United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-001

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.6100:

Environmental 

Chemistry Methods 

and Associated 

Independent 

Laboratory Validation
i

NOTICE

This guideline is one of a series of test guidelines established by the United States 

Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention 

(OCSPP) for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a). 

Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic 

Substances (OPPTS). To distinguish these guidelines from guidelines issued by other 

organizations, the numbering convention adopted in 1994 specifically included OPPTS as 

part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use 

the new acronym (OCSPP) in their title.

The OCSPP harmonized test guidelines serve as a compendium of accepted scientific 

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the 

test, and is also used by EPA, the public, and the companies that are subject to data 

submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance 

document, these guidelines are not binding on either EPA or any outside parties, and the 

EPA may depart from the guidelines where circumstances warrant and without prior notice. 

At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than 

mandatory. The procedures contained in this guideline are strongly recommended for 

generating the data that are the subject of the guideline, but EPA recognizes that departures 

may be appropriate in specific situations. You may propose alternatives to the 

recommendations described in these guidelines, and the Agency will assess them for 

appropriateness on a case-by-case basis.

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.6100: Environmental chemistry methods and associated independent 

laboratory validation.

(a) Scope—

(1) Applicability. This guideline is intended to be used to help develop data to submit to 

EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and 

the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

(2) [Reserved]

(b) Purpose. This guideline is intended to provide guidance on demonstrating the performance 

of environmental chemistry methods (ECMs) used in field dissipation and ground water 

monitoring studies under the OCSPP 835 Environmental Fate Test Guidelines, or used in field 

studies of terrestrial and aquatic organisms and plants under the OCSPP 850 Ecological Effects 

Test Guidelines. In addition guidance is provided on the conduct and performance of an ECM’s 

associated independent laboratory validation (ILV). When reporting an ECM and its 

performance to the Agency in support of study results, registrants are required under 40 CFR 

Part 158 to provide to the Agency an ILV of the ECM(s) used to generate the laboratory and/or 

field residue data. An ILV is a part of a process to verify that a submitted ECM is suitable for its 

intended purpose and will be able to generate laboratory and/or field residue data when used 

routinely that are accurate and precise at the set reportable residue range. This includes all single 

or multianalyte methods for identification and quantification of the parent compound, 

toxicologically significant metabolites and degradates in each environmental matrix sampled in 

the specific guideline study.

(c) General considerations. 

(1) Registrant(s) provide to the Agency a report of a finalized ECM with appropriate 

revisions or changes recommended by an independent laboratory based on their ILV 

along with the laboratory or field residue study results. 

(2) Registrants need to use an independent laboratory to validate the environmental 

chemistry methods used to generate laboratory and/or field residue data. If that 

laboratory is located in the registrant’s organization, or is in anyway associated with the 

development of the original ECM, the same people, equipment, instruments, and supplies 

(i.e., glassware, solvents, reagents, standard reference materials, etc.) should not be 

utilized to validate the ECM. The Agency does not expect the registrant to synthesize or 

purchase another lot of authentic analytical grade standard but it does expect the 

independent laboratory would need to use a new aliquot of that standard to prepare 

spiking solutions for the ILV in order for the results to be meaningful.

(3) If the methods to be used to generate study data use conventional gas chromatography 

(GC), liquid chromatography (LC), etc., confirmatory methods include using a mass 

spectrometer (MS) in conjunction with the method (e.g., GC/MS, LC/MS) or a second 

column or other suitable procedures. It is not necessary typically to perform another 

confirmatory procedure where GC/MS and LC/MS methods are used as the primary 
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method(s) to generate study data.

(4) Regulatory chemists should be able to validate practical and rapid analytical methods 

using a set of samples in twenty-four hours (e.g., three eight-hour working days); 

however, the Agency recognizes that methods may require additional time. 

(5) Performance data submitted to the Agency should demonstrate that an adequate 

number of samples for each test level were extracted, cleaned up, and analyzed. Each set 

should have an appropriate number of samples with quality control samples intermingled. 

