DP Barcode: D424270 MRID : 495 11702

1 Reference

Gould, T., Bowers, L., Dyer, D., and Jerkins, E. (2014). Determination of the Residues of Imidacloprid

and its Metabo lites 5

- Hydroxy Imidacloprid a nd Imidacloprid Olefin in Bee Relevant Matrices Collected

from Treated Cotton During Two Successiv e Years and in White Clover Planted after Treated Cotton

. Study ID EBNTY010

. Bayer Crop Science, Research Triangle Park, NC. MRID 495 11702.



### 1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material #1 Gaucho 600 FL Admire Pro

Systemic P rotectant (SC) Purity 48.59

Test Material #2 Admire Pro Systemic

Protectant Purity 42.9%

Study Type: Non

- Guideline field residue study on c otton to establish imidacloprid and

metabolite levels in pollen, nectar , flowers, leaves and soil after two succe s sive

annual foliar applications in addition to residues in clover rotational crop.

Sponsor: Bayer Crop Science AG

2 T. W. Alexander Drive

Research Triangle Park, NC

USA 27709 Performing Laboratories:

( Analytical ; Pollen, Nectar ): Bayer Crop Science

A G, 2 T. W. Alexander Drive Research Triangle

Park, NC USA 27709

( Analytical; Leaf, Flower, Soil): SynTech Research

Laboratory Services, 17745 S. Metcalf Ave,

Stillwell, KS 66085

( Trial NT0 13, NT014, NT015): Shoffner Farm

Research, Inc

, 4809 Highway FF, Fi sk, MO Sponsor Study

ID: EBNTY0 10

Study

Completion

Date: 6 N ovember 2014

Experiment
Start/End Date: 2 May 2012

– 3 Sept. 2014

Study Location: 3 Field Trials:

Maiden, MO (NT0 13 ) ; Glennonville, MO (NT014 ) ; Fisk, MO (NT01 5 )

GLP Status: GLP

- co mpliant ; protocol reviewed by EPA, PMRA, CDPR



### 2. REVIEWER INFORMATION

Primary Reviewer: Keith Sappington, M.S., Senior Biologist/Science Advisor, ERB5,

Environmental Fate and Effects Division, Office of Pesticide Programs, USEPA

Signature: Date: 1 /28/2016



### 3. EXECUTIVE SUMMARY

This study was designed to measure the magnitude of imidacloprid residues in bee

- relevant pollen and

nectar (floral and extrafloral) samples and in/on flowers, leaves, and soil in cotton grown from

imidacloprid

- treated seed s ( Gaucho ) and pre

- bloom, foliar applications of imidacloprid (Admire Pro) over
two successive years . In addition, this study examined the occurrence of imidacloprid residues in bee

relevant matrices of white clover planted after foliar application to seed treated cotton harvested the

previous year. Six separate trials were conducted in three study sites in MO (NT0 13 , NT0 14, NT015); threeDP Barcode: D424270 MRID : 495 11702

2 of the trials focused on residues in cotton over two years (TRTDA) and the other half focused on residues

in cotton afte r 1 year and rotationally

- planted white clover during the second year (TRTDB). Each trial

included an untreated control (UTC) plot. Each year, five foliar applications (0. 06 lb a.i./ A x 5 ) were made

5

- 8 days apart to seed treated cotton (0.375 mg a.i. / seed; 0 .05 lb a.i./ A) for a total application rate of 0.35

lb a.i./ A. Foliar applications to cotton were made between BBCH growth stages 19 and 60. Five

composite s amples of cotton flowers, pollen, nectar (foliar and extrafloral) and leaves were made at

various sam pling times during the flowering period of the growing season between 12

- 55 days after the

last yearly foliar application. Soil samples were also collected. QA/QC included field and lab

storage/stability samples, blanks, and matrix spikes to validate anal yte recovery.

A summary of the key findings is as follows:

1. Results indicate that pre

- bloom, foliar applications of approximately 0.06 lb a.i./A x 5 (5

- 8 day

interval) to seed

- treated cotton (0.05 lb a.i./ A) over two successive years results in a maximum of

39.5 and 30 ppb of total IMI in individual composite samples of cotton floral and extrafloral

nectar, respectively, beginning about 13 days following the last application. The maximum daily

average concentration of total IMI in cotton floral and extraf loral nectar is 29.0 and 15.7 ppb

. 2. These same pre

- bloom, foliar applications of imidacloprid resulted in a maximum of 56.7 ppb of

total IMI in individual composite samples of cotton pollen beginning about 13 days following the

last application. The maxim um daily average concentration of total IMI in cotton pollen is 25.2

ppb

. 3. Yearly mean concentrations of total IMI in cotton floral nectar, extrafloral nectar , and pollen

measured after the second year of foliar applications to seed treated cotton were 1. 2X

- 2.9X those

measured during the first year. Most of the increases in total IMI concentrations during year 2

occurred within 13

- 20 days following the last foliar application. It is not clear if the increase in

total IMI concentrations during year 2 refl ect different environmental conditions or pesticide

carryover from year 1, since residues in soil were not measured following year 1 applications.

4. Total IMI residues in pollen were approximately 1

- 2 orders of magnitude greater than those

observed in nectar

. 5. Yearly mean concentrations of total IMI measured in nectar (floral and extrafloral) were typically

greater than those measure in pollen. This differs from other similar residue studies conducted

on other crops where concentrations in pollen were typica lly 10X greater than those found in

nectar after foliar applications ( e.g., cherry, citrus).

6. Two metabolites of IMI (IMI

- olefin and 5

- OH

- IMI) were present below the limits of quantification

in nectar and pollen ( 0.7 ppb ) in the vast majority of samples.

7. D T 50 values for total IMI in floral and extrafloral nectar varied from 19

- 68 days , except for one trial

year where residues were relatively constant over the 40

- d sampling period. Three DT 50 values for

total IMI in cotton pollen varied from 14 to 58 days , w ith one trial year exhibiting low but

relatively constant concentrations of total IMI over time.

