Method Validation Guide for Qualifying

Methods Used by Radiological 

Laboratories Participating in Incident 

Response Activities

Office of Radiation and Indoor Air 

National Air and Radiation

Environmental Laboratory

EPA 402-R-09-006

June 2009

www.epa.gov/narel

United States

Environmental Protection

Agency
 
EPA 402-R-09-006

www.epa.gov

June 2009

Revision 0

Method Validation Guide for

Qualifying Methods Used by Radiological

Laboratories Participating in Incident

Response Activities

U.S. Environmental Protection Agency

Office of Air and Radiation

Office of Radiation and Indoor Air

National Air and Radiation Environmental Laboratory

Montgomery, AL 36115
This report was prepared for the National Air and Radiation Environmental Laboratory of the Office of Radiation

and Indoor Air, United States Environmental Protection Agency. It was prepared by Environmental Management

Support, Inc., of Silver Spring, Maryland, under contracts 68-W-03-038, work assignment 35, and EP-W-07-037,

work assignments B-33 and I-33, all managed by David Garman. Mention of trade names or specific applications

does not imply endorsement or acceptance by EPA.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

1 Departments of Agriculture, Commerce, Defense, Energy, Health and Human Services, Homeland Security, Interior,

Justice, and State, and the U.S. Environmental Protection Agency.

i

Preface

This document describes project method validation guidance that a radioanalytical laboratory should

comply with in order to validate methods used to process samples submitted during a radiological

or nuclear incident, such as that caused by a terrorist attack. EPA laboratories using radioanalytical

processes consistent with the guidance provided in the Radiological Laboratory Sample Analysis

Guide for Incidents of National Significance should first validate their methods according to the

guidance provided in this document. The use of the guidance in this document, as well as in the

Radiological Laboratory Sample Analysis Guide for Incidents of National Significance, will assist

in fulfilling EPA’s responsibilities as outlined in the National Response Framework Nuclear/

Radiological Incident Annex. These responsibilities include response and recovery actions to detect

and identify radioactive substances, and to coordinate federal radiological monitoring and assessment

activities. Additionally this document identifies a formalized process for the development (Section

4.0) and testing (Section 5.0) of a new method so that there is confidence that radioanalytical results

meet project-specific data requirements.

The need to ensure adequate laboratory infrastructure to support response and recovery actions

following a major radiological incident has been recognized by a number of federal agencies. The

Integrated Consortium of Laboratory Networks (ICLN), created in 2005 by 10 federal agencies1

,

consists of existing laboratory networks across the Federal Government. The ICLN is designed to

provide a national infrastructure with a coordinated and operational system of laboratory networks

that provide timely, high quality, and interpretable results for early detection and effective

consequence management of acts of terrorism and other events requiring an integrated laboratory

response. It also designates responsible federal agencies (RFAs) to provide laboratory support across

response phases for chemical, biological, and radiological agents. To meet its RFA responsibilities

for environmental samples, EPA has established the Environmental Response Laboratory Network

(ERLN) to address chemical, biological, and radiological threats. For radiological agents, EPA is the

RFA for monitoring, surveillance, and remediation, and will share responsibility for overall incident

response with the U.S. Department of Energy (DOE). As part of the ERLN, EPA’s Office of

Radiation and Indoor Air is leading an initiative to ensure that sufficient environmental radioanalytical capability and competency exists across a core set of laboratories to carry out EPA’s designated

RFA responsibilities.

Laboratories that support EPA’s incident-response mission will undergo training and should adopt

the use of the material presented in this document, with emphasis on validating methods for expected

radionuclide and matrix combinations in the event of a terrorism incident involving radioactive

materials. As soon as reasonably possible, rapid radioanalytical methods expected to be used to

process anticipated radionuclide and matrix combinations from the early to intermediate phases of

a radiological incident should be validated according to the guidance of the document. During these

early phases of an incident response, there may be insufficient time to validate methods. Therefore,

it is prudent to validate the applicable radioanalytical methods for various sample matrices as part

of the preparatory actions that are necessary to respond properly to a possible radiological incident.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

ii

Laboratories developing new methods and operational protocols should review the detailed guidance

on recommended radioanalytical practices found in the Multi-Agency Radiological Laboratory

Analytical Protocols Manual (MARLAP) referenced in this document. Familiarity with Chapters

6 and 7 of MARLAP will benefit readers of this document.

This document is one in a planned series designed to present radioanalytical laboratory personnel,

Incident Commanders (and their designees), and other field response personnel with key laboratory

operational considerations and likely radioanalytical requirements, decision paths, and default data

quality and measurement quality objectives for samples taken after a radiological or nuclear incident,

including incidents caused by a terrorist attack. Documents currently completed or in preparation

include:

 ! Radiological Laboratory Sample Analysis Guide for Incidents of National Significance –

Radionuclides in Water (EPA 402-R-07-007, January 2008) 

 ! Radiological Laboratory Sample Analysis Guide for Incidents of National Significance –

Radionuclides in Air (EPA 402-R-09-007, June 2009)

 ! Radiological Laboratory Sample Screening Analysis Guide for Incidents of National

Significance (EPA 402-R-09-008, June 2009)

 ! Method Validation Guide for Qualifying Methods Used by Radiological Laboratories

Participating in Incident Response Activities (EPA 402-R-09-006, June 2009) 

 ! Guide for Radiological Laboratories for the Identification, Preparation, and Implementation of

Core Operations for Radiological Incident Response (in preparation)

 ! Guide for Radiological Laboratories for the Control of Radioactive Contamination and

Radiation (in preparation)

 ! Radiological Laboratory Sample Analysis Guide for Incidents of National Significance –

Radionuclides in Soil (in preparation)

Comments on this document, or suggestions for future editions, should be addressed to:

Dr. John Griggs

U.S. Environmental Protection Agency

Office of Radiation and Indoor Air

National Air and Radiation Environmental Laboratory

540 South Morris Avenue

Montgomery, AL 36115-2601

(334) 270-3450

Griggs.John@epa.gov
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

iii

Acknowledgments

This manual was developed by the National Air and Radiation Environmental Laboratory (NAREL)

of EPA’s Office of Radiation and Indoor Air (ORIA).

Dr. John Griggs served as project lead for this document. Several individuals provided valuable

support and input to this document throughout its development. Special acknowledgment and

appreciation are extended to Dr. Keith McCroan, ORIA/NAREL; Mr. Daniel Mackney for his

support of instrumental analysis, ORIA/NAREL; Dr. Lowell Ralston and Mr. Edward Tupin, CHP,

both of ORIA/Radiation Protection Division; Ms. Schatzi Fitz-James, Office of Emergency

Management, Homeland Security Laboratory Response Center; and Mr. David Garman,

ORIA/NAREL. We also wish to acknowledge the peer reviews conducted by Carolyn Wong, David

Burns, and Jack Bennett, whose thoughtful comments contributed greatly to the understanding and

quality of the report. Numerous other individuals both inside and outside of EPA provided peer

review of this document, and their suggestions contributed greatly to the quality and consistency of

the final document. Technical support was provided by Dr. N. Jay Bassin, Dr. Carl V. Gogolak, Dr.

Robert Litman, Dr. David McCurdy, Mr. Robert Shannon, and Dr. Anna Berne of Environmental

Management Support, Inc.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

iv

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Acronyms, Abbreviations, Units, and Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Radiometric and General Unit Conversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1.0 Introduction ...............................................................1

2.0 Method Validation Description ................................................1

3.0 Method Description .........................................................2

4.0 Method Performance Characteristics ............................................3

4.1 Method Uncertainty ......................................................3

4.2 Detection Capability .....................................................5

4.3 Bias and Trueness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4.4 Analyte Concentration Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.4.1 Derived Radionuclide Concentrations Corresponding to Established Action 

Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.4.2 Default Analytical Action Levels ......................................7

4.5 Method Specificity .......................................................7

4.6 Method Ruggedness ......................................................8

5.0 Incident Response Method Validation Guidance, Tests, and Requirements ..............9

5.1 Method Specificity .......................................................9

5.2 Analyte Concentration Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5.3 Matrix Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5.4 Method Validation Levels for Testing the Required Method Uncertainty . . . . . . . . . . . 13

5.4.1. Method Validation Requirements Based on MARLAP Concepts . . . . . . . . . . . . 13

5.4.2 Required Method Uncertainty Acceptance Criteria . . . . . . . . . . . . . . . . . . . . . . . 15

5.4.2.1 Level B Method Validation: Same, Similar, or Slightly Different Matrix . . 16

5.4.2.2 Level C Method Validation: New Application of an Existing Method to a

Different Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.4.2.3 Level D Method Validation: Adapted or Newly Developed Methods, Including

Rapid Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5.4.2.4 Level E Method Validation: Adapted or Newly Developed Methods, Including

Rapid Methods, Using Method Validation Reference Materials . . . . . . . . . . 18

5.5 Verification of Required Detection Limit (MDC) Specification . . . . . . . . . . . . . . . . . . . 18

5.5.1 Calculation of the Critical Net Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.5.2 Testing for the Required MDC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.6 Method Bias Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.6.1 Absolute Bias Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.6.2 Relative Bias Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.6.2.1 Test Level Samples with Same Known Value . . . . . . . . . . . . . . . . . . . . . . . . 24
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

v

5.6.2.2 Test Level Samples with Slightly Different Known Values . . . . . . . . . . . . . 25

6.0 Method Validation Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

7.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Appendix A: Tables Summarizing the Derived Radionuclide Concentrations and Required Method

Uncertainties Corresponding to PAGs or Risks for the Water, Air, and Soil Matrices . . . . . 27

Appendix B: Examples of the Method Validation Process for Required Method Uncertainty

Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Appendix C: Example of the Method Validation Process for Verification of the Required MDC

Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Appendix D: Example of the Effect of Bias on the Probability of Failing the Method Validation

Acceptance Criteria for Required Method Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Detecting Bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Appendix E: An Alternative Method Validation Criterion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Alternative Method Validation Criterion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

The Holst-Thyregod Test for Mean Squared Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Theoretical Comparison of Statistical Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Appendix F: Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Figures

Figure 1 – Method Validation Process for the Required Method Uncertainty MQO . . . . . . . . . 19

Figure 2 – Validation Process for Verifying the Required MDC MQO . . . . . . . . . . . . . . . . . . . 20

Figure D1 – Probability of a validation sample failing at concentration 100 pCi/L with and without

bias at various values of the method standard uncertainty. . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Figure D2 – Level D validation (21 samples) failing at test level as a function of relative method bias

for relative method uncertainties of 5%, 7.5%, 10%, and 12.5%. . . . . . . . . . . . . . . . . . . . . . 40

Figure E1a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Figure E1b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Figure E1c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Figure E1d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Tables

Table 1 – Default Analytical Action Levels for General Matrix Categories . . . . . . . . . . . . . . . . 11

Table 2 – Method Validation Test Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

vi

Table 3 – Method Validation Requirements and Applicable to Required Method Uncertainty . 15

Table 4 – Required Method Uncertainty (uMR and nMR) Values for Default AAL Test Levels . . 16

Table 5 – Method Validation Requirements Applicable to Required MDC Verification . . . . . . 22

Table A1 – Alpha-Emitting Radionuclide Concentrations and Required Method Uncertainties in

Water Corresponding to 500- and 100-mrem AAL Derived Water Concentrations . . . . . . . 27

Table A2 – Beta/Gamma-Emitting Radionuclide Concentrations in Water and Required Method

Uncertainties Corresponding to 500- and 100-mrem AAL Derived Water Concentrations . 28

Table A3 – Alpha-Emitting Radionuclide Concentrations in Air and Required Method Uncertainties

Corresponding to 2-rem and 500-mrem AAL Derived Air Concentrations . . . . . . . . . . . . . 29

Table A4 – Beta/Gamma-Emitting Radionuclide Concentrations in Air and Required Method

Uncertainties Corresponding to 2-rem and 500-mrem AAL Derived Air Concentrations . . 30

Table A5 – Alpha-Emitting Radionuclide Concentrations in Air and Required Method Uncertainties

Corresponding to AAL Derived Air Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Table A6 – Beta/Gamma-Emitting Radionuclide Concentrations in Air and Required Method

Uncertainties Corresponding to AAL-Derived Air Concentrations (DACs) . . . . . . . . . . . . . 32

Table A7 – Alpha and Beta/Gamma-Emitting Radionuclide Concentrations in Soil and Required

Method Uncertainties Corresponding to Derived Soil Concentrations . . . . . . . . . . . . . . . . . 33

Table B1 – Required Method Uncertainty for Am-241 in Potable Water . . . . . . . . . . . . . . . . . . 35

Table B2 – Required Method Uncertainty for Am-241 in Street Runoff Water . . . . . . . . . . . . . 36

Table C1 – Results of Blank Sample Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Table C2 – Results of MDC Test Sample Analyses; Test Concentration = 2.0 pCi/L . . . . . . . . 38

Table E1 – Method Validation Measurement Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Table E2 – Acceptance Limits, MARLAP Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Table E3 – Method Validation Results, Alternative Test (W Test) . . . . . . . . . . . . . . . . . . . . . . . 48
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

vii

Acronyms, Abbreviations, Units, and Symbols

(Excluding chemical symbols and formulas)

α . . . . . . . . . . . alpha particle

α . . . . . . . . . . . probability of a Type I decision error

AAL . . . . . . . . analytical action level

AL . . . . . . . . . action level

APS . . . . . . . . analytical protocol specification

β . . . . . . . . . . . beta particle

β . . . . . . . . . . . probability of a Type II decision error

Bq . . . . . . . . . . becquerel (1 dps)

CLNC . . . . . . . . critical net concentration level

CSU . . . . . . . . combined standard uncertainty

d . . . . . . . . . . . day

DAC . . . . . . . . derived air concentration

DP . . . . . . . . . decay product(s)

dpm . . . . . . . . disintegration per minute

dps . . . . . . . . . disintegration per second

DQO . . . . . . . . data quality objective

DRC . . . . . . . . derived radionuclide concentration

DWC . . . . . . . derived water concentration

EPA . . . . . . . . [United States] Environmental Protection Agency

γ . . . . . . . . . . . gamma ray

g . . . . . . . . . . . gram

Gy . . . . . . . . . . gray [unit of absorbed radiation dose in materials; 1 gray = 100 rad]

h . . . . . . . . . . . hour

IC . . . . . . . . . . Incident Commander [or designee]

ISO . . . . . . . . . International Organization for Standardization

keV . . . . . . . . . thousand electron volts

L ........... liter

m . . . . . . . . . . meter

MARLAP . . . . Multi-Agency Radiological Laboratory Analytical Protocols Manual

MARSSIM . . . Multi-Agency Radiation Survey and Site Investigation Manual

MDB . . . . . . . minimum detectable bias

MDC . . . . . . . minimum detectable concentration

MeV . . . . . . . . million electron volts

min . . . . . . . . . minute

mL . . . . . . . . . milliliter (10–3 L)

MQO . . . . . . . measurement quality objective

mrem . . . . . . . millirem (10–3 rem)

MSE . . . . . . . . mean squared error

MV . . . . . . . . . method validation

MVRM . . . . . . method validation reference material

PAG . . . . . . . . protective action guide

pCi . . . . . . . . . picocurie (10–12 Ci)

PE . . . . . . . . . . performance evaluation
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

viii

PT . . . . . . . . . . proficiency test/testing

QC . . . . . . . . . quality control

rad . . . . . . . . . unit of absorbed radiation dose in materials; 100 rad = gray

RDD . . . . . . . . radiological dispersal device (i.e., “dirty bomb”)

rem . . . . . . . . . roentgen equivalent man (traditional units; 1 rem = 0.01 Sv)

RSD . . . . . . . . relative standard deviation

s . . . . . . . . . . . second

sBlanks . . . . . . . standard deviation of blank sample net results

SI . . . . . . . . . . International System of Units

Sv . . . . . . . . . . sievert (1 sievert = 100 rem)

uMR . . . . . . . . . . required method uncertainty

nMR . . . . . . . . . relative required method uncertainty

y . . . . . . . . . . . year
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

ix

Radiometric and General Unit Conversions

To Convert To Multiply by To Convert To Multiply by

years (y) seconds (s)

minutes (min)

hours (h)

days (d)

3.16 × 107

5.26 × 105

8.77 × 103

3.65 × 102

s

min

h

d

y 3.17 × 10–8

1.90 × 10–6

1.14 × 10–4

2.74 × 10–3

disintegrations

per second (dps) becquerels (Bq) 1 Bq dps 1

Bq

Bq/kg

Bq/m3

Bq/m3

picocuries (pCi)

pCi/g

pCi/L

Bq/L

27.0

2.70 × 10–2

2.70 × 10–2

10–3

pCi

pCi/g

pCi/L

Bq/L

Bq

Bq/kg

Bq/m3

Bq/m3

3.70 × 10–2

37.0

37.0

103

microcuries per

milliliter

(μCi/mL)

pCi/L 109 pCi/L μCi/mL 10–9

disintegrations

per minute (dpm)

μCi

pCi

4.50 × 10–7

4.50 × 10–1 pCi dpm 2.22

cubic feet (ft3

) cubic meters

(m3

)

2.83×10–2 cubic meters

(m3

)

cubic feet

(ft3)

35.3

gallons (gal) liters (L) 3.78 L gal 0.264

gray (Gy) rad 102 rad Gy 10–2

roentgen equivalent man (rem) sievert (Sv) 10–2 Sv rem 102

NOTE: Traditional units are used throughout this document instead of International System of Units

(SI) units. Protective Action Guides (PAGs) and their derived concentrations appear in official

documents in the traditional units and are in common usage. Conversion to SI units will be aided by

the unit conversions in this table. Conversions are exact to three significant figures, consistent with

their intended application.
 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

1

1.0 Introduction

The United States Environmental Protection Agency (EPA) is responsible for assessing the extent

of environmental contamination and human health consequences in the event of a radiological

incident such as a terrorist incident involving radioactive materials. Although EPA will be mainly

involved in the intermediate and recovery phases of an incident response, there also may be

involvement in some activities in the early phase. For a terrorist event such as a radiological

dispersion device, the radionuclide(s) and the types and number of sample matrices that may be

collected and analyzed can vary dramatically depending on the type of device used and radioactive

material incorporated. The radioanalytical laboratories used to process the samples must not only be

capable of identifying and quantifying the radionuclide(s) in various matrices, but they must also

have the capacity to process a large number of samples in a short time (thousands of samples per

week). Sufficient laboratory capacity is a balance of adequate facility processing areas and nuclear

instrumentation, validated radioanalytical methods available, and trained staff. 

