Pvrethrins (PC 069001) MRIDs 50475402/50475401 Analytical method for pyrethrins in water Reports: Document No. Guideline: Statements: Classification: PC Code: EFED Final Reviewers: CDM/CSS- Dynamac JV ECM: EPAMRID No. 50475402. DeVellis, S.R. 2017. Validation of an Environmental Chemistry Method for the Determination of Pyrethrins in Groundwater and Surface Water. Smithers Viscient Study No.: 14118.6100. Report prepared by Smithers Viscient, Wareham, Massachusetts; sponsored and submitted by Pyrethrin Steering Committee/Joint Venture (PJV) c/o Consumer Specialty Products Association, Inc., Washington, D.C.; 65 pages. Final report issued June 9, 2017. ILV: EPAMRID No. 50475401. Jutson, J.I. 2017. Independent Laboratory Validation of: Validation of an Environmental Chemistry Method for the Determination of Pyrethrins in Groundwater, Surface Water, Soil and Sediment. Concord Biosciences Study No.: 036079 and Document No.: 036079-1. Report prepared by Concord Biosciences, LLC, Concord, Ohio; sponsored and submitted by Pyrethrin Steering Committee/Joint Venture (PJV) c/o Consumer Specialty Products Association, Inc., Washington, D.C.; 154 pages. Final report issued December 15, 2017. MRIDs 50475402 & 50475401 850.6100 ECM: The study was conducted in accordance with the USEPA FIFRA (40 CFR Part 160) and OECD Good Laboratory Practices (GLP; p. 3 of MRID 50475402). Signed and dated No Data Confidentiality, GLP and Quality Assurance statements were provided (pp. 2-4). A certification of the authenticity of the report was included with the QA statement. ILV: The study was conducted in accordance with the USEPA FIFRA GLP (p. 3 of MRID 50475401). Signed and dated No Data Confidentiality, GLP and Quality Assurance statements were provided (pp. 2-4). A certification of the authenticity of the report was not included. This analytical method is classified as unacceptable. An updated ECM was not submitted incorporating the fact that the source of the acetonitrile was critical. ILV performance data at the LOQs was unacceptable in both water matrices. ILV linearity was not satisfactory for Pyrethrin I in ground water. ECM linearity was not satisfactory for all analyses. 069001 Zoe Ruge, Physical Scientist Mohammed Ruhman, Ph.D., Senior Scientist Lisa Muto, M.S., Environmental Scientist Signature: Date: 9/27/18 Signature: Date: 9/27/18 Signature: Date: 4/26/18 Page 1 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Reviewers: Kathleen Ferguson, Ph.D., Signature: Environmental Scientist Date: / --'7° / 4/26/18 This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JVpersonnel. The CDM/CSS-Dynamac Joint Venture role does not include establishing Agency policies. Executive Summary This analytical method, Smithers Viscient Study No. 14118.6100, is designed for the quantitative determination of Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) at the LOQ of 0.10 |ig/L and of Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II) at the LOQ of 0.0818 |ig/L in water using LC/MS/MS. Analytes were identified using one ion transition; a confirmation method is usually not required when LC/MS or GC/MS is used as the primary method for quantifying residues. The LOQs are less than the currently known lowest toxicological level of acute concern in water (0.76 |ig /L), however they are greater than the currently known chronic levels of concern (0.04 |ig /L). It is suggested that the registrant adjust the ECM so that the LOQ is lower than the currently known chronic value of 0.04 |ig/L in the new submitted study. The