MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

1 Reference

Fischer, D.R., and Jerkins, E. (2015) Determination of the Residues of Imidacloprid, 5

- Hydroxy

Imidacloprid, and Imidacloprid Olefin in Bee Relevant Matrices Collected from Cotton During Two

Successive Years: Final Report. Project Number: EBNTN011. Unpublished study prepared by Bayer

CropScience 632pg. MRID 4 9665202, CDPR study ID 285681, Data Volume 51950

- 0900,

Tracking ID# 270950



### 1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material : Admire Pro Systemic

Protectant (SC) Purity : 43.50% a.i. w.w.

Study Type: Non

- Guideline field residue study on cotton to establish imidacloprid and

metabolite levels in pollen, nectar (floral and extra floral )

, and leaves following

four applications in each of two successive years and three different soil types

(fine, medium, and coarse)

.

Sponsor: Bayer CropScience

2T.W. Alexander Drive

Research Triangle Park, NC

USA 27709 Experiment Start and

End Date: April 12, 2013

-

April 9, 2015

Sponsor Study

ID: EBNTN011

Study Location s : 9 Field Trials in the

cities of :

Davis, Fresno, Kerman,

Sanger, Wheatland, and

Yuba City , California Study

Completion

Date: June 19, 2015

GLP Status: GLP

- compliant; protocol reviewed by EPA, PMRA, CDPR , CDPR study ID 266879

, Data Volume 51950

- 0 808 , Tracking ID# 25 4696

. ]



### 2. REVIEWER INFORMATION

Primary Reviewer s :

California Department

of Pesticide

Regulation John Troiano, Ph.D. , Research Scientist III, Environmental Monitoring

Branch

Richard Bireley , Senior Environmental Scientist (Specialist), Ecotoxicology

Group, Pesticide Registration Branch

Denise Alder , Senior Environmental Scientist (Specialist), Lead Reevaluation

Coordinator, Pesticide Registration Branch

Russell Darling , Environmental Scientist, Reevaluation Coordinato r,

Pesticide Registration Branch



### 3. EXECUTIVE SUMMARY

A total of nine (9) field trials were conducted to measure the magnitude of imidacloprid residues in bee

relevant cotton pollen

, nectar

, and in/on leaves following four applications of Admire Pro Systemic

Protectant

, EPA Reg. No. 264

- 827 in each of two successive years. Admire Pro Systemic Protectant i s a

suspension concentrate formulation c ontaining 550 g/L imidacloprid. Three (3) trials have one year of

data and six (6) trials have two years of data.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

2 Treated p lot s received one soil (in

- furrow) spray application of Admire Pro at planting (BBCH 00: dr y

seed) followed by 3 equivalent Admire Pro foliar spray applications per planting season. Individual soil

application rates ranged from 0.37 to 0.38 kg imidacloprid/ha per application (0.33 to 0.34 lb/A), and

spray volumes were 13 to 15 gal/A. The interva l between the soil and first foliar application was 75 to

99 days. Individual foliar application rates ranged from 0.063 to 0.067 kg imidacloprid/ha per application

(0.056 to 0.060 lb/A). Foliar and soil applications were made using ground

- based equipment . Also, the

adjuvant Dyne

- Amic (0.25% v/v) was used in all foliar applications. Moreover, all foliar applications were

made between BBCH growth stages 61 and 72 (BBCH 61: beginning of flowering; BBCH 72: about 20% of

bolls have attained their final size). The interval between foliar applications was 6 to 7 days. The foliar

spray volumes ranged from 14 to 20 gal/A. Total seasonal application rates ranged from 0.56 to 0.57 kg

imidacloprid/ha (0.50 to 0.51 lb/A).

Cotton leaf and flower samples were collected at three sampling intervals: 4 to 5 days prior to the first

foliar application (70 to 95 days after the soil application), 4 to 5 days after the last foliar application,

and 12 to 14 days after the last foliar application. At each sampling interval, duplic ate composite

samples (two separate runs through the plot) of cotton flowers and cotton leaves were collected from

the treated plots when the plants were at bloom, BBCH 61 (begin flowering, early bloom) to BBCH 73

(about 30% of bolls have attained their fi nal size). Single composite samples of cotton leaves and flowers

were collected from the control plot of each trial on the same days that samples were collected from

the treated plots.

After their collection, cotton flowers were hand

- processed at the fiel d site to obtain the bee

- relevant

samples of cotton pollen, floral nectar, and extra floral nectar. The processed flowers were discarded.

The residues of Admire Pro Systemic Protectant (imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid

olefin) were qu antitated by high performance liquid chromatography/triple stage quadrupole mass

spectrometry (LC/MS/MS) and LC/high resolution mass spectrometry (LC/HRMS) using stable

isotopically labeled internal standards. The individual analyte residues were summed to give a total

imidacloprid residue.

Storage stability studies indicate that the imidacloprid residues would have been stable during frozen

storage for at least 1

, 080 days (36 months) in cotton leaves prior to analysis. Transit spikes showed that

imidaclop rid residues were stable in pollen and nectar for the duration of the study. The maximum

storage period of frozen samples in this study for Admire Pro Systemic Protectant was 569 days for

cotton leaves, 226 days for cotton pollen, and 211 days for cotton f loral and extrafloral nectar.



### 4. STUDY VALIDITY

Guideline Followed: Non

- guideline study (protoco l was reviewed by EPA/PMRA/CDPR

Guideline Deviations: N/A

Other Deviations: N/A

Classification: Acceptable/ Quantitative

Rationale: The data from the study will provide a basis for developing a

quantitative assessment of exposure levels to bees that can be used in a

risk assessment scenario.

Reparability: N/AMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

3 

### 5. MATERIALS AND METHODS

Test Material Characterization

Test item: Admire Pro Systemic Protectant

(Imidacloprid) 550 g a

. i

. /L SC CAS #: 138261

- 41

- 3

Description: Suspension concentrate (SC) Purity: 43.50% w/w

Lot No./Batch No. Batch No. NK41CX0578 Density: 1.41

– 1.54 g/mL

Material Source: Bayer CropScience Cert. # 213CJ2446

Material Receipt

Date: Not Reported Analysis

Date: 12/03/2012

Expiration Date: 12/03/2014 Solubility: 0.51 to 0.61 g/L

Storage of Test

Mat erial : Ambient (35

- 86oF)

except trials NT001

- 13ZB,

NT002

- 13ZB, and NT005

- 13ZB

when the temperature briefly

reached 94oF. Sample

Storage: - 47oC to

- 11oC

- 116oF to

- 52 oF

5A. S TUDY D ESIGN

This study requirement was part of the imidacloprid special review at the California Department of

Pesticide Regulation (CDPR). The study design and protocol were approved by the CDPR prior to study

initiation. This study was conducted using GLP standards and following an approved protocol. The study

initiation date was April 12, 2013. The experimental start date was May 10, 2013 (first application), and

the experimental end date was April 09, 2015 (l ast analysis).

Two plots were included in each trial, to be planted and treated in each of two consecutive years. Only

the first year of trial NT002

- 13ZB could be completed and reported because the plot location was no

longer available. Trials NT001

- 13ZB and NT005

- 13ZB were restarted in 2014, so only first year data from

these trials are reported. The second year of data for the two fine soil trials is anticipated in January

2016.

5 B

. A PPLICATION T IMING AND R ATES

The full study report provides (1) C hronological listing of significant study dates ( Appendix 1 ); (2) F ield

report summaries for each trial detailing the actual amount of test substance applied, plot sizes, dates of

treatment, dates of sample collection, maintenance chemicals, climatic data, and irrigation data

( Appendix 2 ); and (3) Q uality assurance statements for each trial ( Appendix 3 )

. Information on

application timing is provided in T able 1

. Soil and meteorological characteristics of the study sites are

provided in Table 2 and Table 3

. Table 4 provides the sampling dates and cotton developmental stages

.