In order to make proper assessments and decisions in the event of a radiological incident, EPA will

utilize only qualified radioanalytical laboratories that have the capability, capacity and quality needed

to process samples taken from affected areas. Analytical protocol specifications (APSs), including

measurement quality objectives (MQOs), will be preestablished to define the expected quality of the

data for incident situations. The objective of this document is to establish systematic and objective

methodologies and acceptance criteria for validating analytical methods, based on the stated quality

requirements of a specific incident-response project, such as recovery from a radiological dispersal

device. Laboratories developing new methods and operational protocols should review the detailed

guidance on recommended radioanalytical practices found in current editions of MARLAP and

MARSSIM.

Several radiological sample analysis guides for incident response have been developed that provide

information on the expected radionuclides of concern and MQOs to make decisions relative to

sample processing priorities for the water, air particulate filter, and soil/solid matrices. As part of the

laboratory qualifying process, laboratories must demonstrate their ability to meet the APSs and

MQOs for the methods used to analyze each radionuclide and sample-matrix combination. EPA will

require an initial project method validation and a subsequent participation in a performance evaluation (PE) program as a means to demonstrate that the methods used by a laboratory are capable of

meeting the MQOs for incident response applications. For incident-response applications, project

method validation will be required and applied to methods currently being used by the laboratories,

including EPA Safe Drinking Water Act required methods, as well as to newly developed methods

and methods that have been modified for incident response. Project method validation and

participation in a PE program will be required for gross alpha and beta screening methods as well.

In this document, the term “project method validation” is synonymous with “incident response

method validation.”

2.0 Method Validation Description

The Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP) Chapter 6

discusses two distinct applications of method validation: general method validation and project
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

2

method validation. General method validation is the process of demonstrating that a method is

suitable for its general intended use, such as routine radioanalytical processing of samples for the

determination of environmental levels of a given radionuclide. For general method validation, the

methods would address internal measurement quality objectives, and typical sample matrix

constituents and nominally interfering concentrations of expected chemical and radionuclide

interferences. EPA has developed a draft general method validation process document (EPA 2006,

Validation and Peer Review of U.S. EPA Radiochemical Methods of Analysis) that covers the

method validation parameters for radioanalytical methods. That document provides guidance to

satisfy EPA requirements for general method validation for measurement uncertainty, method bias

and trueness, precision, detection capability, analyte concentration range, specificity and ruggedness.

In contrast, this document provides guidance on project method validation applicable to methods for

processing samples during a response to a radiological incident, including radiological incidents of

national significance. Project method validation demonstrates that a method is capable of meeting

project-specific MQOs (in other words, a required method uncertainty at a specific radionuclide

concentration). The method selected for a project needs to address specific sample matrix

characteristics, chemical and radionuclide interferences, special sample preparation requirements,

sample-processing turnaround times, and MQOs defined in an analytical protocol specification

(APS). This document addresses the method validation expectations for an incident response for the

MQOs of the required method uncertainty and the required minimum detectable concentration

(MDC). The method validation procedures for the method uncertainty MQO follow the guidance

provided in MARLAP Chapter 6. As discussed in MARLAP, the principal MQO is the required

method uncertainty at an action level. Although the MDC MQO normally would not be specified as

an MQO for incidence response applications, this document provides method validation guidance

for a “required MDC” MQO. 

Even though a laboratory has a method that has undergone general method validation, use of the

method for the incident response application will require project method validation. The degree of

effort and required level of project method validation will depend on the degree of method

development or use, and the MQOs of the project, as included in the APSs.

Proper planning is critical for successful method validation because many method validation

parameters must be considered, evaluated and documented. Method development and method

validation generally are not separate processes. The types of experiments conducted during method

development and the types of tests performed during method validation have many similarities.

3.0 Method Description

The components of a method or measurement process requiring validation should be clearly

described. Generally, a laboratory method includes all physical, chemical and radiometric processes

conducted at a laboratory in order to provide an analytical result. The processes for radiochemical

methods may include sample preparation or dissolution, chemical separations, preparation of sample

test sources, nuclear counting, analytical calculations, data review and qualification, and data

reporting (MARLAP Chapter 6). Method validation efforts should evaluate all process components

combined. Some radiochemical methods may also include procedures for sampling (e.g., methods

for radon in air analysis or for volatile radioactive organic compounds in soils and other solid

matrices), in which case the sampling procedures should be included in the validation tests. The
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

3

measurement process components validated, and the combination of procedures comprising a

method, must be clearly and completely stated.

The purpose of a method (i.e., measurement objectives) and the intended use of the data must be

clearly defined. In addition, method scope and applicability must be well defined and clearly

described and consistent with the documented performance of the method. These measures will help

minimize misapplication by the users. Method scope and applicability include the following:

 • The measurement process components validated (e.g., sample preparation, dissolution, chemical

isolation, precipitation, final product for counting, radiation measurement process, etc.) 

 • The nature (chemical-physical form, type of radiation and quantity measured) of the radionuclides and matrices (chemical and physical form) studied

 • The range of analyte concentration levels for which the method is claimed to be suitable

 • A description of any known limitations and any assumptions upon which a method is based (e.g.,

radiological and non-radiological interferences, minimum sample size, etc.)

 • A description of how the method and analytical parameters chosen meet the measurement quality

objectives for the intended application, when applicable

 • Aliquant sample size for processing 

4.0 Method Performance Characteristics

The performance characteristics of a radiochemical method that may be evaluated in method

validation include:

 • Method uncertainty at a specific radionuclide concentration (action level)

 • Detection capability (minimum detectable concentration)

 • Bias/trueness

 • Analyte concentration range

 • Method specificity

 • Method ruggedness

A brief discussion of each of these performance characteristics will be covered in the following

sections. For more detailed information on a characteristic, the reader is referred to MARLAP

(Chapters 3 and 6); EPA (2006) Validation and Peer Review of U.S. EPA Radiochemical Methods

of Analysis; EURACHEM Guide (1998) The Fitness for Purpose of Analytical Methods, A

Laboratory Guide to Method Validation and Related Topics; ISO 17025; and ANSI N42.23. 

4.1 Method Uncertainty

MARLAP defines method uncertainty as follows:

Method uncertainty refers to the predicted uncertainty of the result that would be measured if the

method were applied to a hypothetical laboratory sample with a specified analyte concentration.

Although individual measurement uncertainties will vary from one measured result to another, the

required method uncertainty is a target value for the individual measurement uncertainties, and is

an estimate of uncertainty (of measurement) before the sample is actually measured.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

2

 Throughout this guide, the term “Incident Commander” (or “IC”) includes his or her designee. 

4

Method uncertainty can be thought of as an estimate of the expected analytical standard deviation

at a specified radionuclide concentration. For certain projects, including incident response, a required

method uncertainty should be specified. An example of a required method uncertainty specification

would be “...at a 137Cs soil concentration of 10 pCi/g, the required method uncertainty is 1 pCi/g.”

In many applications, including incident response laboratory analyses, the specified radionuclide

concentration is referred to as the analytical action level (AAL) and may be based on either incidentspecific, risk-based or regulatory mandated value, such as a protective action guide (PAG) as

presented in the Radiological Laboratory Sample Analysis Guides for Incidents of National

Significance. Radioanalytical results from an incident response are compared to action level

concentrations, and thus it is very important to have results that are of sufficient quality to support

decisions to be made. Specifying a required method uncertainty at the AAL ensures the data quality

needed to make decisions.

To be consistent with MARLAP, certain nomenclature for the required method uncertainty is used

for incident response applications. The notation “uMR” is specified for the absolute required method

uncertainty at or below the action level and has units of activity or activity concentration that match

the AAL value. Above the action level, a relative required method uncertainty nMR, defined as the

uMR/AAL, is specified (nMR is unitless). For the 137Cs soil example provided above, nMR would be

equal to: 

Method uncertainty should not be confused with measurement uncertainty, which MARLAP and the

International Organization for Standardization (ISO) (1993a) defined as:

“Parameter associated with the result of a measurement that characterizes the dispersion of the

values that could reasonably be attributed to the measurand.”

Each radioanalytical method that will be used by a laboratory processing samples from an incident

response will be evaluated to determine if its uncertainty meets the required method uncertainty. The

result of each test sample processed during the method validation process is compared to the limits

of acceptability established for the specific validation level, i.e., a multiple of the required method

uncertainty. A method will be considered acceptable if it meets the method validation criteria

provided in Section 5.4 for the appropriate level of validation. Derived radionuclide concentrations

(DRCs) corresponding to the AAL for the water, air filter and soil matrices can be found in the

Radiological Laboratory Sample Analysis Guide for Incidents of National Significance (see

Appendix A for summary tables). For project method validation, MARLAP recommends that the

uncertainty of a method be evaluated at or near an action level radionuclide concentration. In the

absence of defined AALs and required method uncertainties (either by the Incident Commander [IC]2

or other project manager), default AALs and corresponding required method uncertainties for the

method validation test samples provided in Sections 5.2 and 5.4 can be used. The default values in

Appendix A may be considered “acceptable” starting levels. The IC may develop and require other
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

3

 Here, β means the probability of a Type II decision error.

5

AALs and required method uncertainties. If so, the IC should verify whether the laboratory can meet

the new method uncertainty requirements for the updated AALs.

4.2 Detection Capability

In some cases, the detection capability of a method, rather than the required method uncertainty, is

the important MQO of a project. Detection capability for this guide uses the concept of the minimum

detectable concentration (MDC) or minimum detectable value. MARLAP defines the minimum

detectable value of the analyte concentration in a sample as:

An estimate of the smallest true value of the measurand that ensures a specified high probability,

1 ! β, of detection.3

For radioanalytical processes, the probability of detection (1 ! β) of 0.95 is commonly used. The

definition of the minimum detectable value presupposes that an appropriate detection criterion has

been specified, i.e., “critical net concentration” for this document. This approach assumes that the

measured radionuclide net concentration in a sample will be above the critical net concentration 95%

of the time if the true concentration is equal to the MDC.

MARLAP (Chapter 20) provides a detailed discussion on how to calculate the critical net

concentration and MDC using a number of equations for various applications. The equations

provided in MARLAP calculate estimates of these method detection parameters for a given method

based on either a measured signal response of a single blank sample or from a population of sample

blanks that have been processed by the method under evaluation. For those applications when a

required MDC for a method has been specified as an MQO, the detection capability of the method

should be evaluated during method validation.

4.3 Bias and Trueness

Bias refers to the overall magnitude of systematic errors associated with the use of an analytical

method. The presence of systematic errors can be determined only by comparison of the average of

many results with a reliable, accepted reference value. Method bias may be estimated by measuring

materials whose composition is reasonably well known, such as reference materials, by comparing

results to those from at least one alternate method or procedure, or by analyzing spiked materials.

ISO (1993a) defines bias as:

 “[T]he mean value that would result from an infinite number of measurements of the same

measurand carried out under repeatability conditions minus a true value of the measurand.”

According to MARLAP (Chapter 6), bias typically cannot be accurately determined from a single

result or a few results because of the uncertainty in the measurement process to determine the

measurand. Bias is normally expressed as the absolute or relative deviation of the average of a group

of samples from the “true” or “known” value. Since it is a calculated estimate, a bias should be
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

6

reported with a combined standard uncertainty and include the number of data points used to

calculate the bias.

It is assumed that the mean response of the method is essentially a linear function of analyte

concentration over the useful range of the method. As defined in MARLAP, “this function can be

characterized by its y-intercept, which reflects the mean response at zero concentration, and its slope,

which reflects the ratio of the change in the mean response to a change in sample analyte

concentration.” The “absolute bias” of a method can be thought of as the difference between the

average concentration of the radionuclide at the y-intercept and the true concentration of zero. 

The IC will specify a method bias limit as an APS when method bias is considered an important

method performance characteristic for the method or a quality parameter for the project. Method bias

must be evaluated during method development, general and project method validation processes, and

subsequently, the processing of batch quality control (QC) samples processed with the incident

response samples (MARLAP Chapter 7). 

The method uncertainty acceptance criteria provided in MARLAP (Chapter 6), as well as for project

method validation in Section 5.4, assume that laboratories would not use a method that has a

significant bias. When a method has excessive bias, the method validation test results for the

required method uncertainty will be unacceptable. Appendices D and E provide information on bias

evaluation methods as related to the method validation acceptance criteria.

4.4 Analyte Concentration Range

The analyte concentration range of a method is a method performance characteristic that defines the

span of radionuclide activity levels, as contained in a sample matrix, for which method performance

has been tested and data quality deemed acceptable for their intended use. However, not all sample

matrices encountered during an incident response will have preestablished analytical action levels

with corresponding required method uncertainty values or required MDCs. Therefore, incident

response method validation must be sufficiently flexible to address not only those typical sample

matrices (liquids, air filters, swipes, and soil/solids) for which there are action levels, but also those

matrices for which there are no specified action levels. The subsequent subsections discuss the

analytical concentration range options for method validation for both situations. For both options,

the method is to be tested at a low, mid and upper validation test concentration/activity except when

noted. 

4.4.1 Derived Radionuclide Concentrations Corresponding to Established Action Levels 

MARLAP (Chapter 6) recommends that a method be validated at the expected action level for a

radionuclide and matrix combination. Therefore, an analyte concentration range should include either

an established regulatory limit or a defined action level, typically near the midpoint of the

radionuclide activity (concentration) range for a project. For a radiological incident response

application, the established AAL would normally be a derived radionuclide concentration

corresponding to a PAG or a risk-based dose as designated by an agency representative. There may

be four or five action levels for the various matrices contaminated, with the range of concentrations

as great as four orders of magnitude. Derived radionuclide concentrations for the various established

AALs have been generated for water and air-filter matrices (AALs for soils/sediments and building
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

7

materials are being developed), and can be found in the Radiological Laboratory Sample Analysis

Guide for Incidents of National Significance series. Summary tables of the established AALs for

these three matrices can be found in Appendix A. AALs vary according to the matrix, phase of the

incident and applied PAG.

At most laboratories, samples that have been screened and found to contain very high radionuclide

activities probably will be subdivided (when possible) prior to specific radioanalytical processing.

Also, samples requiring multiple radionuclide analyses should be subdivided. Both situations will

result in lower-activity subsamples (aliquants) for processing. For example, when a sample that has

a very high radionuclide concentration or activity is received by a laboratory, the sample likely will

be subdivided (possibly into five parts) so that a different radioanalytical pathway for each

radionuclide may be performed in parallel. For aqueous and soil samples, the radionuclide

concentration of the radionuclides in the aliquants would be the same as the original aqueous and

soil concentrations, but the aliquant activity available for processing will be reduced proportionally

from the original sample size. For air-filters, the total activity on the filter matrix represents the

activity in a volume of air collected. For swipe samples, the activity on the sample represents the

activity removed from a surface area swiped. Air filter and swipe samples may be digested prior to

radiochemical processing and the digestate volume generated represents the total activity on the

original sample for the air volume collected or surface area swiped. Aliquanting these digestates to

obtain a lower subsample activity or for multiple analyses is also a common practice. Thus, it is

important to know the exact fraction of the original sample taken so that the analysts know that a

sufficient sub-sample quantity has been processed to ensure that the MQOs have been met.

When developing incident response methods for high activity samples, it is important to note that

the analytical concentration range and detection capability specifications will be significantly higher

than what is usually found in normal procedures for environmental monitoring sample processing.

This difference in concentration range should be emphasized in the procedure’s scope.

4.4.2 Default Analytical Action Levels

Established AALs based on PAG-derived radionuclide concentrations may not be available for all

matrices encountered in an incident response, such as concrete or asphalt. In the absence of

established PAG action levels, default AALs may be used for the validation test concentration or

activity levels. Section 5.4 provides guidance on selecting default AALs applicable to method

validation for the three general matrix categories of liquids, air sampling media/swipes and solids.

