39-3
7906 (NIOSH)
5.2.73
Hydrogen sulfide
2148878
6013 (NIOSH)
5.2.69
Isopropyl methylphosphonic acid
(IMPA)
1832-54-8
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Kerosene
64742-81-0
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8015D (EPA SW-846)
5.2.33
8290A Appendix A (EPA SW-846)
5.2.36
Lead arsenate (analyze as total
arsenic)
7645-25-2
200.7 (EPAOW)
5.2.1
200.8 (EPAOW)
5.2.2
3015A (EPA SW-846)
5.2.16
3050B (EPA SW-846)
5.2.17
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
Analyze as lewisite I, 2, 3 or lewisite oxide
EPA/600/R-15/258 (EPA NHSRC)
5.2.54
Analyze as total arsenic
Lewisite 1 (L-1)
200.7 (EPA OW)
5.2.1
[2-chlorovinyldichloroarsine]
541-25-3
200.8 (EPA OW)
5.2.2
Lewisite 2 (L-2)
[bis(2-chlorovinyl)chloroarsine]
3015A (EPA SW-846)
5.2.16
40334-69-8
3050B (EPA SW-846)
5.2.17
Lewisite 3 (L-3)
[tris(2-chlorovinyl)arsine]
40334-70-1
3051A (EPA SW-846)
5.2.18
1306-02-1
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
Lewisite oxide
IO-3.1 (EPAORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
Mercuric chloride (analyze as total
mercury)
245.1 (EPAOW)
5.2.3
7487-94-7
7473 (EPA SW-846)
5.2.31
9102 (NIOSH)
5.2.75
245.1 (EPAOW)
5.2.3
Mercury, Total
7439-97-6
7473 (EPA SW-846)
5.2.31
IO-5 (EPA ORD)
5.2.46
9102 (NIOSH)
5.2.75
538 (EPA OW)
5.2.11
Methamidophos
10265-92-6
Chromatographia (2006) 63(5/6): 233-237
5.2.103
J. Chromatogr. A (2007) 1154(1): 3-25
5.2.109
531.2 (EPAOW)
5.2.10
3570 (EPA SW-846)
5.2.24
Methomyl
16752-77-5
8290A Appendix A (EPA SW-846)
5.2.36
8318A (EPA SW-846)
5.2.39
5601 (NIOSH)
5.2.65
D7645-16 (ASTM)
5.2.92
245.1 (EPAOW)
5.2.3
Methoxyethylmercuric acetate
151-38-2
7473 (EPA SW-846)
5.2.31
(analyze as total mercury)
IO-5 (EPA ORD)
5.2.46
9102 (NIOSH)
5.2.75
524.2 (EPA OW)
5.2.7
3570 (EPA SW-846)
5.2.24
Methyl acrylonitrile
126-98-7
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
8290A Appendix A (EPA SW-846)
5.2.36
PV2004 (OSHA)
5.2.84
SAM 2017
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September 2017
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
3-Methyl fentanyl
42045-87-4
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
J. Chromatogr. B (2014) 962: 52-58
5.2.115
Methyl fluoroacetate (analyze as
fluoroacetate ion)
453-18-9
S301-1 (NIOSH)
5.2.78
J. Chromatogr. A (2007) 1139: 271-278
5.2.108
J. Chromatogr. B (2010)878: 1045-1050
5.2.114
Methyl hydrazine
60-34-4
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
3510 (NIOSH)
5.2.63
J. Chromatogr. (1993)617: 157-162
5.2.106
Methyl isocyanate
624-83-9
OSHA 54
5.2.80
Methyl paraoxon
Methyl parathion
950-35-6
298-00-0
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Methylamine
74-89-5
OSHA 40
5.2.79
N-Methyldiethanolamine (MDEA)
105-59-9
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
EPA/600/R-11/143 (EPA NHSRC / CDC)
5.2.50
3509 (NIOSH)
5.2.62
D7599-16 (ASTM)
5.2.90
1 -Methylethyl ester
ethylphosphonofluoridic acid (GE)
1189-87-3
TO-17 (EPA ORD)
5.2.49
EPA/600/R-16/115 (EPA NHSRC)
5.2.56
Methylphosphonic acid (MPA)
993-13-5
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Mevinphos
7786-34-7
525.2 (EPA OW)
5.2.8
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Monocrotophos
6923-22-4
3535A (EPA SW-846)
5.2.21
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8270E (EPA SW-846)
5.2.35
8290A Appendix A (EPA SW-846)
5.2.36
TO-10A (EPA ORD)
5.2.47
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
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]
538-07-8
51-75-2
555-77-1
TO-17 (EPA ORD)
5.2.49
EPA/600/R-12/653 (EPA NHSRC)
5.2.51
Mustard, sulfur / Mustard gas (HD)
505-60-2
TO-17 (EPA ORD)
5.2.49
EPA/600/R-16/115 (EPA NHSRC)
5.2.56
Nicotine compounds
54-11-5
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Octahydro-1,3,5,7-tetranitro-1,3,5,7-
tetrazocine (HMX)
2691-41-0
3535A (EPA SW-846)
5.2.21
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
Osmium tetroxide (analyze as total
osmium)
20816-12-0
3015A (EPA SW-846)
5.2.16
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
Oxamyl
23135-22-0
531.2 (EPA OW)
5.2.10
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8318A (EPA SW-846)
5.2.39
5601 (NIOSH)
5.2.65
D7645-16 (ASTM)
5.2.92
Paraoxon
311-45-5
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Paraquat
4685-14-7
549.2 (EPA OW)
5.2.13
J. Chromatogr. A (2008) 1196-97: 110-116
5.2.110
Parathion
56-38-2
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Pentaerythritol tetranitrate (PETN)
78-11-5
3535A (EPA SW-846)
5.2.21
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
Phencyclidine
77-10-1
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
9106 (NIOSH)
5.2.76
9109 (NIOSH)
5.2.77
Phorate
298-02-2
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Phorate sulfone
Phorate sulfone oxon
Phorate sulfoxide
Phorate sulfoxide oxon
2588-04-7
2588-06-9
2588-03-6
2588-05-8
540 (EPA OW)
5.2.12
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Phosgene
75-44-5
OSHA61
5.2.81
Phosphamidon
13171-21-6
525.3 (EPA OW)
5.2.9
3520C (EPA SW-846)
5.2.20
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
TO-10A (EPA ORD)
5.2.47
Phosphine
7803-51-2
6002 (NIOSH)
5.2.67
Phosphorus trichloride
2125683
6402 (NIOSH)
5.2.71
Pinacolyl methyl phosphonic acid
(PMPA)
616-52-4
TO-10A (EPA ORD)
5.2.47
EPA/600/R-13/224 (EPA NHSRC)
5.2.52
D7597-16 (ASTM)
5.2.88
E-2866-12 (ASTM)
5.2.95
Propylene oxide
75-56-9
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
1612 (NIOSH)
5.2.59
R 33 (VR) [methylphosphonothioic
acid, S-[2-(diethylamino)ethyl] 0-2-
methylpropyl ester]
159939-87-4
TO-17 (EPA ORD)
5.2.49
EPA/600/R-12/653 (EPA NHSRC)
5.2.51
Sarin (GB)
Soman (GD)
107-44-8
96-64-0
TO-17 (EPA ORD)
5.2.49
EPA/600/R-16/115 (EPA NHSRC)
5.2.56
Sodium arsenite (analyze as total
arsenic)
7784-46-5
200.7 (EPA OW)
5.2.1
200.8 (EPA OW)
5.2.2
3015A (EPA SW-846)
5.2.16
3050B (EPA SW-846)
5.2.17
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
Sodium azide (analyze as azide ion)
26628-22-8
300.1, Rev 1.0 (EPA OW)
5.2.4
ID-211 (OSHA)
5.2.82
J. Forensic Sci. (1998) 43(1): 200-202
5.2.116
Strychnine
57-24-9
3535A (EPA SW-846)
5.2.21
8270E (EPA SW-846)
5.2.35
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Tabun (GA)
77-81-6
TO-17 (EPA ORD)
5.2.49
EPA/600/R-12/653 (EPA NHSRC)
5.2.51
Tetraethyl pyrophosphate (TEPP)
107-49-3
3511 (EPA SW-846)
5.2.19
8270E (EPA SW-846)
5.2.35
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Tetramethylenedisulfotetramine
(TETS)
80-12-6
TO-10A (EPA ORD)
5.2.47
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Thallium sulfate (analyze as total
thallium)
10031-59-1
200.7 (EPA OW)
5.2.1
200.8 (EPA OW)
5.2.2
3015A (EPA SW-846)
5.2.16
3050B (EPA SW-846)
5.2.17
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
Thiodiglycol (TDG)
111-48-8
TO-10A (EPA ORD)
5.2.47
D7598-16 (ASTM)
5.2.89
E2787-11 (ASTM)
5.2.93
E2838-11 (ASTM)
5.2.94
Thiofanox
39196-18-4
538 (EPA OW)
5.2.11
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
3570 (EPA SW-846)
5.2.24
8290A Appendix A (EPA SW-846)
5.2.36
5601 (NIOSH)
5.2.65
D7645-16 (ASTM)
5.2.92
1,4-Thioxane
15980-15-1
EPA/600/R-16/114 (EPA NHSRC)
5.2.55
Titanium tetrachloride (analyze as
total titanium)
7550-45-0
3051A (EPA SW-846)
5.2.18
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
Triethanolamine (TEA)
102-71-6
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
EPA/600/R-11/143 (EPA NHSRC / CDC)
5.2.50
3509 (NIOSH)
5.2.62
D7599-16 (ASTM)
5.2.90
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
3541 (EPA SW-846)
5.2.22
3545A (EPA SW-846)
5.2.23
Trimethyl phosphite
121-45-9
3570 (EPA SW-846)
5.2.24
8270E (EPA SW-846)
5.2.35
8290A Appendix A (EPA SW-846)
5.2.36
TO-10A (EPA ORD)
5.2.47
3535A (EPA SW-846)
5.2.21
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
3570 (EPA SW-846)
5.2.24
2,4,6-Trinitrotoluene (2,4,6-TNT)
118-96-7
8290A Appendix A (EPA SW-846)
5.2.36
8330B (EPA SW-846)
5.2.40
200.7 (EPA OW)
5.2.1
200.8 (EPA OW)
5.2.2
3015A (EPA SW-846)
5.2.16
3050B (EPA SW-846)
5.2.17
Vanadium pentoxide (analyze as total
vanadium)
3051A (EPA SW-846)
5.2.18
1314-62-1
6010D (EPA SW-846)
5.2.27
6020B (EPA SW-846)
5.2.28
IO-3.1 (EPA ORD)
5.2.43
IO-3.4 (EPA ORD)
5.2.44
IO-3.5 (EPA ORD)
5.2.45
9102 (NIOSH)
5.2.75
VE [phosphonothioic acid, ethyl-, S-
(2-(diethylamino)ethyl) O-ethyl ester]
VG [phosphonothioic acid, S-(2-
(diethylamino)ethyl) O.O-diethyl
ester]
21738-25-0
78-53-5
TO-17 (EPA ORD)
5.2.49
VM [phosphonothioic acid, methyl-,S-
(2-(diethylamino)ethyl) O-ethyl ester]
VX [0-ethyl-S-(2-
diisopropylaminoethyl) methyl-
phosphonothiolate]
21770-86-5
50782-69-9
EPA/600/R-16/116 (EPA NHSRC)
5.2.57
3570 (EPA SW-846)
5.2.24
White phosphorus
12185-10-3
7580 (EPA SW-846)
5.2.32
8290A Appendix A (EPA SW-846)
5.2.36
7905 (NIOSH)
5.2.72
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
107-18-6
TO-10A (EPA ORD)
5.2.47
3-Chloro-1,2-propanediol
96-24-2
TO-15 (EPA ORD)
5.2.48
Chlorosarin
Chlorosoman
1445-76-7
7040-57-5
TO-15 (EPA ORD)
5.2.48
Diisopropyl methylphosphonate
(DIMP)
1445-75-6
TO-15 (EPA ORD)
5.2.48
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Section 5.0 - Selected Chemical Methods
Analyte
CAS RN
Method
Section
Mercuric chloride (analyze as total
mercury)
Mercury, Total
7487-94-7
7439-97-6
7470A (EPA SW-846)
5.2.29
7471B (EPA SW-846)
5.2.30
Methamidophos
10265-92-6
5600 (NIOSH)
5.2.64
Methoxyethylmercuric acetate
(analyze as total mercury)
151-38-2
7470A (EPA SW-846)
5.2.29
7471B (EPA SW-846)
5.2.30
1 -Methylethyl ester
ethylphosphonofluoridic acid (GE)
1189-87-3
TO-15 (EPA ORD)
5.2.48
Sarin (GB)
Soman (GD)
107-44-8
96-64-0
TO-15 (EPA ORD)
5.2.48
1,4-Thioxane
15980-15-1
5030C (EPA SW-846)
5.2.25
5035A (EPA SW-846)
5.2.26
8260D (EPA SW-846)
5.2.34
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
Publisher
Reference
National Environmental Methods Index
(NEMI)
EPA, U.S. Geological
Survey (USGS)
http://www.nemi.qov
EPA Contract Laboratory Program
(CLP) Methods
EPA, CLP
http://www.epa.qov/superfund/proqrams
/clp/
EPA Office of Water (OW) Methods
EPAOW
https://www.epa.qov/dwanalvticalmetho
ds
EPA Solid Waste (SW)-846 Methods
EPA Office of Land and
Emergency Management
(OLEM)
https://www.epa.qov/hw-sw846/sw-846-
compendium
EPA Office of Research and
Development (ORD) Methods
EPA ORD
https://www.epa.qov/aboutepa/about-
office-research-and-development-ord
EPA Air Toxics Methods
EPA Office of Air and
Radiation (OAR)
http://www.epa.qov/ttn/amtic/airtox.html
EPA Analytical Protocols
EPA National Homeland
Security Research Center
(NHSRC)
https://www.epa.qov/homeland-securitv-
research/forms/contact-us-about-
homeland-securitv-research
Occupational Safety and Health
Administration (OSHA) Methods
OSHA
http://www.osha.qov/dts/sltc/methods/in
dex.html
NIOSH Methods
NIOSH
http://www.cdc.qov/niosh/nmam/
Standard Methods for the Examination
of Water and Wastewater (SM), 22nd
Edition, 2005*
American Public Health
Association (APHA)
http://www.standardmethods.orq
Annual Book of ASTM Standards*
ASTM International
http://www.astm.orq
International Organization for
Standardization (ISO) Methods*
ISO
http://www.iso.orq
Official Methods of Analysis of AOAC
International*
AOAC International
http://www.aoac.orq
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Section 5.0 - Selected Chemical Methods
Name
Publisher
Reference
Analyst*
Royal Society of Chemistry
httD://www.rsc.ora/Publishina/Journals/
AN/
Journal of Chromatography A and B*
Elsevier Science Publishers
httD://www.iournals.elsevier. com/iournal
-of-chromatoaraDhv-a/
Journal of Forensic Sciences*
ASTM International
https://www.astm.orq/DIGITAL LIBRAR
Y/JOURNALS/FORENSIC/index.html
Chromatographia*
Vieweg+Teubner
httD://www,SDrinaer. com/chemistrv/anal
vtical+chemistrv/iournal/10337
Encyclopedia of Analytical Chemistry*
Wiley
httD://www.wilev.com/WilevCDA/WilevTi
tle/DroductCd-0470973331 .html
European Journal of Lipid Science and
Technology*
Wiley
http://www.wNev-
vch.de/Dublish/en/iournals/alDhabeticlnd
ex/2114/
EPAWCIT
EPA OW Water Security
Division (WSD)
h tt p s: //www. e p a. q o v/wate rd at a/wat e r-
contaminant-information-tool-wcit
Analytical Chemistry*
American Chemical
Society(ACS)
http://pubs.acs.orq/iournal/ancham
* 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.5 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:
https://www.epa.gov/emergencv-response/environmental-response-laboratorv-network.
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
5 Information regarding EPA's DQO process, considerations, and planning is available at:
https://www.epa. gov/aualitv.
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Section 5.0 - Selected Chemical Methods
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
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
(https://www.epa.gov/compliance/good-laboratorv-practices-standards-compliance-monitoring-program)
and be run as frequently as necessary to ensure the reliability of analytical results. Additional guidance
can be found at: https://www.epa.gov/qualitv: in Chapter 1 of EPA SW-846 "Test Methods for Evaluating
Solid Waste, Physical/Chemical Methods"
(https://www.epa.gov/sites/production/files/2015-10/documents/chap 1 1 .pdf); and in EPA's 2005
"Manual for the Certification of Laboratories Analyzing Drinking Water" (EPA 815-R-05-004)
(https://nepis.epa.gov/Exe/ZvPDF.cgi/30006MXP.PDF?Dockev=30006MXP.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 4.0 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 incident to ensure QA/QC procedures are performed consistently across
laboratories. EPA program offices will be responsible for ensuring that the QA/QC practices are
implemented.
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Section 5.0 - Selected Chemical Methods
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.
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
EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit Program
OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories
OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response
Please note that the Electronic Code of Federal Regulations (e-CFR) is available at:
http://\\\\\\.ccfr.gov/cgi-bin/ECFR'.)pagc=bro\\sc
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Section 5.0 - Selected Chemical Methods
5.2 Method Summaries
Summaries for the analytical methods listed in Appendix A are provided in Sections 5.2.1 through
5.2.116. These sections contain summary information extracted from the selected methods. Each method
summary contains a table identifying the contaminants listed 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) can be found in Appendix A. The full version of the method
should be consulted prior to sample analysis. For information regarding sample collection considerations
for samples to be analyzed by these methods, see the companion Sample Collection Information
Document for Chemicals, Radiochemicals and Biotoxins - Companion to SAM 2017.6
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)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
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 aqueous and solid samples.
Method Selected for: This method has been selected for preparation and analysis of drinking water
samples to address the analytes listed in the table above as total arsenic, thallium or vanadium. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: Method detection limits (MDLs) in aqueous samples are reported for
arsenic (8 (.ig/L). vanadium (3 |ig/L) and thallium (1 (.ig/L).
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
6 EPA's Sample Collection Information Document for Chemicals, Radiochemicals and Biotoxins - Companion to
SAM2017 can be found at: https://www.epa.gov/homeland-securitv-research/sam-compamon-documents-and-
sample-collection-procedures
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Section 5.0 - Selected Chemical Methods
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: If laboratories are approved for storing and handling the appropriate standards,
lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can be detected
and measured using EPA/600/R-15/258 (see Section 5.2.54).
Source: Martin, T.D., Brockhoff, C.A., Creed, J.T. and EMMC Methods Work Group. 1994. "Method
200.7: Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-
Atomic Emission Spectrometry," Revision 4.4. Cincinnati, OH: U.S. EPA.
https://www.epa.gOv/sites/production/files/2015-06/documents/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)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
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: This method has been selected for preparation and analysis of drinking water
samples to address the analytes listed in the table above as total arsenic, thallium or vanadium. See
Appendix A for corresponding method usability tiers.
Detection and Quantitation: MDLs for arsenic, thallium and vanadium in aqueous samples are reported
as 1.4, 0.3 and 2.5 (ig/L, respectively (in scanning mode) and 0.4, 0.02 and 0.9 |ig/L. respectively (in
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Section 5.0 - Selected Chemical Methods
selected ion monitoring (SIM) mode). The recommended calibration range is 10-200 (ig/L (scanning
mode) and may be lower depending on the sensitivity of the instrument.
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: If laboratories are approved for storing and handling the appropriate standards,
lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can be detected
and measured using EPA/600/R-15/258 (see Section 5.2.54).
Source: Creed, J.T., Brockhoff, C.A. and Martin, T.D. 1994. "Method 200.8: Determination of Trace
Elements in Waters and Wastes by Inductively Coupled Plasma-Mass Spectrometry," Revision 5.4.
Cincinnati, OH: U.S. EPA. https://www.epa.gOv/sites/production/files/2015-06/documents/epa-200.8.pdf
5.2.3 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic
Absorption Spectrometry
Analyte(s)
CAS RN
Mercuric chloride (analyze as total mercury)
7487-94-7
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
151-38-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Acid digestion
Determinative Technique: Cold vapor atomic absorption
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: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above as total mercury. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: Applicable concentration range is 0.2-10.0 |ag Hg/L. The detection limit
for this method is 0.2 |_ig Hg/L.
Description of Method: This method will determine mercuric chloride and methoxyethylmercuric
acetate as total mercury. If dissolved mercury is targeted, the sample is filtered prior to acidification. To
detect total mercury (inorganic and organic mercury), the sample is treated with potassium permanganate
and potassium persulfate to oxidize organic mercury compounds prior to analysis. Inorganic mercury is
reduced to the elemental state (using stannous chloride) and aerated from solution. The mercury vapor
passes through a cell positioned in the light path of a cold vapor atomic absorption spectrophotometer.
The concentration of mercury is measured using the spectrophotometer.
Special Considerations: If problems occur during analysis of aqueous samples other than drinking
water, refer to Method 7470A (EPA SW-846).
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Source: O'Dell, J.W., Potter, B.B., Lobring, L.B. and Martin, T.D. 1994. "Method 245.1: Determination
of Mercury in Water by Cold Vapor Atomic Absorption Spectrometry," Revision 3.0. Cincinnati, OH:
U.S. EPA. https://www.epa.gOv/sites/production/files/2015-06/documents/epa-245.l.pdf
5.2.4 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking
Water by Ion Chromatography
Analyte(s)
CAS RN
Fluoride
16984-48-8
Fluorosilicic acid (analyze as fluoride)
16961-83-4
Sodium azide (analyze as azide ion)
26628-22-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: For fluoride and fluorosilicic acid, use direct injection. For sodium
azide in water and solid samples, use water extraction, filtration and acidification steps from the Journal
of Forensic Science, 1998. 43(1): 200-202 (Section 5.2.116).
Determinative Technique: Ion chromatography (IC) with conductivity detection
Method Developed for: Inorganic anions in reagent water, surface water, ground water and finished
drinking water
Method Selected for: This method has been selected for preparation and analysis of water samples for
fluoride and fluorosilicic acid (as fluoride). It also has been selected for analysis of prepared solid
samples for sodium azide (as azide ion). 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 will determine fluoride ion, fluorosilicic acids as fluoride ion, and
sodium azide as azide ion. It was developed for analysis of aqueous samples, and can be adapted for
analysis of sodium azide in solid and air samples when appropriate sample preparation techniques have
been applied (see Appendix A). A small volume of a water sample (10 (iL or 50 |_iL) is introduced into an
ion chromatograph. The volume selected depends on the concentration of fluoride or azide 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.
Source: Hautman, D.P. and Munch, D.J. 1997. "Method 300.1: Determination of Inorganic Anions in
Drinking Water by Ion Chromatography," Revision 1.0. Cincinnati, OH: U.S. EPA.
https://www.epa.gOv/sites/production/flles/2015-06/documents/epa-300.l.pdf
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Section 5.0 - Selected Chemical Methods
5.2.5 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated
Colorimetry
Analyte(s)
CAS RN
Cyanide, Total
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: This method has been selected for preparation and analysis of drinking water
samples to address total cyanide. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The applicable range is 5-500 (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: 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: U.S. EPA. 1993. "Method 335.4: Determination of Total Cyanide by Semi-automated
Colorimetry," Revision 1.0. Cincinnati, OH: U.S. EPA. https://www.epa.gov/sites/production/files/2Q15-
06/documents/epa-335,4.pdf
5.2.6 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate)
Analyte(s)
CAS RN
Ammonia
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: This method has been selected for preparation and analysis of drinking water
samples to address ammonia. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range for ammonia is 0.01-2.0 mg/L.
Description of Method: This method identifies and determines the concentration of ammonia in
drinking water samples by spectrophotometry. Samples are buffered at a pH of 9.5 with borate buffer to
decrease hydrolysis of cyanates and organic nitrogen compounds, and are distilled into a solution of boric
acid. Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is proportional
to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside and
measured spectrophotometrically.
Special Considerations: Reduced volume distillation techniques, such as midi-distillation or micro-
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Section 5.0 - Selected Chemical Methods
distillation, can be used in place of traditional macro-distillation techniques.
Source: U.S. EPA. 1993. "Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate),"
Revision 2.0. Cincinnati, OH: U.S. EPA. https://www .epa.gov/sites/production/files/2015 -
06/documents/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)
CAS RN
Acrylonitrile
107-13-1
Carbon disulfide
75-15-0
1,2-Dichloroethane
107-06-2
Methyl acrylonitrile
126-98-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Purge-and-trap
Determinative Technique: GC-MS
Method Developed for: Purgeable volatile organic compounds (VOCs) in surface water, ground water
and drinking water in any stage of treatment
Method Selected for: This method has been selected for preparation and analysis of drinking water
samples to address carbon disulfide and 1,2-dichloroethane, and preparation and analysis of drinking and
non-drinking water samples to address acrylonitrile and methyl acrylonitrile. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: The method reports detection levels for acrylonitrile, carbon disulfide, 1,2-
dichloroethane and methyl acrylonitrile in reagent water of 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 0.02-200 (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 0.02-20 (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 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 more recent versions of this method (Methods 524.3 or 524.4) may be
used in place of Method 524.2, provided the laboratory has the necessary equipment (e.g., cryogenic auto
samplers).
Source: Eichelberger, J.W., Munch, J.W. and Bellar, T.A. 1995. "Method 524.2: Measurement of
Purgeable Organic Compounds in Water by Capillary Column Gas Chromatography/Mass Spectrometry,"
Revision 4.1. Cincinnati, OH: U.S. EPA. https: //www .epa. gov/site s/production/file s/2015 -
06/documents/epa-524.2 .pdf
Additional Resources:
Prakash, A.D., Zaffiro, A.D., Zimmerman, M., Munch, D.J. and Pepich, B.V. 2009. "Method 524.3:
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Measurement of Purgeable Organic Compounds in Water by Capillary Column Gas
Chromatography/Mass Spectrometry," Revision 1.0. Cincinnati, OH: U.S. EPA. EPA 815-B-09-009.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J75C.PDF?Dockev=P 100J75C.PDF
U.S. EPA. 2013. "Method 524.4: Measurement of Purgeable Organic Compounds in Water by Gas
Chromatography/Mass Spectrometry," Revision 1. Cincinnati, OH: U.S. EPA. EPA 815-R-13-002.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7EE.PDF?Dockev=P100J7EE.PDF
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)
CAS RN
Chlorpyrifos
2921-88-2
Dichlorvos
62-73-7
Disulfoton*
298-04-4
Disulfoton sulfone oxon*
2496-91-5
Disulfoton sulfoxide*
2497-07-6
Disulfoton sulfoxide oxon*
2496-92-6
Fenamiphos
22224-92-6
Mevinphos
7786-34-7
* 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://vwvw.sciencedirect.com/science/article/pii/S0Q43135408004995
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: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above. Note.
EPA NHSRC EPA/600/R-16/114 (Section 5.2.55) has been selected for preparation and analysis
of non-drinking water samples to address chlorpyrifos and fenamiphos.
SW-846 Method 3535A (Section 5.2.21) and Method 8270E (Section 5.2.35) have been selected
for preparation and analysis of non-drinking water samples to address dichlorvos and mevinphos.
See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The applicable concentration range for most analytes is 0.1-10 (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.
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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. SPE using Ci8 resin may not work for certain
compounds having high water solubility. In these cases, other sample preparation techniques or different
SPE resins may be required. The more recent version of this method (Method 525.3) may be used in place
of Method 525.2, provided the laboratory has the necessary equipment and expertise.
Source: Munch, J.W. 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. Cincinnati, OH: U.S. EPA. https://www.epa.gov/sites/production/files/2015-06/documents/epa-
525.2.pdf
Additional Resource: Munch, J.W., Grimmett, P.E., Munch, D.J., Wendelken, S.C., Domino, M.M.,
Zaffiro, A.D. and Zimmerman, M.L. 2012. "Method 525.3: Determination of Semivolatile Organic
Chemicals in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography/
Mass Spectrometry (GC/MS)," Revision 1.0. Cincinnati, OH: U.S. EPA. EPA/600/R-12/010.
https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=241188
5.2.9 EPA Method 525.3: Determination of Semivolatile Organic Chemicals in Drinking
Water by Solid Phase Extraction and Capillary Column Gas Chromatography/
Mass Spectrometry (GC/MS)
Analyte(s)
CAS RN
Phosphamidon
13171-21-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: SPE
Determinative Technique: GC-MS
Method Developed for: Semivolatile organic compounds in drinking water
Method Selected for: This method has been selected for preparation and analysis of drinking water
samples to address phosphamidon. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The applicable concentration range for most analytes is 0.1-10 (ig/L.
Description of Method: A 1-L sample is fortified with surrogates and passed through an SPE apparatus.
Phosphamidon and surrogates are eluted from the solid phase with a small amount of two or more organic
solvents. The solvent extract is dried by passing it through a column of anhydrous sodium sulfate,
concentrated by nitrogen gas blow-down, and adjusted to a 1-mL volume with ethyl acetate after adding
internal standards. A splitless injection is made into a GC equipped with a high-resolution fused silica
capillary column interfaced to an MS. 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 GC-MS conditions. The GC-MS can be operated in the full scan, SIM, or
selected ion storage (SIS) mode. Analyte concentrations are calculated using their integrated peak area
and the internal standard technique.
Special Considerations: Phosphamidon was observed to exhibit matrix induced chromatographic
response enhancement during method development, determined by comparing the peak area response of a
standard prepared in solvent compared to a matrix-matched standard, both at a concentration of 0.2
ng/|iL. The method includes use of matrix-matched standards as an option to evaluated matrix
interferences. If the peak area of a matrix-matched standard is > 130% of the of the peak area produced by
the solvent-prepared standard, matrix enhancement is likely to be present.
Source: Munch, J.W., Grimmett, P.E., Munch, D.J., Wendelken, S.C., Domino, M.M., Zaffiro, A.D.
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Section 5.0 - Selected Chemical Methods
and Zimmerman, M.L. 2012. "Method 525.3 Determination of Semivolatile Organic Chemicals in
Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography/ Mass
Spectrometry (GC/MS)," Version 1.0. Cincinnati, OH: U.S. EPA.
https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=241188
5.2.10 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and N-
Methylcarbamates in Water by Direct Aqueous Injection HPLC With Postcolumn
Derivatization
Analyte(s)
CAS RN
Aldicarb (Temik)
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
Carbofuran (Furadan)
1563-66-2
Methomyl
16752-77-5
Oxamyl
23135-22-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Direct injection
Determinative Technique: High performance liquid chromatography (HPLC)-fluorescence (FL)
Method Developed for: N-methylcarbamoyloximes and N-methylcarbamates in finished drinking water
Method Selected for: This method has been selected for preparation and analysis of drinking water
samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: Detection limits range from 0.026 to 0.115 (ig/L. The concentration range
for target analytes in this method was evaluated between 0.2 |_ig/L and 10 (ig/L.
Description of Method: An aliquot of sample is measured in a volumetric flask. Samples are preserved,
spiked with appropriate surrogates and then filtered. Analytes are chromatographically separated by
injecting a sample aliquot (up to 1000 |_iL) into a HPLC system equipped with a reverse phase (Ci8)
column. After elution from the column, the analytes are hydrolyzed in a post column reaction to form
methylamine, which is in turn reacted to form a fluorescent isoindole that is detected by an FL detector.
Analytes also are quantitated using the external standard technique.
Source: Bassett, S.C., Wendelken, S.C., Pepich, B.V., Munch, D.J. and Henry, L. 2001. "Method 531.2:
Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates in Water by Direct Aqueous
Injection HPLC With Postcolumn Derivatization," Revision 1.0. Cincinnati, OH: U.S. EPA. EPA/815/B-
01/002. https://www.epa.gOv/sites/production/files/2015-06/documents/epa-531.2.pdf
5.2.11 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)
CAS RN
Acephate
30560-19-1
Diisopropyl methylphosphonate (DIMP)
1445-75-6
Methamidophos
10265-92-6
Thiofanox
39196-18-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
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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: This method has been selected for preparation and analysis of drinking water
samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
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 Concentration
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-(jL or larger injection is made into a liquid chromatograph (LC) equipped with a Ci8 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: Shoemaker, J.A. 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. Cincinnati, OH: U.S. EPA. EPA/600/R-09/149.
https://www.epa.gOv/sites/production/files/2015-06/documents/epa-538.pdf
5.2.12 EPA Method 540: Determination of Selected Organic Chemicals in Drinking Water
by Solid Phase Extraction and Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
Analyte(s)
CAS RN
Chlorpyrifos oxon
5598-15-2
Phorate sulfone
2588-04-7
Phorate sulfone oxon
2588-06-9
Phorate sulfoxide
2588-03-6
Phorate sulfoxide oxon
2588-05-8
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: SPE
Determinative Technique: LC-MS-MS
Method Developed for: Chlorpyrifos oxon, phorate sulfone and phorate sulfoxide in drinking water
samples
Method Selected for: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Depending on the SPE cartridge used, detection limits were calculated to
be 0.77 and 1.0 ng/L (chlorpyrifos oxon), 0.32 and 0.57 ng/L (phorate sulfone) and 0.46 and 0.70 ng/L
(phorate sulfoxide). The LCMRLs in reagent water were calculated to be 2.0 and 2.7 ng/L (chlorpyrifos
oxon), 0.86 and 1.0 ng/L (phorate sulfone) and 0.99 and 1.1 ng/L (phorate sulfoxide).
Description of Method: A 250-mL water sample is preserved with Trizmaฎ (Millipore Sigma, St. Louis,
MO, or equivalent), 2-chloroacetamide and ascorbic acid. The sample is fortified with surrogates and
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passed through a SPE cartridge. Compounds are eluted from the solid phase with a small amount of
methanol, and the extract is concentrated by evaporation with nitrogen in a heated water bath, internal
standards are added, and the volume is adjusted to 1 mL with methanol. A 10-jj.L injection is made into an
LC equipped with a Ci8 column that is interfaced to an MS-MS. 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 using the internal standard technique.
Source: Shoemaker, J.A. and Tettenhorst, D.R. 2013. "Method 540: Determination of Selected Organic
Chemicals in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass
Spectrometry," Revision 1.0. Cincinnati, OH: U.S. EPA. EPA/600/R-13/119.
httos://nepis.epa.gov/Exe/ZvPDF.cgi?Dockev=P 100H0Z5.txt
5.2.13 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by
Liquid-Solid Extraction and High Performance Liquid Chromatography With
Ultraviolet Detection
Analyte(s)
CAS RN
Paraquat
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: This method has been selected for preparation and analysis of water samples to
address paraquat. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The 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: Munch, J.W. and Bashe, W.J. 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. Cincinnati, OH: U.S. EPA.
https://www.epa.gOv/sites/production/files/2015-06/documents/epa-549.2.pdf
5.2.14 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts,
Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by
Liquid-Liquid Extraction and Gas Chromatography With Electron-Capture
Detection
Analyte(s)
CAS RN
Chloropicrin
76-06-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
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Section 5.0 - Selected Chemical Methods
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-electron capture detector (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: This method has been selected for preparation and analysis of water samples to
address chloropicrin. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The estimated detection limit (EDL) using methyl tert-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 jxL 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 using either a secondary GC
column or an MS.
Source: Munch, D.J. and Hautman, D.P. 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.
Cincinnati, OH: U.S. EPA. http://www.epa.gov/sites/production/files/2015-06/documents/epa-551.1 .pdf
5.2.15 EPA Method 556.1: Determination of Carbonyl Compounds in Drinking Water by
Fast Gas Chromatography
Analyte(s)
CAS RN
Formaldehyde
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: This method has been selected for preparation and analysis of drinking water
samples to address formaldehyde. See Appendix A for the corresponding method usability tier.
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 5-40 (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
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Section 5.0 - Selected Chemical Methods
(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: Wendelken, S.C., Pepich, B.V. and Munch, D.J. 1999. "Method 556.1: Determination of
Carbonyl Compounds in Drinking Water by Fast Gas Chromatography," Revision 1.0. Cincinnati, OH:
U.S. EPA. http://www.epa.gov/sites/production/files/2015-06/documents/epa-556.1 .pdf
5.2.16 EPA Method 3015A (SW-846): Microwave Assisted Acid Digestion of Aqueous
Samples and Extracts
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
Analysis Purpose: Sample preparation
Sample Preparation Technique: Microwave assisted acid digestion
Determinative Technique: ICP-AES / ICP-MS
Determinative Method: EPA SW-846 Method 6010D (Section 5.2.27) or Method 6020B (Section
5.2.28). Refer to Appendix A for which of these determinative methods should be used for a particular
analyte.
Method Developed for: Metals in water, mobility-procedure extracts, and wastes that contain suspended
solids
Method Selected for: This method has been selected for preparation of non-drinking water samples to
address the analytes listed in the table above as total arsenic, osmium, thallium or vanadium. See
Appendix A for corresponding method usability tiers.
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; osmium tetroxide as osmium; and ammonium
metavanadate and vanadium pentoxide as total vanadium. A 45-mL aliquot of a well-shaken,
homogenized sample is transferred to a fluorocarbon polymer or quartz microwave vessel or vessel liner,
equipped with a controlled pressure relief mechanism. 5 mL of concentrated nitric acid or 4 mL of
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Section 5.0 - Selected Chemical Methods
concentrated nitric acid plus 1 mL of concentration hydrochloric acid are added to the vessel. The vessel
is sealed, placed into the microwave system, and heated. The temperature of each sample should rise to
170 ฑ 5ฐC in approximately 10 minutes and remain at that temperature for 10 minutes, or for the
remainder of the 20-minute digestion period. After cooling, the vessel contents are filtered, centrifuged, or
allowed to settle and then diluted to volume. Samples are analyzed for total arsenic, total thallium, or total
vanadium by SW-846 Method 6010D (Section 5.2.27) or 6020B (Section 5.2.28); use Method 6010D for
total osmium.
Special Considerations: Digestion of samples that contain organics may create high pressures due to
the evolution of gaseous digestion products. This may cause venting of the vessels with potential loss of
sample components and/or analytes. In these cases, a smaller sample size should be used, but the volume
prior to addition of acid(s) should be adjusted to 45 mL with deionized water. Highly reactive samples
may also require pre-digestion in a hood to minimize the danger of thermal runaway and excessively
vigorous reactions. 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.
However, the addition of hydrochloric acid can limit the quantitation techniques when using some ICP-
MS instruments. If laboratories are approved for storing and handling the appropriate standards, lewisites
1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can be detected and
measured using EPA/600/R-15/258 (Section 5.2.54).
Source: U.S. EPA. 2007. "Method 3015A (SW-846): Microwave Assisted Acid Digestion of Aqueous
Samples and Extracts," Revision 1. Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2015-12/documents/3Q15a.pdf
5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: ICP-AES / ICP-MS
Determinative Method: EPA SW-846 Method 6010D (Section 5.2.27) or Method 6020B (Section
5.2.28). Refer to Appendix A for which of these determinative methods should be used for a particular
analyte.
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Method Developed for: Metals in sediments, sludges, and soil samples
Method Selected for: This method has been selected for preparation of solid samples to address the
analytes listed in the table above as total arsenic, thallium or vanadium. See Appendix A for
corresponding method usability tiers.
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; 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 6010D (Section 5.2.27) or 6020B (Section 5.2.28);
use Method 6010D for total osmium.
Special Considerations: If laboratories are approved for storing and handling the appropriate standards,
lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can be detected
and measured using EPA/600/R-15/258 (Section 5.2.54).
Source: U.S. EPA. 1996. "Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils,"
Revision 2. Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-
06/documents/epa-3 05 Ob .pdf
5.2.18 EPA Method 3051A (SW-846): Microwave Assisted Acid Digestion of Sediments,
Sludges, and Oils
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Titanium tetrachloride (analyze as total titanium)
7550-45-0
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
Analysis Purpose: Sample preparation
Sample Preparation Technique: Microwave assisted acid digestion
Determinative Technique: ICP-AES / ICP-MS
Determinative Method: EPA SW-846 Method 6010D (Section 5.2.27) or Method 6020B (Section
5.2.28). Refer to Appendix A for which of these determinative methods should be used for a particular
analyte.
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Method Developed for: Metals in sediments, sludges, soils, and oils
Method Selected for: This method has been selected for:
Preparation of solid samples to be analyzed for the total arsenic component of arsenic trioxide
arsine, lewisite, lewisite degradation products, calcium and lead arsenate and sodium arsenite
Preparation of solid samples to be analyzed for the total thallium component of thallium sulfate
Preparation of solid samples to be analyzed for the total vanadium component of ammonium
metavanadate and vanadium pentoxide
Preparation of solid and wipe samples to be analyzed for the total osmium component of osmium
tetroxide
Preparation of solid and wipe samples to be analyzed for the total titanium component of titanium
tetrachloride
NIOSH Method 9102 (see Section 5.2.75) should be used for the preparation of wipes to be analyzed for
all other analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Description of Method: A well-mixed sample (no more than 0.500 g for soils, sediments and sludges,
and no more than 0.250 g for oil or oil contaminated soil) to the nearest 0.001 g is weighed into a
fluorocarbon polymer or quartz microwave vessel or vessel liner equipped with a controlled pressure
relief mechanism. 10 mL of concentrated nitric acid or 9 mL of concentrated nitric acid plus 3 mL of
concentration hydrochloric acid are added to the vessel, and the vessel is sealed, placed into the
microwave system, and heated. After cooling, the vessel contents are filtered, centrifuged, or allowed to
settle and then diluted to volume. Samples are analyzed for total arsenic, total thallium, total titanium or
total vanadium by Method 6010D or 6020B (SW-846); use Method 6010D (SW-846) fortotal osmium.
Special Considerations: Digestion of samples that contain organics or carbonates can create high
pressures due to the evolution of gaseous digestion products. This can cause venting of the vessels with
potential loss of sample components and/or analytes. Samples that are highly reactive or contaminated
might require dilution or pre-digestion in a hood to minimize the danger of thermal runaway and
excessively vigorous reactions. 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. However, the addition of hydrochloric acid can limit the quantitation techniques when using
some ICP-MS instruments. If laboratories are approved for storing and handling the appropriate
standards, lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can
be detected and measured using EPA/600/R-15/258 (see Section 5.2.54).
Source: U.S. EPA. 2007. "Method 3051A (SW-846): Microwave Assisted Acid Digestion of Sediments,
Sludges, and Oils," Revision 1. Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2015-12/documents/3Q51a.pdf
5.2.19 EPA Method 3511 (SW-846): Organic Compounds in Water by Microextraction
Analyte(s)
CAS RN
Tetraethyl pyrophosphate (TEPP)
107-49-3
Analysis Purpose: Sample preparation
Sample Preparation Technique: Microextraction
Determinative Technique: GC-MS
Determinative Method: EPA SW-846 Method 8270E (Section 5.2.35)
Method Developed for: Organic compounds in aqueous samples
Method Selected for: This method has been selected for preparation of water samples to address TEPP.
See Appendix A for corresponding method usability tiers.
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Description of Method: A measured volume of water sample, usually 35 mL, is placed into a 40-mL
volatile organic analysis (VOA) vial. Surrogates (10 |_ig of each), 2 mL of methylene chloride, and 12 g of
sodium chloride are added to the vial, and the vial is capped and shaken vigorously for 5 minutes or until
the sodium chloride is completely dissolved. After the contents are allowed to settle (centrifuging if
necessary), 1.5 mL of the lower (methylene chloride) layer is transferred to a 2-mL vial. The extract is
then dried with sodium sulfate and a 1-mL aliquot is transferred to a GC vial. Samples are analyzed for
TEPP by SW-846 Method 8270E (Section 5.2.35).
Source: U.S. EPA. 2014. "Method 3511 (SW-846): Organic Compounds in Water by Microextraction,"
Revision 1. Washington, DC: U.S. EPA. httos://www .epa. gov/sites/production/files/2015-
12/documents/3511 .pdf
5.2.20 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction
Analyte(s)
CAS RN
Carfentanil
59708-52-0
Chlorfenvinphos
470-90-6
Diesel range organics
NA
Fentanyl
437-38-7
Kerosene
64742-81-0
3-Methyl fentanyl
42045-87-4
Paraoxon
311-45-5
Parathion
56-38-2
Phosphamidon
13171-21-6
Analysis Purpose: Sample preparation
Sample Preparation Technique: Continuous liquid-liquid extraction (CLLE)
Determinative Technique: GC-flame ionization detector (FID) / GC-MS / LC-MS-MS
Determinative Method: EPA SW-846 Method 8015D (Section 5.2.33), Method 8270E (Section 5.2.35),
adapted from J. Chromatogr. B, 962: 52-58 (Section 5.2.115), or adapted from J. Chromatogr. A, 1218:
1620-1649 (Section 5.2.111). 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: This method has been selected for preparation of water samples to address the
analytes listed in the table above. Note. Drinking water samples to be analyzed for phosphamidon should
be prepared and analyzed using EPA Method 525.3 (Section 5.2.9). 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
dependent on various factors such as sample pH and temperature.
Source: U.S. EPA. 1996. "Method 3520C (SW-846): Continuous Liquid-Liquid Extraction." Revision 3.
http://www.epa.gov/sites/production/files/2015-06/documents/epa-352Qc.pdf
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5.2.21 EPA Method 3535A (SW-846): Solid-Phase Extraction
Analyte(s)
CAS RN
4-Aminopyridine
504-24-5
Carfentanil
59708-52-0
Chlorfenvinphos
470-90-6
Dichlorvos
62-73-7
Dicrotophos
141-66-2
Diesel range organics
NA
Ethyldichloroarsine (ED)
598-14-1
Fentanyl
437-38-7
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
121-82-4
Kerosene
64742-81-0
3-Methyl fentanyl
42045-87-4
Methyl paraoxon
950-35-6
Methyl parathion
298-00-0
Mevinphos
7786-34-7
Monocrotophos
6923-22-4
Nicotine compounds
54-11-5
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)
2691-41-0
Paraoxon
311-45-5
Parathion
56-38-2
Pentaerythritol tetranitrate (PETN)
78-11-5
Ph orate
298-02-2
Phosphamidon
13171-21-6
Strychnine
57-24-9
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
2,4,6-Trinitrotoluene (2,4,6-TNT)
118-96-7
Analysis Purpose: Sample preparation
Sample Preparation Technique: SPE
Determinative Technique: GC-FID / GC-MS / LC-MS-MS / HPLC
Determinative Method: EPA SW-846 Method 8015D (Section 5.2.33), Method 8270E (Section 5.2.35),
Method 8330B (Section 5.2.40), adapted from Analyst, 126:1689-1693 (Section 5.2.102), adapted from J.
Chromatogr. B, 962: 52-58 (Section 5.2.115), or adapted from J. Chromatogr. A, 1218: 1620-1649
(Section 5.2.111). 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: This method has been selected for preparation of water samples to address the
analytes listed in the table above. Note.
EPA Method 525.2 (Section 5.2.8) has been selected for preparation and analysis of drinking
water samples to address dichlorvos and mevinphos.
EPA Method 525.3 (Section 5.2.9) has been selected for preparation and analysis of drinking
water samples to address phosphamidon.
All other drinking water samples and all non-drinking water 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
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samples using SPE media. Sample preparation procedures vary by analyte group. Following 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: Some of the target compounds will hydrolyze in water, with hydrolysis rates
dependent on various factors such as sample pH and temperature.
Source: U.S. EPA. 2007. "Method 3535A (SW-846): Solid-Phase Extraction (SPE)," Revision 1.
Washington, DC: U.S. EPA. https://www.epa.gov/sites/production/files/2015-12/documents/3535a.pdf
5.2.22 EPA Method 3541 (SW-846): Automated Soxhlet Extraction
Analyte(s)
CAS RN
Brodifacoum
56073-10-0
Bromadiolone
28772-56-7
BZ [Quinuclidinyl benzilate]
6581-06-2
Carfentanil
59708-52-0
Diesel range organics
NA
Diphacinone
82-66-6
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
73207-98-4
Ethyldichloroarsine (ED)
598-14-1
N-Ethyldiethanolamine (EDEA)
139-87-7
Fentanyl
437-38-7
Kerosene
64742-81-0
3-Methyl fentanyl
42045-87-4
Methyl hydrazine
60-34-4
N-Methyldiethanolamine (MDEA)
105-59-9
Monocrotophos
6923-22-4
Thiofanox
39196-18-4
Triethanolamine (TEA)
102-71-6
Trimethyl phosphite
121-45-9
Analysis Purpose: Sample preparation
Sample Preparation Technique: Automated Soxhlet extraction
Determinative Technique: GC-FID / GC-MS / LC-MS-MS
Determinative Method: EPA SW-846 Methods 8015D (Section 5.2.33) and 8270E (Section 5.2.35),
EPA/600/R-15/097 (Section 5.2.53), EPA/600/R-11/143 (Section 5.2.50), ASTM Methods D7644
(Section 5.2.91) and D7645 (Section 5.2.92), adapted from J. Chromatogr. 617: 157-162 (Section
5.2.106), adapted from J. Chromatogr. B, 874: 42-50 (Section 5.2.112), adapted from J. Chromatogr. A,
1218: 1620-1649 (Section 5.2.111), or adapted from J. Chromatogr. B, 962: 52-58 (Section 5.2.115).
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: This method has been selected for preparation of solid samples to address the
analytes listed in the table above. 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,
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concentrated and exchanged, as necessary, into a solvent compatible with the cleanup or determinative
procedure for analysis.
Special Considerations: Some of the target compounds will hydrolyze in water, with hydrolysis rates
dependent on various factors such as sample pH and temperature.
Source: U.S. EPA. 1994. "Method 3541 (SW-846): Automated Soxhlet Extraction," Revision 0.
Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-06/documents/epa-3541.pdf
5.2.23 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)
Analyte(s)
CAS RN
Brodifacoum
56073-10-0
Bromadiolone
28772-56-7
BZ [Quinuclidinyl benzilate]
6581-06-2
Carfentanil
59708-52-0
Diesel range organics
NA
Diphacinone
82-66-6
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
73207-98-4
Ethyldichloroarsine (ED)
598-14-1
N-Ethyldiethanolamine (EDEA)
139-87-7
Fentanyl
437-38-7
Kerosene
64742-81-0
3-Methyl fentanyl
42045-87-4
Methyl hydrazine
60-34-4
N-Methyldiethanolamine (MDEA)
105-59-9
Monocrotophos
6923-22-4
Thiofanox
39196-18-4
Triethanolamine (TEA)
102-71-6
Trimethyl phosphite
121-45-9
Analysis Purpose: Sample preparation
Sample Preparation Technique: Pressurized fluid extraction (PFE)
Determinative Technique: GC-FID / GC-MS / LC-MS-MS / HPLC-UV
Determinative Method: EPA SW-846 Methods 8015D (Section 5.2.33) and 8270E (Section 5.2.35),
EPA/600/R-15/097 (Section 5.2.53), EPA/600/R-11/143 (Section 5.2.50), ASTM Method D7644 (Section
5.2.91) and D7645 (Section 5.2.92), adapted from J. Chromatogr. 617: 157-162 (Section 5.2.106),
adapted from J. Chromatogr. B, 874: 42-50 (Section 5.2.112), adapted from J. Chromatogr. A, 1218:
1620-1649 (Section 5.2.111), or adapted from J. Chromatogr. B, 962: 52-58 (Section 5.2.115). 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: This method has been selected for preparation of solid samples to address the
analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: This method has been validated for solid matrices containing 250 to
12,500 (ig/kg of semivolatile organic compounds, 250 to 2500 (ig/kg of organophosphorus pesticides, 5 to
250 jxg/kg of organochlorine pesticides, and 50 to 5000 jxg/kg of chlorinated herbicides.
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
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extracted for 5 minutes (or as recommended by the instrument manufacturer). The extract may be
concentrated, if necessary, and exchanged into a solvent compatible with the cleanup or determinative
step being employed.
Special Considerations: Sodium sulfate can cause clogging, and air-drying or pelletized diatomaceous
earth may be preferred for drying samples. Some of the target compounds will hydrolyze in water, with
hydrolysis rates dependent on various factors such as sample pH and temperature.
Source: U.S. EPA. 2007. "Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)," Revision 1.
Washington, DC: U.S. EPA. https://www.epa.gov/sites/production/files/2015-12/documents/3545a.pdf
5.2.24 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSE)
Analyte(s)
CAS RN
Acrylamide
79-06-1
Acrylonitrile
107-13-1
Aldicarb (Temik)
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
4-Aminopyridine
504-24-5
BZ [Quinuclidinyl benzilate]
6581-06-2
Brodifacoum
56073-10-0
Bromadiolone
28772-56-7
Carbofuran (Furadan)
1563-66-2
Carfentanil
59708-52-0
Diesel range organics
NA
Diphacinone
82-66-6
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
73207-98-4
Fentanyl
437-38-7
Formaldehyde
50-00-0
Gasoline range organics
NA
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
121-82-4
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
Kerosene
64742-81-0
Methomyl
16752-77-5
Methyl acrylonitrile
126-98-7
3-Methyl fentanyl
42045-87-4
Methyl hydrazine
60-34-4
Monocrotophos
6923-22-4
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)
2691-41-0
Oxamyl
23135-22-0
Pentaerythritol tetranitrate (PETN)
78-11-5
Thiofanox
39196-18-4
Trimethyl phosphite
121-45-9
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
2,4,6-Trinitrotoluene (2,4,6-TNT)
118-96-7
White phosphorus
12185-10-3
Analysis Purpose: Sample preparation
Sample Preparation Technique: Microscale solvent extraction (MSE)
Determinative Technique: GC - nitrogen-phosphorus detector (NPD) / GC-FID / GC-MS / HPLC-UV /
LC-MS-MS
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Determinative Method: EPA SW-846 Methods 7580 (Section 5.2.32), 8015D (Section 5.2.33), 8260D
(Section 5.2.34), 8270E (Section 5.2.35), 8315A (Section 5.2.37), 8316 (Section 5.2.38), 8318A (Section
5.2.39), and 8330B (Section 5.2.40); EPA/600/R-15/097 (Section 5.2.53); ASTM Methods D7644-16
(Section 5.2.91) and D7645-16 (Section 5.2.92); adapted from Analyst, 126:1689-1693 (Section 5.2.102);
adapted from J. Chromatogr. 617: 157-162 (Section 5.2.106); adapted from J. Chromatogr. B, 874: 42-50
(Section 5.2.112); adapted from J. Chromatogr. A, 1218: 1620-1649 (Section 5.2.111); or adapted from J.
Chromatogr. B, 962: 52-58 (Section 5.2.115). 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: This method has been selected for preparation of wipe samples to address the
analytes listed in the table above. 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
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 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.
Special Considerations: Method 3570 is not amenable for analysis of samples that have been preserved
in the field using methanol.
Source: U.S. EPA. 2002. "Method 3570 (SW-846): Microscale Solvent Extraction (MSE)," Revision 0.
Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-07/documents/epa-3570.pdf
5.2.25 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples
Analyte(s)
CAS RN
Allyl alcohol
107-18-6
Carbon disulfide
75-15-0
2-Chloroethanol
107-07-3
1,2-Dichloroethane
107-06-2
Ethylene oxide
75-21-8
2-Fluoroethanol
371-62-0
Gasoline range organics
NA
Propylene oxide
75-56-9
The following analyte should be prepared by this method (and determined by the corresponding SW-846 Method
8260D) 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 8015D (Section 5.2.33) or Method 8260D (Section
5.2.34). Refer to Appendix A for which of these determinative methods should be used for a particular
analyte.
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Method Developed for: VOCs in aqueous and water miscible liquid samples
Method Selected for: This method has been selected for preparation of water samples to address the
analytes listed in the table above. Note: For carbon disulfide and 1,2-dichloroethane, EPA Method 524.2
(Section 5.2.7) 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: U.S. EPA. 2003. "Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples," Revision
3. Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-07/documents/epa-
5030c.pdf
5.2.26 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples
Analyte(s)
CAS RN
Acrylonitrile
107-13-1
Allyl alcohol
107-18-6
Carbon disulfide
75-15-0
2-Chloroethanol
107-07-3
1,2-Dichloroethane
107-06-2
Ethylene oxide
75-21-8
2-Fluoroethanol
371-62-0
Gasoline range organics
NA
Methyl acrylonitrile
126-98-7
Propylene oxide
75-56-9
The following analyte should be prepared by this method (and determined by the corresponding SW-846 Method
8260D) 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 8015D (Section 5.2.33) or Method 8260D (Section
5.2.34). 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: This method has been selected for preparation of solid samples to address the
analytes listed in the table above. 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 (ig/kg) of VOCs. The method also provides
specific procedures for preparation of samples containing high levels (>200 |ig/kg) of VOCs. For low-
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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 40ฐC. 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 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., SW-846 Methods 8015D (Section
5.2.33) or 8260D (Section 5.2.34)].
Source: U.S. EPA. 2002. "Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for
Volatile Organics in Soil and Waste Samples," Draft Revision 1. Washington, DC: U.S. EPA.
http://www.epa.gov/sites/production/files/2015-07/documents/epa-5Q35a.pdf
5.2.27 EPA Method 6010D (SW-846): Inductively Coupled Plasma - Optical Emission
Spectrometry
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Titanium tetrachloride (analyze as total titanium)
7550-45-0
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
Analysis Purpose: Analyte determination and measurement
Determinative Technique: ICP-AES
Sample Preparation Method: EPA SW-846 Methods 3015A (Section 5.2.16) for non-drinking water
samples, EPA SW-846 Methods 3050B (Section 5.2.17) and 3051A (Section 5.2.18) for solid samples,
and NIOSH Method 9102 (Section 5.2.75) for wipe samples
Sample Preparation Technique: Acid digestion
Method Developed for: Trace elements in solution
Method Selected for: This method has been selected for analysis of non-drinking water, solid and wipe
samples to address the analytes listed in the table above as total arsenic, osmium, thallium, titanium or
vanadium. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Detection limits vary with each analyte and the specific instrument used.
Instrument manufacturer documentation should be consulted for appropriate wavelengths, estimated
instrument detection limits (IDLs) and analytical ranges. The upper end of the analytical range may be
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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. Non-drinking water samples (prepared using SW-846 Method
3015A [Section 5.2.16]), soil samples (prepared using SW-846 Method 3050B [Section 5.2.17] or 3051A
[Section 5.2.18]) and wipe samples (prepared using NIOSH Method 9102 [Section 5.2.75] or SW-846
Method 3051A [Section 5.2.18]) are analyzed by ICP-AES.
Special Considerations: This method uses hydrofluoric acid, which is highly toxic and penetrates the
skin and tissues deeply if not treated immediately. Boric acid and/or other complexing reagents and
appropriate treatment agents (e.g., benzalkonium chloride or calcium gluconate) should be administered
immediately.7 Concerns also 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.
However, the addition of hydrochloric acid can limit quantitation techniques when samples are analyzed
using some ICP-MS instruments. If laboratories are approved for storing and handling the appropriate
standards, then lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide)
can be detected and measured using EPA/600/R-15/258 (Section 5.2.54).
Source: U.S. EPA. 2014. "Method 6010D (SW-846): Inductively Coupled Plasma-Atomic Emission
Spectrometry," Revision 4. Washington, DC: U.S. EPA. https://www.epa.gov/sites/production/files/2015-
12/documents/601 Od .pdf
5.2.28 EPA Method 6020B (SW-846): Inductively Coupled Plasma - Mass Spectrometry
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze a total arsenic)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Titanium tetrachloride (analyze as total titanium)
7550-45-0
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (see Section 5.2.54)
Analysis Purpose: Analyte determination and measurement
Determinative Technique: ICP-MS
7 Medical management guidelines for hydrofluoric acid exposure are provided on the U.S. Centers for Disease and
Prevention (CDC) National Institute for Occupational Safety and Health (NIOSH) Emergency Response Safety and
Health Database at: https://www.cdc.gov/niosh/ershdb/emergencvresponsecard 29750030.html
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Sample Preparation Method: EPA SW-846 Methods 3015A (Section 5.2.16) for non-drinking water
samples, EPA SW-846 Methods 3050B (Section 5.2.17) and 3051A (Section 5.2.18) for solid samples,
and NIOSH Method 9102 (Section 5.2.75) for wipe samples
Sample Preparation Technique: Acid digestion
Method Developed for: Elements in water samples and in waste extracts or digests
Method Selected for: This method has been selected for analysis of non-drinking water, solid and wipe
samples to address the analytes listed in the table above as total arsenic, osmium, thallium, titanium or
vanadium. It has also been selected for analysis of osmium tetroxide in drinking water. See Appendix A
for corresponding method usability tiers.
Detection and Quantitation: IDLs, sensitivities and linear ranges vary with sample type,
instrumentation and operation conditions. 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
osmium tetroxide as total osmium, thallium sulfate as total thallium, titanium tetrachloride as titanium,
and ammonium metavanadate and vanadium pentoxide as total vanadium. Water samples (prepared using
SW-846 Method 3015A [Section 5.2.16]), soil samples (prepared using SW-846 Method 3050B [Section
5.2.17] or 3051A [Section 5.2.18) and wipe samples (prepared using NIOSH Method 9102 [Section
5.2.75] or SW-846 Method 3051A [Section 5.2.18]) are analyzed by ICP-MS.
Special Considerations: If laboratories are approved for storing and handling the appropriate standards,
then lewisites 1, 2 and 3 and their degradation products (CVAOA, CVAA and lewisite oxide) can be
detected and measured using EPA/600/R-15/258 (see Section 5.2.54).
Source: U.S. EPA. 2014. "Method 6020B (SW-846): Inductively Coupled Plasma-Mass Spectrometry,"
Revision 2. Washington, DC: U.S. EPA. https://www.epa.gov/sites/production/files/2Q15-
12/documents/6020b .pdf
5.2.29 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor
Technique)
Analyte(s)
CAS RN
This method has been selected to address the following analytes as total mercury if problems occur when using
EPA Method 245.1 for preparation and analysis of non-drinking water samples.
Mercuric chloride (analyze as total mercury)
7487-94-7
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
151-38-2
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Acid digestion (solid and non-drinking water samples) and acid
digestion by NIOSH Method 9102 (wipe samples)
Determinative Technique: Cold vapor atomic absorption spectrophotometry
Method Developed for: Mercury in mobility-procedure extracts, aqueous wastes and ground waters
Method Selected for: This method has been selected for use if problems occur when using EPA Method
245.1 for preparation and analysis of non-drinking water samples to address the analytes listed in the table
above as total mercury. (See Footnote 11 of Appendix A.)
Detection and Quantitation: The detection limit for total mercury 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
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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 cold vapor atomic absorption
spectrometry.
Special Considerations: Chloride and copper are potential interferences.
Source: U.S. EPA. 1994. "Method 7470A (SW-846): Mercury in Liquid Waste (Manual Cold-Vapor
Technique)," Revision 1. Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-
07/documents/epa-7470a.pdf
5.2.30 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-
Vapor Technique)
Analyte(s)
CAS RN
This method has been selected to address the following analytes as total mercury if problems occur when using
EPA SW-846 Method 7473 for preparation and analysis of solid and wipe samples.
Mercuric chloride (analyze as total mercury)
7487-94-7
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
151-38-2
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Acid digestion for solid and non-drinking water samples, and acid
digestion by NIOSH Method 9102 (Section 5.2.75) for wipe samples
Determinative Technique: Cold vapor atomic absorption spectrophotometry
Method Developed for: Total mercury in soils, sediments, bottom deposits and sludge-type materials
Method Selected for: This method has been selected for use if problems occur when using EPA SW-
846 Method 7473 (Section 5.2.31) during preparation and analysis of solid and wipe samples to address
the analytes listed in the table above as total mercury. (See Footnote 11 of Appendix A.)
Detection and Quantitation: Depending on the instrument used, the IDL for mercury is 0.2 j^ig/L.
Description of Method: A 0.5-g to 0.6-g sample is digested with aqua regia, permanganate solution and
heat, then cooled and reduced with hydroxylamine-sodium chloride solution. Just prior to analysis, the
sample is treated with Sn(II), reducing mercury to Hg(0). The reduced sample is sparged and the mercury
vapor is analyzed by cold vapor atomic absorption spectrophotometry at a wavelength of 253.7 nm.
Special Considerations: Sulfides, chloride and copper are potential interferences.
Source: U.S. EPA. 2007. "Method 7471B (SW-846): Mercury in Solid or Semisolid Waste (Manual
Cold-Vapor Technique)," Revision 2. Washington, DC: U.S. EPA.
https: //www .epa. gov/site s/production/file s/2015 -12/documents/7471b .pdf
5.2.31 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry
Analyte(s)
CAS RN
Mercuric chloride (analyze as total mercury)
7487-94-7
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
151-38-2
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Thermal decomposition (solid samples) and acid digestion by NIOSH
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Method 9102 (wipe samples)
Determinative Technique: Visible spectrophotometry
Method Developed for: Total mercury in solids, aqueous samples and digested solutions
Method Selected for: This method has been selected for preparation of solid samples and for the
analysis of prepared solid and wipe samples to address the analytes listed in the table above as total
mercury. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The IDL for total mercury is 0.01 ng. The typical working range is 0.05-
600 ng, depending on the instrument used.
Description of Method: Controlled heating in an oxygenated decomposition furnace is used to liberate
mercury from 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 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 nanometers (nm) as a function of mercury
concentration.
Special Considerations: If equipment is not available, use Method 7471B (EPA SW-846) for analysis
of solid samples.
Source: U.S. EPA. 2007. "Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal
Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry," Revision 0. Washington,
DC: U.S. EPA. https://www.epa.gov/sites/production/files/2015-07/documents/epa-7473.pdf
5.2.32 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas
Chromatography
Analyte(s)
CAS RN
White phosphorus
12185-10-3
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Solvent extraction (solid samples and water samples) and MSE /
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: This method has been selected for preparation and analysis of solid, water and
wipe samples to address white phosphorus. 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, sandy loam soil, and soil from the Rocky
Mountain Arsenal 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 j^ig/kg for soil samples.
Description of Method: Method 7580 can be used to determine the concentration of white phosphorus
in soil, sediment and water samples using solvent extraction and GC. Water samples are extracted by one
of two procedures, depending on the sensitivity required. For the more sensitive procedure, a 500-mL
water sample is extracted with 50 mL of diethyl ether. The extract is concentrated by back extraction with
reagent water, yielding a final extract volume of approximately 1.0 mL. A 1.0 (iL aliquot of this extract is
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injected into a GC equipped with an NPD. Wet soil or sediment samples are analyzed by extracting a 40 g
wet-weight aliquot of the sample with a mixture of 10.0 mL degassed reagent water and 10.0 mL
isooctane. The extraction is performed in a glass jar on a platform shaker for 18 hours. A 1,0-|_iL aliquot
of the extract is analyzed by GC-NPD.
Special Considerations: The presence of white phosphorus should be confirmed using either a
secondary GC column or an MS.
Source: U.S. EPA. 1996. "Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and
Gas Chromatography," Revision 0. Washington, DC: U.S. EPA.
http://www.epa.gov/sites/production/files/2015-07/documents/epa-758Q.pdf
5.2.33 EPA Method 8015D (SW-846): Nonhalogenated Organics Using GC/FID
Analyte(s)
CAS RN
Diesel range organics
NA
Gasoline range organics
NA
Kerosene
64742-81-0
Analysis Purpose: Analyte determination and measurement
Determinative Technique: GC-FID
Sample Preparation Method: EPA SW-846 Methods 3541 (Section 5.2.22)/3545A (Section 5.2.23) or
5035A (Section 5.2.26) for solid samples, 3520C (Section 5.2.20)/3535A (Section 5.2.21) or 5030C
(Section 5.2.25) for water samples, and 3570 (Section 5.2.24)/8290A Appendix A (Section 5.2.36) for
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 (water samples), and MSE / solvent extraction (wipe samples).
Method Developed for: Nonhalogenated VOCs and semivolatile organic compounds in water and soil
samples
Method Selected for: This method has been selected for analysis of solid, water and wipe samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The method reports that estimated MDLs vary with each analyte and range
from 2 to 48 (ig/L for aqueous samples. 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 samples, and solvent extraction. An appropriate GC column and temperature
program is used to separate the organic compounds, and the compounds are detected and measured by an
FID.
Special Considerations: The presence of the analytes listed in the table above should be confirmed
using either a secondary GC column or an MS.
Source: U.S. EPA. 2003. "Method 8015D (SW-846): Nonhalogenated Organics Using GC/FID,"
Revision 4. Washington, DC: U.S. EPA. https://www .epa. gov/sites/production/files/2015-
12/documents/8015d r4.pdf
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5.2.34 EPA Method 8260D (SW-846): Volatile Organic Compounds by Gas
Chromatography-Mass Spectrometry (GC/MS)
Analyte(s)
CAS RN
Acrylonitrile
107-13-1
Allyl alcohol
107-18-6
Carbon disulfide
75-15-0
2-Chloroethanol
107-07-3
1,2-Dichloroethane
107-06-2
Ethylene oxide
75-21-8
2-Fluoroethanol
371-62-0
Methyl acrylonitrile
126-98-7
Propylene oxide
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 Methods 5035A (Section 5.2.26) for solid samples, 5030C
(Section 5.2.25) for water samples, 3570 (Section 5.2.24)/8290A Appendix A (Section 5.2.36) for wipes.
Sample Preparation Technique: Purge-and-trap (solid samples and water samples) and MSE / solvent
extraction (wipes).
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: This method has been selected for analysis of solid, water and/or wipe samples to
address the analytes listed in the table above. Note. EPA Method 524.2 (Section 5.2.7), rather than 8260D
(Section 5.2.34) should be used for analysis of acrylonitrile, carbon disulfide, 1,2-dichloroethane and
methyl acrylonitrile in drinking water samples. EPA Method 524.2 also should be used for analysis of
acrylonitrile and methyl acrylonitrile in non-drinking water samples. See Appendix A for corresponding
method usability tiers.
Detection and Quantitation: The method reports estimated quantitation limits (EQLs) of 5 jj.g/kg (wet
weight) for soil/sediment samples and 5 (ig/L for ground water, when using quadrupole instrumentation
and purge-and-trap. The method also reports a lower limit of quantitation (LLOQ) of 0.02 (ig/L for 1,2-
dichloroethane. 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 a 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 of this method). 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: U.S. EPA. 2006. "Method 8260D (SW-846): Volatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS)." Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2017-Q4/documents/method 8260d update vi final 03-13-
2017.pdf
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5.2.35 EPA Method 8270E (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC-MS)
Analyte(s)
CAS RN
Chlorfenvinphos
470-90-6
Dichlorvos
62-73-7
Dicrotophos
141-66-2
Ethyldichloroarsine (ED)
598-14-1
Methyl paraoxon1
950-35-6
Methyl parathion
298-00-0
Mevinphos
7786-34-7
Monocrotophos
6923-22-4
Nicotine compounds
54-11-5
Paraoxon
311-45-5
Parathion
56-38-2
Phorate1
298-02-2
Phosphamidon
13171-21-6
Strychnine
57-24-9
Tetraethyl pyrophosphate (TEPP)
107-49-3
Trimethyl phosphite2
121-45-9
1 If problems occur during measurement of oxon compounds, analysts should consider use of procedures included in
Kamal, A. et al., "Oxidation of selected organophosphate pesticides during chlorination of simulated drinking water."
Water Research. 2009. 43(2): 522-534. http://vwvw.sciencedirect.eom/science/article/pii/S0043135408004995.
2 If problems occur with analyses, lower the injection temperature.
Analysis Purpose: Analyte determination and measurement
Determinative Technique: GC-MS
Sample Preparation Method: EPA SW-846 Methods 3541/3545A/3570 (solid samples),
3511/3520C/3535A (water samples), and 3570/8290A Appendix A or NIOSH Method 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), continuous
liquid-liquid extraction/SPE/MSE (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: This method has been selected for analysis of solid, water and/or wipe samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Note.
EPA Method 525.2 (Section 5.2.8) should be used to prepare and analyze drinking water samples
for dichlorvos, disulfoton, fenamiphos and mevinphos; it also should be used to prepare and
analyze non-drinking water samples for disulfoton.
EPA/600/R-16/114 (Section 5.2.55) should be used to prepare and analyze solid and wipe
samples for chlorfenvinphos, dichlorvos, dicrotophos, methyl paraoxon, methyl parathion,
mevinphos, nicotine compounds, paraoxon, parathion, phorate, phosphamidon, strychnine and
TEPP.
Detection and Quantitation: The method reports LLOQs in water ranging from 10 to 100 j^ig/L,
depending on the analyte. EQLs reported in the method vary with each analyte and range between 10 and
40 |ig/L for aqueous samples. Ranges are not provided for these analytes in soil samples. The analytical
range depends on the target analyte(s) and the instrument used.
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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: Lower injection temperatures can alleviate problems that can occur with
analysis of trimethyl phosphite.
Source: U.S. EPA. 2014. "Method 8270E (SW-846): Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS)." Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2017-04/documents/method 8260d update vi final 03-13-
2017 O.pdf
5.2.36 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,
Analysis, and Reporting of Wipe Tests Performed Within the Laboratory
Analyte(s)
CAS RN
Acrylamide
79-06-1
Acrylonitrile
107-13-1
Aldicarb (Temik)
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
4-Aminopyridine
504-24-5
BZ [Quinuclidinyl benzilate]
6581-06-2
Brodifacoum
56073-10-0
Bromadiolone
28772-56-7
Carfentanil
59708-52-0
Carbofuran (Furadan)
1563-66-2
Diesel range organics
NA
Diphacinone
82-66-6
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
73207-98-4
Fentanyl
437-38-7
Formaldehyde
50-00-0
Gasoline range organics
NA
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
121-82-4
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
Kerosene
64742-81-0
Methomyl
16752-77-5
Methyl acrylonitrile
126-98-7
3-Methyl fentanyl
42045-87-4
Methyl hydrazine
60-34-4
Monocrotophos
6923-22-4
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)
2691-41-0
Oxamyl
23135-22-0
Pentaerythritol tetranitrate (PETN)
78-11-5
Thiofanox
39196-18-4
Trimethyl phosphite
121-45-9
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
2,4,6-Trinitrotoluene (2,4,6-TNT)
118-96-7
White phosphorus
12185-10-3
Analysis Purpose: Sample preparation
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Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-NPD / GC-FID / GC-MS / HPLC
Determinative Method: EPA SW-846 Methods 7580 (Section 5.2.32), 8015D (Section 5.2.33), 8260D
(Section 5.2.34), 8270E (Section 5.2.35), 8315A (Section 5.2.37), 8316 (Section 5.2.38), 8318A (Section
5.2.39), 8330B (Section 5.2.40); NHSRC EPA/600/R-15/097 (Section 5.2.53); ASTM Methods D7644-16
(Section 5.2.91) and D7645-16 (Section 5.2.92); adapted from Analyst, 126:1689-1693 (Section 5.2.102),
adapted from J. Chromatogr. 617: 157-162 (Section 5.2.106); adapted from J. Chromatogr. A, 1218:
1620-1649 (Section 5.2.111); adapted from J. Chromatogr. B, 874 (Section 5.2.112): 42-50; or adapted
from J. Chromatogr. B, 962: 52-58 (Section 5.2.115). 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 polychlorinated
dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF) congeners
Method Selected for: This method has been selected for preparation of wipe samples to address the
analytes listed in the table above. 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: The solvent systems described in this method extraction have been evaluated
for PCDD and PCDF congeners only. Other analytes may require different solvent systems for optimal
sample extraction.
Source: U.S. 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.
Washington, DC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-07/documents/epa-8290a.pdf
5.2.37 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC)
Analyte(s)
CAS RN
Formaldehyde
50-00-0
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Solvent extraction (solid and non-drinking water 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: This method has been selected for preparation and analysis of solid, non-drinking
water and wipe samples to address formaldehyde. 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 reagent water.
Description of Method: A measured volume of aqueous sample (approximately 100 mL), or 100 mL of
extract from an appropriate amount of solids (approximately 25 g), is buffered to pH 5 for analysis of
formaldehyde, and derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH). Using the appropriate
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 by absorbance detection at 360 nm.
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Source: U.S. EPA. 1996. "Method 8315A (SW-846): Determination of Carbonyl Compounds by High
Performance Liquid Chromatography (HPLC)," Revision 1. Washington, DC: U.S. EPA.
http://www.epa.gov/sites/production/files/2015-Q7/documents/epa-8315a.pdf
5.2.38 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC)
Analyte(s)
CAS RN
Acrylamide
79-06-1
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Direct injection (water samples), water extraction (solid), and
MSE/solvent extraction by EPA SW-846 Method 3570/8290A Appendix A (wipe samples)
Determinative Technique: HPLC-UV
Method Developed for: Acrylamide, acrylonitrile and acrolein in water samples
Method Selected for: This method has been selected for preparation and analysis of water samples, and
for analysis of prepared solid and wipe samples to address acrylamide. See Appendix A for corresponding
method usability tiers.
Detection and Quantitation: The MDL for acrylamide is 10 (ig/L.
Description of Method: Samples are analyzed by HPLC. A 200-(iL aliquot is injected onto a Ci8
reverse-phase column, and compounds in the effluent are detected with a UV detector. Water samples can
be injected directly into the HPLC; solid samples must be extracted in water prior to injection.
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: U.S. EPA. 1994. "Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High
Performance Liquid Chromatography (HPLC)," Revision 0. Washington, DC: U.S. EPA.
http://www.epa.gov/sites/production/files/2015-Q7/documents/epa-8316.pdf
5.2.39 EPA Method 8318A (SW-846): A/-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)
Analyte(s)
CAS RN
Aldicarb (Temik)
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
Carbofuran (Furadan)
1563-66-2
Methomyl
16752-77-5
Oxamyl
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-FL
Method Developed for: /v'-mcthylcarbamatcs in soil, water and waste matrices
Method Selected for: This method has been selected for preparation and/or analysis of solid and wipe
samples to address the analytes listed in the table above. See Appendix A for corresponding method
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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 j^ig/kg for soil samples.
Description of Method: 7V-methylcarbamates are extracted from aqueous 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: U.S. EPA. 2007. "Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid
Chromatography (HPLC)," Revision 1. Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2015-12/documents/8318a.pdf
5.2.40 EPA Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by
High Performance Liquid Chromatography (HPLC)
Analyte(s)
CAS RN
4-Aminopyridine
504-24-5
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
121-82-4
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)
2691-41-0
Pentaerythritol tetranitrate (PETN)
78-11-5
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
2,4,6-Trinitrotoluene (2,4,6-TNT)
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 (water samples), and MSE / solvent extraction by EPA SW-846 Method
3570/8290A Appendix A (wipe samples)
Determinative Technique: HPLC-UV
Method Developed for: Trace analysis of explosives and propellant residues in water, soil or sediment
Method Selected for: This method has been selected for preparation and/or analysis of solid, water and
wipe samples to address the analytes listed in the table above. Note. Methods 3535A (Section 5.2.21) and
8330B have been selected for preparation of water samples to address these analytes. For HMTD,
procedures adapted from Analyst (2001) 126:1689-1693 (Section 5.2.102) have been selected 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 either are used in the manufacture of explosives and propellants, or are
the degradation products of compounds used for that purpose. Samples are prepared for analysis by
HPLC-UV detection using the appropriate sample preparation technique (SPE by Method 3535A or
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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: U.S. EPA. 2006. "Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by
High Performance Liquid Chromatography (HPLC)," Revision 2. Washington, DC: U.S. EPA.
http://www.epa.gOv/sites/production/files/2015-07/documents/epa-8330b.pdf
5.2.41 EPA ISM02.3 Cyanide: Analytical Methods for Total Cyanide Analysis
Analyte(s)
CAS RN
Cyanide, Total
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: Total cyanide in water, sediment, sludge and soil
Method Selected for: This method has been selected for preparation and analysis of solid, non-drinking
water and wipe samples to address total cyanide. See Appendix A for corresponding method usability
tiers.
Detection and Quantitation: The method quantitation limits for total cyanide 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 nm. To obtain colors of comparable intensity, it is essential to
have the same salt content in both the sample and the standards.
Special Considerations: Midi-distillation is recommended for soil samples to mitigate low analyte
recoveries that can occur when analyzing these sample types. If the appropriate equipment is available,
the in-line distillation procedure described in EPA-821-B-01-009 (Section 5.2.58) can be used when
preparing and analyzing aqueous samples, to shorten analysis time and reduce matrix interferences.
Source: U.S. EPA. "ISM02.3: Exhibit D - Part D: Analytical Methods for Total Cyanide Analysis."
Washington, DC: U.S. EPA. https://www.epa.gov/sites/production/files/2015-10/documents/ism23d.pdf
5.2.42 EPA Method 3135.21: Cyanide, Total and Amenable in Aqueous and Solid Samples
Automated Colorimetric With Manual Digestion
Analyte(s)
CAS RN
Cyanide, Amenable to chlorination
NA
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Acid digestion followed by distillation
Determinative Technique: Visible spectrophotometry
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Method Developed for: Cyanide in drinking, ground and surface waters, domestic and industrial
wastewaters, sediments and solid waste
Method Selected for: This method has been selected for preparation and analysis of solid, water and
wipe samples to address cyanide amenable to chlorination. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: The applicable range is 0.003-0.500 mg/L cyanide in the distillate. This
range can be expanded either by using less sample for distillation or by 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 is released as hydrocyanic acid by refluxing a
sample with strong acid. The hydrocyanic acid is distilled and collected in an absorber-scrubber
containing sodium hydroxide solution. The cyanide ion in the absorbing solution is then determined by
automated colorimetry. For determination of cyanide amenable to chlorination, a portion of the sample is
chlorinated using sodium hypochlorite at a pH > 11 to decompose the cyanide. Cyanide levels are then
determined in both the chlorinated sample portion of the sample and a portion of the sample that has not
been chlorinated using the total cyanide method. Cyanides amenable to chlorination are then calculated
by difference between unchlorinated and the chlorinated aliquots of the sample.