ECM and ILV used one characterized surface water and one characterized ground water matrices; matrices differed between the ECM and ILV. The ILV validated the ECM in the second trial for pyrethrins in water with insignificant modifications of the analytical instrumentation and parameters; however, the failure of the first ILV trial was due to Sigma-Aldrich acetonitrile, not Fisher acetonitrile, being used. The ECM did not contain a statement that the use of Fisher-brand acetonitrile was critical; an Updated ECM should be submitted specifying the source of the acetonitrile as Fisher as critical. All ECM data was satisfactory regarding accuracy and precision for all analytes, but ILV performance data at the LOQs was unacceptable in both water matrices. All ECM and ILV data was satisfactory regarding specificity for all analytes; however, linearity was not satisfactory for some of the ECM and ILV analyses. Page 2 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Table 1. Analytical Method Summary. Analyte(s) by Pesticide1 MRID Limit of Quantitation (LOQ) Environmental Chemistry Method Independent Laboratory Validation EPA Review Matrix Method Date Registrant Analysis Pyrethrin I 50475402 50475401 Unacceptable Water2-3 9/6/17 Pyrethrin Steering Committee/J oint Venture (PJV) c/o LC/MS/ 0.10 ng/L Consumer MS Pyrethrin II Specialty Products Association Inc. 0.0818 ng/L 1 Pyrethrins = Pyrethrin Concentrate (BAS 383 HB I), which consists of six different esters grouped as such: Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II). Pyrethrin Concentrate (% purities) = 29.46% Pyrethrin I, 24.02% Pyrethrin II, 53.48% Total Pyrethrins. 2 In the ECM, surface (river) water (pH 7.6, 85 mg equiv. CaCCh/L. 0.53 mmhos/cm conductivity) obtained from Taunton River, Taunton, Massachusetts, and ground (well) water (pH 6.24, 84 mg equiv. CaCCh/L. 20 mg/L alkalinity, 642 |iS/cm conductivity) obtained from Rock Creek, Ohio, were used. Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. 3 In the ILV, surface (river) water (EFS-629; pH 8.3, 104 mg equiv. CaCCh/L. 24 ppm total dissolved solids) obtained from Grand River, Madison, Ohio, and ground (well) water (EFS-625; pH 8.0, 242 mg equiv. CaCO.VL. 348 ppm total dissolved solids) obtained from Rock Creek, Ohio, were used. Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. I. Principle of the Method Samples (40.0 mL) were fortified as necessary with 0.04 mL or 0.40 mL of the fortification solution pyrethrins (0.100 mg/L:0.0818 mg/L of Pyrethrin LPyrethrin II) in acetonitrile (pp. 13-17 of MRID 50475402). The water samples were extracted twice with 5.00 mL of dichloromethane. The extraction procedure was not described. The dichloromethane layers were removed, placed in a 45- mL glass vials, and evaporated under a gentle stream of nitrogen set at 40°C to ca. 100 |iL. The residue was reconstituted with 7.50 mL of acetonitrile via vortex-mixing for 30 seconds and sonication for five minutes. An aliquot of purified water (7.50 mL) was added in the same manner, bringing the final composition of the samples to acetonitrile:water (50:50, v:v). LOQ samples were analyzed via LC/MS/MS. For lOxLOQ fortifications, the samples were further diluted 1:10 with acetonitrile:water (50:50, v:v) before analysis via LC/MS/MS. Samples were analyzed by an Agilent 1200 HPLC system coupled with an MDS Sciex 4000 QTRAP® MS (Version 1.6.2; Agilent Poroshell 120 EC-C8 column, 3.0 mm x 50 mm, 2.7 |am column; column temperature 25°C) using a mobile phase gradient of (A) 0.1% formic