The default AALs approximate the expected derived radionuclide activity level for a sample volume

or mass for a 100-mrem or 10-4 risk-based AAL. These AAL levels were chosen because they can

be conveniently scaled to other possible project-specific AALs for the various matrices. For example,

if a specific project had an AAL at 20 mrem (one-fifth of a 100 mrem AAL), the table values for the

AALs can be scaled down simply by dividing the listed values by five. 

4.5 Method Specificity

MARLAP defines “method specificity” as “the ability of the method to measure the analyte of

concern in the presence of interferences.” EURACHEM (1998) defines selectivity or specificity as

“the ability of a method to determine accurately and specifically the analyte of interest in the

presence of other components in a sample matrix under the stated conditions of the test.”
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

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By extension, this guide defines method specificity as:

The ability to correctly identify and quantify the radionuclide(s) of interest in the presence of other

interferences in a sample under stated conditions of the test.

Method specificity should be evaluated during method development and the general and project

method validation processes for the applicable matrices, radionuclide(s) of interest and known

interfering radionuclides. Method specificity may be evaluated during method validation by

analyzing:

 • Matrix samples that have been characterized in terms of radionuclide and chemical constituent

content;

 • Appropriate matrix blanks; and

 • Matrix blanks spiked with interferences.

Each specific sample matrix should be tested for method specificity, e.g., concrete, asphalt, soil, etc.

Matrix samples and blanks should be chosen to be as representative of the target matrix as is

practical. When possible, matrix blanks should contain the chemical species and potential interfering

radionuclides, other than the radionuclide(s) of interest, at concentrations that are reasonably

expected to be present in an actual sample. Each of the three options to determine method specificity

may provide insight into the relative degree of expected quantitative effect that the interferences will

have on the identification and quantification of the radionuclide(s) of interest at different

concentrations. 

Method specificity is typically expressed qualitatively and quantitatively. A radiochemical method

specificity statement would include descriptions of parameters, such as:

 • Expected radionuclide and chemical interferences

 • Effects of the interfering substances on the measurement process

 • Measurement information that substantiates the identity of the analyte (e.g., half-life, or

decay emission and energy)

 • Effects of oxidation or molecular state of the target or interfering radionuclides

 • Chemical processes that can remove interfering materials (e.g., ion exchange, solvent

extraction)

 • Summary of results from analysis of standards, reference materials and matrix blanks

4.6 Method Ruggedness

MARLAP defines “method ruggedness” as “the relative stability of method performance for small

variations in method parameter values.” EURACHEM (1998) discusses the concept of method

ruggedness and robustness interchangeably. Ruggedness is a measure of how well a method’s

performance stands up to less than perfect implementation. In any method there are certain steps

which if not carried out sufficiently, exactly or carefully may have a significant effect on method

performance and the reliability of the results. Typically, these critical steps are identified during the

method development process, and annotations are made in the method description that provide

limiting conditions and an allowable range of application. It is advantageous to identify the variables

in the method that have the most significant effect on the analytical results so that they are closely
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

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controlled. Ruggedness or robustness tests have been developed which involve experimental designs

for examining method performance when minor changes are made in operating steps or in some

cases environmental conditions (EPA 2006, Validation and Peer Review of the U.S. EPA

Radiochemical Methods of Analysis). The tests involve making deliberate variations to the method,

and investigating the subsequent effect on performance.

An example of method ruggedness is the adjustment of pH during the separation of strontium from

calcium in the analysis of milk for 90Sr. The pH of the milk is buffered at 5.4 and disodium EDTA

is added prior to passing the solution through a cation exchange column. The calcium will effectively

complex with EDTA at this pH, forming an anion, while the strontium remains a cation. A pH lower

than about 5.2 will not provide enough EDTA anion to effectively complex calcium, and a pH

greater than about 5.5 will begin to effectively complex strontium. Thus for this analysis method,

ruggedness deals with pH control in the range of 5.2 to 5.5.

Method ruggedness is typically evaluated during method development and prior to method

validation. Therefore, no specific tests for ruggedness will be included in this document for project

method validation of the radioanalytical methods used for incident response. 

5.0 Incident Response Method Validation Guidance, Tests, and Requirements

This section provides guidance, specific tests and minimum requirements for project method

validation for methods used to process samples from a radiological incident. This section addresses

the following selected method performance characteristics:

 • Method specificity

 • Analyte concentration range 

 • Method validation levels for testing the required method uncertainty

 • Verification of required detection limit specification

Discussion of matrix considerations and method bias tests are also included in this section. Before

initiating the method validation process, a validation plan should be prepared that incorporates the

various guidance and requirements specified in this section and Section 6, Method Validation

Documentation.

5.1 Method Specificity

Method specificity is evaluated during general method validation for normal routine applications,

e.g., environmental surveillance programs. During general method validation, the method should be

evaluated for the applicable matrices and radionuclide(s) of interest and known interfering chemical

constituents and radionuclides over a typical expected range. For some incident response

applications, method validation testing for method specificity may be more focused than general

method validation. Incident response scenarios may involve one or many radionuclides and a

multitude of matrices. To ensure method specificity for the incident response application have been

met, the proficiency testing (PT) samples used for incident response method validation should

contain the known or expected concentration levels of the matrix chemical species and potential

interfering radionuclides. Adequate method specificity during project method validation should be

evaluated by analyzing:
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

4

 The term “decontamination factor” is defined as the amount of interferent in the sample before chemical separation

divided by the measured amount in the sample after chemical separation.

5

 The IC may develop and require other AALs and required method uncertainties. If so, the IC should verify whether the

laboratory can meet the new method uncertainty requirements for the updated AALs. Calculating the test levels for

method validation should be consistent with Table 2.

10

 • Matrix PT samples that have been characterized in terms of expected radionuclide and chemical

constituent content;

 • Appropriate matrix blanks containing the applicable radionuclide and chemical interferences;

and

 • Matrix blanks spiked with interferences.

During method development, decontamination factors4

 should be evaluated for the more commonly

expected radionuclide interferences so that the final method can improve method performance and

adequately address radionuclide interferences. Also, the concentration of the interfering radionuclides should be added during method development at their 100 mrem AAL-derived radionuclide

concentrations. Matrix blank results having no absolute bias would indicate adequate method

specificity. Excessive absolute or relative bias, erroneous chemical or radiotracer yields, or possibly

excessive method uncertainty may be indications of inadequate method specificity. 

5.2 Analyte Concentration Range

The radionuclide concentration range applicable to method validation for radiological incident

response should extend from a lower bound (~0.5 AAL) to an upper bound (3 AAL) that are both

a multiple of an incident response action level. For radiological incident response applications, the

analyte concentration range for the method validation process and validation test levels should be

established based on the established PAG or risk-based derived radionuclide concentrations as

designated by a representative of the responsible government agency. If the laboratory has not been

provided with action levels by the IC, default values listed in Table 1 may be used. Also, derived

radionuclide concentrations for the various AALs for water, air filter and soil matrices can be found

in the Radiological Laboratory Sample Analysis Guide for Incidents of National Significance.

Summary tables of the AAL-derived radionuclide concentrations for the water and air filter matrices

can be found in Appendix A5 of this document. These tables list the expected AALs for various

media mainly for the late intermediate and recovery phases, but also for the early phase. The AALderived radionuclide concentrations will vary according to the matrix, phase of the incident and

applied PAG. For air filters, an activity per sample corresponding to an AAL concentration for an

assumed air volume sampled should be used.

The validation test concentration/activity values should be adjusted to reflect the typical sample

aliquant size that would be analyzed. In some cases, the original sample may be aliquanted directly,

but in other cases the sample must be completely digested before sample aliquanting. When the

radionuclide(s) identity is known, the number of aliquants may be small, but when the identity is not

known, the number of aliquants may be three or more depending on the decay particle emission type.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

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In the absence of established PAG AALs, default AALs may be used. Default AALs (activity per

sample aliquant) for three general matrix categories and radionuclide emission type are provided in

Table 1. Default AALs can be used for similar matrix categories, as discussed in Section 5.3 (for all

solutions, use water; for swipes, use air-filter materials; and for pulverized concrete, use soil). It

should be noted that these default AALs and associated required method uncertainty values (Table

4) for the stated general matrix categories do not have a dosimetric basis but may be considered

adequate for method validation purposes.

TABLE 1 – Default Analytical Action Levels for General Matrix Categories

Default Test Level Activity in Each Sample

Aliquant (Total pCi) [1]

Matrix

Category Size Assumptions for Values Alpha

(

241Am)

Pure Beta

(

90Sr)

Gamma

(

60Co)

Liquids 5-mL Screen 2.0 12 33

100-mL Nuclide-Specific 40 240 660

Air Sampling

Media/Swipe

68 m3

 Screen 22 1,900 8,400 

68 m3

 (4 aliquants [2])

Nuclide-Specific 5.5 480 2,100

Solids – soil,

etc. 

2 g

TBD [3] 100 g 

500 g

[1] Test-level activity corresponds closely to 100-mrem dose-derived concentration values for water and 10–4 risk-based

DAC for air (Appendix A). The table values were calculated for the noted radionuclides. To calculate air sampling

default AALs for 10–6 risk-based applications, the 10–4 risk-based values in the table can be scaled down by a factor

of 100. Table values for the solids and soil are pending.

[2] Test-level activity assumes that the air filter has been split into four aliquants after sample digestion.

[3] TBD: To be determined pending development of Radiological Laboratory Sample Analysis Guide for Incidents of

National Significance–Radionuclides in Soils and Solids.

For each matrix category, default AALs are provided for both screening and specific radionuclide

methods requiring validation. The test level values stated in Table 1 were calculated using the DRC

for the 100-mrem AAL values for the gross alpha, beta, and gamma screening levels and for 241Am,

90Sr, and 60Co for the specific alpha and beta/gamma radionuclide categories. The default AALs have

been adjusted to reflect the typical sample aliquant size (column 2 of Table 1) that would be analyzed

by a laboratory.

For practical reasons and to prevent potential laboratory/instrumentation contamination and

radiological safety issues, the test levels for incident response method validation purposes are limited

to three levels related to the designated (established) PAG AALs (derived radionuclide

concentrations) or default AALs. The use of three test levels is consistent with the specifications

indicated in Table 3 and Section 5.4. For incident response method validation, the validation test

levels are denoted as lower, mid, and upper. For method validation levels B, C, D, and E (Section

5.4), the recommended three concentration test levels for the replicate PT samples are presented in

Table 2. The lower level test level of ~0.5 AAL was chosen to avoid detectability issues that could

occur at lower test concentrations. The mid test level corresponds to the established PAG AAL or

default AAL test level. It is assumed that a laboratory will use the same sample aliquant size and
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

12

counting time to analyze the test samples for all three test-level concentrations (values typically

selected to meet the required method uncertainty at the mid test level, or AAL).

 TABLE 2 – Method Validation Test Concentrations

 Test Level Relative Concentration

Upper ~3 AAL

Mid ~1 AAL 

Lower ~ 0.5 AAL

5.3 Matrix Considerations

For method validation, the method under consideration shall address a specific radionuclide and

matrix combination. In many applications, a matrix may be described by a general name or type,

such as water or air particulate. However, when developing and documenting the applicability of a

method, a description of the sample matrix should be specific and address possible variations in the

matrix that may be encountered when such will impact method performance. In addition, validation

of a method applies only to the specifically defined matrix described in the method validation plan,

which must be consistent with the matrix description in the method applicability statement. Listed

below are some specific matrices that may be encountered for radioanalytical processing during an

incident:

 • Liquids

– Fresh water

– Surface water

– Groundwater

– Rain

– Salt/brackish water

– Aqueous suspensions

– Aqueous solutions

– Sewer and water treatment effluents or discharges

– Collection of volatiles

– Organic liquids

– Liquids generated during decontamination activities

 • Air sampling media

– Glass fiber, cellulose, acetate filters

– Charcoal canisters or loose particles

– Molecular sieve

– Silica gel

 • Swipes

– Glass fiber, cellulose, acetate filter paper

 • Solids

– Soil, sediment, stone, sod, vegetation, wood

– Manufactured/construction
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

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– Concrete, asphalt, brick, ceramics, plaster, plastics, metals, clothes, paper, stone, wood, etc.

– Sludges

– Sewer and water treatment

– Solids generated during decontamination activities

5.4 Method Validation Levels for Testing the Required Method Uncertainty

The primary method validation approach used in this document follows the concepts presented in

MARLAP Chapter 6 for the required method uncertainty MQO. The MARLAP method validation

approach and validation acceptance criteria assume that the laboratory method being validated has

no significant bias. However, this may not always be the case. Appendix D provides an insight into

the effect of method bias on the probability of failing the MARLAP validation acceptance criteria.

An alternate approach that may be used to determine if a method has acceptable method validation

performance is presented in Appendix E. This approach is based on the mean squared error (MSE)

or root mean squared error concept and has a greater power to detect excessive imprecision or bias

in many cases. 

If a method fails to meet the method validation acceptance criteria as presented in the subsequent

sections, the laboratory should:

 • Evaluate the possible reasons for the failure; 

 • Identify the root causes for the failure; and

 • Update the method with the appropriate corrections or additions to ensure the method will meet

the specified MQOs.

The updated method must go through another validation process using the same requirements

applied to the first attempt at method validation. 

5.4.1. Method Validation Requirements Based on MARLAP Concepts

Similar to the MARLAP (Chapter 6) graded approach to project method validation, there are four

proposed tiers or “levels” of method validation (Levels B, C, D, E) to demonstrate a method’s

capability of meeting the required method uncertainty MQO applicable to a radiological incident.

For this guide, the MARLAP method validation Level A for the same radionuclide and matrix

combination has been combined with validation Level B (see Table 3). The level(s) of method

validation needed should be designated by the IC. The laboratory will select a method based on

various operational aspects and the status of it’s existing methods to meet the required method

uncertainty uMR or nMR specification for a designated (established) AAL (Appendix A) or a required

method uncertainty for a default AAL (Section 5.2). The uMR is specified in the units of the AAL. The

nMR is a fractional unitless value (e.g., 0.13) and is calculated by dividing the uMR by the AAL.

Appendix A contains tables listing the required method uncertainties for screening and nuclidespecific methods for certain established AALs and sample matrices related to a potential radiological

incident. 

The four levels (B-E) of method validation for testing compliance with the required method

uncertainty using specified PT samples cover the following: 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

14

Level B – Existing methods for radionuclide and matrix combinations for same, similar or

slightly different matrices (internal PT samples); 

Level C – Existing methods that require modification to accommodate matrix differences

(internal or external PT samples);

Level D – Adapted or newly developed methods (internal and external PT samples); and

Level E – Adapted or newly developed methods using method validation reference materials

(method validation reference materials).

During the method validation process, the laboratory shall evaluate the method as to the required

method uncertainty and relative bias for the three test concentrations (Table 2) for the specified

method validation level, as well as the absolute bias through the use of at least seven blanks (Section

5.6 and Appendix E). The acceptable performance of a method to meet the required method

uncertainty will vary according to the level of validation as described in subsequent subsections. It

should be noted that the probability of acceptable performance for meeting a required method

uncertainty specification is dependent on the magnitude of existing method bias. The greater the

magnitude of the method bias, the more likely the method will not meet the required method

uncertainty specification. If excessive bias is measured during the method validation process, the

method should be revised to eliminate the bias as much as possible. 

For radiological incident response applications, the analyte concentration range for the method

validation process, and thus the validation test levels, should be established based on the established

PAG or risk-based derived radionuclide concentrations as designated by an agency representative.

Derived radionuclide concentrations corresponding to the various established AALs (PAG or riskbased) for water, air particulate and soil matrices have been provided in the Radiological Laboratory

Sample Analysis Guide for Incidents of National Significance and summarized in the Appendix A.

In the absence of validation test activity levels based on established AALs (PAG or risk-based),

default AALs specified in Section 5.2 may be used. Validation test activity/sample levels (designated

established AALs or default AALs) are to be used in conjunction with all method validation levels

stated in Table 3 and the required method uncertainty values for the radionuclide and matrix

combinations provided in the Radiological Laboratory Sample Analysis Guide for Incidents of

National Significance (Appendix A) or in Section 5.2. 

A method is considered validated for a project when it has met the required method uncertainty

acceptance criteria stated in Table 3 and the acceptance criteria for other method characteristics such

as bias and required MDC, as may be stated by the IC. When the required method uncertainty

specifications in Table 3 are met for default AALs, it will be assumed that the method has met the

required method uncertainty acceptance criteria for all PAG or risk-based action levels above the

default AALs. 

