Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Analytical method for pyrethrins in soil and sediment

Reports:	ECM: EPA MRID No. 50475403. DeVellis, S.R. 2017. Validation of an

Environmental Chemistry Method for the Determination of Pyrethrins in
Soil and Sediment. Smithers Viscient Study No.: 14118.6101. 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.; 63 pages.
Final report issued June 8, 2017.

ILV: EPA MRID 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 50475403 & 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
50475403). 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. The specificity of the method was not supported by ILV
representative chromatograms. Linearity was not satisfactory for most of the
ILV and ECM analyses. It could not be determined if the ILV was provided
with the most difficult matrices with which to validate the method.

069001

Zoe Ruge, Physical Scientist Signature:

Document No.

Guideline:

Statements:

Classification:

PC Code:
EFED Final
Reviewers:

CDM/CSS-
Dynamac JV

Mohammed Ruhman, Ph.D.,
Senior Scientist

Lisa Muto, M.S.,
Environmental Scientist

Date:
Signature:

Date:

Signature:

Date:

9/27/2018
9/27/2018

4/26/18

Page 1 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Reviewers:

Kathleen Ferguson, Ph.D., Signature:
Environmental Scientist

Date:

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. 14118.6101, is designed for the quantitative
determination of Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) at the LOQ of 8.00 |ig/kg and of
Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II) at the LOQ of 6.54 |ig/kg 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
acceptability of the LOQs cannot be determined because the lowest toxicological level of concern in
soil is currently unknown. At this time, LOD/LOQ should be within the values specified in the
terrestrial field study guidance.1 The ECM and ILV used one characterized sediment and one
characterized soil matrices; matrices differed between the ECM and ILV. It could not be determined
if the ILV was provided with the most difficult matrices with which to validate the method since
terrestrial field dissipation studies were not referenced. The ILV validated the ECM in the first trial
for pyrethrins in soil with insignificant modifications of the analytical instrumentation and
parameters, as well as the use of Fisher acetonitrile; this was identified through previous ILV
experience as a critical component of the method. 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 ILV and ECM data was satisfactory regarding
accuracy and precision for all analytes; however, all ILV data regarding linearity and specificity
was unsatisfactory for all analytes.

1 URL: https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/nafta-guidance-document-conducting-
terrestrial-field

Page 2 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Table 1. Analytical Method Summary.

Analyte(s)

by
Pesticide1

MRID

EPA Review

Matrix

Method
Date

Registrant

Analysis

Limit of
Quantitation
(LOQ)

Environmental
Chemistry
Method

Independent
Laboratory
Validation

Pyrethrin I

50475403

50475401

Unacceptable

Soil/
Sediment2,3

8/6/17

Pyrethrin
Steering
Committee/
Joint
Venture
(PJV) c/o
Consumer
Specialty
Products
Association
Inc.

LC/MS/
MS

8.00 ng/kg

Pyrethrin II

6.54 ng/kg

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, natural freshwater sand sediment (Smithers Viscient Batch No. 102915-M-l; 88% sand 10% silt 2%
clay; pH 5.4 in 1:1 matrix:water, 3.3% organic carbon) obtained from Glen Charlie Pond, Wareham, Massachusetts,
and sand soil (Smithers Viscient Batch No. 012616A; 94% sand 6% silt 0% clay; pH 6.9 11:1 matrix:water, 0.70%
organic carbon) obtained from Sunny Nook Farms, Rochester, Massachusetts, were used (USDA soil textural
classification). Soil/sediment characterization was performed by Agvise Laboratories, Northwood, North Dakota.
Sand soil was labelled sandy loam soil.

3	In the ILV, and sediment (Golden Lake 0-5 cm; 93% sand 7% silt 0% clay; pH 8.1 in 1:1 soil:water; 2.7% organic
matter) obtained from Golden Lake, North Dakota, and loamy sand soil (DU#2 0-6"; 79% sand 12% silt 9% clay; pH
6.6 in 1:1 soil:water; 2.2% organic matter) obtained from Grand Forks, North Dakota, were used. Soil
characterization was performed by Agvise Laboratories, Northwood, North Dakota.