8. The predictability of total IMI residues in floral nectar and extrafloral nectar based on measured

concentrations in other plant matrices (flowers, leaves) is fair, with most of the predicted values

being within a factor of 2X of the measured values. Overall, residues of total IMI in flowers were

about 2X those measured in nectar (floral and extrafloral) while those in leaves were about 4

- 5X

those measured in ne ctar. The predictability of total IMI concentrations in pollen based on

measured concentrations in flowers and leaves is poor.

9. Residues of total IMI were detected in white clover nectar and pollen planted after foliar

application to seed treated cotton ha rvested the previous year. In most samples, total IMIDP Barcode: D424270 MRID : 495 11702

3 residues were at or below the level of detection (0.7 ppb). The maximum concentrations of total

IMI measured in clover nectar and pollen were 2.7 and 8.6 ppb, respectively.

Among the strengths of th is study are the evaluation of residues across two successive years of pesticide

application, use of sites widely different soil textures, high sensitivity and quality of analytical methods,

and a reasonable amount of replication of samples. Among the lim itations are lack of measurement of

IMI residues in soil after application during year 1 and use of sites with relatively similar weather

conditions (all three located in MO).



### 4. STUDY VALIDITY

Guideline Followed: Non

- guideline study (protocol was review ed by EPA/PMRA/CDPR)

Guideline Deviations: N/A

Other Deviations:

Classification: ACCEPTABLE

Rationale: No major deviations were identified in this study that would affect the

scientific integrity of this study

Reparability: N/A



### 5. MATERIALS AND M ETHODS

Test Material Characterization

Test item #1 (foliar) Admire Pro Systemic

Protectant SC (Imidacloprid)
550 g a.i./L CAS #: 138261

- 41

- 3 Test item #2 (seed

treatment) Gaucho 600 FL Flowable

concentrate for seed
treatment (FS), 600 g a.i./L

Descrip tion: N/A Purity: 42.9 %. (Admire Pro)

4 8.59 % ( Gaucho )

Lot No./Batch No. Batch No. NK41BX0170
(Admire Pro)

NK41BX0356 ( Gaucho 600) Density: 1.41

– 1.54 g/mL

Material Source: Bayer Crop Science Cert. # 212PG1962 (Admire Pro) ;

DEH028: 128 ( Gaucho )

Mater ial Receipt

Date: Not reported Analysis

Date: 03/30/2012 (Admire Pro) ;

03/13 /2012 ( Gaucho )

Expiration Date: 03/3 1 /2014 (Admire Pro) ;

3 / 1 3/2014 ( Gaucho ) Solubility: 0.51

- 0.61 g/L

Storage of Test Mat’l : Ambient ( 56

- 87 o F) Sample

Storage:

- 39 o to 3 o F

5A

. S TUDY D ESIGN

This study was designed to measure the magnitude of imidacloprid residues in bee

- relevant pollen , floral

and extrafloral nectar samples and in/on flowers, leaves, and soil from cotton plants grown from

imidacloprid

- treated seeds (Gaucho 600 Flowable) followed by five foliar applications per year of ADMIRE
PRO Systemic Protectant containing 550 g/L imidacloprid for two consecutive years. In addition, a secondDP Barcode: D424270 MRID : 495 11702

4 series of trials was conducted to measure the same residues in/on bee

- relevant white clover pollen and

nectar samples and in blossoms, leaves, and soil from white clover plants grown at locations where cotton

plants were grown from Gaucho 600 Flowable treated seed and sprayed with Admire Pro the previous

year

. Besides quantifying the mag nitude of residues in bee

- relevant matrices of cotton and white clover

, this study was also intended to evaluate the following attributes:

1) The extent of year

- to

- year ‘ carry over ’ of pesticide residues measured in plant and soil

matrices
2) The dissipation rat es of IMI /metabolite residues over time in plant matrices

3) The extent to which toxicological relevant metabolites (IMI

- olefin, 5

- hydroxy

- IMI) occurred in

these matrices

4) The relationship of IMI/metabolite residues among different plant tissues (pollen, necta r, leaf,

flower).

At each of three study sites, two treated trials were conducted (TRTDA and TRTDB). The TRTDA trials

consisted of seed treatment (0.375 mg a.i./ seed or 0.047 lb a.i./ A ) and 5 x 0.06 lb a.i./ A foliar applications

(5

- 8 days apart) each year ove r two successive years. The TRTDB trials consisted of seed treatment and 5

x 0.06 lb a.i./ A foliar applications (5

- 8 days apart) during year 1 followed by planting of white clover and

residue sampling of clover during year 2. In addition to the treated cro p study sites, two control sites

were included for each trial (UTCA and UTCB). The year 1 foliar applications (TRTDA and TRTDB were

made between BBCH growth stage 19 (nine or more leaves unfolded) and BBCH 60 (first flowers open).

The year 2 foliar applic ations were made between BBCH growth stage 24 (four side shoots detectable)

and BBCH 59 (first petals visible and many flower buds closed).

Five composite samples of flowers, pollen, floral nectar, extrafloral nectar and leaves were made at

various samp ling times during the flowering period from each TRTD plot/year. For TRTDB sites i n year 2,

clover seeds were planted on plots UTCB and TRTDB p reviously treated in year 1 . H owever, no clover

grew in trial NT013

- 12ZA, so no clover samples could be collect ed and no bees were used. In trials NT014

-
12HA and NT015

- 12ZA, clover samples were collected f rom within erected bee tents; one tent was placed

per plot, and inside each tent one honey bee hive was placed several days prior to clover sampling

. Composite samples of white clo ver leaves and blossoms for direct analysis were collected by hand, and

composite sa mples of nectar and pollen were collected from the bee hives.

Nine soil samples were collected from all plots using a soil sampling device before cotto n planting in year

1, before cotton planting in year 2, and after all sampling was complete in year 2

. QA/QC included field

and lab storage/stability samples, blanks, and matrix spikes to validate analyte recovery. A schematic

showing the application and residue sampling design is shown in Figure 1

.DP Barcode: D424270 MRID : 495 11702

5 Figure 1 . Timeline of residue sampling of nectar, pollen, flower, leaf relative to foliar pesticide

applications (double arrows) and planting of treated seeds for study trial TRT DA (panel A) and TRTDB

(panel B).

