Test Material: Nicosulfuron

MRID: 48790912

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

MRID: 48790914

Title: Independent laboratory validation of DuPont-33143, “Analytical method for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in soil using 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 48790912 (ECM)/ 48790914 (ILV)

Page 2 of 8 Analytical method for nicosulfuron in soil

Reports: ECM: EPA MRID No. 48790912. Cabusas, M.E.Y. 2012. Analytical method for the determination of nicosulfuron (DPX-V9360), IN-V9367 and INJ0290 in soil using HPLC/ESI-MS/MS. Laboratory Project ID: DuPont33143. 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; 50 pa ges. Final report issued March 16, 2012. ILV: EPA MRID No. 48790914. Rogers, P. 2012. Independent laboratory validation of DuPont-33143, “Analytical method for the determination of nicosulfuron (DPX-V9360), IN-V 9367 and IN-J0290 in soil using HPLC/ESI-MS/MS”. DuPont Project ID: DuPont-33031. Alliance Pharma Project No.: 120103. Report prepared by Alliance Pharma, Inc., Malvern, Pennsylvania, sponsored and submitted by E.I. du Pont de Nemours and Company, Wilmington, Delaware; 52 pa ges. Final report issued March 22, 2012. Document No.: MRIDs 48790912 & 48790914 Guideline: 850.6100 Statements: ECM: The study was not conducted with the restriction of compliance with USEPA FIFRA GLP standards, 40 CFR, Part 160, or OECD GLP (p. 3). Signed and dated Data Confidentiality , No Data Confidentiality, and GLP statements were provided (pp. 2-3). Ce rtification of Authenticity and Quality Assurance statements were not provided. ILV: The study was conducted in accordance with the USEPA FIFRA Good Laboratory Practice (GLP) standards ( 40 CFR Part 160) and OECD GLP (p. 3). Signed and dated Data Confidential ity, 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 fo r soil were not low enough to resolve nicosulfuron at toxicologically rele vant concentrations for plants. PC Code: 129008 Reviewer: Gabriel Rothman Signature: Environmental Scientist Date: May 5, 2014

All page citations refer to MRID 48790912 (ECM) unless otherwise noted

. Executive Summary

This analytical method, Laboratory Project ID: DuPont-33143, is designed for the quantitative determination of nicosulfuron, IN-V9367 and IN -J0290 in soil using LC/MS/MS. The method is quantitative for nicosulfuron and it s transformation products at the stated LOQ of 1.0 μg/kg. The lowest toxicological level of concern in soil was not reported. No major issues were discovered by the independent laboratory.Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV)

Page 3 of 8 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 48790912 48790914 Soil 3/16/2012 E.I. du Pont de Nemours and Company LC/MS/MS 1.0 μg/kg

I. Principle of the Method

Samples (10 g) were extracted twice by shak ing for 15-20 minutes with 0.1 M ammonium carbonate:acetone (9:1, v:v) then centrifuge d (pp. 8, 10, 12-13, 17-19). The supernatant was decanted and filtered. One-fourth of the combin ed extract was purified by passing through an Isolute ® NH 2 solid-phase extraction (SPE) cartridge . The acetone was removed from the partially-purified extract by evapor ation using nitrogen-evaporator (N 2 -vap) system at 30°C. The reduced extract was acid ified to pH 3 with 1M formic acid then purified by passing through stacked Oasis TM HLB and ENV+ ® SPE cartridges. The analytes we re eluted with methanol:1 M ammonium hydroxide (9:1, v:v) solution a nd methanol. The eluent was reduced to ca . 3 mL in an N 2 -vap system at 30°C. The final volume was adjusted to 5 mL with 5 mM ammonium formate:methanol (19:1, v:v). The resulting so lution was filtered (25-mm, 0.45-μm PTFE filter) prior to analysis.

Samples were acidified (10 μL of 1M formic acid added to 990 μL of sample) then analyzed for nicosulfuron, IN-V9367 and IN-J0290 by reversed-phase HPLC (Phenomenex ® Luna, 4.6 mm x 150 mm, 3 μm phenyl-hexyl column) using a mobile phase gradient of (A) 0.1 mM formic acid in 0.01 mM ammonium formate and (B) methanol [percent A:B at 0.0 min. 95.0:5.0 (v:v), 10-13 min. 5.0:95.0, 13.1-17 min. 95.0:5.0] with MS/MS-ESI + detection and Multiple Reaction Monitoring (MRM; pp. 8, 10, 13, 19-21). The ratios of two MRM parent-to-daughter ions were monitored (quantitative and confirmatory) pe r analyte. Injection volumes were 75 μL.

