&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-001
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.6100:
Environmental
Chemistry Methods
and Associated
Independent
Laboratory Validation
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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.qov/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,
lexicologically 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 lexicologically 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
reregi strati on 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,
reregi strati on, 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
(lOxLOQ) 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 lOxLOQ. The independent
laboratory will use the predetermined values from the registrant for the
LOQ and lOxLOQ 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, lexicologically 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)(l) through (f)(10) of this
guideline. Paragraphs (f)(l) 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) Lab ware.
(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 IQxLOQ).
(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.
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