i March 2007

 

Solutions to Analytical Chemistry 

Problems with Clean Water Act Methods 

March 2007 

 
ii March 2007

Solutions to Analytical Chemistry Problems 

with Clean Water Act Methods 

Prepared by 

Analytical Methods Staff 

Engineering and Analysis Division 

Office of Science Technology 

Office of Water 

U. S. Environmental Protection Agency 

Washington, DC 

EPA 821-R-07-002 

March 2007 
 

iii March 2007

Disclaimer

This technical document recommends ways to document and resolve analytical chemistry 

problems encountered in the analysis of wastewater samples. This advice is not a substitute for the Clean 

Water Act (CWA) or EPA regulations, nor is this document a regulation. The advice in this document 

does not alter any otherwise applicable statutory or regulatory requirements and does not, and may not, 

impose legally binding requirements on EPA, States, Tribes, or the regulated community. 

Our advice may not apply to your case-specific circumstances. EPA, State and other decision 

makers retain the discretion to adopt other approaches on a case-by-case basis where appropriate, or when 

additional information is available to them. It is recommended that commercial laboratories convey 

analytical problems to their customers and permittees communicate problems to their regulatory authority 

and regional EPA water program offices. 

Staff of the Engineering and Analytical Support Branch within the Engineering and Analysis 

Division of the EPA Office of Water have reviewed this document. Mention of trade names or 

commercial products does not constitute endorsement or recommendation for use. 
iv March 2007

Foreword

“Solutions to Analytical Chemistry Problems with Clean Water Act Methods“ is an update of the 

document titled “Guidance on Evaluation, Resolution, and Documentation of Analytical Problems 

Associated with Compliance Monitoring”, which was published in 1993. The 1993 document has been 

referred to as the APumpkin Book@ because of its pumpkin-colored cover. The material and technical 

advice in the Pumpkin Book, and this document are based on questions and situations directed to EPA’s 

Clean Water Act chemists by the EPA regions, state agencies and other users of our methods. The 

questions and situations discussed in this document concern analytical challenges encountered in the 

conduct of compliance monitoring under the Clean Water Act (CWA) for chemical pollutants. 

The purpose of this document is to recommend ways to document the existence of a matrix or 

analytical problem with a CWA sample analysis, and mitigate these problems. 

 

This document is organized as follows: 

Chapter 1 Introduction 

Chapter 2 Sampling Requirements 

Chapter 3 Flexibility to Modify an Analytical Method 

Chapter 4 Data Required to Document Matrix Interference 

Chapter 5 Case Histories of Reports of Matrix Interferences 

Chapter 6 Solutions to Matrix Interference Problems 

Chapter 7 Review of Data from Analysis of Samples 

Chapter 8 When a Matrix Interference Is Demonstrated 

Chapter 9 Sources of Additional Help 

EPA's Engineering and Analysis Division (EAD) is solely responsible for the content of this 

document. Comments and suggestions should be directed to: 

CWA Analytical Methods Staff 

Engineering and Analysis Division (4303T) 

Office of Science and Technology 

U.S. Environmental Protection Agency 

1200 Pennsylvania Avenue, N.W. 

Washington, DC 20460 

E-mail: OSTCWAMethods@epa.gov
 

v March 2007

Table of Contents

Disclaimer .....................................................................................................................................iii 

Foreword....................................................................................................................................... iv 

Chapter 1 Introduction................................................................................................................. 1 

Pollutants Regulated Under the Clean Water Act....................................................................... 1 

Analytical Methods Approved Under the Clean Water Act ....................................................... 1 

Scope of This Document............................................................................................................. 2 

Chapter 2 Sampling Requirements............................................................................................. 4 

Sample Collection....................................................................................................................... 4 

NPDES Sampling Requirements ............................................................................................ 4 

Pretreatment Program Sampling Requirements...................................................................... 5 

Trace Metals Sampling Guidance........................................................................................... 5 

Compositing Samples for Volatiles ........................................................................................ 5 

Sample Preservation and Holding Times.................................................................................... 5 

Chapter 3 Flexibility to Modify an Analytical Method............................................................. 7 

Balancing Flexibility and Performance ...................................................................................... 7 

EPA's Alternate Test Procedure (ATP) Program........................................................................ 7 

Flexibility in the EPA Methods .................................................................................................. 8 

Demonstrating Equivalency of a Method Modification ............................................................. 9 

Initial Demonstration of Method Performance ....................................................................... 9 

Application of a Method Modification to a Sample Matrix ................................................. 10 

Suggested QC Acceptance Criteria for Criteria Not Stated in Approved Methods.............. 10 

Intractable Samples................................................................................................................... 10 

Chapter 4 How to Document Matrix Interference .................................................................. 11 

Chapter 5 Reports of Matrix Interferences.............................................................................. 15 

Case Histories ........................................................................................................................... 16 

Chapter 6 Solutions to Matrix Interferences............................................................................ 20 

Solving Matrix Problems .......................................................................................................... 20 

Solutions Applicable to Nearly All Analytes............................................................................ 20 

Selective Reaction and/or Removal of the Interferent.......................................................... 20 

Method of Standard Additions (MSA) ................................................................................. 20 

Solutions Applicable to Classical Pollutants ............................................................................ 21 

Oil and Grease....................................................................................................................... 21 

Cyanide ................................................................................................................................. 21 

Solutions Applicable to Metals Pollutants................................................................................ 24 

Clean Room .......................................................................................................................... 24 

General Matrix Interferences ................................................................................................ 25 

Chromium VI........................................................................................................................ 25 

Mercury................................................................................................................................. 25 

Solutions Applicable to Organic Pollutants.............................................................................. 25 

Volatiles ................................................................................................................................ 25 

Semivolatiles......................................................................................................................... 26 

Determination of Phenol as a Specific Example....................................................................... 27 
vi March 2007

Chapter 7 Review of Data from Analysis of Samples.............................................................. 29 

Standardized Quality Control ................................................................................................... 29 

Provision of QC Data................................................................................................................ 30 

Review of Data from the 600- and 1600-Series Methods......................................................... 30 

Chapter 8 When a Matrix Interference Is Demonstrated....................................................... 41 

Poor Recovery or Precision of the Matrix Spikes..................................................................... 41 

Inability to Meet the Method Detection Limit (MDL) ............................................................. 41 

Allowance for a Matrix Interference......................................................................................... 42 

Chapter 9 Sources of Additonal Help and Information.......................................................... 43 

Web Sites .................................................................................................................................. 43 

Method Indices.......................................................................................................................... 43 

Office of Water CD-ROMs....................................................................................................... 43 

Water Docket ............................................................................................................................ 44 

Federal Register ........................................................................................................................ 44 

Code of Federal Regulations..................................................................................................... 44 

Approval of an Alternate Test Procedure ................................................................................. 45 

Sources for Supporting Documents .......................................................................................... 46 

Appendices

A) Text of October 26, 1984 Preamble, pp. 7-11 

B) Text of Preamble to March 12, 2007 Methods Update Rule (Note flexibility only 

applies to 40 CFR Part 136 methods) 

C) Recommended Approved Modifications to EPA Method 625
 

1 March 2007

Chapter 1

Introduction 

Pollutants Regulated Under the Clean Water Act 

 The Federal Water Pollution Control Act (FWPCA) Amendments of 1972, later amended as the 

Clean Water Act (CWA), classifies each pollutant as a “conventional pollutant,” “toxic pollutant,” or 

“non-conventional pollutant.” The five “conventional pollutants” are codified at Title 40, Part 401.16 of 

the Code of Federal Regulations (40 CFR Part 401.16). (For information on how to access the CFR, see 

Chapter 9 of this document). The five conventional pollutants are: 

$ biological oxygen demand (BOD) 

$ total suspended solids (TSS) 

$ fecal coliform 

$ pH 

$ oil and grease 

 There are 65 “toxic pollutants” listed at 40 CFR Part 401.15 and this group of pollutants has been 

further refined to a list of 126 “priority pollutants” at 40 CFR Part 423, Appendix A. The priority 

pollutants can be subdivided into: 

$ cyanide 

$ asbestos 

$ 13 metals pollutants 

$ 25 pesticide/PCB pollutants 

$ 86 non-pesticide/non-PCB organic pollutants 

 By definition, all pollutants other than “conventional pollutants” or “toxic pollutants” are “nonconventional pollutants.” Examples of non-conventional pollutants are: 

$ toxicity (acute or chronic) 

$ chemical oxygen demand (COD) 

$ metals and organic compounds not on the priority pollutant list 

$ radioactivity 

$ color 

Analytical Methods Approved Under the Clean Water Act 

 CWA Section 304(h) requires EPA to publish test procedures (analytical methods) appropriate for 

the measurement of pollutants. These methods are commonly known as the “304(h) methods”. 

 CWA Section 402 establishes a National Pollutant Discharge Elimination System (NPDES) to 

control the discharge of pollutants to surface waters of the U.S. NPDES is implemented through 

regulations at 40 CFR Parts 100 - 135 and the effluent guidelines and pretreatment regulations at 40 CFR 

Parts 400 - 500. The CWA prohibits any discharger of a pollutant except in compliance with the Act, 

including Section 402. EPA regulations implementing Section 402 generally require facilities that 

discharge wastewater directly to surface waters of the U.S. to obtain an NPDES permit. The regulations 

refer to the facility or person that discharges pollutants as “discharger,” “permittee,” or “applicant”. 
2 March 2007

Facilities that discharge wastewater to a publicly owned treatment works (POTW) are known as indirect 

dischargers and subject to pretreatment requirements. EPA’s pretreatment program regulations are found 

at 40 CFR Part 403. The term “discharger” will be used in this document to mean a discharger, permittee, 

applicant, or other entity regulated under EPA's wastewater regulations. Under the regulations, each 

discharger is required to monitor its effluent for compliance with any and all relevant Federal and State 

discharge limitations, and use the 304(h) methods to demonstrate compliance with NPDES and 

pretreatment program limitations. Regulatory authorities have accepted primacy for implementing the 

Clean Water Act and with that responsibility have authority to be more restrictive than the federal 

regulations. 

 The 304(h) methods are published or incorporated by reference at 40 CFR Part 136 or 40 CFR 

Parts 405 – 500 and are commonly known as the “Part 136 methods.” For many analytes, these methods 

include methods published by EPA, by the U.S. Geological Survey (USGS), by voluntary consensus 

standards bodies such as ASTM International (formerly known as the American Society for Testing and 

Materials International), and by manufacturers of instruments and testing devices. 

 The Part 136 methods include methods approved for use in all of EPA's wastewater and ambient 

water programs (e.g., methods for general use). Methods approved for general use are listed in tables at 

40 CFR Part 136.3(a), including: 

$ Table IA - List of approved biological methods 

$ Table IB - List of approved inorganic test procedures 

$ Table IC - List of approved test procedures for non-pesticide organic compounds 

$ Table ID - List of approved test procedures for pesticides 

$ Table IE - List of approved radiological test procedures 

$ Table IF - List of approved methods for pharmaceutical pollutants 

$ Table IG – Test methods for pesticide active ingredients 

$ Table IH – List of approved microbiological methods for ambient water 

 Methods approved for use in a single industrial category are published in tables at 40 CFR Part 

136 or at 40 CFR Parts 405 - 500. If the methods are not published in tables at 40 CFR Part 136, they are 

not approved for general use, and may only be used for discharges from the industrial category for which 

they are approved. These special category methods are for uses when the nature of the discharge from a 

particular industry poses unique analytical challenges, or when the pollutants to be regulated are specific 

to that industry. For example, methods approved for use in the Pharmaceuticals industrial category are 

listed in Table IF at 40 CFR Part 136, while methods approved for use in the Pesticides Manufacturing 

industrial category are listed in Table 7 at 40 CFR Part 455. 

 At present, there are 75 pollutants listed in Table IB, including common inorganic anions, metals, 

and many of the conventional pollutants named above. To simplify discussions in the remainder of this 

document, the term “classical pollutant” will refer to all the pollutants listed in Table IB, except the 

metals (i.e., the conventional pollutants listed in Table IB and all other non-metals in the table). 

Scope of This Document 

 We presume that you have knowledge of, and access to the relevant Part 136 analytical methods. 

These methods cover a wide range of pollutants and analytical technologies. The method descriptions 

range from a few pages for simple tests to lengthy and detailed documents covering hundreds of analytes. 

Many of these methods and accompanying documents are available on various CD-ROM products or at 

the Office of Science and Technology (OST) web site at www.epa.gov/waterscience/methods. Methods 

from other organizations are often available from those organizations for a fee (see Chapter 9.) 
 

3 March 2007

 Our goal is to address a broad range of analytical problems and sample types. As a result, some 

level of detail was sacrificed and some situations have not been addressed in this document. However, 

the approaches to resolve matrix interferences that are described in this document may be applied to 

issues not specifically addressed in this publication. States and EPA have laboratories with experts to 

answer some questions regarding analytical problems. 

 This document does not cover analyses of oil and grease, metals requiring the use of “clean” 

sampling and analysis techniques, whole-effluent toxicity (WET), biological (microbiological), or 

radiological pollutants. EPA has provided guidance for some of these categories, including: 

$ Analytical Method Guidance for EPA Method 1664A Implementation and Use (40 CFR Part 136),

EPA 821-R-00-003, February 2000, (Oil & Grease)

$ Guidance for Implementation and Use of EPA Method 1631B (40 CFR Part 136), EPA 821-R-01-

023, March 2001, (Mercury)

$ Method 1669: Sampling Ambient Water for Trace Metals at EPA Water Quality Criteria Levels, EPA 

821-R-96-011, July 1996,

$ Guidance on Establishing Trace Metal Clean Rooms in Existing Facilities, EPA 821-B-95-001, 

January, 1996, 

$ Trace Metal Cleanroom, prepared by the Research Triangle Institute, RTI/6302/04/02 F, 

$ Evaluating Field Techniques for Collecting Effluent Samples for Trace Metals Analysis, EPA-821-R98-008, June 1998, 

$ Guidance on the Documentation and Evaluation of Trace Metals Data Collected for Clean Water Act 

Compliance Monitoring, EPA 821-B-96-004, July, 1996, 

$ Water Quality-Based Permitting for Trace Metals Fact Sheet, April 1996 (no EPA number),

$ Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR Part 

136) (EPA 821-B-00-004, July 2000; the “WET Methods guidance”), and 

$ Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications Under 

the National Pollutant Discharge Elimination System Program (June, 2000). 

 Guidance for microbiological methods can be found in Section 9000 of Standard Methods for 

Examination of Water and Wastewater. 

 Guidance for radiochemistry measurements can be found in the Multi-Agency Radiological 

Laboratory Analytical Protocols Manual (EPA 402-B-04-001A to C (in three volumes); the “MARLAP 

Manual”) published in Volume 69, page 77228 of the Federal Register (69 FR 77228) on December 27, 

2004. 

