Test Material: Nicosulfuron

MRID: 48790911

Title: Analytical method for the determin ation of nicosulfuron (DPX-V9360), IN-V9367 and IN-J0290 in wate r using HPLC/ESI-MS/MS.

MRID: 48790913

Title: Independent laboratory validation of DuPont-32132, “Analytical method for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in water by HPLC/ESI-MS/MS”.

EPA PC Code: 129008

OCSPP Guideline: 850.6100

For CDM Smith

Primary Reviewer: Lisa Muto Signature:

Date: 3/20/14

Secondary Reviewer: Dan Hunt Si g nature:

Date: 3/20/14

QC/QA Manager: Joan Gaidos Signature:

Date: 3/20/14Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 2 of 7 Analytical method for nicosulfuron in water

Reports: ECM: EPA MRID No. 48790911. Morgan, E.A. and Cabusas, M.E.Y. 2012. Analytical method for the determina tion of nicosulfuron (DPX-V9360), INV9367 and IN-J0290 in water using HP LC/ESI-MS/MS. Laboratory Project ID: DuPont-32132. Report prepared by E.I. du Pont de Nemours and Company, Newark, Delaware; sponsored and submitted by E.I. du Pont de Nemours and Company, Wilmington, Delaware; 54 pages. Final report issued March 16, 2012. ILV: EPA MRID No. 48790913. Robaugh, D.A. 2012. Independent laboratory validation of DuPont-32132, “Analytical method for the determination of nicosulfuron (DPX-V9360), IN-V9367 and IN-J0290 in water by HPLC/ESI-MS/MS”. DuPont Study No: DuPont-32578. Pyxant Labs Project ID: 2388. Report prepared by Pyxant Labs Inc., Colorado Springs, Colorado; sponsored and submitted by E.I. du Pont de Nemours and Company, Newark, Delaware; 92 pages. Final report issu ed March 30, 2012. Document No.: MRIDs 48790911 & 48790913 Guideline: 850.6100 Statements: ECM: The study was conducted in a GLP compliant facility following standard operating procedures but not w ith the restriction of compliance with USEPA FIFRA GLP standards, 40 CFR, Part 160 (p. 3). Signed and dated Data Confidentiality, No Data Confidentiality, GLP, and Certification of Authenticity statements were provided (pp. 2-4). A Quality Assurance statement was not provided. ILV: The study was conducted in acco rdance with the USEPA FIFRA GLP standards (40 CFR Part 160) and OECD Principles of GLP (p. 3). Signed and dated Data Confidentiality, No Data Confidentiality, GLP, Quality Assurance and Certification of Authenticity statements were provided (pp. 2- 5). Classification: This analytical method is classified as Acceptable . However, the LOD and LOQ of nicosulfuron in this method for water may not be low enough to resolve nicosulfuron at toxicologically relevant concentrations for aquatic plants. There appears to be other me thods available with lower LOQs for nicosulfuron in water. PC Code: 129008 Reviewer: Gabriel Rothman Signature: Environmental Scientist Date: May 5, 2014

All page citations refer to MRID 48790911 (ECM) unless otherwise noted

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Executive Summary

This analytical method, Laboratory Project ID: DuPont-32132, is designed for the quantitative determination of nicosulfuron, IN-V9367 and IN -J0290 in water using LC/MS/MS. The method is quantitative for nicosulfuron and its transfor mation products at the stated LOQ of 0.10 ng/mL.Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 3 of 7 The lowest toxicological level of concern in water was not reported. No major issues were discovered by the independent laboratory.

Table 1. Analytical Method Summary

Analyte(s) by

Pesticide MRID EPA Review Matrix Method Date Registrant Analysis Limit of Quantitation (LOQ) Environmental Chemistry

Method Independent Laboratory Validation Nicosulfuron (DPX-V9360), IN-V9367 and IN-J0290 48790911 48790913 Water 3/16/2012 E.I. du Pont de Nemours and Company LC/MS/MS 0.10 ng/mL

I. Principle of the Method

Samples (20 g) were acidified to a pH of 3.5 to 4.0 with 1 M formic acid then filtered through an Oasis ® HLB SPE cartridge (pp. 9, 14, 18). The anal ytes were eluted with methanol:0.50 M ammonium hydroxide (9:1, v:v). The el uent was acidified to a pH of ca . 4.5 with 1 mL of 1 M formic acid and reduced to ≤ 2 mL under a stream of nitrogen at ca . 25-30°C. The volume was adjusted to 2 mL with water, if necessary, then to 5 mL with 5 mM am monium formate:methanol (19:1, v:v). The resulting solution was filtered (13- mm, 0.45-μm PTFE filter) prior to analysis.

