**Planning for and Responding to**

**Drinking Water Contamination Threats and Incidents**

**Response Protocol Toolbox**

**Module 4: Analytical Guide**

**December 12, 2003**

![](media/image1.wmf)

**OTHER RESPONSE PROTOCOL TOOLBOX MODULES**

> **Module 1: Water Utility Planning Guide** *(December 2003)*
>
> Module 1 provides a brief discussion of the nature of the
> contamination threat to the public water supply. The module also
> describes the planning activities that a utility may undertake to
> prepare for response to contamination threats and incidents.
>
> **Module 2: Contamination Threat Management Guide** *(December 2003)*
>
> Module 2 presents the overarching framework for management of
> contamination threats to the drinking water supply. The threat
> management process involves two parallel and interrelated
> activities: 1) evaluating the threat, and 2) making decisions
> regarding appropriate actions to take in response to the threat.
>
> **Module 3: Site Characterization and Sampling Guide** *(December
> 2003)*
>
> Module 3 describes the site characterization process in which
> information is gathered from the site of a suspected contamination
> incident at a drinking water system. Site characterization activities
> include the site investigation, field safety screening, rapid field
> testing of the water, and sample collection.
>
> **Module 4: Analytical Guide** *(December 2003)*
>
> Module 4 presents an approach to the analysis of samples collected
> from the site of a suspected contamination incident. The purpose of
> the Analytical Guide is **not** to provide a detailed protocol.
> Rather, it describes a framework for developing an approach for the
> analysis of water samples that may contain an unknown contaminant. The
> framework is flexible and will allow the approach to be crafted based
> on the requirements of the specific situation. The framework is also
> designed to promote the effective and defensible performance of
> laboratory analysis.
>
> **Module 5: Public Health Response Guide** *(available March 2004)*
>
> Module 5 deals with the public health response measures that would
> potentially be used to minimize public exposure to potentially
> contaminated water. It discusses the important issue of who is
> responsible for making the decision to initiate public health response
> actions, and considers the role of the water utility in this decision
> process. Specifically, it examines the role of the utility during a
> public health response action, as well as the interactions between the
> utility, the drinking water primacy agency, the public health
> community, and other parties with a public health mission.
>
> **Module 6: Remediation and Recovery Guide** *(available March 2004)*
>
> Module 6 describes the planning and implementation of remediation and
> recovery activities that would be necessary following a confirmed
> contamination incident. The remediation process involves a sequence of
> activities, including: system characterization; selection of remedy
> options; provision of an alternate drinking water supply during
> remediation activities; and monitoring to demonstrate that the system
> has been remediated. Module 6 describes the types of organizations
> that would likely be involved in this stage of a response, and the
> utility's role during remediation and recovery.

**TABLE OF CONTENTS**

1 INTRODUCTION [14](#introduction)

1.1 OBJECTIVES AND ORGANIZATION OF THIS MODULE
[14](#objectives-and-organization-of-this-module)

1.2 LABORATORY GOALS FOR RESPONDING TO CONTAMINATION THREATS TO WATER

> SYSTEMS IN THE CONTEXT OF THE RESPONSE PROTOCOL TOOLBOX
> [15](#laboratory-goals-for-responding-to-contamination-threats-to-water-systems-in-the-context-of-the-response-protocol-toolbox)

1.2.1 SAFETY [16](#safety)

1.2.2 ANALYTICAL GOALS [17](#analytical-goals)

1.3 ROLE OF LABORATORIES IN RESPONSE TO CONTAMINATION THREATS
[18](#role-of-laboratories-in-response-to-contamination-threats)

2 DESCRIPTION OF LABORATORY INFRASTRUCTURE IN U.S.
[19](#description-of-laboratory-infrastructure-in-u.s.)

2.1 CHEMISTRY LABORATORIES [19](#chemistry-laboratories)

2.1.1 ENVIRONMENTAL CHEMISTRY LABORATORIES
[19](#environmental-chemistry-laboratories)

2.1.2 RADIOCHEMISTRY LABORATORIES [20](#radiochemistry-laboratories)

2.1.3 BIOTOXIN LABORATORIES [20](#biotoxin-laboratories)

2.1.4 CHEMICAL WEAPON LABORATORIES [21](#chemical-weapon-laboratories)

2.2 MICROBIOLOGICAL LABORATORIES [21](#microbiological-laboratories)

2.2.1 LABORATORY RESPONSE NETWORK [22](#laboratory-response-network)

2.2.2 ENVIRONMENTAL MICROBIOLOGICAL LABORATORIES
[24](#environmental-microbiological-laboratories)

2.3 INTEGRATION OF LABORATORY RESOURCES
[24](#integration-of-laboratory-resources)

3 CONSIDERATIONS FOR LABORATORY ANALYSIS OF EMERGENCY SAMPLES
[26](#considerations-for-laboratory-analysis-of-emergency-samples)

3.1 SAFETY [26](#safety-1)

3.1.1 HEALTH AND SAFETY PLAN (HASP) [27](#health-and-safety-plan-hasp)

3.1.2 PERSONAL PROTECTIVE EQUIPMENT (PPE)
[27](#personal-protective-equipment-ppe)

3.1.3 COMMUNICATION TO INCREASE INFORMATION QUALITY ABOUT HAZARDOUS
SAMPLES
[28](#communication-to-increase-information-quality-about-hazardous-samples)

3.1.4 SPECIAL PROCEDURES FOR SAFE HANDLING OF HAZARDOUS WATER
CONTAMINANTS
[28](#special-procedures-for-safe-handling-of-hazardous-water-contaminants)

3.2 LABORATORY INFRASTRUCTURE [31](#laboratory-infrastructure)

3.3 DATA ANALYSIS AND REPORTING [33](#data-analysis-and-reporting)

3.4 QUALITY ASSURANCE AND QUALITY CONTROL
[35](#quality-assurance-and-quality-control)

3.5 ADMISSIBILITY OF SCIENTIFIC EVIDENCE
[35](#admissibility-of-scientific-evidence)

4 SITE CHARACTERIZATION AND SAMPLING
[38](#site-characterization-and-sampling)

4.1 SITE CHARACTERIZATION [39](#site-characterization)

4.2 SAMPLING [40](#sampling)

4.3 SAMPLE TRANSPORT AND/OR STORAGE
[40](#sample-transport-andor-storage)

4.4 SITE CHARACTERIZATION REPORT AND CHAIN OF CUSTODY
[40](#site-characterization-report-and-chain-of-custody)

4.5 COMMUNICATION [41](#communication)

5 GENERAL CONSIDERATIONS FOR AN ANALYTICAL APPROACH FOR UNIDENTIFIED
CONTAMINANTS IN WATER
[42](#general-considerations-for-an-analytical-approach-for-unidentified-contaminants-in-water)

5.1 FRAMEWORK FOR DEVELOPMENT OF AN ANALYTICAL APPROACH
[43](#framework-for-development-of-an-analytical-approach)

5.2 INITIAL ASSESSMENT OF AVAILABLE INFORMATION
[45](#initial-assessment-of-available-information)

5.3 OVERVIEW OF ANALYTICAL APPROACH FOR UNKNOWNS
[46](#overview-of-analytical-approach-for-unknowns)

5.3.1 SCREENING FOR CHEMICALS, INCLUDING "UNKNOWNS"
[47](#screening-for-chemicals-including-unknowns)

5.3.2 SCREENING FOR MICROBIOLOGICALS, INCLUDING "UNKNOWNS"
[49](#screening-for-microbiologicals-including-unknowns)

6 ANALYTICAL APPROACH FOR CHEMICAL CONTAMINANTS
[51](#analytical-approach-for-chemical-contaminants)

6.1 OVERVIEW OF CONTAMINANT ISSUES [51](#overview-of-contaminant-issues)

6.1.1 TYPES OF CHEMICAL CONTAMINANTS COVERED BY THE ANALYTICAL APPROACH
[51](#types-of-chemical-contaminants-covered-by-the-analytical-approach)

6.1.2 SELECTION OF APPROPRIATE METHODS FOR CONTAMINANT ANALYSIS
[52](#selection-of-appropriate-methods-for-contaminant-analysis)

6.1.3 INITIAL PROCESSING OF CONTAMINANTS PRIOR TO LABORATORY ANALYSIS
[52](#initial-processing-of-contaminants-prior-to-laboratory-analysis)

6.2 CONFIRMATORY ANALYSIS [54](#confirmatory-analysis)

6.3 LABORATORY ANALYTICAL SCREENING FOR CHEMICAL CONTAMINANTS, INCLUDING
"UNKNOWNS"
[56](#laboratory-analytical-screening-for-chemical-contaminants-including-unknowns)

6.4 BASIC SCREENING USING ESTABLISHED TECHNIQUES WITH STANDARDIZED
METHODS FOR TARGET ANALYTES
[58](#basic-screening-using-established-techniques-with-standardized-methods-for-target-analytes)

6.4.1 VOLATILES (ORGANIC) [60](#volatiles-organic)

6.4.2 SEMIVOLATILES (ORGANIC) [61](#semivolatiles-organic)

6.4.3 CARBAMATE PESTICIDES (ORGANIC) [61](#carbamate-pesticides-organic)

6.4.4 QUATERNARY NITROGEN COMPOUNDS (ORGANIC)
[62](#quaternary-nitrogen-compounds-organic)

6.4.5 TRACE METALS (INORGANIC) [62](#trace-metals-inorganic)

6.4.6 TOTAL MERCURY (INCLUDING ORGANOMERCURY COMPOUNDS)
[64](#total-mercury-including-organomercury-compounds)

6.4.7 CYANIDES [64](#cyanides)

6.4.8 RADIONUCLIDES [65](#radionuclides)

6.5 EXPANDED SCREENING USING ESTABLISHED AND/OR EXPLORATORY TECHNIQUES
WITH STANDARDIZED OR NON-STANDARDIZED METHODS
[66](#expanded-screening-using-established-andor-exploratory-techniques-with-standardized-or-non-standardized-methods)

6.5.1 EXPANDED SCREENING FOR ORGANIC CHEMICALS
[68](#expanded-screening-for-organic-chemicals)

6.5.2 EXPANDED SCREENING OF INORGANIC CHEMICALS
[74](#expanded-screening-of-inorganic-chemicals)

6.5.3 EXPANDED SCREENING OF CYANIDES
[76](#expanded-screening-of-cyanides)

6.5.4 EXPANDED SCREENING OF BIOTOXINS
[76](#expanded-screening-of-biotoxins)

6.5.5 EXPANDED SCREENING OF RADIONUCLIDES
[77](#expanded-screening-of-radionuclides)

6.5.6 EXPANDED SCREENING OF CHEMICAL WEAPONS
[78](#expanded-screening-of-chemical-weapons)

7 EXAMPLES OF THE ANALYTICAL APPROACH TO SITE-SPECIFIC SITUATIONS FOR
CHEMICAL CONTAMINANTS
[80](#examples-of-the-analytical-approach-to-site-specific-situations-for-chemical-contaminants)

7.1 EXAMPLES FOR UTILITY LABORATORIES
[80](#examples-for-utility-laboratories)

7.2 EXAMPLES FOR LABORATORIES WITH A WIDE CLIENT BASE
[83](#examples-for-laboratories-with-a-wide-client-base)

8 ANALYTICAL APPROACH FOR BIOLOGICAL CONTAMINANTS
[87](#analytical-approach-for-biological-contaminants)

8.1 OVERVIEW OF CONTAMINANT ISSUES
[87](#overview-of-contaminant-issues-1)

8.1.1 TYPES OF BIOLOGICAL CONTAMINANTS COVERED BY THE ANALYTICAL
APPROACH
[87](#types-of-biological-contaminants-covered-by-the-analytical-approach)

8.1.2 SELECTION OF APPROPRIATE ANALYTICAL APPROACH FOR CONTAMINANT
ANALYSIS
[87](#selection-of-appropriate-analytical-approach-for-contaminant-analysis)

8.1.3 MICROBIAL SAMPLE COLLECTION CONSIDERATIONS
[88](#microbial-sample-collection-considerations)

8.1.4 MICROBIAL PROCESSING AND ANALYSIS CONSIDERATIONS
[89](#microbial-processing-and-analysis-considerations)

8.2 ESTABLISHED TECHNIQUES FOR SAMPLE COLLECTION OF KNOWN WATERBORNE
CONTAMINANTS
[89](#established-techniques-for-sample-collection-of-known-waterborne-contaminants)

8.2.1 BACTERIA COLLECTION WITH ESTABLISHED TECHNIQUES
[90](#bacteria-collection-with-established-techniques)

8.2.2 VIRUS COLLECTION WITH ESTABLISHED TECHNIQUES
[90](#virus-collection-with-established-techniques)

8.2.3 PROTOZOA COLLECTION WITH ESTABLISHED TECHNIQUES
[91](#protozoa-collection-with-established-techniques)

8.3 ESTABLISHED TECHNIQUES FOR ANALYSIS OF KNOWN WATERBORNE CONTAMINANTS
[91](#established-techniques-for-analysis-of-known-waterborne-contaminants)

8.3.1 BACTERIOLOGICAL ANALYSIS WITH ESTABLISHED TECHNIQUES
[91](#bacteriological-analysis-with-established-techniques)

8.3.2 VIROLOGICAL ANALYSIS WITH ESTABLISHED TECHNIQUES
[92](#virological-analysis-with-established-techniques)

8.3.3 PARASITOLOGICAL ANALYSIS WITH ESTABLISHED TECHNIQUES
[93](#__RefHeading___Toc58462052)

8.4 METHOD FOR SAMPLE COLLECTION, FIELD PROCESSING, AND LABORATORY
ANALYSIS FOR UNKNOWN MICROBIAL CONTAMINANTS
[94](#__RefHeading___Toc58462053)

8.4.1 SAMPLE COLLECTION AND CONCENTRATION FOR UNKNOWN MICROBIALS
[94](#sample-collection-and-concentration-for-unknown-microbials)

8.4.2 FIELD SCREENING AND PROCESSING OF SAMPLE
[96](#field-screening-and-processing-of-sample)

8.4.3 PRESUMPTIVE TESTING FOR MICROBIALS WITH EXPLORATORY PCR TECHNIQUES
[97](#presumptive-testing-for-microbials-with-exploratory-pcr-techniques)

8.4.4 CONFIRMATORY TESTING FOR MICROBIALS BY CULTURE AND CONVENTIONAL
ASSAYS
[99](#confirmatory-testing-for-microbials-by-culture-and-conventional-assays)

9 REFERENCES AND RESOURCES [102](#references-and-resources)

10 APPENDIces [106](#appendices)

10.1 CHEMICALS THAT APPEAR IN CWC SCHEDULES
[106](#chemicals-that-appear-in-cwc-schedules)

10.2 METHODS USED FOR THE BASIC SCREEN
[108](#methods-used-for-the-basic-screen)

10.3 ANALYTE LISTS FOR BASIC SCREEN
[109](#analyte-lists-for-basic-screen)

**LIST OF TABLES**

TABLE 4-1: SUMMARY OF TYPES OF CHEMICAL CONTAMINANTS, THEIR ANALYTICAL
GROUP, AND

> THE CLASS OF CONTAMINANTS DETERMINED BY THE ANALYSIS 51

TABLE 4-2: SOURCES OF STANDARDIZED METHODS 55

TABLE 4-3: SUGGESTED ANALYTICAL TECHNIQUES FOR PERFORMING THE BASIC
CHEMICAL SCREEN, BROKEN OUT BY THE CLASSES GIVEN IN TABLE 4-1 59

TABLE 4-4: EXPANDED SCREENING FOR CONTAMINANTS, BROKEN OUT BY CLASSES OF
CONTAMINANTS AS FOUND IN TABLE 4-1 67

TABLE 4-5: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #1 81

TABLE 4-6: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #2 82

TABLE 4-7: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #3 82

TABLE 4-8: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #4 83

TABLE 4-9: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #5 83

TABLE 4-10: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #6 84

TABLE 4-11: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #7 85

TABLE 4-12: BASIC SCREEN FOR CHEMICAL ANALYSIS FOR EXAMPLE #8 86

TABLE 4-13: GENERAL CLASSES OF PATHOGENS, SPECIFIC ORGANISMS, AND SELECT
AGENT STATUS 87

TABLE 4-14: KNOWN WATERBORNE PATHOGENS WITH ESTABLISHED TECHNIQUES FOR
SAMPLE COLLECTION AND ANALYSIS 90

TABLE 4-15: EXPLORATORY FIELD SCREENING TECHNIQUES FOR WATERBORNE
CONTAMINANTS

AND SELECT AGENTS 97

TABLE 4-16: EXPLORATORY ANALYTICAL TECHNIQUES FOR PRESUMPTIVE TESTING OF

WATERBORNE CONTAMINANTS AND SELECT AGENTS 98

TABLE 4-17: ESTABLISHED CONFIRMATORY ANALYTICAL TECHNIQUES FOR
WATERBORNE CONTAMINANTS AND SELECT AGENTS 100

TABLE 4-18: LIST OF CHEMICALS THAT APPEAR IN CWC SCHEDULES 1, 2, AND 3
106

TABLE 4-19: TITLES OF METHODS USED IN TABLE 4-3 FOR THE BASIC SCREEN 108

TABLE 4-20: ANALYTE LISTS CORRESPONDING TO TABLE 4-3 109

**LIST OF FIGURES**

FIGURE 4-1: SUMMARY OF TYPES OF LABORATORIES BY CONTAMINANT CLASS 19

FIGURE 4-2: DIAGRAMMATIC REPRESENTATION OF THE LABORATORY RESPONSE
NETWORK 23

FIGURE 4-3: SITE CHARACTERIZATION AND SAMPLING PROCESS 38

FIGURE 4-4: DECISION PROCESS FOR THE DEVELOPMENT OF AN ANALYTICAL
APPROACH FOR POTENTIALLY CONTAMINATED WATER SAMPLES 43

FIGURE 4-5: INITIAL ANALYTICAL APPROACH TO PROCESSING SAMPLES WITH
CHEMICAL CONTAMINANTS 53

FIGURE 4-6: OVERVIEW OF SCREENING AND CONFIRMATORY ANALYSES FOR CHEMICAL
CONTAMINANTS 57

FIGURE 4-7: SCHEMATIC FOR BASIC RADIONUCLIDE SCREENING 65

FIGURE 4-8: ANALYTICAL APPROACH FOR THE COLLECTION OF MICROBIAL
CONTAMINANTS 88

FIGURE 4-9: APPROACH FOR THE PROCESSING AND ANALYSIS OF MICROBIAL
CONTAMINANT

GROUPS 89

FIGURE 4-10: BACTERIOLOGICAL SCREENING WITH ESTABLISHED TECHNIQUES 92

FIGURE 4-11: VIROLOGICAL ANALYSIS WITH ESTABLISHED TECHNIQUES 93

FIGURE 4-12: PARASITOLOGICAL ANALYSIS WITH ESTABLISHED TECHNIQUES 94

FIGURE 4-13: SAMPLE COLLECTION AND PROCESSING FOR UNKNOWN MICROBIAL
CONTAMINANTS 95

FIGURE 4-14: FIELD CONCENTRATOR SCHEMATIC 96

FIGURE 4-15: FIELD PROCESSING FOR MICROBIALS 96

FIGURE 4-16: SAMPLE PROCESSING FOR EXPLORATORY PCR PRESUMPTIVE TESTING
99

FIGURE 4-17: SAMPLE PROCESSING FOR CONFIRMATORY MICROBIOLOGICAL ASSAYS
100

**DISCLAIMER**

The mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

**Acronyms**

AA Atomic absorption

AMDIS Automated mass spectral deconvolution and identification system

AOAC Association of Analytical Communities (formerly Association of
Official Analytical Chemists)

APCI Atmospheric pressure chemical ionization

APHL Association of Public Health Laboratories

API Atmospheric pressure ionization

ASTM American Society for Testing and Materials

BSL Biosafety level

BT Biological terrorism

CDC Centers for Disease Control

CLIA Clinical Laboratory Improvement Amendments

CVAA Cold-vapor atomic absorption

CW Chemical warfare

CWA Clean Water Act

CWC Chemical Weapons Convention

DNA Deoxyribonucleic acid

DOD Department of Defense

DOE Department of Energy

DOJ Department of Justice

DOT Department of Transportation

ELCD Electrolytic conductivity detector

EPA Environmental Protection Agency

ESI Electrospray ionization

ETV Environmental Technology Verification

FA Immunofluorescence assay microscopy

FBI Federal Bureau of Investigation

FDA Food and Drug Administration

FEMA Federal Emergency Management Agency

FERN Food Emergency Response Network

FRMAC Federal Radiological Management Center

GC Gas chromatography

GFAA Graphite furnace atomic absorption

HASP Health and safety plan

HazMat Hazardous materials

HHS Health and Human Services

HPLC High performance liquid chromatography

HRMS High resolution mass spectrometry

IC Ion chromatography

ICAP Inductively coupled argon plasma

ICP-AES Inductively coupled plasma atomic emission spectroscopy

ICP-MS Inductively coupled plasma mass spectrometry

ICP-OES Inductively coupled plasma optical emission spectroscopy

ICR Information Collection Rule

IFA Immunofluorescence assay microscopy

IMS Immunomagnetic separation

IOC Intergovernmental Oceanographic Commission

ISE Ion-selective electrode

ISO International Organization for Standardization

LC Liquid chromatography

LLE Liquid-liquid extraction

LRN Laboratory Response Network

MARLAP Multi-Agency Radiological Laboratory Analytical Protocols

MCL Maximum Contaminant Level

MeV Megaelectron volts

MS Mass spectrometry

MWCO Molecular weight cut-off

NEMI National Environmental Methods Index

NIST National Institute of Standards and Technology

NRC National Research Council

ORD Office of Research and Development

OSHA Occupational Safety and Health Administration

OSW Office of Solid Waste

OW Office of Water

PCR Polymerase chain reaction

PDA Photodiode array detector

PID Photoionization detector

PPE Personal protective equipment

PSI Pounds per square inch

QA Quality assurance

QC Quality control

RF Response factor

RIFA Radioimmunofocus assay

RNA Ribonucleic acid

RT-PCR Reverse Transcriptase-PCR

SDWA Safe Drinking Water Act

SM Standard Methods for the Analysis of Water and Wastewater

SOP Standard operating procedure

SPE Solid-phase extraction

SPME Solid-phase microextraction

SQM Semi-quantitative mode

TBD To be determined

UCMR Unregulated Contaminant Monitoring Rule

URL Uniform Resource Locator

USAMRIID United States Army Medical Research Institute of Infectious
Diseases

USDA United States Department of Agriculture

UV Ultraviolet

VEE Venezuelan equine encephalitis virus

VHF Viral hemorrhagic fever

**GLOSSARY**

Definitions in this glossary are specific to the Response Protocol Tool
Box but conform to common usage as much as possible.

**Analyte** B the name assigned to a substance or feature that describes
it in terms of its molecular composition, taxonomic nomenclature, or
other characteristic.

**Analytical Approach** B a plan describing the specific analyses that
are performed on the samples collected in the event of a water
contamination threat. The analytical approach is based on the specific
information available about a contamination threat.

**Analytical Confirmation** B the process of determining an analyte in a
defensible manner.

**Analytically Confirmed** B in the context of the *analytical
approach*, a contaminant is considered to be analytically confirmed if
it has undergone analytical confirmation, as defined herein.

**Basic Screen** - utilizes standardized methods based on established
analytical techniques for the analysis of samples collected in response
to a contamination threat.

**Biosafety Level 1** B suitable for work involving well-characterized
biological agents not known to consistently cause disease in healthy
adult humans, and of minimal potential hazard to laboratory personnel
and the environment. Work is generally conducted on open bench tops
using standard microbiological practices.

**Biosafety Level 2** B suitable for work involving biological agents of
moderate potential hazard to personnel and the environment. Laboratory
personnel should have specific training in handling pathogenic agents
and be directed by competent scientists. Access to the laboratory should
be limited when work is being conducted, extreme precautions should be
taken with contaminated sharp items, and certain procedures should be
conducted in biological safety cabinets or other physical containment
equipment if there is a risk of creating infectious aerosols or
splashes.

**Biosafety Level 3** B suitable for work done with indigenous or exotic
biological agents that may cause serious or potentially lethal disease
as a result of exposure by inhalation. Laboratory personnel must have
specific training in handling pathogenic and potentially lethal agents
and be supervised by competent scientists who are experienced in working
with these agents. All procedures involving the manipulation of
infectious materials are conducted within biological safety cabinets or
other physical containment devices, or by personnel wearing appropriate
personal protective clothing and equipment. The laboratory must have
special engineering and design features.

**Biosafety Level 4** B suitable for work with the most infectious
biological agents. Access to the two Biosafety Level 4 laboratories in
the U.S. is highly restricted.

**Chain of Custody** B the tracking and documentation of physical
control of evidence.

**Chemical Weapons** B the chemicals that the CWC has placed on its
Schedule 1 list.

**CLIA Laboratory** B a laboratory regulated under the CLIA program.

**Clinical Laboratory Improvement Amendments (CLIA)** B regulation that
covers all laboratory testing (except research) performed on humans in
the U.S. Any laboratory participating in the CLIA program that does
diagnostic testing, verification, or proficiency testing is exempt from
select agent regulation, but the laboratory must follow specific
procedures.

**'Confirmed'** B in the context of the *threat evaluation* process, a
water contamination incident is 'confirmed' if the information collected
over the course of the threat evaluation provides definitive evidence
that the water has been contaminated.

**Core Field Testing** B analysis performed at the investigation site
for radiation, cyanide, residual chlorine, and pH. Core field testing is
performed as part of *site characterization* and is composed of two
elements, a *field safety screen* and *rapid field testing*.

**'Credible'** B in the context of the *threat evaluation* process, a
water contamination threat is characterized as 'credible' if information
collected during the threat evaluation process corroborates information
from the *threat warning*.

**Environmental Chemistry Laboratory** B any laboratory that is set up
to perform analysis of water samples for compliance with the Safe
Drinking Water, Clean Water Acts, or other applicable environmental
regulation, as well as other chemical parameters that are important to
system operation and overall water quality. These laboratories have the
instrumentation necessary to implement methods for chemical analysis of
a water sample.

**Established Analytical Techniques** B these techniques are commonly
employed in a large number of laboratories and form the basis of many
standardized analytical methods.

**Expanded Field Testing** B analysis of water at the site of a
suspected contamination incident for parameters beyond those covered
under core field testing (e.g., VOCs, chemical weapons, biotoxins,
etc.).

**Expanded screen** B application of exploratory techniques for chemical
contaminants that do not have standardized methods. Additionally, the
expanded screen provides laboratories with additional options regarding
the instrumentation used to implement both established and exploratory
techniques; however, the results may not be considered definitive and
thus may require confirmation.

**Exploratory Techniques** B techniques capable of detecting chemicals
that are not included in existing standardized methods. They may employ
less common instrumentation than those in the standardized methods, or
they may simply not yet be specifically used in standardized drinking
water methods.

**Field Safety Screening** B screening performed to detect any
environmental hazards (i.e., in the air and on surfaces) that might pose
a threat to the *site characterization* team. Monitoring for
radioactivity as the team approaches the site is an example of field
safety screening.

**Field Sample Concentrate --** the term used for the retentate from the
ultrafiltration device used for the sampling/concentration of unknown
microbial contaminants.

**Filtrate --** in ultrafiltration, the water that passes through the
filtration membrane.

**Food Emergency Response Network (FERN)** B a laboratory network for
the analysis of contaminants in food developed through integration with
the existing LRN for pathogen analysis and established forensic
chemistry laboratories that serve as reference laboratories for other
FDA laboratories.

**Grab Sample -** a sample collected at random from the source.

**Hazard Assessment** B the process of evaluating available information
about the site to identify potential hazards that might pose a risk to
the site characterization team. The hazard assessment results in
assigning one of four levels to risk: low hazard, radiological hazard,
high chemical hazard, or high biological hazard.

**Incident Command System** B a standardized on-scene emergency
management concept specifically designed to allow its user(s) to adopt
an integrated organizational structure equal to the complexity and
demands of single or multiple incidents, without being hindered by
jurisdictional boundaries.

**Incident Commander** B the individual responsible for the management
of all incident operations.

**Infectious --** capable of causing infection. In the context of
waterborne pathogens, infection is caused by exposure to water.

**Laboratory Analytical Screening** B application of multiple analytical
techniques in a laboratory setting to screen for a wide range of
analytes and to confirm tentative results. (Laboratory analytical
screening is different and separate from *laboratory safety screening*,
*field safety screening*, and *rapid field testing*.)

**Laboratory Compendium** B a comprehensive, web-based, searchable
database of laboratory capability for environmental analysis in water,
air, soil, sediments, and other media.

**Laboratory Guide** -- a plan prepared by a specific laboratory
detailing their approach and capabilities for the 24/7 processing of
emergency water samples.

**Laboratory Response Network (LRN)** B a network of laboratories
developed by the CDC, APHL, and FBI for the express purpose of dealing
with bioterrorism threats, including pathogens and some biotoxins.

**Laboratory Safety Screening** B screening for various hazards that is
conducted when samples are received at the laboratory, and which is
designed to reduce the risks to laboratory personnel that may handle the
samples.

**Non-standardized method** B a method that does not meet the definition
of a standardized method.

**Opportunity Contaminant B** contaminants that might be readily
available in a particular area, even though they may not be highly toxic
or infectious or easily dispersed and stable in treated drinking water.

**Pathogen -** an infectious microorganism that is capable of causing
disease.

**Personal protective equipment (PPE)** B equipment and supplies
designed to protect employees from serious injuries or diseasees
resulting from contact with chemical, radiological, biological, or other
hazards. PPE includes face shields, safety glasses, goggles, laboratory
coats, gloves, and respirators.

**'Possible'** B in the context of the *threat evaluation* process, a
water contamination threat is characterized as 'possible' if the
circumstances of the *threat warning* appear to have provided an
opportunity for contamination.

**Presumptive results** B results of chemical and/or biological field
testing that need to be confirmed by further laboratory analysis.
Typically used in reference to the analysis of pathogens.

**Priority Contaminants** B list of chemicals, biotoxins, and
radionuclides that were ranked highly due to their availability,
properties and potential to harm public health if introduced into the
drinking water supply.

**Rapid field testing** - analyzing the water at the site of
contamination using rapid field water testing technology to tentatively
identify any chemicals or pathogens. Rapid field testing is performed as
part of site characterization.

**Reference laboratory** B the core, advanced technology, LRN
laboratories that can provide analytical confirmatory testing of
contaminants.

**Retentate -** in ultrafiltration, the retentate contains the particles
that do not pass through the filtration membrane.

**Select Agent** B biological contaminants, including some pathogens and
biotoxins, that are regulated by HHS. Only certain laboratories,
registered with the CDC in accordance with the Select Agent Act, are
permitted to confirm the presence of, and maintain cultures of, select
agents.

**Sentinel Laboratory** B an LRN laboratory that reports unusual results
that might indicate a possible outbreak, and refers specimens that may
contain select biological agents to *Reference laboratories* within the
LRN.

**Site Characterization** -- the process of collecting information from
an *investigation site* in order to support the evaluation of a drinking
water contamination threat. Site characterization activities include the
site investigation, *field safety screening*, *rapid field testing* of
the water, and sample collection.

**Standardized Method** B a method that has been produced as a standard
by a recognized method development body (EPA, ASTM, AOAC, ISO, etc.) or
applicable regulatory authority. A standardized method has been
subjected to review and validation and is capable of generating data of
sufficient quality for its intended use. Standardized methods often
contain steps to defensibly confirm the presence and/or quantity of
specific contaminants.

**Tentative Identification** B the contaminant identity is hypothesized
based on available information from the site characterization report.
Examples of situations in which tentative identification might occur
include: a specific contaminant named in a threat; tentatively positive
results for a specific contaminant during field safety screening or
rapid field testing; physical evidence at the site pointing to a
specific contaminant; and clinical evidence of the identity of the
disease-causing agent.

**Threat** B an indication that a harmful *incident*, such as
contamination of the drinking water supply, may have occurred. The
threat may be direct, such as a verbal or written threat, or
circumstantial, such as a security breach or unusual water quality.

**Threat Evaluation** B part of the threat management process in which
all available and relevant information about the threat is evaluated to
determine if the threat is 'possible' or 'credible', or if a
contamination *incident* has been 'confirmed.' This is an iterative
process in which the threat evaluation is revised as additional
information becomes available. The conclusions from the threat
evaluation are considered when making *response decisions*.

**Threat Management** B the process of evaluating a contamination threat
and making decisions about appropriate response actions. The threat
management process includes the parallel activities of the *threat
evaluation* and making *response decisions*. The threat management
process is considered in three stages: 'possible', 'credible,' and
'confirmatory.' The severity of the threat and the magnitude of the
response decisions escalate as a threat progresses through these stages.

**Ultrafiltration --** a filtration process for water that uses
membranes to preferentially separate very small particles that are
larger than the membrane's molecular weight cut-off, typically greater
than 10,000 daltons.

**Water Contamination Incident** B a situation in which a contaminant
has been successfully introduced into the system. A water contamination
incident may or may not be preceded by a water contamination threat

**Water Contamination Threat** B a situation in which the introduction
of a contaminant into the water system is threatened, claimed, or
suggested by evidence. Compare *water contamination threat* with *water
contamination incident.* Note that even a threat against a water system
is a crime under the Safe Drinking Water Act as amended by the
Bioterrorism Act.

**Water Utility Emergency Response Manager (WUERM)** B the individual(s)
within the drinking water utility management structure that has the
responsibility and authority for managing certain aspects of the
utility=s response to an emergency (e.g., a contamination threat)
particularly during the initial stages of the response. The
responsibilities and authority of the WUERM are defined by utility
management and will likely vary based on the circumstances of a specific
utility.

# Introduction

## Objectives and Organization of This Module

The primary intended users of this module include laboratory personnel
and planners who would provide analytical support to a water utility in
the case of a contamination threat to the water supply. This module is
intended to be a planning tool for laboratories that may need to provide
an analytical response in the case of a contamination threat, **not** a
"how-to" manual for use during the actual incident. As part of planning
for such an incident, laboratories may want to prepare such a manual
specific to their needs and capabilities. They should also exercise and
improve the manual from lessons learned via conducting drills, which may
encourage laboratories to "think outside the box" in responding to
contamination threats.

While this module is not based expressly on regulatory requirements, it
should be recognized that **failure to plan for an emergency
contamination incident might lead to tragic public health
consequences.** Accordingly, the objectives of this document are to:

-   Describe special laboratory considerations for handling and
    processing emergency water samples suspected of contamination with a
    harmful substance.

-   Present model approaches and procedures for analysis of water
    samples suspected of contamination with a known or unknown
    substance. These approaches and procedures are developed to take
    advantage of existing methodologies and infrastructure.

-   Encourage planners to develop a site-specific *analytical approach*
    and *laboratory guide* that conform to the spirit and general
    principles of the model approaches. Sometimes these models may
    represent the best and/or only way of dealing with the analytical
    issues involved. Frequently, they provide an example of the most
    comprehensive approach.

**Planners and laboratory analysts are encouraged to review this module
in its entirety, as well as the other modules in the Response Protocol
Toolbox*,* to obtain a more comprehensive understanding of the
analytical response approach for water contamination threats.** As
suggested in the Overview and Application to the Response Protocol
Toolbox, the modules that perhaps are most relevant to laboratories are
Modules 3 and 4, but depending on the nature of the laboratory, the
other modules also may prove helpful. Because of the varied audience for
Module 4, particularly certain sections in Module 4, an attempt has been
made to provide explanations of the subject matter in the various
sections that are directed toward certain audiences. For instance, the
bulk of the technical material for the analytical approach is found in
Sections 6 and 8. Section 5 describes the framework for the development
of an analytical approach, so planners may find Section 5 more useful,
whereas laboratory analysts may find the technical material in Sections
6 and 8 more relevant. Furthermore, different laboratories may also
require different levels of sophistication in the technical material.

It is hoped that the presentation of the material represents an
effective compromise among the needs and capabilities of the wide
audience of utilities and laboratories that wish to develop analytical
capability to support the evaluation of water contamination threats.
This module is organized into ten sections as described below.