 The documents listed above are available from the sources in Chapter 9. 
4 March 2007

Chapter 2 

Sampling Requirements 

Sample Collection 

 The collection of the sample can have significant effects on the overall analytical process. In 

addition, to ensure some degree of consistency and representativeness, EPA requires that a sample for 

compliance monitoring be collected in a prescribed fashion. Sampling requirements for the NPDES and 

pretreatment programs are spelled out in Parts 122 and 403 of Title 40 of the CFR. 

 Even when the analyst or other laboratory personnel are not responsible for collecting the sample, 

it is important for them to understand EPA’s sampling requirements in order to provide acceptable and 

cost-effective analytical results (e.g., there may be little point in analyzing an improperly collected sample 

if the results may not be used for compliance monitoring). Ideally, laboratory personnel will have ready 

access to the relevant sections of the CFR. However, recognizing that this is not always practical, a 

reasonable level of detail is provided below. 

NPDES Sampling Requirements 

 The sampling requirements under NPDES are given at 40 CFR Part 122, as part of the 

requirements for applying for an NPDES discharge permit. The requirements are broken out by type of 

industry and discharge. For example: 

$ 40 CFR Part 122.21(g)(7) provides the requirements for sampling existing manufacturing, commercial, 

mining, and silvicultural dischargers, 

$ 40 CFR Part 122.21(h)(4)(i) provides the requirements for sampling manufacturing, commercial, 

mining and silvicultural facilities that discharge only non-process wastewater. 

$ 40 CFR Part 122.21(j)(4)(viii) provides the requirements for sampling new and existing publicly 

owned treatment works (POTWs). 

Although many of the other permit application requirements differ among these types of dischargers, they 

have in common the requirements for collecting grab samples for certain pollutants and how composite 

samples for other pollutants must be collected, namely: 

“Grab samples must be used for pH, temperature, cyanide, total phenols, residual 

chlorine, oil and grease, fecal coliform, fecal streptococcus, E. coli, Enterococci, and 

volatile organics, unless specified otherwise at 40 CFR Part 136. For all other pollutants, 

a 24-hour composite sample, using a minimum of four (4) grab samples, must be used 

unless specified otherwise at 40 CFR Part 136.”

 Providing acceptable data for NPDES compliance samples requires that the sample be collected in 

the required fashion. Therefore, laboratory personnel should recognize that grab samples are required for 

the 12 pollutants listed above and 24-hour composite samples are to be used for all other pollutants 

monitored under an NPDES permit. 
 

5 March 2007

Pretreatment Program Sampling Requirements 

 Sampling requirements for the pretreatment program are found at 40 CFR Part 403, specifically at 

40 CFR Part 403.7(b)(2)(iii), 40 CFR Part 403(b)(2)(iv), 40 CFR Part 403.12(b)(5)(iii), and 40 CFR Part 

403, Appendix E. Similar to NPDES program requirements, these sections list the pollutants for which 

grab sampling is required. Appendix E to Part 403 gives details on the collection of grab and composite 

samples for the pretreatment program. 

Trace Metals Sampling Guidance 

 Sampling for trace metals presents a unique challenge to avoid sample contamination. EPA has 

guidelines for sampling ambient water for trace metals (See Chapter 9). 

Compositing Samples for Volatiles 

 As specified in 40 CR 122.21 and noted above, samples to be analyzed for volatile organics must 

be collected as grab samples and not with an automated compositing device. This stands to reason, since 

the compositing equipment is at least partially open to the atmosphere and volatile contaminants could be 

lost from the equipment during the lengthy sample collection period. While using grab samples for 

volatiles preserves the integrity of the individual sample, it raises the overall analytical cost when multiple 

samples of the same discharge have to be collected and analyzed. 

 EPA has studied the differences between the analysis of individual grab samples, and analysis of a 

composite sample prepared at the laboratory from grab samples collected in the field. The study was not 

conclusive so the EPA has not recommended VOA compositing procedures. 

Sample Preservation and Holding Times 

 Sample preservation and holding time requirements are listed by analyte or analyte group in Table 

II at 40 CFR Part 136, and are detailed in the analytical methods. The information listed in Table II is 

often generic, as it applies to a large group of analytes, e.g., metals. The information in the methods is 

often more specific because preservation and holding times generally are studied as a part of method 

development. However, in some cases, there are footnotes in the table for specific analytes that provide 

additional information or requirements that are critical to compliance monitoring. The footnotes to Table 

II at 40 CFR Part 136 often are quite detailed and address, but are not limited to: 

• Sample containers, 

• Sample holding times, 

• Sample preservation, including instances in which the sample must be held for a shorter time than the 

stated holding time if the shorter time is necessary to maintain sample stability, 

• Department of Transportation (DOT) shipping requirements, 

• Interferences specific to certain parameters; e.g., interferences specific to cyanide. 

 Because the footnotes may change with each update to Part 136, the current version of the CFR 

should be consulted for the latest information. The order of precedence for the sample preservation and 

holding time requirements is: 

$ Table II with footnotes, 

$ The individual method 
6 March 2007

Conflicts between these requirements can arise, particularly if new methods are brought into use and the 

generic requirements in Table II are inadvertently not revised. If you discover a potential conflict between 

the holding time requirements in Table II and in a method, please notify the Engineering and Analysis 

Division at the e-mail address given in Chapter 9, and your permitting authority or your client. 
 

7 March 2007

Chapter 3 

Flexibility to Modify an Analytical Method 

Balancing Flexibility and Performance 

 EPA provides analysts with the flexibility to deal with interferences, or otherwise improve method 

performance. This flexibility dates back to the inception of EPA’s wastewater method approval program. 

In December 1979, when EPA proposed the majority of the test methods for organic pollutants, the 

Agency requested comments on the relationship between flexibility in methods and the approach to quality 

control. After reviewing those comments, EPA decided to allow limited flexibility in both the sample 

preparation and analysis portions of its methods. The major flexibility options are discussed in the 

preamble of the October 26, 1984 final rule promulgating the organic methods at 40 CFR Part 136. That 

discussion, which is reproduced in the appendix of this document, specifically cites the ability to change 

chromatographic conditions such as column packings and detectors and changes to sample concentration 

procedures. The preamble also states that: 

“However, the primary objective underlying this flexibility is to enhance precision and 

accuracy for each analysis. Flexibility should not be permitted if the altered technique 

would be less precise or less accurate than the standard approved analytical method. 

Thus, a corollary of increased flexibility was an increased need for a rigorous and 

unambiguous quality control procedure.”

All of the EPA methods approved at 40 CFR 136 since 1984 have incorporated a rigorous and 

standardized approach to quality control. If unsure, the permittee should contact the regulatory authority 

with questions and guidance on what constitutes allowable flexibility. 

EPA's Alternate Test Procedure (ATP) Program 

 In addition to balancing limited flexibility in the methods against a more rigorous quality control 

procedure, EPA included a process for obtaining approval of an alternate test procedure (ATP) on a 

nationwide basis or on a site- or discharge-specific basis (40 CFR Parts 136.4 and 136.5). 

 The ATP program is intended to encourage development of new or improved analytical methods 

and to give analysts options for resolving analytical problems that may be unique to specific wastewaters. 

If you want to use a method other than those specified at 40 CFR Part136, you should apply to the 

Engineering and Analysis Division for approval of a nationwide ATP, or to the State or Regional EPA 

authority for approval of a limited-use ATP e.g. an approval for method changes from those listed in 40 

CFR Part 136 which are granted to a specific site/facility as opposed to all permittees. 

 As part of the ATP program, EPA developed protocols to assist applicants seeking EPA approval 

of alternate test procedures or new methods for use in monitoring wastewater, ambient water, and drinking 

water. There are protocols for organic and inorganic contaminants, and microbiological contaminants. The 

changes instituted in 1999 made the process simpler. These protocols are available at 

www.epa.gov/waterscience/methods. (EPA 821-B-98-002 Protocol for EPA Approval of Alternate Test 

Procedures for Organic and Inorganic Analytes in Wastewater and Drinking Water March 1999) 
8 March 2007

Flexibility in the EPA Methods 

 As noted above, flexibility is permitted in many EPA analytical methods. For example, the 

methods for organic pollutants published at 40 CFR Part 136, Appendix A note that the analyst is 

permitted to “improve separations or lower the costs of analyses” provided that the results obtained are as 

or more accurate than the results obtained using the unmodified method. Recent EPA methods for other 

analytes may also include specific allowances for flexibility. 

 The flexibility to make changes in approved methods without prior approval from EPA is 

described at 40 CFR Part 136.6. The full text of Part 136.6 is reproduced in the appendix of this 

document. It is strongly recommended that analysts consult the full text of 40 CFR Part 136.6 before 

undertaking method modifications. Briefly, Part 136.6 (b)(1) describes allowable method modifications, 

including: 

$ Changes between automated and manual discrete instrumentation, 

$ Changes between automated and manual sample preparations such as digestions, distillations, and 

extractions (provided that the temperatures and/or exposure times are maintain same as manual method 

to achieve same performance), 

$ Changes in the calibration range (provided that the modified range covers any relevant regulatory 

limit), 

$ Changes in equipment such as using similar equipment from a vendor other than that mentioned in the 

method, 

$ Changes in equipment operating parameters such as minor changes in the monitoring wavelength of a 

colorimeter or modifying the temperature program for a specific GC column, 

$ Changes to chromatographic columns, including the use of a capillary (open tubular) GC column with 

EPA Methods 601-613, 624, 625, and 1624B, and 

$ Increases in purge-and-trap sample volumes, 

$ Adjusting sample sizes or changing extraction solvents to optimize method performance in meeting 

regulatory requirements. 

Such changes are only allowed if the modified method produces equivalent performance for the analyte(s) 

of interest, and the equivalent performance is documented. Part 136.6 provides detailed requirements for 

both the demonstration and documentation of the performance of a modified method. 

Note: The allowance for modifications does not apply to a method for a method-defined analyte or a 

change that would result in measurement of a different form or species of an analyte (e.g., a 

change to a metals digestion or total cyanide distillation). It also does not apply to changes in 

sample preservation and/or holding time. 

 In addition to the flexibility provided by the ATP program and in the analytical methods, EPA 

suggests that regulatory authorities allow flexibility in the spirit of method improvement. Because it is not 

possible to address all matrix interferences in all wastewaters, it may be necessary to tailor a method 

modification to a specific matrix interference problem. For example, the solid-phase and continuous 

liquid/liquid extraction have been shown to be effective in reducing emulsions formed with separatory 

funnel extraction, and microwave and bomb digestions have been shown to be more effective in 

solubilizing some metals than mineral acid digestions. The spirit of allowing a method modification is that 

the change results in improved method performance such as accuracy (e.g. recovery) a lower detection 

limits, or better precision. 
 

9 March 2007

Demonstrating Equivalency of a Method Modification 

 Your objective in modifying a method should be to make it more specific for a given pollutant, 

more sensitive, more accurate, or to improve the method in some other way without compromising the 

performance of the method for the intended use. Such improvements could include reducing the overall 

cost of the analysis, or reducing the volumes of wastes produced by the analysis. However, some 

laboratories have interpreted the provision to modify a method solely as a means of increasing the speed of 

analysis, thus reducing the analysis time, or taking other “shortcuts” to reduce cost. This is not EPA’s 

approach. 

 EPA has addressed this issue by: 

1. providing limited flexibility within the methods, so that improvements can be made, and 

2. requiring the analyst to demonstrate that the results produced by a modification will be equal 

or superior to results produced by the unmodified method. 

The yardsticks by which this performance is to be measured are precision and recovery, but can be 

extended to include detection limit, chromatographic resolution, mass spectral resolution, and other 

measures of method performance. For compliance analyses, clearly note that the method is modified and 

communicate the modifications to the regulatory authority. If in doubt contact your local regulatory 

authority. 

Initial Demonstration of Method Performance 

 To prove the modification is appropriate, the laboratory should first perform an initial precision 

and recovery test (IPR) with the unmodified method, and record the results. The initial demonstration 

provides validation of the performance of a method by a specific laboratory. The procedure is described in 

detail in Section 8 or 9 of the 600-series and 1600-Series wastewater methods and also is in ASTM 

International methods and other methods systems. For some methods systems, the IPR may be termed an 

“initial demonstration of method performance” or “initial demonstration of capability” (IDC). A typical 

test consists of an analysis of four or more replicate volumes of reagent water, or other appropriate 

reference matrix, spiked with the pollutants of interest at the concentration specified in the method or at 5–

10 times the detection limit of the method. The final demonstration should be done in the actual 

wastewater matrix of concern. 

 For each analyte, the precision of analysis of the replicates, as determined by the standard 

deviation or relative standard deviation (RSD) of the measurements, should be less than the standard 

deviation or RSD specified in quality control (QC) acceptance criteria in the method. Similarly, for each 

analyte, the average percent recovery of the measurements should fall within the range of percent recovery 

specified in the method. If either the precision or recovery test is failed, the test is repeated until the 

laboratory is able to meet precision and recovery requirements. 

 Include a minimum of one blank in the initial demonstration, and the concentration of the 

analyte(s) in the blank should be less than the level(s) specified in the method. 

 If you modify a method, repeat the initial demonstration with the modification as an integral part 

of the method, until the QC acceptance criteria in the method for precision and recovery and for the blank 

are met. Otherwise, the modification is not permitted. Maintain records that document that the initial 

demonstration was performed on the modified method and those requirements for precision and recovery 

and the blank were met. 
10 March 2007

Application of a Method Modification to a Sample Matrix 

 In addition to the initial demonstration in a reference matrix such as reagent water, the method 

modification is applied to the specific discharge or sample matrix to which the modified method will be 

applied in monitoring. The modified method is tested by spiking the analytes of interest into duplicate 

aliquots of the sample matrix at a concentration of 5 - 10 times the background concentration of the 

pollutant in the sample, 1 - 5 times the quantitation limit, or 1 -5 times the regulatory limit, whichever is 

greatest. The recoveries of the analytes from these matrix spike/matrix spike duplicate (MS/MSD) tests 

are compared to the QC acceptance limits in the original method. Likewise, the relative percent difference 

(RPD) of the MS/MSD results is calculated and compared to the QC limits for RPD in the approved 

method. The modification is acceptable if the recovery and RPD meet the respective limits. Methods 

from some sources may use terms other than MS/MSD for these QC samples, but the concept and use 

remain the same. 