Special Considerations: Alternate cyanide analyzer equipment may be used, provided it is used
according to the procedures described and the laboratory can demonstrate equivalent performance. If
preferred, Standard Method 4500-CN-G (Section 5.2.97) can be used in place of this method for the
analysis of cyanide amenable to chlorination in water samples.
Source: Greenlee, A. 2008. "RLAB Method 3135.21: Cyanide, Total and Amenable in Aqueous and Soil
Samples Automated Colorimetric With Manual Digestion." Lenexa, KS: U.S. EPA Region 7 Laboratory.
http://www.epa.gOv/sites/production/files/2015-07/documents/epa-3135.2i.pdf
5.2.43 EPA 10 [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and
Extraction of Filter Material
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
40334-70-1
Lewisite oxide (analyze as total arsenic)
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid extraction
Determinative Technique: ICP-AES / ICP-MS
Determinative Method: EPA Method 10-3.4 (osmium tetroxide) or Methods 103.4/10-3.5 (all other
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analytes)
Method Developed for: Particulate metals in air
Method Selected for: This method has been selected for preparation of air samples to address the
analytes listed in the table above as total arsenic, osmium, thallium or vanadium. 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, ammonium metavanadate and vanadium pentoxide are determined as total
vanadium, and osmium tetroxide is determined as total osmium. 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 10-3.4 (Section 5.2.44) or Method 10-3.5 (Section 5.2.45).
Source: Mainey, A. and Winberry, W.T. 1999. "10 Compendium Method 10-3.1: Compendium of
Methods for the Determination of Inorganic Compounds in Ambient Air: Selection, Preparation and
Extraction of Filter Material." Cincinnati, OH: U.S. EPA. EPA/625/R-96/010a.
http://www.epa.gOv/sites/production/files/2015-07/documents/epa-io-3.l.pdf
5.2.44 EPA 10 [Inorganic] Compendium Method 10-3.4: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
40334-70-1
Lewisite oxide (analyze as total arsenic)
1306-02-1
Osmium tetroxide (analyze as total osmium)
20816-12-0
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
Analysis Purpose: Analyte determination and measurement
Determinative Technique: ICP-AES
Sample Preparation Method: EPA Method 10-3.1
Sample Preparation Technique: Acid extraction
Method Developed for: Metals in ambient particulate matter
Method Selected for: This method has been selected for analysis of prepared air samples to address the
analytes listed in the table above as total arsenic, osmium, thallium or vanadium. See Appendix A for
corresponding method usability tiers.
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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 10-3.1 (Section 5.2.43).
Detection limits, ranges and interference corrections are dependent on the analyte and the instrument
used.
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.
Sources: Winberry, W.T. 1999. "10 Compendium Method 10-3.4: 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." Cincinnati, OH: U.S. EPA. EPA/625/R-
96/010a. http://www.epa.gOv/sites/production/files/2015-07/documents/epa-io-3.4.pdf
Winberry, W.T. 1999. "10 Compendium Method 10-2.1: Compendium of Methods for the Determination
of Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate
Matter (SPM) and PMi0 Using High Volume (HV) Sampler." Cincinnati, OH: U.S. EPA. EPA/625/R-
96/010a. http://www.epa.gov/sites/production/files/2015-07/documents/epa-io-2.1 .pdf
5.2.45 EPA 10 [Inorganic] Compendium Method 10-3.5: Determination of Metals in
Ambient Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry
(ICP-MS)
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Calcium arsenate (analyze as total arsenic)
7778-44-1
2-Chlorovinylarsonic acid (CVAOA) (analyze as total arsenic)
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)
85090-33-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)
40334-70-1
Lewisite oxide (analyze as total arsenic)
1306-02-1
Sodium arsenite=(analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
Analysis Purpose: Analyte determination and measurement
Determinative Technique: ICP-MS
Sample Preparation Method: EPA Method 10-3.1
Sample Preparation Technique: Acid extraction
Method Developed for: Metals in ambient particulate matter
Method Selected for: This method has been selected for analysis of prepared air samples to address the
analytes listed in the table above as total arsenic, thallium or vanadium. See Appendix A for
corresponding method usability tiers.
Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation
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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 10-3.1 (see Section 5.2.43). Detection limits, ranges and interference
corrections are dependent on the analyte and the instrument used.
Source: Winberry, W.T. 1999. "10 Compendium Method 10-3.5: 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)." Cincinnati, OH: U.S. EPA.
EPA/625/R-96/010a. http://www.epa.gov/sites/production/files/2015-07/documents/epa-io-3.5 .pdf
Winberry, W.T. 1999. "10 Compendium Method 10-2.1: Compendium of Methods for the Determination
of Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate
Matter (SPM) and PMi0 Using High Volume (HV) Sampler." Cincinnati, OH: U.S. EPA. EPA/625/R-
96/010a. http://www.epa.gov/sites/production/files/2015-07/documents/epa-io-2.1 .pdf
5.2.46 EPA 10 [Inorganic] Compendium Method 10-5: Sampling and Analysis for Vapor
and Particle Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic
Fluorescence Spectrometry (CVAFS)
Analyte(s)
CAS RN
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
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: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above as total mercury. 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, and the desorbed
mercury 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, and 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: Keele, G. and Barres, J. 1999. "10 Compendium Method 10-5: 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)."
Cincinnati, OH: U.S. EPA. EPA/625/R-96/010a. http://www.epa.gov/sites/production/files/2015-
07/documents/epa-io-5 .pdf
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5.2.47 EPA Air Method, Toxic Organics - 10A (TO-10 A): 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)
CAS RN
BZ [Quinuclidinyl benzilate]1
6581-06-2
Chlorfenvinphos
470-90-6
3-Chloro-1,2-propanediol2
96-24-2
Chlorpyrifos
2921-88-2
Chlorpyrifos oxon
5598-15-2
Dichlorvos
62-73-7
Dicrotophos
141-66-2
Diisopropyl methylphosphonate (DIMP)2
1445-75-6
Dimethylphosphite
868-85-9
Dimethylphosphoramidic acid1
33876-51-6
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]1
73207-98-4
Ethyl methylphosphonic acid (EMPA)1
1832-53-7
Fenamiphos
22224-92-6
Isopropyl methylphosphonic acid (IMPA)1
1832-54-8
Methyl paraoxon
950-35-6
Methyl parathion
298-00-0
Methylphosphonic acid (MPA)1
993-13-5
Mevinphos
7786-34-7
Monocrotophos
6923-22-4
Paraoxon
311-45-5
Parathion
56-38-2
Phencyclidine
77-10-1
Phorate
298-02-2
Phorate sulfone
2588-04-7
Phorate sulfone oxon
2588-06-9
Phorate sulfoxide
2588-03-6
Phorate sulfoxide oxon
2588-05-8
Phosphamidon
13171-21-6
Pinacolyl methyl phosphonic acid (PMPA)1
616-52-4
Tetraethyl pyrophosphate (TEPP)
107-49-3
Tetramethylenedisulfotetramine
80-12-6
Thiodiglycol (TDG)
111-48-8
Trimethyl phosphite
121-45-9
The following analyte should be determined by this method only if problems (e.g., insufficient recovery,
interferences) occur when using Method TO-15.
Allvl 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 Method TO-15 be used.
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS orHPLC-UV
Method Developed for: Pesticides and polychlorinated biphenyls in ambient air
Method Selected for: This method has been selected for preparation and analysis of air samples to
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address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The limit of detection (LOD) will depend on the specific compound
measured, the concentration level, and the degree of specificity required. This method is applicable to
multicomponent atmospheres, 0.001-50 (ig/m3 concentrations, and 4-24-hour sampling periods.
Description of Method: A low-volume sample collection rate (1-5 L/minute) is used to collect vapors
on a sorbent cartridge containing polyurethane foam (PUF) in combination with another solid sorbent.
Airborne particles also are collected, but the sampling efficiency for particulates 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 is preferable. HPLC-
UV is also the preferred technique for BZ, dimethylphosphoramidic acid, EA2192, EMPA, IMPA, MPA
and PMPA. If problems occur when using HPLC-UV, these analytes can be analyzed by GC-MS but
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. Also see Special Considerations for EPA SW-
846 8270E (Section 5.2.35) for information regarding derivatization of compounds.
Source: Lewis, R.G. 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)." Cincinnati, OH: U.S. EPA.
EPA 625/R-96/010b. https://www.epa.gov/sites/production/files/2015-07/documents/epa-to-10a.pdf
Additional Resource: Karmel, A., Byrne, C., Bigo, C., Ferrario, J., Stafford, C., Verdin, G., Siegelman,
F., Knizner, S. and Hetrick, J.. 2009. "Oxidation of Selected Organophosphate Pesticides During
Chlorination of Simulated Drinking Water." Water Research. 43(2): 522-534.
http://www.sciencedirect.com/science/article/pii/S00431354080Q4995
5.2.48 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)
CAS RN
Allyl alcohol
107-18-6
Carbon disulfide
75-15-0
Cyanogen chloride
506-77-4
1,2-Dichloroethane
107-06-2
Ethyldichloroarsine (ED)
598-14-1
Ethylene oxide
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 or TO-17.
3-Chloro-1,2-propanediol
96-24-2
Chlorosarin
1445-76-7
Chlorosoman
7040-57-5
Diisopropyl methylphosphonate (DIMP)
1445-75-6
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
1189-87-3
Sarin (GB)
107-44-8
Soman (GD)
96-64-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Samples are collected using canisters.
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Determinative Technique: GC-MS
Method Developed for: VOCs in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. 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
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 sample volume 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 (Section 5.2.47) be used. In cases where lower detection levels are
needed, use procedures included in the supplement to 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: McClenny, W.A. and Holdren, M.W. 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)." Cincinnati, OH: U.S.
EPA. EPA 625/R-96/010b.
http://wipp.energv.gov/librarv/Information Repository A/Supplemental Information/EPA%201999/TQ-
15.pdf
5.2.49 EPA Air Method, Toxic Organics - 17 (TO-17): Determination of Volatile Organic
Compounds in Ambient Air Using Active Sampling Onto Sorbent Tubes
Analyte(s)
CAS RN
Chlorosarin*
1445-76-7
Chlorosoman*
7040-57-5
Cyclohexyl sarin (GF)
329-99-7
1-Methylethyl ester ethylphosphonofluoridic acid (GE)*
1189-87-3
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
538-07-8
Mustard, nitrogen (HN-2)
[2,2'-dichloro-N-methyldiethylamine N,N-bis(2-chloroethyl) methylamine]
51-75-2
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
555-77-1
Mustard sulfur / Mustard gas (HD)
505-60-2
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
159939-87-4
Sarin (GB)*
107-44-8
Soman (GD)*
96-64-0
Tabun (GA)
77-81-6
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Analyte(s)
CAS RN
VE [phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
21738-25-0
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) O.O-diethyl ester]
78-53-5
VM [phosphonothioic acid, methyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
21770-86-5
VX [0-ethyl-S-(2-diisopropylaminoethyl)methyl-phosphonothiolate]
50782-69-9
*lf problems occur when using this method, it is recommended that Method TO-15 be used.
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Thermal desorption
Determinative Technique: GC-MS
Method Developed for: VOCs
Method Selected for: This method has been selected for preparation and analysis of CWAs in air
samples. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The LOD will depend on the specific compounds measured, the
concentration level, and the degree of specificity required. This method is applicable to multicomponent
atmospheres, 2.86 to 275 (ig/m3 concentrations, and 1 to 24-hour sampling periods.
Description of Method: A low-volume (10 to 200 mL/minute) sample collection rate is used to collect
vapors on a sorbent tube. Airborne particles also are collected, but the sampling efficiency for particulates
is not known. Compounds are then thermally desorbed from the sorbent tube and determined by GC-MS.
Special Considerations: Refer to the footnote provided in analyte table above for special considerations
that should be applied when measuring specific analytes. Higher volume sampling flow rates can be used
for high boiling materials such as the V-agents.
Source: Woolfenden, E.A. and McClenny, W.A. 1999. "Air Method, Toxic Organics-17 (TO-17):
Compendium of Methods for the Determination of Toxic Organic Compounds in Ambient Air:
Determination of Volatile Organic Compounds in Ambient Air Using Active Sampling Onto Sorbent
Tubes." Cincinnati, OH: U.S. EPA. EPA/625/R-96/010b.
https://www3 .epa.gov/ttnamti 1/files/ambient/airtox/to-17r.pdf
5.2.50 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)
CAS RN
/V-Ethyldiethanolamine (EDEA)
139-87-7
/V-Methyldiethanolamine (MDEA)
105-59-9
Triethanolamine (TEA)
102-71-6
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Extracted using sonication, and filtered using a syringe-
polyvinylidene fluoride (PVDF) filter unit
Determinative Technique: LC-MS-MS
Method Developed for: TEA, EDEA and MDEA in wipe surfaces
Method Selected for: This method has been selected for preparation and analysis of wipe samples and
for the analysis of prepared solid samples to address the analytes listed in the table above. See Appendix
A for corresponding method usability tiers. Note. SW-846 Methods 3541/3545A should be used for
preparation of solid samples.
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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 quantitations (LOQs) for EDEA, MDEA and TEA are 0.63, 0.69, and
1.23 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 0-6ฐC 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 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.
Special Considerations: A more recent procedure based on this method is provided in the additional
resource cited below. This procedure uses a lower calibration curve and modified LC-MS-MS instrument
conditions.
Source: U.S. 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)."
Cincinnati, OH: U.S. EPA. EPA/600/R-11/143.
https://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc%2F&dirEntrvId=238641
Additional Resource: Dynamac Corporation. 2012. "Standard Operating Procedure for the
Determination of Ethanolamines," Dynamac SOP L-A-303 Rev. 2. Copies of this analytical protocol may
be requested from NHSRC at https://www.epa.gov/homeland-securitv-research/forms/contact-us-about-
homeland-securitv-research
5.2.51 EPA/600/R-12/653: Verification of Methods for Selected Chemical Warfare Agents
(CWAs)
Analyte(s)
CAS RN
Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine]
538-07-8
Mustard, nitrogen (HN-2) [2,2'-dichloro-N-methyldiethylamine N,N-bis(2-chloroethyl) methylamine]
51-75-2
Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine]
555-77-1
R 33 (VR) [methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester]
159939-87-4
Tabun (GA)
77-81-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Microscale extraction
Determinative Technique: GC-MS
Method Developed for: HN-1, HN-3, VR and GA in solid, water and/or wipe samples
Method Selected for: This method has been selected for preparation and analysis of solid, water and/or
wipe samples to address the analytes listed in the table above.
Detection and Quantitation: IDLs for GA, HN-1, HN-3 and R-33, respectively, are: 0.8, 0.025, 0.025
and 0.025 ng/^L (GC-MS full scan); 0.1, 0.01, 0.01 and 0.01 ng/^L (GC-MS SIM); and 0.1, 0.025, 0.005
and 0.02 ng/(.iL (GC-MS time of flight [TOF]). IDLs for GC-MS full-scan using the total ion
chromatogram (TIC) for GA, HN-1, HN-3, and R 33, respectively, are: 0.4, 0.05, 0.025 and 0.025 ng/|_iL.
MDLs for GC-MS full-scan in soils ranged from: 8 to 106 jj.g/kg for GA, 35 to 81 (ig/kg for HN-1, 57
to 243 jj.g/kg for HN-3, and 81 to 213 (ig/kg for R 33.
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MDLs for GC-MS TOF in soils ranged from 0.33 to 0.39 (ig/kg for GA, 0.57 to 2.3 (ig/kg for HN-1,
1.6 to 12 (ig/kg for HN-3, and 15 to 49 (ig/kg for R 33.
MDLs for GA, HN-1, HN-3 and R 33 in reagent water using GC-MS full-scan are 16, 1.8, 20 and 69
(ig/L, respectively.
MDLs for GA, HN-1, HN-3 and R 33 in reagent water using GC-MS TOF are 0.13, 0.084, 0.72 and
22 (ig/L, respectively.
MDLs for GA, HN-1, HN-3 and R 33 in wipes using GC-MS full-scan are 0.11, 0.023, 0.35, and 4.41
ng/cm2, respectively.
Calibration ranges for GC-MS full scan are 0.025-1.0 ng/|_iL for GA, HN-1 and HN-3 and 0.8-3.0 ng/(.iL
for R 33. Calibration ranges for GC-MS SIM are 0.01-0.25 ng/(iL for GA, HN-1 and HN-3 and 0.1-1.0
ng/(.iL for R 33. Calibration ranges for GC-MS TOF are 0.01-1 for GA, HN-1 and HN-3 and 0.10-5 for R
33.
Description of Method: Water samples are extracted by adding -8.8 g of sodium chloride to 35-mL of
water sample. Surrogates and 2 mL of methylene chloride are added. The samples are extracted on a
shaker table for 2 minutes and the layers are allowed to separate. The methylene chloride layer is
collected, dried with anhydrous sodium sulfate, and 1 mL is transferred to an autosampler vial. Solid
samples are extracted by mixing 10 g of solid, 2.5 g of anhydrous sodium sulfate, 5-10 glass beads, and
25 mL of a 25/50/25 (v/v/v) mixture of acetone/methylene chloride/ethyl acetate. The solid samples are
then sonicated in a water bath for 1 hour. The extract is retained, and dried with an additional 1-2 g of
sodium sulfate. The sample is then re-extracted by water bath sonication for an additional hour using 25
mL of 5% TEA/95% ethyl acetate. The second extract is retained and dried with 1-2 g of sodium sulfate.
The first and second extracts are separately reduced in volume under nitrogen, and 1 mL of each extract is
transferred to a separate autosampler vial. Internal standards are added to both extracts, and the extracts
are analyzed by GC-MS.
Special Considerations: During method development studies for the analytical protocol described in the
EPA/600/R-16/114 (Section 5.2.55), ethyl acetate and TEA were found to produce chromatographic
interferences. In addition, ethyl acetate has a higher boiling point than other solvents (e.g., methylene
chloride), resulting in a longer nitrogen blowdown step than if other solvents are used. Alternative solvent
systems used for similar compounds (see methods in Sections 5.2.56 and 5.2.57) may result in improved
chromatography. The method has been single-laboratory tested in reagent water, sand, soil and wipes. The
procedures are specifically for use by laboratories with EPA approval for handling and analysis of
samples and standards containing CWAs.
Source: U.S. EPA. 2013. "Verification of Methods for Selected Chemical Warfare Agents (CWAs)."
Washington, DC: U.S. EPA. EPA/600/R-12/653.
https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=248575
5.2.52 EPA/600/R-13/224: Surface Analysis of Nerve Agent Degradation Products by
Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
CAS RN
Diisopropyl methylphosphonate (DIMP) (degradation product of GB)
1445-75-6
Dimethylphosphoramidic acid (degradation product of GA)
33876-51-6
Ethyl methylphosphonic acid (EMPA)
1832-53-7
Isopropyl methylphosphonic acid (IMPA)
1832-54-8
Methylphosphonic acid (MPA)
993-13-5
Pinacolyl methyl phosphonic acid (PMPA)
616-54-4
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Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Extracted using sonication and filtered using a syringe- PVDF filter
unit
Determinative Technique: LC-MS-MS
Method Developed for: DIMP, EMPA, IMPA, MPA and PMPA in wipe samples
Method Selected for: This method has been selected for analysis of wipe samples to address the
analytes listed in the table above.
Detection and Quantitation: The working range of this method is 0.10-3.0 ng/cm2 for EMPA; 0.25-
7.50 ng/cm2 for IMPA and MPA; and 0.05-1.5 ng/cm2 for DIMP and MPA. MDLs obtained from wiping
a laminate surface for EMPA, IMPA, MPA and PMPA are reported as 0.05, 0.04, 0.07 and 0.02 ng/cm2,
respectively. Method reporting limits for EMPA, IMPA, MPA and PMPA are 0.1, 0.25, 0.25, 0.05
ng/cm2, respectively.
Description of Method: Wipe samples are spiked with surrogates and 5-mL of LC-MS grade water. The
sample solution is then sonicated and extracted with a syringe filter unit, and the extract is analyzed
directly by LC-MS-MS operated simultaneously in positive and negative ESI modes. Each target
compound is separated chromatographically and identified by retention time and by comparison of the
primary MRM transition for the sample to the reference spectra of MRM transition for known standards.
The concentration of each analyte is determined using external calibration. Surrogates are used to monitor
extraction efficiency.
Special Considerations: This procedure uses cotton gauze wipes, which were determined to provide the
highest analyte recoveries with the least interference. Other wipes, such as filter paper or glass fiber filters
had comparable recoveries and could be appropriate alternatives, but are not as sturdy. Data described in
this procedure refer to ESI- mode because some complications can occur in ESI+ mode. Recoveries of
DIMP may be problematic due to the volatility or rapid decomposition. Because wood surfaces resulted in
poor recoveries (likely due to surface porosity), the method recommends that it should not be used to
identify these analytes on wood surfaces. The method does not include analysis of
dimethylphosphoramidic acid, and method modifications (e.g., pH adjustment) may be needed when
analyzing samples for this compound.
Source: U.S. EPA and CDC. 2013. "Surface Analysis of Nerve Agent Degradation Products by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS)." Cincinnati, OH: U.S. EPA. EPA/600/R-
13/224. https://www.hsdl.org/?abstract&did=746488
5.2.53 EPA/600/R-15/097: Adaptation of the Conditions of U.S. EPA Method 538 for the
Analysis of a Toxic Degradation Product of Nerve Agent VX (EA2192) in Water by
Direct Aqueous Injection- Liquid Chromatography/Tandem Mass Spectrometry
Analyte(s)
CAS RN
EA2192 [S-2-(diisopropylamino)ethyl methylphosphonothioic acid]
73207-98-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Direct injection (water samples), EPA SW-846 Method 3541/3545A
(solid samples) and EPA SW-846 Method 3570/8290A Appendix A (wipes)
Determinative Technique: LC-MS-MS
Method Developed for: EA2192 in water
Method Selected for: This method has been selected for preparation and analysis of water samples, and
for analysis of prepared solid samples and wipes to address EA2192.
Detection and Quantitation: The detection limit for EA2192 in deionized water is 0.0130 (ig/L. The
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minimum reporting level in deionized water is 0.125 j^ig/L. The suggested calibration range is 0.05-20
l-ig/L-
Description of Method: A 40-mL water sample is collected in a bottle containing sodium omadine
(antimicrobial agent) and ammonium acetate. An aliquot of sample is placed in an autosampler vial with
the internal standard added. A 50-f.iL injection is made into an LC equipped with a Ci8 column interfaced
to an MS-MS operated in ESI+ mode. 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.
Special Considerations: The method has been tested in deionized water and various drinking waters,
including chlorinated and chloraminated surface and ground waters. EA2192 is highly toxic; therefore,
the procedures are specifically for use by laboratories with EPA approval for handling and analysis of
samples and standards containing CWAs.
Source: U.S. EPA. 2016. "Adaptation of the Conditions of U.S. EPA Method 538 for the Analysis of a
Toxic Degradation Product of Nerve Agent VX (EA2192) in Water by Direct Aqueous Injection- Liquid
Chromatography/Tandem Mass Spectrometry." Cincinnati, OH: U.S. EPA. EPA/600/R-15/097.
https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=311259
5.2.54 EPA/600/R-15/258: Extraction and Analysis of Lewisite 1, by its Degradation
Products, Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
CAS RN
2-Chlorovinylarsonic acid (CVAOA)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA)*
85090-33-1
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine]*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine]*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine]*
40334-70-1
Lewisite oxide*
1306-02-1
* In cases where standards are not available or increased sample throughput is needed, these compounds also can
be addressed by analyzing samples for total arsenic (see Appendix A for appropriate ICP-AES or -MS methods).
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: LC-MS-MS
Method Developed for: Extraction and analysis of lewisite I by its degradation products (CVAA and
CVAOA)
Method Selected for: This method has been selected for preparation and analysis of solid, water and
wipe samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: This method detects both CVAA and CVAOA as total CVAOA. Detection
limits for CVAOA are 0.041 mg/L for water, 0.38 (ig/wipe for wipes, 0.073 jj.g/g for sand, 0.032 jj.g/g for
Nebraska soil, 0.028 jj.g/g for Virginia soil and 0.055 jj.g/g for Georgia soil. The suggested calibration
range for CVAOA is 0.02-0.2 (ig/mL.
Description of Method: Phenyl arsonous acid is added to all samples as a surrogate prior to sample
extraction and analysis. Water samples are mixed thoroughly. An acidified (with hydrochloric acid)
methanolic solution is added to soil samples, followed by agitation on a shaker table for 30 minutes. Soil
samples are then allowed to settle by gravity before an aliquot of the extract (liquid layer) is taken. A
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Section 5.0 - Selected Chemical Methods
dilute aqueous solution of hydrochloric acid is added to wipe samples followed by agitation on a shaker
table for 30 minutes. Prior to analysis by LC-MS-MS, hydrogen peroxide is added to all extracts to
completely degrade target analytes to CVAOA and the surrogate to phenyl arsenic acid.
Special Considerations: In cases where standards are not available or increased sample throughput is
needed, these compounds also can be addressed by analyzing samples for total arsenic (see Appendix A
for appropriate ICP-AES or -MS methods and corresponding method usability tiers).
Source: U.S. EPA. 2015. "Extraction and Analysis of Lewisite 1, by its Degradation Products, Using
Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)," Revision 1. Washington, DC: U.S.
EPA. EPA/600/R-15/258. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=310272
5.2.55 EPA/600/R-16/114: Analytical Protocol for Measurement of Extractable
Semivolatile Organic Compounds Using Gas Chromatography/Mass Spectrometry
Analyte(s)
CAS RN
Chlorfenvinphos
470-90-6
Chloropicrin1
76-06-2
Chlorpyrifos
2921-88-2
Chlorpyrifos oxon
5598-15-2
Crimidine
535-89-7
Dichlorvos
62-73-7
Dicrotophos
141-66-2
Dimethylphosphite
868-85-9
Disulfoton
298-04-4
Disulfoton sulfone oxon
2496-91-5
Disulfoton sulfoxide
2497-07-6
Disulfoton sulfoxide oxon
2496-92-6
1,4-Dithiane
505-29-3
Fenamiphos
22224-92-6
Methyl paraoxon
950-35-6
Methyl parathion
298-00-0
Mevinphos
7786-34-7
Nicotine compounds
54-11-5
Paraoxon
311-45-5
Parathion
56-38-2
Phencyclidine
77-10-1
Phorate
298-02-2
Phorate sulfone
2588-04-7
Phorate sulfone oxon
2588-06-9
Phorate sulfoxide
2588-03-6
Phorate sulfoxide oxon
2588-05-8
Phosphamidon
13171-21-6
Strychnine
57-24-9
Tetraethyl pyrophosphate (TEPP)
107-49-3
T etramethylenedisulfotetramine
80-12-6
1,4-Thioxane2
15980-15-1
1 If problems occur with analyses, lower the injection temperature.
2 If problems occur when using this method, it is recommended that SW-846 Method 8260D and appropriate
corresponding sample preparation procedures (i.e., Method 5035A [Section 5.2.26] for solid samples and Method
5030C [Section 5.2.25] for water samples) be used.
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Sample Preparation Technique: Microscale extraction
Determinative Technique: GC-MS
Method Developed for: Semivolatile organic compounds in extracts prepared from solid waste matrices,
soils, air sampling media and water samples
Method Selected for: This method has been selected for analysis of solid, water and/or wipe samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Note.
EPA Method 525.2 (Section 5.2.8) has been selected for preparation and analysis of drinking
water samples for chlorpyrifos, dichlorvos, fenamiphos and mevinphos.
EPA Method 525.2 (Section 5.2.8) also has been selected for preparation and analysis of water
samples for disulfoton, disulfoton sulfone oxon, disulfoton sulfoxide and disulfoton sulfoxide
oxon.
EPA Method 525.3 (Section 5.2.9) has been selected for preparation and analysis of drinking
water samples for phosphamidon.
EPA Method 540 (Section 5.2.12) has been selected for preparation and analysis of water samples
for chlorpyrifos oxon, phorate sulfone, phorate sulfone oxon, phorate sulfoxide and phorate
sulfoxide oxon.
EPA Method 551.1 (Section 5.2.14) has been selected for preparation and analysis of water
samples for chloropicrin.
SW-846 Method 8270E (Section 5.2.35) has been selected for analysis of prepared water samples
for chlorfenvinphos, dicrotophos, methyl paraoxon, methyl parathion, nicotine compounds,
paraoxon, parathion, phorate, strychnine and TEPP (see Appendix A for the appropriate sample
preparation methods).
SW-846 Method 8270E (Section 5.2.35) also has been selected for analysis of prepared non-
drinking water samples for dichlorvos, mevinphos and phosphamidon (see Appendix A for the
appropriate sample preparation methods).
All other analyte/sample type combinations should be prepared and analyzed by this method.
Detection and Quantitation: MDL and Quantitation Limit (QL) ranges, when performing full-scan
analysis of aqueous samples, are 0.79-4.0 and 28.6-286 |ig/L. respectively; and 1.6-93.1 |ig/L and 50-
1200 (ig/kg, respectively, for soil samples. MDL and QL ranges when performing SIM analysis of
aqueous samples are 0.030-1.45 and 0.23-114 |ig/L. respectively; and 0.047-15.4 and 0.4-80 (.ig/kg.
respectively, for soil samples. The analytical range depends on the target analyte(s) and the mode of
analysis used (i.e., full-scan or SIM).
Description of Method: Prior to analysis, surrogates, sodium chloride and methylene chloride are added
to aqueous, soil and wipe samples and prepared by MSE. Extracts are dried by the addition of sodium
sulfate, concentrated (if necessary in order to achieve appropriate detection and quantitation) by nitrogen
evaporation, and then analyzed by GC-MS in full-scan or SIM mode.
Special Considerations: Laboratory results indicate that improved recovery of alkaline compounds
(e.g., strychnine, nicotine compounds, crimidine, and phencyclidine) from water may result when
extracting samples under acidic conditions (e.g., pH <2) during the first extraction, followed by back
extraction under basic conditions. If problems occur with the analysis of chloropicrin, lower the injection
temperature. If problems occur when analyzing for 1,4-thioxane, it is recommended that SW-846 Method
8260D and appropriate corresponding sample preparation procedures (i.e., Method 5035A [Section
5.2.26] for solid samples or Method 5030C [Section 5.2.25] for water samples) be used.
Source: U.S. EPA. 2016. "Analytical Protocol for Measurement of Extractable Semivolatile Organic
Compounds Using Gas Chromatography/Mass Spectrometry." Cincinnati, OH: U.S. EPA. EPA/600/R-
16/114. https://cfbub.epa.gov/si/si public file download.cfm?p download id=532353
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Section 5.0 - Selected Chemical Methods
5.2.56 EPA/600/R-16/115: Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and
Sulfur Mustard Using Gas Chromatography/Mass Spectrometry
Analyte(s)
CAS RN
Chlorosarin
1445-76-7
Chlorosoman
7040-57-5
Cyclohexyl sarin (GF)
329-99-7
1-Methylethyl ester ethylphosphonofluoridic acid (GE)
1189-87-3
Mustard, sulfur / Mustard gas (HD)
505-60-2
Sarin (GB)
107-44-8
Soman (GD)
96-64-0
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 and HD in water, soil and wipes
Method Selected for: This method has been selected for preparation and analysis of water, solid and
wipe samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: The calibration ranges in full scan mode are 11.4-114 j^ig/L (GB and GF)
and 5.7-57 (ig/L (GD and HD) for water samples; 20-200 j^ig/kg (GB and GF) and 10-100 j^ig/kg (GD
and HD) for soil samples; and 0.02-0.2 jig/cnr (GB and GF) and 0.01-0.1 |_ig/cnr (GD and HD) for
wipes.
Description of Method: The method involves solvent extraction of the sample followed by GC-MS
analysis to determine cyclohexyl sarin, sarin, soman and HD in water, soil and wipes. Prior to analysis,
samples must be prepared using sample preparation techniques appropriate for each sample type.
Aqueous, solid and wipe samples are spiked with surrogates and extracted by microscale extraction, using
methylene chloride. Water is removed from extracts with anhydrous sodium sulfate, and the extracts are
concentrated (solids and wipe extracts only) by nitrogen evaporation, then analyzed by GC-MS using a
mass selective detector in either full scan mode or TOF.
Special Considerations: This method has been tested in multiple laboratories for analysis of cyclohexyl
sarin, sarin, soman and HD in reagent water, drinking water, groundwater, sand and wipes. The
procedures are specifically for use by laboratories with EPA approval for handling and analysis of
samples and standards containing CWAs.
Source: U.S. EPA. 2016. "Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard
Using Gas Chromatography/Mass Spectrometry." Cincinnati, OH: U.S. EPA. EPA/600/R-16/115.
https://cfbub.epa.gov/si/si public file download.cfm?p download id=532354
5.2.57 EPA/600/R-16/116: Analytical Protocol for VX Using Gas Chromatography/Mass
Spectrometry (GC/MS)
Analyte(s)
CAS RN
VE [phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
21738-25-0
VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) O.O-diethyl ester]
78-53-5
VM [phosphonothioic acid, methyl-, S-(2-(diethylamino)ethyl) O-ethyl ester]
21770-86-5
VX [0-ethyl-S-(2-diisopropylaminoethyl)methyl-phosphonothiolate]
50782-69-9
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Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Microscale extraction
Determinative Technique: GC-MS
Method Developed for: Determination of VX in water, soil and wipes
Method Selected for: This method has been selected for preparation and analysis of water, solid and
wipe samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: The calibration ranges for analysis of VX using full scan mode are 11.4-
114 (ig/L for water samples; 20-200 jj.g/kg for soil samples; and 0.02-0.2 (ig/cm2 for wipes.
Description of Method: The method involves micro-scale solvent extraction of samples followed by
GC-MS analysis. Prior to analysis, samples must be prepared using sample preparation techniques
appropriate for each sample type. Aqueous and wipe samples are spiked with surrogates and extracted
using methylene chloride. Solid samples are spiked with surrogates, then extracted first using a Tris
buffer solution, followed by extraction with methylene chloride. Water is removed from extracts using
anhydrous sodium sulfate, and the extracts are 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 TOF.
Special Considerations: The method has been tested in multiple laboratories for analysis of VX in
reagent water, drinking water, groundwater, soil and wipes. Laboratory data indicate some difficulties
with analyte recoveries in soil; modifications might be needed for application of the procedures to various
soil types. The procedures are specifically for use by laboratories with EPA approval for handling and
analysis of samples and standards containing CWAs.
Source: U.S. EPA. 2016. "Analytical Protocol for VX Using Gas Chromatography/Mass Spectrometry
(GC/MS)." Cincinnati, OH: U.S. EPA. EPA/600/R-16/116.
https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=337633
5.2.58 EPA-821 -B-01 -009: Method Kelada-01: Kelada Automated Test Methods for Total
Cyanide, Acid Dissociable Cyanide, and Thiocyanate
Analyte(s)
CAS RN
Cyanide, Total
57-12-5
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: On-line UV irradiation followed by flash distillation
Determinative Technique: Visible spectrophotometry
Method Developed for: Total cyanide, acid dissociable cyanide and thiocyanate in water, sediment,
sludge and soil
Method Selected for: This method has been selected as an alternative to ISM02.3 for preparation and
analysis of non-drinking water samples to address total cyanide.
Detection and Quantitation: The LOD is 0.5 |_ig/L. The working range is 0-100 (ig/L.
Description of Method: The method uses a combined on-line UV irradiation and flash distillation
system in place of manual cyanide distillation procedures to determine total cyanide. Strongly-bound
cyanide complexes (excluding thiocyanate) are degraded into free cyanide by irradiating the sample in a
glass coil. The free cyanide is distilled from the sample matrix and detected using an on-line colorimeter.
The concentration of dissociable cyanide complexes is determined by omitting the UV-irradiation step.
Thiocyanate can also be determined by using a glass irradiation coil instead of a quartz coil.
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Special Considerations: The method was evaluated under EPA's Alternate Test Procedure (ATP)
program and can be used in place of ISM02.3 to prepare and analyze aqueous samples for total cyanide.
Source: U.S. EPA. 2001. "Method Kelada-01: Kelada Automated Test Methods for Total Cyanide, Acid
Dissociable Cyanide, and Thiocyanate." Washington, DC: U.S. EPA. EPA-821-B-01-009.
http://webappl.dlib.indiana.edu/virtual disk librarv/index.cgi/5315321/FID2672/kelada.pdf
5.2.59 NIOSH Method 1612: Propylene Oxide
Analyte(s)
CAS RN
Propylene oxide
75-56-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Coconut shell charcoal solid sorbenttube
Determinative Technique: GC-FID
Method Developed for: Propylene oxide in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address propylene oxide. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range is 8-295 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.
Special Considerations: No interferences have been found. The presence of propylene oxide should be
confirmed using either a secondary GC column or an MS.
Source: NIOSH. 1994. "Method 1612: Propylene Oxide," Issue 2. NIOSH Manual of Analytical
Methods, 4th Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-Q7/documents/niosh- 1612.pdf
5.2.60 NIOSH Method 2016: Formaldehyde
Analyte(s)
CAS RN
Formaldehyde
50-00-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: HPLC-UV
Method Developed for: Formaldehyde in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address formaldehyde. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The detection limit for formaldehyde is 0.07 (ig/sample. The working
range is 0.015-to 2.5 mg/m3 (0.012-2.0 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.
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Special Considerations: 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. NIOSH Manual of Analytical Methods,
Third Supplement. DHHS (NIOSH) Publication No. 2003-154. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-2Q16.pdf
5.2.61 NIOSH Method 2513: Ethylene Chlorohydrin
Analyte(s)
CAS RN
2-Chloroethanol
107-07-3
2-Fluoroethanol
371-62-0
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-FID
Method Developed for: Ethylene chlorohydrin (2-chloroethanol) in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The working range of the method is 0.5-15 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.
Special Considerations: No interferences have been identified. Humidity may decrease the
breakthrough volume during sample collection. The presence of 2-chloroethanol should be confirmed
using either a secondary GC column or an MS.
Source: NIOSH. 1994. "Method 2513: Ethylene Chlorohydrin," Issue 2. NIOSH Manual of Analytical
Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gOv/sites/production/files/2015-07/documents/niosh-2513.pdf
5.2.62 NIOSH Method 3509: Aminoethanol Compounds II
Analyte(s)
CAS RN
A/-Ethyldiethanolamine (EDEA)
139-87-7
W-Methyldiethanolamine (MDEA)
105-59-9
Triethanolamine (TEA)
102-71-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Samples are collected with an impinger containing 15 mL of 2 mM
hexane sulfonic acid
Determinative Technique: IC with conductivity detection
Method Developed for: Triethanolamine in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
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Detection and Quantitation: The LOD and LOQ for triethanolamine are 0.067 and 0.2 j^ig/L.
respectively.
Description of Method: Samples are collected into an impinger containing 15 mL of 2 mM
hexanesulfonic acid using a sampling pump, at a flow rate 0.5 to 1 L/minute for a total sample size of 5 to
300 L. After sampling, the impinger is filled to the 15-mL mark with distilled water and transferred to a
vial for shipment. A portion of the sample is filtered through an in-line membrane filter into an
autosampler vial. The autosampler injects 50 |aL of sample into an ion chromatograph equipped with an
ion-pairing guard, cation separator and cation suppressor. Conductivity is set at 3 (.iS full scale and the
eluent used is 2 mM hexanesulfonic acid.
Special Considerations: If high sample throughput is needed, 2 mM hexanesulfonic acid/0.5% v/v
acetonitrile can be used as the eluent to reduce run time.
Source: NIOSH. 1994. "Method 3509: Aminoethanol Compounds II," Issue 2. NIOSHManual of
Analytical Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS
(NIOSH). https://www.cdc.gov/niosh/docs/2003-154/pdfs/35Q9.pdf
5.2.63 NIOSH Method 3510: Monomethylhydrazine
Analyte(s)
CAS RN
Methyl hydrazine (monomethylhydrazine)
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: This method has been selected for preparation and analysis of air samples to
address methyl hydrazine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 0.027-2.7 ppm for a 20-L 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, then transferred to a 25-mL flask, mixed with phosphomolybdic acid solution,
diluted with 0.1 M hydrochloric acid, and transferred to a large test tube for spectrophotometric analysis.
Special Considerations: Positive interferences include other hydrazines, as well as stannous and ferrous
ion, zinc, sulfur dioxide and hydrogen sulfide. Negative interferences may occur by oxidation of mono-
methylhydrazine by halogens, oxygen (especially in the presence of copper (I) ions) and hydrogen
dioxide.
Source: NIOSH. 1994. "Method 3510: Monomethylhydrazine," Issue 1. NIOSH Manual of Analytical
Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-351Q.pdf
5.2.64 NIOSH Method 5600: Organophosphorus Pesticides
Analyte(s)
CAS RN
Disulfoton
298-04-4
Disulfoton sulfone oxon
2496-91-5
Disulfoton sulfoxide
2497-07-6
Disulfoton sulfoxide oxon
2496-92-6
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Analyte(s)
CAS RN
The following analyte should be prepared by this method only if problems (e.g., insufficient recovery,
interferences) occur when using the sample preparation/determinative techniques identified for these analytes in
Appendix A
Methamidophos
10265-92-6
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: GC-flame photometric detector (FPD)
Method Developed for: Organophosphorus pesticides in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. 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 a
GC-FPD. Samples are prepared by desorbing the sampler 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 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. The presence of the analytes listed in the table above should be confirmed using either a
secondary GC column or an MS.
Source: NIOSH. 1994. "Method 5600: Organophosphorus Pesticides," Issue 1. NIOSHManual of
Analytical Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS
(NIOSH). http://www.epa.gov/sites/production/files/2015-07/documents/niosh-5600.pdf
5.2.65 NIOSH Method 5601: Organonitrogen Pesticides
Analyte(s)
CAS RN
Aldicarb (Temik)
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
Carbofuran (Furadan)
1563-66-2
Methomyl
16752-77-5
Oxamyl
23135-22-0
Thiofanox
39196-18-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC-UV
Method Developed for: Organonitrogen pesticides in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
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Detection and Quantitation: The method reports detection limits of 1.2 (.ig for aldicarb and 0.6 (.ig for
carbofuran, methomyl and oxamyl for collected air sample volumes of 240 L. The working ranges for
aldicarb, carbofuran and oxamyl are listed in Table 2 of the method, and range from 0.5 to 10 times the
OSHA PEL.
Description of Method: This method can be used for the determination of organonitrogen pesticides
using HPLC with a UV detector. Samples are prepared by desorbing the sampler 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.
Special Considerations: The presence of analytes listed in the table above should be confirmed using
either a secondary HPLC column or an MS. 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, caffeine), HPLC reagent impurities (e.g., in triethylamine), other pesticides (2,4-
dichlorophenoxyacetic acid [2,4-D], atrazine, parathion), and pesticide hydrolysis products (1-naphthol).
Source: NIOSH. 1998. "Method 5601: Organonitrogen Pesticides," Issue 1. NIOSHManual of
Analytical Methods, Second Supplement. DHHS (NIOSH) Publication No. 98-119. Washington, DC:
DHHS (NIOSH). http://www.cdc.gov/niosh/docs/20Q3-154/pdfs/5601.pdf
5.2.66 NIOSH Method 6001: Arsine
Analyte(s)
CAS RN
Arsine
7784-42-1
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Coconut shell charcoal solid sorbenttube
Determinative Technique: Graphite furnace atomic absorption (GFAA)
Method Developed for: Arsine in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address arsine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 0.001-0.2 mg/m3 for a 10-L sample.
Description of Method: Arsine is determined as arsenic. 0.1 to 10 L 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. NIOSH Manual ofAnalytical Methods, Fourth
Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-6Q01.pdf
5.2.67 NIOSH Method 6002: Phosphine
Analyte(s)
CAS RN
Phosphine
7803-51-2
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Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption with hot acidic potassium permanganate solution
Determinative Technique: Visible spectrophotometry
Method Developed for: Phosphine in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address phosphine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 0.02-0.9 mg/m3 for a 16-L sample.
Description of Method: Phosphine is determined as phosphate. 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. 1998. "Method 6002: Phosphine," Issue 2. NIOSH Manual of Analytical Methods,
Second Supplement. DHHS (NIOSH) Publication No. 98-119. Washington, DC: DHHS (NIOSH).
http://www.epa.gOv/sites/production/files/2015-07/documents/niosh-6002.pdf
5.2.68 NIOSH Method 6010: Hydrogen Cyanide
Analyte(s)
CAS RN
Cyanide, Total
57-12-5
Hydrogen cyanide
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: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The working range of the method is 3-260 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. NIOSH Manual of Analytical
Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-601Q.pdf
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5.2.69 NIOSH Method 6013: Hydrogen Sulfide
Analyte(s)
CAS RN
Hydrogen sulfide
7783-06-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction
Determinative Technique: IC with conductivity detection
Method Developed for: Hydrogen sulfide in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address hydrogen sulfide. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 0.9-20 mg/m3 for a 20-L sample.
Description of Method: This method determines hydrogen sulfide as sulfate. 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 IC.
Special Considerations: The method is subject to interference from sulfur dioxide.
Source: NIOSH. 1994. "Method 6013: Hydrogen Sulfide," Issue 1. NIOSH Manual of Analytical
Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-6Q13.pdf
5.2.70 NIOSH Method 6015: Ammonia
Analyte(s)
CAS RN
Ammonia
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: This method has been selected for preparation and analysis of air samples to
address ammonia. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 0.15-300 mg/m3 for a 10-L sample.
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. NIOSH Manual ofAnalytical Methods,
Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-6Q15.pdf
5.2.71 NIOSH Method 6402: Phosphorus Trichloride
Analyte(s)
CAS RN
Phosphorus trichloride
7719-12-2
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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: This method has been selected for preparation and analysis of air samples to
address phosphorus trichloride. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The working range of the method is 1.2-80 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.
Special Considerations: Phosphorus (III) compounds can interfere with analysis of phosphorus
trichloride, by increasing the amount of phosphorus that is measured. Phosphorus (V) compounds do not
interfere.
Source: NIOSH. 1994. "Method 6402: Phosphorus Trichloride," Issue 2. NIOSH Manual of Analytical
Methods, Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-64Q2.pdf
5.2.72 NIOSH Method 7905: Phosphorus
Analyte(s)
CAS RN
White phosphorus
12185-10-3
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: GC solid sorbenttube and solvent extracted (desorbed)
Determinative Technique: GC-FPD
Method Developed for: Phosphorus in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address white phosphorus. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The LOD for samples analyzed by GC-FPD is 0.005 (ig per sample. The
working range for samples analyzed by GC-FPD is 0.056-0.24 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 can be used in the sampling train.
Special Considerations: The presence of white phosphorus should be confirmed using either a
secondary GC column or and MS.
Source: NIOSH. 1994. "Method 7905: Phosphorus," Issue 2. NIOSH Manual ofAnalytical Methods,
Fourth Edition. DHHS (NIOSH) Publication No. 94-113. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-79Q5.pdf
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5.2.73 NIOSH Method 7906: Particulate Fluorides and Hydrofluoric Acid by Ion
Chromatography
Analyte(s)
CAS RN
Hydrogen fluoride
7664-39-3
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Aqueous solution extraction
Determinative Technique: IC with conductivity detection
Method Developed for: Fluorides in aerosol and gas
Method Selected for: This method has been selected for preparation and analysis of air samples to
address hydrogen fluoride. (See Footnote 10 of Appendix A.)
Detection and Quantitation: The working range of the method is 0.04-8 mg/m3 for 250-L samples.
Description of Method: Hydrogen fluoride is determined as fluoride ion by this method. A volume of 15
to 1000 L of air is drawn through a 0.8-(.un cellulose nitrate prefilter (to trap particulate fluorides) and a
cellulose nitrate filter treated with sodium carbonate (to trap gaseous fluoride). The filter is extracted with
an aqueous solution of 8 mM sodium carbonate /I mM sodium bicarbonate and the extract is analyzed for
fluoride by IC.
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. 2014. "Method 7906: Particulate Fluorides and Hydrofluoric Acid 7906 by Ion
Chromatography," Issue 2. NIOSH Manual of Analytical Methods, Fifth Edition. Washington, DC: DHHS
(NIOSH). https://www.cdc.gov/niosh/docs/2003-154/pdfs/7906.pdf
5.2.74 NIOSH Method 7907: Volatile Acids by Ion Chromatography (Hydrogen Chloride,
Hydrogen Bromide, Nitric Acid)
Analyte(s)
CAS RN
Hydrogen bromide
10035-10-6
Hydrogen chloride
7647-01-0
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Aqueous solution extraction
Determinative Technique: IC with conductivity detection
Method Developed for: Hydrogen bromide, hydrogen chloride and nitric acid in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address hydrogen bromide and hydrogen chloride.
Detection and Quantitation: The working range is 0.04-8 mg/m3 for hydrogen bromide and hydrogen
chloride in 240-L samples.
Description of Method: Hydrogen bromide and hydrogen chloride are determined as bromide and
chloride ions, respectively. A volume of 30 to 600 L of air is drawn through a 37-mm diameter quartz
fiber prefilter (to trap potentially interfering particulate chlorides) and a 37-mm diameter quartz fiber
filter treated with sodium carbonate (to trap gaseous hydrogen bromide and hydrogen chloride). After
discarding the prefilters, the filter is extracted with an aqueous solution of 3.1 mM sodium carbonate/0.35
mM sodium carbonate and the extract is analyzed for bromide and chloride by IC.
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Special Considerations: Inorganic acids can react with co-sampled particulate matter on the pre-filter,
leading to low results (e.g., zinc oxide reacting with hydrochloric acid). Potentially interfering particulate
chlorides and nitrates removed by the pre-filter can react with the sampled acids and liberate hydrochloric
acid that is subsequently collected on the sampling filter, leading to high results. Silica gel sorbent tubes
can be used instead of treated filters, but each sorbent tube must be preceded by a pre-filter.
Source: NIOSH. 2014. "Method 7907: Volatile Acids by Ion Chromatography (Hydrogen Chloride,
Hydrogen Bromide, Nitric Acid)," Issue 1. NIOSH Manual ofAnalytical Methods, Fifth Edition.
Washington, DC: DHHS (NIOSH). https://www.cdc.gov/niosh/docs/2003-154/pdfs/7907.pdf
5.2.75 NIOSH Method 9102: Elements on Wipes
Analyte(s)
CAS RN
Ammonium metavanadate (analyze as total vanadium)
7803-55-6
Arsenic, Total
7440-38-2
Arsenic trioxide (analyze as total arsenic)
1327-53-3
Arsine (analyze as total arsenic in non-air samples)
7784-42-1
Calcium arsenate (analyze as total arsenic)
7778-44-1
Chlorovinyl arsonic acid (CVAOA) (analyze as total arsenic)*
64038-44-4
2-Chlorovinylarsonous acid (CVAA) (analyze as total arsenic)*
85090-33-1
Ethyldichloroarsine (ED)
598-14-1
Lead arsenate (analyze as total arsenic)
7645-25-2
Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] (analyze as total arsenic)*
541-25-3
Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze as total arsenic)*
40334-69-8
Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze as total arsenic)*
40334-70-1
Lewisite oxide (analyze as total arsenic)*
1306-02-1
Mercuric chloride (analyze as total mercury)
7487-94-7
Mercury, Total
7439-97-6
Methoxyethylmercuric acetate (analyze as total mercury)
151-38-2
Sodium arsenite (analyze as total arsenic)
7784-46-5
Thallium sulfate (analyze as total thallium)
10031-59-1
Vanadium pentoxide (analyze as total vanadium)
1314-62-1
* If laboratories are approved for storing and handling the appropriate standards, these analytes can be detected and
measured using EPA/600/R-15/258 (Section 5.2.54)
Analysis Purpose: Sample preparation
Sample Preparation Technique: Acid digestion
Determinative Technique: ICP-AES / ICP-MS / Spectrophotometry
Determinative Method: EPA SW-846 Methods 6010D, 6020B, 7473 and 8270E. 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: This method has been selected for preparation of wipe samples to address the
analytes listed in the table above as total arsenic, mercury, thallium or vanadium. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: The working ranges are: 0.261-105 (ig/wipe (arsenic); 0.136-50.0
(ig/wipe (thallium); and 0.0333-25.0 (ig/wipe (vanadium). A working range is not provided for mercury.
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 150ฐC 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
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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. If laboratories are approved for
storing and handling the appropriate standards, lewisites 1, 2 and 3 and their degradation products
(CVAOA, CVAA and lewisite oxide) can be detected and measured using EPA/600/R-15/258 (Section
5.2.54).
Source: NIOSH. 2003. "Method 9102, Issue 1: Elements on Wipes." NIOSH Manual of Analytical
Methods, 3rd Supplement 2003-154. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-91Q2.pdf
5.2.76 NIOSH Method 9106: Methamphetamine and Illicit Drugs, Precursors and
Adulterants on Wipes by Liquid-Liquid Extraction
Analyte(s)
CAS RN
Phencyclidine
77-10-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS
Method Developed for: Phencyclidine in wipe samples
Method Selected for: This method has been selected for the preparation and analysis of wipe samples to
address phencyclidine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The MDLs for phencyclidine on spiked cotton gauze in full-scan and SIM
mode are 0.3 and 0.2 ng/cm2, respectively. The working range of the method is 1-100 ng/ cm2 for both
full-scan and SIM modes.
Description of Method: Internal standards and desorption solution (0.1M sulfuric acid) are added to
each sample contained in a centrifuge tube. The tubes are capped and the samples and solution are mixed
with a rotatory mixer at 10-30 rpm for at least one hour. If necessary, the pH is adjusted to <4 with 3M
sulfuric acid. Sample extracts are transferred to a glass centrifuge tube and cleaned by adding 10 mL of
hexane to 10 mL of extract. The contents are mixed by rotary mixer for at least one hour, then allowed to
stand for 15-20 minutes. If an emulsion forms, extracts are centrifuged for a few minutes at 1500-2000
rpm. The upper organic layer is aspirated off as waste, and 1-2 drops of pH indicator (phenolphthalein
and bromothymol blue) and 0.5 mL of 10M sodium hydroxide are added to the aqueous fraction, as
needed, to turn the solution to purple or magenta. Once this color change is achieved, 10 mL of methylene
chloride is added, the sample container is capped and the contents remixed on a rotary mixer for 1 hour.
The mixture is allowed to stand for 15-30 minutes, and the centrifuge procedure is repeated if an
emulsion forms. Any remaining water is removed using packed potassium carbonate-sodium sulfate
drying columns. The methylene chloride is evaporated under nitrogen, and 100 (.iL of chlorodifluoroacetic
anhydride is added followed by additional mixing. The tube is then placed into an oven at 70-75ฐC and
heated for 20-30 minutes. After cooling, the extract is evaporated to dryness under nitrogen until a blue
or violet color is visible. Reconstituting solution (1 mL) is added and the solution is transferred to an
amber-colored GC vial containing 200-250 mg anhydrous sodium sulfate. Vials are capped and analyzed
by GC-MS.
Special Considerations: If an oil-like residue or film persists, the sample may contain contaminants that
were not removed during the cleanup step or were introduced following sample cleanup. In such cases,
the cleanup step is repeated on another 10-mL sample aliquot, using methylene chloride instead of hexane
as the cleanup solvent. Analyte losses have been experienced during the derivatization step if blowing is
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continued for more than 2 minutes beyond the appearance of a blue or violet color.
Source: NIOSH. 2011. "Method 9106: Methamphetamine and Illicit Drugs, Precursors and Adulterants
on Wipes by Liquid-Liquid Extraction," Issue 1. NIOSH Manual ofAnalytical Methods, Fifth Edition.
Washington, DC: DHHS (NIOSH). https://www.cdc.gov/niosh/docs/2003-154/pdfs/91Q6.pdf
5.2.77 NIOSH Method 9109: Methamphetamine and Illicit Drugs, Precursors, and
Adulterants on Wipes by Solid Phase Extraction
Analyte(s)
CAS RN
Phencyclidine
77-10-1
Analysis Purpose: Sample preparation and analysis
Sample Preparation Technique: Solvent extraction
Determinative Technique: GC-MS
Method Developed for: Phencyclidine in wipe samples
Method Selected for: This method has been selected for preparation and analysis of wipe samples to
address phencyclidine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The MDLs for phencyclidine on spiked cotton gauze and AlphaWipesฎ
wipes (All-Spec, Wilmington, NC, or equivalent) are 1 and 5 ng/cm2, respectively. The working range of
the method is 3-300 ng/ cm2.
Description of Method: Internal standards and desorption solution (0.1 M sulfuric acid) are added to
wipe samples contained in a centrifuge tube. The tubes are capped and samples mixed with a rotatory
mixer at 10-30 rpm for at least one hour. If necessary, the pH is adjusted to <4 with 2.5 to 3M sulfuric
acid. An SPE column is attached to a vacuum capable of 25-30 psi pressure and conditioned with 3 mL of
methanol, followed by 1 mL of ASTM Type II deionized water. The SPE column is loaded with 10 mL of
sample and the sample is pulled through the column via vacuum. The column is then washed with 3 mL
of 0.1M hydrochloric acid, followed by 3 mL of methanol, and all effluent is discarded. The vacuum is
increased to remove all traces of water, and the analytes are eluted with 3 mL of 80:20:2 methylene
chloride:isopropanol:concentrated ammonium hydroxide (v/v) into a collection tube. About 5 |_iL of
crystal violet solution and 100 |_iL of 0.3M hydrochloric acid in methanol are added to the tube, and the
samples are evaporated to dryness under nitrogen. Acetonitrile containing internal standard (100 (.iL) and
derivatizing agents is added to the collection tube, and the tubes are capped, vortexed for 4-5 seconds,
and a 300-500 |_iL aliquot is transferred to an autosampler vial for analysis by GC-MS.
Special Considerations: No chromatographic interferences were observed during method development;
however, water, surfactants and polyols can inhibit derivatization. The color of the reconstituted solution
should be deep blue to violet. If the color turns light blue or turquoise upon standing, moisture may be
present. Such samples need to be reprocessed beginning at the SPE extraction step, since the derivatives
are not stable in the presence of moisture.
Source: NIOSH. 2011. "Method 9109: Methamphetamine and Illicit Drugs, Precursors, and Adulterants
on Wipes by Solid Phase Extraction," Issue 1. NIOSH Manual of Analytical Methods, Fifth Edition.
Washington, DC: DHHS (NIOSH). http://www.cdc.gov/niosh/docs/2003-154/pdfs/9109.pdf
5.2.78 NIOSH Method S301-1: Fluoroacetate Anion
Analyte(s)
CAS RN
Fluoroacetic acid and fluoroacetate salts
NA
Methyl fluoroacetate
453-18-9
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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
Method Selected for: This method has been selected for preparation of air samples to address the
analytes listed in the table above. 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-(iL aliquot of a 0.2-|ig/mL standard. The analytical range of this method
is estimated to be 0.01-0.16 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." NIOSHManual of Analytical
Methods, Second Edition, Volume 5. Washington, DC: DHHS (NIOSH).
http://www.epa.gov/sites/production/files/2015-07/documents/niosh-s301-1 .pdf
5.2.79 OSHA Method 40: Methylamine
Analyte(s)
CAS RN
Methylamine
74-89-5
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC-FL/vis
Method Developed for: Methylamine in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address methylamine. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The detection limit is 0.35 (.ig per sample (28 ppb or 35 (.ig/nr3).
Quantitation limits of 28 ppb (35 (.ig/ni3) 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 sampler 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 HPLC-FL or high performance liquid chromatography-visible (HPLC-vis).
Source: OSHA. 1982. "Method 40: Methylamine." Method originally obtained from
https://www.osha.gov. but is provided here for reference. Salt Lake City, UT: OSHA.
http://www.epa.gov/sites/production/files/2015-07/documents/osha-method40.pdf
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5.2.80 OSHA Method 54: Methyl Isocyanate (MIC)
Analyte(s)
CAS RN
Methyl isocyanate
624-83-9
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC
Method Developed for: Methyl isocyanate in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address methyl isocyanate. See Appendix A for the corresponding method usability tier.
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
sampler tubes containing resin 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
https://www.osha.gov. but is provided here for reference. Sandy, UT: OSHA.
https://www.osha.gov/dts/sltc/methods/organic/org054/org054.html
5.2.81 OSHA Method 61: Phosgene
Analyte(s)
CAS RN
Phosgene
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: This method has been selected for preparation and analysis of air samples to
address phosgene. See Appendix A for the corresponding method usability tier.
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
resin 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 using either a secondary GC
column or an MS.
Source: OSHA. 1986. "Method 61: Phosgene." Method originally obtained from https://www.osha.gov.
but is provided here for reference. Salt Lake City, UT: OSHA.
http://www.epa.gov/sites/production/files/2015-07/documents/osha-method61.pdf
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5.2.82 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres
Analyte(s)
CAS RN
Sodium azide (analyze as azide ion)
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
Method Selected for: This method has been selected for preparation and analysis of air and wipe
samples to address sodium azide as azide ion. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The detection limit for sodium azide was found to be 0.003 mg/m3 for a 5-
L air sample. The quantitation limit was found to 0.011 mg/m3, also for a 5-L air sample.
Description of Method: This method describes sample collection and analysis of airborne azides (as
sodium azide and hydrazoic acid. Particulate sodium azide is collected on a polyvinyl chloride (PVC)
filter or in the glass wool plug of the sampling tube. Gaseous hydrazoic acid is collected and converted to
sodium azide by the impregnated silica gel (ISG) sorbent within the sampling tube. The collected azide on
either media is desorbed in a weak buffer solution, and the resultant azide anion is analyzed by IC using a
variable wavelength UV detector at 210 nm. A gravimetric conversion is used to calculate the amount of
sodium azide or hydrazoic acid collected.
Source: OSHA. 1992. "Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace
Atmospheres." Sandy, UT: OSHA. http://www.epa.gov/sites/production/files/2015-07/documents/osha-
id-211 .pdf
5.2.83 OSHA Method ID216SG: Boron Trifluoride (BF3)
Analyte(s)
CAS RN
Boron trifluoride
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: This method has been selected for preparation and analysis of air samples to
address boron trifluoride. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The detection limit is 10 |ag 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 0.1M ammonium fluoride. The bubbler solution is diluted and
analyzed with a fluoroborate ISE.
Source: OSHA. 1989. "Method ID216SG: Boron Trifluoride (BF3)." Method originally obtained from
https://www.osha.gov. but is provided here for reference. Sandy, UT: OSHA.
http://www.epa.gov/sites/production/files/2015-Q7/documents/osha-id216sg.pdf
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5.2.84 OSHA Method PV2004: Acrylamide
Analyte(s)
CAS RN
Acrylamide
79-06-1
Acrylonitrile
107-13-1
Methyl acrylonitrile
126-98-7
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent desorption
Determinative Technique: HPLC-UV
Method Developed for: Acrylamide in air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
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 sample).
Applicable working ranges for 1-mL and 5-mL desorption volumes 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 adsorbent. Samples are
desorbed with a solution of 5% methanol/95% water, and analyzed by HPLC-UV.
Special Considerations: The presence of acrylamide, acrylonitrile and methyl acrylonitrile should be
confirmed using either a secondary HPLC column or an MS.
Source: OSHA. 1991. "Method PV2004: Acrylamide." Sandy, UT: OSHA.
http://www.epa.gov/sites/production/files/2015-07/documents/osha-pv2004.pdf
5.2.85 OSHA Method PV2103: Chloropicrin
Analyte(s)
CAS RN
Chloropicrin
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: This method has been selected for preparation and analysis of air samples to
address chloropicrin. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The detection limit is 0.01 ng, with a l-(iL injection volume. This is the
smallest amount that could be detected under normal operating conditions. The working range is 33.2-
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 adsorbent tubes in series. Samples
are desorbed with ethyl acetate and analyzed by GC-ECD.
Special Considerations: The presence of chloropicrin should be confirmed using either a secondary GC
column or an MS. Chloropicrin is light sensitive, and samples should be protected from light.
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Source: OSHA. 1991. "Method PV2103: Chloropicrin." Salt Lake City, UT: OSHA.
http://www.epa.gov/sites/production/files/2015-07/documents/osha-pv21Q3.pdf
5.2.86 ASTM Method D5755-09: Standard Test Method for Microvacuum Sampling and
Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos
Structure Number Surface Loading
Analyte(s)
CAS RN
Asbestos
1332-21-4
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Direct transfer
Determinative Technique: Transmission electron microscopy (TEM)
Method Developed for: Asbestos in dust
Method Selected for: This method has been selected for preparation and analysis of solid (e.g., soft
surfaces-microvac) samples to address asbestos. See Appendix A for the corresponding method usability
tier.
Description of Method: This method describes procedures to identify asbestos in dust and provide an
estimate of surface loading reported as the number of asbestos structures per unit area of sampled surface.
Samples are 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. Once collected,
samples are 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 20,000X.
Source: ASTM. 2014. "Method D5755-09(el): Standard Test Method for Microvacuum Sampling and
Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos Structure Number Surface
Loading." West Conshohocken, PA: ASTM International, http://www.astm.or g/Standards/D5755.htm
5.2.87 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)
CAS RN
Asbestos
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: This method has been selected for preparation and analysis of wipe (e.g., hard
surfaces-wipes) samples to address asbestos. See Appendix A for the corresponding method usability tier.
Description of Method: This method describes a procedure to identify asbestos in samples wiped from
surfaces and to provide an estimate of the concentration reported as the number of asbestos structures per
unit area of sampled surface. Samples are collected by wiping a surface of known area with a wipe
material. Once collected, samples are transferred from the wipe material to an aqueous suspension of
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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 20,000X.
Source: ASTM. 2010. "Method D6480-05: Standard Test Method for Wipe Sampling of Surfaces,
Indirect Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission
Electron Microscopy." West Conshohocken, PA: ASTM International.
http://www.astm.org/Standards/D6480.htm
5.2.88 ASTM Method D7597-16: 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)
CAS RN
Diisopropyl methylphosphonate (DIMP)
1445-75-6
Dimethylphosphoramidic acid
33876-51-6
Ethyl methylphosphonic acid (EMPA)
1832-53-7
Isopropyl methylphosphonic acid (IMPA)
1832-54-8
Methylphosphonic acid (MPA)
993-13-5
Pinacolyl methyl phosphonic acid (PMPA)
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: DIMP, EMPA, IMPA, MPA and PMPA in surface water
Method Selected for: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above. Note. EPA Method 538 (Section 5.2.11) has been selected
for sample preparation and analysis of DIMP in drinking water samples. See Appendix A for
corresponding method usability tiers.
Detection and Quantitation: The detection verification levels (DVLs) and reporting range vary for each
analyte and range from 0.25 to 20 (ig/L and 5 to 1500 (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 6ฐC, 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.
Special Considerations: Method modifications (e.g., pH adjustment) may be needed when analyzing for
dimethyphosphoramidic acid.
Source: ASTM. 2016. "Method D7597-16: 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." West Conshohocken. PA: ASTM International.
http://www.astm.org/Standards/D75 97 .htm
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5.2.89 ASTM Method D7598-16: Standard Test Method for Determination of Thiodiglycol
in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass
Spectrometry
Analyte(s)
CAS RN
Thiodiglycol
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
Method Selected for: This method has been selected for preparation and analysis of water samples to
address thiodiglycol. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The DVL for thiodiglycol is 20 |_ig/L: the reporting range is 100-10000
l-ig/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 6ฐC, 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. 2016. "Method D7598-16: Standard Test Method for Determination of Thiodiglycol in
Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry." West
Conshohocken, PA: ASTM International, htto://www.astm,org/Standards/D7598.htm
5.2.90 ASTM Method D7599-16: Standard Test Method for Determination of
Diethanolamine, Triethanolamine, W-Methyldiethanolamine and N-
Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
CAS RN
N-Ethyldiethanolamine (EDEA)
139-87-7
N-Methyldiethanolamine (MDEA)
105-59-9
Triethanolamine (TEA)
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, MDEA and EDEA in surface water samples
Method Selected for: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above. 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 25-500
(ig/L, respectively. The DVL and reporting range for MDEA are 10 |_ig/L and 50-500 (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 6ฐC, 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
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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. 2016. "Method D7599-16: Standard Test Method for Determination of Diethanolamine.
Triethanolamine. iV-Methyldiethanolamine and iV-Ethyldiethanolamine in Water by Single Reaction
Monitoring Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)." West Conshohocken.
PA: ASTM International, http://www.astm.org/Standards/D75 99 .htm
5.2.91 ASTM Method D7644-16: Standard Test Method for Determination of
Bromadiolone, Brodifacoum, Diphacinone and Warfarin in Water by Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
Analyte(s)
CAS RN
Brodifacoum
56073-10-0
Bromadiolone
28772-56-7
Diphacinone
82-66-6
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
Sample Preparation Technique: Filtered using a syringe-driven PVDF filter unit for water samples;
automated Soxhlet or pressured fluid extraction for solid samples, and solvent extraction for wipes.
Determinative Technique: LC-MS-MS
Method Developed for: Bromadiolone, brodifacoum and diphacinone in reagent, surface and drinking
water
Method Selected for: This method has been selected for preparation and analysis of water samples, and
for analysis of prepared solid and wipe samples to address the analytes listed in the table above. Note.
EPA SW-846 Methods 3541/3545A (Sections 5.2.22 and 5.2.23) and Methods 3570/8290A Appendix A
(Sections 5.2.24 and 5.2.36) have been selected for preparation of solid and wipe samples, respectively.
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 6ฐC, 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. 2016. "Method D7644-16: Standard Test Method for Determination of Bromadiolone,
Brodifacoum, Diphacinone and Warfarin in Water by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)." West Conshohocken, PA: ASTM International.
http ://www.astm ,org/Standards/D7644 .htm.
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5.2.92 ASTM Method D7645-16: 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)
CAS RN
Aldicarb
116-06-3
Aldicarb sulfone
1646-88-4
Aldicarb sulfoxide
1646-87-3
Carbofuran
1563-66-2
Methomyl
16752-77-5
Oxamyl
23135-22-0
Thiofanox
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
Method Developed for: Aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, oxamyl, methomyl
and thiofanox in water
Method Selected for: This method has been selected for preparation and analysis of non-drinking water
samples to address aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbofuran, methomyl, oxamyl and
thiofanox. It has also been selected for the analysis of prepared solid and wipe samples to address
thiofanox. See Appendix A for corresponding method usability tiers. Note.
SW-846 Methods 3541 (see Section 5.2.22) or 3545A (see Section 5.2.23) have been selected for
preparation of solid samples to be analyzed for thiofanox.
SW-846 Methods 3570 (see Section 5.2.24) and 8290 (see Section 5.2.36) have been selected for
preparation of wipe samples to be analyzed for thiofanox.
Detection and Quantitation: A DVL is reported as 250 ng/L for all compounds listed in the table above.
The reporting range for these compounds is 1-100 (ig/L.
Description of Method: Samples are spiked with surrogates, filtered using a syringe-driven filter unit,
and analyzed directly by LC-MS-MS within 14 days. 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 must fall within ฑ 5% of the retention time of the analytes in standard
solution. Analytes are measured using the primary SRM transition and external standard calibration.
Source: ASTM. 2016. "Method D7645-16: Standard Test Method for Determination of Aldicarb,
Aldicarb Sulfone, Aldicarb Sulfoxide, Carbofuran, Methomyl, Oxamyl and Thiofanox in Water by Liquid
Chromatography/Tandem Mass Spectrometry." West Conshohocken, PA: ASTM International.
http://www.astm.org/Standards/D7645 .htm.
5.2.93 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)
CAS RN
Thiodiglycol
111-48-8
Analysis Purpose: Sample preparation, and/or analyte determination and measurement
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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: This method has been selected for preparation and analysis of solid samples to
address thiodiglycol. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL is 54 j^ig/kg. The reporting range is 200-16,000 ng/kg.
Description of Method: 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 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. 2016. "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." West Conshohocken, PA: ASTM International.
http: //www. astm. org/Standards/E2787. htm.
5.2.94 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)
CAS RN
Thiodiglycol
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: This method has been selected for preparation and analysis of wipe samples to
address thiodiglycol. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The MDL 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 6ฐC, 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. Thiodiglycol is 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. 2016. "Method E2838-11: Standard Test Method for Determination of Thiodiglycol on
Wipes by Solvent Extraction Followed by Liquid Chromatography/Tandem Mass Spectrometry." West
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Conshohocken, PA: ASTM International, http://www.astm,org/Standards/E2838.htm.
5.2.95 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)
CAS RN
Diisopropyl methylphosphonate (DIMP)
1445-75-6
Dimethylphosphoramidic acid
33876-51-6
Ethyl methylphosphonic acid (EMPA)
1832-53-7
Isopropyl methylphosphonic acid (IMPA)
1832-54-8
Methylphosphonic acid (MPA)
993-13-5
Pinacolyl methyl phosphonic acid (PMPA)
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: DIMP, EMPA, IPMA, MPA and PMPA in soil
Method Selected for: This method has been selected for preparation and analysis of solid samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The reporting range for all analytes is 40-2000 (.ig/kg. MDLs range from
1.3 to 8.7 (ig/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 6ฐC 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.
Special Considerations: Method modifications (e.g., pH adjustment) may be needed when analyzing for
dimethyphosphoramidic acid.
Source: ASTM. 2016. "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." West Conshohocken, PA:
ASTM International, http://www.astm.org/Standards/E2866.htm.
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5.2.96 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-
Transfer Transmission Electron Microscopy Method
Analyte(s)
CAS RN
Asbestos
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 ambient air
Method Selected for: This method has been selected for preparation and analysis of air samples to
address asbestos. See Appendix A for the corresponding method usability tier.
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 50-7,000 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/cataloguc detail.htm?csnumber=18358
5.2.97 Standard Method 4500-CN G: Cyanides Amenable to Chlorination after Distillation
Analyte(s)
CAS RN
Cyanide, Amenable to chlorination
NA
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Acid digestion followed by distillation
Determinative Technique: Visible spectrophotometry, titrimetry or cyanide-selective electrode
Method Developed for: Cyanide in drinking water, groundwater, surface water, domestic and industrial
wastewaters, and solid waste
Method Selected for: This method has been selected for preparation and analysis of water samples to
address cyanide amenable to chlorination. The method has been selected as an alternative to EPA
Regional Laboratory (RLAB) Method 3135.21 (see Section 5.2.42), for use by laboratories more familiar
with its procedures.
Detection and Quantitation: The method has been evaluated in the ranges of 0.008-0.191 mg/L
(colorimetric procedure) and 1-4 mg/L (titrimetric procedure). These ranges can be expanded by sample
dilution, by either distilling less sample or diluting the distillate.
Description of Method: This method is applicable to the determination of cyanides amenable to
chlorination (also known as available cyanide). After part of the sample is chlorinated to decompose the
cyanides, both the chlorinated and the untreated sample are distilled as described in Standard Method
(SM) 4500-CN C. The difference between the cyanide concentrations in the two samples is expressed as
cyanides amenable to chlorination. The sample is divided into two equal portions of 500 mL (or equal
portions diluted to 500 mL). Chlorinate one of the portions using the procedure in the next paragraph.
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Both portions are analyzed for cyanide and the difference in determined concentrations is the cyanide
amenable to chlorination.
One portion is placed in a 1-L beaker covered with aluminum foil or black paper. The beaker is kept
covered with a wrapped watch glass during chlorination. Calcium hypochlorite solution is added dropwise
to the sample while agitating and maintaining pH between 11 and 12 by adding sodium hydroxide
solution. The sample is then tested for chlorine by placing a drop of treated sample on a strip of Kl-starch
paper. A distinct blue color indicates sufficient chlorine (approximately 50 to 100 mg chlorine/L). The
sample is agitated for 1 hour, while adding more calcium hypochlorite if necessary to maintain the
chlorine concentration. After agitating for 1 hour, residual chlorine is removed by the dropwise addition
of sodium arsenite solution (2g/100 mL) or by the addition of 8 drops of hydrogen peroxide (3% solution)
followed by 4 drops of sodium thiosulfate (500 g/L). The sample is tested with potassium iodide-starch
paper by adding a drop or two of sample to the paper. The dechlorinating solutions are to be added until
there is no color change. Both the chlorinated and unchlorinated samples are distilled as described in
4500-CN C. The samples are tested according to the procedures in SM 4500-CN D (titrimetric), E
(colorimetric) or F (cyanide-selective electrode).
Special Considerations: Samples should be protected from exposure to UV radiation. All sample
manipulations should be performed under incandescent light, to prevent photodecomposition of some
metal-cyanide complexes by UV light. Some unidentified organic chemicals may oxidize or form
breakdown products during chlorination, giving higher results for cyanide after chlorination than before
chlorination. This may lead to a negative value for cyanides amenable to chlorination after distillation for
wastes from, for example, the steel industry, petroleum refining, and pulp and paper processing. Where
such interferences are encountered use SM 4500-CN I for determining dissociable cyanide.
Source: APHA, AWWA and WEF. 2011. "Method 4500-CN G: Cyanides Amenable to Chlorination
after Distillation." Standard Methods for the Examination of Water and Wastewater. 22nd Edition.
Washington, DC: APHA. htto://www.standardmethods.org/
5.2.98 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step
Analyte(s)
CAS RN
Ammonia
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: This method has been selected for preparation of non-drinking water samples to
address ammonia. See Appendix A for the corresponding method usability tier.
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 SM 4500-NH3 G.
Source: APHA, AWWA and WEF. 2011. "Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary
Distillation Step." Standard Methods for the Examination of Water and Wastewater. 22nd Edition.
Washington, DC: APHA. htto: //www. standardmethods .org/
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5.2.99 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method
Analyte(s)
CAS RN
Ammonia
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
Method Developed for: Nitrogen (ammonia) in drinking waters, clean surface or groundwater, and
good-quality nitrified wastewater effluent
Method Selected for: This method has been selected for analysis of non-drinking water samples to
address ammonia. See Appendix A for the corresponding method usability tier.
Detection and Quantitation: The range of the method in drinking water, surface, and domestic and
industrial wastewaters is 0.02-2.0 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 [Section 5.2.98]) 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.
Special Considerations: Remove interfering turbidity by filtration. Color in the samples that absorbs in
the photometric range (630-660 nm) can interfere with analysis.
Source: APHA, AWWA and WEF. 2011. "Method 4500-NH3 G: Nitrogen (Ammonia) Automated
Phenate Method." Standard Methods for the Examination of Water and Wastewater. 22nd Edition.
Washington, DC: APHA. htto://www.standardmethods.org/
5.2.100 Standard Method 4500-CI G: Chlorine (Residual) DPD Colorimetric Method
Analyte(s)
CAS RN
Chlorine
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 is used for preparation of air samples.
Determinative Technique: Visible spectrophotometry
Method Developed for: Chlorine in water and wastewater
Method Selected for: This method has been selected for preparation and analysis of water samples to
address chlorine. It also has been selected 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 10-mL portion of buffered aqueous sample is reacted with N.N-diethyl-/;-
phenylenediamine (DPD) color agent. The resulting free chlorine is determined by colorimetry. If total
chlorine (including chloramines 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. Color and
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turbidity in the sample can cause interference and can be compensated for by first zeroing the photometer
using the sample. Chromate interferences are minimized by using thioacetamide blank correction.
Source: APHA, AWWA and WEF. 2011. "Method 4500-C1 G: DPD Colorimetric Method." Standard
Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
htto: //www. standardmethods .org/
5.2.101 Literature Reference for Chlorine in Air (Analyst, 1999. 124(12): 1853-1857)
Analyte(s)
CAS RN
Chlorine
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: This method has been selected for preparation of air samples to address chlorine.
See Appendix A for the corresponding method usability tier.
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., molecular chlorine, hypochlorous acid, 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
5.2.102 Literature Reference for Hexamethylenetriperoxidediamine (HMTD) (Analyst,
2001. 126:1689-1693)
Analyte(s)
CAS RN
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
Analysis Purpose: Analyte determination and measurement
Sample Preparation Technique: SW-846 Methods 8330B/3535A (solid samples and 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.
Determinative Technique: LC-MS-MS
Method Developed for: Trace quantities of HMTD in explosives or explosive mixtures
Method Selected for: This method has been selected for analysis of solid, water and wipe samples to
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address HMTD. 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 inner diameter [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 procedures described in SW-846 Methods 8330B and 3535A (Sections 5.2.40 and
5.2.21) be used to prepare solid and water samples, and the procedures described in SW-846 Methods
3570 and 8290A Appendix A (Sections 5.2.24 and 5.2.36) be used to prepare wipe samples.