I.	Principle of the Method

Samples (5.00 g, dry weight) 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 or 10.0 mg/L:8.18 mg/L of Pyrethrin
LPyrethrin II) in acetonitrile (pp. 13-15 of MRID 50475403). The soil/sediment samples were
extracted twice with 20.0 mL of acetonitrile. The samples were shaken for 20 minutes on a shaker
table at 150 rpm then centrifuged at 3000 rpm for 10 minutes and transferred to a 50-mL volumetric
flask. The volume of the combined extracts was brought to 50 mL with acetonitrile. The samples
were further diluted 1:80 for LOQ and 1:267 for lOxLOQ with acetonitrile:water (50:50, v:v)
before analysis via LC/MS/MS.

Samples were analyzed by 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, 15-16 of MRID 50475403). 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

Page 3 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

retention times were reported as 4.2 minutes for pyrethrin I, 4.5 minutes for jasmolin 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 soil and sediment. 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. 17-18, 24; Table A-9, p. 36 of MRID 50475401).
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 of the water method validation (ILV
performed soil/sediment and water method validations in the same study report - water validations
were performed before soil/sediment validations; 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
for both water and soil/sediment methods.

In the ECM and ILV, the method Limits of Quantification (LOQs) in water were 8.00 |ig/kg for
Pyrethrin I (pyrethrin I, jasmolin I, and cinerin I) and 6.54 |ig/kg for Pyrethrin II (pyrethrin II,
jasmolin II, and cinerin II; pp. 18-20, 22-24 of MRID 50475403; pp. 6, 25 of MRID 50475401). In
the ECM and ILV, the method Limits of Detection (LOD) were 1.21 |ig/kg in soil and 1.03 |ig/kg in
sediment for Pyrethrin I and 1.46 |ig/kg in soil and 0.963 |ig/kg in sediment for Pyrethrin II.

II. Recovery Findings

ECM (MRID 50475403): 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 8.00 |ig/kg (LOQ) and 80.0 |ig/kg (lOxLOQ) and for analysis of
Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II) at 6.54 |ig/kg (LOQ) and 65.4 |ig/kg
(lOxLOQ) in soil and sediment matrices (Tables 1-4, pp. 27-30). 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. Natural freshwater sand sediment (Smithers Viscient
Batch No. 102915-M-l; 88% sand 10% silt 2% clay; pH 5.4 in 1:1 matrix:water, 3.3% organic
carbon) obtained from Glen Charlie Pond, Wareham, Massachusetts, and sand soil (Smithers
Viscient Batch No. 012616A; 94% sand 6% silt 0% clay; pH 6.9 I 1:1 matrix:water, 0.70% organic
carbon) obtained from Sunny Nook Farms, Rochester, Massachusetts, were used (USDA soil
textural classification; pp. 12-13). Soil/sediment characterization was performed by Agvise
Laboratories, Northwood, North Dakota. Sand soil was labelled sandy loam soil.

ILV (MRID 50475401): Mean recoveries and RSDs were within guideline requirements for
analysis of Pyrethrin I at 8.00 |ig/kg (LOQ) and 80.0 |ig/kg (lOxLOQ) and for analysis of Pyrethrin
II at 6.54 |ig/kg (LOQ) and 65.4 |ig/kg (lOxLOQ) in soil and sediment matrices (Tables 3-4, pp. 22-
23; Tables A-5 to A-8, pp. 32-35). LC/MS/MS Analytes were identified using one ion transition.
Sand sediment (Golden Lake 0-5 cm; 93% sand 7% silt 0% clay; pH 8.1 in 1:1 soil:water; 2.7%
organic matter) obtained from Golden Lake, North Dakota, and loamy sand soil (DU#2 0-6"; 79%
sand 12%) silt 9% clay; pH 6.6 in 1:1 soil: water; 2.2% organic matter) obtained from Grand Forks,
North Dakota, were used (p. 14; Appendix A, pp. 93-94). Soil characterization was performed by

Page 4 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Agvise Laboratories, Northwood, North Dakota. The ILV validated the ECM in the first trial for
pyrethrins in soil with insignificant modifications of the analytical instrumentation and parameters,
as well as the use of only Fisher-brand acetonitrile (pp. 17-18, 24-25; Appendix E, pp. 127-130).
ILV performed soil/sediment and water method validations in the same study report - water
validations were performed before soil/sediment validations. After the failure of the LOQ
fortification in the first trial of the water method validation, 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 for both the water and
soil/sediment methods. 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 Soil and Sediment.1'2'3