Suggested QC Acceptance Criteria for Criteria Not Stated in Approved Methods 

 Many of the older methods listed in the tables at 40 CFR Part 136 do not contain standardized QC 

or QC acceptance criteria. To fill this gap, EPA proposed standardized QC tests and analyte-specific QC 

acceptance criteria for all 75 contaminants in Table IB at 40 CFR Part 136 in a “Streamlining Initiative” in 

1997. The initiative was proposed on March 28, 1997 (62 FR 14975) and a correction was published on 

June 26, 1997 (62 FR 34573). The QC acceptance criteria published in the Streamlining Initiative were 

developed from interlaboratory data or from single-laboratory data with an allowance for interlaboratory 

variability (see Section III.B.2 of the proposal at 62 FR 14983). Although that initiative was not 

completed, EPA remains committed to the intent of this initiative. EPA suggests use of the QC tests and 

QC acceptance criteria in the Streamlining Initiative as a starting point for evaluating method 

modifications when the approved method is absent of such tests and criteria. 

Intractable Samples 

 Method flexibility permits pollutant identities and concentrations to be determined in nearly all 

wastewaters, but EPA recognizes that there may be a few intractable sample matrices that do not yield 

readily to extensive analytical efforts. Please let EPA or your regulatory authority know about 

modifications that you have made that have worked or not worked with difficult matrices. Reporting to the 

permitting authority that “the sample couldn't be analyzed” is not sufficient and will not be accepted as 

justification for a claim of matrix interference. See Chapter 4 for the information that will document a 

matrix interference and Chapter 8 for possible relief when a matrix interference is shown. 
 

11 March 2007

Chapter 4 

How to Document Matrix Interference 

 This chapter outlines the analytical data and other information that the EPA recommends be 

provided to evaluate a discharger's claim that a complex matrix precludes measurement of a pollutant. 

Generally, the data are the same as data gathered by EPA in developing the Agency's regulations. 

 Because different analytical techniques provide different data (e.g., gas chromatography/mass 

spectrometry (GC/MS) procedures produce plots of mass intensities while colorimetric procedures do not), 

the specific form of the data will differ according to the method. The following items describe the 

minimum data that should be developed to support a claim of compliance. 

1. The identity of the method used for the measurement. 

In order to support a claim of a matrix interference, the analyst should, of course, use a method that is 

approved for the pollutant of interest for NPDES compliance monitoring. Therefore, the most basic 

information an analyst should submit is the identity of the method used for the measurement e.g., 

separatory funnel or continuous liquid/liquid extraction. This information should include the source of 

the method (e.g., EPA, Standard Methods, or ASTM), the method number, complete with any letter 

suffixes or “point” designations (e.g., 1613B or 350.1), and the date of issue of the method (for EPA 

methods) or the edition of the method compilation (e.g., Standard Methods, 18th edition). The tables 

at 40 CFR Part 136 illustrate the level of detail required to unambiguously identify a particular 

method. The date of an EPA method revision or the edition from which a Standard Method is drawn 

are often critical because not all EPA method revisions are approved at 40 CFR Part 136 and different 

editions of Standard Methods may use different letter suffixes for the same technique as methods are 

added or removed from the manual (e.g., SM 4500-S-2 E in the 18th edition is the iodometric method, 

but in the 19th and 20th editions, the iodometric method is SM 4500-S-2 F). 

2. A detailed narrative discussing the problems with the analysis, corrective actions taken, and the 

changes made to the approved method identified. 

The discharger should describe the reasons for the change to the approved method, the supporting 

logic behind the technical approach to the change, and the result of the change. 

Many compliance monitoring analyses are performed by contract laboratories on behalf of the 

discharger. However, the responsibility for providing the information to EPA rests with the 

discharger. The discharger should, therefore, impress upon its contract laboratory the need for detailed 

technical communication of problems experienced and solutions attempted. The narrative should be 

authored by an analyst and written in terms that another analyst can understand. 

3. A summary level report or data reporting forms giving the pollutants for which analyses were 

conducted and the concentrations detected. For the pollutants that were not detected, the 

detection limits or estimated detection limits should be provided. 

Such results should be provided for each field sample analyzed, including any dilutions and 

reanalyses. If not specified in the approved method, the means for estimating the detection limit of 

each pollutant should be provided in the narrative. If the laboratory uses “flags” in its data reporting, 

the definition of each flag should be provided with the data. 
12 March 2007

4. A summary of all quality control results required by the approved method. 

These results include, but are not limited to the following: 

• Instrument tuning, if applicable 

• Calibration 

• Calibration verification 

• Initial precision and recovery test, as described in Chapter 3 

• Ongoing demonstration of laboratory capability (i.e., ongoing precision and recovery, laboratory 

control sample, laboratory fortified blank) 

• Matrix spike/matrix spike duplicate (MS/MSD) or equivalent spiked sample matrices 

• Surrogate recovery, if applicable 

• Labeled compound recovery (isotope dilution methods) 

• Blank results 

5. Raw data that will allow an independent reviewer to validate (reconstruct) each determination 

and calculation performed by the laboratory. 

This validation would consist of tracing the instrument output (peak height, area, or other signal 

intensity) to the final result reported. The raw data are method specific and may include any of the 

following: 

• Sample numbers or other identifiers used by the both the discharger and the laboratory 

• Extraction or digestion date 

• Analysis date and time 

• Sequence of analyses or run log 

• Sample volume 

• Extract volume prior to each cleanup step 

• Extract volume after each cleanup step 

• Final extract volume prior to injection 

• Digestion volume 

• Titration volume 

• Percent solids or percent moisture 

• Dilution data, differentiating between dilution of a sample and dilution of an extract or digestate 

• Instrument and operating conditions 

• GC and/or GC/MS operating conditions, including detailed information on 

-columns used for determination and confirmation (column length and diameter, stationary 

phase, solid support, film thickness, etc.) 

 -analysis conditions (temperature program, flow rate, etc.) 

 -detector (type, operating conditions, etc.) 

• Chromatograms, extracted ion current profiles, bar graph spectra, library search results 

• Quantitation reports, data system outputs, and other data to link the raw data to the results 

reported. (Where these data are edited manually, explanations of why manual intervention was 

necessary should be included.) 

• Direct instrument readouts; i.e., strip charts, printer tapes, etc., and other data to support the final 

results 

• Laboratory bench sheets and copies of all pertinent logbook pages for all sample preparation and 

cleanup steps, and for all other parts of the determination 
 

13 March 2007

The raw data required should be provided not only for the analysis of samples, but also for all 

calibrations, calibration verifications, blanks, matrix spikes and duplicates, and other QC analyses 

required by the approved method. Data should be organized so that an analyst can clearly understand 

how the analyses were performed. 

6. Example calculations that will allow the data reviewer to determine how the laboratory used the 

raw data to arrive at the final results. 

Useful examples include both detected compounds and undetected compounds. If the laboratory or the 

method employs a standardized reporting level for undetected compounds, this should be made clear in 

the example, as should adjustments for sample volume, dry weight reporting (solids only), dilutions, 

etc. 

7. Possible submission of raw data in electronic format. 

For GC/MS and other instruments involving data systems, the discharger should be prepared to submit 

raw data in electronic format or current permanent format upon request by EPA. 

8. The names, titles, addresses, and telephone numbers of the analysts that performed the analyses 

and of the quality control officer that assured and will attest to the results. 

If a contract laboratory collected the data, it is the discharger's responsibility to see that the contract 

laboratory met all of the requirements in the methods and that the pertinent data listed above are 

provided. 

9. Describe attempts to minimize interference. 

 

 It is important that the laboratory describe all attempts to eliminate or minimize the interference, e.g., 

use of simple dilution or use of a totally different 40 CFR Part 136 method that still allows reliable 

measurements at the permit level. 

10. Document modifications. 

The lab should also lists how any modifications made were demonstrated by the supporting data to 

give equivalent performance over the reference method as written. 

 

15 March 2007

Chapter 5 

Reports of Matrix Interferences 

 Chapter 4 described the kind of information that in the EPA’s view should be provided to 

demonstrate that a matrix problem precluded measurement of a pollutant regulated under a NPDES permit 

limitation. This chapter provides case histories of selected reports of matrix interference problems 

submitted by dischargers regulated under the Organic Chemicals, Plastics, and Synthetic Fibers (OCPSF) 

rule. Additional details of matrix interferences reported by dischargers and others, and how to overcome 

these interferences, are given in Chapter 6 of this document. 

 In the early 1990s, the Engineering and Analysis Division (EAD) of EPA reviewed data provided 

by at least 15 dischargers regulated under the categorical pretreatment standards for the OCPSF industry. 

In each instance, the discharger reported that the facility's wastewater could not be monitored for 

compliance with the pretreatment standards because of interferences. EAD was asked by either the Region 

or State permitting authority to review these reports of matrix interferences. Over the years, dischargers 

have reported similar matrix interferences in other industrial categories and many of the documents cited 

in this document were developed by EPA to address these reports. 

 EAD's review focused on each facility's reported inability to determine the organic analytes in its 

wastewater because of interferences. This chapter presents six case histories of EAD's review of data 

submitted by dischargers reporting interference problems and provides further detail as to how these 

dischargers might resolve matrix interference problems. None of the dischargers nor any of the 

laboratories involved are identified in this document. 

 

 Prior to EAD reviewing the data, each of the permitting authorities was provided with: 

• A draft checklist of laboratory data required to support a claim that the discharger was unable to 

measure pollutants due to matrix problems. That draft checklist resembled the Data Required to 

Document a Matrix Interference in Chapter 4 of this document. 

• Draft guidance for analysts attempting to identify and quantify pollutants in wastewaters discharged 

from plants manufacturing organic chemicals, plastics, and synthetic fibers. That draft guidance was 

ultimately incorporated into the 1993 “Pumpkin” book. 

• Draft guidance for permit writers and others reviewing data from the analysis of organic compounds 

determined using the 600- and 1600-Series methods, similar to that found in Chapter 7 of this 

document. 

 It was EAD's intention that these draft documents be provided to the dischargers and in turn to 

their laboratories, as needed. However, the review revealed that the States and Regions had either not 

provided the draft documents or had not followed them. In general, EAD's review of the reports submitted 

by the dischargers revealed the following: 

• In nearly all instances where data were submitted, the dischargers and/or their contract laboratories 

used incorrect analytical methods or did not follow the procedures required in 40 CFR Part 136. 

• In other instances, the dischargers and/or their contract laboratories did not submit data necessary to 

document that the methods were followed. 
16 March 2007

• Finally, the dischargers and/or their contract laboratories did not submit documentation regarding the 

nature of interferences and the attempts (if any) to resolve these interferences. 

Case Histories 

Case #1: This discharger used a contract laboratory for its analytical work. Information submitted by the 

laboratory revealed inconsistencies with the stated analytical methods. 

 The discharger allowed the laboratory to either: 

 

(1) Use methods other than the 40 CFR Part 136 methods, or 

(2) Modify methods 624 and 625. 

 At the time the data were evaluated by EAD, alternative methods were allowed under the ATP 

program described at 40 CFR Part 136.4 and 136.5, but required prior approval from EPA. Otherwise, 

alternative methods were not allowed. EAD found no reference to the approval of the laboratory’s 

modified methods. 

 If Methods 624 and 625 were modified under the spirit of the 40 CFR Part 136.6, the laboratory 

did not document these modifications and did not demonstrate their equivalence. Modifications that the 

laboratory made to Methods 624 and 625 included: 

• Combining acid and base/neutral fractions for samples analyzed by Method 625, 

• Using a fused-silica capillary column for the analysis of acid and base/neutral fractions (since 

approved by EPA Appendix B and C) 

• Using alternative internal standards, 

• Using alternative surrogates, 

• Using higher detection limits, 

• Using fewer matrix spike compounds, and 

• Using matrix spike amounts inconsistent with regulatory compliance, background, or method-specified 

levels. 

 The October 26, 1984 preamble to the 40 CFR Part 136 methods states that a method is considered 

to be equivalent, if its performance has been demonstrated to meet or exceed the specifications in the 

original method. None of the submitted data provided any evidence supporting method equivalence. 

 The use of multiple internal standards and a fused-silica capillary column for the base/neutral and 

acid fractions represent improvements. EPA has provided letters recommending approving of these 

modifications. 40 CFR Part 136.6 explicitly allows for changes to the chromatographic column and use of 

alternative internal standards and surrogates without prior approval from EPA. 

 However, at that time EPA did not accept combining fractions, higher detection limits, alternative 

matrix spike compounds, and matrix spike amounts inconsistent with background or regulatory 

compliance levels represent improvements. It was determined also at that time these changes degrade 

method performance and are therefore in violation of both the spirit and letter of the flexibility permitted in 

the 600- and 1600-Series 40 CFR Part 136 organic methods. 

 The matrix spike compounds and spiking levels used by the laboratory appeared to have been from 

Office of Solid Waste (OSW) SW-846 methods or from Superfund Contract Laboratory Program (CLP) 

methods. The 600- and 1600-Series wastewater methods require the matrix spike compounds to be the 
 

17 March 2007

compounds regulated in the discharge (e.g., 40 CFR Part 136, Appendix A: Method 624, Section 8.3) and 

require that the spike levels be at: 

(1) The regulatory compliance level, 

(2) 1–5 times the background level of the analyte in the sample, or 

(3) The level specified in the method (e.g., Method 624, Section 8.3.1). 

The compounds spiked were not those regulated, and the spikes were not at the levels required. Therefore, 

the results were not useful in demonstrating performance of the method for the problem analytes. 

 The matrix spike was performed on a diluted sample. Had the matrix spike been performed as 

specified in Method 624 or 625, the spike would likely have failed the specifications in the method and the 

associated sample result could not have been reported for regulatory compliance purposes. This should 

have triggered cleanup procedures, the use of alternative methods, or modification of Method 624 or 625 

to improve method performance. 

 The QC specifications for matrix spike recovery used by the laboratory were not the specifications 

given in Methods 624 and 625. The specifications in the wastewater methods (40 CFR Part 136, Appendix 

A: Method 624, Table 5; and Method 625, Table 6) must be used for compliance monitoring. While 

tighter specifications from a documented source may be acceptable if met, use of wider limits without 

documentation is not acceptable. 

 The detection limits reported for semivolatiles were, for the most part, twice the minimum levels 

given in Method 625 and were approximately 10–20 times the method detection limits (MDLs) given in 

Method 625. No explanation for the increased detection limits was given, nor could the limits be derived 

from the data provided. 

 The laboratory made no attempt to clean up the samples using pH change, gel permeation 

chromatography, or the other techniques described in the 600- and 1600-Series methods or in the draft 

guidance provided by EPA. 

 Even with the increased and explicit allowance for flexibility provided at 40 CFR Part 136.6, the 

majority of the modifications made by this laboratory did not improve performance; did not see the 

analytes at the regulatory limits, and thus the modifications were not acceptable. 