Samples were analyzed for nicosulfuron, IN-V9367 and IN-J0290 by reversed-phase HPLC (Phenomenex ® Luna, 4.6 mm x 150 mm, 3 μm phenyl-h exyl column) using a mobile phase gradient of (A) 0.1 mM formic acid in 0.1 mM ammonium formate and (B) methanol [percent A:B at 0.00 min. 95:5 (v:v), 4.00 min. 50: 50, 4.10 min. 35:65, 7.00 min. 10:90, 7.10-10.00 min. 5:95, 10.10-12.00 min. 95:5] with MS/MS-ESI + detection and Multiple Reaction Monitoring (MRM; pp. 9, 10, 14, 20). The ratios of two MRM parent-to-daughter ions were monitored (quantitative and confirmatory) per analyte. Injection volumes were 10-20 μL; an injection volume of 10 μL was indicted for pond wate r in order to reduce matrix effects.

The LOQ was the same in the ECM and ILV (0.10 ng/mL; p. 24; p. 11 of MRID 48790913). In the ECM, the LOD was estimated to be one-third of the LOQ, ca . 0.03 ng/mL. The LOD was not defined in the ILV.

II. Recovery Findings

ECM (MRID 48790911): Mean recoveries and relative standard devi ations (RSD) were within guideline requirements (mean 70-120%; RSD ≤ 20%) for analysis of nicosulfuron, IN-V9367 and IN-J0290 in tap (drinking) water, pond water and well water (p. 24; Table 1, pp. 28-36). Confirmation of the identified peak s was based on the relative rati o of its two parent-to-daughter ion transitions; all of these ratios were accurate w ithin the range of the equivalent ion ratios of the calibration standards (pp. 25-27).

ILV (MRID 48790913): Mean recoveries and RSDs were within guideline requirements for analysis of nicosulfuron, IN-V 9367 and IN-J0290 in pond water (p. 18; Tables 4-6, pp. 24-26).Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 4 of 7 Quantitative ion and confirmatory ion results were comparable. The method was validated with the second trial (p. 11; see Comment #2).

Table 2. Initial Validation Method Recoveries for Analytes in Water

Analyte Fortification Level (ng/mL) Number of Tests Recovery Range (%) Mean Recovery (%) Standard Deviation (%) Relative Standard Deviation (%)
Well Water (Kimblesville, Pennsylvania)
Nicosufluron (DPX-V9360) 0.10 (LOQ) 5 90-94 92 1.5 1.5
1.0 5 84-96 91 4.3 4.7

IN-V9367 0.10 (LOQ) 5 93-104 98 4.7 4.8
1.0 5 100-112 106 4.7 4.4

IN-J0290 0.10 (LOQ) 5 86-91 88 2.1 2.4
1.0 5 89-95 93 2.4 2.6 Lums Pond Water (Lums Pond, Delaware)
Nicosufluron (DPX-V9360) 0.10 (LOQ) 5 71-94 82 8.5 10.4
1.0 5 91-109 98 6.9 7.1

IN-V9367 0.10 (LOQ) 5 74-92 82 8.4 10.3
1.0 5 90-112 97 9.3 9.6

IN-J0290 0.10 (LOQ) 5 79-88 83 3.8 4.6
1.0 5 71-82 77 5.5 7.1 Tap (Drinking) Water (Newark, Delaware)
Nicosufluron (DPX-V9360) 0.10 (LOQ) 5 82-89 85 2.5 2.9
1.0 5 88-102 95 5.3 5.5

IN-V9367 0.10 (LOQ) 5 88-94 92 3.1 3.4
1.0 5 98-110 106 5.2 4.9

IN-J0290 0.10 (LOQ) 5 68-83 77 6 7
1.0 5 74-87 83 5.3 6.4 Data were obtained from p. 24 and Table 1, pp. 28-36 in the study report.