BBCH or Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie

, identifies the specific

phenological development stages of cotton

. Plot TRTD received one soil (in

- furrow) spray application of

Admire Pro at planting (BBCH 00: dry seed) followed by 3 equivalent Admire Pro foliar spray applications

per planting season. Individual soil application rates ranged from 0.37 to 0.38 kg imidacloprid/ha per

application (0.33 to 0.34 l b/A), and spray volumes were 13 to 15 gal/A. The interval between the soil and

first foliar application was 75 to 99 days. Individual foliar application rates ranged from 0.063 to 0.067 kg

imidacloprid/ha/application (0.056 to 0.060 lb/A). All foliar appli cations were made between BBCH

growth stages 61 and 72 (BBCH 61: beginning of flowering; BBCH 72: about 20% of bolls have attainedMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

4 their final size). The interval between foliar applications was 6 to 7 days. The foliar spray volumes ranged

from 14 to 20 ga l/A. Total seasonal application rates ranged from 0.56 to 0.57 kg imidacloprid/ha

(0.50 to 0.51 lb/A).

Temperature and precipitation data were recorded for each trial and are summarized in Appendix 2 of

the study report EBNTN011 . Temperatures recorded du ring the field phase of the study were similar to

average historical records. Recorded rainfall was lower than historical records across all trials, but this

would not affect the conclusions of the study. Irrigation supplemented normal ra infall as needed i n

some trials

. CDPR requested that the trial sites be distributed as three coarse, three medium, and three

fine textured soils [ per USDA’s Soil Survey Geographic database ( SSURGO ) mapping units ] ; however, due

to various issues, the final trials contained four coarse, three medium and two fine textured sites (in

some cases, the plots were shifted a short distance within the designated field, but this placed the plot

in a coarse texture mapping unit instead of the intended medium texture area). The two

- year study

includes one year of residue data from each of two fine texture fields, one to two years of data from the

three medium texture field s , and one to two years of data from four coarse texture fields

. See Table 3

for more site specific soil information

Table 1. Summary of foliar and soil application rates and timing *

.TrialIdentificationLocation(City,State,NAFTARegion)FormulationPlotNameYear Application aMethodTiming/GrowthStage(BBCH)ActualSprayVolume,GPA(L/ha)Rate,lba.i./A(kga.i./ha)RetreatmentInterval(days)TotalRate,lba.i./A(kga.i./ha)Adjuvant

N T 001

-

13 Z B b Y u b a C i t y

, CA

Reg i on 10 A d m i r e

P r o T R T D 2014 S o i l

S p r a y 00 14

( 133) 0.3 2 8

( 0.368) N A c 0.50

( 0.56) NA

F o l i ar

S p r a y 61 16

( 150) 0.0 5 8

( 0.065) 93 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 16

( 147) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 16

( 148) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

N T 002

-

13 Z B b W h eat l an d

, C A

Reg i on 10 A d m i r e

P r o T R T D 2014 S o i l

S p r a y 00 15

( 142) 0.3 3 1

( 0.371) NA 0.50

( 0.56) NA

F o l i ar

S p r a y 65 15

( 142) 0.0 5 7

( 0.064) 92 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 69 15

( 144) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 72 15

( 140) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / vMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

5TrialIdentificationLocation(City,State,NAFTARegion)FormulationPlotNameYear Application aMethodTiming/GrowthStage(BBCH)ActualSprayVolume,GPA(L/ha)Rate,lba.i./A(kga.i./ha)RetreatmentInterval(days)TotalRate,lba.i./A(kga.i./ha)Adjuvant

N T 003

-

13 Z A Fr e s no, CA

Reg i on 10 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 14

( 132) 0.3 3 0

( 0.370) NA 0.51

( 0.57) NA

F o l i ar

S p r a y 65 15

( 142) 0.0 5 8

( 0.066) 81 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 140) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 142) 0.0 5 8

( 0.066) 7 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 14

( 132) 0.3 3 0

( 0.370) 254 0.51

( 0.57) NA

F o l i ar

S p r a y 61 15

( 141) 0.0 5 8

( 0.065) 77 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 142) 0.0 5 9

( 0.066) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 16

( 145) 0.0 6 0

( 0.067) 7 D y n e

- A m ic

0.25 % v / v

N T 004

-

13 Z A Da v i s

, CA

Reg i on 10 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 14

( 127) 0.3 3 2

( 0.372) NA 0.51

( 0.57) NA

F o l i ar

S p r a y 61 15

( 142) 0.0 5 8

( 0.065) 91 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 15

( 140) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 14

( 136) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 15

( 139) 0.3 2 7

( 0.367) 253 0.50

( 0.56) NA

F o l i ar

S p r a y 61 14

( 135) 0.0 5 7

( 0.064) 99 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 17

( 156) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 16

( 148) 0.0 5 7

( 0.063) 7 D y n e

- A m ic

0.25 % v / v

N T 005

-

13 Z B b Y u b a C i t y

, CA

Reg i on 10 A d m i r e

P r o T R T D 2014 S o i l

S p r a y 00 15

( 143) 0.3 3 7

( 0.377) NA 0.51

( 0.57) NA

F o l i ar

S p r a y 61 15

( 142) 0.0 5 6

( 0.063) 91 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 144) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 142) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / vMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

6TrialIdentificationLocation(City,State,NAFTARegion)FormulationPlotNameYear Application aMethodTiming/GrowthStage(BBCH)ActualSprayVolume,GPA(L/ha)Rate,lba.i./A(kga.i./ha)RetreatmentInterval(days)TotalRate,lba.i./A(kga.i./ha)Adjuvant

N T 006

-

13 Z A S a n ge r

, C A

Reg i on 10 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 13

( 118) 0.3 2 8

( 0.368) NA 0.50

( 0.56) NA

F o l i ar

S p r a y 61 16

( 146) 0.0 5 8

( 0.065) 95 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 15

( 142) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 144) 0.0 5 8

( 0.065) 6 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 13

( 125) 0.3 3 2

( 0.372) 249 0.51

( 0.57) NA

F o l i ar

S p r a y 61 19

( 179) 0.0 5 8

( 0.065) 77 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 19

( 181) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 20

( 185) 0.0 5 7

( 0.064) 6 D y n e

- A m ic

0.25 % v / v

N T 007

-

13 Z A Fr e s no, CA

Reg i on 10

2013 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 14

( 132) 0.3 3 1

( 0.372) NA 0.50

( 0.56) NA

F o l i ar

S p r a y 61 15

( 141) 0.0 5 7

( 0.064) 81 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 140) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 14

( 132) 0.3 3 0

( 0.369) 262 0.50

( 0.56) NA

F o l i ar

S p r a y 61 15

( 140) 0.0 5 7

( 0.064) 75 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 15

( 140) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / vMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

7TrialIdentificationLocation(City,State,NAFTARegion)FormulationPlotNameYear Application aMethodTiming/GrowthStage(BBCH)ActualSprayVolume,GPA(L/ha)Rate,lba.i./A(kga.i./ha)RetreatmentInterval(days)TotalRate,lba.i./A(kga.i./ha)Adjuvant

N T 008

-

13 Z A K e r m an, CA

Reg i on 10 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 14

( 130) 0.3 2 8

( 0.368) NA 0.50

( 0.56) NA

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 83 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 142) 0.0 5 8

( 0.065) 7 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 14

( 130) 0.3 2 6

( 0.365) 226 0.50

( 0.56) NA

F o l i ar

S p r a y 61 15

( 138) 0.0 5 6

( 0.063) 83 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 140) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

N T 009

-

13 Z A K e r m an, CA

Reg i on 10 A d m i r e

P r o T R T D 2013 S o i l

S p r a y 00 14

( 132) 0.3 3 1

( 0.371) NA 0.50

( 0.56) NA

F o l i ar

S p r a y 61 15

( 139) 0.0 5 6

( 0.063) 83 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

2014 S o i l

S p r a y 00 14

( 131) 0.3 3 0

( 0.369) 225 0.50

( 0.56) NA

F o l i ar

S p r a y 61 15

( 141) 0.0 5 7

( 0.064) 83 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 61 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

F o l i ar

S p r a y 65 15

( 141) 0.0 5 7

( 0.064) 7 D y n e

- A m ic

0.25 % v / v

a Values for spray volume, rate, and total rate have been rounded using values provided in Appendix 2

of the study report.

b Trials NT001

- 13ZB and NT005

- 13ZB were restarted in 2014, so only one year of test substance

application data are recorded. Only the first year of trial NT002

- 13ZB will be reported because the

plot location was no longer available.

c NA = Not applicable.