This data should support the limit of detection (LOD) or the method detection limit 

(MDL), the established limit of quantitation (LOQ), and the precision and accuracy for 

each ECM. 

(d) Environmental chemistry method—

(1) Method Details. 

(i) The ECM should be clearly written with complete analytical methods that 

describe the exact procedure, materials, and equipment in sufficient detail to be 

used by laboratory chemists to review the ECM and its associated ILV results. 

All environmental chemistry methods should be stamped non-confidential.

(ii) Analytical environmental chemistry methods used should be practical, and be 

able to quantitate analytes in the study matrix (or matrices) at the data quality

objective(s) set for the laboratory or field residue study. Equipment used to 

perform the method(s) should be commercially available in the United States.

(iii) The analytical methods should meet acceptable performance objectives 

described in paragraph (d)(2) of this guideline. 

(iv) During the Agency evaluation of an ECM or ECMs, the registrant may be 

requested to supply to the Agency an active ingredient analytical grade standard 

and/or toxicologically significant metabolite(s) and degradate(s) to the Agency. 

In addition stable derivative(s) that were used to identify and quantitate the target 

analytes may also be requested to support the evaluation of the ECM and ILV by

the Agency. 

(2) Data quality objectives. 

(i) The mean recovery at each spiking level, at or above the LOQ, should lie 

between 70 and 120% of the known quantity of the pesticide/metabolite/ 

degradate spiked into the matrix blanks during the method validation. 

(ii) The relative standard deviation (RSD) of replicate measurements of recoveries 

should not exceed the target level of plus or minus 20%. 

(iii) The Agency recognizes that some methods may not be able to meet these 

precision objectives. The Agency will review the results of those methods to 
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determine their significance on the acceptability of the ECM. Methods will not be 

rejected outright for failure to comply with each and every aspect of the data 

quality objectives but will be reviewed on a case-by-case basis to determine their 

suitability by science reviewers and chemists in the Agency.

(iv) For an acceptable ECM, reported sample values should not be corrected for 

recoveries.

(e) Independent laboratory validation procedures—

(1) Independent laboratory. 

(i) Validation of the ECM is conducted by a laboratory operating independently 

from that of the originator of the method. The independent laboratory conducting 

the validation may be privately or publicly owned and under certain conditions 

may belong to the same organization as the originating laboratory or in the 

registrant’s own organization.

(ii) The originating and validation laboratories may belong to the same 

organization, but in order for the validation to be independent the analysts, 

equipment, instruments, and supplies (e.g., glassware, solvents, reagents, standard 

reference materials) should be distinct and operate separately and without 

collusion for the validation. Furthermore, the personnel conducting the 

independent validation should not report to the same study director who was 

involved in developing the original method or who may have used the method to 

develop data for laboratory field studies to support pesticide registration or 

reregistration actions. For the validation to be independent, the laboratory 

chemists chosen to conduct the ILV would be expected to be unfamiliar with the 

method both in its development and subsequent use in analyzing samples 

collected from field studies. These individuals, however, should be trained and 

experienced pesticide residue chemists.

(iii) ILV trials are conducted under FIFRA GLP (40 CFR Part 160). Alternatively 

ILV trials conducted under OECD Principles of Good Laboratory Practice are 

also considered in compliance with FIFRA GLP.

(2) Test substance. An aliquot of the test substance used in the method development 

should be used to prepare spiking solutions for the ILV aliquot of that standard in order to 

prepare spiking solutions for the ILV.

(3) Test matrix. 

(i) To assess the ECM, the ILV test includes normal test conditions which include 

the presence of other compounds expected to be present. Matrix or matrices (e.g., 

soil, water, plant or animal tissue) to be sampled as part of the laboratory and/or 

field study to generate residue data should be identified and/or supplied to the 

independent lab by the registrant. Chemists at the independent lab should use 

these samples to prepare matrix spikes for the validation study.
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(ii) An ILV is conducted for each ECM for a given sample matrix for each parent 

compound, significant metabolites, and/or degradates. A method used for more 

than one study should meet the same performance standards as the original 

method. For a given sample matrix, the registrant should select the most difficult 

analytical sample condition from the study (e.g., high organic content versus low 

organic content in a soil matrix) to analyze from the study to demonstrate how 

well the method performs.