 EAD has since recommended (Appendix C) allowing several acceptable modifications to EPA 

Method 625 for environmental permitting and compliance monitoring under the EPA’s CWA program. 

Case #2: Information provided with data submitted by this discharger was insufficient for a detailed 

review. 

 Despite the general lack of data, it appeared the discharger submitted samples to a contract 

laboratory for analyses by a GC/MS method which failed to produce useful results. The discharger and/or 

the laboratory attributed the problems to large concentrations of acetone in the discharge, though this 

problem could not be confirmed from the information provided. The analytical contractor proposed to the 

discharger that Methods 601 and 602 be used for the volatiles analysis in an attempt to overcome the 

interference problems. Both of these methods are approved at Part 136 methods and they are more 

sensitive and more selective than a GC/MS method. Therefore, the regulated analytes should be 

measurable in the presence of a large concentration of acetone. The discharger ignored the laboratory's 
18 March 2007

proposal and submitted a report of matrix interferences. EPA reported that the approach proposed by the 

laboratory was workable and appropriate, and should have been attempted. 

Case #3: This discharger used several contract laboratories for analyses. The reports from these 

laboratories consisted of summary reporting forms showing detection limits that were 10–50 

times greater than the MDLs in Methods 624 and 625. 

 There were no QC results, no details of how the analyses were performed, and no documentation 

of interference problems or steps taken to overcome interference problems, and therefore, no 

documentation that an interference existed. The laboratory may have chosen to dilute samples for 

convenience. The discharger and its laboratory should have provided the data listed in Chapter 4 of this 

document, and attempted to solve interference problems using the techniques discussed in Chapter 6 of this 

document. 

Case #4: This discharger submitted a report from one contract laboratory that contained insufficient 

information for evaluation; and two letters from a second contract laboratory describing a 

problem with 4,6-dinitro-o-cresol. 

 The report provided by the first laboratory indicated no results for spikes of the OCPSF-regulated 

analytes into samples, no details of how the analyses were performed, what interference problems were 

encountered, or what steps were taken to overcome interference problems. In addition, it appeared that the 

contract laboratory combined acid and base/neutral extracts, thus exacerbating interference effects. 

 The letters from the second laboratory describing the problem with 4,6-dinitro-o-cresol asked for 

suggestions on how to determine this compound in the presence of interferences. Chapter 6 of this 

document provides general suggestions for overcoming matrix interference problems and specific 

suggestions for determination of phenol. The specific suggestions for determination of phenol can be 

applied to 4,6-dinitro-o-cresol. 

 Other reports by the contract laboratory showed high detection limits for the substituted phenols 

because of a huge quantity of phenol in the sample. The analytical laboratory should have used the 

procedures for determination of phenol detailed in the Chapter 6 of this document. 

Case #5: This discharger submitted letters and reports from several contract laboratories. 

 Data items that were present and are required for a thorough review were instrument tunes, run 

chronologies, chromatograms, calibration data, calibration verification data, results for blanks, quantitation 

reports for samples, and matrix spike data run against the QC limits for Methods 624 and 625. The initial 

precision and recovery (IPR) data that demonstrate method equivalence were missing. 

 

 The semivolatile matrix spike data were inconsistent. Results of analysis of unspiked samples 

indicated that some of the acids and base/neutrals were not detected, yet results for the spiked samples 

showed large concentrations of some analytes that were not spiked into the samples. 

 The volatiles matrix spike had been diluted by a factor of 200 and spiked after dilution. Diluting 

and spiking will not show matrix interferences, and thus these data are of no value in evaluating the 

undiluted sample results. 
 

19 March 2007

Cases #6: Several dischargers simply submitted summary reports from their laboratories. 

 None of the materials contained the information required in Chapter 4 of this document, and none 

contained explanations of the nature of the interferences found or descriptions of attempts to overcome 

these interferences. These facilities should have followed the guidance in Chapters 4 and 6 of this 

document, and reviewed the data produced using the data review guidance provided in Chapter 7 of this 

document. 
20 March 2007

Chapter 6 

Solutions to Matrix Interferences 

Solving Matrix Problems 

 The inability to measure the concentration of a pollutant in a specific wastewater is often attributed 

to a “matrix problem.” Matrix problems are caused by substances in the water that interfere in some way 

in the analysis. These substances can be suspended materials, dissolved salts, polymeric materials, and 

highly acidic or caustic waters. Examples of solutions to matrix problems are described below and given 

in references in this chapter. The examples are not intended to be exhaustive, but rather representative 

enough to help the analyst understand how to overcome typical matrix interference problems. 

 In addition to the information below and in the guidance documents referenced in this document, 

the means to overcome matrix interferences can often be found in the technical literature and in sets of 

methods and individual methods published by other EPA Offices and by other organizations, most notably 

Standard Methods for the Examination of Water and Wastewater (Standard Methods) and methods 

published by ASTM International. Both of these method sets contain extensive means for overcoming 

matrix interference problems, and Standard Methods and ASTM methods should be consulted before 

contacting EPA for a solution to a matrix interference problem. 

 

Solutions Applicable to Nearly All Analytes 

Selective Reaction and/or Removal of the Interferent 

 The best solution to a matrix interference problem caused by a particular substance is to first 

identify the substance, then selectively remove it from the sample or from the sample extract or digestate. 

Selective removal can be accomplished by reaction with another substance that will not interfere or by 

physical separation from the analyte of interest by adsorption on an ion exchange or chromatographic 

column. The selective reaction/removal technique is described in some of the suggested solutions to 

matrix interference problems below, and is described in further detail in Standard Methods and ASTM 

methods. 

Method of Standard Additions (MSA) 

 A common means of resolving matrix interferences that can be applied to nearly all analytes in all 

matrices is the “method of standard additions” (MSA). MSA for metals is described in Methods for 

Chemical Analysis of Water and Wastes (MCAWW Revised March 1983, NTIS PB 84-128677). MSA for 

organics is described in ASTM Standard D 5788. Also, instrument manufacturers may provide MSA 

procedures in instruction manuals and/or application notes. In MSA, increasing concentrations, typically 

at factors of 2, 4, and 8 times the concentration of the analyte in the sample, are added to separate aliquots 

of the sample. The aliquots are analyzed and a regression or plot of response versus concentration is used 

to determine the concentration of the analyte in the sample. 
 

21 March 2007

Solutions Applicable to Classical Pollutants 

Oil and Grease 

 Oil and grease is the pollutant for which the most matrix interferences have been reported to EPA, 

and nearly all of the reports have been about the formation of emulsions in the extraction of oil and grease. 

EPA has published advice on these problems in Analytical Method Guidance for EPA Method 1664A 

Implementation and Use, EPA 821-R-00-003, February 2000 (see Chapter 9). 

Cyanide 

 Next to oil and grease, cyanide is the pollutant for which the most matrix interferences have been 

reported to EPA. Cyanide chemistry is very complex, and resolving matrix interferences with cyanides 

may involve considerable investigation. Fortunately, companies that work with cyanides are usually very 

familiar with the cyanide chemistry used in their products/processes and wastewaters, and have addressed 

cyanide interference issues. Suggested means for mitigating or overcoming cyanide interferences are 

presented in Section 4500-CN-

 of Standard Methods for the Examination of Water and Wastewater, in 

ASTM D2036, Standard Test Methods for Cyanides in Water, and in OIA Method 1677, approved for use 

at 40 CFR Part 136. Standard Method 4500-CN-

 and ASTM D2036 devote large sections to overcoming 

cyanide interferences. 

 The most common interfering species in the determination of cyanides is sulfur, primarily in the 

form of sulfide. Footnotes to Table II at 40 CFR Part 136.3 address cyanide interferences other than 

sulfide, including: 

$ sulfur, 

$ sulfite, 

$ oxidants (including chlorine and hypochlorite), 

$ thiocyanate, 

$ aldehydes, and 

$ carbonate. 

The footnotes also address the preservatives that may be used and how to deal with particulate matter in 

the sample. The following text is based on footnotes to Table II at 40 CFR Part 136.3 and input from 

ASTM: 

“Add a reducing agent only if an oxidant (e.g., chlorine) is present. Reducing agents shown to be 

effective are sodium thiosulfate (Na2S2O3), ascorbic acid, sodium arsenite (NaAsO2), or sodium 

borohydride (NaBH4). However, some of these agents have been shown to produce a positive or 

negative cyanide bias, depending on other substances in the sample and the analytical method 

used. Therefore, do not add an excess of reducing agent. Methods recommending ascorbic acid 

(e.g., EPA Method 335.4) specify adding ascorbic acid crystals, 0.1 - 0.6 g, until a drop of sample 

produces no color on potassium iodide (KI) starch paper, then adding 0.06 g (60 mg) for each liter 

of sample volume. If NaBH4 or NaAsO2 is used, 25 mg/L NaBH4 or 100 mg/L NaAsO2 will 

reduce more than 50 mg/L of chlorine (see method “Kelada-01" and/or Standard Method 4500-

CN–

 for more information). After adding reducing agent, test the sample using KI paper, a test 

strip (e.g. for chlorine, SenSafeTM Total Chlorine Water Check 480010) moistened with acetate 

buffer solution (see Standard Method 4500-Cl.C.3e), or a chlorine/oxidant test method (e.g., EPA 

Method 330.4 or 330.5), to make sure all oxidant is removed. If oxidant remains, add more 
22 March 2007

reducing agent. Whatever agent is used, it should be tested to assure that cyanide results are not 

affected adversely. 

6

 Sample collection and preservation: Collect a volume of sample appropriate to the analytical 

method in a bottle of the material specified. If the sample can be analyzed within 48 hours and 

sulfide is not present, adjust the pH to >12 with sodium hydroxide solution (e.g., 5 % w/v), 

refrigerate as specified, and analyze within 48 hours. Otherwise, to extend the holding time to 14 

days and mitigate interferences, treat the sample immediately using any or all of the following 

techniques, as necessary, followed by adjustment of the sample pH to >12 and refrigeration as 

specified. There may be interferences that are not mitigated by approved procedures. Any 

procedure for removal or suppression of an interference may be employed, provided the 

laboratory demonstrates that it more accurately measures cyanide. Particulate cyanide (e.g., ferric 

ferrocyanide) or a strong cyanide complex (e.g., cobalt cyanide) are more accurately measured if 

the laboratory holds the sample at room temperature and pH >12 for a minimum of 4 hours prior 

to analysis. 

(1) Sulfur: To remove elemental sulfur (S8), filter the sample immediately. If the 

filtration time will exceed 15 minutes, use a larger filter or a method that requires 

a smaller sample volume (e.g., EPA Method 335.4 or Lachat Method 01). Adjust 

the pH of the filtrate to 12-13 with NaOH, refrigerate the filter and filtrate, and 

ship or transport to the laboratory. In the laboratory, extract the filter with 100 mL 

of 5% NaOH solution for a minimum of 2 hours. Filter the extract and discard the 

solids. Combine the 5% NaOH-extracted filtrate with the initial filtrate, lower the 

pH to approximately 12 with concentrated hydrochloric or sulfuric acid, and 

analyze the combined filtrate. Because the detection limit for cyanide will be 

increased by dilution by the filtrate from the solids, test the sample with and 

without the solids procedure if a low detection limit for cyanide is necessary. Do 

not use the solids procedure if a higher cyanide concentration is obtained without 

it. Alternatively, analyze the filtrates from the sample and the solids separately, 

add the amounts determined (in Φg or mg), and divide by the original sample 

volume to obtain the cyanide concentration. 

(2) Sulfide: If the sample contains sulfide as determined by lead acetate paper, or if 

sulfide is known or suspected to be present, immediately conduct one of the 

volatilization treatments or the precipitation treatment as follows: Volatilization - 

Headspace expelling. In a fume hood or well-ventilated area, transfer 0.75 liter of 

sample to a 4.4-L collapsible container (e.g., CubitainerTM). Acidify with 

concentrated hydrochloric acid to pH < 2. Cap the container and shake vigorously 

for 30 seconds. Remove the cap and expel the headspace into the fume hood or 

open area by collapsing the container without expelling the sample. Refill the 

headspace by expanding the container. Repeat expelling a total of five headspace 

volumes. Adjust the pH to >12, refrigerate, and ship or transport to the laboratory. 

Scaling to a smaller or larger sample volume must maintain the air to sample 

volume ratio. A larger volume of air will result in too great a loss of cyanide (> 

10%). Dynamic stripping: In a fume hood or well-ventilated area, transfer 0.75 

liter of sample to a container of the material specified and acidify with 
 

23 March 2007

concentrated hydrochloric acid to pH < 2. Using a calibrated air sampling pump 

or flowmeter, purge the acidified sample into the fume hood or open area through 

a fritted glass aerator at a flow rate of 2.25 L/min for 4 minutes. Adjust the pH to 

12 - 13, refrigerate, and ship or transport to the laboratory. Scaling to a smaller or 

larger sample volume must maintain the air to sample volume ratio. A larger 

volume of air will result in too great a loss of cyanide (> 10%). Precipitation: If 

the sample contains particulate matter that would be removed by filtration, filter 

the sample prior to treatment to assure that cyanide associated with the particulate 

matter is included in the measurement. Ship or transport the filter to the 

laboratory. In the laboratory, extract the filter with 100 mL of 5% NaOH solution 

for a minimum of 2 hours. Filter the extract and discard the solids. Combine the 

5% NaOH-extracted filtrate with the initial filtrate, lower the pH to approximately 

12 with concentrated hydrochloric or sulfuric acid, and analyze the combined 

filtrate. Because the detection limit for cyanide will be increased by dilution by 

the filtrate from the solids, test the sample with and without the solids procedure if 

a low detection limit for cyanide is necessary. Do not use the solids procedure if a 

higher cyanide concentration is obtained without it. Alternatively, analyze the 

filtrates from the sample and the solids separately, add the amounts determined (in 

Φg or mg), and divide by the original sample volume to obtain the cyanide 

concentration. For removal of sulfide by precipitation, raise the pH of the sample 

to >12 with NaOH solution, then add approximately 1 mg of powdered cadmium 

chloride for each mL of sample. For example, add approximately 500 mg to a 

500-mL sample. Cap and shake the container to mix. Allow the precipitate to 

settle and test the sample with lead acetate paper. If necessary, add cadmium 

chloride but avoid adding an excess. Finally, filter through 0.45 micron filter. 