Analyte

Fortification
Level (jig/kg)

Number
of Tests

Recovery
Range (%)

Mean
Recovery (%)

Standard
Deviation (%)

Relative Standard
Deviation (%)



Natural Freshwater Sand Sediment

Pyrethrin I

8.00 (LOQ)

7

77.9-88.4

82.3

4.09

4.96

80.0

5

84.3-101

90.1

6.67

7.40

Pyrethrin II

6.54 (LOQ)

7

85.6-95.2

80.3

5.88

7.32

65.4

5

81.6-94.0

91.3

5.02

5.50



Sand Soil

Pyrethrin I

8.00 (LOQ)

7

73.4-89.7

91.3

3.83

4.20

80.0

5

85.5-97.9

88.4

4.80

5.43

Pyrethrin II

6.54 (LOQ)

7

76.3-97.2

89.7

7.11

7.92

65.4

5

89.3-97.5

92.0

3.34

3.63

Data (uncorrected recovery results, pp. 18-19) were obtained from Tables 1-4, pp. 27-30 of MRID 50475403.

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	Natural freshwater sand sediment (Smithers Viscient Batch No. 102915-M-l; 88% sand 10% silt 2% clay; pH 5.4 in
1:1 matrix:water, 3.3% organic carbon) obtained from Glen Charlie Pond, Wareham, Massachusetts, and sand soil
(Smithers Viscient Batch No. 012616A; 94% sand 6% silt 0% clay; pH 6.9 I 1:1 matrix:water, 0.70% organic carbon)
obtained from Sunny Nook Farms, Rochester, Massachusetts, were used (USDA soil textural classification; pp. 12-
13). Soil/sediment characterization was performed by Agvise Laboratories, Northwood, North Dakota. Sand soil was
labelled sandy loam soil.

Page 5 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Table 3. Independent Validation Method Recoveries for Pyrethrins as Pyrethrin I and
^rethrin II in Soil and Sediment.1'2'3 				

Analyte

Fortification
Level (jig/kg)

Number
of Tests

Recovery
Range (%)

Mean
Recovery (%)

Standard
Deviation (%)

Relative Standard
Deviation (%)



Sand Sediment

Pyrethrin I

8.00 (LOQ)

5

83.7-103

95.8

9.3

9.7

80.0

5

77.0-93.9

85.8

6.8

7.9

Pyrethrin II

6.54 (LOQ)

5

75.2-99.8

90.1

9.1

10.0

65.4

5

69.8-89.4

79.8

7.9

10.0



Loamy Sand Soil

Pyrethrin I

8.00 (LOQ)

5

71.5-89.8

83.7

7.4

8.8

80.0

5

89.4-103

96.9

4.9

5.0

Pyrethrin II

6.54 (LOQ)

5

81.2-85.5

82.7

1.7

2.0

65.4

5

93.4-103

98.2

3.8

3.9

Data (uncorrected recovery results, Tables A-5 to A-8, pp. 32-35) were obtained from Tables 3-4, pp. 22-23 and Tables

A-5 to A-8, pp. 32-35 of MRID 50475401 and DER Attachment 2.

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	Sand sediment (Golden Lake 0-5 cm; 93% sand 7% silt 0% clay; pH 8.1 in 1:1 soil:water; 2.7% organic matter)
obtained from Golden Lake, North Dakota, and loamy sand soil (DU#2 0-6"; 79% sand 12% silt 9% clay; pH 6.6 in
1:1 soil:water: 2.2% organic matter) obtained from Grand Forks, North Dakota, were used (p. 14; Appendix A, pp.
93-94). Soil characterization was performed by Agvise Laboratories, Northwood, North Dakota.

