Propargite (PC 097601) MRIDs 50356002/ 50356003 Analytical method for propargite in water Reports: Document No. Guideline: Statements: Classification: PC Code: EFED Final Reviewer: CDM/CSS- Dynamac JV Reviewers: ECM: EPAMRID No.: 50356002. Keenan, D. 2016. Development and Validation of a Method for the Determination of Propargite in Surface and Drinking Water. Report prepared by PTRL West (now doing business as EAG Laboratories), Hercules, California, sponsored and submitted by Arysta LifeScience, Waterbury, Connecticut; 80 pages. PTRL Study No.: 2823W. Arysta Project No.: 16176-X5-03. Final report issued August 19, 2016. ILV: EPAMRID No.: 50356003. MacGregor, J.A., and E.S. Bodle. 2017. INDEPENDENT LABORATORY VALIDATION OF METHODS FOR THE DETERMINATION OF PROPARGITE IN SURFACE AND DRINKING WATER. Report prepared by Wildlife International (now doing business as EAG Laboratories), Easton, Maryland, sponsored and submitted by Arysta LifeScience, Waterbury, Connecticut; 140 pages. Wildlife International Project No: 443C-128. Arysta Study No.: 2016-006. Final report issued May 1, 2017. MRIDs 50356002 & 50356003 850.6100 ECM: The study was conducted in compliance with USEPA FIFRA Good Laboratory Practice (GLP) standards (40 CFR 160; p. 3 of MRID 50356002). Signed and dated Data Confidentiality, GLP and Quality Assurance statements were provided (pp. 2-4). The statement of authenticity was included with the Quality Assurance statement. ILV: The study was conducted in compliance with USEPA FIFRA GLP standards, with the exception that periodic analyses of the test water for potential contaminants was not performed according to GLP standards (p. 3 of MRID 50356003). Signed and dated Data Confidentiality, GLP and Quality Assurance statements were provided (pp. 2-4). The statement of authenticity was not included. This analytical method is classified as Acceptable. However, an LOD was not reported in the ILV report. 097601 Lewis R. Brown, Env. Bio Lisa Muto, Environmental Scientist Kathleen Ferguson, Ph.D., Environmental Scientist Signature: Date: Signature: Date: Signature: Date: Lewis Ross Brown, 05/17/18 Env. Biologist 11/13/17 11/13/17 This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel. Page 1 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 Executive Summary The analytical method, PTRL Study No. 2823W (Arysta Project No. 16176-X5-03), is designed for the quantitative determination of propargite in water at the stated LOQ of 0.01 |ig/L. The LOQ is less than the lowest toxicological level of concern in water (7 |ig/L, the lowest aquatic life benchmark; DP 4164791). Characterized surface and drinking water matrices were used for the ECM and ILV validations. Propargite was identified using two ion transitions in the ECM and ILV. The ECM method for propargite in water was validated by the ILV with insignificant modifications to the analytical equipment and parameters. The number of trials was not specified, but the reviewer assumed that the method was validated in the first trial based on the insignificant modifications to the method. Communication between the ILV and ECM was not reported or discussed. All ILV and ECM data regarding repeatability, accuracy, precision, linearity and specificity were satisfactory for propargite. The LOD was not reported in the ILV. Table 1. Analytical Method Summary Analyte(s) by Pesticide mrii) EPA Review Matrix Method Date (dd/mm/yyyy) Registrant Analysis Limit of Quantitation (LOQ) Environmental Chemistry Method Independent Laboratory Validation Propargite 503560021 503560032 Water 19/08/2016 Arysta LifeScience LC/MS/MS 0.01 ng/L 1 In the ECM, the drinking water (2706W-055; pH 7.6, hardness 29 mg/L as CaCCb, 58 ppm total dissolved solids) and surface water (2706W-049; pH 8.5, hardness 262 mg/L as CaCCb, 204 ppm total dissolved solids) were characterized by Agvise Laboratories, Northwood, North Dakota (p. 13; Appendix C, pp. 65-66 ofMRID 50356002). Water matrix sources were not reported. 2 In the ILV, the drinking (well) water (pH 8.1, hardness 142 mg/L as CaCCb, 336 |iS/cm specific conductance), obtained from a well at Easton Laboratories-Easton testing facility in Easton, Maryland, and surface (lake) water (pH 7.0, hardness 64.0 mg/L as CaCCb, 188 |iS/cm specific conductance), obtained from Tuckahoe Lake located in Tuckahoe State Park in Ridgely, Maryland, were characterized internally (pp. 11-12; Appendices III-IV, pp. 131-132 ofMRID 50356003). 