Cool the sample as specified and ship or transport the filtrate and filter to the 

laboratory. In the laboratory, extract the filter with 100 mL of 5% NaOH solution 

for a minimum of 2 hours. Filter the extract and discard the solids. Combine the 

5% NaOH-extracted filtrate with the initial filtrate, lower the pH to approximately 

12 with concentrated hydrochloric or sulfuric acid, and analyze the combined 

filtrate. Because the detection limit for cyanide will be increased by dilution by 

the filtrate form the solids, test the sample with and without the solids procedure if 

a low detection limit for cyanide is necessary. Do not use the solids procedure if a 

higher cyanide concentration is obtained without it. Alternatively, analyze the 

filtrates from the sample and the solids separately, add the amounts determined (in 

Φg or mg), and divide by the original sample volume to obtain the cyanide 

concentration. If a ligand-exchange method is used (e.g., ASTM D6888), it may 

be necessary to increase the ligand-exchange reagent to offset any excess of 

cadmium chloride 

(3) Sulfite, thiosulfate, or thiocyanate: If thiocyanate is known or suspected to be 

present, use UV digestion with a glass coil (Method Kelada-01) or ligand 

exchange (Method OIA-1677) to preclude cyanide loss or positive interference. If 

sulfite and thiosulfate are present there is no way to accurately determine cyanide 

if heat is applied. In these situations a non-distillation method such as D6888-04, 

or method OI-1677 may be used. 
24 March 2007

(4) Aldehyde: If formaldehyde, acetaldehyde, or another water-soluble aldehyde is 

known or suspected to be present, treat the sample with 20 mL of 3.5% 

ethylenediamine solution per liter of sample. 

(5) Carbonate: Carbonate interference is evidenced by noticeable effervescence upon 

acidification in the distillation flask, a reduction in the pH of the absorber solution, and 

incomplete cyanide spike recovery. When significant carbonate is present, adjust the 

pH to ∃12 using calcium hydroxide instead of sodium hydroxide. Allow the precipitate 

to settle and decant or filter the sample prior to analysis (also see Standard Method 

4500-CN.B.3.d)”. 

Solutions Applicable to Metals Pollutants 

 In environmental testing, samples analyzed for metals are digested with strong mineral acid(s), or 

the metal is chelated and extracted, followed by determination of the metals by: 

$ flame atomic absorption spectrophotometry (FLAA), 

$ graphite furnace atomic absorption spectrophotometry (GFAA), 

$ cold-vapor atomic absorption spectrophotometry (CVAA), 

$ hydride generation atomic absorption spectrophotometry (HGAA), 

$ direct-current plasma atomic absorption spectrophotometry (DCPAA), 

$ inductively coupled plasma/optical emission spectrometry or mass spectrometry (ICP/OES and ICPMS), 

$ atomic fluorescence spectrophotometry (AF) 

$ or colorimetric, titrimetric, voltammetric, or gravimetric techniques. 

The full list of techniques approved for metals analysis is listed in Table IB at 40 CFR Part 136. 

 The most commonly used of these techniques for determination of metals in wastewater are 

GFAA, HGAA, and ICP-MS. HGAA is only approved at 40 CFR Part 136 for arsenic and selenium. 

CVAA is used exclusively for determination of mercury. The introduction of EPA Method 1631, an 

atomic fluorescence method, has caused a shift in technology because of the ability to measure to lower 

levels than CVAA, and with fewer interferences. 

Clean Room 

 Although not strictly a matrix interference problem, contamination of metals samples, particularly 

at or near ambient water quality criteria (WQC) levels, can be a significant problem in sampling and in 

some laboratories. The problem is particularly common for mercury, which is a volatile metal and, 

therefore, can be transported throughout a building through heating, ventilating, and air conditioning 

systems. However, clean room techniques have been successful at eliminating contamination sources for 

many other metals, including lead and zinc. 

 To mitigate laboratory contamination problems with mercury and other metals, EPA published 

Guidance on Establishing Trace Metal Clean Rooms In Existing Facilities, EPA-821-B-96-001, April, 

1995 and Trace Metal Cleanroom, RTI/6302/04-02F, Research Triangle Institute, October, 1995. These 

guidance documents detail how the laboratory can modify existing facilities to reduce contamination to the 

lowest levels and, thereby, prevent contamination from interfering in the analysis. 
 

25 March 2007

General Matrix Interferences 

 Matrix interferences in metals determinations by AA, ICP, and other techniques result in a 

decrease or increase in the signal (response) from what the signal would be if the interference were not 

present. For analysis of environmental samples, the most common forms of matrix interference are caused 

by dissolved materials in the sample digestate. These interferents change the characteristics of the solution 

that is injected into the instrument. 

 One means of resolving matrix problems with metals is use of MSA, described in the section on 

MSA above. Another means of overcoming matrix interferences from dissolved materials is to match the 

blank and the matrix containing the standard used for instrument calibration with the characteristics of the 

sample or digestate. Matrix matching can involve matching the pH, acid concentration, and dissolved 

solids content of the blank and standards. If chelation is used, or if the sample contains significant 

concentrations of organic compounds, the standards and blank should be chelated and/or otherwise 

matched to contain the organic compounds also. 

Chromium VI 

 Although chromium VI (also known as “hexavalent chromium,” and colloquially known as 

“chrome 6") can be considered a metal pollutant, it has historically been treated as a classical pollutant 

because it is usually determined using classical wet-chemistry techniques. Interferences in the 

determination of chromium VI have been overcome by use of ion chromatography with methods such as 

EPA Method 218.6. 

Mercury 

 The dual amalgam purge-and-trap and fluorescence system in EPA Method 1631 is less 

susceptible to matrix interferences, particularly at low levels, than cold vapor atomic absorption and other 

mercury analysis techniques. Therefore, if a matrix interference is encountered in the determination of 

mercury, EPA Method 1631 should be used. Generally, use of this method will resolve most matrix 

interferences. Recommended approaches for addressing any remaining interferences can be found in 

Chapter 3 of EPA's Guidance for Implementation and Use of EPA Method 1631B, EPA 821-R-01-023, 

March 2001, which specifically addresses matrix interferences in the determination of mercury. This 

guidance is applicable to subsequent versions of the method, e.g. 1631E. 

Solutions Applicable to Organic Pollutants 

 Many of these solutions focus on the pollutants regulated under the OCPSF rule, but are applicable 

to pollutants in other effluent guidelines as well. 

Volatiles - The 304(h) methods for volatiles include Methods 601, 602, 603, 624, and 1624. 

1. Use of Selective GC Detectors 

The effluent limits in the OCPSF regulation and any other industry regulations involving volatiles are 

all greater than 10 μg/L (10 ppb). The selective GC detectors in Methods 601 and 602 cover all 

OCPSF volatile pollutants regulated, and allow detection at levels well below the effluent limits in the 

OCPSF regulation. The specificity provided by the electrolytic conductivity detector and by the 

photoionization detector allows detection of the halogenated and aromatic analytes, respectively, in 

complex matrices. 
26 March 2007

2. Micro-extraction and Gas Chromatography with Selective Detectors 

The selective GC detectors in Methods 601 and 602 provide sensitivity that is 10–100 times greater 

than that required to detect the volatile analytes of interest. Some of this sensitivity can be used to 

substitute micro-extraction in place of purge-and-trap. The advantage of micro-extraction is that the 

pH of the water can be adjusted to attempt to keep the interferences in the water while the analytes of 

interest are extracted.1

3. Sample Dilution 

Methods 601 and 602 can achieve method detection limits of less than 1 μg/L (ppb) for all volatile 

analytes in the OCPSF regulation, and of less than 0.1 μg/L (ppb) for many of these analytes. The 

added sensitivity of the selective GC detectors can be used to overcome matrix problems by diluting 

the sample by a factor of 10–100. Even with this dilution, the pollutants can be detected at the levels 

required, and the effects of the interferences will be reduced or eliminated. 

4. Isotope Dilution 

Method 1624 employs stable, isotopically labeled analogs of the pollutants as internal standards in the 

analysis. The use of these labeled compounds frequently permits the pollutant to be determined in the 

presence of interferences because the unique spectrum of the labeled compound can be located in the 

presence of these interferences, and the pollutant can then be located by reference to the labeled 

compound. 

Semivolatiles

1. Use of Selective GC Detectors 

Methods 604 through 612 employ gas chromatography with selective detectors and high-performance 

liquid chromatography with an ultraviolet (UV) or electrochemical detector to detect pollutants in the 

presence of interferences. In addition, Method 604 employs derivatization and a halogen-specific 

detector for the determination of phenols. As with volatiles, the added sensitivity of the selective 

detectors permits the sample to be diluted by a factor of 10–100 while allowing detection of the 

analytes at the effluent limits specified in the OCPSF regulation. 

2. pH Change 

A very powerful means of separating the pollutants of interest from interferences is to adjust the pH of 

the sample to keep the interferences in solution while allowing the pollutants to be extracted in an 

organic solvent. For example, neutral pollutants can be extracted at either low or high pH. Therefore, 

if the main interferences are acidic, the pH can be adjusted to >13 and the acidic interferences will 

remain in the water in ionic form while the neutral pollutants are extracted using an organic solvent. 

Phenol and 2,4-dimethylphenol can be extracted at high pH (11–13) using continuous liquid/liquid 

extractors, as described in Method 1625. This permits phenol and 2,4-dimethylphenol to be extracted 

in the presence of other, stronger acids.2

 Continuous liquid-liquid extraction at low pH is also an 

 

1

 Rhodes, J.W., and Nulton, C.P., J. Env. Sci. and Health, vol. A15, no. 5, (1980).

2

 Jackson, C.B. et. al., J. Env. Sci. and Health, vol. A15, no. 5, (1980).
 

27 March 2007

effective means of extracting phenols and overcoming poor recovery “matrix interferences” caused by 

separatory funnel extraction. 

In a manner analogous to the pH change described above, the extract from the primary extraction can 

be back-extracted with water of the opposite pH to remove other interferences. To keep the organic 

pollutants in the extract, the water used for back-extraction can be saturated with salt (sodium sulfate 

and/or sodium chloride). Aqueous solutions containing 2% of each of these salts have been shown to 

be effective in keeping the pollutants of interest in the extract. 

3. Gel-permeation (Size-exclusion) Chromatography (GPC) 

This technique is described in Revision C of Method 1625. The same technique is used in the 

Superfund Contract Laboratory Program (CLP) methods and SW-846 method 3640, and has been 

shown to be effective for removing lipids and high-molecular-weight interferences that can degrade 

GC and mass spectrometer performance. 

4. Solid-phase Extraction (SPE) 

Although SPE has not been fully evaluated as a cleanup technique, SPE may be effective as a cleanup 

for acidic, basic and neutral organic species. It has been shown to be effective in removing 

interferences from extracts containing pesticides3

 and in its use for the extraction of pollutants from 

drinking water in EPA Method 525. Method 525 is the drinking water analog to method 625. The 

principle has been extensively used to remove interferences in HPLC by adding a short C18 or other 

appropriate column as a guard column in front of the HPLC analytical column. 

5. Florisil, Alumina, and Silica Gel Chromatography 

These adsorbents are effective in separating neutral species from polar interferences. For polar 

analytes of interest, the adsorbent should be evaluated to determine if the analyte will be recovered. 

The level of activation of the adsorbent plays a major role in this recovery process. Techniques can be 

found in SW-846 methods 3610, 3620 and 3630. 

6. Isotope Dilution 

Method 1625 permits determination of pollutants in the presence of interferences in semivolatile 

samples in the same way described above for volatiles. In addition, the wide range of recovery of the 

labeled analogs permitted in the method allows good quantitation of the pollutant when interferences 

reduce the efficiency of the extraction. 

Determination of Phenol as a Specific Example 

 Phenol is a commonly occurring pollutant in OCPSF wastewaters. The protocols below are suggested 

as approaches to the determination of phenol in a complex sample matrix. After a protocol has been found 

to be effective, the laboratory must demonstrate that the modification has equivalent performance to the 

original method. This demonstration involves the tests described in Chapter 3 of this document. The QC 

acceptance criteria in the approved method must be met before proceeding with analysis of a sample for 

compliance monitoring. 

 

3

 Tessari, J.D., 12th Annual EPA Conference on Analysis of Pollutants in the Environment, Norfolk, Virginia, May 

1989. 
28 March 2007

1. Base/neutral extraction, acid back extraction, and isotope dilution GC/MS (based on Method 1625)

 1.1 Measure 1.0 L of well-mixed sample into a graduated cylinder and spike with labeled phenol per 

Section 10 of Method 1625. Stir and equilibrate per this method. Quantitatively transfer the 

sample to a continuous liquid/liquid extractor. Adjust the pH of the sample to 11–13 and extract 

with methylene chloride as described in the method. 

 1.2 Remove the extract from the extractor and place in a 1–2 L separatory funnel. Back-extract the 

extract sequentially three times with 500-mL portions of salt-saturated reagent water (pH <2), 

discarding the reagent water after each back-extraction. 

1.3 Concentrate the extract to 10 mL and clean up using gel-permeation chromatography (GPC) per 

Section 10 of Method 1625. 

1.4 After GPC, concentrate the extract to 0.5 mL and analyze by isotope dilution GC/MS, as described 

in Method 1625. 

1.5 Calculate the recovery of labeled phenol and compare to the performance specifications in Method 

1625. 

2. Dilution, acid extraction, back-extraction with base, derivatization, silica gel cleanup, and gas 

chromatography with an electrolytic conductivity detector (based on Method 604)

2.1 Measure two 100-mL aliquots of well-mixed sample into 1000-mL graduated cylinders. Spike one 

of the aliquots with phenol at the level specified in Section 8 of Method 604. This aliquot serves 

as the matrix spike sample. Dilute both aliquots to 1.0 L with reagent water. Adjust the pH of 

each aliquot to less than 2 with HCl. 

2.2 Pour each aliquot into a separate 1–2 L separatory funnel and sequentially extract three times with 

methylene chloride per Method 604. Discard the aqueous phase and return the extract to the 

separatory funnel. It is recommended that the use of continuous liquid-liquid extraction in place of 

separatory funnel extraction be used. The recoveries of the analyte of interest are usually better. 

2.3 Back-extract the extract sequentially three times with salt-saturated reagent water, discarding the 

reagent water after each back extraction. 

2.4 Concentrate, derivatize, and clean up the extract per Method 604. 

2.5 Analyze using the electrolytic conductivity detector. This detector is less susceptible to 

interferences than the electron capture detector used in Method 604. Newer models have 

sensitivity nearly equivalent to the electron capture detector. 

2.6 Calculate the recovery of phenol in the matrix spike aliquot and compare this recovery to the 

specifications in Method 604. 
 