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/cn/Contcnt/Articlc Landing/2001/AN/b 1073 54k
5.2.103 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233-
237)
Analyte(s)
CAS RN
Acephate
30560-19-1
Methamidophos
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: This method has been selected for preparation and analysis of non-drinking water
samples to address the analytes listed in the table above. 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(.un particle size) coupled with a Ci8 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/
5.2.104 Literature Reference for Cyanogen Chloride (Encyclopedia of Anal. Chem. 2006
DOI: 10.1002/9780470027318.a0809)
Analyte(s)
CAS RN
Cyanogen chloride
506-77-4
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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: This method has been selected for preparation and analysis of water and solid
samples to address cyanogen chloride. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: In drinking water, the MDL is 0.13 j^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
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
sodium sulfate, 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.a08Q9/abstract
5.2.105 Literature Reference for 3-Chloro-1,2-propanediol (Eur. J. Lipid Sci. Technol.
2011. 113: 345-355)
Analyte(s)
CAS RN
3-Chloro-1,2-propanediol
96-24-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction, followed by SPE cleanup and derivatization
Determinative Technique: GC-MS
Method Developed for: Trace quantities of 3-chloro-l,2-propanediol in foodstuffs
Method Selected for: This method has been selected for preparation and analysis of solid and wipe
samples to address 3-chloro-l,2-propanediol. 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 jj.g/kg. The working range is 4-4,000 j^ig/kg.
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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 heptafluorobutyrylimidazole (HFBI) at 70ฐC 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.com/doi/10.1002/eilt.vl 13.3/issuetoc
5.2.106 Literature Reference for Methyl Hydrazine (Journal of Chromatography. 1993.
617(1): 157-162)
Analyte(s)
CAS RN
Methyl hydrazine
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: This method has been selected for preparation and analysis of water samples, and
for the analysis of solid and wipe samples to address methyl hydrazine. 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
l-ig/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 water, solid and wipe 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
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5.2.107 Literature Reference for 3-Chloro-1,2-propanediol (Journal of Chromatography A.
2000. 866(1): 65-77)
Analyte(s)
CAS RN
3-Chloro-1,2-propanediol
96-24-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Solvent extraction followed by derivatization
Determinative Technique: GC-ECD
Method Developed for: Determination of 3-chloro-l,2-propanediol in water
Method Selected for: This method has been selected for preparation and analysis of water samples to
address 3-chloro-l,2-propanediol. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The MDL is 0.73 j^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 |_iL 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 |aL 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 shaken 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. The presence of 3-chloro-l,2-
propanediol should be confirmed using either a secondary GC column or an MS.
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.sciencedirect.com/science/article/pii/S002196739901Q81X
5.2.108 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl
Fluoroacetate (Journal of Chromatography A. 2007. 1139: 271-278)
Analyte(s)
CAS RN
Fluoroacetic acid and fluoroacetate salts (analyze as fluoroacetate ion)
NA
Methyl fluoroacetate (analyze as fluoroacetate ion)
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: This method has been selected for preparation and analysis of solids and wipes
and for the analysis of air samples to address the analytes listed in the table above as fluoroacetate ion.
See Appendix A for corresponding method usability tiers.
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Detection and Quantitation: The LOD is 0.8 j^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 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 65ฐC 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.sciencedirect.com/science/article/pii/S0021967306021388
5.2.109 Literature Reference for Methamidophos (Journal of Chromatography A. 2007.
1154: 3-25)
Analyte(s)
CAS RN
Acephate
30560-19-1
Methamidophos
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: This method has been selected for preparation and analysis of solid, air and wipe
samples to address the analytes listed in the table above. See Appendix A for corresponding method
usability tiers.
Detection and Quantitation: The LOD for this method is 0.01 mg/kg.
Description of Method: An 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(.un particle size)
coupled with a Ci8 guard cartridge system (4 mm x 3.0 mm I.D.).
Special Considerations: The procedure has been developed for the analysis of various pesticides
(methamidophos) in crops using LC-MS-MS; modifications will be needed for application to
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environmental samples such as soils, wipes and air samples collected on sorbent/filters. If problems occur
when using this method to analyze for methamidophos in air samples, NIOSH Method 5600 (Section
5.2.64) should be used.
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/article/pii/S00219673070Q5845
5.2.110 Literature Reference for Paraquat (Journal of Chromatography A. 2008,1196-
1197, 110-116)
Analyte(s)
CAS RN
Paraquat
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: This method has been selected for preparation and analysis of solid and wipe
samples to address paraquat. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: Limits of detection are 10 j^ig/kg by digestion and 50 j^ig/kg by MAE when
using LC-UV and 1.0 j^ig/kg by digestion and 3.0 j^ig/kg by MAE when using LC-MS-MS. EQLs are 20
(ig/kg and 100 j^ig/kg when using LC-UV and 2.0 jj.g/kg by digestion and 7.5|_ig/kg by MAE when using
LC-MS-MS.
Description of Method: Soil matrices can be extracted using one of the following three procedures: (1)
digestion with an acidic mcthanol/tEDTA solution, (2) shaking in an EDTA/ammonium formate solution,
or (3) using a MAE 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.sciencedirect.com/science/article/pii/S00219673080Q5335
5.2.111 Literature Reference for Fentanyl (Journal of Chromatography A. 2011. 1218:
1620-1649)
Analyte(s)
CAS RN
Fentanyl
437-38-7
Analysis Purpose: Analyte determination and measurement
Sample Preparation Technique: SW-846 Methods 3541 and 3545A (solid samples), 3520C and 3535A
(water samples), and 3570 and 8290A Appendix A (wipes)
Determinative Technique: LC-MS-MS
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Method Developed for: Fentanyl in wastewater and surface water
Method Selected for: This method has been selected for analysis of water, solid and wipe samples to
address fentanyl. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: MDLs in surface water and wastewater (effluent and influent) are 0.05 and
0.2 ng/L, respectively. Method quantitation limits in surface water, wastewater effluent and wastewater
influent are 0.10, 0.6 and 0.7 ng/L, respectively. The reportable range for fentanyl in surface water is
0.08-750 ng/L.
Description of Method: Water samples are vacuum filtered, first through two sequential glass fiber
filters (2.7 (jm, then 0.7 |im). After filtration, samples are acidified with 31 % hydrochloric acid to pH
1.8-1.9. The SPE cartridge (60 mg) is conditioned with 2 mL of methanol and equilibrated with 2%
formic acid in water (2 mL, pH 2), both at a flow rate of 3 mL/minute. Acidified samples are spiked with
50 ng of each surrogate and internal standard and then passed through the cartridge at a rate of 6
mL/minute. Immediately following loading, cartridges are washed with 2% formic acid in water (2 mL at
pH 2) at a flow rate of 3 mL/minute, then washed again with 2 mL of 0.6% formic acid in methanol (pH
2) at a flow rate of 3 mL/minute, followed by elution with 3 mL of 7% ammonium hydroxide in methanol
at a flow rate of 1 mL/minute into silanized vials. Extracts are evaporated to dryness under nitrogen,
reconstituted with 500 |iL of 0.3% acetic acid/5% methanol in reagent-grade water, and filtered through
0.2 (mi polytetrafluoroethylene (PTFE) filters before being transferred to deactivated maximum recovery
vials with PTFE septa. Extracts are analyzed by LC-MS-MS equipped with an ethylene-bridged hybrid
(BEH) column.
Source: Baker, D. and Kaxprzyk-Hordern, B. 2011. "Multi-residue analysis of drugs of abuse in
wastewater and surface water by solid-phase extraction and liquid chromatography-positive electrospray
ionisation tandem mass spectrometry." Journal of Chromatography A. 1218(12): 1620-1659.
http://www.sciencedirect.com/science/article/pii/S0021967311001312
5.2.112 Literature Reference for BZ (Journal of Chromatography B. 2008. 874: 42-50.
Analyte(s)
CAS RN
BZ [Quinclidinyl benzylate]
6581-06-2
Analysis Purpose: Sample preparation, and analyte determination and measurement
Sample Preparation Technique: Direct injection for water samples, SW-846 Methods 3541 or 3545 for
solid samples, and SW-846 Methods 3570 and 8290A Appendix A for wipes.
Determinative Technique: LC-MS-MS
Method Developed for: Benzodiazepines in human plasma
Method Selected for: This method has been selected for preparation and analysis of water samples and
for the analysis of prepared solid and wipe samples to address BZ. See Appendix A for corresponding
method usability tiers.
Detection and Quantitation: The limits of detection for the benzodiazepines in human plasma ranged
from 0.1 to 1 ng/mL, the LOQs ranged from 0.25 to 5 ng/mL, and the working range for the
benzodiazepines in human plasma is 0.25-1000 ng/mL (depending on the analyte). This method was
developed for compounds similar in structure to BZ; therefore, no detection or quantification information
is available for BZ.
Description of Method: Water samples are filtered and directly injected into an LC-MS-MS for
analysis. Soil samples are extracted by PFE or Soxhlet extraction using SW-846 Method 3541/3545A
(Sections 5.2.22 and 5.2.23), prior to filtration and injection into the LC-MS-MS. Wipe samples are
solvent extracted prior to filtration and injection into the LC-MS-MS. In all cases, an internal standard is
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Section 5.0 - Selected Chemical Methods
added prior to extraction or filtration. The triple quadrupole LC-MS-MS is equipped with a C8 column
and operated in positive ion mode with MRM. Instrument parameters specific for BZ can be found in
Schaer, 2012 (see Additional Resource).
Special Considerations: The procedure has been developed for the analysis of benzodiazepines in
human plasma; modifications will be needed for application to BZ in environmental samples. An
overview of a strategy for detection and identification of BZ, including information regarding ESI-source,
fragmentation, precursor-ion, collision energies, and scanning time, is provided in a poster presentation
cited in the Additional Resource cited below.
Source: Abbara, C., Bardot, I., Cailleux, A., Lallement, G., Le Bouil, A., Turcant, A., Clair, P. and
Diquet, B. 2008. "High-performance liquid chromatography coupled with electrospray tandem mass
spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and
pralidoxime in human plasma." Journal of Chromatography B. 874: 42-50.
http://www.sciencedirect.com/science/article/pii/S15700232080Q6545
Additional Resource: Schaer, M. 2012. "Rapid Screening and Identification of Chemical Warfare
Agents in Environmental Samples using LC/MS and a MS/MS-Library." Spiez Laboratory.
https: //www. labor-spiez. ch/pdf/ en/dok/fas/LC-
MS Screening and Identification of CWA in Environmental Samples using LCMS and a MSMS-
Librarv 2008.pdf
5.2.113 Literature Reference for Fluoroacetamide (Journal of Chromatography B. 2008.
876(1): 103-108)
Analyte(s)
CAS RN
Fluoroacetamide
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: This method has been selected for preparation and analysis of solid, water, air and
wipe samples to address fluoroacetamide. See Appendix A for corresponding method usability tiers.
Detection and Quantitation: The detection limit of this method for fluoroacetamide is 0.01 jj.g/g.
Description of Method: Samples are extracted by microscale liquid-liquid extraction using acetonitrile,
ENVI-Carb (Sigma-Aldrich, St. Louis, MO) 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
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5.2.114 Literature Reference for Fluoroacetic Acid/Fluoroacetate Salts/Methyl
Fluoroacetate (Journal of Chromatography B. 2010. 878: 1045-1050)
Analyte(s)
CAS RN
Fluoroacetic acid and fluoroacetate salts (analyze as fluoroacetate ion)
NA
Methyl fluoroacetate (analyze as fluoroacetate ion)
453-18-9
Analysis Purpose: Sample preparation (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: This method has been selected for preparation and analysis of water samples to
address the analytes listed in the table above as fluoroacetate ion. 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 50-5000 (ig/L.
Description of Method: Fluoroacetate, in the form of MFA, 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, is 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 aqueous
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-MS-Flouroacetate-Water-AN7Q478 E.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.sciencedirect.com/science/article/pii/S15700232100Q142X
5.2.115 Literature Reference for Carfentanil and 3-Methyl Fentanyl (J. Chromatogr. B.
2014. 962: 52-58
Analyte(s)
CAS RN
Carfentanil
59708-52-0
3-Methyl fentanyl
42045-87-4
Analysis Purpose: Analyte determination and measurement
Sample Preparation Technique: SW-846 Methods 3541 and 3545A (solid samples), 3520C and 3535A
(water samples), and 3570 and 8290A Appendix A (wipe samples)
Determinative Technique: LC-MS-MS
Method Developed for: Carfentanil, fentanyl and 3-methyl fentanyl in human urine
Method Selected for: This method has been selected for analysis of water, solid and wipe samples to
address the analytes listed in the table above. See Appendix A for corresponding method usability tiers.
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Detection and Quantitation: LODs for carfentanil, fentanyl and 3-methyl fentanyl in human urine when
using off-line SPE are estimated to be 0.008, 0.007 and 0.020 ng/mL, respectively. The reportable range
is 0.010-10 ng/mL for carfentanil and fentanyl, and 0.050-10 ng/mL for 3-methyl fentanyl.
Description of Method: Carfentanil, fentanyl and 3-methylfentanyl are extracted from samples by
placing aliquots off-line on a 96-well plate or using an on-line SPE system. In both cases, internal
standards are added to sample aliquots prior to loading onto the SPE cartridge. For on-line SPE,
cartridges are conditioned with acetonitrile and aqueous ammonium hydroxide solutions. Sample aliquots
are loaded onto the SPE unit, washed with aqueous ammonium hydroxide:acetonitrile solution and eluted
with LC gradient onto the HPLC column. When using the 96-well plate, sample aliquots are diluted with
aqueous ammonium hydroxide and loaded onto a plate conditioned with acetonitrile and aqueous
ammonium hydroxide solutions, washed with a water:acetonitrile:ammonium hydroxide mixture, and
eluted with acetonitrile. The extracts are evaporated to dryness, reconstituted in water and injected into
the HPLC for analysis. Target compounds are identified using ESI tandem mass spectrometry. The
retention time for all three compounds is expected to be -3.4 minutes. The analytes can be measured as
individual compounds due to the use of different monitoring ions.
Special Considerations: This procedure has been developed for urine samples; modifications may be
needed for application to environmental samples.
Source: Shaner, R. L., Kaplan, P., Hamelin, E. I., William A. Bragg, W. A., Johnson, R. C. 2014.
"Comparison of two automated solid phase extractions for the detection of ten fentanyl analogs and
metabolites in human urine using liquid chromatography tandem mass spectrometry." Journal of
Chromatography B. 962: 52-55.
http://www.sciencedirect.com/science/article/pii/S157002321400333X7via%3Dihub
5.2.116 Literature Reference for Sodium Azide (Journal of Forensic Sciences. 1998. 43(1):
200-202)
Analyte(s)
CAS RN
Sodium azide (analyze as azide ion)
26628-22-8
Analysis Purpose: Sample preparation
Sample Preparation Technique: Water extraction, filtration and/or acidification
Determinative Technique: IC with conductivity detection
Determinative Method: EPA Method 300.1, Revision 1.0
Method Developed for: Sodium azide in blood
Method Selected for: This method has been selected for preparation of solid, water samples to address
sodium azide as azide ion. 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 IC 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 was developed for 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
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Section 6.0: Selected Radiochemical Methods
A list of analytical methods to be used in analyzing environmental and outdoor building and infrastructure
material 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 a radiological or nuclear incident. The isotopes included are
based on selection criteria that address the needs and priorities of EPA as well as other federal agencies
(see Section 1.0).
Please note: This section provides guidance for selecting radiochemical methods that have a high
likelihood of assuring analytical consistency when laboratories are tasked with analyzing samples
following a large scale radiological or nuclear contamination incident. Although the majority of methods
have been validated for the analyte/sample type combination for which they have been selected,
validation is still needed for a few of the methods that have been selected for analysis of vegetation.
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 4.0.
Appendix B1 (environmental samples) 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
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presence of a radionuclide. Although quantitative, these methods have been selected as qualitative
methods since they can be utilized with shorter counting times, at greater uncertainty, when increased
sample throughput and more rapid reporting of results are 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.
Appendix B2 (outdoor building and infrastructure materials) 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.
Asphalt shingle sample methods. The recommended methods/procedures for sample preparation
and analysis to measure the analyte of interest in asphalt roofing materials. Methods have been
identified for sample preparation and confirmatory determination.
Asphalt matrices sample methods. The recommended methods/procedures for sample preparation
and analysis to measure the analyte of interest in asphalt paving material samples. Methods have been
identified for sample preparation and confirmatory determination.
Concrete sample methods. The recommended methods/procedures for sample preparation and
analysis to measure the analyte of interest in concrete samples. Methods have been identified for
sample preparation and confirmatory determination.
Brick sample methods. The recommended methods/procedures for sample preparation and analysis
to measure the analyte of interest in brick samples. Methods have been identified for qualitative and
confirmatory determination.
Sample preparation method identifier. A unique identifier or number assigned to an analytical
method by the method publisher. The identified method is intended to digest solid samples into a
liquid form suitable for analysis.
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.
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 or on
buildings and infrastructure 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
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resource. EPA's Radiation Protection Program (http://www.epa.gov/radiation/radionuclides/index.html')
and the Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP)
(https://www.epa.gov/radiation/marlap-manual-and-supporting-documents') (last accessed 07/2017)
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 https://www.epa.gov/radiation/radiation-
protection-document-librarv (last accessed 07/2017; enter "incident guides" in the search bar for quick
access).
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 B1: 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.
To determine the appropriate method to be used on an outdoor building or infrastructure material sample,
locate the analyte of concern in Appendix B2: 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 sample
preparation and/or confirmatory method for the sample type of interest (asphalt shingles, asphalt
materials, concrete or brick).
Once a method has been identified in Appendix B1 or B2, Table 6-1 can be used to locate the method
summary. Sections 6.2.1 through 6.2.59 provide summaries of the qualitative and confirmatory methods
listed in Appendix B1 for analysis of environmental samples. Sections 6.3.1 through 6.3.8 provide
summaries of the sample preparation and confirmatory methods listed in Appendix B2 for analysis of
outdoor building and infrastructure material samples.
Table 6-1. Radiochemical Methods and Corresponding Section Numbers
Analyte / Analyte
Class
CAS RN
Method
Section
900.0 (EPA)
6.2.2
Gross Alpha
NA
FRMAC, Vol 2, pg. 33 (DOE)
6.2.39
Gross Beta
NA
AP1 (ORISE)
6.2.43
7110 B (SM)
6.2.53
Gamma
901.1 (EPA)
6.2.3
Select Mixed Fission
Products
NA
Ga-01-R (HASL-300)
6.2.31
Total Activity
Screening
NA
Y-12 Preparation of Samples for Total Activity
Screening (DOE)
6.2.59
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Analyte / Analyte
Class
CAS RN
Method
Section
Alpha Spectrometry:
Rapid Radiochemical Method for Am-241 (EPA)
6.2.12
Rapid methods for acid or fusion digestion (EPA)
6.2.17 and 6.2.18
Rapid Method for Fusion of Soil and Soil-Related
Matrices (EPA)
6.2.20
Am-01-RC (HASL-300)
6.2.28
Am-04-RC (HASL-300)
6.2.29
Am-06-RC (HASL-300)
6.2.30
Pu-12-RC (HASL-300)
6.2.33
Actinides and Sr-89/90 in Vegetation (SRS)
6.2.42
AP11 (ORISE)
6.2.47
Americium-241
14596-10-2
D3084-05 (ASTM)
6.2.49
Rapid Method for Sodium Hydroxide Fusion of
Concrete and Brick (EPA)
6.3.3
Rapid Method for Americium-241 in Building
Materials (EPA)
6.3.6
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Matrices (EPA)
6.3.7
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Roofing Materials (EPA)
6.3.8
Gamma Spectrometry:
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
7120 (SM)
6.2.54
Rapid Radiochemical Method for Californium-
252 (EPA)
6.2.23
Californium-252
13981-17-4
Am-06-RC (HASL-300)
6.2.30
AP11 (ORISE)
6.2.47
D3084-05 (ASTM)
6.2.49
Cesium-137
10045-97-3
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
Cobalt-60
10198-40-0
7120 (SM)
6.2.54
Rapid Radiochemical Method for Curium-244 in
Water (EPA)
6.2.24
Curium-244
13981-15-2
Rapid Radiochemical Method for Curium-244 in
Air Particulate Filters, Swipes and Soil (EPA)
6.2.25
Am-06-RC (HASL-300)
6.2.30
AP11 (ORISE)
6.2.47
D3084-05 (ASTM)
6.2.49
901.1 (EPA)
6.2.3
Europium-154
15585-10-1
Ga-01-R (HASL-300)
6.2.31
7120 (SM)
6.2.54
lndium-111
15750-15-9
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
lodine-125
14158-31-7
Procedure #9 (ORISE)
6.2.48
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Analyte / Analyte
Class
CAS RN
Method
Section
lodine-131
10043-66-0
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
901.1 (EPA)
6.2.3
lridium-192
14694-69-0
Ga-01-R (HASL-300)
6.2.31
7120 (SM)
6.2.54
Molybdenum-99
14119-15-4
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
907.0 (EPA)
6.2.7
Neptunium-237
13994-20-2
SOP for Actinides in Environmental Matrices
(EPA-NAREL)
6.2.27
901.1 (EPA)
6.2.3
Neptunium-239
13968-59-7
Ga-01-R (HASL-300)
6.2.31
7120 (SM)
6.2.54
Rapid Radiochemical Method for P-32 (EPA)
6.2.9
Phosphorus-32
14596-37-3
R4-73-014 (EPA)
6.2.10
RESL P-2 (DOE)
6.2.40
EMSL-33 (EPA)
6.2.8
Rapid Radiochemical Method for Pu-238 and -
239/240 (EPA)
6.2.13
Rapid methods for acid or fusion digestion (EPA)
6.2.17 and 6.2.18
Rapid Method for Fusion of Soil and Soil-Related
Matrices (EPA)
6.2.20
Rapid Method for Sodium Hydroxide/Sodium
Peroxide Fusion of Radioisotope Thermoelectric
Generator Materials in Water and Air Filter
Matrices (EPA)
6.2.22
Plutonium-238
13981-16-3
SOP for Actinides in Environmental Matrices
(EPA-NAREL)
6.2.27
Plutonium-239
15117-48-3
Am-06-RC (HASL-300)
6.2.30
Actinides and Sr-89/90 in Vegetation (SRS)
6.2.42
AP11 (ORISE)
6.2.47
D3084-05 (ASTM)
6.2.49
Rapid Method for Sodium Hydroxide Fusion of
Concrete and Brick (EPA)
6.3.3
Rapid Method for Plutonium-238, -239/240 in
Building Materials (EPA)
6.3.5
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Matrices (EPA)
6.3.7
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Roofing Materials (EPA)
6.3.8
Polonium-210
13981-52-7
Method 111 (EPA)
6.2.1
Po-02-RC (HASL-300)
6.2.32
Radium-223
15623-45-7
Rapid Radiochemical Method
for Ra-226 in Water (EPA)
6.2.14
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Analyte / Analyte
Class
CAS RN
Method
Section
903.1 (EPA)
6.2.4
Rapid Radiochemical Method for Ra-226 in
Water (EPA)
6.2.14
Rapid methods for acid or fusion digestion (EPA)
6.2.17 and 6.2.18
Rapid Method for Radium in Soil (EPA)
6.2.19
Ra-03-RC (HASL-300)
6.2.34
AP7 (ORISE)
6.2.46
Radium-226
13982-63-3
7500-Ra B (SM)
6.2.55
7500-Ra C (SM)
6.2.56
Rapid Radiochemical Method for Radium-226 in
Building Materials (EPA)
6.2.26 and 6.3.2
Rapid Method for Sodium Hydroxide Fusion of
Concrete and Brick (EPA)
6.3.3
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Matrices (EPA)
6.3.7
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Roofing Materials (EPA)
6.3.8
Rhenium-188
14378-26-8
901.1 (EPA)
6.2.3
Rubidium-82
14391-63-0
Ga-01-R (HASL-300)
6.2.31
Ruthenium-103
13968-53-1
901.1 (EPA)
6.2.3
Ruthenium-106
13967-48-1
Ga-01-R (HASL-300)
6.2.31
Selenium-75
14265-71-5
7120 (SM)
6.2.54
905.0 (EPA)
6.2.5
Strontium-89
14158-27-1
Strontium in Food and Bioenvironmental
Samples (EPA)
6.2.11
Actinides and Sr-89/90 in Soil Samples (SRS)
6.2.41
Actinides and Sr-89/90 in Vegetation (SRS)
6.2.42
905.0 (EPA)
6.2.5
Rapid Radiochemical Method for Radiostrontium
(EPA)
6.2.15
Rapid methods for acid or fusion digestion (EPA)
6.2.17 and 6.2.18
Rapid Method for Sodium Carbonate Fusion of
Soil and Soil-Related Matrices Prior to Strontium-
90 Analyses (EPA)
6.2.21
Sr-03-RC (HASL-300)
6.2.35
Strontium-90
10098-97-2
Actinides and Sr-89/90 in Vegetation (SRS)
6.2.42
D5811-08 (ASTM)
6.2.51
Rapid Method for Total Radiostrontium in
Building Materials (EPA)
6.3.1
Rapid Method for Sodium Hydroxide Fusion of
Concrete and Brick (EPA)
6.3.3
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Matrices (EPA)
6.3.7
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Roofing Materials (EPA)
6.3.8
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Analyte / Analyte
Class
CAS RN
Method
Section
Tc-01-RC (HASL-300)
6.2.36
Technetium-99
14133-76-7
Tc-02-RC (HASL-300)
6.2.37
AP5 (ORISE)
6.2.45
D7168-05 (ASTM)
6.2.52
Technetium-99m
378784-45-3
901.1 (EPA)
6.2.3
Ga-01-R (HASL-300)
6.2.31
Thorium-227
15623-47-9
907.0 (EPA)
6.2.7
Thorium-228
14274-82-9
Thorium-230
Thorium-232
14269-63-7
7440-29-1
SOP for Actinides in Environmental Matrices
(EPA-NAREL)
6.2.27
Tritium (Hydrogen-3)
10028-17-8
906.0 (EPA)
6.2.6
AP2 (ORISE)
6.2.44
Rapid Radiochemical Method for Isotopic
Uranium in Water (EPA)
6.2.16
Rapid methods for acid or fusion digestion (EPA)
6.2.17 and 6.2.18
Rapid Method for Fusion of Soil and Soil-Related
Matrices (EPA)
6.2.20
U-02-RC (HASL-300)
6.2.38
Actinides and Sr-89/90 in Vegetation(SRS)
6.2.42
AP11 (ORISE)
6.2.47
Uranium-234
13966-29-5
D3972-02 (ASTM)
6.2.50
Uranium-235
15117-96-1
7500-U B (SM)
6.2.57
7500-U C (SM)
6.2.58
Uranium-238
7440-61-1
SOP for Actinides in Environmental Matrices
(EPA-NAREL)
6.2.27
Rapid Method for Sodium Hydroxide Fusion of
Concrete and Brick (EPA)
6.3.3
Rapid Method for Isotopic Uranium in Building
Materials (EPA)
6.3.4
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Matrices (EPA)
6.3.7
Rapid Method for Sodium Hydroxide Fusion of
Asphalt Roofing Materials (EPA)
6.3.8
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.
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Table 6-2. Sources of Radiochemical Methods
Name
Publisher
Reference
National Environmental Methods
Index (NEMI)
EPA, U.S. Geological
Survey (USGS)
httD: //www. n em i. a ov
Code of Federal Regulations
(CFR) Promulgated Test Methods
EPA, Emission
Measurement Center (EMC)
https://www.epa.qov/emc/emc-promulqated-
test-methods
Prescribed Procedures for
Measurement of Radioactivity in
Drinking Wafer (EPA-600 4-80-
032, August 1980)
EPA, ORD, Environmental
Monitoring and Support
Laboratory (EMSL)
https://nepis.epa.qov/Exe/ZvPDF.cqi/30000Q
HM.PDF?Dockev=30000QHM.PDF
Also available from National Technical
Information Service (NTIS)*, U.S. Department
of Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
Rapid Radiochemical Methods for
Selected Radionuclides in Water
for Environmental Restoration
Following Homeland Security
Events (EPA 402-R-10-001)
EPA, ORIA, National
Analytical and Radiation
Environmental Laboratory
(NAREL)
https://www.epa.qov/radiation/rapid-
radiochemical-methods-selected-
radionuclides
Rapid Radiochemical Methods for
Selected Radionuclides
EPA, ORIA, NAREL
https://www.epa.qov/radiation/rapid-
radiochemical-methods-selected-
radionuclides
Radiochemical Analytical
Procedures for Analysis of
Environmental Samples, March
1979. EMSL-LV-0539-17
EPA, EMSL
Available NTIS*, U.S. Department of
Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
EML Procedures Manual, Health
and Safety Laboratory (HASL-
300), 28th Edition, February, 1997
Department of Energy
(DOE), Environmental
Measurements Laboratory
(EML) / Now DHS
http://www.wipp.enerqv.qov/NAMP/EMLLeqa
cy/
Also available from NTIS*, U.S. Department
of Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
Federal Radiological Monitoring
and Assessment Center (FRMAC)
Laboratory Manual
DOE, National Nuclear
Security Administration
(NNSA)
https://www.epa.qov/sites/production/files/201
5-06/documents/frmac-vol2-pq33.pdf
Y-12 National Security Complex
(Y-12)
DOE, NNSA
http://www.v12.doe.qov/
Radiological and Environmental
Sciences Laboratory (RESL)
Analytical Chemistry Branch
Procedures Manual
DOE, RESL
Available from NTIS, U.S. Department of
Commerce, 5285 Port Royal Road,
Springfield, VA 22161, (703)605-6000.
Savannah River Site (SRS)
Methods
DOE, SRS
Savannah River National Laboratory
Savannah River Site
Aiken, SC 29808,
(803) 725-6211
Annual Book of ASTM Standards,
Vol. 11.02*
ASTM International
http://www.astm.orq
Standard Methods for the
Examination of Water and
Wastewater, 22nd Edition, 2005*
American Public Health
Association (APHA)
http://www.standardmethods.orq
* Subscription and/or purchase required.
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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 the effectiveness of those activities.
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.
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 used. Implementation of the analytical methods for evaluation of environmental
and outdoor building and infrastructure material samples during site assessment through site clearance,
such as those identified in this document, might require increased QC frequency and more stringent QC
criteria.
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: https ://www. epa. gov/radiation/radiation-
protection-document-librarv (last accessed 07/2017; enter "MARLAP" in the search for quicker access).
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 4.0 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.
8 Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/OUALITY/daos.html (last accessed 09/2017).
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6.1.3 Safety and Waste Management
It is imperative that safety precautions be used during collection, processing and analysis of
environmental and outdoor building and infrastructure material 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 and Section 6.3 contain specific requirements, guidelines or
information regarding safety precautions that should be followed when handling or processing 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:
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 (last accessed 9/2017)
EPA - 40 CFR part 260. Hazardous Waste Management System: General. Available at:
https://www.gpo.gov/fdsvs/granule/CFR-2012-title40-vol27/CFR-2012-title40-vol27-part260 (last
accessed 9/2017)
EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit Program.
Available at: https://www.gpo.gov/fdsvs/granule/CFR-2012-title40-vol28/CFR-2012-title4Q-vol28-
part270 (last accessed 9/2017)
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 OO.html (last
accessed 9/2017)
DOE. Order O 435.1: Radioactive Waste Management. January 1, 2007. Available at:
https://www.directives.doe.gov/directives-documents/400-series/Q435.l-BOrder-chgl (last accessed
9/2017)
DOE. M 435.1-1. Radioactive Waste Management Manual. Office of Environmental Management.
June 8, 2011. Available at: https://www.directives.doe.gov/directives-documents/400-series/0435.l-
DManual-1 -chg2-AdmChg (last accessed 9/2017)
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/DQE/Misc/radmeth3.pdf (last accessed 9/2017)
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 (last accessed 9/2017)
EPA. 2001. Changes to 40 CFR 266 (Storage, Treatment, Transportation, and Disposal of Mixed
Waste), Federal Register 66.21211-21266, May 16, 2001. Available at:
https://www.federalregister.gov/documents/2001/05/16/01-11411/hazardous-waste-identification-
rule-hwir-revisions-to-the-mixture-and-derived-from-rules (last accessed 9/2017)
EPA. 2014. Resource Conservation and Recovery Act (RCRA) Orientation Manual. Office Of
Resource Conservation And Recovery (ORCR), Washington, DC. EPA530-F-11-003. 242 pp.
Available at: https://www.epa.gov/sites/production/files/2015-07/documents/rom.pdf (last accessed
09/2017)
MARLAP Manual. 2004. Chapter 17. Waste Management in a Radioanalytical Laboratory. EPA 402-
B-04-001B. Available at: https://www.epa.gov/sites/production/files/2015-05/documents/402-b-Q4-
001b-17-final.pdf (last accessed 09/2017)
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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 (last accessed 09/2017)
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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6.2 Method Summaries (Environmental Samples)
Summaries corresponding to the methods selected for analysis of environmental samples listed in
Appendix B1 are provided in Sections 6.2.1 through 6.2.59. These summaries contain information that
has been extracted from the selected methods. Each method summary contains a table identifying the
contaminants listed in Appendix B1 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. For information regarding sample collection considerations for samples to be analyzed
by these methods, see the companion Sample Collection Information Document for Chemicals,
Radiochemicals and Biotoxins, Companion to SAM 2017.
6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary
Sources
Analyte(s)
CAS RN
Polonium-210
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: This method has been selected 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 onto the surface of metals that are more electropositive than polonium.
Polonium-209 tracers should be added to each sample to determine the chemical yield.
Special Considerations: Compounds, such as clays that are present in some decontamination agents,
can contain iron, magnesium and/or calcium, which can potentially be released as ions via ion exchange
in the presence of certain radionuclides, and cause analytical interferences. Although iron (III), a major
interference in the analysis of polonium-210 by alpha spectrometry, is extracted from the concentrated
hydrochloric solution using liquid-liquid extraction with diisopropylether, high concentrations of iron
may not be completely removed. Chelators, also present in some decontamination agents, can tightly
complex iron that may be present in the sample, preventing its removal.
Source: U.S. EPA. EMC, prepared by the Office of Air Quality Planning and Standards (OAQPS).
2000. "Method 111: Determination of Polonium-210 Emissions from Stationary Sources." Research
Triangle Park, NC: U.S. EPA. http://www.epa.gov/sites/production/files/2015-06/documents/epa-111.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: This method has been selected for gross alpha and gross beta determination in
drinking water samples.
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Description of Method: The method provides an indication of the presence of alpha and beta emitters,
including the following analytes:
Americium-241 (CAS
Californium-252 (CAS
Cesium-137 (CAS
Cobalt-60 (CAS
Curium-244 (CAS
Europium-154 (CAS
Iridium-192 (CAS
Plutonium-23 8 (CAS
Plutonium-23 9 (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
An aliquot of a preserved drinking water sample is evaporated to a small volume (3 to 5 mL) and
transferred quantitatively to a tared 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.9 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-137).
Special Considerations: Long counting time and increased sample size may be required to meet
detection limits. Sensitivity is limited by the concentration of solids in the sample. 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. Compounds
containing carbonate, fluoride, hydroxide, or phosphate, such as those present in some decontamination
agents, can precipitate radionuclides out of solution prior to analysis. This precipitation can result in a
lesser amount of radionuclides in cases where an aliquot of a water sample is transferred and analyzed
separately from the entire sample.
Source: U.S. EPA, EMSL. 1980. "Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking
Water " Prescribed Procedures for Measurement of Radioactivity in Drinking Water. Cincinnati, OH:
U.S. EPA. EPA/600/4/80/032, http://www.epa.gov/sites/production/files/2015-06/documents/epa-
900.0.pdf
6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water
Analyte(s)
CAS RN
Americium-241
14596-10-2
Cesium-137
10045-97-3
Cobalt-60
10198-40-0
9 EPA lists standards for use when analyzing drinking water in the table at 40 CFR 141.25 (Footnote 11).
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Analyte(s)
CAS RN
Europium-154
15585-10-1
Gamma
NA
lodine-131
10043-66-0
lndium-111
15750-15-9
lridium-192
14694-69-0
Molybdenum-99
14119-15-4
Neptunium-239
13968-59-7
Rhenium-188
14378-26-8
Rubidium-82
14391-63-0
Ruthenium-103
13968-53-1
Ruthenium-106
13967-48-1
Selenium-75
14265-71-5
Select Mixed Fission Products
NA
Technetium-99m
378784-45-3
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Gamma spectrometry
Method Developed for: Gamma emitting radionuclides in drinking water
Method Selected for: This method has been selected for qualitative and confirmatory analysis of select
gamma emitters in drinking water samples.
Description of Method: This method is applicable for analysis of water samples that contain
radionuclides that emit gamma photons with energies ranging from approximately 60 to 2000 keV. The
method uses gamma spectroscopy for measurement of gamma photons emitted from radionuclides
without separating them from the sample matrix. A homogeneous aliquot of water is placed into a
standard geometry (normally a Marinelli beaker) for gamma counting, typically using a high purity
germanium 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 poorer 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.
Special Considerations: The presence of reducing agents, such as those contained in some
decontamination agents, can convert radionuclides to an insoluble zero-valent state that can precipitate
out of solution. Although the addition of nitric acid can prevent this precipitation from occurring, iridium,
molybdenum and ruthenium would likely still precipitate in the presence of these agents. Compounds
such as clays, which are also present in some decontamination agents, can sequester cesium-137, which
would only be released upon complete dissolution when using this method. Compounds containing
carbonate, fluoride, hydroxide or phosphate also can precipitate radionuclides out of solution. All of these
are a concern in cases where an aliquot of water sample is transferred and analyzed separately from the
entire sample.
Source: U.S. EPA, EMSL. 1980. "Method 901.1: Gamma Emitting Radionuclides in Drinking Water."
Prescribed Procedures for Measurement of Radioactivity in Drinking Water. Cincinnati, OH: U.S. EPA.
EPA/600/4/80/032, http://www.epa.gov/sites/production/files/2015-06/documents/epa-901.1 .pdf
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6.2.4 EPA Method 903.1: Radium-226 in Drinking Water - Radon Emanation Technique
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha counting
Method Developed for: Radium-226 in drinking water
Method Selected for: This method has been selected 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 (EDTA) reagent, placed in a sealed bubbler and stored for
ingrowth of radon-222. After ingrowth, the radon-222 gas is purged into a scintillation cell. When the
short-lived radon-222 daughters are in equilibrium with the parent (after ~4h), the scintillation cell is
counted for activity. The absolute measurement of radium-226 is effected by calibrating the scintillation
cell system with a standard solution of the nuclide. There are no radioactive interferences in this method.
Based on a 1000-mL sample and 100-minute counting time, the minimum detectable level for this method
is 0.5 picocuries/liter (pCi/L).
Special Considerations: The presence of compounds contained in various decontamination agents can
impact the results of analysis using this procedure due to precipitation. Precipitation can result in a lesser
amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed separately
form the entire sample. Such compounds include:
Permanganate and permanganic acid can be reduced to insoluble manganese (IV) oxide which
can remove radium.
Certain chelating agents may compromise the collection of radionuclides prior to analysis, by
preventing them from being precipitated out of solution. Dispersants and corrosion inhibitors can
have this chelating ability as well.
Clays contain iron, magnesium and calcium that can be released as ions via ion exchange, in the
presence of certain radionuclides, and cause interference in the analysis of the water.
Compounds containing sulfate, carbonate, oxalate, or phosphate can precipitate radium out of
solution prior to analysis, resulting in a lesser amount of radium in cases where an aliquot of
water sample is transferred prior to the precipitation step, and analyzed separately from the entire
sample.
Source: U.S. EPA, EMSL. 1980. "Method 903.1: Radium-226 in Drinking Water - Radon Emanation
Technique." Prescribed Procedures for Measurement of Radioactivity in Drinking Water. Cincinnati,
OH: U.S. EPA. EPA/600/4/80/032, http://www.epa.gov/sites/production/files/2015-06/documents/epa-
903.1.pdf
6.2.5 EPA Method 905.0: Radioactive Strontium in Drinking Water
Analyte(s)
CAS RN
Strontium-89
14158-27-1
Strontium-90
10098-97-2
Analysis Purpose: Qualitative and confirmatory analysis
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Technique: Beta counting
Method Developed for: Strontium-89, strontium-90 and total strontium in drinking water
Method Selected for: This method has been selected 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.
Special Considerations: Certain chelating compounds found in decontamination agents can tightly
complex barium, iron, lead, magnesium and potassium, causing interference when analyzing for
strontium-89 and -90. Compounds containing carbonate, fluoride, phosphate or sulfate, which are also
present in some decontamination agents, can precipitate radionuclides out of solution prior to analysis.
This precipitation can result in a lesser amount of strontium in cases where an aliquot of water sample is
transferred and analyzed separately from the entire sample.
Source: U.S. EPA, EMSL. 1980. "Method 905.0: Radioactive Strontium in Drinking Water."
Prescribed Procedures for Measurement of Radioactivity in Drinking Water. Cincinnati, OH: U.S. EPA.
EPA/600/4/80/032. http://www.epa.gOv/sites/production/files/2015-06/documents/epa-905.0.pdf
6.2.6 EPA Method 906.0: Tritium in Drinking Water
Analyte(s)
CAS RN
Tritium (Hydrogen-3)
10028-17-8
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Tritium (as T20 or HTO) in drinking water
Method Selected for: This method has been selected 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
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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.
Special Considerations: Some compounds present in decontamination agents, such as organic
compounds containing oxygen, reductants, halogenated compounds or elevated levels of nitrates or
nitromethane, can cause chemical quenching. Color quenching compounds, such as dyes and pigments
also contained in some decontamination agents, can have a significant impact when using liquid
scintillation methods.
Source: U.S. EPA, EMSL. 1980. "Method 906.0: Tritium in Drinking Water " Prescribed Procedures
for Measurement of Radioactivity in Drinking Water. Cincinnati, OH: U.S. EPA. EPA/600/4/80/032.
http://www.epa.gOv/sites/production/files/2015-06/documents/epa-906.0.pdf
6.2.7 EPA Method 907.0: Actinide Elements in Drinking Water - Thorium, Uranium,
Neptunium, Plutonium, Americium and Curium
Analyte(s)
CAS RN
Neptunium-237
13994-20-2
Thorium-227
15623-47-9
Thorium-228
14274-82-9
Thorium-230
14269-63-7
Thorium-232
7440-29-1
Analysis Purpose: Qualitative and confirmatory determination
Technique: Alpha spectrometry
Method Developed for: Alpha emitting actinide elements in drinking water
Method Selected for: This method has been selected for qualitative and confirmatory analysis of
drinking water samples.
Description of Method: Actinide elements are concentrated by coprecipitation using ferric hydroxide.
The ferric hydroxide is dissolved and thorium, neptunium, plutonium, americium and curium are
coprecipitated with bismuth phosphate. The bismuth phosphate is dissolved in 8M hydrochloric acid and
plutonium and neptunium are extracted in tri-isooctylamine (TIOA). The thorium is separated from
americium and curium by extraction with trioctylphosphine oxide (TOPO). All separated and purified
elements are coprecipitated on lanthanum fluoride and alpha counted.
Special Considerations: The technical contacts in Section 4.0 should be consulted regarding use of
alpha spectrometry for analysis of samples prepared using this method to detect and measure specific
isotopes. Ammonium ions interfere in the precipitation of neptunium with ferric hydroxide. If ammonium
ions are present, adding sodium hydroxide to raise the pH should result in complete recovery of
neptunium. Chelating agents, which are present in some decontamination agents, will interfere to varying
extents by totally or partially complexing actinide elements. Dispersants and corrosion inhibitors, also
present in decontaminating agents, can have chelating ability as well. When chelating agents are present,
alternate methods, such as coprecipitation from acid solutions (Section 6.2.27), should be considered.
Clays that are present in some decontamination agents can contain iron, magnesium and calcium that can
be released as ions via ion exchange, in the presence of certain radionuclides, and cause interferences.
Source: U.S. EPA, EMSL. 1980. "Method 907.0: Actinide Elements in Drinking Water - Thorium,
Uranium, Neptunium, Plutonium, Americium and Curium." Prescribed Procedures for Measurement of
Radioactivity in Drinking Water. Cincinnati, OH: U.S. EPA. EPA/600/4/80/032.
https://nepis.epa.gov/Exe/ZvPDF.cgi/30000QHM.PDF?Dockev=30000QHM.PDF
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6.2.8 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and
Thorium in Water, Soil, Air, and Biological Tissue
Analyte(s)
CAS RN
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
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: This method has been selected for confirmatory analysis of plutonium-238 and -
239 in drinking water samples.
Description of Method: This method is appropriate for the analysis of isotopic plutonium, uranium and
thorium, together or individually, by alpha spectrometry. Plutonium-236, uranium-232 and thorium-234
tracer standards are added for the determination of chemical yields. Samples are decomposed by nitric-
hydrofluoric acid digestion or ignition to assure that all of the plutonium is dissolved and chemically
separated from the sample by coprecipitation with sodium and ammonium hydroxide, anion exchange and
electrodeposition. The residues are dissolved in dilute nitric acid and successive sodium and ammonium
hydroxide precipitations are performed in the presence of boric acid to remove fluoride and soluble salts.
The hydroxide precipitate is dissolved, the solution is pH-adjusted with hydrochloric acid, and plutonium
and uranium are adsorbed on an anion exchange column, separating them from thorium. Plutonium is
eluted with hydrobromic acid. The actinides are electrodeposited on stainless steel discs from an
ammonium sulfate solution and subsequently counted by alpha spectrometry. This method is designed to
detect environmental levels of activity as low as 0.02 pCi per sample. To avoid possible cross-
contamination, sample aliquot activities should be limited to 25 pCi or less.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1 (Section 6.2.47). The presence of compounds contained in various decontamination
agents can impact the results of analysis using this procedure due to precipitation. Precipitation can result
in a lesser amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed
separately form the entire sample. Such compounds include:
Chelating compounds can compromise the collection of radionuclides prior to analysis by
preventing them from being trapped on the ion exchange column or from being precipitated out
of solution. Some chelators also can tightly complex barium that may be present in the sample
causing interferences when analyzing for plutonium-238. Dispersants and corrosion inhibitors can
have chelating ability as well.
Compounds containing carbonate, fluoride, hydroxide or phosphate can precipitate radionuclides
out of solution prior to analysis.
The presence of higher valence anions can lead to lower yields when using the evaporation
option, due to competition with active sites on the resin used to collect the radionuclides.
Source: U.S. 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. Cincinnati, OH: U.S. EPA. http://www.epa. gov/sites/production/files/2015-
06/documents/epa-emsl-33 .pdf
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6.2.9 EPA Method Rapid Radiochemical Method for Phosphorus-32 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
CAS RN
Phosphorus-32
14596-37-3
Analysis Purpose: Qualitative analysis
Technique: Liquid scintillation
Method Developed for: Phosphorus-32 in water
Method Selected for: This method has been selected 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: This method has been selected for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water. Organic
compounds containing oxygen, reductants, halogenated compounds or elevated levels of nitrates or
nitromethane, such as those contained in some decontamination agents, can cause chemical quenching.
Chemical and color quenching compounds, such as dyes and pigments, also contained in some
decontamination agents, can have a significant impact when using liquid scintillation methods. This
method also can be impacted by high levels of phosphates or phosphorus compounds.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2011. "Rapid
Radiochemical Method for Phosphorus-32 in Water for Environmental Remediation Following Homeland
Security Events." Montgomery, AL: U.S. EPA. EPA/600/R-11/181. https://www.epa.gov/radiation/rapid-
radiochemical-methods-selected-radionuclides
6.2.10 EPA Method R4-73-014: Radioactive Phosphorus
Analyte(s)
CAS RN
Phosphorus-32
14596-37-3
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Low background alpha/beta counter
Method Developed for: Phosphorus-32 in nuclear reactor solutions
Method Selected for: This method has been selected for qualitative and confirmatory analysis of
aqueous and liquid phase samples, and for confirmatory analysis of drinking 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
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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.
Special Considerations: Chelating compounds, such as those present in some decontamination agents,
can compromise the collection of scavenging carriers that are added to the sample solution prior to
analysis by preventing them from being precipitated out of solution, affecting the chemical yield of
phosphorus-32. This method also can be impacted by the presence of high levels of phosphates or
phosphorus compounds.
Source: U.S. EPA. EMSL. 1980. "Method R4-73-14: Radioactive Phosphorus." Prescribed Procedures
for Radiochemical Analysis of Nuclear Reactor Solutions. Cincinnati, OH: U.S. EPA.
http://www.epa.gOv/sites/production/files/2015-06/documents/epa-r4-73-014.pdf
6.2.11 EPA Method: Determination of Radiostrontium in Food and Bioenvironmental
Samples
Analyte(s)
CAS RN
Strontium-89
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: This method has been selected for qualitative analysis of strontium-89 in wipes
and air filters and confirmatory analysis strontium-89 in 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. At this time, there are no known interferences posed by decontamination agents that might be
present in a sample.
Source: U.S. EPA, National Environmental Research Center. 1975. "Determination of Radiostrontium
in Food and Bioenvironmental Samples " Handbook of Radiochemical Methods. Washington, DC: U.S.
EPA. EPA-680/4-75-001. http://www.epa.gov/sites/production/files/2Q15-
06/documents/radiostrontium in food.pdf
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6.2.12 EPA Method: Rapid Radiochemical Method for Americium-241 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
CAS RN
Americium-241
14596-10-2
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241 in water
Method Selected for: This method has been selected 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: This method has been selected for rapid qualitative screening of drinking water
samples. It is not intended for use in compliance monitoring of drinking water. The presence of higher
valence anions such as phosphates can lead to lower yields when using the evaporation option in this
method, due to competition with active sites on the resin. High levels of iron, manganese, calcium or
magnesium can also have an impact on exchange site availability and or poison the extraction resin used
in this method.
The presence of compounds contained in various decontamination agents can impact the results of
analysis using this procedure due to precipitation. Precipitation can result in a lesser amount of
radionuclide in cases where an aliquot of water sample is transferred and analyzed separately form the
entire sample. Such compounds include:
Reducing or oxidizing compounds can result in lower measured concentrations when using this
method, which requires specific valence states for radionuclides.
Compounds, such as clays that are also present in some decontamination agents, contain iron,
magnesium and calcium, which can be released as ions via ion exchange in the presence of
radionuclides, and cause interference when analyzing water samples.
Compounds containing carbonate, fluoride, hydroxide or phosphate can precipitate radionuclides
out of solution prior to analysis.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 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. Montgomery, AL: U.S. EPA. EPA 402-R-10-001.
httos://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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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)
CAS RN
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Plutonium-238 and -239 in water
Method Selected for: This method has been selected 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.
Special Considerations: This method has been selected for rapid qualitative screening of drinking water
samples. It is not intended for use in compliance monitoring of drinking water. The presence of
compounds contained in various decontamination agents can impact the results of analysis using this
procedure:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on the ion exchange column or from being precipitated out
of solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Compounds such as clays can contain iron, magnesium or calcium, which can be released as ions
via ion exchange, and cause interference when analyzing water samples.
High levels of iron, manganese, calcium or magnesium can also have an impact on exchange site
availability and/or poison the extraction resins used in this method.
Higher valence anions may lead to lower yields when using the evaporation option due to
competition with active sites on the resin used to collect the radionuclides.
The presence of fluoride can precipitate out plutonium prior to measurement.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 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. Montgomery, AL: U.S. EPA. EPA
402-R-10-001. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.14 EPA Method: Rapid Radiochemical Method for Radium-226 in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
CAS RN
Radium-223
15623-45-7
Radium-226
13982-63-3
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Analysis Purpose: Qualitative and confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Radium-226 in water
Method Selected for: This method has been selected for qualitative analysis of radium-226 in drinking
water samples and for the qualitative and confirmatory analysis of radium-223 in drinking water, aqueous
and liquid phase, soil and sediment, surface wipes, air filters and vegetation 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 and -223) 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 and -223 region of interest is corrected for chemical yield based
on the observed activity of the alpha peak at 7.07 mega-electron volts [MeV].
Special Considerations: This method has been selected for rapid qualitative screening of drinking water
samples. It is not intended for use in compliance monitoring of drinking water. Although the method has
not been validated in sample types other than water, it is likely be applicable to the additional sample
types for qualitative and confirmatory analyses of radium-223. The presence of compounds contained in
various decontamination agents can impact the results of analysis using this procedure:
Chelating compounds can compromise the collection of radionuclides prior to analysis, by
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Permanganate and permanganic acid can be reduced to insoluble manganese (IV) oxide, which
could remove radium.
Clays can contain iron, magnesium or calcium, which can be released as ions via ion exchange in
the presence of certain radionuclides, and cause interference in the analysis of the water.
High levels of iron, manganese, calcium or magnesium can impact exchange site availability
and/or poison the extraction resin used in this method.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 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. Montgomery, AL: U.S. EPA. EPA 402-R-10-001.
http://www.epa.gov/sites/production/files/2015-07/documents/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)
CAS RN
Strontium-90
10098-97-2
Analysis Purpose: Qualitative analysis
Technique: Beta counting
Method Developed for: Strontium-90 in water
Method Selected for: This method has been selected for qualitative analysis of drinking water samples.
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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 8M nitric acid to solubilize strontium. The solution is passed through a
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 0.05M 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: This method has been selected for rapid qualitative screening of drinking water
samples. It is not intended for use in compliance monitoring of drinking water. High levels of radioactive
cesium or cobalt (>1000 times the activity of strontium being measured) may not be completely removed
during ion exchange and can cause interferences. Chelating compounds, such as those present in some
decontamination agents, can compromise the collection of strontium prior to analysis, by preventing it
from being trapped on an ion exchange column or from being precipitated out of solution. Compounds
containing carbonate, fluoride, phosphate or sulfate, also present in some decontamination agents, can
precipitate radionuclides out of solution prior to analysis. This precipitation can result in a lesser amount
of strontium in cases where an aliquot of water sample is transferred and analyzed separately from the
entire sample.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 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. Montgomery, AL: U.S. EPA. EPA 402-R-10-001.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.16 EPA Method: Rapid Radiochemical Method for Isotopic Uranium in Water for
Environmental Restoration Following Homeland Security Events
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Uranium-234, -235 and -238 in water
Method Selected for: This method has been selected 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.
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Special Considerations: This method has been selected for rapid qualitative screening of drinking water
samples. The method is not intended for use in compliance monitoring of drinking water. Higher valence
anions may lead to lower yields when using the evaporation option due to competition with active sites on
the resin. The presence of compounds contained in various decontamination agents can impact the results
of analysis using this procedure due to precipitation. Precipitation can result in a lesser amount of
radionuclide in cases where an aliquot of water sample is transferred and analyzed separately form the
entire sample. Such compounds include:
Chelating compounds can compromise the collection of radionuclides prior to analysis, by
preventing them from being trapped on the ion exchange column or from being precipitated out
of solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Clays can contain iron, magnesium or calcium that can be released as ions via ion exchange, in
the presence of certain radionuclides, and cause interference in the analysis of water. High levels
of iron, manganese, calcium or magnesium can also impact exchange site availability and/or
poison the extraction resins used in this method.
Compounds containing carbonate, fluoride, hydroxide or phosphate can precipitate uranium out
of solution.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2010. "Rapid
Radiochemical Method for Isotopic Uranium in Water for Environmental Restoration Following
Homeland Security Events." Rapid Radiochemical Methods for Selected Radionuclides in Water for
Environmental Restoration Following Homeland Security Events. Montgomery, AL: U.S. EPA. EPA
402-R-10-001. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Radium-226
13982-63-3
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
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: This method has been selected 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 450ฐC
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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
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
Rapid Radiochemical Method for Americium-241 in Water for Environmental Remediation
Following Homeland Security Events (Section 6.2.12)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Water for
Environmental Remediation Following Homeland Security Events (Section 6.2.13)
Rapid Radiochemical Method for Radium-226 in Water for Environmental Remediation
Following Homeland Security Events (Section 6.2.14)
Rapid Radiochemical Method for Total Radiostrontium (Sr-90) in Water for Environmental
Remediation Following Homeland Security Events (Section 6.2.15)
Rapid Radiochemical Method for Isotopic Uranium in Water for Environmental Remediation
Following Homeland Security Events (Section 6.2.16)
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
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.
Some concrete or brick materials can have native concentrations of uranium, radium, thorium, strontium
or barium, all of which can have an effect on the chemical separations used following sample fusion. In
some cases (e.g., radium or strontium analysis), elemental analysis of the digest prior to chemical
separation may be necessary to determine native concentrations of carrier elements. Lanthanum is used in
this method to pre-concentrate actinides, along with lanthanum (III) fluoride, to eliminate matrix
interferences including silica, which can cause column flow problems. Compounds contained in
decontamination agents are not expected to cause interferences during sample preparation; see the
sections corresponding to the analytical methods listed in the description of this method for potential
interferences caused by constituents of decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2012. "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." Montgomery, AL: U.S. EPA. EPA 402-R-12-009.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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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)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Radium-226
13982-63-3
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
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: This method has been selected 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 (Section 6.2.12)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Water for
Environmental Remediation Following Homeland Security Events (Section 6.2.13)
Rapid Radiochemical Method for Radium-226 in Water for Environmental Remediation
Following Homeland Security Events (Section 6.2.14)
Rapid Radiochemical Method for Total Radiostrontium (Sr-90) in Water for Environmental
Remediation Following Homeland Security Events (Section 6.2.15)
Rapid Radiochemical Method for Isotopic Uranium in Water for Environmental Remediation
Following Homeland Security Events (Section 6.2.16)
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.
Some surface materials (e.g., concrete or brick) can contain native concentrations of uranium, radium,
thorium, strontium or barium, all of which may have an effect on the chemical separations used following
the fusion of the sample. In some cases (e.g., radium or strontium analysis), elemental analysis of the
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digest prior to chemical separation may be necessary to determine concentrations of carrier elements.
Trace levels of radium-226 may be present in sodium carbonate used in the pre-concentration step used in
this method. Compounds contained in decontamination agents are not expected to cause interferences
during sample preparation; see the sections corresponding to the analytical methods listed in the
description of this method for potential interferences caused by constituents of decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2012. "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," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-
R-12-008.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.19 Rapid Method for Radium in Soil Incorporating the Fusion of Soil and Soil-Related
Matrices with the Radioanalytical Counting Method for Environmental
Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Radium-226 in soil samples
Method Selected for: This method has been selected for qualitative analysis of soil/sediment samples.
Description of Method: This method is based on the complete fusion of a representative, finely ground
1-g aliquot of dried sample with no insoluble residue remaining after dissolution of the fused melt in acid.
For organic soils, the sample is dry ashed at 600ฐC in an appropriate vessel prior to fusion, then dissolved
in a crucible with hydrofluoric acid and evaporated to dryness. Dry flux mix (equal weight of dried
sodium carbonate, potassium carbonate and boric acid) is added, and the crucible is warmed under a
flame until a reaction initiates. The crucible is then heated under full flame until the reaction subsides and
the melt is completely liquid and homogeneous. After cooling, the solidified melt is dissolved in
hydrochloric acid, and transferred to a digestion container while rinsing the crucible with 6M
hydrochloric acid. The barium content is determined for a small aliquot of the dissolved flux by adding
sufficient amount of barium so that the final mass (native plus added) is not more than 90 |ig and then
analyzing by ICP-AES. A manganese (IV) solution and phenolphthalein indicator are added to this
mixture, and the pH is adjusted with sodium hydroxide until the solution turns pink. Hydrogen peroxide is
slowly added forming insoluble manganese (II) oxide. The manganese (IV) oxide precipitate is
centrifuged, rinsed with water and dissolved in a manganese (IV) oxide stripping agent. Ascorbic acid is
added and the solution is passed through a resin column, rinsing with hydrochloric acid. The rinse
solution is collected, ammonium sulfate and isopropanol are added, and the solution is placed in a cold
water ultrasonic bath for 20 minutes, after which it is filtered, rinsing with a solution of ammonium
sulfate in isopropanol. The filter is then placed in a Petri dish, dried, and stored for at least 24 hours. The
sample is then counted by alpha spectrometry.
Special Considerations: The presence of discrete radioactive particles or particles larger than 150 |im
can require additional sample preparation, as described in Sections A4 and A5.2.3 of the method
(Interferences and Hot Particles, respectively). Soils with high silica content may require either additional
fusing reagent and boric acid or a longer fusion melt. Platinum crucibles must be used when digesting
samples with hydrofluoric acid. If platinum crucibles are not available, effective alternate methods are
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available that use zirconium crucibles (see Rapid Method for Sodium Hydroxide Fusion of Concrete and
Brick Matrices Prior to Am, Pu, Sr, Ra, and U Analyses [Section 6.3.3] and Rapid Radiochemical Method
for Total Radiostrontium (Sr-90) in Building Materials for Environmental Remediation Following
Radiological Incidents [Section 6.3.1]). At this time, there are no known interferences posed by
decontamination agents that might be present in a sample.
In some cases, elemental analysis of the digest prior to chemical separations may be necessary to
determine native concentrations of carrier elements present in the sample. Trace levels of radium-226
might be present in the sodium carbonate used in the pre-concentration step.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2012. "Rapid Method
for Radium in Soil Incorporating the Fusion of Soil and Soil-Related Matrices with the Radioanalytical
Counting Method for Environmental Remediation Following Radiological Incidents," Revision 0.
Montgomery, AL: U.S. EPA. EPA-600-R-12-635.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.20 Rapid Method for Fusion of Soil and Soil-Related Matrices Prior to Americium,
Plutonium, and Uranium Analyses for Environmental Remediation Following
Radiological Incidents
Analyte(s)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241, plutonium-238, plutonium-239, uranium-234, uranium-235
and uranium-238 in soil samples
Method Selected for: This method has been selected for qualitative analysis of soil/sediment samples
Description of Method: The method is based on the complete fusion of a representative finely ground
1-g aliquot of dried sample with no insoluble residue remaining after dissolution of the fused melt in acid.
For organic soils, the sample is dry ashed at 600ฐC in an appropriate vessel prior to fusion. The sample is
dissolved in a crucible with hydrofluoric acid, and evaporated to dryness on a hotplate at medium to high
heat (~300ฐC). Dry flux mix (equal weight of dried sodium carbonate, potassium carbonate and boric
acid) is added, and the crucible is warmed under a flame until a reaction initiates. The crucible is then
heated under full flame until the reaction subsides and the melt is completely liquid and homogeneous.
After cooling, the solidified melt is dissolved in nitric acid. The dissolved sample is transferred to an
appropriately sized beaker, and the crucible is rinsed with nitric acid to ensure a quantitative transfer of
material. The sample is then processed using one of the following methods:
Rapid Radiochemical Method for Americium-241 in Water (Section 6.2.12)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Water (Section
6.2.13)
Rapid Radiochemical Method for Isotopic Uranium in Water (Section 6.2.16)
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Special Considerations: The presence of discrete radioactive particles or particles larger than 150 |im
can require additional sample preparation as described in Sections A4 and A5.2.3 of the method
(Interferences and Hot Particles, respectively). Soils with high silica content may require either additional
fusing reagent and boric acid or a longer fusion melt. Platinum crucibles must be used when digesting
samples with hydrofluoric acid. If platinum crucibles are not available, effective alternate methods are
available that use zirconium crucibles (see Rapid Method for Sodium Hydroxide Fusion of Concrete and
Brick Matrices Prior to Am, Pu, Sr, Ra, and U Analyses [Section 6.3.3] and Rapid Radiochemical Method
for Total Radiostrontium (Sr-90) in Building Materials for Environmental Remediation Following
Radiological Incidents [Section 6.3.1]). Compounds contained in decontamination agents are not
expected to cause interferences during sample preparation; see the sections corresponding to the
analytical methods listed in the description of this method for potential interferences caused by
constituents of decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2012. "Rapid Method
for Fusion of Soil and Soil-Related Matrices Prior to Americium, Plutonium, and Uranium Analyses for
Environmental Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA.
EPA-600-R-12-636, EPA-600-R-12-637 and EPA-600-R-12-638.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.21 Rapid Method for Sodium Carbonate Fusion of Soil and Soil-Related Matrices
Prior to Strontium-90 Analyses for Environmental Remediation Following
Radiological Incidents
Analyte(s)
CAS RN
Strontium-90
10098-97-2
Analysis Purpose: Qualitative analysis
Technique: Beta counting
Method Developed for: Strontium-90 in soil samples
Method Selected for: This method has been selected for qualitative analysis of soil/sediment samples
Description of Method: The method is based on the complete fusion of a representative, finely ground
1-g aliquot of dried sample with no insoluble residue remaining after dissolution of the fused melt in acid.
For organic soils, the sample is dry ashed at 600ฐC prior to fusion. The sample is dissolved in a crucible
with hydrofluoric acid and evaporated to dryness on a hotplate at medium to high heat (~300ฐC). Dry flux
mix (equal weight of dried sodium carbonate, potassium carbonate and boric acid) is added, and the
crucible is heated under a flame until a reaction initiates. The crucible is then heated under full flame until
the reaction subsides and the melt is completely liquid and homogeneous. After cooling, the solidified
melt is dissolved in nitric acid. Calcium solution and phenolphthalein indicator are added to this mixture,
and the pH is adjusted to 8.3 with sodium hydroxide. The sample will become pinkish-orange due to the
indicator color change and the formation of hydroxide precipitate. Sodium carbonate and heat are added
to complete precipitation. After cooling and allowing the precipitate to settle, the supernatant is decanted
and the precipitate is transferred to a centrifuge tube and dissolved in nitric acid. The sample is then
processed for strontium-90 determination using Rapid Radiochemical Method for Total Radiostrontium
(Sr-90) in Water for Environmental Restoration Following Homeland Security Events (Section 6.2.15).
Special Considerations: The presence of discrete radioactive particles or particles larger than 150 |im
can require additional sample preparation as described in Sections A4 and A5.2.3 of the method
(Interferences and Hot Particles, respectively). Soils with high silica content may require either additional
fusing reagent and boric acid or a longer fusion melt. Platinum crucibles must be used in this method
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when digesting samples with hydrofluoric acid. If platinum crucibles are not available, an effective,
alternate method is available that uses zirconium crucibles (see Rapid Method for Sodium Hydroxide
Fusion of Concrete and Brick Matrices Prior to Am, Pu, Sr, Ra, and U Analyses [Section 6.3.3] and
Rapid Radiochemical Method for Total Radiostrontium (Sr-90) in Building Materials for Environmental
Remediation Following Radiological Incidents [Section 6.3.1]). Compounds contained in
decontamination agents are not expected to cause interferences during sample preparation; see the
sections corresponding to the analytical methods listed in the description of this method for potential
interferences caused by constituents of decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2012. "Rapid Method
for Sodium Carbonate Fusion of Soil and Soil-Related Matrices Prior to Strontium-90 Analyses for
Environmental Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA.
EPA-600-R-12-640.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.22 Rapid Method for Sodium Hydroxide/Sodium Peroxide Fusion of Radioisotope
Thermoelectric Generator Materials in Water and Air Filter Matrices Prior to
Plutonium Analyses for Environmental Remediation Following Radiological
Incidents
Analyte(s)
CAS RN
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry
Method Developed for: Plutonium-238 and -239 in water and air filters
Method Selected for: This method has been selected as a pre-treatment technique supporting analysis of
refractory radioisotopic forms of plutonium in drinking water and air filters using the following
qualitative techniques:
Rapid methods for acid or fusion digestion (Sections 6.2.17 and 6.2.18)
Rapid Radiochemical Method for Plutonium-238 and Plutonium 239/240 in Building Materials
for Environmental Remediation Following Radiological Incidents (Section 6.3.5)
Description of Method: This method is a pre-treatment technique for qualitative analysis of water and air
filters, and has been validated together with the chemical separation and analysis process described in
EPA's Rapid Radiochemical Method for Plutonium-238 and Plutonium 239/240 in Building Materials for
Environmental Remediation Following Radiological Incidents (Section 6.3.5). The method is based on
total dissolution of radioisotope thermoelectric generator (RTG). Air filters are fused using rapid sodium
hydroxide/sodium peroxide at 700ฐC. For water samples, refractory RTG particles are collected on a
0.45(im filter using a vacuum, and RTG activity in the filtrate is preconcentrated using calcium phosphate
precipitation. Solid fractions and filtrate fractions are processed separately by fusing with sodium
hydroxide/sodium peroxide prior to subsequent chemical separation and alpha spectrometric analysis.
Pre-concentration steps are needed to eliminate the alkaline fusion matrix and collect the radionuclides.
Plutonium is separated from the fusion matrix using a lanthanum/calcium fluoride matrix removal step in
preparation for separation and analysis using the rapid separation method cited above. Assuming a 68 m3
air volume, the method is capable of meeting a required minimum detectable concentration (MDC) of
0.003 pCi/m3 or below for air filters (240-minute count time) and an uncertainty of 1.9 pCi/filter at and
below the analytical action level of 15.0 pCi/filter (360-minute count time). Assuming a 1-L volume and
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360-minute count time, the method is capable of satisfying an uncertainty of 2.1 pCi/L at and below an
analytical action level of 16.3 pCi/L, and meeting a required MDC of 0.23 pCi/L for water samples
(filtered solids, filtrate, or combined result) with a 240-minute count time.
Special Considerations: Organic-based materials, such as cellulose nitrate or cellulose acetate filters,
may react vigorously upon addition of peroxide or during charring steps. Wet ashing with nitric acid and
hydrogen peroxide is needed to destroy organic constituents prior to fusion. Samples with elevated
activity or samples that require multiple analyses may need to be split after dissolution. Reducing or
oxidizing compounds, such as those present in some decontamination agents, can impact this method,
which requires specific valence states for radionuclides. All plutonium must be reduced to plutonium (+3
or +4) before isotopic exchange with the tracer can be achieved with reasonable certainty. Additionally,
only plutonium (+3 or +4) will precipitate in the lanthanum fluoride/calcium fluoride pre-concentration
step. Although peroxide may reduce plutonium +6 to +4, the valence must be controlled with certainty.
Valence controls also ensure that plutonium will be present in the plutonium +4 form prior to separation.
Although this method was validated using plutonium-242 tracer, plutonium-236 tracer can be used
assuming it can be obtained with sufficient purity.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Method for Sodium Hydroxide/Sodium Peroxide Fusion of Radioisotope Thermoelectric Generator
Materials in Water and Air Filter Matrices Prior to Plutonium Analyses for Environmental Remediation
Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-003.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.23 EPA Method: Rapid Radiochemical Method for Californium-252 in Water, Air
Particulate Filters, Swipes and Soil for Environmental Restoration Following
Homeland Security Events
Analyte(s)
CAS RN
Californium-252
13981-17-4
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Californium-252 in water, air particulate filters, swipes and soil samples
Method Selected for: This method has been selected for confirmatory analysis of californium-252 in
drinking water, aqueous liquid, air filter, surface wipes and soil matrices.
Description of Method: This method is based on the use of extraction chromatography resins to isolate
and purify californium by removing interfering radionuclides as well as other matrix components. The
method utilizes vacuum-assisted flow to improve the speed of the separations. Americium-243 tracer
equilibrated with the sample is used as a yield monitor.
Water samples are concentrated using a calcium phosphate coprecipitation. The calcium
phosphate precipitate is dissolved in a load solution containing ~3M nitric acid/lM aluminum
nitrate before continuing with chemical separations.
Glass-fiber or cellulose-based air particulate filter samples are wet ashed with repeated additions
of nitric and hydrofluoric acids and hydrogen peroxide. The residues are treated with nitric-boric
acid, and dissolved in a load solution containing 3M nitric acid/lM aluminum nitrate before
continuing with chemical separations.
Cotton-twill swipe and organic-polymer-based air particulate filter samples are dry ashed in a
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beaker for 30-60 minutes using a ramped program to minimize the risk of flash-ignition. The
residue is transferred to a polytetrafluoroethylene (PTFE) beaker with nitric acid and hydrogen
peroxide, digested with hydrofluoric acid, and taken to dryness. The residues are wet ashed with
nitric acid and hydrogen peroxide and taken to dryness before being treated with nitric-boric acid
and dissolved in a load solution containing 3M nitric acid/lM aluminum nitrate for chemical
separations.
Soils are finely ground before being fused with sodium hydroxide in zirconium crucibles. The
fusion cake is dissolved in water and californium preconcentrated from the alkaline matrix using
an iron/titanium hydroxide precipitation (enhanced with calcium phosphate precipitation)
followed by a lanthanum fluoride matrix removal step. The fluoride precipitate is dissolved with
nitric-boric acid and diluted in nitric acid and aluminum nitrate to yield a load solution containing
~3M nitric acid/lM aluminum nitrate.
Extraction chromatography resins (TEVAฎ + DGAฎ resins [Eichrom Technologies, Lisle, IL, or
equivalent]) are then used to isolate and purify californium and americium by removing interfering
radionuclides and other matrix components. Following chemical separation of curium and americium, the
sample test source is prepared by microprecipitation with cerium (III) fluoride.
Water: This method is capable of achieving a required method uncertainty for californium-252 of 2.0
pCi/L at an analytical action level of 15.3 pCi/L and a required MDC of 1.5 pCi/L, using a
sample volume of 0.2 L and count time of at least 4 hours.
Air Particulate Filter. This method is capable of achieving a required method uncertainty for
californium-252 of 0.57 pCi/filter at an analytical action level of 4.37 pCi/filter and a required
MDC of 0.44 pCi/filter, using a sample aliquant of one filter and count time of at least 4 hours.
Swipe or Organic-Polymer-Based Air Particulate Filter. This method is capable of achieving a required
method uncertainty for californium-252 of 0.12 pCi/swipe or filter at an analytical action level of
0.12 pCi/ swipe or filter and a required MDC of 0.15 pCi/ swipe or filter, using a sample aliquant
of one swipe or filter and count time of at least 4 hours.
Soil: This method is capable of achieving a required method uncertainty for californium-252 of 0.18
pCi/g at an analytical action level of 1.38 pCi/g and a required MDC of 0.14 pCi/g, using a
sample weight of 1 g and count time of at least 4 hours.
Special Considerations: Alpha emissions from californium-250 fall in the same region as californium-
252, and cannot be differentiated from those of californium-252 using alpha spectrometry. Measurements
should be reported in terms of the activity of californium-250/252. Since alpha spectrometry does not
differentiate between californium-250 and californium-252, decay corrections based on the half-life of
californium-252 will impart a positive bias to results as mixtures age. The effect can be minimized by
keeping the time between the activity reference date (i.e., collection or standard reference date) short, or
by reporting the activity at the time of the measurement.
Other radionuclides (or their short-lived progeny) that emit alpha particles that are isoenergetic with
californium-252 (e.g., bismuth-212 at 6.1 MeV supported by thorium-228 and/or radium-224) must be
chemically separated to prevent positive interference with the measurement. This method effectively
separates these radionuclides.
The use of the americium-243 tracer assumes that both californium and americium are removed from the
column at the time of elution. The separation scheme is designed to ensure that nitrates and lanthanum
will not interfere with this elution. High levels of calcium in soil samples can have an adverse impact on
the retention of californium and americium on the DGA resin. The method is designed to minimize
calcium interference and enhance californium and americium affinity by increasing the nitrate
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concentration in the load and initial rinse solutions. Non-radiological anions, including fluoride and
phosphate, can complex californium and americium and lead to depressed yields. Boric acid added to the
load solution will complex residual fluoride ions, while aluminum in the load solution will complex
phosphate ions.
Chelating compounds, such as those present in some decontamination agents, can compromise the
collection of radionuclides prior to analysis, preventing them from being trapped on the ion exchange
column or from being precipitated out of solution. Dispersants and corrosion inhibitors can have chelating
ability as well. Clays, which are also present in some decontamination agents can contain iron,
magnesium and calcium that can be released as ions via ion exchange, in the presence of certain
radionuclides, and cause analytical interferences. High levels of iron, manganese, calcium or magnesium
can impact exchange site availability and/or poison extraction resins used in this method.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. 2017. "[Validation of]
Rapid Radiochemical Method for Cf-252 in Water, Air Particulate Filters, Swipes and Soils for
Environmental Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA.
EPA 402-S17-003. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides.
6.2.24 EPA Method: Rapid Radiochemical Method for Curium-244 in Water Samples for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Curium-244
13981-15-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Curium-244 in water samples
Method Selected for: This method has been selected for confirmatory analysis of curium-244 in water
samples.
Description of Method: This method is based on the use of extraction chromatography resins to isolate
and purify curium by removing interfering radionuclides and other matrix components and preparing the
curium fraction for counting by alpha spectrometry. The method utilizes vacuum-assisted flow to improve
the speed of the separations. An americium-243 tracer is equilibrated with the water sample and used as a
yield monitor. Following chemical separation of curium and americium, the sample test source is
prepared by microprecipitation with cerium (III) fluoride. Alpha emissions from the source are measured
using an alpha spectrometer and used to calculate the activity of curium-244 in the sample.
Using a 0.2 L sample and a count time of four hours, this method is capable of achieving an uncertainty of
2.0 pCi/L californium-252 at an analytical action level of 15 pCi/L and a required MDC of 1.515 pCi/L.
Special Considerations: The alpha emissions from curium-243 fall in the same region as curium-244
and cannot be differentiated using alpha spectrometry. Although curium-243 and curium-244 alpha
emissions overlap, monitoring the region of the spectrum between 5.8 and 6.0 MeV for less intense
emissions of curium-243 can qualitatively indicate the presence of curium-243 in a sample. Alpha
spectrometry measurements that show activity in the region of interest for curium-244 should be reported
as curium-243/244. Americium and californium are chemical analogs of curium in the separations scheme
used for this analysis. Several isotopes of californium or americium emit alpha particles within the region
of interest for curium-244. These include californium-249 and californium-251. If high levels of
californium could be present in samples, alpha spectrometry results should be monitored for other
isotopes of californium. Radionuclides of other elements (or their short-lived progeny) that emit alpha
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particles which are isoenergetic with curium-244 (e.g., thorium-227 or actinium-225 5.8 MeV) must be
chemically separated using the method procedures to prevent positive interference.
Non-radiological anions that can complex curium, including fluoride and phosphate, can lead to
depressed yields. Aluminum in the load solution will complex both fluoride and residual phosphate. High
levels of calcium can have an adverse impact on curium and americium retention on DGA resin. Calcium
retention is minimized, and curium and americium affinity is enhanced, by increasing nitrate
concentrations in the load and initial rinse solutions. A dilute nitric acid rinse is performed on DGA resin
to remove calcium that could otherwise end up in the sample test source as the fluoride. For samples
containing elevated concentrations of calcium, it may be advisable to increase the volume of this rinse
step slightly to better remove calcium ions and possibly improve alpha peak resolution.
Chelating compounds, such as those present in some decontamination agents, can compromise the
collection of radionuclides prior to analysis, preventing them from being trapped on the ion exchange
column or from being precipitated out of solution. Dispersants and corrosion inhibitors can have chelating
ability as well. Clays, which are also present in some decontamination agents, can contain iron,
magnesium or calcium that can be released as ions via ion exchange, in the presence of certain
radionuclides, and cause analytical interferences. High levels of iron, manganese, calcium or magnesium
can impact exchange site availability and/or poison extraction resins used in this method.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. May 2017. "Rapid
Radiochemical Method for Curium-244 in Water Samples for Environmental Remediation Following
Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-S17-001.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides.
6.2.25 EPA Method: Rapid Radiochemical Method for Curium-244 in Air Particulate
Filters, Swipes and Soil
Analyte(s)
CAS RN
Curium-244
13981-15-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Curium-244 in air particulate filters, swipes and soil samples
Method Selected for: This method has been selected for confirmatory analysis of curium-244 in air
particulate filters, swipes and soil matrices.
Description of Method: This method is based on the use of extraction chromatography resins to isolate
and purify curium by removing interfering radionuclides and matrix components in order to prepare the
curium fraction for counting by alpha spectrometry. The method utilizes vacuum-assisted flow to improve
the speed of the separations. An americium-243 tracer is equilibrated with the sample as a yield monitor.
Glass-fiber or cellulose-based air particulate filter samples are wet ashed with repeated additions
of nitric acid and hydrofluoric acid, and hydrogen peroxide. The residues are treated with nitric-
boric acid and dissolved in a load solution containing ~3M nitric acid/lM aluminum nitrate
before continuing with chemical separations.
Cotton-twill swipe and organic-polymer-based air particulate filter samples are dry ashed in a
beaker for 30-60 minutes using a ramped program to minimize the risk of flash-ignition. The
residue is transferred to a PTFE beaker with nitric acid and hydrogen peroxide, digested with
hydrofluoric acid, and taken to dryness. The residues are then wet ashed with nitric acid and
hydrogen peroxide and taken to dryness before being treated with nitric-boric acid and dissolved
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in a load solution containing ~3M nitric acid/lM aluminum nitrate before continuing with
chemical separations.
Soils are finely ground before being fused with sodium hydroxide in zirconium crucibles. The
fusion cake is dissolved in water and curium preconcentrated from the alkaline matrix using an
iron/titanium hydroxide precipitation (enhanced with calcium phosphate precipitation) followed
by a lanthanum fluoride matrix removal step. The fluoride precipitate is dissolved with nitric-
boric acid and diluted in nitric acid and aluminum nitrate to yield a load solution containing ~3M
nitric acid/lM aluminum nitrate before continuing with chemical separations.
Extraction chromatography resins are then used to isolate and purify curium by removing interfering
radionuclides and other matrix components. The method utilizes vacuum-assisted flow to improve the
speed of the separations. Following chemical separation of curium and americium, the sample test source
(STS) is prepared by microprecipitation with cerium (III) fluoride.
Air Particulate Filters. This method is capable of achieving a required method uncertainty for curium-
244 of 1.4 pCi/filter at an analytical action level of 10.5 pCi/filter and a required MDC of 0.25
pCi/filter, using a sample aliquant of one filter and a count time of at least four hours.