*Table 4 of the study reportMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

8 Table 2. Soil and meteorological characteristics of the study sites*.

T r ial

ID a T r ial Lo ca t i o n ( Ci t y

, C ount r y/ S t a t e

, Y ea r

, G P S

C oo r d i n a t es b ) S o il C h a r a c t e r i s t ics c M e t e o r o l og ical Da t a d

Variety OM ( % ) p H C E C

( me q /

100 g

s o il) %

S a n d %

S ilt %

Cl a y T y p e T ot al

Rai nf all

( i n ) T e mp . Ra ng e

( ° F )

N T 001

-

13 Z B Y u b a C i t y

, C A

, 2 0 14

( 38°5 9 .0 5 5’ N, 1 2 1° 3 6. 1 15’ W) 1.0

- 2.0 6.6

-

8.4 20

- 25 35 34 31 C l a y

l oam 0.92 47 97 Pima

N T 002

-

13 Z B W h eat l an d

, C A

, 2 014

( 39°0 0 .2 2 ’ N, 1 2 1° 2 7.59 W ) 0.5

- 2.0 6.1

-

7.8 5

- 15 71 17 12 S a ndy

Loam 0.92 47 97 Pima

N T 003

-

13 Z A Fr e s no, C A

, 2 0 13 †

- 2 014 ‡

( 36.7 3 62 8

, - 11 9 .8 7 515) 0.7 7.7 9.1 71 20 9 S a ndy

Loam 3.92 30 102 DP 358 RF †

PHY 804 RF ‡

N T 004

-

13 Z A Da v i s

, C A

, 2 0 13

- 2 014

( 38.5 3 37,

- 1 21

. 77 9 3) 1.8 7.4 14.4 35 40 25 Loam 14. 9 3 32 93 Pima

N T 005

-

13 Z B Y u b a C i t y

, C A

, 2 0 14

( 39°0 2 .2 4 ’ N, 1 2 1° 3 7.6 3 ’ W ) 1.0

- 2.0 6.6

-

8.4 20

- 25 35 34 31 C l a y

l oam 0.92 47 97 Pima

N T 006

-

13 Z A S a n ge r

, C A

, 2 0 1 3

- 20 1 4

( 36.6 9 98 2

, - 11 9 .4 6 196) 0.3 4.8 0.7 66 23 11 S a ndy

Loam 5.69 31 99 Pima

N T 007

-

13 Z A Fr e s no, C A

, 2 0 13

- 2 014

( 36.7 3 91 6

, - 11 9 .8 7 599) 0.5 7.2 11.2 65 24 11 S a ndy

Loam 3.92 30 102 PHY 802 RF

N T 008

-

13 Z A K e r m an, C A

, 20 1 3

- 20 1 4

( 36.7 9 55 2

, - 12 0 .0 5 406) 0.5 6.9 5.2 77 20 3 Loa m y

s and 2.74 29 99 Delta Pine

358 RF

N T 009

-

13 Z A K e r m an, C A

, 20 1 3 †

- 20 1 4 ‡

( 36.7 9 51 6

, - 12 0 .0 5 676) 0.6 7.6 4.9 81 18 1 Loa m y

s and 2.74 29 99 Delta Pine

358 RF †

PHY 802 RF ‡

a Site conditions listed are for the TRTD plot. For UTC plot conditions, see Appendix 2 of the study report.

b GPS coordinates are in the form (latitude, longitude).

c The soil characteristics for trials NT001

- 13ZB, NT002

- 13ZB, and NT005

- 13ZB were pulled from the USDA

- NRCS

web soil survey of the respective county by the principal field investigator (PFI). For all other trials, the soil

characteristics are based on analyses of soil samples collected from within approximately 500 ft of the treated

plot. Abbreviations used: %OM = percent organic matter; CEC = cation exchange capacity.

d Data is for the interval of the month of first application through the month of last sampling.

Meteorological data were obtained from nearby government weather stations.

* Combined table f rom Table 3 A and Table 8 o f the study report.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

9 Table 3 . S SURGO soil characteristics of the study sites*.

Tri a l ID Tri a l

Lo ca tion G e n e r a l T e x ture

D esc ription

(SSUR GO ) CDPR

T ex ture

C a t e go r y Co m pon e nt

% a Dr a in a ge

Cl ass H y drologic

G roup Runoff P a rti c le

Si z e

N T 001

-

13 ZB Y ub a C i t y

, CA M a r cum

- G r idley

C la y Loa m

, 0

- 1 % slo p es F in e 4 5 / 4 0 M ode r a t el y

W el l Dr a in ed C / D Lo w /

M edium F in e

N T 002

-

13 ZB W he a t lan d

, CA C olu m bi a F ine

S and y Lo a m

, 0

- 1 % slo p es (~ 10 % K im b al l

Loam ) Medium 85 S ome w hat

P oo r l y Dr ained A V e ry Low C oa r se

loam y

N T 003

-

13 ZA Fr es n o , CA R amon a L o am Medium 80 W el l Dr a in ed C Low F ine

loam y

N T 004

-

13 ZA D a v is , CA R ei ff V e ry F i ne

S and y Lo a m Medium 85 W el l Dr a in ed A V e ry Low C oa r se

loam y

N T 005

-

13 ZB Y ub a C i t y

, CA M a r cum

- G r idley

C la y Loa m

, 0

- 1 % slo p es F in e 4 5 / 4 0 M ode r a t el y

W el l Dr a in ed C / D Lo w /

M edium F in e

N T 006

-

13 ZA S ange r , CA H an f o rd f ine

sand y l oa m

,

g r a v ell y

su b s tr a t um C oa r s e 85 W el l Dr a in ed A V e ry Low C oa r se

loam y

N T 007

-

13 ZA Fr es n o , CA G r een f i e l d

S and y Lo a m

, 0

- 3 % slo p es C oa r s e 85 W el l Dr a in ed A V e ry Low C oa r se

loam y

N T 008

-

13 ZA K e r ma n , CA H an f o rd C o a r s e

S and y Lo a m C oa r s e 85 W el l Dr a in ed A V e ry Low C oa r se

loam y

N T 009

-

13 ZA K e r ma n , CA H an f o rd S andy

Loa m

, s il ty

su b s tr a t um C oa r s e 85 W el l Dr a in ed A V e ry Low C oa r se

loam y

a Ma j or c o m ponent (s ) of the s o i l as a p e rc ent a ge of to t al s o il ; f or Ma rc u m

- Gr i d l e y : M a rc um i s

45 %

, h y d r o l o g i c g r o up C, a nd Lo w r u no ff ; Gr i d l e y i s 40 %

, h y d r o l o g i c g r o up B , and M e d i um r unof f

. * From Table 3B of the study report

.

5D. S AMPLE C OLLECTION

, H ANDLING

, P ROCESSING

Cotton Plant Matrices.

Cotton leaf and flower samples were collected at three sampling intervals. The first samples were

collected at early bloom, prior to any foliar sprays, to measure residues in bee

- relevant matrices as a

result of the at

- plant soil application. The second and third sampling intervals measured residues that

were a result of the soil application at planting plus three additional at

- bloom sprays. The first, second,

and third sampling intervals corresponded to 4 to 5 days prior to the first foliar application (70 to 95

days after the soil application), 4 to 5 days after the last foliar application, and 12 to 14 days after the

last foliar application, respectively ( Table 4 ). At each sampling interval, duplicate composite samples

(two separate runs through the plot) of cot ton flowers and cotton leaves were collected from the

treated plots when the plants were at bloom, BBCH 61 (begin flowering, early bloom) to BBCH 73 (about

30% of bolls have attained their final size). Single composite samples of cotton leaves and flowers wereMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

10 collected from the control plot of each trial on the same days that samples were collected from the

treated plots.

Cotton flowers and leaves were collected by hand into Ziplock bags. Each composite flower sample

contained a minimum of 125 g (minimum 250 blossoms) collected from at least 12 different areas of the

plot, avoiding the edges. Each composite leaf sample contained a minimum of 100 g.