29 March 2007

Chapter 7 

Review of Data from Analysis of Samples 

 This chapter describes how a responsible party should review data submitted for compliance 

monitoring under the National Pollutant Discharge Elimination System (NPDES) and data submitted to 

EPA and State authorities under the Clean Water Act. This data should be maintained on file in an 

organized fashion available for inspection. The 1993 Pumpkin Book focused on the review of data for 

organic compounds regulated under the OCPSF Rule that was collected with the 600-series and 1600-

Series wastewater methods. Although this revision of the Pumpkin Book now includes references to EPA 

documents for review of other data, the data from the 600- and 1600-Series methods has been described in 

this chapter so that the data reviewer can see details of the information reviewed. EPA uses these data 

reviews in data gathering to support development of effluent guidelines and standards under Sections 301, 

304, 307, 308, 402, and 501 of the Clean Water Act and for other purposes. The principles of data review 

described in this Chapter would also be applicable to data from the 500-series drinking water methods, the 

SW-846 (RCRA) methods, and any method that contains the standardized quality control elements that are 

contained in these methods; e.g., recent ASTM International Committee D19 (Water) methods. 

 The following example is technically detailed and is intended for data reviewers familiar with the 

EPA methods and similar analytical methods. Reviewers unfamiliar with these methods should review the 

methods and the supporting background materials provided in the preamble to the promulgation of the 

600- and 1600-Series methods for a full understanding of the philosophy behind these documents. 

Standardized Quality Control 

 In developing methods for the determination of organic pollutants in wastewater, EPA sought 

scientific and technical advice from many sources, including EPA's Science Advisory Board, scientists at 

EPA's environmental research laboratories, scientists in industry and academia, and scientists, managers, 

and legal staff. The result of discussions held among these groups was the standardized quality assurance 

and quality control (QA/QC) approach that is an integral Part of the 600- and 1600-Series methods. This 

QA/QC takes the form of performance specifications for each method and contains the following elements: 

• Purity and traceability of reference standards 

• Number of calibration points 

• Linearity of calibration 

• Calibration verification 

• Method detection limit (MDL) and minimum level of quantitation 

• Initial precision and recovery 

• Analysis of blanks 

• Recovery of analytes spiked into the sample matrix (e.g., an matrix spike or laboratory-fortified matrix 

aliquot) or recovery of labeled compounds spiked into samples 

• Statements of data quality for recovery of spikes of analytes or labeled compounds into samples 

• Ongoing precision and recovery 

• Statements of data quality for the laboratory 

 In reviewing data submitted for compliance, the permit writer or other individual or organization 

has the authority and responsibility to assure that the test data submitted contain the elements listed above. 

Otherwise, the data may be considered unacceptable for compliance monitoring. 
30 March 2007

Provision of QC Data 

 Dischargers and other organizations submitting test data under CWA or other acts may use their 

own laboratories or contract the testing to laboratories that meet the requirements specified in the methods. 

The permit writer may require that the supporting QA/QC data described above be submitted with results 

or that it be on record at the discharger's facility or at the testing laboratory. 

 EPA strongly recommends that the supporting QA/QC data be submitted along with the analytical 

results, so that the quality of the data can be evaluated directly, and so that these supporting data are not 

lost between the time of submission of the analytical results and the time that the QA/QC data are required. 

 In many of its early analytical programs, EPA relied upon laboratories to maintain records of 

QA/QC data. This practice was cumbersome for the laboratories, because many of the QA/QC data were 

common to the analytical results for a variety of clients. Retrieving these data from the laboratory to 

resolve questions of permit compliance was time-consuming for the discharger and the permit writer. 

More importantly, this practice occasionally resulted in unscrupulous laboratories failing to perform the 

necessary QA/QC testing, or performing the QA/QC testing “after the fact” to satisfy an audit or data 

submission request. In particular, many laboratories did not perform the initial precision and recovery test 

(the “start-up” test) prior to practice of the method and did not perform a spike of the analytes into the 

sample matrix to prove that the method would work on a particular sample. Therefore, while the data 

provided by those laboratories may have been compliant, there was no way to prove the data was 

acceptable for compliance purposes. 

 When collecting data for the development of a regulation, EPA requires that supporting QA/QC 

data be provided along with the results for the sample analyses. If an individual or organization submits 

analytical results for inclusion into EPA's regulations, EPA similarly requires submission of the QA/QC 

data. Sample results are evaluated relative to the QA/QC specifications in the method, and those results 

that pass the QA/QC requirements are included for consideration. Submission of QA/QC data at the time 

of submission of analytical results is essential to timely and effective evaluation of permit compliance 

issues. 

Review of Data from the 600- and 1600-Series Methods 

 Details of the data review process depend to a great extent upon the specific analytical methods 

being employed for compliance monitoring. Even for data from the same methods, there are probably as 

many specific approaches as there are reviewers. However, given the standardized QA/QC requirements 

of the 600- and 1600-Series EPA methods, a number of basic concepts apply. The following sections 

provide the basic details for reviewing data submitted and provide some of EPA's rationale for the QA/QC 

tests. 

1. Purity and Traceability of Reference Standards 

The accuracy of any non-absolute empirical measurement is dependent on the reference for that 

measurement. In determining pollutants in water or other sample matrices, the analytical instrument 

and analytical process should be calibrated with a known reference material. The 600- and 1600-

Series analytical methods, as well as other EPA methods, require that the standards used for calibration 

and other purposes be of known purity and traceable to a reliable reference source. 

The ultimate source for reference materials is National Institute for Standards and Technology (NIST). 

Dischargers and their supporting laboratories submitting analytical data should be able to prove 
 

31 March 2007

traceability of the reference standards used in the analysis to EPA or NIST. The proof of this 

traceability is a written certification from the supplier of the standard. 

Documentation of the purity and traceability of the standards need not be provided with every sample 

analysis. Rather, it should be maintained on file at the laboratory and provided on request. When 

analyses are conducted in a contract laboratory, such documentation ought to be provided to the 

discharger the first time that a laboratory is employed for specific analyses and then updated as 

needed. 

2. Number of Calibration Points 

The 600-series methods specify a minimum of three calibration points. The lowest of these points is 

required to be near the MDL. The highest is required to be near the upper linear range of the analytical 

system, and the third point is approximately midway between the two. Some methods, such as 

Methods 1624 and 1625, require calibration at five specific concentrations for nearly all analytes, and 

three or four specific concentrations for the remaining analytes for which the methods are not as 

sensitive. 

The lowest calibration point should be below the action level and the high standard should still be 

within the calibration range of the instrument. 

The flexibility in selecting the levels of the calibration points in the 600-series methods has led to a 

wide variety of calibration ranges as each laboratory may determine its own calibration range. Some 

laboratories may establish a relatively narrow calibration range, for instance a five-fold concentration 

range such as 10 to 50 μg/L (ppb), because it makes it simpler to meet the linearity specifications of 

the 600-series methods. Other laboratories may choose wider calibration ranges, e.g., 10 to 200 μg/L 

(ppb), in order to minimize the number of samples that should be diluted and reanalyzed because the 

concentration of one or more analytes exceeds the calibration range. 

The data reviewer will need to make certain that all measurements are within the calibration range of 

the instrument. Samples with analyte concentrations above the calibration range should have been 

diluted and reanalyzed. The diluted sample results need only apply to those analytes that were out of 

the calibration range in the initial analysis. In other words, it is acceptable to use results for different 

analytes from different levels of dilution within the same sample. Some flexibility may be exercised in 

acceptance of data that are only slightly above (<10%) the calibration range. Such data are generally 

acceptable as calculated. 

If data from an analysis of the diluted sample are not provided, limited use should be made of the data 

that are above the calibration range (>10%). The response of the analytical instrument to 

concentrations of analytes will eventually level off at concentrations above the calibration range. 

While it is not possible to specify at what concentration this will occur from the calibration data 

provided, it is generally safe to assume that the reported concentration above the calibrated range is a 

lower limit of the actual concentration. Therefore, if concentration above the calibration range is also 

above a regulatory limit, it is highly likely that the actual concentration would also be above that limit. 
32 March 2007

3. Linearity of Calibration 

The relationship between the response of an analytical instrument to the concentration or amount of an 

analyte introduced into the instrument is referred to as the “calibration curve.” An analytical 

instrument can be said to be calibrated in any instance in which an instrumental response can be 

related to a single concentration of an analyte. The response factor (GC/MS methods) or calibration 

factor (GC, HPLC methods) is the ratio of the response of the instrument to the concentration (or 

amount) of analyte introduced into the instrument. The response factor and calibration factor concepts 

are used in many methods for organic contaminants, while methods for metals and some other analytes 

may employ different concepts such as linear regressions. 

While the shape of calibration curves can be modeled by quadratic equations or higher order 

mathematical functions, most analytical methods focus on a calibration range where the response is 

essentially a linear function of the concentration of the analyte. An advantage of linear calibration is 

that the response factor or calibration factor represents the slope of the calibration line and is relatively 

constant, simplifying the calculations and data interpretation. Whichever approach is used, all the 600- 

and 1600-Series methods specify some criterion for determining linearity of calibration. When this 

criterion is met, the calibration is sufficiently linear to permit the laboratory to use an average response 

factor or calibration factor, and it is assumed that the calibration is a straight line that passes through 

the zero/zero calibration point. Linearity is determined by calculating the relative standard deviation 

(RSD) of the response factor or calibration factor for each analyte and comparing this RSD to the limit 

specified in the method. If the RSD does not exceed the specification, linearity is assumed. 

In the 600- and 1600-Series methods, the linearity specification varies from method to method, 

depending on the quantitation technique. The typical limits on the RSD are as follows: 

$ 15% for the gas chromatography (GC) and high-performance liquid chromatography (HPLC) 

methods 

$ 20% for analytes determined by the internal standard technique in the gas chromatography/mass 

spectrometry (GC/MS) methods (624, 625, 1624, and 1625) 

$ 20% for analytes determined by isotope dilution in Methods 1613, 1624, and 1625 

$ 15% for mercury determined by atomic fluorescence in Method 1631 

Metals methods that employ a linear regression specify a criterion for the correlation coefficient, r, 

such as 0.995. 

If the calibration is not linear, as determined by the RSD of the response factor or calibration factor, a 

calibration curve should be used. This means that a regression line or other mathematical function 

should be employed to relate the instrument response to the concentration. However, properly 

maintained and operated lab instrumentation should have no difficulty in meeting linearity 

specifications for 600- and 1600-Series methods. Linear regression emphasizes the importance of 

higher concentration standards and that the correlation coefficient is little impacted by poor 

performance of calibration standards with low concentrations. 

For determination of nearly all of the organic analytes using the 600- and 1600-Series methods, 

calibration curves are linear over a concentration range of 20–100 times the nominal concentration, 

depending on the detector being employed. Whatever calibration range is used, the laboratory should 

provide the RSD results by which one can judge linearity, even in instances where the laboratory is 

using a calibration curve. In instances where the laboratory employs a curve rather than an average 
 

33 March 2007

response or calibration factor, the data reviewer should review each calibration point to assure that the 

response increases as the concentration increases. If it does not, the instrument is not operating 

properly, or the calibration curve is out of the range of that instrument, and data are not considered 

usable. 

4. Calibration Verification 

Calibration verification involves the analysis of a single standard, typically in the middle of the 

calibration range, at the beginning of each analytical shift. The concentration of each analyte in this 

standard is determined using the initial calibration data and compared to specifications in the method. 

If the results are within the specifications, the laboratory is allowed to proceed with analyses without 

recalibrating and to use the multi-point calibration data to quantify sample results. It is also 

recommended that a calibration verification at the action level is periodically analyzed. 

Specifications for calibration verification are generally given as a range of concentrations, as a 

recovery range, or as a percentage difference from the test concentration. For the 600-series 

semivolatile GC and HPLC methods, the difference must be within 15%. For Method 625, the 

difference must be within 20%. The GC and GC/MS methods for volatiles and the 1600-Series 

methods specify a range of concentrations or recoveries for each analyte. These ranges are based on 

interlaboratory method validation studies. 

If calibration cannot be verified, the laboratory may either recalibrate the instrument or prepare a fresh 

calibration standard and make a second attempt to verify calibration. If calibration cannot be verified 

with a fresh calibration standard, the instrument should be recalibrated. If calibration is not verified, 

subsequent data are considered to be invalid until the instrument is recalibrated. 

5. Method Detection Limit or Minimum Level 

Although this requirement is not explicitly stated in EPA wastewater methods (e.g., 600 and 1600-

Series methods) we recommend use of the method detection limit (MDL) concept to establish 

detection capabilities. Detailed procedures for determining the MDL are provided at 40 CFR Part 136, 

Appendix B. Although exact frequencies vary by method, most methods require that, at a minimum, 

laboratories conduct an MDL study as part of their initial demonstration of capability and whenever a 

modification is made to the method that might affect the detection limit and amends thereafter. Data 

reviewers should consult the methods used for specific requirements, or the requirements of their 

customers, auditors, etc. 

The Minimum Level (ML) is used as a quantitation level, and is defined in most of the 1600-Series 

methods as the lowest level at which the entire analytical system gives a recognizable signal and 

acceptable calibration point. Therefore, each 1600-Series method specifies that the calibration range 

for each analyte encompass the method-specified ML. 

Many of the EPA wastewater methods provide specific requirements regarding reporting results that 

are below the ML or the method-specified quantitation limit when these data will be used for 

compliance monitoring. Since these requirements vary slightly, data reviewers should consult the 

specific method for details. 

If the sample results are above the ML, but are below the facility’s regulatory compliance level, then 

the laboratory should report the results to indicate that the pollutant has been detected but is compliant 

with a facility’s permit, assuming all QC criteria are met. If sample results are above the regulatory 
34 March 2007

compliance level, the data reviewer may wish to evaluate the laboratory QC sample results to verify 

that the reported concentration is not attributable to analytical bias. In addition, the data reviewer 

should evaluate all blank results to determine if the level of pollutant detected may be attributable to 

contamination. 

6. Initial Precision and Recovery 

Part 136 methods require this Initial Precision Recovery (IPR) test before use of a method. It is 

sometimes termed the “start-up test.” The laboratory should demonstrate that it can meet the 

specifications in the method for the recovery of analytes spiked into a reference matrix (reagent water). 

EPA’s experience has been that laboratories that have difficulty passing the start-up test have such 

marginal performance that they will have difficulty in the routine practice of the method. 

The start-up test consists of spiking the analytes of interest into reagent water and analyzing four 

aliquots. The mean concentration and the standard deviation of the concentration are calculated for 

each analyte and compared to the specifications in each method. If the mean and standard deviation 

are within the limits, the laboratory may use the method to analyze field samples. For some methods, 

a repeat test is allowed because of the large number of analytes being tested simultaneously. 

If start-up test data fail to meet the specifications in the method, none of the data produced by that 

laboratory using that method should be considered usable. As with the documentation of the purity of 

the standards, the start-up test data need not be submitted with each set of sample results, but should be 

submitted the first time a laboratory is employed for analyses, and updated as changes to the method 

necessitate (see below) in order to allow the data reviewer to determine the adequacy of the 

laboratory’s performance. 