Swipes (or Organic-Polymer-Based Air Particulate Filters). This method is capable of achieving a
required method uncertainty for curium-244 of 0.051 pCi/swipe at an analytical action level of
0.39 pCi/swipe and a required MDC for of 0.065 pCi/swipe, using one swipe and a count time of
at least four hours.
Soil. This method is capable of achieving a required method uncertainty for curium-244 of 0.66 pCi/g at
an analytical action level of 5.09 pCi/g and a required MDC of 0.66 pCi/g, using a sample weight
of 1 gram and a count time of at least four hours.
Special Considerations: The alpha emissions from curium-243 fall in the same region as curium-244
and cannot be differentiated using alpha spectrometry. Although curium-243 and curium-244 alpha
emissions overlap, monitoring the region of the spectrum between 5.8 and 6.0 MeV for less intense
emissions of curium-243 can qualitatively indicate the presence of curium-243 in a sample. Alpha
spectrometry measurements that show activity in the region of interest for curium-244 should be reported
as curium-243/244. Americium and californium are chemical analogs of curium in the separations scheme
used for this analysis. Several isotopes of californium or americium emit alpha particles within the region
of interest for curium-244. These include californium-249 and californium-251. If high levels of
californium could be present in samples, alpha spectrometry results should be monitored for other
isotopes of californium. Radionuclides of other elements (or their short-lived progeny) that emit alpha
particles which are isoenergetic with curium-244 (e.g., thorium-227 or actinium-225 5.8 MeV) must be
chemically separated using the method procedures to prevent positive interference.
Non-radiological anions that can complex curium, including fluoride and phosphate, can lead to
depressed yields. Aluminum in the load solution will complex both fluoride and residual phosphate. High
levels of calcium can have an adverse impact on curium and americium retention on DGA resin. Calcium
retention is minimized, and curium and americium affinity is enhanced, by increasing nitrate
concentrations in the load and initial rinse solutions. A dilute nitric acid rinse is performed on DGA resin
to remove calcium that could otherwise end up in the sample test source as the fluoride. For samples
containing elevated concentrations of calcium, it may be advisable to increase the volume of this rinse
step slightly to better remove calcium ions and possibly improve alpha peak resolution.
Chelating compounds, such as those present in some decontamination agents, can compromise the
collection of radionuclides prior to analysis, preventing them from being trapped on the ion exchange
column or from being precipitated out of solution. Dispersants and corrosion inhibitors can have chelating
ability as well. Clays, which are also present in some decontamination agents, can contain iron,
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magnesium or calcium that can be released as ions via ion exchange, in the presence of certain
radionuclides, and cause analytical interferences. High levels of iron, manganese, calcium or magnesium
can impact exchange site availability and/or poison extraction resins used in this method.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. May 2017. "Rapid
Radiochemical Method for Curium-244 in Air Particulate Filters, Swipes and Soil for Environmental
Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-S17-
004. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides.
6.2.26 Rapid Radiochemical Method for Radium-226 in Building Materials for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Radium-226 in building materials
Method Selected for: This method is selected for confirmatory analysis of radium-226 in surface wipes
and air filters (also see Section 6.3.2 for application of this method to building materials).
Description of Method: A known quantity of radium-225 is used as the yield tracer in this analysis.
Samples are fused using procedures in Rapid methods for acid or fusion digestion (Sections 6.2.17 and
6.2.18), and the radium isotopes are removed from the fusion matrix using a carbonate precipitation step.
The sample is acidified and loaded onto a cation exchange resin to remove interferences such as calcium,
and radium is eluted from the cation resin with 8M nitric acid. After evaporation of the eluate, the sample
is dissolved in a minimal amount of 3M nitric acid and passed through Sr Resin to remove any barium.
This solution is then evaporated to dryness, redissolved in 0.02M hydrochloric acid, and passed through
Ln Resin to remove interferences such as calcium, and to remove the initial actinium-225. The radium
(including radium-226) is prepared for counting by micro-precipitation with barium sulfate. Activity
measured in the radium-226 region of interest is corrected for chemical yield based on observed activity
of the alpha peak at 7.07 MeV (astatine-217, the third progeny of radium-225).
This method is suited for low-level measurements for radium-226 using alpha spectrometry and is capable
of satisfying a method uncertainty of 0.83 pCi/g at an analytical action level of 6.41 pCi/g, using a sample
aliquant of ~lg and count time of at least eight hours.
Special Considerations: Depending on actual spectral resolution, method performance may be
compromised if samples contain high levels of other radium isotopes (e.g., ~3 times the radium-226
activity concentration) due to ingrowth of interfering decay progeny. Calcium, iron (+3 oxidation state),
and radionuclides with overlapping alpha energies, such as thorium-229, uranium-234, and neptunium-
237, will interfere if they are not removed effectively. Delaying the count significantly longer than one
day may introduce positive bias in results near the detection threshold due to the decay progeny from the
radium 225 tracer. If radium-226 measurements close to detection levels are required and sample
counting cannot be performed within -36 hours of tracer addition, the impact of tracer progeny tailing
into the radium-226 may be minimized by reducing the amount of the tracer that is added to the sample.
This will aid in improving the signal-to-noise ratio for the radium-226 peak by minimizing the amount of
tailing from higher energy alphas of the radium-225 progeny. If actinium-225 is present prior to the final
separation time and the flow rate through the column is too fast (>1.5 drops/second), then actinium-225
will break through the resin, resulting in a high bias in the tracer yield. Additional information regarding
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procedures to remove or minimize interferences is provided in Section 4.0 of the method.
Clays that are present in some decontamination agents can contain iron, magnesium or calcium, which
can be released as ions via ion exchange in the presence of certain radionuclides, and cause interferences.
High levels of iron, manganese, calcium or magnesium might have an impact on exchange site
availability and or poisoning of the extraction resins used. Chelators, also present in some
decontamination agents, can tightly complex barium, calcium, iron and magnesium that may be present in
the sample causing interferences when analyzing for radium-226.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Radium-226 in Building Materials for Environmental Remediation Following
Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-002.
httos://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.2.27 EPA Method: NAREL Standard Operating Procedure for Actinides in
Environmental Matrices by Extraction Chromatography
Analyte(s)
CAS RN
Neptunium-237
13994-20-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Thorium-227
15623-47-9
Thorium-228
14274-82-9
Thorium-230
14269-63-7
Thorium-232
7440-29-1
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Alpha spectrometry / Beta counting (analysis of tracer)
Method Developed for: Americium-241; neptunium-237; plutonium-238 and -239; thorium-227, -228, -
230 and -232; and uranium-234, -235 and -238 in water, soil, vegetation, air filters, swipes and tissue
Method Selected for: This method has been selected for qualitative and confirmatory analysis of
aqueous and liquid phase, soil and sediment, surface wipes, air filters and vegetation samples.
Standard Methods 7500-U B (Section 6.2.57) and 7500-U C (Section 6.2.58) should be used for
qualitative (7500-U B) and confirmatory (7500-U C) analysis of uranium-234, -235 and -238 in
aqueous and liquid phase samples.
ASTM Method D3084-05 (Section 6.2.49) should be used for qualitative analysis of plutonium-
238 and -239 in aqueous and liquid phase samples.
EPA NAREL Rapid Method for Fusion of Soil and Soil-Related Matrices (Section 6.2.20) should
be used for qualitative analysis of plutonium-238 and -239 and uranium-234, -235 and -238, in
soil and sediment samples.
EPA NAREL Rapid Methods for Acid or Fusion Digestion (Sections 6.2.17 and 6.2.18) should
be used for qualitative analysis of plutonium-238 and -239 and uranium-234, -235 and -238, in
surface wipes and air filters.
DOE SRS Actinides and Sr-89/90 in Vegetation (Section 6.2.42) should be used for qualitative
analysis of plutonium-238 and -239 and uranium-234, -235 and -238 in vegetation.
EML HASL-300 Methods Am-06-RC (Section 6.2.30) and U-02-RC (Section 6.2.38) should be
used for confirmatory analysis of plutonium -238 and -239 and uranium-234, -235 and -238 in
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vegetation, respectively.
Description of Method: This method involves the use a tandem arrangement of cartridges containing
extraction chromatographic resins connected in series, which effectively separate and isolate americium,
plutonium, thorium, uranium and neptunium from a variety of environmental matrices. The oxidation
states of the elements of interest in the load solution are as follows: americium (III), neptunium (IV),
plutonium (III), thorium (IV) and uranium (VI). The sample is first loaded onto a TEVAฎ-resin (Eichrom
Technologies, Lisle, IL, or equivalent) cartridge. Any thorium or neptunium present in the sample will be
retained on the cartridge. The effluent passes through this cartridge and onto a transuranic (TRU)-resin
cartridge, which will retain any americium, plutonium or uranium. The tandem cartridge arrangement is
then separated and the elements of interest are selectively eluted, then co-precipitated as a fluoride
complex and radio-assayed by alpha-particle spectrometry.
Special Considerations: Prior to adding americium-243 or neptunium-239 tracers, the sample should be
analyzed for native neptunium-239 by the appropriate method (EPA Method 901.1 for drinking water;
Standard Method 7120 for aqueous and liquid phase; and HASL-300 Method Ga-01-R for surface wipes,
air filters and vegetation).
If present, iron (III) can interfere with retention of the actinides on the TRU resin, and must be reduced
with ascorbic acid to iron (II) so that it will not interfere with desired chemical reactions. Phosphates,
sulfates, and oxalates can cause interferences by forming insoluble complexes with actinides. These
anions can be complexed to aluminum (III) so that they do not interfere with the analysis. In fact, the
presence of aluminum (III) actually increases the retention factor of americium to the TRU resin. There
may be instances when increasing the aluminum (III) concentration in the nitric acid/aluminum nitrate
load solution can improve radiochemical separation and recovery.
Neptunium and americium cannot be determined sequentially and each must be analyzed using a separate
sample aliquot. The tracer used for americium analysis is americium-243, while the one used for
neptunium analysis is neptunium-239, which is in equilibrium with americium-243. The neptunium-239
yield is determined by beta counting using approximately the amount of neptunium-239 equivalent to -50
dpm of americium-243. The calcium phosphate preparation option described may give low plutonium
recoveries in unpreserved or weakly preserved samples. Until the calcium phosphate procedure is revised,
analysts must use the evaporation/digestion option when preparing water samples for plutonium analysis.
High levels of iron, manganese, calcium or magnesium, which are present in some decontamination
agents, can impact exchange site availability and/or poison the extraction resins. The presence of
compounds contained in various decontamination agents can impact the results of analysis using this
procedure due to precipitation. Precipitation can result in a lesser amount of radionuclide in cases where
an aliquot of water sample is transferred and analyzed separately form the entire sample. Such compounds
include:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on the ion exchange column or from being precipitated out
of solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Clays can contain iron, magnesium and calcium that can be released as ions via ion exchange, in
the presence of certain radionuclides, and cause analytical interferences.
Compounds containing carbonate, fluoride, hydroxide, or phosphate, present in some
decontamination agents can precipitate radionuclides out of solution prior to analysis of water
samples, resulting in a lesser amount of polonium in cases where an aliquot of water sample is
transferred prior to the precipitation step.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. "NAREL Standard
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Operating Procedure for Actinides in Environmental Matrices by Extraction Chromatography." AM/SOP-
1. Revision 7. Montgomery, AL: U.S. EPA. For access to this procedure, consult the appropriate contact
in Section 4.0.
6.2.28 EML HASL-300 Method Am-01-RC: Americium in Soil
Analyte(s)
CAS RN
Americium-241
14596-10-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Americium in soil
Method Selected for: This method has been selected for confirmatory analysis of soil/sediment samples.
Description of Method: This method uses alpha spectrometry for determination of americium-241 in
soil. 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. After source preparation by
microprecipitation, americium-241 is 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 milli Becquerel (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 (Section 6.2.47). High levels of ammonium compounds or polyacrylamides (which
can degrade into ammonium) present in some decontamination agents can potentially cause interference
and should be removed during sample preparation by heating the extracts in nitric acid for 1 to 1.5 hours.
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/sites/production/files/2015-06/documents/eml-am-01-rc.pdf
6.2.29 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters -
Eichrom's TRU Resin
Analyte(s)
CAS RN
Americium-241
14596-10-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Americium (but not lanthanides) in water and air filters
Method Selected for: This method has been selected for confirmatory analysis of drinking water,
aqueous/liquid samples, surface wipes and air filters.
Description of Method: This method is specific to measurement of americium isotopes in samples that
do not contain lanthanides, but also can be used for measurement of californium and curium. The method
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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 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 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 (Section 6.2.47). Lanthanides, if present, will not be removed by this method and
will significantly reduce the resolution of the alpha spectrograph. At this time, there are no known
interferences posed by decontamination agents that might be present in a sample.
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/sites/production/files/2015-06/documents/eml-am-04-rc.pdf
6.2.30 EML HASL-300 Method Am-06-RC: Americium and/or Plutonium in Vegetation
Analyte(s)
CAS RN
Americium-241
14596-10-2
Californium-252
13981-17-4
Curium-244
13981-15-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Americium and/or plutonium in vegetation
Method Selected for: This method has been selected 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
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 recovery. Americium (and californium-252 and curium-244, if
present) is collected with 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: PTFE beakers must be used when digesting samples with hydrofluoric acid.
Clays that are present in some decontamination agents can contain iron, magnesium or calcium, which
can be released via ion exchange in the presence of certain radionuclides, and cause analytical
interferences. High levels of iron, manganese, calcium or magnesium can impact exchange site
availability and/or poison the extraction resins used in this method.
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/sites/production/files/2015-06/documents/eml-am-Q6-rc.pdf
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6.2.31 EML HASL-300 Method Ga-01-R: Gamma Radioassay
Analyte(s)
CAS RN
Americium-241
14596-10-2
Cesium-137
10045-97-3
Cobalt-60
10198-40-0
Europium-154
15585-10-1
Gamma
NA
lndium-111
15750-15-9
Iodine-131
10043-66-0
lridium-192
14694-69-0
Molybdenum-99
14119-15-4
Neptunium-239
13968-59-7
Rhenium-188
14378-26-8
Rubidium-82
14391-63-0
Ruthenium-103
13968-53-1
Ruthenium-106
13967-48-1
Selenium-75
14265-71-5
Select Mixed Fission Products
NA
Technetium-99m
378784-45-3
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: This method has been selected 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 measurement of gamma photons
emitted from radionuclides without separating them from the sample matrix. Samples are placed into a
standard geometry for counting, typically using a high purity Germanium [HP(Ge)] detector. Ge(Li) or
Nal(Tl) detectors 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
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. 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. Because of electronic limitations, count rates higher than 2000 counts per second
(cps) should be avoided. High activity samples can be diluted, reduced in size, or moved away from the
detector (a limited distance) to reduce the count rate. 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 for Nal(Tl) detectors.
Special Considerations: Clays and hydrated alumina, which are present in some decontamination
agents, can sequester cesium-137 and iodine-131, respectively. Each would be released only upon
complete dissolution and, therefore, not measured when using this method for analysis of water samples.
Compounds containing carbonate, fluoride, hydroxide or phosphate, also present in some
decontamination agents, can precipitate radionuclides out of solution prior to analysis, resulting in a lesser
amount of gamma emitting radionuclides in cases where a water sample aliquot is transferred and
analyzed separately from the entire sample.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Ga-01-R: Gamma
Radioassay." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sites/production/files/2015-Q6/documents/eml-ga-01-r.pdf
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6.2.32 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air
Filters
Analyte(s)
CAS RN
Polonium-210
13981-52-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Polonium in water, vegetation, soil and air filters
Method Selected for: This method has been selected 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.
Special Considerations: This method requires specific valence states for radionuclides; oxidizing and
reducing agents, which are present in decontamination agents, can impact the analysis. Oxidizers can
oxidize nickel or lead to form soluble metal ions that can cause interferences. Chelating compounds, such
as those present in some decontamination agents, can complex tightly to nickel and lead that may be
present in a sample, preventing their precipitation during sample preparation. These metal ions can
potentially cause interferences when analyzing for polonium-210.
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/sites/production/files/2015-07/documents/eml-po-Q2-rc.pdf
6.2.33 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or
Sediments
Analyte(s)
CAS RN
Americium-241
14596-10-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Plutonium and americium in soil
Method Selected for: This method has been selected 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.
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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 (Section 6.2.47). High levels of iron, manganese, calcium or
magnesium can impact exchange site availability and/or poison the extraction resins used in this method.
The presence of compounds contained in various decontamination agents can impact the results of
analysis using this procedure due to precipitation. Precipitation can result in a lesser amount of
radionuclide in cases where an aliquot of water sample is transferred and analyzed separately form the
entire sample. Such compounds include:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Clays can contain iron, magnesium or calcium that can be released via ion exchange in the
presence of certain radionuclides, and cause analytical interferences.
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/sites/production/files/2015-Q7/documents/eml-pu-12-rc.pdf
6.2.34 EML HASL-300 Method Ra-03-RC: Radium-226 in Soil, Vegetable Ash, and Ion
Exchange Resin
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Radon emanation / Gamma spectroscopy (analysis of tracer)
Method Developed for: Radium-226 in soil, vegetation ash and ion exchange resin
Method Selected for: This method has been selected 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. Certain chelating
compounds, which are present in some decontamination agents, can compromise the collection of
radionuclides prior to analysis, by preventing them from being precipitated out of solution during
precipitation procedures. Other chelators can tightly complex barium or strontium that may be present in
the sample, causing interference when analyzing for radium-226. Dispersants and corrosion inhibitors,
also present in decontaminating agents, can have chelating ability as well.
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/sites/production/files/2015-07/documents/eml-ra-Q3-rc.pdf
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6.2.35 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples
Analyte(s)
CAS RN
Strontium-90
10098-97-2
Analysis Purpose: Confirmatory analysis
Technique: Beta counting / Gamma spectroscopy (analysis of tracer)
Method Developed for: Strontium-90 in vegetation, water, air filters and soil
Method Selected for: This method has been selected 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 gas
proportional beta counter. Chemical yield is determined with strontium-85 tracer by counting in a gamma
well detector.
Special Consideration: If analyzing highly calcareous soils, or if carbonate compounds found in some
decontamination agents are present in the sample, an additional quantity of hydrochloric acid should be
added to replace the acid required to decompose the carbonates. At this time, there are no known
interferences posed by decontamination agents that might be present in a sample.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method Sr-03-RC:
Strontium-90 in Environmental Samples." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sites/production/files/2015-07/documents/eml-sr-Q3-rc.pdf
6.2.36 EML HASL-300 Method Tc-01-RC: Technetium-99 in Water and Vegetation
Analyte(s)
CAS RN
Technetium-99
14133-76-7
Analysis Purpose: Confirmatory analysis
Technique: Beta counting / Gamma spectrometry
Method Developed for: Technetium-99 in water and vegetation
Method Selected for: This method has been selected 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 cannot be obtained, technetium-99m tracer may be substituted.
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Compounds containing carbonate, hydroxide, phosphate or sulfate, which are present in some
decontamination agents, can precipitate radionuclides out of solution prior to analysis. This precipitation
can result in a lesser amount of technetium in cases where an aliquot of water sample is transferred and
analyzed separately from the entire sample.
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/sites/production/files/2015-Q7/documents/eml-tc-01-rc.pdf
6.2.37 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water - TEVAฎ Resin
Analyte(s)
CAS RN
Technetium-99
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Technetium-99 in water
Method Selected for: This method has been selected 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 TEVA-resin column,which is highly specific for technetium
in the pertechnetate form. The resin is washed with dilute 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.
Special Considerations: Chemical and color quenching can have a significant impact when using liquid
scintillation methods. Several compounds contained in decontamination agents can cause this quenching,
such as organic compounds containing oxygen; halogenated compounds; elevated levels of nitrates or
nitromethane; and dyes, pigments or other colored compounds. Chelators can compromise the collection
of radionuclides prior to analysis, by causing them to avoid being precipitated out of solution during
precipitation procedures.
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/sites/production/files/2015-07/documents/eml-tc-Q2-rc.pdf
6.2.38 EML HASL-300 Method U-02-RC: Isotopic Uranium in Biological and
Environmental Materials
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Confirmatory analysis
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Technique: Alpha spectrometry
Method Developed for: Isotopic uranium in biological and environmental materials
Method Selected for: This method has been selected 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.
Chelating or complexing compounds, such as those present in some decontamination agents, can
compromise the collection of radionuclides prior to analysis, by preventing them from being trapped on
the ion exchange column or from being precipitated out of solution during precipitation procedures.
Dispersants and corrosion inhibitors can have chelating ability as well.
Source: EML, DOE (EML is currently part of the DHS). 1997. "HASL-300 Method U-02-RC: Isotopic
Uranium in Biological and Environmental Materials." EML Procedures Manual, HASL-300, 28th Edition.
http://www.epa.gov/sites/production/files/2015-07/documents/eml-u-Q2-rc.pdf
6.2.39 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: This method has been selected for gross alpha and gross beta determination in air
filters, and for direct counting of surface wipes.
Description of Method: A 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 analytes:
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
Americium-241
Californium-252
Cesium-137
Cobalt-60
Curium-244
Europium-154
Iridium-192
Plutonium-23 8
Plutonium-23 9
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Strontium-90
Uranium-234
Uranium-235
Uranium-238
(CAS RN 14596-10-2)
(CAS RN 13981-17-4)
(CAS RN 10045-97-3)
(CAS RN 10198-40-0)
(CAS RN 13981-15-2)
(CAS RN 15585-10-1)
(CAS RN 14694-69-0)
(CAS RN 13981-16-3)
(CAS RN 15117-48-3)
(CAS RN 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)
(CAS RN 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
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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.
Special Considerations: High levels of particulate loading on the air filter or swipe will affect the alpha
efficiency. Accurate results for radionuclides, other than cesium-137 and thorium-230, may be difficult
because of the difference in efficiencies for the uncalibrated radionuclides. At this time, there are no
known interferences posed by decontamination agents that might be present in a sample.
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. Las Vegas,
NV: U.S. DOE. http://www.epa.gov/sites/production/files/2015-06/documents/frmac-vol2-pg33.pdf
6.2.40 DOE RESL Method P-2: P-32 Fish, Vegetation, Dry Ash, Ion Exchange
Analyte(s)
CAS RN
Phosphorus-32
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: This method has been selected 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 550ฐC 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 of fish 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 milligram quantities of both elements.
Chemical and color quenching can have a significant impact when using liquid scintillation methods.
Several compounds contained in decontamination agents can cause this quenching, such as organic
compounds containing oxygen; halogenated compounds; elevated levels of nitrates or nitromethane; and
dyes, pigments or other colored compounds. Chelators also can tightly complex calcium that may be
present in the sample causing interference when analyzing for phosphorus-32.
Source: RESL, DOE. 1977. "Method P-2: P-32 Fish, Vegetation, Dry Ash, Ion Exchange." RESL
Analytical Chemistry Branch Procedures Manual, IDO-12096.
http://www.epa.gov/sites/production/files/2015-Q7/documents/resl-p-2.pdf
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6.2.41 DOE SRS Actinides and Sr-89/90 in Soil Samples
Analyte(s)
CAS RN
Strontium-89
14158-27-1
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: This method has been selected for qualitative analysis of strontium-89 in soil and
sediment samples.
Description of Method: Radioactive tracers are added to samples prior to sample fusion at 600ฐC 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 TEVA, TRU and DGA resins is applied to separate the actinides into
four fractions: thorium, 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. Actinide tracers are not needed when analyzing samples only for Sr-89. The
various fractions of actinides are eluted from the resin columns and precipitated with cerium fluoride,
dried, and counted by alpha spectrometry. Strontium resin is used to separate strontium-89/90 for
measurement by beta counting.
Special Considerations: The presence of compounds contained in various decontamination agents can
impact the results of analysis using this procedure:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Clays can contain iron, magnesium or calcium, which can be released as ions via ion exchange in
the presence of certain radionuclides, and cause interference in the analysis of the water.
High levels of iron, manganese, calcium or magnesium can impact exchange site availability
and/or poison extraction resins used in this method.
Reducing agents also can impact this method, which requires specific valence states for
radionuclides.
Source: SRS, DOE. 2011. "Actinides and Sr-89/90 in Soil." SRS Manual L3.23, Procedure L3.23-
10054. http://www.epa.gov/sites/production/files/2015-07/documents/13.23-10054 .pdf
6.2.42 DOE SRS Actinides and Sr-89/90 in Vegetation: Fusion Method
Analyte(s)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15517-48-3
Strontium-89
14158-27-1
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
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Analysis Purpose: Qualitative analysis
Technique: Alpha spectrometry / Beta counting
Method Developed for: Actinides and strontium-89 and -90 in vegetation
Method Selected for: This method has been selected for qualitative analysis of vegetation.
Description of Method: Radioactive tracers are added to samples prior to sample fusion at 600ฐC 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 (TEVA, 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 cerium 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. If uranium-232 is present in a sample, the
procedure of standard addition can be used to determine the amount of uranium-232 contamination. The
presence of compounds contained in various decontamination agents can impact the results of analysis
using this procedure:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution. Dispersants and corrosion inhibitors can have chelating ability as well.
Clays can contain iron, magnesium or calcium, which can be released as ions via ion exchange in
the presence of certain radionuclides, and cause interference in the analysis of the water.
High levels of iron, manganese, calcium or magnesium can impact exchange site availability
and/or poison extraction resins used in this method.
Reducing agents also can impact this method, which requires specific valence states for
radionuclides.
Source: SRS, DOE. 2011. "Actinides and Sr-89/90 in Vegetation: Fusion Method." SRSManual L3.23,
Procedure L3.23-10055. http://www.epa.gov/sites/production/files/2015-07/documents/13.23-10Q55.pdf
6.2.43 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: This method has been selected 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 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
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Cobalt-60
(CAS
RN
10198-40-0)
Beta emitter
Curium-244
(CAS
RN
13981-15-2)
Alpha emitter
Europium-154
(CAS
RN
15585-10-1)
Beta emitter
Iridium-192
(CAS
RN
14694-69-0)
Beta emitter
Plutonium-23 8
(CAS
RN
13981-16-3)
Alpha emitter
Plutonium-23 9
(CAS
RN
15117-48-3)
Alpha emitter
Polonium-210
(CAS
RN
13981-52-7)
Alpha emitter
Radium-226
(CAS
RN
13982-63-3)
Alpha emitter
Ruthenium-103
(CAS
RN
13968-53-1)
Beta emitter
Ruthenium-106
(CAS
RN
13967-48-1)
Beta emitter
Strontium-90
(CAS
RN
10098-97-2)
Beta emitter
Uranium-234
(CAS
RN
13966-29-5)
Alpha emitter
Uranium-235
(CAS
RN
15117-96-1)
Alpha emitter
Uranium-238
(CAS
RN 7440-16-1)
Alpha emitter
This procedure provides screening measurements to indicate whether specific 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 homogenized, 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.
At this time, there are no known interferences posed by decontamination agents that might be present in a
sample.
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/sites/production/files/2015-06/documents/orise-apl.pdf
6.2.44 ORISE Method AP2: Determination of Tritium
Analyte(s)
CAS RN
Tritium (Hydrogen-3)
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: This method has been selected 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.
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Chemical and color quenching can have a significant impact when using liquid scintillation methods.
Some decontamination agents include organic compounds that contain oxygen, halogenated compounds,
elevated levels of nitrates or nitromethane, or dyes, pigments or other colored compounds that can cause
chemical or color quenching.
Source: ORISE, ORAU. 2001. "Method AP2: Determination of Tritium " Laboratory Procedures
Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sites/production/files/2015-06/documents/orise-ap2.pdf
6.2.45 ORISE Method AP5: Determination of Technetium-99
Analyte(s)
CAS RN
Technetium-99
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
Method Selected for: This method has been selected 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 TEVA-resin column, 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, sulfiir-35, strontium-90, yttrium-90 and thorium-234)
are effectively removed using TEVA resin under the conditions in this procedure.
Special Considerations: Tritium may follow technetium due to the absorption of some tritium-labeled
compounds by the resin. Possible tritium interferences are eliminated by setting the technetium counting
window above the maximum energy of tritium beta particles. Chelating compounds, such as those
contained in some decontamination agents, can compromise the collection of radionuclides prior to
analysis, by preventing them from being precipitated out of solution during precipitation procedures.
Some decontamination agents include organic compounds that contain oxygen, halogenated compounds,
elevated levels of nitrates or nitromethane, or dyes, pigments or other colored compounds can cause
chemical or color quenching.
Source: ORISE, ORAU. 2001. "Method AP5: Determination of Technetium-99 "Laboratory
Procedures Manual for the Environmental Survey and Site Assessment Program.
http://www.epa.gov/sites/production/files/2015-06/documents/orise-ap5.pdf
6.2.46 ORISE Method AP7: Determination of Radium-226 in Water and Soil Samples
Using Alpha Spectroscopy
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry / Gamma spectroscopy (analysis of tracer)
Method Developed for: Radium-226 in water and soil
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Method Selected for: This method has been selected for confirmatory analysis of radium-226 in
soil/sediment samples.
Description of Method: The tracer (barium-133) and potassium hydrogen fluoride are added to a soil
sample aliquot in a platinum crucible. The sample is heated until the potassium hydrogen fluoride has
completely dried. Heating is continued at 900ฐC until total dissolution of the sample. After allowing the
sample to cool slightly, sulfuric acid is added and the mixture is heated to dissolve the fluoride cake.
Sodium sulfate is added to the slurry and the temperature is slowly raised until the slurry melts
completely. Water and 12M hydrochloric acid are added to dissolve the pyrosulfate cake. Sulfuric acid
(9M), potassium sulfate and sodium sulfate are added and the solution is evaporated to 35-40 mL. Three
portions of lead (II) in solution are added while stirring, waiting 5 minutes between each addition. After
centrifuging, the supernatant is discarded and the precipitate is dissolved in diethylenetriaminepentaacetic
acid (DTPA). Barium (II) in solution is added to form barium sulfate, which acts to separate radium-226
from possible interfering radionuclides. The barium precipitate is filtered, and radium-226 is counted by
alpha spectroscopy. Barium-133 is used to quantify the yield by gamma spectroscopy.
Special Considerations: High levels of barium will add mass to the final sample causing self-
attenuation and degradation of the alpha spectrum. If the amount of barium in the sample can be
predetermined, it may be possible to adjust sample size and not add the barium (II) in step 4.2.11 of the
method. Contamination with barium-133 will interfere with the yield determination. This may be
corrected by gamma counting before analysis and adjusting the barium yield accordingly.
Chelating compounds, such as those contained in some decontamination agents, can compromise the
collection of radionuclides prior to analysis, by preventing them from being precipitated out of solution
during precipitation procedures. Other chelators can tightly complex barium or strontium that may be
present in the sample causing interference when analyzing for radium-226. Dispersants and corrosion
inhibitors, also present in decontaminating agents, can have chelating ability as well.
Source: ORISE, ORAU. 2001. "Method AP7: Determination of Radium-226 in Water and Soil
Samples Using Alpha Spectroscopy." Laboratory Procedures Manual for the Environmental Survey and
Site Assessment Program.
https://www.epa.gov/sites/production/files/2Q17-10/documents/ap7 ra-226 water soil alpha spec.pdf
6.2.47 ORISE Method AP11: Sequential Determination of the Actinides in Environmental
Samples Using Total Sample Dissolution and Extraction Chromatography
Analyte(s)
CAS RN
Americium-241
14596-10-2
Californium-252
13981-17-4
Curium-244
13981-15-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
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
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Method Selected for: This method has been selected 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, this method is
recommended 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
of americium from the lanthanides. All actinides are coprecipitated on cerium fluoride and counted with
an alpha spectrometer system.
Special Considerations: Chelating compounds, such as those present in some decontamination agents,
can compromise the collection of radionuclides prior to analysis by preventing them from being trapped
on the ion exchange column or from being precipitated out of solution. Dispersants and corrosion
inhibitors can have chelating ability as well. Clays, which are also present in some decontamination
agents, can contain iron, magnesium or calcium that can be released via ion exchange in the presence of
certain radionuclides, and cause analytical interferences. High levels of iron, manganese, calcium or
magnesium can impact exchange site availability and/or poison extraction resins used in this method.
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/sites/production/files/2015-06/documents/orise-apll.pdf
6.2.48 ORISE Method Procedure #9: Determination of 1-125 in Environmental Samples
Analyte(s)
CAS RN
Iodine-125
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: This method has been selected 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" sodium iodide (Nal(Tl)) 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
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Section 6.0 - Selected Radiochemical Methods
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. Chelating
compounds, such as those contained in some decontamination agents, can compromise the collection of
radionuclides prior to analysis, preventing them from being trapped on an ion exchange column. Hydrated
alumina, also present in certain decontamination agents, can sequester iodine-125 and iodine-131, which
would only be released upon complete dissolution and therefore not be measured when using this method.
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/sites/production/files/2015-06/documents/orise-procedure9-1995.pdf
6.2.49 ASTM Method D3084-05 (2012): Standard Practice for Alpha Spectrometry in Water
Analyte(s)
CAS RN
Americium-241
14596-10-2
Californium-252
13981-17-4
Curium-244
13981-15-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Analysis Purpose: Qualitative determination
Technique: Alpha spectrometry
Method Developed for: Alpha particle spectra in water
Method Selected for: This method has been selected 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
and curium-244 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 Section
10.1 of the method), 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 (Section 6.2.47) for sample preparation instead of the methods referenced in
ASTM Method D3084. At this time, there are no known interferences posed by decontamination agents
that might be present in a sample.
Source: ASTM. 2012. "Method D3084-05 (2012): Standard Practice for Alpha Spectrometry in Water."
Annual Book of ASTM Standards, Vol. 11.02. West Conshohocken, PA: ASTM International.
http://www.astm.org/Standards/D3084.htm
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6.2.50 ASTM Method D3972-09 (2015): Standard Test Method for Isotopic Uranium in
Water by Radiochemistry
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Alpha-particle-emitting isotopes of uranium in water
Method Selected for: This method has been selected for confirmatory analysis of drinking water
samples.
Description of Method: Uranium is chemically separated from a water sample by coprecipitation with
ferrous hydroxide followed by anion exchange, and electrodeposition. When suspended matter is present,
an acid dissolution step is added to ensure that all of the uranium dissolves. The sample is acidified, and
uranium-232 is added as an isotopic tracer to determine chemical yield. Uranium is coprecipitated from
the sample with ferrous hydroxide. This precipitate is dissolved in concentrated hydrochloric acid, or is
subjected to acid dissolution with concentrated nitric and hydrofluoric acids, if the hydrochloric acid fails
to dissolve the precipitate. Uranium is separated from other radionuclides by adsorption on anion
exchange resin, followed by elution with hydrochloric acid. The uranium is finally electrodeposited onto a
stainless steel disc and counted using alpha spectrometry.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1 (Section 6.2.47). The presence of compounds contained in various decontamination
agents can impact the results of analysis using this procedure due to precipitation. Precipitation can result
in a lesser amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed
separately from the entire sample. Such compounds include:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution during precipitation procedures. Dispersants and corrosion inhibitors can have chelating
ability as well.
Compounds containing carbonate, hydroxide, or phosphate can precipitate uranium out of
solution prior to analysis.
Source: ASTM. 2015. "Method D3972-09 (2015): Standard Test Method for Isotopic Uranium in Water
by Radiochemistry." Annual Book of ASTM Standards, Vol. 11.02. West Conshohocken, PA: ASTM
International. http://www.astm.org/Standards/D3972.htm
6.2.51 ASTM Method D5811-08 (2013): Standard Test Method for Strontium-90 in Water
Analyte(s)
CAS RN
Strontium-90
10098-97-2
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Beta counting
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Method Developed for: Strontium-90 in water samples
Method Selected for: This method has been selected 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. The presence of compounds contained in various decontamination agents
can impact the results of analysis using this procedure due to precipitation. Precipitation can result in a
lesser amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed
separately from the entire sample. Such compounds include:
Chelating compounds can compromise the collection of radionuclides prior to analysis,
preventing them from being trapped on an ion exchange column or from being precipitated out of
solution during precipitation procedures. Dispersants and corrosion inhibitors can have chelating
ability as well.
Compounds containing carbonate, fluoride, phosphate or sulfate can precipitate radionuclides out
of solution prior to analysis, resulting in a lesser amount of strontium in cases where an aliquot of
water sample is transferred and analyzed separately from the entire sample.
Source: ASTM. 2013. "Method D5811-08 (2013): Standard Test Method for Strontium-90 in Water."
Annual Book of ASTM Standards, Vol. 11.02. West Conshohocken, PA: ASTM International.
http://www.astm.org/Standards/D5811 .htm
6.2.52 ASTM Method D7168-16: Standard Test Method for Technetium-99 in Water by
Solid Phase Extraction Disk
Analyte(s)
CAS RN
Technetium-99
14133-76-7
Analysis Purpose: Qualitative and confirmatory analysis
Technique: Liquid scintillation
Method Developed for: Technetium-99 in water samples
Method Selected for: This method has been selected 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
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. High levels of iodate. iron (III) and antimony can interfere with the measurement
of technetium-99 and lead to a positive bias in sample results.
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Chelating compounds, such as those contained in some decontamination agents, can compromise the
collection of radionuclides prior to analysis, preventing them from being precipitated out of solution
during precipitation procedures. Chemical and color quenching can have a significant impact when using
liquid scintillation methods. Some decontamination agents include organic compounds that contain
oxygen, halogenated compounds, elevated levels of nitrates or nitromethane, or dyes, pigments or other
colored compounds that can cause chemical or color quenching, which can significantly impact liquid
scintillation methods.
Source: ASTM. 2016. "Method D7168-16: Standard Test Method for Technetium-99 in Water by Solid
Phase Extraction Disk." Annual Book of ASTM Standards, Vol. 11.02. West Conshohocken, PA: ASTM
International. http://www.astm.org/Standards/D7168.htm
6.2.53 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: This method has been selected 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 analytes:
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. Compounds containing carbonate, fluoride, hydroxide, or
SAM 2017 176 September 2017
Americium-241
Californium-252
Cesium-137
Cobalt-60
Curium-244
Europium-154
Iridium-192
Plutonium-23 8
Plutonium-23 9
Polonium-210
Radium-226
Ruthenium-103
Ruthenium-106
Strontium-90
Uranium-234
Uranium-235
Uranium-238
(CAS RN 14596-10-2) Alpha emitter
(CAS RN 13981-17-4) Alpha emitter
(CAS RN 10045-97-3) Beta emitter
(CAS RN 10198-40-0) Beta emitter
(CAS RN 13981-15-2) Alpha emitter
(CAS RN 15585-10-1) Betaemitter
(CAS RN 14694-69-0) Beta emitter
(CAS RN 13981-16-3) Alpha emitter
(CAS RN 15117-48-3) Alpha emitter
(CAS RN 13981-52-7) Alpha emitter
(CAS RN 13982-63-3) Alpha emitter
(CAS RN 13968-53-1) Beta emitter
(CAS RN 13967-48-1) Beta emitter
(CAS RN 10098-97-2) Beta emitter
(CAS RN 13966-29-5) Alpha emitter
(CAS RN 15117-96-1) Alphaemitter
(CAS RN 7440-16-1) Alpha emitter
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Section 6.0 - Selected Radiochemical Methods
phosphate, which are present in some decontamination agents, can precipitate radionuclides out of
solution prior to analysis. This precipitation can result in a lesser amount of radionuclides in cases where
an aliquot of water sample is transferred and analyzed separately from the entire sample. The presence of
hydroxide, in particular, can have a significant impact. At a pH above 5, it will precipitate out iron,
carrying actinides (e.g., uranium and plutonium) with it.
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.
22nd Edition. Washington, DC: APHA. http://www.standardmethods.org/
6.2.54 Standard Method 7120: Gamma-Emitting Radionuclides
Analyte(s)
CAS RN
Americium-241
14596-10-2
Cesium-137
10045-97-3
Cobalt-60
10198-40-0
Europium-154
15585-10-1
lridium-192
14694-69-0
Neptunium-239
13968-59-7
Ruthenium-103
13968-53-1
Ruthenium-106
13967-48-1
Selenium-75
14265-71-5
Analysis Purpose: Qualitative and confirmatory determination
Technique: Gamma spectrometry
Method Developed for: Gamma emitting radionuclides in water
Method Selected for: This method has been selected 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.
Special Considerations: The presence of compounds contained in various decontamination agents can
impact the results of analysis using this procedure due to precipitation. Precipitation can result in a lesser
amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed separately
from the entire sample. Such compounds include:
Reducing agents can potentially convert radionuclides into an insoluble zero-valent state that can
precipitate out of solution. The addition of nitric acid during sample collection can prevent this
precipitation from occurring. Iridium and ruthenium would likely still precipitate in the presence
of reducing agents.
Clays can sequester cesium-137, which would only be released upon complete dissolution when
using this method.
Compounds containing carbonate, fluoride, hydroxide, or phosphate can precipitate radionuclides
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out of solution prior to analysis.
Source: APHA, AWWA and WEF. 2005. "Method 7120: Gamma-Emitting Radionuclides." Standard
Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
http: //www. standardmethods .org/
6.2.55 Standard Method 7500-Ra B: Radium: Precipitation Method
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Qualitative determination
Technique: Alpha counting
Method Developed for: Alpha-emitting isotopes of radium in water
Method Selected for: This method has been selected for qualitative determination in aqueous/liquid
samples.
Description of Method: This method is for determination of all alpha-emitting radium isotopes by alpha
decay analysis. Lead and barium carriers are added to the sample containing alkaline citrate, then sulfuric
acid is added to precipitate radium, barium and lead as sulfates. The precipitate is purified by washing
with nitric acid, dissolving in alkaline EDTA, and re-precipitating as radium-barium sulfate after pH
adjustment to 4.5. This slightly acidic EDTA keeps other naturally occurring alpha-emitters and the lead
carrier in solution. Radium-223, -224 and -226 are identified by the rate of ingrowth of their daughter
products in barium sulfate precipitate. The results are corrected by the rate of ingrowth of daughter
products to determine radium activity. This method involves alpha counting by a gas-flow internal
proportional counter, scintillation counter or thin end-window gas-flow proportional counter.
Special Considerations: The presence of compounds contained in various decontamination agents can
impact the results of analysis using this procedure due to precipitation. Precipitation can result in a lesser
amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed separately
from the entire sample. Such compounds include:
Chelating compounds, such as those contained in some decontamination agents, can complex
tightly to calcium, nickel, lead, magnesium or strontium that may be present in a sample,
preventing their precipitation during sample preparation. These metal ions can potentially cause
interferences when analyzing for radium-226.
Compounds containing sulfate, carbonate, oxalate or phosphate, which are also present in some
decontamination agents, can precipitate radium out of solution prior to analysis.
Source: APHA, AWWA and WEF. 2005. "Method 7500-Ra B: Radium: Precipitation Method."
Standard Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
http: //www. standardmethods .org/
6.2.56 Standard Method 7500-Ra C: Radium: Emanation Method
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Confirmatory determination
Technique: Alpha counting
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Method Developed for: Soluble, suspended and total radium-226 in water
Method Selected for: This method has been selected 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 MDC
depends on counter characteristics, background-counting rate of scintillation cell, cell efficiency, length
of the counting period, and contamination of the 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.
Special Considerations: The presence of compounds contained in various decontamination agents can
impact the results of analysis using this procedure due to precipitation, Precipitation can result in a lesser
amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed separately
from the entire sample. Such compounds include:
Chelating compounds can complex tightly to calcium, nickel, lead, magnesium or strontium that
may be present in a sample, preventing their precipitation during sample preparation. These metal
ions can potentially cause interferences when analyzing for radium-226.
Compounds containing sulfate, carbonate, oxalate or phosphate can precipitate radium out of
solution prior to analysis. This precipitation can result in a lesser amount of radium in cases
where an aliquot of water sample is transferred and analyzed separately from the entire sample.
Oxidizers present in these agents can oxidize nickel or lead to form soluble metal ions that can
also cause interferences.
Source: APHA, AWWA and WEF. 2005. "Method 7500-Ra C: Radium: Emanation Method." Standard
Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
htto: //www. standardmethods.org/
6.2.57 Standard Method 7500-U B: Uranium: Radiochemical Method
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Qualitative determination
Technique: Alpha counting
Method Developed for: Total uranium alpha activity in water
Method Selected for: This method has been selected for qualitative determination in aqueous/liquid
samples.
Description of Method: The sample is acidified with hydrochloric or nitric acid and boiled to eliminate
carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and subsequently
separated. The ferric hydroxide is dissolved, passed through an anion-exchange column, washed with
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Section 6.0 - Selected Radiochemical Methods
acid, and the uranium is eluted with dilute hydrochloric acid. The acid eluate is evaporated to near
dryness, the residual salt is converted to nitrate, and the alpha activity is counted by a gas-flow
proportional counter or alpha scintillation counter.
Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible
or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use
ORISE Method API 1 (Section 6.2.47). The presence of compounds contained in various decontamination
agents can impact the results of analysis using this procedure due to precipitation, Precipitation can result
in a lesser amount of radionuclide in cases where an aliquot of water sample is transferred and analyzed
separately from the entire sample. Such compounds include:
Certain chelating agents, such as those contained in some decontamination agents, can
compromise the collection of radionuclides prior to analysis, preventing them from being trapped
on an ion exchange column or from being precipitated out of solution during precipitation
procedures.
Compounds containing carbonate, hydroxide, or phosphate, which are present in some
decontamination agents can precipitate uranium out of solution.
Source: APHA, AWWA and WEF. 2005. "Method 7500-U B: Uranium: Radiochemical Method."
Standard Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
http: //www. standardmethods .org/
6.2.58 Standard Method 7500-U C: Uranium: Isotopic Method
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
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: This method has been selected 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 (Section 6.2.47). Chelating compounds, such as those present in some
decontamination agents, can compromise the collection of radionuclides prior to analysis, preventing
them from being trapped on an ion exchange column or from being precipitated out of solution during
precipitation procedures. Dispersants and corrosion inhibitors can have chelating ability as well.
Compounds containing carbonate, hydroxide or phosphate, which are also present in some
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Section 6.0 - Selected Radiochemical Methods
decontamination agents, can precipitate uranium out of solution prior to analysis, resulting in a lesser
amount of uranium in cases where an aliquot of water sample is transferred and analyzed separately from
the entire sample.
Source: APHA, AWWA and WEF. 2005. "Method 7500-U C: Uranium: Isotopic Method." Standard
Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: APHA.
http: //www. standardmethods .org/
6.2.59 Y-12 (DOE) Preparation of Samples for Total Activity Screening
Analyte(s)
CAS RN
Total Activity Screening
NA
Analysis Purpose: Total activity screening
Technique: Liquid scintillation
Method Developed for: Total activity screening
Method Selected for: This method has been selected 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.
Chemical and color quenching can have a significant impact when using liquid scintillation methods.
Organic compounds containing oxygen; halogenated compounds; elevated levels of nitrates or
nitromethane; and dyes, pigments or other colored compounds present in certain decontamination agents,
can cause quenching. Chelators, also present in decontamination agents, can tightly complex calcium that
may be present in a sample, causing analytical interference (e.g., excessive levels of calcium can interfere
with detection and measurement of phosphorus-32).
Source: Y-12 (DOE). 2005. "Preparation of Samples for Total Activity Screening." Procedure Y50-AC-
65-7230. http ://www .epa.gov/sites/production/files/2015-07/documents/v50-ac-65-7230.pdf
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6.3 Method Summaries (Outdoor Infrastructure and Building Material Samples)
Summaries corresponding to the methods selected for analysis of outdoor infrastructure and building
material samples listed in Appendix B2 are provided in Sections 6.3.1 through 6.3.8. These summaries
contain information that has been extracted from the selected methods. Each method summary contains a
table identifying the contaminants listed in Appendix B2 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. For information regarding sample collection considerations
for samples to be analyzed by these methods, see the companion Sample Collection Information
Document for Chemicals, Radiochemicals and Biotoxins - Companion to SAM 2017.
6.3.1 Rapid Radiochemical Method for Total Radiostrontium (Sr-90) In Building
Materials for Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Strontium-90
10098-97-2
Analysis Purpose: Confirmatory analysis
Technique: Beta counting
Method Developed for: Strontium-89 and -90 in building materials
Method Selected for: This method has been selected for confirmatory analysis of strontium-90 in asphalt
singles, asphalt paving materials, concrete and brick.
Description of Method: Strontium is solubilized and purified by sodium hydroxide fusion using
procedures described in Section 6.3.3 for concrete and brick matrix samples, Section 6.3.7 for asphalt
matrix samples and Section 6.3.8 for asphalt shingles, and purified from potentially interfering
radionuclides and matrix constituents using a strontium-specific, rapid chemical separation method. The
sample is equilibrated with strontium carrier, and preconcentrated by strontium/calcium carbonate
coprecipitation from the alkaline fusion matrix. The carbonate precipitate is dissolved in hydrochloric
acid and strontium is precipitated with calcium fluoride to remove silicates. The strontium fluoride
precipitate is dissolved in strong nitric acid and the solution is passed through an Sr Resin extraction
chromatography column. 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. The
method is capable of satisfying a method uncertainty for total strontium-90 of 0.31 pCi/g at an analytical
action level of 2.4 pCi/g, using a sample weight of 1.5 g and a count time of approximately 1.5 hours.
If differentiating between strontium-89 and strontium-90 is needed, then the same prepared sample can be
recounted after -10 days. If the initial and second counts agree (based on the expected ingrowth of
yttrium-90) then strontium-89 is not present in significant amounts relative to strontium-90.
Computational methods are available for resolving the concentration of strontium-89 and strontium-90
from two sequential counts of the sample (see Appendix B of the method). If significant amounts of
strontium-89 are suspected, it can be determined more rapidly using Cerenkov counting; however, the
minimum detectable activity levels will be higher than that of determination with gas proportional
counting and may or may not meet measurement quality objectives.
Special Considerations: Count results should be monitored for detectable alpha activity and appropriate
corrective actions should be taken when this is observed. Failure to address the presence of alpha emitters
in the sample test source may lead to high bias in the results, due to alpha-to-beta crosstalk.
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Elevated levels of tetravalent plutonium, neptunium, cerium or ruthenium in the sample may hold up on
the column and co-elute with strontium. The method uses an oxalic acid rinse that should address low to
moderate levels of these interferences. Significant levels of strontium-90 also will interfere with the total
radiostrontium analysis (see Appendix B of the method for an alternative approach should this situation
arise). High levels of lead-210 can interfere with low level strontium analysis due to ingrowth of short-
lived bismuth-210 during chemical separations, where lead is retained by the resin, but is not eluted. If
lead-210 is known to be present in samples, minimizing the time between the final rinse and the elution of
strontium to less than 15 minutes will minimize the levels of interfering bismuth-210.
The presence of compounds contained in various decontamination agents can impact the results of
analysis using this procedure:
Clays and other compounds containing iron, magnesium and calcium, which can be released as
ions via ion exchange in the presence of certain radionuclides, can cause interferences.
High levels of iron or magnesium can impact exchange site availability and/or poison the
extraction resins.
Chelating compounds can tightly complex barium, iron, lead, magnesium and potassium causing
interference when analyzing for strontium-89 or -90.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Total Radiostrontium (Sr-90) In Building Materials for Environmental
Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-
001. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.3.2 Rapid Radiochemical Method for Radium-226 in Building Materials for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Radium-226
13982-63-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Radium-226 in building materials
Method Selected for: This method has been selected for confirmatory analysis of radium-226 in asphalt
shingle, asphalt paving materials, concrete and brick.
Description of Method: A known quantity of radium-225 is used as the yield tracer in this analysis. The
sample is fused using the procedures described in Section 6.3.3 for concrete and brick matrix samples,
Section 6.3.7 for asphalt matrix samples and Section 6.3.8 for asphalt roofing matrix samples. Radium
isotopes are removed from the fusion matrix using a carbonate precipitation step. The sample is acidified
and loaded onto a cation exchange resin to remove interferences, such as calcium. Radium is eluted from
the cation resin with 8M nitric acid. After evaporation of the eluate, the sample is dissolved in a minimal
amount of 3M nitric acid and passed through Sr Resin to remove any barium present. This solution is then
evaporated to dryness, redissolved in 0.02M hydrochloric acid, and passed through Ln Resin to remove
interferences such as residual calcium, and to remove the initial actinium-225 present. The radium
(including radium-226) is prepared for counting by microprecipitation with barium sulfate. 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 (astatine-217, the third progeny of radium-225).
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This method is suited for low-level measurements for radium-226 using alpha spectrometry and is capable
of satisfying a method uncertainty of 0.83 pCi/g at an analytical action level of 6.41 pCi/g, using a sample
aliquant of approximately 1 g and count time of 8 hours (or longer).
Special Considerations: Depending on actual spectral resolution, method performance may be
compromised if samples contain high levels of other radium isotopes (e.g., ~3 times the radium-226
activity concentration) due to ingrowth of interfering decay progeny. Calcium, iron (+3 oxidation state),
and radionuclides with overlapping alpha energies, such as thorium-229, uranium-234, and neptunium-
237, will interfere if they are not removed effectively. Delaying the count significantly longer than one
day may introduce positive bias in results near the detection threshold due to the decay progeny from the
radium 225 tracer. If radium-226 measurements close to detection levels are required and sample
counting cannot be performed within -36 hours of tracer addition, the impact of tracer progeny tailing
into the radium-226 may be minimized by reducing the amount of the tracer that is added to the sample.
This will aid in improving the signal-to-noise ratio for the radium-226 peak by minimizing the amount of
tailing from higher energy alphas of the radium-225 progeny. If actinium-225 is present prior to the final
separation time and the flow rate through the column is too fast (>1.5 drops/second), then actinium-225
will break through the resin, resulting in a high bias in the tracer yield. Additional information regarding
procedures to remove or minimize interferences is provided in Section 4.0 of the method.
The presence of compounds contained in various decontamination agents can impact the results of
analysis using this procedure:
Clays can contain iron, magnesium or calcium, which can be released as ions via ion exchange in
the presence of certain radionuclides, and cause interferences.
High levels of iron, manganese, calcium or magnesium might have an impact on exchange site
availability and or poisoning of the extraction resins used.
Chelators can tightly complex barium, calcium, iron and magnesium that may be present in the
sample causing interferences when analyzing for radium-226.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Radium-226 in Building Materials for Environmental Remediation Following
Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-002.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.3.3 Rapid Method for Sodium Hydroxide Fusion of Concrete and Brick Matrices Prior
to Americium, Plutonium, Strontium, Radium, and Uranium Analyses for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Radium-226
13982-63-3
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Sample Preparation
Sample Preparation Technique: Fusion
Determinative Technique: Alpha spectrometry/beta counting
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Method Developed for: Americium-241, plutonium-238, plutonium-239, radium-226, strontium-90,
uranium-234, uranium-235 and uranium-238 in concrete and brick samples.
Method Selected for: This method has been selected for preparation of concrete and brick samples to be
analyzed for americium-241, plutonium-238, plutonium-239, radium-226, strontium-90, uranium-234,
uranium-235 and uranium-238.
Description of Method: Concrete and brick samples may be received as core samples, pieces of various
sizes, dust or particles (wet or dry) from scabbling, or powder. The method is based on the rapid fusion, in
zirconium crucibles, of a representative, finely ground (5-100 mesh sized) 1-1.5 gram aliquant using
rapid sodium hydroxide fusion at 600ฐC. Plutonium, uranium and americium are separated from the
alkaline matrix using an iron/titanium hydroxide precipitation (enhanced with calcium phosphate
precipitation) followed by a lanthanum fluoride matrix removal step. Strontium is separated from the
alkaline matrix using a carbonate precipitation, followed by calcium fluoride precipitation to remove
silicates. Radium is separated from the alkaline matrix using a carbonate precipitation. These sample
preparation procedures are performed prior to the chemical separation procedures described in the
following:
Rapid Radiochemical Method for Total Radiostrontium (Strontium-90) in Building Materials for
Environmental Remediation Following Radiological Incidents (Section 6.3.1)
Rapid Radiochemical Method for Radium-226 in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.2)
Rapid Radiochemical Method for Isotopic Uranium in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.4)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-23 9/240 in Building Materials
for Environmental Remediation Following Radiological Incidents (Section 6.3.5)
Rapid Radiochemical Method for Americium-241 in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.6)
Special Considerations: In samples where native constituents may be present that could interfere with
determination of the chemical yield (e.g., strontium for strontium-90 analysis) or with the creation of a
sample test source (e.g., barium for radium-226 analysis by alpha spectrometry), it may be necessary to
determine the concentration of the native constituents in advance of chemical separation (using a separate
aliquant of fused material) and make appropriate adjustments to the yield calculations or amount of
carrier added. Concrete and brick can contain native barium or radium, which can cause interferences
with the analysis of radium-226. Compounds contained in decontamination agents are not expected to
cause interferences during sample preparation; see the sections corresponding to the analytical methods
listed in the description of this method for potential interferences caused by constituents of
decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Method for Sodium Hydroxide Fusion of Concrete and Brick Matrices Prior to Americium, Plutonium,
Strontium, Radium, and Uranium Analyses for Environmental Remediation Following Radiological
Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R-14-004.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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6.3.4 Rapid Radiochemical Method for Isotopic Uranium in Building Materials for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Uranium-234, -235 and -238 in concrete and brick samples
Method Selected for: This method has been selected for confirmatory analysis of uranium-234, uranium-
235 and uranium-238 in asphalt shingles, asphalt building materials, concrete and brick.
Description of Method: This method is based on the use of extraction chromatography resins to isolate
and purify uranium isotopes by removing interfering radionuclides as well as other components of the
sample matrix in order to prepare the uranium fraction for counting by alpha spectrometry. The method
utilizes vacuum-assisted flow to improve the speed of the separations. Uranium-232 tracer, added to the
building materials sample, is used as a yield monitor. A 1.0- to 1.5-gram sample is fused using the
procedure described in Section 6.3.3 for concrete and brick samples, Section 6.3.7 for asphalt samples and
Section 6.3.8 for asphalt roofing materials. The uranium isotopes are then removed from the fusion matrix
using iron hydroxide and lanthanum fluoride precipitation steps. The sample test source is prepared by
microprecipitation with cerium (III) fluoride. The method is capable of achieving a method uncertainty
for uranium-234, uranium-235, and uranium-238 of 1.9 pCi/g at an analytical level of 14.7 pCi/g, using a
sample weight of approximately 1 g and count time of at least 3 to 4 hours.
Special Considerations: Alpha-emitting radionuclides with peaks at energies that cannot be adequately
resolved from the tracer or analyte (e.g., polonium-210 [5.304 MeV], thorium-228 [5.423 MeV, 5.340
MeV] and americium-243 [5.275 MeV, 5.233 MeV]) must be chemically separated to enable
radionuclide-specific measurements (see Section 4.0 of the method for procedures to remove specific
interferences). Nonradiological anions such as fluoride and phosphate that complex uranium ions may
cause lower chemical yields. Aluminum that is added in the column load solution complexes fluoride, as
well as any residual phosphate that may be present. Lanthanum, added to preconcentrate uranium from
the sample matrix as lanthanum fluoride, can have a slight adverse impact on uranium retention on TRU
resin, but this impact is minimal at the level added. Iron (3+) can also have an adverse impact on uranium
retention on TRU resin, but the residual iron levels after preconcentration steps are acceptable.
Clays, which are present in some decontamination agents, can contain iron, magnesium or calcium that
can be released as ions via ion exchange, in the presence of certain radionuclides, and cause analytical
interferences. High levels of iron, manganese, calcium or magnesium can also have an impact on
exchange site availability and/or poison extraction resins used in alpha spectrometry. Higher valence
anions such as phosphates may lead to lower yields when using the evaporation option due to competition
with active sites on the resin. Concrete and brick can contain native uranium isotopes, which can cause
interferences with the analysis of uranium isotopes.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Isotopic Uranium in Building Materials for Environmental Remediation
Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-005.
httos://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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6.3.5 Rapid Radiochemical Method for Plutonium-238 and Plutonium-239/240 in
Building Materials for Environmental Remediation Following Radiological
Incidents
Analyte(s)
CAS RN
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Plutonium-238 and -239 in building materials
Method Selected for: This method has been selected for confirmatory analysis of plutonium-238 and -
239 in asphalt shingles, asphalt paving materials, concrete and brick.
Description of Method: This method is based on the use of TEVAresin to isolate and purify plutonium
by removing interfering radionuclides as well as other components of the sample 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. The sample may be fused using the procedure described in
Section 6.3.3 for concrete and brick matrix samples, Section 6.3.7 for asphalt matrix samples and Section
6.3.8 for asphalt roofing matrix samples. The plutonium isotopes are then removed from the fusion matrix
using iron hydroxide and lanthanum fluoride precipitation steps. Plutonium-242 or plutonium-236 tracer,
added to the sample, is used as a yield monitor. The sample test source is prepared by microprecipitation
with cerium (III) fluoride. The method is capable of achieving a required method uncertainty of 0.25
pCi/g for plutonium-238, -239/240, at an analytical action level of 1.89 pCi/g, using a sample weight of
approximately 1 g and a count time of at least 3 to 4 hours.
Special Considerations: Alpha-emitting radionuclides with irresolvable alpha energies, such as
plutonium-238 (5.50 MeV), americium-241 (5.48 MeV) and thorium-228 (5.42 MeV) must be chemically
separated to enable measurement. This method separates these radionuclides effectively. The significance
of peak overlap is determined by the detector's alpha energy resolution characteristics and the quality of
the final precipitate that is counted.
Non-radiological interferences include very high levels of anions such as phosphates, which may lead to
lower yields due to competition with active sites on the resin and/or complexation with plutonium ions.
The presence of fluoride (e.g., from hydrofluoric or fluoroboric acids) can precipitate out plutonium prior
to sample measurement. Aluminum is added in the column load solution to complex interfering anions
such as fluoride and phosphate. Compounds such as clays containing iron, magnesium or calcium, which
are present in some decontamination agents, can release these elements as ions via ion exchange in the
presence of certain radionuclides, and cause interference. High levels of iron, manganese, calcium or
magnesium can also have an impact on exchange site availability and/or poison extraction resins used in
alpha spectrometry.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Plutonium-238 and Plutonium-239/240 in Building Materials for
Environmental Remediation Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA.
EPA 402-R14-006. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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6.3.6 Rapid Radiochemical Method for Americium-241 in Building Materials for
Environmental Remediation Following Radiological Incidents
Analyte(s)
CAS RN
Americium-241
14596-10-2
Analysis Purpose: Confirmatory analysis
Technique: Alpha spectrometry
Method Developed for: Americium-241 in building materials
Method Selected for: This method has been selected for confirmatory analysis of americium-241 in
asphalt shingles, asphalt paving materials, concrete and brick.
Description of Method: This method is based on the use of extraction chromatography resins (TEVA
and DGA resins) to isolate and purify americium by removing interfering radionuclides as well as other
matrix components to prepare the americium fraction for counting by alpha spectrometry. The method
uses vacuum-assisted flow to improve the speed of separations. The sample is fused using procedures
described in Section 6.3.3 for concrete and brick, Section 6.3.7 for asphalt matrix samples and Section
6.3.8 for asphalt roofing matrix samples. The americium isotopes removed from the fusion matrix using
iron hydroxide and lanthanum fluoride precipitation steps. Americium-243 tracer, added to the sample, is
used as a yield monitor. The STS is prepared by microprecipitation with cerium (III) fluoride.
The method is capable of achieving a required method uncertainty for Am-241 of 0.20 pCi/g at an
analytical action level of 1.5 pCi/g, using a sample weight of approximately 1 g and a count time of at
least 4 hours.
Special Considerations: Alpha-emitting radionuclides with irresolvable alpha energies, such as
plutonium-238 (5.50 MeV) and thorium-228 (5.42 MeV) can interfere with measurement of americium-
241, and must be chemically separated to enable measurement. This method separates these radionuclides
effectively. The significance of peak overlap is determined by the detector's alpha energy resolution
characteristics and the quality of the final precipitate that is counted. A thorium removal rinse is
performed on DGA resin in the event that any thorium ions pass through TEVA resin onto DGA resin. A
dilute nitric acid rinse is performed to remove calcium and lanthanum ions that could end up on the final
alpha source filter as fluoride solids.
Non-radiological interferences include anions that can complex americium, such as fluoride and
phosphate, and lead to lower yields. Higher valence anions (e.g., phosphate) may lead to lower yields
when using the evaporation option due to competition with active sites on the resin. Boric acid added in
the load solution complexes fluoride ions, while aluminum complexes both fluoride as well as any
residual phosphate that may be present. Clays that are present in some decontamination agents can
contain iron, magnesium and calcium, which can be released as ions, via ion exchange, in the presence of
certain radionuclides and cause interferences. High levels of calcium can have an adverse impact on
americium retention on DGA resin. This interference is minimized by increasing the nitrate concentration
to lower calcium retention and increase americium affinity. High levels of iron, manganese or magnesium
can also have an impact on exchange site availability and/or poisoning of the Eichrom extraction resins
used in this method.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. April 2014. "Rapid
Radiochemical Method for Americium-241 in Building Materials for Environmental Remediation
Following Radiological Incidents," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R14-007.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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6.3.7 Rapid Method for Sodium Hydroxide Fusion of Asphalt Matrices Prior to
Americium, Plutonium, Strontium, Radium, and Uranium Analyses
Analyte(s)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Radium-226
13982-63-3
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Sample Preparation
Sample Preparation Technique: Fusion
Determinative Technique: Alpha spectrometry/beta counting
Method Developed for: Americium-241, plutonium-238, plutonium-239, radium-226, strontium-90,
uranium-234, uranium-235 and uranium-238 in asphalt samples
Method Selected for: This method has been selected for preparation of americium-241, plutonium-238,
plutonium-239, radium-226, strontium-90, uranium-234, uranium-235 and uranium-238 in asphalt
matrices.
Description of Method: The method is based on heating a representative, finely milled 1- to 1.5-g
aliquant asphalt sample to remove organic components, followed by rapid fusion using sodium hydroxide
fusion at 600ฐC. Plutonium, uranium and americium are separated from the alkaline matrix using an
iron/titanium hydroxide precipitation (enhanced with calcium phosphate precipitation), followed by a
lanthanum fluoride matrix removal step. Strontium is separated from the alkaline matrix using a
phosphate precipitation, followed by a calcium fluoride precipitation to remove silicates. Radium is
separated from the alkaline matrix using a carbonate precipitation. The method is applicable to the sodium
hydroxide fusion of asphalt samples, prior to the chemical separation procedures described in the
following procedures:
Rapid Radiochemical Method for Total Radiostrontium (Strontium-90) in Building Materials for
Environmental Remediation Following Radiological Incidents (Section 6.3.1)
Rapid Radiochemical Method for Radium-226 in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.2)
Rapid Radiochemical Method for Isotopic Uranium in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.4)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-23 9/240 in Building Materials
for Environmental Remediation Following Radiological Incidents (Section 6.3.5)
Rapid Radiochemical Method for Americium-241 in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.6)
Special Considerations: Asphalt samples with larger particle size may require a longer fusion time.
Information regarding the elemental composition of the sample may be helpful to determine any native
concentrations of uranium, radium, thorium, strontium or barium, all of which may have an effect on the
chemical separations used following the fusion of the sample. In those samples where native constituents
are present that could interfere with the determination of the chemical yield (e.g., strontium for strontium-
90 analysis) or with the creation of a sample test source (e.g., barium for radium-226 analysis by alpha
spectrometry), it may be necessary to determine the concentration of these constituents in advance of
chemical separation (using a separate aliquant of fused material) and to make appropriate adjustments to
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the yield calculations or amount of carrier added. Aluminum nitrate reagent typically contains trace levels
of uranium contamination. To achieve the lowest possible blanks for isotopic uranium measurements, the
aluminum nitrate reagent can be passed through ~7 mL TRU resin or UTEVA resin. Compounds
contained in decontamination agents are not expected to cause interferences during sample preparation;
see the sections corresponding to the analytical methods listed in the description of this method for
potential interferences caused by constituents of decontamination agents.
Source: U.S. EPA, National Analytical and Radiation Environmental Laboratory. May 2017. "Rapid
Method for Sodium Hydroxide Fusion of Asphalt Matrices Prior to Americium, Plutonium, Strontium,
Radium, and Uranium Analyses," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R16-001.
https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
6.3.8 Rapid Method for Sodium Hydroxide Fusion of Asphalt Roofing Material Matrices
Prior to Americium, Plutonium, Strontium, Radium, and Uranium Analyses
Analyte(s)
CAS RN
Americium-241
14596-10-2
Plutonium-238
13981-16-3
Plutonium-239
15117-48-3
Radium-226
13982-63-3
Strontium-90
10098-97-2
Uranium-234
13966-29-5
Uranium-235
15117-96-1
Uranium-238
7440-61-1
Analysis Purpose: Sample Preparation
Sample Preparation Technique: Fusion
Determinative Technique: Alpha spectrometry/beta counting
Method Developed for: Americium-241, plutonium-238, plutonium-239, radium-226, strontium-90,
uranium-234, uranium-235 and uranium-238 in asphalt roofing material samples
Method Selected for: This method has been selected for preparation of americium-241, plutonium-
238, plutonium-239, radium-226, strontium-90, uranium-234, uranium-235 and uranium-238 in asphalt
shingles.
Description of Method: Asphalt roofing material samples should be cut into very small pieces prior to
taking a representative aliquant for furnace heating and fusion. The method is based on ashing a 25-g
subsample of asphalt roofing material sample in a furnace to remove organic components, followed by
taking a representative aliquant from the ashed sample. A 1- to 1.5-g aliquant is fused using sodium
hydroxide fusion at 600ฐC. Plutonium, uranium and americium are separated from the alkaline matrix
using an iron/titanium hydroxide precipitation (enhanced with calcium phosphate precipitation) followed
by a lanthanum fluoride matrix removal step. Strontium is separated from the alkaline matrix using a
phosphate precipitation, followed by a calcium fluoride precipitation to remove silicates. Radium is
separated from the alkaline matrix using a carbonate precipitation. The method is applicable to the sodium
hydroxide fusion of asphalt shingle samples, prior to the chemical separation procedures described in the
following procedures:
Rapid Radiochemical Method for Total Radiostrontium (Strontium-90) in Building Materials for
Environmental Remediation Following Radiological Incidents (Section 6.3.1)
Rapid Radiochemical Method for Radium-226 in Building Materials for Environmental Remediation
Following Radiological Incidents (Section 6.3.2)
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Rapid Radiochemical Method for Isotopic Uranium in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.4)
Rapid Radiochemical Method for Plutonium-238 and Plutonium-23 9/240 in Building Materials for
Environmental Remediation Following Radiological Incidents (Section 6.3.5)
Rapid Radiochemical Method for Americium-241 in Building Materials for Environmental
Remediation Following Radiological Incidents (Section 6.3.6)
Special Considerations: The term "asphalt roofing materials" is used in this procedure to mean asphalt
organic shingles or asphalt fiberglass shingles typically used for residential or commercial roofs. This
roofing material procedure was validated with asphalt fiberglass shingles. Roofing material samples
should be cut into very small pieces prior to taking a representative aliquant for furnace heating and
fusion.
Bitumen components, which may have affinity for the radionuclides, are destroyed in this method.
Radionuclides deposited on the surface of the asphalt roofing material are effectively digested, including
refractory radionuclide particles. A small amount of mineralized granules may remain after the fusion.
Information regarding the elemental composition of the sample may be helpful. For example, asphalt
roofing materials may have native concentrations of uranium, radium, thorium, stable strontium or stable
barium, all of which may have an effect on the chemical separations used following the fusion of the
sample. In those samples where constituents are present that could interfere with the determination of the
chemical yield (e.g., strontium for strontium-90 analysis) or with the creation of a sample test source (e.g.,
barium for radium-226 analysis by alpha spectrometry), it may be necessary to determine the
concentration of these constituents in advance of chemical separation (using a separate aliquant of fused
material) and make appropriate adjustments to the yield calculations or amount of carrier added.
Compounds contained in decontamination agents are not expected to cause interferences during sample
preparation; see the sections corresponding to the analytical methods listed in the description of this
method for potential interferences caused by constituents of decontamination agents.
Source: U.S. EPA, National Air and Radiation Environmental Laboratory. August 2016. "Rapid Method
for Sodium Hydroxide Fusion of Asphalt Roofing Material Matrices Prior to Americium, Plutonium,
Strontium, Radium, and Uranium Analyses," Revision 0. Montgomery, AL: U.S. EPA. EPA 402-R16-
003. https://www.epa.gov/radiation/rapid-radiochemical-methods-selected-radionuclides
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Section 7.0: Selected Pathogen Methods
Following a microbial contamination incident, it is assumed that the identification, confirmation and
strain-level characterization of the pathogen have been completed before the U.S. Environmental
Protection Agency's (EPA) remediation actions begin. The first phase of EPA's actions includes site
characterization, to determine the extent and magnitude of contamination and to 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.
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
contamination incident. Emphasis is given to the following environmental sample types: aerosols,
particulates (wipes or swabs), soils, 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 incident. As depicted in
Figure 7-1, for Pathogens, site characterization refers to the assessment phase, decontamination refers to
the cleanup phase and post decontamination refers to the clearance phase.
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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
Selected Analytical Methods fa EnvitonmentaI
Remediation and Recovety
Assessment^) (^"cieanup^ (^Clearance^
For Pathogens, site characterization refers to the assessment phase and post decontamination refers
to the clearance phase. Methods included in Section 7 and Appendix C may be used during the
cleanup phase, if needed.
Sample
Aerosol, Particulate, Drinking Water, Post Decontamination Waste Water
Solid
BSL-2 or BSL-3 Bioaaent
Drinking Water/
Post Decontamination
Waste Water
Aerosol, Particulate, Solid
Concentration
(As necessary)
Sample Processing
Characterization
Post Decontamination
Culture Followed by
Rapid Confirmation
(Real-time PCR, ELISA, other
immunoassay)
OR
RV-PCR (if available)
Rapid Analytical Method
(Real-time PCR, ELISA, other
immunoa ssay)
Neutralization of decontamination agents may be required for post decontamination phase samples.
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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. Therefore, in most cases, the analytical methods selected
for the site characterization phase may not have to determine the viability of the pathogen. The methods
chosen 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 incident and response, culture methods could be
appropriate for use during site characterization. In certain cases, the determination of the extent of
pathogen contamination 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 to minimize false negative results. 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. Vox Bacillus anthracis (Letant etal.
201110, U.S. EPA 201111, U.S. EPA 201212) and Yersiniapestis (U.S. EPA 201613) the Rapid Viability-
PCR (RV-PCR) method may be used because it provides rapid and high throughput results in addition to
viability determination.
A list of methods that have been selected by the 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 contamination incident. 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 4.0.
10 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.
11 U.S. EPA. 2011. "Development and Verification of Rapid Viability Polymerase Chain Reaction (RV-PCR)
Protocols for Bacillus anthracis - For Application to Air Filters, Water and Surface Samples." EPA/600/R-10/156.
12 U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA. EPA/600/R-12/577.
13 U.S. EPA. 2016. "Protocol for Detection of Yersinia pestis in Environmental Samples During the Remediation
Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA. EPA/600/R-16/109.
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Pathogens that require biosafety level (BSL)-4 containment and practices, such as hemorrhagic fever
viruses and Variola major (smallpox) will be handled only by reference laboratories with BSL-4
capability and are not included in 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.
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 and/or confirmation 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.
Analytical technique. An analytical procedure used to determine the identity, quantity and/or
viability of a pathogen.
Method type. Two method types (sample preparation and analytical) are used to complete sample
analysis. In some cases, a single method contains information for both sample preparation and the
analytical procedure. In most instances, however, two separate methods may need to be used.
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, Sponge-Sticks, vacuum socks, 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.
Soil. The recommended method/procedure for the pathogen of interest in soils.
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 included, 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, deoxyribonucleic acid/ribonucleic acid [DNA/RNA], antigen/protein)
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Section 7.0 - Selected Pathogen Methods
from the samples, but also prevent accurate quantitation and high throughput. Samples should not be
stored indefinitely, and should be processed and analyzed as soon as possible upon receipt. Note. For post
decontamination samples it may be necessary to neutralize the decontamination agent.
The methods listed 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, emphasis is given to the environmental sample types that are predominately used to fulfill
EPA's responsibilities following a contamination incident (e.g., aerosols, particulates [wipes or swabs],
soils, drinking water, post decontamination waste water). Other sample types may have to be analyzed,
and for those sample types, specific requests should be sent to the Pathogen Methods Lead and Alternate
Lead (see Section 4.0).
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 the Centers for Disease Control and Prevention's (CDC's) Emergency
Preparedness and Response website (https://emergencv.cdc.gov/bioterrorism/index.asp') and the U.S.
Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN) 2012,
"Bad Bug Book" (https://www.fda.gov/food/foodborneillnesscontaminants/causesofillnessbadbugbook/).
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 (aerosol, particulate [wipes or swabs], soil, drinking water or post decontamination waste
water).
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 pathogen methods have been assigned tiers
(below) to indicate a level of method usability for the specific analyte and sample type. The assigned tiers
pertain only to technical aspects of method usability, and do not pertain to aspects such as cost, equipment
availability and sample throughput.
Tier I: The method was developed for the pathogen and sample type. The method has been evaluated
by multiple laboratories, a detailed protocol has been developed, and suitable QC measures
and checks are provided. (Examples: EPA Method 1623.1 [Cryptosporidium in drinking
water]; Standard Methods 9260 E [Shigella culture method].)
Tier II: The pathogen is the target of the method, and the method has been evaluated by one or more
laboratories. The available data and/or information indicate that additional testing and/or
modifications will likely be needed. (Example: Cunningham etal. 2010 [Shigella molecular
method].)
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Tier III: The pathogen is not the target of the method but the method is for the specific sample type
and the pathogen is similar to the target of the method (i.e., vegetative bacteria, spore-
forming bacteria, virus or protozoan). Data and expert opinion suggest, however, that the
method(s) may be applicable with modifications. (Example: EPA Yersiniapestis protocol for
Salmonella enterica serovar Typhi (S. Typhi) in 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
Publisher
Reference
Antimicrobial Agents and
Chemotherapy*
American Society for
Microbiology (ASM)
httD://aac.asm.ora/
Applied and Environmental
Microbiology*
ASM
httD://aem.asm.ora/
Applied Biosafety*
SAGE Journals
http://apb. saaepub.com/
Archives of Virology*
Springer
http://link.sprinaer.com/iournal/705
Bacteriological Analytical Manual (BAM)
FDA CFSAN
http://www.fda.aov/Food/FoodScienceRese
arch/LaboratorvMethods/ucm2006949.htm
BMC Microbiology
Springer
http://www.sprinaer. com/I ife+sciences/micr
obioloav/iournal/12866
Canadian Journal of Microbiology*
NRC Research Press
http://www.nrcresearchpress.com/loi/cim
Clinical Chemistry*
American Association for
Clinical Chemistry
http://www.clinchem.ora/content/bv/vear
Clinical Infectious Diseases*
Oxford
https://cid.oxfordiournals.ora/
Current Protocols in Microbiology*
Wiley
http://onlinelibrarv.wilev.eom/book/10.1002/
9780471729259
Emerging Infectious Diseases
CDC
http://wwwnc.cdc.aov/eid/
Environmental Regulations and
Technology: Control of Pathogens and
Vector Attraction in Sewage and Sludge
EPA, National Risk
Management Research
Laboratory (NRMRL)
https://www.epa.aov/biosolids/control-
pathoaens-and-vector-attraction-sewaae-
sludae
Environmental Science and
Technology*
American Chemical
Society (ACS)
http://pubs.acs.ora/iournal/esthaa
EPA Analytical Protocols
EPA, National Homeland
Security Research
Center (NHSRC)
https://www.epa.aov/homeland-securitv-
research
EPA Microbiology Home Page
EPA
https://www.epa.aov/cwa-methods
FEMS Immunology & Medical
Microbiology*
Oxford
http://femsim.oxfordiournals.ora/
International Organization for
Standardization (ISO) Methods*
ISO
http://www.iso.ora/iso/home.html
Journal of Clinical Microbiology*
ASM
http://icm.asm.ora/
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Section 7.0 - Selected Pathogen Methods
Name
Publisher
Reference
Journal of Environmental Monitoring*
Royal Society of
Chemistry
httD://Dubs.rsc.ora/en/iournals/iournalissues
/em?issueid=em014011&issnDrint=1464-
0325#!issueid=em014011&tvDe=archive&is
snDrint=1464-0325
Journal of Food Protection*
International Association
for Food Protection
httDs://www. food protection. ora/Dublications/
iournal-of-food-Drotection/
Journal of Medical Virology*
Wiley
httD://onlinelibrarv.wilev.com/iournal/10.100
2/(ISSN)1096-9071
Journal of Microbiological Methods*
Elsevier
httD://www. sciencedirect.com/science/iourn
al/01677012
Journal of Parasitology*
American Society of
Parasitologists
httD://www.iournalofDarasitoloav.ora/
Journal of Parasitology Research*
Hindawi Publishing
Corporation
https://www. hindawi.com/iournals/ipr/conte
nts/
Journal of Virological Methods*
Elsevier
httD://www. sciencedirect.com/science/iourn
al/01660934
Legionella: Methods and Protocols,
Methods in Molecular Biology
Springer
httD://link.sDrinaer.com/book/10.1007%2F9
78-1-62703-161-5
Methods in Molecular Biology
Springer
httD://link.sDrinaer.com/search?facet-
series=7651&facet-content-tvDe=Book
Molecular and Cellular Probes*
Elsevier
http://www. sciencedirect.com/science/iourn
al/08908508
National Institute for Occupational
Safety and Health (NIOSH) Methods
CDC NIOSH
httD://www.cdc.aov/niosh/nmam/
Neglected Tropical Diseases
PLOS
httD://iournals.Dlos.ora/Dlosntds/
Occupational Safety and Health
Administration (OSHA) Methods
OSHA
http://www.osha.qov
Parasitology Research*
Springer
httD://www.sDrinaer.com/biomed/medical+
microbioloav/iournal/436
Sentinel Level Clinical Microbiology
Laboratory Guidelines for Suspected
Agents of Bioterrorism and Emerging
Infectious Diseases
ASM
httD://www.asm.ora/index.DhD/auidelines/se
ntinel-auidelines
Standard Methods for the Examination
of Water and Wastewater, 22nd Edition,
2012*
American Public Health
Association (APHA)
http://www. standard methods, ora
Transactions of the Royal Society of
Tropical Medicine and Hygiene*
Oxford
httD://trstmh.oxfordiournals.ora/
U.S. Department of Agriculture (USDA)
Food Safety and Inspection Service
(FSIS) Microbiology Laboratory
Guidebook
USDA FSIS
httD://www.fsis.usda.aov/wDs/Dortal/fsis/toDi
cs/science/laboratories-and-
Drocedures/auidebooks-and-
methods/microbioloav-laboratorv-
auidebook/microbioloav-laboratorv-
auidebook
U.S. Department of Health and Human
Services Procedures for the Recovery
of Legionella from the Environment
CDC
httDs://www.cdc.aov/leaionella/health-
depts/inv-tools-cluster/lab-inv-
tools/procedures-manual.pdf
Subscription and/or purchase required. Note. ASM does not require a subscription or purchase 6 months after the
publication date.