> Section 1: [Introduction]{.underline}: describes the overall
> organization of the document and discusses the concept of due
> diligence as applied to laboratories in terms of meeting two main
> goals: the safety of laboratory personnel and provision of quality
> analytical data.
>
> Section 2: [Description of Laboratory Infrastructure in the
> U.S.]{.underline}: describes existing laboratory resources that might
> be involved in the implementation of the analytical approaches
> presented in this module.
>
> Section 3: [Considerations for Laboratory Analysis of Emergency
> Samples]{.underline}: briefly discusses infrastructure, staffing,
> personnel safety, and sample capacity considerations for laboratory
> analysis of water samples suspected to be contaminated with an unknown
> substance.
>
> Section 4: [Field Screening and Sample Collection]{.underline}:
> provides an overview of the field screening and sampling procedures
> presented in Module 3 and describes how these activities are linked to
> laboratory analysis.
>
> Section 5: [Analytical Approach for Unidentified Contaminants in
> Water]{.underline}: presents a framework for developing an analytical
> approach for emergency water samples in response to a specific
> contamination threat.
>
> Section 6: [Analytical Approach for Chemical
> Contaminants]{.underline}: presents a tiered approach for the analysis
> of unknown chemical contaminants, including biotoxins and
> radionuclides, in water samples.
>
> Section 7: [Examples of the Analytical Approach to Site-Specific
> Situations for Chemical Contaminants]{.underline}: presents examples
> of how several, hypothetical utility laboratories with differing
> capabilities have chosen to plan their analytical approach, based on
> the discussion in Section 6. **Laboratories that choose to perform
> analysis of emergency water samples and serve a broader client base,
> such as Federal, State, and commercial labs, may need to adopt a more
> comprehensive approach, such that they maintain flexibility to provide
> analytical support in a variety of situations.**
>
> Section 8: [Analytical Approach for Microbiological
> Contaminants]{.underline}: presents a tiered approach for the analysis
> of unknown microbiological agents in water samples.
>
> Section 9: [References and Resources]{.underline}: lists the
> references used in the development of this module as well as
> additional information resources.
>
> Section 10: [Appendices]{.underline}: provides additional information
> and materials that may be of value to the reader.

## Laboratory Goals for Responding to Contamination Threats to Water Systems in the Context of the Response Protocol Toolbox

The analytical approach discussed in this module is directed at any
laboratory potentially involved in analysis of emergency water samples.
Water utilities are most familiar with their own systems, and they are
often interested in dealing with incidents of all sorts at the local
level (i.e., through the use of their own personnel and their own
laboratories). In fact, it is not practical for HazMat responders and
hazard materials laboratories to become involved in every incident that
occurs at the thousands of water systems across the country. The
responsibility falls on water utilities, who routinely investigate water
quality complaints, whether they originate from intentional
contamination or not. Accordingly, during intentional water
contamination events, it should be the general goal of non-utility
laboratories to support the utilities.

For any laboratory to exercise due diligence in responding to
contamination threats to water systems, it must meet two specific,
essential goals. The first goal is ensuring the safety of laboratory
personnel. The second goal is the timely generation of comprehensive,
quality analytical results to support difficult decisions that must be
made during an emergency situation.

### Safety

Specific safety considerations for laboratories wishing to receive and
analyze emergency water samples are discussed in more depth in Section
3. It is important to realize that details important for laboratory
safety are integrated into the threat evaluation (Module 2) and the site
characterization (Module 3) processes, even though they occur outside of
the laboratory setting. Information in these other modules impact
laboratory safety in several ways. First, the *site characterization*
and *threat evaluation* processes help define the hazard conditions at
the site of sample collection, which govern who should collect the
samples and which laboratories should analyze them. For highly hazardous
samples, samples should be collected and analyzed by specifically
trained personnel, namely HazMat responders and laboratories that
specialize in the handling of specific hazardous substances. This
mitigates potential exposure to unprepared laboratory personnel. As an
example, if the site characterization results indicate a potential
radiological hazard, samples would be delivered only to laboratories
that specialize in the analysis and handling of radiological materials.

Second, in the *threat management* process, water contamination
incidents range from *'possible'* to *'confirmed'*. The principle set
forth in Module 2 is that, to date, there are many, perhaps thousands,
of 'possible' incidents (ones that are not really intentional
contamination incidents) for every 'confirmed' incident (one in which
contamination has occurred). Those incidents falling into the
'confirmed' category are those that generate samples containing a
harmful contaminant. Even among those samples, it is expected that the
vast majority will not contain highly hazardous materials (e.g.,
chemical or biological warfare agents or radionuclides). Naturally,
there is a continuum, described in Module 2 as *'credible'*, between the
'possible' and 'confirmed' categories. A contamination threat is deemed
'credible' if additional information collected during the evaluation
supports the likelihood that contamination has occurred. However, the
number of 'credible' cases is expected to be just a few more than the
'confirmed' ones, which is still vastly less than the number of
'possible' ones.

In summary, it is likely that most "emergency" water samples will be
sent on the basis of a 'probable' contamination threat. Samples sent to
a laboratory as a result of a 'probable' contamination threat should be
treated as if they contain a potentially harmful substance. However, the
site characterization, along with the threat evaluation process, should
result in most highly hazardous samples being screened out before they
reach the laboratory. From a safety standpoint, it is important for a
laboratory to realize that it will **not be expected to determine every
potential contaminant**. For instance, utility laboratories typically
may expect to receive samples from 'possible' incidents. The utility
laboratories will need additional laboratory support for 'credible'
incidents, and specialty laboratories would be called into service for
'confirmed' incidents.

[]{.mark}

### Analytical Goals

Utilities and laboratories familiar with regulatory compliance
monitoring are accustomed to thinking about water analysis in terms of
contaminant concentrations that may cause chronic (long-term) toxicity.
In responding to intentional contamination incidents, an important
paradigm shift is that one goal of the analysis may be to help rule in
or rule out the presence of significantly elevated levels of certain
types/classes of contaminants. Other goals may exist, and their
significant features are discussed below.

[]{.mark}

[Analytical goals for the three levels of the threat evaluation
process]{.underline}

The threat evaluation process is important to a laboratory's analytical
goals, because part of providing timely, accurate results is the proper
allocation of analytical resources. The allocation of laboratory
resources to a threat will be determined by the analytical goals of the
laboratory and the incident's credibility category. Regardless of
category, it is important to remember that even if one contaminant is
identified during the analysis, the presence of additional contaminants
should also be investigated.

-   'Possible' --- For the vast majority of cases, because it is
    unlikely that there will be an actual contaminant, it is very
    important to report accurate results and to not misidentify an
    instrumental response. These results need to be rapid, but not
    instantaneous. Keep in mind that many of the decisions about water
    system operations will have been made before the analytical results
    are back from the laboratory. While speed and accuracy are necessary
    analytical goals for any scenario, they take on a special meaning
    during the evaluation of 'possible' incidents.

-   'Credible' --- In the few 'credible' cases, laboratories may receive
    water samples containing potentially harmful contaminants; however,
    the activities performed during the threat evaluation and site
    characterization processes should reduce the likelihood that samples
    containing high hazard materials (as defined in Module 3, Section
    4.1.2) reach the laboratory. Thus, laboratories should exercise due
    diligence to meet the goals of protecting their personnel and
    providing timely, accurate, analytical results. The laboratory
    investigation should be focused on those types of contaminants
    discussed in Sections 6 (toxic industrial chemicals, biotoxins, and
    radionuclides) and Section 8 (some waterborne pathogens, etc.).

-   'Confirmed'--- For the rare 'confirmed' incidents, the laboratories
    receiving these materials should be ones with specific capabilities
    for the contaminants (chemical, biological, or radiological), which
    will be suspected or known as a result of the site characterization.
    Environmental laboratories will be capable of many analyses, but are
    prohibited from handling materials such as Schedule 1 chemical
    warfare agents. This Module should **not** be construed to suggest
    that laboratories intentionally handle materials for which they are
    not licensed.

[Data Interpretation]{.underline}

Another goal of analysis is to interpret data in an appropriate manner.
Often, part of the interpretation involves understanding baseline
concentrations. **The importance of knowing baseline levels of
contaminants at a location cannot be overemphasized.** Not only does
this affect the site characterization (Module 3) and threat management
process (Module 2) in terms of the proper use of analytical data, but it
may serve the larger goal of creating greater public acceptance of water
from a distribution system that was once contaminated. Most laboratories
currently do not retain this information because they do not necessarily
know the sampling location. However, the laboratory may be aware of
typical background levels through analysis of routine samples.
Laboratories should be aware of issues regarding background data,
particularly if they are asked to render an opinion on the presence of
an unusual contaminant.

[]{.mark}

## Role of Laboratories in Response to Contamination Threats

While this Toolbox is aimed primarily at utilities, very few utility
labs will be able to independently implement Module 4 in its entirety.
However, they may find the information in Module 4 useful in
understanding their analytical options, planning their analytical
approach, and developing their *laboratory guide*.

Federal, State, and commercial laboratories will play a critical support
role in the response to contamination threats -- specifically, some of
these laboratories will be responsible for implementing the analytical
approaches presented in this module. These laboratories may already be
familiar with some of the material in Module 4. However, they should
work with their utility clients to ensure that the utilities are
familiar with the capabilities of the laboratory. In working with the
utility, the laboratory should be aware that response to a contamination
threat is led by the incident commander, who may be from the water
utility. Please see Module 1, Section 4.4 for more details on the
Incident Command System and the role of the laboratory within it.

Utilities need to have confidence that laboratories that agree to
process emergency water samples operate according to the following
guidelines:

-   Apply the analytical approach presented in this module according to
    the circumstances of a particular incident and the needs of the
    client.

-   Maintain facilities and implement procedures for ensuring the
    security and integrity of samples and analytical results that may be
    considered as evidence for use in prosecution.

-   Receive and process emergency samples 24/7. The laboratory should
    develop an appropriate plan for staffing, sample receipt, and
    internal chain of custody.

-   Provide results to the client in a time frame stipulated by the
    client. The laboratory should be prepared to provide the client with
    an estimate of the time frame in which results may be available. As
    discussed in Module 2, the utility may need to take certain response
    measures before analytical results are available. Accordingly, the
    time frame may be dictated by site-specific factors, such as the
    hydraulic residency time within a segment of a distribution system.

-   Implement appropriate *quality assurance* and *quality control*
    (QA/QC) procedures, and report the QC data along with the analytical
    results. See Section 3 for additional discussion.

-   Use proper channels for reporting results.

-   Provide support in the analysis and interpretation of analytical
    results.

-   Have a back-up plan for processing samples should the laboratory's
    facility become unusable or unavailable.

Laboratories **may** wish to develop their laboratory guide in
accordance with these guidelines, and to share these plans with their
clients. Timely and accurate results from the laboratory may provide
valuable input for making decisions about how to proceed with a response
to a contamination threat. Identification of a harmful contaminant in a
water sample would likely trigger additional public health measures,
including additional sampling to characterize the spread of the
contaminant, and possibly some initial remediation efforts. Likewise, if
laboratory results reveal nothing out of the ordinary, the response
would likely be terminated, and any precautionary public health measures
could be cancelled or scaled down.

# Description of Laboratory Infrastructure in U.S.

The analytical approach described in the module was developed under the
assumption that it would be implemented using existing laboratory
infrastructure. If an environmental laboratory response network,
designed specifically to analyze environmental samples, is developed in
the future, the approaches presented in this document may be revised to
take advantage of the new resource. Figure 4-1 summarizes laboratory
infrastructure, as it currently exists, for the analysis of
environmental samples. Discussion of both access to and roles of these
laboratories during the response to an intentional contamination threat
or incident follows.

![](media/image2.wmf)

Figure 4-1. Summary of Types of Laboratories by Contaminant Class

The following two sections provide a general description of the
laboratory infrastructure for the analysis of chemical and
microbiological contaminants in a water matrix. A comprehensive,
web-based, searchable database of laboratory capability for
environmental analysis in water, air, soil, sediments, and other media
will be provided in the *Laboratory Compendium* when available. It is
not a listing of laboratories approved, certified, or recommended to
analyze samples from intentional contamination incidents. Rather, the
Compendium is designed to be a tool for searching for laboratories and
determining their ability to perform various analytical techniques, such
as those presented in this module.

## Chemistry Laboratories

In addition to laboratories within water utilities, standard and
specialized chemistry laboratories within Federal, State, local, city,
and municipal government agencies, as well as commercial laboratories,
may support analysis of chemicals in water samples. Some laboratory
resources may also be available from the academic and industrial
sectors. For example, a major chemical manufacturer in an area might
want to bring their laboratory operations to bear during the evaluation
of an incident in which their products are suspected in a water
contamination threat. Many academic and industrial laboratories,
however, may not necessarily be set up to rapidly respond to water
contamination threats without extensive planning. Regardless of their
origin, it is anticipated that four broad categories of analytical
chemistry laboratories would play a role in implementing the chemistry
procedures in the analytical approach: environmental chemistry,
radiochemistry, biotoxins, and chemical weapons.

### Environmental Chemistry Laboratories

This group forms the largest sector of the laboratory infrastructure for
analysis of chemicals in water, and includes many EPA, State, and
commercial water analysis laboratories. Environmental chemistry
laboratories are typically set up to perform analysis of water samples
for compliance with the Safe Drinking Water or Clean Water Acts, as well
as other chemical parameters that are important to system operation and
overall water quality. It is important to realize that these
laboratories are typically involved in the analysis of contaminants at
concentrations associated with chronic (long-term) toxicity, not the
acutely (short-term) toxic levels potentially associated with an
intentional contamination incident. While it may seem intuitive that
laboratories capable of determining contaminants at low concentrations
should not experience difficulties at high concentrations, this is not
necessarily the case for a number of technical and practical reasons.

These laboratories typically have the instrumentation necessary to
implement *standardized methods* for chemical analysis in a water
matrix. Because many of these laboratories are involved in regulatory
compliance, the laboratory and staff may already be accredited and
certified to implement these methods. However, unless the laboratory
tests for the particular chemical *analyte* on a routine basis, the
laboratory will not necessarily be able to run the associated method
without advanced notice. This includes maintaining an inventory of
standards and reagents, setting up the instrument for a particular
method, and having staff trained to run the method. This may be
particularly relevant in the context of chemical analysis in the case of
a suspected intentional contamination incident since many of the
chemicals of greatest concern are not routinely analyzed for in water,
even though standardized methods are available.

Some environmental chemistry laboratories may have unique capabilities
for analysis of select radionuclides or biotoxins, but this is not the
expected norm. Analysis for these chemicals may need to be performed by
a specialty laboratory as discussed below. Further, there are a number
of research laboratories within the government and academic sectors that
may be available on a limited basis. These laboratories are typically
involved in method development, and thus are equipped with advanced
equipment and highly trained analysts that provide capability for
implementation of *exploratory techniques* that are currently beyond the
means of other environmental chemistry laboratories.

### Radiochemistry Laboratories

If a radioactive contaminant is suspected, analyses should be performed
by a laboratory specifically equipped to handle such material and
analyze for a range of radionuclides. EPA, DOE, States, and commercial
firms have laboratories dedicated to the analysis of radioactive and/or
nuclear material. For further information about EPA's laboratory
services and radiological emergency response programs, see
[http://www.epa.gov/radiation/programs.htm -
er](http://www.epa.gov/radiation/programs.htm#er).

Another source of support in the case of an attack utilizing a
radiological contaminant is the Federal Radiological Management Center
(FRMAC) operated by the Federal Emergency Management Agency (FEMA).
FRMAC is set up to provide rapid response teams and emergency management
services related to incidents involving radioactive materials. This
center works with a number of Federal and State agencies and draws on a
variety of resources including fixed and mobile laboratories. FRMAC also
maintains databases of national laboratory resources and radiological
capabilities. Information regarding FEMA's Radiological Emergency
Preparedness Program can be found at
<http://www.fema.gov/rrr/rep/index.shtm>.

### Biotoxin Laboratories

Currently, few laboratories are set up specifically for the analysis of
biotoxins. Those in existence primarily focus on the analysis of marine
biotoxins in coastal waters and seafood products. Some *Laboratory
Response Network* (LRN) laboratories (see Section 2.2.1) may have the
capability to analyze for select biotoxins in water samples, assuming
proper sample preparation. In addition, there are a number of
laboratories in government and academia that perform biotoxin analysis,
usually for other matrices than water (e.g., seafood or agricultural
products). It is possible that some biotoxin analyses could be performed
in qualified environmental chemistry laboratories using techniques such
as GC/MS, HPLC, immunoassay, and possibly LC/MS; however, such
capability may not be currently widespread.

### Chemical Weapon Laboratories

For the purposes of the Response Protocol Toolbox, a "chemical weapon"
refers to those chemicals that the Chemical Weapons Convention (CWC) has
placed on its Schedule 1
(<http://www.cwc.gov/Regulations/cfr-15/part-712-s1_html>). This list
includes toxic chemicals with few or no legitimate other purposes, that
were developed or used primarily for military purposes
(<http://www.cwc.gov/Industry_Outreach/Publications/002/cwc-b0001_html>).
CWC also monitors chemicals on two other Schedules and certain
"unscheduled discrete organic chemicals." A list of chemicals in the CWC
Schedules appears in the appendix in Table 4-18. Some of these other
chemicals (not on Schedule 1) may be present in water through a number
of routes other than intentional contamination. For instance, there are
some relatively non-toxic chlorinated hydrocarbons listed on Schedule 2
because they can be precursors to Schedule 1 chemicals. Coincidentally,
these chemicals may also be disinfection byproducts, a term which refers
to the hundreds of substances formed in very small amounts by reactions
between drinking water disinfectants and substances naturally present in
the water.

Only a few laboratories are qualified and permitted to work with
concentrated Schedule 1 chemical weapons surety material. In fact, the
only two chemical weapons surety laboratories in the U.S. are the U.S.
Army's Edgewood laboratory and Lawrence Livermore National Laboratories.
These laboratories can only be accessed through specific channels (e.g.,
through certain Federal agencies such as FBI). A broader group of
laboratories can work with dilute chemical weapons materials, such as
might be encountered in a water contamination incident; however, the
ability to access these laboratories through normal channels is
uncertain, and even if such capability can be accessed, analysis may not
be widely available. In contrast, many environmental laboratories work
routinely with Schedule 2, Schedule 3, and unscheduled chemicals, and
analytical standards for many of these are readily available.

## Microbiological Laboratories

The analysis of waterborne pathogens will likely be performed either by
an environmental microbiology laboratory or a laboratory that is part of
the Laboratory Response Network (LRN). This may include hospital
laboratories, medical laboratories, public health laboratories, and
environmental microbiology laboratories. However, the missions and
capabilities of these two distinct sets of laboratories are
significantly different, and neither may be particularly well prepared
for the analysis of all biological terrorism (BT) contaminants of
concern in a water matrix. The potential role of each of these types of
laboratories in responding to a water contamination threat involving
pathogens is discussed in the following two sections.

### Laboratory Response Network

The Laboratory Response Network (LRN) was developed by the Centers for
Disease Control (CDC), the Association of Public Health Laboratories
(APHL), and the FBI for the express purpose of dealing with bioterrorism
(BT) threats, including pathogens and some biotoxins
(<http://www.cdc.gov/cic/functions-specs/function4Docs/nLRNvision-summary.doc>).
Various laboratories within each State participate in the LRN (see
<http://www.bt.cdc.gov/emcontact/index.asp> for contact information).
Laboratories that are part of the LRN can analyze the *select agents*
(<http://www.bt.cdc.gov/Agent/agentlist.asp>) subject to legislative
requirements set forth in the Select Agent Regulation (42 CFR 72,
<http://www.cdc.gov/od/sap>). The legislation requires that, subject to
certain exemptions, entities possessing biological agents that are
listed as select agents must register with CDC and/or USDA's Animal and
Plant Inspection Service (for veterinary purposes,
<http://www.aphis.usda.gov/>) and demonstrate compliance with specific
safety and security standards for handling these agents.

Two details of select agent regulation
(<http://www.cdc.gov/od/sap/faq.htm>) that might be of immediate
interest are: 1) The USA Patriot Act places restrictions on persons who
possess select agents and provides criminal penalties for possession of
such agents that cannot be justified for specified peaceful purposes
(<http://www.cdc.gov/od/sap/addres.htm>). 2) Any diagnostic or *Clinical
Laboratory Improvement Amendments (CLIA) laboratory* that does
diagnostic testing, verification, or proficiency testing is exempt from
the regulation. However, the director of such a laboratory must notify
the Department of Health and Human Services (HHS, see contact info
above) immediately upon identifying specific select agents, and it must
transfer the agents to a registered facility or destroy them (unless
directed otherwise by law enforcement or HHS) within 7 calendar days of
identification of the select agent, subject to certain exemptions.
Retention of any select agent as a positive control or reference sample
is not permitted in this case.

Thus, by legislation, confirmatory analysis of samples containing select
pathogens, such as *Bacillus anthracis*, *Brucella* spp., *Yersinia
pestis*, *Francisella tularensis*, and *C. botulinum* toxins, among
others, must be performed through the LRN. It should be noted that the
LRN also performs analysis of biological samples other than the "select"
agents, and many of the waterborne pathogens of concern are not select
agents. Since the LRN will be involved in an analytical response to any
bioterrorism incident, it is important to understand the structure and
organization of this network. The LRN, composed of city, county, State,
and Federal public health laboratories, is in a perpetual state of
evolution as capabilities are matched with current needs. Figure 4-2
provides a schematic overview of the current LRN structure.

![](media/image3.png)

**Figure 4-2. Diagrammatic Representation of the Laboratory Response
Network**

The LRN membership is organized into "*Sentinel Labs*," which recognize
an agent, rule it out, and/or refer the sample to the next level for
confirmatory testing, performed in "*Reference Labs*". At the top of the
pyramid are "*National Labs*" (namely CDC and USAMRIID), which are
capable of definitive characterization of even the most hazardous
biological agents.

Although the LRN is set up to identify and characterize dangerous
pathogens through an upward referral system, most LRN laboratories are
not equipped to process water samples that may contain these pathogens
or their toxic byproducts. Specifically, they do not have appropriate
protocols to process the relatively large sample volumes needed for
analysis of pathogens at low concentrations that are still of public
health concern. For example, some Reference laboratories have received
standardized environmental protocols for handling dry samples that might
contain *Bacillus anthracis*, *Brucella* spp., *Yersinia pestis*,
*Francisella tularensis*, *C. botulinum* toxins, and *Staphylococcus*
enterotoxins; however, protocols are not yet in place for handling water
samples containing these agents.

The LRN can accept specimens and samples from hospitals, clinics, the
FBI and other law enforcement groups, emergency medical services, the
military, and other agencies. Thus, it may be reasonable to expect that
water utilities could work through their local or State health
departments to deliver samples to LRN laboratories. However, this is
complicated by regulations governing the transport of samples that are
known or suspected of containing select pathogens. Specifically, such
samples can be shipped only in an approved and appropriately marked
container (see Section 6 in Module 3 for details, including the use of
technical escort services). While these containers are adequately sized
to ship clinical specimens and culture tubes, none are of sufficient
size or integrity to ship the large volumes of water (a minimum of 10
liters) that are necessary to achieve the desired detection limit. These
issues need to be resolved before LRN laboratories can be engaged in the
response to a BT incident at a water system. The protocols presented in
this module are one proposed solution.

Another challenge arises from the fact that public health and
environmental protection are legislated and managed differently by each
state. Only four states have a single state agency that is responsible
for both public and environmental health. All other states have one
agency responsible for public health issues and another for
environmental issues. In addition, some states also separate their state
public health (clinical) laboratories from the state environmental
laboratories. Only 14 state public health laboratories also serve as the
state environmental laboratories (Arkansas, Colorado, Connecticut, Iowa,
Kansas, Louisiana, Maine, New Mexico, North Carolina, South Carolina,
South Dakota, Tennessee, Virginia, and Wisconsin). Personnel at these 14
laboratories, however, rarely handle **both** clinical and environmental
samples. Some states even have strict rules that prohibit cross-training
of public health (clinical) microbiologists and environmental
microbiologists. To further complicate interactions and communications
among agencies, the individual responsible for drinking water is located
in the health agency in half of the states and in the environmental
agency in the other half of the states.

### Environmental Microbiological Laboratories

Environmental microbiological laboratories, including those of EPA,
state environmental agencies, and the commercial sector, typically
perform analyses for waterborne pathogens. Most of these laboratories
have the equipment and staff necessary to perform classical
microbiological methods, and routinely analyze for indicators of fecal
contamination such as fecal and total coliforms and *E. coli*. Culture
techniques are available for many of the more common waterborne
pathogens such as *Vibrio cholerae, Salmonella enteriditis* Typhi, and
*Shigella* spp.; however, analyses for these pathogens are not routinely
performed in most environmental microbiological laboratories. While some
environmental microbiological laboratories have expanded capabilities to
analyze for parasites such as *Cryptosporidium* and *Giardia* or to
perform molecular assays for some organisms, these capabilities are not
widespread.

While many environmental microbiological laboratories are well equipped
to analyze for microbiological contaminants in a water matrix, they
generally lack the infrastructure, training, and methods to analyze for
many pathogens of concern. Furthermore, as discussed in Section 2.2.1,
only laboratories registered for the analysis of select agents are
legally permitted to analyze for those agents, and currently most
registered labs reside in the LRN. Thus, even if environmental
microbiological laboratories develop additional capabilities for
pathogen analysis, they could not perform such analyses without
registering for select agents.

## Integration of Laboratory Resources

Sections 2.1 and 2.2 presented a brief overview of the laboratory
infrastructure that will likely be called upon to implement the
procedures presented in this module. While the core infrastructure may
exist for both chemical and microbiological analysis, no mechanism
currently exists to provide coordination in a manner conducive to
optimal analytical response. At a minimum, this will create a greater
logistical burden on the organization coordinating sampling and shipment
to qualified laboratories. In the worst case, these inefficiencies may
result in an incomplete analysis of an unknown, shipment to the wrong
laboratory, or delays in receiving time sensitive information.

Formation of environmental laboratory response networks would help to
address these coordination issues. Such networks are in existence for
the analysis of clinical samples (CDC's LRN) and food samples (FDA's
*Food Emergency Response Network* \[FERN\]). The FERN was developed
through integration with the existing LRN for pathogen analysis and
establishment of regional forensic chemistry laboratories that serve as
reference laboratories for other FDA laboratories. In the absence of a
formal network, the analytical response to water contamination threats
may be supported by the laboratory infrastructure as it currently
exists.

Accordingly, the approaches described in this module were developed for
implementation by the existing laboratory infrastructure. Some states
have established/will establish network-like entities to coordinate
laboratory efforts. The following steps may help states better integrate
laboratory resources and provide a more coordinated response to water
contamination threats:

-   Establish environmental chemistry laboratories that are capable of
    implementing both basic and expanded screens (see Sections 6.3 and
    6.4) for unknown chemicals in water samples.

-   Establish environmental microbiology laboratories within the LRN
    that are capable of performing Sentinel testing for pathogens of
    concern in a water matrix.

-   Determine those biotoxins that will likely be analyzed for in
    environmental chemistry laboratories and those that will be analyzed
    for in LRN laboratories. Considering the range of techniques used to
    measure biotoxins, there may be some overlap in biotoxin capability.

-   Establish a clear sample referral system for the *analytical
    confirmation* of tentatively identified contaminants, in cases where
    the *environmental chemistry laboratory* cannot perform it. This
    concept is integrated into the LRN for microbiological analysis but
    is not formally defined for chemical analyses.

# Considerations for Laboratory Analysis of Emergency Samples 

Laboratories participating in the analysis of emergency water samples
that may contain an unknown and potentially dangerous substance have
additional responsibilities beyond those associated with routine
analyses. This section briefly discusses some of the special issues
related to safety, infrastructure, responsiveness, data reporting,
quality assurance, and legal admissibility of scientific evidence that
laboratories should consider before engaging in the analysis of
emergency samples. This discussion is not intended to comprehensively
address all issues that laboratories may face, but focuses on issues
specifically dealing with the analysis of emergency water samples. This
discussion applies largely to fixed laboratories, not mobile
laboratories. Mobile laboratories often are designed to meet specific
needs, so it is anticipated that most analyses will be performed by
fixed laboratories, which are likely capable of a wider range of
analytical methodologies.

This section primarily deals with operations within the laboratory
during the analysis of emergency water samples. It is also important,
however, for the laboratory to keep in mind its role within the
*incident command structure*, which, as discussed in Module 1, is
through the "laboratory point of contact." The incident command
structure, which must be developed at the local level, establishes and
clarifies the roles of each of the various participants (*water utility
emergency response manager*, first responders, state drinking water
program personnel, laboratory personnel, etc.) and how they should
interact with each other.

## Safety

This section seeks to promote the safety of laboratory personnel during
the analysis of samples arising from the suspected contamination of the
water supply, which is subject to regulations with which most
laboratories should be familiar. In addition, the entire Toolbox,
including the analytical approach presented in Sections 6 and 8 of this
module, is intended to enhance the safety of laboratory personnel. For
instance, it is important to realize that during the site
characterization process, *field safety screening* and *rapid field
testing* have occurred. This may identify potentially hazardous samples
before a decision is made regarding the laboratory that will receive the
samples. In fact, part of site characterization is the *site hazard
assessment*, in which the site is categorized as low hazard,
radiological hazard, high chemical hazard, or high biological hazard.
The results of this site hazard assessment should dictate the laboratory
that will be used for analysis, and they should also help assure that
only laboratories equipped to deal with highly hazardous materials will
receive samples potentially containing such materials.

Considering that the vast majority of evaluated threats that generate
laboratory samples will likely **not** prove to be intentional
contamination incidents (see discussion in Section 1.2 of this module),
additional risks may be manageable. Due to site characterization
procedures, environmental chemistry laboratories may have increased
confidence that they will not be processing hazardous biological agents.
In the unlikely case that such a sample does reach the laboratory, the
measures described below and the analytical approach in Sections 6 and 8
are designed to reduce the risk to laboratory personnel. Accordingly,
many more laboratories may be willing and able to help respond to
potential intentional contamination incidents.

### Health and Safety Plan (HASP)

Under current regulations, laboratories are required to have a plan in
place to ensure worker safety. Some laboratories may wish to treat
certain emergency water samples as hazardous material as defined in
Module 3, whether it be chemical, biological or radiological in nature,
and to develop a specific health and safety plan (HASP) to address this
potential risk, although there is currently no requirement to do so in
most cases. Information on HASPs is available at
<http://www.ertresponse.com/health_safety/index.htm>, along with an
electronic expert system jointly developed by EPA and OSHA
(<http://www.osha.gov/dts/osta/oshasoft/ehasp/>) to help determine the
appropriate controls of health and safety hazards for a specific
situation.

Laboratory personnel involved in the handling and analysis of water
samples should have appropriate, current safety training that will allow
them to conform to applicable regulations. Laboratories, although not
required, may wish to explore some of the measures contained in
regulations for the handling of hazardous materials, such as OSHA
1910.120 ([http://www.osha.gov](http://www.osha.gov/)). If planners and
laboratories do not conclude that these regulations are applicable to
them, they may still wish to adopt some of the principles in these
regulations. For instance, laboratory personnel may work in cooperation
with a designated "buddy" and maintain visual and/or vocal contact with
the buddy at all times during the analysis. This system may provide an
additional level of protection compared to regulations applicable for
most environmental chemistry laboratories, which do not necessarily rely
on the "buddy" system.

### Personal Protective Equipment (PPE)

Analysis of potentially hazardous samples during an emergency situation
may require additional *personal protective equipment (PPE)* above that
normally used in the laboratory. Such PPE requirements should be
determined during the creation of the site-specific HASP described in
Section 3.1.1. The requirements should be contained in the HASP, and PPE
should be freely available to laboratory personnel. Personnel should be
trained, competent, and medically certified (particularly for
respiratory PPE) to use all necessary levels of PPE, in accordance with
applicable regulations (i.e., OSHA 1910.120 or OSHA 1910.132
([http://www.osha.gov](http://www.osha.gov/)), along with local or State
requirements).

Conventional PPE may be used in conjunction with hand-held
"sniffer-type" instruments, which can monitor for a variety of
preselected volatile organic and inorganic compounds. References such as
[http://www.chrismanual.com](http://www.chrismanual.com/),
<http://www.osha.gov/SLTC/personalprotectiveequipment/index.html>, and
<http://www.cdc.gov/niosh/npptl/default.html>, should be consulted for
more detail on special PPE. For instance, butyl gloves and full-face
shields should be considered for optimal protection, particularly during
pouring and splitting of non-volatile samples when maximum risk of
accidental exposure could occur.

Although not necessarily a personal protection issue, care should be
used when using PPE to preserve the forensic integrity of the samples.
For example, dirty laboratory coats and gloves could result in
cross-contamination of samples. Hair bonnets, aside from some safety
benefit, may prevent loose or attached hair from contacting samples,
which could potentially introduce a variety of contaminants into the
sample.

### Communication to Increase Information Quality about Hazardous Samples

Information about the collection site may be an **invaluable** component
in personnel safety. In general, laboratories may be asked to analyze
samples that originate from two types of sources: 1) those with rigorous
sample collection procedures and 2) those using less-than-perfect sample
collection procedures. In either case, and particularly the latter, it
is recommended that the laboratory be fully informed about the sample
collection and site investigation procedures, including any *field
safety screening* and *rapid field testing* results, to ensure not only
personnel safety but also analytical integrity. The laboratory may wish
to take several steps to become more fully informed. One such step is to
inquire if the field personnel followed the procedures and performed the
tests suggested in Module 3 for site characterization. Another possible
step is to establish a system to review procedures used to collect the
samples and assess the reliability of the source of the samples. A third
step might be for laboratories to enhance their communication with the
field sampling teams.

With respect to enhancing communications, representatives from the
laboratory ideally would be present during site characterization and
sampling to provide as much information as possible to the laboratory
analysts regarding sample collection, field screening, sample transport,
and eventual analysis. Representatives from the laboratory might then
accompany samples to the laboratory and interact with the laboratory
personnel while maintaining contact with the field personnel. A less
ideal case might be that the laboratory can readily communicate
interactively in real time with the field sampling team so that they can
provide details of sampling strategies, environmental conditions, and
other pertinent information to the laboratory. In any case, the written
site characterization report (see Module 3) should be provided to the
laboratory at the time of sample delivery or faxed separately. Direct
verbal communication, such as a conference call to discuss the site
characterization findings, should improve the quality and clarity of the
information in such a report.

It is important to note that different organizations may use different
terminology when discussing issues related to potential contamination
events, ranging from a description of the contaminants, to what is meant
by field testing, to descriptions of laboratory results (i.e., is a
"negative" result good or bad?). The glossary to this module attempts to
standardize these terms, but it is imperative that laboratories ensure
that necessary and effective communication is **not** impeded by
inconsistent terminology.

### Special Procedures for Safe Handling of Hazardous Water Contaminants

[Examination of incoming packages]{.underline}

Infrastructure requirements for the safe receipt of packages are
discussed in the next section. In addition, the staff in the receiving
area should be trained in the identification of safety hazards
associated with packages. Sometimes these hazards are indicated by
warning labels required by DOT regulations (<http://hazmat.dot.gov/>)
and/or information contained on the shipping papers. Other hazards may
result from improper packaging and shipment. They include, but are not
limited to: obvious leaks, breaks in packaging tape or custody seals,
damage to packaging, unusual stains on shipping container, strange
odors, etc. (Note: It is not advisable to smell or taste the package).

[Laboratory safety screening upon receipt]{.underline}

During the site characterization process, samples from a site are
subjected to field safety screening and/or rapid field testing, as
described in Section 4.1 of this module. To reduce risks associated with
potential, undetected hazards, laboratories may wish to screen the
sample for various hazards upon receipt at the laboratory, regardless of
the reported field safety screening results. Specifically, some of the
safety screening techniques employed in the field (see Module 3) may
also be used in the *laboratory safety screening*. Some of these
techniques may, in fact, be performed better in the controlled
conditions of the laboratory.

If the safety screening tests indicate the presence of a previously
undetected contaminant, the sample should be referred to the appropriate
laboratory, depending on the type of hazard (radiological, biological,
high hazard chemical, low hazard chemical), as described in Section 4,
Module 3. For instance, a positive test for radiation would prompt
referral of the sample to a radionuclide laboratory, whereas a positive
test for cyanide, a low hazard chemical, would allow the sample to
remain at the environmental chemistry laboratory and be treated as a
cyanide sample.