All method validation levels require replicate samples at three different validation test concentration/

activity levels below, at, and above the derived radionuclide concentration corresponding to an AAL

(designated, established, or default). To ensure testing for sufficient method specificity, the known

concentration levels of potentially interfering radionuclides should be included in the test samples.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

15

TABLE 3 – Method Validation Requirements and Applicable to Required Method Uncertainty

Validation

Level [1] Application

Sample

Type

Acceptance

Criterion [2]

Levels [4]

(Concentration) Replicates [3]

# of 

Analyses

B

Existing Method 

Radionuclide – Same,

Similar or Slightly

Different Matrix

Internal

PT 

Measured Value

Within ±2.8 uMR or ±

2.8 nMR of

Validation Value

3 39

C Similar Matrix:

New Application

Internal or

External

PT 

Measured Value

Within ±2.9 uMR or ±

2.9 nMR of

Validation Value

3 5 15

D

 Adapted, Newly

Developed, Rapid

Methods

Internal or

External

PT

Measured Value

Within ±3.0 uMR or ±

3.0 nMR of

Validation Value

3 7 21

E

Adapted, Newly

Developed, Rapid

Methods

Method

Validation

Reference

Materials

Measured Value

Within ±3.0 uMR or ±

3.0 nMR of

Validation Value

3 7 21

[1] MARLAP method validation Level A for the same radionuclide and matrix combination has been included into

validation Level B.

[2] The acceptance criterion is applied to each analysis/test sample used for method validation, not the mean of the

analyses. uMR and nMR values are the required absolute and relative method uncertainty specifications stipulated in

the Radiological Laboratory Sample Analysis Guide for Incidents of National Significance for gross screening

concentrations and quantification of individual radionuclide concentrations in various matrices. The acceptance

criteria are chosen to give a false rejection rate of ~5% when the measurement process is unbiased, with a standard

deviation equal to the required method uncertainty (uMR or nMR). The stated multiplier (k = 2.8, 2.9, 3.0) for the

required method uncertainty was calculated using the formula where N is the number of

measurements, α is the desired false rejection rate, and, for any p, zp denotes the p-quantile (0 < p < 1) of the

standard normal distribution (MARLAP Appendix G, Table G.1). The uMR or nMR values are provided in Appendix

A or Table 4.

[3] For certain matrices, not all samples in a given test level can be spiked with the same known radionuclide activity

or concentration. In such cases, the measured activity or concentration in the test sample should be compared to the

known value for that test sample.

[4] At least seven blank samples should be analyzed as part of method validation but are not considered part of the three

required concentration test levels.

5.4.2 Required Method Uncertainty Acceptance Criteria

For all four method validation levels for method uncertainty, acceptable method validation is

determined by comparing each test sample result for a given test concentration or activity with the

required method uncertainty specification (k × uMR or k ×nMR) provided in Table 3. The values for uMR

or nMR are stipulated in the Radiological Laboratory Sample Analysis Guide for Incidents of National

Significance (Appendix A) for the three basic incident response matrices of water, air filter/swipes,

and soil. A “k” value can be either 2.8, 2.9, or 3.0. It should be noted that the required method

uncertainty specification and AAL-derived radionuclide concentrations may vary according to the

matrix, the phase of the incident response and applied PAG.

Appendix B provides examples for testing a method’s acceptability to meet validation Level D for

an established PAG AAL and a default AAL test level in a water matrix. 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

16

5.4.2.1 Level B Method Validation: Same, Similar, or Slightly Different Matrix

Most qualified laboratories will have existing methods to analyze for the radionuclides of interest

in the three most common matrices of water, air particulate filters and soil/sediment. Under method

validation Level B, a method that has been previously validated for a different project and used for

one matrix may be used for that same matrix or modified for use for a very similar matrix. An

example of a slightly different matrix might be a method used for water samples having low

dissolved solids, modified for water samples containing high dissolved solids. Level B requires the

laboratory to conduct a method validation study for the radionuclide and matrix combination where

three replicate samples from each of the three concentration levels are analyzed according to the

method. Table 2 is to be used to determine the lower, middle, and upper testing levels for the

replicate analyses. The test samples are internal PT samples prepared at the laboratory. In order to

determine if a proposed method meets the project MQO requirements for the required method

uncertainty, each internal PT sample result is compared with the method uncertainty acceptance

criteria in Table 3. The acceptance criteria in Table 3 for Level B validation stipulate that, for each

test sample analyzed, the measured value must be within ±2.8 uMR for test-level concentrations at or

less than the AAL or ± 2.8 nMR for the test-level concentration above the AAL. These acceptance

criteria apply to either established AALs stated in Appendix A or default AALs (Section 5.2). The

values of uMR and nMR for select radionuclide and matrix combinations are provided in Appendix A

for established PAG AALs or in Table 4 for default AALs. The required method uncertainty values

for the established PAG AALs are based on the Radiological Laboratory Sample Analysis Guide for

Incidents of National Significance for the three basic matrices addressed: water, air and soil. The

Table 4 values for uMR are base on the default AALs stated in Table 1, and the nMR values are taken

from the air and water editions of the Radiological Laboratory Sample Analysis Guide for Incidents

of National Significance. For example, in Table 4 the uMR value of 5.2 pCi/sample for specific alphaemitting nuclides in a water matrix (column two, row two) is calculated by multiplying the relative

required method uncertainty (nMR) of 0.13 (column three, row two) for this radionuclide and matrix

combination by the default AAL in Table 1 (column three, row two) of 40 pCi/sample value. The

nMR values used in Table 4 are 0.30 for screening measurements and 0.13 for specific radionuclide

analyses. The uMR values listed are in units of pCi per sample, assuming the aliquant sample sizes that

will be used in the method validation process given in Table 1. 

TABLE 4 – Required Method Uncertainty (uMR and nMR) Values for Default AAL Test Levels

Radionuclide [1]

Water [2,6]

Air Sampling

Media/Swipes [3,6] Solids [5,6]

uMR (pCi/

sample) nMR

uMR (pCi/

sample) nMR

uMR (pCi/

sample) nMR

Gross α Screen 0.60 0.30 6.7 0.30 TBD TBD

Specific Alpha-Emitting[4]

Nuclides - based on 241Am

5.2 0.13 0.73 0.13 TBD TBD

Gross β Screen 3.6 0.30 580 0.30 TBD TBD

Specific Beta-Emitting[4]

Nuclides - based on 90Sr 31 0.13 63 0.13 TBD TBD

Gamma Screen 9.9 0.30 2,500 0.30 TBD TBD

Specific GammaEmitting[4]

 Nuclides - based on 60Co

86 0.13 270 0.13 TBD TBD
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

17

Notes:

[1] For each radionuclide category, the absolute required method uncertainty (uMR) is applied to the lower and mid test

levels. The relative required method uncertainty (nMR) is to be used for the upper test level.

[2] Required method uncertainty values for water correspond to the 100-mrem dose-derived concentration values from

Scenario 1 of the Radiological Laboratory Sample Analysis Guide for Incidents of National Significance–

Radionuclides in Water (EPA 2008). 

[3] Required method uncertainty values for air sampling media correspond to the 10–4 risk-based derived air concentration values from Scenario 1 of the Radiological Laboratory Sample Analysis Guide for Incidents of National

Significance–Radionuclides in Air (EPA 2009a).

[4] The default values stated in the specific emitting nuclide rows apply to all radionuclides in the designated emission

category. The reference to a radionuclide is presented only as information indicating the basis for the specific

emission category. 

[5] TBD: To be determined pending development of Radiological Laboratory Sample Analysis Guide for Incidents of

National Significance–Radionuclides in Soils (In preparation).

[6] Table values have been rounded after calculations.

5.4.2.2 Level C Method Validation: New Application of an Existing Method to a Different

Matrix

When a laboratory has a validated method for a radionuclide in one matrix but not others, the method

may require modification to accommodate a completely different media/matrix (water versus soil)

or a very different similar matrix (two soils of different physicochemical compositions). The degree

of adaptation or modification needed will vary according to chemical and physical differences in the

two matrices. Because of the extent of these differences, a laboratory may choose to validate the

method for the radionuclide and matrix combination through method validation Level C. Level C

method validation requires the laboratory to conduct a method validation study wherein five replicate

samples from each of the three concentration levels are analyzed according to the method. Table 2

is to be used to determine the lower, mid and upper testing levels for the replicate analyses. The test

samples are internal PT samples prepared at the laboratory. In order to determine if a proposed

method meets the project MQO requirements for the required method uncertainty, each internal PT

sample result is compared with the method uncertainty acceptance criteria of Table 3. The

acceptance criteria stated in Table 3 for Level C for new applications stipulate that, for each test

sample analyzed, the measured value must be within ±2.9 uMR for test level concentrations at or less

than the AAL or ± 2.9nMR for the test level concentration above the AAL. These acceptance criteria

apply to either established PAG AALs stated in Appendix A or default AALs (see Section 5.2). The

values of uMR and nMR for select radionuclide and matrix combinations are provided in Appendix A

for established PAG AALs or in Table 4 for default AALs. 

5.4.2.3 Level D Method Validation: Adapted or Newly Developed Methods, Including Rapid

Methods

In some cases, a laboratory may not have a method for a certain radionuclide and matrix combination. For such situations, the laboratory may either develop a new method internally or adapt a

method from the literature. In this case, the new method should undergo general method validation

first and then incident response method validation. A laboratory would validate the new method for

the radionuclide and matrix combination through method validation Level D or E (Section 5.4.2.4).

Level D method validation requires the laboratory to conduct a method validation study wherein

seven replicate samples from each of the three concentration levels are analyzed according to the

method. Table 2 is to be used to determine the lower, mid and upper testing levels for the replicate
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

18

analyses. For validation Level D, the test samples are internal PT samples prepared at the laboratory.

In order to determine if a proposed method meets the project MQO requirements for the required

method uncertainty, each internal PT sample result is compared with the method uncertainty

acceptance criteria in Table 3. The acceptance criteria stated in Table 3 for Level D validation

stipulate that, for each test sample analyzed, the measured value must be within ±3.0 uMR for test

level concentrations at or less than the AAL or ± 3.0 nMR for the test level concentration above the

AAL. These acceptance criteria apply to either established PAG AALs stated in Appendix A or

default AALs (Section 5.2). The values of uMR and nMR for select radionuclide and matrix combinations are provided in Appendix A for established AALs or in Table 4 for default AALs. 

5.4.2.4 Level E Method Validation: Adapted or Newly Developed Methods, Including Rapid

Methods, Using Method Validation Reference Materials

Methods developed by the laboratory or adapted from the literature that have undergone general

method validation but not project method validation for an incident response are to be validated

according to Levels D or E of Table 3. Both of these method validation levels have the same number

of required test sample replicates and validation acceptance criteria. However, validation Level E

is used when the sample matrix under consideration is unique and the IC determines that the method

should be validated using the same matrix as the expected sample matrix. In this case, special

method validation reference material (MVRM) would be used in the method validation process. The

use of MVRM may be important for unique non-potable water matrices, soils or sediments, and

manufactured type sample matrices. 

Level E method validation requires the laboratory to conduct a method validation study wherein

seven replicate samples from each of the three concentration levels are analyzed according to the

method. Table 2 is to be used to determine the lower, mid and upper testing levels for the replicate

analyses. The test samples are external MVRM samples prepared for the laboratory. In order to

determine if a proposed method meets the project MQO requirements for the required method

uncertainty, each MVRM sample result is compared with the method uncertainty acceptance criteria

of Table 3. The acceptance criteria stated in Table 3 for Level E validation stipulate that, for each

test sample analyzed, the measured value must be within ±3.0 uMR for test level concentrations at or

less than the AAL or ± 3.0nMR for the test level concentration above the AAL. These acceptance

criteria apply to either established PAG AALs stated in Appendix A or default AALs (See Section

5.2). The values of uMR and nMR for select radionuclide and matrix combinations are provided in

Appendix A for established AALs or in Table 4 for default AALs. 

Figure 1 (page 20) identifies the general approach to the method validation path that needs to be

taken for a specific combination of radionuclide and matrix. Laboratories should use this chart to see

where they are in the method validation process. It may be helpful for laboratories to create a similar

flowchart in their method validation documentation to assist reviewers, auditors, and training

personnel in recognizing the thought process used by the laboratory to validate methods.

5.5 Verification of Required Detection Limit (MDC) Specification

This section provides specifications for the method validation process to verify the required MDC

specification MQO. Figure 2 (page 20) illustrates the process used to verify the required MDC for

a method. The specifications presented are separate requirements with respect to the project method
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

19

Figure 1 – Method Validation Process for the Required Method Uncertainty MQO

validation process for the required method uncertainty MQO. General method validation requirements are to be met prior to the initiation of this verification process. The specifications given in this

section are distinct from those given for the required method uncertainty MQO method-validation

process. 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

20

5.5.1 Calculation of the Critical Net Concentration

The critical net concentration shall be calculated for the required MDC method validation process.

The calculation of the critical net concentration for the method is based on the analytical results of

the blank matrix samples used in the MDC validation process. A minimum of seven blank samples

is required. To ensure testing for sufficient method specificity, the matrix blanks should contain the

anticipated concentration levels of chemical interferences and the potential interfering radionuclides

(naturally occurring and incident response-related).

The critical net concentration (CLNC), with a Type I error probability of α = 0.05, is calculated using

the following equation (consistent with MARLAP, Chapter 20, Equation 20.35):

(1)

Analyze at least 7 blanks to

establish critical net 

concentration, CLNC

Has method 

been established

and tested for 

radionuclide-matrix 

combination 

?

Analyze 10 replicate samples

spiked at the required MDC

Are the

number of

replicate results

below CLNC

< 2

?

Required MDC is

the MQO

Revise or develop method

chemical-separations or 

counting techniques

Yes

Yes

Evaluate method

for matrix effects

MDC MQO

has been

verified

No

No

Figure 2 – Validation Process for Verifying the Required MDC MQO
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

21

where sBlanks is the standard deviation of the n blank-sample net results (corrected for instrument

background) in radionuclide concentration units of pCi/sample, and t1!α(n!1) is the (1!α)-quantile

of the t-distribution with n!1 degrees of freedom (see MARLAP Table G.2 in Appendix G).

Although the Type I error rate of 0.05 is routinely used and accepted, it is possible that other error

rates may be used in incident response situations.

For seven (minimum) blank results (six degrees of freedom) and a Type I error probability of 0.05,

Equation (1) reduces to: 

(2)

If the number of blank samples is different than the minimum value of seven, refer to MARLAP

Chapter 20, Attachment 20A for appropriate guidance. Care must be taken to ensure that all samples

and blanks are analyzed under conditions that are typical of those used for routine analyses using the

same sample weight or volume and with the same instruments with representative counting efficiencies and background levels. The calculated critical net concentration will be used in the verification

process to determine if a method is capable of meeting the required MDC specification as described

in Section 5.5.2. 

5.5.2 Testing for the Required MDC

When a required MDC specification for a radionuclide and matrix combination is given as an MQO

rather than the required method uncertainty, the method should be validated by verifying that the

method can meet the required MDC. As noted in Table 5, method validation for the required MDC

specifies that ten replicate samples, each spiked at the required MDC, should be analyzed and

evaluated. In addition, the results of at least seven blank samples are used to determine the critical

net concentration of the method (Section 5.5.1). The ten replicate spiked samples and seven blanks

should contain the chemical species and potential interfering radionuclides which are reasonably

expected to be present in an actual sample. To ensure the testing for sufficient method specificity,

the expected concentration levels of the chemical species and potential interfering radionuclides

should be used during testing. Figure 2 (page 20) provides an overview of the method validation

process for verifying the required MDC MQO.

Testing for the required MDC verification is based on the null hypothesis that the true MDC for the

method is at or below the required MDC. If the true MDC of the method has been calculated

properly and is equal to or less than the required MDC, the probability of failing to detect the

radionuclide at or above the critical net concentration is at most β. For project method validation

related to incident response, β is assumed to be 0.05. The number of “non-detects” (sample results

below the critical net concentration) for a set of n samples spiked at the required MDC is assumed

to have a binomial distribution with parameters β and n. For a set of ten samples spiked at the

required MDC, the number of Y sample results expected to be below the critical net concentration

is not more than two (2) for a β of 0.05. If Y is greater than two, the null hypothesis is rejected.

The following protocol should be used to verify a method’s capability to meet the required method

MDC for each radionuclide (including gross screening)-matrix combination:
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

22

 1. Analyze a minimum of seven blank samples (representing the matrix of interest) for the

radionuclide under consideration.

 2. From the blank sample net results, calculate the estimated Critical Net Concentration

(Section 5.5.1), CLNC.

 3. Analyze ten replicate samples (representing the matrix of interest) spiked at the required

MDC for the radionuclide under consideration.

 4. From the results of the ten replicate samples spiked at the required MDC, determine the

number (Y) of sample results at or below the estimated Critical Net Concentration.

 5. If Y # 2, the method evaluated at the required MDC passes the test for the required MDC

specification.

 6. If Y > 2, the method evaluated at the required MDC fails the test for the required MDC

specification.

Appendix C provides an example for testing a method’s capability to meet a required MDC

specification.

TABLE 5 – Method Validation Requirements Applicable to Required MDC Verification

Method

Characteristic Application

Sample

Type

Acceptance

Criterion

Levels

(Concentrations) Replicates

# of 

Analyses

Detection

Capability

Required

MDC

Specification

Internal

PT

Number of Sample

Results Below

Critical Net

Concentration Value

# 2

Single Concentration

at the Required MDC

Value

10 10

Note: At least seven blank samples should be analyzed to estimate the critical net concentration as part of the required

MDC verification.