Table 3. Independent Validation Method R ecoveries for Analytes in Pond Water

Analyte Fortification Level (ng/mL) Number of Tests Recovery Range (%) Mean Recovery (%) Standard Deviation (%) Relative Standard Deviation (%) 1

Water Recoveries – Quantitative ion
Nicosufluron (DPX-V9360) 0.10 (LOQ) 5 82-94 89 4.6 5
1.0 5 84-92 88 3.2 4

IN-V9367 0.10 (LOQ) 5 85-92 90 3.1 3
1.0 5 94-99 97 2.2 2

IN-J0290 0.10 (LOQ) 5 79-95 87 6.0 7
1.0 5 75-80 78 1.9 2 Confirmation ion
Nicosufluron (DPX-V9360) 0.10 (LOQ) 5 86-100 94 5.6 --
1.0 5 81-91 86 4.0 --

IN-V9367 0.10 (LOQ) 5 90-93 92 1.1 --
1.0 5 94-103 99 3.2 --

IN-J0290 0.10 (LOQ) 5 72-84 77 4.4 --
1.0 5 75-82 77 2.9 -- Data were obtained from p. 18; Tables 4-6, pp. 24-26 of MRID 48790913.Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 5 of 7 1 Reviewer-calculated for quantitative ion; confirmation ion raw data not supplied in ILV study report (see DER Attachment 2).

III. Method Characteristics

The LOQ was the same in the ECM and ILV (0.10 ng/mL). In the ECM, the LOQ was defined as the lowest fortification level which obtained av erage recoveries of 70-110% and a RSD <20% (p. 24). The LOD was not determined experimentally; however, it was estimated as one-third of the LOQ ( ca . 0.03 ng/mL). In the ILV, the LOQ was reported from the ECM, and no justification was provided (p. 11 of MRID 48790913) . The LOD was not defined.

Table 4. Method Characteristics
Nicosulfuron IN-V9367 IN-J0290

Limit of Quantitation (LOQ) 0.1 ng/mL 0.1 ng/mL 0.1 ng/mL Limit of Detection (LOD) ca . 0.03 ng/mL ca . 0.03 ng/mL ca . 0.03 ng/mL Linearity (calibration curve r 2 and concentration range) r 2 = 0.9996 1 (0.25-10 ng/mL) r 2 = 0.9951-0.9999 1 (0.25-10 ng/mL) r 2 = 0.9929-0.9993 1 (0.25-10 ng/mL) Repeatable Yes Yes Yes Reproducible Yes 2 Yes 2 Yes 2 Specific Yes Yes Yes Data were obtained from p. 24; Figure 1, p. 37; Appendices 1-2, pp. 46-54. 1 ILV calibration curves yielded similar linearity, r 2 = 0.9974-0.9993, for concentration range of 0.25-10 ng/mL (see p. 17 and Figures 1-3, pp. 27-29 of MRID 48790913). 2 The ILV successfully validated the method at the LOQ in th e second trial, after decreasing the injection volume to 10 μL as specified in the ECM for pond water (see Comment #2).

IV. Method Deficiencies and Reviewer’s Comments

1. It may be possible to obtain lower LOCs and LOQs for nicosulfuron in water using other methods. The reviewer found references to other LOQs determined for nicosulfuron of 0.01 ppb, approximately one order of one magnitude lower than this evaluated analytical method (Battaglin et al., 2001).

2. In general, toxicologically relevant concen trations of nicosulfur on are > 1.0 ppm range for aquatic animals. However, there is uncerta inty with the toxicity of aquatic plants as this study has not been submitted to the Agenc y, but has been requested as part of the Registration Review program. Given nicosu lfuron’s phytotoxic mode of action, it is likely that aquatic plants will be far more se nsitive to nicosulfuron exposure than aquatic animals. Therefore, the analytical met hod should possess the abil ity to measure the lowest concentrations possible such as i ndicated by Battaglin et al., 2001 to account for nicosulfuron’s pot ential toxicity to aquatic plants.