[Avoiding aerosols]{.underline}

The water solubility of potential contaminants sometimes contributes to
their safe handling. Namely, as long as the contaminant remains in the
bulk aqueous solution, the principal risk involves ingestion. Therefore,
**steps should be taken to avoid volatilizing or aerosolizing water
samples**, which would then increase the inhalation risk. For instance,
the pressurized portion of the *ultrafiltration* device used for
concentrating microbial samples (Section 8) is a closed system which
prevents the formation of aerosols, assuming there are no leaks.
Analytical methods should be appropriately selected to reduce
volatilization and the formation of aerosols (e.g., performing
extractions in sealed vials). Accordingly, separatory funnel
liquid-liquid extraction, which may release aerosols when vented, is not
recommended unless laboratories can utilize appropriate hoods or other
precautions.

Some aerosol generation is unavoidable. Samples, particularly biological
ones, may produce aerosols when shaken vigorously for homogenization,
and these aerosols should be contained. Purge-and-trap technology for
analysis of volatile organic compounds produces aerosols, which are
contained within the instrument, provided that the purge vessel is not
accidentally broken during analysis. Also, inductively coupled plasma
and some atomic absorption techniques for trace metal analysis must
produce aerosols to function properly. For optimal protection, these
instruments have to be properly vented to minimize inhalation risk.
Manufacturers of modern instruments have designed their instruments to
minimize aerosol release (many of these instruments are used in
industrial clean rooms), but laboratories should ensure that the venting
on their instruments is adequate. If this is not possible, because of
the short instrument run times for metal analysis, laboratories may
consider performing these analyses after other procedures have been used
to screen for the presence of other volatilizable or aerosolizable
contaminants.

[Dilution]{.underline}

As a general principle, dilution of a hazardous water sample with
laboratory-grade water helps reduce risks associated with handling of
the sample and its analysis for chemical contaminants. Dilution,
however, may have undesirable effects on the ability to detect and
quantify the contaminants. Accordingly, it should be used carefully only
as part of an overall handling (and/or analysis) strategy that balances
contaminant effects (e.g., health and safety concerns) with instrument
response and timeliness of the results. Contaminant effects may be
important primarily for extremely hazardous chemicals, such as Schedule
1 chemical warfare agents and biological warfare agents. The need to
dilute may be reduced by safety screening performed both during site
characterization (Module 3) and also upon receipt by the laboratory.

If dilution is desired, "log dilutions" may be attempted. For instance,
first, a 1/1,000 dilution may be analyzed, followed by a 1/100 dilution
if nothing is detected in the highest dilution, followed by a 1/10
dilution, and finally the undiluted sample. In deciding on a dilution
scheme, it is important to keep in mind that most drinking water methods
are designed to detect contaminants in the low parts-per-billion range.
Concentrations that are hundreds of times this level may be present in
samples from the site of an intentional contamination incident.
Appropriate QC measures should be implemented, particularly with regard
to the purity of the dilution water. Also, before diluting samples in
the laboratory, it should be verified that samples were not diluted in
the field prior to shipment to the laboratory.

Although not directly related to safety, dilution may also protect the
analytical instrument from becoming saturated with a contaminant.
Procedures for reducing the saturation are usually possible, but they
take time, limiting the availability of the instrument. Working out the
correct dilution requires time, which could be an important
consideration. On the other hand, the presence of sufficient analyte to
saturate the instrumentation may indicate a significant contamination
incident. The decision to implement dilution should accordingly be made
during planning.

[Reduction of sample volumes used]{.underline}

Like dilution, reducing the volumes of sample handled may help minimize
exposure for both chemical and biological contaminants. Some techniques
(like those discussed in Section 6) may involve using smaller volumes of
sample. For example, direct aqueous injection typically uses a few
microliters of sample. Micro-liquid extraction uses about 40 milliliters
(mL), and large volume extractions may use 1 liter (L) or more. A
sufficient volume of sample is required to achieve the desired detection
limits, but it may be helpful to plan the analytical approach based on
the desired goals. For example, if the goal is to quantify a high
concentration of contaminant (high micrograms per liter), then less
sample may be required than for a low concentration (low to
mid-micrograms per liter).

[Irradiation and other means to reduce pathogenic infectivity,
particularly of samples contaminated with both chemicals and
pathogens]{.underline}

Some laboratories may consider irradiating (UV or gamma) the samples
prior to chemical analysis to reduce the risk of possible exposure to
pathogens. There may be other ways of achieving the goal of reducing
pathogenic infectivity, such as pasteurization, other heat
sterilization, and/or direct filtration. However, all of these
approaches to reduce pathogen infectivity may alter the identity and/or
quantity of some chemicals by thermal, photochemical, or physical means.
Also, in the case of filtration, significant leaching of interfering
substances from the filter material can occur. Therefore, when using
techniques to reduce pathogenic infectivity, it is important to keep in
mind the goals of the analysis, such as analysis of chemicals, detection
of viable organisms, etc.

**Currently there is no general consensus on proper use of irradiation
and/or other techniques to reduce risks associated with sample handling
and analysis while maintaining the integrity of the sample and the
analysis. Accordingly, these techniques for reducing pathogenic
infectivity are not generally recommended** (Module 3, Section 4.4.1).
The laboratory may wish to explore these techniques, however, if the
authorities in charge of the threat evaluation process (Module 2)
believe there is very good reason to suspect that the sample is
contaminated with both biological and chemical contaminants. In this
case, one way to proceed would be to split the sample prior to
irradiation. One portion could be irradiated and analyzed for chemicals,
and a non-irradiated portion could be analyzed for pathogens, following
confirmation that there are no highly hazardous volatile chemicals in
the sample. (Note that an irradiated sample could be used for the
pathogens, provided the analysis did not require the presence of viable
organisms, as is the case with many molecular assays.) Informed
decisions could then be made about how to safely proceed with other
portions of the original, non-irradiated sample. Appropriate hoods and
other physical control measures should be used when handling such
samples.

## Laboratory Infrastructure

Infrastructure must be adequate not only to support the analysis of the
target analyte in a water matrix, but also must be sufficient to ensure
the safety of laboratory staff and the security of samples and data. The
following subsections describe infrastructure considerations applicable
to all laboratories, followed by those applicable to specific analytical
classes. Details of laboratory infrastructure are discussed in more
detail in this section, particularly as they relate to safety, security,
and analytical quality. This section is not intended to be a laboratory
design manual, but rather to highlight areas in which infrastructure
considerations may play an important role when handling emergency
samples.

[General considerations for laboratory capability]{.underline}

The infrastructure must be consistent with the goals of emergency
analysis. Ample instrumentation and laboratory equipment should be
available to meet analysis goals. This infrastructure should be
compatible with relevant laboratory accreditation requirements, quality
assurance plans, and requirements of auditing groups. This may help
ensure that laboratories function well during an emergency and do not
forego sound laboratory practice due to emergency conditions.

[Safety equipment]{.underline}

Infrastructure includes any safety equipment such as eyewashes, safety
showers, spill containment devices/supplies, etc. It also includes first
aid kits, which should include inexpensive antidote kits for various
poisons, such as cyanide and organophosphate compounds. Personnel should
be trained on the availability, indications, and use of these antidotes.
Safety supplies and training materials are readily available from
numerous companies servicing industrial safety needs.

[Chemical/biological hoods and physical control measures]{.underline}

Appropriate chemical hoods, biosafety cabinets, containment glove boxes,
and/or other physical control measures should be incorporated into
laboratory infrastructure and may be required by the HASP. Exhaust and
pollution control systems must be evaluated to determine proper
operation prior to every analysis (i.e., through the use of appropriate
monitors for hood face velocity, etc). The chemical hood, biosafety
cabinet, and/or containment glove box should be cleared of all other
equipment, samples, reagents, supplies, etc., except those required to
open and process the sample itself. Some laboratories may wish to
deactivate any contaminants that exist on the outside of the sample
container or the sample opening equipment. In this case, the
deactivating solution (10% bleach, chemical neutralizers, etc) may be
kept in the hood or glove box. Note that deactivating solutions,
including bleach, have finite shelf-lives and must be refreshed
accordingly.

Ventilation and physical control requirements for chemical samples vary
with hazard classification. Most laboratories should currently have
sufficient controls for low hazard samples. In general, occupational
exposure to hazardous chemicals in laboratories is described in OSHA
standard 1910.1450 ([http://www.osha.gov](http://www.osha.gov/)). This
standard also forms the basis for laboratory safety guidelines for
laboratories possessing one milliliter or less of a pure Schedule 1
chemical warfare agent
(<http://www.usapa.army.mil/pdffiles/p385_61.pdf>). This link is
provided for reference, and should not imply that a laboratory with a
liter of water that contains only 10 microliters of pure agent and that
has the appropriate physical controls should consider itself an approved
laboratory capable of analyzing these substances.

For biological samples, appropriate equipment is required based on the
desired *biosafety level* (BSL). For BSL-2, biosafety hoods may be
required, while BSL-3 requires a specially designed facility. See
[http://www.cdc.gov/od/ohs/pdffiles/4th
BMBL.pdf](http://www.cdc.gov/od/ohs/pdffiles/4th BMBL.pdf) for design
considerations for biosafety hoods and for more information on BSL
requirements for particular biological agents in various forms, e.g.
dried or in solution. For example, although *B. anthracis* is considered
an organism that may be safely contained using Biosafety Level 2 (BSL-2,
<http://www.bt.cdc.gov/documents/PPTResponse/table3abiosafety.pdf>)
conditions, if the procedure is likely to generate aerosols, it should
be handled using BSL-3 conditions.

[Sample receipt, handling, and security]{.underline}

Adequate infrastructure is required to achieve the goals of safety for
laboratory personnel charged with the receipt, handling, and analysis of
potentially hazardous forensic samples. In some cases, the same
infrastructure used for receipt of routine samples may be sufficient,
but depending on the existing facilities, some upgrades may be necessary
to safely handle hazardous samples.

Infrastructure-related safety issues begin with initial sample receipt.
The sample receipt area should be isolated from the analysis area and
include features that allow effective handling and security of samples.
Furthermore, samples should be handled and secured in a manner that will
support the admissibility of the results into a court of law. This may
include electronic sample tracking and impenetrable storage areas.

Samples should be opened in a manner to maximize personnel safety and
minimize laboratory contamination. For example, samples may be opened in
a dedicated glove box or hood which is physically and geographically
separate from the hoods used in the analytical laboratory. Laboratories
may wish to take pictures of samples and/or install automatic video
surveillance equipment in order to document sample receipt, handling,
and security. Likewise, laboratories may wish to conduct physical
measurements (e.g., determine the mass and/or volume of the water
sample, upon receipt and at various stages of analysis). This may be
important for safety as well as forensic reasons.

[Dual-use facilities for routine and emergency samples]{.underline}

For routine analysis, laboratories often can anticipate the
concentration range of certain chemicals in water samples and plan
accordingly. However, in the case of an unknown, there may not be any
information regarding the potential concentration of a particular
chemical, and some samples could contain very high concentrations. This
can lead to significant problems, such as a laboratory becoming
contaminated when unusually high concentrations of a contaminant are
present. To minimize the potential for laboratory contamination when
working with unknown samples, precautions should be taken, such as
working in a dedicated sample prep area, carrying samples in buckets
with gas-tight, friction-fit lids containing absorbent material in the
bottom, and other measures to physically prevent the accidental
introduction of contaminants (including vapors) from water samples into
the laboratory.

Aside from safety issues, accurate analytical determinations are related
to good laboratory practices, particularly in the area of
cross-contamination of samples. (Note that radioactive contamination may
be preventable by identifying the presence of radioactivity during
initial sample screening procedures.) In addition to degrading the
validity of analytical results, cross-contamination can affect aspects
of the quality assurance features of a method. For example,
contamination of the reagent blank can occur. The reagent blank is an
aliquot of reagent water or other blank matrix that is treated exactly
as a sample. The reagent blank is used to determine if method analytes
or other interferences are present in the laboratory environment, the
reagents, or the apparatus. If a chemical from an unknown sample
contaminates the reagent blank, the accuracy of all subsequent
measurements to identify and quantify that chemical in a sample may be
compromised. In principle, concerns related to the reagent blank and
other related quality assurance issues are procedurally dealt with by
the quality control section of the chemical or biological analysis
method. However, it is still possible for difficulties to arise,
especially with volatile material that may easily pass through the air
from one sample to another or to the reagent blank.

In the case of microbiological contaminants, the high sensitivity of
molecular techniques makes them susceptible to contamination by trace
amounts of biological materials. For example, molecular methods
utilizing the polymerase chain reaction (PCR) are extremely sensitive to
the presence of any target DNA. It is imperative that PCR product
analysis be carried out in an area physically separated, with separate
airflow, from the areas where the samples are prepared for analysis. To
avoid contaminating reagents, it is desirable to have a separate reagent
preparation facility. These considerations warrant a specialized
laboratory facility, specifically designed to deal with the demands of
molecular methods. Frequently, a core facility strategy is used to
minimize the impact of this space requirement. In this case, there
should be separate preparation areas for suspected biological
contaminants, to prevent possible cross contamination with routine
environmental samples, and to minimize the opportunity for contamination
of a laboratory with biological contaminants.

[Sample capacity and turn-around time]{.underline}

Laboratory infrastructure involves elements that are desirable for
ensuring sufficient analytical capacity and rapid turn-around time in
the case of an emergency incident, while maintaining quality standards.
This includes elements of laboratory staffing, planning of work
schedules, and creating provisions for the availability of staff during
an emergency. In addition, the facility should allow for 24/7 access and
control of safety measures. For instance, laboratory personnel should be
able to override systems that reduce automatic hood ventilation at
preset times to save energy. Laboratories may choose to perform frequent
exercises to meet the combined goals of analysis speed and accuracy.

[Sample disposal]{.underline}

Laboratories should have waste disposal plans in place, subject to
applicable regulations. As discussed previously, most samples will
likely contain no or little contamination, and hence existing laboratory
waste disposal practices should suffice. In the relatively rare instance
that the sample does contain a hazard, then it is likely that law
enforcement will want the sample retained as evidence and provide
guidance as to its storage and ultimate disposition when no longer
needed. In any case, all regulations governing the disposal of hazardous
substances should be adhered to when disposing of samples.

## Data Analysis and Reporting

The responsibility of the laboratory during an emergency does not end
with sample analysis. At a minimum, the laboratory should report the
results in a timely manner to the designated recipients. Furthermore,
the laboratory may be called upon to assist in analysis and
interpretation of the data. While specific arrangements for the
reporting and evaluation of analytical results need to be made between
the laboratory and the client, the following general guidelines will
apply in most situations:

1.  The laboratory and the client (e.g., the water utility emergency
    response manager or the designated incident commander) should agree
    on the format and content of the report as part of their planning.
    In this manner, incident command will know what to expect and can
    practice interpreting the report. There are efforts in the forensic
    community to provide standardized guidance on report writing (i.e.,
    the minimum types of information that should be contained in such a
    report). When it is available, this guidance may be included in this
    module.

An important issue in the content of the report arises when the
laboratory is asked to provide an analysis of a water sample for which
the contaminant is unknown (e.g., applying a laboratory analytical
screening procedure to a water sample, as described in Section 6.3).
Environmental laboratories are often familiar with determining specific
analytes and providing reports regarding those analytes. For example,
the laboratory may apply a standardized method and report on the
analytes listed in the method. However, if the laboratory observes
non-method analytes during the analysis, several questions arise about
how theses should be reported, such as:

-   What is a significant result in terms of the concentration of the
    non-target analytes?

-   Should all non-detects be reported?

-   How much detail is appropriate in identifying and/or quantifying the
    analytes?

> In principle, the report should be thorough enough so that one does
> not miss anything important, but if too much information is reported,
> the laboratory may confuse the client. These are complex issues that
> are likely to be situation-specific and may also be dependent on local
> policy. Also, reporting priorities may change during the course of the
> evaluation of a water contamination threat/incident. For example,
> during the initial phases of the evaluation, the water utility
> emergency response manager may set a high priority on identifying any
> unexpected substances in the water, but only above certain
> concentrations (e.g., normal background levels). Later on, as critical
> decisions may need to be made, management may be more concerned about
> analytical confirmation of identification and accurate quantification.
> Planning and table-top exercises may help the water utility and
> laboratory decide on what report content is best for them,
> particularly at various stages of the evaluation.

2.  During a suspected contamination incident, it is important that all
    relevant information be managed through the incident command. This
    is especially true for analytical results that could be subject to
    misinterpretation if not placed into the context of the situation.
    Thus, **analytical results should be reported only to those
    individuals designated by incident command**, and it will be their
    responsibility to inform other stakeholders. The water utility
    emergency response manager (or the designated incident commander)
    and laboratory contact should be aware of the various applicable
    Federal, State and local legal requirements, especially for
    reporting *infectious* diseases. If they are not, the laboratory
    should inform incident command of these requirements when reporting
    the results to incident command, provided such reporting is
    permitted by regulatory requirements.

3.  In a crisis situation, the laboratory may be asked to provide
    tentative results (sometimes called a "rolling report") prior to
    complete review and confirmation, especially in cases where more
    than a couple of hours is required for confirmation. **The
    laboratory may need to provide appropriate caveats regarding the
    validity of the data at this stage of the analysis.** Depending on
    the analytical methods used, such caveats, perhaps in the form of a
    clearly presented narrative summary, might include: methods and
    techniques used, the probability of false negative/false positive
    results, limit of detection, method accuracy/precision, quantitative
    versus qualitative results, and the time necessary to confirm
    tentative results. It is the responsibility of incident command to
    weigh all of this information and make decisions about appropriate
    response actions.

4.  The laboratory should be available to assist in the analysis and
    interpretation of the results. Not only will the laboratory staff
    have a unique perspective regarding the reliability of the method
    and interpretation of results, but they also may have substantial
    experience with the application of the methodology to other samples.
    Thus, the analyst may have the ability to discriminate between
    results that fall within the normal range of occurrence and those
    likely to be indicative of an actual contamination incident. As an
    example, chloropicrin and cyanogen chloride are potentially
    hazardous chemicals at high concentrations in water. However, these
    same compounds can occur at very low levels in disinfected drinking
    water as disinfection by-products, resulting from the reaction of
    the disinfectant with materials naturally present in the water. An
    analyst with experience determining these compounds would likely
    recognize elevated levels outside of the range typically encountered
    in treated drinking waters. **The issue of baseline (background)
    levels is critical to the interpretation of analytical results and
    thus to the threat evaluation (Module 2)**. A more difficult
    interpretation of the results would occur if low levels of such
    "naturally occurring yet potentially hazardous contaminants" are
    detected, since these levels could represent typical background
    **or** the tail of a transient contaminant slug, in which case
    additional sampling and analysis may be necessary.

## Quality Assurance and Quality Control

Standardized methods available in fixed laboratories are designed and
verified to provide accurate and defensible results. However, methods
for analysis are only as good as the people who perform them, assuming
suitable analytical technology is at their disposal. It is common
experience that some individuals obtain better results from analytical
methods compared to others due to the skill level of the analyst, and
this may be particularly true during the analysis of an unidentified
contaminant using *non-standardized methods*, as discussed in Section 6.
To address these types of concerns, EPA has developed extensive
guidelines regarding the quality of data generated internally and for
non-EPA organizations. For more details, see
<http://www.epa.gov/quality1/exmural.html#genreqts>[.]{.underline}

There are currently specific QA plans/guidance being developed for
forensic analysis of unknowns for both biological and chemical samples.
This QA guidance may be included in a future version of this module.
Many laboratories may already have quality assurance plans that meet
these requirements. Until (and after) this QA guidance is formalized,
laboratories should be aware of legal issues involving admissibility of
scientific evidence, as discussed in the following section.

One consideration with respect to QA/QC for analytical methods used
during the evaluation of suspected water contamination incidents is the
objective of the analysis. For instance, one goal may be to
qualitatively confirm the presence of a particular contaminant. Another
goal would be to quantitatively determine the concentration of a
contaminant. Selecting the objectives of a particular analysis is part
of planning for a site-specific response to a contamination threat. In
many cases, the threat evaluation process discussed in Module 2 will
drive the purpose of the analysis toward quantifying chemicals at
concentrations from milligrams per liter to low micrograms per liter of
chemicals, and pathogens at their infective doses. The analytical
approach presented in Sections 6 and 8 reflects this goal. However,
depending on the specific plan, other goals may be applicable.

## Admissibility of Scientific Evidence

If criminal or terrorist activity is the suspected cause of a
contamination incident, any samples collected and analyses performed may
be considered evidence by law enforcement agencies. Many environmental
chemistry laboratories are currently performing water analyses to
support regulatory compliance, and they are familiar with the need to
treat samples with precautions necessary for legal defensibility.
Analysis for regulatory compliance purposes, however, may operate
somewhat differently than analysis of emergency samples in response to a
contamination threat, which has the dual role of both providing an
emergency response to protect public health and also supporting a
criminal investigation.

The objective of the emergency response is to provide the water utility
emergency response manager (or the designated incident commander) with
timely and accurate scientific results (Module 1). Supporting a criminal
investigation may involve additional time and effort. Improper decisions
made during emergency response may also result in public outcry and/or
legal action, whether criminal or civil, so the quality of data required
for a response action may rival that of the criminal investigation.

Meeting these dual goals requires careful planning and analysis of the
public health needs and legal requirements in place for a specific
location. Nevertheless, utility planners should be aware that most of
the groundwork is already in place at laboratories, and may be readily
built upon through appropriate selection of the analytical approach, as
well as training exercises. Some of this groundwork is described below.

To maintain the credibility of evidence throughout the sampling and
analysis process, the laboratory must demonstrate an unbroken *chain of
custody* and ensure that basic principles of admissibility of scientific
evidence are met. Chain of custody begins with sample collection and
shipment to the laboratory. Sample custody typically becomes the
responsibility of the laboratory upon sample receipt. Documentation
should clearly track the sample and analytical results from the point of
sample receipt through reporting of results. Furthermore, when
designated laboratory staff are not in possession of the sample or
related information, it should be secured. If there are gaps in the
chain of custody or periods during which the sample or results could
have been compromised, the analytical results could be dismissed by the
court. In cases in which the analysis leads to prosecution of a
perpetrator, chain of custody should continue until law enforcement has
determined that any remaining sample is not needed as evidence.

Certain principles govern the admissibility of scientific evidence in a
court of law, and laboratories processing samples considered as evidence
should adhere to these principles to improve the legal defensibility of
their results. There are no universally accepted criteria for the
admissibility of scientific evidence, although State and Federal courts
typically use one of three standards: Federal (or State) Rules of
Evidence, the Frye standard, or the Daubert standard. A more detailed
discussion of the admissibility of scientific evidence can be found in
the *Reference Manual on Scientific Evidence, 2^nd^ ed.*, which can be
downloaded from the Federal Justice Center's web site at
<http://www.fjc.gov/public/pdf.nsf/lookup/sciman00.pdf/$file/sciman00.pdf>.

Note that in the case of scientific evidence, states may develop their
own criteria, other than Frye or Daubert, which are more stringent than
the relevancy issue considered in the rule of evidence.

In considering the principles that govern the admissibility of
scientific evidence in the context of the analysis of water samples for
known or unknown contaminants, it is most useful to consider the most
stringent standard -- Daubert. It is clear that a laboratory would play
a crucial role in the selection and implementation of an analytical
approach that would result in data that would pass the Daubert tests.
There are currently efforts underway in the forensic community to
prepare guidance for proper implementation, particularly as it refers to
the validity of the analytical results. While this guidance is not
available at this time, it may be included in future versions of this
module. In the meantime, a few example areas that should be addressed
that would aid in meeting the Daubert standard include:

-   Quantification of Type I (false positive) and Type II (false
    negative) error rates through appropriate quality assurance and
    quality control (QA/QC).

-   Use of standardized methods, or methods that have been subject to
    rigorous peer review if standardized methods are unavailable.

-   Participation in a certification or accreditation program for the
    relevant methodologies.

-   Participation in interlaboratory studies (or third-party performance
    evaluation studies).

-   Supporting documentation for methods, SOPs, QA plans, etc.

-   Training and competency testing for analysts.

-   Special training for scientists and analysts required to give expert
    testimony.

# Site Characterization and Sampling

While this module focuses on the analytical approach, laboratory
personnel need to have a basic understanding of the processes
implemented prior to transfer of sample custody to the analytical
laboratory. This understanding may be of value to the laboratory for the
purpose of developing procedures for sample receipt that will ensure the
safety of laboratory personnel, a meaningful analytical approach, the
timeliness and quality of the analysis, and the legal defensibility of
the results.

The processes leading up to sample analysis, namely site
characterization and sampling, are covered in detail in Module 3, "Site
Characterization and Sampling Guide," but it is worthwhile to briefly
discuss a few points here. Readers are encouraged to refer to Module 3
for more complete explanations and extensive description of site
characterization activities, including a general discussion of **who**
will perform the activities under various hazard conditions. Figure 4-3
illustrates the steps leading up to sample shipment. The glossary
provides definitions of terms used.

![](media/image4.wmf)

**Figure 4-3: Site Characterization and Sampling Process**

## Site Characterization

The left side of Figure 4-3 shows the *threat evaluation* process
conducted in parallel with the site characterization and sampling
procedures. Throughout the entire evaluation of the incident, the
credibility of the threat is continually reevaluated as new information
becomes available. If at any point the threat is determined to be not
credible, the process is discontinued. Thus, samples might only be sent
to the laboratory if the threat is still deemed credible following
sample collection, as indicated in Figure 4-3. The threat evaluation
protocol is discussed in more detail in Module 2.

The site characterization process begins with an evaluation of the site
hazards. Based on the available data and initial threat evaluation, a
determination will be made regarding the need to implement special
hazardous material handling techniques. This may dictate the
organization or team that is sent to the site to perform field screening
and sampling. While it may be necessary to consider the details of a
specific threat/incident when determining the level of protection
required for the response team, planning for a response to various
scenarios should occur well in advance of an actual threat.
Specifically, response plans should document who would be called to
respond to contamination threats under different hazard conditions.

Upon site entry, *field safety screening* protocols may be implemented.
The purpose of field safety screening is to detect any immediate threats
to the response team from contaminants in the atmosphere or on surfaces.
Core safety screening focuses on threats from radiation, but the safety
screening may be expanded to include volatile chemicals, chemical
weapons, and biological weapons as appropriate for the situation. If any
of these threats are detected, it would likely be necessary to
immediately evacuate the site and send in teams properly equipped to
deal with the hazard tentatively identified during the safety screening.
The site characterization team should have training in the uses of
safety screening equipment (see section 4.2.2 of Module 3) and be
familiar with its capabilities and limitations.

After the field safety screen has been completed, the appropriate team
may continue the site characterization, including *rapid field testing*
of the water. Recommended *core field testing* consists of radiation
monitoring (in the water), cyanide, chlorine residual, and pH. Section
4.3.2 of Module 3 discusses options for *expanded field testing*. Note
that these core tests are based on historically reliable or verified
technology. **Few of these technologies for rapid field water testing
beyond the core tests, however, have undergone a thorough and
independent performance evaluation. Without reliable performance data,
any field testing technology for chemicals, pathogens and/or
radionuclides should be used with caution. The same is true for
technology for safety screening, particularly for biological
contaminants.** This document is somewhat forward looking, and it
assumes that some of the existing kits will provide some useful data;
however, these kits have not yet been verified. Verification of several
of these technologies is being carried out through EPA's Environmental
Technology Verification program in 2003, and the verification reports
can be found, when available, at <http://www.epa.gov/etv/>.

There are three objectives of field testing the water: 1) provide
additional information to assess the credibility of the incident; 2)
provide *tentative identification* of contaminants that would need to be
*analytically confirmed* in the laboratory; and 3) determine if hazards
tentatively identified in the water require special precautions during
sampling. Note that these goals are distinct from the analytical testing
that occurs at the laboratory, even if the same technology is employed
at the laboratory as part of their safety screen upon sample receipt
(Section 3.1.4).

## Sampling

After field testing is completed, samples may be collected using those
precautions appropriate for the hazards identified. A sampling kit that
contains bottles and supplies for the proper collection and
identification of each sample should be available. In Section 4.2.2 of
Module 3, Table 3-1 describes such a sampling kit. Note also that Table
3-1 does not contain any personal protective equipment, except that
required to add the chemicals used to preserve the samples. If any
personal protective equipment is required, it should be included as part
of the health and safety plan (HASP) included in the sample kit (Table
3-1).

In general, samples for chemical analyses may be collected in clean
glass or plastic containers according to sampling procedures appropriate
for the analysis. The specific sample containers suggested for chemical
analytes are listed in Table 3-2 of Module 3, and correspond to the
particular analysis in the analytical approach set forth in Sections 6
and 8 of this module. The table lists the number, type, and volume of
each sample container, as well as any preservatives or dechlorinating
agents appropriate for the sample. Laboratories may find the information
in these tables useful to increase their familiarity with incoming
samples.

## Sample Transport and/or Storage

The process flow chart in Figure 4-3 indicates that samples would
typically be collected after field testing regardless of threat
credibility; however, a decision must be made whether or not to send
samples to the laboratory immediately following sample collection. This
decision should largely be based on the outcome of the threat
evaluation. If the threat is deemed credible, the samples may be sent to
the appropriate laboratory for immediate analysis. Packaging and
transport of the sample to the laboratory is discussed in Section 6 of
Module 3. If the threat is not deemed credible, the process is halted;
however, some analysis may still be performed on the samples at the
discretion of the utility or other parties involved in the response.

If samples are not sent to the laboratory for analysis, they should be
held in cold storage (protected from freezing at 4° C) under safe and
secure conditions for at least one week following collections, and
possibly longer if dictated by other parties that could be involved in
the response (FBI, CDC, EPA, etc.). If the samples are stored, then the
samples must be securely stored and appropriately preserved (Section
4.2). Proper chain-of-custody procedures should be maintained, and
holding times must be within acceptable bounds to meet the analytical
goals for the contaminant of interest.

## Site Characterization Report and Chain of Custody

Chain of custody is initiated at the time of sampling, along with other
sampling documentation. If samples are sent to the laboratory, all
relevant documentation should be submitted at the same time including:

-   Contact information for the sampling organization.

-   Sample documentation, including chain-of-custody forms.

-   Details about any pretreatment performed on the samples in the
    field, such as dilution, preservation, dechlorination, etc., if not
    already included elsewhere.

-   Site characterization report, including site investigation forms,
    field safety screening results, and results from rapid field testing
    conducted on the water.

-   The current stage of the threat evaluation. Note: the laboratory
    should be cautioned **against** reducing handling precautions for
    lower threat evaluation stages without good reason.

-   Instructions on data reporting (i.e., to whom, what format, special
    reporting requirements, etc.).

This information may be of tremendous value in the development of an
analytical approach for a water sample that may be contaminated with a
harmful substance, as discussed in greater detail in Section 5. The
laboratory should be aware that the site characterization report may be
incomplete and/or inaccurate. If the laboratory has any questions,
communication (see section 4.5 below) becomes extremely important. The
laboratory may also wish to verify the preservation and/or
dechlorination of some arriving samples (e.g., through the use of test
strips for free chlorine and pH). Deviations from the preservation
and/or dechlorination described on the sample documentation should be
noted by the laboratory, and the laboratory should adjust their sample
processing accordingly.

In addition, chain-of-custody procedures should be followed carefully to
support future criminal and/or civil legal action regarding the
incident. Chain-of-custody should not stop with sample receipt but
should also include an internal chain-of-custody procedure. Internal
chain-of-custody should be established for all samples regardless of any
questions about chain-of-custody maintained prior to receipt by the
laboratory.

## Communication 

Multiple laboratories may be involved, depending on the nature of the
analytes. **All relevant laboratories should be included in the
communication process.** Communication between the laboratory(ies) and
sampling team should begin no later than the time of sample collection,
especially in the case of a highly credible threat. In some cases, a
laboratory may choose to send personnel to the site to ensure that
proper sampling techniques are followed, assist in the interpretation of
field test results, and facilitate chain of custody. This interaction
may help laboratories identify any potential hazards associated with the
samples and consider site-specific information in developing an
analytical approach for the specific incident. If laboratory staff
cannot be present during sample collection, a conference call might be
scheduled to discuss the information outlined above, as it relates to
laboratory safety and development of an analytical approach, which is
discussed in more detail in the following section.

Communication should be appropriately documented for future reference.
Written notes may be taken, and electronic recording devices may be
used, but only to the extent allowed by privacy laws, particularly for
telephone conversations.

# General Considerations for an Analytical Approach for Unidentified Contaminants in Water

In the case of a water contamination threat, it will likely be necessary
to make rapid decisions, based on limited information, about the
contaminants that will be evaluated by the laboratory. These decisions
may impact the samples that are collected, selection of the laboratory,
and the specific analyses that would ultimately be performed (defined as
the *analytical approach*). In some cases, evidence from the site or
results from field screening may provide some indication about the
identity of the contaminant, and the analytical approach can be adjusted
accordingly. However, it is generally assumed that the presence and
identity of a contaminant in the water sample would need to be
*analytically confirmed* with limited evidence to guide the analysis.
Furthermore, even if a tentative identification of the contaminant has
been made in the field, circumstances may warrant analysis for other
contaminants in addition to those tentatively identified.

A number of officials from various organizations, including the
laboratory, may need to be involved in the development of an analytical
approach **in response to a specific contamination threat**. During the
response to a contamination threat, the laboratory would need to
establish clear lines of communication with incident command, the
affected utility, sampling teams, and any other stakeholders that are
not coordinated directly through incident command. This collective group
of decision officials would need to evaluate available information and
work together to develop an analytical approach that is appropriate for
the circumstances of the specific threat.

The analytical approach may need to be developed rapidly -- in a time
frame of minutes to hours. **Planning, preparation, and communication
are key to making such decisions in a rapid and effective manner.**
After careful planning, laboratories may wish to formalize their
analytical and management approach in a **laboratory guide** that is
analogous to the **response guide** (Module 1) prepared by utilities
that summarizes the actions the utilities plan to take during an
emergency.

This section lays out a framework for developing an analytical approach
in response to a specific contamination threat. The technical aspects of
the analytical approach for chemicals and pathogens are described in
Sections 6 and 8, respectively. Hypothetical examples of specific
approaches developed to meet site-specific objectives are given in
Section 7. While this framework may be used in real-time during a
response, it should also be used for preparation and planning for such a
response. Laboratories and their customers (utilities, States, Federal
agencies, etc.) should work though this process under various test
scenarios to become familiar with the process, and to understand the
capabilities and limitations of a laboratory in implementing the
approaches described in Sections 6 and 8. Laboratories may find they
need to expand their capability in a particular area to provide adequate
coverage for target analytes, or it may become apparent that limitations
may require use of other laboratories for some specialty analyses.

This section is intended not necessarily for laboratories, which may be
more interested in Sections 6 and 8, but for planners and managers, who
may be more interested in the concepts involved. Section 5 is divided
into three subsections. The first subsection presents a decision tool
that may serve as a model for the development of an analytical approach
to a specific contamination threat, based on different levels of
tentative identification. The next subsection, "Initial Assessment of
Available Information" describes potentially useful information from a
specific contamination threat to support the development of an
analytical approach. The final subsection describes the general approach
used for the analysis of unknown contaminants.

## Framework for Development of an Analytical Approach

Once the decision to send samples to a laboratory for analysis has been
made (see Section 4), it will be necessary to develop an analytical
approach that is appropriate for a specific contamination threat or
incident. Figure 4-4 presents a decision tool that is intended to aid in
the development of the analytical approach. Figure 4-4 is geared towards
the analysis of water samples potentially containing an unknown chemical
or biological contaminant that was collected from a site characterized
as having low hazard conditions. It is assumed that radiation screening
has been performed prior to the start of Figure 4-4. In particular, if
the field testing results for radiation were positive, one would go down
an entirely different, and fairly well-defined, analytical path (see
Section 6.4.8).

![](media/image5.wmf)

**Figure 4-4. Decision Process for the Development of an Analytical
Approach for Potentially Contaminated Water Samples**

It is important to emphasize that the decision process depicted in
Figure 4-4 should be used as a planning tool, not just during an
incident. Laboratories, in conjunction with their clients, should work
through this process **prior** to an emergency situation to develop and
refine their own analytical plans and procedures. The decision process
begins with an evaluation of available information about the
contamination threat that might be of value in developing an analytical
approach. The information used to support this initial assessment is
described further in Section 5.2 and in greater detail in Module 2.