If the laboratory did not perform the start-up tests, the data should not be considered usable, unless all 

other QC criteria have been met and the laboratory has submitted IPR (and associated instrument QC) 

data that were generated after-the-fact on the same instrument. If these conditions are met, then the 

data reviewer may consider the data to be acceptable for most purposes. 

Note: Discussion of this alternative should not in any way be construed as EPA approval of the 

practice of performing IPR analyses after the analysis of field samples. Rather, EPA regards 

the demonstration of laboratory capability prior to sample analysis as an essential QC 

component. This suggestion provides a tool to permitting authorities when data have already 

been collected without the required IPR samples. Once the missing IPR data has been 

identified as a problem, all responsible parties should implement corrective action necessary to 

ensure that it is not repeated. 

It is important to remember that if a change is made to a method, the start-up test will need to be 

repeated with the change as an integral part of the method. Such changes may involve alternative 

extraction, concentration, or cleanup processes; alternative GC columns, GC conditions, or detectors; 

or other steps designed to address a particular matrix problem. If the start-up test is not repeated when 

these steps are modified or added, then laboratory data produced by the modified method should not be 

considered reliable and thus should not be used. Many laboratories report the configuration of their 

GCs (instrument number, column, detector) as a part of their report header. If a configuration change 

is made and new IPR data are not supplied, EPA recommends requesting the new IPR data from the 

laboratory. 
 

35 March 2007

7. Analysis of Blanks 

Blanks should be analyzed on a routine basis, when any part of the analytical process has been 

changed, and when contamination of the analytical system is suspected. Most recent EPA methods 

require that a blank be prepared and analyzed with each batch (set) of samples. The size of a batch is 

usually limited to a maximum of 20 field samples. In practice, this means that on each day that a 

laboratory prepare samples, they should also prepare a blank, even if fewer than 20 samples are 

prepared. The purpose of analyzing a blank with each set of samples is to determine the extent of 

possible contamination of the samples while in the laboratory. If the blank is handled by the same 

analysts in the same way as the samples and the blank shows no contamination, it is likely that the 

samples will not have been contaminated. Analyzing a blank when the analytical process has been 

changed is consistent with requiring a repeat of the start-up tests, because the change introduces a new 

possibility for contamination of samples through the use of the new materials or procedures. 

Contamination in the laboratory is a common problem, though there are many opinions on what 

constitutes contamination. In more recent EPA methods, a concentration above the minimum level of 

quantitation of the method is a cause for concern. In reality, it is not unusual to find low levels of 

common laboratory solvents, phthalates, and other ubiquitous compounds in laboratory blanks. 

Controlling laboratory contamination is an important aspect of each laboratory's quality assurance 

plan. The laboratory should maintain records, typically in the form of control charts, of blank 

contaminants. These records should prompt corrective action by the laboratory, including reanalysis 

of any affected samples, when concentration of an analyte in a blank rises above a historical level. The 

reviewer in evaluating sample results may request control charts; however, they are not required in 

EPA methods and are not routinely submitted with sample data. 

Unfortunately, by the time that results have been found to be contaminated, it is usually too late for 

corrective action. Therefore, the reviewer has several options in making use of the sample data. First, 

if a contaminant is present in a blank, but not present in a sample, then there is little need for concern 

about the sample result, though it may be useful to occasionally review the raw data for samples 

without the contaminant to ensure that the laboratory did not edit the results for this compound. 

The second approach deals with instances where the contaminant is also reported in a sample. Some 

general guidance will help determine the degree to which the contaminant is affecting sample results: 

• If the sample contains the contaminant at levels of at least 10 times that in the blank, then the 

likely contribution to the sample from the contaminant in the laboratory environment is at most 

10%. Since most of the methods in question are no more accurate than that level, the possible 

contamination is negligible. 

• If the sample contains the contaminant at levels of at least 5 times but less than 10 times the blank 

result, the compound is probably present in the sample, but the numerical result should be 

considered an upper limit of the true concentration. 

• If the sample contains the contaminant at levels below 5 times the level in the blank, there is no 

adequate means by which to judge whether or not the sample result is attributable to laboratory 

contamination. The results for that compound in that sample should be considered unacceptable 

for compliance monitoring. 
36 March 2007

There are two difficulties in evaluating sample results relative to blank contamination. First, the 

reviewer should be able to associate the samples with the correct blanks. For analysis of volatiles by 

purge-and-trap techniques, where no sample extraction is required, the blanks and samples are 

associated by analysis date and time, and specific to the instrument as well. For methods involving the 

extraction of organic compounds from the samples, the blanks and samples are primarily associated by 

the date on which they were extracted, and by the batch of samples and associated lab equipment 

(glassware, reagents, cleanup media). 

The second difficulty involves samples that have been diluted. Dilution of a sample with reagent 

water or dilution of an extract with solvent represents an additional potential source of contamination 

that will not be reflected in the results for the blank unless the blank was similarly diluted. Therefore, 

in applying the 10-times rule, the concentration of the sample is compared to the blank result 

multiplied by the dilution factor of the sample or sample extract. For instance, if 12 ppb of a 

contaminant are found in the blank, and the associated sample extract was diluted by a factor of 6 

relative to the extract from the blank prior to analysis, then the sample result would have to be greater 

than 12x6x10, or 720 ppb, to be acceptable. Between 360 ppb and 720 ppb, the sample result would 

best be considered an upper limit of the actual concentration. Below 360 ppb, the sample result is not 

acceptable for compliance monitoring. 

In general, practitioners of analytical methods do not subtract the concentration of the analyte in the 

blank from the concentration of the analyte in the sample to determine the true concentration of the 

analyte in the sample. Experience indicates that this practice is not reliable. The obvious problem 

occurs when the blank concentration is higher than that in the sample, and subtraction would yield a 

negative concentration. Using the 10-times rule above provides a more appropriate means of 

evaluating the results and does not require that the reviewer alter the results reported by the laboratory. 

8. Ongoing Precision and Recovery

The 1600-Series methods require that an “ongoing precision and recovery” (OPR) sample be analyzed 

with each sample set, and the results of this OPR sample should meet the acceptance criteria in the 

method prior to the analysis of blanks and samples. Most other methods approved at 40 CFR Part 136 

contain a similar requirement, but may use different terminology, such as a laboratory control sample 

(LCS), laboratory fortified blank (LFB), or QC check sample. For this purposes of this discussion, all 

such samples are referred to as OPR samples. 

The OPR samples are used to ensure that laboratory performance is in control during analysis of the 

associated batch of field samples. The data reviewer should determine if the OPR sample has been run 

with each sample set and if all criteria have been met. For methods that do not require sample 

digestion or extraction, such as volatile analyses by Method 1624, the OPR analysis is associated with 

the samples on the basis of the analysis date and time and the specific GC/MS system. For other 

analyses, such as semivolatile analyses by Method 1625, OPR results are associated with samples 

extracted (or digested) at the same time as the OPR. In addition to defining sample batches by date 

and time of extraction or analysis, each method specifies a maximum batch size (generally no more 

than 10 or 20 samples) that can be associated with a single OPR. The reviewer should verify that OPR 

samples were run at the proper frequency. 

Because of the large number of compounds being tested simultaneously in the 600- and 1600-Series 

methods, there is a small probability that the OPR analysis will occasionally fail to meet the 

specifications. While the laboratory is supposed to correct any problems and analyze another OPR 

aliquot, it may still be possible to utilize the data associated with an OPR aliquot that does not meet all 
 

37 March 2007

of the method specifications. The following guidelines may be useful to data reviewers when 

evaluating the usability of data: 

 

• If the concentration of an analyte in the OPR is above the method specifications, but that 

compound is not detected in an associated sample, then it is unlikely that the sample result is 

affected by the failure in the OPR. 

• If the concentration of the analyte in the OPR is above method specifications and the analyte is

detected in the sample, then the numerical result may represent an upper limit of the true 

concentration, and data users should be cautioned when using the data for enforcement purposes. 

• If the concentration in the OPR is below the method specifications, and that analyte is detected in 

an associated sample, then the sample result is likely a lower limit of the true concentration for that 

analyte. 

• If the concentration of the analyte in the OPR is below method specification and that analyte is not

detected in the associated sample, then the sample data are suspect and are not usable for 

regulatory compliance purposes because the analysis does not demonstrate the absence of the 

analyte. 

If the OPR sample was not run, there is no way to verify that the laboratory processes were in control. 

In such cases, a data reviewer may be able to utilize the field sample data by examining labeled 

compound or matrix spike recovery results, the IPR results, OPR results from previous and subsequent 

batches, and any available historical data from both the laboratory and the sample site. If the matrix 

spike or labeled compound results associated with the sample batch do not meet the performance 

criteria in the methods, then the results for that set of samples cannot be considered usable. 

If the laboratory’s IPR results and the matrix spike or labeled compound results associated with the 

sample batch in question meet all applicable performance criteria in the methods, then the data 

reviewer may be reasonably confident that laboratory performance was in control during field sample 

analysis. This level of confidence may be further increased if there is a strong history of both 

laboratory performance with the method and method performance with the sample matrix in question, 

as indicated by additional OPR and matrix spike data collected from the laboratory and samples from 

the same site. 

Note: The preceding discussion of maximizing use of failed OPR data is not an EPA endorsement of 

the practice of proceeding with uncontrolled laboratory analyses. Rather, laboratories failing to 

meet OPR specifications should identify and correct the problem and re-analyze affected 

samples whenever possible. This preceding discussion is provided only to describe a tool for 

permitting authorities when re-analysis is not possible due to sample holding times, insufficient 

sample volumes, or other reasons. 

9. Recovery of Analyte Spiked into the Sample Matrix or Recovery of Labeled Compound Spiked 

into Samples

The majority of the 600- and 1600-Series methods were developed to analyze effluent samples, and 

may not be appropriate for in-process samples. While many of the methods were tested using effluents 

from a wide variety of industries, samples from some sources may not yield acceptable results. It is, 

therefore, important to evaluate method performance in the sample matrix of interest. 
38 March 2007

The non-isotope dilution wastewater methods require a spike of the analytes of interest into a second 

aliquot of the sample for analysis with the sample. The purpose of spiking the sample (often termed a 

“matrix spike (MS)”) is to determine if the method is applicable to the sample in question. Most of 

these wastewater methods also require that laboratories prepare and analyze a duplicate aliquot of the 

matrix spike (often called a “matrix spike duplicate (MSD)”), or a duplicate aliquot of an unspiked 

field sample (usually called a “duplicate”). Generally, one matrix spike/matrix spike duplicate 

(MS/MSD) pair or one MS and one duplicate sample is required for every 10 or 20 field samples, 

depending on the requirements of the specific method being used. 

In evaluating method performance in the sample matrix, data reviewers should examine both the 

precision and accuracy of the analysis. Precision is evaluated by comparing the relative percent 

difference (RPD) of results obtained from the MS/MSD pair or from the duplicate and its 

corresponding field sample. Accuracy is assessed by examining the recovery of compounds in the 

matrix spike sample (and if applicable, the matrix spike duplicate sample). In evaluating matrix spike 

results, the data reviewer should verify that: 

• The unspiked sample has been analyzed. 

• The spiked sample has been analyzed, and that the analytes were spiked at an appropriate 

concentration (generally 5 - 10 times the background concentration of the analyte in the sample or 

1 - 5 times the regulatory compliance limit, whichever is greater). If the analytes are spiked too 

high or too low, it is not usually possible to differentiate recovery of the spiked concentration from 

recovery of the analyte in the unspiked sample. 

• The recovery of the spike is within the range specified. 

If the RPD and recoveries of the MS/MSD or MS and duplicate samples are within the limits specified 

in the method, the method is judged to be applicable to that sample matrix. If, however, the RPD or 

recoveries in these samples are not within the range specified, either the method does not work on the 

sample, or the sample preparation process which includes sample collection is out of control. 

If the method is not appropriate for the sample matrix, changes to the method or use of an alternative 

method would be needed. Matrix spike results are necessary in evaluating a modified method. If the 

analytical process is out of control, the laboratory should take immediate corrective action before any 

more samples are analyzed. 

To separate indications of method performance from those of laboratory performance, the laboratory 

should prepare and analyze a quality control check standard (laboratory control sample) or an OPR 

sample, as described in Items 4 and 8 of this chapter. If the results for either of these analyses are not 

within the range specified, the analytical system or process should be repaired. After verifying the 

performance of the repaired system and processes through successful analysis of calibration 

verification and OPR samples, the sample and spiked sample analysis should be repeated. If 

recoveries and RPD of the repeated matrix spike and duplicate analyses are within the ranges 

specified, the analytical process is judged to be in control. If, however, the repeated analysis results 

are still outside the specified ranges, then sample results generally are not useful for regulatory 

compliance purposes because the matrix spike and duplicate results indicate that the method is not 

applicable to the sample. 

In rare cases, it may be possible to make use of such data while efforts are being made to identify a 

method that works on the matrix in question. The following guidelines may be applicable as a 

temporary measure in such circumstances: 
 

39 March 2007

• If the recovery of the matrix spike and duplicate are above the method specifications, but the 

regulated analyte was not detected in the associated sample or was detected below the regulatory 

compliance limit, it is unlikely that the sample result was affected by the failure in the matrix spike 

because the factors that caused the analysis to over-estimate the concentration in the spiked sample 

would not likely have resulted in an under-estimate in the unspiked sample. In other words, it is 

likely that the sampled effluent is in compliance with the permit limit in such cases. 

• If the recovery of the matrix spike and duplicate are below method specifications, but the regulated 

analyte was detected above the regulatory compliance limit in an associated sample, the sample 

result may represent the lower limit of the true concentration for that pollutant and it is likely that 

true concentration in the effluent is in violation of the permit limit. 

• If the recovery of the matrix spike and duplicate are above the method specifications and the 

regulated pollutant was detected in an associated sample, the sample result may represent the 

upper limit of the true concentration and the data cannot be considered useable for regulatory 

compliance purposes. 

• If the recovery of the matrix spike and duplicate are below the method specifications and the 

regulated pollutant was either not detected or was detected below the regulatory compliance limit, 

the sample result may represent a lower limit of the true concentration and cannot be considered 

usable for regulatory compliance purposes. 

Note: The preceding discussion of maximizing use of failed matrix spike or duplicate data is not an 

EPA endorsement of the practice of using methods that do not work on the sample matrix. The 

preceding discussion is provided only to describe a tool for permitting authorities for use in evaluating 

compliance monitoring results pending the permittees’ successful identification and use of an alternate 

method or method modifications such as those described in Chapter 6 of this document. 