After their collection, cotton flowers were hand

- processed at the field site to obtain the bee

- relevant

sam ples of cotton pollen, floral nectar, and extrafloral nectar. Processing occurred the same day as

flower collection, and all available matrix were collected. Extrafloral nectar from the sub bracteal and

inner bracteal nectaries was removed using a micropip ette and placed into a pre

- weighed amber glass

collection vial. Nectar from the floral nectary was removed by micropipette and placed in a separate

vial. Pollen was removed from the cotton blossoms either by vacuum aspiration with collection in filter

tips or by tapping the pollen from the blossoms onto wax paper and collection of the accumulated

pollen into a vial. All resulting nectar and pollen samples were labeled and placed in the freezer

immediately after they were generated. After processing was comp leted, the flowers were discarded.

Figures 4 through 6 provide trends in total imidacloprid residue measured in extrafloral nectar, pollen,

and leaf tissue at each site. Figure 7 depicts the relationship between concentration of total

imidacloprid residue s measured in leaves and floral nectar parsed out by soil type.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

11 Table 4 . Sampling dates and cotton develop mental stages for plant residue analysis *

Pollen, Nectar

, Extrafloral Nectar, and Leaf Sampling

Year 1 Year 2 Year 1 Year 2

BBCH † Dates ‡ BBCH † Dates ‡ BBCH † Dates ‡ BBCH † Dates ‡

Davis 1 = NT004

- 13ZA Sanger 1 = NT006

- 13ZA

61 86 DASA,

- 5 DA1FA 61 95 DASA,

- 4 DA1FA 61 90 DASA,

- 5 DA1FA 61 73 DASA,

- 4 DA1FA

61 5 DA3FA 65 4 DA3FA 65 4 DA3FA 65 4 DA3FA

61 13 DA3FA 67 13 DA3FA 65 14 DA3FA 67 14 DA3FA

Fresno 1 = NT003

- 13ZA Wheatland = NT002

- 13ZB

65 7 6 DASA,

- 5 DA1FA 61 72 DASA,

- 5 DA1FA 65 88 DASA,

- 4 DA1FA NC 1 NC

67 5 DA3FA 65 5 DA3FA 69 5 DA3FA NC NC

67 14 DA3FA 67 14 DA3FA 73 14 DA3FA NC NC

Fresno 2 = NT007

- 13ZA Yuba City 1 = NT001

- 13ZB

65 7 6 DASA,

- 5 DA1FA 61 70 DASA,

- 5 DA1FA 61 88 DASA,

- 5 DA1FA TBC 2 TBC

65 5 DA3FA 65 5 DA3FA 61 5 DA3FA TBC TBC

67 14 DA3FA 67 14 DA3FA 61 14 DA3FA TBC TBC

Kerman 1 = NT008

- 13ZA Yuba City 2 = NT005

- 13ZB

65 78 DASA,

- 5 DA1FA 61 78 DASA,

- 5 DA1FA 61 86 DASA,

- 5 DA1FA TBC TBC

65 4 DA3FA 65 4 DA3FA XX 3 5 DA3FA TBC TBC

67 14 DA3FA 67 12 DA3FA 67 14 DA3FA TBC TBC

Kerman 2 = NT009

- 13ZA

65 78 DASA,

- 5 DA1FA 61 78 DASA,

- 5 DA1FA

65 4 DA3FA 65 5 DA3FA

67 14 DA3FA 67 14 DA3FA

† BBCH = B iologische Bundesanstalt, B undessortenamt und CH emische Industrie growth stage scale for c otton

. ‡ DASA = Days after the soil (in

- furrow) application; DA1FA = days after the first foliar application; DA3FA = days

after the third foliar (and last) application. A negative number designates days prior to the indicated application.

1 NC = Not collected; no year 2 samples from trial NT002

- 13ZB can be collected.

2 TBC = To Be Collected; the samples will be collecte d when the second year of the trial is performed.

3 BBCH XX indicates no growth stage reported in the field data summary.

* Combination of Appendix 1 and Appendix 4 (Section 6) of the study report.

Sample Storage

.

Composite samples of cotton leaves, pollen, floral nectar, and extrafloral nectar were placed into

labeled (study number and sample number) containers for shipment. All samples were frozen within

4 hours of collection with the exception of certain samples in trial NT004

- 13ZA, which were frozen

within 9 hours and 1 minute. Samples remained frozen until receipt at Bayer CropScience in RTP, NC.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

12 5E. A NALYTICAL M ETHODS

The analytical methods 1

- 2 used in this study measured the residues of imidacloprid, 5

- hydroxy

imidacloprid, and imidacloprid olefin in cotton leaves, floral nectar, extra floral nectar, and pollen. These

data are reported in Appendix 4 of the full study report titled, “ Analytical Report for EBNTN011

Determination of the Residues of Imidacloprid, 5

- Hydroxy Imidacloprid, and Imidacloprid Olefin in Bee

Relevant Matrices Collected from Cotton During Two Successive Years.”

For the cotton leaves, a 2.5

- g sample was weighed into a 50

- mL polypropylene conical centrifuge tube,

and 10 mL HPLC

- grade water was added. The tube was mixed manually for 1 min, followed by adding

20 mL of acetonitrile and shaking for an additional 1 min. Then, 3 g of MgSO4 and 1.5 g of NaCl were

added. The sample w as amended with a mixed internal standard solution and mixed manually for 1 min.

For leaf samples which were found to contain high residues of imidacloprid (>2 ppm), as determined by

an initial run in which the response exceeded the calibration curve, the sample was amended with a 10X

mixed internal standard solution before the salts were added. The sample was centrifuged. For samples

containing low levels of imidacloprid residue, 20 mL of organic supernatant was transferred into a

separate 50

- mL polypropyl ene conical centrifuge tube containing 0.3 g of Bondesil

- PSA and 1.8 g of

MgSO4. For samples containing high levels of imidacloprid residue, 2.0 mL of organic supernatant and

18.0 mL of acetonitrile were transferred into a separate 50

- mL polypropylene coni cal centrifuge tube

containing 0.3 g of Bondesil

- PSA and 1.8 g of MgSO4, which was manually mixed for 1 min. The sample

extract was centrifuged, and a 1.25 mL aliquot of supernatant was transferred into a clean culture tube.

The sample aliquot was evaporat ed to near dryness using a Turbo

- Vap (Biotage, Charlotte, NC). The solid

was reconstituted with 1.25 mL of 9:1 water/MeOH containing 10 mM NH4HCO3 by vortexing, and the

resulting solution was transferred into a 2

- mL sample vial for LC/MS/MS analysis.

For nectar, a 0.1

- mL sample was weighed into a 20 x 150 mm culture tube and dissolved in 4 mL of

water. If the total sample volume was less than 0.1 mL, the entire sample was weighed and recorded.

The mixture was amended with isotopically labeled internal stan dards, mixed well, and applied to an

Agilent BondElut SPE cartridge (50 mg resin; previously conditioned with methanol then water). The

cartridge was washed with 1 mL of MeOH/H2O (1:19 v/v), and the combined eluates were discarded.

The analytes were eluted from the cartridge with 0.5 mL of MeOH/H2O (1:4 v/v). The eluate was

collected in a 2 mL sample vial for analysis by LC/MS/MS.

For pollen, a 0.1

- g sample was weighed into a 2

- mL centrifuge tube containing 2.8 mm steel balls. If an

individual sample volum e was not sufficient for analysis, the two samples collected at the same interval

were composited and analyzed. The composite sample was weighed and recorded. A 1

- mL portion of

methanol/water (3:1 v/v) was added, and the mixture was homogenized with a bead mixer at 5000

beats/min for 1 min on a Precellys homogenizer (Bertin Technologies, Rockville, MD). The isotopically

labeled internal standards were added and mixed, and the mixture was centrifuged at 12,000 rpm for 2

min. The supernatant was transferred i nto a clean culture tube containing 2.5 mL of water. The sample

was evaporated to an aqueous remainder and applied to a 3

- mL ChemElut SPE cartridge. After 10 to 15

min, the cartridge was washed three times with 4 mL of hexane/ethyl acetate (1:1 v/v), and t he eluates

were collected in a clean culture tube. The combined eluates were evaporated to dryness. The analytes

were dissolved in 0.5 mL of MeOH/H2O (1:4 v/v), and the resulting solution was transferred into a 2 mL

sample vial for analysis by LC/high reso lution mass spectrometry (LC/HRMS).