5 B

. A PPLICATION T IMING AND R ATES

During each of the two successive years, five foliar applications of approximately 0. 06 lb a.i./A were

applied at intervals from 5

- 8 days apart at each of the three treatment sites for a total annual application

rate of 0.35 lb a.i./ A (annual foliar application rate = 0. 3 lb a.i./A + 0.05 lb a.i./ A from seed treatment ). All

applications were made with adjuvant (Dyne

- Amic, 0. 25 % ) using a ground

- based equipment

. Plot

seeding rates ranged from 57 ,088 to 60,002 seeds/A. Information on the application rates and timing of

applications is provided in Appendix A

.

5C. S TUDY S ITE L OCATION AND C HARACTERISTICS

Various aspects of the study sites are summarized in Table 2. The soil type differed across the three trials

but overall plot areas and type of cotton were similar.DP Barcode: D424270 MRID : 495 11702

6 Table 1 . Summary of C otton study site characteristics (treated sites only)

. Attribute NT0 13

- 12ZA

Malden, MO Trial NT0 14

- 12ZA

Glennonville , MO Trial NT01 5

- 12ZA

Fisk, MO

Plot ID TRTDA / (TRTDB) TRTDA / (TRTDB) TRTDA / (TRTDB)

Variety Pima cotton

(Landino clover) Pima cotton

(Landino clover) Pima cotton

(Landino clover)

Planting Date 9

- May

- 2012,

8

- May 2013
(15 Apr. 2013) 24

- May

- 2012,

8

- May 2013
(15 Apr. 2 013) 22

- May

- 2012,

26

- May 2013
(15 Apr. 2013)

Soil Type Sand y loam Silty loam Sand

Plot Length 1150 ft 600 ft 625 ft

Plot Width 90 ft 180 ft 173 ft

Plot Area 103,500 ft 2 108,000 ft 2 108,331 ft 2

Row Spacing 40 in 40 in 40 in

Plant Spacing

within Row 2.7 in 2.7 in 2.7 in

GPS Lat . Long. 36.60981,

- 90.00092 36. 56850

, - 90. 13092 36. 70934

, - 90.1 9482

A summary of soil and meteorological data from the three study sites is shown in Table 3. Soil organic

carbon varied from 0.7 to 1% across all three trial s. Soils from NT015 contained the highest proportion as

sand (89%) followed by NT013 (66%). NT014 contained very little sand (19%) and mostly consisted of silt

(66 %). Temperatures and t otal p recipitation were similar among sites . Monthly precipitation patterns
follow similar temporal patterns which is expected given the relatively close proximity of study sites

( Figure 2 ). All three sites were irrigated to some extent during the summer months (0.5

– 3 in/month).

Maps showing the locations of the trial s are found in Appendix B.

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

* (from Table 3 of study report)DP Barcode: D424270 MRID : 495 11702

7 Figure 2

. Monthly precipitation for the 3 study trial sites (NT0 13, NT014, NT015).

5D. S AMPLE C OLLECTION
, H ANDLING
, P ROCESSING

Cotton Plant Matrices. Cotton sample collection began 13 to 15 days after the last application (DAA) each

year. Samples were collected at five sampling intervals per year of collection. The sampling intervals were

targeted for growth stages of BBCH 60, 61, 65, 67, and 69 (cotton BBCH 60: first flowers opened,

sporadically within the population; BBCH 69: end of flowering ; Table 4 ). Five composite samples of cotton

blossoms for direct analysis , cotton blossoms for pollen and nectar processing, and cotton leaves were

collected by hand from at least 12 different areas of the treated plot ( avoiding the edges ) at each

sampling interval . Two composite samples were taken from the control plots in the same manner.

Table 3 . Sampling dates and cotton developmental stages for plant residue analysis

Pollen, Nectar and Flower Sampling Leaf Sampling

Year 1

(TRTDA & B) Year 2

(TRTDA) Year 1

(TRTDA & B) Year 2

(TRTDA)

BBCH* DAA* BBCH DAA BBCH DAA BBCH DAA

NT0 13

60 14 60 15 60 15 60 15

61 21 61 22 61 22 61 22

65 28 65 29 65 29 65 29

67 36 67 35 67 35 67 35

--- --- 69 42 69 42 69 42

NT0 14

60 13 60 14 60 13 60 14

61 20 61 21 61 20 61 21

65 26 65 33 65 26 65 33

67 33 67 40 67 33 67 40

69 41 69 50 69 41 69 50DP Barcode: D424270 MRID : 495 11702

8 Pollen, Nectar and Flower Sampling Leaf Sampling

Year 1

(TRTDA & B) Year 2

(TRTDA) Year 1

(TRTDA & B) Year 2

(TRTDA)

BBCH* DAA* BBCH DAA BBCH DAA BBCH DAA

NT01 5

60 13 60 14 60 13 60 14

61 20 61 21 61 20 61 21

65 26 65 28 65 26 65 28

67 33 67 35 67 33 67 35

69 41 69 43 69 41 69 43

*BBCH = B iologische Bundesanstalt, B undessortenamt und CH emische Industrie growth sta ge scale for c otton ; DAA
= days after last application

Clover Plant Matrices. Samples of white clover leaves, blossoms, nectar (hive

- collected), and pollen (hive

collected) were collected at four sampling periods in study year 2. Sample collection was tar geted for

growth stages of BBCH 61, 63, 65, and 67 (clover BBCH 61: flowers open on first raceme; BBCH 67:

flowering declining). For trials NT014

- 12HA and NT015

- 12ZA, two bee

- tight, ventilated mesh

- covered

tents (tunnel) were constructed on plot UTCB. Five tunnels were constructed on plot TRTDB. The tents

were constructed after the last test substance application (to plot TRTDA) in year 2 and around the start

of clover blooming. Normally developed, apparently healthy and queen

- right honey bees, Apis mellife ra (purchased from local sources), were used to collect the clover pollen and nectar samples. One bee colony

housed in a standard 10 frame Langstroth hive was placed in each tent 3

- 4 days prior to sample collection.