The first decision point in the process is an assessment of whether or
not there is sufficient information to make a tentative identification
of the contaminant as chemical and/or biological. If this is possible,
then an entire class of contaminants is eliminated from consideration,
allowing focus on the tentatively identified contaminant class. If the
information is not sufficient to make a determination between chemical
and biological contaminants, then the sample may be treated as a
complete unknown. The recommended analytical approach for a complete
unknown is described in Section 5.3.

The second decision point involves tentative identification of the
specific contaminant. At this point, the contaminant identity is
hypothesized based on available information from the site
characterization report. Examples of situations in which tentative
identification might occur include: a specific contaminant named in a
threat; tentatively positive results for a specific contaminant during
field safety screening or rapid field testing; physical evidence at the
site pointing to a specific contaminant; and clinical evidence of the
identity of the disease causing contaminant. It is important to note,
however, that each of these situations has a different level of
credibility for the purpose of tentative identification. In general,
tentative identification may focus the analytical approach on the
specific contaminant subclass, leading to confirmatory analyses for the
particular contaminant. For example, tentative identification of a class
of pesticides (e.g., organophosphates) may be based on results from a
test kit, and this information might be used to focus the analytical
approach on specific pesticides within that class. Depending on the
strength of the tentative identification, as determined with due
diligence by the water utility emergency response manager, it may be
wise to consider heading down both paths simultaneously (Module 2). If
the evidence for tentative identification is sufficiently strong, then
screening may be delayed. Otherwise, screening for a broad array of
potential contaminants should begin simultaneously with the confirmatory
analysis for the tentatively identified contaminant.

The third decision point is based on the results of the confirmatory
analysis used for the tentatively identified contaminant. If the
presence of the contaminant was analytically confirmed, then the results
are reported to incident command. In the case of potential multiple
contaminants, multiple analyses may be required before incident command
decides if the analysis is complete. (Note: For the purpose of this
document, analytical confirmation refers to the legally defensible
identification of an analyte. There are efforts in the analytical
forensic community to rigorously define what constitutes confirmation,
and results of these efforts may be included in later versions of this
module. In the interim, laboratories should pay careful attention to
principles that govern the admissibility of scientific evidence
\[Section 3.5\].)

The fourth decision point involves the possibility of additional
screening for chemicals or biologicals if the presence of the
tentatively identified contaminant was not confirmed or additional
contaminants are suspected. At this point, communication between the
laboratory and incident command will likely be necessary, because the
decision to perform additional screening may be based on recently
available information regarding the threat. For example, new evidence
may come to light indicating that, despite initial indications, the
threat is no longer credible. However, in case the threat is still
deemed credible, it may be necessary to revise and/or expand the
analytical approach, possibly in conjunction with a fresh review of the
site characterization report.

In any situation involving tentative identification of a contaminant,
the reliability of the information should be carefully evaluated. In
some cases, it may be determined that the source of information is too
unreliable to provide the basis for a tentative identification. For
example, if a specific contaminant is named in a threat made over the
phone, the reliability of the information might be considered suspect.
In this case, the analytical approach might start with the named
contaminant, but additional screening might be warranted depending on
other considerations such as the credibility of the incident. For this
reason, development of the analytical approach may be iterative, as
indicated by the decision point "Is additional screening necessary?" If
the answer to this question is "yes," available information would be
reevaluated to refine or broaden the analytical approach.

## Initial Assessment of Available Information

While previous water contamination threats may indicate that the most
likely scenario is an unknown contaminant, it is possible that there may
be some information from the incident that may inform the development of
the analytical approach. For example, negative results from reliable
field tests may eliminate some contaminant classes from further
consideration. Likewise, information from the site characterization
report may be useful in tailoring the screening to the specific
situation. Even if the available information is insufficient to make a
tentative identification at the grossest level (i.e., chemical or
microbiological), it still may be of value during application of the
screening.

The first step of the process shown in Figure 4-4 is an evaluation of
available information from the current threat that might provide clues
about the identity of the suspected contaminant. Information that should
be considered in developing an analytical approach for a specific
contamination threat includes the following:

-   **Public health information** may be available if exposed
    individuals seek medical attention, and the clinical data for these
    patients may provide insight regarding the identity of the
    contaminant. Such information may be used to make a tentative
    identification of the contaminant or contaminant class, and in some
    cases might provide definitive identification. It is important to
    note that even though exposure has occurred, it does not necessarily
    imply that drinking water is the source of the contamination; other
    routes of exposure, such as food, air, or surfaces might be
    considered depending on the situation.

-   **Tentatively positive results** from field testing may be used to
    tentatively identify a specific contaminant or contaminant class.
    Negative results may be used to exclude certain contaminants from
    further consideration. The credibility of the field test results
    **must** be evaluated in the context of the reliability of the field
    test equipment and procedures.

-   **Physical evidence** at the site may provide insight regarding the
    identity of the contaminant. For example, empty containers might
    have markings indicating the nature of the contaminant, and analysis
    of the residual material in the container may confirm the identity
    of the substance. Other physical evidence from the site, such as
    dead animals or plants, might be used to formulate a hypothesis
    about the identity of the substance, and thus inform the analytical
    approach.

-   **Information about a specific contaminant** from a database or fact
    sheets may be used to make a tentative identification of the suspect
    contaminant. For example, field screening results and/or physical
    evidence might be used in conjunction with detailed information
    about potential contaminants to make a tentative identification of
    the contaminant or contaminant class.

-   **Location-specific contamination threats** should also be
    considered during development of an analytical approach for a
    specific contamination threat. For example, utilities may have
    identified potential contaminant sources (e.g., chemical warehouses)
    during their vulnerability assessment, and such opportunity
    contaminants might be explicitly included in the analytical
    approach.

-   **Background concentrations of the specific contaminant at the
    site** may be extremely important in determining if a contamination
    incident has occurred. In some cases and for some contaminants,
    background levels may be at detectable concentrations. **[If
    unrecognized, background concentration of a contaminant may be
    confused with an actual contamination incident.]{.underline}**

-   **Threat credibility** may provide an indication of the severity of
    the incident, and thus may indirectly impact the analytical
    approach. For example, if a threat is deemed highly credible, then
    complete screening for both chemical and biological contaminants
    might serve as the analytical approach regardless of other
    information collected from the site.

-   **Consequences** of failing to properly identify an unknown
    contaminant (or not confirming the absence of harmful contaminants)
    should also be considered in developing an analytical approach. The
    higher the potential consequences of a misdiagnosis of the incident,
    the more rigorous the analytical approach should be.

## Overview of Analytical Approach For Unknowns

In the case of a complete unknown, the problem of confirming the
presence/absence of a contaminant or identifying and quantifying a
specific contaminant presents a significant challenge. The difficulty
arises from the large number of potential contaminants of concern, and
the impracticality of screening for them all. To address this issue, the
analytical approach for unknowns was based on contaminant classes
derived from a prioritization of chemicals and pathogens of public
health concern if present in the drinking water supply. The approach
used to prioritize potential contaminants is briefly discussed in
Section 2.1 of Module 1.

The analytical approach for unknown contaminants in water presented in
this module is comprehensive for selected, *priority contaminants* and
provides coverage for hundreds of additional contaminants not on this
list. The following assumptions and principles were used in the
development of these approaches:

-   Selection of target analytes was based on an assessment of
    contaminants likely to pose a threat to **public health** if
    introduced into the drinking water supply.

-   Existing laboratory infrastructure and analytical methodologies were
    utilized when possible.

-   Both chemical and microbiological procedures are tiered, with a
    progression through field safety screening and rapid field testing
    to laboratory testing to confirmatory analysis.

-   Samples analyzed in a laboratory that cannot be confirmed by the
    laboratory performing the test are referred to laboratories that can
    perform a confirmatory analysis. This upward referral system is
    formalized for microbiological analysis, through the Laboratory
    Response Network, while it is not as well defined for chemical
    analysis.

-   The entire approach relies on the systematic elimination of
    potential contaminants, both to ensure the safety of sampling and
    laboratory personnel, and to aid in identification of the unknown
    contaminant.

Considering the principles and assumptions upon which the comprehensive
analytical approach is founded, it is likely that the approach may
evolve as new methodologies are developed and refined. Furthermore,
laboratories with advanced capabilities may wish to implement
alternative procedures that either expand coverage or streamline the
analysis. Such modifications would be acceptable assuming that they
provide at least equivalent coverage of target analytes compared to the
standard approach and meet the data quality objectives of the analysis,
such as legal defensibility of data. The comprehensive analytical
approach in this module should serve as a baseline against which any
alternate approach should be compared.

It is also important to realize that identification of unknown
contaminants in water samples is not an exact science. There should be
no expectation than any combination of technology and analytical
personnel will guarantee successful identification of unknown
contaminants. However, application of appropriate technologies and
training of laboratory personnel will likely increase the probability of
success. The successful identification of an unknown is dependent upon
the skill of the laboratory personnel and the laboratory having
appropriate analytical technology. With the many types of analytical
techniques and methods in existence, it is likely that no one analyst,
no matter how skilled, can use all these techniques and methods. A more
thorough identification of unknown contaminants may involve a team of
chemists and microbiologists to provide consultation and/or perform
various analyses.

Since the procedures and laboratories used for the analysis of chemical
and microbiological contaminants are significantly different, discrete
analytical screening procedures have been developed for each. The
screening for chemical contaminants is presented in Section 6, while the
screening for microbiological contaminants is presented in Section 8.
These procedures are briefly discussed in the following subsections.

Not only may different laboratories be involved in the analysis of
chemical and microbiological contaminants, but some select analyses or
extreme hazards should be handled only by a limited number of
specialized laboratories. Since different laboratories may be involved
in implementation of the analytical approach for a specific water
contamination threat, communication and coordination among the various
laboratory personnel involved in the analysis of an unknown sample is
critical. For example, other laboratories should be made aware of
potentially harmful contaminants revealed by other laboratories. This
may be vital to the safety of the laboratory personnel as well as the
expeditious identification of unknowns.

### Screening for Chemicals, Including "Unknowns"

Screening for chemicals, including "unknowns," is described in detail in
Section 6. Because the target audience for Section 6 is laboratories,
the following overview is presented for the benefit of planners and
managers. In summary, the chemical screen integrates several analytical
techniques to cover a broad range of chemical classes. These analytical
techniques include not only wet chemistry and instrumental analysis,
with which laboratories are typically familiar, but also various
hand-held equipment and commercially available test kits, such as those
based on immunoassays.

The overall screen is broken up into two parts, one referred to as a
*basic screen* and the other as an *expanded screen*. The suite of
techniques utilized in the expanded chemical screen is comprehensive for
all prioritized contaminants of concern discussed in Section 6.1.1.
Furthermore, the screen may be capable of detecting hundreds of
additional chemicals that were not identified as high priority, but
could still pose some problem if used in an intentional contamination
threat or incident. It is important to note that the screens are not
prescriptive and labs have a great deal of flexibility in building an
analytical approach that is consistent with their existing capabilities
and experience while meeting the needs of their clients. However, it is
strongly recommended that screening for chemicals encompass a wide range
of possible contaminants given the large number of chemicals potentially
available. See Section 6.1 for more details.

The *basic screen* utilizes *established analytical techniques* in
conjunction with legally defensible, *standardized methods* for the
analysis of contaminants in water. However, these established techniques
do not provide complete coverage for all target analytes. For instance,
currently there are no standardized methods for analysis of the
biotoxins of concern in water. To address these gaps, exploratory
techniques, which do not have standardized methods, are used in the
*expanded screen*. For instance, immunoassays or liquid
chromatography-mass spectrometry may be incorporated into the expanded
screen to cover biotoxins. Of the many analytical techniques available,
the ones utilized in the expanded screen are those that may show the
most promise for water analysis. Particularly, those that have
established applications in media other than water provide a basis for
inclusion in the expanded screen.

In the screening procedure, analysis of contaminants is divided into
chemical classes, such as organic, inorganic, and radionuclides:

-   Organic analyses utilized in this approach are comprised of some
    combination of the following three steps: 1) extraction or recovery
    of the contaminant from the aqueous matrix, 2) separation of the
    compounds through gas chromatography or liquid chromatography;
    and/or 3) detection and identification of the analyte. Preparatory
    and extraction techniques for organic constituents should be broad
    enough to recover a variety of compound classes (e.g., a range of
    hydrophilic properties and molecular weights). A variety of
    techniques are used for detection of organic constituents. When mass
    spectrometry is used for detection, qualitative identification may
    be realized through electron ionization mass spectral library
    comparisons.

-   The inorganic analyses include several analytical techniques:
    classical wet chemistry and instrumental techniques such as
    inductively coupled plasma mass spectrometry, inductively coupled
    atomic emission spectrometry, atomic absorption spectrometry for
    trace metals, and ion chromatography for anionic and cationic
    contaminants.

-   The analysis of radionuclides during an emergency incident relies on
    conventional radiological techniques, but falls into a separate
    paradigm than other chemicals because radionuclides may be
    characterized by both the type of radioactivity they emit as well as
    specific radioisotopes.

Figure 4-4 illustrates an important issue in the screening for unknown
chemicals, namely that confirmatory analysis may be required in the case
of a tentatively identified chemical. In general, a positive result from
a rapid field test or safety screening (performed in the field or
laboratory) would be considered tentative identification and require
independent confirmation. By contrast, chemicals identified through the
application of standardized methods typically do not require independent
analytical confirmation because recommended confirmatory steps are often
incorporated into the methods themselves. In some cases, another
laboratory with specialized capability may need to perform the
confirmatory analysis. For instance, confirmatory analyses for chemical
weapons would be performed only in few established laboratories designed
and permitted to work with these substances.

When possible, confirmatory analyses should be performed using existing
standardized methods accepted for analysis of the target analyte in a
water matrix. When a standardized method is unavailable, confirmatory
analysis may need to be performed through application of methods that
rely upon different separation and/or detection techniques as a means of
independently verifying the identity of a chemical contaminant.

### Screening for Microbiologicals, Including "Unknowns"

The microbiological screen is designed to recover several classes of
organisms, including parasites, bacteria, and viruses. Furthermore, the
approach may recover some high molecular weight biotoxins, such as
botulinum toxins. The four basic steps of the microbiological screen
include: 1) concentration and recovery in the field; 2) rapid field
testing; 3) testing at a Sentinel laboratory; and, if needed, 4) testing
at a Reference laboratory.

To obtain the detection limits necessary to provide results from
microbiological analysis that are meaningful from a public health
perspective, it is often necessary to concentrate the water sample by
several orders of magnitude (e.g., 100 liters concentrated to 100
milliliters). In the microbiological screen (Section 8), concentration
is performed by *ultrafiltration*, a physical separation process capable
of complete retention of parasites, bacteria, viruses, and potentially
some large molecular weight biotoxins. Ultrafiltration is capable of
processing large volumes of finished water in a short period of time,
while continuously concentrating pathogens and other suspended solids in
the retentate. The accumulated material is then removed from the
membrane through mechanical or chemical means and collected as a
retentate. This low volume retentate may be then subjected to
microbiological analysis, so it should be handled with appropriate
caution to ensure the safety of the sampler and the viability of any
organisms collected.

The rationale for performing this concentration step in the field as an
integral part of sample collection is based on two considerations.
First, containers approved for the shipment of large volumes of liquid
suspected of containing select pathogens do not exist; thus there may be
no effective means of quickly transferring a large water sample to
another facility for processing. Second, concentration of
microbiological contaminants in the field allows for the useful
application of field screening devices (e.g., pathogen field test kits),
the next step of the microbiological screen.

After concentration, the concentrated sample is divided into three
aliquots. Two of these aliquots are packaged for shipment to the
laboratory, while the third may be used for field testing. The method
for field testing will depend on the technology utilized, but in the
case of pathogen field test kits, a small volume of the concentrate may
be directly applied to the pathogen field test kit, followed by a short
reaction period and evaluation of the test result. A positive result
should be considered tentative, and the information used accordingly in
the context of the overall threat evaluation.

The remaining two aliquots are sent to a laboratory, such as an LRN
Sentinel Lab, capable of performing tests for tentative identification
of target pathogens. One aliquot may be used to perform culture
techniques for the target pathogens. If the initial results of the
culture method are analyzed, and colonies of a particular morphology are
considered tentatively positive, they are forwarded to an LRN Reference
laboratory. The second aliquot may be used for PCR and subsequent
molecular analysis for target pathogens, which will provide more rapid,
yet still tentative, results compared to the culture technique. However,
molecular assays do not necessarily produce specimens that can be
forwarded on to LRN Reference laboratories for confirmatory analysis. It
is important to note that LRN PCR techniques for select agents may not
be performed by Sentinel labs, although Sentinel labs can perform
certain non-LRN PCR techniques.

Upon tentative identification of a target pathogen, the suspect cultures
are isolated and processed for referral to higher level LRN laboratories
capable of confirming the presence of select pathogens through a
combination of culture and PCR techniques. Once the sample enters this
level of the LRN, the existing network, and methodologies will be
utilized to further characterize the pathogen.

# Analytical Approach for Chemical Contaminants

## Overview of Contaminant Issues

### Types of Chemical Contaminants Covered by the Analytical Approach

Module 1 of this Toolbox provides an overview of the nature of
intentional contamination threats that drinking water facilities might
face. Water from the drinking water distribution systems is considered
the most significant vulnerability, as opposed to raw source water or
wastewater. The focus of the analytical approach in this section is on
approximately 35 **specific** contaminants, selected from a list of
chemicals, biotoxins, and radionuclides, prioritized based on the
contaminant's potential threat to public health from a national
perspective (see Module 1). **General** water quality parameters from
distribution systems (pH, turbidity, chlorine residual, total organic
carbon, etc.) are discussed in terms of site characterization (Module 3,
Table 3-2). These general water quality parameters are potential
indicators of chemical contamination at best, and may not cover all of
the contaminants of concern.

The approximately 35 specific contaminants are not necessarily listed in
this module or in Table 1-1 of Module 1. There are several reasons for
this. First, it may not be advisable to specifically list these, since
such information could be used with malicious intent. Second, a
particular contaminant may be very important in a particular locality,
but not have been included on the national priority list. Third, as part
of fulfilling the goal of due diligence in providing a comprehensive
screen (Section 1.2.2), it is necessary to include other chemicals in
the analytical approach that encompass a broad range of effects, not
just those that may have the highest public health impact.

The approach taken in this module is to divide potential contaminants
into classes based on their analytical grouping (i.e., how contaminants
with similar properties are analytically determined). This helps to
limit the number of methods to those that encompass the largest number
of analytes. The chemical types, resulting analytical groups, and
examples of specific contaminants, are shown in Table 4-1. Suitable
analytical methods are discussed further in Sections 6.4 and 6.5.

[]{.mark}

Table 4-1. Summary of Types of Chemical Contaminants, their Analytical
Group, and the Class of Contaminants Determined by the Analysis

  --------------- -------------------- ---------------------------------------
  **Chemical      **Analytical Group** **Example Contaminants\***
  types**                              

  Organic         Volatiles            Acetone, acrylonitrile, chloroform,
                                       methyl *t-*butyl ether,
                                       tetrachloroethene, toluene,

                  Semivolatiles        Organophosphates (e.g., malathion,
                                       mevinphos, dichlorvos, etc.),
                                       cyanazine, chlorinated insecticides,
                                       chlordane, pentachlorophenol

                  Non-volatiles        Sodium trifluroacetates, surfactants

                  Carbamate compounds  Aldicarb, carbofuran, oxamyl

                  Quaternary nitrogen  Diquat, paraquat
                  compounds            

                  Pharmaceuticals      Nicotine, illicit drugs

  Inorganic       Trace metals         Mercury, lead, cobalt

                  Nonmetals            Arsenic salts

                  Organometallics      Organomercury compounds

  Cyanides        Cyanides             Cyanide salts, cyanogen chloride

  Radionuclides   Radiologicals        Cesium-137, Cobalt-60, Strontium-92

  CW Agents       Schedule 1 only\*\*  (e.g., VX, sarin, nitrogen and sulfur
                                       mustards, Lewsites)
  --------------- -------------------- ---------------------------------------

\* Not every contaminant in a particular analytical group is listed in
this column.

\*\*See Section 2.1.4 for a discussion of Schedule 1 agents.

### Selection of Appropriate Methods for Contaminant Analysis

As described in Section 5 of this module, there are two general
approaches to the chemical screen. One approach is referred to as the
"basic" screen, while the other is referred to as the "expanded" screen.
Together, they are a comprehensive screen for the 35 priority
contaminants and also provide coverage for hundreds of other potential
water contaminants, although it is not feasible (or necessary) to
determine all of the hundreds of thousands of chemicals in existence.
Fewer laboratories may be capable of implementing the expanded screen
(Section 6.5).

It must be emphasized that this module along with Sections 6 and 8 are
**not** intended to represent a prescriptive "how-to" laboratory manual.
Rather, the model screening procedure (Sections 6.3-6.5, 8.1) is
intended to be a planning tool for laboratories to formulate a
laboratory guide specific to their needs and capabilities. For instance,
the basic screen presented in Section 6.4 is composed of relatively
reliable and accessible techniques, which hopefully will encourage more
laboratories to develop some screening capability for potential harmful
chemical contaminants (i.e., beyond regulatory requirements). However,
planners may not wish to employ any elements of the basic screen, but
rather rely only on techniques used in the expanded screen (Section
6.5), backed up by suitable analytical confirmation to ensure
defensibility of the results. Planners must exercise due diligence in
planning their analytical approach, as well as all other response
activities, to protect public health and safeguard the water supply.

For the selection of appropriate methods to include in the screen, it is
important to realize that planning is a vital part of site-specific
response to a contamination incident. In many cases, planning for the
site characterization (Module 3) will drive the selection of methods. In
some cases, it may be desired only to identify the contaminant, but
accurate quantification may be necessary in others. Depending on the
specific plan, various goals and analytical methods may be applicable.

An intentional contamination incident may produce contaminant
concentrations ranging from extremely high (milligrams per liter), when
much contaminant is added to a relatively small amount of water, to very
low (low to mid microgram per liter), as in the tail of a transient
contaminant slug in a drinking water distribution pipe. Accordingly, the
analytical methods described in this approach were selected to be
conservative and reflect the goal of defensibly determining lower
concentrations of chemicals. This great variability in analytical needs
that may arise in each threat/incident is another reason that this
section is not a prescriptive "how-to" manual.

### Initial Processing of Contaminants prior to Laboratory Analysis 

Regardless of the type of screening that the laboratory uses, it is
likely that laboratories will receive samples that have been
characterized to some extent, and perhaps tentatively identified, at the
site. Figure 4-5 indicates the types of laboratories that might analyze
emergency water samples. The site hazard assessment performed during
site characterization will help the water utility emergency response
manager (or the designated incident commander) decide which laboratories
to use for a particular sample set. If a chemical contaminant has been
tentatively identified in the field, it may aid in the selection of an
appropriate laboratory. It will be necessary to then apply specific
analytical techniques to confirm its identity and/or quantify the
concentration. Otherwise, the sample may be subjected to a screening
procedure, which is described in more detail below. Note that the
screening procedure box does not explicitly list radionuclides, since
radionuclide analysis involves a different paradigm, as discussed in
Section 6.4.8, below. Also, some radionuclide screening is performed
upon receipt by the laboratory as discussed in Section 3, in addition to
the field screening in Section 4.

[]{.mark}

![](media/image6.wmf)

**Figure 4-5. Initial Analytical Approach to Processing Samples with
Chemical Contaminants**

*\*Specialty labs include the LRN and those only accessible through law
enforcement (e.g., CW labs).*

At the starting point for Figure 4-5, the contaminant identity is
hypothesized based on available information from the site
characterization report. Examples of situations in which tentative
identification might occur include: a specific contaminant named in a
threat; tentatively positive results for a specific contaminant during
field safety screening or rapid field testing; physical evidence at the
site pointing to a specific contaminant; and clinical evidence of the
identity of the disease-causing contaminant.

It is important to note, however, that each of these situations has a
different level of credibility for the purpose of tentative
identification (Module 2) and that additional contaminants may be
present. In general, tentative identification may focus the analytical
approach on the specific contaminant subclass, leading to confirmatory
analysis for the particular contaminant. For example, tentative
identification of a class of pesticides (e.g., organophosphates) may be
based on results from a test kit, and this information might be used to
focus the analytical approach on specific pesticides within that class.

In most cases, it is anticipated that a specific chemical contaminant
will not be identified with sufficient reliability to be considered
tentatively identified, and it will be necessary to conduct screening
for potential chemical contaminants. Depending on the strength of the
tentative identification, it may be wise to consider heading down both
paths of Figure 4-5 simultaneously. If the evidence for tentative
identification is sufficiently strong, and if there are no other
contaminants suspected, then screening may be delayed. Otherwise,
screening should begin simultaneously with the confirmatory analysis, as
described in the Section below. At the other extreme, during a
'confirmed' incident, the contaminant will likely be known, and the
point of analysis in this case is to further characterize the
contaminated area to support the planning stage of remediation and
recovery.

## Confirmatory analysis

It is necessary to confirm the identity and/or concentration of a
contaminant identified either in the field or in the laboratory. In the
case of a tentatively identified chemical contaminant, Figure 4-5
indicates that one of several types of distinct, specialized
laboratories might be involved in conducting confirmatory analysis. The
functions of these laboratories are described in more detail in Section
2.1 as part of the infrastructure discussion. Some confirmatory analysis
may only take place in specialty laboratories, such as those for
Schedule 1 CW agents. A discussion of these techniques and the
associated equipment is beyond the scope of this document since these
laboratories presumably have established procedures for these specialty
analytes, and an overview of these techniques would not be particularly
helpful to the target audience for this document. In addition, access to
these laboratories is restricted and must be made by the appropriate
authorities.

For radionuclides, analytical confirmation may be viewed differently
than most chemicals because radionuclide analysis can take the form of
screens for gross alpha, beta, or gamma radiation, and/or it can take
the form of analysis for the specific radionuclides themselves, such as
strontium-90. Thus, analytical confirmation may involve the process of
confirming quantity of gross radiation or identifying the radionuclide.
The type of analysis that is performed will be based on the analytical
goals. For example, radionuclides in drinking water regulation are often
characterized by their gross alpha and gross beta emissions. For the
purpose of emergency analysis of water contaminants, a tiered approach
is suggested, with screening for gross radiation in the field or upon
receipt by the laboratory followed by identification of the
radionuclide, which may be important for a more complete understanding
of risks to public health as well as potential criminal actions against
perpetrators.

On the other hand, many environmental chemistry laboratories can confirm
a significant number of chemical contaminants through the use of a
variety of standardized methods. In the context of this module,
standardized methods are produced as a standard by a recognized method
development organization (EPA, ASTM, AOAC, ISO, etc.) and contain valid
steps to defensibly confirm the presence and/or quantity of specific
contaminants. For the analysis of emergency water samples, a
standardized method need not be promulgated for compliance monitoring of
drinking water samples.

Environmental chemistry laboratories are often familiar with
promulgated, standardized methods and may have the equipment and
expertise to perform confirmatory analysis using such standardized
methods. A number of standardized methods are published by various
organizations, and may be selected from an appropriate method database,
such as the National Environmental Methods Index (NEMI,
[http://www.nemi.gov](http://www.nemi.gov/)). NEMI contains methods
reviewed and selected by the National Methods and Data Comparability
Board (<http://wi.water.usgs.gov/pmethods/>), a partnership of
water-quality experts from Federal agencies, States, Tribes,
municipalities, industry, and private organizations. The Board provides
a framework and forum for comparing, evaluating, and promoting
monitoring approaches for water quality. Methods are available in NEMI
to support the analytical approach for the priority chemical
contaminants, and a laboratory can use the NEMI database to select a
confirmatory method for a particular chemical contaminant based on its
particular expertise and equipment. NEMI is searchable by analyte, and a
search will often return several methods for the same analyte, developed
to meet a variety of needs. For example, some methods are EPA drinking
water methods, some are EPA SW-846 methods, and some were developed by
USGS or DOE for their environmental monitoring programs, etc.

Although consulting NEMI first is recommended, there are other sources
of standardized methods, some of which are shown in Table 4-2. There are
other comparable standardized methods available from standards setting
organizations like ASTM ([http://www.astm.org](http://www.astm.org/)),
AOAC ([http://www.aoac.org](http://www.aoac.org/)), and ISO
(<http://www.iso.org/>). In choosing a method, keep in mind that some of
these standardized methods may be prescribed for a specific intended
purpose, such as drinking water compliance monitoring. However, in the
context of this module, the methods are **not** prescribed, because
there is no regulatory driver for doing so. Rather, all of these methods
resources are to be used as **planning tools** to assist laboratories in
selecting methods for use in implementing the analytical approach
presented in this module **prior** to analyzing actual emergency water
samples. For many laboratories, planning may be simplified through the
use of methods they are already familiar with, such as EPA drinking
water and SW-846 methods.

Table 4-2. Sources of Standardized Methods

+----------------+-------------------+-----------+--------------------+
| **Name**       | **Description**   | **Pu      | **How to obtain**  |
|                |                   | blisher** |                    |
+================+===================+===========+====================+
| National       | Contains methods  | US EPA    | [http:/            |
| Environmental  | compiled from     | and USGS  | /www.nemi.gov](htt |
| Methods Index  | many sources.     |           | p://www.nemi.gov/) |
| (NEMI)         | **May be          |           |                    |
|                | consulted         |           |                    |
|                | first.**          |           |                    |
+----------------+-------------------+-----------+--------------------+
| Recently       | Contains current  | US EPA    | <http://www.epa.   |
| developed US   | revisions of      | Office of | gov/safewater/meth |
| EPA methods    | promulgated       | Water     | ods/sourcalt.html> |
|                | methods for       |           |                    |
|                | drinking water    | US EPA    | <http              |
|                | analysis with     | Office of | ://www.epa.gov/ner |
|                | notes             | Research  | lcwww/ordmeth.htm> |
|                |                   | and       |                    |
|                |                   | De        |                    |
|                |                   | velopment |                    |
|                |                   | (ORD)     |                    |
+----------------+-------------------+-----------+--------------------+
| EPA SW-846     | Mainly for solid  | US EPA    | [[http://w         |
| methods        | waste, but many   | Office of | ww.epa.gov/epaoswe |
|                | are applicable    | Solid     | r/hazwaste/test/ma |
|                | for drinking      | Waste     | in.htm]{.underline |
|                | water, using      |           | }](http://www.epa. |
|                | appropriate       |           | gov/epaoswer/hazwa |
|                | preparation       |           | ste/test/main.htm) |
|                | techniques.       |           |                    |
+----------------+-------------------+-----------+--------------------+
| EPA drinking   | Printed versions  | US EPA    | For ordering       |
| water manuals  | of drinking water | ORD       | details, see       |
|                | methods           |           | <http:/            |
|                |                   |           | /www.epa.gov/nerlc |
|                |                   |           | www/methmans.html> |
+----------------+-------------------+-----------+--------------------+
| 40 CFR Parts   | Promulgated list  |           | <http              |
| 136 and 141    | of defensible     |           | ://www.epa.gov/doc |
|                | methods widely    |           | s/epacfr40/chapt-I |
|                | accepted in the   |           | .info/subch-D.htm> |
|                | analytical        |           |                    |
|                | community for     |           |                    |
|                | water and         |           |                    |
|                | wastewater.       |           |                    |
+----------------+-------------------+-----------+--------------------+
| Standard       | Various methods   | American  | Available from     |
| Methods for    | covering the      | Public    | most booksellers.  |
| the            | range of water    | Health    |                    |
| Examination of | analytes.         | As        | ISBN: 0875532357   |
| Water and      |                   | sociation |                    |
| Wastewater,    |                   | and       |                    |
| 20^th^ edition |                   | American  |                    |
| (January 1999) |                   | Water     |                    |
|                |                   | Works     |                    |
|                |                   | As        |                    |
|                |                   | sociation |                    |
+----------------+-------------------+-----------+--------------------+
| Other          | May provide       | AOAC      | [http:/            |
| recognized     | comparable        |           | /www.astm.org](htt |
| method         | methods           | ASTM      | p://www.astm.org/) |
| development    |                   |           |                    |
| organizations  |                   | ISO       | [http:/            |
|                |                   |           | /www.aoac.org](htt |
|                |                   | others    | p://www.aoac.org/) |
|                |                   |           |                    |
|                |                   |           | <ht                |
|                |                   |           | tp://www.iso.org/> |
+----------------+-------------------+-----------+--------------------+

If standardized methods are not available for the contaminant of
interest, then analytical confirmation may involve the techniques
described below for basic and/or expanded screening (Sections 6.3 and
6.4). In this situation, the requirements for analytical confirmation
are generally not well defined and are further complicated by the issue
of legal defensibility of scientific data. Section 3.5 of this module
provides an overview of rules that govern the admissibility of
scientific evidence into a court of law and points out the pitfalls
associated with using methods that have not been standardized. There are
currently efforts underway in the forensic community to resolve these
issues and develop standard approaches for the analysis (including
confirmation) of samples contaminated with an unknown substance. While
guidance from the forensic community is not available at this time, it
may be included in future versions of this module. Ultimately, it may be
the responsibility of laboratories to take appropriate steps to support
the admissibility of data produced using exploratory techniques that
have not yet been widely accepted in the scientific community (see
Section 3.5).

## Laboratory Analytical Screening for Chemical Contaminants, Including "Unknowns"

The *laboratory analytical screening* described here is **different and
separate** from the laboratory safety screening (Section 3.1.4 of this
Module) and the field safety screening and rapid field testing (briefly
described in Section 4 of this Module and more completely in Module 3).
Briefly, the field safety screening and rapid field testing were
performed during characterization of the contamination site, before any
laboratory work is performed. The entire site characterization process
is designed to reduce risk to the site characterization team and any
individuals coming into contact with samples collected from the site.
Site characterization activities are not limited to only field safety
screening and rapid water testing, which may only capture a few
contaminants, but also include a site investigation that may identify
other signs of hazards. These activities aid in the site hazard
assessment and help ensure that samples are sent to a laboratory
prepared to deal with both the analysis and any safety issues. For
example, if pathogens are suspected, the samples should not be sent to
an environmental chemistry lab.

The overall concept of laboratory analytical screening for an unknown
chemical in a water sample is presented in Figure 4-6. This chemical
screen consists of two core elements: 1) application of multiple
analytical techniques to screen for a wide range of analytes; and 2)
analytical confirmation of tentative results. In the first core element,
the combination of analytical techniques used to achieve broad coverage
of the contaminant classes of concern includes both standardized methods
and exploratory techniques. The standardized methods are derived from
those same sources as listed in Section 6.2. Exploratory techniques are
those that do not necessarily form the basis of standardized methods for
water analysis, but are capable of detecting chemicals that are not
included in existing standardized methods. Both of these elements are
implemented in the laboratory.

Two different chemical screens were constructed from the established and
exploratory techniques. The first, indicated in Figure 4-6 as the "Basic
Screen," (Section 6.4) uses only standardized methods, and the method
analytes include a significant percentage of the priority chemical
contaminants as well as many other chemicals. The second, referred to as
the "Expanded Screen," (Section 6.5) combines established techniques and
exploratory techniques to achieve the broadest coverage, including all
priority chemical contaminants. Additionally, the Expanded Screen
provides laboratories with additional options regarding the
instrumentation used to implement both established and exploratory
techniques; however, the results may not be as definitive as those for
established techniques.

The second core element in Figure 4-6 is analytical confirmation of the
results. As shown in the figure, some methods utilized in the "Basic"
screen contain required steps for defensible confirmation. Some do not,
and other confirmatory analysis should be performed, as described in
Section 6.2. As shown in Figure 4-6, it also would be necessary to
confirm the results of most exploratory techniques, unless the technique
is applied within a methodology that utilizes suitable validation
procedures. The strategy of using the variety of techniques in the
"Expanded Screen" may lend itself to fulfilling the requirements
discussed in Section 6.2 for confirming an analyte, particularly one for
which no standard method exists. For example, analytical confirmation
may sometimes be accomplished through determination of the contaminant
by two or more analytical techniques that operate on independent
principles (e.g., LC/UV and GC/MS). In comparison, analytical techniques
that operate on the same principles (e.g., different immunoassays) may
be unreliable for confirmation purposes and should be used with caution.