(iii) It is recommended that the registrant or their representative keep a reasonable 

amount of each solid matrix tested from a site for two years after registration, 

reregistration, or until the Agency has completed its evaluation of the studies.

(4) Environmental chemistry methods. 

(i) The ECMs given to the independent laboratory should be the same ones that 

were used or to be used by the registrant to generate laboratory and/or field study

residue data. For the ILV, the laboratory conducting the ILV uses the methods 

exactly as they are written by the registrant or its method development laboratory 

and should only contact the registrant or developer to clarify minor deficiencies in 

the methods. For example, if the characteristics of the clean up column were not 

adequately described in a method, the independent laboratory should contact the 

registrant or developer for clarification prior to running the first sample set of ILV 

samples (see paragraph (e)(5)(i)(A) of this guideline).

(ii) The laboratory conducting the ILV trial may contact the developers or 

previous users of the method prior to running the first set of samples, but all 

communications should be logged and reported to the Agency documenting that 

such communication did not compromise the independent evaluation. The ILV 

itself is likely to be invalid if anyone from the petitioner, developer, or any 

previous users are allowed to visit the laboratory during the ILV trial to observe, 

offer help, or assist the chemists or technicians.

(5) ILV. The independent laboratory establishes the relationship between the instrument 

responses and concentrations of analytes and verifies that matrix control samples are free 

of interferences at the appropriate retention time, wavelength or detector setting. All 

quality control conditions should be satisfied in order to demonstrate that the method is 

under control and before the independent laboratory analyzes any performance samples 

to be reported to the Agency. Any contact with the registrant or developers during the 

establishment of the method should be documented in writing in the final report 

submitted by the independent laboratory. This should be conducted following FIFRA 

GLP (40 CFR Part 160) or alternatively conducted following OECD Principles of GLP, 

which are considered in compliance with FIFRA GLP.

(i) Performance samples—

(A) Sample sets. A maximum of three sample sets are used by an 

independent laboratory to validate the method as written. A complete 
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sample set consists at a minimum of a reagent blank, two unspiked matrix 

control samples, five matrix control samples spiked at the LOQ and

another five matrix control samples spiked at ten times the LOQ 

(10xLOQ) for each distinct matrix. A complete set may include more than 

thirteen samples depending on the number of reagent, unspiked and spiked 

matrix control samples. It may be necessary, however, to divide a 

complete set into two subsets for efficient handling. Each subset should 

contain a reagent blank, two unspiked matrix control samples, and five 

matrix control samples spiked at the LOQ or 10xLOQ. The independent 

laboratory will use the predetermined values from the registrant for the 

LOQ and 10xLOQ to establish appropriate spiking levels.

(B) Validation. 

(1) A successful ILV includes performance data on at least one 

complete set of samples that meets those data quality objectives 

described in paragraph (d)(2) of this guideline. 

(a) Data from matrix control samples (blanks) are not used 

to correct values from spiked matrix controls for 

recoveries.

(b) Interferences with peak areas that are less than 50 

percent (%) at the MDL or LOD, are considered not 

significant.

(2) If the performance data on the first set of samples at any of the 

spiking levels are unsuccessful, the independent laboratory may

contact the registrant or developer to clarify the directions given in 

the method. Any contact with the registrant or developers during 

the method validation should be documented in writing in the final 

report submitted by the independent laboratory.

(3) If the independent laboratory cannot generate performance data 

that is similar to the registrant’s or developer’s after running the 

second sample set, the independent laboratory may contact the 

registrant, developer, or other users of the method to further clarify

the directions given in the method. All communications should be 

recorded.

