MRID 49665202 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 Unpublished study prepared by Bayer 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 49665202, 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 extrafloral), 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 Locations:

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 51950-0808, Tracking ID# 254696.]

2. REVIEWER INFORMATION

Primary Reviewers:

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 Coordinator, 

Pesticide Registration Branch

3. EXECUTIVE SUMMARY

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

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

data and six (6) trials have two years of data. 
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

2

Treated plots 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 lb/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 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, duplicate 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 final 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 field 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 quantitated 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 

imidacloprid 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 floral and extrafloral nectar.

4. STUDY VALIDITY

Guideline Followed: Non-guideline study (protocol 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/A
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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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 

Material:

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 -52oF

5A. STUDY DESIGN

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 (last 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.

5B. APPLICATION TIMING AND RATES

The full study report provides (1) Chronological listing of significant study dates (Appendix 1); (2) Field 

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) Quality assurance statements for each trial (Appendix 3). Information on 

application timing is provided in Table 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 lb/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 applications were made between BBCH 

growth stages 61 and 72 (BBCH 61: beginning of flowering; BBCH 72: about 20% of bolls have attained 
MRID 49665202 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 gal/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 during 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 rainfall as needed in 

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 fields, 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*.

Trial Id

entificatio

n

Locatio

n (City,

State, 

NAFT

A Re

gion)

Form

ulatio

n

Plot Na

m

e

Y

ear

Applicationa

Metho

d

Timing

/

Growth

Stag

e (BB

CH)

Actual

S

pray 

V

olume,

GPA (L

/

ha)

Rate, lb a.i./A (kga.i./

ha)

Retreatm

e

nt

Int

erval (days)

Total Rate, lb a.i.

/A 

(kg a.i./

ha)

A

djuva

nt

NT001-

13ZBb

Yuba City, CA

Region 10

Admire

Pro

TRTD 2014 Soil

Spray

00 14

(133)

0.328

(0.368)

NAc 0.50

(0.56)

NA

Foliar

Spray

61 16

(150)

0.058

(0.065)

93 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 16 

(147)

0.057 

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 16

(148)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

NT002-

13ZBb

Wheatland, CA

Region 10

Admire

Pro

TRTD 2014 Soil

Spray

00 15

(142)

0.331

(0.371)

NA 0.50

(0.56)

NA

Foliar

Spray

65 15

(142)

0.057

(0.064)

92 Dyne-Amic

0.25 % v/v

Foliar

Spray

69 15

(144)

0.058

(0.065)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

72 15 

(140)

0.057 

(0.064)

7 Dyne-Amic

0.25 % v/v
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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Trial Id

entificatio

n

Locatio

n (City,

State, 

NAFT

A Re

gion)

Form

ulatio

n

Plot Na

m

e

Y

ear

Applicationa

Metho

d

Timing

/

Growth

Stag

e (BB

CH)

Actual

S

pray 

V

olume,

GPA (L

/

ha)

Rate, lb a.i./A (kga.i./

ha)

Retreatm

e

nt

Int

erval (days)

Total Rate, lb a.i.

/A 

(kg a.i./

ha)

A

djuva

nt

NT003-

13ZA

Fresno, CA

Region 10

Admire

Pro

TRTD 2013 Soil

Spray

00 14

(132)

0.330

(0.370)

NA 0.51

(0.57)

NA

Foliar

Spray

65 15

(142)

0.058

(0.066)

81 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(140)

0.058

(0.065)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(142)

0.058

(0.066)

7 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 14

(132)

0.330

(0.370)

254 0.51

(0.57)

NA

Foliar

Spray

61 15 

(141)

0.058 

(0.065)

77 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(142)

0.059

(0.066)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 16

(145)

0.060

(0.067)

7 Dyne-Amic

0.25 % v/v

NT004-

13ZA

Davis, CA

Region 10

Admire

Pro

TRTD 2013 Soil

Spray

00 14

(127)

0.332

(0.372)

NA 0.51

(0.57)

NA

Foliar

Spray

61 15

(142)

0.058

(0.065)

91 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 15 

(140)

0.058 

(0.065)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 14

(136)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 15 

(139)

0.327 

(0.367)

253 0.50 

(0.56)

NA

Foliar

Spray

61 14

(135)

0.057

(0.064)

99 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 17

(156)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 16

(148)

0.057

(0.063)

7 Dyne-Amic

0.25 % v/v

NT005-

13ZBb

Yuba City, CA

Region 10

Admire

Pro

TRTD 2014 Soil

Spray

00 15

(143)

0.337

(0.377)

NA 0.51

(0.57)

NA

Foliar

Spray

61 15 

(142)

0.056 

(0.063)

91 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(144)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(142)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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Trial Id

entificatio

n

Locatio

n (City,

State, 

NAFT

A Re

gion)

Form

ulatio

n

Plot Na

m

e

Y

ear

Applicationa

Metho

d

Timing

/

Growth

Stag

e (BB

CH)

Actual

S

pray 

V

olume,

GPA (L

/

ha)

Rate, lb a.i./A (kga.i./

ha)

Retreatm

e

nt

Int

erval (days)

Total Rate, lb a.i.