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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;
(6) conduct training and proficiency testing; and (7) maintain personnel training and proficiency testing
records.14
The level or amount of QC needed depends on the intended purpose of the data generated. 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 2004 Quality Assurance/Quality Control Guidance for
Laboratories Performing PCR Analyses on Environmental Samples (Cincinnati,OH: U.S. EPA. EPA 815-
B-04-001) at: https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf.
Ongoing analysis of control samples to ensure the continued reliability of the analytical results should
also be performed. At a minimum, the following QC analyses should be conducted on an ongoing basis:
Media and reagent sterility checks
Positive and negative controls
Method blanks
Reference matrix spikes to evaluate initial and ongoing method/analyst performance, if available
Matrix spikes (where possible) to evaluate method performance in the sample type of interest
Matrix spike duplicates (MSD) and/or sample replicates to assess method precision
Sample processing controls to evaluate processing procedures (e.g., extraction, concentration) in the
sample type of interest
Instrument calibration checks and temperature controls
QC procedures and proper calibration and maintenance of ancillary laboratory equipment (e.g.,
thermometers, autoclaves, pipettors) should be performed as frequently as necessary to ensure the
reliability of analytical results.
Please note: The appropriate points of contact identified in Section 4.0 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. 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.
14 Information regarding EPA's DQO process, considerations, and planning is available at:
http://www.epa.gov/OUALITY/daos.html
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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 and waste handling 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. Sample disposal should follow federal and local regulations.
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/biosafetv/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
Select Agent Rules and Regulations found at the National Select Agent Registry. Available at:
http://www.selectagents. gov/ and http://www. selectagents.gov/regulations .html
The following sources provide information regarding waste management:
U.S. EPA - Hazardous Waste Management (40 CFR part 260) and U.S. EPA Administered Permit
Programs (40 CFR part 270). Available at: http://www.ecfr.gov/
U.S. 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/sites/production/files/2015-Q6/documents/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?p table=STANDARDS&p id=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?p table=STANDARDS&p id=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://www.ecfr.gov/cgi-bin/text-idx?gp=&SID=994b04d45ee6d584ce676138929280b3&mc=true&t
pl=/ecfrbrowse/Title49/49tab 02.tpl
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7.1.4 Laboratory Response Network (LRN)
The LRN is a national network of local, state, federal, military, food, agricultural, veterinary and
environmental laboratories that was created in accordance with Presidential Decision Directive 39, which
established terrorism preparedness responsibilities for federal agencies. The CDC provides technical and
scientific support to member laboratories as well as secure access to standardized procedures (e.g.,
immunoassay, PCR) and reagents for rapid (4-6 hours) presumptive detection of select 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. However, EPA has published methods for the
analysis of Bacillus anthracis and Yersinia pestis in environmental matrices that are included 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. Except for Coxiella burnetii,
culture methods are available for all of these pathogens as American Society for Microbiology's (ASM)
Sentinel Laboratory Guidelines. Available at: https://www.asm.org/index.php/guidelines/sentinel-
guidelines
The pathogens 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 CFRpart 121,
available at http://www.ecfr.gov/cgi-bin/ECFR?page=browse) and safety and BSL requirements (see
CDC's BMBL, 5th Edition, available at: http://www.cdc.gov/biosafetv/publications/bmbl5/)
Select Agents Listed in Appendix C
Pathogen(s) [Disease]
Agent Category
Bacillus anthracis [Anthrax]
Bacteria
Brucella spp. [Brucellosis]
Bacteria
Burkholderia mallei [Glanders]
Bacteria
Burkholderia pseudomallei [Melioidosis]
Bacteria
Coxiella burnetii [Q-fever]
Bacteria
Francisella tularensis [Tularemia]
Bacteria
Yersinia pestis [Plague]
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
https://emergencv.cdc.gov/lrn/.
Centers for Disease Control and Prevention (CDC)
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
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E-mail: lrn@cdc.gov
Website: https://emergencv.cdc.gov/lrn/contact.asp
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@aph1 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/PSAB/Novel Influenza Julv2013.pdf
Brucella: http://www.asm.org/images/PSAB/LRN/Brucella316 .pdf
Burkholderia mallei and B. pseudomallei:
http://www.asm.org/images/PSAB/LRN/Burkholderia316.pdf
Coxiella burnetii: http://www.asm.org/images/PSAB/LRN/Coxiella316.pdf
Fransicella tularensis:
http://www.asm.org/images/PSAB/LRN/Tularemia316.pdf
Yersiniapestis: http://www.asm.org/images/PSAB/LRN/Ypestis316.pdf
Sources:
ASM. 2010. Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of
Bioterrorism and Emerging Infectious Diseases.
https://www.asm.org/index.php/guidelines/sentinel-guidelines
CDC. 2009. "Biosafety in Microbiological and Biomedical Laboratories" (BMBL), 5th Edition.
http://www.cdc.gov/biosafetv/publications/bmbl5/
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7.2 Method Summaries for Bacteria
Summaries for the analytical methods listed in Appendix C are provided in Sections 7.2.1 through 7.2.17.
Each summary contains a brief description of the analytical methods selected for each bacterial pathogen,
and links to, or sources for, obtaining full versions of the methods. Summaries are provided for
informational use. Tiers that have been assigned to each method/analyte pair (see Section 7.1.1) can be
found in Appendix C. The full version of the method should be consulted prior to sample analysis. For
information regarding sample collection considerations for samples to be analyzed by these methods, see
the companion Sample Collection Information Document for Pathogens - Companion to SAM 2017.15
7.2.1 Bacillus anthracis [Anthrax] - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.1.1
Post Decontamination
Rapid Viability-PCR (RV-PCR)
7.2.1.2
Culture and Real-Time PCR
7.2.1.3
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols (filters), particulates (wipes
or swabs), soils, drinking water and post decontamination waste water samples.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to EPA's "Protocol
for Detection of Bacillus anthracis in Environmental Samples During the Remediation
Phase of an Anthrax Event" (U.S. EPA 2012), referred to as the "EPA BA Protocol."
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA BA Protocol, Section 9.2 (U.S. EPA 2012).
Analytical Technique: Use real-time PCR (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 [U.S. EPA 2012]), and analyzed using the referenced target-specific PCR
primers, 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:
15 EPA's Sample Collection Information Document for Pathogens - Companion to SAM 2017 can be found at:
https://www.epa.gov/homeland-securitv-research/sam-compamon-documents-and-sample-collection-procedures
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https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-pcr.pdf. or consult the
points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/.
Sources:
Silvestri, E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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: This method is listed for analysis of aerosols (filters), particulates (wipes
or swabs), soils, drinking water and post decontamination waste water samples.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA BA
Protocol (U.S. EPA 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA BA Protocol, Section 10.4 (U.S. EPA 2012).
Analytical Technique: Use RV-PCR (U.S. EPA 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 (e.g., aerosols, particulates, soils, water) are processed using
multiple extraction and wash steps. After brain heart infusion broth is added to the spores, an
aliquot (Time 0 [T0]) is removed and stored at 4ฐC. The remaining broth is then incubated for 9 to
15 hours at 37ฐC. After the incubation, an aliquot is removed (Time Final [Tf]). Both T0 and Tf
aliquots then go through DNA extraction and purification followed by real-time PCR analysis.
The cycle threshold (CT) values for the T0 and Tf aliquots are then compared. The difference in CT
values between the T0 and T9 is used to detect viable Bacillus anthracis spores. A change
(decrease) in the PCR CT > 6 represents increased DNA amplification in the Tf aliquot relative to
the T0 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.
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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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Silvestri, E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577.
https://www.epa.gov/homeland-securitv-research/protocol-detection-bacillus-anthracis-
environmental-samples
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: This method is listed for analysis of aerosols (filters), particulates (wipes
or swabs), soils, drinking water and post decontamination waste water samples.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA BA
Protocol (U.S. EPA 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA BA Protocol, Section 11.6 (U.S. EPA 2012).
Analytical Technique: Use culture and real-time PCR (U.S. EPA 2012).
Description of Method: 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 35ฐC to 37ฐC for 18-24 hours. Isolated typical colonies are
resuspended in sterile distilled water. The bacterial suspensions are then heated at 95ฐC to 98ฐC
to release the DNA from the cells (EPA BA Protocol, Section 11 [U.S. EPA 2012]). 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
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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:
https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-pcr.pdf. or consult the
points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Silvestri, E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
7.2.2 Brucella spp. [Brucellosis] - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.2.1
Post Decontamination
Real-Time PCR/lmmunoassay
7.1.4 2
Culture and Real-Time PCR
7.2.2.2
See Appendix C for corresponding method usability tiers.
2 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories (see Section 7.1.4).
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to EPA's "Protocol
for Detection of Yersinia pestis in Environmental Samples During the Remediation Phase
of a Plague Incident" (U.S. EPA 2016), referred to as the EPA YP Protocol.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Hinic et al. 2008 or the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Hinic et al. 2008).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Hinic et al.
2008 or EPA YP Protocol, Section 9.6 [U.S. EPA. 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Hinic et al. 2008). 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:
https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf. or consult the
points of contact identified in Section 4.0.
Special Considerations: Brucella spp. are select agents requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also
be followed see (BMBL, 5th Edition [CDC 2009]).
http://www.cdc.gov/biosafetv/publications/bmbl5/
Sources:
Silvestri, E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersinia pestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=329170
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/S01677012080Q2522
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: This method is listed for analysis of aerosols, particulates (wipes or
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swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Hinic et al. 2008 or the EPA YP Protocol, Section 10.5 (U.S. EPA. 2016).
Analytical Technique: Use culture (ASM 2004) and real-time PCR (Hinic et al. 2008).
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 35ฐC (5-10% carbon dioxide) for up to 7 days. Confirmation is performed using
real-time PCR. Target nucleic acid should be extracted, purified (Hinic et al. 2008 or EPA YP
Protocol, Section 10.5 [U.S. EPA. 2016]), and analyzed using the referenced target-specific PCR
primers, probes and assay parameters (Hinic et al. 2008). 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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Brucella spp. are select agents requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should be
followed (BMBL, 5th Edition [CDC 2009]). http://www.cdc.gov/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Silvestri, E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). EPA/600/R-16/109. Cincinnati,
OH: U.S. EPA. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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/S01677012080Q2522
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ASM. 2004. "Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents
of Bioterrorism and Emerging Infectious Diseases: Brucella species."
https://www.asm.org/images/PSAB/LRN/Brucella316.pdf
7.2.3 Burkholderia mallei [Glanders] - BSL-3 and Burkholderia pseudomallei
[Melioidosis] - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7 2 3 1
Post Decontamination
Real-Time PCR/lmmunoassay
7.1.42
Culture and Real-Time PCR
7.2.3.2
See Appendix C for corresponding method usability tiers.
2 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories (see Section 7.1.4).
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Limmathurotsakul et al. 2013.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the analytical technique references cited below or the EPA YP Protocol,
Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Tomaso et al. 2006 and Novak et al. 2006).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (analytical
technique references cited above or the EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and
analyzed using the referenced target-specific PCR primers, probes and assay parameters (Tomaso
et al. 2006 and Novak et al. 2006). 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:
https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf. or consult the
points of contact identified in Section 4.0.
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Special Considerations: Burkholderia mallei and Burkholderiapseudomallei 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/biosafetv/publications/bmbl5/
Sources:
Limmathurotsakul, D., Dance, D. A.B., Wuthiekanun, V., Kaestli, M., Mayo, M., Warner, J.,
Wagner, D.M., Tuanyok, A., Wertheim, H., Cheng, T.Y., Mukhopadhyay, C., Puthucheary, S.,
Day, N.P.J., Steinmetz, I., Currie, B.J. and Peacock, S. 2013. "Systematic Review and Consensus
Guidelines for Environmental Sampling of Burkholderia pseudomallei." PLoS Neglected
Tropical Diseases. 7(3): e2105.
http://iournals.plos.org/plosntds/article?id=10.1371/journal.pntd. 0002105
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
Tomaso, H., Scholz, H.C., A1 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
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: //i cm. asm. org/content/44/1/85. 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Limmathurotsakul et al. 2013.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the real-time PCR analytical techniques cited below or the EPA YP Protocol,
Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (ASM 2008) and real-time PCR (Tomaso et al. 2006 and
Novak et al. 2006).
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
35ฐC-37ฐC for 48 hours. Confirmation is performed using real-time PCR. Target nucleic acid
should be extracted, purified (real-time PCR analytical techniques cited above or the EPA YP
Protocol, Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced target-specific PCR
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primers, probes and assay parameters (Tomaso etal. 2006 and Novak etal. 2006). 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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Limmathurotsakul, D., Dance, D. A. B., Wuthiekanun, V., Kaestli, M., Mayo, M., Warner, J.,
Wagner, D. M., Tuanyok, A., Wertheim, H., Cheng, T.Y., Mukhopadhyay, C., Puthucheary, S.,
Day, N. P. J., Steinmetz, I., Currie, B.J. and Peacock, S. 2013. "Systematic Review and
Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei." PLoS
Neglected Tropical Diseases. 7(3): e2105.
http://iournals.plos.org/plosntds/article?id=10.1371/journal.pntd. 0002105
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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., A1 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
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: //i cm. asm. org/content/44/1/85. full .pdf+html
7.2.4 Campylobacter jejuni [Campylobacteriosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.4.1
Post Decontamination
Culture and Real-Time PCR
7.2.4.2
See Appendix C for corresponding method usability tiers.
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Cunningham et al. 2010 or the EPA YP Protocol, Section 9.6 (U.S. EPA
2016).
Analytical Technique: Use real-time PCR (Cunningham et al. 2010).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Cunningham
et al. 2010 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Cunningham et al. 2010). The use of
real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 ://j cm .asm .org/content/48/8/2929 .full .pdf+html
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Cunningham et al. 2010 or the EPA YP Protocol, Section 10.5 (U.S. EPA
2016).
Analytical Technique: Use culture (ISO 2005) and real-time PCR (Cunningham et al. 2010).
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 (Cunningham et al. 2010 or EPA YP Protocol, Section 10.5 [U.S.
EPA 2016]), and analyzed using the referenced target-specific PCR primers, probes and assay
parameters (Cunningham et al. 2010). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 ://i cm .asm .org/content/48/8/2929 .full .pdf+html
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ISO. 2005. ISO 17995: Water quality - Detection and Enumeration of Thermotolerant
Campylobacter Species.
http://www.iso.org/iso/iso catalogue/catalogue tc/catalogue detail.htm?csnumber=42082
7.2.5 Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci) -
BSL-2; BSL-3 for Aerosols and Large Volumes
Remediation Phase
Analytical Technique 1
Section
Site Characterization
PCR
7.2.5.1
Post Decontamination
Tissue Culture and PCR
7.2.5.2
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Madico et al. 2000 orthe EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use PCR (Madico et al. 2000).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Madico et al.
2000 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Madico et al. 2000). The use of 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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
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U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntryId=329170
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Madico et al. 2000 orthe EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use tissue culture and PCR (Madico et al. 2000).
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 (Madico et
al. 2000 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Madico et al. 2000). The use of PCR
analyses directly on isolates allows for rapid confirmation of Chlamydophilapsittaci.
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2016. "Protocol for Detection of Yersinia pestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
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EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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
7.2.6 Coxiella burnetii [Q-fever] - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.6.1
Post Decontamination
Real-Time PCR/lmmunoassay
7.1.4 2
Tissue Culture and Real-Time PCR
7.2.6.2
See Appendix C for corresponding method usability tiers.
2 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories (see Section 7.1.4).
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Silvestri et al. 2016.
Water samples should be prepared according to the EPA and CDC Ultrafiltration (UF)
Report (U.S. EPA and CDC 2011), referred to as the EPA and CDC UF Report.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Panning et al. 2008 orthe EPA BA Protocol, Section 9.2 (U.S. EPA 2012)
Analytical Technique: Use real-time PCR (Panning et al. 2008).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Panning et
al. 2008 or EPA BA Protocol, Section 9.2 [U.S. EPA 2012]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Panning et al. 2008). The use of real-
time PCR analyses directly on samples (e.g., no culture component) allows for rapid detection of
Coxiella 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
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Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on
Environmental Samples (EPA 815-B-04-001) document at:
http://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf. or consult the
points of contact identified in Section 4.0.
Special Considerations: Coxiella burnetii is a select agent requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should be
followed (BMBL, 5th Edition [CDC 2009]). http://www.cdc.gov/biosafetv/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/S016770121000Q692
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. fiill.pdf+html
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Panning, M., Kilwinski, J., Greiner-Fischer, S., Peters, M., Kramme, S., Frangoulidis, D., Meyer,
H., Henning, K. and Drosten, C. 2008. "High Throughput Detection of Coxiella burnetii by Real-
Time PCR With Internal Control System and Automated DNA Preparation." BMC Microbiology.
8:77. http://www.biomedcentral.eom/1471-2180/8/77
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
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Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, or Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Silvestri et al. 2016.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Panning et al. 2008 or the EPA BA Protocol, Section 11.6 (U.S. EPA 2012).
Analytical Technique: Use tissue culture (Raoult et al. 1991) and real-time PCR (Panning et al.
2008).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto human erythroleukemia cells and
incubated for 6 days at 37ฐC. Target nucleic acid should be extracted, purified (Panning et al.
2008 or EPA BA Protocol, Section 11.6 [U.S. EPA 2012]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Panning et al. 2008). The use of real-
time PCR analyses directly on isolates allows for rapid confirmation of Coxiella 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Special Considerations: Coxiella burnetii is a select agent requiring regulatory compliance (42
CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also
be followed (see BMBL, 5th Edition [CDC 2009]).
http://www.cdc.gov/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
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/S016770121000Q692
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. fiill.pdf+html
U.S. EPA. 2012 "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
SAM 2017
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Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Panning, M., Kilwinski, J., Greiner-Fischer, S., Peters, M., Kramme, S., Frangoulidis, D., Meyer,
H., Henning, K. and Drosten, C. 2008. "High Throughput Detection of Coxiella burnetii by Real-
Time PCR With Internal Control System and Automated DNA Preparation." BMC Microbiology.
8:77. http://www.biomedcentral.eom/1471-2180/8/77
Raoult, D. Torres, H. and Drancourt, M. 1991. "Shell-Vial Assay: Evaluation of a New
Technique for Determining Antibiotic Susceptibility, Tested in 13 Isolates of Coxiella burnetii."
Antimicrobial Agents and Chemotherapy. 35(10): 2070-2077.
http: //aac. asm .org/content/3 5/10/2070. long
7.2.7 Escherichia coli 0157:H7 - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.7.1
Post Decontamination
Culture and Real-Time PCR
7.2.7.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
Drinking water samples should be prepared according to the EPA Escherichia coli
0157:H7 Protocol (referred to as the EPA EC Protocol [U.S. EPA 2010]) or the EPA YP
Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Sen et al. 2011 or the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Sen et al. 2011).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Sen et al.
2011 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Sen et al. 2011). The use of real-time
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PCR analyses directly on samples (e.g., no culture component) allows for rapid detection of E.
coli 0157: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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. September 2010. "Standard Analytical Protocol for Escherichia coli 0157:H7 in
Water" (EPA EC Protocol). Cincinnati, OH: U.S. EPA. EPA/600/R-10/056.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=498725
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
Sen, K., Sinclair, J.L., Boczek, L. and Rice, E.W. 2011. "Development of a Sensitive Detection
Method for Stressed E. coli 0157: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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
Drinking water samples should be prepared according to the EPA EC Protocol (U.S. EPA
2010) or the EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Sen et al. 2011 or the EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (EPA EC Protocol [U.S. EPA 2010]) and real-time PCR
(Sen et al. 2011).
Description of Method: Following the appropriate sample preparation procedure (see Sample
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Preparation procedures above), samples are cultured using multiple media and immunomagnetic
separation (IMS) (EPA EC Protocol [U.S. EPA 2010]). 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 (Sen el al. 2011 or EPA
YP Protocol, Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced target-specific
PCR primers, probes and assay parameters (Sen et al. 2011). The use of real-time PCR analyses
allows for rapid confirmation of E. coli 0157: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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. September 2010. "Standard Analytical Protocol for Escherichia coli 0157:H7 in
Water" (EPA EC Protocol). Cincinnati, OH: U.S. EPA. EPA/600/R-10/056.
http://oaspub.epa.gov/eims/eimscomm.getfile7p download id=498725
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=329170
Sen, K., Sinclair, J.L., Boczek, L. and Rice, E.W. 2011. "Development of a Sensitive Detection
Method for Stressed E. coli 0157: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
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.8.1
Post Decontamination
Real-Time PCR/Immunoassay
7.1.4 2
Culture and Real-Time PCR
7.2.8.2
1 See Appendix C for corresponding method usability tiers.
2 Standardized procedures, reagents and agent-specific algorithms are available to LRN member
laboratories (see Section 7.1.4).
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
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needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Versage et al. 2003 orthe EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Versage et al. 2003).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Versage et
al. 2003 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Versage et al. 2003). 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/sites/production/files/2015-07/documents/epa-aaac-pcr.pdf. or consult the
points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 of Francisella tularensis in Complex
Specimens." Journal of Clinical Microbiology. 41(12): 5492-5499.
http: //i cm. asm. org/content/41/12/5492. full .pdf+html
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Versage et al. 2003 orthe EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (CDC et al. 2001) and real-time PCR (Versage et al. 2003).
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 (Versage et al.
2003 or EPA YP Protocol, Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Versage et al. 2003). The use of real-
time PCR analyses directly on isolates (e.g., no biochemical/serological component) allows for
rapid confirmation of Francisella 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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
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EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 of Francisella tularensis in Complex
Specimens." Journal of Clinical Microbiology. 41(12): 5492-5499.
http: //i cm. asm. org/content/41/12/5492. 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."
Washington, DC: ASM. http://www.asm.org/images/pdf/Clinical/Protocols/tularemia.pdf
Humrighouse, B.W., Adcock, N.J. and Rice, E.W., 2011. "Use of Acid Treatment and a Selective
Medium to Enhance the Recovery of Francisella tularensis from Water." Applied and
Environmental Microbiology. 77(18): 6729-6732.
http://aem.asm.Org/content/77/l 8/6729.full.pdf+html
7.2.9 Legionella pneumophila [Legionellosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.9.1
Post Decontamination
Culture and Real-Time PCR
7.2.9.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to U.S. DHHS 2005.
All other environmental sample types should be prepared according to Kozak el. al. 2013.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Benitez and Winchell 2013 or the EPA YP Protocol, Section 9.6 (U.S. EPA
2016).
Analytical Technique: Use real-time PCR (Benitez and Winchell 2013).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Benitez and
Winchell 2013 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the
referenced target-specific PCR primers, probes and assay parameters (Benitez and Winchell
2013). The use of real-time PCR analyses directly on samples (e.g., no culture component) allows
for rapid detection of Legionella pneumophila.
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
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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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. Department of Health and Human Services. 2005. "Procedures for the Recovery of
Legionella from the Environment." Atlanta, GA: CDC.
http://www.cdc.gov/legionella/health-depts/inv-tools-cluster/lab-inv-tools/procedures-
manual.html
Kozak, N.A., Lucas, C.E. and Winchell, J.M. 2013. "Identification of Legionella in the
Environment." Legionella: Methods and Protocols, Methods in Molecular Biology. 954: 3-25.
https: //www .ncbi .nlm .nih. gov/pubmed/23150387
Benitez, A.J. and Winchell, J.M. 2013. "Clinical Application of a Multiplex Real-Time PCR
Assay for Simultaneous Detection of Legionella Species, Legionella pneumophila,
and Legionella pneumophila Serogroup 1." Journal of Clinical Microbiology. 51(1): 348-351.
https: //www .ncbi .nlm .nih. gov/pubmed/23135949
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
7.2.9.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to U.S. DHHS 2005.
All other environmental sample types should be prepared according to Kozak el al. 2013.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Benitez and Winchell 2013 or the EPA YP Protocol, Section 10.5 (U.S. EPA
2016).
Analytical Technique: Use culture (Kozak et al. 2013) and real-time PCR (Benitez and
Winchell 2013).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are cultured using multiple media (buffered charcoal yeast
extract [BCYE] with polymyxin B, cycloheimide and vancomycin [BCYE PCV]; or BCYE with
glycine, polymyxin B, cycloheximide and vancomycin [BCYE GPCV]). Typical isolates are then
confirmed using serological tests. To expedite time to results, isolates should be confirmed using
real-time PCR analyses. Target nucleic acid should be extracted, purified (Benitez and Winchell
2013 or EPA YP Protocol, Section 10.5 [U.S. EPA 2016]) and analyzed using the referenced
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target-specific PCR primers, probes and assay parameters (Benitez and Winchell 2013). The use
of real-time PCR analyses allows for rapid confirmation of Legionella pneumophila.
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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. Department of Health and Human Services. 2005. "Procedures for the Recovery of
Legionella from the Environment." Atlanta, GA: CDC.
http://www.cdc.gov/legionella/health-depts/inv-tools-cluster/lab-inv-tools/procedures-
manual.html
Kozak, N.A., Lucas, C.E. and Winchell, J.M. 2013. "Identification of Legionella in the
Environment." Legionella: Methods and Protocols, Methods in Molecular Biology. 954: 3-25.
https: //www .ncbi .nlm .nih. gov/pubmed/23150387
Benitez, A.J. and Winchell, J.M. 2013. "Clinical Application of a Multiplex Real-Time PCR
Assay for Simultaneous Detection of Legionella Species, Legionella pneumophila, and
Legionella pneumophila Serogroup 1." Journal of Clinical Microbiology. 51(1): 348-351.
https: //www .ncbi .nlm .nih. gov/pubmed/23135949
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
7.2.10 Leptospira interrogans [Leptospirosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.10.1
Post Decontamination
Culture and Real-Time PCR
7.2.10.2
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
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Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Palaniappan etal. 2005 orthe EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Palaniappan et al. 2005).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Palaniappan
et al. 2005 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Palaniappan et al. 2005). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=329170
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 Leptospiras." Molecular and
Cellular Probes. 19(2): 111-117.
http://www.sciencedirect.eom/science/article/pii/S0890850804000970
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures in Palaniappan et al. 2005 orthe EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
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Analytical Technique: Use culture (Standard Method 9260 I [APHA etal. 2012]) and real-time
PCR (Palaniappan et al. 2005).
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 30ฐC. Confirmation is performed using real-time PCR. Target nucleic acid
should be extracted, purified (Palaniappan etal. 2005 or EPA YP Protocol, Section 10.5 [U.S.
EPA 2016]), and analyzed using the referenced target-specific PCR primers, probes and assay
parameters (Palaniappan et al. 2005). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 Leptospiras." Molecular and
Cellular Probes. 19(2): 111-117.
http://www.sciencedirect.com/science/article/pii/S089085080400097Q
APHA, AWWA and WEF. 2012. "Method 9260 I: Leptospira.'" Standard Methods for the
Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public Health
Association, http://www.standardmethods.org/
7.2.11 Listeria monocytogenes [Listeriosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR1
7.2.11.1
Post Decontamination
Culture and Real-Time PCR
7.2.11.2
See Appendix C for corresponding method usability tiers.
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.
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Method Selected for: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within USDA FSIS 2009 or the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (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 (USDA FSIS
2009 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (USDA FSIS 2009). The use of real-
time PCR analyses directly on samples (e.g., no culture component) allows for rapid detection of
Listeria 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
USDA, FSIS. 2009. "FSIS Procedure for the Use of a Listeria monocytogenes Polymerase Chain
Reaction (PCR) Screening Test." Chapter MLG 8A.04 in Microbiology Laboratory Guidebook.
Athens, GA: USDA. http://www.epa.gov/sites/production/files/2015-07/documents/usda-mlg-
8a.03.pdf
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
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Section 7.0 - Selected Pathogen Methods
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the USDA FSIS 2009 or the EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (Hitchins et al. 2003) and real-time PCR (USDA FSIS 2009).
Description of Method: Following appropriate sample preparation (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 (USDA FSIS 2009 or EPA YP Protocol, Section 10.5 [U.S. EPA
2016]), and analyzed using the referenced target-specific PCR primers, probes and assay
parameters (USDA FSIS 2009). The use of real-time PCR analyses directly on isolates (e.g., no
biochemical/serological component) allows for rapid confirmation of Listeria 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/SO 167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
USDA, FSIS. 2009. "FSIS Procedure for the Use of a Listeria monocytogenes Polymerase Chain
Reaction (PCR) Screening Test." Chapter MLG 8A.04 in Microbiology Laboratory Guidebook.
Athens, GA: USDA. http://www.epa.gov/sites/production/files/2015-07/documents/usda-mlg-
8a.03.pdf
Hitchins, A.D., Jinneman, K. and Chen, Y., FDA, CFSAN. 2003. "Chapter 10 - Detection and
Enumeration of Listeria monocytogenes in Foods." Bacteriological Analytical Manual Online.
http://www.fda.gov/Food/FoodScienceResearch/LaboratorvMethods/ucm071400.htm
SAM 2017
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Section 7.0 - Selected Pathogen Methods
7.2.12 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.12.1
Post Decontamination
Culture and Real-Time PCR
7.2.12.2
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to EPA Method 1682 (U.S. EPA 2006).
Drinking water samples should be prepared according to EPA Method 1200 (U.S. EPA
2012), Standard Method 9260 B (APHA etal. 2012) or the EPA YP Protocol (U.S. EPA
2016).
Post decontamination waste water samples should be prepared according to EPA Method
1200 (U.S. EPA 2012), EPA Method 1682 (U.S. EPA 2006), Standard Method 9260 B
(APHA etal. 2012) orthe EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to procedures within
the EPA YP Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jyoti et al. 2011 or the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Jyoti etal. 2011).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Jyoti et al.
2011 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Jyoti et al. 2011). The use of real-time
PCR analyses directly on samples (e.g., no culture component) allows for rapid detection 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2006. "Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis (MSRV) Medium." Washington, DC: U.S. EPA. EPA-821-R-
06-14. http://www.epa.gov/sites/production/files/2015-07/documents/epa-1682.pdf
SAM 2017
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Section 7.0 - Selected Pathogen Methods
U.S. EPA. 2012. "Method 1200: Analytical Protocol forNon-Typhoidal Salmonella in Drinking
Water and Surface Water." Washington, DC: U.S. EPA. EPA 817-R-12-004.
https://www.epa.gov/sites/production/files/2015-08/documents/epa817rl20Q4.pdf
APHA, AWWA and WEF. 2012. "Method 9260 B: Salmonella." Standard Methods for the
Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public Health
Association, http://www.standardmethods.org/
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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/l 0.102 l/es2018994
7.2.12.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to EPA Method 1682 (U.S. EPA 2006).
Drinking water samples should be prepared according to EPA Method 1200 (U.S. EPA
2012), Standard Method 9260 B (APHA etal. 2012) or the EPA YP Protocol (U.S. EPA
2016).
Post decontamination waste water samples should be prepared according to EPA Method
1200 (U.S. EPA 2012), EPA Method 1682 (U.S. EPA 2006), Standard Method 9260 B
(APHA etal. 2012) orthe EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jyoti etal. 2011 orthe EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (Method 1682 [U.S. EPA 2006] or EPA Method 1200 [U.S.
EPA 2012]) and real-time PCR (Jyoti et al. 2011).
Description of Method: Following appropriate sample preparation (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 (Jyoti et al. 2011 or EPA YP Protocol, Section 10.5
[U.S. EPA 2016]), and analyzed using the referenced target-specific PCR primers, probes and
assay parameters (Jyoti et al. 2011). The use of real-time PCR analyses directly on isolates (e.g.,
no biochemical/serological component) allows for rapid confirmation of non-typhoidal
Salmonella.
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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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2006. "Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis (MSRV) Medium." Washington, DC: U.S. EPA. EPA-821-R-
06-14. http://www.epa.gov/sites/production/files/2015-Q7/documents/epa-1682.pdf
U.S. EPA. 2012. "Method 1200: Analytical Protocol forNon-Typhoidal Salmonella in Drinking
Water and Surface Water." Washington, DC: U.S. EPA. EPA 817-R-12-004.
https://www.epa.gov/sites/production/files/2015-08/documents/epa817rl20Q4.pdf
APHA, AWWA and WEF. 2012. "Method 9260 B: Salmonella." Standard Methods for the
Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public Health
Association, http://www.standardmethods.org/
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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
7.2.13 Salmonella enterica serovar Typhi (S. Typhi) [Typhoid fever] - BSL-2; BSL-3 for
Aerosol Release
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.13.1
Post Decontamination
Culture and Real-Time PCR
7.2.13.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
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Section 7.0 - Selected Pathogen Methods
Drinking water samples should be prepared according to the Salmonella Typhi Protocol
(referred to as the EPA ST Protocol [U.S. EPA 2010]), Standard Method 9260 B (APHA
et al. 2012) or the EPA YP Protocol (U.S. EPA 2016).
Post decontamination waste water samples should be prepared according to Standard
Method 9260 B (APHA et al. 2012) or the EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the CDC Laboratory Assay or the EPA YP Protocol, Section 9.6 (U.S. EPA
2016).
Analytical Technique: Use real-time PCR (CDC Laboratory Assay).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (CDC
Laboratory Assay or the EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the
referenced target-specific PCR primers, 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2010. "Standard Analytical Protocol for Salmonella Typhi in Drinking Water" (EPA
ST Protocol). Washington, DC: U.S. EPA. EPA 600/R-10/133.
https://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntryId=230138
APHA, AWWA and WEF. 2012. "Method 9260 B: Salmonella." Standard Methods for the
Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public Health
Association, http://www.standardmethods.org/
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
CDC Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using SmartCyclerฎ." Contact:
Dr. Eija Trees, Foodborne and Diarreheal Diseases Branch, CDC, Atlanta, GA.
https://www.nemi.gov/methods/method summary/10303/
SAM 2017
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Section 7.0 - Selected Pathogen Methods
7.2.13.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
Drinking water samples should be prepared according to the EPA ST Protocol (U.S. EPA
2010), Standard Method 9260 B (APHA ei al. 2012) or the EPA YP Protocol (U.S. EPA
2016).
Post decontamination waste water samples should be prepared according to Standard
Method 9260 B (APHA et al. 2012) or the EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the CDC Laboratory Assay or the EPA YP Protocol, Section 10.5 (U.S. EPA
2016).
Analytical Technique: Use culture (EPA ST Protocol [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 (CDC Laboratory Assay or EPA
YP Protocol, Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced target-specific
PCR primers, 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2010. "Standard Analytical Protocol for Salmonella Typhi in Drinking Water" (EPA
ST Protocol). Washington, DC: U.S. EPA. EPA 600/R-10/133.
https://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=230138
SAM 2017
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Section 7.0 - Selected Pathogen Methods
APHA, AWWA and WEF. 2012. "Method 9260 B: SalmonellaStandard Methods for the
Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public Health
Association, http://www.standardmethods.org/
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
CDC Laboratory Assay. "Triplex PCR for Detection of S. Typhi Using SmartCyclerฎ." Contact:
Dr. Eija Trees, Foodborne and Diarreheal Diseases Branch, CDC, Atlanta, GA.
https://www.nemi.gov/methods/method summary/10303/
7.2.14 Shigella spp. [Shigellosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.14.1
Post Decontamination
Culture and Real-Time PCR
7.2.14.2
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Cunningham et al. 2010 or the EPA YP Protocol, Section 9.6 (U.S. EPA
2016).
Analytical Technique: Use real-time PCR (Cunningham et al. 2010).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Cunningham
et al. 2010 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Cunningham et al. 2010). 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
SAM 2017
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Section 7.0 - Selected Pathogen Methods
Guidance for Laboratories Performing PCR Analyses on Environmental Samples (EPA 815-B-
04-001) document at: http://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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 ://j cm .asm .org/content/48/8/2929 .full .pdf+html
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: This method is listed for analysis of aerosols, particulates (wipes and
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Cunningham et al. 2010 or the EPA YP Protocol, Section 10.5 (U.S. EPA
2016).
Analytical Technique: Use culture (Standard Method 9260 E [APHA et al. 2012]) and real-time
PCR (Cunningham et al. 2010).
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 media. Confirmation is performed using real-time PCR.
Target nucleic acid should be extracted, purified (Cunningham et al. 2010 or EPA YP Protocol,
Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced target-specific PCR primers,
probes and assay parameters (Cunningham et al. 2010). The use of real-time PCR analyses
directly on isolates (e.g., no biochemical/serological component) allows for rapid confirmation 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.
SAM 2017
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Section 7.0 - Selected Pathogen Methods
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/SO 167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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: //i cm. asm. org/content/48/8/2929. full .pdf+html
APHA, AWWA and WEF. 2012. "Method 9260 Detection of Pathogenic Bacteria E: Shigella."
Standard Methods for the Examination of Water and Wastewater. 22nd Edition. Washington, DC:
American Public Health Association, http://www.standardmethods.org/
7.2.15 Staphylococcus aureus - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.15.1
Post Decontamination
Culture and Real-Time PCR
7.2.15.2
See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Chiang et al. 2007 orthe EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Chiang et al. 2007).
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Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Chiang et al.
2007 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Chiang et al. 2007). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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://ifoodprotection.org/doi/abs/10.4315/0362-028X-70.12.2855
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Chiang et al. 2007 orthe EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture (Standard Method 9213 B: Staphylococcus aureus [APHA et
al. 2012]) and real-time PCR (Chiang et al. 2007).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are inoculated into broth media, incubated for 24 hours,
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and then plated onto selective media. Confirmation is performed using real-time PCR. Target
nucleic acid should be extracted, purified (Chiang et al. 2007 or EPA YP Protocol, Section 10.5
[U.S. EPA 2016]), and analyzed using the referenced target-specific PCR primers, probes and
assay parameters (Chiang et al. 2007). 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.
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=329170
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://ifoodprotection.org/doi/abs/10.4315/0362-028X-70.12.2855
APHA, AWWA and WEF. 2012. "Method 9213 B: Staphylococcus aureus." Standard Methods
for the Examination of Water and Wastewater. 22nd Edition. Washington, DC: American Public
Health Association, http://www.standardmethods.org/
7.2.16 Vibrio cholerae [Cholera] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.16.1
Post Decontamination
Culture and Real-Time PCR
7.2.16.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
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Water samples should be prepared according to the EPA Vibrio cholerae Protocol
(referred to as the EPA VC Protocol [U.S. EPA 2010]) or the EPA YP Protocol (U.S.
EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Blackstone et al. 2007 or the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (Blackstone et al. 2007).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), the target nucleic acid should be extracted, purified (Blackstone et
al. 2007 or EPA YP Protocol, Section 9.6 [U.S. EPA 2016]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Blackstone et al. 2007). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. 2010. "Standard Analytical Protocol for Vibrio cholerae 01 and 0139 in Drinking
Water and Surface Water" (EPA VC Protocol). Washington, DC: U.S. EPA. EPA 600/R-10/139.
http://nepis.epa.gov/Adobe/PDF/P100978K.pdf
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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/S01677012060Q248X
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Soil samples should be prepared according to Silvestri et al. 2016.
Water samples should be prepared according to the EPA VC Protocol (U.S. EPA 2010)
or the EPA YP Protocol (U.S. EPA 2016).
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Blackstone et al. 2007 or the EPA YP Protocol, Section 10.5 (U.S. EPA 2010).
Analytical Technique: Use culture (EPA VC Protocol [U.S. EPA 2010]) and real-time PCR
(Blackstone et al. 2007).
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 (Blackstone et al. 2007 or EPA YP Protocol,
Section 10.5 [U.S. EPA 2010]), and analyzed using the referenced target-specific PCR primers,
probes and assay parameters (Blackstone et al. 2007). 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-
04-001) document at: http://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S0167701216302238
U.S. EPA. October 2010. "Standard Analytical Protocol for Vibrio cholerae 01 and 0139 in
Drinking Water and Surface Water" (EPA VC Protocol). Cincinnati, OH: U.S. EPA. EPA 600/R-
10/139. http://nepis.epa.gov/Adobe/PDF/P100978K.pdf
U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=329170
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/S01677012060Q248X
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7.2.17 Yersinia pestis [Plague] - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.2.17.1
Post Decontamination
RV-PCR (Water Samples)
7.2.17.2
Culture and Real-Time PCR
7.2.17.3
1 See Appendix C for corresponding method usability tiers.
7.2.17.1 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA YP Protocol, Section 9.6 (U.S. EPA 2016).
Analytical Technique: Use real-time PCR (EPA YP Protocol [U.S. EPA 2016]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (EPA YP
Protocol, Section 9.6), and analyzed using the referenced target-specific PCR primers, probes and
assay parameters. 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:
https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf. or consult the
points of contact identified in Section 4.0.
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 be
followed (BMBL, 5th Edition [CDC 2009]). http://www.cdc.gov/biosafetv/publications/bmbl5/
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
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U.S. EPA. 2016. "Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfbub.epa.gov/si/si public record report.cfm?dirEntryId=329170
7.2.17.2 Post Decontamination Sample Analyses (RV-PCR) Water Samples
Note: Laboratories without RV-PCR capability should analyze samples according to the culture
procedure provided in Section 7.2.17.3.
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: This method is listed for analysis of drinking water and post
decontamination waste water samples. Further research is needed to develop comprehensive
pathogen-specific procedures for different environmental sample types.
Sample Preparation: Water samples should be prepared according to the EPA YP Protocol
(U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA YP Protocol, Section 11.3 (U.S. EPA 2016).
Analytical Technique: Use RV-PCR (EPA YP Protocol [U.S. EPA 2016]).
Description of Method: The RV-PCR procedure serves as an alternative to the traditional
culture-based methods for detection of viable pathogens. The RV-PCR procedure integrates high-
throughput sample processing, short-incubation broth culture, and highly sensitive and specific
real-time PCR assays to detect low concentrations of viable Y. pestis. Specifically, the procedure
uses the change in real-time PCR response, referred to as the change in cycle threshold, or ACT,
between the initial cycle threshold (CT) at time 0 (T0) (just before sample incubation) and the final
CT after incubation (CT Tf).
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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Sources:
U.S. EPA. 2016. "Protocol for Detection of Yersinia pestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
7.2.17.3 Post Decontamination Sample Analyses (Culture and Real-Time PCR)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
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Method Selected for: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils and drinking water and post decontamination waste water samples. Further research
is needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Soil samples should be prepared according to Silvestri el al. 2016.
All other environmental sample types should be prepared according to the EPA YP
Protocol (U.S. EPA 2016).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within the EPA YP Protocol, Section 10.5 (U.S. EPA 2016).
Analytical Technique: Use culture and real-time PCR (EPA YP Protocol [U.S. EPA 2016]).
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 YP Protocol, Section 10.5 [U.S. EPA 2016]), and analyzed using the referenced target-
specific PCR primers, probes and assay parameters. 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-
04-001) document at: https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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/biosafetv/publications/bmbl5/
Some laboratories may not have access to a positive control for this agent for culture analyses.
For laboratories that may not have access to a virulent strain for the positive control, an avirulent
strain may be used to meet the laboratory's BSL.
Sources:
Silvestri E.E., Feldhake, D., Griffin, D., Lisle, J., Nichols, T.L., Shah, S.R., Pemberton, A. and
Schaefer, F.W. III. 2016. "Optimization of a Sample Processing Protocol for Recovery of
Bacillus anthracis Spores from Soil." Journal of Microbiological Methods. 130: 6-13.
http://www.sciencedirect.com/science/article/pii/S01677012163Q2238
U.S. EPA. 2016. "Protocol for Detection of Yersinia pestis in Environmental Samples During the
Remediation Phase of a Plague Incident" (EPA YP Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-16/109. https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=329170
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7.3 Method Summaries for Viruses
Summaries for the analytical methods listed in Appendix C for analysis of viral pathogens are provided in
Sections 7.3.1 through 7.3.10. Each summary contains a brief description of the analytical methods
selected for each viral pathogen, and links to, or sources for, obtaining full versions of the methods.
Summaries are provided for informational use. Tiers that have been assigned to each method/analyte pair
(see Section 7.1.1) can be found in Appendix C. The full version of the method should be consulted prior
to sample analysis. For information regarding sample collection considerations for samples to be analyzed
by these methods, see the companion Sample Collection Information Document for Pathogens -
Companion to SAM 2017.
7.3.1 Adenoviruses: Enteric and Non-enteric (A-F) - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.3.1.1
Post Decontamination
Tissue Culture and Real-Time PCR
7.3.1.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
adenovirus using the Nanoceramฎ filter (Argonide Corporation, Sanford, FL) has not
been optimized, use of a 1MDS filter is recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2005 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time PCR (Jothikumar et al. 2005).
Description of Method: Following appropriate sample preparation (see Sample Preparation
Procedures above), the target nucleic acid should be extracted, purified (Jothikumar et al. 2005 or
EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced target-specific real-time
PCR primers, probes and assay parameters (Jothikumar et al. 2005). Use of real-time PCR
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
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Section 7.0 - Selected Pathogen Methods
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M.,and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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/71/6/3131 .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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
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and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note: Since the concentration of
adenovirus using the Nanoceram filter has not been optimized, use of the 1MDS filter is
recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2005 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (Boczek et al. 2016 or Green and Loewenstein 2005)
and real-time PCR (Jothikumar et al. 2005).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples should be cultured to assess viability (Boczek et al. 2016
or Green and Loewenstein 2005). For confirmation, target nucleic acid should be extracted,
purified (Jothikumar et al. 2005 or EPA Method 1615 [Fout et al. 2012]), and analyzed using the
referenced target-specific real-time PCR primers, probes and assay parameters (Jothikumar et al.
2005).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZyPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
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/71/6/3131 .full.pdf+html
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Boczek, L.A., Rhodes, E.R., Cashdollar, J.L., Ryu, J., Popovici, J., Hoelle, J.M., Sivaganesan,
M., Hayes, S.L., Rodgers, M.R. and Ryu, H. 2016. "Applicability of UV Resistant Bacillus
pumilus Endospores as a Human Adenovirus Surrogate for Evaluating the Effectiveness of Virus
Inactivation in Low-pressure UV Treatment Systems." Journal of Microbiological Methods. 122:
43-49. http://www.sciencedirect.com/science/article/pii/S016770121630Q124
Green, M., and Loewenstein, P.M. 2005. "UNIT 14C.1 Human Adenoviruses: Propagation,
Purification, Quantification, and Storage." Current Protocols in Microbiology. 00:C:14C.1.1-
14C.1.19. http://onlinelibrarv.wilev.com/doi/10.1002/9780471729259.mcl4c01sQ0/abstract
7.3.2 Astroviruses - BSL not specified
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.2.1
Post Decontamination
Integrated Cell Culture and Real-Time Reverse Transcription-PCR
7.3.2.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
astrovirus using the Nanoceram filter has not been optimized, use of the 1MDS filter is
recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Grimm et al. 2004 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Grimm et al. 2004).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Grimm et al.
2004 or EPA Method 1615 [(Fout et al. 2012]), and analyzed using the referenced target-specific
PCR primers, probes and assay parameters (Grimm et al. 2004). 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
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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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate ribonuclease (RNAse) inhibitors should be included
during sample processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
Grimm, A.C., Cashdollar, J.L., Williams, F.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/10.113 9/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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water. Further research is needed to
develop comprehensive pathogen-specific procedures for different environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
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Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
astrovirus using the Nanoceram filter has not been optimized, use of the 1MDS filter is
recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Grimm et al. 2004 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use integrated cell culture and real-time reverse transcription-PCR
(Grimm et al. 2004).
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, probes and assay parameters (Grimm et al. 2004). For confirmation, target nucleic acid
should be extracted, purified (Grimm et al. 2004 or EPA Method 1615 [Fout et al. 2012]), and
analyzed using the referenced target-specific real-time PCR primers, probes and assay parameters
(Grimm et al. 2004).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
SAM 2017
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Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
Grimm, A.C., Cashdollar, J.L., Williams, F.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/10.113 9/w04-012
7.3.3 Caliciviruses: Noroviruses - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.3.1
Post Decontamination
No method available to determine viable virus after
decontamination
7.3.3.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 Fout et al. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (EPA Method 1615 [Fout et al.
2012]).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (U.S. EPA
2012), and analyzed using the referenced target-specific PCR primers, probes and assay
parameters (EPA Method 1615 [Fout et al. 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
SAM 2017
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Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F. M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldiv Abstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.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
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.4.1
Post Decontamination
Tissue Culture and Real-Time Reverse Transcription-PCR
7.3.4.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout etal. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Oka et al. 2006 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Oka et al. 2006).
Description of Method: The method is a TaqManฎ (Thermo Fisher Scientific, Waltham, MA, or
equivalent)-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 (Oka et al. 2006 or EPA Method 1615 [Fout et al. 2012]), and
analyzed using the referenced target-specific PCR primers, probes and assay parameters (Oka et
al. 2006).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F. M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
SAM 2017
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1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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://onlinelibrarv.wilev.com/doi/10.1002/imv.2Q699/abstract
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout etal. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Oka et al. 2006 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (Parwani et al. 1991) and real-time reverse
transcription-PCR (Oka et al. 2006).
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 (Parwani et al. 1991). For confirmation,
target nucleic acid should be extracted, purified (Oka et al. 2006 or EPA Method 1615 [Fout et al.
2012]), and analyzed using the referenced target-specific real-time PCR primers, probes and
assay parameters (Oka et al. 2006).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
SAM 2017
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an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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://onlinelibrarv.wilev.com/doi/10.1002/imv.2Q699/abstract
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/
7.3.5 Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human
Coronavirus (HCoV) - BSL-2; BSL-3 for Propagation
Remediation Phase
Analytical Technique1
Section
Site Characterization
Reverse Transcription-PCR
7.3.5.1
Post Decontamination
Tissue Culture and Reverse Transcription-PCR
7.3.5.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Adachi et al. 2004 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use reverse transcription-PCR (Adachi et al. 2004).
Description of Method: This method uses a conventional single-tube reverse transcription-PCR
procedure using the Stratagene Robocyclerฎ (Stratagene Corporation, La Jolla, California, or
equivalent). 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
(Adachi et al. 2004 or EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Adachi et al. 2004).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" ( EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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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/S01660934040Q2162
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Adachi et al. 2004 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (Pagat et al. 2007) and reverse transcription-PCR
(Adachi et al. 2004).
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 (Pagat et al. 2007). For confirmation,
target nucleic acid should be extracted, purified (Adachi et al. 2004 or EPA Method 1615 [Fout et
al. 2012]), and analyzed using the referenced target-specific PCR primers, probes and assay
parameters (Adachi et al. 2004).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdfi or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
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Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntryId=238310
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/S01660934040Q2162
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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://iournals.sagepub.eom/doi/pdf/10.l 177/153567600701200206
7.3.6 Hepatitis E Virus (HEV) - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.6.1
Post Decontamination
Tissue Culture and Real-Time Reverse
Transcription-PCR
7.3.6.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
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Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
hepatitis E virus using the Nanoceram filter has not been evaluated, use of the 1MDS
filter is recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2006 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Jothikumar ei al. 2006).
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 (Jothikumar et al. 2006 or EPA
Method 1615 [Fout et al. 2012]), and analyzed using the referenced target-specific PCR primers,
probes and assay parameters (Jothikumar et al. 2006).
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
SAM 2017
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Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.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://www.sciencedirect.com/science/article/pii/S01660934050024177via%3Dihub
7.3.6.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao et al. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
hepatitis E virus using the Nanoceram filter has not been evaluated, use of the 1MDS
filter is recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2006 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (Zaki et al. 2009) and real-time reverse transcription-
PCR (Jothikumar et al. 2006).
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 (Zaki et al. 2009). For confirmation, target nucleic acid should be
extracted, purified (Jothikumar et al. 2006 or EPA Method 1615 [Fout et al. 2012]), and analyzed
using the referenced target-specific PCR primers, probes and assay parameters (Jothikumar et al.
2006).
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
SAM 2017
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Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntryId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.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://www.sciencedirect.com/science/article/pii/S01660934050Q24177via%3Dihub
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://onlinelibrarv.wilev.eom/doi/10.l 11 l/i.l574-695X.2009.00552.x/pdf
7.3.7 Influenza H5N1 virus - BSL-3
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.7.1
Post Decontamination
Tissue Culture and Real-Time Reverse
Transcription-PCR
7.3.7.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
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needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Hermann et al. 2006.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Ng et al. 2005 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Ng et al. 2005).
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
(Ng et al. 2005 or EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced
target-specific PCR primers, probes and assay parameters (Ng et al. 2005).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Hermann, J.R., Hoff, S.J., Yoon, K.J., Burkhardt, A.C., Evans, R.B. and Zimmerman, J.J. 2006.
"Optimization of a Sampling System for Recovery and Detection of Airborne Porcine
Reproductive and Respiratory Syndrome Virus and Swine Influenza Virus." Applied and
Environmental Microbiology. 72(7): 4811-4818. http://aem.asm.Org/content/72/7/4811
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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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/pmc/articles/PMC3320469/
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
7.3.7.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Hermann et al. 2006.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Ng et al. 2005 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (Krauss et al. 2012) and real-time reverse
transcription-PCR (Ng et al. 2005).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are inoculated onto Madin-Darby Canine Kidney Cells
(MDCK); the cells are examined for CPEs to assess viability (Krauss et al. 2012). For
confirmation, target nucleic acid should be extracted, purified (Ng et al. 2005 or EPA Method
1615 [Fout et al. 2012]), and analyzed using the referenced target-specific PCR primers, probes
and assay parameters (Ng et al. 2005).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
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Sources:
Hermann, J.R., Hoff, S.J., Yoon, K.J., Burkhardt, A.C., Evans, R.B. and Zimmerman, J.J. 2006.
"Optimization of a Sampling System for Recovery and Detection of Airborne Porcine
Reproductive and Respiratory Syndrome Virus and Swine Influenza Virus." Applied and
Environmental Microbiology. 72(7): 4811-4818. http://aem.asm.Org/content/72/7/4811
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntryId=238310
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/pmc/articles/PMC3320469/
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
Krauss, S., Walker, D. and Webster, R.G. 2012. "Influenza Virus Isolation." Methods in
Molecular Biology. 865: 11-24. https://www.ncbi.nlm.nih.gov/pubmed/22528151
7.3.8 Picornaviruses: Enteroviruses - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.8.1
Post Decontamination
Tissue Culture
7.3.8.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
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Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout etal. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (EPA Method 1615 [Fout et al.
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, probes and assay parameters (Method
1615 [Fout et al. 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G. W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K. E., Radosevich, M. and Wommack, K. E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZyPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), drinking water and post decontamination waste water samples. Further research is needed
to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout etal. 2012).
Analytical Technique: Use tissue culture (EPA Method 1615 [Fout etal. 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 (EPA Method 1615 [Fout et al. 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.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
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7.3.9 Picornaviruses: Hepatitis A Virus (HAV) - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.9.1
Post Decontamination
Integrated Cell Culture and Real-Time Reverse Transcription-PCR
7.3.9.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout etal. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Hyeon et al. 2011 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Hyeon et al. 2011).
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 (Hyeon et al. 2011 or
EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced target-specific PCR
primers, probes and assay parameters (Hyeon et al. 2011).
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
SAM 2017
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Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
htto: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih. gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 100LX19.PDF?Dockev=P 100LX19.PDF
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
7.3.9.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Hyeon et al. 2011 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use integrated cell culture and real-time reverse transcription-PCR
(Hyeon etal. 2011).
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 (Hyeon etal. 2011 or EPA Method 1615 [Fout et al. 2012]),
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Section 7.0 - Selected Pathogen Methods
and analyzed using the referenced target-specific PCR primers, probes and assay parameters
(Hyeon et al. 2011).
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F. M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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
Analytical Technique1
Section
Site Characterization
Real-Time Reverse Transcription-PCR
7.3.10.1
Post Decontamination
Tissue Culture and Real-Time Reverse Transcription-PCR
7.3.10.2
1 See Appendix C for corresponding method usability tiers.
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park et al. 2015).
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615: Enterovirus and Norovirus Occurrence in Water
by Culture and RT-qPCR" (Fout et al. 2012). Note. Since the concentration of reoviruses
using the Nanoceram filter has not been optimized, use of the 1MDS filter is
recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2009 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use real-time reverse transcription-PCR (Jothikumar et al. 2009).
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 (Jothikumar
et al. 2009 or EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced target-
specific PCR primers, probes and assay parameters (Jothikumar et al. 2009).
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
SAM 2017
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Williamson, K.E., Radosevich, M. and Wommack, K.E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus andNorovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZyPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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/S01660934080Q3571
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples should be prepared according to Cao etal. 2011.
Particulate samples (swabs and wipes) should be prepared according to Park el al. 2015.
Soil samples should be prepared according to Williamson et al. 2005.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or EPA Method 1615 (Fout et al. 2012). Note. Since the concentration of
reoviruses using the Nanoceram filter has not been optimized, use of the 1MDS filter is
recommended.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Jothikumar et al. 2009 or EPA Method 1615 (Fout et al. 2012).
Analytical Technique: Use tissue culture (EPA Method 1615 [Fout et al. 2012]) and real-time
reverse transcription-PCR (Jothikumar et al. 2009).
Description of Method: This method describes procedures for determining infectivity and
quantifying enteroviruses using BGMK cells. Following appropriate sample preparation (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 (EPA Method 1615
[Fout et al. 2012]). For confirmation, target nucleic acid should be extracted, purified (Jothikumar
et al. 2009 or EPA Method 1615 [Fout et al. 2012]), and analyzed using the referenced target-
specific PCR primers, probes and assay parameters (Jothikumar et al. 2009).
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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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Special Considerations: Appropriate RNAse inhibitors should be included during sample
processing and analysis.
Sources:
Cao, G., Noti, J.D., Blachere, F.M., Lindsley, W.G. and Beezhold, D.H. 2011. "Development of
an Improved Methodology to Detect Infectious Airborne Influenza Virus Using the NIOSH
Bioaerosol Sampler." Journal of Environmental Monitoring. 13: 3321-3328.
http://pubs.rsc.Org/en/content/articlelanding/2011/em/cleml0607d#ldivAbstract
Park, G.W., Lee, D., Treffiletti, A., Hrsak, M., Shugart, J. and Vinje, J. 2015. "Evaluation of a
New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate
Surfaces." Applied and Environmental Microbiology. 81(17): 5987-5992.
http: //aem. asm .org/content/81/17/5987. full .pdf+html
Williamson, K. E., Radosevich, M. and Wommack, K. E. 2005. "Abundance and Diversity of
Viruses in Six Delaware Soils." Applied and Environmental Microbiology. 71(6): 3119-3125.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMCl 151856/
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., McMinn, B.R., Rhodes, E.R.,
Varughese, E.A., Karim, M.R., Grimm, A.C., Spencer, S.K. and Borchardt, M.A. 2012. "Method
1615: Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR." Cincinnati,
OH: U.S. EPA. EPA/600/R-10/181.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1OOLX19.PDF?Dockev=P 1OOLX19.PDF
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/S01660934080Q3571
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7.4 Method Summaries for Protozoa
Summaries for the analytical methods listed in Appendix C for analysis of protozoa are provided in
Sections 7.4.1 through 7.4.5. Each summary contains a brief description of the analytical methods
selected for each protozoan, and links to, or sources for, obtaining full versions of the methods. Tiers that
have been assigned to each method/analyte pair (see Section 7.1.1) can be found in Appendix C. The full
version of the method should be consulted prior to sample analysis. For information regarding sample
collection considerations for samples to be analyzed by these methods, see the companion Sample
Collection Information Document for Pathogens - Companion to SAM 2017.
7.4.1 Cryptosporidium spp. [Cryptosporidiosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.4.1.1
IMS/immunofluorescence
assay(FA)
7.4.1.2 2
IMS/FA
7.4.1.32
Post Decontamination
Cell Culture
Immunofluorescence
Procedure
7.4.1.4
1 See Appendix C for corresponding method usability tiers.
2 Methods 1622 and 1623.1 include the same sample processing and analytical procedures for
Cryptosporidium; 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011, or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1622 (U.S. EPA 2005),
EPA Method 1623.1 (U.S. EPA 2012b), or the EPA and CDC UF Report (U.S. EPA and
CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Guy et al. 2003, Jiang et al. 2005 or the EPA BA Protocol, Section 9.2 (U.S.
EPA 2012a).
Analytical Technique: Use real-time PCR (Guy et al. 2003 and Jiang et al. 2005).
Description of Method: Following appropriate sample preparation (see Sample Preparation
Procedures above), the target nucleic acid should be extracted, purified (Guy et al. 2003, Jiang et
al. 2005 or EPA BA Protocol, Section 9.2 [U.S. EPA 2012a]), and analyzed using the referenced
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target-specific real-time PCR primers, probes and assay parameters (Guy et al. 2003). 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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." Washington,
DC: U.S. EPA. EPA 815-R-05-001. https://www.epa.gov/sites/production/files/2015-
07/documents/epa-1622 .pdf
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P 100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Guy, R.A., Payment, P., Krull, U.J. and Horgen, P.A. 2003. "Real-Time PCR for Quantification
of Giardia 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
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7.4.1.2 Site Characterization Sample Analyses (Immunomagnetic
Separation/lmmunofluorescence Assay [IMS/FA])
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges el al.
2010, Rose et al. 2011, or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1622 (U.S. EPA 2005),
Method 1623.1 (U.S. EPA 2012b) or the EPA and CDC UF Report (U.S. EPA and CDC
2011).
Analytical Technique: Use IMS and FA microscopy (EPA Method 1622 [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 (DIC)
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:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples during
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. httos://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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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. fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem. asm. org/content/77/18/6476. abstract
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." Washington,
DC: U.S. EPA. EPA 815-R-05-001. https://www.epa.gov/sites/production/files/2015-
07/documents/epa-1622 .pdf
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P 100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water. Further research is needed to
develop comprehensive pathogen-specific procedures for environmental sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges el al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1622 (U.S. EPA 2005),
EPA Method 1623.1 (U.S. EPA 2012b) or the EPA and CDC UF Report (U.S. EPA and
CDC 2011).
Analytical Technique: Use IMS and FA microscopy (EPA Method 1623.1 [U.S. EPA 2012b]).
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 anti-Cryptosporidium and anti-Giardia
antibodies conjugated to magnetic beads is added to the pellet and mixed. The oocyst and cyst
magnetic bead complex is separated from the extraneous materials using a magnet, and the
extraneous materials are discarded. The magnetic bead complex is then detached from the oocysts
and cysts. The oocysts and cysts are stained on well slides with fluorescently labeled mAbs and
DAPI. The stained sample is examined using fluorescence and DIC microscopy. Qualitative
analysis is performed by scanning each slide well for objects that meet the size, shape, and
fluorescence characteristics of Cryptosporidium oocysts and Giardia cysts. Quantitative analysis
is performed by counting the total number of objects on the slide confirmed as oocysts or cysts.
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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:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." Washington,
DC: U.S. EPA. EPA 815-R-05-001. https://www.epa.gov/sites/production/files/2015-
07/documents/epa-1622 .pdf
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P 100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water." Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntryId=238310
7.4.1.4 Post Decontamination Sample Analyses (Cell Culture
Immunofluorescence Procedure)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (surface swabs and wipes) should be prepared according to Hodges el
al. 2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012a).
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Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1622 (U.S. EPA 2005),
EPA Method 1623.1 (U.S. EPA 2012b) or the EPA and CDC UF Report (U.S. EPA and
CDC 2011).
Analytical Technique: Use cell culture immunofluorescence procedure (Bukhari etal. 2007).
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 immunofluorescence to determine the
number of viable oocysts present in the sample. The use of cell culture immunofluorescence
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:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/SO 167701210000692
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. fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA. 2005. "Method 1622: Cryptosporidium in Water by Filtration/IMS/FA." Washington,
DC: U.S. EPA. EPA 815-R-05-001. https://www.epa.gov/sites/production/files/2015-
07/documents/epa-1622 .pdf
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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."
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Canadian Journal of Microbiology. 53(5): 656-663.
http://www.nrcresearchpress.com/doi/abs/10.1139/W07-032?url ver=Z39.88-
2003&rfr id=ori:rid:crossref.org&rfr dat=cr pub%3dpubmed
7.4.2 Entamoeba histolytica - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.4.2.1
Post Decontamination
Cell Culture
7.4.2.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Roy et al. 2005 or the EPA BA Protocol, Section 9.2 (U.S. EPA 2012).
Analytical Technique: Use real-time PCR (Roy et al. 2005).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Roy et al.
2005 or EPA BA Protocol, Section 9.2 [U.S. EPA 2012]), and analyzed using the referenced
target-specific real-time PCR primers, probes and assay parameters (Roy et al. 2005). 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
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
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EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Roy, S., Kabir, M., Mondal, D., Ali, I.K.M., Petri Jr., W.A. and Haque, R. 2005. "Real-Time-
PCR Assay for Diagnosis of Entamoeba histolytica Infection." Journal of Clinical Microbiology.
43(5): 2168-2172. http: //i cm .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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protol (U.S. EPA 2012).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011).
Analytical Technique: Use cell culture (Stringert 1972).
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.
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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:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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. fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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
Analytical Technique
Section
Site Characterization
Real-Time PCR
7.4.3.1
IMS/FA
7.4.3.2
Post Decontamination
Cell Culture
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1623.1 (U.S. EPA 2012b)
or the EPA and CDC UF Report (U.S. EPA and CDC 2011).
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Guy et al. 2003 or the EPA BA Protocol, Section 9.2 (U.S. EPA 2012a).
Analytical Technique: Use real-time PCR (Guy et al. 2003).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Guy et al.
2003 or the EPA BA Protocol [U.S. EPA 2012a]), and analyzed using the referenced target-
specific real-time PCR primers, probes and assay parameters (Guy et al. 2003). The use of real-
time PCR analyses directly on samples allows for rapid detection of Giardia.
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/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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. fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
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U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
Guy, R.A., Payment, P., Krull, U.J. and Horgen, P.A. 2003. "Real-Time PCR for Quantification
of Giardia 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types included.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges el al.
2010, Rose et al. 2011, or EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1623.1 (U.S. EPA 2012b)
or the EPA and CDC UF Report (U.S. EPA and CDC 2011).
Analytical Technique: Use IMS and FA microscopy (Method 1623.1 [U.S. EPA 2012b]).
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 anti-Cryptosporidium and anti-Giardia
antibodies conjugated to magnetic beads is added to the pellet and mixed. The oocyst and cyst
magnetic bead complex is separated from the extraneous materials using a magnet, and the
extraneous materials are discarded. The magnetic bead complex is then detached from the oocysts
and cysts. The oocysts and cysts are stained on well slides with fluorescently labeled mAbs and
DAPI. The stained sample is examined using fluorescence and DIC microscopy. Qualitative
analysis is performed by scanning each slide well for objects that meet the size, shape, and
fluorescence characteristics of Cryptosporidium oocysts and Giardia cysts. Quantitative analysis
is performed by counting the total number of objects on the slide confirmed as oocysts or cysts.
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:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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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.eom/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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem. asm. org/content/77/18/6476. abstract
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to EPA Method 1623.1 (U.S. EPA 2012b)
or the EPA and CDC UF Report (U.S. EPA and CDC 2011).
Analytical Technique: Use cell culture (Keister 1983).
Description of Method: Procedures are described for analysis of cell culture samples and may
be adapted for assessment of aerosols, 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 36ฐC 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
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results should also be performed.
Sources:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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/00359203839012Q7
7.4.4 Naegleria fowleri [Naegleriasis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.4.4.1
Post Decontamination
Culture and Real-Time PCR
7.4.4.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
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Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Mull et al. 2013.
Water samples should be prepared according to Cope et al. 2015 or the EPA and CDC
UF Report (U.S. EPA and CDC 2011).
Nucleic acid extraction should be performed according to procedures within Mull et al. 2013 or
the EPA BA Protocol, Section 9.2 (U.S. EPA 2012).
Analytical Technique: Use real-time PCR (Mull et al. 2013).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), samples are concentrated by centrifugation. The pellet is then
resuspended and further concentrated using IMS. Target nucleic acid should be extracted, purified
(Mull et al. 2013 or EPA BA Protocol, Section 9.2 [U.S. EPA 2012]) and analyzed using the
referenced target-specific PCR primers, probes and assay parameters (Mull et al. 2013). The use
of real-time PCR analyses directly on samples (e.g., no culture component) allows for rapid
detection of Naegleria fowleri.
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:
https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-pcr.pdf. or consult the
points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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.eom/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. fiill.pdf+html
Mull, B.J., Jothikumar, N. and Hill, V.R. 2013. "Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation
and Real-Time PCR." Journal of Parasitology Research. Article ID 608367: 8 pages.
https://www.hindawi.com/iournals/ipr/2013/6Q8367/
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Cope, J.R., Ratard, R.C., Hill, V.R., Sokol, T., Causey, J.J., Yoder, J.S., Mirani, G., Mull, B.
Mukeijee, K.A., Narayanan, J., Doucet, M., Qvarnstrom, Y., Poole, C.N., Akingbola, O.A.,
Ritter, J.M., Xiong, Z., da Silva, A.J., Roellig, D., Van Dyke, R.B., Stern, H., Xiao, L. and
Beach, M.J. 2015. "The First Association of a Primary Amebic Meningoencephalitis Death With
Culturable Naegleria fowleri in Tap Water From a US Treated Public Drinking Water System."
Clinical Infectious Diseases, 60(8): e36-42. https://www.ncbi.nlm.nih.gov/pubmed/25595746
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfbub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
7.4.4.2 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to procedures within the EPA BA
Protocol (U.S. EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Mull et al. 2013.
Water samples should be prepared according to Cope et al. 2015 or the EPA and CDC
UF Report (U.S. EPA and CDC 2011).
Nucleic acid extraction should be performed according to Mull et al. 2013 or the EPA BA
Protocol, Section 11.6 (U.S. EPA 2012).
Analytical Technique: Use culture and real-time PCR (Mull et al. 2013).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation procedures above), samples are centrifuged to pellet the trophozoites and cysts and a
portion of the pellet is plated with E. coli. The remaining pellet is further concentrated using IMS
and then plated with E. coli. Plates are incubated for seven days. Confirmation is performed using
real-time PCR. Target nucleic acid should be extracted, purified (Mull et al. 2013 or EPA BA
Protocol, Section 11.6 [U.S. EPA 2012]) and analyzed using the referenced target-specific PCR
primers, probes and assay parameters (Mull et al. 2013). The use of real-time PCR analyses
directly on isolates (e.g., no biochemical/serological component) allows for rapid confirmation of
Naegleria fowleri.
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: https://www.epa.gov/sites/production/files/2015-07/documents/epa-qaqc-
pcr.pdf. or consult the points of contact identified in Section 4.0.
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Sources:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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.eom/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. fiill.pdf+html
Mull, B.J., Jothikumar, N. and Hill, V.R. 2013. "Improved Method for the Detection and
Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation
and Real-Time PCR." Journal of Parasitology Research. Article ID 608367: 8 pages.
https://www.hindawi.com/iournals/ipr/2013/6Q8367/
Cope, J.R., Ratard, R.C., Hill, V.R., Sokol, T., Causey, J.J., Yoder, J.S., Mirani, G., Mull, B.
Mukeijee, K.A., Narayanan, J., Doucet, M., Qvarnstrom, Y., Poole, C.N., Akingbola, O.A.,
Ritter, J.M., Xiong, Z., da Silva, A. J., Roellig, D., Van Dyke, R.B., Stern, H., Xiao, L. and
Beach, M.J. 2015. "The First Association of a Primary Amebic Meningoencephalitis Death With
Culturable Naegleria fowleri in Tap Water From a US Treated Public Drinking Water System."
Clinical Infectious Diseases. 60(8): e36-42. https://www.ncbi.nlm.nih.gov/pubmed/25595746
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
7.4.5 Toxoplasma gondii [Toxoplasmosis] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.4.5.1
Post Decontamination
Cell Culture
7.4.5.2
1 See Appendix C for corresponding method usability tiers.
7.4.5.1 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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
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EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011, or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to Villegas et al. 2010 or EPA Method
1623.1 (U.S. EPA 2012b).
Nucleic acid extraction should be performed according to procedures within Yang et al. 2009 or
the EPA BA Protocol, Section 9.2 (U.S. EPA 2012a).
Analytical Technique: Use real-time PCR (Yang et al. 2009).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Yang et al.
2009 or EPA BA Protocol, Section 9.2 [U.S. 2012a]), and analyzed using the referenced target-
specific real-time PCR primers, probes and assay parameters (Yang et al. 2009). 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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
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 Toxoplasma gondii Oocysts to Chemical
Sanitizers." Journal of Microbiological Methods. 81(3): 219-225.
http://www.sciencedirect.eom/science/article/pii/S0167701210001107
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U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
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 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 1/3477.fiill.pdf+html
7.4.5.2 Post Decontamination Sample Analyses (Cell Culture)
Method: This method includes a combination of sample preparation and analysis procedures as
summarized below.
Method Selected for: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012a).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011) or the EPA BA Protocol (U.S. EPA 2012a).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to Villegas et al. 2010 or EPA Method
1623.1 (U.S. EPA 2012b).
Analytical Technique: Use cell culture (Villegas et al. 2010).
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 quantitative
polymerase chain reaction (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.
Sources:
U.S. EPA. 2012a. "Protocol for Detection of Bacillus anthracis in Environmental Samples
During the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S.
EPA. EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem. asm. org/content/77/18/6476. abstract
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 Toxoplasma gondii Oocysts to Chemical
Sanitizers." Journal of Microbiological Methods. 81(3): 219-225.
http://www.sciencedirect.eom/science/article/pii/S0167701210001107
U.S. EPA. 2012b. "Method 1623.1: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA." Washington, DC: U.S. EPA. EPA 816-R-12-001.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100J7G4.PDF?Dockev=P100J7G4.PDF
7.5 Method Summaries for Helminths
Summaries for the analytical methods listed in Appendix C for analysis of helminths are provided in
Section 7.5.1. The section contains a brief description of the analytical methods selected, and links to, or
sources for, obtaining full versions of the methods. Tiers that have been assigned to each method/analyte
pair (see Section 7.1.1) can be found in Appendix C. The full version of the method should be consulted
prior to sample analysis. For information regarding sample collection considerations for samples to be
analyzed by these methods, see the companion Sample Collection Information Document for Pathogens -
Companion to SAM 2017.
7.5.1 Baylisascaris procyonis [Raccoon roundworm fever] - BSL-2
Remediation Phase
Analytical Technique1
Section
Site Characterization
Real-Time PCR
7.5.1.1
Post Decontamination
Embryonation of Eggs and Microscopy
7.5.1.2
1 See Appendix C for corresponding method usability tiers.
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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges el al.
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2010, Rose etal. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or Gatcombe et al. 2010.
Nucleic acid extraction and purification for all sample types should be performed according to
procedures within Gatcombe etal. 2010 or the EPA BA Protocol, Section 9.2 (U.S. EPA 2012).
Analytical Technique: Use real-time PCR (Gatcombe et al. 2010).
Description of Method: Following the appropriate sample preparation procedure (see Sample
Preparation Procedures above), the target nucleic acid should be extracted, purified (Gatcombe et
al. 2010 or EPA BA Protocol, Section 9.2 [U.S. EPA 2012]), and analyzed using the referenced
target-specific real-time PCR primers, probes and assay parameters (Gatcombe etal. 2010). The
use of real-time PCR analyses directly on samples (e.g., no embryonation or microscopic
examination) allows for rapid detection of Baylisascaris procyonis.
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/sites/production/files/2015-07/documents/epa-aaac-
pcr.pdf. or consult the points of contact identified in Section 4.0.
Sources:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem.asm.org/content/77/18/6476.abstract
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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
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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: This method is listed for analysis of aerosols, particulates (wipes or
swabs), soils, drinking water and post decontamination waste water samples. Further research is
needed to develop comprehensive pathogen-specific procedures for different environmental
sample types.
Sample Preparation:
Aerosol samples (filters) should be prepared according to the EPA BA Protocol (U.S.
EPA 2012).
Particulate samples (swabs and wipes) should be prepared according to Hodges et al.
2010, Rose et al. 2011 or the EPA BA Protocol (U.S. EPA 2012).
Soil samples should be prepared according to Liang and Keeley 2011.
Water samples should be prepared according to the EPA and CDC UF Report (U.S. EPA
and CDC 2011) or Gatcombe et al. 2010.
Analytical Technique: Use microscopy and embryonation of eggs (U.S. EPA. 2003).
Description of Method: The protocol describes procedures for analysis of soil and wastewater
samples. Samples are processed by blending with buffered water containing a surfactant. The
blend is screened to remove large particles, the soils 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 A scar is 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:
U.S. EPA. 2012. "Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Event" (EPA BA Protocol). Cincinnati, OH: U.S. EPA.
EPA/600/R-12/577. https://www.epa.gov/homeland-securitv-research/protocol-detection-
bacillus-anthracis-environmental-samples
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/S016770121000Q692
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.fiill.pdf+html
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Liang, Z. and Keeley, A. 2011. "Detection of Viable Cryptosporidium parvum in Soil by Reverse
Transcription-Real-Time PCR Targeting hsp70 mRNA." Applied and Environmental
Microbiology. 77(18): 6476-6485. http://aem. asm. org/content/77/18/6476. abstract
U.S. EPA and CDC. 2011. "Comparison of Ultrafiltration Techniques for Recovering Biothreat
Agents in Water" (EPA and CDC UF Report). Cincinnati, OH: U.S. EPA. EPA 600/R-l 1/103.
http://cfpub.epa.gov/si/si public record report.cfm?address=nhsrc/&dirEntrvId=238310
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.
htto://www.springer1ink com/content/k8t3581t07n82562
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. Cincinnati, OH: U.S. EPA
EPA/625/R-92/013. https://www.epa.gov/sites/production/files/2015-07/documents/epa-625-r-92-
013.pdf
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Section 8.0: Selected Biotoxin Methods
Appendix D provides a list of methods to be used in analyzing environmental samples for biotoxin
contaminants during remediation activities following a contamination incident. For the purposes of this
document, biotoxins are defined as poisonous chemicals or group of related chemicals that are derived
from plants or animals, and include those that can be artificially produced in sufficient quantities as to
represent a substantial hazard. Procedures and methods have been selected for each biotoxin that may
need to be identified and/or quantified during remediation. Analytical procedures are not currently
available for all the biotoxin/sample type combinations included in this document, and on-going research
efforts include identification of additional methods, as well as development and testing of some of the
procedures listed. Appendix D is sorted alphabetically by biotoxin.
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 biotoxin/sample type combination listed
in Appendix D, and method usability tiers have been assigned to indicate the fitness of each method
for its intended use. Users should refer to the specified methods and reference citations provided
throughout Sections 8.2 and 8.3 to identify biotoxin/sample type combinations that have been verified,
and should also consider the possibility of analytical interferences inherent to environmental sample
types (e.g., sample composition, the presence and concentrations of additional or competing biotoxins
or biotoxin variants. Any questions regarding this information should be addressed to the appropriate
contact(s) listed in Section 4.0.
Appendix D provides the following information:
Analyte(s). The biotoxin of interest.
Chemical Abstracts Service Registry Number (CAS RN) / Description. A unique identifier for
substances that provides an unambiguous way to identify a biotoxin or biotoxin isoform when there
are many possible systematic, generic or trivial names, and/or a brief statement describing the
biotoxin.
Analysis type. Tests are either for presumptive identification, confirmatory identification or
biological activity determination; test types are described below.
Analytical technique. Type of analytical instrumentation or assay used to determine the quantity and
identity 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.
Solid (soil, powder). The recommended method/procedure to measure the analyte of interest in solid
samples.
Particulate (swabs, wipes, filters). The recommended method/procedure to measure the analyte of
interest in particulate sample collection media.