(4) If the independent laboratory cannot generate performance data 

that is similar to the registrant’s or developer’s after the third set, 

the method should be failed and a report sent to the registrant 

explaining why the method failed. The registrant should then 

decide whether to repeat the independent laboratory validation at 

another laboratory, further develop the method, or withdraw it.
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(5) Any significant changes that are made to the method by the 

chemists at the independent lab should be incorporated into the 

original method and reported in writing immediately to the 

registrant. If those changes impact the performance of the original 

method, such as its precision, accuracy or limit of quantitation, the 

registrant should report those changes to the Agency. The revised 

or rewritten method, performance data, chromatograms and 

computations from all sets and/or subsets are sent to the registrant. 

The registrant, with the assistance of the IL, will decide how to 

present that information to the Agency.

(ii) ILV report to registrant. The independent laboratory should prepare a well 

documented laboratory report and send it to the registrant as the ILV of the ECM 

and that report with appropriate changes by the registrant is then submitted to the 

Agency with the laboratory or field study. The Agency highly recommends that 

the ILV report follow the format in paragraph (f) of this guideline.

(A) The independent laboratory should submit performance data to the 

registrant demonstrating that an adequate number of samples for each test 

level were extracted, cleaned up, and analyzed. Each set should have an 

appropriate number of spiked matrix control samples with method control 

and matrix control samples intermingled.

(B) Results reported should include:

(1) Results of each performance sample set — LOD or the MDL, 

the established LOQ, and accuracy and precision measured. 

(2) The mean and individual values for recoveries and standard 

deviations for the pesticide parent, toxicologically significant 

metabolites and/or degradates in fortified matrix control samples at 

each spiking level.

(3) The confidence intervals (95 or 99%) for the true average 

recoveries at each spiking level.

(4) Individual values for recoveries at each spiking level.

(C) Any matrix or solvent effects that result in signal enhancement, 

masking or suppression and the impact those effects have on the test 

results is described. 

(f) Reporting and reporting format. Information to be supplied in the report for each ECM for 

a given matrix includes the information items listed in paragraphs (f)(1) through (f)(10) of this 

guideline. Paragraphs (f)(1) through (f)(10) are also the recommended reporting format to aid 

the applicant/registrant in generating reports which are compatible with the Agency’s review 

process. Recommend for the ILV report that the IL also follow this reporting format, excluding 
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information in paragraph (f)(7)(ix) and with modifications applicable to the IL, for submittal to 

the registrant.

(1) Title/cover page.

(2) Certification. 

(i) For each ECM, the identification of those laboratories that developed the data 

submitted to the Agency. For each ECM ILV, the identification of the laboratory 

that conducted the ILV and a statement that the laboratory was requested to 

perform the ILV for the registrant.

(ii) Certification of authenticity by the Study Director and Lead Chemist for the 

laboratory that developed the method (including signature, typed name, title, 

affiliation, address, telephone number, and date).

(iii) Statement of adherence to the FIFRA GLP (40 CFR part 160) or OECD 

Principles of Good Laboratory Practice, as appropriate. 

(iv) Statement of claims for non-confidentiality and that the method contains no 

trade secrets or proprietary data. 

(3) Table of contents.

(4) Summary. Provide a brief description of paragraphs (f)(6)(ii) and (f)(6)(iii) of this 

guideline. 

(5) Materials—

(i) Equipment. A list and description of equipment used.

(ii) Reagents and standards. 

(A) List and describe the source, purity and stability of analytical grade 

standards. 

(B) Describe the source and preparation of reagents.

(iii) Safety and Health. 

(A) Provide material safety data sheets (MSDS).

(B) Describe any special precautions that need to be taken with standards, 

solvents or reagents and any procedural steps that require special 

precautions to avoid safety or health hazards.

(6) Methods.