/A 

(kg a.i./

ha)

A

djuva

nt

NT006-

13ZA

Sanger, CA

Region 10

Admire

Pro

TRTD 2013 Soil

Spray

00 13

(118)

0.328

(0.368)

NA 0.50

(0.56)

NA

Foliar

Spray

61 16

(146)

0.058

(0.065)

95 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 15

(142)

0.058

(0.065)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15 

(144)

0.058 

(0.065)

6 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 13

(125)

0.332

(0.372)

249 0.51

(0.57)

NA

Foliar

Spray

61 19

(179)

0.058

(0.065)

77 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 19

(181)

0.058

(0.065)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 20

(185)

0.057

(0.064)

6 Dyne-Amic

0.25 % v/v

NT007-

13ZA

Fresno, CA 

Region 10

2013

Admire

Pro

TRTD 2013 Soil

Spray

00 14 

(132)

0.331 

(0.372)

NA 0.50 

(0.56)

NA

Foliar

Spray

61 15

(141)

0.057

(0.064)

81 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(140)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 14 

(132)

0.330 

(0.369)

262 0.50 

(0.56)

NA

Foliar

Spray

61 15

(140)

0.057

(0.064)

75 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 15

(140)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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Trial Id

entificatio

n

Locatio

n (City,

State, 

NAFT

A Re

gion)

Form

ulatio

n

Plot Na

m

e

Y

ear

Applicationa

Metho

d

Timing

/

Growth

Stag

e (BB

CH)

Actual

S

pray 

V

olume,

GPA (L

/

ha)

Rate, lb a.i./A (kga.i./

ha)

Retreatm

e

nt

Int

erval (days)

Total Rate, lb a.i.

/A 

(kg a.i./

ha)

A

djuva

nt

NT008-

13ZA

Kerman, CA

Region 10

Admire

Pro

TRTD 2013 Soil

Spray

00 14

(130)

0.328

(0.368)

NA 0.50

(0.56)

NA

Foliar

Spray

65 15 

(141)

0.057 

(0.064)

83 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(142)

0.058

(0.065)

7 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 14

(130)

0.326

(0.365)

226 0.50

(0.56)

NA

Foliar

Spray

61 15

(138)

0.056

(0.063)

83 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15 

(140)

0.057 

(0.064)

7 Dyne-Amic

0.25 % v/v

NT009-

13ZA

Kerman, CA

Region 10

Admire

Pro

TRTD 2013 Soil

Spray

00 14

(132)

0.331

(0.371)

NA 0.50

(0.56)

NA

Foliar

Spray

61 15

(139)

0.056

(0.063)

83 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

2014 Soil

Spray

00 14 

(131)

0.330 

(0.369)

225 0.50 

(0.56)

NA

Foliar

Spray

61 15

(141)

0.057

(0.064)

83 Dyne-Amic

0.25 % v/v

Foliar

Spray

61 15

(141)

0.057

(0.064)

7 Dyne-Amic

0.25 % v/v

Foliar

Spray

65 15

(141)

0.057

(0.064)

7 Dyne-Amic

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 report
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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

Trial

IDa

Trial Location (City, 

Country/State, Year, GPS 

Coordinatesb)

Soil Characteristicsc MeteorologicalDatad

OM (%) pH Variety

CEC

(meq/

100 g

soil)

% 

Sand

% 

Silt

% 

Clay Type

Total 

Rainfall 

(in)

Temp. Range

(°F)