The LOQ was the same in the ECM and ILV ( 1.0 μg/kg; p. 24; p. 11 of MRID 48790914). In the ECM, the LOD was not experimentally determined (p. 24); however, was reported as one-third of the LOQ or 0.33 μg/kg for the Drummer clay loam soil (Appendix 4, pp. 47-49). In the ILV, the LOD was estimated to be 0.3 μg/kg (p. 11 of MRID 48790914).

II. Recovery Findings

ECM (MRID 48790912): 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 clay loam, loam, silty loam and sandy loam soils (p. 23; Table 1, pp. 28-39). 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. 25-26).Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV)

Page 4 of 8 ILV (MRID 48790914): Mean recoveries and RSDs were within guideline requirements for analysis of nicosulfuron, IN-V 9367 and IN-J0290 in one soil (pp. 18-19; Tables 1-6, pp. 22-27). Quantitative ion and confirmatory ion results were comparable. The relative ratios of the two parent-to-daughter ion transiti ons were similar between the LOQ and 10xLOQ sample sets (Tables 7-9, pp. 28-30). The method was valid ated with the first trial (p. 10).

Table 2. Initial Validation Method Recoveries for Analytes in Soil

Analyte Fortification Level (μg/kg) Number of Tests Recovery Range (%) Mean Recovery (%) Standard Deviation (%) Relative Standard Deviation (%)
Clay loam (Drummer #7 from Illinois)
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 99-115 104 6.1 5.8
10.0 5 95-106 101 4.3 4.2

IN-V9367 1.0 (LOQ) 5 85-100 90 7.1 7.9
10.0 5 82-93 88 4.8 5.5

IN-J0290 1.0 (LOQ) 5 70-85 77 5.7 7.4
10.0 5 71-78 75 3.0 4.0 Loam (Mattapex #25 from Maryland)
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 94-107 100 5.8 5.8
10.0 5 96-110 101 5.9 5.9

IN-V9367 1.0 (LOQ) 5 78-94 87 6.5 7.6
10.0 5 82-94 87 5.1 5.9

IN-J0290 1.0 (LOQ) 5 72-89 80 6.4 8.0
10.0 5 76-85 81 3.8 4.8 Silty loam (Nambsheim from France)
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 92-104 99 4.5 4.6
10.0 5 96-105 99 3.7 3.7

IN-V9367 1.0 (LOQ) 5 85-105 97 7.8 8.0
10.0 5 98-100 98 0.9 0.9

IN-J0290 1.0 (LOQ) 5 73-86 82 5.5 6.7
10.0 5 83-85 84 1.0 1.2 Sandy loam (Speyer 2.2 from Germany)
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 94-113 106 8.3 7.8
10.0 5 97-120 104 9.1 8.7

IN-V9367 1.0 (LOQ) 5 82-94 89 4.8 5.3
10.0 5 82-96 88 5.8 6.6

IN-J0290 1.0 (LOQ) 5 78-92 88 5.8 6.6
10.0 5 74-95 82 8.1 9.9 Data were obtained from p. 23; Table 1, pp. 28-39; and Appendix 4, pp. 47-49 in the study report.Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV)

Page 5 of 8 Table 3. Independent Validation Method Recoveries for Analytes in Soil

Analyte Fortification Level (μg/kg) Number of Tests Recovery Range (%) Mean Recovery (%) Standard Deviation (%) Relative Standard Deviation (%)
Soil Recoveries – Quantitative ion
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 105-114 109 3.8 3
10.0 5 97-108 103 4.8 5

IN-V9367 1.0 (LOQ) 5 72-84 77 5.4 7
10.0 5 74-82 78 3.1 4

IN-J0290 1.0 (LOQ) 5 84-91 89 3.1 4
10.0 5 72-83 80 4.5 6 Confirmation ion
Nicosufluron (DPX-V9360) 1.0 (LOQ) 5 100-120 106 8.8 8
10.0 5 93-102 98 4.2 4

IN-V9367 1.0 (LOQ) 5 58-86 71 10.1 14
10.0 5 70-80 75 3.7 5

IN-J0290 1.0 (LOQ) 5 82-84 83 0.8 1
10.0 5 77-88 83 4.2 5 Data were obtained from pp. 11, 19; and Tables 1-6, pp. 22-27 of MRID 48790914.

III. Method Characteristics

The LOQ was the same in the ECM and ILV (1.0 μg/kg). 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 reported as one-third of the LOQ (0.33 ng/g) for the Drummer clay loam so il (Appendix 4, pp. 47-49). In the ILV, the LOQ was reported from the ECM, and no justifi cation was provided (p. 11 of MRID 48790914). The LOD was estimated to be 0.3 μg/kg; no justification was provided.