Non-drinking water. The recommended method/procedure to measure the analyte of interest in
water samples other than drinking water.
Drinking water. The recommended method/procedure to measure the analyte of interest in drinking
water samples.
Depending on site- and incident-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
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contaminant occurrence. For example, periodic episodes of some of the biotoxins (such as microcystins
[MC] and other algal biotoxins) have been detected and measured in surface waters throughout the United
States (U.S.) by methods similar to those in Appendix D (see, for example,
htto ://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.
The "analysis types" identified in Appendix D are intended to address: (1) the level of certainty of results
needed and (2) the remediation phase during which analytical support is needed (e.g., site assessment,
clearance). Many of the presumptive methods that have been selected are immunoassays, which can be
adapted for large-scale sample analysis 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 higher level of certainty than those provided by presumptive methods. 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. 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) or any other organization's use of these terms.
A tiered approach, or algorithm, can be used when implementing the analysis types, particularly when
needed to address a large number of samples. For example, methods identified as presumptive are
generally more rapid than confirmatory methods, and might be used during the initial stages of
remediation to evaluate the extent of contamination. Presumptive methods also might be used to identify
samples that should be analyzed using the more extensive confirmatory methods. Confirmatory methods
should be considered for use when: (1) presumptive analysis indicates the presence of the biotoxin, (2) a
smaller subset of samples requires analysis, or (3) as required for a tiered approach to remediation.
Depending on the goals of the remediation phase, biological activity methods may be needed because
biotoxins are sometimes 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, both biological
availability (i.e., biotoxin accessibility to site of action) and activity are required to elicit toxicity, and
some in vitro methods may not address both parameters.
Numerous analytical techniques using a variety of instrumentation have been selected and are cited in
Appendix D. It is recognized that advances in procedures for analysis of biotoxins are frequently reported
in the literature, and commercially available equipment for these analyses is evolving rapidly.
Accordingly, the individual techniques and methods listed in Appendix D are to be regarded as a starting
point - the user is encouraged to consult the SAM website (https://www.epa.gov/homeland-securitv-
research/sam) for updates. The availability of critical reagents (e.g., antibodies) and reference standards
required for the selected analytical methods might be limited; in cases where specific information
regarding their availability is known, it is provided in the method summaries throughout Section 8.2. The
biotoxin methods points of contact listed in Section 4.0 should be contacted for additional information.
Additional research on biotoxin contaminants is ongoing within the U.S. Environmental Protection
Agency (EPA) and includes the impact of disinfectants and preservatives. The presence of disinfectants
(e.g., chlorine) and/or preservatives added during water sample collection (e.g., pH adjustors, de-
chlorinating agents) may affect analytical results. When present, the impact of these agents on method
performance should be evaluated, if not previously determined. EPA's National Homeland Security
Research Center (NHSRC) continues to maintain sample collection information documents that are
intended as companions to SAM. These sample collection documents provide information regarding
sampling container/media, preservation, holding time, sample sizes and shipping, and are available at:
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https://www.epa.gov/homeland-securitv-research/sam-companion-documents-and-sample-collection-
procedures.
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, users are referred to
the 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.
There are other biotoxins that may be of interest in environmental samples, in addition to those listed in
Appendix D. It is beyond the scope of this section and Appendix D to discuss every possible biotoxin.
Some examples are included in the resources listed below; these resources also contain additional general
information that may be of use to laboratories performing biotoxin analysis.
Additional resources:
Defense Against Toxin Weapons, published by the U.S. Army Medical Research Institute of
Infectious Diseases (http://www.usamriid.armv.mil/education/defensetox/toxdefbook.pdf) contains
information regarding sample collection, biotoxin analysis and identification, as well as
decontamination and water treatment.
The Centers for Disease Control and Prevention (CDC) has additional information regarding select
agent biotoxins (http://www.cdc.gov/od/sap/sap/toxinamt.htm).
CDC's "Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition"
(https://www.cdc.gov/biosafetv/publications/bmbl5/') includes some biotoxins.
INCHEM contains both chemical and toxicity information (http: //www. inchem. org/).
The Registry of Toxic Effects of Chemical Substances (RTECS) database, 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), accessed via http://www.fbi.gov/about-us/lab/forensic-
science -communications.
The U.S. National Response Team publishes Quick Reference Guides on a number of biotoxins
(https://www.nrt.org/Main/Resources.aspx?ResourceType=Hazards&ResourceSection=2').
8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods
To determine the appropriate method for analysis of 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 row 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, drinking water or non-drinking water).
The fitness of a method for its intended use is related to data quality objectives (DQOs) for a particular
remediation activity. The tiers below have been assigned to the methods selected for each biotoxin/sample
type pair to indicate a level of method usability for the specific biotoxin and sample type for which it has
been selected. 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), and
appropriate QC. The sample types reflect representative examples and are not necessarily inclusive of all
sample types that might be encountered by laboratories following a contamination incident.
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Tier I: The biotoxin and sample type are both targets of the method(s). Data are available for all
aspects of method performance and QC measures supporting its use without modifications.
Tier II: The biotoxin is a target of the method, and the method has been evaluated by one or more
laboratories. The sample type may or may not be a target of the method, and available data
and/or information regarding sample preparation indicate that analyses of similar sample
types were successful. However, additional testing and/or modifications may be needed.
Tier III: The sample type is not a target of the method, and no reliable data supporting the method's
fitness for its intended use are available. Data suggest, however, that the method(s) may be
applicable with significant modification.
Once a procedure has been identified in Appendix D, the corresponding procedure summary can be found
in Section 8.2. Section 8.2 follows the organization of Appendix D, with biotoxins listed in alphabetical
order and method summaries provided for each analysis type. Where available, a direct link to the
references cited or to a source to obtain the reference cited is provided with the method summary. For
additional information on sample preparation procedures and methods available through consensus
standards organizations, please use the reference contact information provided in Table 8-1.
Table 8-1. Sources of Biotoxin Methods
Name
Publisher
Reference
EPA Analytical Methods
EPA Office of Water (OW)
EPA NHSRC
https://www.epa.qov/homeland-securitv-
research/sam
Food and Drug Administration (FDA),
Bacteriological Analytical Manual (BAM)
Online
FDA
httD://www.fda.aov/Food/FoodScienceRe
search/LaboratorvMethods/ucm2006949.
htm
Official Methods of Analysis of AOAC
International*
AOAC International
http://www.aoac.orq
American Public Health Association
(APHA) Press Compendium
American Public Health
Association
httD://www.aDha.ora
Analytical Biochemistry*
Science Direct
http://www. sciencedirect.com/
Analytical Chemistry*
American Chemical Society
(ACS)
http ://www. acs. o rq/
Analytical and Bioanalytical Chemistry*
Springer
httD://www. SDrinaer.com/chemistrv/analvti
cal+chemistrv/iournal/216
Analytical Methods*
Royal Society of Chemistry
http://www.rsc.orq/iournals-books-
databases/about-iournals/analvtical-
methods/
Applied and Environmental
Microbiology*
American Society for
Microbiology (ASM)
http://aem.asm.orq/
Austin Immunology*
Austin Publishing Group
http://austinpublishinqqroup.com/austin-
immunoloqv/
Biosecurity and Bioterrorism:
Biodefense Strategy, Practice, and
Science*
Health Security
http://online.liebertpub.com/bsp
Canadian Journal of Microbiology*
NRC Research Press
http://www.nrcresearchpress.com/loi/cim
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Name
Publisher
Reference
Environmental Pollution*
Elsevier Science Publishers
https://www. journals, elsevier. com/environ
mental-pollution/
Environmental Science and
Technology*
ACS Publications
httD://Dubs.acs.ora/Daae/esthaa/about.htm
J
Food Chemistry*
Elsevier Science Publishers
https://www. journals. elsevier.com/food-
chemistrv
Food Research International*
Canadian Institute of Food
Science and Technology
http://www. sciencedirect.com/science/iour
nal/09639969
Forensic Science International*
Elsevier Science Publishers
httDs://www. iournals.elsevier.com/Forens
ic-Science-lnternational
Harmful Algae*
Elsevier Science Publishers
https://www. iournals.elsevier.com/harmfu
l-alaae
International Journal of Food
Microbiology*
Science Direct
http://www. sciencedirect.com/science/iou
rnal/01681605
Journal of Agricultural and Food
Chemistry*
ACS Publications
httD://Dubs.acs.ora/iournal/iafcau
Journal of AOAC International*
AOAC International
http://aoac.orq/aoac prod imis/AOAC/Pu
blications/Journal Of AOAC/AOAC Me
mber/Publications/Journal of AOAC/The
Journal of AOAC.aspx
Journal of Chromatography A*
Elsevier Science Publishers
https://www. journals, elsevier. com/iournal
-of-chromatoaraphv-a/
Journal of Clinical Immunology
Austin Publishing Group
https://www. journals, elsevier. com/clinical
-immunoloqv
Journal of Food Protection*
International Association for
Food Protection
http://vwvw.foodprotection.ora/
Journal of the Mexican Chemical
Society*
Mexican Chemical Society
http://www.imcs.ora.mx/index.php/imcs
Journal of Pharmaceutical and
Biomedical Analysis*
Elsevier Science Publishers
https://www.iournals.elsevier. com/iournal
-of-pharmaceutical-and-biomedical-
analvsis
Journal of the Science of Food and
Agriculture*
Society of Chemical
Industry
http://onlinelibrarv.wilev.eom/iournal/10.1
002/(ISSNH 097-0010/issues?vear=2014
Journal of Shellfish Research*
National Shellfisheries
Association
http://www.bioone.ora/toc/shre/current
Letters in Applied Microbiology
Wiley Online Library
http://onlinelibrarv.wilev.eom/iournal/10.1
111/(ISSN)1472-765X
Rapid Communications in Mass
Spectrometry*
John Wiley And Sons Ltd.
http://www.wilev.com/WilevCDA/WilevTitl
e/productCd-RCM.html
Toxicon*
Elsevier Science Publishers
http://www.iournals.elsevier.com/toxicon/
Toxins*
Molecular Diversity
Preservation
International (MDPI)
http://www.mdpi.com/iournal/toxins
* Subscription and/or purchase required.
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8.1.2 General QC Guidelines for Biotoxin Methods
Public officials must have data of known and documented quality to 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.16 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,
and on the need to support timely decisions. 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 DQOs should be derived based on those needs
and 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 additional QC (e.g.,
demonstrations of method sensitivity, precision and accuracy). It is also important to consider that, during
the course of remediation, the concentration of biotoxins and these interferences are expected to change,
potentially affecting certain analytical systems. For example, some immunologically based approaches
and mass spectrometer designs - due to the underlying chemistry and physics - can yield false negatives
or inaccurately low results if the amount of biotoxins and/or interferences exceeds the test's design
criteria. Procedural and QC steps (e.g., dilution, matrix spikes) should be applied to ensure appropriate
method performance for concentratinos in sample taken throughout the remediation, including higher
initial concentrations than might normally be expected in environmental samples.
The following describes a minimum set of QC samples and procedures that should be conducted for all
analyses. Method-specific QC requirements may be included in some of the procedures cited in this
document, and will be referenced in any EPA methods that are developed to address specific analytes and
sample types of concern. Individual methods, sampling and analysis protocols, or contractual statements
of work 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:
Blanks (e.g., method blanks, matrix blanks, solvent blanks, calibration blanks, reagent blanks)
Positive test samples / negative test samples
Calibration check samples
Use of test kits and reagents prior to expiration date
Accurate temperature controls (sample and reagent storage)
Examples of sufficient QC for the confirmatory tests listed in Appendix D include:
Demonstration that the measurement system is operating properly
~ Initial calibration
~ Method blanks
Demonstration of measurement system suitability for intended use
~ Precision and recovery (verify measurement system has adequate accuracy)
~ Analyte/sample type/level of concern-specific QC samples (verify that measurement system has
adequate sensitivity at levels of concern)
16 Information regarding EPA's DQO process, considerations, and planning is available at:
https://www.epa. gov/aualitv.
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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 4.0 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
All appropriate safety precautions should 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. 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. Additional information may be found in the following resources:
American Biological Safety Association, Risk Group Classifications for Infectious Agents. Available
at: https://my.absa.org/Riskgroups
CDC. 2009. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition.
Available at: http://www.cdc.gov/biosafetv/publications/bmbl5/index.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 - Title 42 of the Code of Federal Regulations part 72 (42 CFR part 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.ecfr.gov/
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 1910.1450. Occupational
Exposure to Hazardous Chemicals in Laboratories. Available at:
http://www.gpo.gov/fdsvs/pkg/CFR-2014-title29-vol6/pdf/CFR-2014-title29-vol6-secl910-1450.pdf
OSHA - 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response
U.S. Department of Agriculture (USDA) - 9 CFR part 121. Possession, Use, and Transfer of Select
Agents and Toxins
The Electronic Code of Federal Regulations (e-CFR). Available at: http://www.ecfr.gov/
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8.2 Method Summaries
Summaries of the analytical methods for the biotoxins listed in Appendix D are provided in Sections 8.2.1
through 8.2.17. Each section contains a brief description of the analytical methods selected, and links to,
or sources for, obtaining full versions of the methods. For information regarding sample collection
considerations for samples to be analyzed by these methods, see the companion Sample Collection
Information Document for Chemicals, Radiochemicals and Biotoxins - Companion to SAM 2017.
8.2.1 Abrin/Abrine
CAS RN (Abrin): 1393-62-0
CAS RN (Abrine): 526-31-8
Description (Abrin): Toxin found in the seeds of jequirity pea (or rosary pea) plants. Contains
glycoproteins, and consists of deadenylase (25-32 kDa A chain) and lectin (35 kDa B chain); an
agglutinin (A2B2) may be present in crude preparations.
Description (Abrine): Small molecule, indole alkaloid marker for abrin.
Selected Methods
Analysis Type
Analytical Technique
Section
Biosecurity and Bioterrorism:
Biodefense Strategy, Practice, and
Science. 2014. 12(1): 49-62
Presumptive
Lateral Flow Immunoassay
(LFA)
8.2.1.1
EPA/600-R-13/022. 2013. Version 1.0
Presumptive
Liquid Chromatography-Tandem
Mass Spectrometry
(LC-MS-MS)
8.2.1.2
Journal of Food Protection. 2008.
71(9): 1868-1874
Confirmatory
Immunoassays (Enzyme-Linked
Immunosorbent Assay [ELISA]
and Electrochemiluminescence
[ECLD
8.2.1.3
Analytical Biochemistry. 2008.
378: 87-89
Biological Activity
Enzyme activity
8.2.1.4
8.2.1.1 Presumptive Analysis
Analytical Technique: LFA
Method Developed for: Abrin in buffer, aerosol filters, and food powders
Method Selected for: These procedures have been selected for presumptive analysis of abrin in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This assay involves a lateral flow immunochromatographic device that
uses two antibodies in combination to specifically detect target antigen in solution. One of the
specific antibodies is labeled with a colloidal gold derivative. Samples applied to the test strips
mix with the colloidal gold-labeled antibody and move along the strip membrane by capillary
action. The second specific antibody captures the colloidal gold-labeled antibody and bound
target. When a sufficient amount of target antigen is present, the colloidal gold label accumulates
in the sample window on the test strip, forming a visible reddish-brown colored line. As an
internal control, a second band in the control window indicates that the test strip functioned
properly. Two bands or colored lines (in the sample and control windows) are required for a
positive result determination. To complete a test on a liquid sample, the sample is mixed with the
provided buffer, and five or six drops are added to the sample well of the test strip. A positive
result is indicated by the appearance of a colored line in the test window of the test strip and can
be read visually or with a reader.
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The method source (below) describes a multicenter evaluation of the sensitivity, specificity,
reproducibility, and limitations of the LFA for abrin that can be used as a rapid qualitative test to
detect the presence of abrin in environmental samples. Samples analyzed in this study included
various powders of food and non-food substances (prepared in buffer) and aerosol samples (filter
extracts). Using the test strip and the manufacturer's recommended threshold, the estimated limit
of detection (LOD) of the LFA is approximately lOng/mL or 1.5ng/reaction, which is well below
clinically relevant levels (median lethal dose [LD50] 3.3mg/kg inhaled and 20mg/kg ingested).
Because this assay does not discriminate between abrin and Abrus precatorius agglutinin-1
(APA-1), it can only be used as a qualitative screening assay when testing unknown samples.
Special Considerations: This LFA is listed as Tier I for presumptive analysis of abrin in solid
and aerosol samples, and Tier II for presumptive analysis of abrin in other environmental sample
types. Like some other types of immunoassays, this assay is subject to the "hook effect," which is
an interference that occurs when analyte is present in amounts significantly higher than the
amounts for which the assay was designed. The end result is a decreased response and, under
extreme conditions, a false-negative. The incorporation of a serial dilution step in the sample
protocol can eliminate such potential errors.
Source: Ramage, J.G., Prentice, K.W., Morse, S.A., Carter, A.J., Datta, S., Drumgoole, R.,
Gargis, S.R., Griffin-Thomas, L., Hastings, H.P., Masri, H.P., Reed, M.S., Sharma, S.K., Singh,
A.K., Swaney, E., Swanson, T., Gauthier, C., Toney, D., Pohl, J., Shakamuri, P., Stuchlik, O.,
Elder, I.A., Estacio, P.L., Farber, E.A.E., Hojvat, S., Kellogg, R.B., Kovacs, G., Stanker, L.,
Weigel, L., Hodge, D.R. and Pillai, S.P. 2014. "Comprehensive Laboratory Evaluation of a
Specific Lateral Flow Assay for the Presumptive Identification of Abrin in Suspicious White
Powders and Environmental Samples." Biosecurity and Bioterrorism: Biodefense Strategy,
Practice, and Science. 12(1): 49-62.
htto: //online. liebertpub. com/bsp
8.2.1.2 Presumptive Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Abrine in drinking water
Method Selected for: This method has been selected for presumptive analysis of abrin by abrine
detection in aerosol, solid, particulate 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 other than drinking water.
Description of Method: This method involves solid-phase extraction (SPE) of samples,
followed by analysis of the extracts for abrine by liquid chromatography and LC-MS-MS.
Samples are combined with isotopically-labeled internal standards and sample extracts are
concentrated to dryness under nitrogen and heat, then adjusted to a I ()0-|llL volume in high
performance liquid chromatography (HPLC)-grade water. Accuracy and precision data are
provided for application of the method to reagent water, as well as finished ground water and
surface waters containing residual chlorine and/or chloramine. The method has a detection limit
(DL) of 0.06 (ig/mL and a minimum reporting level (MRL) of 0.50 (ig/mL for abrine. The
stability of 50 (ig/mL abrine was tested in ground water and surface water samples stored at 4ฐC
for up to 28 days. Percent recoveries of abrine were significantly reduced in chlorine-containing
samples after five hours. Percent recoveries for chlorine-containing samples at five hours ranged
from 9 to 21; percent recoveries at 28 days ranged from 8 (ฑ 0) to 19% (ฑ 1). Abrine was much
more stable in monochloramine-containing samples. Percent recoveries for monochloramine-
containing samples at 5 days ranged from 103 (ฑ 3) to 107 (ฑ 3); percent recoveries at 28 days
ranged from 90 (ฑ 4) to 93 (ฑ 5).
Special Considerations: This method is listed as Tier I for presumptive analysis of abrin (as
abrine) in drinking water and Tier II for presumptive analysis of abrin (as abrine) in all other
environmental sample types. Performance data were generated using preserved water samples. If
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Section 8.0 - Selected Biotoxin Methods
concentrations of abrine are unexpectedly low (or absent), additional QC steps may be needed.
The biotoxin methods points of contact listed in Section 4.0 should be consulted for additional
information regarding analysis of sample types other than drinking water. While abrine can be
used to indicate the presence of abrin, it can also be found alone, which can limit the usefulness
of this determination.
Source: U.S. EPA and CDC. August 2013. "High Throughput Determination of Ricinine,
Abrine, and Alpha-Amanitin in Drinking Water by Solid Phase Extraction and High Performance
Liquid Chromatography Tandem Mass Spectrometry (HPLC/MS/MS)," Version 1.0. Cincinnati,
OH: EPA/Atlanta, GA: CDC. EPA 600/R-13/022.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100I5I0.PDF?Dockev=P 100I5I0.PDF
Additional Resource: Knaack, J.S., Pittman, C.T., Wooten, J.V., Jacob, J.T, Magnuson, M.,
Silvestri, E. and Johnson, R.C. 2013. "Stability of ricinine, abrine, and alpha-amanitin in finished
tap water." Analytical Methods. 20(5): 5804-5811.
8.2.1.3 Confirmatory Analysis
Analytical Technique: Immunoassay (ELISA and ECL-based immunoassay)
Method Developed for: Abrin in food
Method Selected for: These procedures have been selected for confirmatory analysis of abrin in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: These commercially available immunoassays are used for detecting
abrin in various food products. The procedures use mouse monoclonal antibodies (mAbs) and
rabbit-derived polyclonal antibodies prepared against a mixture of abrin isozymes for three
separate ELISA and ECL-based assays. 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 has been shown to pose a health concern.
Special Considerations: These procedures are listed as Tier II for confirmatory analysis of
abrin in aerosol, solid, particulate and water samples. Sample preparation procedures used for
foods suggest that similar aqueous extraction procedures may be applicable to environmental
samples. 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 of Artifacts Associated
With Wheat Lectin." Journal of Immunological Methods. 336(2): 251-254.)
http: //www. sciencedirect. com/science/i ournal/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://ifoodprotection.Org/doi/abs/10.4315/0362-028X-71.9.1868
8.2.1.4 Analysis of Biological Activity
Analytical Technique: Enzyme activity
Method Developed for: Jequirity seed (abrin) and castor bean (ricin) extracts in buffer
Method Selected for: These procedures have been selected for biological activity analysis of
abrin in aerosol, solid, particulate and water samples. Further research is needed to adapt and
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Section 8.0 - Selected Biotoxin Methods
verify the procedures for environmental sample types.
Description of Method: This in vitro assay is a ribonucleic acid (RNA) N-glycosidase enzyme
activity assay that can be used for the detection of purified abrin or abrin in jequirity seed
extracts. The method can be applied to both abrin and ricin, due to the similarity in enzyme
activities of the two toxins. Synthetic biotinylated RNA substrates with varied loop sequences are
cleaved by abrin toxin and the RNA products are hybridized to ruthenylated-
oligodeoxynucleotides to generate an ECL signal. Assays require incubation for 2 hours at 48ฐC.
Commercially available ECL-based reagents and ribonuclease (RNAse) inactivators are used.
Control experiments for the jequirity seed experiments demonstrate lack of non-specific cleavage
for the assay. The undiluted jequirity seed extract was 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.
Undiluted jequirity seed extract contained a calculated level of 740 ฑ 50 (ig/mL ricin activity
equivalents, which greatly exceeded the immunoassay-based value.
Special Considerations: These procedures are listed as Tier II for analysis of the biological
activity of abrin in aerosol, solid, particulate and water samples. The enzyme activity assay does
not test for cell binding; other cell binding assays are in development, but are not currently
available. At this time, the mouse bioassay is the only readily available assay to test for both cell
binding and enzymatic activity of the intact (whole) toxin.
Source: Keener, W.K., Rivera, V.R., Cho, C.R., Hale, M L., Garber, E.A.E. and Poli, M.A.
2008. "Identification of the RNA N-glycosidase Activity of Ricin in Castor Bean Extracts by an
Electrochemiluminescence-Based Assay." Analytical Biochemistry. 378(1): 87-89.
htto://www.sciencedirect.com/science/iournal/00032697
8.2.2 Aflatoxin
CAS RN: 27261-02-5 (Bl), 22040-96-6 (B2), (1385-95-1 (Gl), 7241-98-7 (G2)
Considered Variants: Aflatoxins Bl, B2, Gl and G2
Description: Toxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus.
Composed of difiiranocoumarin molecules; B-group aflatoxins (Bl and B2) have a cyclopentane
ring, while the G-group aflatoxins (Gl and G2) contain a lactone ring
Selected Methods
Analysis Type
Analytical Technique
Section
AOAC Method 991.31
Presumptive and Confirmatory
Immunoaffinity (column)
purification / LC-FL (detection)
8.2.2.1
8.2.2.1 Presumptive and Confirmatory Analysis
Analytical Technique: Immunoaffinity column purification and LC-fluorescence detector (FL)
Method Developed for: Aflatoxins in corn, raw peanuts and peanut butter
Method Selected for: These procedures have been selected for presumptive and confirmatory
analyses of aflatoxins in aerosol, solid, particulate and water samples. Further research is needed
to adapt and verify the procedures for environmental sample types.
Description of Method: This method involves extraction of samples with methanol/water,
followed by sample filtration, dilution with water, and application to a commercially available
affinity column containing mAbs specific for aflatoxins Bi, B2, Gi and G2 Antibody-bound
aflatoxins are eluted from the column with methanol. Reaction with bromine solution and
subsequent fluorescence measurement is performed for detection and quantitation of total
aflatoxins. Post-column iodine derivatization and LC-FL are performed for quantitation of
individual aflatoxins. Method performance was characterized using various commodities (e.g.,
corn) for aflatoxin levels ranging from 10 to 30 ng/g.
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Special Considerations: These procedures are listed as Tier II for presumptive and
confirmatory analysis of aflatoxins in aerosol, solid, particulate and water samples. The 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. Research results
indicate that clean up and analyte concentration in water samples can be accomplished using an
immunoaffinity column containing a mAbs specific for aflatoxin Bi, B2, Gi and G2 serotypes
(Paterson, 2007, see additional resource citation below). Additional research indicates that soil
samples can be extracted overnight in water/ethyl acetate and the supernatant evaporated to
dryness, reconstituted in methanol/water, then filtered and eluted through a mini-column packed
with aluminum oxide (Accinelli, 2008, see additional resource citation below). The method notes
that AOAC Official Method 994.08: Aflatoxin in Corn, Almonds, Brazil Nuts, Peanuts, and
Pistachio Nuts, which uses derivatization and a multifunctional clean-up column, can be used as a
complementary LC-FL procedure.
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/
Additional Resources:
AOAC International. 1998. AOAC Official Method 994.08: Aflatoxin in Corn, Almonds,
Brazil Nuts, Peanuts, and Pistachio Nuts. Official Methods of Analysis of AOAC
International, 16th Edition. 4th Revision, Vol. II. http://www.aoac.org/.
Paterson, R. R. M., Kelly, J. and Gallagher, M. 2007. "Natural occurrence of aflatoxins and
Aspergillus flavus (Link) in water." Letters in Applied Microbiology. 25: 435-436.
Accinelli, C., Abbas, H. K., Zablotowsicz, R. M. and Wilkinson, J. R. 2008. "Asperigjullus
flavus aflatoxin biosynthesis genes in soil." Canadian Journal of Microbiology. 54: 371-379.
8.2.3 a-Amanitin
CAS RN: 23109-05-9
Considered Variants: NA
Description: Toxins produced by the poisonous death cap mushroom, Amanitaphalloides. One
of a group of thermostable bicyclic octapeptides
Selected Methods
Analysis Type
Analytical Technique
Section
Journal of Food Protection. 2005.
68(6): 1294-1301
Presumptive
Immunoassay (ELISA)
8.2.3.1
EPA 600/R-13/022
Confirmatory
LC-MS-MS
8.2.3.2
8.2.3.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: a-Amanitin, ricin and T-2 mycotoxin in food and beverages
Method Selected for: These procedures have been selected for presumptive analysis of a-
amanitin and T-2 toxin (see Section 8.2.16.1) in aerosol, solid, particulate and water samples.
Further research is needed to adapt and verify the procedures for environmental sample types.
Description of Method: These commercially available ELISAs detect a-amanitin in a variety of
food and beverage samples at levels below those shown to be a health concern. Solid food
samples are extracted with sodium phosphate buffer, then diluted with phosphate-buffered saline.
Liquid beverage samples are diluted in sodium phosphate buffer. Prepared samples are analyzed
according to the kit 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
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37ฐC. a-Amanitin can be detected in food products at 1 jxg/g. Background responses are noted for
some samples, but at less than the equivalent of 0.5 jxg/g for most samples.
Special Considerations: These procedures are listed as Tier II for presumptive analysis of a-
amanitin in aerosol, solid, particulate and water samples. Sample preparation procedures used for
foods and beverages suggest that similar procedures may be applicable to environmental samples.
If appropriate concentrations are present, sample dilution can be used to minimize background
interferences.
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://ifoodprotection.Org/doi/abs/10.4315/0362-028X-68.6.1294?=
8.2.3.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Determination of ricinine, abrine, and a-amanitin in drinking water
Method Selected for: This method has been selected for presumptive analysis of a-amanitin in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for sample types other than drinking water.
Description of Method: An isotope dilution LC-MS-MS method is used for the determination of
a-amanitin in drinking water. Sample aliquots are combined with mixture containing an internal
quantification standard for a-amanitin, pipetted into a 96-well SPE plate, and extracted. Extracts
are concentrated to dryness under nitrogen and heat, adjusted to 100 |iL with HPLC-grade water,
and injected onto an HPLC-MS-MS operated in MRM mode. a-Amanitin is identified by
comparing the acquired mass spectra, including ion ratios and retention times, to reference
spectra and retention times for calibration standards acquired under identical HPLC-MS-MS
conditions. Quantitation is performed using the internal standard technique.
Special Considerations: These procedures are listed as Tier I for confirmatory analysis of a-
amanitin in drinking water and Tier II for confirmatory analysis of a-amanitin in all other
environmental sample types. Extraction of non-aqueous samples prior to SPE may be required
(Kaya, 2013). These procedures might be modified for application to soil, air filter and particulate
samples. Isotopically labeled a-amanitin internal standards were not available during method
development, but efforts to synthesize these standards are in progress. The biotoxin contacts
listed in Section 4.0 should be consulted for current status and availability of a-amanitin internal
standards.
Source: U.S. EPA and CDC. August 2013. "High Throughput Determination of Ricinine,
Abrine, and Alpha Amanitin in Drinking Water by Solid Phase Extraction and High Performance
Liquid Chromatography Tandem Mass Spectrometry (HPLC/MS/MS)," Version 1.0. Cincinnati,
OH: EPA/Atlanta, GA: CDC. EPA 600/R-13/022.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100I5I0.PDF?Dockev=P 100I5I0.PDF
Additional Resource: Kaya, E., Yilmaz, I., Sinirlioglu, Z.A., Karahan, S., Bayram, R.,
Yaykasli, K.O., Colakoglu, S., Saritas, A. and Severoglu, Z. 2013. "Amanitin and pallotoxin
concentration in Amanita phalloides var. alba mushroom." Toxicon. 76: 225-233.
http://www.sciencedirect.eom/science/article/pii/S0041010113003942
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8.2.4 Anatoxin-a
CAS RN: 64285-06-9
Considered Variants: NA
Description: Tropane-related bicyclic alkaloid produced by a variety of cyanobacteria species
Selected Methods
Analysis Type
Analytical Technique
Section
See Source Citation
Presumptive
Immunoassay (ELISA)
8.2.4.1
EPA Method 545
Confirmatory
LC-MS-MS
8.2.4.2
8.2.4.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Anatoxin-a in drinking and recreational water
Method Selected for: This immunoassay procedure has been selected for presumptive analysis
of anatoxin-a in aerosol, solid, particulate and water samples. Further research is needed to adapt
and verify the procedures for environmental sample types.
Description of Method: This commercially available immunoassay is a direct competitive
ELISA based on the recognition of anatoxin-a by a mAbs. When present in a sample, anatoxin-a
and an anatoxin-a-enzyme conjugate compete for the binding sites of mouse anti-anatoxin-a
antibodies in solution. The anatoxin-a antibodies are then bound by a second antibody (anti-
mouse) immobilized on the microtiter plate. After a wash step and addition of the substrate
solution, a color signal is generated. The color reaction is stopped after a specified time and the
color is evaluated using an ELISA reader. The intensity of the blue color is inversely proportional
to the concentration of anatoxin-a present in the sample. The concentrations of the samples are
determined by interpolation using the standard curve constructed with each run. A DL of
approximately 0.1 (ig/L, based on anatoxin-a, is reported by the manufacturer of this kit.
Special Considerations: This commercial ELISA is currently listed as Tier III for presumptive
analysis of anatoxin-a in aerosol, solid, particulate and water samples, pending results of ongoing
assay verification in water samples. Please consult with the technical contacts provided in Section
4.0 for information regarding the status of verification efforts.
Source: A commercially available ELISA immunoassay kit is currently undergoing testing and
verification. Updates will be provided once the results are available. Please consult technical
contacts listed in Section 4.0 for information regarding the status of these results and availability
of corresponding procedures.
8.2.4.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Anatoxin-a in potable water
Method Selected for: This method has been selected for confirmatory analysis of anatoxin-a in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types other than drinking water.
Description of Method: This method is used to detect anatoxin-a in water samples. Samples are
frozen and thawed three times, then filtered. A 1-mL sample aliquot from the supernatant is
combined with internal standards and analyzed using LC-MS-MS with ESI. Anatoxin-a is
identified by comparing retention times and signals produced by unique mass to those of
procedural calibration standards acquired under identical LC-MS-MS conditions. The
concentration of each analyte is determined using the integrated peak area and the internal
standard technique. The method reports a lowest concentration minimum reporting level
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(LCMRL) of 0.018 (ig/L for anatoxin-a in fortified reagent water. The working range reported in
the method is 0.029-5.87 (ig/L.
Special Considerations: This method is listed as Tier I for confirmatory analysis of anatoxin-a
in drinking water and Tier II for confirmatory analysis of anatoxin-a in all other environmental
sample types. Samples containing intact cyanobacteria must be treated to disrupt the cells in order
to recover intracellular toxins. It may be possible to analyze relatively clean water samples by
direct injection into the LC-MS-MS. Extraction of anatoxin-a from cyanobacterial biocrusts has
been achieved (Chrapusta, 2015). These procedures might be modified for application to soil, air
filter and particulate samples, for analysis using the LC-MS-MS conditions described in EPA
Method 545. LC-MS-MS methods for anatoxin-a, cylindrospermopsin and microcystins also have
been developed by EPA's Office of Research and Development, and are currently undergoing
review. Once published, these methods will be provided on the SAM website
https://www.epa.gov/homeland-securitv-research/sam.
Source: U.S. EPA. April 2015. Method 545: Determination of Cylindrospermopsin and
Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem Mass
Spectrometry (LC/ESI-MS/MS). Cincinnati, OH: U.S. EPA. EPA 815-R-15-009.
https://www .epa.gov/sites/production/files/2017-1O/documents/epa 815 -r-15 -
009 method 545.pdf
Additional Resource: Chrapusta, E., Wegrzyn, M., Zabaglo, K., Kaminski, A., Adamski, M.,
Wietrzyk, P. and Bialczyk, J. 2015. "Microcystins and anatoxin-a in Arctic biocrust
cyanobacterial communities." Toxicon. 101: 35-40.
http://www.sciencedirect.com/science/article/pii/S0041010115001130
8.2.5 Botulinum Neurotoxins (BoNTs)
CAS RN: 93384-43-1 (BoNT-A), 93384-44-2 (BoNT-B), 93384-45-3 (BoNT-C), 93384-46-4
(BoNT-D), 93384-47-5 (BoNT-E), 107231-15-2 (BoNT-F), 107231-16-3 (BoNT-G)
Considered Variants: A-G
Description: Protein neurotoxin produced by Clostridium botulinum and related species.
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.
Selected Methods
Analysis Type
Analytical Technique
Section
EPA Environmental Technology
Verification (ETV) Program Reports
Presumptive
Immunoassay (LFA)
8.2.5.1
Analytical Biochemistry. 2011. 411
(2): 200-209
Presumptive
Immunocapture-Forster
Resonance Energy Transfer
(FRET)-based activity assay
8.2.5.2
U.S. Department of Homeland
Security (DHS) Report
Presumptive
Immunoassay (fluorescent
bead-based)
8.2.5.3
Journal of the Science of Food and
Agriculture. 2014. 94: 707-712
Presumptive
Immunoassay (ECL)
8.2.5.4
LC-MS-MS
Journal of Agricultural and Food
Chemistry. 2015. 63(4): 1133-1141
Confirmatory
Matrix-assisted laser desorption
ionization-time-of-flight mass
spectrometry (MALDI-TOF-MS)
8.2.5.5
APHA Press Compendium of
Methods, Chapter 32
Biological Activity
Mouse bioassay
8.2.5.6
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8.2.5.1 Presumptive Analysis
Analytical Technique: Immunoassay (LFA)
Method Developed for: BoNTs (Types A, B) in buffer or water samples
Method Selected for: These procedures have been selected for presumptive analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types other than water.
Description of Method: The commercial test strip is a lateral flow immunochromatographic
device that uses two antibodies in combination to specifically detect target antigen in solution.
One of the specific antibodies is labeled with a colloidal gold derivative. Samples applied to the
test strips mix with the colloidal gold-labeled antibody and move along the strip membrane by
capillary action. The second specific antibody captures the colloidal gold-labeled antibody and
bound target. When a sufficient amount of target antigen is present, the colloidal gold label
accumulates in the sample window on the test strip, forming a visible reddish-brown colored line.
As an internal control, a second band in the control window indicates that the test strip functioned
properly. Two bands or colored lines (in the sample and control windows) are required for a
positive result determination. For a liquid sample, the sample is mixed with the provided buffer,
and five or six drops are added to the sample well of the test strip. A positive result is indicated
by the appearance of a colored line in the test window of the test strip and is read visually or with
a reader.
The LFA kits have been evaluated by the EPA ETV Program for the detection of BoNTs Types A
and B in concentrated (ultrafiltration [UF]) and unconcentrated drinking water. The reference
source reports the lowest detectable concentration of BoNT Type A as 0.01 mg/L and Type B as
0.05 mg/L, with no false negatives detected when interferents are present. Reports and
information associated with these evaluations are available at:
http://www.epa.gov/sites/production/files/2015-07/documents/etv-biothreat092104.pdf.
Special Considerations: This assay is listed as Tier I for presumptive analysis of BoNTs in
drinking water samples and Tier II for presumptive analysis of BoNTs in other environmental
sample types. Like some other types of immunoassays, this assay is subject to the "hook effect,"
which is an interference that occurs when analyte is present in amounts significantly higher than
the amounts for which the assay was designed. The end result is a decreased response and, under
extreme conditions, a false-negative.The incorporation of a serial dilution step can eliminate such
potential errors.
Source: Environmental Technology Verification Report. 2004. Anthrax, Botulinum Toxin, and
Ricin Immunoassay Test Strips; available at:
http://www.epa.gov/sites/production/files/2015-07/documents/etv-biothreat092104.pdf.
8.2.5.2 Presumptive Analysis
Analytical Technique: Immunocapture FRET-based activity assay
Method Developed for: BoNTs Serotypes A, B, D, E, F and G
Method Selected for: These procedures have been selected for presumptive analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercial FRET-based assay detects BoNT serotypes A and E,
and serotypes B, D, F and G, using separate kits for each serotype group. The assays measure the
ability of BoNTs to proteolytically cleave synthetic substrates that mimic the natural BoNT
substrates (SNAP25 or VAMP2) in a sensitive, FRET-based format using most standard
fluorescent plate readers. The substrates used in the assay encompass both the exosite binding
sites and cleavage site of BoNT, resulting in high BoNT affinity for the substrate with
femtomolar to picomolar detection sensitivities within a few minutes to a few hours. The FRET-
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based nature of the assays allows for real-time detection of BoNT proteolytic activity, enabling
the determination of kinetic constants and enzymatic activity.
Special Considerations: This assay is listed as Tier II for presumptive analysis of BoNTs in
aerosol, solid, particulate and water samples. Application of the assay to complex or dilute
sample types may require a preliminary antibody capture/enrichment procedure using magnetic
beads conjugated to serotype-specific antibodies. Non-liquid samples such as soils, powders and
aerosol filters will require an aqueous extraction step prior to antibody capture. The FRET-based
detection assay and antibody-coated beads are both commercially available.
Source: Ruge, D.R., Dunning, F.M., Piazza, T.M., Molles, B.E., Adler, M., Zeytin, F.N. and
Tucker, W.C. 2011. "Detection of six serotypes of BoNT using fluorogenic reporters." Analytical
Biochemistry. 411: 200-209.
httos: //www .ncbi .nlm .nih. gov/pubmed/21216216
8.2.5.3 Presumptive Analysis
Analytical Technique: Immunoassay (fluorescent bead-based)
Method Developed for: BoNT Serotypes A, B, C, D, E, F and G
Method Selected for: These procedures have been selected for presumptive analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This multiplexed immunoassay is based on a commercially available
technology that uses antibody-coated fluorescent polystyrene microspheres or beads as an
immunoassay reaction surface. The beads are optically encoded internally with two spectrally
distinct fluorescent dyes which identify each of the beads. Using a fluidics system, individual
beads pass by a red laser that identifies each bead set based on their unique internal dye
signatures and, hence, the antigen specificity assigned to each bead set. A green laser is used to
detect a third spectrally distinct fluorescent dye that quantifies the extent of antibody-antigen
reactions on the surface of each bead. Distinct bead sets, coupled with unique capture antibodies
with specific reactivity to each of the seven BoNT Serotypes (A-G), are added as a mixture to an
unknown sample. As specific beads contact specific antigenic components in the sample, the
BoNTs are captured on the surface of the beads. The mixture is washed to remove unbound
sample; then a biotin-labeled detection antibody is added and allowed to bind to the complex,
followed by addition of fluorescent reporter streptavidin phycoerythrin (SA-PE). The fluorescent
intensity of the reporter as read by the fluorescence reader is proportional to the amount of toxin
bound to the bead. LODs ranged from 20 to 200 pg/mL for each serotype, which is similar to the
definition of 1 mouse LD50 by the mouse bioassay.
Special Considerations: This method is listed as Tier II for presumptive analysis of BoNTs in
aerosol, solid, particulate and water samples. Non-liquid samples such as soils, powders and
aerosol filters will require aqueous extraction and clarification (e.g., centrifugation) prior to assay.
Source: DHS. 2015. Multi-agency Report. Rapid Botulinum Toxin Assay Test, Evaluation and
Validation Study Report. [Note: This document is available only to select government agencies.
Please consult technical contacts listed in Section 4.0 for additional information regarding this
report. ]
8.2.5.4 Presumptive Analysis
Analytical Technique: Immunoassay (ECL)
Method Developed for: BoNT A in milk products
Method Selected for: These procedures have been selected for presumptive analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
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procedures for environmental sample types.
Description of Method: This commercial imaging-based ECL immunoassay detects BoNT-A.
The imaging-based ECL detection method is a sandwich-format immunoassay that uses a target-
specific immobilized capture antibody and a biotinylated detection antibody. The ECL assay uses
96-well multiarray assay plates with integrated screen-printed carbon electrodes. The electrodes
act both as solid phase supports to capture reagents used in the solid phase binding assays, and as
the source of electrical energy for inducing ECL. The basis of the ECL measurement is the ability
of these labels to emit light when oxidized at an electrode surface in the presence of tertiary
amine (i.e. tripropylamine). During the ECL measurement, the plate reader applies a voltage to
electrodes in the wells of the multiarray plates and measures the resulting ECL from labeled
detector antibodies incorporated in sandwich complexes on each spot in the well with a cooled
charge-coupled device (CCD) sensor. The LOD of this ECL assay is 40 pg/mL for BoNT-A
complex. The additional resource listed below (Cheng, L.W. and Stanker, L.H. 2013) describes
the use of a similar ECL immunoassay for detection of both BoNT-A and BoNT-B in different
liquids, liquified solid foods, and horse serum.
Special Considerations: This method is listed as Tier II for presumptive analysis of BoNTs in
aerosol, solid, particulate and water samples. Sample preparation procedures described in both the
source and additional resource citations, suggest that similar aqueous extraction may be
applicable to environmental samples. Soils, powders and aerosol filters will require aqueous
extraction followed by clarification (e.g., centrifugation) prior to assay.
Source: Sachdeva, A., Singh, A.K. and Sharma, S.K. 2014. "An electrochemiluminescence
assay for the detection of bio threat agents in selected food matrices and in the screening of
Clostridium botulinum outbreak strains associated with type A botulism." Journal of the Science
of Food and Agriculture. 94: 707-712.
http://onlinelibrarv.wilev.com/doi/10.1002/isfa.6310/abstract
Additional Resource: Cheng, L.W. and Stanker, L.H. 2013. "Detection of Botulinum
Neurotoxin Serotypes A and B Using a Chemiluminescent versus Electrochemiluminescent
Immunoassay in Food and Serum." Journal of Agricultural and Food Chemistry. 61: 755-760.
https://www.ncbi.nlm.nih.gov/pubmed/23265581
8.2.5.5 Confirmatory Analysis
Analytical Technique: LC-MS-MS (BoNT Serotypes A, B, E and F) and MALDI-TOF-MS
(BoNT Serotypes A-G)
Method Developed for: BoNT Serotypes A, B, C, D, E, F and G in serum, stool and food
Method Selected for: These procedures have been selected for confirmatory analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to validate the
procedures for environmental sample types.
Description of Method: This Endopep-MS assay has been developed to detect specific activities
of all seven BoNT (serotypes A-G). Peptide products are cleaved by the enzymatic action of the
BoNTs on four target peptide substrates in a reaction buffer created to maximize the enzymatic
activity of the BoNT toxins. An inert peptide substrate is added as the internal standard, and the
reaction mixture is incubated at 37ฐC for a minimum of 2 hours. If present, BoNT A-G will react
with the target peptides to form cleaved peptide products that can be measured by LC-MS-MS
with ESI or MALDI-TOF-MS, although the source cited below evaluated application of LC-MS-
MS to measurement of only A, B, E and F. For MALDI-TOF-MS analysis, the reaction is
quenched by the addition of a solution of a-cyano-4-hydroxycinnamic acid (CHCA) matrix. For
LC-MS-MS analysis, the reaction is quenched by adding 5 |_iL of 10% acetic acid to the sample.
A sample aliquot is then injected into and analyzed by LC-MS-MS, or spotted onto a MALDI
sample plate and analyzed by MALDI-TOF-MS.
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Quantification of the toxins is performed by comparing the area ratios of the unknowns to those
of calibration standards. LOD concentrations are given in units of U/mL, where 1U is defined as
the enzyme catalysis of 1 micromole (fimole) of substrate per minute, and niL represents the
sample volume. LODs varied for all serotypes and ranged from 1.25 to 6.25 U/mL (MALDI-
TOF-MS) and 0.078 to 1.25 U/mL (LC-MS-MS) after four hours of incubation in the reaction
buffer; and 0.313 to 6.25 U/mL (MALDI-TOF-MS) and 0.039 to 1.00 U/mL (LC-MS-MS) after
incubation for 10 or 17 hours. The additional resource listed below (Kalb et al., 2015) describes a
modified Endopep-MS assay, using antibody-based toxin capture followed by synthetic peptide
cleavage and MALDI-TOF-MS detection. The MALDI-TOF-MS analysis required less than one
minute to record the mass spectrum, and LODs in food matrices ranged from (depending on the
serotype) 0.01 mouse lethal dose (mLD50) to 0.75 mLD50.
Special Considerations: This assay is listed as Tier II for confirmatory analysis of BoNTs in
aerosol, solid, particulate and water samples. Only qualitative information (presence/absence)
using MALDI-TOF-MS exists for BoNT-C, -D and -G, and LODs for these serotypes have not
been reported using either LC-MS-MS or MALDI-TOF-MS. The sample preparation procedures
used for food products also may be applicable to environmental sample types. Non-water samples
such as soils, powders and aerosol filters will require aqueous extraction followed by clarification
(e.g., centrifugation) prior to assay. Although procedures are not provided, the reference source
notes that this method has also been used to test human stool and serum; and results indicate no
false positives.
Source: Kalb, S.R., Krilich, J.C., Dykes, J.K., Luquez, C., Maslanka, S.E. and Barr, J.R. 2015.
"Detection of Botulinum Toxins A, B, E, and F in Foods by Endopep-MS." Journal of
Agricultural and Food Chemistry. 63(4): 1133-1141.
htto: //pubs. acs. org/doi/abs/ 10.1021/if505482b
8.2.5.6 Analysis of Biological Activity
Analytical Technique: Mouse bioassay
Method Developed for: BoNTs in food products and clinical samples
Method Selected for: These procedures have been selected for confirmatory analysis of BoNTs
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: The mouse bioassay is the standard for government agencies testing for
BoNT-containing food and clinical samples. Mice are injected with 0.5 mL sample, each sample
dilution, or a heat-inactivated control sample. Mice are observed over a period of 1 to 7 days for
symptoms of botulism and/or death. Results for the mouse bioassay are reported in median lethal
dose (LD50) units, where 1 LD50 is the amount of BoNT required to kill 50% of injected mice
after a defined time interval. The mouse bioassay has a DL of 5 - 10 pg for BoNT serotype A.
Some BoNTs are produced by non-proteolytic strains of C. botulinum (group II) and require
trypsin activation prior to testing in the mouse bioassay. It is important to observe for symptoms
of botulism in the test mice since death without clinical symptoms of botulism is not sufficient
evidence that the material injected contained BoNT. BoNT typing can be conducted by injecting
pairs of test mice with monovalent antitoxins prior to injecting with suspected toxin sample and
noting a protective or neutralizing effect of the initial sample toxicity.
Special Considerations: This assay is listed as Tier I for analysis of the biological activity of
BoNTs in aerosol, solid, particulate and water samples. It is a low-throughput, labor intensive
assay and is not suitable for screening a large number of samples.
Source: Maslanka, S.E, Solomon, H.M., Sharma, S. and Johnson, E.A. 2015. "Clostridium
botulinum and Its Toxins" APHA Press Compendium of Methods for the Microbiological
Examination of Foods. Fifth Edition, Chapter 32: 397. Washington, DC: APHA Press.
https://secure.apha.org/imis/ItemDetail?iProductCode=978-087553-2738&CATEGORY=BK
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8.2.6 Brevetoxins (BTX)
CAS RN: 98112-41-5 (A Form), 79580-28-2 (B Form)
Considered Variants: BTX-B1, BTX-B2, BTX-B5
Description: Suite of cyclic polyether neurotoxin compounds produced by a species of
dinoflagellate, Karenia brevis.
Selected Methods
Analysis Type
Analytical Technique
Section
Toxicon. 2015. 96: 82-88
Presumptive
Immunoassay (ELISA)
8.2.6.1
Toxicon. 2015. 96: 82-88
Confirmatory
LC-MS
8.2.6.2
8.2.6.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Determination of brevetoxins (BTX) in shellfish and salt water
Method Selected for: These procedures have been selected for presumptive analysis of
brevetoxins in aerosol, solid, particulate and water samples. Further research is needed to adapt
and verify the procedures for environmental sample types.
Description of Method: This commercially available ELISA is used for determination of
brevetoxin in shellfish and sea water. It is a direct competitive assay in which the antibody is
directed against the brevetoxin B-type backbone, responding to B-type brevetoxin analogues with
intact epitopic region. Brevetoxin in the sample and brevetoxin conjugated to peroxidase enzyme
compete for the binding sites of sheep antibrevetoxin antibodies that have been immobilized in
the wells of the microtiter plate. BTX-3 is used as the standard and composite toxins are
expressed as mg BTX-3 equivalents. Results are determined by measuring absorbance at 450 nm
using a microplate ELISA photometer. The LOD was calculated as 0.05 ng/mL in water. The
concentration of residue necessary to cause 50% inhibition is approximately 0.16 ng/mL.
Special Considerations: This ELISA is listed as a Tier II procedure for presumptive analysis of
brevetoxin in aerosol, solid, particulate and water samples. Sample preparation procedures used
for shellfish and sea water suggest that similar extraction procedures may be applicable to
environmental samples. Numerous organic and inorganic compounds commonly found in
samples have been tested and found not to interfere with this test. However, due to the high
variability of compounds that might be found in samples, interferences caused by matrix effects
cannot be completely excluded.
Source: Abraham, A., El Said, K.R., Wang, Y., Jester, E.L.E., Plakas, S.M., Flewelling, L.J.,
Henry, M.S., Pierce, R.H. 2015. "Biomarkers of brevetoxin exposure and composite toxin levels
in hard clam (Mercenaira sp.) exposed to Karenia brevis blooms." Toxicon. 96: 82-88.
https://www.ncbi.nlm.nih.gov/pubmed/25620222
8.2.6.2 Confirmatory Analysis
Analytical Technique: LC-MS
Method Developed for: Determination of brevetoxins in shellfish
Method Selected for: These procedures have been selected for confirmatory analysis of
brevetoxins in aerosol, solid, particulate and water samples. Further research is needed to adapt
and verify the procedures for environmental sample types.
Description of Method: This method involved extraction of clam tissue homogenates with
either acetone or 80% methanol in water. Extracts are then defatted using 95% n-hexane,
followed by SPE using a Ci8 column. The SPE eluent is evaporated to dryness under nitrogen,
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redissolved in methanol at a concentration of 0.5 g tissue/mL of solution, filtered through a
syringe filter and analyzed by LC-MS equipped with a C8 column.
Special Considerations: This method is listed as a Tier II procedure for confirmatory analysis
of brevetoxin in aerosol, solid, particulate and water samples. The procedures described in the
source method are for extraction of clam homogenates (Abraham, 2015). Additional resource
citation listed below (Abraham, 2006) includes extraction procedures for aerosol filters, shellfish,
laboratory cultures and natural blooms of Karenia brevis, which may be applicable to water, soil,
aerosol filter and particulate sample types.
Source: Abraham, A., El Said, K.R., Wang, Y., Jester, E.L.E., Plakas, S.M., Flewelling, L.J.,
Henry, M.S. and Pierce, R.H. 2015. "Biomarkers of brevetoxin exposure and composite toxin
levels in hard clam (Mercenaira sp.) exposed to Karenia brevis blooms." Toxicon. 96: 82-88.
httos: //www .ncbi .nlm .nih. gov/pubmed/25 620222
Additional Resource: Abraham, A., Plakas, S.M., Wang, Z., Jester, E.L.E., El Said, K.R.,
Granade, H.R., Henry, M.S., Blum, P.C., Pierce, R.H. and Dickey, R.W. 2006. "Characterization
of polar brevetoxin derivatives isolated from Karenia brevis cultures and natural blooms."
Toxicon. 48: 104-115.
http://www.sciencedirect.eom/science/article/pii/S0041010106001632
8.2.7 Cylindrospermopsin
CAS RN: 143545-90-8
Considered Variants: NA
Description: Polycyclic uracil derivative containing guanidino and sulfate groups produced by a
variety of freshwater cyanobacteria.
Selected Methods
Analysis Type
Analytical Technique
Section
Environ. Sci. Technol. 44: 7361-7368
Presumptive
Immunoassay (ELISA)
8.2.7.1
EPA Method 545
Confirmatory
LC-MS-MS
8.2.7.2
8.2.7.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Cylindrospermopsin in ground water, surface water and well water
Method Selected for: These procedures have been selected for presumptive analysis of
Cylindrospermopsin in aerosol, solid, particulate and water samples. Further research is needed to
adapt and verify the procedures for environmental sample types.
Description of Method: Cylindrospermopsin is detected using a commercially available
colorimetric immunoassay (competitive ELISA) procedure. Cyanobacterial cells in the sample
are lysed by three sequential freeze-thaw cycles to allow determination of total toxin. Sample
(0.05 mL), enzyme conjugate (cylindrospermopsin-horseradish peroxidase [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. 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
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calibrators 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.
Special Considerations: This method is listed as Tier II for presumptive analysis of
cylindrospermopsin in aerosol, solid, particulate and water samples. The source citation listed
below (Graham, 2010) details the use of this ELISA for assessing cylindrospermopsin in naturally
occurring freshwater cyanobacterial blooms. Non-aqueous samples will require an aqueous
extraction procedure prior to assay. Samples containing intact cyanobacteria must be treated to
disrupt the cells in order to recover intracellular toxins.
Source: Graham, J.L., Loftin, K.A., Meyer, M.T. and Ziegler, A.C. 2010. "Cyanotoxin Mixtures
and Taste-and-Odor Compounds in Cyanobacterial Blooms from the Midwestern United States."
Environmental Science and Technology. 44: 7361-7368. {Note. Descriptive lake data, analytical
details for LC/MS/MS, cyanobacterial community composition data, and dissolved toxin data are
available at: http://pubs.acs.org/.')
8.2.7.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Cylindrospermopsin in potable water
Method Selected for: This method has been selected for confirmatory analysis of
cylindrospermopsin in aerosol, solid, particulate and water samples. Further research is needed to
adapt and verify the procedures for environmental sample types other than drinking water.
Description of Method: This method is used for detection of cylindrospermopsin in water
samples. Samples are frozen and thawed three times, then filtered. A 1-mL sample aliquot from
the supernatant is combined with internal standards and analyzed using LC-MS-MS with ESI.
Cylindrospermopsin is identified by comparing retention times and signals produced by unique
mass transitions to those of procedural calibration standards acquired under identical LC-MS-MS
conditions. The concentration of each analyte is determined using the integrated peak area and the
internal standard technique. The method reports an LCMRL of 0.063 (ig/L for anatoxin-a in
fortified reagent water. The working range reported in the method is 0.050-10.0 j^ig/L.
Special Considerations: This method is listed as Tier I for confirmatory analysis of
cylindrospermopsin in drinking water and Tier II for confirmatory analysis of cylindrospermopsin
in all other environmental sample types. Non-aqueous samples will require additional extraction
procedures. SPE also may be needed for additional cleanup and concentration of these sample
types. Extraction of cylindrospermopsin from ground up cyanobacterial mat material has been
achieved (Wood, 2008). These procedures might be modified for analysis of soil, air filter and
particulate samples using the LC-MS-MS conditions described in this method. LC-MS-MS
methods for anatoxin-a, cylindrospermopsin and microcystins also have been developed by
EPA's Office of Research and Development, and are currently undergoing review. Once
published, these methods will be provided on the SAM website https://www.epa.gov/homeland-
securitv-research/sam.
Source: U.S. EPA. April 2015. Method 545: Determination of Cylindrospermopsin and
Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem Mass
Spectrometry (LC/ESI-MS/MS). Washington, DC: U.S. EPA. EPA 815-R-15-009.
https://www.epa.gov/sites/production/files/2017-10/documents/epa 815 -r-15 -
009 method 545.pdf Additional Resource: Wood, S., Mountfourt, D., Selwood, A., Holland,
P., Puddick, J. and Cary, S. 2008. "Widespread Distribution and Identification of Eight Novel
Microcystins in Antarctic Cyanobacterial Mats." Applied Environmental Microbiology. 74(23):
7243-7251. https://www nchi nlm nih gov/pmc/articles/PMC2592942/
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8.2.8 Diacetoxyscirpenol (DAS)
CAS RN: 2270-40-8
Considered Variants: NA
Description: Trichothecene mycotoxin produced by Fusarium spp.
Selected Methods
Analysis Type
Analytical Technique
Section
International Journal of Food
Microbiology. 1988. 6(1): 9-17
Presumptive
Immunoassay (ELISA)
8.2.8.1
Food Research International.
2015. 72: 247-255
Confirmatory
LC-MS-MS
8.2.8.2
8.2.8.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: DAS in food
Method Selected for: These procedures have been selected for presumptive analysis of DAS in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This 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 DL for DAS using this assay is approximately 10 pg/mL. This assay will cross-react with
related toxins.
Special Considerations: This immunoassay is listed as Tier III for presumptive analysis of
DAS in aerosol, solid, particulate and water samples due to concerns regarding the availability of
the rabbit polyclonal antibodies and DAS enzyme conjugate. Efforts to identify and verify a
readily available immunoassay for analysis of DAS in environmental sample types are ongoing.
Please consult the technical contacts listed in Section 4.0 for information regarding the status of
these efforts.
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/016816058890Q797
8.2.8.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Mycotoxins in brewed coffee
Method Selected for: These procedures have been selected for confirmatory analysis of DAS in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This liquid-liquid extraction procedure using either triple quadrupole or
ion trap LC-MS/MS is used for determination of DAS in brewed coffee. Samples are first
clarified with a diluted Carrez solution and filtration, extracted, and evaporated to dryness. The
residue is reconstituted, filtered, and analyzed by LC-MS-MS. The limits of detection using an
ion trap and triple quadrupole are 1.38 and 18.70 (ig/kg, respectively. The limits of quantification
using an ion trap and triple quadrupole are 5.99 and 39.64, respectively.
Special Considerations: This method is listed as Tier II for all confirmatory analysis of DAS in
aerosol, solid, particulate and water samples. The procedures described will need to be modified
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for water, soil, air filter and particulate samples. Non-aqueous samples will require additional
extraction, cleanup and concentration procedures (e.g., SPE). Non-drinking water samples or
water samples containing low concentrations of DAS may require additional concentration and
clean up.
Source: Garcia-Moraleja, A., Font, G., Manes, J. and Ferrer, E. 2015. "Development of anew
method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid
chromatography tandem mass spectrometry." Food Research International. 72: 247-255.
http://www.sciencedirect.com/science/iournal/09639969/72
8.2.9 Domoic Acid (DA)
CAS RN: 14277-97-5
Considered Variants: NA
Description: Water-soluble, nonprotein amino acid neurotoxin produced by microscopic algae,
specifically the diatom species Pseudo-nitzschia. Composed of a proline ring, one imino group
and three carboxyl groups
Selected Methods
Analysis Type
Analytical Technique
Section
Journal of AOAC International. 2007.
90(4): 1011-1027
Presumptive
Immunoassay (ELISA)
8.2.9.1
Journal of Shellfish Research. 2008.
27(5): 1301-1310
Presumptive
Immunoassay (ELISA)
8.2.9.2
Journal ofthe Mexican Chemical
Society. 2011. 55(2): 65-71
Confirmatory
Liquid
chromatography-
ultraviolet detection
(LC-UV)
8.2.9.3
8.2.9.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Determination of DA in shellfish
Method Selected for: These procedures have been selected for presumptive analysis of DA in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercially available ELISA assay is used to determine DA in
shellfish. The ELISA assay is a direct competitive format, where free DA in the sample competes
with DA-conjugated protein coated on plastic wells for binding to anti-DA antibodies free in the
solution. Resulting color intensity is measured spectrophotometrically on a plate reader at 450
nm, and is inversely proportional to the concentration of DA in the sample solution. The assay is
calibrated using dilutions of a DA standard. The LOD for shellfish extracts is 0.01 mg/kg. Based
on the results of a collaborative study (Kleivdal, 2007), the ELISA kit is under consideration as
an official AOAC method.
Special Considerations: These procedures are listed as Tier II for presumptive analysis of DA
in aerosol, solid, particulate and water samples. The methanol/water extraction method used for
shellfish may be applicable to non-aqueous environmental sample types. Liquid samples may
only require dilution prior to assay while those containing intact organisms (e.g., phytoplankton),
may require cell disruption prior to dilution and assay. Results of a multi-laboratory study are
described in the source reference (Kleivdal, 2007) and indicate that the ELISA method may
slightly overestimate DA levels in shellfish when compared to LC-MS (see additional resource
citation, Quilliam, 1995).
Source: Kleivdal, H., Kristiansen, S.I., Nilsen, M.V., Goksoyr, A., Briggs, L., Holland, P. and
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McNabb, P. 2007. "Determination of Domoic Acid Toxins in Shellfish by Biosense ASP ELISA
- A Direct Competitive Enzyme-Linked Immunosorbent Assay: Collaborative Study." Journal of
AO AC International. 90(4): 1011-1027.
http://www.ingentaconnect.eom/content/aoac/iaoac/2007/00000090/00000004/art00016
Additional Resource: Quilliam, M.A., Xie, M. and Hardstaff, W.R. 1995. "Rapid extraction and
cleanup for liquid chromatographic determination of domoic acid in unsalted seafood." Journal of
AOAC International. 78(2): 543-554.
http://www.aoac.org/aoac ref imis/AOAC Member/PUBSCF/JAOACCF/JN M.aspx?&Journal
Tnformation=2
8.2.9.2 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: DA in shellfish
Method Selected for: These procedures have been selected for presumptive analysis of DA in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercially available ELISA test kit is used for detecting DA
using a mAbs. The sequential competitive ELISA gives equivalent results to those obtained using
standard HPLC, fluorenylmethoxycarbonyl HPLC, or liquid chromatography-mass spectrometry
(LC-MS) methods. It has a linear range from 0.1 to 3 ppb and was used to measure DA in razor
clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete
and has a standard 96-well format.
Special Considerations: These procedures are listed as Tier II for presumptive analysis of DA
in aerosol, solid, particulate and water samples. The methanol/water extraction method used for
shellfish may be applicable to non-aqueous environmental sample types. Liquid samples may
only require dilution prior to assay while those containing intact organisms (e.g., phytoplankton),
may require cell disruption prior to dilution and assay.
Source: Litaker, R.W., Stewart, T. N., Eberhart, B-T. L., Wekell, J.C., Trainer, V.L., Kudela,
R.M., Miller, P.E., Roberts, A., Hertx, C., Johnson, T.A., Frankfurter, G., Smith, G.J., Schnetzer,
A., Schumacker, J., Bastian, J.L., Odell, A., Gentien, P., Le Gal, D., Hardison, D.R. and Tester,
P. A. 2008. "Rapid Enzyme-Linked Immunosorbent Assay for Detection of the Algal Toxin
Domoic Acid." Journal of Shellfish Research. 27(5): 1301-1310.
http://www.bioone.Org/doi/abs/10.2983/0730-8000-27.5.1301
8.2.9.3 Confirmatory Analysis
Analytical Technique: LC-Ultraviolet Detection (UV)
Method Developed for: DA in seawater
Method Selected for: These procedures have been selected for confirmatory analysis of DA in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This modified LC-UV detection procedure is based on a previous study
(Mafra, 2009), and was used to detect DA in seawater. An on-line SPE step was added to the off-
line SPE step in order to allow for the use of larger sample volumes to increase sensitivity. Off-
line SPE was performed using a Ci8 column. On-line SPE is performed on the diluted sample
extract using a polymeric reversed phase (PLRP-S) pre-column for sample concentration,
followed by elution onto a reverse-phase chromatography column. Detection is performed using a
variable wavelength UV detector set at 239 nm. The method detection limit (MDL) for DA in
synthetic saline water is estimated to be 0.04 j^ig/L.
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Special Considerations: This method is listed as Tier II for confirmatory analysis of DA in
aerosol, solid, particulate and water samples. The procedures described in this method may need
to be modified for water, soil, air filter and particulate samples. The use of LC-MS (Tor, 2003)
and LC-MS-MS (Barbara, 2013) might provide additional sensitivity without requiring an online
SPE step. It may be possible to analyze relatively clean water samples by direct injection into the
LC-MS/MS; however, dirty water samples or water samples with low concentrations of DA may
require cleanup and concentration.
Source: Vera-Avila, L.E., Marin-Perez, D.Y. and Covarrubias-Herrera, R. 2011. "Trace Level
Determination of Domoic Acid in Seawater by Off-line/on-line Solid-phase Extraction Coupled
to HPLC-UV." Journal of the Mexican Chemical Society. 55(2): 65-71.
http://www.scielo.org.mx/pdf/imcs/v55n2/v55n2al.pdf
Additional Resources:
Mafia Jr., L.L. Leger, C., Bates, S.S. and Quilliama, M.A. 2009. "Analysis of trace levels of
domoic acid in seawater and plankton by liquid chromatography without derivatization, using
UV or mass spectrometry detection." Journal of Chromatography A. 1216: 6003-6011.
http://www.sciencedirect.com/science/article/pii/S0021967309009303?np=v&npKev=bd9c94
4498548a090bb6el724cdf4d78a00bc25122771553803186158373 lea7
Tor, E.R., Puschner, B. and Whitehead, W.E. 2003. "Rapid Determination of Domoic Acid in
Serum and Urine by Liquid Chromatography-Electrospray Tandem Mass Spectrometry."
Journal of Agriculture and Food Chemistry. 51: 1791-1796.
http://pubs.acs.org/doi/abs/10.1021/ifD20947f
Barbara, E., Zangrando, R, Rossi, S., Cairns, W.R.L., Piazza, R., Corami, F., Barbante, C.
and Gambaro, A. 2013. "Domoic acid at trace levels in lagoon waters: assessment of a
method using internal standard quantification." Analytical and Bioanalytical Chemistry.
405(28): 9113-9123. http://link.springer.com/article/10.1007/s00216-Q13-7348-5
8.2.10 Microcystins
CAS RNs: 96180-79-9 (LA), 154037-70-4 (LF), 101043-37-2 (LR), 123304-10-9 (LY), 111755-
37-4 (RR), 101064-48-6 (YR)
Considered Variants: LA, LF, LR, LY, RR, YR
Description: Cyclic heptapeptide hepatotoxins produced by a variety of cyanobacteria
Selected Methods
Analysis Type
Analytical Technique
Section
EPA Method 546
Presumptive
Immunoassay (ELISA)
8.2.10.1
EPA Method 544
Confirmatory
LC-MS-MS
8.2.10.2
8.2.10.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Microcystins (MC) in water
Method Selected for: This method has been selected for presumptive analysis of microcystins in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: EPA Method 546 determines total microcystins (MC) and nodularins
(NOD) in finished drinking water and in ambient water using a commercially available indirect
competitive ELISA. The term "total microcystins and nodularins" is defined as the sum of the
congener-independent, intracellular and extracellular microcystins and nodularins that are
measurable in a sample. Cyanobacterial cells in the sample are lysed by three sequential freeze-
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thaw cycles to allow determination of total toxin, and the total concentration of toxin is measured
based on detection of a characteristic feature common to microcystin and nodularin congeners
(structural variants), specifically, the Adda amino acid side chain (see Section 1.1 of Method
546). To ensure comparability between laboratories, the ELISA is calibrated against one
congener, MC-LR. The minimum reporting level (MRL) for MC-LR is 0.30 (ig/L. The method
describes required laboratory QC and demonstration of capability procedures.
Special Considerations: This method is listed as Tier I for presumptive analysis of
microcystins in water samples and Tier II for presumptive analysis of microcystins in all other
environmental sample types. Non-aqueous samples will require aqueous extraction prior to
analysis. Samples containing intact cyanobacteria must be treated to disrupt the cells in order to
recover intracellular MC.
Source: Zaffiro, A., Rosenblum, L. and Wendelken, S. August 2016. Method 546: Determination
of Total Microcystins and Nodularins in Drinking Water and Ambient Water by Adda Enzyme-
Linked Immunosorbent Assay. Washington, DC: U.S. EPA.
https://www.epa.gov/sites/production/files/2016-09/documents/method-546-determination-total-
microcvstins-nodularins-drinking-water-ambient-water-adda-enzvme-linked-immunosorbent-
assav.pdf
8.2.10.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Determination of microcystins and nodularin in drinking water
Method Selected for: This method has been selected for confirmatory analysis of microcystins
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types other than drinking water.
Description of Method: Method 544 determines six microsytins (including MC-LR) and
nodularin in drinking water using SPE and LC-MS-MS. Samples are fortified with a surrogate,
then filtered, and the filter is placed in a methanol solution to release toxin. The filtered sample
and methanol solution are combined, passed through an SPE cartridge, eluted, evaporated to
dryness, and redissolved in methanol solution. A 10-jj.L aliquot of extracted sample is analyzed
by LC-MS-MS equipped with a C8 column. Microcystins are identified by comparing the
acquired mass spectra and retention times to those of calibration standards acquired under
identical LC-MS-MS conditions. The concentration of each analyte is determined by external
standard calibration. DLs for analytes in this method range from 1.2 to 4.6 ng/L.
Special Considerations: This method is listed as Tier I for confirmatory analysis of
microcystins in drinking water, Tier II for confirmatory analysis of microcystins in non-drinking
water, and Tier III for confirmatory analysis of microcystins in all other environmental sample
types. Non-aqueous samples will require aqueous extraction prior to analysis. For example, a
procedure resulting in 90% recovery of microcystins in soil and sediment has been reported by
Chen, 2006 (see additional resource citation below). These procedures may need to be modified
for application to air filters and particulates and for analysis using the LC-MS-MS conditions
described in the EPA method selected for confirmatory analysis of these toxins. It may be
possible to analyze relatively clean water samples for extracellular toxins by direct injection into
an LC-MS-MS; however, dirty water samples or water samples with low concentrations of toxin
may require cleanup and concentration using SPE. LC-MS-MS methods for anatoxin-a,
cylindrospermopsin and microcystins have been developed by EPA's Office of Research and
Development, and are currently undergoing review. Once published, these methods will be
provided on the SAM website https://www.epa.gov/homeland-securitv-research/sam.
Source: Shoemaker, J., Tettenhorst, D. and Delacruz, A. 2015. Method 544: Determination of
Microcystins and Nodularin in Drinking Water by Solid Phase Extraction and Liquid
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Chromatography/Tandem Mass Spectrometry (CL/MS/MS), Version 1.0. Cincinnati, OH: EPA.
EPA/600/R-14/474. https://cfbub.epa.gov/si/si public record report.cfm?dirEntrvId=306953
Additional Resource: Chen, W., Li, L., Gan, N. and Song, L. 2006. "Optimization of an
effective extraction procedure for the analysis of microcystins in soils and lake sediments."
Environmental Pollution. 143: 241-246.
http://www.sciencedirect.com/science/article/pii/S02697491050Q6172
8.2.11 Picrotoxin
CAS RN (Picrotoxin): 124-87-8
CAS RN (Picrotin): 21416-53-5
CAS RN (Picrotoxinin): 17617-45-7
Considered Variants: NA
Description: Alkaloid toxin produced by the climbing plant, Anamirta cocculus, and consisting
of picrotin and picrotoxinin.
Selected Methods
Analysis Type
Analytical Technique
Section
To be determined (TBD)
Presumptive
TBD
NA
Journal of Pharmaceutical and Biomedical
Analysis. 1989. 7(3): 369-375
Confirmatory
LC-UV
8.2.11.1
8.2.11.1 Confirmatory Analysis
Analytical Technique: LC-UV
Method Developed for: Picrotoxin in serum
Method Selected for: These procedures have been selected for confirmatory analysis of
picrotoxin in aerosol, solid, particulate and water samples. Further research is needed to adapt and
verify the procedures for environmental sample types.
Description of Method: Picrotoxin (picrotin and picrotoxinin) is quantified in serum by
reversed phase HPLC. 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 ammonium acetate buffer (pH 6.4) 34:66 (v/v) for assay. 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
of 0.2 to 20.0 (ig/mL with mean recoveries > 94.2%. Increased sensitivity may be possible using
LC-TOF-MS analyses (Ogawa, 2016).
Special Considerations: This method is listed as Tier II for confirmatory analysis of picrotoxin
in aerosol, solid, particulate and water samples. The procedures described may need to be
modified for water, soils, air filters and particulates, such as use of agitation (e.g., shaking,
ultrasonication). The extraction solvent specified (chloroform) may be suitable for these sample
types, but has not been evaluated. Extracts also may require cleanup and concentration by SPE.
Although it may be possible to analyze relatively clean water samples by direct injection into a
reversed-phase LC, dirty water samples or water samples with low levels of picrotoxin may
require cleanup and concentration using SPE.
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
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Additional Resource: Ogawa, T., Tada, M., Hattori, H., Shiraishi, Y., Suzuki, T., Iwai, M.,
Kusano, M., Zaitsu, K., Ishii, A. and Seno, H. May 2016. "Sensitive determination of picrotoxin
by liquid chromatography-quadrupole time-of-flight mass spectrometry." Letter to the Editor.
Legal Medicine. 20: 8-11. http://www.legalmedicineiournal.eom/article/S 1344-6223(16)30018-
9/abstract
8.2.12 Ricin (Ricinine)
Ricin - CAS RN: 9009-86-3.
Description: Toxic lectin (carbohydrate-binding protein) found in the seeds of the castor oil
plant, Ricinus communis. 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 castor bean
preparations.
Ricinine - CAS RN: 5254-40-3.
Description: Small molecule, alkaloid marker for ricin.
Selected Methods
Analysis Type
Analytical Technique
Section
Biosecurity and Bioterrorism:
Biodefense Strategy, Practice, and
Science. 2013. 11 (4): 237-250
Presumptive
LFA
8.2.12.1
Journal of Food Protection. 2005.
68(6): 1294-1301
Presumptive
Immunoassay (ELISA)
8.2.12.2
Journal of AOAC International.
2008. 91(2): 376-382
Presumptive
Immunoassay (ECL)
8.2.12.3
EPA 600/R-13/022 (EPA/CDC)
Presumptive
LC-MS-MS
8.2.12.4
Analytical Chemistry, 2011. 83:
2897-2905
Confirmatory
Immunocapture /
LC-MS-MS
8.2.12.5
Analytical Chemistry, 2011. 83:
2897-2905
Biological Activity
Immunocapture /
MALDI-TOF-MS
8.2.12.6
8.2.12.1 Presumptive Analysis
Analytical Technique: LFA
Method Developed for: Ricin in buffer, food products, powders and aerosol filter extracts
Method Selected for: These procedures have been selected for presumptive analysis of ricin in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This lateral flow immunochromatographic device uses two antibodies
in combination to specifically detect target antigen in solution. One of the specific antibodies is
labeled with a colloidal gold derivative. Samples applied to the test strips mix with the colloidal
gold-labeled antibody and move along the strip membrane by capillary action. The second
specific antibody captures the colloidal gold-labeled antibody and bound target. When a sufficient
amount of target antigen is present, the colloidal gold label accumulates in the sample window on
the test strip, forming a visible reddish-brown colored line. As an internal control, a second line in
the control window indicates that the test strip functioned properly. Two colored lines (in the
sample and control windows) are required for a positive result determination. To perform a test
on a liquid sample, the sample is mixed with the provided buffer, and five or six drops are added
to the sample well of the test strip. A positive result is indicated by the appearance of a colored
line in the test window of the test strip and can be read visually or with a reader.
The source reference (below) details a multicenter evaluation of the sensitivity, specificity,
reproducibility, and limitations of a LFA for ricin that can be used in the field or in the laboratory
to qualitatively screen for ricin in environmental samples. Using the recommended test strip
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reader, the LFA could reproducibly detect >3.6 ng ricin/mL (0.54 ng/test) in various 'white
powder' samples and aerosol filter extracts. Because this assay does not discriminate among
Ricinus communis agglutinin (RCA) 60, RCA 120, and ricin A chain, it can be used only as a
qualitative screening assay when testing unknown samples. The test strips have also been
evaluated by the EPA ETV Program for the detection of ricin in water samples. Reports and
information associated with these evaluations are in the additional resource cited below.
Special Considerations: This LFA is listed as Tier I for solid, aerosol and water samples, and
Tier II for all other environmental sample types. Crude preparations of ricin may also contain
agglutinins that are unique to castor beans and that can cross-react in the immunoassays. A hook
effect in the response of LFAs occurs when the amount of antigen in a sample overwhelms the
amount of detector antibody present in the LFA. The resultant free antigen competes with the
antigen-detector antibody complex for the capture antibody, which results in a decrease in the
response and, under extreme conditions, can produce false-negative results. Like some other
types of immunoassays, this assay is subject to the "hook effect," which is an interference that
occurs when analyte is present in amounts significantly higher than the amounts for which the
assay was designed. The end result is a decreased response and, under extreme conditions, a
false-negative. False-negative results due to the hook effect were not observed with the
commercial LFA evaluated, although a quantitative decrease in the response has been observed at
high ricin concentrations. The incorporation of a serial dilution step in the sample protocol can
eliminate such potential errors.
Source: Hodge, D.R., Prentice, K.W., Ramage, J.G., Prezioso, S., Gauthier, C., Swanson, T.,
Hastings, R., Basavanna, U., Datta, S., Sharma, S.K., Garber, E.A.E., Staab, A., Pettit, D.,
Drumgoole, R., Swaney, E., Estacio, P.L., Elder, I.A., Kovacs, G., Morse, B.S., Kellogg, R.B.,
Stanker, L., Morse, S. and Pillai, S.P. 2013. "Comprehensive Laboratory Evaluation of a Highly
Specific Lateral Flow Assay for the Presumptive Identification of Ricin in Suspicious White
Powders and Environmental Samples." Biosecurity and Bioterrorism: Biodefense Strategy,
Practice, and Science. 11(4): 237-250.
httos: //www .ncbi .nlm .nih. gov/pubmed/24320219
Additional Resource: Environmental Technology Verification (ETV) Report. 2004. Anthrax,
Botulinum Toxin, and Ricin Immunoassay Test Strips. Cincinnati, OH: EPA.
http://www.epa.gov/sites/production/files/2015-07/documents/etv-biothreat0921Q4.pdf
8.2.12.2 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Ricin in various foods and beverages
Method Selected for: This method has been selected for presumptive analysis of ricin in
aerosol, solid, particulate and drinking water samples. Further research is needed to adapt and
verify the procedures for environmental sample types.
Description of Method: This commercial antigen-capture ELISA detects antigens in samples by
capturing them between a sandwich of antibodies. Positive and negative capture antibody
reagents are applied to alternating wells of a 96-well plate, where they are passively adsorbed.
Sample is then applied to the wells; if the target antigen is present in the sample, it will bind to
the capture antibody. A detector antibody forms the top of the sandwich and binds to any bound
antigen in the sample. The conjugate, to which the enzyme is covalently bound, is the third
reagent added and binds to the detector antibody. The substrate, added after the conjugate,
changes color in the presence of HRP. The amount of color change is directly proportional to the
amount of HRP present, which correlates to the amount of ricin. Forty-eight samples can be
processed in approximately 5 hours. The reference source reports an LOD of less than or equal to
0.02 jj.g/g. The additional resource provided below (ETV report 2004) describes the performance
of this ELISA for water samples, and reports an LOD of 0.0075 mg/L.