Quantitation of each analyte was based on the daughter ion transitions of the analyte and the

respective internal standard analog. The responses of the LC/MS/MS and LC/HRMS systems to eachMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

13 analyte and its internal stand ard were measured in samples and in standards, and a relative response

was calculated (as the ratio of the analyte and the stable isotopically labeled internal standard

responses). The relative response of the analyte in each sample was compared to the rel ative response

of the analyte in the standards provided in Appendix 4 of the study report

.

The relative responses of imidacloprid were measured over the range of 0.00012 to 4 ppm. The

correlation coefficients (R) were calculated using linear regression an alysis with 1/x weighting.

All data are reported in parent equivalents, and the individual measured residues of imidacloprid,

5

- hydroxy imidacloprid, and imidacloprid olefin are summed to give a total imidacloprid residue.

5F. Q UALITY A SSURANCE R ESULTS

The responses of the LC/MS/MS and LC/HRMS systems to imidacloprid, 5

- hydroxy imidacloprid, and

imidacloprid olefin were linear in solvent over the range of 0.00012 to 4 ppm. The correlation

coefficients were >0.99. The response data and analytical data su mmaries are located in Appendix 4.

Control interferences for cotton matrices are discussed in this paragraph; no total imidacloprid residue

was calculated for the UTC samples, so the levels of imidacloprid as an individual analyte are described.

Imidacloprid (parent) residues in UTC cotton floral nectar ranged from below the analyte LOD to

0.067 ppm. Imidacloprid residues in UTC cotton extrafloral nectar ranged from below the analyte LOD to

0.053 ppm. Imidacloprid residues in UTC cotton pollen ranged from below the analyte LOD to

0.017 ppm. Imidacloprid residues in UTC cotton leaves ranged from below the analyte LOD to

0.052 ppm.

The recovery data for imidacloprid (parent), 5

- hydroxy imidacloprid, and imidacloprid olefin residues are

summarized in Table 5 . All recoveries were cor rected for any interferences in corresponding controls.

The overall means of the recoveries for each matrix at each fortification level were within the

acceptable range of 70 to 120%, and standard deviation values were below 20%.

The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which

acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values

assigned to the individual analytes for a particular matrix. The limit of detecti on (LOD) is defined as the

lowest concentration of an analyte that can be determined to be statistically different from a blank. The

LODs were determined from method validation data obtained from control samples fortified at the

respective analyte LOQs. Th e LODs were calculated by multiplying the standard deviation of recovery

measurements at the LOQ by t0.99 [where t0.99 is the one

- tailed t

- statistic at the 99% confidence level

for the number of replicates (n)]. 3 The LOD for the total imidacloprid residue in each matrix is the

highest LOD value of any one individual analyte for that particular matrix.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

14 Table 5 . Limits of quantification and detection for imidacloprid and metabolites *.

* From page 10 of the study rep ort

.



### 6. RESULTS:

6. A. C OMPARISON OF C ONCENTRATIONS M EASURED FOR P ARENT AND D EGRADATES

Comparison of the relative concentrations measured for parent imidacloprid and degradation products

in floral nectar, extrafloral nectar, pollen, and leaves are presented in Table 6 through Table 8

. The LOD

of values were very low ranging from 0.3 ppb to 1.2 ppb so data reported as below the LOD were

assigned one

- half the LOD value. Comparison of the contribution of each chemical to the total residue

indicates that parent imidacloprid represented the majorit y of the total residue measured in each plant

part. For floral nectar, extrafloral nectar, and pollen bee relevant tissue, parent imidacloprid comprised

over 90% of the total at all but one of the sampling intervals where it was at 87%. Since the degradati on

products comprised a small portion of the measured residue and since they are considered as toxic as

parent imidacloprid, the following discussions will focus on comparing total imidacloprid residue

measured in plant tissues.

6. B

. M AGNITUDE OF R ESIDUES IN B EE

- R ELEVANT M ATRICES

Figure 1 explains the statistical aspects relayed in the Box

- and

- Whisker plots used to compare the

distribution of concentrations calculated for comparison of total imidacloprid residue at each sampling

int erval. For each data set analyzed, the box graphic presents values for the mean, median, minimum,

maximum, and 25 th and 75 th percentiles. The LOD of values were very low ranging from 0.3 ppb to

1.2 ppb so data reported as below the LOD were assigned one

- h alf the LOD value.

Floral Nectar

. Comparison of overall statistics for total imidacloprid residue indicated much greater

concentrations in all plant parts at the second sampling interval than at the other two sampling intervals

( Figure 2 ). The first soil application occurred at planting. Sampling for the first interval occurred at a

mean of 81 days after the soil application with a range of values from 70 to 95 days ( Table 4 ). Mean

total imidacloprid residue was 23.1 ppb in floral nectar ( Table 6 A ). The se cond sampling interval was

conducted after 3 foliar applications of imidacloprid and was approximately 23 days after the first

interval. Concentrations in floral nectar at the second sampling interval increased to a mean of 83.9 ppb.

The third sampling int erval occurred approximately 9 days after the second interval with totalMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

15 imidacloprid residue concentration dropping to a mean of 38.7 ppb. Table 6C compares the total

imidacloprid residues in floral nectar between soil types . At interval 1, coarse mean va lues appear

higher. At interval 2, the fine mean value is higher than for coarse soil. At interval 3, coarse and medium

values are similar.

Extrafloral Nectar

. Although a similar increase in concentrations were observed at sampling interval 2

for extrafloral nectar, the increase in concentration measured was relatively much larger than observed

for floral nectar ( Table 6B ) . The mean value was 438 ppb, which was approximately 500% greater than

the value measured for floral nectar. There were a few h igh values measured in the extrafloral nectar

which tend to bias the mean value. Comparison of the median values, which puts less weight on the

large values, still indicated a large increase in residues in the extrafloral nectar where the median was

296 pp b as compared to 84 ppb for the floral nectar. Comparison of median values still indicated a

substantial increase in concentration in extrafloral compared to floral nectar of approximately 350%.

Table 6D compares the total imidacloprid residues in extraflo ral nectar between soil types. At interval 1,

coarse mean values are similar to medium mean value s . At interval 2, coarse mean value s are higher

than other two types but all values are extremely high. At interval 3, mean values are similar.

Table 6 A . Magnitude of imidacloprid and related metabolites in cotton floral nectar (ppb).

Interval 1 Interval 2 Interval 3

Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total

N 15 1 5 15 15 15 15 15 15 15 15 15 15

Mean 0.7 0.7 21.8 23.1 1.4 2.4 80.2 83.9 1.2 1.2 36.4 38.7

SD 0.5 0.4 27 .0 27.7 0.8 1.1 38.9 40.5 0.7 0.7 25.8 26.9

CV (%) 70% 60% 124% 120% 56% 45% 49% 48 % 61% 57% 71% 69%

Min. 0.3 0.4 0.4 1.0 0.5 1.0 19.2 20.8 0.3 0.6 9.3 10.5

Median 0.5 0.5 5.8 6.7 1.3 2.2 80.1 83.6 0.8 0.8 23.5 25.1

75 th 1.1 0.9 41.4 43.1 1.9 3.5 107.6 113.4 1.7 1.6 54.1 57.4

90 th 1.5 1.5 71.0 74.1 3.0 3.9 139.0 144.0 1.9 2.4 75.8 78.8

Max. 1.6 1.5 80.9 83.1 3.2 4.1 146.9 153.3 2.9 2.9 99.3 103.9

% of

Total 2.9% 2.9% 94.4% 1.7% 2.8% 95.5% 3.0% 3.2% 93.9%

N OTES : IMI= imidacloprid, olefin = imidacloprid olefin, 5

- OH = 5 hydroxy imidacloprid, Total = sum of IMI, olefin and

5

- OH (in bold). SD=standard deviation, CV = coefficient of variation.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