1 https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/aauatic-life-benchmarks-and-ecological-risk Page 2 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 I. Principle of the Method Water (100 mL) was fortified then extracted three times with 25 mL of hexane (pp. 14- 15; Figure 1, p. 31; Appendix D, pp. 72-73 of MRID 50356002). 10 g of sodium chloride was added to induce the separation of the aqueous and organic layers. After vigorously shaking by hand for 1 minute, the layers were allowed to separate for 10 minutes. The organic layer was transferred to a 125-mL concentration flask via a filter funnel containing glass wool and 5 g of sodium sulfate. The combined organic extracts were reduced to near dryness under increased pressure (230 mbar) in a water bath set at 40°C. The residue was reduced to dryness under a stream of nitrogen. The residue was reconstituted to 10 mL with acetonitrile via sonication. Approximately 500 |iL of the final sample was transferred to a 0.45 |im microfilterfuge tube and centrifuged. The supernatant was transferred to an autosampler vial for analysis by LC/MS/MS. Samples were analyzed for propargite using an Agilent 1260 Series HPLC coupled with an Applied Biosystems MDS/Sciex API 4000 Tandem Mass Spectrometer using a Turbo-Ion Spray source operated in the positive ion, multiple reaction monitoring (MRM) mode (300°C; pp. 12, 15; Appendix D, pp. 73-74 of MRID 50356002). The following LC conditions were used: Phenomenex Gemini 3 ju. C18 110 column (2.0 mm x 50 mm, 3 |i; column temperature 55°C), mobile phase of (A) 10 mM ammonium acetate in HPLC grade water and (B) 10 mM ammonium acetate in HPLC grade methanol [mobile gradient phase of percent A:B (v:v) at 0.00 min. 70:30, 1.0-5.0 min. 5:95, 5.1-8.0 min. 70:30] and injection volume of 12 |iL. Two ion pair transitions were monitored for propargite: m/z 368.1—>231.6 (quantitation) and m/z 368.1—>81.4 (confirmation). Reported retention times were ca. 5.0 minutes for propargite. The ILV performed the ECM methods for each analyte as written, except insignificant modifications to the analytical equipment and parameters (pp. 13-14; Table 1, p. 20 of MRID 50356003). The LC/MS/MS instrument and parameters were similar to those of the ECM. Samples were analyzed for propargite using an Agilent Technologies 1200 Infinity Series HPLC coupled with an Applied Biosystems API 5000 Tandem Mass Spectrometer using Turbo-V Ion Spray Source, electrospray ionization (ESI) operated in the positive ion, and MRM. The other LC conditions were the same as those reported in the ECM. Two ion pair transitions were monitored for propargite: m/z 368.1—>231.1 (quantitation) and m/z 368.1—>81.1 (confirmation). Reported retention times were ca. 5.0 minutes for propargite. Other than the insignificant modifications to the analytical equipment, no other method modifications were reported. In the ECM and ILV, the Limit of Quantification (LOQ) was 0.01 |ig/L for propargite in water (equivalent to 0.4 ng/mL using the current methodology; p. 11; Appendix D, pp. 68, 74 of MRID 50356002; p. 10 of MRID 50356003). In the ECM, the Limit of Detection (LOD) for propargite was determined to be 0.002 |ig/L for drinking water and 0.003 |ig/L for surface water. In the ILV, the LOD was not reported. Page 3 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 II. Recovery Findings ECM (MRID 50356002): Mean recoveries and relative standard deviations (RSDs) were within guidelines (mean 70-120%; RSD <20%) for analysis of propargite at fortification levels of 0.01 |ig/L (LOQ) and 0.1 |ig/L (lOxLOQ) in two water matrices (p. 11; Tables III-IV, pp. 24-25). Two ion pair transitions were monitored for propargite using LC/MS/MS in positive ESI mode; the quantification and confirmation ion data was comparable. Drinking water (2706W-055; pH 7.6, hardness 29 mg/L as CaCCb, 58 ppm total dissolved solids) and surface water (2706W-049; pH 8.5, hardness 262 mg/L as CaCCb, 204 ppm total dissolved solids) were characterized by Agvise Laboratories, Northwood, North Dakota (p. 13; Appendix C, pp. 65-66). Water matrix sources were not reported. ILV (MRID 50356003): Mean recoveries and RSDs were within guidelines for analysis of propargite at fortification levels of 0.01 |ig/L (LOQ) and 