United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7101) EPA 712

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- 001 January 2012 

# Ecological Effects **Test Guidelines** OCSPP 850.6100 : Environmental Chemistry Methods and Associated Independent Laboratory Validationi **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, e t 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, Pestici des 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 A pril 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 guideline s 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

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- 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

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- OPPT

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- 0150 through EPA

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- 0159, and EPA

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**OCSPP 850.**  **61 0**  **0 :**  **Environmental chemistry methods and associated independent**  **laboratory**

**v alidation** 

**.** (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, Fungic ide, 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 m ethods (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 multianaly

te methods for identification and quantification of the parent compound, toxicologically

s ignificant metabolites and degradates in each environmental matrix sampled in the specific guideline study

. (c) **General considerations.** (1) Registrant(s) provide to the Agenc y a report of a finalized ECM with appropriate revisions or changes recommended by an independent laboratory b ased 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 m ethods used to generate laborato ry and/or field residue data. I f that laboratory

i s located in the registrant’s organization, or is in anyw ay 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 sy nthesize or purchase another lot of authentic analy tical 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 ., conf

irmatory 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 primaryPage **2** of **10**

method(s) to generate study data. (4) Regulatory

chemists should be able to validate practical and rapid analy tical 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 Agenc y 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 f or 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 l aboratory

chemists to review the ECM and its ass ociated ILV results. All environmental chemistry methods should be stamped non

- confidential. (ii) Analy tical environmental chemistry m ethods used should be practical, and be able to quantitate analy

tes in the study

matrix (or matrices) at the data quality objective(s) set for the laboratory o r field residue study

. E quipment used to perform the method(s) should be commercially available in the United States. (iii) The analy

tical 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 b e requested to supply to the Agency an active ingredient analytical grade standard and/or toxicologically

s ignificant 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 toPage **3** of **10** 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 suitabi lity 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)**  **I ndependent laboratory.**  (i) Validation of the ECM is conducted by a l aboratory

operating independently from that of the originator of the method. The independent laboratory c onducting the validation may

b e privately

o r publicly

o wned and under certain conditions may b elong to the same organization as the originating laboratory or in the registrant’s own organization. (ii) The originating and validation laboratories may b elong to the same organization, but in order for the validation to be independent the analysts, equipment, instruments, and supplies ( e.g., gl assware, 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 f ield studies to support pesticide registration or reregistration actions. F or the validation to be independent, the laboratory chemists chos en to conduct the ILV would be expected to be unfamiliar with the method both in its development and subsequent use in analyz ing 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 conditi ons 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 suppli ed to the independent lab by

the registrant. Chemists at the independent lab should use these samples to prepare matrix spikes for the validation study

.Page **4** of **10** (ii) An ILV is conducted for each ECM for a given sample matrix for each parent compound, significant metabolites, and/or degradates. Amethod used for more than one study

should meet the same performance standards as the original method. F or 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 analy ze from the study

to demonstrate how well the method performs. (iii) Itis 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. F or the ILV, the laboratory c onducting 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. F or example, if the characteristics of the clean up column were not adequately described in a method, the independent laboratory should contact the registrant or devel oper for clarification prior to running the first sample set of ILV samples (see paragraph (e)(5)(i)(A) of this guideline). (ii) The laboratory

c onducting the ILV tri al may c ontact the developers or previous users of the method prior to running the first s et of samples, but all communications should be logged and reported to the Agenc y 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 e stablishes the relationship between the instrument responses and concentrations of analy tes and verifies that matrix control samples are free of interferences at the appropriate retention time, wavelength or detector setting. All quality c ontrol conditions should be satisfied in order to demonstrate that the method is under control and be fore the independent laboratory analyz es any performance samples to be reported to the Agency

. Any c ontact 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. Ac ompletePage **5** of **10** 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 b e necessary, however, to divide a complete set into two subsets for efficient handling. E ach subset should contain a reagent blank, two unspiked matrix control samples, and five matrix control samples spiked at the LOQ or 10 xLOQ. The independent laboratory will use the predetermined values from the registrant for the LOQ and 10 xLOQ 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 ob jectives described in paragraph (d)(2) of this guideline. (a ) D ata 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 o f the spiking levels are unsuccessful, the independent laboratory m ay contact the registrant or developer to clarify the directions given in the method. Any

c ontact 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 c ontact 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.Page **6** of **10** (5 ) Any significant changes that are made to the method by the chemists at the independent lab should be incorp orated into the original method and reported in writing immediately to the registrant. I f those changes impact the performance of the original method, such as its precision, accuracy o r 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 Agenc y. (ii) **IL V**  **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 analyz ed. E ach 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 s ignificant metabolites and/or degradates in fortified matrix control samples at each spiking level. (3 ) The confidence intervals (95 or 99%) f or 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** r**eporting 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, excludingPage **7** of **10**

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 i dentification 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 b y the Study

D irector and Lead Chemist for the laboratory

that developed the method (including signature, ty ped 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 o f analy tical grade standards. (B) Describe the source and preparation of reagents. (iii) **Safety and Healt h.**  (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.Page **8** of **10** (ii) Analytical procedures. D escribe the analy tical 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. F or 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. D emonstrate efficiency in specific soil samples from field sites. (D) Fortifications. D escribe fortifications used during method validation runs. (E) Clean

-up. (F) Derivization (if any). (iii) Instrumentation. (A) Description of the instruction including make and model, ty pe 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). D escribe 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. D escribe 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 analy

tical procedure, including sample preparation,Page **9** of **10** 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. S pecify any allowable variances and describe any unique steps where little variation is allowed in the method. Describe any potential problems and/or modifi cations that were made to the analytical procedures. (viii) Methods of calculation. D escribe 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 analys is, 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 s et 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. D escribe 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.Page **10** of **10** (viii) Limitations. (ix) Interference/calibration. (x) Independent laboratory validation. (8) **Conclusion**
**.** Discuss applicability o f 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 b e consulted for additional background material on this test guideline. (1) Organization for Economic Cooperation and Development, 1998. S eries 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.