![](media/image7.wmf)

**Figure 4-6. Overview of Screening and Confirmatory Analyses for
Chemical Contaminants**

## Basic Screening using Established Techniques with Standardized Methods for Target Analytes 

The basic screen is designed to capture many of the chemical
contaminants of concern using a relatively small number of well-defined
analytical techniques. The techniques chosen for this basic screening
analysis are summarized in Table 4-3. This is to say, if the methods in
Table 4-3 are performed, then the screen may cover a large percentage of
the priority chemical contaminants (Table 4-1). Furthermore, many other
contaminants of concern, but of lower priority, may be screened for as
well. To increase confidence in the results, only standardized methods
(e.g., EPA drinking water, SW-846, or comparable methods) are used for
the basic screen. Comparability involves analytes determined, detection
limits, QA/QC, etc. See Section 6.2 for information about comparable
methods.

As discussed previously, EPA standardized methods are suggested mainly
because many labs may be familiar with some or all of these methods, and
many laboratories are certified and/or accredited to determine water
contaminants using these methods. This increases the defensibility of
the results, since the laboratories would have experience in performing
all of the required QC within acceptable ranges. Also, in most cases,
the use of EPA methods also means that if a method analyte is determined
by these methods, no further analytical confirmation would be necessary
since many of these methods contain confirmatory steps. In choosing a
method, keep in mind that some of these standardized methods may be
prescribed for a specific intended purpose, such as drinking water
compliance monitoring. However, in the context of this module, the
methods are not prescribed, because there is no regulatory driver for
doing so.

**The basic screen may serve as a springboard for more complete
characterization of the sample,** in an "outside-the-box" manner.
Namely, a significant advantage to using the techniques in the Table
4-2, or comparable ones, is that many rely on chromatography and/or mass
spectrometry, so the data should be capable of being evaluated for the
presence of not only target analytes, but also other compounds. If a
contaminant is discovered that is not in the analyte list of the method
used, the identification should be considered tentative until confirmed
(Section 6.2). In the absent of confirmation, expanded screening of the
sample should be applied (Section 6.5).

Information from the basic screen may also be used to inform the
selection of exploratory techniques. For instance, if a target analyte
or unknown peak is not found in a Method 524.2 analysis, the compound is
likely not a volatile organic; if no peak is present in a Method 8270D
analysis, the unknown may be inorganic in nature or thermally unstable.

Table 4-3. Suggested Analytical Techniques for Performing the Basic
Chemical Screen, broken out by the classes given in Table 4-1.

+--------------------+--------------------+--------------+-------------+
| **Chemical         | **Analytical       | **EPA        | **Analyte   |
| (general class)**  | Technique**        | Method^1^**  | List^4^**   |
+--------------------+--------------------+--------------+-------------+
| Volatiles          | Purge-and-trap     | 502.2, 8021B | A           |
| (organic)          | PID/ELCD           |              |             |
|                    |                    | 524.2, 8260B |             |
|                    | Purge-and-trap     |              |             |
|                    | GC/MS              |              |             |
+--------------------+--------------------+--------------+-------------+
| Semivolatiles      | Solid-phase        | 525.2,       | B           |
| (organic, includes | extraction GC/MS   | 8270D/3535A  |             |
| many pesticides)   |                    |              |             |
+--------------------+--------------------+--------------+-------------+
| Carbamate          | HPLC --            | 531.1, 531.2 | C           |
| pesticides         | fluorescence       |              |             |
| (organic)          | detection          |              |             |
+--------------------+--------------------+--------------+-------------+
| Quaternary         | HPLC -- UV         | 549.2        | D           |
| nitrogen compounds |                    |              |             |
| (organic)          |                    |              |             |
+--------------------+--------------------+--------------+-------------+
| Trace metals       | ICP-AES, ICP-MS,   | 200.7,       | E           |
| (inorganic)        |                    | 200.8,^2^    |             |
|                    | graphite furnace   | 200.9        |             |
|                    | AA                 |              |             |
+--------------------+--------------------+--------------+-------------+
| Total mercury      | Cold vapor AA      | 245.1, 245.2 | F           |
| (inorganic,        |                    | with         |             |
| includes           |                    | persulfate   |             |
| organomercury      |                    |              |             |
| compounds)         |                    |              |             |
+--------------------+--------------------+--------------+-------------+
| Cyanides           | Wet chemistry      | 335.2,       | G           |
|                    |                    | 335.3, 335.4 |             |
+--------------------+--------------------+--------------+-------------+
| Radionuclides      | Gross alpha, gross | 900, or      | H           |
|                    | beta, Gross gamma  | technique    |             |
|                    |                    | preferred by |             |
|                    |                    | lab^3^       |             |
+--------------------+--------------------+--------------+-------------+

*1. Method titles are listed in Table 4-19 in the appendix (Section 10)*

*2. Method 200.8 may be applied in the time-saving "direct analysis"
mode that does not require digestion, if the NTU of the water sample is
\<1. Note that the NTU of the water may be quickly determined in-house
and/or may be available in the site characterization report supplied
with the sample. The use of the scanning mode in Method 200.8 is also
suggested.*

*3. Many radionuclide laboratories have developed in-house methods for
rapid screening of radionuclides. These may be similar to EPA 900-Series
methods, which were developed for the measurement of very low,
environmental levels of radioactivity in drinking water but may require
several days to obtain a result. See Section 6.4.8 for further
discussion. Analytical confirmation may be required via EPA Method or
other defensible methods.*

*4. Standard may contain these. See Table 4-20 in the appendix (Section
10).*

As mentioned in Section 1, this module is primarily intended for the
purpose of planning an analytical response. The users may wish to
develop their own basic screen after evaluation of their particular
capabilities and experience as well as the needs and concerns of their
clients. (**Some hypothetical examples of basic screens are given in
Section 7 of this module.**) For a utility laboratory, the basic screen
would be based on the utility's perception and knowledge about threats
in their area and the capabilities of their laboratories. Because there
is no regulatory requirement to apply any basic screen, a laboratory
may, in principle, choose not to employ any of the standardized methods
in Table 4-3. In this case, however, it would be prudent from a public
health perspective to employ an expanded screen based on exploratory
techniques capable of determining the analytes of interest, such as
those listed in Table 4-20.

There are several important notes about Table 4-3:

-   Screening of radionuclides involves a different paradigm than
    conventional chemicals. See Section 6.4.8 for details.

-   The preparation method for 8270D listed is 3535A, based on
    solid-phase extraction. Separatory funnel preparation for 8270D, or
    any other separatory funnel method, is not included because
    separatory funnels may release aerosols when vented, potentially
    increasing the risk of exposure for laboratory personnel, unless
    appropriate hoods and/or other control measures are employed.
    Granted, separatory funnel techniques are safely used every day but,
    considering that other sample preparation methods (such as 3535A)
    are available, it may be prudent for laboratories to consider not
    using the separatory funnel preparation method.

-   The methods encompass many analytes. In terms of obtaining standards
    to use in conjunction with the respective methods, it may be helpful
    to focus on certain analytes. The analytes that may be included in
    the standard are identified in the appendix (Table 4-20) by the list
    number included in Table 4-3. Laboratories may wish to add analytes
    to those listed in the appendix, and may already be doing so (e.g.,
    to meet local regulation). These analytes must meet the same QC
    acceptance criteria and be part of the overall quality systems in
    the laboratory, so that the added analytes are as defensible as the
    method analytes.

-   The methods listed in Table 4-3 determine a number of analytes that
    probably would not be involved in a contamination incident (in
    addition to those that are priority contaminants). However, for the
    purposes of a screen, it is valuable to include some of the
    additional analytes that are picked up by the method, especially
    since there is no way to predict which contaminants might be used in
    a threat or incident.

-   The standardized methods listed in Table 4-3 do **not** include some
    analytes of concern, such as the biotoxins, chemical warfare agents,
    and pharmaceuticals. This may be due to the fact that since these
    are not regulated compounds in drinking water, there has been little
    impetus to develop methods for these analytes. This may change, due
    to the inclusion of some algal biotoxins, namely microcystins, in
    the Contaminant Candidate List
    (<http://www.epa.gov/safewater/ccl/cclfs.html>) and Unregulated
    Contaminant Monitoring Rule
    (<http://www.epa.gov/safewater/standard/ucmr/main.html>).

-   The methods in Table 4-3 are not always the same ones that are
    required for compliance with the Safe Drinking Water Act as amended.
    For instance, Method 551.1, which is used for compliance monitoring
    of some disinfection by-products, does not encompass a sufficient
    number of chemicals considered as priority contaminants from an
    acute health effect standpoint. After all, disinfection by-products
    largely produce a long-term health risk, not the acute risk to human
    health that would be the most pressing concern during an intentional
    contamination threat/incident.

The following is a brief discussion of the analytical methods utilized
in the Basic Screen, broken down by the chemical classes listed in Table
4-1. Many laboratories may be familiar with the individual methods
listed, as these are typically the EPA drinking water compliance
methods. However, the purpose of the module is not to insist that only
EPA drinking water compliance methods may be used. As mentioned above,
there are other fine method sources, including, but not limited to,
those in Table 4-3. If these other methods are employed, they should be
capable of determining the analytes of interest (i.e., Table 4-20), and
they should conform to the spirit and intent of the methods described
briefly below.

### Volatiles (Organic)

Purge-and-trap concentration and analyses by GC/MS (based on EPA Method
524.2 or 8260B) may be utilized to identify organic compounds that are
volatile in nature. To summarize the method, volatile organic compounds
with low water solubility are extracted (purged) from the sample matrix
by bubbling an inert gas through the aqueous sample. Purged sample
components are trapped in a tube containing suitable sorbent materials.
When purging is complete, the sorbent tube is heated and backflushed
with helium to desorb the trapped sample components into a capillary gas
chromatography (GC) column interfaced to a mass spectrometer (MS). The
column is temperature-programmed to facilitate the separation of the
method analytes, which are then detected with the MS. Compounds eluting
from the GC column are identified by comparing their measured mass
spectra and retention times to reference spectra and retention times in
a database. Reference spectra and retention times for analytes are
obtained by the measurement of calibration standards under the same
conditions used for samples. The instrument should be calibrated for the
analytes of interest, using analytes similar to those found in Analyte
List A (Table 4-20).

Some laboratories possess purge-and-trap instrumentation with a PID
detector in series with an ELCD detector. This combination provides the
ability to determine most of the analytes in list A. Because of the
nature of the PID and ELCD detectors, however, they cannot necessarily
provide the additional level of information and analytical confidence as
a mass spectrometer (see Section 6.5.1 below). Accordingly, Methods
502.2 and 8021B are included primarily to increase the number of
laboratories capable of this analysis.

### Semivolatiles (Organic)

Solid-phase extraction involves the removal of analytes from water onto
a solid sorbent material (based on EPA Method 525.2). Analytes, internal
standards, and surrogates are extracted from a water sample by passing 1
liter of sample water through a cartridge or disk containing a solid
matrix with a chemically bonded C18 organic phase (solid-phase
extraction, SPE). The organic compounds are eluted from the SPE
cartridge or disk with small quantities of ethyl acetate followed by
methylene chloride, and this extract is concentrated further by
evaporation of some of the solvent. The 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 gas
chromatography/mass spectrometry (GC/MS) system.

Compounds eluting from the GC column are identified by comparing their
measured mass spectra and retention times to reference spectra and
retention times in a database. Reference spectra and retention times for
analytes are obtained by the measurement of calibration standards under
the same conditions used for samples. The instrument should be
calibrated for the analytes of interest, using analytes similar to those
found in list B (Table 4-20).

### Carbamate Pesticides (Organic)

EPA Methods 531.1 and 531.2 can be utilized to detect carbamate
pesticides and degradation products, namely the N-methylcarbamolyoximes
and N‑methylcarbamates. (Note that Method 531.1 requires a different
preservative.) Compared to the other EPA methods in the basic screening
analysis, EPA Methods 531.1 and 531.2 do not encompass many analytes. It
may be worth noting that at least one chemical (aldicarb) in this class
has an immunoassay test available for screening purposes.

This immunoassay is not recognized for compliance monitoring, so it is
not included in the basic screening analysis, but might be useful as a
field test. In Methods 531.1 and 531.2, the water sample is filtered and
a 400-µL aliquot is injected into a reversed-phase HPLC column.
Separation of analytes is achieved using gradient elution
chromatography. After elution from the HPLC column, the analytes are
hydrolyzed to form a methyl amine, which is further reacted to form a
highly fluorescent derivative that is detected by a fluorescence
detector. The derivatization procedure imparts specificity to the
analysis, and a sample component is identified by comparison of its
retention time to the retention time of a reference chromatogram. If the
retention time of an unknown compound corresponds, within limits, to the
retention time of a standard compound, then identification is considered
positive. Because of specificity limitations of fluorescent detectors,
identification requires expert judgment when sample components are not
resolved chromatographically.

When peaks obviously represent more than one sample component, or any
time doubt exists over the identification of a peak on a chromatogram,
appropriate alternate techniques should be applied to identify the peak,
such as the use of a second chromatography column, several of which are
contained in the method. SW-846 Method 8321B also employs HPLC
instrumentation for the analysis of carbamates, and it also describes
the use of thermospray mass spectrometric detection, possibly providing
additional information about the target compound. However, for technical
reasons revolving around data interpretation, the thermospray interface
is considered obsolete, and is in the process of being replaced
(<http://www.epa.gov/epaoswer/hazwaste/test/new-meth.htm>).

### Quaternary Nitrogen Compounds (Organic)

EPA Method 549.2 is a high performance liquid chromatography (HPLC)
method for the determination of diquat and paraquat in drinking water
sources and finished drinking water. In the method, a volume of water
sample is subjected to solid-phase extraction. The eluate from the
extraction device is separated through the use of liquid chromatography
with UV detection. A photodiode array detector may be utilized to
provide simultaneous detection and analytical confirmation of the method
analytes.

### Trace Metals (Inorganic)

a\) **Inductively Coupled Plasma Mass Spectrometry (ICP-MS).** EPA
Method 200.8 provides procedures for the determination of 21 elements in
a single run and provides mass spectral qualitative identification with
significantly better detection limits than most standard instrumental
elemental analysis techniques. The method involves a sample preparation
procedure with a digestion step using nitric and hydrochloric acids,
although there is an alternative "direct analysis" for samples with
turbidity less than 1 NTU, which is applicable to many treated drinking
water samples as they typically have turbidity less than 0.3 NTU. The
method describes the multi-element determination of trace elements by
inductively coupled plasma mass spectrometry (ICP-MS). Sample material
in solution is introduced by pneumatic nebulization into a
radiofrequency plasma where energy transfer processes cause desolvation,
atomization, and ionization. The ions are extracted from the plasma
through a vacuum interface and detected on the basis of their
mass-to-charge ratio by a mass spectrometer. The mass spectrometer may
be operated in the "scanning" or the "single ion monitoring" mode. While
detection limits are slightly less in the scanning mode, the use of the
scanning mode offers benefits in terms of identifying non-method
analytes. Details are discussed below in Section 6.5.2a, as part of the
expanded screen.

It should be noted that Method 200.8 is designed for compliance
monitoring, and if acidification of the sample is not performed in the
field, the method recommends that the sample be held 16 hours after
acidification, prior to analysis. This may not be practical in
responding to contamination incidents. (See Module 3, Section 3.2, for
more details on preservation issues.)

Acid addition in the field may result in the release of toxic gases,
such as hydrogen cyanide, depending on the nature of the contamination.
As a safeguard, sample preservation in the field should occur only after
the potential presence of cyanide has been eliminated through field
screening using devices such as portable colorimeters or probes.
According to the procedures in Module 3, sample collection should always
follow field testing (including cyanide), so this check should be
automatic. If cyanide is found, the speed of reporting of Method 200.8
analytes may not be an issue since discovery of elevated cyanide levels
would elevate the credibility of the threat and prompt immediate
response actions, which would provide additional time for careful
forensic analysis.

Because of the specificity and detection limits of ICP-MS for all the
elements in Analyte List E (Table 4-20), it may be considered a superior
technique for trace metal analysis. However, in the interest of more
laboratories being capable of performing trace metal screening, it
should be recognized that inductively coupled plasma atomic emission
spectrometry and graphite furnace atomic absorption spectrometry are
widely used for trace metal determination. Practitioners should be
familiar with the comparative advantages and disadvantages of the
techniques for trace metal determination listed in Table 4-3,
particularly regarding detection limits and interferences for some of
the analytes in Analyte List E (Table 4-20).

b\) **Inductively Coupled Plasma Atomic Emission Spectrometry
(ICP-AES).** While ICP-MS is a trace elemental technique with excellent
specificity and detection limits for some of the contaminants of
concern, other trace elemental techniques are available in many
laboratories and may be used as part of the expanded screening
procedure. EPA Method 200.7 provides procedures for the determination of
32 elements in a single run through the use of ICP-AES. ICP-AES is
sometimes referred to as ICAP (for inductively coupled argon plasma) or
ICP-OES (for optical emission spectrometry). The instruments measure the
atomic line emission spectra by optical spectrometry. The aqueous sample
is acidified prior to instrument analysis. The analysis begins with a
nebulization and the aerosol stream is transported to the plasma torch,
which may be viewed by an optical spectrometer either head on (radially)
or from the side (axially) depending on the instrument. Each orientation
provides different performance characteristics, particularly in terms of
interferences and detection limits, but each of these is dependent on
the matrix as well as the instrument design. The speed of the analysis
is also influenced by instrument design. For example, the optical system
may allow either sequential or simultaneous determination of the
analytes, with the simultaneous determination allowing faster analysis;
it also may allow tentative identification of non-method analytes,
similar to ICP-MS (above).

c\) **Atomic Absorption (AA) Spectrometry.** Graphite furnace atomic
absorption spectrometry (EPA Method 200.9) can also be used for a select
number of metals. The acid-digested sample is placed in the instrument
and is pretreated prior to atomization. The atomization occurs after a
purging with an inert gas and drying. The atomization is performed by
rapid heating and produces an atomic cloud. The atomic cloud then
absorbs the element-specific atomic emission from a hollow cathode lamp
or electrodeless discharge lamp. Instrument design influences
performance characteristics, like detection limits and number of
elements that may be analyzed at one time. The atomic absorption
technique may be used with other introduction techniques than graphite
furnace, as shown in the next section for total mercury.

### Total Mercury (Including Organomercury Compounds)

EPA Methods 245.1 and 245.2 allow the determination of total mercury,
which includes inorganic mercury and many organomercury compounds,
following digestion with potassium persulfate, which oxidizes the
organomercury compound to mercuric ion before measurement. The total
mercury is determined through the use of cold vapor atomic absorption.
This technique greatly enhances the detection limit for mercury
determination and offers other benefits as well. Total mercury may be
determined as part of the trace metal analysis using methods in Table
4-3 (Section 6.4.5), except GFAA, Method 200.9. However, Methods 245.1
and 245.2, which form the basis for the dedicated mercury analysis
instruments currently found in many laboratories, were included here.

### Cyanides

Ideally, the core field testing recommended in Module 3 will detect the
presence of cyanide, so laboratory analysis of cyanide would most likely
serve a confirmatory role. Depending on the nature of the field test, it
may use similar chemistry to the EPA Methods 335.2, 335.3, and 335.4, as
described below, but typically the field test will not employ
distillation. Thus, the field tests primarily detect free cyanide ion
(not cyanide complexes).

It should be noted that most cyanide field tests are designed for
quantitative analysis of cyanide at low levels (near the MCL), and they
typically provide only a qualitative indication of the presence of high
cyanide concentrations. Accordingly, it may not be necessary to perform
redundant tests in the laboratory, although it may be deemed prudent to
do so. Such decisions may be based on the concentration of cyanide in
the water or the form of cyanide known or suspected. For instance, a
titration procedure for cyanides is used when the concentrations exceed
1 mg/L (EPA Method 335.2). A reflux distillation is used to release the
cyanides, which are then absorbed in a scrubber solution. The titration
uses a silver nitrate solution to titrate cyanide in the presence of a
silver sensitive indicator. For lesser concentrations, cyanide in water
is determined through the use of colorimetry (EPA Method 335.3 and
335.4). UV digestion and distillation are used to convert the cyanides
to hydrogen cyanide. Cyanides are then converted to cyanogen chloride
and then subsequent reactions with pyridine and barbituric acid give a
red colored complex.

The form of cyanide added to water may be important in deciding on which
cyanide test is appropriate for use. Free cyanide in water may result
from the addition of soluble cyanide salts or water-soluble organic
cyanogenic compounds. Methods 335.2, 335.3, and 335.4 measure total
cyanide, which could be a mix of free cyanide, complexed cyanide, and
some organic cyanogenic compounds. Because free cyanide is the most
toxic, without knowing the concentrations of the other forms, which have
widely varying toxicity, the use of the total cyanide concentration
would be conservative with respect to public health implications.

### Radionuclides

Basic screening for radionuclides represents a somewhat different
paradigm than conventional chemicals because radionuclides can be
characterized by both the type of radiation they emit as well as their
exact chemical identity. Accordingly, initial screening for
radionuclides may involve an examination of gross radioactivity.
However, any initial screening that indicates the presence of a
radionuclide should be followed by analytical confirmation of the
chemical identity. A schematic for radionuclide screening is shown in
Figure 4-7. Note that in Section 3, screening for radioactivity upon
sample receipt is recommended. Accordingly, the decision point in Figure
4-7 that asks "Does lab policy require screening for radioactivity?"
provides flexibility for local planning decisions.

![](media/image8.wmf)

**Figure 4-7. Schematic for Basic Radionuclide Screening**

The results of field testing for radioactivity (Figure 4-7) must be
compared to background levels to determine whether the site may have
been contaminated with radioactive material. Although discussed in
Module 1, it is worth noting here that EPA has developed protective
action guidelines for radiation levels in water
(<http://www.epa.gov/radiation/docs/readytorespond/ready3.htm>).

The analysis for gross alpha and beta radiation (EPA Method 900.0) may
be conducted as a screening method for alpha and beta particle
activities in water and used to determine if specific radiological
analyses are needed. Preliminary analysis can first be conducted in the
field using appropriate field portable or hand-held devices, but may be
verified in the laboratory. EPA Method 900.0 can measure alpha particles
with energies above 3.9 megaelectronvolts (MeV) and beta particles with
energies above 0.1 MeV. In this method, the water sample is evaporated
and dried, and the dried residue is counted for alpha and beta by a gas
flow proportional counting system or scintillation detector system. Note
that as part of their safety plan, laboratories may wish to screen
samples upon arrival for gamma radiation using appropriate technologies.

If the presence of radioactive material is indicated by the initial
screening, specific radioisotopes may be determined by radiochemical
specific procedures, using EPA 900-Series methods or other legally
defensible techniques. These procedures often involve separation of the
radionuclide from the sample by precipitation procedures and subsequent
determination by gas flow proportional counting system or scintillation
detector system for alpha and beta emitters and an appropriate gamma
detector for gamma emitters. For example, strontium-89 and strontium-90
can be measured by EPA Method 905.0. Strontium-89 and strontium-90, are
precipitated as carbonates from the sample. Additional precipitation
steps allow separation from other radionuclides and interferences. The
daughter product of strontium-90, ytrium-90 is separated by a hydroxide
precipitation step and is beta counted by a low background beta counting
system. The combined strontium-89 and strontium-90 residue is also beta
counted. The results for each radionuclide are determined.

Due to the unique nature of radionuclide analysis, some laboratories
have developed in-house procedures for radionuclide analysis that make
use of their special skills and capabilities to enhance the speed of
analysis, especially since some EPA 900-Series methods are not rapid
methods. For example, EPA 905.0 for radioactive strontium in drinking
water recommends a two-week in-growth period for obtaining the yttrium
isotope from the purified strontium. Modification of the method results
in much faster results. Reduction in analysis time could be accomplished
by measuring the total amount of an element's radionuclide, not the
isotopic distribution. Also, for some isotopes, faster results may be
obtained by simply reducing the volume of water sample processed.

In summary, many laboratories may choose to utilize a specialized method
or technique for initial sample screening that may not be necessarily
identical to those described in EPA 900-Series methods, although these
methods must be used when reporting drinking water compliance monitoring
results.

## Expanded Screening using Established and/or Exploratory Techniques with Standardized or Non-standardized Methods 

The purpose of the expanded screen is to capture chemical contaminants
not picked up by the basic screen, namely those analytes listed in Table
4-20. The expanded screen may also detect those analytes covered by the
basic screen more rapidly. It should be a sufficiently broad screen to
"go fishing" for many possible contaminants. The expanded screen can be
used with a combination of standardized or non-standardized methods.
Note that in the case of standardized methods, no additional analytical
confirmation may be required because the method used may itself be
considered definitive if applied correctly. In other cases, please see
Section 6.2 for a further discussion of confirmation of analytical
results.

In practice, the expanded screen can be used **in addition** **to** the
basic screen, because the results of the basic screen may provide a
"springboard" to guide the selection of techniques for the expanded
screen. **Alternatively,** some laboratories may choose to utilize only
the expanded screen, comprised of potentially sensitive techniques,
including those summarized in Table 4-4. In the latter case, preliminary
results can be cautiously used to make response decisions, but must be
followed up with confirmatory analysis since screening techniques are
not necessarily definitive or legally defensible.

Table 4-4. Expanded Screening for Contaminants, broken out by classes of
contaminants as found in Table 4-1.

+--------------------+-------------------------------------------------+
| **Contaminant      | **Expanded Screening Technique**                |
| Type**             |                                                 |
+--------------------+-------------------------------------------------+
| Organic            | GC, GC/MS, HPLC, LC/MS, Immunoassay test kits\* |
+--------------------+-------------------------------------------------+
| Inorganic          | IC, AA, ICP, ICP-MS                             |
+--------------------+-------------------------------------------------+
| Cyanides           | Wet chemistry\*                                 |
+--------------------+-------------------------------------------------+
| Biotoxin           | Immunoassay test kits,\* GC/MS, HPLC, and LC/MS |
+--------------------+-------------------------------------------------+
| Radiological       | Handheld equipment\*                            |
+--------------------+-------------------------------------------------+
| Chemical Warfare   | GC/MS with direct injection, purge & trap, and  |
| Agents             | SPE/SPME,                                       |
|                    |                                                 |
|                    | test kits,\* handheld equipment\*               |
+--------------------+-------------------------------------------------+

\*See Section 4.1 for important disclaimers and information about the
use of these technologies. Note that when these technologies as being
used in the expanded screen, they are being applied in the laboratory,
not the field.

Given the variety of choices presented in Table 4-4, the question
naturally arises about how to select suitable methods and equipment for
the expanded screen. This section attempts to address this in a number
of ways:

-   Although noted in Section 6.4, it is worth repeating here that
    information gained during the basic screen may help guide the
    selection of techniques and methods for use in an expanded screen.
    For example, many of the techniques in the basic screen rely on
    chromatography and/or mass spectrometer, so the data should be
    capable of being evaluated for the presence of not only target
    analytes, but also other compounds. In addition, clues about the
    identity of the contaminant may be provided by the mass spectra or
    chromatographic retention behavior (e.g., early eluting compounds
    may indicate a lower boiling point, poor peak shape on a non-polar
    chromatography column may suggest certain (polar) functional groups,
    etc).

-   Combining observations from multiple basic screening techniques may
    be helpful. For instance, if no target analyte or unknown peak is
    found in a Method 524.2 analysis, the unknown compound is likely not
    a volatile organic; if no peak is present in a Method 8270D
    analysis, the unknown may be inorganic in nature or thermally
    unstable. This reduces the possibilities, although requires
    knowledge of the properties of potential contaminants. Some of these
    many properties may be obvious to skilled analysts. One resource to
    help with this is the Water Contaminant Information Tool (see Module
    2), currently under development.

-   Many EPA standardized drinking water compliance methods are not
    included in Table 4-4, but may be used to implement the analysis
    summarized in Table 4-4 for expanded screening. This increases the
    number of analytes covered and, depending on the method, may
    **decrease** analysis time. Some standardized methods (see Section
    6.2), including drinking water and SW-846 methods, may be useful to
    apply directly in the expanded screen, even though they are not
    necessarily intended for Table 4-20 analytes. In fact, by their
    design, SW-846 methods may allow for some \"quick and dirty\"
    approaches to screening that might be significant time savers when
    faced with the short turnaround times associated with the initial
    phases of an incident.

-   A discussion of the expanded screening techniques listed in Table
    4-4 is provided below, along with some appropriate **precautionary**
    notes. Many readers will recognize some of these precautionary notes
    as being components of standardized methods. The discussion below
    should not be considered an exhaustive technical discussion on the
    subject of analysis of unknowns, but is intended to be an overview
    of those that are more widely used, or highly promoted. Thus, this
    information is primarily intended for planners and some
    laboratories, in terms of investing in resources and techniques that
    may be very useful for expanding the screening capability to look
    for unknown chemicals. Skilled analysts may wish to pursue other
    sources, such as technical books, scientific journals, and
    peer-interaction, for detailed insight and application of these
    techniques to specific technical challenges.

### Expanded Screening for Organic Chemicals

Contaminants are often extracted from water samples to facilitate the
analysis (e.g., to obtain a suitable amount of contaminant for
analysis). Regardless of the detector system employed, there are a few
widely used sample preparation techniques. Following sample preparation,
a variety of detection techniques are used to analyze the sample. Figure
4-6 illustrates which preparation techniques are commonly used with
which detection techniques.

[Sample Preparation]{.underline}

**NOTE:** **Large Volume Liquid/Liquid Extraction (LLE) generally may
not be recommended for aerosolizable samples**. This technique, although
utilized in some EPA methods, requires operation of separatory funnels
that may release aerosols when vented. The generation of these aerosols
may represent a larger health hazard than other techniques, unless
laboratories take precautions, such as appropriate hoods and sampling
handling techniques. Many of the same goals of large volume
liquid/liquid extraction may be accomplished through the use of the
techniques described below. The techniques below may also have other
benefits, such as reducing the consumption of sample and solvent.

a\) **Direct Aqueous Injection.** Although a potentially powerful
analytical technique, the use of direct aqueous injection with gas
chromatography may present technical difficulties in chromatographic
separation and may reduce the lifetime of the GC column and the
detector. However, some modern GC capillary columns are designed to
survive the rigors of direct aqueous injection, provided appropriate QC
is applied to instrument operations and required maintenance is
performed. For the high concentrations of contaminants that may be
present during an emergency incident, the use of direct injection of
aqueous samples with GC/MS library matching may prove valuable,
particularly for initial and rapid screening of analytes for which
laboratories do not possess the equipment utilized in a standardized
method. For all but a few analytes, analytical confirmation procedures
may be necessary, and laboratories should plan carefully, in terms of
developing skills to applying this technique, verifying their GC columns
are suitable, implementing QC requirements, and preparing for additional
maintenance requirements of both column and detector.

b\) **Micro Liquid-Liquid Extraction (micro-LLE).** Liquid micro
extraction involves the use of small volumes of solvent (\~ 2 mL) to
extract analytes from a small volume (\~ 40 mL) of water (for an
example, see EPA Method 551.1). This approach is usually much quicker
than the typical LLE because of the ease of the microextraction and
because no concentration/solvent exchange is required. If the analyte is
known, then a calibration standard should be analyzed along with other
requisite quality control samples to quantify the analyte. If the
analyte is unknown, then a screening approach should be taken. The
extraction may be immediately followed by GC/MS analysis, which can
provide qualitative identification from the mass fragmentation library
search. Analytical confirmation procedures may be necessary, and
laboratories should plan carefully in terms of QC requirements and
additional maintenance requirements of both column and detector. For the
high, perhaps acutely toxic, concentrations of contaminants that may be
present during an emergency incident, the use of liquid-liquid
microextraction of aqueous samples with a suitable solvent, such as
methylene chloride (dichloromethane) **may prove particularly
valuable**, particularly for initial and rapid screening of analytes for
which laboratories do not possess the equipment utilized in a
standardized method. Micro-LLE may not provide adequate detection limits
for lower concentrations, which may occur if the water sample represents
the tail of a transient contaminant slug.

c\) **Continuous Liquid-Liquid Extraction (Cont LLE).** This technique,
as described in EPA Method 3520C, is applicable to the isolation and
concentration of water-insoluble and slightly soluble organics. Its use
can result in excellent detection limits, like solid-phase extraction
described below, although detection limits for specific analytes may be
different.

d\) **Solid-Phase Extraction (SPE).** Solid-phase extraction (sometimes
referred to as liquid-solid extraction) is described in Section 6.4.2
for EPA Method 525.2 as one of the techniques for basic screening
analysis. Like micro liquid-liquid extraction, SPE extracts many
contaminants, but can achieve larger concentration factors compared with
the former technique, which may be necessary for some analytical goals
(Section 1.3). However, many variants of the solid-phase extraction
technique are possible. For instance, instead of using a C18 adsorbent,
there are many other adsorbents available. The adsorbents have markedly
different properties and may be used to extract contaminants that are
not amenable to C18 adsorbents. Different elution solvents can also be
used as an experimental variable. As a safety note, SPE should produce
few aerosols.

e\) **Solid-Phase Microextraction (SPME).** A relatively recent
variation of solid-phase adsorption is solid-phase micro extraction
(SPME). Like SPE, SPME should produce few aerosols. SPME involves the
use of a fiber coated with sorbent material, and the sorbent coated
fiber is exposed to either the aqueous sample or the headspace from the
sample. The analytes then adsorb to the coating on the fiber. After
exposure to the sample, the fiber can be introduced into the detection
system, although the technical details depend on the detector (i.e., GC
or HPLC). For instance, after exposure to the sample, the SPME fiber is
inserted into the injector of a GC, and contaminants are released to the
column by thermal desorption. Fibers are available with several types of
coating to enable extraction of analytes of different properties, such
as volatiles or semivolatiles. Accordingly, SPME could be used for a
quick screen. As with micro-LLE, another quick screen, the detection
limits may only be useful in the case of elevated, perhaps acutely
toxic, contaminant concentrations. Lower concentrations like those found
in the tail of a transient contaminant slug, might not be detected using
SPME.

f\) **Headspace Collection.** The headspace above an aqueous sample may
be injected in a GC. Commercial equipment, interfaced with the GC, is
designed to facilitate this analysis, although manual injections may be
made.

g\) **Flow Injection.** In the flow injection analysis experiment, an
aqueous sample or sample extract is injected directly into an LC/MS in
such a manner that it bypasses the LC column. Thus, the analytes are not
chromatographically separated, but the technique can prove useful if
high concentrations of a single analyte are present, or if sample
preparation is employed that is selective of particular analytes.

[Detection Techniques]{.underline}

a\) **Multidetector GC in Screening Mode.** In Table 4-3, a
multidetector GC is utilized for specific analytes as an alternative to
a mass spectrometer. However, the intent of using multidetector GC in
the analysis of unknowns is primarily as a screening tool, not
necessarily to quantify or identify, but rather to determine if the
sample contains a component not normally found in the background.
Multidetector (sometimes called multispectral) GC refers to the use of
GC with a variety of detectors (there are over a dozen on the market)
and then comparing the data from all the detectors. Examples of
detectors include atomic emission, electron capture, infrared, flame
ionization, nitrogen-phosphorus specific, thermal conductivity, etc.

Various GC detectors respond to contaminants in different ways, and the
evaluation of all the data from the various detectors increases the
selectivity of the analysis. For example, detectors like flame
ionization detectors tend to respond to most contaminants (but not with
high sensitivity), while electron capture detectors respond with great
sensitivity (but mostly to halogenated compounds and are less sensitive
to certain other compounds). The detectors may be used in series with
one GC, or in parallel through the use of multiple GCs. In the latter
case, the peaks are correlated through the use of relative retention
times, i.e., the retention time of the peak of interest compared to a
standard. Multidetector GC should be applied carefully because of
differing response of different detectors to coeluting contaminants, and
confirmatory/supplemental analysis may be necessary. For the expanded
screen, because methods are used other than the standardized EPA methods
for drinking water compliance monitoring, it may be possible to utilize
other detectors than specified in the standardized methods.

b\) **Gas Chromatography with Electron Impact Ionization Mass
Spectrometry**. The subsequent analysis of contaminants extracted from
water may be conducted by the use of gas chromatography/mass
spectrometry. When the mass spectrometry is performed using electron
impact ionization, eluting peaks may show distinctive fragmentation
patterns, which may be used in identification, particularly through the
use of a variety of computerized tools for library matching to
ionization patterns of known compounds. Thus, the use of electron
ionization in full scan mode (i.e., *m/z* 35 to the upper scan limit of
the particular instrument) may be particularly useful when performing
the analysis for unknowns using the GC/MS for analyte separation and
detection. Note that for the analysis of small molecules, it may be
necessary to start the scan below *m/z* 35.