16 Table 6 B . Magnitude of imidacloprid and related metabolites in cotton extrafloral nectar (ppb).

Interval 1 Interval 2 Interval 3

Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total

N 14 14 14 14 15 15 15 15 15 15 15 15

Mean 0.9 0.5 7.3 8.4 2.5 1 2. 1 423.8 438.3 0.6 1.5 3 0

. 8 32.9

SD 1.3 0.3 9.5 9 .7 2.7 1 2 .1 461.0 475.2 0.7 0.8 19.5 20.2

CV (%) 142% 65% 129% 1 16 % 112 % 97 % 109 % 108% 1 16 % 57% 63 % 6 1 %

Min. 0.3 0.4 0.5 1.2 0.9 2

. 9 50.5 54.3 0.3 0.5 5

. 2 7

. 6

Median 0.4 0.4 4.5 5

. 4 1.4 8 .2 284.7 296.6 0.3 1.5 2 6.0 2 6

. 8

75 th 0.8 0.5 9.1 9

. 7 2

. 5 1 3. 2 524.3 538.8 0.5 2.0 5 7.8 60.7

90 th 1.8 1.0 18.1 19

. 3 4

. 8 26

. 0 904.0 932.5 0 .9 2.6 5 9

. 0 63.1

Max. 5.0 1.4 34.1 3 5

. 9 11.6 4 8

. 9 1891.0 1951.5 3

. 1 3.6 64.8 66.7

% of

Total 10.5% 6.3% 87.1% 0

. 6 % 2.8% 9 6

. 7 % 1

. 8 % 4

. 5 % 93.6 %

N OTES : IMI= imidacloprid, olefin = imidacloprid olefin, 5

- OH = 5 hydroxy imidacloprid, Total = sum of IMI, olefin and

5

- OH (in bold). SD=standard deviation, CV = coefficient of variation.

Table 6 C

. Comparison of total imidacloprid residues between soil types at each sampling period for

floral nectar (ppb).

Interval 1 Interval 2 Interval 3

Coarse Medium Fine Coarse Medium Fine Coarse Medium Fine

N 10 3 2 10 3 2 10 3 2

Mean 32 9 2 88 51 114 45 30 20

SD 31 8 0.4 39 32 43 31 9 2

CV (%) 96 88 19 45 62 38 69 31 12

Min. 1 3 2 32 21 84 11 24 18

Median 24 6 2 86 48 114 40 25 20

75 th 53 18 2 113 84 144 64 41 22

90 th 79 18 2 144 84 144 91 41 22

Max. 83 18 2 153 84 144 104 41 22

SD=standard deviation, CV = coefficient of variation

Table 6 D

. Comparison of total imidacloprid residues between soil types at each sampling period for

extrafloral nectar (ppb).

Interval 1 Interval 2 Interval 3

Coarse Medium Fine Coarse Medium Fine Coarse Medium Fine

N 10 3 2 10 3 2 10 3 2

Mean 10 5 1 567 216 135 31 32 41

SD 11 4 0 540 140 9 19 25 31

CV (%) 101 96 8 96 65 6 61 79 76

Min. 1 5 1 179 54 129 8 12 19

Median 7 8 1 373 297 135 28 24 41

75 th 16 8 1 624 297 141 35 61 63

90 th 28 8 1 1442 297 141 64 61 63

Max. 36 8 1 1952 297 141 67 61 63

SD=standard deviation, CV = coefficient of variationMRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

17 Pollen. Mean residues of total imidacloprid in cotton pollen also followed the pattern noted in floral

nectar where concentrations were greater at sampling interval 2 than at the other two sampling

intervals ( Table 7 )

. The mean values appeared similar to those measured for floral nectar but the

maximum value at sampling interval 2 was twice as large as measured for floral nectar

.

Table 7 . Magnitude of imidacloprid and related metabolites in cotton pollen (ppb).

Interval 1 Interval 2 Interval 3

Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total

N 15 15 15 15 15 15 15 15 15 15 15 15

Mean 0.2 0.3 5.6 6.1 0.6 1.2 85.0 86.9 0.5 0.8 39.7 41.0

SD 0.1 0.1 10.7 10.8 0.8 1.3 102.3 104.1 0.5 1.0 48.7 50.0

CV (%) 52% 46% 190% 178% 125% 104% 120% 120% 99% 113% 123% 122%

Min. 0.2 0.3 0.2 0.6 0.2 0.3 3.8 4.2 0.2 0.3 2.8 3.2

Median 0.2 0.3 0.6 1.0 0.2 0.8 36.3 39.9 0.2 0.4 12.0 12.6

75 th 0.2 0.3 10.4 10.8 0.8 2.0 154.1 156.7 0.8 0.8 85.2 87.5

90 th 0.2 0.25 13.9 14.3 2.0 3.3 280.0 285.2 1.5 2.7 123.7 126.9

Max. 0.5 0.8 40.2 41.1 2.4 4.3 317.7 324.4 1.5 2.9 127.2 131.6

% of

Total 2.8% 4.6% 92.6% 0.7 % 1.4 % 97.8 % 1.3% 2.0% 96.8%

N OTES : IMI= imidacloprid, olefin = imidacloprid olefin, 5

- OH = 5 hydroxy imidacloprid, Total = sum of IMI, olefin and

5

- OH (in bold). SD=standard deviation, CV = coefficient of variation.

6.C. M AGNITUDE OF R ESIDUES IN L EAVES

As expected, direct foliar application of imidacloprid to plants between the first and second sampling

interval greatly increased the magnitude of resid u es of total imidacloprid in leaves measured at the

second sampling interval ( Table 8 ). At around 80 days after the s oil application mean total residues in

leaves was at 49 ppb. The concentration at sampling interval 2 was 30 times greater at 1621 ppb. At

interval 3 the levels in leaves were greatly decreased measured at 327 ppb, which was still greater than

measured at the first interval.

Table 8 . Magnitude of imidacloprid and related metabolites in cotton leaves (ppb).

Interval 1 Interval 2 Interval 3

Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total Olefin 5

- OH IMI Total

N 15 15 15 15 15 15 15 15 15 15 15 15

Mean 2.7 3.7 42.1 48.5 42.7 105.9 1472.7 1621.2 26.5 41.8 258.5 326.7

SD 4.3 4.3 49.5 56.8 17.3 35.9 678.9 711.4 10.5 15.7 140.4 156.6

CV (%) 124% 114% 118% 117% 41% 34% 46% 44% 40% 38% 54% 48%

Min. 0.4 0.4 0.6 1.3 14.9 62.5 437.8 524.5 4.8 16.4 40.2 61.4

Med ian 0.7 1.5 25.5 27.4 43.8 97.9 1371.5 1509.6 28.3 39.1 233.6 305.0

75 th 4.4 5.3 55.4 62.2 47.7 121.3 1780.7 2043.7 34.7 52.2 333.5 416.1

90 th 9.5 11.6 118.0 140.1 65.2 159.1 2653.6 2860.3 36.3 57.9 418.0 503.0

Max. 10.5 13.7 169.7 192.9 78.4 184.6 2956.7 3098.2 46.2 79.6 606.0 698.7

% of

Total 5.6 % 7.7 % 86.8% 2.6% 6.5% 90.8% 8.1% 12.8% 79.1%

N OTES : IMI= imidacloprid, olefin = imidacloprid olefin, 5

- OH = 5 hydroxy imidacloprid, Total = sum of IMI, olefin and 5

- OH

(in bold). SD=standard deviation, CV = coefficient of variation.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

18 6. D

. S ITE S PECIFIC T RENDS

Temporal patterns in residue concentration for the 9 separate sites are depicted in Figures 3 through 6

for flora l nectar, extraflora l nectar, pollen, and leaves, respectively. The general pattern noted above

where concentrations rise steeply at the second sampling interval is reflected in floral nectar samples at

13 of the 15 curves (Figure 3). Curves at two sites, one Fresno1

– 2013 and Ker m an2

– 2013, reflect

different patterns where a rise in concentration over time is noted at the Fresno1 Site and where

concentrations were relatively stable across sampling intervals at the Ker m an2 site. The cause for these

differences is unknown, but both were observed in data obtained in the first year of sampling in 2013.

Greater variability in results in the first year of the study could be caused by familiarity with the study

design, sampling protocol, an d analytical methodology. All sites sampled in 2014 exhibited the increase

in concentration from sampling interval 1 to 2. Decreases in concentration from interval 2 to 3 were

predominant but there were subtle differences in slopes where some indicated dee p decreases in

concentrations between the intervals while others indicated only a shallow, slight decrease in

concentration over time. Data for 2014 for the two Ker m an sites (green lines) exemplify the potential

differences in slopes measured between inter vals 2 and 3 where a large decrease is indicated at

Ker m an1 (solid dots) and only a small decrease at Ker m an2 (open squares).