The colonies were equipped with pollen t raps. Colonies and tents were removed after the end of pollen

and nectar sampling. The bee colonies were reportedly in good condition, with no visible signs of infection

or infestation, throughout their use in the study.

All clover blossom and leaf samp les were collected by hand into cloth sample bags from at least 12

different areas of the tent, avoiding the edges. Each sample contained a target of 100 g collected from all

areas of the plant. All clover pollen and nectar samples were collected from the tented bee colonies,

which were used as a sampling device for nectar (hive

- deposited) and pollen (from pollen traps). After 3

- 4
days of bee foraging, nectar was sampled from uncapped cells within a newly placed bee hive frame using

a disposable spoon and s ample vial. Pollen from the blossoms in the tent was sampled from the pollen

traps affixed to the colonies. All available pollen and nectar was collected (up to 40

- mL), and the samples

were individually placed in 40

- mL amber glass containers.

Soil. Nine s oil samples were collected from all plots using a soil sampling device before cotton planting in

year 1, before cotton planting in year 2, and after all sampling was complete in year 2. Soil samples were

collected from the surface of the plots (6 inches [1 5 cm] deep) by bulb planter or soil probe and placed in

a plastic bag. In each sampling period, sample locations were randomly selected from the inner half of the

plots (e.g., the inner 100 ft x 200 ft [30 x 60 m] area of a 200 ft x 400 ft [60 x 120 m] plo t), and individual

sample locations were separated by at least 10 feet (3 m).

Sample Storage

. All samples were protected from sunlight and placed in field coolers containing ice

substitute and the transit stability samples. Upon arrival at the site facili ty, all composite samples were

placed directly into frozen storage except the flowers to be processed for nectar and pollen, which were

processed for pollen and nectar by the field investigators

.DP Barcode: D424270 MRID : 495 11702

9 5E. A NALYTICAL M ETHODS

The residues of imidacloprid, 5

- h ydroxy 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 stan dards. Details of

the analytical methods are provided in the study report.

The limit of detection (LOD) for the total residue is the highest LO D value for an individual analyte in a

particular matrix. The limit of quantification (LOQ) for the total resid ue is the highest LOQ value for an

individual analyte in a particular matrix. The LOD and LOQ values for each matrix are shown below in

Table 5

.

Table 4 . Limits of quantification and detection for imidacloprid and metabolitesDP Barcode: D424270 MRID : 495 11702

10 5F. Q UALITY A SSURANCE

Storage Stability

. Storage stability studies and transit spikes indicate that the imidacloprid residues would

have been stable during frozen storage for at least 1080 days (36 months) in cotton flower and leaf

matrices and for at le ast 1281 days (42 months) in soil matrices prior to analysis

.

Transit Stability. To ensure that imidacloprid and its metabolites 5

- hydroxy imidacloprid and imidacloprid

olefin were stable in nectar and pollen, sample sets of two pollen unfortified control s, four fortified pollen

samples, two nectar surrogate controls, and four fortified nectar surrogate samples, were prepared yearly

at Bayer CropScience and shipped frozen via FedEx to each test site. The transit stability samples, fortified

with approximat ely 0.10 ppm each of imidacloprid and its metabolites 5

- hydroxy imidacloprid and

imidacloprid olefin, accompanied the respective study sample types throughout the sampling, shipping,

and storage events of the field phase of the study. They were analyzed af ter all sample analyses of a given

matrix and served to demonstrate stability of imidacloprid residues through the storage intervals and

transport conditions encountered by the samples.

Spike Recovery. The method for determining imidacloprid, 5

- hydrox y i midacl oprid, and i midacloprid

olefin residues in/on cotton and soil samples was validated by measuring the recovery of these analytes

from control matrices fortified at their respective matrix LOQs. A dditional recoveries at higher

fortification levels val idated the method for the highest residues observed in individual matrices.

Concurrent recoveries of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin from fortified

samples were measured with each set of samples to verify method performance

.



### 6. RESULTS:

6.A. Q UALITY A SSURANCE R ESULTS

Sample Storage. The maximum storage period of frozen samples in this study for imidacloprid was 621
days for cotton flowers, 595 days for c otton leaves, 413 for cotton nectar, and 544 days for cotton pollen.

Ma ximum storage times for clover samples was 425 days and that for soil samples was 734 days. These

storage times are within the bounds of the storage stability results described earlier and indicate that

storage times are acceptable.

Transit Stability. T he transit stability analyses indicate that the imidacloprid residues are stable (<30%

decomposition) during concurrent freezer storage with the samples from this study.

Matrix Spike Recoveries. As indicated in Table 5 of the study report, all matrix sp ike recoveries (poll en, nectar, flower, leaf, soil were within the acceptable range of 70 to 120%, and the standard deviation

values were below 20%.

6. B

. M AGNITUDE OF R ESIDUES IN B EE

- R ELEVANT M ATRICES

Cotton Nectar. Summary statistics of t he overall magnitude of total IMI residues in cotton extrafloral and

floral nectar are shown in Table 6

. These statistics in Table 6 reflect analysis of individual composite

samples (usually 5 replicates per sampling date) among the sampling times within each year o f each trial.

Plots of the raw data for each trial are found in Appendix C

. Since IMI

- olefin and 5

- OH IMI are considered

of similar toxicity as parent IMI, this discussion focuses on total IMI residues (sum of IMI, IMI

- olefin and 5

-DP Barcode: D424270 MRID : 495 11702

11 OH

- IMI). The underlying data for nectar and pollen were taken from Appendix 7 of the study report. As

conducted by the study authors, the total IMI reside value for each sample was calculated by summing

the results for all three individual analytes. This total residue value was considered <LOD if all three

summed analytes were <LOD, <LOQ if at least one of the summed analytes was >LOD but none of the

analytes were >LOQ, and quantifiable if at least one analyte was >LOQ. As a result, residue values of total

IMI at or below 1.2 pp b essentially reflect these assumptions regarding <LOD and <LOQ.