NT001-

13ZB

Yuba City, CA, 2014 

(38°59.055’N, 121°36.115’ W) 1.0-2.0

6.6-

8.4 20-25 35 34 31

Clay 

loam 0.92 47 97

Pima

NT002-

13ZB

Wheatland, CA, 2014 

(39°00.22’ N, 121°27.59 W) 0.5-2.0

6.1-

7.8 5-15 71 17 12

Sandy

Loam 0.92 47 97

Pima

NT003-

13ZA

Fresno, CA, 2013†-2014‡

(36.73628, -119.87515) 0.7 7.7 9.1 71 20 9

Sandy

Loam 3.92 30 102

DP 358 RF†

PHY 804 RF‡

NT004-

13ZA

Davis, CA, 2013-2014 

(38.5337, -121.7793) 1.8 7.4 14.4 35 40 25 Loam 14.93 32 93

Pima

NT005-

13ZB

Yuba City, CA, 2014 

(39°02.24’ N, 121°37.63’ W) 1.0-2.0

6.6-

8.4 20-25 35 34 31

Clay 

loam 0.92 47 97

Pima

NT006-

13ZA

Sanger, CA, 2013-2014 

(36.69982, -119.46196) 0.3 4.8 0.7 66 23 11

Sandy

Loam 5.69 31 99

Pima

NT007-

13ZA

Fresno, CA, 2013-2014 

(36.73916, -119.87599) 0.5 7.2 11.2 65 24 11

Sandy

Loam 3.92 30 102

PHY 802 RF

NT008-

13ZA

Kerman, CA, 2013-2014 

(36.79552, -120.05406) 0.5 6.9 5.2 77 20 3

Loamy 

sand 2.74 29 99

Delta Pine 

358 RF

NT009-

13ZA

Kerman, CA, 2013†-2014‡

(36.79516, -120.05676) 0.6 7.6 4.9 81 18 1

Loamy 

sand 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 from Table 3A and Table 8 of the study report.
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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Table 3. SSURGO soil characteristics of the study sites*.

Trial ID Trial

Location

General Texture 

Description 

(SSURGO)

CDPR 

Texture 

Category

Component

%a

Drainage

Class

Hydrologic

Group

Runoff Particle

Size

NT001-

13ZB

Yuba City, 

CA

Marcum-Gridley

Clay Loam,

0-1% slopes

Fine 45 / 40 Moderately

Well Drained

C / D Low / 

Medium

Fine

NT002-

13ZB

Wheatland, 

CA

Columbia Fine

Sandy Loam,

0-1% slopes

(~10% Kimball

Loam)

Medium 85 Somewhat 

Poorly Drained

A Very Low Coarseloamy

NT003-

13ZA

Fresno, CA Ramona Loam Medium 80 Well Drained C Low Fineloamy

NT004-

13ZA

Davis, CA Reiff Very Fine

Sandy Loam

Medium 85 Well Drained A Very Low Coarseloamy

NT005-

13ZB

Yuba City, 

CA

Marcum-Gridley

Clay Loam,

0-1% slopes

Fine 45 / 40 Moderately

Well Drained

C / D Low / 

Medium

Fine

NT006-

13ZA

Sanger, CA Hanford fine 

sandy loam, 

gravelly 

substratum

Coarse 85 Well Drained A Very Low Coarseloamy

NT007-

13ZA

Fresno, CA Greenfield

Sandy Loam,

0-3% slopes

Coarse 85 Well Drained A Very Low Coarseloamy

NT008-

13ZA

Kerman, CA Hanford Coarse

Sandy Loam

Coarse 85 Well Drained A Very Low Coarseloamy

NT009-

13ZA

Kerman, CA Hanford Sandy 

Loam, silty 

substratum

Coarse 85 Well Drained A Very Low Coarseloamy

a Major component(s) of the soil as a percentage of total soil; for Marcum-Gridley: Marcum is 

45%, hydrologic group C, and Low runoff; Gridley is 40%, hydrologic group B, and Medium runoff.

* From Table 3B of the study report.

5D. SAMPLE COLLECTION, HANDLING, PROCESSING

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 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 final size). Single composite samples of cotton leaves and flowers were 
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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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 

samples 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 micropipette 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 completed, 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 residues measured in leaves and floral nectar parsed out by soil type.
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Table 4. Sampling dates and cotton developmental 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

76 DASA,

-5 DA1FA

61

72 DASA,

-5 DA1FA

65 88 DASA,

-4 DA1FA

NC1 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

76 DASA,

-5 DA1FA

61

70 DASA,

-5 DA1FA

61 88 DASA,

-5 DA1FA

TBC2 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 XX3 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 = Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie growth stage scale for cotton. ‡

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 collected 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 49665202 CDPR IMI Soil and Foliar Cotton Study

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5E. ANALYTICAL METHODS

The analytical methods1-2 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 was 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 polypropylene 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 conical 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 evaporated 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 standards, 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 volume 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 into 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 the 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 resolution 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 each 
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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analyte and its internal standard 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 relative 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 analysis 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. QUALITY ASSURANCE RESULTS

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 summaries 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 corrected 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 detection (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. The 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.
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Table 5. Limits of quantification and detection for imidacloprid and metabolites*.

* From page 10 of the study report.