3. In the ILV, Method Validation Trial 1 wa s unsuccessful (pp. 10, 17-18 and Appendix 3, p. 92 of MRID 48790913). After consultation w ith the Sponsor, the ILV decreased the HPLC injection volume to 10 μL, as sugge sted in the ECM method for pond water (p. 20), and successfully validate d the method with Trial 2.Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 6 of 7 4. The calibration standards in the ECM and ILV included only samples in the range of 0.25 ng/mL to 10 ng/mL when the LOQ for method validation was a 0.10 ng/mL spiking level; however, the linear regressions of th e peak area versus concentration of the calibration standards were not used to calcu late the analyte found (p. 22). The calibration standards were used to calculate Response F actors (concentration of standard/peak area counts) which were used to calculate the analyte found. Also, the peak areas of the samples were within range of the peak areas of th e calibration standard s, most likely due to the concentration procedures used dur ing the method (Appendices 1-2, pp. 46-54).

5. Matrix characterization of the pond, well and drinking water in the ECM (p. 17) and pond water in the ILV was reported (Appendix 1, p. 84 of MRID 48790913).

6. The linear regression equations of the revi ewer-generated calibration curves did not exactly match those reported in the EC M and ILV; however, the equations and r 2 values were similar, in general.

7. The reviewer was unable to verify the recove ries for the ECM from data and equations provided in Appendices 1-2, pp. 46-54 of the st udy report. The equation [% recovery = (analyte found *100)/(fo rtification level)] was provide d by the study authors; however, all of the significant figures of the “analyte found” data we re not reported. For example, in the recoveries of IN-V9367 in the well wa ter samples dosed at the LOQ, the test samples LOQ 1 and LOQ 3 had the same “analyte found” (0.10 ng/mL) and sample weight (20.00 g), but differing percent r ecoveries of 93% and 95%, respectively (Appendix 2, p. 53). The reviewer did not calcu late “analyte found” from the raw data.

8. It was reported for the ILV that a single an alyst completed a sample set consisting of 15 samples in ca . 8-10 hours or 2 calendar days (p. 19 of MRID 48790913).

V. References

Battaglin W.A., Furlong E.T., and M.R. Bu rkhardt., 2001. Concentration of Selected Sulfonylruear Sulfonamide, and Imidazolinon e Herbicides, Other Pesticides, and Nutrients in 71 Streams, 5 Reservoir Outflo ws, and 25 Wells in the Midwestern United States, 1998. U.S. Geological Survey, Denver, CO.

U.S. Environmental Protection Agency. 2012. Ecological Effects Test Guidelines, OCSPP 850.6100, Environmental Chemistry Methods an d Associated Independent Laboratory Validation. Office of Chemi cal Safety and Pollution Prev ention, Washington, DC. EPA 712-C-001.

40 CFR Part 136. Appendix B. Definition and Pro cedure for the Determination of the Method Detection Limit-Revision 1.11, pp. 317-319.Nicosulfuron (PC 129008) MRID 48790911 (ECM)/ 48790913 (ILV)

Page 2 of 7 Attachment 1: Chemical Names and Structures Nicosulfuron; DPX-V9360 IUPAC Name: 1-(4,6-Dimethoxypyrimidin-2-yl)-3-(3-dimethylcarbamoyl-2- pyridylsulfonyl)urea. 2-[(4,6-Dimethoxypyrimidin-2-ylc arbamoyl)sulfamoyl]-N,Ndimethylnicotinamide. CAS Name: 2-[[[[(4,6-Dimethoxy-2-pyrimidinyl )amino]carbonyl]a mino]sulfonyl]- N

, N -dimethyl-3-pyridinecarboxamide monohydrate. CAS Number: 111991-09-4. SMILES String: c1(c(nccc1)S(=O)(=O)NC(=O)Nc 2nc(cc(n2)OC)OC)C(=O)N(C)C

IN-V9367 IUPAC Name: Not reported. CAS Name: 2-(Aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide. CAS Number: 112006-75-4. SMILES String: c1(cccnc1S(=O)(=O)N)C(=O)N(C)C

IN-J0290 IUPAC Name: Not reported. CAS Name: 4,6-Dimethoxy-2-pyrimidinamine. CAS Number: 36315-01-2. SMILES String: Nc1nc(cc(n1)OC)OC