Across all three trials, yearly m ean residues of total IMI in c otton extrafloral nectar r ange from

approximately 3.4

– 11 ppb while yearly maxima range from 21

– 30 pp b

. Results for floral nectar are
somewhat greater than those for extrafloral nectar, with yearly average total IMI residues ranging from

4.3 to 16.2 ppb and yearly maxima ranging from 10.7 to 39.5 ppb

. Notably, the highest mean (and

maximum) values of total IMI in floral and extrafloral nectar occurred in trials NT013 and NT015 (year 2 of

TRTDA). The percent sand in soils from trials NT013 and NT015 (66% and 89%) are 3X and 4X that found in

trial NT014 (19%)

. This suggests that the greater accumulation in nectar in trials NT01 3 and NT015 may

be due to their coarser soils.

Table 5 . Magnitude of t otal IMI in cotton extrafloral and floral nectar

Extrafloral Nectar (ppb)

Trial NT013 * Trial NT014 * Trial NT015 *

TRTDA TRTDB TRTDA TRTDB TRTDA TRTDB

2012 2013 2012 2012 2013 2012 2012 2013 2012

Mean 4.1 11 3.4 4.2 5.1 4.2 4.2 7.1 2.5

Min. 1 2.8 1.8 1.2 1.2 1.2 1.2 2.2 1.2

Med. 3.2 12 3.1 2.4 2.6 1.8 4.2 5.1 2.5

90th 7.5 19 7.2 12 14 11 9.3 11 4.9

Max. 9.1 24 8.9 16 21 16 14 30 6.2

Floral Nectar (ppb)

Mean 5.2 14.4 9.0 5.2 7.4 4.3 9.8 16.2 6.6

Min. 2.4 5.9 2.3 2.0 1.9 1.8 2.7 4.2 3.3

Med. 4.6 11.0 5.0 3.3 2.6 2.6 9.6 11.7 6.0

90th 8.8 26.3 19.9 12.9 18.0 10.1 14.3 29.8 9.9

Max. 11.6 28.9 38.4 15.7 21.9 17.7 19.8 39.5 10.7

N OTES : Total IMI = sum of i midacloprid, imidacloprid olefin and hydroxy imidacloprid

. The h ighest measured

concentration is shown in bold

. * V alues below the LOD were assumed equal to 1⁄2 the LOD . Values below the LOQ (but above the LOD) were

assumed equal to 1⁄2 the LOQ.

Plots of the daily average residue values of total IMI (and daily ranges) in cotton floral nectar are shown in

Figure 3. It is apparent that mean daily residues of total IMI measured following the second application

year are greater than those during the first yea r . This occurred throughout year 2 in trial NT013 and

during 1 3

- 20 DAA for year 2 in trials NT014 and NT015. Furthermore, mean daily concentrations of total

IMI in floral nectar from year 1 of TRTDA and TRTDB are very similar within each trail. Since ye ar 1 of

TRTDA and TRTDB are essentially replicate study plots, this similarity in residue profiles between TRTDA

and TRTDB suggests the elevated residues in year 2 of TRTDA are not simply spurious results. The

maximum daily average concentration of total IMI in cotton floral nectar is 29.0 ppb.DP Barcode: D424270 MRID : 495 11702

12 Plots of the daily average residue values of total IMI (and daily ranges) in cotton extrafloral nectar are

shown in Figure 4

. With the exception of the latter sampling dates in Trial NT013, average daily residue s of total IMI from year 2 are similar to those from year 1. As observed with floral nectar, the mean daily

concentrations of total IMI in extrafloral nectar from year 1 of TRTDA and TRTDB are similar within each

trail. The maximum daily average concentr ation of total IMI in extra floral nectar is 1 5.7 ppb.DP Barcode: D424270 MRID : 495 11702

13 Figure 3

. Total imidacloprid residues measured in cotton floral nectar for trials NT013, NT014 and NT015

( symbols = mean, error bars = range)

.DP Barcode: D424270 MRID : 495 11702

14 Figure 4

. Total imidacloprid residues measured in cotton extrafloral nectar for trials NT013, NT014 and

NT015 ( symbols = mean, error bars = range)

.

Pollen. Yearly m ean and maximum residues of total IMI in cotton pollen in each trial are shown in Table 6

. Yearly m ean residues of total I MI pollen vary from 1.5

– 6.9 ppb while maxima range from 3.9

– 56.7 ppb

. As observed with floral nectar, the highest yearly mean (and maximum) values of total IMI in cotton

pollen occurred in year 2 of the TRTDA trials, which were 1.5

– 3X the respective yearly means from year

1. The relationship between total IMI residues in pollen and soil type (% sand vs. % silt) was not as

evident as that observed with floral and extrafloral nectar. Specifically, the lowest mean and maxima

residues of total IMI occu rred with trial NT015 (89 % sand) while mean and maxima residues in trials

NT013 and NT014 are similar despite their large difference in soil coarseness (66% and 19% sand,

respectively).

Table 6

. Magnitude of total IMI residues in c otton pollen (ppb).

Concentration in Pollen (ppb)

Trial NT013 Trial NT014 Trial NT015

TRTDA TRTDB TRTDA TRTDB TRTDA TRTDB

2012 2013 2012 2012 2013 2012 2012 2013 2012

Mean 2.4 6.9 2.7 4.0 6.1 5.1 1.5 3.0 1.5

Min. 0.9 0.6 0.9 0.6 0.6 0.6 0.6 0.6 0.6

Med. 2.1 2.8 1.7 1.7 0.6 1.9 0.9 0.9 0.9

90th 3.9 16.8 6.3 12.2 19.4 5.2 3.2 6.2 3.5

Max. 5.3 36.0 11.7 17.4 47.4 56.7 3.9 19.4 4.4

N OTES : Total IMI = sum of i midacloprid, imidacloprid olefin and hydroxy imidacloprid . The h ighest measured

concentrati on shown is in bold

. * Values below the LOD were assumed equal to 1⁄2 the LOD. Values below the LOQ (but above the LOD) were

assumed equal to 1⁄2 the LOQ.