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

Limit of Quantitation (LOQ) 1.0 μg/kg 1.0 μg/kg 1.0 μg/kg Limit of Detection (LOD) 0.33 μg/kg 1 0.33 μg/kg 1 0.33 μg/kg 1 Linearity (calibration curve r 2 and concentration range) r 2 = 1.0000 2 (0.25-20 ng/mL) r 2 = 0.9996 2 (0.25-20 ng/mL) r 2 = 0.9999 2 (0.25-20 ng/mL) Repeatable Yes Yes Yes Reproducible Yes Yes Yes Specific Yes Yes Yes Data were obtained from pp. 22, 24; Figure 1, p. 40; and Appendix 4, pp. 47-79. 1 Reported for Drummer clay loam soil. The LOD was reported as 0.3 μg/kg in the ILV (p. 11 of MRID 48790914). 2 ILV calibration curves were linear, r 2 = 0.9940-0.9952, for concentration range of 0.25-20 ng/mL (see p. 19 and Figure 4, pp. 40-42 of MRID 48790914).

IV. Method Deficiencies and Reviewer’s CommentsNicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV)

Page 6 of 8 1. The LOD and LOQ arrived at with the analytic al method used is not sufficient to address the concern for nicosulfuron’s potential to adversely impact plants at very low exposure levels. The EC25 determined for the most se nsitive endpoint for plant effects is very low at 0.00045 lbs./A (seedling emergence endpoi nt for mustard seeds, MRID No. 42220001). This endpoint can be triggered with a concentration of < 0.1 ppb of nicosulfuron in soil. The method evaluated established the LOD an order of magnitude higher at 0.33 ppb and LOQ two order of ma gnitude higher at 1.0 ppb. While these are low concentrations, an analytical method for nicosulfuron extractions from soil should be precise enough to resolve nicosu lfuron residues at toxicologica lly relevant concentrations for plants.

2. The reviewer is aware of one other prev iously developed analytical method for nicosulfuron. This method has demonstrated the ability to meas ure nicosulfuron and other sulfonylurea pesticide residue s in soils at very low concen trations. This analytical method utilizing LC-MS possessed LOQs as low as 6 ppt in soil (Marek and Koskinen, 1997), approximately 2 orders of magnitude lower than this reviewed analytical method.

3. In the ECM, the extraction efficiency of the method was evaluated in an aged soil (clay loam from Japan) fortified at 50 μg/kg (pp. 13, 16, 26; Appendix 5, p. 50). The original method was modified to eliminate the pur ification and concentration with SPE. Recoveries in the aged soil were 94- 99%, 93-96% and 89-90% of the applied for nicosulfuron, IN-V0367 and IN -J0290, respectively. These recoveries were normalized using fresh soil recoveries; normalized r ecoveries were 101%, 97% and 102% for nicosulfuron, IN-V0367 a nd IN-J0290, respectively.

4. A reagent blank was not included in the ILV (p. 20; Tables 1-6, pp. 22-27 of MRID 48790914).

5. The linear regression equations of the revi ewer-generated calibration curves matched those reported in the ECM; however, the li near regression equati ons of the reviewergenerated calibration curves indicated greater linearity than those reported in the ILV.

6. The reviewer was unable to verify the rec overies for the ECM from data provided in Tables 1-6, pp. 28-39 of the study report. Th e equation [% recovery = (analyte found *100)/(fortification level)] was provided by the study authors on pp. 20-21; however, the study author noted that only two significan t figures of the “analyte found” data were reported. For example, in the recoveries of nicosulfuron in the clay loam soil samples dosed at the LOQ, the test samples LOQ A and LOQ B had the same “analyte found” of 1.0 μg/kg, but differing percent r ecoveries of 101% and 103%, respectively (Table 1, p. 28). The reviewer did not calculate “analyte found” from the raw data; raw data was only provided for one soil, the clay loam.

7. It was reported for the ILV that a single analyst completed two sample sets consisting of 6 samples per set in 1.5 working days (p. 20 of MRID 48790914).Nicosulfuron (PC 129008) MRID 48790912 (ECM)/ 48790914 (ILV)

Page 7 of 8 8. For the ILV, a Phenomenex® Luna Phenyl-H exyl 3.0 x 150 mm, 3-μm column was used at a flow rate of 0.425 mL/min, instead of a Phenomenex® Luna Phenyl-Hexyl 4.6 x 150 mm, 3-μm column at a flow rate of 1.0 mL/min as described in the ECM (MRID 48790914, pp. 14-15). The study author stated th at the study monitor approved of the substitution and that the substitution was demons trated to be equivalent to that specified in the method.

9. Characterization of the test soils should have included informa tion regarding organic carbon content, cation exchange capacity, a nd soil taxonomic classification as these properties may influence recoveries pesticide residues at different spiking levels in the analytical method evaluation.

V. References

Marek L.J. and Koskinen W.C., 1997. LC-MS Analys is of Polar Pesticides in Soil. RJ Reynolds Industry Publication, Richmond, VA.

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 48790912 (ECM)/ 48790914 (ILV)

Page 2 of 8 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