Trends in total imidacloprid residue measured in extrafloral nectar also reflected a large increase in

concentration at sampling interval 2 followed by reductions at sampling interval 3 (Figure 4). The

exception was data for Fresno site 1 in 2013. Data in 2014 reflected the pattern measured at the rest of

the sites. The increase at some sites was rather large where, for example at Kerman2 in 2013 the

averag e concentration was 932 ppb.

For pollen samples, there were noticeable differences in the trend and magnitude of concentrations

measured between 2013 and 2014 data (Figure 5). Concentrations in Pollen at all sites in 2013 were

lo wer than measured in 2014 with extremely low values reported at the Fresno, Davis, and Sanger sites.

Since concentrations in other plant parts did not reflect similar large differences between the years, lack

of significant detections could be due to devel opment of sampling procedures and chemical analyses for

pollen samples in 2014. In 2014 trends for 8 of the 9 curves exhibited the previously described overall

trend of increased concentration from sampling interval 1 to 2 and then decrease in values from interval

2 to 3.

Lastly, all curves for leaf concentration exhibited an increase at interval 2 and a sharp decline at interval

3 (Figure 6). The sharp decrease in concentration between intervals 2 and 3 does not appear to be due

to washoff from rainfall or irrigation. For example, at the Fresno sites and the Sanger sites there was no

recorded rainfall during this time interval. Also, the irrigation systems used had a low potential for

wetting the leaves because drip irrigation was used at the Fresno sites and a combination of drip and

furrow methods at the Sanger site.

6.E. Potential Carry

- over of Residues

The site specific curves provide comparison for levels measured between 2013 and 2014 at 6 of the sites

(Figure 3 through F igure 6) . There is variation in the comparison between years. Some curves are very

similar between the years, such as for floral nectar at the Sanger site and l eaves at the Ker m an and Davis

sites. S ome indicate greater concentration measured at sampling interval 2 in 2013 than in 2014, such as

for floral nectar at the Davis site and Ker m an site 1 an d for leaves at Fresno site 1. T hese results provide

no evidence for a carry

- over effect.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

19 6. F

. L EAF AND N ECTAR C ONCENTRATION IN R ELATION TO S OIL T YPE

Originally, the p roposed study design suggested sampling sites located in coarse, medium, and fine

textured soils with 3 replicates assigned to each soil type. There is an inherent difficulty in fulfilling the

proposed design when the study is conducted after fields have b een planted and then attempting to

procure cooperating growers. The reported soils in this study were biased towards coarse soils where in

2013 four of six sites were located in coarse soil, two in medium textured soil s and none in the fine

textured catego ry. In 2014, two sites were located in the fine textured category, three site s in medium,

and four sites in the coarse textured category. In Figure 7 the concentration of total imidacloprid residue

in floral nectar is plotted in relation to the levels in corresponding leaf samples. Data for both years are

plotted with data for interval 1 indicated as black markers, interval 2 as red markers, and interv al 3 as

blue markers. The overlap in marker types for each sampling interval does not indicate a clear difference

in soil type, e.g. squares and circles. Figure 8 and Figure 9 present the relationship between extrafloral

nectar and leaves, and pollen and l eaves, respectively. The pattern is the same as discussed for floral

nectar where there is some overlap in soil type as indicated between the types of markers, e.g. squares

and circles. A m ore robust representative sampling of the soil groups is needed to prove or disprove

differences in concentration due to soil type. The greater range in total imidacloprid residue values

noted for extrafloral nectar provided a clearer contrast for the values measured at sampling interval 2

with respect to the first and third intervals.



### 7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS

In the context of documenting the magnitude of imidacloprid residues in bee

- related matrices of cotton

, the following strengths are obs erved with this study.

1. Data provide quantitative values of total imidacloprid residues expected in pollen, nectar,

extrafloral nectar, and leaves of cotton.

2. Measurements were taken at 3 time intervals in an attempt to quantify levels expected in plant

tissues first following a period of time after a soil application at planting, then soon after a third

and final foliar application, and finally at a longer period after the final foliar application.

3. Measurements were taken for two consecutive years at six sites, four coarse and two medium

soil s

, of the nine sites providing data to determine whether carry

- over was evident.

L i mitations noted in this study includ e :

1. The study did not follow the residue data protocol calling for three replicates each in fine,

medium and coarse soils resulting in uncertainty in regards to the effects of soil type on

imidacloprid concentration in cotton flower parts and leaves

.

2. An inade quate sample of extrafloral nectar to analyse was collected following the soil

application and prior to the foliar application in replicate NT003

- 13ZA.

3. The values most likely do not reflect a maximal exposure because bee relevant tissue was not

sampled du ring the period of foliar application.

4. Study was conducted for two years at 6 sites. Data to determine whether or not carry

- over

occurred was not adequate

.

5. Pollen analysis appeared problematic in 2013, as reflected in Figure 5 , where in 2013

concentration of imidacloprid residues at some sites were extremely low but residues at the

same sites in the following year in 2014 were at much higher concentrations.

6. Data are inadequate to characterize residues in cotton matrices in medium or fine soils because

these two soil types were not adequately represented in the sample sites.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

20 Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:

1. Maximum concentrations of total imidacloprid residues a t approximately 81 days after s oil

application of approximately 0.34 lb ai/A were 83, 36, and 43 ppb in floral nectar, extrafloral

nectar, and pollen , respectively.

2. For sample s taken approximately 5 days after a 3 rd foliar application of 0.056

– 0.067 lb ai/A,

maximum total imidacloprid residues were measured at 153, 3615, and 320 ppb , in floral nectar,

extrafloral nectar, and pollen

, respectively

.

3. Subsequent s ampling 14 days after the 3 rd foliar application resulted in maximum residues of

104, 67, and 130 ppb in nectar, extrafloral nectar, and pollen, respectively.

4. M ean total imidacloprid residue for samples taken 81 days after the soil application was 23.1,

8.4, and 6.1 ppb i n floral nectar, extrafloral nectar and pollen, respectively.

5. Mean total imidacloprid residues were 83.9, 438.3, and 86.1 ppb in floral nectar, extrafloral

nectar, and pollen, respectively, when sampled 5 days after the 3 rd foliar application.

6. Mean total imidacloprid residues were 38.7, 32.9, and 41.0 in floral nectar, extrafloral nectar,

and pollen, respectively, when sampled 13 or 14 days after the 3 rd foliar application.

7. There was no evidence of year to year carryover within the data.

8. Generally, resi dues were high e r when sampling occurred following 4 or 5 days after the 3 rd foliar

application and lower 10 days later at the final sampling interval.



### 8. STUDY VALIDITY/CLASSIFICATION

The data from this study provide an expected distribution of the concentration of imidacloprid residues

that bees are exposed to in nectar and pollen in flowers and extrafloral nectaries of cotton plants grown

under actual agronomic practices in California . Relating concentrations measured in flower parts to bee

health is possible by comparing the concentrations measured in bee relevant plant parts to target values

that define acute or chronic exposure scenarios. These data, however, would represent a minim al

exposure assessment to foliar applications because samples were not taken during the period of foliar

ap plication. Therefore, there is uncertainty as to how reflective the values obtained at the 3 sampling

interval s represent maximum exposure scenarios. The study is considered scientifically sound and useful

for risk assessment purposes. The study is classified as ACCEPTABLE for quant itative use in risk

assessment.



### 9. REFERENCES

1 . Brungardt, J. 2010. An Analytical Method for the Determination of Residu es of Imidacloprid and its

Metabolites Imidacloprid Olefin and 5

- Hydroxy Imidacloprid in Bee Relevant Matrices Using LC/MS/MS.

Bayer Method No. NT

- 005

- P10

- 01.

2 . Miller, A. 2014. An Analytical Method for the Determination of Residues of Imidacloprid and i ts

Metabolites Imidacloprid Olefin and 5

- Hydroxy Imidacloprid in Bee Relevant Matrices Using LC/MS/MS.

Bayer CropScience Report Number: NT

- 006

- A13

- 01.