5.6 Method Bias Tests

In order to provide quality data, a method should not have a significant bias. Depending on the

radiological incident, acceptable absolute and relative bias criteria for a method may be specified by

the IC. Since the degree of acceptability of method bias depends on many parameters and circumstances, specific acceptance criteria for method bias have not been included for this method

validation process. However, because the acceptance criteria for method uncertainty and required

MDC verification will not tolerate a significant method bias or measurement uncertainty, acceptable

method bias is indirectly evaluated when evaluating method uncertainty and the required MDC.

Appendix D provides an example of the effect of bias on the probability of failing the required

method uncertainty validation acceptance criteria for method validation Level D.

Method bias is initially evaluated during method development, general and project method validation

processes, and then continuously during the processing of incident response samples using batch QC

samples (MARLAP Chapter 7). Tests for absolute and relative biases shall be made for the method
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

6

 Notice that the sum under the radical in equation 4 is divided by the number of degrees of freedom, N – 1, not the

number of results, N. When calculated in this manner, sX

2

 is an unbiased estimator for the variance of the results. If the

true mean of the results, μX, were known, a better estimate of the variance would be , but because the 1

N 1

Xi x i

N

( ) − ∑ =μ

mean is estimated from the data, the number of degrees of freedom is reduced by 1. Notice also that the expression in

the denominator of the right-hand side of Equation 5 gives the experimental standard deviation of the mean, more

commonly known as the “standard error of the mean.” The division by in this case accounts for the effect of N

averaging N independent results.

23

validation level specified by the IC. The absolute bias shall be evaluated using the blank sample

results and the relative bias evaluated for each test level (lower, mid and upper) using the results of

the replicates.

When there is a significant absolute or relative bias, the probability of failing the required method

uncertainty acceptance criteria of Section 5.4.2 may become significant depending on the magnitude

of the actual method uncertainty. Appendix D provides an example of the probability of failing

method validation Level D for three actual method uncertainty values (as compared to the required

method uncertainty) as a function of relative bias up to 20%. In general, to avoid failure to meet the

method validation acceptance criteria, it is best to have an actual method uncertainty at the AAL that

is a fraction of the required method uncertainty. 

The following equations, taken from MARLAP Chapter 6 (Attachment 6A) and other statistical

references, are used to test for absolute and relative biases. 

5.6.1 Absolute Bias Testing

The protocol for testing for absolute bias is the following:

1. Calculate the mean ( ) for “N” (at least seven) blank sample net results using Equation 3.

X (3) NXi i

N

= ∑ =

1

1

where N should be at least seven blank sample results.

2. Calculate the experimental standard deviation (sx) of the same results6

 using Equation 4.

(4)

S

N X i X X i

N

= − − ∑ =

1

1

2

1

( )

3. Use Equation 5 to calculate the |T| value:

(5)
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

24

4. An absolute bias in the measurement process is indicated if

(6)

where, t1!α/2 (N-1) represents the (1 - α/2)-quantile of the t-distribution with N-1 degrees of

freedom. For seven blanks, an absolute bias is identified at a significance level of 0.05, when

|T| > 2.447.

5.6.2 Relative Bias Testing 

5.6.2.1 Test Level Samples with Same Known Value

When the samples for a test level have the same concentration (e.g., water) or activity, the protocol

for testing relative bias for each method validation test level is the following:

1. Calculate the mean ( ) and estimated standard deviation (sX) of the replicate results for each

method validation test level using Equations 3 and 4, respectively.

2. Use Equation 7 to calculate the |T| value

(7)

where:

is the average measured value

sX is the experimental standard deviation of the measured values

N is the number of replicates

K is the reference value

u(K) is the standard uncertainty of the reference value

3. A relative bias in the measurement process is indicated if

(8)

The number of effective degrees of freedom for the T statistic is calculated as follows:

(9)

νeff as calculated by the equation generally is not an integer so νeff should be truncated (rounded

down) to an integer. Then, given the significance level of 0.05, the critical value for is defined

to be t1!α/2(νeff), the (1 ! α/2)-quantile of the t-distribution with νeff degrees of freedom (see MARLAP

Appendix G, Table G.2 ).
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

7

 During the preparation of the proficiency test samples for a test level, the spread in activity deposited on the samples

of the test level should be controlled so that the coefficient of variation of the test-sample activities does not exceed 3%.

25

5.6.2.2 Test Level Samples with Slightly Different Known Values

When the PT samples for a test level have slightly different concentrations or activities (e.g.,

independently prepared7

 water samples, air filters, or swipes), the following protocol (paired t-test)

for testing relative bias for each method validation test level is:

1. Calculate the average difference ( ) between the measured value and the known spiked

value using Equation 10:

D (10) NX K i i i

N

= − ∑ =

1

1

( )

where 

Xi is the measured value for the i

th sample at a particular test level

Ki is the known value for the same sample

N is the number of samples at that test level

2. Calculate the standard deviation of the differences, SD, as:

S (11) N D i D D i

N

= − − ∑ =

1

1

2

1

( )

where Di = Xi – Ki.

3. Calculate the absolute value of the t statistic as:

T (12)

D

S N D

= /

4. A relative bias in the measurement process for a test level is indicated if 

(13)

6.0 Method Validation Documentation

The information and data to be retained should be specified in the method validation plan for each

radionuclide and matrix combination. When the laboratory conducts project method validation for

incident response applications, the detailed analytical method and all records, laboratory workbooks,

and matrix spike data used to validate the analytical method should be retained on file and be

retrievable for a specified length of time after the method has been discontinued. Data evaluations

such as comparison of individual results to the validation acceptance criteria and absolute bias in
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

26

blanks and, when available, method precision and bias, should be part of the data validation package

retained as part of the documentation related to the laboratory’s quality system. In addition, for each

radionuclide and matrix combination, a synoptic method validation report containing the analytical

method identification, method validation acceptance criteria, test levels, validation results and a

method acceptability decision should be generated and retained.

7.0 References 

American National Standards Institute (ANSI) N42.23. Reapproved 2003. Measurement and

Associated Instrumentation Quality Assurance for Radioassay Laboratories. 

U.S. Environmental Protection Agency (EPA). 2006. Validation and Peer Review of U.S.

Environmental Protection Agency Radiochemical Methods of Analysis. FEM Document Number

2006-01, November 8.

U.S. Environmental Protection Agency (EPA). 2008. Radiological Laboratory Sample Analysis

Guide for Incidents of National Significance–Radionuclides in Water. Revision 0. Office of Air

and Radiation, Washington, DC. EPA 402-R-07-007, January. Available at: www.epa.gov/

narel/recent_info.html.

U.S. Environmental Protection Agency (EPA). 2009a. Radiological Laboratory Sample Analysis

Guide for Incidents of National Significance–Radionuclides in Air. Revision 0. Office of Air and

Radiation, Washington, DC. EPA 402-R-09-007, June. Available at: www.epa.gov/narel/recent_

info.html.

U.S. Environmental Protection Agency (EPA). 2009b. Radiological Laboratory Sample Screening

Analysis Guide for Incidents of National Significance. Revision 0. Office of Air and Radiation,

Washington, DC. EPA 402-R-09-008, June. Available at: www.epa.gov/narel/recent_info.html.

EURACHEM Guide. 1998 The Fitness for Purpose of Analytical Methods, A Laboratory Guide to

Method Validation and Related Topics. Available at: http://www.eurachem.org/.

International Standard ISO/IEC 17025. 2005. General Requirements for the Competence of Testing

and Calibration Laboratories. ISO, Geneva, Switzerland.

International Organization for Standardization (ISO). 1993. International Vocabulary of Basic and

General Terms in Metrology. 2nd Edition. ISO, Geneva, Switzerland.

Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP). 2004. EPA 402-

B-04-001A, July. Volume I, Chapters 6, 7, 20, Glossary; Volume II and Volume III, Appendix

G. Available at: www.epa.gov/radiation/marlap.

Multi-Agency Radiation Survey and Site Investigation Manual (MARSSIM), Revision 1. 2000.

NUREG-1575 Rev 1, EPA 402-R-97-016 Rev1, DOE/EH-0624 Rev1. August. Available from

www.epa.gov/radiation/marssim/.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

27

Appendix A: 

Tables Summarizing the Derived Radionuclide Concentrations and Required

Method Uncertainties Corresponding to PAGs or Risks for the Water, Air, and Soil

Matrices

TABLE A1 – Alpha-Emitting Radionuclide Concentrations and Required Method Uncertainties in

Water Corresponding to 500- and 100-mrem AAL Derived Water Concentrations (DWCs)

Radionuclide

 pCi/L

500 mrem 100 mrem

AAL

DWC

[1][2]

Screening

Methods

Required

Method

Uncertainty

 (uMR) [6]

AAL

DWC

[1][2][3]

Screening

Methods

Required

Method

Uncertainty

 (uMR) [6]

NuclideSpecific

Required

Method

Uncertainty

 (uMR) [6]

Gross α Screen [5] 2.0×103 610 400 120 —

Am-241 2.0×103 610 400 120 50

Cm-242 1.4×104 4.3×103 2.8×103 850 350

Cm-243 2.5×103 760 500 150 63

Cm-244 2.9×103 880 580 180 73

Np-237 3.9×103 1.2×103 780 240 98

Po-210 130 40 26 7.9 3.3

Pu-238 1.8×103 550 360 110 45

Pu-239 1.7×103 520 340 100 43

Pu-240 1.7×103 520 340 100 43

Ra-226 [4] 910 280 180 55 23

Th-228 [4] 2.6×103 790 520 160 65

Th-230 1.8×103 550 360 110 45

Th-232 1.6×103 490 320 97 40

U-234 6.3×103 1.9×103 1.3×103

 400 160

U-235 6.6×103 2.0×103 1.3×103

 400 160

U-238 7.0×103 2.1×103 1.4×103 430 180

Notes:

[1] Values are based on the dose conversion factors in Federal Guidance Report No.13, CD Supplement, 5-

year-old child and the 50th percentile of water consumption.

[2] 365-day intake. 

[3] Values obtained by dividing 500-mrem DWC values by 5. 

[4] Includes the dose from the decay products originating from the 226Ra or 228Th in the body.

[5] Values for gross alpha screening are based on 241Am.

[6] The required relative method uncertainty (nMR) for values greater than the AALs in this table is obtained

by dividing the uMR value by the corresponding AAL.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

28

TABLE A2 – Beta/Gamma-Emitting Radionuclide Concentrations in Water and Required Method

Uncertainties Corresponding to 500- and 100-mrem AAL Derived Water Concentrations (DWCs)

Radionuclide

pCi/L

500 mrem 100 mrem

NuclideSpecific

Required

Method

Uncertainty

(uMR )

AAL DWC [5] [1][2]

Screening

Methods

Required

Method

Uncertainty

(uMR )

[5] AAL DWC

[1][2][3]

Screening

Methods

Required

Method

Uncertainty

(uMR )

[5]

Gross β/γ

Screen[4] 5.8×104 1.8×104 1.2×104 3.6×103 —

Ac-227DP[6] 1.1×103 330 220 67 28

Ce-141 2.2×105 6.7×104 4.4×104 1.3×104 5.5×103

Ce-144 2.9×104 8.8×103 5.8×103 1.8×103 730

Co-57 6.3×105 1.9×105 1.3×105 4.0×104 1.6×104

Co-60 3.3×104 1.0×104 6.6×103 2.0×103 830

Cs-134 4.3×104 1.3×104 8.6×103 2.6×103 1.1×103

Cs-137 5.8×104 1.8×104 1.2×104 3.6×103 1.5×103

H-3 7.7×106 2.3×106 1.5×106 4.6×105 1.9×105

I-125 1.3×104 4.0×103 2.6×103 790 320

I-129 3.3×103 1.0×103 660 200 83

I-131 5.4×103 1.6×103 1.1×103 330 140

Ir-192 1.2×105 3.6×104 2.4×104 7.3×103 3.0×103

Mo-99 3.2×105 9.7×104 6.4×104 1.9×104 8.1×103

P-32 5.9×104 1.8×104 1.2×104 3.6×103 1.5×103

Pd-103 7.8×105 2.4×105 1.6×105 4.9×104 2.0×104

Pu-241 1.0×105 3.0×104 2.0×104 6.1×103 2.5×103

Ra-228[6] 160 49 32 9.7 4.0

Ru-103 2.3×105 7.0×104 4.6×104 1.4×104 5.8×103

Ru-106 2.2×104 6.7×103 4.4×103 1.3×103 550

Se-75 6.7×104 2.0×104 1.3×104 4.0×103 1.6×103

Sr-89 6.3×104 1.9×104 1.3×104 4.0×103 1.6×103

Sr-90 1.2×104 3.6×103 2.4×103 730 300

Tc-99 2.4×105 7.3×104 4.8×104 1.5×104 6.0×103

Notes:

[1] Values are based on the dose conversion factors in Federal Guidance Report No.13, CD Supplement, 5-

year-old child and the 50th percentile of water consumption.

[2] 365-day intake. 

[3] Values obtained by dividing 500-mrem DWC values by 5. 

[4] Gross beta screening values are based on 137Cs.

[5] The required relative method uncertainty (nMR) for values greater than the AALs is obtained by dividing

the uMR value in this table by the corresponding AAL value.

[6] Includes the dose from the decay products originating from the 228Ra or 227Ac in the body.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

29

TABLE A3 – Alpha-Emitting Radionuclide Concentrations in Air and Required Method

Uncertainties Corresponding to 2-rem and 500-mrem AAL Derived Air Concentrations (DACs)

Radionuclide

pCi/m3

2 rem 500 mrem

 AAL

DAC

[1]

Screening

Method

 Required

Method

Uncertainty

(uMR) 

[3]

NuclideSpecific

Required

Method

Uncertainty

(uMR) 

[3]

 AAL

DAC 

[1]

Screening

Method

Required

Method

Uncertainty

(uMR) 

[3]

NuclideSpecific

Required

Method

Uncertainty

(uMR)

[3]

Gross α

Screen[4] 0.70 0.21 — 0.17 0.052 —

Am-241 0.70 0.21 0.088 0.17 0.052 0.021

Cm-242 11 3.3 1.4 2.8 0.85 0.35

Cm-243 0.97 0.29 0.12 0.24 0.073 0.030

Cm-244 1.2 0.36 0.15 0.29 0.088 0.037

Np-237 1.3 0.40 0.16 0.34 0.10 0.043

Po-210 16 4.9 2.0 3.9 1.2 0.49

Pu-238 0.62 0.19 0.081 0.15 0.046 0.020

Pu-239 0.56 0.17 0.071 0.14 0.043 0.018

Pu-240 0.56 0.17 0.071 0.14 0.043 0.018

Ra-226 [2] 7.0 2.1 0.88 1.8 0.55 0.23

Th-228 [2] 1.7 0.52 0.21 0.42 0.13 0.053

Th-230 0.66 0.20 0.083 0.17 0.052 0.021

Th-232 0.61 0.19 0.077 0.15 0.046 0.019

U-234 7.1 2.2 0.89 1.8 0.55 0.23

U-235 7.9 2.4 0.99 2.0 0.61 0.25

U-238 8.3 2.5 1.0 2.1 0.64 0.26

Notes:

[1] Morbidity for long-term inhalation. Child as receptor. Value corresponds to solubility class having

lowest value.

[2] Includes the dose from the decay products originating from the 226Ra or 228Th in the body.

[3] Required method uncertainty values are based on a sampled aerosol volume of 68 m3

 at the 2 rem or 500-

mrem DAC. The required relative method uncertainty (nMR) for values greater than the AALs in this table

is obtained by dividing the uMR value in this table by the corresponding AAL value.

[4] The gross α screening values are not related to a specific radionuclide.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

30

TABLE A4 – Beta/Gamma-Emitting Radionuclide Concentrations in Air and Required Method

Uncertainties Corresponding to 2-rem and 500-mrem AAL Derived Air Concentrations (DACs)

Radionuclide

pCi/m3

2 rem 500 mrem 

AAL

DAC

[1,4]

Screening

Method

Required

Method

Uncertainty

 (uMR) 

[3]

NuclideSpecific 

Required

Method

Uncertainty

(uMR) 

[3]

 AAL

DAC

[1,4]

Screening

Method

Required

Method

Uncertainty

(uMR) 

[3]

NuclideSpecific 

Required

Method

Uncertainty

(uMR) 

[3]

Gross β

Screen[5] 420 130 — 110 33 —

Ac-227+DP [2] 0.43 0.13 0.054 0.11 0.033 0.014

Ce-141 1.8×104 5.5×103 2.3×103 4.5×103 1.4×103 570

Ce-144 1.3×103 400 160 320 97 40

Co-57 6.7×104 2.0×104 8.4×103 1.7×104 5.2×103 2.1×103

Co-60 2.2×103 670 280 540 170 69

Cs-134 3.3×103 1.0×103 420 820 250 100

Cs-137 1.7×103 520 210 430 130 54

H-3 2.6×105 7.9×104 3.3×104 6.4×104 1.9×104 8.1×103

I-125 [6] 1.3×104 4.0×103 1.6×103 3.2×103 970 400

I-129 [6] 1.9×103 580 240 470 140 59

I-131 [6] 9.1×103 2.8×103 1.1×103 2.3×103 700 290

Ir-192 1.0×104 3.0×103 1.3×103 2.5×103 760 310

Mo-99 6.8×104 2.1×104 8.6×103 1.7×104 5.2×103 2.1×103

P-32 1.7×104 5.2×103 2.1×103 4.3×103 1.3×103 540

Pd-103 1.5×105 4.6×104 1.9×104 3.8×104 1.2×104 4.8×103

Pu-241 29 8.8 3.7 7.3 2.2 0.92

Ra-228 [2] 4.2 1.3 0.53 1.0 0.30 0.13

Ru-103 2.3×104 7.0×103 2.9×103 5.7×103 1.7×103 720

Ru-106 1.0×103 300 130 250 76 31

Se-75 5.0×104 1.5×104 6.3×103 1.3×104 4.0×103 1.6×103

Sr-89 8.4×103 2.6×103 1.1×103 2.1×103 640 260

Sr-90 420 130 53 110 33 14

Tc-99 5.0×103 1.5×103 630 1.3×103 400 160

Notes:

[1] Derived air concentration yielding stated committed effective dose assuming a 365-day year. Child as receptor.