III. Method Characteristics

In the ECM and ILV, the method LOQs in water were 8.00 |ig/kg for Pyrethrin I (pyrethrin I,
jasmolin I, and cinerin I) and 6.54 |ig/kg for Pyrethrin II (pyrethrin II, jasmolin II, and cinerin II;
pp. 18-20, 22-24 of MRID 50475403; 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
1.21 |ig/kg in soil and 1.03 |ig/kg in sediment for Pyrethrin I and 1.46 |ig/kg in soil and 0.963 |ig/kg
in sediment 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 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Table 4. Method Characteristics for Pyrethrins in Soil and Sediment.

Analyte1

Pyrethrin I

Pyrethrin II

Limit of

Quantitation

(LOQ)

ECM

8.00 ng/kg

6.54 ng/kg

ILV

Limit of

Detection (LOD)

ECM

Nominal

Not reported



Calculated

1.21 |ig/kg (soil)
1.03 ng/kg (sediment)

1.46 ng/kg (soil)
0.963 ng/kg (sediment)

ILV2

1.21 |ig/kg (soil)
1.03 ng/kg (sediment)

1.46 ng/kg (soil)
0.963 ng/kg (sediment)

Linearity
(calibration curve
r2 and

concentration
range)

ECM

Soil

r2 = 0.99562

r2 = 0.99128



Sediment

r2 = 0.99642

r2 = 0.99394

ILV

Soil

r2 = 0.9948

r2 = 0.9939



Sediment

r2 = 0.9915

r2 = 0.9904

Range

0.05-0.50 ng/L

0.0409-0.409 (ig/L

Repeatable

ECM3

Yes at LOQ and lOxLOQ in one sediment and one soil matrices
(characterized).

ILV4-5

Reproducible

Yes at LOQ and lOxLOQ

Specific

ECM

Soil

Yes, matrix interferences were < 20% of the LOQ (based on peak area) for
each of the three components. Baseline noise interference at the LOQ was
significant in chromatograms of pyrethrin I/II and jasmolin I/II.

Sediment

Yes, matrix interferences were < 30% of the LOQ (based on peak area) for
each of the three components. Baseline noise interference at the LOQ was
significant in chromatograms of cinerin I/II and jasmolin I/II.

ILV

Soil

No, baseline noise interference at the LOQ was significant in
chromatograms of cinerin I/II and very significant (height ca. 50% of
LOQ peak height) in chromatograms of jasmolin I/II. Matrix interferences
were < 30% of the LOQ (based on peak area) for each of the three
components.

Sediment

No, baseline noise interference at the LOQ was very significant (height ca.
40-70% of LOQ peak height) in chromatograms of cinerin I/II, and
jasmolin I/II was barely distinguishable from the baseline noise at the
LOQ.6 Matrix interferences were < 6% of the LOQ (based on peak area)
for each of the three components.

Data were obtained from pp. 18-20, 22-24 (LOQ/LOD); Tables 1-4, pp. 27-30 (recovery results); Figures 1-20, pp. 31-
50 (chromatograms); Figures 21-24, pp. 51-54 (calibration curves) of MRID 50475403; pp. 6, 25 (LOQ/LOD); Tables
3-4, pp. 22-23 and Tables A-5 to A-8, pp. 32-35 (recovery results); Figures 5-8, pp. 42-45 (calibration curves); Figures
29-48, pp. 66-85 (chromatograms) of MRID 50475401. 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	In the ECM, natural freshwater sand sediment (Smithers Viscient Batch No. 102915-M-l; 88% sand 10% silt 2%
clay; pH 5.4 in 1:1 matrix:water, 3.3% organic carbon) obtained from Glen Charlie Pond, Wareham, Massachusetts,
and sand soil (Smithers Viscient Batch No. 012616A; 94% sand 6% silt 0% clay; pH 6.9 11:1 matrix:water, 0.70%
organic carbon) obtained from Sunny Nook Farms, Rochester, Massachusetts, were used (USDA soil textural
classification; pp. 12-13 of MRID 50475403). Soil/sediment characterization was performed by Agvise Laboratories,
Northwood, North Dakota. Sand soil was labelled sandy loam soil.