Plots of the daily average residue values of total IMI (and daily ranges) in cotton pollen are shown in

Figure 5

. Generally, average daily residues of total IMI in pollen between 1 3

- 20 DAA from year 2 are

greater than those from year 1 across all three trials . As observed with floral and extrafloral nectar, theDP Barcode: D424270 MRID : 495 11702

15 mean daily concentrations of total IMI in pol len from year 1 of TRTDA and TRTDB are similar within each

trail. The maximum daily average concentration of total IMI in pollen is 2 5. 2 ppb.

Figure 5

. Total imidacloprid residues measured in cotton pollen for trials NT01 3, NT014 and NT015

( symbols = mean, error bars = range)

.DP Barcode: D424270 MRID : 495 11702

16 Flowers

. Across all trials and years , m ean residues of total IMI in flowers range from 6.0

- 13.6 ppb in 201 2

and 6.0

- 10.2 ppb in 2014 ( Table 7 ). No consistent trend is observed between mean residues measured in

flowers during 2012 vs. 2013 for TRTDA. Mean concentrations of total IMI in flowers are higher by up to

5X those measured in pollen ( Table 7 vs. Table 6 ).

Table 7

. Mean concentrations of total IMI in cotton flowers (ppb).

Treatment ( Year ) Residue Trial

NT013 NT014 NT015

TRTDA (2012 ) 8.8 13.6 8.8

TRTDA (2013 ) 10.2 6.0 8.4

TRTDB (2012) 9.6 12.0 6.0

6. C

. M AGNITUDE OF R ESIDUES IN L EAVES AND S OIL

Mean concentrations of total IMI in cotton leaves within each trial/study year are shown in Table 8.

In terestingly, mean residues in year 2 (2013) are substantially lower (by 2X

- 3X) than those measured

during year 1 (2012). This is in contrast to total IMI residues measured in nectar and pollen, Mean

concentrations of total IMI in leaves are higher by up t o 2X

- 20X those measured in pollen ( Table 8 vs.

Table 6 ).

Table 8

. Mean concentrations of total IMI in cotton leaves (ppb).

Treatment ( Year ) Residue Trial

NT013 NT014 NT015

TRTDA (2012 ) 29.3 54.0 27.8

TRTDA (2013 ) 16.4 14.6 12.0

TRTDB (2012) 28.2 41.4 17.8

Imidacloprid concentrations (parent only) measured in soils of all three study trials are shown in Table 9

. Concentrations are less than 100 ppb at all sampling times. Based on samples taken prior to planting

during years 1 and 2, the concentration of IMI in soil increases from 0.3

- 12 ppb (year 1) to 24

- 45 ppb prior

to planting in year 2. No samples were taken at the end of the 1 st growing season, so the extent of

pesticide carryover could not be determined immediately aft er applications.

Table 9

. Magnitude of IMI residues in soil (ppb).

Year Date Event DAFA 1 Mean IMI in Soil

TRTDA (ppb) 2 Mean IMI in Soil

TRTDB (ppb) 2

NT 013

1 05/09/12 Prior to planting 0 1 2 12

1 7/9/12

– 7/30/12 Foliar applicatio ns 61

- 82

1 NA End of 1 st growing season NA NA NA

2 4/25/13 Prior to planting 351 34 42

2 6/27/13

– 7/18/13 Foliar applications 414

- 435

2 11/21/13 End of 2 nd growing season 561 79 23

NT0 14

1 05/10/12 Prior to planting

- 14 5 2

1 7/5/12

– 7/27/12 F oliar applications 42

- 64DP Barcode: D424270 MRID : 495 11702

17 Year Date Event DAFA 1 Mean IMI in Soil

TRTDA (ppb) 2 Mean IMI in Soil

TRTDB (ppb) 2

1 NA End of 1 st growing season NA NA NA

2 4/25/13 Prior to planting 336 45 41

2 6/27/13

– 7/18/13 Foliar applications 399

- 420

2 11/21/13 End of 2 nd growing season 546 98 33

NT015

1 5/21/12 Prior to planting

- 1 0.3 0.3

1 7/ 3 /1 3

– 7/2 6 /1 3 Foliar applications 42

- 65

1 NA End of 1 st growing season NA NA NA

2 4/25/13 Prior to planting 338 24 36

2 7/ 3 /1 3

– 7/2 4 /1 3 Foliar applications 407

- 428

2 11/21/13 End of 2 nd growing season 548 79 32

1 DAFA = Days After First Applic ation in Year 1

2 Mean of 3 soil composite samples for IMI only

6. D

. Y EAR TO Y EAR A CCUMULATION IN P OLLEN
, N ECTAR
, AND S OIL

A qualitative evaluation of the potential for ‘year

- to

- year’ carryover in the accumulation of total IMI

residues in nectar and pol len is conducted here for the three trials based on results presented in Tables 5

and 6 and Figures 4

- 6 . Based on yearly mean values of total IMI in floral and extrafloral nectar, year 2

means increase by 1.2X to 2.7X over year 1 means ( Table 5 ). With cot ton pollen, yearly averages of total

IMI increase by 1.5 X to 2.9X from year 1 to year 2. As noted previously, the majority of increase in year 2

residues of total IMI occurred sooner after application (1 3

- 20 days) across the trials. Interestingly, the two

trials with the greatest % sand in soils (NT013 and NT015) show the greatest relative increase in yearly

average total IMI from year 1 to year 2 in nectar and pollen (1.7X to 2.9X) compared to NT014 which

contained mostly silt (1.2

- 1.5X). It is not certa in whether this differential increase is related to differences

in soil composition, but all three trials had similar amounts of IMI in soil prior to the 2 nd year planting (24

- 42 ppb).