Value corresponds to solubility class having lowest value. 

[2] Includes the dose from the decay products originating from the 228Ra or 227Ac in the body. DP refers to “decay

products.”

[3] Required method uncertainty values are based on a sampled aerosol volume of 68 m3

 at the 2 rem or 500-mrem

DAC. The required relative method uncertainty (nMR) for values greater than the AALs in this table is obtained by

dividing the uMR value in this table by the corresponding AAL value.

[4] All nuclides can be collected on a fibrous or membrane air filter media except 3

H, 125I, 129I, and 131I in the vapor

states.

[5] Gross beta screening values are based on 90Sr.

[6] These values are based on the vapor plus particulate dose rate.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

31

TABLE A5 – Alpha-Emitting Radionuclide Concentrations in Air and Required Method

Uncertainties Corresponding to AAL Derived Air Concentrations (DACs)

Radionuclide

pCi/m3

10–4 Risk

 AAL DAC

[1]

10–4 Risk AAL

 Required

Method

Uncertainty (uMR)

[3]

10–6 Risk

 AAL DAC

[1]

10–6 Risk AAL 

Required Method

Uncertainty (uMR)

[3]

Gross α Screen [4] 0.33 0.042 3.3×10–3 4.2×10–4

Am-241 0.33 0.042 3.3×10–3 4.2×10–4

Cm-242 0.62 0.078 6.2×10–3 7.8×10–4

Cm-243 0.34 0.043 3.4×10–3 4.3×10–4

Cm-244 0.35 0.044 3.5×10–3 4.4×10–4

Np-237 0.43 0.054 4.3×10–3 5.4×10–4

Po-210 0.86 0.11 8.6×10–3 1.1×10–3

Pu-238 0.24 0.030 2.4×10–3 3.0×10–4

Pu-239 0.22 0.028 2.2×10–3 2.8×10–4

Pu-240 0.22 0.028 2.2×10–3 2.8×10–4

Ra-226 [2] 0.44 0.055 4.4×10–3 5.5×10–4

Th-228 [2] 0.094 0.012 9.4×10–4 1.2×10–4

Th-230 0.36 0.045 3.6×10–3 4.5×10–4

Th-232 0.30 0.038 3.0×10–3 3.8×10–4

U-234 0.45 0.057 4.5×10–3 5.7×10–4

U-235 0.49 0.062 4.9×10–3 6.2×10–4

U-238 0.52 0.065 5.2×10–3 6.5×10–4

Notes:

[1] Morbidity for long-term inhalation. Value corresponds to solubility class having lowest value.

[2] Includes the dose from the decay products originating from the 226Ra or 228Th in the body.

[3] Required method uncertainty values are based on a sampled aerosol volume of 1,600 m3

 at the 10–4 and

10–6 risk DACs, respectively. The required relative method uncertainty (nMR) for values greater than the

AALs in the table is obtained by dividing the uMR value by the corresponding AAL value.

[4] The gross α screening values are not related to a specific radionuclide.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

32

TABLE A6 – Beta/Gamma-Emitting Radionuclide Concentrations in Air and Required

Method Uncertainties Corresponding to AAL-Derived Air Concentrations (DACs)

Radionuclide

pCi/m3

10–4 Risk

 AAL DAC

[1,4]

10–4 Risk AAL

Required Method

Uncertainty (uMR)

[3]

10–6 Risk

 AAL DAC

[1,4]

10–6 Risk AAL

 Required

Method

 Uncertainty (uMR)

[3]

Gross β Screen

(Sr-90) 29 3.8 0.29 0.038

Ac-227+DP [2] 0.083 0.010 8.3×10–4 1.0×10–4

Ce-141 920 120 9.2 1.2

Ce-144 69 8.7 0.69 0.087

Co-57 3.3×103 420 33 4.2

Co-60 120 15 1.2 0.15

Cs-134 180 23 1.8 0.23

Cs-137 110 14 1.1 0.14

H-3 Vapor 1.5×104 1.9×103 150 19

I-125 1.2×103 150 12 1.5

I-129 200 25 2 0.25

I-131 640 81 6.4 0.81

Ir-192 510 64 5.1 0.64

Mo-99 2.6×103 330 26 3.3

P-32 890 110 8.9 1.1

Pd-103 7.0×103 880 70 8.8

Pu-241 14 1.8 0.14 0.018

Ra-228 [2] 0.28 3.5×10–2 2.8×10–3 3.5×10–4

Ru-103 1.2×103 150 12 1.5

Ru-106 56 7.1 0.56 0.071

Se-75 2.5×103 310 25 3.1

Sr-89 410 52 4.1 0.52

Sr-90 29 3.7 0.29 0.037

Tc-99 330 42 3.3 0.42

Notes:

[1] Morbidity for long-term inhalation. Value corresponds to solubility class having lowest value.

[2] Includes the dose from the decay products originating from the 228Ra or 227Ac in the body.

[3] Required method uncertainty values are based on a sampled aerosol volume of 1,600 m3

 at the 10–4 and

10–6 risk DAC, respectively. The required relative method uncertainty (nMR) for values greater than the

AALs in the table is obtained by dividing the uMR value by the corresponding AAL value.

[4] All nuclides can be collected on a fibrous or membrane air filter media except 3

H, 125I, 129I, and 131I in the

vapor states.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

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TABLE A7 – Alpha and Beta/Gamma-Emitting Radionuclide Concentrations in Soil and

Required Method Uncertainties Corresponding to Derived Soil Concentrations

Table to be determined following publication of Radiological Laboratory Sample Analysis Guide

for Incidents of National Significance–Radionuclides in Soil.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

8

 EPA 2008. Radiological Laboratory Sample Analysis Guide for Incidents of National Significance – Radionuclides

in Water, Table 9A. 

34

Appendix B: 

Examples of the Method Validation Process for 

Required Method Uncertainty Specifications

Two examples are provided to demonstrate the method validation process when the MQO involves

a required method uncertainty (uMR or nMR) specification. The first example is when an Incident

Commander (IC) specifies a required method uncertainty for a method and an AAL (PAG or riskbased derived radionuclide concentration) for a typical radionuclide and matrix combination as

provided in Appendix A. The radionuclide and matrix combination for this first example is 241Am

in potable water. Values for the derived radionuclide concentration AAL and required method

uncertainty were obtained from the Radiological Laboratory Sample Analysis Guide for Incidents

of National Significance–Radionuclides in Water (EPA 2008). The three testing level concentrations

were determined using the AAL concentration value and the lower, mid and upper test level

multipliers in Table 2. Acceptable validation criteria are established in Table 3 for Validation Level

D. 

The second example is when an Incident Commander specifies a required method uncertainty for

a method used to analyze a radionuclide or matrix not provided in Appendix A. For this example,

the radionuclide and matrix combination is 241Am in street runoff water. Values for the derived

AALs and required method uncertainty were obtained from Tables 1 and 4, respectively. The three

testing level concentrations were determined using the AAL concentration value and the lower, mid

and upper test level multipliers in Table 2. Acceptable validation criteria are established in Table 3

for validation Level D. 

Example 1. Method Validation for Am-241 in Potable Water; Established AALs

Nuclide: 241Am

Matrix: Water

Analytical Action Level: 400 pCi/L (Appendix A, Table A1, 100 mrem)

Proposed Method: Radiochemistry with alpha spectrometry

Required Method Validation Level: D

Required Method Uncertainty8

: 50 pCi/L at AAL or below; 13% above AAL

Acceptance Criteria (Table 3): Measured value within ±3 uMR (± 150 pCi/L) of known value #AAL

and ±3 nMR (± 39%) of known value > AAL.

Test levels (Table 2): Lower (0.5 × AAL = 200 pCi/L; Mid (AAL) = 400 pCi/L; Upper (3 × AAL)

= 1,200 pCi/L 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

35

Data Evaluation:

TABLE B1 – Required Method Uncertainty for Am-241 in Potable Water

Lower Test Level

Concentration

200 pCi/L

Acceptable Range:

 50 to 350 pCi/L

Mid Test Level

Concentration[1]

400 pCi/L[2]

Acceptable Range:

 250 to 550 pCi/L

Upper Test Level

 Concentration

1,200 pCi/L

Acceptable Range:

 732 to 1,670 pCi/L

Test

Sample

Measured

Value

± 1 CSU[3]

Acceptable

Value

(Y/N)

Measured

Value

± 1 CSU[3]

Acceptable

Value

(Y/N)

Measured

Value

± 1 CSU[3]

Acceptable

Value (Y/N)

1 221 ± 27 Y 429 ± 40 Y 1,283 ± 87 Y

2 179 ± 24 Y 381 ± 37 Y 1,117 ± 78 Y

3 210 ± 26 Y 405 ± 39 Y 1,241 ± 85 Y

4 190 ± 25 Y 304 ± 32 Y 1,159 ± 80 Y

5 169 ± 25 Y 362 ± 36 Y 1,262 ± 86 Y

6 225 ± 27 Y 458 ± 42 Y 1,138 ± 79 Y

7 213 ± 26 Y 390 ± 38 Y 994 ± 72 Y

Notes:

[1] Mid test level is at the AAL (Table 2)

[2] AAL taken from Table 9A, Radiological Laboratory Sample Analysis Guide for Incidents of National

Significance– Radionuclides in Water (EPA 2008).

[3] Approximate combined standard uncertainty for a 100-minute count on an alpha detector having a typical

detector efficiency plus another 5% uncertainty for other method parameters at the action level. Counting

time was estimated so that the required method uncertainty would be met at the AAL. All samples would

be counted for the same length of time regardless of the test level.

Example 2. Method Validation for Am-241 in Street Runoff Water - Default AAL and

Required Method Uncertainty

Nuclide: 241Am

Matrix: Street runoff water 

Default AAL: 40 pCi/sample (Table 1, liquid, specific nuclide)

Proposed Method: Radiochemistry with alpha spectrometry. Specific nuclide measurement.

Required Method Validation Level: D, new matrix

Required Method Uncertainty: 5.2 pCi/test sample at AAL or below; 13% above AAL

(Table 4)

Acceptance Criteria (Table 2): Measured Value within ±3 uMR (±15.6 pCi/sample) of known value

#AAL and ±3 nMR (± 39%) of known value > AAL
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

36

Method Validation Test Levels (Table 2): Lower (0.5 × AAL) = 20 pCi/sample; Mid (AAL) = 40

pCi/sample; Upper (3 × AAL) = 120 pCi/sample 

Data Evaluation:

TABLE B2 – Required Method Uncertainty for Am-241 in Street Runoff Water

Lower Test Level Concentration

20 pCi/sample

Acceptable Range: 

4.4 to 35.6 pCi

Mid Test Level Concentration[1]

40 pCi/sample[2]

Acceptable Range: 

24.4 to 55.6 pCi 

Upper Test Level Concentration

120 pCi/sample

Acceptable Range:

 73.2 to 167 pCi

Test

Sample

Measured Value

± 1 CSU[3]

Acceptable

Value (Y/N)

Measured Value

± 1 CSU[3]

Acceptable

Value (Y/N)

Measured

Value

± 1 CSU[3]

Acceptable

Value (Y/N)

1 22.3 ± 2.3 Y 44.2 ± 3.6 Y 128.6 ± 8.0 Y

2 17.6 ± 2.0 Y 36.7 ± 3.2 Y 112.2 ± 7.2 Y

3 20.9 ± 2.2 Y 42.4 ± 3.5 Y 124.7 ± 7.8 Y

4 23.4 ± 2.4 Y 38.1 ± 3.2 Y 117.0 ± 7.4 Y

5 15.8 ± 1.9 Y 50.5 ± 3.9 Y 140.0 ± 8.6 Y

6 21.7 ± 2.2 Y 41.5 ± 3.4 Y 122.0 ± 7.7 Y

7 18.8 ± 2.1 Y 31.1 ± 2.8 Y 113.4 ± 7.2 Y

Notes:

[1] Mid test level is at the AAL, (Table 2)

[2] Table 1, liquid, specific nuclide

[3] Approximate combined standard uncertainty for a 15-minute count on an alpha detector having a typical

detector efficiency plus another 5% uncertainty for other method parameters at the action level. Counting

time was estimated so that the required method uncertainty would be met at the AAL. All samples would

be counted for the same length of time regardless of the test level. Sample volume ~ 100 mL.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

37

Appendix C: 

Example of the Method Validation Process 

for Verification of the Required MDC Specification

Refer to Section 5.5.2 (page 21) for the protocol to follow for verifying that a method’s MDC meets

the required MDC specification.

Nuclide: 90Sr

Matrix: Street runoff water

Required MDC = 2 pCi/L (MQO designated by Incident Commander)

Proposed Method: Radiochemistry with beta counting on gas proportional counter. Sample volume

= 1 L, counting time = 240 minutes. Analytical result calculations to include detector efficiency,

detector background (cpm) and 90Y ingrowth factor.

Number of Blanks: 7 

Number of Spiked Test Samples: 10

Testing Level: 2 pCi/L of 90Sr

Calculations:

a) 90Sr concentration and associated combined standard uncertainty for the blanks and test samples.

b) Critical Net Concentration = 1.94 × standard deviation of the seven blank results.

c) Number (Y) of sample results at or below the estimated Critical Net Concentration

Test: Does the number (Y) of sample results at or below the estimated Critical Net Concentration

exceed 2? 

 • If Y # 2, the method tested at the required MDC passes the test for the required MDC

specification.

 • If Y > 2, the method tested at the required MDC fails the test for the required MDC specification.

TABLE C1 – Results of Blank Sample Analyses

Blank Number Result (pCi/L)

1 –0.21 ± 0.44

2 0.10 ± 0.45

3 0.44 ± 0.46

4 0.82 ± 0.46

5 –0.40 ± 0.44

6 –0.75 ± 0.44

7 0.61 ± 0.46

Average 0.09

Standard Deviation of Results 0.57

Critical Net Concentration 1.11
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

38

TABLE C2 – Results of MDC Test Sample Analyses; Test Concentration = 2.0 pCi/L

Test Sample Number Result (pCi/L) Result #Critical Net

Concentration (1.11 pCi/L)

1 2.57 ± 0.50 N

2 1.00 ± 0.47 Y

3 2.43 ± 0.50 N

4 1.57 ± 0.48 N

5 2.29 ± 0.50 N

6 1.71 ± 0.48 N

7 2.01 ± 0.49 N

8 3.14 ± 0.52 N

9 0.86 ± 0.46 Y

10 1.43 ± 0.48 N

Average 1.90

Standard Deviation of Results 0.72

Y - Number of Results #Critical Net

Concentration

2

Conclusion: The hypothesis that the true MDC for the method is at or below the required MDC

cannot be rejected. Therefore, the method is assumed to be capable of meeting the required MDC

specification.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

39

Appendix D: 

Example of the Effect of Bias on the Probability of Failing the Method Validation

Acceptance Criteria for Required Method Uncertainty

Suppose one is validating a method for water using Level D acceptance criteria, so tests should be

made at three concentration levels with seven samples at each level. Consider that the action level

is 100 pCi/L, and that this is one of the test levels. Also suppose that the required method uncertainty

at 100 pCi/L is uMR = 10 pCi/L, i.e. the relative required method uncertainty is φMR = 10%. The

acceptance bounds are then 100 ± 30 pCi/L. For the purpose of this illustration, only a positive

method bias will be considered (although the same effect would occur for negative biases). 

In Figure D1, the area under the

curves above 130 pCi/L is the

probability that a single sample will

fail validation for the given method

uncertainty and bias. If the method

just meets the criterion and there is

no bias, the figure shows that the

probability of an individual sample

failing is very small (<0.01%). Now

suppose there is a bias of +10%.

What is the probability of failing a

single sample? When the uncertainty

is 10 pCi/L it is 2.28%. Of course, if

the method uncertainty already

exceeds the required 10%, (e.g., 12.5

pCi/L), this probability is even

higher, 5.48%. However, if the

method uncertainty is less than that

required (e.g., 7.5 pCi/L or 5 pCi/L), then this probability becomes lower, 0.38% and 0.13%,

respectively.