(i) Principles of the analytical methods.
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(ii) Analytical procedures. Describe the analytical procedure(s) for an 

environmental matrix in a detailed step wise fashion:

(A) Sample matrix. Identify the source of the sample and characterize the 

sample matrix. For example, characterization of a soil sample might 

include a description of its textural class and percent organic matter, and 

characterization of a water sample might include pH, turbidity, specific 

conductance, etc.

(B) Preparation of samples. 

(C) Extraction. Demonstrate efficiency in specific soil samples from field 

sites.

(D) Fortifications. Describe fortifications used during method validation 

runs.

(E) Clean-up.

(F) Derivization (if any). 

(iii) Instrumentation. 

(A) Description of the instruction including make and model, type and 

specificity of detector(s), column(s) (packing materials, size), carrier 

gas(es), etc.

(B) Operating conditions of the instruments including flow rate(s), 

temperature(s), voltage, etc.

(C) Calibration procedures (frequency and stability). 

(iv) Potential interference(s). Describe the effects of the following on signal 

enhancement, masking or suppression of signal and their impact on the test 

results: 

(A) Sample matrices.

(B) The use of other pesticides on the test site.

(C) Solvents.

(D) Labware.

(v) Confirmatory techniques. Describe confirmatory techniques (i.e., GC/MS, 

LC/MS, second column, etc.)

(vi) Time required for analysis. Give the time required to take one sample set 

completely through the analytical procedure, including sample preparation, 
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extraction, cleanup, derivatization, and determination steps. Identify those steps 

that could significantly increase the time required to complete the method.

(vii) Modifications or potential problems. Specify any allowable variances and 

describe any unique steps where little variation is allowed in the method. 

Describe any potential problems and/or modifications that were made to the 

analytical procedures. 

(viii) Methods of calculation. Describe calculations in a step wise fashion for a 

specific individually spiked matrix control sample at the LOQ which would 

include the raw data of the analysis, calibration factors, calibration curves with the 

raw data of all of the standards used to generate the curve for the parent 

compound, metabolites, and degradates, etc. with the calculations used to generate 

the final concentration and recovery of the spiked matrix control sample.

(ix) Copies of chromatograms/spectra. Representative sample 

chromatograms/spectra should be submitted for each analyte measured in each 

matrix at all spiking levels, including method and matrix blanks. In addition, 

chromatograms/spectra with the highest levels of background and poorest 

resolution and copies of the chromatograms/spectra for the standards used to 

quantitate the analyte(s) in the representative matrix are submitted in the report to 

the Agency. Also recommend submitting a set of representative sequential 

chromatograms. Analyte peaks of interest should be correctly labeled to show 

appropriate retention times, peak areas, and heights on each chromatogram.

(x) Other. Describe any and all additional information the registrant considers 

appropriate and relevant to provide a complete and thorough description of the 

soil and water method. 

(7) Results/Discussion. 

(i) Method validation results (include tables of test levels and results of analysis). 

(ii) Accuracy (mean, range of recoveries, standard deviations and confidence 

limits for specific concentration levels, such as the LOQ or 10×LOQ). 

(iii)) Precision. Provide the RSD at each specific concentration level.

(iv) Limit of detection. Provide a clearly written explanation of how this value is 

calculated and cite the reference.

(v) Limit of quantitation. Provide a clearly written explanation of how this value 

is calculated and cite the reference.

(vi) Ruggedness testing (if performed). 

(vii) Discussion of selectivity and specificity of method. 
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(viii) Limitations. 

(ix) Interference/calibration. 

(x) Independent laboratory validation. 

(8) Conclusion. Discuss applicability of the ECM for measuring specific test 

compound(s) in the study matrix or various matrices, ranges, expected recoveries, 

interference(s), etc.

(9) Tables/Figures.

(10) References.

(g) References. The following reference should be consulted for additional background material 

on this test guideline.

(1) Organization for Economic Cooperation and Development, 1998. Series on 

Principles of Good Laboratory Practice and Compliance Monitoring No. 1 OECD 

Principles of Good Laboratory Practice, ENV/MC/CHEMC(98)17, Revised in 1997.