It is clear from Table 9 that IMI residues in soil measured prior to planting in year 2 ( 24

- 45 ppb ) are

elevated compared to those measured prior to planting in year 1 ( 0.3

- 12 ppb ). This could explain some of

the higher residues of total IMI in pollen and nectar observed in year 2. Unfortunately, soil samples at the

end of the 1 st growing season were not taken; therefore, it is not known whether post application

residues in soil increase from year to year or remain similar from year to year. Other factors (weather)

may also contribute to these observed differences.

6. E

. R ESIDUE D ECLINE (DT 50 ) IN P OLLEN AND N ECTAR

The rate of residue decline of total IMI in cotton extrafloral nectar, floral nectar and pollen was

determined via linear regression of total IMI residues (log transformed) vs. time (days after last pesticid e application). The resulting DT 50 values were determined for those trial years with 4 or more sampling

days with residues measured above the LOD. Results are shown in Table 10

. Plots of the residue decline

determinations are shown in Appendix D

. For r esidues of total IMI in cotton floral nectar, DT 50 values

vary from 19

- 68 days while those for extrafloral nectar vary from 27

– 373 days . With floral and

extrafloral nectar, 1 4 /16 of the DT 50 values of total IMI are between 19 and 51 days . The large DT 5 0 valueDP Barcode: D424270 MRID : 495 11702

18 of 373 days for year 2 of trial NT013 is unusual among the cotton trials in that concentrations of total IMI

remain above or near 10 ppb for 42 days following application with little apparent decline.

With pollen, most of the trials contained in sufficient data for reliable determination of DT 50 values (<4

sampling points with detectable residues of total IMI). Three DT 50 values for total IMI in pollen varied from

14 to 58 days . The negative DT 50 values of

- 450 represents slightly increasing conc entrations of total IMI

over time in pollen, but residue levels were close to the LOD and therefore, this finding may be less

reliable than other DT 50 values.

Trial Trial Year DT 50 (days) r 2 P

- Value

Floral Nectar

NT013 2012A 32 0.75 <0.001

2013A 39 0. 65 <0.001

2012B 19 0.67 <0.001

NT014 2012A 44 0.32 0.003

2013A 24 0.86 <0.001

2012B 41 0.32 0.004

NT015 2012A 50 0.43 <0.001

2013A 33 0.50 <0.001

2012B 68 0.37 0.001

Extrafloral Nectar

NT013 2012A 28 0.63 <0.001

2013A 373 0.00 0.75

2012B ND

NT014 2012A 27 0.52 <0.001

2013A 27 0.71 <0.001

2012B ND

NT015 2012A 27 0.65 <0.001

2013A 33 0.61 <0.001

2012B 51 0.31 0.01

Floral Pollen

NT013 2012A

- 450 0.00 0.83

2013A 14 0.76 <0.001

2012B ND

NT014 2012A 25 0.33 0.003

2013A N D

2012B 58 0.06 0.25

NT015 2012A ND

2013A ND

2012B ND

ND = not determined because <4 sampling events contained detectable residues.DP Barcode: D424270 MRID : 495 11702

19 6. F

. O CCURRENCE OF R ELEVANT IMI M ETABOLITES

Very few samples contained concentrations of IMI

- olefin or 5

- OH

- IMI above the limits of quantitation in

pollen and nectar (0.7 ppb). Therefore a quantitative analysis of the fraction of total IMI represented by

these two metabolites was not conducted.

6. G

. O CCURRENCE OF IMI IN R OTATIONAL C ROP ( WHITE C LOVER )

Th e concentrations of total IMI measured in white clover nectar and pollen planted following foliar

application to seed treated cotton harvested the previous year (trials NT014 and NT015) were near or

below the level of detection (0.7 ppb) in the majority of samples analyzed (detection frequency = 38% for

clover nectar and 53% for clover pollen) . The maximum concentration s of total IMI measured in clover

nectar in trials NT014 and NT015 are 1.6 and 2.7 ppb , respectively

. The maximum concentrations of total

I MI measured in clover pollen in trials NT014 and NT015 are 8 and 8.6 ppb , respectively.

6. H

. R ELATIONSHIP OF R ESIDUES A MONG P LANT T ISSUE S

Predictability of Flower Residues. The relationship between mean residues of total IMI measured in

flowers and tot al IMI measured in cotton floral nectar, extrafloral nectar and pollen on the same sampling

day is shown in Figure 6 A

, 6 B

, and 6C, respectively. Total IMI residues in floral nectar are predicted within

2X of measured residues for 36/43 samples using simpl e linear regression with flower data (r 2 = 0.48).

Total IMI residues in extrafloral nectar are predicted within 2X for 34/42 paired samples (r 2 = 0.59). Based

on the slopes, concentrations of total IMI in nectar are about 1⁄2 those in flowers on average. A weaker

relationship is seen between total IMI in pollen vs. flower and much more scatter is seen in the

relationship (Figure 6C). It is likely that assumptions regarding total IMI in pollen measured below the

LOD or LOQ confounded this relationship , as many values are near or below the LOD and LOQ

. Only

about 1/3 of total IMI residues in pollen are predicted within a factor of 2 of measured values.DP Barcode: D424270 MRID : 495 11702

20 Figure 6

. Relationship between total IMI residues in cotton nectar (A), extrafloral nectar (B) and p ollen

( C ) to those measured in flowers

Predictability of Leaf Residues

. The relationship between mean residues of total IMI measured in c otton

leaves and total IMI measured in floral nectar, extrafloral nectar, and pollen on t he same sampling day is

shown in Figure 7 A, 7B, and 7C

. With flora and extrafloral nectar, predicted concentrations of total IMI

using leaf data are within a factor of 2X of measured values for 41 and 35 of the 43 comparisons made,

respectively. Overall, total IMI residues in cotton leaves are about 4

- 5X those in floral nectar and

extrafloral nectar. A s observed with flower data, residues of total IMI in leaves produce a poorer

prediction of total IMI residues in pollen ( Figure 7C )

.DP Barcode: D424270 MRID : 495 11702

21 Figure 7

. Relationship between total IMI residues in cotton floral nectar (A), extrafloral nectar (B) and

pollen (C) to those measured in leavesDP Barcode: D424270 MRID : 495 11702

22 

### 7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS

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

- related matrices of c otton , the

following strengths are observed with this study.