0.1 |ig/L (lOxLOQ) in two water matrices (Tables 2-5, pp. 21-24). Two ion pair transitions were monitored for propargite using LC/MS/MS in positive ESI mode; the quantification and confirmation ion data was comparable, except for the LOQ analysis in drinking water. For the LOQ quantitation and confirmation ion analyses in drinking water, the original result for one sample was deviant; therefore, the mean of duplicate re- analyses were reported for this sample. For the confirmation ion analysis, measured concentrations were corrected for extrapolated mean matrix blank contributions of 0.0024033 |ig/L (ca. 24% of the LOQ) in surface water and 0.0026306 |ig/L (ca. 26% of the LOQ) in drinking water. Drinking (well) water (pH 8.1, hardness 142 mg/L as CaC03, 336 |iS/cm specific conductance), obtained from a well at Easton Laboratories-Easton testing facility in Easton, Maryland, and surface (lake) water (pH 7.0, hardness 64.0 mg/L as CaC03, 188 |iS/cm specific conductance), obtained from Tuckahoe Lake located in Tuckahoe State Park in Ridgely, Maryland, were characterized internally (pp. 11-12; Appendices III-IV, pp. 131-132). The method for propargite was validated with insignificant modifications to the analytical equipment and parameters; the number of trials was not specified, but the reviewer assumed that the method was validated in the first trial (pp. 10, 13-14). Page 4 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 Table 2. Initial Validation Method Recoveries for Propargite in Water Analyte Fortification Number Recovery Mean Standard Relative Standard Deviation (%) Level (jig/L) of Tests Range (%) Recovery (%) Deviation (%) Drinking Water1 Quantitation ion2 Propargite 0.01 (LOQ) 5 98-109 104 4.3 4.2 0.1 5 96-118 102 9.5 9.4 Confirmation ion2 Propargite 0.01 (LOQ) 5 101-110 106 3.4 3.2 0.1 5 92-115 100 9.3 9.3 Surface Water1 Quantitation ion2 Propargite 0.01 (LOQ) 5 84-105 99 8.5 8.6 0.1 5 82-102 93 9.6 10.4 Confirmation ion2 Propargite 0.01 (LOQ) 5 91-110 97 7.5 7.7 0.1 5 80-101 92 9.5 10.4 Data (uncorrected recovery results; Tables III-IV, pp. 24-25) were obtained from p. 11; Tables III-IV, pp. 24-25 of MRID 50356002. 1 Drinking water (2706W-055; pH 7.6, hardness 29 mg/L as CaCCb, 58 ppm total dissolved solids) and surface water (2706W-049; pH 8.5, hardness 262 mg/L as CaCCb, 204 ppm total dissolved solids) were characterized by Agvise Laboratories, Northwood, North Dakota (p. 13; Appendix C, pp. 65-66). Water matrix sources were not reported. 2 Two ion pair transitions were monitored for propargite: m/z 368.1—>231.6 (quantitation) and m/z 368.1—>81.4 (confirmation). Page 5 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 Table 3. Independent Validation Method Recoveries for Propargite in Water Analyte Fortification Number Recovery Mean Standard Relative Standard Deviation (%) Level (jig/L) of Tests Range (%) Recovery (%) Deviation (%) Drinking Water1 Quantitation ion2 Propargite 0.01 (LOQ) 53 110-114 112 1.48 1.32 0.1 5 104-108 106 1.52 1.43 Confirmation ion2,4 Propargite 0.01 (LOQ) 53 86.5-90.9 88.4 1.76 1.99 0.1 5 101-103 102 1.00 0.980 Surface Water1 Quantitation ion2 Propargite 0.01 (LOQ) 5 96.2-117 105 7.88 7.50 0.1 5 97.7-101 98.9 1.34 1.35 Confirmation ion2,4 Propargite 0.01 (LOQ) 5 100-109 104 3.91 3.75 0.1 5 98.1-101 99.5 1.06 1.07 Data (uncorrected recovery results, except where noted; Tables 2-5, pp. 21-24) were obtained from Tables 2-5, pp. 21- 24 of MRID 50356003. 1 Drinking (well) water (pH 8.1, hardness 142 mg/L as CaCCb, 336 |iS/cm specific conductance), obtained from a well at Easton Laboratories-Easton testing facility in Easton, Maryland, and surface (lake) water (pH 7.0, hardness 64.0 mg/L as CaCCb, 188 |iS/cm specific conductance), obtained from Tuckahoe Lake located in Tuckahoe State Park in Ridgely, Maryland, were characterized internally (pp. 11-12; Appendices III-IV, pp. 131-132). 2 Two ion pair transitions were monitored for propargite: m/z 368.1—>231.1 (quantitation) and m/z 368.1—>81.1 (confirmation). 3 The original result for one sample was deviant; the mean of duplicate re-analyses were reported for this sample. 