It is desirable to review peaks that do not match the retention time of
analytes listed in the method. Water samples may contain hundreds or
thousands of compounds, so each laboratory must decide a threshold for
examining unidentified peaks. For instance, as a starting point, the
laboratory may attempt to identify an unknown peak if it is present at
\>10% (height threshold) of the internal standard using the automated
programs that are available with many instrument software packages. The
threshold may need to be reduced (e.g., to 1%) or increased (e.g., to
20%) depending on the sampling location, as water samples vary in the
number and intensity of their normal background peaks.

Usually, the program performs a spectral search using a user-defined
library (such as NIST, EPA, Wiley, etc), and will report the compound
with the best spectral match as the tentatively identified compound with
an estimated concentration (based on height, assuming a response factor
of 1.0). Some of the guidelines in evaluating the spectra are:

-   The major ions in the reference spectrum (ions greater than 10% of
    the most abundant ion) should be present in the sample spectrum.

-   The relative intensities of the major ions should agree within +/-
    20%.

-   The base mass ion present in the reference spectrum should be
    present in the sample spectrum.

-   Ions present in the sample spectrum but not in the reference
    spectrum should be reviewed for possible coelution or background
    contamination.

Identification of unknown compounds relies on more than the efficiency
of the library search program. The expertise of the analyst in
evaluating the mass spectra and library matches is critical for
assigning a tentative identification to the unknown. Electron impact
mass spectrometers may vary slightly by manufacturer, vintage, and mass
analyzer design. An analyst's familiarity with the nuances of a
particular instrument may greatly enhance a laboratory's ability to
confidently identify an unknown. In addition, there are a number of
options within library search software, such as fitting algorithm,
threshold parameters, result constraints, etc., and a particular analyst
may be more proficient with one of these options for performing
library-enhanced identification. For example, one approach may be that
if the mass spectral library "reverse fit" is greater than 800 (of a
possible 1,000), the peak is reported as identified in the report. If
the fit is 600-800, the analyst should review the library search to
determine if the identification was skewed by a coelution or high
background. If the analyst is not able to improve upon the initial
identification (fit between 600-800), the unknown should be listed by
the class of compound assigned to that peak (e.g., "alkylated aromatic",
"halogenated aliphatic", etc.) and an approximate molecular weight. If
the report finds an unknown with a fit below 600, and the analyst cannot
improve on the identification, the peak should simply be listed as
"unknown." The library search report should be scrutinized by an
experienced GC/MS analyst to determine how the "unknowns" should be
reported.

The above procedures are based on the library match and represent only a
tentative identification. To increase confidence in the identification,
a reference standard can be purchased, when available, to help confirm
tentatively identified compounds. Primary dilution standards may be
prepared and analyzed using the same procedure as for samples.
Comparison of the retention time and mass spectrum of the known
reference standard with the data from the unknown may verify or negate
the tentative identification.

c\) **High Performance Liquid Chromatography-UV Detector.** Analogous to
multidetector GC, HPLC with UV detection can be used to determine if
organic compounds not amenable to gas chromatographic procedures (e.g.,
nonvolatile or thermally unstable compounds) are present in amounts
greater than background. Calibration and quality control samples should
be included to provide an accurate analysis. Analytical confirmation may
be necessary using established techniques, such as GC/MS. Due to
technical issues with different HPLC methods, some laboratories
currently dedicate HPLC instruments to standardized methods currently
being run in the laboratory. Accordingly, it may be desirable, albeit
not always practical, to dedicate an HPLC instrument to screening
activities.

Analysis for specific pesticides and other chemicals are possible, and
various HPLC methods for specific chemicals appear in the sources of
methods described in Section 6.2. Screening procedures using HPLC may be
more successful if targets are selected. For instance, a HPLC analysis
for pharmaceutical compounds of concern in water might be developed to
supplement existing methods. This method would probably involve some
type of extraction (micro-LEE, SPE, SPME, etc.) and analysis by HPLC
with UV detection. Contaminants in the extracts can be separated with
HPLC and detected through the use of a UV photodiode array detector
(PDA), which can record a UV spectrum that can be used for qualitative
identification, although not always definitely. If peaks cannot be
identified from the UV spectrum, LC/MS, described below, may be
attempted under the exact same conditions (e.g., chromatography column
and elution solvent program), provided such conditions are consistent
with LC/MS operation.

d\) **High Performance Liquid Chromatography-Mass Spectrometry
(LC/MS).** Many polar hydrophilic compounds cannot be easily extracted
from an aqueous sample. In addition, there are contaminants of large
molecular weight (e.g., biotoxins) or thermally unstable compounds that
are not amenable to GC analysis but can sometimes be analyzed by LC/MS.
Direct aqueous injection HPLC allows analysis of a sample without
extraction or concentration. SPME and SPE (and other extraction
procedures) may be utilized for compounds that can be extracted.
Identification of unknowns can be performed but there are no
standardized (EPA, NIST, Wiley, etc.) mass spectral libraries, as in
GC/MS. Analyst interpretation can help identify possible compound
fragments and structure. There are several ionization techniques
available. Two of the more popular ones that fall into the category of
atmospheric pressure ionization (API) are atmospheric pressure chemical
ionization (APCI), which allows the analysis of neutral analytes, and
electrospray ionization (ESI), which allows the analysis of ionic
analytes.

Analysis of samples by LC/MS is often not a straightforward or
conclusive endeavor, due to complexities of the ionization process. For
instance, depending on the composition of the sample, when ESI-MS is
operated in the positive ion mode, it can produce \[M+H\]^+^,
\[M+Na\]^+^, and other adduct ions, which sometimes can be useful to the
analyst and sometimes confounding. For this and other reasons inherent
to ESI-MS, quantification may not be always as straightforward as with
other LC detectors. Qualitative analysis of unknowns by LC/MS may be
hampered because the technique is not readily amenable to the
development of libraries, such as those available with electron impact
ionization GC/MS. For qualitative analysis, LC/MS may be considered best
suited to providing an estimate of the molecular weight of the compound.

In summary, more than a decade after its commercialization, LC/MS is not
standardized for drinking water analysis, although it has proved
extremely useful for analysis of target analytes in other industries.
Nonetheless, LC/MS can be an added tool in an expanded screen for
unknown chemicals in specific cases, and may be useful for certain
classes of pesticides, such as carbamates. For the analysis of specific
target compounds, a skilled analyst may be able to establish methods to
analyze for pesticides and pharmaceuticals in a particular water matrix,
thereby eliminating the need for a combination of HPLC detectors and
derivatization schemes. However, these methods may not be as robust as
standardized methods in terms of the analysis of waters from various
sources. In addition, there may be many opportunities for
misinterpretation and misapplication of LC/MS data, so additional
analytical confirmation steps may be necessary to ensure confidence in
and legal defensibility of the analytical results.

e\) **Tandem Mass Spectrometry (MS/MS).** Both GC and HPLC may be
utilized in conjunction with tandem mass spectrometry, also known as
MS/MS. Different MS/MS instruments operate under different principles to
achieve similar results, but in essence can be considered to be like two
mass spectrometers connected by a collision cell. The first mass
spectrometer separates ionized molecules, which are broken apart in the
collision cell, and the resulting fragments are separated in the second
mass spectrometer. This produces a great deal of information that can be
used to identify the original molecules, but even when used with
electron impact ionization, this technique does not necessarily produce
searchable libraries. Like other advanced mass spectrometric techniques,
MS/MS is not as widely available as MS and requires a high degree of
skill. However, it can produce unique and powerful results in the right
hands.

f\) **High Resolution Mass Spectrometry (HR/MS).** GC or HPLC, combined
with a high-resolution mass spectrometer, may provide exact mass data of
an eluting compound, allowing for calculation of elemental composition
of both molecular and fragmentation ions. This information is useful in
the identification of unknown organic compounds, especially when the
result of mass spectral library research is not conclusive or when the
standard of a tentatively identified compound is not available. Careful
quality control procedures are required, and the technique is not always
definite, especially for unknown compounds, because many compounds may
produce fragments with the same exact masses. Due to high capital cost
and complexity of use, HR/MS is not widely available.

g\) **Immunoassays.** There are a large number of immunoassay tests kits
for organic chemicals, such as pesticides and biotoxins. These may be
useful for screening a sample for specific unknowns either in the field
or in the laboratory. These kits may be used for speed or if
instrumental methods are not available in a laboratory. However, use of
these kits requires that the goals of the analysis be planned because
some kits are slower than the instruments, especially if analytical
confirmation time is considered. Also, appropriate training is necessary
in the use of these tests. Not all are the same, or easy to use.
Laboratories should be aware of the kits' reliability and levels of
detection before using.

A large collection of test kits for chemicals, such as carbamate and
quaternary ammonium pesticides, and many other contaminants is
summarized at <http://www.aoac.org/testkits/TKDATA2.HTM>. This is by no
means a complete list, and there are a number of vendors with products
not in this listing. It is important to note that most of these test
kits are not recognized by any standard setting organization. Not all of
these products have been studied in detail as to their efficacy for
drinking water, which may contain interfering and/or cross-reacting
substances. Efforts are underway to confirm the efficacy of some of
these kits for particular purposes, and as results become available,
they may be reported in the Module 3, Section 3.2. In the meantime, it
may be important to keep in mind the trade-offs these kits offer in
terms of sample throughput and false negatives/positive results. In
general, a positive result from one of these test kits should be
considered tentative and confirmed through a more rigorous laboratory
analysis.

Immunoassays have been developed for the analysis of urine for chemicals
that may be classed as illicit pharmaceuticals (e.g., narcotics and
psychedelics). While it seems unlikely that these substances would be
used as water contaminants, there is some historical precedence for
concern. Immunoassays represent a very sensitive approach to screening
of these compounds, and they are available from a variety of sources.
Because many of these tests were designed for urine analysis, the
performance of these tests for detecting the parent compound in water
should be verified, due to the potential for cross reactivity with
substances in water, which may result in a false positive detection.
Because immunoassays may be interfered with by chlorine-based
disinfectants, the residual should be quenched before use. Established
procedures also exist for the analytical confirmation of many of these
substances from urine using techniques such as GC/MS.

### Expanded Screening of Inorganic Chemicals

Like the determination of organic chemicals, there are often a number of
similar preparation steps that are used in the analysis of inorganic
chemicals. These are numerous and vary with the methodology being
employed. As a starting place to select a sample preparation approach,
it may be useful to refer to relevant standardized methods. For
instance, if the goal is to look for trace metals not listed in a
particular method, it may be useful to refer to a method in which the
sample is prepared for metal analysis. This is not necessarily an exact
process, and some metals have certain characteristics that may cause
them to not be amenable to a preparation technique applicable to
another. For instance, a digestion method for nickel may not be
applicable for mercury analysis. Following preparation, the samples may
be analyzed by a number of techniques:

a)  **ICP-AES or ICP-MS in Semiquantitative Mode.** Analogous to
    multi-detector GC and HPLC with UV detection, the ICP-AES and ICP-MS
    methods (EPA Methods 200.7 and 200.8, respectively) can also be
    expanded to provide a broad screen approach to identifying unknown
    trace metals. Under the semiquantitative mode (SQM, referred to by
    various names depending on instrument manufacturer), the ICP-MS
    instrument, operated in scanning mode, may be capable of providing
    semiquantitative results for more than 60 elements including major
    atomic cations, metals, semi-metals, rare earth elements and
    selected radionuclides (uranium and thorium) without actually
    including all of the elements being analyzed in the calibration
    standards. If an element is suspected, then standards may be
    obtained to perform quantitative analysis. (Note: radioactive
    materials should be handled by a specialized laboratory.) Some
    ICP-AES instruments can provide semiquantitative results for many,
    albeit fewer, elements than ICP-MS instruments, most of which are
    capable of SQM through the use of the correct software. The
    following paragraph discusses ICP-MS operated in scanning mode, and
    SQM for appropriate ICP-AES instruments functions analogously.

> The original calculation algorithm of SQM may be applicable to all
> naturally occurring elements. Additional information may be obtained
> for elements that are not considered in the default algorithm, such as
> man-made radioisotopes or stable fission products with long
> half-lives, especially for isotopes in mass range between 210 and 238.
> Under the SQM mode, the concentrations of the naturally occurring
> elements are calculated based upon instrument response factors (RF)
> for each element. No internal standards are necessarily added when
> using the SQM method. Following the external calibration procedure, a
> blank and a single calibration standard of 20-30 elements are
> typically used to update the RF before each analytical run. Increasing
> the number of elements in the standard generally may improve the
> accuracy of the analysis. For elements not included in the calibration
> standard, the RFs are updated indirectly by software interpolation
> from the calibrated elements. SQM identifies and assigns signal
> intensity based upon the natural abundance distributions for each
> isotope, corrects the molecular interferences by subtracting the
> signal of the assigned polyatomic species from the total signal,
> further corrects for polyatomic and isobaric interferences given in
> the elemental equation, and calculates the final concentration.

b)  **Ion Chromatography (IC).** Ion chromatography forms the basis of
    several EPA methods to determine ions of regulatory interest, such
    as EPA Method 300.1 for several common drinking water ions and
    disinfection byproducts. By the correct choice of operating
    conditions and ion chromatography columns, determination of many
    different types of ions have appeared in the literature, including
    those for some potential, albeit not high priority, drinking water
    contaminants of concern from a drinking water security perspective.

c)  **Wet Chemistry.** Wet chemistry forms the basis of many types of
    chemical test kits. The chemistry and detectors for test kits
    approved for compliance monitoring are traceable to EPA methods. Wet
    chemistry techniques, through the use of so-called "autoanalyzers,"
    form the basis of many types of chemical analysis, for environmental
    and clinical applications. Manufacturers of these devices often
    provide full detailed methodology for defensible application of wet
    chemistry to a variety of analytes. Titrimetric methods are also
    available and can be used to analyze background water quality
    parameters such as alkalinity.

d)  **Ion Selective Electrodes.** Ion selective electrodes (ISE, also
    known as electrochemical probes) can be utilized to analyze for some
    background water quality parameters. A simple example of an ISE is
    the familiar pH probe for the hydrogen ion. Other ISEs for a variety
    of ions (such as ammonia, calcium, chloride, fluoride, nitrate,
    potassium, silver, sodium, and sulfide) also may be available from a
    variety of manufacturers and may be considered. Some parameters that
    can be monitored by ISE may be useful in characterizing the extent
    of contamination or verifying credibility of a contamination threat
    (see the discussion on field testing in Module 3).

### Expanded Screening of Cyanides

Expanded screening of cyanides typically involves the use of sample
preparation techniques and wet chemical detection chemistries that are
not used in standard EPA methods. For instance, the distillation step
may be omitted, as it is for cyanide methods based on the Conway
diffusion technique. Note that distillation is required for
determination of total cyanide concentration, which, as discussed in
Section 6.4.7, may be the most conservative approach with respect to
public health implications. However, free cyanide concentration measured
without distillation, may still be useful, particularly in detecting
high, acutely toxic concentrations

Distillation also is not used in the rapid field tests for cyanide
(Section 4.1), but may be applicable for expanded cyanide screening. As
discussed in Section 3, some laboratories may consider using these rapid
field tests as safety screening tests upon receipt of the sample by the
laboratory. Note that several rapid field tests for cyanide have been
evaluated as part of EPA's Environmental Technology Verification
program, including some ion selective electrodes. The reports, available
at <http://www.epa.gov/etv/verifications/vcenter1-23.html> indicate that
some of these rapid field test kits, although acceptable for field use,
may perform better when used under controlled laboratory conditions than
in the field, especially in cold weather.

### Expanded Screening of Biotoxins

Some biotoxins have been monitored routinely for quite a while,
particularly in conjunction with naturally occurring outbreaks of
biotoxins in marine environments. There are hundreds of biotoxins from
dozens of different plant and animal species. The broad screening for
microbiological contaminants (Section 8) may capture some of the higher
molecular weight biotoxins, and analysis of some biotoxins may be
supported by LRN laboratories, as indicated in Table 4-1. In addition,
the LRN currently may utilize immunoassays for biotoxin analysis of
botulism, ricin, and other biotoxins where applicable. The results of
the LRN analysis should be further communicated to any other
laboratories to which chemical samples are sent.

**Immunoassays.** Many/all of the biotoxins listed in Table 1-1 have
commercially available immunoassay kits. A large collection of test kits
is summarized at <http://www.aoac.org/testkits/TKDATA2.HTM>. This is by
no means a complete list, and there are a number of vendors with
products not in this listing. It is important to note that most of these
test kits are not recognized by any standard setting organization, and
the applicability of these products to disinfected drinking water, which
may contain interfering and/or cross-reacting substances, is not well
studied. Efforts are underway to confirm the efficacy of some of these
kits for particular purposes, and as results become available, they may
be reported in the Appendix to Module 3 that lists field test equipment.
In the meantime, it may be important to keep in mind the trade-offs
these kits offer in terms of sample throughput and false negative/false
positive results. In general, a positive result from one of these test
kits should be considered tentative and confirmed through a more
rigorous laboratory analysis.

A key part of laboratory analytical screening for a biotoxin is the
ability to confirm its tentative identification (e.g., via an
immunoassay test kit). Confirmatory analyses are discussed in more
detail below, and usually involve GC/MS, LC, or LC/MS. The IOC Manual on
Harmful Marine Microalgae, available at
<http://unesdoc.unesco.org/images/0012/001220/122021eo.pdf>, contains
many examples of these types of analyses, chiefly intended for analysis
of algal biotoxins. One type of assay that has been widely used for the
determination of biotoxins in seafoods over the past 60 years is the
mouse bioassay. The applicability of this technique to water samples is
not well investigated, and most laboratories do not have facilities for
the care of laboratory animals. In addition, immunoassay techniques are
less sensitive than instrumental analysis techniques.

The biotoxins may be considered as organic chemicals, and the same types
of sample preparation and instrumental analysis techniques may be
applicable, depending on the molecular weight of the biotoxin. Low
molecular weight toxins may be treated much like any organic chemical
described in Section 6.5, and may be analyzed by the same type of
analytical techniques (e.g., GC/MS) as described therein. Because
biotoxins tend to be very water soluble, liquid chromatography
techniques have been used to determine biotoxins in water. When LC/MS is
used, the same precautions may be necessary as those for other organic
chemicals (see Section 6.5.1). The analysis of biotoxins is one area
where LC/MS has proved particularly valuable, especially if the
molecular weight of the biotoxin precludes its analysis by GC/MS. Again,
the skill of the analyst is critical in obtaining meaningful results for
LC/MS analysis of biotoxins.

### Expanded Screening of Radionuclides 

Radiological analysis, as noted above, should be performed only by
licensed, specialty laboratories, and the need for such analysis should
be indicated by the field screening equipment for alpha, beta, and gamma
emitters. As discussed in Module 3, the site characterization will
determine the appropriate laboratory to receive radioactive samples
(e.g., high levels of radiation would indicate a radiation hazard, and
only the appropriate radiation laboratory would receive the sample). If
the laboratory should wish to screen samples before accepting them or
analyzing them, then inspection of samples for alpha, beta, and gamma
radioactivity using probes for these emitters may be prudent (e.g., the
devices used by the first responder/sample collector as described in
section 3.1.4). Again, a positive test from the screening devices should
be sent to specialty laboratories for confirmatory analysis.

The basic screen discussed in Section 6.4.8 is rather comprehensive
because it requires identification of the specific radionuclide if
indicated by the screens for gross alpha, beta, and gamma radiation.
Therefore, the expanded screen is designed to capture radionuclides that
do not fall into the energy range of the gross radionuclide screen for
gross alpha and beta. Fortunately, these radionuclides have specific EPA
900-Series methods designed for their analysis, and radionuclide
laboratories may be familiar with these few radionuclides and have other
reliable methods at their disposal for their analysis.

Two other techniques that may be particularly useful for radionuclide
analysis are gamma spectroscopy, which can directly identify the gamma
emitting radionuclide and inductively coupled plasma mass spectrometry
(ICP-MS). Principal considerations in the use of both of these
techniques include detection limits and availability of instrumentation.
Often, these considerations are based on the particular radionuclide
involved. To better understand and deal with the needs and roles of the
wide variety of analytical radiochemistry techniques at the disposal of
radionuclide laboratories, the Multi-Agency Radiological Laboratory
Analytical Protocols (MARLAP) Manual has been developed by the
Environmental Protection Agency, the Department of Energy, the Nuclear
Regulatory Commission, the Department of Defense, the National Institute
of Standards and Technology, along with some states
(<http://www.epa.gov/radiation/marlap/>). This effort is but one
component in the EPA's Radiological Emergency Response Plan
(<http://www.epa.gov/radiation/rert/docs/rerp-1-00.pdf>) in support of
EPA's responsibilities for protecting the environment and in support of
the Federal Radiological Emergency Response Plan
(<http://www.nrt.org/production/nrt/home.nsf/0/5c23c5d58074d6e48525660c005b56b5?OpenDocument>).

### Expanded Screening of Chemical Weapons

As discussed in Section 2.1.4, the term *chemical weapons* refers to the
substances, such as those shown in Table 1.1, that appear on Schedule 1
of the Chemical Weapons Convention (CWC,
<http://www.cwc.gov/Regulations/cfr-15/part-712-s1_html>). There are
other substances, such as chemical weapon precursors, that are monitored
by the CWC under Schedules 2 or 3, which also may be present in water at
very low concentrations (typically less than 10 g/L) due to industrial
pollution or the formation of disinfection byproducts. Table 4-18 in the
Appendix (Section 10) lists the Scheduled chemicals. Chemicals on
Schedules 2 or 3 have fewer restrictions compared with Schedule 1, since
many have legitimate industrial applications. Thus, environmental
laboratories could potentially analyze for many Schedule 2 or 3
chemicals. This section focuses on the analysis of Schedule 1 chemicals,
which are most restricted.

The Schedule 1 agents used as chemical weapons are extremely hazardous
to handle and most environmental chemistry laboratories do not have the
facilities or the procedures in place to handle these agents. In
addition, most of the agents are not available commercially to prepare
analytical standards for quantification. The chemical weapons agents
will need to be analyzed by special chemical weapons laboratories for
confirmatory analysis. However, in the unlikely case that an
environmental chemistry laboratory receives a sample containing a
chemical weapon, screening techniques can be used by environmental
chemistry laboratories to identify the presence of the agents in water.

The best analytical approach may be to utilize the preparatory
procedures (direct injection, micro-LLE, SPE, and/or SPME) as discussed
above, followed by GC/MS for identification. This may only be able to
determine the presence, not concentration, of the agent because an
analytical standard would not be available. However, the standard
electron impact mass spectral libraries frequently contain mass spectra
of these compounds and can be used for tentative identification. As an
aid to increasing confidence in chemical warfare agents' GC/MS library
matches, the National Institute of Standards and Technology has
developed the Automated Mass Spectral Deconvolution and Identification
System (AMDIS), <http://chemdata.nist.gov/mass-spc/amdis/>.

In the unlikely case CW agents are present, the expanded screen for
organic chemicals may be procedurally designed to reduce risk, namely
through reduction of aerosols. As with any organic chemical, an
additional way to reduce risk would be through sample dilution. The
laboratory may first start with the most dilute sample (1/1,000) and if
nothing is detected may proceed to analyze the next dilution (1/100),
followed by the 1/10 dilution, and lastly the undiluted sample. If the
laboratory proceeds through the undiluted sample and nothing is
detected, the sample may be deemed non-detect for the chemical weapons
that would be captured by the screen. If the chemical weapons agents are
identified in the screen, proper notifications should be made to law
enforcement to gain access to laboratory resources that can confirm the
presence of the chemical weapons agent.

#   {#section .unnumbered}

# Examples of the Analytical Approach to Site-Specific Situations for Chemical Contaminants

Section 6 describes an analytical approach based on chemical
contaminants considered important from a national perspective. As noted
above, local authorities may wish to develop their own analytical
approach based on their perceived needs and concerns. Here are some
brief examples of analytical approaches based on the concept of basic
and expanded screening. The first five (Section 7.1) are derived from
the needs of specific, hypothetical utilities, and the final three
(Section 7.2) are geared toward laboratories that serve a wider base of
clients.

The overriding principle in these examples is the flexibility that
laboratories have in developing and updating an analytical plan for the
analysis of unknowns in water. Utility laboratories may have the
greatest level of flexibility with respect to the comprehensiveness of
their screen **if** they have made arrangements with other labs to
perform more comprehensive screening as necessary. For example, utility
laboratories may choose to perform some level of basic screening that
may be applied in the case of a **'possible'** threat; however, analysis
may need to be performed at another laboratory in response to a threat
deemed **'credible'**. On the other hand, laboratories that serve a
wider customer base should have a more comprehensive and robust
screening procedure to cover the range of potential scenarios that might
be encountered in working with various utilities.

## Examples for Utility Laboratories

These five examples are targeted at **utility** laboratories that chose
to develop an analytical approach to support analysis of their own
emergency water samples, and make arrangements to engage other
laboratories with broader capabilities when necessary. This is to say,
it is part of upfront planning for a utility to decide when they will
handle samples in-house and when they will send them to external
laboratories. In many of these examples, the utility laboratories often
take a "springboard" approach to screening, using the results of the
basic screen to make decisions in the expanded screen.

**Example #1:** Based on the results of a vulnerability assessment, a
local water utility has concluded that their consumers are at only a
very small risk from water contamination incidents, and so do not want
to invest much of their limited resources in overly detailed analytical
approach. They possess a purge-and-trap-GC instrument with conventional
photoionization and electrolytic conductivity detectors in series, and
are certified to perform analysis of Method 502.2 analytes. They possess
a GC/MS for another purpose, so they choose to expand their laboratories
capabilities by making a small purchase of a solid-phase extraction
manifold and supplies suitable for Method 525.2 analytes. They allow
their laboratory staff the time to become proficient with Method 525.2
to ultimately obtain certification by their state. In the meantime, they
plan on using the technique in the expanded screen. They decide to
exclude carbamate pesticides and quaternary ammonium compounds from
their analytical approach because they are not available anywhere
nearby, and the utility does not believe that they will be important
because there are *opportunity contaminants* available locally. One of
these opportunity contaminants is glyphosate, which, although not very
toxic to humans, is widely used in the area. They search NEMI, or
another source such as those listed in Table 4-2, and find a suitable
method. They do not possess any capability for trace metal analysis, so
they contract this out to a third party who performs Method 200.7. In
looking at the analyte list for Method 200.7, the laboratory sees that
mercury can be analyzed by Method 200.7 and seeks data from the
contracted third party that their instrument provides sufficient
detection limits for arsenic and mercury. The utility laboratory
realizes Method 200.7 most likely will determine many organomercury
compounds (as total mercury), so they decide that the benefit of
enhancing their mercury screening capabilities through the addition of
Method 245.2 to their basic screen is not worth the expense. In their
locality, the first responders to contamination incidents possess, and
are trained in, the use of portable cyanide detection systems and
radiation detectors. They buy a low-cost gamma detector to screen
samples in the laboratory as a safety precaution. Thus, instead of Table
4-3, their basic analysis may be summarized in Table 4-5. Clearly, this
is not as rigorous as Table 4-3, but does meet many of the same needs.

During an incident, if the threat evaluation and site characterization
indicated that greater analytical capability is required for expanded
screening or confirmatory analysis, they plan to rely on state
resources, although there is a four-hour delay in getting the samples to
the state laboratories. If the water utility emergency response manager
(or the designated incident commander) must have results faster, they
plan to perform direct aqueous injection into their GC/MS with the hope
of getting a successful GC/MS library match. They also consider
performing a micro-liquid liquid extraction using methylene chloride for
the same purpose. However, due to the resulting additional instrument
maintenance requirements, which may make the instrument unavailable for
its intended purpose, they do not want to do this routinely.

Table 4-5. Basic Screen for Chemical Analysis for Example #1

  -------------------------- -------------------------- -----------------
  **Chemical (general        **Analytical Technique**   **EPA Method**
  class)**                                              

  Volatiles (organic)        Purge-and-trap PID/ELCD    502.2

  Semivolatiles (organic)    Solid-phase extraction     525.2
                             GC/MS                      

  Trace metals (inorganic)   Contracted out             200.7

  Cyanides                   By first responders        N/A

  Radionuclides              By first responders        N/A

  Local contaminant          HPLC-UV                    547
  (Glyphosate)                                          
  -------------------------- -------------------------- -----------------

**Example #2:** A utility laboratory has a great number of legally
defensible protocols (some of which are EPA methods) already in place,
except for radionuclides, which are handled by a separate department.
They examine the analytes in the methods in Table 4-3, and determine to
the best of their ability and interest that their protocols already
cover all the contaminants about which they are concerned. Thus, their
version of an analytical approach for chemicals may appear as shown in
Table 4-6.

Table 4-6. Basic Screen for Chemical Analysis for Example #2

  --------------------------- --------------------------- ----------------
  **Chemical (general         **Analytical Technique**    **EPA Method**
  class)**                                                

  Volatiles (organic)         Purge-and-trap GC/MS        In-house
                                                          protocol

  Semivolatiles (organic)     Solid-phase extraction      In-house
                              GC/MS                       protocol

  Carbamate pesticides        Derivatization GC/MS        In-house
  (organic)                                               protocol

  Quaternary nitrogen         HPLC -- UV                  In-house
  compounds (organic)                                     protocol

  Trace metals (inorganic)    ICP-MS                      In-house
                                                          protocol

  Total mercury (including    Cold vapor AA               In-house
  organomercury compounds)                                protocol

  Cyanides                    Wet chemistry               In-house
                                                          protocol

  Radionuclides               First responders            In-house
                                                          protocol
  --------------------------- --------------------------- ----------------

In addition, the laboratory possesses a significant capability in terms
of instrumentation and analytical expertise to perform the expanded
screening analysis, such as interpreting electron impact mass spectra
and performing immunoassays. Thus, Table 4-6 is consistent with their
needs and may comprise their analytical approach.

**Example #3:** A water utility decides its water security concerns are
reflected in the approach presented in Section 6. The utility has access
to laboratories that are capable of performing the methods in Table 4-3,
except radionuclide analysis, which is handled by emergency responders
in accordance with state policy. However, they do not have access to
expanded screening techniques, and choose not to include in-house
expanded screening, since their local emergency operations policy does
not allow time for detailed expanded screening. Thus, decisions would
typically be based on the results from the basic analytical screen.
However, the utility has made arrangements with a commercial laboratory
to perform some expanded screening if deemed necessary based on the
circumstances of the incident.

Table 4-7. Basic Screen for Chemical Analysis for Example #3

  --------------------------- --------------------------- ----------------
  **Chemical (general         **Analytical Technique**    **EPA Method**
  class)**                                                

  Volatiles (organic)         Purge-and-trap GC/MS        524.2

  Semivolatiles (organic)     Solid-phase extraction      525.2
                              GC/MS                       

  Carbamate pesticides        HPLC -- fluorescence        531.1
  (organic)                   detection                   

  Quaternary nitrogen         HPLC -- UV                  549.2
  compounds (organic)                                     

  Trace metals (inorganic)    Graphite furnace AA         200.9

  Total mercury (including    Cold vapor AA               245.1 with
  organomercury compounds)                                persulfate

  Cyanides                    Wet chemistry               335.3

  Radionuclides               Emergency responders        
  --------------------------- --------------------------- ----------------

**Example #4:** A water utility only has capability for basic water
quality parameters, such as pH, turbidity, and chlorine residual. The
utility has always sent samples to the state laboratory for routine
analysis and does not have resources to do differently, even in the case
of an emergency. They do, however, make plans for how to most
efficiently deliver the samples to the state laboratory, and recruit
employees from other city and county offices to help implement the plan
if the regular staff of two persons is not available. Note that the
techniques used by the state laboratory (shown in Table 4-8) are the
same as in Table 4-3. However, from the utility's perspective, the state
laboratory governs the selection of the techniques.

Table 4-8. Basic Screen for Chemical Analysis for Example #4

+--------------------------+--------------------------+----------------+
| **Chemical (general      | **Analytical Technique** | **EPA Method** |
| class)**                 |                          |                |
+--------------------------+--------------------------+----------------+
| Volatiles (organic)      | Purge-and-trap GC/MS     | 524.2          |
+--------------------------+--------------------------+----------------+
| Semivolatiles (organic)  | Solid-phase extraction   | 2525A/8270D    |
|                          | GC/MS                    |                |
+--------------------------+--------------------------+----------------+
| Carbamate pesticides     | HPLC -- fluorescence     | 531.1          |
| (organic)                | detection                |                |
+--------------------------+--------------------------+----------------+
| Quaternary nitrogen      | HPLC -- UV               | 549.2          |
| compounds (organic)      |                          |                |
+--------------------------+--------------------------+----------------+
| Trace metals (inorganic) | ICP-MS                   | 200.8          |
+--------------------------+--------------------------+----------------+
| Cyanides                 | Wet chemistry            | 335.3          |
+--------------------------+--------------------------+----------------+
| Radionuclides            | Gross alpha, gross beta, | technique      |
|                          |                          | referred by    |
|                          | gross gamma radiation    | state lab      |
+--------------------------+--------------------------+----------------+

**Example #5:** A utility laboratory has a purge-and-trap GC/MS, a
regular GC/MS, and an ICP-MS. Their basic screen is represented in Table
4-9.

Table 4-9. Basic Screen for Chemical Analysis for Example #5

  --------------------------- --------------------------- ----------------
  **Chemical (general         **Analytical Technique**    **EPA Method**
  class)**                                                

  Volatiles (organic)         Purge-and-trap GC/MS        524.2

  Semivolatiles (organic)     Solid-phase extraction      525.2
                              GC/MS                       

  Trace metals (inorganic)    ICP-MS                      200.8
  --------------------------- --------------------------- ----------------

While this leaves some gaps in their analytical coverage, they plan to
address these deficiencies through an expanded screen. They acquired
immunoassay test kits that have undergone some level of validation, such
as ETV (<http://www.epa.gov/etv>). For these and other kits, the
laboratory understands the limitations of these test kits (see Section
6.5.1 and 6.5.4) and has arranged analytical confirmation of the
analytes with state laboratories should the need arise. The laboratory
also has an agreement with a local university that has a mass
spectrometry facility including an LC/MS. The utility laboratory takes
steps to ensure that the university laboratory/staff will be available
in the case of an emergency and all chain-of-custody rules and legal
defensibility issues have been met. The utility also has made
arrangements to contract out the confirmatory analysis, if necessary.
Although the utility recognizes there are potential problems with this
arrangement, it believes this plan meets its needs.

## Examples for Laboratories with a Wide Client Base

The concept of basic screening takes on a different dimension for
laboratories that serve a wide client base because utility laboratories
are most likely faced with 'possible' incidents. Accordingly, they may
feel reasonably confident in the comprehensiveness of the screening
procedures, illustrated in examples #1-#5. On the other hand,
laboratories that serve a wide client base have a larger opportunity to
receive a sample from a 'credible' incident. There are two reasons for
this. First, these laboratories serve more clients, so on a strictly
mathematical basis, they have a greater chance. Second, utility
laboratories in 'credible' situations may be more likely to refer
samples to another laboratory, which may have greater capability to
apply a more focused analytical approach in response to a specific
incident (as discussed in Section 5).

Accordingly, laboratories that choose to perform analysis of emergency
water samples and serve a broader client base, such as Federal, State,
and commercial labs, may need to adopt a more comprehensive approach
such that they maintain flexibility to provide analytical support in a
variety of situations. However, as the following three examples
illustrate, this goal can be met in a variety of ways.

The first two examples represent extreme cases of laboratories preparing
to respond to a 'possible' and/or 'credible' incident. The first example
represents a laboratory that does not define a basic screen and will
perform analyses as part of a comprehensive expanded screening program.
The second example depicts a laboratory that attempts to be
comprehensive by including a very large number of standard methods in
their basic screen. The third example blends the first two examples and
adopts a screening approach that aligns closely with the complete "Basic
Screen" (Table 4-3) and has some capability for the "Expanded Screen"
(Table 4-4), as well.