1. Analytical methods were sufficiently accurate and precise based on QA data

2. Concentrations were measured for toxicologically

- relevant metabolites in multiple plant matrices

3. Application methods and rates were well documented

4. Sampling contained a reasonable amount of replication and compositing to account for natural

variability

5. Residues were measured over two growing seasons to enable carry over to be assessed

. 6. A wide range of soil characteristics (% sand, % silt) was represented in the 3 trials

A number of limitations are also noted with this study, including:

1. The three trials were relatively close in proximity as indicated by the similarity in precipitation

p atterns, temperature and maps of the study locations. This limits the extent to which

differences in weather conditions could be assessed on residues in cotton matrices.

2. IMI was not measured in soil samples immediately after the growing season in year 1. This

substantially limits the ability to assess year to year carryover of IMI in soils.

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

1. P re

- bloom, foliar application s of approximately 0. 06 lb a .i./A x 5 (5

- 8 day interval) to seed

- treated

cotton (0.05 lb a.i./ A) over two successive years result s in a maximum of 39.5 and 30 ppb of total

IMI in individual composite samples of cotton floral and extrafloral nectar , respectively, beginning

about 1 3 days following the last application. The maximum daily average concentration of total

IMI in cotton floral and extrafloral nectar is 29.0 and 15.7 ppb

. 2. These same pre

- bloom, foliar application s of imidacloprid result ed in a maximum of 56.7 ppb of

total IMI in individual composite samples of cotton pollen beginning about 13 days following the

last application. The maximum daily average concentration of total IMI in cotton pollen is 25.2
ppb

. 3. Yearly m ean concentrations of total IMI in cotton floral nectar, e xtrafloral nectar , and pollen

measured after the second year of foliar applications to seed treated cotton were 1.2X

- 2.9X those

measured during the first year. Most of the increases in total IMI concentrations during year 2

occurred within 1 3

- 20 days foll owing the last foliar application. It is not clear if the increase in

total IMI concentrations during year 2 reflect different environmental conditions or pesticide

carryover from year 1, since residues in soil were not measured following year 1 applicati ons.

4. Total IMI residues in pollen were approximately 1

- 2 orders of magnitude greater than those

observed in nectar

. 5. Yearly m ean concentrations of total IMI measured in nectar (floral and extrafloral) were typically

greater than those measure in pollen. T his differs from other similar residue studies conducted

on other crops where concentrations in pollen were typically 10X greater than those found in

nectar after foliar applications ( e.g., cherry, citrus).

6. Two metabolites of IMI (IMI

- olefin and 5

- OH

- IMI) were present below the limits of quantification

in nectar and pollen ( 0.7 ppb ) in the vast majority of samples.

7. DT 50 values for total IMI in floral and extrafloral nectar varied from 19

- 68 days, except for one trial

year where residues were relatively cons tant over the 40

- d sampling period. Three DT 50 values for

total IMI in cotton poll en varied from 14 to 58 days , with one trial year exhibiting low but

relatively constant concentrations of total IMI over time.

8. The predictability of t otal IMI residues in f loral nectar and extrafloral nectar based on measuredDP Barcode: D424270 MRID : 495 11702

23 concentrations in other plant matrices (f lowers , leaves) is fair, with most of the predicted values

being within a factor of 2X of the measured values. Overall, residues of total IMI in flowers were

abo ut 2X those measured in nectar (floral and extrafloral) while those in leaves were about 4

- 5X
those measured in nectar. The predictability of total IMI concentrations in pollen based on

measured concentrations in flowers and leaves was poor.

9. Residues of t otal IMI were detected in white clover nectar and pollen planted after foliar

application to seed treated cotton harvested the previous year . In most samples, total IMI

residues were at or below the level of detection ( 0.7 ppb ). The maximum concentration s of total

IMI measured in clover nectar and pollen were 2.7 and 8.6 ppb, respectively.



### 8. STUDY VALIDITY/CLASSIFICATION

Data from the three trials (NT0 13 , NT0 14 , NT01 5 ) are considered scientifically sound and useful for risk

assessment purposes, alth ough these trials were located in relatively close proximity with similar weather

conditions, a range of soil types are represented. Overall, this study is classified as ACCEPTABLE for

quantitative use in risk assess ment.DP Barcode: D424270 MRID : 495 11702

24 

### Appendix A . Details of Imidac loprid Application Rates and TimingDP Barcode: D424270 MRID : 495 11702

25DP Barcode: D424270 MRID : 495 11702

26DP Barcode: D424270 MRID : 495 11702

27 Appendix B . Location of C otton Residue Trials.

Figure B 1. Locations of Trial NT0 13 ( Maiden, MO), NT014 (Glennonville, MO) and NT015 (Fisk, MO)

NT013

NT014 NT015DP Barcode: D424270 MRID : 495 11702

28 

### Appendix C. Plots Of Raw Data For Total IMI I n Cotton Floral Nectar, Extrafloral Nectar And

Pollen.

Figure C

- 1. Total imidacloprid concentrations in cotton floral nectar for TRTDA and TRTDB trialsDP Barcode: D424270 MRID : 495 11702

29 Figure C

- 2 . Total imidacloprid concentrations in cotton extrafloral nectar for TRTDA and T RTDB trialsDP Barcode: D424270 MRID : 495 11702

30 Figure C

- 3. Total imidacloprid concentrations in cotton pollen for TRTDA and TRTDB trialsDP Barcode: D424270 MRID : 495 11702

31 Appendix D. Plots of Total IMI Residue Decline Curves for Cotton

Figu r e D

- 1. Residue decline curves for total IMI in cotton floral nec tarDP Barcode: D424270 MRID : 495 11702

32 Figu r e D

- 2 . Residue decline curves for total IMI in cotton extrafloral nectarDP Barcode: D424270 MRID : 495 11702

33 Figu r e D

- 3 . Residue decline curves for total IMI in cotton pollen (Trials NT013 and NT014 only)