4	In the ILV, sand sediment (Golden Lake 0-5 cm; 93% sand 7% silt 0% clay; pH 8.1 in 1:1 soil:water; 2.7% organic
matter) obtained from Golden Lake, North Dakota, and loamy sand soil (DU#2 0-6"; 79% sand 12% silt 9% clay; pH
6.6 in 1:1 soikwater; 2.2% organic matter) obtained from Grand Forks, North Dakota, were used (p. 14; Appendix A,

Page 7 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

pp. 93-94 of MRID 50475401). Soil characterization was performed by Agvise Laboratories, Northwood, North
Dakota.

5	The ILV validated the ECM in the first trial for pyrethrins in soil with insignificant modifications of the analytical
instrumentation and parameters, as well as the use of only Fisher-brand acetonitrile (pp. 17-18, 24-25; Appendix E,
pp. 127-130 of MRID 50475401). ILV performed soil/sediment and water method validations in the same study
report - water validations were performed before soil/sediment validations. After the failure of the LOQ fortification
in the first trial of the water method validation, 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
for both the water and soil/sediment methods. 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.

6	Based on Figure 42, p. 79 and Figure 47, p. 84 of MRID 50475401.

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. ILV performed soil/sediment and water method validations in the same study
report - water validations were performed before soil/sediment validations. After the failure
of the LOQ fortification in the first trial of the water method, 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 for both the water (second trial) and soil/sediment (first
trial) methods. 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.	The specificity of the method was not supported by ILV representative chromatograms
(Figures 29-48, pp. 66-85 of MRID 50475401). Although matrix interferences were < 30%
of the LOQ (based on peak area) for each of the three components in both the soil and
sediment matrices, the height of baseline noise interference at the LOQ ranged from ca. 30-
90% of the LOQ peak height for cinerin I/II and jasmolin I/II. This level of baseline noise
interference at the LOQ interfered with peak integration confidence, especially when the
analyte peak was barely distinguishable from the baseline noise. The reviewer noted that the
extraction procedure was fairly simple; the reviewer did not know if the use of additional
SPE clean-up processes had been attempted in order to increase resolution of the analyte
signals.

3.	ILV linearity was not satisfactory for any analyses: Pyrethrin I in soil (r2 = 0.9948) and
sediment (r2 = 0.9915); and Pyrethrin II in soil (r2 =0.9939) and sediment (r2 = 0.9904;
Figures 5-8, pp. 42-45 of MRID 50475401; DER Attachment 2).

Page 8 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

ECM linearity was not satisfactory for Pyrethrin II in soil (r2 = 0.99128) and sediment (r2 =
0.99394; Figures 21-24, pp. 51-54 of MRID 50475403).

Linearity is satisfactory when r2 > 0.995.

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 CFRPart 136 (pp. 18-20, 22-24 of MRID 50475403;
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 significant typographical error in the ECM: LODs were
not reported correctly in the method summary tables on pages 23-24 (pp. 23-24 of MRID
50475403). These LODs were not placed correctly according to p. 20 of MRID 50475403
(ECM) and p. 6 of MRID 50475401 (ILV); the reviewer reported the LODs based on these
values.

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.

Page 9 of 13


-------
Pvrethrins (PC 069001)	MRIDs 50475403/50475401

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 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

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-l-enyl)cyclopropane-l-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 11 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Jasmolin I

IUPAC Name:

CAS Name:
CAS Number:
SMILES String:

Pyrethin 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)-2,2-

dimethyl-3-(2-methylprop-l-enyl)cyclopropane-l-carboxylate

Not reported

4466-14-2

CC/C=C\CC 1=C([C@H](CC 1=0)0C(=0)[C@@H]2[C@H](C2(C)C)C=C(
C)C)C

[(lS)-2-methyl-4-oxo-3-[(2E)-penta-2,4-dienyl]cyclopent-2-en-l-yl]

(lR,3R)-3-[(E)-3-methoxy-2-methyl-3-oxoprop-l-enyl]-2,2-

dimethyl cyclopropane-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)0C)C/C=C/C=C

Page 12 of 13


-------
Pvrethrins (PC 069001)

MRIDs 50475403/50475401

Cinerin II
IUPAC Name:

CAS Name:
CAS Number:
SMILES String:

[(1 S)-3-[(Z)-but-2-enyl]-2-methyl-4-oxocyclopent-2-en-1 -yl] (lR,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 -methoxy-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 13 of 13


-------