acid in water (B) 0.1% formic acid in acetonitrile [percent A:B at 0.01-0.50 min. 98.0:2.0, 2.00 min. 30.0:70.0, 5.00-6.00 min. 2.0:98.0, 6.10-7.00 min. 98.0:2.0] with MS/MS detection and Multiple Reaction Monitoring (MRM; TEM 550°C; pp. 11, 17-18 of MRID 50475402). Ion source was positive ESI for all analytes. One ion transition was monitored for each analyte: m/z 329.0—>-161.30 for pyrethrin I, m/z 331.40—>163.20 for jasmolin I, m/z 317.40—>149.30 for cinerin I, m/z 373.40—>-161.10 for pyrethrin II, m/z 375.30—>163.20 for jasmolin II, and m/z 361.30—>149.00 for cinerin II. Approximate retention times were reported as 4.2 minutes for pyrethrin I, 4.5 minutes for jasmolin Page 3 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 I, 4.2 minutes for cinerin I, 3.6 minutes for pyrethrin II, 3.8 minutes for jasmolin II, and 3.6 minutes for cinerin II for ground water and surface water. Injection volume was 100 |iL. In the ILV, the ECM was performed as written, except for insignificant modifications of the analytical instrumentation and parameters (pp. 15-16, 24; Table A-9, p. 36 of MRID 50475401). Details for the dichloromethane extraction procedure were provided. The ILV reported that Shimadzu Nexera UPLC system (System X) coupled with an AB Sciex 4000 MS was used (TEM 500°C). All monitored ion transitions were the same as those of the ECM. Retention times were not reported. A critical component of the method was elucidated by the ILV through the failure of the LOQ fortification in the first trial (p. 18; Appendix E, pp. 127-130). The Study Monitor informed the ILV that Fisher acetonitrile must be used for the sample processing since problems have occurred when other sources of acetonitrile have been used for pyrethrins. After switching from Sigma-Aldrich to Fisher brand acetonitrile, the ILV successfully validated the ECM. In the ECM and ILV, the method Limits of Quantification (LOQs) in water were 0.10 |ig/L for Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and 0.0818 |ig/L for Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II; pp. 19-22, 25-26 of MRID 50475402; pp. 6, 25 of MRID 50475401). In the ECM and ILV, the method Limits of Detection (LOD) were 0.0132 |ig/L in ground water and 0.0138 |ig/L in surface water for Pyrethrin I and 0.0181 |ig/L in ground water and 0.00986 |ig/L in surface water for Pyrethrin II. II. Recovery Findings ECM (MRID 50475402): Mean recoveries and relative standard deviations (RSD) were within guideline requirements (mean 70-120%; RSD <20%) for analysis of Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) at 0.10 |ig/L (LOQ) and 1.0 |ig/L (lOxLOQ) and for analysis of Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II) at 0.0818 |ig/L (LOQ) and 0.818 |ig/L (lOxLOQ) in water matrices (Tables 1-4, pp. 29-32). Analytes were identified using one ion transition; a confirmation method is usually not required when LC/MS or GC/MS is used as the primary method for quantifying residues. Surface (river) water (pH 7.6, 85 mg equiv. CaC03/L, 0.53 mmhos/cm conductivity) obtained from Taunton River, Taunton, Massachusetts, and ground (well) water (pH 6.24, 84 mg equiv. CaC03/L, 20 mg/L alkalinity, 642 |iS/cm conductivity) obtained from Rock Creek, Ohio, were used (p. 12). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. ILV (MRID 50475401): Mean recoveries and RSDs were within guideline requirements for analysis of Pyrethrin I at 1.0 |ig/L (lOxLOQ) and for analysis of Pyrethrin II at 0.818 |ig/L (lOxLOQ) in water matrices (Tables 1-2, pp. 20-21; Tables A-l to A-4, pp. 29-31; DER Attachment 2). Analysis of Pyrethrin I at 0.10 |ig/L (LOQ) and Pyrethrin II at 0.0818 |ig/L (LOQ) were unacceptable in both water matrices: Pyrethrin I (ground water, mean 67.8% and RSD 34.6%; surface water, RSD 44%) and Pyrethrin II (ground water, RSD 28.1%; surface water RSD 35%). LOQ recovery statistics were reviewer-calculated based on all reported recovery values (n = 6 or 7). The study author only used five values for statistics. Recovery values which were deemed outliers were omitted, and the samples were replaced with additional samples. LC/MS/MS Analytes were identified using one ion transition. Surface (river) water (EFS-629; pH 8.3, 104 mg equiv. CaC03/L, 24 ppm total dissolved solids) obtained from Grand River, Madison, Ohio, and ground (well) water (EFS-625; pH 8.0, 242 mg equiv. CaC03/L, 348 ppm total dissolved solids) obtained from Rock Page 4 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Creek, Ohio, were used (p. 14; Appendix A, pp. 91-92). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. The ILV validated the ECM in the second trial for pyrethrins in water with insignificant modifications of the analytical instrumentation and parameters; however, through the failure of the LOQ fortification in the first trial, the ILV learned that Fisher acetonitrile must be used for the sample processing since problems have occurred when other sources of acetonitrile have been used for pyrethrins (pp. 15-16, 18, 24-25; Appendix E, pp. 127-130). After switching from Sigma-Aldrich to Fisher brand acetonitrile, the ILV successfully validated the ECM. The ECM did not contain a statement that the use of Fisher-brand acetonitrile was critical; an Updated ECM should be submitted specifying the source of the acetonitrile as Fisher as critical. Table 2. Initial Validation Method Recoveries for Pyrethrins as Pyrethrin I and Pyrethrin II in Water.1'2'3 Analyte Fortification Number Recovery Mean Standard Relative Standard Level (jig/L) of Tests Range (%) Recovery (%) Deviation (%) Deviation (%) Ground (Well) Water Pyrethrin I 0.10 (LOQ) 7 86.4-98.6 91.8 4.21 4.59 1.0 5 92.3-94.1 93.2 0.763 0.819 Pyrethrin II 0.0818 (LOQ) 7 81.9-99.6 92.3 7.03 7.61 0.818 5 93.1-102 95.9 3.76 3.92 Surface (River) Water Pyrethrin I 0.10 (LOQ) 7 80.6-94.4 87.3 4.39 5.03 1.0 5 65.3-92.8 80.8 10.9 13.5 Pyrethrin II 0.0818 (LOQ) 7 88.7-99.5 92.8 3.84 4.13 0.818 5 78.7-94.9 86.2 7.46 8.66 Data (uncorrected recovery results, pp. 20-21) were obtained from Tables 1-4, pp. 29-32 of MRID 50475402. 1 Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II). 2 One ion transition was monitored for each analyte: m/z 329.0—>161.30 for pyrethrin I, m z 331.40—>163.20 for jasmolin I, m/z 317.40—>149.30 for cinerin I, m/z 373.40—>161.10 for pyrethrin II, m/z 375.30—>163.20 for jasmolin II, and m/z 361.30—>149.00 for cinerin II. 3 Surface (river) water (pH 7.6, 85 mg equiv. CaCCh/L. 0.53 mmhos/cm conductivity) obtained from Taunton River, Taunton, Massachusetts, and ground (well) water (pH 6.24, 84 mg equiv. CaCCh/L. 20 mg/L alkalinity, 642 |iS/cm conductivity) obtained from Rock Creek, Ohio, were used (p. 12). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. Page 5 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Table 3. Independent Validation Method Recoveries for Pyrethrins as Pyrethrin I and pyrethrin II in Water.1'2'3 Analyte Fortification Level (jig/L) Number of Tests Recovery Range (%) Mean Recovery (%) Standard Deviation (%) Relative Standard Deviation (%) Ground (Well) Water Pyrethrin I 0.10 (LOQ)4 6 20.2-81.9 67.8 23.5 34.6 1.0 5 94.6-100 97.7 2.4 2.5 Pyrethrin II 0.0818 (LOQ)4 6 30.6-80.3 70.7 19.9 28.1 0.818 5 87.9-107 95.3 8.1 8.5 Surface (River) Water Pyrethrin I 0.10 (LOQ)4 7 67.0-213 112 49 44 1.0 5 87.1-104 93.6 7.8 8.3 Pyrethrin II 0.0818 (LOQ)4 7 76.2-202 117 41 35 0.818 5 91.5-105 98.9 5.8 5.9 Data (uncorrected recovery results, Tables A-l to A-4, pp. 29-31) were obtained from Tables 1-2, pp. 20-21 and Tables A-l to A-4, pp. 29-31 of MRID 50475401 and DER Attachment 2. Red values indicate discrepancies with meeting guideline requirements. 1 Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II). 2 One ion transition was monitored for each analyte: m/z 329.0—>161.30 for pyrethrin I, m z 331.40—>163.20 for jasmolin I, m/z 317.40—>149.30 for cinerin I, m/z 373.40—>161.10 for pyrethrin II, m/z 375.30—>163.20 for jasmolin II, and m/z 361.30—>149.00 for cinerin II. 3 Surface (river) water (EFS-629; pH 8.3, 104 mg equiv. CaCCh/L. 24 ppm total dissolved solids) obtained from Grand River, Madison, Ohio, and ground (well) water (EFS-625; pH 8.0, 242 mg equiv. CaCO.VL. 348 ppm total dissolved solids) obtained from Rock Creek, Ohio, were used (p. 14; Appendix A, pp. 91-92). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. 4 Means, standard deviations and RSDs were reviewer-calculated based on all reported recovery values. Rules of significant figures were followed when reporting results. The study author only used five values for statistics. Recovery values which were deemed outliers were omitted, and the samples were replaced with additional samples. III. Method Characteristics In the ECM and ILV, the method LOQs in water were 0.10 |ig/L for Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and 0.0818 |ig/L for Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II; pp. 19-22, 25-26 of MRID 50475402; pp. 6, 25 of MRID 50475401). In the ECM and ILV, the LOQ was defined as the lowest fortification level successfully tested. In the ECM, it was noted that background noise should not exceed 20% of the LOQ. In the ECM and ILV, the method LODs were 0.0132 |ig/L in ground water and 0.0138 |ig/L in surface water for Pyrethrin I and 0.0181 |ig/L in ground water and 0.00986 |ig/L in surface water for Pyrethrin II. In the ECM, the LOD was calculated as the standard deviation at the LOQ multiplied by to.99, where to.99 equalled 3.143 for n-1 degrees of freedom where n = 7. The LOD was not justified in the ILV. Page 6 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Table 4. Method Characteristics for Pyrethrins in Water. Analyte1 Pyrethrin I Pyrethrin II Limit of Quantitation (LOQ) ECM 0.10 ng/L 0.0818 ng/L ILV Limit of Detection (LOD) ECM Nominal Not reported Calculated 0.0132 |ig/L (ground) 0.0138 |ig/L (surface) 0.0181 |ig/L (ground) 0.00986 ng/L (surface) ILV2 0.0132 |ig/L (ground) 0.0138 |ig/L (surface) 0.0181 |ig/L (ground) 0.00986 ng/L (surface) Linearity (calibration curve r2 and concentration range) ECM Ground r2 = 0.99625 r2 = 0.99435 Surface r2 = 0.99290 r2 = 0.99768 ILV Ground r2 = 0.99393 r2 = 0.9983 Surface r2 = 0.99643 r2 = 0.99853 Range 0.05-0.50 ng/L 0.0409-0.409 (ig/L 0.0409-0.327 jig/L (ILV/surface) Repeatable ECM4 Yes at LOQ and lOxLOQ in one surface water and one ground water matrices (characterized). ILV5-6 Yes at lOxLOQ in one surface water and one ground water matrices (characterized). No at LOQ [mean 67.8% and RSD 34.6% (ground); surface water, RSD 44% (surface)]. No at LOQ [RSD 28.1% (ground); RSD 35% (surface)]. Reproducible No at LOQ Yes at lOxLOQ Specific ECM Yes, matrix interferences were < 7% of the LOQ (based on peak area) for each of the three components. Baseline noise interference was more significant in chromatograms of jasmolin I/II. ILV Yes, matrix interferences were < 6% of the LOQ (based on peak area) for each of the three components. Baseline noise interference was more significant in chromatograms of jasmolin I/II and cinerin I/II. Data were obtained from pp. 19-22, 25-26 (LOQ/LOD); Tables 1-4, pp. 29-32 (recovery results); Figures 1-20, pp. 33- 52 (chromatograms); Figures 21-24, pp. 53-56 (calibration curves) of MRID 50475402; pp. 6, 25 (LOQ/LOD); Tables 1-2, pp. 20-21 and Tables A-l to A-4, pp. 29-31 (recovery results); Figures 1-4, pp. 38-41 (calibration curves); Figures 9-28, pp. 46-65 (chromatograms) of MRID 50475401; DER Attachment 2. Analytes were identified using one ion transition; a confirmation method is usually not required when LC/MS or GC/MS is used as the primary method for quantifying residues. Red values indicate discrepancies with meeting guideline requirements. 1 Pyrethrins = Pyrethrin Concentrate (BAS 383 HB I), which consists of six different esters grouped as such: Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II). Pyrethrin Concentrate (% purities) = 29.46% Pyrethrin I, 24.02% Pyrethrin II, 53.48% Total Pyrethrins. 2 ILV LODs were reported from the ECM. 3 ILV coefficient of determination (r2) values were reviewer-generated from reported regression data from Figures 1-4, pp. 38-41 of MRID 50475402 since the study author did not report these values (DER Attachment 2). Linear regression equations were used. 4 In the ECM, surface (river) water (pH 7.6, 85 mg equiv. CaCO.VL. 0.53 mmhos/cm conductivity) obtained from Taunton River, Taunton, Massachusetts, and ground (well) water (pH 6.24, 84 mg equiv. CaCO.VL. 20 mg/L alkalinity, 642 |iS/cm conductivity) obtained from Rock Creek, Olio, were used (p. 12 of MRID 50475402). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. 5 In the ILV, surface (river) water (EFS-629; pH 8.3, 104 mg equiv. CaCO.VL. 24 ppm total dissolved solids) obtained from Grand River, Madison, Ohio, and ground (well) water (EFS-625; pH 8.0, 242 mg equiv. CaCOs/L, 348 ppm total dissolved solids) obtained from Rock Creek, Ohio, were used (p. 14; Appendix A, pp. 91-92 of MRID 50475401). Water characterization was performed by Agvise Laboratories, Northwood, North Dakota. Page 7 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 6 The ILV validated the ECM in the second trial for pyrethrins in water with insignificant modifications of the analytical instrumentation and parameters; however, through the failure of the LOQ fortification in the first trial, the ILV learned that Fisher acetonitrile must be used for the sample processing since problems have occurred when other sources of acetonitrile have been used for pyrethrins (pp. 15-16, 18, 24-25; Appendix E, pp. 127-130 of MRID 50475401). After switching from Sigma-Aldrich to Fisher brand acetonitrile, the ILV successfully validated the ECM. The ECM did not contain a statement that the use of Fisher-brand acetonitrile was critical; an Updated ECM should be submitted specifying the source of the acetonitrile as Fisher as critical. Linearity is satisfactory when r2 > 0.995. IV. Method Deficiencies and Reviewer's Comments 1. An updated ECM was not submitted incorporating the fact that the source of the acetonitrile was critical. After the failure of the LOQ fortification in the first trial, the ILV presumed that