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Special Considerations: This method is listed as Tier II for presumptive analysis of ricin in
aerosol, solid, particulate and water samples. Non-liquid samples such as soils, powders, and
aerosol filters will require aqueous extraction followed by clarification (e.g., centrifugation) prior
to assay.
Source: Garber, E.A., Eppley, R.M., Stack, M.E., McLaughlin, M.A. and Park, D.L. 2005.
"Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in
Food." Journal of Food Protection. 68(6): 1294-1301.
http://ifoodprotection.Org/doi/abs/10.4315/0362-028X-68.6.1294
Additional Resource: James, R., Dinal, A., Willenberg, Z. and Riggs, K. Environmental
Technology Verification (ETV) Report. 2004. "Anthrax, Botulinum Toxin, and Ricin Enzyme-
Linked Immunosorbent Assay (ELISA)." Cincinnati, OH: EPA.
https://archive.epa.gov/nrmrl/archive-etv/web/pdf/01 vr elisa.pdf
8.2.12.3 Presumptive Analysis
Analytical Technique: Immunoassay (ECL)
Method Developed for: Ricin in various foods and beverages
Method Selected for: This method has been selected for presumptive analysis of ricin in
aerosol, solid, particulate and drinking water samples. Further research is needed to adapt and
verify the procedures for environmental sample types.
Description of Method: This commercial ECL-based immunoassay detects ricin in various food
and beverage samples. The ECL immunoassay uses 96-well plates with integrated screen-printed
carbon electrodes. The electrodes act both as solid phase supports to capture reagents used in the
solid phase binding assays, and as the source of electrical energy for inducing ECL. The basis of
the ECL measurement is the ability of these labels to emit light when oxidized at an electrode
surface in the presence of tertiary amine (i.e., tripropylamine). During the ECL measurement, the
plate reader applies a voltage to electrodes in the wells of the multi-array plates and measures the
resulting ECL from labeled detector antibodies incorporated in sandwich complexes on each spot
in the well with a cooled CCD sensor. The reference reports LODs of 0.04 ng/mL (sequential
assay protocol) and 0.2-10 ng/mL (simultaneous assay protocol).
Special Considerations: This method is listed as Tier II for presumptive analysis of ricin in
aerosol, solid, particulate and water samples. Sample preparation procedures used for food and
beverages suggest that similar aqueous extraction procedures may be applicable to environmental
samples. Soils, powders and aerosol filters may require aqueous extraction followed by
clarification (e.g., centrifugation) prior to assay.
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.
https://www.ncbi.nlm.nih.gov/pubmed/18476351
8.2.12.4 Presumptive Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Ricinine in drinking water samples
Method Selected for: This method has been selected for presumptive analysis of ricin by
ricinine detection in aerosol, solid, particulate 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: This method involves sample extraction by SPE, followed by LC-MS-
MS analysis, using an isocratic LC gradient and detection by ESI-MS-MS. Samples are combined
with isotopically-labeled internal standards, and sample extracts are concentrated to dryness
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under nitrogen and heat, then adjusted to a 100-|_iL volume in HPLC-grade water. Accuracy and
precision data are provided for application of the method to reagent water, finished ground water
and surface waters containing residual chlorine and/or chloramine. The method has DL of 0.09
(ig/mL and an MRL of 0.50 (ig/mL for ricinine. Storage stability was tested at 4ฐC for up to 28
days for ricinine (50 (ig/mL in ground water and surface water containing either chlorine or
monochloramine). Analyte percent recoveries at five hours ranged from 94.2% (ฑ 2) to 110.4%
(ฑ 9.5); percent recoveries at 28 days ranged from 86% (ฑ 10) to 92% (ฑ 7).
Special Considerations: This method is listed as Tier I for presumptive analysis of ricin (as
ricinine) in drinking water and Tier II for presumptive analysis of ricin (as ricinine) in all other
environmental sample types. Non-liquid samples such as soils, powders and aerosol filters will
require aqueous extraction followed by clarification (e.g., centrifugation) prior to assay. While
ricinine can be used to indicate the presence of ricin, it can also be found alone, which is a
limitation of this method.
Source: U.S. EPA and CDC. August 2013. "High Throughput Determination of Ricinine,
Abrine, and Alpha-Amanitin in Drinking Water by Solid Phase Extraction and High Performance
Liquid Chromatography Tandem Mass Spectrometry (HPLC/MS/MS)," Version 1.0. Cincinnati,
OH: EPA/Atlanta, GA: CDC. EPA 600/R-13/022.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P100I5I0.PDF?Dockev=P 100I5I0.PDF
Additional Resource: Knaack, J.S., Pittman, C.T., Wooten, J.V., Jacob, J.T, Magnuson, M.,
Silvestri, E. and Johnson, R.C. 2013. "Stability of ricinine, abrine, and alpha-amanitin in finished
tap water." Analytical Methods. 20(5): 5804-5811.
http://pubs.rsc.Org/en/content/articlelanding/2013/av/c3av40304a#ldivAbstract
8.2.12.5 Confirmatory Analysis
Analytical Technique: Immunocapture / LC-MS-MS
Method Developed for: Ricin in beverages and tap water
Method Selected for: These procedures have been selected for confirmatory analysis of ricin in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types other than water.
Description of Method: MS is used in a two-pronged approach for detection and quantification
of ricin: LC-MS-MS or multiple reaction monitoring (MRM)-MS for absolute quantification of
toxin using isotope dilution MS, combined with an enzymatic assay and MALDI-TOF-MS
detection to determine functional activity. Both approaches include an antibody capture step
(polyclonal anti-ricin antibodies immobilized on magnetic beads) for ricin extraction.
Quantitative analysis is achieved by trypsin digestion to generate peptides that are analyzed by
LC-MS-MS and quantified relative to isotope internal peptide standards. Ricin can be quantified
down to 10 finol/mL in tap water, milk, apple juice and orange juice.
Special Considerations: These procedures are listed as Tier I for confirmatory analysis of ricin
in drinking water and Tier II for confirmatory analysis of ricin in all other environmental sample
types. Non-liquid samples such as soils, powders and aerosol filters will require aqueous
extraction followed by clarification (e.g., centrifugation) prior to assay. Some information
regarding the analysis of ricin in supernatants obtained from wipe samples is provided in the
additional resource cited below. The quantification assay and enzymatic activity assay can be run
either in parallel (to return results faster when sample is plentiful), or the quantification can be
performed on ricin-bound beads after the enzymatic activity has been assessed (when sample is
limited). It should be noted that the antibody capture step does not distinguish between ricin A
chain and intact ricin (both A and B chains).
Source: McGrath, S.C., Schieltz, D.M., McWilliams, L.G., Pirkle, J.L. and Barr, J.R. 2011.
"Detection and Quantification of Ricin in Beverages Using Isotope Dilution Tandem Mass
Spectrometry." Analytical Chemistry. 83: 2897-2905.
http: //pubs. acs. org/doi/abs/ 10.1021/acl02571f
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8.2.12.6 Analysis of Biological Activity
Analytical Technique: Immunocapture / MALDI-TOF-MS
Method Developed for: Ricin activity in beverages and tap water
Method Selected for: These procedures have been selected for biological activity analysis of
ricin in aerosol, solid, particulate and water samples. Further research is needed to adapt and
verify the procedures for environmental sample types other than water.
Description of Method: MS is used in a two-pronged approach for detection and quantification
of ricin: LC-MS-MS for absolute quantification of toxin, combined with an enzymatic assay and
MALDI-TOF-MS detection to determine functional activity. The MS measurement of ricin's
deadenylase activity is conducted using immunocaptured ricin. The magnetic bead/ricin complex
is incubated with a synthetic deoxyribonucleic acid (DNA) mimic of the toxin's natural ribosomal
ribonucleic acid (rRNA) target, and the product of the reaction is detected by MALDI-TOF-MS.
Ricin deadenylase activity is determined by MALDI-TOF detection of the DNA oligomer
reaction product. The immunopurification step prior to MS analysis allows application to
complex matrices. The DL is approximately 1 pmol/mL (64 ng/mL) for the immunopurified ricin,
using 500 pmol of DNA substrate and a reaction time of between 4-6 hours.
Special Considerations: These procedures are listed as Tier I for analysis of the biological
activity of ricin in drinking water and Tier II for analysis of the biological activity of ricin in all
other environmental sample types. Non-liquid samples such as soils, powders and aerosol filters
will require aqueous extraction followed by clarification (e.g., centrifiigation) prior to assay. The
additional resource cited below describes procedures for MALDI-TOF-MS analysis of ricin in
supernatants obtained from wipe samples. Neither the antibody capture step or the deadenylase
activity assay distinguish between ricin A chain and intact ricin (both A and B chains).
Source: McGrath, S.C., Schieltz, D.M., McWilliams, L.G., Pirkle, J.L. and Barr, J.R. 2011.
"Detection and Quantification of Ricin in Beverages Using Isotope Dilution Tandem Mass
Spectrometry." Analytical Chemistry. 83: 2897-2905.
http: //pubs. acs. org/doi/abs/ 10.1021/acl02571f
Additional Resource: Schieltz, D M., McGrath, S C., McWilliams, L.G., Rees, J., Bowen,
M.D., Kools, J.J., Dauphin, L.A., Gomez-Saladin, E., Newton, B.N., Stang, H.L., Vick, M.J.,
Thomas, J., Pirkle, J.L. and Barr, J.R. 2011. "Analysis of active ricin and castor bean proteins in a
ricin preparation, castor bean extract, and surface swabs from a public health investigation."
Forensic Science International. 209: 70-79.
http://www.sciencedirect.com/science/article/pii/S03790738100Q5554
8.2.13 Saxitoxins
CAS RNs: 35523-89-8 (STX) 64296-20-4 (NEO), 58911-04-9 (dcSTX), 68683-58-9
(dcNEOSTX), 143084-69-9 (doSTX), 77462-64-7 (GTX), 122075-86-9 (dcGTX)
Considered Variants: Saxitoxins (STX), Neosaxitoxins (NEO), Gonyautoxins (GTX)
Description: A suite of more than 50 structurally related neurotoxins produced by algae and
cyanobacteria, including STX, NEO, GTX and decarbamoylsaxitoxin (dcSTX). Composed of
3,4-propinoperhydropurine tricyclic carbamates
Selected Methods
Analysis Type
Analytical Technique
Section
AOAC Method 2011.27
Presumptive
Receptor Binding Assay (RBA)
8.2.13.1
Toxicon. 2009. 54: 313-320
Presumptive
Immunoassay (ELISA)
8.2.13.2
Harmful Algae. 2016. 77-90
Presumptive
Immunoassay (ELISA)
8.2.13.3
J. Chromatogr. A. 2015. 1387: 1-12
Confirmatory
LC-MS-MS
8.2.13.4
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8.2.13.1 Presumptive Analysis
Analytical Technique: RBA
Method Developed for: Determination of paralytic shellfish toxins (PSTs) in shellfish
Method Selected for: This method has been selected for presumptive analysis of saxitoxins in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: An RBA is used to determine saxitoxins in shellfish tissue
homogenates. The procedure is a competitive binding assay in which [3H] STX competes with
unlabeled STX standards or samples containing PSTs for a finite number of receptor sites in a rat
brain membrane preparation. Tissue samples are extracted with 0.1 M hydrochloric acid at a pH
of 3.0-4.0, followed by heating, cooling to room temperature, decantation and centrifugation to
obtain clarified supernatant. Bound [3H] STX is quantified by liquid scintillation counting. A
standard curve is generated using unlabeled STX standards, which results in reduction in bound
[3H] STX that is proportional to the amount of unlabeled toxin. The concentration of STX in
samples is determined in reference to the standard curve. Incubations are carried out in a
microplate format to minimize sample handling and the amount of radioactivity used.
Special Considerations: This method is listed as Tier II for presumptive analysis of saxitoxins
in aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types. Samples must be extracted under acidic (pH 3-4)
conditions to prevent toxin degradation. Samples containing intact cyanobacteria must be treated
to disrupt the cells in order to recover intracellular toxins.
Source: AOAC International. 2011. Official Method 2011.27: Paralytic Shellfish Toxins (PSTs)
in Shellfish, Receptor Binding Assay, First Action.
http://\vww.coma.aoac.org/mcthods/info.asp'.)ID=49771
8.2.13.2 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Determination of PSTs in shellfish
Method Selected for: This method has been selected for presumptive analysis of saxitoxins in
aerosol, solid and particulate samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: A commercially available ELISA kit is used for detection of saxitoxins
in shellfish. The test is a direct competitive ELISA based on the recognition of saxitoxin by
specific antibodies. Shellfish homogenates are heat extracted in 1% acetic acid and diluted prior
to assay. When present in the sample, saxitoxin and a saxitoxin-enzyme-conjugate compete for
the binding sites of rabbit anti-saxitoxin antibodies in solution. The saxitoxin antibodies are then
bound by a second antibody (sheep anti-rabbit) immobilized on the plate. After a washing step
and addition of the substrate solution, a color signal is produced. The intensity of the blue color is
inversely proportional to the concentration of the saxitoxin present in the sample. The color is
evaluated using an ELISA reader. The concentrations of the samples are determined by
interpolation using the standard curve constructed with each run. The kit allows the determination
of 42 samples in duplicate, with a total analysis time of 60 minutes. The assay LOD for saxitoxin
in shellfish homogenates is reported as 0.02 ppb (0.02 (.ig/L). Cross-reactivity of other PSTs (e.g.,
NEO, GTX 1-4) are considerably less than STX in this ELISA, and their presence may be
underestimated in samples containing complex toxin profiles.
Special Considerations: The ELISA assay is listed as Tier II for presumptive analysis of
saxitoxins in aerosol, solid, particulate and water samples. The extraction procedure used for
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shellfish may be applicable to non-aqueous environmental sample types. Samples containing
intact cyanobacteria must be treated to disrupt the cells in order to recover intracellular toxins.
Source: Costa, P.R., Baugh, K.A., Wright, B., RaLonde, R., Nance, S.L., Tatarenkova, N.,
Etheridge, S.M. and Lefebvre, K.A. 2009. "Comparative determination of paralytic shellfish
toxins (PSTs) using five different toxin detection methods in shellfish species collected in the
Aleutian Islands, Alaska." Toxicon. 54(3): 313-320.
http://www.sciencedirect.com/science/article/pii/S00410101090024Q2
8.2.13.3 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Determination of PSTs in water
Method Selected for: This method has been selected for presumptive analysis of saxitoxins in
liquid and water samples. Further research is needed to adapt and verify the procedures for
environmental sample types.
Description of Method: A commercially available immunoassay (ELISA) is used to detect
saxitoxins in water samples. The test is a direct competitive ELISA based on the recognition of
saxitoxin by specific antibodies. Water samples are prepared by three successive freeze-thaw
cycles followed by filtration prior to assay. Saxitoxin and a saxitoxin-enzyme-conjugate compete
for the binding sites of rabbit anti-saxitoxin antibodies in solution. The saxitoxin antibodies are
then bound by a second antibody (sheep anti-rabbit) immobilized on the plate. After a washing
step and addition of the substrate solution, a color signal is produced. The intensity of the blue
color is inversely proportional to the concentration of the saxitoxin present in the sample. The
color is evaluated using an ELISA reader. The concentrations of the samples are determined by
interpolation using the standard curve constructed with each run. The kit allows the determination
of 42 samples in duplicate, with a total analysis time of 60 minutes. The MRL reported for
saxitoxin in water samples is 0.02 (ig/L. Cross-reactivities of other PSTs (e.g., NEOSTX, GTX 1-
4) are considerably less than STX in this ELISA.
Special Considerations: The ELISA method is listed as Tier I for presumptive analysis of
saxitoxins in water samples. Non-aqueous samples require extraction prior to assay (see Section
8.2.13.2). Samples containing intact cyanobacteria must be treated to disrupt the cells in order to
recover intracellular toxins.
Source: Loftin, K.A., Graham, J.L., Hilborn, E.D., Lehmann, S.C., Meyer, M.T., Dietze, J.E.
and Griffith, C.B. 2016. "Cyanotoxins in inland lakes of the United States: Occurrence and
potential recreational health risks in the EPA National Lakes Assessment 2007." Harmful Algae.
56: 77-90. http: //www. science direct .com/science/article/pii/S 1568988315300883
8.2.13.4 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Determination of saxitoxins (STX, NEO, GTX, dcGTX, dcSTX) and
saxitoxin analogues in shellfish
Method Selected for: These procedures have been selected for confirmatory analysis of
saxitoxins and analogues in aerosol, solid, particulate and water samples. Modification and
further research are needed to adapt and verify the procedures for environmental sample types.
Description of Method: This method uses multiple analogues of saxitoxin in shellfish are
detected using LC-MS-MS. The method includes a single dispersive extraction of shellfish
homogenates followed by SPE using graphitized carbon cartridges for sample cleanup and LC-
MS-MS, with ahydrophilic interaction liquid chromatography (HILIC) column, for analysis.
Validation study results included in Turner el al. (2015) are provided for specificity, linearity,
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recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly
improved in comparison to other currently available fluorescence-based detection methods.
Special Considerations: This method is listed as Tier II for confirmatory analysis of saxitoxin in
aerosol, solid, particulate and water samples. The sample extraction and cleanup (SPE)
procedures described for shellfish homogenates may be suited for other sample types with slight
modifications. For example, relatively clean samples (e.g., drinking water and liquid samples)
may not require extensive extraction procedures prior to sample cleanup and analyte
concentration using SPE. In addition, adaptation of online SPE in conjunction with LC-MS-MS,
as reported by Bragg et al. (2015) for STX and NEO, and by Coleman et al. (2016) for
tetrodotoxin analysis, may provide additional streamlining and high-throughput capacity for
saxitoxin analysis.
Source: Boundy, M.J., Selwood, A.I., Harwood, D.T., McNabb, P.S. and Turner, A.D. 2015.
"Development of a sensitive and selective liquid chromatography-mass spectrometry method for
high-throughput analysis of paralytic shellfish toxins using graphitized carbon solid phase
extraction." Journal of Chromatography A. 1387: 1-12.
http://www.sciencedirect.com/science/article/pii/S00219673150Q1995
Additional Resources:
Turner, A.D., McNabb, P.S., Harwood, A.J. and Boundy, M.J. 2015. "Single-Laboratory
Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS
Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish." Journal of
AOAC International. 98(3): 609-621.
http://www.ingentaconnect.eom/content/aoac/iaoac/2015/00000098/00000003/art00011
Bragg, W.A., Lemire, S.W., Coleman, R.M., Hamelin, E. I. and Johnson, R.C. 2015.
"Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase
extraction-liquid chromatography-tandem mass spectrometry." Toxicon. 99: 118-124.
https://www.ncbi.nlm.nih.gov/pubmed/25817003
Coleman, R., Lemire, S.W., Bragg, W., Garrett, A., Ojeda-Torres, G., Hamelin, E., Johnson,
R. C. and Thomas, J. 2016. "Development and validation of a high-throughput online solid
phase extraction-liquid chromatography-tandem mass spectrometry method for the detection
of tetrodotoxin in human urine." Toxicon. 119: 64-71.
http://www.sciencedirect.com/science/article/pii/S0041010116301386
8.2.14 Shiga and Shiga-like Toxins (Stx)
CAS RN: 75757-64-1 (Stx)
Considered Variants: Stx-la, lc, Id and le, Stx-2ato 2g
Description: Protein produced by Shigella dysenteriae and the shiga toxin-producing
Escherichia coli (STEC). Composed of one ~32 kDa A chain and five 7.7 kDa B chains
Selected Methods
Analysis Type
Analytical Technique
Section
Austin Immunology. 2016. 1(2), id1007: 1-7
Presumptive
Immunoassay (ELISA)
8.2.14.1
Analytical Chemistry. 2014. 86: 4698-4706
Confirmatory
LC-MS-MS
8.2.14.2
8.2.14.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Shiga-like toxins (Stx-1 and Stx-2) in foods and bacterial enrichment
broth
Method Selected for: These procedures have been selected for presumptive analysis of Shiga
and Shiga-like toxins in aerosol, solid, particulate and water samples. Further research is needed
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to adapt and verify the procedures for these sample types.
Description of Method: Shiga-like toxins (Stx-1, including 4 Stx-la, lc, Id and le four
subtypes and Stx-2, including Stx-2a to 2g seven subtypes) are produced by various STEC. Two
commercial ELISA kits (Stx-1 ELISA and Stx-2 ELISA) are used to detect these toxins, using
type-specific, polyclonal and mAbs to differentiate between the Stx-1 and Stx-2 toxin types.
Samples are incubated with a medium that encourages E. coli (EC) growth and Stx production. If
Stx-1 or Stx-2 is present, it is bound by an immobilized polyclonal antibody on the wells of a
microtiter plate. After a washing step, a mixture of mAbs is added which binds to the Stx-1 or
Stx-2. A second washing step is followed by the addition of an HRP-labeled antibody that binds
to the existing antigen/antibody complex in the wells. After a final wash and addition of substrate
solution, a color signal is generated. The color intensity is evaluated using an ELISA plate reader
and is proportional to the amount of Stx-1 or Stx-2 present. The result is compared to a known
value to determine whether the sample is positive or below the limit of Stx-1 or Stx-2 detection
(25 pg/mL). The additional source citation (below) describes the evaluation of these two ELISA
kits for STEC-inoculated and enriched ground beef, romaine lettuce, recreational pond water, and
pasteurized milk samples. Results indicate that Stx-1 and Stx-2 were readily detected and
distinguished for all tested sample types.
Special Considerations: This assay is listed as Tier II for presumptive analysis of Shiga-like
toxins in aerosol, solid, particulate and water samples. Appropriate standards are available.
Sample preparation procedures used for foods and bacterial broth suggest that similar aqueous
extraction procedures may be applicable to environmental samples. The Stx-1 ELISA may also be
applicable for analysis of Stx produced by Shigella bacteria.
Source: Kong, Q., Patfield, S., Skinner, C., Stanker, L.H., Gehring, A. G., Fratamico, P.M.,
Rubio, F., Qi, W., and He, X. 2016. "Validation of Two New Immunoassays for Sensitive
Detection of a Broad Range of Shiga Toxins." Austin Immunology. 1(2), id 1007: 1-7.
http://austinpublishinggroup.eom/austin-immunology/#
Additional Resource: Gehring, A G., Fratamico, P.M., Lee, J., Ruth, L.E., He, X., He, Y., Paoli,
G.C., Stanker, L.H. and Rubio, F.M. 2017. "Evaluation of ELISA Tests Specific for Shiga Toxin
1 and 2 in Food and Water Samples." Food Control. 77: 145-149.
http://linkinghub.elsevier.com/retrieve/pii/S095671351730049X?showall=true
8.2.14.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: Stx-1 and Stx-2 in plasma and enrichment broth
Method Selected for: These procedures have been selected for confirmatory analysis of Shiga
and Shiga-like toxins in aerosol, solid, particulate and water samples. Further research is needed
to adapt and verify the procedures for these sample types.
Description of Method: This MRM method is based on analyzing conserved peptides, derived
from the tryptic digestion of the toxin B subunits. Stable isotope-labeled analogues are prepared
and used as internal standards to identify and quantify these characteristic peptides. The method
detects and quantifies Shiga toxins (Stx) and Shiga-like toxins type 1 (Stx-1) and type 2 (Stx-2),
and also distinguishes among most of the known Stx-1 and Stx-2 subtypes. The LOD for digested
pure standards is approximately 10 attomole range/injection, which corresponds to a
concentration of 1.7 femtomol/mL. Samples and standards are reduced using dithiothreitol, then
alkylated using iodoacetamide, and finally subjected to proteolysis by the addition of a trypsin
solution and incubation at 37ฐC for 16 hours. Digested samples are filtered through a 10000
molecular weight cut-off (MWCO) filter. For quantitative analysis, an aliquot of filter-sterilized
sample is digested with trypsin and a fixed amount of the appropriate trypsin digested 15N-labled
internal standard is added. An MS equipped with a linear ion trap and a nanoelectrospray source
was used to perform LC-MS-MS, operated in MRM mode, and alternating between detection of
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Section 8.0 - Selected Biotoxin Methods
the nine peptides and the corresponding 15N-labeled internal standards. The additional source
citation (see below) provides a more rapid (3-hour) approach to reduce/alkylate and trypsin digest
serum samples, resulting in equivalent or improved detection compared to the procedure
described above.
Special Considerations: This method is listed as Tier II for confirmatory analysis of Shiga-like
toxins in aerosol, solid, particulate and water samples. Appropriate standards are available.
Sample preparation procedures used for plasma and bacterial broth suggest that aqueous
extraction may be applicable to environmental samples. Matrix effects were observed when dilute
samples were digested in buffer, Luria broth, or mouse plasma (LOD ~30 attomol/injection = 5
femtomol/mL). This method may also be applicable for analysis of Stx produced by Shigella
bacteria, using an appropriate standard.
Source: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Dynin, I., Hui, C., Patfield, S.A.,
Carter, J.M. and He, X. 2014. "Safe and Effective Means of Detecting and Quantitating Shiga-
Like Toxins in Attomole Amounts." Analytical Chemistry. 86(10): 4698-4706.
http: //pubs. acs. org/doi/abs/10.1021 /ac40293 Or
Additional Resource: Silva, C.J., Erickson-Beltran, M.L., Skinner, C.B., Patfield, S.A. and He,
X. 2015. "Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2
Shiga-Like Toxins in Human Serum." Toxins. 7(12): 5236-5253.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690125/
8.2.15 Staphylococcal Enterotoxins (SETs)
CAS RNs: 37337-57-8 (staphylococcal enterotoxin type A [SEA]), 39424-53-8 (staphylococcal
enterotoxin type B [SEB]), 39424-54-9 (staphylococcal enterotoxin type C [SEC]), 12788-99-7
(staphylococcal enterotoxin type D [SED]), (staphylococcal enterotoxin type E [SEE])
Considered Variants: SEA - SEE
Description: Heat stable, basic, single-chain proteins with molecular weights of 26,000 to
29,000 kDa, produced by certain Staphylococcus strains.
Selected Methods
Analysis Type
Analytical Technique
Section
AOAC Official Method 2007.06
Presumptive
Immunoassay (Enzyme-
linked fluorescent
immunoassay [ELFA])
8.2.15.1
Journal of AOAC International.
2014. 97(3): 862-867
Presumptive
Immunoassay (ECL)
8.2.15.2
Letters in Applied Microbiology.
2011. 52: 468-474
Confirmatory
Immunoassay (ELISA)
8.2.15.3
8.2.15.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELFA)
Method Developed for: Staphylococcal enterotoxins in selected foods
Method Selected for: This method has been selected for presumptive analysis of SETs in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercial test is an ELFA used with automated instrumentation
for the specific detection of SEA - SEE. The solid-phase-receptacle (SPR) serves as the solid
phase as well as the pipetting device for the assay. Reagents for the assay are ready-to-use and
predispensed in the sealed reagent strips. The instrument performs all of the assay steps
automatically. The user places the sample extract into the reagent strip, and the sample is cycled
in and out of the SPR for a specific length of time. SET present in the sample will bind to the
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Section 8.0 - Selected Biotoxin Methods
anti-SET mAbs, which are coated on the interior of the SPR. Unbound sample components are
washed away. Alkaline phosphatase-labeled antibodies are cycled in and out of the SPR and will
bind to any SET captured on the SPR wall. Further wash steps remove unbound conjugate.
During the final detection step, the substrate (4-methyl-umbelliferyl phosphate) is cycled in and
out of the SPR. The bound enzyme conjugate catalyzes the hydrolysis of this substrate into a
fluorescent product (4-methyl-umbelliferone), the fluorescence of which is measured at 450 nm.
The test value is calculated by the instrument and is equal to the sample relative fluorescence
value (RFV)/standard RFV. A "negative" result has a test value less than the threshold (0.13),
indicating that the sample does not contain SET or contains SET at a concentration below the DL.
A "positive" result has a test value equal to or greater than the threshold and indicates that the
sample is contaminated with SET. DLs for SETs in various foods range from 0.25 ng/g to 0.5
ng/g (solids) or ng/mL (liquids).
Special Considerations: These procedures are listed as Tier II for presumptive analysis of
staphylococcal neurotoxins in aerosol, solid, particulate and water samples. Non-liquid samples
such as soils, powders and aerosol filters will require aqueous extraction followed by clarification
(e.g., centrifugation) prior to assay.
Source: AOAC International. 2008. "Method 2007.06: "VIDAS SET 2 for Detection of
Staphylococcal Enterotoxins in Selected Foods" Journal of AOAC International. 91: 164.
htto: //www .aoac. org/
8.2.15.2 Presumptive Analysis
Analytical Technique: Immunoassay (ECL)
Method Developed for: Determination of SEB in foods
Method Selected for: These procedures have been selected for presumptive analysis of SEB in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercial ECL immunoassay is used with automated
instrumentation for the specific detection of SEB. The source reference describes a comparison of
its use to ELISA and LFD assays, in conjunction with an outbreak of staphylococcal food
poisoning, and notes demonstrated cross-reactivity with SED present in the food samples as well
as interferences from the food matrices tested.
Special Considerations: These procedures are listed as Tier III for presumptive analysis of SEB
in aerosol, solid, particulate and water samples. Non-liquid samples such as soils, powders and
aerosol filters will require aqueous extraction followed by clarification (e.g., centrifugation) prior
to assay. Please consult the technical contacts listed in Section 4.0 for information regarding the
status and availability of assay reagents and analytical instrumentation.
Source: Tallent, S. M., Hait, J., and Bennet, R., J. 2014. "Staphylococcal Enterotoxin B-Specific
Electrochemiluminescence and Lateral Flow Device Assays Cross-React with Staphylococcal
Enterotoxin D." Journal of AOAC International. 97(3): 862-867.
8.2.15.3 Confirmatory Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: Determination of SETs in naturally contaminated cheese
Method Selected for: These procedures have been selected for confirmatory analysis of SETs in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This commercially available sandwich type enzyme immunoassay
(ELISA) detects the combined SET types SEA through SEE. The surface of a microtitre plate is
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Section 8.0 - Selected Biotoxin Methods
coated with specific purified antibodies that bind enterotoxins. The immobilized toxins are then
bound by a mixture of toxin-specific detector antibodies (conjugated to biotin) forming a
sandwich complex (antibody-antigen-antibody complex). Addition of specific antibodies forms a
sandwich complex (antibody-antigen-antibody). The presence of enterotoxins is revealed by
adding an enzyme substratc/ch romogen solution containing tetramethylbenzidine, which results in
a blue color in the presence of SETs. Addition of a sulphuric acid solution leads to a color change
from blue to yellow, allowing for the confirmation of the presence of SEs using a
spectrophotometer at a double wavelength of 45CK630 nm. The sample is considered to be
contaminated by SEs if the absorbance test value is greater than or equal to the threshold value
(negative control value plus 0.15). Solid food samples (e.g., cheese) are homogenized,
centrifiiged and concentrated by dialysis prior to assay. DLs reported for cheese samples range
from 0.012 ng/g (SEA) to 0.05 ng/g (SED).
Special Considerations: These procedures are listed as Tier II for confirmatory analysis of
SETs in aerosol, solid, particulate and water samples. Non-liquid samples such as soils, powders
and aerosol filters will require aqueous extraction followed by clarification (e.g., centrifugation)
prior to assay. All samples may require a concentration step (e.g., dialysis) prior to assay.
Source: Ostyn, A., Fuillier, F., Prufer, A.L., Messio, S., Krys, S., Lombard, B. and Hennekinne,
J. A. 2011. "Intra-laboratory validation of the Ridascreenฎ SET Total kit for detection
staphylococcal enterotoxins SEA to SEE in cheese." Letters in Applied Microbiology. 52(5): 468-
474. http://onlinelibrarv.wilev.eom/doi/10.l 111/i. 1472-765X.2011.03025.x/abstract
8.2.16 T-2 Mycotoxin
CAS RN: 21259-20-1 (T-2), 26934-87-2 (HT-2)
Considered Variants: T-2 and HT-2
Description: Trichothecene toxins produced by Fusarium spp.
Selected Methods
Analysis Type
Analytical Technique
Section
Journal of Food Protection. 2005. 68(6):
1294-1301
Presumptive
Immunoassay (ELISA)
8.2.16.1
Rapid Communications in Mass
Spectrometry. 2006. 20(9): 1422-1428
Confirmatory
LC-MS
8.2.16.2
8.2.16.1 Presumptive Analysis
Analytical Technique: Immunoassay (ELISA)
Method Developed for: T-2 mycotoxin in food and beverages
Method Selected for: These procedures have been selected for presumptive analysis of a-
amanitin (see Section 8.2.3.1) and T-2 toxin in aerosol, solid, particulate and water samples.
Further research is needed to adapt and verify the procedures for environmental sample types.
Description of Method: A commercially available ELISA is used to detect T-2 mycotoxin at
levels below those described as a health concern in food samples. Solid food samples are
prepared by extracting the sample with methanol/water followed by dilution with phosphate-
buffered saline. Liquid beverage samples are prepared by dilution in sodium phosphate buffer.
The prepared samples are analyzed using 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 37ฐC.
Special Considerations: This assay is listed as Tier II for presumptive analysis of T-2
mycotoxins in aerosol, solid, particulate and water samples. Non-aqueous samples will require
extraction prior to analysis. The ELISA kit successfully detects T-2 toxin at targeted levels of 0.2
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Section 8.0 - Selected Biotoxin Methods
jxg/g; the immunoassay for T-2 toxin, however, shows variable background responses up to 0.1
jxg/g. Detection thresholds of 0.2 jxg/g for T-2 toxin avoided the background problems but were
low enough to allow detection at concentrations below those associated with serious health
effects. The additional resource citation (Tima, 2016) describes the application of this ELISA for
monitoring swine feedstuff for T-2 mycotoxin. The LOD for T-2 mycotoxin in this study was 12
Mg/kg-
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://ifoodprotection.Org/doi/abs/10.4315/0362-028X-68.6.1294?=
Additional Resource: Tima, H., Racz, A., Guld, Z., Mohacsi-Farkas, C. and Kisko, G. 2016.
"Deoxynivalenol, zearalenone and T-2 in grain based swine feed in Hungary." Food Additives &
Contaminants: PartB. 9(4):275-280. DOI: 10.1080/19393210.2016.1213318.
http://dx.doi.org/10.1080/19393210.2Q16.1213318
8.2.16.2 Confirmatory Analysis
Analytical Technique: LC-MS
Method Developed for: DAS and mycotoxins in food
Method Selected for: These procedures have been selected for confirmatory analysis of T-2 and
HT-2 mycotoxins in aerosol, solid, particulate and water samples. Further research is needed to
adapt and verify the procedures for environmental sample types.
Description of Method: A LC-TOF-MS with atmospheric pressure chemical ionization (APCI)
and time-of-flight (TOF)-MS with a real-time reference mass correction is used for simultaneous
determination of Fusarium mycotoxins (including T-2 and HT-2 mycotoxins) in food. Samples
are extracted and centrifuged, and the supernatant is applied to a MultiSep #226 column for
cleanup. Prepared samples are separated by reversed-phase LC and detected by APCI-MS. LODs
range from 0.1 to 0.3 ng/g and 0.5 to 0.9 ng/g for T-2 and HT-2 in analyzed foodstuffs,
respectively. The additional resource citation below (Garcia-Moraleja, 2015) describes a liquid-
liquid extraction procedure using either triple quadrupole or ion trap LC-MS-MS for
determination of various mycotoxins in brewed coffee (see Section 8.2.8.2).
Special Considerations: This method is listed as Tier II for confirmatory analysis of T-2
mycotoxins in aerosol, solid, particulate and water samples. The procedures described may need
to be modified for water, soils, air filters and particulates. It may be possible to analyze relatively
clean water samples by direct injection into an LC-MS-MS. Dirty water samples or water samples
with low concentrations of toxin may require cleanup and concentration.
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 ://onlinelibrarv .wilev.com/doi/10.1002/rcm ,2460/abstract
Additional Resource: Garcia-Moraleja, A., Font, G., Manes, J. and Ferrer, E. 2015.
"Development of a new method for the simultaneous determination of 21 mycotoxins in coffee
beverages by liquid chromatography tandem mass spectrometry." Food Research International.
72: 247-255. http://www.sciencedirect.com/science/article/pii/S0963996915Q01167
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Section 8.0 - Selected Biotoxin Methods
8.2.17 Tetrodotoxin (TTX)
CAS RN: 9014-39-5
Considered Variants: NA
Description: Neurotoxin produced by certain infecting or symbiotic bacteria like Pseudomonas
and Vibrio, found in animals from the order Tetraodontiformes, which includes pufferfish,
porcupinefish, ocean sunfish, and triggerfish. Composed of heterocyclic heat-stable (except in
alkaline environments) and water-soluble neurotoxin.
Selected Methods
Analysis Type
Analytical Technique
Section
AOAC Official Method 2011.27. 2011
Presumptive
RBA
8.2.17.1
Toxicon. 2016. 119: 64-71
Confirmatory
LC-MS-MS
8.2.17.2
8.2.17.1 Presumptive Analysis
Analytical Technique: RBA
Method Developed for: PSTs in shellfish
Method Selected for: This method has been selected for confirmatory analysis of TTX in
aerosol, solid, particulate and water samples. Further research is needed to adapt and verify the
procedures for environmental sample types.
Description of Method: This assay is used for determination of PSTs in shellfish tissue
homogenates. The procedure is a competitive binding assay in which [3H] STX competes with
unlabeled standards (TTX) or PSTs in samples for a finite number of receptor sites in a rat brain
membrane preparation. Tissue samples are extracted with 0.1 M hydrochloric acid at a pH of 3.0-
4.0, followed by heating, cooling to room temperature, decantation and centrifiigation to obtain
clarified supernatant. Unbound [3H] STX is removed by filtration and bound [3H] STX is
quantified by liquid scintillation counting. A standard curve is generated using unlabeled TTX
standards, which results in reduction in bound [3H] STX that is proportional to the amount of
unlabeled toxin. The concentration of TTX in samples is determined in reference to the standard
curve. Incubations are carried out in a microplate format to minimize sample handling and the
amount of radioactivity used.
Special Considerations: This method is listed as Tier II for presumptive analysis of TTX in
aerosol, solid, particulate and water samples. Samples must be extracted under acidic (pH 3-4)
conditions to prevent toxin degradation. This method was originally developed for STX but can
be used to quantify TTX in samples when TTX standards are used to develop a standard curve
since both toxins compete for the same receptor.
Source: AOAC International. 2011. Official Method 2011.27: Paralytic Shellfish Toxins (PSTs)
in Shellfish, Receptor Binding Assay, First Action.
http://www.eoma.aoac.org/methods/info.asp?ID=49771
8.2.17.2 Confirmatory Analysis
Analytical Technique: LC-MS-MS
Method Developed for: TTX in human urine
Method Selected for: These procedures have been selected for confirmatory analysis in aerosol,
solid, particulate and water samples. Further research is needed to adapt and verify the procedures
for environmental sample types.
Description of Method: This procedure uses online SPE and LC-MS-MS is used for
determination of TTX in human urine. Sample extraction involves online SPE using a mixed
mode cartridge (strong cation exchange/C-18 reverse phase) followed by direct injection and
detection of analytes by LC-MS-MS using a HILIC column. The reportable TTX concentration
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Section 8.0 - Selected Biotoxin Methods
range was 2.80-249 ng/mL in urine with a calculated LOD of 0.302 ng/mL.
Special Considerations: This method is listed as Tier II for confirmatory analysis of TTX in
aerosol, solid, particulate and water samples. The procedures described may need to be modified
for water, soil, air filter and particulate samples. Soil, air filter and particulate extracts may
require cleanup and concentration by SPE. It may be possible to analyze relatively clean water
samples by direct injection into an LC-MS-MS. Dirty water samples or water samples with low
levels of TTX may also require cleanup and concentration using SPE. The additional resources
cited below describe solvent or accelerated extraction of fish tissue and snail shells, followed by
centrifugation or filtration. The resulting supernatant can then be cleaned up and concentrated by
online SPE, and analyzed using LC-MS-MS.
Source: Coleman, R., Lemire, S.W., Bragg, W., Garrett, A., Ojeda-Torres, G., Hamelin, E.,
Johnson, R.C. and Thomas, J. 2016. "Development and validation of a high-throughput online
solid phase extraction-liquid chromatography-tandem mass spectrometry method for the detection
of tetrodotoxin in human urine." Toxicon. 119: 64-71.
http://www.sciencedirect.com/science/article/pii/S0Q41010116301386
Additional Resources:
Rodriguez, P., Alfonso, A., Otero, P., Katikou, P., Georgantelis, D.B and
Botana, L.M. 2012. "Liquid chromatography-mass spectrometry method to detect
Tetrodotoxin and Its analogues in the puffer fish Lagocephalus sceleratus (Gmelin, 1789)
from European waters." Food Chemistry. 132(2): 1103-1111.
http://www.sciencedirect.com/science/article/pii/S0308814611Q16633
Nzoughet, J.K., Campbell, K., Barnes, P., Cooper, K.M., Chevallier, O.P. and Elliott, C.T.
2013. "Comparison of sample preparation methods, validation of an UPLC-MS/MS
procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of
pufferfish." Food Chemistry. 136(3-4): 1584-1589.
http://www.sciencedirect.com/science/article/pii/S03088146120Q1616
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Section 9 - Conclusions
Section 9.0: Conclusions
The information in this document 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 ICLN. The
information also can be found on the SAM website (https://www.epa.gov/homeland-securitv-
rcscarch/sam). which provides searchable links to supporting information based on the 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 incident.
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 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 are provided in Attachment 1. This current
revision (2017) includes the addition of outdoor building and infrastructure materials as sample types in
the radiochemistry sections; the addition of solids as a sample type in the pathogens sections; the
assignment of method applicability tiers for methods selected for pathogen and biotoxin analytes; several
new methods added or replaced for chemical, radiochemical, pathogen and biotoxin analytes; the addition
of several new analytes to each of these technical sections; and considerations regarding the potential
impacts of decontamination agents on the analytical performance of selected radiochemical methods.
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Appendix A - Selected Chemical Methods
Appendix A: Selected Chemical Methods
SAM 2017 - Appendix A
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SAM 2017 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 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. Assigned usability tiers are indicated next to each method or method combination throughout this appendix.
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
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 (e.g., due to potential interferences,
alternate matrices, the need to address different DQOs).
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.
Note: Column headings are defined in Section 5.0.
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Acephate
30560-19-1
LC-MS-MS
Sample Prep
Determinative
Adapted from J.
Chromatogr. A,
(2007) 1154(1):
3-25
n
Adapted from
Chromatographia,
63(5/6): 233-237
II
538
(EPA OW)
I
Adapted from J.
Chromatogr. A,
(2007) 1154(1):
3-25
ni
Adapted from J.
Chromatogr. A,
(2007) 1154(1): 3-25
ni
Acrylamide
79-06-1
HPLC-UV
Sample Prep
Water extraction
in
8316
(EPA SW-846)
II
8316
(EPA SW-846)
n
PV2004
(OSHA)
i
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8316
(EPA SW-846)
8316
(EPA SW-846)
Acrylonitrile
107-13-1
HPLC-UV /
GC-MS
Sample Prep
5035A
(EPA SW-846)
n
524.21
(EPA OW)
II
524.21
(EPA OW)
n
PV2004
(OSHA)
ni
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
Aldicarb (Temik)
116-06-3
HPLC-UV/
HPLC-FL /
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
n
D7645-16
(ASTM)
II
531.2
(EPA OW)
i
5601
(NIOSH)
i
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8318A
(EPA SW-846)
Aldicarb sulfone
1646-88-4
HPLC-UV/
HPLC-FL/
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
n
D7645-16
(ASTM)
II
531.2
(EPA OW)
i
5601
(NIOSH)
ni
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8318A
(EPA SW-846)
Aldicarb sulfoxide
1646-87-3
HPLC-UV/
HPLC-FL/
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
in
D7645-16
(ASTM)
II
531.2
(EPA OW)
i
5601
(NIOSH)
in
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
8318A
(EPA SW-846)
SAM 2017 Appendix A
A -1
September 2017
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Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Allyl alcohol
107-18-6
GC-MS
Sample Prep
5035A
(EPA SW-846)
II
5030C
(EPA SW-846)
II
5030C
(EPA SW-846)
n
TO-152
(EPA ORD)
ni
Not of concern
NA
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
8260D
(EPA SW-846)
4-Aminopyridine
504-24-5
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
in
3535A/8330B
(EPA SW-846)
III
3535A/8330B
(EPA SW-846)
in
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Ammonia
7664-41-7
Visible
spectrophotometry
Sample Prep
Not of concern
NA
4500-NH3 B
(SM)
I
350.1
(EPA OW)
1
6015
(NIOSH)
I
Not of concern
NA
Determinative
45OO-NH3 G
(SM)
Ammonium metavanadate
(analyze as total vanadium)
7803-55-6
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
1
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
1
IO-3.1
(EPA ORD)
1
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Arsenic, Total
7440-38-2
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
I
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
1
IO-3.1
(EPA ORD)
I
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Arsenic trioxide
(analyze as total arsenic)
1327-53-3
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
T
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
1
IO-3.1
(EPA ORD)
T
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Arsine
(analyze as total arsenic in non-air
samples)
7784-42-1
GFAA /
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
T
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
1
6001
(NIOSH)
I
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
Asbestos
1332-21-4
TEM
Sample Prep
Determinative
D5755-03 (soft
surfaces-microvac)
(ASTM)
in
Not of concern
NA
Not of concern
NA
10312:1995
(ISO)
I
D6480-05
(hard surfaces-wipes)
(ASTM)
I
Boron trifluoride
7637-07-2
ISE
Sample Prep
Determinative
Not of concern
NA
Not of concern
NA
Not of concern
NA
ID216SG
(OSHA)
I
Not of concern
NA
Brodifacoum
56073-10-0
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
D7644-16
(ASTM)
II
D7644-16
(ASTM)
II
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
D7644-16
(ASTM)
D7644-16
(ASTM)
SAM 2017 Appendix A
A - 2
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Bromadiolone
28772-56-7
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
D7644-16
(ASTM)
II
D7644-16
(ASTM)
II
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
D7644-16
(ASTM)
D7644-16
(ASTM)
BZ [Quinuclidinyl benzilate]
6581-06-2
HPLC-UV /
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
Adapted from J.
Chromatogr. B (2008)
874: 42-50
III
Adapted from J.
Chromatogr. B (2008)
874: 42-50
ni
TO-10A
(EPA ORD)
in
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
Adapted from J.
Chromatogr. B (2008)
874: 42-50
Adapted from J.
Chromatogr. B (2008)
874: 42-50
Calcium arsenate
(analyze as total arsenic)
7778-44-1
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
i
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
i
IO-3.1
(EPA ORD)
i
9102
(NIOSH)
i
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Carbofuran (Furadan)
1563-66-2
HPLC-UV/
HPLC-FL /
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
ii
D7645-16
(ASTM)
II
531.2
(EPA OW)
i
5601
(NIOSH)
i
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
8318A
(EPA SW-846)
Carbon disulfide
75-15-0
GC-MS
Sample Prep
5035A
(EPA SW-846)
i
5030C
(EPA SW-846)
I
524.21
(EPA OW)
i
TO-15
(EPA ORD)
i
Not of concern
NA
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
Carfentanil
59708-52-0
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
3520C/3535A
(EPA SW-846)
III
3520C/3535A
(EPA SW-846)
iii
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Adapted from J.
Chromatogr. B(2014)
962: 52-58
Adapted from J.
Chromatogr. B(2014)
962: 52-58
Chlorfenvinphos
470-90-6
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
ii
3520C/3535A
(EPA SW-846)
T
3520C/3535A
(EPA SW-846)
i
TO-10A
(EPA ORD)
n
EPA/600/R-16/114
(EPA NHSRC)
ii
Determinative
8270E
(EPA SW-846)
8270E
(EPA SW-846)
Chlorine
7782-50-5
Visible
spectrophotometry
Sample Prep
Not of concern
NA
4500-CI G
(SM)
I
4500-CI G
(SM)
i
Adapted from Analyst
(1999) 124(12) :1853-
1857
n
Not of concern
NA
Determinative
4500-CI G
(SM)
SAM 2017 Appendix A
A - 3
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
2-Chloroethanol
107-07-3
GC-MS/
GC-FID
Sample Prep
5035A
(EPA SW-846)
II
5030C
(EPA SW-846)
n
5030C
(EPA SW-846)
n
2513
(NIOSH)
I
Not of concern
NA
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
8260D
(EPA SW-846)
3-Chloro-1,2-propanediol
96-24-2
GC-MS
Sample Prep
Determinative
Adapted from Eur. J.
Lipid Sci. Technol.
(2011) 113: 345-355
II
Adapted from J.
Chromatogr. A
(2000) 866: 65-77
n
Adapted from J.
Chromatogr. A
(2000) 866: 65-77
n
TO-10A3
(EPA ORD)
ni
Adapted from Eur. J.
Lipid Sci. Technol.
(2011) 113: 345-355
ni
Chloropicrin
76-06-2
GC-MS/
GC-ECD
Sample Prep
Determinative
EPA/600/R-16/11415
(EPA NHSRC)
n
551.1
(EPA OW)
i
551.1
(EPA OW)
i
PV2103 (OSHA)
i
EPA/600/R-16/11415
(EPA NHSRC)
n
Chlorosarin
1445-76-7
GC-MS
Sample Prep
Determinative
EPA/600/R-16/115
(EPA NHSRC)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*m
TO-173
(EPA ORD)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*m
Chlorosoman
7040-57-5
GC-MS
Sample Prep
Determinative
EPA/600/R-16/115
(EPA NHSRC)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*m
TO-173
(EPA ORD)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*m
2-Chlorovinyl-arsonic acid (CVAOA)
(degradation product of Lewisite)
64038-44-4
LC-MS-MS/
ICP-AES /
ICP-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
IO-3.15
(EPA ORD)
*i
EPA/600/R-15/2584
(EPA NHSRC)
*n
Determinative
IO-3.4/IO-3.55
(EPA ORD)
2-Chlorovinylarsonous acid
(CVAA) (degradation product of Lewisite)
85090-33-1
LC-MS-MS/
ICP-AES /
ICP-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
IO-3.15
(EPA ORD)
*i
EPA/600/R-15/2584
(EPA NHSRC)
*n
Determinative
IO-3.4/IO-3.55
(EPA ORD)
Chlorpyrifos
2921-88-2
GC-MS
Sample Prep
Determinative
EPA/600/R-16/114
(EPA NHSRC)
n
EPA/600/R-16/114
(EPA NHSRC)
n
525.26
(EPA OW)
i
TO-10A
(EPA ORD)
i
EPA/600/R-16/114
(EPA NHSRC)
n
Chlorpyrifos oxon
5598-15-2
GC-MS/
LC-MS-MS
Sample Prep
Determinative
EPA/600/R-16/114
(EPA NHSRC)
in
540
(EPA OW)
i
540
(EPA OW)
i
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Crimidine
535-89-7
GC-MS
Sample Prep
Determinative
EPA/600/R-16/114
(EPA NHSRC)
n
EPA/600/R-16/114
(EPA NHSRC)
n
EPA/600/R-16/114
(EPA NHSRC)
n
Not of concern
NA
EPA/600/R-16/114
(EPA NHSRC)
n
Cyanide, Amenable to chlorination
NA
Visible
spectrophotometry
Sample Prep
Determinative
3135.21
(EPA RLAB)
i
3135.2I7
(EPA RLAB)
i
3135.2I7
(EPA RLAB)
i
Not of concern
NA
3135.21
(EPA RLAB)
ni
Cyanide, Total
57-12-5
Visible
spectrophotometry
Sample Prep
Determinative
ISM02.3 CN
(EPA CLP)
i
ISM02.3 CN8
(EPA CLP)
i
335.4
(EPA OW)
i
6010
(NIOSH)
I
ISM02.3 CN
(EPA CLP)
ni
SAM 2017 Appendix A
A - 4
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Cyanogen chloride
506-77-4
GC-MS/
Sample Prep
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.a0809
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.a0809
II
Adapted from
Encyclopedia of Anal.
Chem. (2006)
DOI: 10.1002/9780
470027318.a0809
II
TO-15
ni
Not of concern
NA
GC-ECD
Determinative
11
(EPAORD)
Cyclohexyl sarin (GF)
329-99-7
GC-MS
Sample Prep
EPA/600/R-16/115
*1
EPA/600/R-16/115
*1
EPA/600/R-16/115
*1
TO-17
ni
EPA/600/R-16/115
*1
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPAORD)
(EPA NHSRC)
1,2-Dichloroethane
107-06-2
GC-MS
Sample Prep
5035A
(EPA SW-846)
I
5030C
(EPA SW-846)
I
524.21
I
TO-15
i
Not of concern
NA
(degradation product of HD)
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
(EPA OW)
(EPA ORD)
Dichlorvos
62-73-7
GC-MS
Sample Prep
EPA/600/R-16/114
II
3535A
(EPA SW-846)
I
525.26
I
TO-10A
i
EPA/600/R-16/114
n
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
(EPA OW)
(EPA ORD)
(EPA NHSRC)
Dicrotophos
141-66-2
GC-MS
Sample Prep
EPA/600/R-16/114
II
3535A
(EPA SW-846)
I
3535A
(EPA SW-846)
I
TO-10A
i
EPA/600/R-16/114
n
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
(EPA ORD)
(EPA NHSRC)
Diesel range organics
NA
GC-FID
Sample Prep
3541/3545A
(EPA SW-846)
I
3520C/3535A
(EPA SW-846)
I
3520C/3535A
(EPA SW-846)
I
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
Diisopropyl methylphosphonate (DIMP)
1445-75-6
HPLC-UV /
Sample Prep
E2866-12
II
D7597-16
II
538
I
TO-10A3
ni
EPA/600/R-13/224
ii
(degradation product of GB)
LC-MS-MS
Determinative
(ASTM)
(ASTM)
(EPA OW)
(EPA ORD)
(EPA NHSRC)
Dimethylphosphite
868-85-9
GC-MS
Sample Prep
EPA/600/R-16/114
n
Not of concern
NA
Not of concern
NA
TO-10A
n
EPA/600/R-16/114
ii
Determinative
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
Dimethylphosphoramidic acid (degradation
33876-51-6
HPLC-UV/
Sample Prep
E2866-12
in
D7597-16
III
D7597-16
ni
TO-10A
ni
EPA/600/R-13/224
ni
product of GA)
LC-MS-MS
Determinative
(ASTM)
(ASTM)
(ASTM)
(EPA ORD)
(EPA NHSRC)
Diphacinone
82-66-6
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
D7644-16
(ASTM)
II
D7644-16
(ASTM)
ii
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
D7644-16
(ASTM)
D7644-16
(ASTM)
Disulfoton
298-04-4
GC-MS/
Sample Prep
EPA/600/R-16/114
525.26,9
II
525.26'9
n
5600
I
EPA/600/R-16/114
GC-FPD
Determinative
(EPA NHSRC)
n
(EPA OW)
(EPA OW)
(NIOSH)
(EPA NHSRC)
n
SAM 2017 Appendix A
A - 5
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Disulfoton sulfone oxon
2496-91-5
GC-MS/
Sample Prep
EPA/600/R-16/114
in
525.26'9
III
525.26'9
III
5600
ni
EPA/600/R-16/114
ni
GC-FPD
Determinative
(EPA NHSRC)
(EPA OW)
(EPA OW)
(NIOSH)
(EPA NHSRC)
Disulfoton sulfoxide
2497-07-6
GC-MS/
Sample Prep
E PA/600/R -16/114
in
525.26'9
II
525.26'9
n
5600
ni
EPA/600/R-16/114
ni
GC-FPD
Determinative
(EPA NHSRC)
(EPA OW)
(EPA OW)
(NIOSH)
(EPA NHSRC)
Disulfoton sulfoxide oxon
2496-92-6
GC-MS/
Sample Prep
EPA/600/R-16/114
in
525.26'9
III
525.26'9
iii
5600
ni
EPA/600/R-16/114
ni
GC-FPD
Determinative
(EPA NHSRC)
(EPA OW)
(EPA OW)
(NIOSH)
(EPA NHSRC)
1,4-Dithiane
505-29-3
GC-MS
Sample Prep
EPA/600/R-16/114
n
EPA/600/R-16/114
II
EPA/600/R-16/114
ii
Not of concern
NA
EPA/600/R-16/114
ii
(degradation product of HD)
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
3541/3545A
(EPA SW-846)
3570/8290A
EA 2192 [S-2-(diisopropylamino)ethyl
methylphosphonothioic acid]
(hydrolysis product of VX)
73207-98-4
HPLC-UV /
Sample Prep
in
EPA/600/R-15/097
II
EPA/600/R-15/097
n
TO-10A
ni
Appendix A
(EPA SW-846)
ni
LC-MS-MS
Determinative
EPA/600/R-15/097
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
EPA/600/R-15/097
(EPA NHSRC)
(EPA NHSRC)
Ethyl methylphosphonic acid (EMPA)
1832-53-7
HPLC-UV/
Sample Prep
E2866-12
n
D7597-16
II
D7597-16
iii
TO-10A
ni
EPA/600/R-13/224
ii
(degradation product of VX)
LC-MS-MS
Determinative
(ASTM)
(ASTM)
(ASTM)
(EPA ORD)
(EPA NHSRC)
Sample Prep
3541/3545A
3535A
3535A
9102
Ethyldichloroarsine (ED)
598-14-1
GC-MS
(EPA SW-846)
in
(EPA SW-846)
III
(EPA SW-846)
iii
TO-15
ni
(NIOSH)
ni
Determinative
8270E
8270E
8270E
(EPA ORD)
8270E
(EPA SW-846)
(EPA SW-846)
(EPA SW-846)
(EPA SW-846)
N-Ethyldiethanolamine (EDEA)
(degradation product of HN-1)
LC-MS-MS/
Sample Prep
3541/3545A
(EPA SW-846)
D7599-16
(ASTM)
D7599-16
(ASTM)
3509
(NIOSH)
EPA/600/R-11/143
(EPA NHSRC)
139-87-7
IC- conductivity
detection
Determinative
EPA/600/R-11 /143
(EPA NHSRC)
in
II
ni
ni
ii
Ethylene oxide
75-21-8
GC-MS
Sample Prep
5035A
(EPA SW-846)
ii
5030C
(EPA SW-846)
n
5030C
(EPA SW-846)
n
TO-15
i
Not of concern
NA
Determinative
8260D
8260D
8260D
(EPA ORD)
(EPA SW-846)
(EPA SW-846)
(EPA SW-846)
Fenamiphos
22224-92-6
GC-MS
Sample Prep
EPA/600/R-16/114
n
EPA/600/R-16/114
ii
525.26
i
TO-10A
n
EPA/600/R-16/114
n
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA OW)
(EPA ORD)
(EPA NHSRC)
SAM 2017 Appendix A
A - 6
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Fentanyl
437-38-7
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
3520C/3535A
(EPA SW-846)
3520C/3535A
(EPA SW-846)
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
Determinative
Adapted from J.
Chromatogr. A (2011)
1218:
1620- 1649
Adapted from J.
Chromatogr. A (2011)
1218:
1620-1649
111
Adapted from J.
Chromatogr. A (2011)
1218:
1620-1649
111
Adapted from J.
Chromatogr. A (2011)
1218:
1620-1649
111
Fluoride
16984-48-8
IC-conductivity
detection
Sample Prep
Determinative
Not of concern
NA
300.1, Rev 1.0
(EPA OW)
I
300.1, Rev 1.0
(EPA OW)
I
Not of concern
NA
Not of concern
NA
Fluoroacetamide
640-19-7
GC-MS
Sample Prep
Determinative
Adapted from J.
Chromatogr. B (2008)
876(1):
103-108
n
Adapted from J.
Chromatogr. B (2008)
876(1):
103-108
II
Adapted from J.
Chromatogr. B (2008)
876(1):
103-108
II
Adapted from J.
Chromatogr. B (2008)
876(1):
103-108
in
Adapted from J.
Chromatogr. B (2008)
876(1):
103-108
in
Fluoroacetic acid and fluoroacetate salts
(analyze as fluoroacetate ion)
NA
LC-MS /
LC-MS-MS
Sample Prep
Adapted from J.
Chromatogr. A (2007)
1139:
271-278
hi
Adapted from J.
Chromatogr. B(2010)
878:
1045-1050
III
Adapted from J.
Chromatogr. B (2010)
878:
1045-1050
ni
S301-1
(NIOSH)
in
Adapted from J.
Chromatogr. A (2007)
1139:
271-278
in
Determinative
Adapted from J.
Chromatogr. A (2007)
1139:
271-278
2-Fluoroethanol
371-62-0
GC-MS/
GC-FID
Sample Prep
5035A
(EPA SW-846)
hi
5030C
(EPA SW-846)
III
5030C
(EPA SW-846)
ni
2513
(NIOSH)
in
Not of concern
NA
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
8260D
(EPA SW-846)
Fluorosilicic acid (analyze as fluoride)
16961-83-4
IC-conductivity
detection
Sample Prep
Determinative
Not of concern
NA
300.1, Rev 1.0
(EPA OW)
I
300.1, Rev 1.0
(EPA OW)
i
Not of concern
NA
Not of concern
NA
Formaldehyde
50-00-0
FGC-ECD /
HPLC-UV
Sample Prep
8315A
(EPA SW-846)
I
8315A
(EPA SW-846)
I
556.1
(EPA OW)
i
2016
(NIOSH)
I
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
8315A
(EPA SW-846)
Gasoline range organics
NA
GC-FID
Sample Prep
5035A
(EPA SW-846)
T
5030C
(EPA SW-846)
T
5030C
(EPA SW-846)
i
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
Hexahydro-1,3,5-trinitro-l ,3,5-triazine
(RDX)
121-82-4
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
i
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
SAM 2017 Appendix A
A - 7
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Hexamethylenetriperoxidediamine (HMTD)
283-66-9
LC-MS
Sample Prep
8330B
(EPA SW-846)
11
3535A/8330B
(EPA SW-846)
11
3535A/8330B
(EPA SW-846)
II
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
Adapted from Analyst
(2001) 126:1689-
1693
Adapted from Analyst
(2001) 126:1689-
1693
Adapted from Analyst
(2001) 126:1689-
1693
Adapted from Analyst
(2001) 126:1689-1693
Hydrogen bromide
10035-10-6
IC-conductivity
detection
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
7907
(NIOSH)
I
Not of concern
NA
Determinative
Hydrogen chloride
7647-01-0
IC-conductivity
detection
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
7907
(NIOSH)
I
Not of concern
NA
Determinative
Hydrogen cyanide
74-90-8
Visible
spectrophotometry
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
6010
(NIOSH)
I
Not of concern
NA
Determinative
Hydrogen fluoride
7664-39-3
IC-conductivity
detection
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
7906
(NIOSH)
I
Not of concern
NA
Determinative
Hydrogen sulfide
7783-06-4
IC-conductivity
detection
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
6013
(NIOSH)
I
Not of concern
NA
Determinative
Isopropyl methylphosphonic acid (IMPA)
(degradation product of GB)
1832-54-8
HPLC-UV /
LC-MS-MS
Sample Prep
E2866-12
(ASTM)
n
D7597-16
(ASTM)
II
D7597-16
(ASTM)
III
TO-10A
(EPA ORD)
ni
EPA/600/R-13/224
(EPA NHSRC)
II
Determinative
Kerosene
64742-81-0
GC-FID
Sample Prep
3541/3545A
(EPA SW-846)
i
3520C/3535A
(EPA SW-846)
I
3520C/3535A
(EPA SW-846)
I
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
I
Determinative
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
8015D
(EPA SW-846)
Lead arsenate
(analyze as total arsenic)
7645-25-2
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
i
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
I
IO-3.1
(EPA ORD)
I
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Lewisite 1 (L-1)
[2-chlorovinyldichloroarsine]
541-25-3
ICP-AES /
ICP-MS/
LC-MS-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
EPA/600/R-15/2584
(EPA NHSRC)
*n
IO-3.15
(EPA ORD)
*1
EPA/600/R-15/2584
(EPA NHSRC)
*11
Determinative
IO-3.4/IO-3.55
(EPA ORD)
Lewisite 2 (L-2)
[bis(2-chlorovinyl)chloroarsine]
40334-69-8
ICP-AES /
ICP-MS/
LC-MS-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*ni
EPA/600/R-15/2584
(EPA NHSRC)
*in
EPA/600/R-15/2584
(EPA NHSRC)
*iii
IO-3.15
(EPA ORD)
*1
EPA/600/R-15/2584
(EPA NHSRC)
*111
Determinative
IO-3.4/IO-3.55
(EPA ORD)
SAM 2017 Appendix A
A - 8
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Lewisite 3 (L-3)
[tris(2-chlorovinyl)arsine]
40334-70-1
ICP-AES /
ICP-MS /
LC-MS-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*ni
EPA/600/R-15/2584
(EPA NHSRC)
*in
EPA/600/R-15/2584
(EPA NHSRC)
*111
IO-3.15
(EPA ORD)
*1
EPA/600/R-15/2584
(EPA NHSRC)
*111
Determinative
IO-3.4/IO-3.55
(EPA ORD)
Lewisite oxide
(degradation product of Lewisite)
1306-02-1
ICP-AES /
ICP-MS/
LC-MS-MS
Sample Prep
EPA/600/R-15/2584
(EPA NHSRC)
*ni
EPA/600/R-15/2584
(EPA NHSRC)
*in
EPA/600/R-15/2584
(EPA NHSRC)
*111
IO-3.15
(EPA ORD)
*1
EPA/600/R-15/2584
(EPA NHSRC)
*111
Determinative
IO-3.4/IO-3.55
(EPA ORD)
Mercuric chloride (analyze as total
mercury)
7487-94-7
Visible
spectrophotometry /
CVAA /
CVAFS
Sample Prep
747310
(EPA SW-846)
i
245.111
(EPA OW)
i
245.1
(EPA OW)
I
Not of concern
NA
9102
(NIOSH)
I
Determinative
747310
(EPA SW-846)
Mercury, Total
7439-97-6
Visible
spectrophotometry /
CVAA /
CVAFS
Sample Prep
747310
(EPA SW-846)
i
245.111
(EPA OW)
i
245.1
(EPA OW)
I
IO-5
(EPA ORD)
I
9102
(NIOSH)
I
Determinative
747310
(EPA SW-846)
Methamidophos
10265-92-6
LC-MS-MS
Sample Prep
Adapted from J.
Chromatogr. A (2007)
1154(1):
3-25
n
Adapted from
Chromatographia
(2006) 63(5/6):
233-237
ii
538
(EPA OW)
I
Adapted from J.
Chromatogr. A (2007)
1154(1):
3-25
ni
Adapted from J.
Chromatogr. A (2007)
1154(1): 3-25
ni
Determinative
Methomyl
16752-77-5
HPLC-UV /
HPLC-FL /
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
ii
D7645-16
(ASTM)
ii
531.2
(EPA OW)
I
5601
(NIOSH)
i
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
8318A
(EPA SW-846)
Methoxyethylmercuric acetate
(analyze as total mercury)
151-38-2
Visible
spectrophotometry /
CVAA/
CVAFS
Sample Prep
747310
(EPA SW-846)
i
245.111
(EPA OW)
i
245.1
(EPA OW)
I
IO-5
(EPA ORD)
i
9102
(NIOSH)
i
Determinative
747310
(EPA SW-846)
Methyl acrylonitrile
126-98-7
HPLC-UV/
GC-MS
Sample Prep
5035A
(EPA SW-846)
ii
524.21
(EPA OW)
ii
524.21
(EPA OW)
n
PV2004
(OS HA)
ni
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
Methylamine
74-89-5
HPLC-FL/
HPLC-vis
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
OSHA 40
i
Not of concern
NA
Determinative
N-Methyldiethanolamine (MDEA)
(degradation product of HN-2)
105-59-9
LC-MS-MS/
IC-conductivity
detection
Sample Prep
3541/3545A
(EPA SW-846)
in
D7599-16
(ASTM)
II
D7599-16
(ASTM)
ni
3509
(NIOSH)
in
EPA/600/R-11/143
(EPA NHSRC)
II
Determinative
EPA/600/R-11 /143
(EPA NHSRC)
1-Methylethyl ester
ethylphosphonofluoridic acid (GE)
1189-87-3
GC-MS
Sample Prep
EPA/600/R-16/115
(EPA NHSRC)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*111
EPA/600/R-16/115
(EPA NHSRC)
*m
TO-173
(EPA ORD)
*iii
EPA/600/R-16/115
(EPA NHSRC)
*m
Determinative
SAM 2017 Appendix A
A - 9
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
3-Methyl fentanyl
42045-87-4
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
III
3520C/3535A
(EPA SW-846)
ni
3520C/3535A
(EPA SW-846)
ni
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
III
Determinative
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Adapted from J.
Chromatogr. B (2014)
962: 52-58
Methyl fluoroacetate
(analyze as fluoroacetate ion)
Sample Prep
J. Chromatogr. A
J. Chromatogr. B
J. Chromatogr. B
S301-1
(NIOSH)
J. Chromatogr. A
453-18-9
LC-MS
Determinative
(2007) 1139:
271-278
111
(2010) 878:
1045-1050
in
(2010) 878:
1045-1050
in
J. Chromatogr. A
(2007) 1139:
271-278
111
(2007) 1139:
271-278
111
Methyl hydrazine
60-34-4
Visible
spectrophotometry /
HPLC-UV
Sample Prep
3541/3545A
(EPA SW-846)
III
J. Chromatogr. (1993)
hi
J. Chromatogr. (1993)
in
3510
I
3570/8290A
Appendix A
(EPA SW-846)
III
Determinative
J. Chromatogr.
(1993) 617:
157-162
617: 157-162
617: 157-162
(NIOSH)
J. Chromatogr.
(1993) 617: 157-162
Methyl isocyanate
624-83-9
HPLC-UV
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
OSHA 54
I
Not of concern
NA
Determinative
Methyl paraoxon
950-35-6
GC-MS
Sample Prep
EPA/600/R-16/114
III
3535A
(EPA SW-846)
in
3535A
(EPA SW-846)
in
TO-10A
III
EPA/600/R-16/114
III
Determinative
(EPA NHSRC)
8270E9
(EPA SW-846)
8270E9
(EPA SW-846)
(EPA ORD)
(EPA NHSRC)
Methyl parathion
298-00-0
GC-MS
Sample Prep
E PA/600/R-16/114
n
3535A
(EPA SW-846)
i
3535A
(EPA SW-846)
i
TO-10A
I
EPA/600/R-16/114
II
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
(EPA ORD)
(EPA NHSRC)
Methylphosphonic acid (MPA)
993-13-5
HPLC-UV/
Sample Prep
E2866-12
ii
D7597-16
ii
D7597-16
in
TO-10A
III
EPA/600/R-13/224
II
(degradation product of VX, GB, & GD)
LC-MS-MS
Determinative
(ASTM)
(ASTM)
(ASTM)
(EPA ORD)
(EPA NHSRC)
Mevinphos
7786-34-7
GC-MS
Sample Prep
E PA/600/R -16/114
n
3535A
(EPA SW-846)
i
525.26
i
TO-10A
EPA/600/R-16/114
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
(EPA OW)
(EPA ORD)
11
(EPA NHSRC)
11
Monocrotophos
6923-22-4
GC-MS
Sample Prep
3541/3545A
(EPA SW-846)
i
3535A
(EPA SW-846)
i
3535A
(EPA SW-846)
i
TO-10A
(EPA ORD)
III
3570/8290A
Appendix A
(EPA SW-846)
III
Determinative
8270E
(EPA SW-846)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
Mustard, nitrogen (HN-1)
538-07-8
GC-MS
Sample Prep
EPA/600/R-12/653
*n
EPA/600/R-12/653
*11
EPA/600/R-12/653
*11
TO-17
*111
EPA/600/R-12/653
*n
[bis(2-chloroethyl)ethylamine]
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
SAM 2017 Appendix A
A-10
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Mustard, nitrogen (HN-2)
[2,2'-dichloro-N-methyldiethylamine N, N-
bis(2-chloroethyl)methylamine]
51-75-2
GC-MS
Sample Prep
EPA/600/R-12/653
(EPA NHSRC)
*111
EPA/600/R-12/653
(EPA NHSRC)
*111
EPA/600/R-12/653
(EPA NHSRC)
*m
TO-17
(EPA ORD)
*111
EPA/600/R-12/653
(EPA NHSRC)
*m
Determinative
Mustard, nitrogen (HN-3)
[tris(2-chloroethyl)amine]
555-77-1
GC-MS
Sample Prep
EPA/600/R-12/653
(EPA NHSRC)
=i=II
EPA/600/R-12/653
(EPA NHSRC)
*11
EPA/600/R-12/653
(EPA NHSRC)
*n
TO-17
(EPA ORD)
*m
EPA/600/R-12/653
(EPA NHSRC)
=1=11
Determinative
Mustard, sulfur / Mustard gas (HD)
505-60-2
GC-MS
Sample Prep
EPA/600/R-16/115
(EPA NHSRC)
*1
EPA/600/R-16/115
(EPA NHSRC)
*1
EPA/600/R-16/115
(EPA NHSRC)
*i
TO-17
(EPA ORD)
ni
EPA/600/R-16/115
(EPA NHSRC)
*i
Determinative
Nicotine compounds
(analyze as nicotine)
54-11-5
GC-MS
Sample Prep
E PA/600/R -16/114
(EPA NHSRC)
II
3535A
(EPA SW-846)
II
3535A
(EPA SW-846)
n
Not of concern
NA
EPA/600/R-16/114
(EPA NHSRC)
n
Determinative
8270E
(EPA SW-846)
8270E
(EPA SW-846)
Octahydro-1,3,5,7-tetranitro-1,3,5,7-
tetrazocine (HMX)
2691-41-0
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
i
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Osmium tetroxide
(analyze as total osmium)
20816-12-0
ICP-AES /
ICP-MS
Sample Prep
3051A
(EPA SW-846)
II
3015A
(EPA SW-846)
II
3015A
(EPA SW-846)
n
IO-3.1
(EPA ORD)
II
3051A
(EPA SW-846)
in
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
6010D/6020B
(SW-846)
IO-3.4
(EPA ORD)
6010D/6020B
(EPA SW-846)
Oxamyl
23135-22-0
HPLC-UV/
HPLC-FL /
LC-MS-MS
Sample Prep
8318A
(EPA SW-846)
II
D7645-16
(ASTM)
II
531.2
(EPA OW)
i
5601
(NIOSH)
I
3570/8290A
Appendix A
(EPA SW-846)
TTT
Determinative
8318A
(EPA SW-846)
Paraoxon
311-45-5
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
in
3520C/3535A
(EPA SW-846)
III
3520C/3535A
(EPA SW-846)
ni
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Determinative
8270E
(EPA SW-846)
8270E
(EPA SW-846)
Paraquat
4685-14-7
HPLC-UV/
LC-MS-MS
Sample Prep
Adapted from J.
Chromatogr. A (2008)
1196-1197:
110-116
n
549.2
(EPA OW)
I
549.2
(EPA OW)
i
Not of concern
NA
Adapted from J.
Chromatogr. A (2008)
1196-1197:
110-116
ni
Determinative
Parathion
56-38-2
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
n
3520C/3535A
(EPA SW-846)
I
3520C/3535A
(EPA SW-846)
i
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
n
Determinative
8270E
(EPA SW-846)
8270E
(EPA SW-846)
SAM 2017 Appendix A
A-11
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Pentaerythritol tetranitrate (PETN)
78-11-5
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
I
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Phencyclidine
77-10-1
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
II
EPA/600/R-16/114
(EPA NHSRC)
II
EPA/600/R-16/114
(EPA NHSRC)
II
TO-10A
(EPA ORD)
II
9106/9109
(NIOSH)
n
Determinative
Phorate
298-02-2
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
II
3535A
(EPA SW-846)
I
3535A
(EPA SW-846)
I
TO-10A
(EPA ORD)
II
EPA/600/R-16/114
(EPA NHSRC)
n
Determinative
8270E9
(EPA SW-846)
8270E9
(EPA SW-846)
Phorate sulfone
2588-04-7
GC-MS/
LC-MS-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
in
540
(EPA OW)
I
540
(EPA OW)
I
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Determinative
Phorate sulfone oxon
2588-06-9
GC-MS/
LC-MS-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
in
540
(EPA OW)
III
540
(EPA OW)
III
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Determinative
Phorate sulfoxide
2588-03-6
GC-MS/
LC-MS-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
in
540
(EPA OW)
I
540
(EPA OW)
I
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Determinative
Phorate sulfoxide oxon
2588-05-8
GC-MS/
LC-MS-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
in
540
(EPA OW)
III
540
(EPA OW)
III
TO-10A
(EPA ORD)
ni
EPA/600/R-16/114
(EPA NHSRC)
ni
Determinative
Phosgene
75-44-5
GC-NPD
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
OSHA 61
i
Not of concern
NA
Determinative
Phosphamidon
13171-21-6
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)
II
3520C/3535A
(EPA SW-846)
I
525.3
(EPA OW)
I
TO-10A
(EPA ORD)
n
EPA/600/R-16/114
(EPA NHSRC)
II
Determinative
8270E
(EPA SW-846)
Phosphine
7803-51-2
Visible
spectrophotometry
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
6002
(NIOSH)
i
Not of concern
NA
Determinative
Phosphorus trichloride
7719-12-2
Visible
spectrophotometry
Sample Prep
Not of concern
NA
Not of concern
NA
Not of concern
NA
6402
(NIOSH)
i
Not of concern
NA
Determinative
Pinacolyl methyl phosphonic acid (PMPA)
(degradation product of GD)
616-52-4
HPLC-UV/
LC-MS-MS
Sample Prep
E2866-12
(ASTM)
n
D7597-16
(ASTM)
II
D7597-16
(ASTM)
III
TO-10A
(EPA ORD)
ni
EPA/600/R-13/224
(EPA NHSRC)
II
Determinative
SAM 2017 Appendix A
A-12
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Propylene oxide
75-56-9
GC-MS/
Sample Prep
5035A
(EPA SW-846)
11
5030C
(EPA SW-846)
11
5030C
(EPA SW-846)
II
1612
I
Not of concern
NA
GC-FID
Determinative
8260D
(EPA SW-846)
8260D
(EPA SW-846)
8260D
(EPA SW-846)
(NIOSH)
R 33 (VR)
[methylphosphonothioic acid, S-[2-
159939-87-4
GC-MS
Sample Prep
EPA/600/R-12/653
*11
EPA/600/R-12/653
*n
EPA/600/R-12/653
=i=II
TO-17
*in
EPA/600/R-12/653
*11
(diethylamino)ethyl] O-2-methylpropyl
ester]
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
Sarin (GB)
107-44-8
GC-MS
Sample Prep
EPA/600/R-16/115
*1
EPA/600/R-16/115
*i
EPA/600/R-16/115
*1
TO-173
*ni
EPA/600/R-16/115
*1
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
Sodium arsenite
(analyze as total arsenic)
7784-46-5
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
I
3015A
(EPA SW-846)
i
200.7/200.8
(EPA OW)
I
IO-3.1
(EPA ORD)
i
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Sodium azide
(analyze as azide ion)
26628-22-8
IC-conductivity
detection
Sample Prep
Adapted from J.
Forensic Sci. (1998)
43(1): 200-20212
n
Adapted from J.
Forensic Sci. (1998)
43(1): 200-20212
TT
Adapted from J.
Forensic Sci. (1998)
43(1): 200-20212
TT
ID-211 (OS HA)
i
ID-211 (OSHA)
I
Determinative
300.1, Rev 1.013
(EPA OW)
300.1, Rev 1.013
(EPA OW)
300.1, Rev 1,013
(EPA OW)
Soman (GD)
96-64-0
GC-MS
Sample Prep
EPA/600/R-16/115
*i
EPA/600/R-16/115
*1
EPA/600/R-16/115
*1
TO-173
*ni
EPA/600/R-16/115
*1
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
Strychnine
57-24-9
GC-MS
Sample Prep
EPA/600/R-16/114
ii
3535A
(EPA SW-846)
I
3535A
(EPA SW-846)
I
Not of concern
NA
EPA/600/R-16/114
II
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
(EPA NHSRC)
Tabun (GA)
77-81-6
GC-MS
Sample Prep
EPA/600/R-12/653
*n
EPA/600/R-12/653
'ii
EPA/600/R-12/653
*11
TO-17
*ni
EPA/600/R-12/653
*n
Determinative
(EPA NHSRC)
(EPA NHSRC)
(EPA NHSRC)
(EPA ORD)
(EPA NHSRC)
Tetraethyl pyrophosphate (TEPP)
107-49-3
GC-MS
Sample Prep
EPA/600/R-16/114
n
3511
(EPA SW-846)
II
3511
(EPA SW-846)
II
TO-10A
n
EPA/600/R-16/114
n
Determinative
(EPA NHSRC)
8270E
(EPA SW-846)
8270E
(EPA SW-846)
(EPA ORD)
(EPA NHSRC)
Tetramethylenedisulfotetramine (TETS)
80-12-6
GC-MS
Sample Prep
EPA/600/R-16/114
EPA/600/R-16/114
I
EPA/600/R-16/114
TO-10A
n
EPA/600/R-16/114
Determinative
(EPA NHSRC)
n
(EPA NHSRC)
(EPA NHSRC)
1
(EPA ORD)
(EPA NHSRC)
n
SAM 2017 Appendix A
A-13
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
Thallium sulfate
(analyze as total thallium)
10031-59-1
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
I
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
I
IO-3.1
(EPA ORD)
I
9102
(NIOSH)
I
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
Thiodiglycol (TDG)
(degradation product of HD)
111-48-8
HPLC /
LC-MS-MS
Sample Prep
E2787-11
(ASTM)
n
D7598-16
(ASTM)
II
D7598-16
(ASTM)
ni
TO-10A
(EPA ORD)
in
E2838-11
(ASTM)
n
Determinative
Thiofanox
39196-18-4
HPLC-UV /
LC-MS-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
D7645-16
(ASTM)
II
538
(EPA OW)
i
5601
(NIOSH)
in
3570/8290A
Appendix A
(EPA SW-846)
in
Determinative
D7645-16
(ASTM)
D7645-16
(ASTM)
1,4-Thioxane
(degradation product of HD)
15980-15-1
GC-MS
Sample Prep
EPA/600/R-16/114
(EPA NHSRC)14
n
EPA/600/R-16/114
(EPA NHSRC)14
II
EPA/600/R-16/114
(EPA NHSRC)14
ii
Not of concern
NA
EPA/600/R-16/114
(EPA NHSRC)14
n
Determinative
Titanium tetrachloride
(analyze as total titanium)
7550-45-0
ICP-AES /
ICP-MS
Sample Prep
3051A
(EPA SW-846)
i
Not of concern
NA
Not of concern
NA
Not of concern
NA
3051A
(EPA SW-846)
ni
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
Triethanolamine (TEA)
(degradation product of HN-3)
102-71-6
LC-MS-MS/
IC-conductivity
detection
Sample Prep
3541/3545A
(EPA SW-846)
in
D7599-16
(ASTM)
II
D7599-16
(ASTM)
ni
3509
(NIOSH)
II
EPA/600/R-11/143
(EPA NHSRC)
ii
Determinative
EPA/600/R-11 /143
(EPA NHSRC)
Trimethyl phosphite
121-45-9
GC-MS
Sample Prep
3541/3545A
(EPA SW-846)
in
Not of concern
NA
Not of concern
NA
TO-10A
(EPA ORD)
in
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
8270E15
(EPA SW-846)
8270E15
(EPA SW-846)
1,3,5-Trinitrobenzene (1,3,5-TNB)
99-35-4
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
i
3535A/8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
2,4,6-Trinitrotoluene (2,4,6-TNT)
118-96-7
HPLC-UV
Sample Prep
8330B
(EPA SW-846)
i
3535A/8330B
(EPA SW-846)
I
3535A/8330B
(EPA SW-846)
I
Not of concern
NA
3570/8290A
Appendix A
(EPA SW-846)
i
Determinative
8330B
(EPA SW-846)
8330B
(EPA SW-846)
8330B
(EPA SW-846)
Vanadium pentoxide
(analyze as total vanadium)
1314-62-1
ICP-AES /
ICP-MS
Sample Prep
3050B/3051A
(EPA SW-846)
i
3015A
(EPA SW-846)
I
200.7/200.8
(EPA OW)
I
IO-3.1
(EPA ORD)
I
9102
(NIOSH)
i
Determinative
6010D/6020B
(EPA SW-846)
6010D/6020B
(EPA SW-846)
IO-3.4/IO-3.5
(EPA ORD)
6010D/6020B
(EPA SW-846)
SAM 2017 Appendix A
A-14
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Method Type
Solid Samples
Non-Drinking Water
Samples
Drinking Water Samples
Air Samples
Wipes
VE [phosphonothioic acid, ethyl-, S-(2-
(diethylamino)ethyl) O-ethyl ester]
21738-25-0
GC-MS
Sample Prep
Determinative
EPA/600/R-16/116
(EPA NHSRC)
*ni
EPA/600/R-16/116
(EPA NHSRC)
*in
EPA/600/R-16/116
(EPA NHSRC)
*111
TO-17
(EPA ORD)
*m
EPA/600/R-16/116
(EPA NHSRC)
*111
VG [phosphonothioic acid, S-(2-
(diethylamino)ethyl) 0,0-diethyl ester]
78-53-5
GC-MS
Sample Prep
Determinative
EPA/600/R-16/116
(EPA NHSRC)
*ni
EPA/600/R-16/116
(EPA NHSRC)
*in
EPA/600/R-16/116
(EPA NHSRC)
*111
TO-17
(EPA ORD)
*m
EPA/600/R-16/116
(EPA NHSRC)
*111
VM [phosphonothioic acid,
methyl-, S-(2-(diethylamino)ethyl) O-ethyl
ester]
21770-86-5
GC-MS
Sample Prep
Determinative
EPA/600/R-16/116
(EPA NHSRC)
*ni
EPA/600/R-16/116
(EPA NHSRC)
*in
EPA/600/R-16/116
(EPA NHSRC)
*111
TO-17
(EPA ORD)
*m
EPA/600/R-16/116
(EPA NHSRC)
*111
VX [0-ethyl-S-(2-
diisopropylaminoethyl)methyl-
phosphonothiolate]
50782-69-9
GC-MS
Sample Prep
Determinative
EPA/600/R-16/116
(EPA NHSRC)
*n
EPA/600/R-16/116
(EPA NHSRC)
*i
EPA/600/R-16/116
(EPA NHSRC)
*1
TO-17
(EPA ORD)
*m
EPA/600/R-16/116
(EPA NHSRC)
*11
White phosphorus
12185-10-3
GC-NPD /
GC-FPD
Sample Prep
7580
(EPA SW-846)
i
7580
(EPA SW-846)
i
7580
(EPA SW-846)
I
7905
(NIOSH)
i
3570/8290A
Appendix A
(EPA SW-846)
ni
Determinative
7580
(EPA SW-846)
* 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 Methods 524.3 or 524.4 may be used in place of Method 524.2 provided the laboratory has the necessary equipment and expertise.
2 If problems occur when using this method, TO-1 OA should be used.
3 If problems occur when using this method, Method TO-15 should be used.
4 The following methods can be used to analyze these compounds as total arsenic in situations where high throughput analysis is needed or where standards are not available for the specific compounds: ICP AES/MS Methods
200.7/200.8 for drinking water; Methods 3015A/6010D/6020B for aqueous liquid samples; Methods 3050B/3051 A/6010D/6020B for solid samples; and Methods 9102/6010D/6020B for wipes.
5 TO Methods IO-3.1, IO-3.4 and IO-3.5 address these compounds as total arsenic in air samples.
6 Method 525.3 may be used in place of Method 525.2 provided the laboratory has the necessary equipment and expertise.
7 Standard Method 4500-CN-G may be used in place of RLAB Method 3135.21 for the analysis of cyanide amenable to chlorination in aqueous liquid or drinking water samples.
8 The inline distillation method, EPA-821-B-01-009, may be used to prepare and analyze fortotal cyanide in aqueous liquid samples.
9 If problems occur during measurement of oxon compounds, analysts should consider use of procedures included in Kamal, A. et al., "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.
10 If equipment is not available or problems occur when analyzing solid and wipe samples, use CVAA Method 7471B (EPA SW-846).
11
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).
12 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.
13
If analyses are problematic, refer to column manufacturer for alternate conditions.
14 If problems occur when using this method, SW-846 Method 8260D and appropriate corresponding sample preparation procedures (i.e., 5035A for solid samples, and 5030C for aqueous liquid and drinking water samples) should be
used.
15
If problems occur with analyses, lower the injection temperature.
SAM 2017 Appendix A
A-15
September 2017
-------
This page left intentionally blank
-------
Appendix B1 - Selected Radiochemical Methods
Appendix B1: Selected Radiochemical Methods for
Environmental Samples
SAM 2017 - Appendix B1
September 2017
-------
This page left intentionally blank
-------
SAM 2017 Appendix B1: Selected Radiochemical Methods for Environmental Samples
Note: Column headings are defined in Section 6.0.
Analyte Class
Determinative
Technique
Drinking Water
Aqueous and Liquid Phase
Soil and Sediment
Surface Wipes
Air Filters
Vegetation
Gross Alpha
Alpha / Beta
counting
900.0
(EPA)
7110 B
(SM)
AP1
(ORISE)
FRMAC, Vol 2, pg. 33
(DOE)
FRMAC, Vol 2, pg. 33
(DOE)
AP1
(ORISE)
Gross Beta
Alpha / Beta
counting
900.0
(EPA)
7110 B
(SM)
AP1
(ORISE)
FRMAC, Vol 2, pg. 33
(DOE)
FRMAC, Vol 2, pg. 33
(DOE)
AP1
(ORISE)
Gamma
Gamma
spectrometry
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Select Mixed Fission Products1
Gamma
spectrometry
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Total Activity Screening
Liquid
scintillation
Preparation of Samples for
Total Activity Screening
(Y-12)
Preparation of Samples for
Total Activity Screening
(Y-12)
Preparation of Samples for
Total Activity Screening
(Y-12)
Preparation of Samples for
Total Activity Screening
(Y-12)
Preparation of Samples for
Total Activity Screening
(Y-12)
Preparation of Samples for
Total Activity Screening
(Y-12)
Analyte(s)
CAS RN
Determinative
Technique
Drinking Water
Aqueous and Liquid Phase
Soil and Sediment
Surface Wipes
Air Filters
Vegetation
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Americium-2413
14596-10-2
Alpha
spectrometry
Rapid
Radiochemical
Method for
Am-2414
(EPA)
Am-04-RC
(HASL-300)
D3084-05 (2012)
(ASTM)
Am-04-RC
(HASL-300)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices (EPA)
Am-01-RC5
(HASL-300)
Rapid methods*
for acid or fusion
digestion
(EPA)
Am-04-RC
(HASL-300)
Rapid methods*
for acid or fusion
digestion
(EPA)
Am-04-RC
(HASL-300)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Am-06-RC
(HASL-300)
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Californium-2523
13981-17-4
Alpha
spectrometry
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Californium-252
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Californium-252
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Californium-252
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Californium-252
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Californium-252
(EPA)
D3084-05 (2012)
(ASTM)
Am-06-RC
(HASL-300)
Cesium-137
10045-97-3
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Cobalt-60
10198-40-0
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Curium-2443
13981-15-2
Alpha
spectrometry
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Curium-244 in
Water
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Curium-244 in
Water
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Curium-244 in Air
Particulate Filters,
Swipes and Soil
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Curium-244 in Air
Particulate Filters,
Swipes and Soil
(EPA)
D3084-05 (2012)
(ASTM)
Rapid
Radiochemical
Method for
Curium-244 in Air
Particulate Filters,
Swipes and Soil
(EPA)
D3084-05 (2012)
(ASTM)
Am-06-RC
(HASL-300)
Europium-154
15585-10-1
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
lndium-111
15750-15-9
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
lodine-125
14158-31-7
Gamma
spectrometry
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
Procedure #96
(ORISE)
Procedure #96
(ORISE)
Procedure #9
(ORISE)
Procedure #9
(ORISE)
SAM 2017 Appendix B1
B1 -1
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Drinking Water
Aqueous and Liquid Phase
Soil and Sediment
Surface Wipes
Air Filters
Vegetation
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
lodine-131
10043-66-0
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R6
(HASL-300)
Ga-01-R6
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
lridium-192
14694-69-0
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Molybdenum-99
14119-15-4
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Neptunium-237
13994-20-2
Alpha
spectrometry
907.0
(EPA)
907.0
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for
Actinides in
Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Neptunium-239
13968-59-7
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Phosphorus-32
14596-37-3
Liquid
scintillation / Beta
counting
Rapid
Radiochemical
Method for P-32
in water4
(EPA)
R4-73-014
(EPA)
R4-73-014
(EPA)
R4-73-014
(EPA)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
RESL P-2
(DOE)
Plutonium-2383
13981-16-3
Alpha
spectrometry
Rapid
Radiochemical
Method for
Pu-238 and -
239/2404
(EPA)
EMSL-33
(EPA)
D3084-05 (2015)
(ASTM)
SOP for
Actinides in
Environmental
Matrices
(EPA-NAREL)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for
Actinides in
Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Am-06-RC
(HASL-300)
Plutonium-2393
15117-48-3
Alpha
spectrometry
Rapid
Radiochemical
Method for
Pu-238 and
239/2404
(EPA)
EMSL-33
(EPA)
D3084-05 (2012)
(ASTM)
SOP for
Actinides in
Environmental
Matrices
(EPA-NAREL)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Am-06-RC
(HASL-300)
Polonium-210
13981-52-7
Alpha
spectrometry
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Method 111
(EPA)
Method 111
(EPA)
Method 111
(EPA)
Method 111
(EPA)
Po-02-RC
(HASL-300)
Po-02-RC
(HASL-300)
Radium-223
15623-45-7
Alpha
spectrometry
Rapid Radiochemical Method
for Ra-2264
(EPA)
Rapid Radiochemical Method
for Ra-226
(EPA)
Rapid Radiochemical Method
for Ra-226
(EPA)
Rapid Radiochemical Method
for Ra-226
(EPA)
Rapid Radiochemical Method
for Ra-226
(EPA)
Rapid Radiochemical Method
for Ra-226
(EPA)
Radium-226
13982-63-3
Alpha
spectrometry /
Alpha counting /
Gamma
spectrometry
Rapid
Radiochemical
Method for
Ra-2264
(EPA)
903.1
(EPA)
7500-Ra B
(SM)
7500-Ra C
(SM)
Rapid Method for
Radium in Soil
(EPA)
AP7
(ORISE)
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid Method for
Radium-226 in
Building Materials
(EPA)
Rapid methods*
for acid or fusion
digestion
(EPA)
Rapid Method for
Radium-226 in
Building Materials
(EPA)
Ra-03-RC
(HASL-300)
Ra-03-RC
(HASL-300)
Rhenium-188
14378-26-8
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Rubidium-82
14391-63-0
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ruthenium-103
13968-53-1
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
SAM 2017 Appendix B1
B1 -2
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Drinking Water
Aqueous and Liquid Phase
Soil and Sediment
Surface Wipes
Air Filters
Vegetation
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Ruthenium-106
13967-48-1
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Selenium-75
14265-71-5
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
7120
(SM)
7120
(SM)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Strontium-89
14158-27-1
Beta counting
905.0
(EPA)
905.0
(EPA)
905.0
(EPA)
905.0
(EPA)
Actinides and
Sr-89/90 in
Soil Samples
(DOE SRS)
Strontium in
Food and
Bioenvironmental
Samples
(EPA)
Strontium in Food and
Bioenvironmental Samples
(EPA)
Strontium in Food and
Bioenvironmental Samples
(EPA)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Strontium in
Food and
Bioenvironmental
Samples
(EPA)
Strontium-90
10098-97-2
Beta counting /
alpha
spectrometry
Rapid
Radiochemical
Method for
Radiostrontium4
(EPA)
905.0
(EPA)
D5811-08 (2013)
(ASTM)
D5811-08
(2013)
(ASTM)
Rapid Method for
Sodium
Carbonate Fusion
of Soil and Soil-
Related Matrices
(EPA)
Sr-03-RC
(HASL-300)
Rapid methods*
for acid or fusion
digestion
(EPA)
Sr-03-RC
(HASL-300)
Rapid methods*
for acid or fusion
digestion
(EPA)
Sr-03-RC
(HASL-300)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
Sr-03-RC
(HASL-300)
Technetium-99
14133-76-7
Liquid
scintillation / beta
counting
Tc-02-RC
(HASL-300)
Tc-02-RC
(HASL-300)
D7168-16
(ASTM)
D7168-16
(ASTM)
AP5
(ORISE)
AP5
(ORISE)
AP5
(ORISE)
AP5
(ORISE)
AP5
(ORISE)
AP5
(ORISE)
AP5
(ORISE)
Tc-01-RC
(HASL-300)
Technetium-99m
378784-45-3
Gamma
spectrometry
901.1
(EPA)
901.1
(EPA)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Ga-01-R
(HASL-300)
Thorium-227
15623-47-9
Alpha
spectrometry
907.0
(EPA)
907.0
(EPA)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
Thorium-228
14274-82-9
Alpha
spectrometry
907.0
(EPA)
907.0
(EPA)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
Thorium-230
14269-63-7
Alpha
spectrometry
907.0
(EPA)
907.0
(EPA)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
Thorium-232
7440-29-1
Alpha
spectrometry
907.0
(EPA)
907.0
(EPA)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
SOP for Actinides in Environmental
Matrices
(EPA-NAREL)
SOP for Actinides in
Environmental Matrices
(EPA-NAREL)
Tritium
(Hydrogen-3)
10028-17-8
Liquid
scintillation
906.0
(EPA)
906.0
(EPA)
906.0
(EPA)
906.0
(EPA)
AP2
(ORISE)
AP2
(ORISE)
AP2
(ORISE)
AP2
(ORISE)
Not
applicable7
Not applicable7
AP2
(ORISE)
AP2
(ORISE)
Uranium-2343
13966-29-5
Alpha
spectrometry
Rapid
Radiochemical
Method for
Isotopic Uranium
in Water4
(EPA)
D3972-09
(ASTM)
7500-U B8
(SM)
7500-U C
(SM)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
U-02-RC
(HASL-300)
Uranium-2353
15117-96-1
Alpha
spectrometry
Rapid
Radiochemical
Method for
Isotopic Uranium
in Water4
(EPA)
D3972-09
(ASTM)
7500-U B8
(SM)
7500-U C
(SM)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
U-02-RC
(HASL-300)
SAM 2017 Appendix B1
B1 -3
September 2017
-------
Analyte(s)
CAS RN
Determinative
Technique
Drinking Water
Aqueous and Liquid Phase
Soil and Sediment
Surface Wipes
Air Filters
Vegetation
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Qualitative
Determination2
Confirmatory
Uranium-2383
7440-61-1
Alpha
spectrometry
Rapid
Radiochemical
Method for
Isotopic Uranium
in Water4
(EPA)
D3972-09
(ASTM)
7500-U B8
(SM)
7500-U C
(SM)
Rapid Method for
Fusion of Soil and
Soil-Related
Matrices
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Rapid methods*
for acid or fusion
digestion
(EPA)
SOP for Actinides
in Environmental
Matrices
(EPA-NAREL)
Actinides and
Sr-89/90 in
Vegetation
(DOE SRS)
U-02-RC
(HASL-300)
* 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 analysis of the digested samples.
Footnotes
1 Please note that this category does not cover all fission products. In addition to the specific radionuclides listed in this appendix, gamma-ray spectrometry with a high resolution HP(Ge) detector will identify and quantify 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 If it is suspected that the sample exists in refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use ORISE Method AP11 for qualitative determination or confirmatory analysis of alpha radioactivity.
4 This method is listed for rapid qualitative screening of drinking water samples. The method is not intended for use in compliance monitoring of drinking water.
5
In cases where only small sample volumes (<100 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.
SAM 2017 Appendix B1
B1 -4
September 2017
-------
Appendix B2 - Selected Rapid Radiochemical Methods
Appendix B2: Selected Rapid Radiochemical Methods for
Outdoor Building and Infrastructure Materials
SAM 2017 - Appendix B2
September 2017
-------
This page left intentionally blank
-------
SAM 2017 Appendix B2: Selected Rapid Radiochemical Methods for Outdoor Building and Infrastructure Materials
Note: Column headings are defined in Section 6.0.
An.ilyte(s)
CAS RN
Determinative
Technique
Asph.ilt Shingles
Asph.ilt Matrices
(Paving M.iteri.ils)
Concrete
Brick
Sample Preparation
Confirm.itory
Analysis
Sample Preparation
Confirm.itory
An.ilysis
S.iniple Prep.ir.ition
Confirm.itory
Analysis
S.iniple Prep.ir.ition
Confirm.itory
An.ilysis
Americium-
241
14596-10-2
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for Am-
241 in Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for Am-
241 in Building
Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Americium-241 in
Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Americium-241 in
Building Materials
(EPA)
Plutonium-238
13981-16-3
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for Pu-
238 and Pu-239/240 in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for Pu-
238 and Pu-239/240
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Plutonium-238, -239/240
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Plutonium-238, -239/240
in Building Materials
(EPA)
Plutonium-239
15117-48-3
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for Pu-
238 and Pu-239/240 in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for Pu-
238 and Pu-239/240
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Plutonium-238, -239/240
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Plutonium-238, -239/240
in Building Materials
(EPA)
Radium-226
13982-63-3
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for Ra-
226 in Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for Ra-
226 in Building
Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Radium-226 in Building
Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Radium
226 in Building Materials
(EPA)
Strontium-90
10098-97-2
Beta counting
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for Total
Radiostrontium in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for
Total Radiostrontium
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Total
Radiostrontium in
Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Total
Radiostrontium in
Building Materials
(EPA)
Uranium-234
13966-29-5
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Isotopic Uranium
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Isotopic
Uranium
in Building Materials
(EPA)
Uranium-235
15117-96-1
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Isotopic Uranium
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Isotopic
Uranium
in Building Materials
(EPA)
Uranium-238
7440-61-1
Alpha
spectrometry
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Roofing
Materials
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for Sodium
Hydroxide Fusion of
Asphalt Matrices
(EPA)
Rapid Method for
Isotopic Uranium in
Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for
Isotopic Uranium
in Building Materials
(EPA)
Rapid Method for
Sodium Hydroxide
Fusion of Concrete and
Brick
(EPA)
Rapid Method for Isotopic
Uranium
in Building Materials
(EPA)
SAM 2017 Appendix B2
B2-1
September 2017
-------
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Appendix C - Selected Pathogen Methods
Appendix C: Selected Pathogen Methods
SAM 2017 - Appendix C
September 2017
-------
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SAM 2017 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 (QC)
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).
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 pathogen methods have been assigned tiers (below) to indicate a level of method usability for the
specific analyte and sample type. The assigned tiers pertain only to technical aspects of method usability, and do not pertain to aspects such as cost, equipment availability, and sample throughput. Assigned usability tiers are indicated next to each method or method
combination throughout this appendix.
Tier I: The method was developed for the pathogen and sample type. The method has been evaluated by multiple laboratories, a detailed protocol has been
developed, and suitable QC measures and checks are provided. (Examples: EPA Method 1623.1 [ Cryptosporidium in drinking water]; Standard
Methods 9260 E [Shigella culture method])
Tier II: The pathogen is not the target of the method, and the method has been evaluated by one or more laboratories. The available data and/or information indicate
that additional testing and/or modifications will likely be needed. (Example: Cunningham et al. 2010. [Shigella molecular method])
Tier III: The pathogen is not the target of the method but the method is for the specific sample type and the pathogen is similar to the target of the method (i.e.
vegetative bacteria, spore-forming bacteria, virus or protozoan). Data and expert opinion suggest, however, that the method(s) may be applicable with
modifications. (Example: EPA Yersinia pestis protocol for Salmonella enterica serovar Typhi [S . Typhi] in post decontamination waste water.)
Notes:
Samples should not be stored indefinitely, and should be processed and analyzed as soon as possible upon receipt.
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 the extent and magnitude of contamination (e.g., for site characterization phase samples). Please see Figure 7-1.
Column headings are defined in Section 7.0.
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Bacteria
Bacillus anthracis
[Anthrax]
NA
Sample
Preparation
EPA Bacillus anthracis (BA)
Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
Silvestri etai 2016. J. of
Microbiol. Methods. 130:
6-13
II
EPA BA Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
Culture
Analytical
Technique
EPA BA Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
EPA BA Protocol
(EPA/600/R-12/577)
Real-time PCR/
RV-PCR
Brucella spp.
(S. abortus,
B. melitensis,
B. suis)
[Brucellosis]
NA
Sample
Preparation
EPA Yersinia pestis (YP)
Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etai 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Brucella species
ASM Sentinel Level Clinical
Microbiology Laboratory Guidelines
for Suspected Agents of Bioterrorism
and Emerging Infectious Diseases:
Brucella species
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Brucella species
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Brucella species
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Brucella species
Real-time PCR
Analytical
Technique
Hinicefa/. 2008. J. Microbiol.
Methods. 75(2): 375-378
II
Hinicefa/. 2008. J. Microbiol.
Methods. 75(2): 375-378
II
Hinicefa/. 2008. J. Microbiol.
Methods. 75(2): 375-378
II
Hinicefa/. 2008. J. Microbiol.
Methods. 75(2): 375-378
II
Hinicefa/. 2008. J. Microbiol.
Methods. 75(2): 375-378
II
SAM 2017 Appendix C
C-1
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Burkholderia mallei
[Glanders] and
Burkholderia
pseudomallei
[Melioidosis]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Limmathurotsakul etal. 2013.
PLoS Negl. Trop. Dis. 7(3):
e2105
II
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Burkholderia mallei and B.
pseudomallei
ASM Sentinel Level Clinical
Microbiology Laboratory Guidelines
for Suspected Agents of Bioterrorism
and Emerging Infectious Diseases:
Burkholderia mallei and B.
pseudomallei
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Burkholderia mallei and B.
pseudomallei
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Burkholderia mallei and B.
pseudomallei
ASM Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Burkholderia mallei and B.
pseudomallei
Real-time PCR
Analytical
Technique
Tomaso et al. 2006. Clin.
Chem. 52(2): 307-310
and
Novak et al. 2006. J. Clin.
Microbiol. 44(1): 85-90
II
Tomaso etal. 2006. Clin. Chem.
52(2): 307-310
and
Novak et al. 2006. J. Clin. Microbiol.
44(1): 85-90
II
Tomaso etal. 2006. Clin.
Chem. 52(2): 307-310
and
Novak et al. 2006. J. Clin.
Microbiol. 44(1): 85-90
II
Tomaso etal. 2006. Clin.
Chem. 52(2): 307-310
and
Novak et al. 2006. J. Clin.
Microbiol. 44(1): 85-90
II
Tomaso etal. 2006. Clin.
Chem. 52(2): 307-310
and
Novak et al. 2006. J. Clin.
Microbiol. 44(1): 85-90
II
Campylobacter jejuni
[Campylobacteriosis]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
ISO 17995
ISO 17995
ISO 17995
ISO 17995
ISO 17995
Real-time PCR
Analytical
Technique
Cunningham et al. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Cunningham et al. 2010. J.Clin.
Microbiol. 48(8): 2929-2933
II
Cunningham et al. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Cunningham et al. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Cunningham et al. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Chlamydophila
psittaci
Cformerly known as
Chlamydia psittaci)
[Psittacosis]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Tissue culture
Analytical
Technique
Madico etal. 2000. J. Clin.
Microbiol. 38(3): 1085-1093
II
Madico etal. 2000. J. Clin. Microbiol.
38(3): 1085-1093
II
Madico etal. 2000. J. Clin.
Microbiol. 38(3): 1085-1093
II
Madico etal. 2000. J. Clin.
Microbiol. 38(3): 1085-1093
II
Madico etal. 2000. J. Clin.
Microbiol. 38(3): 1085-1093
II
PCR
SAM 2017 Appendix C
C - 2
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Coxiella burnetii
[Q-fever]
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA and CDC Ultrafiltration
(UF) Report
(EPA 600/R-11/103)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
Tissue Culture
Analytical
Technique
Raoult et al. 1991. Antimicrob.
Agents Chemother. 35(10):
2070-2077
II
Raoult et al. 1991. Antimicrob.
Agents Chemother. 35(10):
2070-2077
II
Raoult et al. 1991. Antimicrob.
Agents Chemother. 35(10):
2070-2077
II
Raoult et al. 1991. Antimicrob.
Agents Chemother. 35(10):
2070-2077
II
Raoult etal. 1991. Antimicrob.
Agents Chemother. 35(10):
2070-2077
II
Real-time PCR
Analytical
Technique
Panning etal. 2008. BMC
Microbiol. 8:77
II
Panning etal. 2008. BMC Microbiol.
8:77
II
Panning etal. 2008. BMC
Microbiol. 8:77
II
Panning etal. 2008. BMC
Microbiol. 8:77
II
Panning etal. 2008. BMC
Microbiol. 8:77
II
Escherichia coli
0157:H7
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA Eschericia coli 0157:H7
(EC) Protocol
(EPA/600/R-10/056)
or
EPA YP Protocol
(EPA/600/R-16/109)
I/III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
EPA EC Protocol
(EPA/600/R-10/056)
EPA EC Protocol
(EPA/600/R-10/056)
EPA EC Protocol
(EPA/600/R-10/056)
EPA EC Protocol
(EPA/600/R-10/056)
EPA EC Protocol
(EPA/600/R-10/056)
Real-time PCR
Analytical
Technique
Sen etal. 2011. Environ. Sci.
Technol. 45(7):
2250-2256
II
Sen etal. 2011. Environ. Sci.
Technol. 45(7): 2250-2256
II
Sen etal. 2011. Environ. Sci.
Technol. 45(7):
2250-2256
II
Sen etal. 2011. Environ. Sci.
Technol. 45(7): 2250-2256
II
Sen etal. 2011. Environ. Sci.
Technol. 45(7):
2250-2256
II
Francisella tularensis
[Tularemia]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
CDC, ASM and APHL:
Sentinel Level Clinical
Microbiology Laboratory
Guidelines for Suspected
Agents of Bioterrorism and
Emerging Infectious Diseases:
Francisella tularensis
CDC, ASM and APHL: Sentinel Level
Clinical Microbiology Laboratory
Guidelines for Suspected Agents of
Bioterrorism and Emerging Infectious
Diseases: Francisella tularensis
CDC, ASM and APHL: Sentinel
Level Clinical Microbiology
Laboratory Guidelines for
Suspected Agents of
Bioterrorism and Emerging
Infectious Diseases:
Francisella tularensis
CDC, ASM and APHL: Sentinel
Level Clinical Microbiology
Laboratory Guidelines for
Suspected Agents of
Bioterrorism and Emerging
Infectious Diseases: Francisella
tularensis
CDC, ASM and APHL: Sentinel
Level Clinical Microbiology
Laboratory Guidelines for
Suspected Agents of
Bioterrorism and Emerging
Infectious Diseases: Francisella
tularensis
Real-time PCR
Analytical
Technique
Versage et al. 2003. J. Clin.
Microbiol. 41(12): 5492-5499
II
Versage etal. 2003. J. Clin.
Microbiol. 41(12): 5492-5499
II
Versage etal. 2003. J. Clin.
Microbiol. 41(12): 5492-5499
II
Versage etal. 2003. J. Clin.
Microbiol. 41(12): 5492-5499
II
Versage etal. 2003. J. Clin.
Microbiol. 41(12): 5492-5499
II
SAM 2017 Appendix C
C - 3
September 2017
-------
Analytical Method
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
NA
Sample
Preparation
US DHHS. 2005. Procedures
for the Recovery of Legionella
from the Environment
Kozak et al. 2013. Identification of
Legionella in the Environment.
Methods Mol. Biol. 954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol.
Biol. 954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol. Biol.
954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol.
Biol. 954: 3-25
Legionella
[Legionellosis ]
Culture
Analytical
Technique
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol.
Biol. 954: 3-25
Kozak et al. 2013. Identification of
Legionella in the Environment.
Methods Mol. Biol. 954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol.
Biol. 954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol. Biol.
954: 3-25
Kozak et al. 2013. Identification
of Legionella in the
Environment. Methods Mol.
Biol. 954: 3-25
Real-time PCR
Analytical
Technique
Benitez and Winchell. 2013. J.
Clin. Microbiol. 51(1):
348-351
II
Benitez and Winchell. 2013. J. Clin.
Microbiol. 51(1): 348-351
II
Benitez and Winchell. 2013. J.
Clin. Microbiol. 51(1):
348-351
II
Benitez and Winchell. 2013. J.
Clin. Microbiol. 51 (1):
348-351
II
Benitez and Winchell. 2013. J.
Clin. Microbiol. 51 (1):
348-351
II
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Leptospira
[Leptospirosis]
Culture
Analytical
Technique
Standard Method 9260 I:
Leptospira
Standard Method 9260 I: Leptospira
Standard Method 9260 I:
Leptospira
Standard Method 9260 I:
Leptospira
Standard Method 9260 I:
Leptospira
Real-time PCR
Analytical
Technique
Palaniappan etal. 2005. Mol.
Cell Probes. 19(2):
111-117
II
Palaniappan etal. 2005. Mol. Cell
Probes. 19(2): 111-117
II
Palaniappan etal. 2005. Mol.
Cell Probes. 19(2):
111-117
II
Palaniappan etal. 2005. Mol.
Cell Probes. 19(2):
111-117
II
Palaniappan etal. 2005. Mol.
Cell Probes. 19(2):
111-117
II
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Listeria
monocytogenes
[Listeriosis]
Culture
Analytical
Technique
Hitchinsetal. 2003.
Bacteriological Analytical
Manual Online
Hitchins et al. 2003. Bacteriological
Analytical Manual Online
Hitchins et al. 2003.
Bacteriological Analytical
Manual Online
Hitchins et al. 2003.
Bacteriological Analytical
Manual Online
Hitchins et al. 2003.
Bacteriological Analytical
Manual Online
Real-time PCR
Analytical
Technique
USDA, FSIS. 2009.
Microbiology Laboratory
Guidebook MLG 8A.04
USDA, FSIS. 2009. Microbiology
Laboratory Guidebook MLG 8A.04
USDA, FSIS. 2009.
Microbiology Laboratory
Guidebook MLG 8A.04
USDA, FSIS. 2009.
Microbiology Laboratory
Guidebook MLG 8A.04
USDA, FSIS. 2009.
Microbiology Laboratory
Guidebook MLG 8A.04
SAM 2017 Appendix C
C - 4
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Non-typhoidal
Salmonella
(Not applicable to
S. Typhi)
[Salmonellosis]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA Method 1682
(EPA-821-R-06-14)
EPA Method 1200
(EPA 817-R-12-004)
or
Standard Method 9260 B:
Salmonella
or
EPA YP Protocol
(EPA/600/R-16/109)
I/I/III
EPA Method 1200
(EPA 817-R-12-004)
or
EPA Method 1682
(EPA-821-R-06-14)
or
Standard Method 9260 B:
Salmonella
or
EPA YP Protocol
(EPA/600/R-16/109)
I/I/I/III
Culture
Analytical
Technique
EPA Method 1682
(EPA-821-R-06-14)
or
EPA Method 1200
(EPA 817-R-12-004)
EPA Method 1682
(EPA-821-R-06-14)
or
EPA Method 1200
(EPA 817-R-12-004)
EPA Method 1682
(EPA-821-R-06-14)
or
EPA Method 1200
(EPA 817-R-12-004)
EPA Method 1682
(EPA-821-R-06-14)
or
EPA Method 1200
(EPA 817-R-12-004)
EPA Method 1682
(EPA-821-R-06-14)
or
EPA Method 1200
(EPA 817-R-12-004)
Real-time PCR
Analytical
Technique
Jyoti etal. 2011. Environ. Sci.
Technol. 45(20):
8996-9002
II
Jyoti eta/. 2011. Environ. Sci.
Technol. 45(20):
8996-9002
II
Jyoti efa/.2011. Environ. Sci.
Technol. 45(20):
8996-9002
II
Jyoti et a/. 2011. Environ. Sci.
Technol. 45(20):
8996-9002
II
Jyoti et a/. 2011. Environ. Sci.
Technol. 45(20):
8996-9002
II
Salmonella Typhi
[Typhoid fever]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA Salmonella Typhi (ST)
Protocol
(EPA 600/R-10/133)
or
Standard Method 9260 B:
Salmonella
or
EPA YP Protocol
(EPA/600/R-16/109)
I/I/III
Standard Method 9260 B:
Salmonella
or
EPA YP Protocol
(EPA/600/R-16/109)
I/III
Culture
Analytical
Technique
EPA ST Protocol
(EPA 600/R-10/133)
EPA ST Protocol
(EPA 600/R-10/133)
EPA ST Protocol
(EPA 600/R-10/133)
EPA ST Protocol
(EPA 600/R-10/133)
EPA ST Protocol
(EPA 600/R-10/133)
Real-time PCR
Analytical
Technique
CDC Laboratory Assay
CDC Laboratory Assay
CDC Laboratory Assay
CDC Laboratory Assay
CDC Laboratory Assay
Shigella spp.
[Shigellosis]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130: 6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
Standard Method 9260 E:
Shigella
Standard Method 9260 E: Shigella
Standard Method 9260 E:
Shigella
Standard Method 9260 E:
Shigella
Standard Method 9260 E:
Shigella
Real-time PCR
Analytical
Technique
Cunningham etal. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Cunningham etal. 2010. J. Clin.
Microbiol. 48(8): 2929-2933
II
Cunningham etal. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
Cunningham etal. 2010. J. Clin.
Microbiol. 48(8):
2929-2933
II
Cunningham etal. 2010. J.
Clin. Microbiol. 48(8):
2929-2933
II
SAM 2017 Appendix C
C - 5
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Staphylococcus
aureus
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Culture
Analytical
Technique
Standard Method 9213 B:
Staphylococcus aureus
Standard Method 9213 B:
Staphylococcus aureus
Standard Method 9213 B:
Staphylococcus aureus
Standard Method 9213 B:
Staphylococcus aureus
Standard Method 9213 B:
Staphylococcus aureus
Real-time PCR
Analytical
Technique
Chiang etal. 2007. J. Food
Prot. 70(12): 2855-2859
II
Chiang etal. 2007. J. Food Prot.
70(12): 2855-2859
II
Chiang etal. 2007. J. Food
Prot. 70(12): 2855-2859
II
Chiang etal. 2007. J. Food
Prot. 70(12): 2855-2859
II
Chiang etal. 2007. J. Food
Prot. 70(12): 2855-2859
II
Vibrio cholerae
[Cholera]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
III
EPA YP Protocol
(EPA/600/R-16/109)
III
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA Vibrio cholerae (VC)
Protocol
(EPA 600/R-10/139)
or
EPA YP Protocol
(EPA/600/R-16/109)
I/III
EPA VC Protocol
(EPA 600/R-10/139)
or
EPA YP Protocol
(EPA/600/R-16/109)
I/III
Culture
Analytical
Technique
EPA VC Protocol
(EPA 600/R-10/139)
EPA VC Protocol
(EPA 600/R-10/139)
EPA VC Protocol
(EPA 600/R-10/139)
EPA VC Protocol
(EPA 600/R-10/139)
EPA VC Protocol
(EPA 600/R-10/139)
Real-time PCR
Analytical
Technique
Blackstoneetal. 2007. J.
Microbiol. Methods. 68(2):
254-259
II
Blackstone etal. 2007. J. Microbiol.
Methods. 68(2): 254-259
II
Blackstone et al. 2007. J.
Microbiol. Methods. 68(2):
254-259
II
Blackstone etal. 2007. J.
Microbiol. Methods. 68(2):
254-259
II
Blackstone et al. 2007. J.
Microbiol. Methods. 68(2):
254-259
II
Yersinia pestis
[Plague]
NA
Sample
Preparation
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
Silvestri etal. 2016. J. of
Microbiol. Methods. 130:
6-13
III
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
Culture
Analytical
Technique
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
EPA YP Protocol
(EPA/600/R-16/109)
Real-time PCR/
RV-PCR
SAM 2017 Appendix C
C - 6
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Viruses
Adenoviruses:
Enteric and
non-enteric (A-F)
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Tissue Culture
Analytical
Technique
Boczek et al. 2016. J.
Microbiol. Methods. 122:
43-49
or
Green and Loewenstein. 2005.
Curr. Protoc. Microbiol. 14C.1.1
14C.1.19
II
Boczek et al. 2016. J. Microbiol.
Methods. 122: 43-49
or
Green and Loewenstein. 2005. Curr.
Protoc. Microbiol. 14C.1.1-14C.1.19
II
Boczek et al. 2016. J.
Microbiol. Methods. 122:
43-49
or
Green and Loewenstein. 2005.
Curr. Protoc. Microbiol. 14C.1.1-
14C.1.19
II
Boczek et al. 2016. J. Microbiol.
Methods. 122:
43-49
or
Green and Loewenstein. 2005.
Curr. Protoc. Microbiol.
14C. 1.1-14C. 1.19
II
Boczek et al. 2016. J. Microbiol.
Methods. 122:
43-49
or
Green and Loewenstein. 2005.
Curr. Protoc. Microbiol. 14C.1.1-
14C.1.19
II
Real-time PCR
Analytical
Technique
Jothikumar et al. 2005. Appl.
Environ. Microbiol. 71(6): 3131 -
3136
II
Jothikumar etal. 2005. Appl. Environ.
Microbiol. 71(6):
3131-3136
II
Jothikumar etal. 2005. Appl.
Environ. Microbiol. 71(6):
3131-3136
II
Jothikumar etal. 2005. Appl.
Environ. Microbiol. 71(6):
3131-3136
II
Jothikumar et al. 2005. Appl.
Environ. Microbiol. 71(6):
3131-3136
II
Astroviruses
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Integrated Cell Culture
Analytical
Technique
Grimm etal. 2004. Can. J.
Microbiol. 50(4): 269-278
II
Grimm et al. 2004. Can. J. Microbiol.
50(4): 269-278
II
Grimm et al. 2004. Can. J.
Microbiol. 50(4): 269-278
II
Grimm etal. 2004. Can. J.
Microbiol. 50(4): 269-278
II
Grimm etal. 2004. Can. J.
Microbiol. 50(4): 269-278
II
Real-time reverse
transcription-PCR
Caliciviruses:
Noroviruses
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Wlliamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III/I
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III/I
Real-time reverse
transcription-PCR
Analytical
Technique
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
SAM 2017 Appendix C
C - 7
September 2017
-------
Analytical Method
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6): 3119-
3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Caliciviruses:
Sapovirus
Tissue Culture
Analytical
Technique
Parwani etal. 1991. Arch.
Virol. 120(1-2): 115-122
II
Parwani etal. 1991. Arch. Virol. 120(1
2): 115-122
II
Parwani et al. 1991. Arch. Virol.
120(1-2): 115-122
II
Parwani etal. 1991. Arch. Virol.
120(1-2): 115-122
II
Parwani etal. 1991. Arch. Virol.
120(1-2): 115-122
II
Real-time reverse
transcription-PCR
Analytical
Technique
Oka et al. 2006. J. Med. Virol.
78(10): 1347-1353
II
Oka et a/. 2006. J. Med. Virol. 78(10):
1347-1353
II
Oka et a/. 2006. J. Med. Virol.
78(10): 1347-1353
II
Oka et al. 2006. J. Med. Virol.
78(10): 1347-1353
II
Oka etal. 2006. J. Med. Virol.
78(10): 1347-1353
II
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6): 3119-
3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
Coronaviruses:
SARS-associated
human coronavirus
Tissue Culture
Analytical
Technique
Pagat etal. 2007. Applied
Biosafety 12(2): 100-108
II
Pagat et al. 2007. Applied Biosafety
12(2): 100-108
II
Pagat et al. 2007. Applied
Biosafety 12(2): 100-108
II
Pagat et al. 2007. Applied
Biosafety 12(2): 100-108
II
Pagat et al. 2007. Applied
Biosafety 12(2): 100-108
II
Reverse
transcription-PCR
Analytical
Technique
Adachi etal. 2004. J. Virol.
Methods. 122(1): 29-36
II
Adachi etal. 2004. J. Virol. Methods.
122(1): 29-36
II
Adachi etal. 2004. J. Virol.
Methods. 122(1): 29-36
II
Adachi etal. 2004. J. Virol.
Methods. 122(1): 29-36
II
Adachi etal. 2004. J. Virol.
Methods. 122(1): 29-36
II
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Wlliamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Hepatitis E virus
(HEV)
Tissue Culture
Analytical
Technique
Zaki etal. 2009. FEMS
Immunol. Med. Mic. 56:
73-79
II
Zaki etal. 2009. FEMS Immunol.
Med. Mic. 56:
73-79
II
Zaki etal. 2009. FEMS
Immunol. Med. Mic. 56:
73-79
II
Zaki etal. 2009. FEMS
Immunol. Med. Mic. 56:
73-79
II
Zaki etal. 2009. FEMS
Immunol. Med. Mic. 56:
73-79
II
Real-time reverse
transcription-PCR
Analytical
Technique
Jothikumar et al. 2006. J. Virol.
Methods. 131(1): 65-71
II
Jothikumar et al. 2006. J. Virol.
Methods. 131(1): 65-71
II
Jothikumar et al. 2006. J. Virol.
Methods. 131(1): 65-71
II
Jothikumar et al. 2006. J. Virol.
Methods. 131(1): 65-71
II
Jothikumar etal. 2006. J. Virol.
Methods. 131(1): 65-71
II
NA
Sample
Preparation
Hermann etal. 2006. Appl.
Environ. Microbiol. 72(7): 4811 -
4818
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Wlliamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
Influenza H5N1 virus
Tissue Culture
Analytical
Technique
Kraussetal. 2012. Influeza
Virus Isolation. Methods Mol.
Biol. 865: 11-24
II
Krauss etal. 2012. Influeza Virus
Isolation. Methods Mol. Biol. 865:
11-24
II
Krauss etal. 2012. Influeza
Virus Isolation. Methods Mol.
Biol. 865: 11-24
II
Krauss et al. 2012. Influeza
Virus Isolation. Methods Mol.
Biol. 865: 11-24
II
Krauss etal. 2012. Influeza
Virus Isolation. Methods Mol.
Biol. 865: 11-24
II
Real-time reverse
transcription-PCR
Analytical
Technique
Ng etal. 2005. Emerg. Infect.
Dis. 11(8): 1303-1305
II
Ng etal. 2005. Emerg. Infect. Dis.
11(8): 1303-1305
II
Ng etal. 2005. Emerg. Infect.
Dis. 11(8): 1303-1305
II
Ng etal. 2005. Emerg. Infect.
Dis. 11(8): 1303-1305
II
Ng etal. 2005. Emerg. Infect.
Dis. 11(8): 1303-1305
II
SAM 2017 Appendix C
C - 8
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Picornaviruses:
Enteroviruses
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III/I
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III/I
Tissue Culture
Analytical
Technique
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
EPA Method 1615
(EPA/600/R-10/181)
Reverse
transcription-PCR
Picornaviruses:
Hepatitis A virus
(HAV)
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Williamson etal. 2005. Appl.
Environ. Microbiol. 71(6):
3119-3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Integrated Cell Culture
Analytical
Technique
Hyeon etal-2011. J. Food
Prot. 74(10): 1756-1761
II
Hyeon etal. 2011. J. Food Prot.
74(10) :1756-1761
II
Hyeon etal. 2011. J. Food
Prot. 74(10) :1756-1761
II
Hyeon etal. 2011. J. Food
Prot. 74(10): 1756-1761
II
Hyeon etal. 2011. J. Food
Prot. 74(10): 1756-1761
II
Real-time Reverse
Transcription-PCR
Reoviruses:
Rotavirus (Group A)
NA
Sample
Preparation
Cao et al. 2011. J. Environ.
Monit. 13: 3321-3328
(NIOSH biosampler)
III
Park etal. 2015. Appl. Environ.
Microbiol. 81(17): 5987-5992
III
Wlliamson etal. 2005. Appl.
Environ. Microbiol. 71(6): 3119-
3125
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
EPA Method 1615
(EPA/600/R-10/181)
III
Tissue Culture
Analytical
Technique
EPA Method 1615
(EPA/600/R-10/181)
III
EPA Method 1615
(EPA/600/R-10/181)
III
EPA Method 1615
(EPA/600/R-10/181)
III
EPA Method 1615
(EPA/600/R-10/181)
III
EPA Method 1615
(EPA/600/R-10/181)
III
Real-time reverse
transcription-PCR
Analytical
Technique
Jothikumar etal. 2009. J. Virol.
Methods. 155(2):
126-131
II
Jothikumar et al. 2009. J. Virol.
Methods. 155(2): 126-131
II
Jothikumar et al. 2009. J. Virol.
Methods. 155(2):
126-131
II
Jothikumar etal. 2009. J. Virol.
Methods. 155(2):
126-131
II
Jothikumar etal. 2009. J. Virol.
Methods. 155(2):
126-131
II
SAM 2017 Appendix C
C - 9
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Protozoa
Cryptosporidium spp.
[Cryptosporidiosis]
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Liang and Keeley. 2011. Appl.
Environ. Microbiol. 77(18):
6476-6485
II
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
or
EPA and CDC UF Report
(EPA 600/R-11/103)
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
or
EPA and CDC UF Report
(EPA 600/R-11/103)
Cell Culture
Immunofluorescence
Procedure
Analytical
Technique
Bukhari etal. 2007. Can. J.
Microbiol. 53(5): 656-663
II
Bukhari etal. 2007. Can. J.
Microbiol. 53(5): 656-663
II
Bukhari etal. 2007. Can. J.
Microbiol. 53(5): 656-663
II
Bukhari etal. 2007. Can. J.
Microbiol. 53(5): 656-663
II
Bukhari etal. 2007. Can. J.
Microbiol. 53(5): 656-663
II
IMS/FA
Analytical
Technique
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1622
(EPA 815-R-05-001)
or
EPA Method 1623.1
(EPA 816-R-12-001)
Real-time PCR
Analytical
Technique
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185
and
Jiang etal. 2005. Appl.
Environ. Microbiol. 71(3): 1135-
1141
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185
and
Jiang etal. 2005. Appl. Environ.
Microbiol. 71(3): 1135-1141
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9):
5178-5185
and
Jiang etal. 2005. Appl.
Environ. Microbiol. 71(3):
1135-1141
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185
and
Jiang etal. 2005. Appl. Environ.
Microbiol. 71(3):
1135-1141
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9):
5178-5185
and
Jiang etal. 2005. Appl.
Environ. Microbiol. 71(3):
1135-1141
II
Entamoeba
histolytica
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Liang and Keeley. 2011. Appl.
Environ. Microbiol. 77(18):
6476-6485
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
EPA and CDC UF Report
(EPA 600/R-11/103)
III
Cell Culture
Analytical
Technique
Stringertetal. 1972. J
Parasitol. 58(2): 306-310
II
Stringert et al. 1972. J Parasitol.
58(2): 306-310
II
Stringert et al. 1972. J
Parasitol. 58(2): 306-310
II
Stringert et al. 1972. J
Parasitol. 58(2): 306-310
II
Stringert et al. 1972. J
Parasitol. 58(2): 306-310
II
Real-time PCR
Analytical
Technique
Roy etal. 2005. J. Clin.
Microbiol. 43(5): 2168-2172
II
Roy etal. 2005. J. Clin. Microbiol.
43(5): 2168-2172
II
Roy etal. 2005. J. Clin.
Microbiol. 43(5): 2168-2172
II
Roy etal. 2005. J. Clin.
Microbiol. 43(5): 2168-2172
II
Roy et al. 2005. J. Clin.
Microbiol. 43(5): 2168-2172
II
SAM 2017 Appendix C
C-10
September 2017
-------
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Analytical Method
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Giardia spp.
[Giardiasis]
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Liang and Keeley. 2011. Appl.
Environ. Microbiol. 77(18):
6476-6485
III
EPA Method 1623.1
(EPA 816-R-12-001)
or
EPA and CDC UF Report
(EPA 600/R-11/103)
EPA Method 1623.1
(EPA 816-R-12-001)
or
EPA and CDC UF Report
(EPA 600/R-11/103)
Cell Culture
Analytical
Technique
Keister. 1983. T. Roy. Soc.
Trop. Med. H. 77(4): 487-488
II
Keister. 1983. T. Roy. Soc. Trop.
Med. H. 77(4): 487-488
II
Keister. 1983. T. Roy. Soc.
Trop. Med. H. 77(4): 487-488
II
Keister. 1983. T. Roy. Soc.
Trop. Med. H. 77(4): 487-488
II
Keister. 1983. T. Roy. Soc.
Trop. Med. H. 77(4): 487-488
II
IMS/FA
Analytical
Technique
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1623.1
(EPA 816-R-12-001)
EPA Method 1623.1
(EPA 816-R-12-001)
Real-time PCR
Analytical
Technique
Guy et al. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9): 5178-5185
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9):
5178-5185
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9):
5178-5185
II
Guy etal. 2003. Appl. Environ.
Microbiol. 69(9):
5178-5185
II
Naegleria fowleri
[Naegleriasis]
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Mull etal. 2013. J. Parasitol.
Res. 2013: 1-8
II
Copeetal. 2015. Clin. Infect.
Dis. 60(8): e36-42
or
EPA and CDC UF Report
(EPA 600/R-11/103)
II/III
Cope etal. 2015. Clin. Infect.
Dis. 60(8): e36-42
or
EPA and CDC UF Report
(EPA 600/R-11/103)
II/III
Cell Culture
Analytical
Technique
Mull et al. 2013. J. Parasitol.
Res. 2013: 1-8
II
Mull etal. 2013. J. Parasitol. Res.
2013: 1-8
II
Mull etal. 2013. J. Parasitol.
Res. 2013: 1-8
II
Mull etal. 2013. J. Parasitol.
Res. 2013: 1-8
II
Mull etal. 2013. J. Parasitol.
Res. 2013: 1-8
II
Real-time PCR
SAM 2017 Appendix C
C-11
September 2017
-------
Analytical Method
Pathogen(s)
[Disease]
Analytical
Technique
Method
Type
Aerosol
(growth media, filter, liquid)
Particulate
(swabs, wipes, Sponge-Sticks, vacuum
socks and filters)
Soil
Drinking Water
Post Decontamination Waste Water
Toxoplasma gondii
[Toxoplasmosis]
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Liang and Keeley. 2011. Appl.
Environ. Microbiol. 77(18):
6476-6485
III
Villegas etal. 2010. J.
Microbiol. Methods. 81(3):
219-225
or
EPA Method 1623.1
(EPA 816-R-12-001)
II/III
Villegas et al. 2010. J.
Microbiol. Methods. 81(3):
219-225
or
EPA Method 1623.1
(EPA 816-R-12-001)
II/III
Cell Culture
Analytical
Technique
Villegas etal. 2010. J.
Microbiol. Methods. 81(3):
219-225
II
Villegas et al. 2010. J. Microbiol.
Methods. 81(3): 219-225
II
Villegas et al. 2010. J.
Microbiol. Methods. 81(3):
219-225
II
Villegas etal. 2010. J.
Microbiol. Methods. 81(3):
219-225
II
Villegas et a/. 2010. J.
Microbiol. Methods. 81(3):
219-225
II
Real-time PCR
Analytical
Technique
Yang etal. 2009. Appl.
Environ. Microbiology. 75(11):
3477-3483
II
Yang efa/,2009. Appl. Environ.
Microbiology. 75(11): 3477-3483
II
Yang etal. 2009. Appl.
Environ. Microbiology. 75(11):
3477-3483
II
Yang etal. 2009. Appl. Environ.
Microbiology. 75(11): 3477-3483
II
Yang etal. 2009. Appl. Environ.
Microbiology. 75(11): 3477-
3483
II
Helminths
Baylisascaris
procyonis
[Raccoon roundworm
NA
Sample
Preparation
EPA BA Protocol
(EPA/600/R-12/577)
III
Hodges etal. 2010. J. Microbiol.
Methods. 81(2): 141-146
or
Rose etal. 2011. Appl. Environ.
Microbiol. 77(23): 8355-8359
or
EPA BA Protocol
(EPA/600/R-12/577)
III
Liang and Keeley. 2011. Appl.
Environ. Microbiol. 77(18):
6476-6485
III
EPA and CDC UF Report
(EPA 600/R-11/103)
or
Gatcombe etal. 2010. Parasitol.
Res. 106: 499-504
III/II
EPA and CDC UF Report
(EPA 600/R-11/103)
or
Gatcombe etal. 2010.
Parasitol. Res. 106: 499-504
III/II
infection]
Real-time PCR
Analytical
Technique
Gatcombe etal. 2010.
Parasitol. Res. 106: 499-504
II
Gatcombe etal. 2010. Parasitol. Res.
106: 499-504
II
Gatcombe et al. 2010.
Parasitol. Res. 106: 499-504
II
Gatcombe etal. 2010. Parasitol.
Res. 106: 499-504
II
Gatcombe etal. 2010.
Parasitol. Res. 106: 499-504
II
Embryonation of Eggs
and Microscopy
Analytical
Technique
Control of Pathogens and
Vector Attraction in Sewage
Sludge
(EPA/625/R-92/013)
II
Control of Pathogens and Vector
Attraction in Sewage Sludge
(EPA/625/R-92/013)
II
Control of Pathogens and
Vector Attraction in Sewage
Sludge
(EPA/625/R-92/013)
II
Control of Pathogens and
Vector Attraction in Sewage
Sludge
(EPA/625/R-92/013)
II
Control of Pathogens and
Vector Attraction in Sewage
Sludge
(EPA/625/R-92/013)
II
SAM 2017 Appendix C
C- 12
September 2017
-------
Appendix D - Selected Biotoxin Methods
Appendix D: Selected Biotoxin Methods
SAM 2017 - Appendix D
September 2017
-------
This page left intentionally blank
-------
SAM 2017 Appendix D: Selected Biotoxin Methods
The fitness of a method for its intended use is related to data quality objectives (DQOs) for a particular environmental remediation activity. The tiers below have been assigned to the methods selected for each biotoxin/sample type pair to indicate a level of method usability for the
specific biotoxin and sample type for which it has been selected. 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), and appropriate quality
control. The sample types indicated reflect respresentative examples and are not necessarily inclusive of all sample types that might be encountered by laboratories following a contamination incident. Assigned usability tiers are indicated next to each method or method combination
throughout this appendix.
Tier I: The biotoxin and sample type are both targets of the method(s). Data are available for all aspects of method performance and QC measures supporting its use without modifications.
Tier II: The biotoxin is a target of the method, and the method has been evaluated by one or more laboratories. The sample type may or may not be a target of the method, and available data and/or
information regarding sample preparation indicate that analyses of similar sample types were successful. However, additional testing and/or modifications may be needed.
Tier III: The sample type is not a target of the method, and no reliable data supporting the method's fitness for its intended use are available. Data suggest, however, that the method(s) may be applicable
with significant modification.
Notes:
The presence of disinfectants (e.g., chlorine) and/or preservatives added during water sample collection to slow degradation (e.g., pH adjustors, 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.
Column headings are defined in Section 8.0.
Analyte(s)
CAS RN
Analysis Type
Analytical Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, dust socks)
Non-Drinking Water
(surface water, waste water)
Drinking Water
Abrin
Abrin (1393-62-0)
Abrine (526-31-8)
Presumptive
Immunoassay
(LFA)
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2014) 12(1): 49-62
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2014) 12(1): 49-62
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2014) 12(1): 49-62
II
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2014) 12(1): 49-62
II
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2014) 12(1): 49-62
II
Presumptive
(Abrine)
LC-MS-MS
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
I
Confirmatory
Immunoassays
(ELISA and ECL)
Adapted from
Journal of Food Protection
(2008)
71(9): 1868-1874
II
Adapted from
Journal of Food Protection
(2008)
71(9): 1868-1874
II
Adapted from
Journal of Food Protection
(2008)
71(9): 1868-1874
II
Adapted from
Journal of Food Protection
(2008)
71(9): 1868-1874
II
Adapted from
Journal of Food Protection
(2008)
71(9): 1868-1874
II
Biological Activity
Enzyme activity
Adapted from Analytical
Biochemistry (2008)
378(1): 87-89
II
Adapted from Analytical
Biochemistry (2008)
378(1): 87-89
II
Adapted from Analytical
Biochemistry (2008)
378(1): 87-89
II
Adapted from Analytical
Biochemistry (2008)
378(1): 87-89
II
Adapted from Analytical
Biochemistry (2008)
378(1): 87-89
II
Aflatoxins
B1 (27261-02-5)
B2 (22040-96-6)
G1 (1385-95-1)
G2 (7241-98-7)
Presumptive and
Confirmatory
(B1, B2, G1, G2)
Immunoaffinity
(column) purification /
LC-FL (detection)
Adapted from 991.31
(AOAC)
II
Adapted from 991.31
(AOAC)
II
Adapted from 991.31
(AOAC)
II
Adapted from 991.31
(AOAC)
II
Adapted from 991.31
(AOAC)
II
a-Amanitin
23109-05-9
Presumptive
Immunoassay
(ELISA)
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Confirmatory
LC-MS-MS
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
II
EPA 600/R-13/022
I
Anatoxin-a
64285-06-9
Presumptive
Immunoassay
(ELISA)
See summary in
Section 8.2.4.1
III
See summary in
Section 8.2.4.1
III
See summary in
Section 8.2.4.1
III
See summary in
Section 8.2.4.1
III
See summary in
Section 8.2.4.1
III
Confirmatory
LC-MS-MS
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
I
SAM 2017 Appendix D
D -1
September 2017
-------
Analyte(s)
CAS RN
Analysis Type
Analytical Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, dust socks)
Non-Drinking Water
(surface water, waste water)
Drinking Water
Botulinum neurotoxins
(Serotoypes A, B, C, D, E, F, and G)
Type A (93384-43-1)
Type B (93384-44-2)
Type C (93384-45-3)
Type D (93384-46-4)
Type E (93384-47-5)
Type F (107231-15-2)
Type G (107231-16-3)
Presumptive
(Types A and B)
Immunoassay
(LFA)
Adapted from EPA
Environmental
Technology Verification
report
II
Adapted from EPA
Environmental
Technology Verification
report
II
Adapted from EPA
Environmental
Technology Verification
report
II
Adapted from EPA
Environmental
Technology Verification
report
II
Adapted from EPA
Environmental
Technology Verification
report
I
Presumptive
(Types A, B, D, E,
F, and G)
Immunocapture
Forster Resonance
Energy Transfer
(FRET)-based activity
assay
Adapted from Analytical
Biochemistry (2011)
411(2): 200-209
II
Adapted from Analytical
Biochemistry (2011)
411(2): 200-209
II
Adapted from Analytical
Biochemistry (2011)
411(2): 200-209
II
Adapted from Analytical
Biochemistry (2011)
411(2): 200-209
II
Adapted from Analytical
Biochemistry (2011)
411(2): 200-209
II
Presumptive
(Types A-G)
Immunoassay
(fluorescent bead-
based)
See summary in
Section 8.2.5.3
II
See summary in
Section 8.2.5.3
II
See summary in
Section 8.2.5.3
II
See summary in
Section 8.2.5.3
II
See summary in
Section 8.2.5.3
II
Presumptive
(Type A)
Immunoassay
(ECL)
Adapted from Journal of
the Science of Food and
Agriculture (2014)
94: 707-712
II
Adapted from Journal of
the Science of Food and
Agriculture (2014)
94: 707-712
II
Adapted from Journal of
the Science of Food and
Agriculture (2014)
94: 707-712
II
Adapted from Journal of
the Science of Food and
Agriculture (2014)
94: 707-712
II
Adapted from Journal of
the Science of Food and
Agriculture (2014)
94: 707-712
II
Confirmatory
(Types A-G)
LC-MS-MS
(Types A, B, E and F)
MALDI-TOF MS
(Types A-G)
Adapted from
J. Agric.Food Chem.
63(4): 1133-1141
II
Adapted from
J. Agric.Food Chem.
63(4): 1133-1141
II
Adapted from
J. Agric.Food Chem.
63(4): 1133-1141
II
Adapted from
J. Agric.Food Chem.
63(4): 1133-1141
II
Adapted from
J. Agric.Food Chem.
63(4): 1133-1141
II
Biological Activity
(Total)
Mouse Bioassay
APHA Press Compendium
of Methods, Chapter 32
I
APHA Press
Compendium of Methods,
Chapter 32
I
APHA Press Compendium
of Methods, Chapter 32
I
APHA Press Compendium
of Methods, Chapter 32
I
APHA Press Compendium
of Methods, Chapter 32
I
Brevetoxins
98112-41-5 (A form)
79580-28-2 (B form)
Presumptive
(B forms)
Immunoassay
(ELISA)
Adapted from Toxicon
(2015)96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Adapted from Toxicon
(2015)96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Confirmatory
(A and B forms)
LC-MS
Adapted from Toxicon
(2015)96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Adapted from Toxicon
(2015)96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Adapted from Toxicon
(2015) 96: 82-88
II
Cylindrospermopsin
143545-90-8
Presumptive
Immunoassay
(ELISA)
Adapted from
Environmental Sciences
and Technology (2010)
44: 7361-7368
II
Adapted from
Environmental Sciences
and Technology (2010)
44: 7361-7368
II
Adapted from
Environmental Sciences
and Technology (2010)
44: 7361-7368
II
Adapted from
Environmental Sciences
and Technology (2010)
44: 7361-7368
II
Adapted from
Environmental Sciences
and Technology (2010)
44: 7361-7368
II
Confirmatory
LC-MS-MS
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
II
Method 545
(EPA)
I
Diacetoxyscirpenol (DAS)
2270-40-8
Presumptive
Immunoassay
(ELISA)
See summary in
Section 8.2.8.1
III
See summary in
Section 8.2.8.1
III
See summary in
Section 8.2.8.1
III
See summary in
Section 8.2.8.1
III
See summary in
Section 8.2.8.1
III
Confirmatory
LC-MS-MS
Adapted from Food
Research International
(2015) 72: 247-255
II
Adapted from Food
Research International
(2015) 72: 247-255
II
Adapted from Food
Research International
(2015) 72: 247-255
II
Adapted from Food
Research International
(2015)72: 247-255
II
Adapted from Food
Research International
(2015)72: 247-255
II
SAM 2017 Appendix D
D - 2
September 2017
-------
Analyte(s)
CAS RN
Analysis Type
Analytical Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, dust socks)
Non-Drinking Water
(surface water, waste water)
Drinking Water
Presumptive
Immunoassay
(ELISA)
Adapted from Journal of
AOAC International (2007)
90(4):
1011-1027
II
Adapted from Journal of
AOAC International
(2007) 90(4):
1011-1027
II
Adapted from Journal of
AOAC International (2007)
90(4):
1011-1027
II
Adapted from Journal of
AOAC International (2007)
90(4):
1011-1027
II
Adapted from Journal of
AOAC International (2007)
90(4):
1011-1027
II
Domoic acid (DA)
14277-97-5
Presumptive
Immunoassay
(ELISA)
Adapted from Journal of
Shellfish Research (2008)
27(5): 1301-1310
II
Adapted from Journal of
Shellfish Research (2008)
27(5): 1301-1310
II
Adapted from Journal of
Shellfish Research (2008)
27(5): 1301-1310
II
Adapted from Journal of
Shellfish Research (2008)
27(5): 1301-1310
II
Adapted from Journal of
Shellfish Research (2008)
27(5): 1301-1310
II
Confirmatory
LC-UV
Adapted from Journal of
the Mexican Chemical
Society (2011)
55(2): 65-71
II
Adapted from Journal of
the Mexican Chemical
Society (2011)
55(2): 65-71
II
Adapted from Journal of
the Mexican Chemical
Society (2011)
55(2): 65-71
II
Adapted from Journal of
the Mexican Chemical
Society (2011)
55(2): 65-71
II
Adapted from Journal of
the Mexican Chemical
Society (2011)
55(2): 65-71
II
Microcystins
96180-79-9 (LA)
154037-70-4 (LF)
101043-37-2 (LR)
Presumptive
(Total Adda-
containing
microcystins)
Immunoassay
(ELISA)
Method 546
(EPA)
II
Method 546
(EPA)
II
Method 546
(EPA)
II
Method 546
(EPA)
I
Method 546
(EPA)
I
123304-10-9 (LY)
111755-37-4 (RR)
101064-48-6 (YR)
Confirmatory (LA,
LF, LR, LY, RR,
YR)
LC-MS-MS
Method 544
(EPA)
III
Method 544
(EPA)
III
Method 544
(EPA)
III
Method 544
(EPA)
II
Method 544
(EPA)
I
Presumptive
TBD
TBD
TBD
TBD
TBD
TBD
Picrotoxin
124-87-8
Confirmatory
LC-UV
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
(1989) 7(3): 369-375
II
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
(1989) 7(3): 369-375
II
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
(1989) 7(3): 369-375
II
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
(1989) 7(3): 369-375
II
Adapted from Journal of
Pharmaceutical and
Biomedical Analysis
(1989) 7(3): 369-375
II
Presumptive
Immunoassay
(LFA)
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2013)
11(4): 237-250
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2013)
11(4): 237-250
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2013)
11(4): 237-250
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2013)
11(4): 237-250
I
Adapted from Biosecurity
and Bioterrorism:
Biodefense Strategy,
Practice, and Science
(2013)
11(4): 237-250
I
Presumptive
Immunoassay
(ELISA)
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Ricin
Ricin (9009-86-3)
Ricinine (5254-40-3)
Presumptive
Immunoassay
(ECL)
Adapted from Journal of
AOAC International (2008)
91(2): 376-382
II
Adapted from Journal of
AOAC International
(2008)91(2): 376-382
II
Adapted from Journal of
AOAC International (2008)
91(2): 376-382
II
Adapted from Journal of
AOAC International (2008)
91(2): 376-382
II
Adapted from Journal of
AOAC International (2008)
91(2): 376-382
II
Presumptive
(Ricinine)
LC-MS-MS
EPA 600/R-13/022
(EPA/CDC)
II
EPA 600/R-13/022
(EPA/CDC)
II
EPA 600/R-13/022
(EPA/CDC)
II
EPA 600/R-13/022
(EPA/CDC)
II
EPA 600/R-13/022
(EPA/CDC)
I
Confirmatory
Immunocapture /
LC-MS-MS
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from-Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
I
Biological Activity
Immuncapture /
MALDI-TOF MS
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
II
Adapted from Analytical
Chemistry (2011)
83: 2897-2905
I
SAM 2017 Appendix D
D - 3
September 2017
-------
Analyte(s)
CAS RN
Analysis Type
Analytical Technique
Aerosol
(filter/cassette, liquid
impinger)
Solid
(soil, powder)
Particulate
(swabs, wipes, dust socks)
Non-Drinking Water
(surface water, waste water)
Drinking Water
Saxitoxins
35523-89-8 (STX)
64296-20-4 (NEO)
58911-04-9 (dcSTX)
68683-58-9 (dcNEOSTX)
143084-69-9 (doSTX)
77462-64-7 (GTX 1 - 6)
122075-86-9 (dcGTX 1 - 4)
Presumptive
(Total)
Receptor Binding
Assay
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Presumptive
(Total)
Immunoassay
(ELISA)
Adapted from Toxicon
(2009)54: 313-320
II
Adapted from Toxicon
(2009) 54: 313-320
II
Adapted from Toxicon
(2009) 54: 313-320
II
Adapted from Harmful
Algae (2016)56: 77-90
I
Adapted from Harmful
Algae (2016)56:77-90
I
Confirmatory
(STXs and GTXs)
LC-MS-MS
Adapted from Journal of
Chromatography A (2015)
1387: 1-12
II
Adapted from Journal of
Chromatography A (2015)
1387: 1-12
II
Adapted from Journal of
Chromatography A (2015)
1387: 1-12
II
Adapted from Journal of
Chromatography A (2015)
1387: 1-12
II
Adapted from Journal of
Chromatography A (2015)
1387: 1-12
II
Shiga and Shiga-like Toxins
Stx (75757-64-1)
Presumptive (Stx,
Stx-1 and Stx-2)
Immunoassay
(ELISA)
Adapted from Austin
Immunology (2016)
1(2): 1007:1-7
II
Adapted from Austin
Immunology (2016)
1(2): 1007:1-7
II
Adapted from Austin
Immunology (2016)
1(2): 1007:1-7
II
Adapted from Austin
Immunology (2016)
1(2): 1007:1-7
II
Adapted from Austin
Immunology (2016)
1(2): 1007:1-7
II
Confirmatory (Stx,
Stx-1 and Stx-2)
LC-MS-MS
Adapted from Analytical
Chemistry (2014)
86: 4698-4706
II
Adapted from Analytical
Chemistry (2014)
86: 4698-4706
II
Adapted from Analytical
Chemistry (2014)
86: 4698-4706
II
Adapted from Analytical
Chemistry (2014)
86: 4698-4706
II
Adapted from Analytical
Chemistry (2014)
86: 4698-4706
II
Staphylococcal enterotoxins
37337-57-8 (SEA)
39424-53-8 (SEB)
39424-54-9 (SEC)
12788-99-7 (SED)
39424-55-0 (SEE)
Presumptive
(SEA - SEE)
Enzyme
Immunoassay
(ELFA)
2007.06
(AOAC)
II
2007.06
(AOAC)
II
2007.06
(AOAC)
II
2007.06
(AOAC)
II
2007.06
(AOAC)
II
Presumptive
(SEB)
Immunoassay
(ECL)
Adapted from Journal of
AOAC International (2014)
97(3): 862-867
III
Adapted from Journal of
AOAC International
(2014)97(3): 862-867
III
Adapted from Journal of
AOAC International (2014)
97(3): 862-867
III
Adapted from Journal of
AOAC International (2014)
97(3): 862-867
III
Adapted from Journal of
AOAC International (2014)
97(3): 862-867
III
Confirmatory
(SEA - SEE)
Immunoassay
(ELISA)
Adapted from Letters in
Applied Microbiology
(2011) 52: 468-474
II
Adapted from Letters in
Applied Microbiology
(2011) 52: 468-474
II
Adapted from Letters in
Applied Microbiology
(2011) 52: 468-474
II
Adapted from Letters in
Applied Microbiology
(2011)52: 468-474
II
Adapted from Letters in
Applied Microbiology
(2011)52: 468-474
II
T-2 Mycotoxin
21259-20-1 (T-2)
26934-87-2 (HT-2)
Presumptive
(T-2)
Immunoassay
(ELISA)
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Adapted from Journal of
Food Protection (2005)
68(6): 1294-1301
II
Confirmatory
(T-2 and HT-2)
LC-MS
Adapted from Rapid
Communications in Mass
Spectrometry (2006)
20(9): 1422-1428
II
Adapted from Rapid
Communications in Mass
Spectrometry (2006)
20(9): 1422-1428
II
Adapted from Rapid
Communications in Mass
Spectrometry (2006)
20(9): 1422-1428
II
Adapted from Rapid
Communications in Mass
Spectrometry (2006)
20(9): 1422-1428
II
Adapted from Rapid
Communications in Mass
Spectrometry (2006)
20(9): 1422-1428
II
Tetrodotoxin
9014-39-5
Presumptive
Receptor Binding
Assay
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Method 2011.27
(AOAC)
II
Confirmatory
LC-MS-MS
Adapted from Toxicon
(2016) 119: 64-71
II
Adapted from Toxicon
(2016) 119: 64-71
II
Adapted from Toxicon
(2016) 119: 64-71
II
Adapted from Toxicon
(2016)119:64-71
II
Adapted from Toxicon
(2016) 119: 64-71
II
SAM 2017 Appendix D
D - 4
September 2017
-------
Attachment 1 - Supporting. Documents
Attachment 1:
SAM Revisions and Supporting Documents
The information in this document is updated periodically to incorporate revisions to the list of target analytes and 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
since publication of Revision 1.0 in September 2004.
SAM Revisions Trackiii" Table
SAM Revision
Publication
Date
Changes incorporated summary
Revision 1.0
EPA/600/R-04/126
September 2004
Standardized Analytical Methods for Use During Homeland Security Events (SAM) 1.0
Included chemical and biological contaminants
Revision 2.0
EPA/600/R-04/126B
September 2005
Added radiochemicals
Added several persistent chemical warfare agent (CWA) degradation products
Added separate drinking water sample type for chemicals and radiochemicals
Added viability determination methods for pathogens
Added separate section for biotoxins
Revision 3.0
EPA/600/R-07/015
February 2007
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) 3.0
Revision 3.1
EPA/600/R-07/136
November 2007
Developed a SAM website, to provide the SAM document and a format for searching and linking to SAM
methods bv analvte and sample tvpe. (See httDs://ww\\ .eDa.gov/homeland-securitv-research/sam)
Revision 4.0
EPA/600/R-04/126D
September 2008
Added wipe samples for chemistry analytes
Added PCR methods for pathogens
Revision 5.0
EPA/600/R-04/126E
September 2009
Added separate drinking water sample type to biotoxins
2010 (Revision 6.0)
EPA/600/R-10/122
October 2010
Removed non-aqueous liquid sample type from chemistry
Temporary removal of pathogens
SAM 2017
Attachment 1-1
September 2017
-------
Attachment 1 - Supporting. Documents
SAM Revisions Tracking Table
SAM Revision
Publication
Date
Changes incorporated summary
2012 (Revision 7.0)
EPA/600/R-12/5 5 5
July 2012
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
2017 (Revision 8.0)
EPA/600/R-17/356
October 2017
Added outdoor building and infrastructure material sample types for radiochemistry analytes
Added soil sample type for pathogens
Assigned applicability tiers to pathogen and biotoxin methods
Added analytes to chemistry, radiochemistry, pathogen and biotoxin sections
Added considerations regarding the potential impacts of decontamination agents on the analytical
performance of selected radiochemical methods
Changed the names of "aqueous liquid" (chemistry methods sample type) and "liquid water" (biotoxin
methods sample type) to "non-drinking water" to clarify that the sample type applies to all non-drinking
water aqueous sample matrices
SAM 2017
Attachment 1-2
September 2017
-------
Attachment 1 - Supporting Documents
The following documents and tools have been developed by EPA to provide information regarding a
contamination incident. The information included in these documents is intended to be complementary to
information provided in the analytical methods that are 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 website at: https://www.epa.gov/homeland-securitv-research/sam
U.S. EPA. 2009. "Guide for Development of Sample Collection Plans for Radiochemical Analytes in
Environmental Matrices Following Homeland Security Events." Cincinnati, OH: U.S. EPA.
EPA/600/R-08/128.
This document provides a framework to assist incident commanders, project managers, state and local
authorities, contractors and enforcement divisions in developing and implementing an approach for
sample collection during the cleanup of an urban environment after a radiological contamination
incident. Information in this document can be used to develop a systematic and integrated
methodology for sample collection to meet data use needs and site disposition objectives.
https://www.epa.gov/sites/production/files/2015-
07/documents/guide for developing sample collection plans for radiochemical analvtes.pdf
U.S. EPA. 2010. "Sample Collection Information Document for Pathogens - Companion to SAM
2017." Cincinnati, OH: U.S. EPA. EPA/600/R-17/374.
This document provides information regarding sample containers/media, preservation, holding time,
sizes, packaging and shipping, pertaining to collection of environmental samples to be analyzed for
the pathogens in SAM 2017.
https://www.epa.gov/homeland-securitv-research/sam-companion-documents-and-sample-collection-
procedures
U.S. EPA. 2010. "Rapid Screening and Preliminary Identification Techniques and Methods -
Companion to SAM Revision 5.0." Cincinnati, OH: U.S. EPA. EPA/600/R-10/090.
This document provides information regarding procedures for use when multiple laboratories are
needed to perform rapid preliminary analysis of environmental samples following a contamination
incident. The information is intended to support the analytical methods listed for chemical and
radiochemical analytes in SAM Revision 5.0.
https://www.epa.gov/sites/production/files/2015-07/documents/rapid screening and preid.pdf
U.S. EPA. 2010. "Laboratory Environmental Sample Disposal Information Document - Companion
to SAM Revision 5.0." Cincinnati, OH: U.S. EPA. EPA/600/R-10/092.
This document addresses laboratory disposal of samples and associated analytical waste unique to
remediation activities following a contamination incident, and assumes specific environmental sample
types (i.e., water, soil, particulates and air collection media) that will be analyzed by laboratories
using the methods listed in SAM.
https://www.epa.gov/sites/production/files/2015-06/documents/lesdid.pdf
U.S. EPA. 2012. "Sample Collection Procedures for Radiochemistry Analytes in Environmental
Matrices." Cincinnati, OH: U.S. EPA. EPA/600/R-12/566.
This document focuses on the Site Characterization, Remediation, and Final Status Survey (site
release) phases of a contamination incident and is not intended to address sample collection needs
SAM 2017
Attachment 1-3
September 2017
-------
Attachment 1 - Supporting Documents
during Initial Response. The procedures are intended for collection of environmental samples in
response to a radiological contamination incident at the point where Federal Radiological Monitoring
and Assessment Center (FRMAC) activities are turned over to EPA.
https://www.epa.gov/sites/production/files/2015-
07/documents/sample collection procedures for radiochemical analvtes.pdf
U.S. EPA. 2017. "Sample Collection Information Document for Chemicals, Radiochemicals and
Biotoxins - Companion to SAM 2017." Cincinnati, OH: U.S. EPA. EPA/600/R-17/389.
This document provides information regarding sample containers/media, preservation, holding time,
sizes, packaging and shipping, pertaining to collection of samples to be analyzed for the chemical,
radiochemical and biotoxin analytes listed in SAM 2017.
https://www.epa.gov/homeland-securitv-research/sam-companion-documents-and-sample-collection-
procedures
U.S. EPA. 2016. "Sample Collection Procedures for Radiochemistry Analytes in Outdoor Building
and Infrastructure Materials." Cincinnati, OH: U.S. EPA. EPA/600/R-16/128.
This document provides instructions regarding the collection of samples from outdoor building and
infrastructure materials to be analyzed for radiological contaminants following a contamination
incident. The document focuses on the Site Characterization, Remediation and Final Status Survey
(site release) phases of an incident and is not intended to address sample collection needs during
Initial Response. The procedures are intended for collection of samples to be analyzed using the
methods in SAM 2017.
https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvId=335065
SAM 2017
Attachment 1-4
September 2017
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oEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development
Homeland Security Research Program
Cincinnati, OH 45268
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