**Example #6.** Utilities in a county are all serviced by the same
central laboratory. However, the needs of the utilities are somewhat
different, so after a meeting between all utility and laboratory
representatives, it is agreed that the plan is to perform no basic
screens. The decision was based on response times for utilities,
mandated by the locality, being too short for the basic screen. The
laboratory will instead perform the expanded screen using exploratory
techniques that have short response time, such as those discussed in
Section 6.5 (Table 4-4). The laboratory has skilled analysts and modern
equipment and is confident of its capability. The laboratory also is
capable of performing confirmatory techniques for some contaminants, if
necessary. The planners realize that this approach may not meet all
desired QC goals for every contaminant, but are prepared to live with
the consequences of basing decisions on this kind of data.

Table 4-10. Basic Screen for Chemical Analysis for Example #6

  --------------------------- --------------------------- ----------------
  **Chemical (general         **Analytical Technique**    **EPA Method**
  class)**                                                

  None                        \--                         \--
  --------------------------- --------------------------- ----------------

**Example #7.** A state laboratory that supports a large number of
utilities. The laboratory performs many EPA drinking water and SW-846
methods routinely, and it wants to include as many techniques as
possible in the basic screen. It chooses to do this since the needs of
water systems it serves vary greatly, and most are small and
understaffed to the point that the utilities generally rely on the state
for all analyses and analytical decisions. The laboratory also realizes
that only a broad screen will be sufficient to cover the majority of
potential emergency scenarios that its utility clients may face. Also,
the laboratory wants to exercise "due diligence" up to the level of a
'credible' threat, as defined by the threat evaluation , and it realizes
that more analytes are captured by these methods than are in the basic
screen in Table 4-3, and that some of the methods are more sensitive for
particular analytes. In all, the laboratory feels that the inclusion of
all these methods would be valuable for their clients.

Note that the laboratory chooses preparation methods for the SW-846
methods to make them compatible with the drinking water methods, so that
it does not have to prepare the sample twice in many cases. The
laboratory feels that its basic screen best meets the needs of its
clients. It does perform some expanded screening to try to identify
unknown chromatographic peaks in its analysis and through combining all
the analytical results from the various techniques.

Table 4-11. Basic Screen for Chemical Analysis for Example #7

  --------------------------- --------------------------- ----------------
  **Chemical (general         **Analytical Technique**    **EPA Method**
  class)**                                                

  Volatiles (organic)         Purge-and-trap GC/MS        8260B

  Semivolatiles (organic)     Solid-phase extraction      3535A/8270D
                              GC/MS                       

  Nitrogen and                GC/NPD                      507
  phosphorus-containing                                   
  pesticides (organic)                                    

  Chlorinated pesticides,     GC/ECD                      508.1
  herbicides, and                                         
  organohalides (organic)                                 

  Chlorinated acids (organic) GC/ECD                      515.1

  Phenols (organic)           solid-phase extraction      528
                              GC/MS                       

  Carbamate pesticides        HPLC -- fluorescence        531.1
  (organic)                   detection                   

  Glyphosate (organic)        HPLC-UV                     547

  Quaternary nitrogen         HPLC -- UV                  549.2
  compounds (organic)                                     

  Chlorination disinfection   GC/ECD                      551.1
  byproducts, chlorinated                                 
  solvents, and halogenated                               
  pesticides/herbicides                                   
  (organic)                                               

  Water soluble               GC/FID with direct aqueous  8015B/5031
  alcohols/non-chlorinated    injection                   
  organics (organic)                                      

  Organochlorine pesticides   GC/MS                       8081A
  (organic)                                               

  Organophosphorus compounds  GC/MS                       8141A
  (organic)                                               

  Chlorinated herbicides      GC/MS after methylation or  8151A
  (organic)                   pentafluorobenzylation      

  Trace metals (inorganic)    ICP-MS in scanning mode     200.8

  Total mercury (including    Cold vapor AA               245.2 with
  organomercury compounds)                                persulfate

  Cyanides                    Wet chemistry               335.3

  Biotoxins                   Immunoassays with           
                              confirmatory techniques     

  Radionuclides               Gross alpha, gross beta,    technique
                              gross gamma radiation       referred by lab
  --------------------------- --------------------------- ----------------

**Example #8:** A laboratory serves an area that has several drinking
water treatment plants located downstream from an agricultural area, an
industrial area, and/or a nuclear power plant. In addition, there are
geological formations that contribute to high inorganic contaminant
levels. Fortunately, the laboratory is very well equipped and staffed,
with strong capabilities and experience in monitoring all the classes of
contaminants in Table 4-3. In addition, the laboratory expands the
analyte list (see Table 4-20) for these methods to include as many
analytes as possible.

Table 4-12. Basic Screen for Chemical Analysis for Example #8

  --------------------------- ------------------------- ------------------
  **Chemical (general         **Analytical Technique**  **EPA Method**
  class)**                                              

  Volatiles (organic)         Purge-and-trap GC/MS      524.2

  Semivolatiles (organic)     Solid-phase extraction    3535A/8270D
                              GC/MS                     

  Carbamate pesticides        HPLC -- fluorescence      531.1
  (organic)                   detection                 

  Quaternary nitrogen         HPLC -- UV                549.2
  compounds (organic)                                   

  Trace metals (inorganic)    ICP-MS                    200.8

  Total mercury (including    Cold vapor AA             245.2 with
  organomercury compounds)                              persulfate

  Cyanides                    Wet chemistry             335.3

  Radionuclides               Gross alpha, gross beta,  technique
                              gross gamma               preferred by lab
  --------------------------- ------------------------- ------------------

Due to sources of potential contaminants other than intentional
contamination, the laboratory also maintains careful records of ranges
of contaminants it normally encounters, so that during an emergency
incident, it does not confuse background levels of contaminants with
intentionally introduced ones.

For expanded screening, the laboratory utilizes its existing
instrumentation with the techniques discussed in Section 6.5. The
laboratory is particularly interested in developing an HPLC screen for
pharmaceuticals (which it was already interested in due to potential
regulatory issues), and would like to explore micro-LLE and SPME for
this purpose. Therefore, the laboratory acquires the required equipment
and supplies, and encourages its staff to investigate these techniques.

# Analytical Approach for Biological Contaminants

## Overview of Contaminant Issues 

### Types of Biological Contaminants Covered by the Analytical Approach

Two broad classes of *pathogens* may be encountered in a contaminated
water sample: 1) traditional waterborne pathogens such as enteric
bacteria, viruses and protozoa; and 2) exotic and select agents that may
be suitable for a water route of transmission. Table 4-13 lists some of
these pathogens. This section is intended as a guide for selecting
analytical approaches for samples suspected of containing known and
unknown microbial contaminants. Specific methodologies will be addressed
in a laboratory analysis guide for biological contaminants (under
development).

**Table 4-13. General Classes of Pathogens, Specific Organisms, and
Select Agent Status**

  ---------------------------- --------------------------- --------------
  **Pathogen general class**   **Organism**                **Select
                                                           Agent\***

  Bacteria (non-spore forming) *Brucella* spp.             Yes

                               *Burkholderia               Yes
                               pseudomallerei*             

                               *Campylobacter* spp.        

                               E. coli 0157:H7             

                               *Francisella tularensis*    Yes

                               *Salmonella* spp.           

                               *Shigella* spp.             

                               *Vibrio cholerae*           

                               *Yersinia pestis*           Yes

  Bacteria (spore forming)     *Bacillus anthracis*        Yes

                               *Clostridium botulinum* A   Yes

  Bacteria (Rickettsia)        *Coxiella burnetti*         Yes

  Protozoa                     *Cryptosporidium parvum*    

                               *Entamoeba histolytica*     

                               *Giardia intestinalis*      

                               *Toxoplasma gondii*         

  Viruses                      Enteroviruses               

                               Hepatitis A                 

                               Hepatitis E                 

                               Noroviruses                 

                               Rotavirus                   

                               Variola                     Yes

                               VEE                         Yes

                               VHF                         Yes
  ---------------------------- --------------------------- --------------

\* See <http://www.cdc.gov/od/sap/docs/salist.pdf>

### Selection of Appropriate Analytical Approach for Contaminant Analysis

The analytical approach for these biological contaminants can be divided
into two distinct steps, sample collection and sample analysis. All
information prior to and during the threat evaluation (Module 2) needs
to be carefully considered to assess the microbial contaminant threat
and to apply a relevant sample collection and sample analysis procedure.
Different sample collection and analysis approaches are described below.

The overall approach for sample collection and analysis for microbial
contaminants is based on the target contaminant. When the target
contaminant is known, then established and validated technologies for
sample collection and methods for analysis are utilized. When the target
contaminant is unknown or when the contaminant is known but established
and validated procedures for collection are not available, then an
ultrafiltration device is used for sample collection and exploratory
methods of analysis are utilized.

### Microbial Sample Collection Considerations

Figure 4-8 illustrates the overall approach for sample collection for
microbial contaminants.

This approach utilizes established and validated sample collection
techniques when the target contaminant has been tentatively identified
based on available information from the site characterization report.
Examples of situations in which tentative identification might occur
include: a specific contaminant named in a threat; physical evidence at
the site pointing to a specific contaminant; and clinical evidence of
the identity of the disease-causing contaminant. In the case where the
contaminant is unknown, sample collection is performed through the use
of an ultrafiltration sampler (see Section 8.3).

![](media/image9.wmf)

**Figure 4-8. Analytical Approach for the Collection of Microbial
Contaminants**

The ultrafiltration approach may also be desirable for a number of
reasons. First, the sample may contain a mixture of microbial
contaminants. Second, due to the low infectious dose of most waterborne
pathogens, a sample of 100 liters of finished water may need to be
concentrated to obtain suitable detection limits. Third, sample
concentration technology for viruses and protozoa require separate field
equipment and procedures and are practical only if the microbial
contaminant is suspected or identified as one amenable for concentration
by each of these specific technologies. Thus, in a contamination threat
or incident, it may be necessary to utilize a more general sample
concentration procedure based on ultrafiltration that targets a wider
range of microorganisms.

### Microbial Processing and Analysis Considerations

Making a determination as to whether the microbial contaminant has been
tentatively identified is important since it will determine the type of
laboratory that can do the processing and analysis. Non-select agents or
BSL levels 1 and 2 contaminants that are regarded as waterborne
pathogens, including the enteric bacteria, viruses and protozoa, can be
sent to an environmental laboratory. If the contaminant is unknown or is
a select agent, the sample is to be shipped to a specialty laboratory
(Figure 4-9).

![](media/image10.wmf)

**Figure 4-9. Approach for the Processing and Analysis of Microbial
Contaminant Groups**

\*The specialty laboratory includes the Laboratory Response Network
Laboratories among other specialty labs.

## Established Techniques for Sample Collection of Known Waterborne Contaminants

If a microbial contaminant has been tentatively identified, the
available and established techniques may be the more direct approach for
sample collection and analysis. The advantage of these techniques is
that a broader base of expertise and more analytical laboratories are
available to water utilities than is available for select agents and for
exploratory techniques.

Established and validated methods for sample collection and analysis of
microbial contaminants in water are limited to a few microorganisms,
mainly some enteric bacteria, viruses and protozoa, and a few other
organisms with a known waterborne transmission route, listed in Table
4-14. If the contaminant is not known or the available site
investigation and intelligence information is not considered
sufficiently reliable for the use of an established method, the
procedure for microbial unknowns (Section 8.4) is to be used.

**Table 4-14. Known Waterborne Pathogens with Established Techniques for
Sample Collection and Analysis**

+-------------+-------------------+-------------------+---------------+
| **Pathogen  | **Organism**      | **Analytical      | **Method^1^** |
| (general    |                   | Technique**       |               |
| class)**    |                   |                   |               |
+-------------+-------------------+-------------------+---------------+
| Bacteria    | *Campylobacter    | Culture in        | SM 9260 G     |
|             | spp.*             | selective media   |               |
|             |                   |                   | SM 9260 F     |
|             | E. coli 0157:H7   |                   |               |
|             |                   |                   | SM 9260 B     |
|             | *Salmonella spp.* |                   |               |
|             |                   |                   | SM 9260 E     |
|             | *Shigella spp.*   |                   |               |
|             |                   |                   | SM 9260 H     |
|             | *Vibrio cholerae* |                   |               |
+-------------+-------------------+-------------------+---------------+
| Protozoa    | *Cryptosporidium  | IMS/IFA^2^        | EPA 1622/23   |
|             | parvum*           |                   |               |
|             |                   |                   | EPA 1623      |
|             | *Giardia          |                   |               |
|             | intestinalis*     |                   |               |
+-------------+-------------------+-------------------+---------------+
| Viruses     | Enteroviruses     | Mammalian cell    | EPA           |
|             |                   | culture           | /600/4-84/013 |
|             | Hepatitis A       |                   |               |
|             |                   |                   | EPA ICR       |
|             |                   |                   | manual        |
+-------------+-------------------+-------------------+---------------+

1\. SM refers to Standard Methods for the Examination of Water and
Wastewater (Clesceri *et. a.l.*, 1999). The EPA methods may be found at
<http://www.epa.gov/nerlcwww/>

2\. Immunomagnetic separation (IMS) and immunofluorescence assay (FA)
microscopy.

### Bacteria Collection with Established Techniques

The established sample collection procedures for bacteria are
essentially *grab samples* that are later processed by membrane
filtration of typically 100 milliliters to two liters. A four liter grab
sample will allow for analysis for different bacteria by plating
multiple membrane filters on separate selective or enrichment media.
Special shipping regulations apply to samples known to contain pathogens
(see Module 3, Section 6 for requirements for shipment of infectious
materials, <http://www.bt.cdc.gov/labissues/PackagingInfo.pdf>). If
select agents are not known or are not suspected to be in the water,
then the sample is considered an environmental sample, and volumes of
four liters or less can be collected and shipped using standard sampling
containers.

### Virus Collection with Established Techniques

The established sample collection procedures for viruses involve
concentration on positively charged filters (ICR Microbial Laboratory
Manual, EPA/600/R-95/178, April 1996, <http://www.epa.gov/nerlcwww/>).
This method is reasonably effective for concentration of enteric viruses
from large volumes of water. Samples in excess of 100 liters can be
easily concentrated by this method. The ICR method has been used to
concentrate 1,200 liters of finished water. The processed filters can be
shipped to the laboratory, or viruses adsorbed to the filter can be
eluted in the field and shipped as a one-liter concentrate to a
laboratory for further processing by conventional reconcentration
procedures.

### Protozoa Collection with Established Techniques

The established procedure for protozoa sampling in water consists of
obtaining a 10-liter grab sample or field concentration by filtration
(Method 1623: Cryptosporidium and Giardia in Water by Filtration by
IMS/FA, EPA-821-R-99-006, April 1999, <http://www.epa.gov/nerlcwww/>).
The ten-liter grab sample or the filter is shipped to the laboratory for
further processing.

## Established Techniques for Analysis of Known Waterborne Contaminants

Established analytical techniques are based on culture assays in
selective media for bacteria, selected cell lines for viruses and IFA
with antibodies for protozoa identification (Table 4-14). Culture based
techniques are not available for some contaminants and these techniques
have long turn-around times for analysis. Thus, it may be necessary to
utilize exploratory methods (Section 8.4.3) due to time restraints or
the absence of an established culture method for a particular pathogen.
Exploratory methods are primarily molecular-based, and involve PCR and
probe hybridization as a *presumptive test*, followed by sequence
analysis to confirm. If necessary, samples could be simultaneously
analyzed by both rapid exploratory methods and established methods.

### Bacteriological Analysis with Established Techniques

Sample collection for laboratory analysis of pathogenic bacteria in
water samples consists of processing a grab sample by membrane
filtration and transferring the membrane onto selective or enrichment
media for presumptive identification (Figure 4-10). The enteric bacteria
and other non-select pathogenic bacteria can be confirmed by
conventional methods based on biochemical profiles, serological
identification, or other culture based methods. The vast majority of
enteric bacteria can be analyzed with BSL-2 containment level; thus an
environmental laboratory with that containment level could proceed with
confirmatory identification of non-select organisms.

If the presumptive tests indicate the possible presence of a select
agent, the sample and the isolated culture must be referred to a
specialty laboratory (Figure 4-10). Federal regulations prohibit the
handling and maintenance of select agents unless the laboratory has
registered with the CDC under the requirements of the Select Agent Rule
(see Section 2.2).

![](media/image11.wmf)

Figure 4-10. Bacteriological Screening with Established Techniques

### Virological Analysis with Established Techniques

Established techniques for virological analysis consist of plating the
sample concentrate on cell lines receptive to the target viruses. The
target viruses anticipated in a water sample are the enteric viruses.
There are cell lines receptive to a number of them (see Table 4-13). It
may be necessary to inoculate a sample on multiple cell lines to cover
the range of possible virus targets. A presumptive positive result of a
cell culture test is the production of cytopathic effects on the cell
monolayer as observed by light microscopy. Established virus
confirmation and identification techniques consist of serological
neutralization with specific antibodies against the various viruses or
by means of pools of antisera with respective identification tables. If
a presumptive identification suggests the presence of a select virus
agent, the sample, culture and other processed sample materials must be
referred to a specialty laboratory (Figure 4-11).

If an established technique is not available for a specific virus or if
the virus is known not to grow in cell culture, exploratory PCR-based
techniques may be the more appropriate approach for presumptive
identification. Presumptive identification is based on PCR amplification
and probing. Analytical confirmation is conducted by sequence analysis
of the amplified material.

![](media/image12.wmf)

**Figure 4-11. Virological Analysis with Established Techniques**

[]{#__RefHeading___Toc58462052 .anchor}

### Parasitological Analysis with Established Techniques

Established sample collection techniques for protozoa in water consist
of obtaining a grab sample of 10 liters or alternatively, performing
filtration in the field (EPA Method 1623,
<http://www.epa.gov/nerlcwww/>). The grab sample or the filter is sent
to an environmental laboratory for processing. In the laboratory,
protozoa retained by the filter are eluted, concentrated by
centrifugation and purified by immunomagnetic separation (IMS).
*Giardia* cysts and *Cryptosporidium* oocysts are identified and counted
by the indirect fluorescent assay with an epifluorescence (or UV)
microscope (Figure 4-12).

Exploratory techniques such as PCR can be readily applied to cysts and
oocysts purified by the immunomagnetic separation step of Method 1623.
In addition, the infectivity of *Cryptosporidium* oocysts can also be
determined by cell culture infectivity after IMS purification.

![](media/image13.wmf)

**Figure 4-12. Parasitological Analysis with Established Techniques**

[]{#__RefHeading___Toc58462053 .anchor}

## Method for Sample Collection, Field Processing, and Laboratory Analysis for Unknown Microbial Contaminants

> Sample collection, processing, and analysis of an unknown microbial
> contaminant are inherently linked because the procedure relies on the
> use of an ultrafiltration device in the field (see Module 3).
> Together, the collection, processing, and analysis may be considered
> to constitute a method for unknown microbial contaminants, and the
> individual parts are discussed below. This method, including a
> suitable ultrafiltration device, is currently under development.

### Sample Collection and Concentration for Unknown Microbials

Ultrafiltration is a size exclusion process, and particles larger than
the molecular weight cut-off (MWCO) of the membrane, are concentrated in
the *retentate* and separated from the *filtrate*. Ultrafiltration can
concentrate viruses, bacteria, spores or parasites as long as the
organisms are larger than the MWCO of the membrane selected. Thus, the
method is suitable for sampling water with an unknown biological
contaminant. Also, ultrafiltration of water samples can be used to
concentrate some biotoxins if the MWCO of the selected membrane is
sufficiently small.

The overall procedure, illustrated in Figure 4-13, consists of
concentrating large (100 liters or more) volumes of water by cross-flow
disposable hollow fiber ultrafiltration cartridges, and subsequently
recovering pathogens and other concentrated microorganism and particles
in the filter retentate. The recovered retentate is referred to as the
*field sample concentrate*. The field sample concentrate will be
separated into three aliquots. Ultrafiltration of 100 liters or more of
finished (e.g., tap) water will result in a retentate volume of
approximately 250 ml. A small aliquot (1/20^th^ of the field sample
concentrate) can be used for field screening (Section 8.4.2).
Approximately one half of the field sample concentrate can be used for
presumptive PCR testing (Section 8.4.3). The remainder can be used for
confirmatory culture techniques (Section 8.4.4). The reason for dividing
the field sample concentrate is that some of the reagents for molecular
analysis that may be added in the field will essentially inactivate any
microbes present in the sample.

The basic elements of an ultrafiltration field concentration apparatus
(Figure 4-14) consist of an input reservoir for sample collection, a
peristaltic pump, a cross-flow hollow fiber membrane cartridge, a
retentate reservoir and a filtrate reservoir. Operating the unit in
cross-flow mode is preferable because "caking" or fouling of membranes
is reduced. Non-reactive membranes are preferable, such as polysulfone
or low protein binding membranes.

Sample filtration is conducted by recirculating water (necessary to
maintain cross-flow conditions) with a 5-10 PSI differential between the
input and retentate pressure. The pressure differential forces water
through the hollow fiber membrane which retains particles (including
microbes) larger than the membrane pores in an increasingly concentrated
liquid retentate volume. The water containing particles that passes
through the filter is known as filtrate. The volume of the retentate is
reduced to approximately 250 ml by recirculating the concentrated sample
through the smaller retentate loop for capture in the retentate
reservoir. The retentate reservoir vessel can be used for sample
shipping if necessary. The sample retentate can also be recovered by
backflushing (reversing the flow through of the hollow fiber membrane
cartridge).

![](media/image14.wmf)

**Figure 4-13. Sample Collection and Processing for Unknown Microbial
Contaminants**

![](media/image15.wmf)

**Figure 4-14. Field Concentrator Schematic**

### Field Screening and Processing of Sample

Field processing of the small aliquot of the field sample concentrate
from the ultrafiltration concentrator consists of field screening for
microbial contaminants (Figure 4-15), which also is a component of site
characterization (see Module 3). This field screening, in turn, may
result in rapid actions to prevent further adverse consequences to
safety and health of the samplers, laboratory personnel, and the general
public. It is important to remember that both field screening by
equipment and field test assays have only limited use, and any results
need to be confirmed by additional laboratory testing.

![](media/image16.wmf)

**Figure 4-15. Field Processing for Microbials**

Field screening equipment encompasses a wide range of technologies
including lateral flow chromatography, antibody identification,
hand-held or field deployable PCR based assays, and others (Table 4-15).
The specific technology chosen will affect the interpretation of field
results. The Office of Justice Programs of the Department of Justice
(DOJ) has provided a list of available technologies for field screening
(<http://www.ojp.usdoj.gov/nij/pubs-sum/190747.htm>). (The DOJ document
contains neither an endorsement of any specific technology nor an
evaluation of their performance.)

Many of the available field screening assay tests (Table 4-15) are less
sensitive than laboratory-based assays by perhaps several orders of
magnitude, and thus there is a potential for false negative results. Due
to possible cross-reactivity, these technologies may also lead to false
positive reactions. The results of field screening should be used as
part of a threat characterization process and not as part of a
conclusive analytical procedure.

Table 4-15. Exploratory Field Screening Techniques for Waterborne
Contaminants and Select Agents

+-------------+-------------------+-------------------+---------------+
| **Pathogen  | **Organism**      | **Screening       | **Commercial  |
| (general    |                   | Technique\***     | Sources**     |
| class)**    |                   |                   |               |
+-------------+-------------------+-------------------+---------------+
| *           |                   |                   |               |
| *Waterborne |                   |                   |               |
| Con         |                   |                   |               |
| taminants** |                   |                   |               |
+-------------+-------------------+-------------------+---------------+
| Bacteria    | *Campylobacter*   | PCR               | Yes           |
|             | spp.              |                   |               |
|             |                   | PCR               |               |
|             | *E. coli* 0157:H7 |                   |               |
|             |                   | PCR               |               |
|             | *Salmonella* spp. |                   |               |
+-------------+-------------------+-------------------+---------------+
| Protozoa    |                   | TBD               | TBD           |
+-------------+-------------------+-------------------+---------------+
| Viruses     |                   | TBD               | TBD           |
+-------------+-------------------+-------------------+---------------+
| **Select    |                   |                   |               |
| Agents**    |                   |                   |               |
+-------------+-------------------+-------------------+---------------+
| Spores      | *Bacillus         | PCR and Smart     | Yes           |
|             | anthracis*        | tickets           |               |
|             |                   |                   |               |
|             | *Clostridium      | PCR               |               |
|             | botulinum A*      |                   |               |
+-------------+-------------------+-------------------+---------------+
| Bacteria    | *Brucella* spp.   | PCR               | Yes           |
|             |                   |                   |               |
|             | *Francisella      |                   |               |
|             | tularensis*       |                   |               |
|             |                   |                   |               |
|             | *Yersinia pestis* |                   |               |
+-------------+-------------------+-------------------+---------------+
| Protozoa    |                   | TBD               | TBD           |
+-------------+-------------------+-------------------+---------------+
| Viruses     |                   | TBD               | TBD           |
+-------------+-------------------+-------------------+---------------+

\* TBD = to be determined

### Presumptive Testing for Microbials with Exploratory PCR Techniques

For presumptive testing of the field sample concentrate from the
ultrafiltration concentrator, exploratory techniques such as PCR testing
may be utilized as a means to obtain faster presumptive results for
critical samples. These presumptive results may be used during the
threat evaluation (Module 2) and site characterization (Module 3)
procedures. Although PCR techniques are widely used in the clinical and
medical fields, these techniques are still regarded as exploratory in
the field of environmental microbiology. PCR has three main benefits in
a presumptive test: 1) results can be obtained faster; 2) multiple
targets can be assayed in a short period of time; and 3) cultural
methods are not available yet for some microbial contaminants of
interest. Another advantage of PCR testing is that samples could be
analyzed concurrently with the conventional established methods. Table
4-16 lists PCR techniques for the biological contaminants of concern.

Table 4-16. Exploratory Analytical Techniques for Presumptive Testing of
Waterborne Contaminants and Select Agents

+-------------+-------------------+---------------+-------------------+
| *           | **Organism**      | **Presumptive | **Confirmation**  |
| *Pathogen** |                   | Testing       |                   |
|             |                   | Technique**   |                   |
| **general   |                   |               |                   |
| class**     |                   |               |                   |
+-------------+-------------------+---------------+-------------------+
| *           |                   |               |                   |
| *Waterborne |                   |               |                   |
| Con         |                   |               |                   |
| taminants** |                   |               |                   |
+-------------+-------------------+---------------+-------------------+
| Bacteria    | *Campylobacter*   | PCR           | Probe or          |
|             | spp.              |               | sequencing        |
|             |                   | PCR           |                   |
|             | E. coli 0157:H7   |               | Probe or          |
|             |                   | PCR           | sequencing        |
|             | *Salmonella* spp. |               |                   |
|             |                   | PCR           | Probe or          |
|             | *Shigella* spp.   |               | sequencing        |
|             |                   | PCR           |                   |
|             | *Vibrio cholerae* |               | Probe or          |
|             |                   |               | sequencing        |
|             |                   |               |                   |
|             |                   |               | Probe or          |
|             |                   |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| Protozoa    | *Cryptosporidium  | PCR           | Probe or          |
|             | parvum*           |               | sequencing        |
|             |                   | PCR           |                   |
|             | *Giardia          |               | Probe or          |
|             | intestinalis*     | PCR           | sequencing        |
|             |                   |               |                   |
|             | *Entamoeba        | PCR           | Probe or          |
|             | histolytica*      |               | sequencing        |
|             |                   |               |                   |
|             | *Toxoplasma       |               | Probe or          |
|             | gondii*           |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| Viruses     | Enteroviruses     | RT-PCR        | Probe or          |
|             |                   |               | sequencing        |
|             | Hepatitis A       | RT-PCR        |                   |
|             |                   |               | Probe or          |
|             | Hepatitis E       | RT-PCR        | sequencing        |
|             |                   |               |                   |
|             | Noroviruses       | RT-PCR        | Probe or          |
|             |                   |               | sequencing        |
|             | Rotavirus         | RT-PCR        |                   |
|             |                   |               | Probe or          |
|             |                   |               | sequencing        |
|             |                   |               |                   |
|             |                   |               | Probe or          |
|             |                   |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| **Select    |                   |               |                   |
| Agents**    |                   |               |                   |
+-------------+-------------------+---------------+-------------------+
| Spores      | *Bacillus         | PCR           | Probe or          |
|             | anthracis*        |               | sequencing        |
|             |                   | PCR           |                   |
|             | *Clostridium      |               | Probe or          |
|             | botulinum* A      |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| Bacteria    | *Brucella* spp.   | PCR           | Probe or          |
|             |                   |               | sequencing        |
|             | *Burkholderia     | PCR           |                   |
|             | pseudomallerei*   |               | Probe or          |
|             |                   | PCR           | sequencing        |
|             | *Francisella      |               |                   |
|             | tularensis*       | PCR           | Probe or          |
|             |                   |               | sequencing        |
|             | *Yersinia pestis* |               |                   |
|             |                   |               | Probe or          |
|             |                   |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| Rickettsia  | *Coxiella         | PCR           | Probe or          |
|             | burnetti*         |               | sequencing        |
+-------------+-------------------+---------------+-------------------+
| Viruses     | Variola\*         | PCR           | Probe or          |
|             |                   |               | sequencing        |
|             | VEE\*             | RT-PCR        |                   |
|             |                   |               | Probe or          |
|             | VHF\*             | RT-PCR        | sequencing        |
|             |                   |               |                   |
|             |                   |               | Probe or          |
|             |                   |               | sequencing        |
+-------------+-------------------+---------------+-------------------+

\*These viruses are not considered to pose a major threat in water

Protozoa and bacteria in the field sample concentrate are further
concentrated by centrifugation of the sample in the laboratory (Figure
4-16). Possible spores, bacteria or protozoa in the sample are recovered
in the pellet whereas viruses will be retained in the supernatant. DNA
from spores, vegetative bacteria or protozoa is extracted from the
pellet. DNA extraction kits designed to remove co-contaminants from
environmental samples are more desirable since ultrafiltration will tend
to concentrate soil-derived PCR inhibitors (e.g., humic and fulvic
acids). The supernatant is further concentrated by a second phase of
ultrafiltration. RNA-containing viruses are the anticipated target in a
water sample; thus, a procedure for RNA extraction and purification is
to be followed. The purified RNA is used for reverse transcriptase-PCR
(RT-PCR) to desired viral RNA targets.

Primers and probes for PCR presumptive testing for waterborne and
non-select agents may be obtained from the scientific literature. Some
commercial vendors provide primers and probes for select agents,
although these are not necessarily utilized by the LRN for its analysis.
The LRN system utilizes a restricted set of primers and probes for
detection of select agents. (If a DNA virus, such as variola, is
strongly suspected, a different technique will be necessary for
extraction and purification of its DNA, although variola is not
considered a priority threat in water).

![](media/image17.wmf)

**Figure 4-16. Sample Processing for Exploratory PCR Presumptive
Testing**

### Confirmatory Testing for Microbials by Culture and Conventional Assays

Processing of the field sample concentrate for culture assays (Figure
4-17) consists of an initial centrifugation step to further concentrate
bacteria (vegetative cells and spores) and protozoa in the sample.
Bacteria and protozoa will be concentrated in the pellet, and viruses
will remain in the supernatant. Protozoa are separated from the bacteria
by IMS. Bacteria will remain in the IMS supernatant (liquid phase) and
the protozoa will be concentrated with the beads (solid phase). Viruses
are further concentrated for cell culture analysis. Table 4-17 lists
established confirmatory analytical techniques for waterborne and select
agents.

![](media/image18.wmf)

**Figure 4-17. Sample Processing for Confirmatory Microbiological
Assays**

Table 4-17. Established Confirmatory Analytical Techniques for
Waterborne Contaminants and Select Agents

+------------+-------------------+----------------+-------------------+
| **Pathogen | **Organism**      | **Confirmatory | **Additional      |
| (general   |                   | Techniques**   | Exploratory       |
| class)**   |                   |                | Techniques**      |
+------------+-------------------+----------------+-------------------+
| **         |                   |                |                   |
| Waterborne |                   |                |                   |
| Cont       |                   |                |                   |
| aminants** |                   |                |                   |
+------------+-------------------+----------------+-------------------+
| Bacteria   | *Campylobacter*   | Biochemical    | PCR and           |
|            | spp.              | and            | Sequencing        |
|            |                   | Serological    |                   |
|            | E. coli 0157:H7   | Tests          |                   |
|            |                   |                |                   |
|            | *Salmonella* spp. |                |                   |
|            |                   |                |                   |
|            | *Shigella* spp.   |                |                   |
|            |                   |                |                   |
|            | *Vibrio cholerae* |                |                   |
+------------+-------------------+----------------+-------------------+
| Protozoa   | *Cryptosporidium  | IFA and        | PCR and           |
|            | parvum*           | Microscopy     | Sequencing        |
|            |                   |                |                   |
|            | *Giardia          |                |                   |
|            | intestinalis*     |                |                   |
|            |                   |                |                   |
|            | *Entamoeba        |                |                   |
|            | histolytica*      |                |                   |
|            |                   |                |                   |
|            | *Toxoplasma       |                |                   |
|            | gondii*           |                |                   |
+------------+-------------------+----------------+-------------------+
| Viruses    | Enteroviruses     | Plaque         | RT-PCR and        |
|            |                   | neutralization | Sequencing        |
|            | Hepatitis A       |                |                   |
|            |                   | N/A            | IFA, RIFA, PCR    |
|            | Hepatitis E       |                |                   |
|            |                   | N/A            | RT-PCR and        |
|            | Noroviruses       |                | Sequencing        |
|            |                   | N/A            |                   |
|            | Rotavirus         |                | RT-PCR and        |
|            |                   | IFA            | Sequencing        |
|            |                   |                |                   |
|            |                   |                | RT-PCR and        |
|            |                   |                | Sequencing        |
+------------+-------------------+----------------+-------------------+
| **Select   |                   |                |                   |
| Agents**   |                   |                |                   |
+------------+-------------------+----------------+-------------------+
| Spores     | *Bacillus         | Biochemical    | PCR and           |
|            | anthracis*        | and            | Sequencing        |
|            |                   | Serological    |                   |
|            | *Clostridium      | Tests          |                   |
|            | botulinum* A      |                |                   |
+------------+-------------------+----------------+-------------------+
| Bacteria   | *Brucella* spp.   | Biochemical    | PCR and           |
|            |                   | and            | Sequencing        |
|            | *Burkholderia     | Serological    |                   |
|            | pseudomallerei*   | Tests          |                   |
|            |                   |                |                   |
|            | *Francisella      |                |                   |
|            | tularensis*       |                |                   |
|            |                   |                |                   |
|            | *Yersinia pestis* |                |                   |
+------------+-------------------+----------------+-------------------+
| Rickettsia | *Coxiella         | Biochemical    |                   |
|            | burnetti*         | and            |                   |
|            |                   | Serological    |                   |
|            |                   | Tests          |                   |
+------------+-------------------+----------------+-------------------+
| Viruses    | Variola           | TBD            | PCR and           |
|            |                   |                | Sequencing        |
|            | VEE, VHF          |                |                   |
|            |                   |                | PCR and           |
|            |                   |                | Sequencing        |
+------------+-------------------+----------------+-------------------+

The procedures outlined in this section are intended for use in water
samples where select agents are suspected to be present, thus these
procedures should be performed in microbiology laboratories that use
appropriate Biological Safety Level practices. For all suspected
pathogens, refer to the CDC publication "Biosafety in Microbiological
and Biomedical Laboratories," 4^th^ edition (HHS Publication No.
93-8395,
[http://www.cdc.gov/od/ohs/pdffiles/4th%20BMBL.pdf](http://www.cdc.gov/od/ohs/pdffiles/4th BMBL.pdf)).
Some BSL requirements are summarized below, broken out by pathogen
class.

[Bacterial assays]{.underline}

Pathogenic enteric bacteria can be handled in an environmental BSL-2
laboratory. Nonetheless, select agents should only be analyzed for
presumptive identification by laboratories approved for presumptive
identification of select agents. *Brucella* spp. (BSL-3), *Burkholderia
mallei* (BSL-2), *B. pseudomallei* (BSL-2), *Yersinia pestis* (BSL-2),
and *Francisella tularensis* (BSL-3) are highly infectious and have
caused laboratory-acquired infections. Because of the highly infectious
nature of these pathogens, consultation with a state public health
laboratory is recommended if any of these are suspected in a sample.