3 . Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/nonquantified pesticide

r esidues in human health food exposure assessments. EPA Docket #OPP

- 00570A.MRID 4 9665202 CDPR IMI Soil and Foliar Cotton Study

21 Figure 1. Explanation of statistical meaning of the Box

- and

- Whisker plots.



## Maximum Observation

Minimum Observation Median Value (50%) Mean Value

25% Quartile 75% QuartileMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

22 Figure 2

. Relative concentration of total imidacloprid residues measured in ( A) Flo ral Nectar; ( B)

Extra f lora l Nectar; ( C) Pollen; and ( D) Leaves compared between sampling intervals. Data were averaged

over all sites.



### A. Floral B. Extrafloral

D. Leaves C. Interval 1 Interval 2 Interval 3 Interval 1 Interval 2 Interval 3

Interval 1 Interval 2 Interval 3 Interval 1 Interval 2 Interval 3MRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

23 Figure 3

. Trend in total imidacloprid residue measured in floral nectar at each site. Within each panel

markers denote the site, for example Fresno site 1 are solid circles and Fresno site 2 are open squares.

Black dashed lines are data collected in 2013 and green solid lines are data collected in 2014. Note the

differences in scale between the graphs.Total Imidacloprid Residue (ppb)



## Days After Imidacloprid Soil Application

0 50 100 150 Fresno1 - 2013

Fresno1 - 2014

Fresno2 - 2013

Fresno2 - 2014

0 50 100 150 200 Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

0 50 100 150 200

60 70 80 90 100 110 120 130 Davis - 2013

Davis - 2014

Sanger - 2013

Sanger - 2014

0 50 100 150 200

60 70 80 90 100 110 120 130 Yuba City1 - 2014

Yuba City2 - 2014

Wheatland - 2014



### Specific Site Information:

Fresno1=NT003

- 13ZA

Fresno2=NT007

- 13ZA

Davis1=NT004

- 13ZA

Sanger1=NT006

- 13ZA Specific Site Information:

Kerman 1=NT00 8

- 13ZA

Kerman 2=NT00 9

- 13ZA

Yuba City 1=NT00 1

- 13Z B

Yuba City2=NT005

- 13ZB

Wheatland =NT00 2

- 13Z BMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

24 Figure 4

. Trend in total imidacloprid residue measured in extrafloral nectar at each site. Within each panel

markers denote the site, for example Fresno site 1 are solid circles and Fresno site 2 are open squares.

Black dashed lines are data collected in 2013 and g reen solid lines are data collected in 2014. Note the

differences in scale between the graphs.

0 500 1000 1500 2000

60 70 80 90 100 110 120 130 Davis - 2013

Davis - 2014

Sanger - 2013

Sanger - 2014Total Imidacloprid Residue (ppb)



## Days After Imidacloprid Soil Application 0 100 200 300 400

60 70 80 90 100 110 120 130 Yuba City1 - 2014

Yuba City2 - 2014

Wheatland - 2014 0 200 400 600 800 1000 Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

0 200 400 600 800 Fresno1 - 2013

Fresno1 - 2014

Fresno2 - 2013

Fresno2 - 2014



### Specific Site Information:

Fresno1=NT003

- 13ZA

Fresno2=NT007

- 13ZA

Davis1=NT004

- 13ZA

Sanger1=NT006

- 13ZA Specific Site Information:

Kerman 1=NT00 8

- 13ZA

Kerman 2=NT00 9

- 13ZA

Yuba City 1=NT00 1

- 13Z B

Yuba City2=NT005

- 13ZB

Wheatland =NT00 2

- 13Z BMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

25 Figure 5

. Trend in total imidacloprid residue measured in pollen at each site. Within each panel markers

denote the site, for example Fresno site 1 are solid circles and Fresno site 2 are open squares. Black

dashed lines are data collected in 2013 and green solid l ines are data collected in 2014. Note the

differences in scale between the graphs.Total Imidacloprid Residue (ppb)



## Days After Imidacloprid Soil Application 0 50 100 150 200 Fresno1 - 2013

Fresno1 - 2014

Fresno2 - 2013

Fresno2 - 2014

0 50 100 150 200 Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

0 100 200 300 400

60 70 80 90 100 110 120 130 Davis - 2013

Davis - 2014

Sanger - 2013

Sanger - 2014

0 20 40 60 80 100

60 70 80 90 100 110 120 130 Yuba City1 - 2014

Yuba City2 - 2014

Wheatland - 2014



### Specific Site Information:

Fresno1=NT003

- 13ZA

Fresno2=NT007

- 13ZA

Davis1=NT004

- 13ZA

Sanger1=NT006

- 13ZA Specific Site Information:

Kerman 1=NT00 8

- 13ZA

Kerman 2=NT00 9

- 13ZA

Yuba City 1=NT00 1

- 13Z B

Yuba City2=NT005

- 13ZB

Wheatland =NT00 2

- 13Z BMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

26 Figure 6

. Trend in total imidacloprid residue measured in leaf tissue at each site. Within each panel

markers denote the site, for example Fresno site 1 are solid circles and Fresno site 2 are open squares.

Black dashed lines are data collected in 2013 and green li nes are data collected in 2014. Note the

differences in scale between the graphs.Total Imidacloprid Residue



## Days After Imidacloprid Soil Application 0 500 1000 1500 2000 2500

Fresno1 - 2013

Fresno1 - 2014

Fresno2 - 2013

Fresno2 - 2014

0 500 1000 1500 2000

Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

0 500 1000 1500 2000 2500 3000 3500

60 70 80 90 100 110 120 130 Davis - 2013

Davis - 2014

Sanger - 2013

Sanger - 2014

0 500 1000 1500 2000

60 70 80 90 100 110 120 130 Yuba City1 - 2014

Yuba City2 - 2014

Wheatland - 2014



### Specific Site Information:

Fresno1=NT003

- 13ZA

Fresno2=NT007

- 13ZA

Davis1=NT004

- 13ZA

Sanger1=NT006

- 13ZA Specific Site Information:

Kerman 1=NT00 8

- 13ZA

Kerman 2=NT00 9

- 13ZA

Yuba City 1=NT00 1

- 13Z B

Yuba City2=NT005

- 13ZB

Wheatland =NT00 2

- 13Z BMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

27 Figure 7

. Relationship between concentration of total imidacloprid residues measured in leaves and floral

nectar parsed out by soil type. Numbers after the texture category indicate data for interval 1, 2, or 3

where black markers are from sampling interval 1, red markers from interval 2, and blue markers from

interval 3.

0.00 50.00 100.00 150.00 200.00

0 500 1000 1500 2000 2500 3000 3500 Coarse -1 Medium -1 Fine -1

Coarse -2 Medium -2 Fine -2

Coarse -3 Medium -3 Fine -3Floral Nectar Total Imidacloprid Residue (ppb)



### Leaf Total Imidacloprid ConcentrationMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

28 Figure 8. Relationship between concentration of total imidacloprid residues measured in leaves and

extrafloral nectar parsed out by soil type. Numbers after the texture category indicate data for interval 1,

2, or 3 where black markers are from sampling interval 1, red markers from interval 2, and blue markers

from interval 3.

0.00 500.00 1000.00 1500.00 2000.00

0 500 1000 1500 2000 2500 3000 3500 Coarse -1 Medium -1 Fine -1

Coarse -2 Medium -2 Fine -2

Coarse -3 Medium -3 Fine -3Extra Floral Nectar Total Imidacloprid Residue (ppb)



### Leaf Total Imidacloprid ConcentrationMRID 4 9665202 CDPR IMI Soil & Foliar Cotton Study

29 Figure 9 . Relationship between concentration of total imidacloprid residues measured in leaves and

pollen parsed out by soil type. Numbers after the texture category indicate data for interval 1, 2, or 3

where black markers are from sampling interval 1, red markers from interval 2, and blue markers from

interval 3.

0.00 100.00 200.00 300.00 400.00 500.00

0 500 1000 1500 2000 2500 3000 3500 Coarse -1 Medium -1 Fine -1

Coarse -2 Medium -2 Fine -2

Coarse -3 Medium -3 Fine -3Pollen Total Imidacloprid Residue (ppb)



### Leaf Total Imidacloprid Concentration