For isotope dilution analyses, data evaluation is simpler because isotopically labeled analogs of the 

pollutants are spiked into every sample. If recovery of a labeled compound spiked into a sample is not

within the range specified in the method, and results of analysis of the ongoing precision and recovery 

standard are within the respective limits, sample results would be considered invalid. When labeledcompound recoveries are outside of method specifications, the problem may be related to the sample 

matrix. The isotope dilution methods specify that, in these instances, the sample should be diluted 

with reagent water and reanalyzed. If the labeled compound recoveries meet the method specifications 

after dilution of the sample, the sample results are acceptable, although the sensitivity of the analysis 

will be decreased by the dilution. 

For some sample matrices, even dilution will not resolve the problem, and for other matrices, the loss 

of sensitivity precludes use of the results for determining compliance. In these instances, additional 

steps need to be taken to achieve acceptable results. 

Steps that may be taken when the results of matrix-spike or labeled-compound recoveries are not 

within the limits specified in the methods are described in Chapter 6 of this document. These steps 

include suggestions for more extensive extraction and cleanup procedures, for sample dilution, and for 

other measures to overcome matrix interference problems. 
40 March 2007

10. Control limits for Recovery of Spiked Analytes or Labeled Compounds in Samples

The 600- and 1600-Series methods specify that after the analyses of five spiked samples, control limit 

is constructed for each analyte. The control limits for each analyte is computed as the mean percent 

recovery plus and minus two times the standard deviation of percent recovery for each analyte. The 

laboratory should then update their control limits after each five to ten subsequent spiked sample 

analyses. 

For non-isotope dilution results, the control limits can be used to estimate the true value of a reported 

result and to construct confidence bounds around the result. For example, if the result reported for 

analysis of phenol is 25 μg/L (ppb), and the statement of data quality for phenol is 70% +30% (i.e., the 

mean recovery is 70% and the standard deviation of the recovery is 15%), the true value for phenol 

will be in the range of 28–43 μg/L (ppb), with 95% confidence. This range is derived as follows: 

Lower limit = [(25 ÷ 0.7) – (25 x 0.3)] = {35.7 – 7.5] = 28 ug/L (ppb) 

Upper limit = [(25 ÷ 0.7) + (25 x 0.3)] = {35.7 + 7.5] = 43 ug/L (ppb) 

 

 Many laboratories do not maintain or provide control limits with sample results, in which case a data 

reviewer should contact the laboratory to determine if the control limits are being maintained for each 

analyte. If necessary, the reviewer can construct a control limits from individual data points if the 

laboratory has records of recoveries for matrix spikes. 

Statements of data quality for isotope dilution methods are based on the recoveries of the labeled 

compounds. Using an isotope dilution method, the sample result has already been corrected for the 

recovery of the labeled analog of the compound. Therefore, for a reported result for phenol of 25 μg/L 

where the standard deviation of the labeled phenol recovery is 15%, the true value for phenol will be in 

the range of 17–32 μg/L, with 95% confidence, derived as follows: 

 Lower limit = [25 – (25 x 0.3)] = 17 μg/L (ppb) 

 Upper limit = [25 + (25 x 0.3)] = 32 μg/L (ppb) 

The lack of control limits does not invalidate results, but makes some compliance decisions more 

difficult. If the laboratory does not maintain control limits there may be increased concern about both 

specific sample results and the laboratory's overall quality assurance program. 

11. Contol limits for the Laboratory (Methods 1624 and 1625) 

In addition to statements of data quality for results of analyses of the labeled compounds spiked into 

the samples, Methods 1624 and 1625 require that control limits be constructed from the initial and 

ongoing precision and recovery data. The purpose of the control limits is to assess laboratory 

performance in the practice of the method, as compared to the assessment of method performance 

made from the labeled compound results for the samples. Ideally, the two limits would be the same. 

Any difference is attributable to either random error or sample matrix effects. 

If the laboratory is practicing isotope dilution methods, the data reviewer should review the control 

chart for the laboratory. If the laboratory does not make these statements available for the reviewer, 

they may be requested. If the laboratory still does not make them available, it does not necessarily 

invalidate any data, but indicates that the laboratory may not be following the method as written.
41 March 2007

When a Matrix Interference Is Demonstrated  The preceding chapters describe how to overcome matrix interference problems and case histories of matrix interfer

were mitigated. This chapter describes help that may be available when all attempts at overcoming matrix interference probexhausted. Poor Recovery or Precision of Matrix Spikes  The most common indication of a matrix problem will be recovery or precision of the matrix spike and matrix spike 

outside of the QC acceptance criteria in the method or QC acceptance criteria suggested in the Streamlining Initiative (see Cdocument). Once a matrix interference is demonstrated to be the cause of a laboratory's inability to meet the QC acceptance 

should document the interference and attempt to overcome it using the procedures suggested in the analytical method, in Ch

document, and other techniques in the test method or technical literature.  If an allowance for matrix effects is warranted or appropriate without a demonstration that a matrix interference exi

attempt to overcome the matrix interference, such an allowance provides a disincentive for addressing interferences that may 

procedures recommended in this document and in the method. The discharger should be familiar with its wastewater and th

to matrix interference problems. However, a site-specific or facility-specific allowance may be warranted after all efforts to 

interference(s) have been exhausted, and should be handled on a case-by-case basis by the regulatory/control authority. Inability to Meet the Method Detection Limit (MDL)  Another common indication of a matrix interference is that measurements cannot be made at low levels because intethese levels.  Statements of the performance in EPA methods, including estimates of MDLs, are estimates based on the Agency's 

in various performance studies, and the method may not achieve all of the stated performance characteristics in all possible 

Scope and Application section of most modern methods approved for use in EPA's wastewater programs states: “The detect

level of quantitation in this method usually are dependent on the level of interferences rather than instrumental limitations.” 

and minimum level of quantitation (ML) should be treated as “presumptive” performance characteristics. These characteris

depending on the sample matrix and on the concentration of interest. The MDL issue has not been resolved and may change The MDL procedure at 40 CFR Part 136, Appendix B allows determination of an MDL in a matrix other than reageand Application section of the MDL procedure). A permit could specify a different detection or quantitation limit when a di
42 March 2007

that a different limit is appropriate for its effluent based on the presumptive statement at the beginning of most modern EPA 

statements in the MDL procedure. After the discharger demonstrates that the approved test method cannot achieve the presuquantitation limit on an effluent-specific basis, the discharger and regulatory authority should work cooperatively to establis

a procedure such as the procedure given at 40 CFR Part 132, Appendix F, Procedure 8. This procedure, titled Water QualityLimitations Below the Quantification Level allows a discharger to establish an effluent-specific ML.  Although EPA has provided the procedure above to develop an effluent-specific ML, EPA recommends that the dis

achieve the MDL and ML stated in the approved method by using the interference-reducing procedures given in this docum

method. Prior to allowing the adjustment of a permit limit because the discharger reports it is unable to achieve the MDL anmethod approved at 40 CFR Part 136, the regulatory authority should review the steps taken by the discharger to reduce inte

all reasonable efforts have been made to achieve the permit limit. It is critical that the permittee be able to measure and accuor above their permit limit and the achievement of method specified MDLs or MLs is of less importance. Allowance for a Matrix Interference  Because every situation is different, EPA has not adopted a rigid protocol for obtaining data that demonstrate that a exists, nor can a hard-and-fast rule be developed to state the conditions under which an allowance for a matrix interference s

all attempts at resolving the matrix interference are unsuccessful, the most common analytical solution to a matrix interferenthe sample with reagent water until the precision and recovery are within normal levels. No more than the minimum amoun

used. The effect of this dilution will be to raise the MDL and ML and may necessitate development of an effluent-specific Mthis situation arise, EPA suggests that the regulatory/control authority solicit and evaluate the following information to demo

allowance for matrix interferences in the form of an effluent-specific MDL and ML may be appropriate: • MDL, IPR, and blank data demonstrating that the laboratory can perform the method; 

• Field, equipment, and reagent blank data demonstrating that the sampling and analysis systems are free from contamina

required for reliable determination of the pollutant; 

• MS/MSD data (where applicable) demonstrating that a potential matrix interference exists because the recovery and or p

the QC acceptance criteria of the method; 

• Confirmation of the out-of-specification MS/MSD recovery or precision by a second laboratory; 

• Identification of the potential interferent(s); • Steps taken to attempt to mitigate the interference (e.g., sample, extract, or digestate concentration; sample dilution; use 

use of cleanup procedures; use of pH change prior to extraction; use of a greater amount of a removal reagent; use of tec

remove the interferent; etc.); and 

• Calculation of an effluent-specific MDL using the procedure at 40 CFR Part 136, Appendix B, and calculation of an ML 

given at 40 CFR Part 132, Appendix F, Procedure 8; 

• Other methods approved for NPDES compliance which utilize different approaches were tried. 
43 March 2007

 Once the regulatory/control authority receives these data, the authority would make a determination that an effluent

are appropriate. 

Sources of Additional Help and Information  Following are several sources of information and EPA contacts related to the issues addressed in this document. Ple(listed below), and/or obtain a copy of the EPA CD-ROMs for reference information on analytical methods and topics discu

Web Sites 

EPA’s home page http://www.epa.gov

EPA’s Office of Science and Technology’s water science analytical methods pages 

http://www.epa.gov/ost/methods

or http://www.epa.gov/waterscience/methods

Effluent Guidelines http://www.epa.gov/ost/guide

Method Indices 

EPA Region 1 Library http://www.epa.gov/epahome/index/

EPA Information Sources http://www.epa.gov/epahome/index/key.htmNational Environmental Methods Index (NEMI) 

http://www.nemi.gov

Office of Water CD-ROMs 

Selected Office of Water Methods and Guidance, Version 5 (EPA 821-C-04-001; September 2004) 
44 March 2007

Water Docket  The Water Docket contains copies of materials that support our rules under the Safe Drinking Water Act (SDWA) a

(CWA). These materials include Federal Register notices, references cited in these notices; health criteria, analytical methotechnology, and economic impact and environmental assessment data; development documents, public comments, and other 

information. 

US Environmental Protection Agency EPA Docket Center (EPA/DC) Public Reading Room Room B102, EPA West Building 1301 Constitution Avenue, NW Washington, DC 20460 

The Docket is open to the public on all Federal government work days from 8:30 a.m. until 4:30 p.m. A reasonable fee may 

photocopying. On-line Docket searches may be performed at http://www.epa.gov/ow/docket.html. 

Federal Register  The Federal Register page is at http://www.gpoaccess.gov/fr/index.html. All issues of the Federal Register from 19

online. Federal Register notices prior to 1994 may be found at a library or through a search service. Search instructions for 

are at the Government Printing Office (GPO) web site at http://www.gpoaccess.gov. 

Code of Federal Regulations  All issues from 1996 to the present are on line. The Code of Federal Regulations (CFR) is at http://www.gpoaccess

CFRs prior to 1996 may be found at a library or through a search service. 
45 March 2007

Approval of an Alternate Test Procedure or Questions Specifically Related to this Guidance 

Procedure for nationwide use (see the regulations at 40 CFR Parts 136.4 and 136.5)

Analytical Methods Staff (4303T) 

 U.S. EPA  Ariel Rios Building  1200 Pennsylvania Avenue, N.W.  Washington, DC 20460  email: OSTCWAMethods@epa.gov 

Procedure for use on a specific discharge (see the regulations at 40 CFR Parts 136.4 and 136.5)
46 March 2007

Sources for Supporting Documents Note: These sources are documents referenced in this document. For a more comprehensive list of guidance and other doc

waterscience, yosemite, and other websites, or perform an online search for the document by title or subject. 

Table 9-1 Sources for Supporting Documents

Subject Title of Guidance Document number Date SMethods Update Rule 

Guidelines Establishing Test Procedures for the Analysis of Pollutants Under the Clean Water Act; National Primary Drinking Water Regulations; and National Secondary Drinking Water Regulations; Analysis and Sampling Procedures; Final Rule 

EPA 821-F-06-005 March 12, 2007 7

hete

Clean spaces guidance 

Guidance on Establishing Trace Metal Clean Rooms in Existing Facilities

EPA 821B96001 January 1996 h

w

Cleanroom guidance Trace Metal Cleanroom, prepared by the 

Research Triangle Institute 

RTI/6302/04/02 F October 1995 R

In

Mercury; Method 1631 guidance 

Method 1631, Revision E: Mercury in Water by Oxidation, Purge and Trap and Cold Vapor Atomic Fluorescence Spectrometry

EPA 821-R-02-019 August 2002 h

eg

Methods, wastewater and drinking water 

EPA Methods and Guidance for Analysis of Water

EPA 821-C-99-004 June 1999 N

Methods, historical Methods for Chemical Analysis of Water and 

Wastes (MCAWW) 

EPA 600/4-79-020 March 1983 N

Methods and guidance 

Selected Office of Water Methods and Guidance, Version 5

EPA 821-C-04-001 September 2004 

Metals sampling techniques evaluation 

Evaluating Field Techniques for Collecting Effluent Samples for Trace Metals Analysis

EPA-821-R-98-008 June 1998 h

w
47 March 2007

Table 9-1 Sources for Supporting Documents

Subject Title of Guidance Document number Date SMetals sampling video 

Office of Water Methods and Guidance, Version 2.0 - Suite (video and CD-ROM; includes methods on CD-ROM EPA 821-C-99-004) 

 2002 N

inm

Metals data evaluation guidance 

Guidance on the Documentation and Evaluation of Trace Metals Data Collected for Clean Water Act Compliance Monitoring

EPA 821-B-96-004 July 1996 h

w

Method flexibility Streamlining Initiative – Guide to Method 

Flexbility and Approval of EPA Water Methods 

EPA-821-D-96-006 December 1996 h

nh

Oil and grease; Method 1664 guidance 

Analytical Method Guidance for EPA Method 1664A Implementation and Use (40 CFR Part 136)

EPA 821-R-00-003 February 2000 h

eg

Radiochemistry method guidance 

Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP 

Manual) 

(EPA 402-B-04-001A to C (in three volumes) 

December 2004 6

Sampling guidance Method 1669: Sampling Ambient Water for 

Trace Metals at EPA Water Quality Criteria Levels

EPA 821-R-96-011 July 1996 

Whole-effluent toxicity (WET) testing guidance 

Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR Part 136)

EPA 821-B-00-004 July 2000 h

e

Whole-effluent toxicity (WET) variability guidance 

Understanding and Accounting for Method Variability in Whole Effluent Toxicity Applications Under the National Pollutant Discharge Elimination System Program

EPA 833-R-00-003 June 2000 h

e

1 National Technical Information Service, http://www.ntis.gov
 

i March 2007

 

Solutions to Analytical Chemistry 

Problems with Clean Water Act Methods 

March 2007