[Protozoa]{.underline}

The solid phase of the IMS procedure is further processed by EPA Method
1623 for IFA identification of *Cryptosporidium* and *Giardia*. The
protozoa *Cryptosporidium parvum*, *Giardia intestinalis*, *Entamoeba
histolytica*, and *Toxoplasma gondii* can be handled in an environmental
BSL-2 laboratory.

[Viruses]{.underline}

Viruses in the supernatant from the centrifugation step above are
further concentrated by a second ultrafiltration to reduce the assay
volume for cell culture analysis. Pathogenic enteric viruses can be
handled in an environmental BSL-2 laboratory. Nonetheless, select agents
should only be analyzed for presumptive identification by laboratories
approved for presumptive identification of select agents. Variola
(BSL-3), VEE (BSL-3) and VHF (BSL-4) are highly infectious, and
suspected samples should be referred to an LRN laboratory. Because
multiple virus targets are pursued, the sample is likely to be
inoculated onto more than one cell line.

Public health laboratories primarily use tube cultures of mammalian
cells which are amenable only for small inoculation volumes. However,
tube culture is a well established method and fairly efficient for virus
detection. Infectious viruses are amplified in the receptive cells and
are used as the source of infectious material for subsequent
identification and confirmation assays. Identification and analytical
confirmation consists of neutralization assays with specific antisera.
PCR and sequencing of positive samples is also an exploratory technique
that could be used to further identify and characterize the isolated
virus. DNA sequencing is used for fingerprinting studies and for
definitive identification of pathogens.

# References and Resources

References and information cited or used to develop this module are
listed below. The URLs of several sources are cited throughout the text.
These URLs were correct at the time of the preparation of this document.
If the document is no longer available at the URL provided, please
search the sponsoring organization's Web site or the World Wide Web for
alternate sources. A copy of referenced documents may also be provided
on the CD version of this module, although readers should consult the
referenced URL for the latest version.

AOAC. 2003a. "Home of AOAC International, The Cornerstone for Online
Analytical Methods" [http://www.aoac.org](http://www.aoac.org/).

AOAC. 2003b. "Rapid Test Kits Test Kit Database"
<http://www.aoac.org/testkits/TKDATA2.HTM>.

ASTM, 2003, "Standards Worldwide -- ASTM International"
[http://www.astm.org](http://www.astm.org/).

CDC. 1999. "Biosafety in Microbiological and Biomedical Laboratories"
[http://www.cdc.gov/od/ohs/pdffiles/4th%20BMBL.pdf](http://www.cdc.gov/od/ohs/pdffiles/4th BMBL.pdf).

CDC. 2002a. "Summary of the Laboratory Response Network"
<http://www.cdc.gov/cic/functions-specs/function4Docs/nLRNvision-summary.doc>.

CDC. 2002b. "Possession, Use, and Transfer of Select Agents and Toxins:
Interim Final Rule; Department of Health and Human Services, 42 CFR Part
1003; December 13, 2002" <http://www.cdc.gov/od/sap/docs/42cfr73.pdf>.

CDC. 2003a. "What to do in an Emergency"
<http://www.bt.cdc.gov/emcontact/index.asp>.

CDC. 2003b. "Biological Agents-Diseases Public Health Emergency"
<http://www.bt.cdc.gov/Agent/agentlist.asp>.

CDC. 2003c. "Office of the Director\--Select Agent Program"
<http://www.cdc.gov/od/sap>.

CDC. 2003d. "Office of the Director\--Select Agent Program FAQ"
<http://www.cdc.gov/od/sap/faq.htm>.

CDC. 2003e. "Office of the Director\--Select Agent Program Additional
Resource Material" <http://www.cdc.gov/od/sap/addres.htm>.

CDC. 2003f. "Summary of Biosafety Level-1&2 for Infectious Agents"
<http://www.bt.cdc.gov/documents/PPTResponse/table3abiosafety.pdf>.

CDC. 2003f. "List of Select Biological Agents"
<http://www.cdc.gov/od/sap/docs/salist.pdf>.

Clesceri, Lenore S., Arnold E. Greenberg, Andrew D. Eaton, Editors.
1999. Standard Methods for the Examination of Water and Wastewater,
20^th^ edition (January 1999). A joint publication of the Water
Environment Federation, the American Water Works Association, and the
American Public Health Association, Washington, DC. ISBN 0-87553-235-7

CWC. 2003a. "CWC Regulations -- Supplement No. 1 to Part 712 --Schedule
1 Chemicals" <http://www.cwc.gov/Regulations/cfr-15/part-712-s1_html>
See also
<http://www.cwc.gov/Industry_Outreach/Publications/002/cwc-b0001_html>
<http://www.cwc.gov/Regulations/cfr-15/index_html>.

CWC. 2003b. "The Chemical Weapons Convention -- A Quick Guide, CWC-002"
<http://www.cwc.gov/Industry_Outreach/Publications/002/cwc-b0001_html>.

CWC. 2003c. "CWC Regulations -- Supplement No. 1 to Part 713 --Schedule
2 Chemicals" <http://www.cwc.gov/Regulations/cfr-15/part-713-s1_html>.

CWC. 2003d. "CWC Regulations -- Supplement No. 1 to Part 714 --Schedule
3 Chemicals" <http://www.cwc.gov/Regulations/cfr-15/part-714-s1_html>.

DOJ/NIJ. 2001. "An Introduction to Biological Agent Detection Equipment
for Emergency First Responders, NIJ Guide 101-00, December 2001"
<http://www.ojp.usdoj.gov/nij/pubs-sum/190747.htm>.

DOT. 2003. "Welcome to the Hazmat Safety Homepage"
<http://hazmat.dot.gov/>.

EPA and USGS. 2003. "National Environmental Methods Index"
[http://www.nemi.gov](http://www.nemi.gov/).

EPA. 2000. "EPA Radiological Emergency Response Plan"

<http://www.epa.gov/radiation/rert/docs/rerp-1-00.pdf>.

EPA. 2002a. "EPA Manual of Manuals -- Laboratory Analytical Methods
Manuals" <http://www.epa.gov/nerlcwww/methmans.html>.

EPA. 2002b. "Setting Guidelines to Protect the Public -- Information
Home (EPA's Radiation Protection Program Information)"
<http://www.epa.gov/radiation/docs/readytorespond/ready3.htm>.

EPA. 2002c. "EPA's Radiation Protection Program"
<http://www.epa.gov/radiation/marlap/>.

EPA. 2003a. "EPA's Radiation Protection Program Information"
[http://www.epa.gov/radiation/programs.htm -
er](http://www.epa.gov/radiation/programs.htm#er).

EPA. 2003b. "EPA Quality System -- Information for non-EPA
Organizations"
*<http://www.epa.gov/quality1/exmural.html#genreqts>[.]{.underline}*

EPA. 2003c. "EPA Environmental Technology Verification Home"
<http://www.epa.gov/etv/>.

EPA. 2003d. "EPA Analytical Methods for Drinking Water and Analytical
Methods Developed by the Office of Ground Water and Drinking Water"
<http://www.epa.gov/safewater/methods/sourcalt.html>.

EPA. 2003e. "EPA Methods from the Office of Research and Development"
<http://www.epa.gov/nerlcwww/ordmeth.htm>.

EPA. 2003f. "Test Methods SW-846 On-line"
[[http://www.epa.gov/epaoswer/hazwaste/test/main.htm]{.underline}](http://www.epa.gov/epaoswer/hazwaste/test/main.htm).

EPA. 2003g. "CFR Title 40 Protection of Environment"
<http://www.epa.gov/docs/epacfr40/chapt-I.info/subch-D.htm>.

EPA. 2003h. "EPA Ground Water and Drinking Water Drinking Water
Contaminant Candidate List"
<http://www.epa.gov/safewater/ccl/cclfs.html>.

EPA. 2003i. "EPA Unregulated Contaminant Monitoring Regulation"
<http://www.epa.gov/safewater/standard/ucmr/main.html>.

EPA. 2003j. "New Test Methods On-line"
<http://www.epa.gov/epaoswer/hazwaste/test/new-meth.htm>.

EPA. 2003k. "ETV -- Verifications"
<http://www.epa.gov/etv/verifications/>.

EPA. 2003l. "EPA Microbiology Home Page" <http://www.epa.gov/nerlcwww/>.

ERT. 2003. "ERT Health and Safety"
<http://www.ertresponse.com/health_safety/index.htm>.

FEMA. 2003. "FEMA Radiological Emergency Preparedness Program"
<http://www.fema.gov/rrr/rep/index.shtm>.

FJC. 2000. "Reference Manual Scientific Evidence, Second Edition"
<http://www.fjc.gov/public/pdf.nsf/lookup/sciman00.pdf/$file/sciman00.pdf>.

ISO. 2003. "ISO -- International Standard for Organization"
[http://www.iso.org](http://www.iso.org/).

NIOSH. 2003. "NIOSH -- National Personal Protective Technology
Laboratory" <http://www.cdc.gov/niosh/npptl/default.html>.

NIST. 2002. "AMDIS Download Page"
<http://chemdata.nist.gov/mass-spc/amdis/>.

NRT. 1996. "Federal Register -- FEMA Federal Radiological Emergency
Response Plan"
<http://www.nrt.org/production/nrt/home.nsf/0/5c23c5d58074d6e48525660c005b56b5?OpenDocument>.

OSHA. 2002. "Safety and Health Topics Personal Protective Equipment"
<http://www.osha.gov/SLTC/personalprotectiveequipment/index.html>.

OSHA. 2003a. "OSHA e-HASP Software --Version 1.0, September 2003"
<http://www.osha.gov/dts/osta/oshasoft/ehasp/>.

OSHA. 2003b. "Occupational Safety and Health Administration Homepage"
[http://www.osha.gov](http://www.osha.gov/).

UNESCO. 1995. "UNESCO's Documentary Resources---Manual on Harmful Marine
Microalgae" <http://unesdoc.unesco.org/images/0012/001220/122021eo.pdf>.

US Army. 2002. "Toxic Chemical Agent Safety Standards"
<http://www.usapa.army.mil/pdffiles/p385_61.pdf>.

US Coast Guard. 2001. "Chemical Hazards Response Information System"
[http://www.chrismanual.com](http://www.chrismanual.com/).

USDA. 2003a. "Animal and Plant Health Inspection Service"
[http://www.aphis.usda.gov](http://www.aphis.usda.gov/).

USGS. 2003. "Methods and Data Comparability Board Home Page"
<http://wi.water.usgs.gov/pmethods/>.

# Appendices

## Chemicals that appear in CWC Schedules

Table 4-18. List of chemicals that appear in CWC Schedules 1, 2, and 3
(<http://www.cwc.gov/Regulations/cfr-15/index_html>)

+-----------------------+-----------------------+-----------------------+
| **Schedule 1 Chemical | **Schedule 2 Chemical | **Schedule 3 Chemical |
| Agents**              | Agents**              | Agents**              |
+=======================+=======================+=======================+
| O-Alkyl               | Amiton: O,O-Diethyl   | Phosgene: carbonyl    |
| ([\<]{.underline}C10, | S-\[2-                | dichloride            |
| incl. cycloalkyl)     | (diethylamino)ethyl\] |                       |
| alkyl (Me, Et, n-Pr   | phosphorothiolate and |                       |
| or                    | corresponding         |                       |
| i-Pr)                 | alkylated or          |                       |
| -phosphonofluoridates | protonated salts      |                       |
|                       |                       |                       |
| (e.g. Sarin:          |                       |                       |
| O-Isopropyl           |                       |                       |
| met                   |                       |                       |
| hylphoshonofluoridate |                       |                       |
|                       |                       |                       |
| Soman: O-Pinacolyl    |                       |                       |
| methy                 |                       |                       |
| lphosphonofluoridate) |                       |                       |
+-----------------------+-----------------------+-----------------------+
| O-Alkyl               | PFIB:                 | Cyanogen chloride     |
| ([\<]{.underline}C10, | 1,1,3,3,3             |                       |
| incl.                 | -Pentafluoro-2-(trifl |                       |
| c                     | uoromethyl)-1-propene |                       |
| ycloalkyl)N,N-dialkyl |                       |                       |
|                       |                       |                       |
| (Me, Et, n-Pr or      |                       |                       |
| I-Pr)-ph              |                       |                       |
| osphoramidocyanidates |                       |                       |
|                       |                       |                       |
| (e.g. Tabun: O-Ethyl  |                       |                       |
| N,N-dimethyl          |                       |                       |
| ph                    |                       |                       |
| osphoramidocyanidate) |                       |                       |
+-----------------------+-----------------------+-----------------------+
| O-alkyl (H or         | BZ: 3-Quinuclidinyl   | Hydrogen cyanide      |
| [\<]{.underline} C10, | benzilate             |                       |
| incl.                 |                       |                       |
| c                     |                       |                       |
| ycloalkyl)S-2-dialkyl |                       |                       |
|                       |                       |                       |
| (Me, Et, n-Pr or      |                       |                       |
| I-Pr)-aminoethyl      |                       |                       |
| alkyl                 |                       |                       |
|                       |                       |                       |
| (Me, Et, n-Pr or      |                       |                       |
| I-Pr)                 |                       |                       |
| phosphonothiolates    |                       |                       |
| and corresponding     |                       |                       |
| alkylated or          |                       |                       |
| protonated salts      |                       |                       |
|                       |                       |                       |
| (e.g. VX: O-Ethyl     |                       |                       |
| S-2-                  |                       |                       |
| diisopropylaminoethyl |                       |                       |
| methyl                |                       |                       |
| phosphonothiolate)    |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Sulfur mustards:      | Chemicals, except for | Chloropicrin:         |
|                       | those listed in       | trichloronitromethane |
| 2-Chloroeth           | Schedule 1,           |                       |
| ylchloromethylsulfide | containing a          |                       |
|                       | phosphorus atom to    |                       |
| Mustard gas:          | which is bonded one   |                       |
| Bis(                  | methyl, ethyl or      |                       |
| 2-chloroethyl)sulfide | propyl (normal or     |                       |
|                       | iso) group but not    |                       |
| Bis(2-ch              | further carbon atoms, |                       |
| loroethylthio)methane | e.g.,                 |                       |
|                       | Methylphosphonyl      |                       |
| Sesquimustard:        | dichloride Dimethyl   |                       |
| 1,2-Bis(2-c           | methylphosphonate     |                       |
| hloroethylthio)ethane |                       |                       |
|                       | Exemption: Fonofos:   |                       |
| 1,3-Bis(2-chlor       | O-Ethyl S-phenyl      |                       |
| oethylthio)-n-propane | ethylph               |                       |
|                       | osphonothiolothionate |                       |
| 1,4-Bis(2-chlo        |                       |                       |
| roethylthio)-n-butane |                       |                       |
|                       |                       |                       |
| 1,5-Bis(2-chlor       |                       |                       |
| oethylthio)-n-pentane |                       |                       |
|                       |                       |                       |
| Bis(2-chloro          |                       |                       |
| ethylthiomethyl)ether |                       |                       |
|                       |                       |                       |
| O-Mustard:            |                       |                       |
| Bis(2-chlor           |                       |                       |
| oethylthioethyl)ether |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Lewisites:            | N,N-Dialkyl (Me, Et,  | Phosphorus            |
|                       | n-Pr or i-Pr)         | oxychloride           |
| Lewisite              | phosphoramidic        |                       |
| 1:2-Chlo              | dihalides             |                       |
| rovinyldichloroarsine |                       |                       |
|                       |                       |                       |
| Lewisite              |                       |                       |
| 2:Bis(2-chl           |                       |                       |
| orovinyl)chloroarsine |                       |                       |
|                       |                       |                       |
| Lewisite 3:Tris       |                       |                       |
| (2-chlorovinyl)arsine |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Nitrogen mustards:    | Dialkyl (Me, Et, n-Pr | Phosphorus            |
|                       | or i-Pr) N,N-dialkyl  | trichloride           |
| HN1:                  | (Me, Et, n-Pr or      |                       |
| Bis(2-c               | i                     |                       |
| hloroethyl)ethylamine | -Pr)-phosphoramidates |                       |
|                       |                       |                       |
| HN2:                  |                       |                       |
| Bis(2-ch              |                       |                       |
| loroethyl)methylamine |                       |                       |
|                       |                       |                       |
| HN3:                  |                       |                       |
| Tri                   |                       |                       |
| s(2-chloroethyl)amine |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Saxitoxin             | Arsenic trichloride   | Phosphorus            |
|                       |                       | pentachloride         |
+-----------------------+-----------------------+-----------------------+
| Ricin                 | 2,2-Dip               | Trimethyl phosphite   |
|                       | henyl-2-hydroxyacetic |                       |
|                       | acid                  |                       |
+-----------------------+-----------------------+-----------------------+
| Alkyl (Me, Et, n-Pr   | Quinuclidine-3-ol     | Triethyl phosphite    |
| or                    |                       |                       |
| I-Pr                  |                       |                       |
| )phoshonyldifluorides |                       |                       |
|                       |                       |                       |
| e.g.,                 |                       |                       |
| DF:Methy              |                       |                       |
| lphosphonyldifluoride |                       |                       |
+-----------------------+-----------------------+-----------------------+
| O-Alkyl(incl.         | N,N-Dialkyl (Me, Et,  | Dimethyl phosphite    |
| C                     | n-Pr or I-Pr)         |                       |
| ycloalkyl)O-2-dialkyl | a                     |                       |
|                       | minoethyl-2-chlorides |                       |
| (Me, Et, n-Pr or      | and corresponding     |                       |
| I-Pr)-aminoethyl      | protonated salts      |                       |
| alkyl                 |                       |                       |
|                       |                       |                       |
| (Me, Et, n-Pr or      |                       |                       |
| I-Pr) phosphonites    |                       |                       |
| and corresponding     |                       |                       |
| alkylated or          |                       |                       |
| protonated salts      |                       |                       |
|                       |                       |                       |
| e.g., QL: O-Ethyl     |                       |                       |
| O-2-                  |                       |                       |
| diisopropylaminoethyl |                       |                       |
| methylphosphonite     |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Chlorosarin:          | N,N-Dialkyl (Me, Et,  | Diethyl phosphite     |
| O-Isopropyl           | n-Pr or I-Pr)         |                       |
| meth                  | aminoethane-2-ols and |                       |
| ylphosphonochloridate | corresponding         |                       |
|                       | protonated salts      |                       |
|                       |                       |                       |
|                       | Exemption:            |                       |
|                       | N,N                   |                       |
|                       | -Dimethylaminoethanol |                       |
|                       | and corresponding     |                       |
|                       | protonated salts      |                       |
|                       | N,                    |                       |
|                       | N-Diethylaminoethanol |                       |
|                       | and corresponding     |                       |
|                       | protonated salts      |                       |
+-----------------------+-----------------------+-----------------------+
| Chlorosoman:          | N,N-Dialkyl (Me, Et,  | Sulfur monochloride   |
| O-Pinacolyl           | n-Pr or I-Pr)         |                       |
| meth                  | aminoethane-2-thiols  |                       |
| ylphosphonochloridate | and corresponding     |                       |
|                       | protonated salts      |                       |
+-----------------------+-----------------------+-----------------------+
|                       | Thiodiglycol:         | Sulfur dichloride     |
|                       | Bis(2-hydroxyethyl)   |                       |
|                       | sulfide               |                       |
+-----------------------+-----------------------+-----------------------+
|                       | Pinacolyl alcohol:    | Thionyl chloride      |
|                       | 3,                    |                       |
|                       | 3-Dimethylbutane-2-ol |                       |
+-----------------------+-----------------------+-----------------------+
|                       |                       | Ethyldiethanolamine   |
+-----------------------+-----------------------+-----------------------+
|                       |                       | Methyldiethanolamine  |
+-----------------------+-----------------------+-----------------------+
|                       |                       | Triethanolamine       |
+-----------------------+-----------------------+-----------------------+

##  Methods Used for the Basic Screen

**Table 4-19. Titles of methods used in Table 4-3 for the basic
screen.** (See Table 4-2 for information on how to obtain these
methods.)

+----------------------+-----------------------------------------------+
| **EPA Method         | **Title**                                     |
| number**             |                                               |
+----------------------+-----------------------------------------------+
| **Drinking water     |                                               |
| methods**            |                                               |
+----------------------+-----------------------------------------------+
| 200.7, May 1994,     | Trace Elements in Water, Solids, and          |
| Revision 4.4\*       | Biosolids by Inductively                      |
|                      |                                               |
|                      | Coupled Plasma-Atomic Emission Spectrometry   |
+----------------------+-----------------------------------------------+
| 200.8, May 1994,     | Determination Of Trace Elements In Waters And |
| Revision 5.4\*       | Wastes By Inductively Coupled Plasma Mass     |
|                      | Spectrometry                                  |
+----------------------+-----------------------------------------------+
| 200.9, May 1994,     | Trace Elements in Water, Solids, and          |
| Revision 2.2\*       | Biosolids by Stabilized                       |
|                      |                                               |
|                      | Temperature Graphite Furnace Atomic           |
|                      | Absorption Spectrometry                       |
+----------------------+-----------------------------------------------+
| 245.1, May 1994,     | Mercury (Manual Cold Vapor Technique)         |
| Revision 3.0\*       |                                               |
+----------------------+-----------------------------------------------+
| 245.2, March 1983\*  | Mercury (Automated Cold Vapor Technique)      |
+----------------------+-----------------------------------------------+
| 335.2, March 1983    | Cyanide, Total (Titrimetric;                  |
|                      | Spectrophotometric)                           |
+----------------------+-----------------------------------------------+
| 335.3, March 1983    | Cyanide, Total (Colorimetric, Automated UV)   |
+----------------------+-----------------------------------------------+
| 335.4, March 1983    | Cyanide, Total (Colorimetric, Semi-automated  |
|                      | UV)                                           |
+----------------------+-----------------------------------------------+
| 502.2, 1995,         | Volatile Organic Compounds in Water By Purge  |
| Revision 2.1\*       | And Trap Capillary Column Gas Chromatography  |
|                      | with Photoionization and Electrolytic         |
|                      | Conductivity Detectors in Series              |
+----------------------+-----------------------------------------------+
| 524.2, 1995,         | Measurement Of Purgeable Organic Compounds In |
| Revision 4.1\*       | Water By Capillary Column Gas                 |
|                      | Chromatography/mass Spectrometry              |
+----------------------+-----------------------------------------------+
| 525.2, 1995,         | Determination of Organic Compounds in         |
| Revision 2.0\*       | Drinking Water By Liquid-solid Extraction and |
|                      | Capillary Column Gas Chromatography/Mass      |
|                      | Spectrometry                                  |
+----------------------+-----------------------------------------------+
| 531.1, 1995,         | Measurement of N-methylcarbamoyloximes and    |
| Revision 3.1\*       | N-methyl-carbamates                           |
|                      |                                               |
| 531.2, September     | in Water by Direct Aqueous Injection HPLC     |
| 2001, Revision 1.0   | with Post-Column Derivatization               |
+----------------------+-----------------------------------------------+
| 549.2, 1997,         | Determination of Diquat and Paraquat in       |
| Revision 1.0\*       | Drinking Water by Liquid-Solid                |
|                      |                                               |
|                      | Extraction and High Performance Liquid        |
|                      | Chromatography with Ultraviolet Detection     |
+----------------------+-----------------------------------------------+
| 900, August 1980\*   | ###### Proscribed Procedur                    |
|                      | es for the Determination of Radioactivity in  |
|                      | Water {#proscribed-procedures-for-the-determi |
|                      | nation-of-radioactivity-in-water .unnumbered} |
+----------------------+-----------------------------------------------+
| **SW-846 methods**   |                                               |
+----------------------+-----------------------------------------------+
| 3535A, January 1998, | Solid-Phase Extraction (SPE) \[preparation    |
| Revision 1           | technique for 8270D\]                         |
+----------------------+-----------------------------------------------+
| 5030B, December      | Purge-and-Trap for Aqueous Samples            |
| 1996, Revision 2     |                                               |
+----------------------+-----------------------------------------------+
| 8021B, December      | Aromatic and Halogenated Volatiles by Gas     |
| 1996, Revision 2     | Chromatography Using Photoionization and/or   |
|                      | Electrolytic Conductivity Detectors           |
+----------------------+-----------------------------------------------+
| 8260B, December      | Volatile Organic Compounds by Gas             |
| 1996, Revision 2     | Chromatography/Mass Spectrometry (GC/MS)      |
+----------------------+-----------------------------------------------+
| 8270D, January 1998, | Semivolatile Organic Compounds by Gas         |
| Revision 4           | Chromatography/Mass Spectrometry (GC/MS)      |
+----------------------+-----------------------------------------------+
| 1998                 |                                               |
+----------------------+-----------------------------------------------+

**\*** These methods are approved at 40 CFR Part 141 for regulatory
compliance monitoring of drinking water.

## Analyte Lists for Basic Screen

**Table 4-20.** **Analyte Lists Corresponding to Table 4-3**.

[Note]{.underline}: Laboratories may recognize these as commercially
available standard mixes or combinations thereof. This does not imply
endorsement of any company, product, or service. The standard mixes do
not necessarily include all analytes that the method is capable of
determining. For example, in list B, only 90 analytes are included,
although commercially available mixes could be selected to encompass
more than 150 compounds. Laboratories should be encouraged to include as
many analytes as possible in the standards mix to increase the breadth
of the screen. Because the analyte lists are intended for the basic
screen, which is designed to produce defensible results, laboratories
may wish to modify the analyte lists to reflect their accreditations and
certifications. However, additional analytes may become part of the
expanded screen, so laboratories may wish to include those anyway.
Analytes added to those listed must meet the same QC acceptance criteria
and be part of the overall quality system in the laboratory, so that the
added analytes will be as defensible as those listed.

The exclusion or inclusion of an analyte in the lists in this table does
not reflect its potential hazard as a water contaminant. Rather, these
analytes were chosen for the convenience of commercial availability of
standard mixes. In practice, site-specific plans and situations may be
used (and are encouraged) to modify these lists.

+-------------+-------------+-----------+----+------+----+----+-----+
| **Analyte   |             |           |    |      |    |    |     |
| Lists       |             |           |    |      |    |    |     |
| Co          |             |           |    |      |    |    |     |
| rresponding |             |           |    |      |    |    |     |
| to Table    |             |           |    |      |    |    |     |
| 4-3**       |             |           |    |      |    |    |     |
+=============+=============+===========+====+======+====+====+=====+
| **A**       | **B**       | **C**     | *  | *    | *  | *  | **  |
|             |             |           | *D | *E** | *F | *G | H** |
|             |             |           | ** |      | ** | ** |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1         | 2,2         | Aldicarb  | di | ars  | m  | fr | c   |
| ,1,2-Tetrac | ',3,3',4,4' |           | qu | enic | er | ee | esi |
| hloroethane | ,6-Heptachl |           | at |      | cu | c  | um- |
|             | orobiphenyl |           |    |      | ry | ya | 137 |
|             |             |           |    |      |    | ni |     |
|             |             |           |    |      |    | de |     |
|             |             |           |    |      |    | (s |     |
|             |             |           |    |      |    | ee |     |
|             |             |           |    |      |    | m  |     |
|             |             |           |    |      |    | et |     |
|             |             |           |    |      |    | ho |     |
|             |             |           |    |      |    | d) |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1,1-Tric  | 2,2',       | aldicarb  | pa | cad  |    |    | ir  |
| hloroethane | 3,3',4,5',6 | sulfone   | ra | mium |    |    | idi |
|             | ,6'-Octachl |           | qu |      |    |    | um- |
|             | orobiphenyl |           | at |      |    |    | 192 |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1         | 2,2',3',4   | aldicarb  |    | chro |    |    | cob |
| ,2,2-Tetrac | ,6-Pentachl | sulfoxide |    | mium |    |    | alt |
| hloroethane | orobiphenyl |           |    |      |    |    | -60 |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1,2-Tric  | 2,2',4,4',5 | c         |    | co   |    |    | str |
| hloroethane | ,6'-Hexachl | arbofuran |    | balt |    |    | ont |
|             | orobiphenyl |           |    |      |    |    | ium |
|             |             |           |    |      |    |    | -90 |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1-Dic     | 2,2',4,     | 3         |    | co   |    |    |     |
| hloroethane | 4'-Tetrachl | -Hydroxyc |    | pper |    |    |     |
|             | orobiphenyl | arbofuran |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1-Dic     | 2,3-Dichl   | m         |    | lead |    |    |     |
| hloroethene | orobiphenyl | ethiocarb |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,1-Dich    | 2           | methomyl  |    | mer  |    |    |     |
| loropropene | ,4,5-Trichl |           |    | cury |    |    |     |
|             | orobiphenyl |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2,3-Trich | 2,4-Din     | Oxamyl    |    |      |    |    |     |
| lorobenzene | itrotoluene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2,3-Trich | 2,6-Din     | Propoxur  |    |      |    |    |     |
| loropropane | itrotoluene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2,4-Trich | 2-Chl       | Sevin     |    |      |    |    |     |
| lorobenzene | orobiphenyl | (         |    |      |    |    |     |
|             |             | carbaryl) |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2,4-Trime | a-BHC       |           |    |      |    |    |     |
| thylbenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2-D       | Ace         |           |    |      |    |    |     |
| ibromo-3-ch | naphthylene |           |    |      |    |    |     |
| loropropane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2-Di      | a-Chlordane |           |    |      |    |    |     |
| bromoethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2-Dich    | Alachlor    |           |    |      |    |    |     |
| lorobenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2-Dic     | Aldrin      |           |    |      |    |    |     |
| hloroethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,2-Dich    | alph        |           |    |      |    |    |     |
| loropropane | a-Chlordane |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,3,5-Trime | Anthracene  |           |    |      |    |    |     |
| thylbenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,3-Dich    | Atrazine    |           |    |      |    |    |     |
| lorobenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,3-Dich    | Azinphos    |           |    |      |    |    |     |
| loropropane | methyl      |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 1,4-Dich    | b-BHC       |           |    |      |    |    |     |
| lorobenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 2,2-Dich    | Benz(a      |           |    |      |    |    |     |
| loropropane | )anthracene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 2-Ch        | Ben         |           |    |      |    |    |     |
| lorotoluene | zo(a)pyrene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 2-N         | Ben         |           |    |      |    |    |     |
| itropropane | zo(a)pyrene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| 4-Ch        | Benzo(b)f   |           |    |      |    |    |     |
| lorotoluene | luoranthene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| ac          | Benzo(g,h   |           |    |      |    |    |     |
| rylonitrile | ,i)perylene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| allyl       | Benzo(k)f   |           |    |      |    |    |     |
| chloride    | luoranthene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Benzene     | bi          |           |    |      |    |    |     |
|             | s(2-Ethylhe |           |    |      |    |    |     |
|             | xyl)adipate |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| B           | bis(        |           |    |      |    |    |     |
| romobenzene | 2-Ethylhexy |           |    |      |    |    |     |
|             | l)phthalate |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Bromoch     | Bolstar     |           |    |      |    |    |     |
| loromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Bromodich   | Butachlor   |           |    |      |    |    |     |
| loromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Bromoform   | Butylbenz   |           |    |      |    |    |     |
|             | ylphthalate |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| B           | Chlo        |           |    |      |    |    |     |
| romomethane | robenzilate |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Butyl       | Chloroneb   |           |    |      |    |    |     |
| chloride    |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Carbon      | Chl         |           |    |      |    |    |     |
| disulfide   | orothalonil |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Carbon      | C           |           |    |      |    |    |     |
| te          | hlorpyrifos |           |    |      |    |    |     |
| trachloride |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Chloroa     | Chrysene    |           |    |      |    |    |     |
| cetonitrile |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Ch          | cis         |           |    |      |    |    |     |
| lorobenzene | -Permethrin |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| C           | Coumaphos   |           |    |      |    |    |     |
| hloroethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Chloroform  | Cyanazine   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Ch          | Dacthal     |           |    |      |    |    |     |
| loromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Cis-1,2-Dic | d-BHC       |           |    |      |    |    |     |
| hloroethene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| C           | Demeton     |           |    |      |    |    |     |
| is-1,3-Dich | (mixed      |           |    |      |    |    |     |
| loropropene | isomers)    |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Dibromoch   | Diazinon    |           |    |      |    |    |     |
| loromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Dib         | Dibenz(a,h  |           |    |      |    |    |     |
| romomethane | )anthracene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| D           | Dichlorvos  |           |    |      |    |    |     |
| ichlorodifl |             |           |    |      |    |    |     |
| uoromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Diethyl     | Dieldrin    |           |    |      |    |    |     |
| ether       |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Ethyl       | Diethyl     |           |    |      |    |    |     |
| m           | phthalate   |           |    |      |    |    |     |
| ethacrylate |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| E           | Dimethyl    |           |    |      |    |    |     |
| thylbenzene | phthalate   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Hexachlo    | Di-n-butyl  |           |    |      |    |    |     |
| robutadiene | phthalate   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Hexac       | Disulfoton  |           |    |      |    |    |     |
| hloroethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Isopr       | Endosulfan  |           |    |      |    |    |     |
| opylbenzene | I           |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Methac      | Endosulfan  |           |    |      |    |    |     |
| rylonitrile | II          |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| methanol    | Endosulfan  |           |    |      |    |    |     |
| (solvent)   | sulfate     |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Methyl      | Endrin      |           |    |      |    |    |     |
| acrylate    |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Methyl      | Endrin      |           |    |      |    |    |     |
| m           | aldehyde    |           |    |      |    |    |     |
| ethacrylate |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Methyl      | Ethoprop    |           |    |      |    |    |     |
| tert-butyl  |             |           |    |      |    |    |     |
| ether       |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Methylene   | Etridiazole |           |    |      |    |    |     |
| chloride    |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| m-Xylene    | Fe          |           |    |      |    |    |     |
|             | nsulfothion |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Naphthalene | Fenthion    |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| n-B         | Fluorene    |           |    |      |    |    |     |
| utylbenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| N           | g-BHC       |           |    |      |    |    |     |
| itrobenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| n-Pr        | g-Chlordane |           |    |      |    |    |     |
| opylbenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| o-Xylene    | Heptachlor  |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Pentac      | Heptachlor  |           |    |      |    |    |     |
| hloroethane | epoxide     |           |    |      |    |    |     |
|             |             |           |    |      |    |    |     |
|             | (Isomer B)  |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| p-Isopr     | Hexach      |           |    |      |    |    |     |
| opyltoluene | lorobenzene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Pr          | Hex         |           |    |      |    |    |     |
| opionitrile | achlorocycl |           |    |      |    |    |     |
|             | opentadiene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| p-Xylene    | Indeno(1,2, |           |    |      |    |    |     |
|             | 3-cd)pyrene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| sec-B       | Lindane     |           |    |      |    |    |     |
| utylbenzene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Styrene     | Merphos     |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| tert-B      | M           |           |    |      |    |    |     |
| utylbenzene | ethoxychlor |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Tetrac      | Methyl      |           |    |      |    |    |     |
| hloroethene | parathion   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Tetr        | Metolachlor |           |    |      |    |    |     |
| ahydrofuran |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Toluene     | Metribuzin  |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| tr          | Mevinphos   |           |    |      |    |    |     |
| ans-1,2-Dic |             |           |    |      |    |    |     |
| hloroethene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| tra         | Naled       |           |    |      |    |    |     |
| ns-1,3-Dich |             |           |    |      |    |    |     |
| loropropene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| trans       | p,p\'-DDD   |           |    |      |    |    |     |
| -1,4-Dichlo |             |           |    |      |    |    |     |
| ro-2-butene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Tric        | p,p\'-DDE   |           |    |      |    |    |     |
| hloroethene |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Trichlorofl | p,p\'-DDT   |           |    |      |    |    |     |
| uoromethane |             |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
| Vinyl       | Pentac      |           |    |      |    |    |     |
| chloride    | hlorophenol |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | P           |           |    |      |    |    |     |
|             | henanthrene |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Phorate     |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Propachlor  |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Pyrene      |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Ronnel      |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Simazine    |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Stirophos   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Tokuthion   |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | tran        |           |    |      |    |    |     |
|             | s-Nonachlor |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+
|             | Tr          |           |    |      |    |    |     |
|             | ichloronate |           |    |      |    |    |     |
+-------------+-------------+-----------+----+------+----+----+-----+