6. RESULTS:

6.A. COMPARISON OF CONCENTRATIONS MEASURED FOR PARENT AND DEGRADATES

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 majority 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 degradation 

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. MAGNITUDE OF RESIDUES IN BEE-RELEVANT MATRICES 

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 

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

maximum, and 25th and 75th 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-half 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 6A). The second 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 interval occurred approximately 9 days after the second interval with total 
MRID 49665202 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 values 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 high 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 ppb 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 extrafloral nectar between soil types. At interval 1, 

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

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

Table 6A. 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 15 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

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

90th 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%

NOTES: 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 49665202 CDPR IMI Soil and Foliar Cotton Study

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Table 6B. 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 12.1 423.8 438.3 0.6 1.5 30.8 32.9

SD 1.3 0.3 9.5 9.7 2.7 12.1 461.0 475.2 0.7 0.8 19.5 20.2

CV (%) 142% 65% 129% 116% 112% 97% 109% 108% 116% 57% 63% 61%

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 26.0 26.8

75th 0.8 0.5 9.1 9.7 2.5 13.2 524.3 538.8 0.5 2.0 57.8 60.7

90th 1.8 1.0 18.1 19.3 4.8 26.0 904.0 932.5 0.9 2.6 59.0 63.1

Max. 5.0 1.4 34.1 35.9 11.6 48.9 1891.0 1951.5 3.1 3.6 64.8 66.7

% of 

Total

10.5% 6.3% 87.1% 0.6% 2.8% 96.7% 1.8% 4.5% 93.6%

NOTES: 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 6C. 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

75th 53 18 2 113 84 144 64 41 22

90th 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 6D. 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

75th 16 8 1 624 297 141 35 61 63

90th 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 variation
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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

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

90th 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%

NOTES: 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. MAGNITUDE OF RESIDUES IN LEAVES

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

interval greatly increased the magnitude of residues of total imidacloprid in leaves measured at the 

second sampling interval (Table 8). At around 80 days after the soil 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

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

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

90th 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%

NOTES: 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.
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6.D. SITE SPECIFIC TRENDS

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

for floral nectar, extrafloral 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 Kerman2 – 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 Kerman2 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, and 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 deep decreases in 

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

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

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

Kerman1 (solid dots) and only a small decrease at Kerman2 (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 

average 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 

lower 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 development 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 Figure 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 leaves at the Kerman and Davis 

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

for floral nectar at the Davis site and Kerman site 1 and for leaves at Fresno site 1. These results provide 

no evidence for a carry-over effect. 
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6.F. LEAF AND NECTAR CONCENTRATION IN RELATION TO SOIL TYPE

Originally, the proposed 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 been 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 soils and none in the fine 

textured category. In 2014, two sites were located in the fine textured category, three sites 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 interval 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 leaves, 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 more 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 observed 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 

soils, of the nine sites providing data to determine whether carry-over was evident.

Limitations noted in this study include:

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 inadequate 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 during 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 49665202 CDPR IMI Soil and Foliar Cotton Study

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

1. Maximum concentrations of total imidacloprid residues at approximately 81 days after soil 

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

nectar, and pollen, respectively.

2. For samples taken approximately 5 days after a 3rd 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 sampling 14 days after the 3rd foliar application resulted in maximum residues of 

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

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

8.4, and 6.1 ppb in 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 3rd 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 3rd foliar application. 

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

8. Generally, residues were higher when sampling occurred following 4 or 5 days after the 3rd 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 minimal 

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

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

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

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

assessment.

9. REFERENCES

1. Brungardt, J. 2010. An Analytical Method for the Determination of Residues 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 its 

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 

residues in human health food exposure assessments. EPA Docket #OPP-00570A.
MRID 49665202 CDPR IMI Soil and Foliar Cotton Study

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Figure 1. Explanation of statistical meaning of the Box-and-Whisker plots.

Maximum Observation

Minimum Observation

Median Value (50%)

Mean Value 

25% Quartile

75% Quartile
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Figure 2. Relative concentration of total imidacloprid residues measured in (A) Floral Nectar; (B) 

Extrafloral 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 3
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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:

Kerman1=NT008-13ZA

Kerman2=NT009-13ZA

Yuba City1=NT001-13ZB

Yuba City2=NT005-13ZB

Wheatland=NT002-13ZB
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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 green 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 - 2014

Total 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:

Kerman1=NT008-13ZA

Kerman2=NT009-13ZA

Yuba City1=NT001-13ZB

Yuba City2=NT005-13ZB

Wheatland=NT002-13ZB
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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 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

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:

Kerman1=NT008-13ZA

Kerman2=NT009-13ZA

Yuba City1=NT001-13ZB

Yuba City2=NT005-13ZB

Wheatland=NT002-13ZB
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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 lines 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:

Kerman1=NT008-13ZA

Kerman2=NT009-13ZA

Yuba City1=NT001-13ZB

Yuba City2=NT005-13ZB

Wheatland=NT002-13ZB
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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 -3

Floral Nectar Total Imidacloprid Residue (ppb)

Leaf Total Imidacloprid Concentration
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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 -3

Extra Floral Nectar Total Imidacloprid Residue (ppb)

Leaf Total Imidacloprid Concentration
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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 -3

Pollen Total Imidacloprid Residue (ppb)

Leaf Total Imidacloprid Concentration