The probabilities above are for a single sample. At each level, seven samples must pass. Because

there are three levels, there are 21 tests, and if the probability of failure for a single sample is F, then

the overall probability of failure is about 1 – (1–F)21. If F is 2%, 1 – (1–F)21 >> 35%. 

Consider an example of method validation Level D for a water matrix. There are 3 test levels with

7 samples each, or 21 total samples: 

 • 0.5 ×AAL level = 50 pCi/L; uReq = 10 pCi/L, nReq = 0.20

 • AAL level = 100 pCi/L; uReq = 10 pCi/L, nReq = 0.10

 • 3.0 ×AAL level = 300 pCi/L; uReq = 30 pCi/L, nReq = 0.10

The acceptance criterion is that each measured value (for all 21 samples) must be within ±3.0 uReq

or ±3.0 nReq of the validation test activity. The probability of passing the acceptance criteria was

calculated for four assumed relative method uncertainty values: 5%, 7.5%, 10%, and 12.5%. The

range of biases evaluated included 0 to 20% at the AAL. 

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

70 80 90 100 110 120 130 140 150

Concentration

Normal Probability Density

u = 12.5

u = 10

u = 7.5

u = 5

u = 10

The area under the curve 

above 130 is the 

probability that a single 

sample will fail validation.

Figure D1 – Probability of a validation sample failing at

concentration 100 pCi/L with and without bias at various

values of the method standard uncertainty.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

40

Figure D2 shows the overall

probability of failing the Level D

validation as a function of bias and

relative method uncertainty. It is clear

that if the method just meets the

required relative method uncertainty,

then there is not much room to

accommodate bias. However, when

the relative method uncertainty is

7.5%, about an equal amount of bias

might be tolerated. If the relative

method uncertainty is half that

required, three times as much bias can

exist without bringing the probability

of failure over 5%.

 • When the actual relative method

uncertainty is one-half or less than

the relative required method uncertainty, biases up to 15% may

be tolerated without substantially

increasing the probability of

failing the acceptance criteria.

 • When the actual relative method uncertainty is equal to the relative required method uncertainty,

it is best not to have a bias in order to maintain a reasonable probability of passing the acceptance

criteria.

 • When the actual relative method uncertainty is greater than the relative required method

uncertainty, the probability of failing the acceptance criteria is extremely high, regardless of the

magnitude of the bias.

Detecting Bias 

Testing for a bias smaller than the standard uncertainty is difficult. There must be at least 16 replicate

measurements to make the minimum detectable bias (MDB) less than σ, the true method standard

deviation, and at least 54 replicate measurements to make the MDB less than σ/2 (see MARLAP

Chapter 6A). This problem seems to be inescapable for absolute bias tests based on method-blank

analyses. For relative bias tests based on spiked samples, statistics can be improved if there are highactivity samples whose reference values have small uncertainties. 

The mean squared error (MSE) is the sum of the squared differences between the measurements and

the true values. The MSE is the sum of the variance, σ2

, and the square of the bias, b2. The root MSE

= . If the root mean squared error is kept below the required method uncertainty, the 2 2 σ + b

MQOs are likely to be met. If the bias is less than one third the relative method standard uncertainty,

bias will only contribute 10% to the MSE. 

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10 12 14 16 18 20

Bias (%)

Probability Method Fails 

12.5%

5%

7.5%

10%

Figure D2 – Level D validation (21 samples) failing at test

level as a function of relative method bias for relative

method uncertainties of 5%, 7.5%, 10%, and 12.5%.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

41

The validation criteria in MARLAP were developed with the presumption that any known biases in

the method will be corrected, and that any remaining bias will be small compared to the method

uncertainty. Thus, the primary focus was placed on detecting an unacceptably high method

uncertainty. Note that if the reverse is true, namely that the method uncertainty is much smaller than

the bias, the method may pass the acceptance criteria while having what might be considered

unacceptably high bias. In the extreme case of zero method uncertainty, a method with bias up to

30% might still pass the criteria. In this case different validation criteria may be desirable. Appendix

E contains alternative method validation criteria which treat the detection of excessive bias and

imprecision more equally using the concept of MSE. 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

42

Appendix E: 

An Alternative Method Validation Criterion

Introduction

The method validation process and acceptance criteria described in Section 5.4 are based on the

criteria recommended in Chapter 6 of the Multi-Agency Radiological Laboratory Analytical

Protocols Manual (MARLAP). This appendix presents alternative method performance acceptance

criteria that may have greater power to detect large imprecision and bias in some situations.

However, the number of test levels and replicates for the appropriate method validation level (B, C,

D, E) as presented in Section 5.4.2 are still to be used.

Every measurement process involves both bias and imprecision to some degree. The MARLAP

method validation criterion is predicated on the assumption that the laboratory has eliminated any

substantial bias in the measurement process, so that measurement results are likely to be evenly

distributed about the true value. If this assumption is not true, use of the MARLAP test alone may

in some cases allow a method with a substantial bias to be accepted for use. Although MARLAP

recommends that the candidate method be evaluated for bias, it does not recommend an objective

criterion for determining whether a detected bias is tolerable. Furthermore, as noted in MARLAP

and in Appendix D of this document, testing for bias tends to be difficult in any case, because of the

number of measurements required to detect a bias that is comparable in magnitude to the standard

deviation.

The assumption of this appendix is that a measurement process may be considered adequate for its

intended use if a certain combination of bias and imprecision, called the “root mean squared error,”

does not exceed the required uncertainty. According to this view, the fact that bias is hard to quantify

is less troublesome, because what one cares about most is not bias alone or imprecision alone but

a combination of the two.

Definitions

Suppose is an estimator for some parameter K. The variance of , denoted by or , X$ X$ V X( $ ) σ X$

2

is defined as the expected value of the square of the deviation of from its mean. X$

σ (E1) $ [( $ ( $ )) ] X E X EX 2 2 = −

where E(•) denotes the expected value (mean) of the operand within the brackets or parentheses. The

square root of the variance, denoted by , is called the σ $ standard deviation. The standard deviation X

of an estimator is commonly used as a measure of its imprecision.

The error of (as an estimator for K) is defined to be the difference between and K. X$ X$

Error X X K ( (E2) $ ) $ = −

The error , like itself, is a random variable. X$ − K X$
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

43

The bias of is defined as the difference between the expected value (or mean) of and the X$ X$

value of the parameter K. In symbols,

Bias X E X K ( (E3) $ ) ( $ = −)

The bias of also equals its mean error. So . X$ Bias X E X K ( $ ) ( $ = − )

If is an unbiased estimator (i.e., if ), then the standard deviation is a good measure of X$ E(Xˆ) = K

the overall quality of as an estimator. However, in the context of laboratory analyses, the X$

estimator is typically the result of a measurement made using a specified method and X$

measurement process, and in this situation, is usually biased to some extent. It is common to X$

evaluate a laboratory method or measurement process in terms of both the bias and imprecision

(standard deviation) of the estimator . During method validation, separate limits may be set for the $

X

maximum allowable bias and for the maximum allowable standard deviation; however, since the

overall quality of a measurement process is affected by both bias and imprecision, one may instead

choose to specify a limit for some combination of the bias and imprecision. If this is done, then a

biased but precise method may be considered to be as good as an essentially unbiased but less precise

method.

Note that although neither the bias nor the standard deviation is ever known exactly, it is possible

to use statistical methods to test hypotheses about their magnitudes or to determine likely bounds for

their values. Note also that acknowledging the existence of bias in a measurement process does not

mean that one should cease trying to find and eliminate the causes of any significant bias.

The “mean squared error” or the “root mean squared error” of an estimator is often used as a measure

of the estimator’s overall quality. The mean squared error of , as the name implies, is the X$

expected value of the squared error of . So: X$

MSE X E X K ( (E4) $ ) [( $ = − ) ] 2

Notice that the definition of resembles that of the variance , but with MSE X( K substituted $ ) σ X$

2

for the mean . It can be shown mathematically that the mean squared error of is equal to E X( $ ) X$

the sum of its squared bias and its variance.

MSE X Bias X (E5) X ( $ ) ( $ ) = + $

2 2 σ

The root mean squared error of is simply the positive square root of . X$ MSE X( $ )

MSE X E X K Bias X (E6) X ( $ ) [( $ )] ( $ ) = −= + $ 2 22σ

So the root mean squared error can be viewed as a mathematical combination of bias and

imprecision. For an unbiased estimator, the root mean squared error is exactly equal to the standard

deviation, but for a biased estimator, the root mean squared error is always larger than the standard

deviation.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

44

The approach to method validation described in this document is based on the concept of a required

uncertainty at each activity level. If one interprets this required uncertainty, u, as a required bound

for , then an unbiased method can have MSE X a standard deviation as large as u, or a ^ ⎛

⎝

⎜ ⎞

⎠

⎟ σ X$

perfectly precise method can have a bias as large as u. In general, both the bias and standard

deviation may be nonzero, but in principle, neither the bias nor the standard deviation is allowed to

exceed the required uncertainty, u, at any level of activity.

Alternative Method Validation Criterion

The validation procedure of Section 5.4 involves making several measurements of samples spiked

at known activity levels. Let L denote the number of activity levels and N the number of

measurements made at each level. Then the test described in Section 5.4 compares each result Xij

(where i denotes the number of the activity level and j denotes the number of the measurement) to

acceptance limits:

K ku (E7) i i ±

where

Ki

 = target value at the i

th activity level (1 # i # L)

k = uncertainty multiplier (from Table 3)

ui

 = required uncertainty at the i

th analyte level

The method is judged acceptable if every result Xij falls within the appropriate acceptance limits for

its activity level.

As noted under Table 3, the uncertainty multiplier, k, may be calculated as follows:

k z = LN (E8) 05 05 1 + − 1 . .( ) / α

where α is the chosen significance level, or the probability of a false rejection (α = 0.05), and for any

p, zp denotes the p-quantile of the standard normal distribution. (Note that k is rounded to two figures

in Table 3.) The multiplier k also equals the square root of the (1 – α)

1/LN -quantile of the chi-squared

distribution with one degree of freedom, and for purposes of exposition, it will be convenient to use

the latter interpretation here.

k = LN (E8') − χ ( ) α / ( ) 1 1

2 1

The required uncertainty, ui, at each activity level equals the required method uncertainty, uMR, if Ki #

AL, and it equals nMR Ki

 if Ki > AL.

u (E9) u if K AL

K if K AL i

MR i

MR i i

= ≤

× >

⎧

⎨

⎩

,

φ ,
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

9

 The test could also be based on a statistic equal to the maximum of the absolute values | Zij |, using k as a critical value.

10 The expected value of Wi

 equals N times the ratio of the mean squared error (MSE) to the square of the required

uncertainty ( ) at this activity level. ui

2

45

A more traditional presentation of the same statistical test would define a test statistic and a critical

value for that statistic. For this test, the statistic can be defined as:

M Z (E10)

i L

j N

ij = ≤ ≤

≤ ≤

max

1

1

2

where for each i and j, Zij denotes the “Z-score” for the measurement:

Z (E11)

X K

u ij

ij i

i

= −

The corresponding critical value for the statistic M is just the square of the uncertainty multiplier,

which equals:

m k C = = LN (E12) −

2

1

2 χ 1 1 ( ) α / ( )

So the method’s performance is considered acceptable if the value of M does not exceed mC.

9

Because the statistic M is derived from only the most extreme value of Zij, it essentially discards

much of the information contained in the measurement data, resulting in reduced power for the test.

A different statistic that makes better use of the same data is the following:

W Z (E13) i L ij

j

N

= ≤ ≤ =max

1 ∑ 2

1

where all the symbols on the right-hand side are as defined above for M. The critical value of the

statistic W is the (1 ! α)

1 / L

-quantile of the chi-squared distribution with N degrees of freedom:

w N (E14) c L = − χ ( ) α / ( ) 1 1

2

where again α = 0.05. This test can also be implemented by calculating a statistic Wi at each activity

level:

W Z (E15) i ij

j

N

= =

∑ 2

1

and comparing Wi to the critical value wC. If Wi at any activity level exceeds wC, the method is

rejected.10

Note that both the MARLAP test and the W test can be viewed as chi-squared tests. The MARLAP

test, which is equivalent to the method validation criterion presented in Section 5.4, uses a chisquared statistic with one degree of freedom for each of the LN measurements, while the W test pools

the data for each activity level to obtain fewer statistics (L of them), each of which has more degrees

of freedom (N).
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

46

Under the assumptions that the root MSE of the measurement process at each activity level does not

exceed the required uncertainty, and that all the measurement results are independent, either test

(MARLAP or W) will incorrectly reject a candidate method at most 5 % of the time (because

α = 0.05). The greatest false rejection rate (5 %) occurs when the bias is zero and the standard

deviation at each activity level exactly equals the required uncertainty. The important differences

between the two tests are the differences in their power to reject methods with larger bias or

imprecision. For example, if a candidate method has negligible imprecision, either test will reject

it if the measurement bias at some activity level is larger than a specified multiple of the required

uncertainty. However, the associated uncertainty multiplier for the W test ( ) is generally w N c /

smaller than the multiplier (k or ) for the MARLAP test. Furt m hermore, the MARLAP test has c

the undesirable property that it actually loses power to detect such biases as the number of

measurements (N) as each activity level increases, because the value of k increases with N. The

power of the W test, on the other hand, improves, because the value of decreases with w N N, c /

approaching 1 as N goes to infinity.

The Holst-Thyregod Test for Mean Squared Error

The W test described above was originally derived as a test of variance given a presumed value for

the mean, but it can be employed as a test of the MSE or root MSE, as was done above. Holst and

Thyregod have also derived a statistical test that explicitly tests hypotheses about the MSE of a

measurement process (Holst and Thyregod, 1999). The Holst-Thyregod statistic is slightly more

complicated to calculate, and tables of percentiles for the statistic are not widely available. However,

the Holst-Thyregod test has an advantage over the W test when the MSE is dominated by bias. In

some situations where the measurement process has good precision and somewhat large bias, the

power of the Holst-Thyregod test far exceeds the power of the W test, just as the power of the W test

in some situations can far exceed the power of the MARLAP test. In other situations, and especially

when the MSE is dominated by variance rather than bias, the W test outperforms the Holst-Thyregod

test. For these reasons, this appendix recommends the W test as the best choice for most method

validation experiments.

Example

Suppose the action level for a certain project is 100 pCi/L and the required method uncertainty is

uMR = 10.0 pCi/L at the action level. The relative required method uncertainty (at or above the action

level) is nMR = 0.10, or 10 %. A Level D validation experiment is performed for a candidate method,

with three activity levels (L = 3) and seven measurements at each activity level (N = 7). Suppose the

measurement results are as shown in Table E1.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

11 Presumably the laboratory was unaware of this bias; otherwise, it would have corrected it.

47

TABLE E1 – Method Validation Measurement Results

Measurement (j)

Target Value (Ki

)

50 pCi/L 100 pCi/L 300 pCi/L

1 36.1 83.2 256.1

2 39 83.7 235.2

3 42.2 84.8 249

4 44.4 75.4 258.5

5 47.5 82.3 265.2

6 40.2 94.7 255.7

7 44 88.4 254.5

Average x = 41.91 84.64 253.46 x = x =

Standard deviation s = 3.81 s = 5.90 s = 9.40

The method appears to have good precision, but it also has a relative bias of approximately !16 %,

which is larger than the required relative method uncertainty (10 %).11 If one performs the validation

test described in Section 5.4, the acceptance limits for the results are as shown below.

TABLE E2 – Acceptance Limits, MARLAP Test

Target Value

Ki

/(pCi/L)

Required Uncertainty

ui/(pCi/L)

Acceptance Limits

(Ki ± kui)/(pCi/L)

50 10 20 – 80

100 (AL) 10 70 – 130

300 30 210 – 390

Note: The “uncertainty multiplier” (k) in this case equals 3.0307, which is rounded to 3.0.

Since all the measured results are within these acceptance limits, the method is judged acceptable

in spite of the obvious negative bias.

If the chi-squared test described in this appendix is used instead, then the critical value for the chisquared statistic is

w N c L = == − χ χ ( ) α / . / ( ) () . 1 1

2

0 951 3

2 7 17 07

and the results are shown in Table E3. 
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

48

TABLE E3 – Method Validation Results, Alternative Test (W Test)

Measurement

(j)

Activity Level (i)

123

Ki

 = 50 pCi/L

ui

 = 10.0 pCi/L

Ki

 = 100 pCi/L

ui

 = 10.0 pCi/L

Ki

 = 300 pCi/L

ui

 = 30.0 pCi/L

Xij Xij Xij Z

X K

u ij

ij i

i

= − Z

X K

u ij

ij i

i

= − Z

X K

u ij

ij i

i

= −

1 36.1 !1.39 83.2 !1.68 256.1 !1.4633

2 39 !1.10 83.7 !1.63 235.2 !2.1600

3 42.2 !0.78 84.8 !1.52 249 !1.7000

4 44.4 !0.56 75.4 !2.46 258.5 !1.3833

5 47.5 !0.25 82.3 !1.77 265.2 !1.1600

6 40.2 !0.98 94.7 !0.53 255.7 !1.4767

7 44 !0.60 88.4 !1.16 254.5 !1.5167

W Z i ij = = ∑ 5.45 2 W Z i ij = = ∑ 2 18.6 W Z i ij = = ∑ 2 17.4

Because the statistics W2 and W3 both exceed the critical value wC = 17.1, the method is judged to

be unacceptable.