4 Measured concentrations were corrected for extrapolated mean matrix blank contributions of 0.0024033 |ig/L (ca. 24% of the LOQ) in surface water and 0.0026306 |ig/L (ca. 26% of the LOQ) in drinking water (Table 3, p. 22; Table 5, p. 24). Page 6 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 III. Method Characteristics In the ECM and ILV, the method LOQ was 0.01 |ig/L for propargite in water (equivalent to 0.4 ng/mL using the current methodology; pp. 11, 18-19; Table VIII, p. 29; Appendix D, pp. 68, 74 of MRID 50356002; p. 10 of MRID 50356003). In the ECM, the LOQ was defined as the lowest concentration validated for each matrix, and the LOQ was reported from the ECM in the ILV. No calculation was provided for the method LOQ in the ECM or ILV. In the ECM, the LOD for propargite was calculated to be 0.002 |ig/L for drinking water and 0.003 |ig/L for surface water. The LOD was calculated using the following equation: LOD = to.99 x S, where to.99 equaled 3.747 (the one-tailed t-statistic at the 99% confidence level for n-1 replicates) and S equaled the standard deviation of n samples fortified at the LOQ. In the ILV, the LOD was not reported. Page 7 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 Table 4. Method Characteristics for Propargite in Water Propargite Limit of Quantitation (LOQ) ECM 0.01 ng/L (equivalent to 0.4 ng/mL using the current methodology) ILV 0.01 ng/L Limit of Detection (LOD) ECM 0.002 |ig/L for drinking water 0.003 |ig/L for surface water ILV Not reported Linearity (calibration curve r2 and concentration range) ECM Drinking r2 = 0.9998 (Q) r2 = 0.9992 (C) Surface r2 = 0.9986 (Q) r2 = 0.9978 (C) ILV1 Drinking r2 = 0.9997 (Q) r2 = 0.9995 (C) Surface r2 = 0.9994 (Q) r2 = 0.9995 (C) Concentration range 0.04-5.0 ng/mL Repeatable ECM2 Yes at LOQ and 10/LOQ. ILV3-4 Drinking5 Yes at LOQ6 and lOxLOQ. Surface Yes at LOQ and lOxLOQ. Reproducible Yes at LOQ and lOxLOQ. Specific ECM Yes, matrix interferences were <5% of the LOQ (based on peak area). Some minor baseline interference was observed in the C chromatogram.7 ILV Q Yes, matrix interferences were less than ca. 14% of the LOQ (based on measured concentration). c Matrix interferences were ca. 24-26% of the LOQ (based on measured concentration).7 Data were obtainedfromp.il; Tables I-II, pp. 22-23 (calibration coefficients); Tables III-IV, pp. 24-25 (recovery results); Figure 2, pp. 32-33 and Figure 11, pp. 42-43 (calibration curve); Figures 3-10, pp. 34-41 and Figures 12-19, pp. 44-51 (chromatograms) of MRID 50356002; pp. 13; Tables 2-5, pp. 21-24 (recovery results); Figures 1-2, pp. 25-26 (calibration curves); Figures 5-8, pp. 29-32 (chromatograms) of MRID 50356003. Q = quantitation ion; C = confirmation ion. All results reported for Q and C ions unless specified otherwise. 1 Correlation coefficients (r2) were reviewer-calculated based on r values (1/x weighted linear regression analysis) reported in the study report (Figures 1-2, pp. 25-26 of MRID 50356003; DER Attachment 2). 2 In the ECM, the drinking water (2706W-055; pH 7.6, hardness 29 mg/L as CaCCb, 58 ppm total dissolved solids) and surface water (2706W-049; pH 8.5, hardness 262 mg/L as CaCCb, 204 ppm total dissolved solids) were characterized by Agvise Laboratories, Northwood, North Dakota (p. 13; Appendix C, pp. 65-66 of MRID 50356002). Water matrix sources were not reported. 3 In the ILV, the drinking (well) water (pH 8.1, hardness 142 mg/L as CaCCb, 336 |iS/cm specific conductance), obtained from a well at Easton Laboratories-Easton testing facility in Easton, Maryland, and surface (lake) water (pH 7.0, hardness 64.0 mg/L as CaC03, 188 |iS/cm specific conductance), obtained from Tuckahoe Lake located in Tuckahoe State Park in Ridgely, Maryland, were characterized internally (pp. 11-12; Appendices III-IV, pp. 131-132 of MRID 50356003). 4 The ECM method for propargite was validated by the ILV with insignificant modifications to the analytical equipment and parameters; the number of trials was not specified, but the reviewer assumed that the method was validated in the first trial (pp. 10, 13-14 of MRID 50356003). 5 For the ILV confirmation ion analysis only, measured concentrations were corrected for extrapolated mean matrix blank contributions of 0.0024033 |ig/L (ca. 24% of the LOQ) in surface water and 0.0026306 |ig/L (ca. 26% of the LOQ) in drinking water (Table 3, p. 22; Table 5, p. 24 of MRID 50356003). 6 The original result for one ILV LOQ sample in drinking water was deviant; the mean of duplicate re-analyses were reported for this sample (Tables 4-5, pp. 23-24 of MRID 50356003). 