matrix effects caused suppression of the analyte signal. After some communication with the Study Monitor and testing, the ILV determined that matrix effects were not the cause. The Study Monitor informed the ILV that Fisher acetonitrile must be used for the sample processing since problems have occurred when other sources of acetonitrile have been used for pyrethrins. The ILV communicated that Sigma-Aldrich acetonitrile had been used in previous trials. After switching from Sigma-Aldrich to Fisher brand acetonitrile, the ILV successfully validated the ECM. The ECM did not contain a statement that the use of Fisher- brand acetonitrile was critical; an Updated ECM should be submitted specifying the source of the acetonitrile as Fisher as critical. The use of the Fisher brand acetonitrile by the ILV was necessary for the successful validation of the ECM method. 2. ILV performance data at the LOQs was unacceptable in both water matrices: Pyrethrin I (ground water, mean 67.8% and RSD 34.6%; surface water, RSD 44%) and Pyrethrin II (ground water, RSD 28.1%; surface water RSD 35%; Tables 1-2, pp. 20-21; Tables A-l to A-4, pp. 29-31; DER Attachment 2). LOQ recovery statistics were reviewer-calculated based on all reported recovery values (n = 6 or 7). The study author only used five values for statistics. Recovery values which were deemed outliers were omitted, and the samples were replaced with additional samples; subsequent statistics calculated by the study author were acceptable. OCSPP guidelines state that acceptable mean recoveries and relative standard deviations (RSD) are 70-120%) and <20%, respectively, at the LOQ and higher fortifications. Since the ILV did not provide acceptable data at the LOQ for the analytes in either water matrix, the reproducibility of the method was not demonstrated in the submitted method validation set. 3. ILV linearity was not satisfactory for Pyrethrin I in ground water, r2 = 0.9939 (Figures 1-4, pp. 38-41 of MRID 50475401; DER Attachment 2). Coefficient of determination (r2) values were reviewer-generated for three of the four calibration curves since the study author did not report these values. Linear regression equations from reported regression data were used. ECM linearity was not satisfactory for Pyrethrin I in surface water, r2 = 0.9929, and was not satisfactory for Pyrethrin II in ground water, r2 = 0.99435 (Figures 21-24, pp. 53-56 of MRID 50475402). Linearity is satisfactory when r2 > 0.995. Page 8 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 4. The communications between the ILV Study Director and the CSPA Study Monitor were documented (p. 26; Appendix E, pp. 127-130 of MRID 50475401). Communications involved trial successes and failures, as well as procedure clarification (specifically the dichloromethane extraction procedure and not allowing the extract to be reduced to dryness). The reviewer noted that the CSPA Study Monitor involved the Smithers Study Director after the ILV Study Director asked him to check with the method developer, and this communication was forwarded to the ILV Study Director. However, the reviewer did not deem this interaction to be collusion since the Smithers Study Director only clarified and re- emphasized points of the method. The Smithers Study Director did not direct supplementary work or modifications to the ILV. The reviewer noted that the solution to the ILV LOQ performance data problem came from the Study Monitor talking to another chemist familiar with analyzing pyrethrins (Appendix E, p. 129 of MRID 50475401). The reviewer would have liked this chemist to have been identified. 