Theoretical Comparison of Statistical Power

The following set of four figures graphically illustrates the power of the MARLAP test and the W

test for the same conditions assumed in Figure D2 of Appendix D. The power of the Holst-Thyregod

(H-T) test is also graphed for comparison. The scenario (as above) involves a Level D validation of

a method for a project where the required method uncertainty is 10 pCi/L at an action level of

100 pCi/L. Each of the following figures assumes a different value for the ratio of the relative

standard deviation (RSD) to the required uncertainty at each activity level. In Figure E1a, the ratio

is 0.5, so that the RSD at the action level equals 5 %. The ratios for Figures E1b, E1c, and E1d are

0.75, 1, and 1.25, respectively. In each graph, the horizontal axis represents possible values for the

relative bias of the method ranging from 0 to 20 %. The vertical axis, labeled P, represents the

probability that a method with the given relative standard deviation and relative bias will be rejected.

In every case, the W test outperforms the MARLAP test, although the differences are most noticeable

when the precision of the method is good but the bias is large. Also note that the power of the HolstThyregod test exceeds that of the W test in Figures E1a and E1b but not in E1c and E1d.

Reference

Holst, Erik and Poul Thyregod. 1999. “A statistical test for the mean squared error,” Journal of

Statistical Computation and Simulation, 63:4, 321–347.
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

49

5 % RSD at 100 pCi/L

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Relative Bias (%)

P

MARLAP

W

H-T

Figure E1a

RSD = 7.5 %

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Relative Bias (%)

P

MARLAP

W

H-T

Figure E1b
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

50

RSD = 12.5 %

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Relative Bias (%)

P

MARLAP

W

H-T

Figure E1d

RSD = 10 %

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Relative Bias (%)

P

MARLAP

W

H-T

Figure E1c
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

51

Appendix F: 

Glossary

accuracy: The closeness of a measured result to the true value of the quantity being measured.

Various recognized authorities have given the word “accuracy” different technical definitions,

expressed in terms of bias and imprecision. Following MARLAP, this document avoids all of

these technical definitions and uses the term “accuracy” in its common, ordinary sense. 

aerosol: A suspension of fine solid or liquid particles within a gaseous matrix (usually air).

aliquant: A representative portion of a homogeneous sample removed for the purpose of analysis

or other chemical treatment. The quantity removed is not an evenly divisible part of the whole

sample. An aliquot, by contrast, is an evenly divisible part of the whole.

analyte: For this document, an analyte is a specific radionuclide or a category of radionuclides that

comprise gross alpha or beta analyses. An analyte may be on the list of radionuclides of interest

or a radionuclide of concern for a project. See target analyte. 

analyte concentration range: (1) Method validation definition – the radionuclide concentration

range corresponding to three test levels (lower, mid and upper) that are used during method

validation. The mid level concentration corresponds to the action level. (2) MQO definition –

the expected concentration range (minimum to maximum) of an analyte expected to be present

in a sample for a given project. While most analytical protocols are applicable over a fairly large

range of concentration for the radionuclide of interest, performance over a required concentration range can serve as a measurement quality objective for the protocol selection process, and

some analytical protocols may be eliminated if they cannot accommodate the expected range of

concentration. 

analytical action level (AAL): The value of a quantity that will cause the decision maker to choose

one of the alternative actions. The action level may be a derived concentration level (such as the

derived water concentration in this document), background level, release criteria, regulatory

decision limit, etc. The AAL is often associated with the type of media, target analyte, and

concentration limit. Some AALs are expressed in terms of a derived radionuclide concentration

corresponding to a dose or risk, such as a protective action guide. MARLAP uses the term

“action level.” 

analytical decision level (ADL): The minimum measured value for the radionuclide concentration

in a sample that indicates the amount of radionuclide present is equal to or greater than the

analytical action level at a specified Type II error rate. (Assumes that method uncertainty

requirements have been met.) Any measurement result equal to or greater than the applicable

ADL is considered to have exceeded the corresponding analytical action level. MARLAP uses

the term “critical level.”

analytical protocol specification (APS): The output of a directed planning process that contains the

project’s analytical data needs and requirements in an organized, concise form. The level of
Method Validation Guide for Radiological Laboratories Participating in Incident Response Activities

52

specificity in the APSs should be limited to those requirements that are considered essential to

meeting the project’s analytical data requirements to allow the laboratory the flexibility of

selecting the protocols or methods that meet the analytical requirements.

background (instrument): Radiation detected by an instrument when no source is present. The

background radiation that is detected may come from radionuclides in the materials of construction of the detector, its housing, its electronics, and the building, as well as the environment and

natural radiation.

background level: A term that usually refers to the presence of radioactivity or radiation in the

environment. From an analytical perspective, the presence of background radioactivity in

samples needs to be considered when clarifying the radioanalytical aspects of the decision or

study question. Many radionuclides are present in measurable quantities in the environment.

bias (of a measurement process): A persistent deviation of the mean measured result from the true

or accepted reference value of the quantity being measured, which does not vary if a measurement is repeated. 

blank (analytical or method): A sample that is assumed to be essentially free of the target analyte

(the “unknown”), which is carried through the radiochemical preparation, analysis, mounting,

and measurement process in the same manner as a routine sample of a given matrix. 

calibration: The set of operations that establish, under specified conditions, the relationship between

values indicated by a measuring instrument or measuring system, or values represented by a

material measure, and the corresponding known value of a parameter of interest. 

calibration source: A prepared source, made from a certified reference material (standard), that is

used for calibrating instruments.

carrier: (1) A stable isotopic form of a tracer element or nonisotopic material added to effectively

increase the quantity of a tracer element during radiochemical procedures, ensuring conventional

behavior of the element in solution. (2) A substance in appreciable amount that, when associated

with a tracer of a specified substance, will carry the tracer with it through a chemical or physical

process, or prevent the tracer from undergoing non-specific processes due to its low

concentration (IUPAC, 1995). A stable isotope of a radionuclide (usually the analyte) added to

increase the total amount of that element so that a measurable mass of the element is present.

chain of custody: Procedures that provide the means to trace the possession and handling of a

sample from collection to data reporting.

check source: A material used to validate the operability of a radiation measurement device,

sometimes used for instrument quality control. See source, radioactive.
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combined standard uncertainty: Standard uncertainty of an output estimate calculated by combining the standard uncertainties of the input estimates. The combined standard uncertainty of

y is denoted by uc(y). See uncertainty (of measurement).

critical level: Termed analytical decision level in this document in the context of evaluating sample

results relative to an analytical action level. In the context of analyte detection, critical level

means the minimum measured value (e.g., of the instrument signal or the radionuclide concentration) that indicates a positive (nonzero) amount of a radionuclide is present in the material within

a specified probable error. The critical level is sometimes called the critical value or decision

level. 

critical net concentration: Similar in concept as the “critical level.”

data quality objective (DQO): Qualitative and quantitative statements that clarify the study

objectives, define the most appropriate type of data to collect, determine the most appropriate

conditions from which to collect the data, and specify tolerable limits on decision error rates.

Because DQOs will be used to establish the quality and quantity of data needed to support

decisions, they should encompass the total uncertainty resulting from all data collection

activities, including analytical and sampling activities.

default AAL test level: Radionuclide test concentration for a given general matrix category to be

used in the method validation process in the absences of PAG or risk-based AALs.

derived air concentration (DAC): The concentration of a radionuclide, in pCi/m3, that would result

in exposure to a specified dose level. Generally refers to a protective action guide or other

specified dose- or risk-based factor related to an analytical action level. In this document, for

example, the “500-mrem DAC for 239Pu” is the concentration of 239Pu, in pCi/m3

, that would

result in an exposure of 500 mrem and would refer to the 500-mrem PAG. The DAC is

radionuclide-specific.

derived radionuclide concentration (DRC): General application term used in discussions involving

both of the terms DAC and DWC.

derived water concentration (DWC): The concentration of a radionuclide, in pCi/L, that would

result in exposure to a specified dose level. Generally refers to a protective action guide or other

specified dose- or risk-based factor related to an analytical action level.

detection capability: The capability of a measurement process to distinguish small amounts of

analyte from zero. 

detection limit: The smallest value of the amount or concentration of analyte that ensures a specified

high probability of detection. Also called “minimum detectable value.”
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discrimination limit (DL): The DL is the point where it is important to be able to distinguish

expected signal from the analytical action level. The boundaries of the gray region.

dose equivalent: Quantity that expresses all radiations on a common scale for calculating the

effective absorbed dose. This quantity is the product of absorbed dose (grays (Gy) or rads)

multiplied by a quality factor and any other modifying factors (MARSSIM, 2000). The quality

factor adjusts the absorbed dose because not all types of ionizing radiation create the same effect

on human tissue. For example, a dose equivalent of one sievert (Sv) requires 1 Gy of beta or

gamma radiation, but only 0.05 Gy of alpha radiation or 0.1 Gy of neutron radiation. Because the

sievert is a large unit, radiation doses often are expressed in millisieverts (mSv). See total

effective dose equivalent and roentgen.

gray (Gy): The International System of Units (SI) unit for absorbed radiation dose. One gray is 1

joule of energy absorbed per kilogram of matter, equal to 100 rad. See sievert.

gray region: The range of possible values in which the consequences of decision errors are relatively

minor. Specifying a gray region is necessary because variability in the analyte in a population and

imprecision in the measurement system combine to produce variability in the data such that the

decision may be “too close to call” when the true value is very near the analytical action level.

The gray region establishes the minimum distance from the analytical action level where it is

most important to control Type II decision errors.

hypothesis testing: The use of statistical procedures to decide whether a null hypothesis should be

rejected in favor of an alternative hypothesis or not rejected.

incident response method validation: Project method validation for incident response applications.

See project method validation and method validation.

interferences: The presence of other chemicals or radionuclides in a sample that hinder the ability

to analyze for the radionuclide of interest.

MARLAP Process: A performance-based approach that develops Analytical Protocol Specifications,

and uses these requirements as criteria for the analytical protocol selection, development, and

evaluation processes, and as criteria for the evaluation of the resulting laboratory data. This

process, which spans the three phases of the data life cycle for a project, is the basis for achieving

MARLAP’s basic goal of ensuring that radioanalytical data will meet a project’s or program’s

data requirements or needs.

measurand: “Particular quantity subject to measurement” (ISO, 1993a).

measurement quality objective (MQO): The analytical data requirements of the data quality

objectives, which are project- or program-specific and can be quantitative or qualitative. These

analytical data requirements serve as measurement performance criteria or objectives of the

analytical process. MARLAP refers to these performance objectives as MQOs. Examples of

quantitative MQOs include statements of required analyte detectability and the uncertainty of
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the analytical protocol at a specified radionuclide concentration, such as the action level.

Examples of qualitative MQOs include statements of the required specificity of the analytical

protocol (e.g., the ability to analyze for the radionuclide of interest [or target analyte] given the

presence of interferences).

measurement uncertainty: See uncertainty.

method blank: A sample assumed to be essentially target analyte-free that is carried through the

radiochemical preparation, analysis, mounting and measurement process in the same manner as

a routine sample of a given matrix.

method performance characteristics: The characteristics of a specific analytical method such as

method uncertainty, method range, method specificity, and method ruggedness. MARLAP

recommends developing measurement quality objectives for select method performance

characteristics, particularly for the uncertainty (of measurement) at a specified concentration

(typically the action level).

method ruggedness: The relative stability of method performance for small variations in method

parameter values.

method specificity: The ability of the method to measure the analyte of concern in the presence of

interferences.

method uncertainty: Refers to the predicted uncertainty of the result that would be measured if the

method were applied to a hypothetical laboratory sample with a specified analyte concentration.

Although individual measurement uncertainties will vary from one measured result to another,

the required method uncertainty is a target value for the individual measurement uncertainties

and is an estimate of uncertainty before the sample is actually measured.

method validation (MV): The demonstration that the method selected for the analysis of a particular

analyte in a given matrix is capable of providing analytical results to meet the project’s measurement quality objectives and any other requirements in the analytical protocol specifications.

minimum detectable concentration (MDC): An estimate of the smallest true value of the analyte

concentration that gives a specified high probability of detection.

nuclide-specific analysis: Radiochemical analysis performed to isolate and measure a specific

radionuclide.

null hypothesis (H0): One of two mutually exclusive statements tested in a statistical hypothesis test

(compare with alternative hypothesis). The null hypothesis is presumed to be true unless the test

provides sufficient evidence to the contrary, in which case the null hypothesis is rejected and the

alternative hypothesis (H1) is accepted. 
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performance evaluation (PE) program: A laboratory’s participation in an internal or external

program of analyzing proficiency-testing samples appropriate for the analytes and matrices under

consideration (i.e., PE program traceable to a national standards body, such as NIST). Referencematerial samples used to evaluate the performance of the laboratory may be called performanceevaluation, performance or proficiency-testing samples or materials. See proficiency test

samples.

precision: The closeness of agreement between independent test results obtained by applying the

experimental procedure under stipulated conditions. Precision may be expressed as the standard

deviation. Conversely, imprecision is the variation of the results in a set of replicate

measurements. 

proficiency test (PT) samples: Samples having a known radionuclide concentration used in a PE

program or internally at the laboratory for method validation and for the measurement of bias.

project method validation: The demonstrated method applicability for a particular project.

protective action guide (PAG): The radiation dose to individuals in the general population that

warrants protective action following a radiological event. In this document, PAGs limit the

projected radiation doses for different exposure periods: not to exceed 2-rem total effective dose

equivalent (TEDE) during the first year, 500-mrem TEDE during the second year, or 5 rem over

the next 50 years (including the first and second years of the incident). See derived water

concentration and analytical action level. 

quality control (QC): The overall system of technical activities that measures the attributes and

performance of a process, item, or service against defined standards to verify that they meet the

stated requirements established by the project; operational techniques and activities that are used

to fulfill requirements for quality. This system of activities and checks is used to ensure that

measurement systems are maintained within prescribed limits, providing protection against outof-control conditions and ensuring that the results are of acceptable quality.

radiochemical analysis: The analysis of a sample matrix for its radionuclide content, both

qualitatively and quantitatively.

radionuclide: A nuclide that is radioactive (capable of undergoing radioactive decay).

relative required method uncertainty (nMR): The required method uncertainty divided by the

analytical action level. The relative required method uncertainty is applied to radionuclide

concentrations above the analytical action level. A key measurement quality objective.

rem: The common unit for the effective or equivalent dose of radiation received by a living

organism, equal to the actual dose (in rads) multiplied by a factor representing the danger of the

radiation. Rem is an abbreviation for “roentgen equivalent man,” meaning that it measures the

biological effects of ionizing radiation in humans. One rem is equal to 0.01 Sv. See sievert.
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replicates: Two or more aliquants of a homogeneous sample whose independent measurements are

used to determine the precision of laboratory preparation and analytical procedures.

required method uncertainty (uMR): Method uncertainty at a specified concentration. A key

measurement quality objective. See relative required method uncertainty.

required minimum detectable concentration (RMDC): An upper limit for the minimum detectable

concentration required by some projects.

sample: (1) A portion of material selected from a larger quantity of material. (2) A set of individual

samples or measurements drawn from a population whose properties are studied to gain information about the entire population.

screening method: An economical gross measurement (alpha, beta, gamma) used in a tiered

approach to method selection that can be applied to analyte concentrations below an analyte

level in the analytical protocol specifications or below a fraction of the specified action level.

sievert (Sv): The SI unit for the effective dose of radiation received by a living organism. It is the

actual dose received (grays in SI or rads in traditional units) times a factor that is larger for more

dangerous forms of radiation. One Sv is 100 rem. Radiation doses are often measured in mSv.

An effective dose of 1 Sv requires 1 gray of beta or gamma radiation, but only 0.05 Gy of alpha

radiation or 0.1 Gy of neutron radiation. 

swipe: A filter pad used to determine the level of general radioactive contamination when it is wiped

over a specific area, about 100 cm2

 in area. Also called “smear” or “wipe.”

target analyte: A radionuclide on the list of radionuclides of interest or a radionuclide of concern

for a project. For incident response applications, typical radionuclides of interest are provided

in Appendix A. 

total effective dose equivalent: The sum of the effective dose equivalent (for external exposure) and

the committed effective dose equivalent (for internal exposure), expressed in units of Sv or rem.

Type I decision error: In a hypothesis test, the error made by rejecting the null hypothesis when it

is true. A Type I decision error is sometimes called a “false rejection” or a “false positive.”

Type II decision error: In a hypothesis test, the error made by failing to reject the null hypothesis

when it is false. A Type II decision error is sometimes called a “false acceptance” or a “false

negative.”

uncertainty: A parameter, usually associated with the result of a measurement, that characterizes the

dispersion of the values that could reasonably be attributed to the measurand.