7 A confirmatory method is not typically required where GC/MS and/or LC/MS methods are used as the primary method(s) to generate study data. Page 8 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 IV. Method Deficiencies and Reviewer's Comments 1. The communications between the ILV staff and study developers and sponsors was not reported or addressed in the ILV. The ECM laboratory (PTRL West) and ILV laboratory (Wildlife International) are both now doing business as EAG Laboratories; however, the ECM and ILV laboratories were located at different facilities and all laboratory personnel and equipment differed between the two laboratories. The registrant, Arysta, sent an email to Wilhelmena Livingston, EPA, dated 05/16/18, with the list of communications between the method development staff and ILV staff. This email indicated that there was no collusion between the ILV staff and the study developers and sponsors. 2. The determinations of the LOD and LOQ in the ECM and ILV were not based on scientifically acceptable procedures as defined in 40 CFRPart 136 (pp. 11, 18-19; Table VIII, p. 29; Appendix D, pp. 68, 74 of MRID 50356002; p. 10 of MRID 50356003). In the ECM, the LOQ was defined as the lowest concentration validated for each matrix, and the LOQ was reported from the ECM in the ILV. No calculation was provided for the method LOQ in the ECM or ILV. In the ECM, the LOD for propargite was calculated using the following equation: LOD = to.99 x S, where to.99 equaled 3.747 (the one-tailed t-statistic at the 99% confidence level for n-1 replicates) and S equaled the standard deviation of n samples fortified at the LOQ. In the ILV, the LOD was not reported. 3. The number of trials required by the ILV to validate the ECM method was not specified; however, the reviewer assumed that the method was validated in the first trial based on the insignificant modifications to the method (pp. 10, 13-14 of MRID 50356003). 4. For the ILV confirmation ion analysis only, measured concentrations were corrected for extrapolated mean matrix blank contributions of 0.0024033 |ig/L (ca. 24% of the LOQ) in surface water and 0.0026306 |ig/L (ca. 26% of the LOQ) in drinking water (Table 3, p. 22; Table 5, p. 24 of MRID 50356003). The ILV study author discussed that the matrix interference was less in the quantitation ion transition because of differences in ionization efficiency of the transitions by the LC/MS/MS instrument (pp. 17-18). These interferences and recovery corrections did not have an effect on the validity of the study since a confirmation method is not required when the primary method is GC/MS or LC/MS/MS. 5. The reviewer noted the following significant typographical error in the ECM: the ionization mode was reported as "negative positive mode" in the analytical method (Appendix D, p. 73 of MRID 50356002). The reviewer assumed that the ionization mode was positive, based on the ILV, despite the fact that "Negative Ion Spray Voltage (IS):" was listed as one of the LC/MS/MS settings (Appendix D, p. 74). 6. In the ECM, matrix effects were assessed and determined to be insignificant in the surface and drinking water matrices (<20% difference; p. 18; Table V, p. 26 of MRID 50356002). Solvent-based calibration standards were used for the recovery results. In the ECM, the stability of the calibration standards and final extracts were assessed and determined to be up to 21 days and up to 18-21 days, respectively, when stored under frozen conditions (p. 18; Tables VI-VII, pp. 27-28). Page 9 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 7. In the ECM, the time required to complete the extraction of one set of 12 samples (two matrix controls and ten fortified samples) was reported as 6 hours for one chemist, followed by ca. 10 hours for LC/MS/MS analysis (unattended), and then ca. 2 hours of data processing for one chemist (Appendix D, p. 75 of MRID 50356002). The time requirements of the method were not reported in the ILV. 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 10 of 11 ------- Propargite (PC 097601) MRIDs 50356002/ 50356003 Attachment 1: Chemical Names and Structures Propargite IUPAC Name: CAS Name: CAS Number: 2-(4-Tert-butylphenoxy)cyclohexyl prop-2-ynyl sulfite 2-[4-(l,l-Dimethylethyl)phenoxy]cyclohexyl 2-propyn-l-yl sulfite 2312-35-8 SMILES String: Not provided (CHJ. Page 11 of 11 ------- |