5. Method LODs were not reported by the ECM or ILV; calculated LODs were provided. The estimations of the LOQ and LOD in ECM and ILV were not based on scientifically acceptable procedures as defined in 40 CFR Part 136 (pp. 19-22, 25-26 of MRID 50475402; pp. 6, 25 of MRID 50475401). In the ECM and ILV, the LOQ was defined as the lowest fortification level successfully tested. In the ECM, it was noted that background noise should not exceed 20% of the LOQ. In the ECM, the LOD was calculated as the standard deviation at the LOQ multiplied by to.99, where to.99 equalled 3.143 for n-1 degrees of freedom where n = 7. The LOD was not justified in the ILV. Detection limits should not be based on arbitrary values. 6. The reviewer noted the following typographical error: PYRI in the Figure Title should have been PYR II (Figure 20, p. 52 of MRID 50475402). 7. It was reported for the ILV that one sample set of 13 samples required ca. 8 hours including calculation of results (p. 25 of MRID 50475401). V. References U.S. Environmental Protection Agency. 2012. Ecological Effects Test Guidelines, OCSPP 850.6100, Environmental Chemistry Methods and Associated Independent Laboratory Validation. Office of Chemical Safety and Pollution Prevention, Washington, DC. EPA 712- C-001. 40 CFR Part 136. Appendix B. Definition and Procedure for the Determination of the Method Detection Limit-Revision 1.11, pp. 317-319. Page 9 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 DER Attachment 1: Chemical Names and Structures. Pyrethrin I IUPAC Name: CAS Name: CAS Number: SMILES String: [(lS)-2-methyl-4-oxo-3-[(2Z)-penta-2,4-dienyl]cyclopent-2-en-l-yl] (lR,3R)-2,2-dimethyl-3 -(2-methylprop-1 -enyl )cyclopropane-1 -carboxylate Not reported 121-21-1 CC1=C(C(=0)C[C@@H]10C(=0)[C@@H]2[C@H](C2(C)C)C=C(C)C) C/C=C\C=C Cinerin I IUPAC Name: CAS Name: CAS Number: SMILES String: [(lR)-3-[(Z)-but-2-enyl]-2-methyl-4-oxocyclopent-2-en-l-yl] (lR,3R)-2,2- dimethyl-3-(2-methylprop-1 -enyl )cyclopropane-1 -carboxylate Not reported 25402-06-6 C/C=C\CC1=C([C@@H](CC1=0)0C(=0)[C@@H]2[C@H](C2(C)C)C=C( C)C)C Page 10 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Jasmolin I IUPAC Name: CAS Name: CAS Number: SMILES String: [(lS)-2-methyl-4-oxo-3-[(Z)-pent-2-enyl]cyclopent-2-en-l-yl] (lR,3R)-2,2- dimethyl-3-(2-methylprop-1 -enyl )cyclopropane-1 -carboxylate Not reported 4466-14-2 CC/C=C\CC1=C([C@H](CC1=0)0C(=0)[C@@H]2[C@H](C2(C)C)C=C( C)C)C / Pyrethin II IUPAC Name: CAS Name: CAS Number: SMILES String: [(1 S)-2-methyl-4-o\o-3-[(2E)-penta-2,4-dienyl]cyclopent-2-en-1 -yl] (lR,3R)-3-[(E)-3-methoxy-2-methyl-3-oxoprop-l-enyl]-2,2- di m ethyl cy cl opropane-1 -carboxylate Not reported Not reported CC1=C(C(=0)C[C@@H]10C(=0)[C@@H]2[C@H](C2(C)C)/C=C(\C)/C (=0)OC)C/C=C/C=C Page 11 of 12 ------- Pvrethrins (PC 069001) MRIDs 50475402/50475401 Cinerin II IUPAC Name: CAS Name: CAS Number: SMILES String: [(1 S)-3-[(Z)-but-2-enyl]-2-methyl-4-o\ocyclopent-2-en-1 -yl] (1 R,3R)-3- [(E)-3 -methoxy-2-methyl-3 -oxoprop-1 -enyl]-2,2-dimethyl cyclopropane-1 - carboxylate Not reported 121-20-0 C/C=C\CC1=C([C@H](CC1=0)0C(=0)[C@@H]2[C@H](C2(C)C)/C=C(\ C)/C(=0)OC)C Jasmolin II IUPAC Name: CAS Name: CAS Number: SMILES String: [(lS)-2-methyl-4-oxo-3-[(Z)-pent-2-enyl]cyclopent-2-en-l-yl] (lR,3R)-3- [(E)-3-metho\y-2-methyl-3 -oxoprop-1 -enyl]-2,2-dimethyl cyclopropane-1 - carboxylate Not reported 1172-63-0 CC/C=C\CC1=C([C@H](CC1=0)0C(=0)[C@@H]2[C@H](C2(C)C)/C=C (\C)/C(=0)OC)C Page 12 of 12 ------- |