DP Barcode: D424267 MRID : 495 11701

1 Reference

Miller, A

., 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 Seed Treated Seed Treated Field Corn During Two Successive Years and in White

Clover Planted after Seed Treated Field Corn

. Study ID EBNTY0 09

. Bayer Crop Science, Research

Triangle Park, NC. MRID 495 1170 1

.



### 1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material #1 Gaucho 600 FL Admire Pro

Systemic Protectant (SC) Purity 48.59

Study Type: Non

- Guideline field residue study on seed treated corn to establish imidacloprid

and metabolite levels in pollen, tassel , leaves and soil after two succes 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
AG, 2 T. W. Alexander Drive Research Triangle

Park, NC USA 27709

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

Laboratory Services, 17745 S. Metcalf Ave,

Stillwell, KS 66085

( Field Trial NT0 1 0) Heartland Ag Research, LLC

, 1

5635 W. 83rd Terrace

, Lenexa, KS 66219
(Field Trial NT01 1): Dozier Ag Research

Services, LLC

, PO Box 308 Springfield, NE 68059
(Field Trial NT012): MidWest Research,

Inc.

, 910 Road 15 York, NE 68467 Sponsor Study

ID: EBNTY 0 0 9
Study

Completion

Date: 3 N ovember 2014

Experiment
Start/End Date: 1 May 2012

– 9 July 2014
Study Location: 3 Field Trials:

Edgerton, KS, (NT010 ) ; Springfield, NE (NT01 1 ) ; York, NE (NT01 2 )

GLP Status: GLP

- compliant ; 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
samples and in/on tassels , leaves, and soil in co rn grown from imidacloprid

- treated seeds ( Gaucho )

. In

addition, this study examined the occurrence of imidacloprid residues in bee relevant matrices of white

clover planted after planting and harvesting seed treated co rn the previous year. Six separate trials were

conducted in three study sites in KS and NE (NT0 1 0 , NT0 1 1

, and NT01 2 ); three of the trials focused on

residues in co rn over two years (TRT ST A) and the other three trials foc used on residues in co rn after 1

year and rotationally

- planted white clover during the second year (TRT ST B). Each trial included anDP Barcode: D424267 MRID : 495 11701

2 untreated control (UTC) plot. Each year

, seed treated co rn (1.34 mg a.i./ seed; 0.12 a.i./ A) was planted at

the beginning of the growing season. Five composite s amples of co rn pollen, tassels, and leaves were

made at various sampling times during t he flowering period of the growing season between 58 and 84

days after planting. Soil samples were also collected. QA/QC included field and lab storage/stability

samples, blanks, and matrix spikes to validate analyte recovery.

A summary of the key find ings is as follows:

1. Planting of seed treated corn (1.34 mg a.i./ seed; 0.12 a.i./ A) over two successive years result s in a

maximum of 3 9

. 7 ppb of total IMI in individual composite samples of corn pollen , which occurred

at the end of the blooming period during the second year of planting. The maximum daily

average concentration of total IMI in co rn pollen is 2 2.3 ppb and the maximum yearly average

concentration is 7.5 ppb

. 2. Yearly average concentrations o f total IMI in corn tassels are about 2X

– 3X the mean residues

reported for corn pollen ( yearly average total IMI residues rang e from 4.8 to 13.7 ppb and yearly

maxima rang e from 9.4 to 26.1 ppb.

3. Temporal trends in the concentration of total IMI in corn pollen are either variable or show
decreases with time during 2012. During 2013 (year 2), concentrations of total IMI in corn pollen

consistently increased with time for each of the three trials. T he reason for this increase in

concentration with time is not clear.

4. Year to year carryover of total IMI residues was not evident based on overall average

concentrations in corn pollen, although measurements in soil from the study plots indicates

increasi ng concentrations from year 1 to year 2.

5. Two metabolites of IMI (IMI

- olefin and 5

- OH

- IMI) were present below the limits of quantification

in corn pollen (0.3 and 0.5 ppb, respectively ) in the vast majority of samples.

6. Due to the highly variable or increasi ng trends seen in concentrations of total IMI in corn pollen,

DT 50 values were not determined.

7. The predictability of t otal IMI residues in corn pollen based on measured concentrations in tassels

is fair, with most of the predicted values being within a f actor of 2X of the measured values. Total

IMI residues in corn leaves provides little predictive capability for corresponding concentrations in

pollen.

8. Residues of total IMI measured in white clover pollen after planting and harvesting seed treated

corn the previous year are low, with a detection frequency of approximately 28%. Residues of

total IMI in white clover nectar were not found above limits of quantitation. The maximum

concentration of total IMI detected in clover pollen is 3.8 ppb

.



### 4. STUDY VALIDITY

Guideline Followed: Non

- guideline study (protocol was reviewed 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/ADP Barcode: D424267 MRID : 495 11701

3 

### 5. MATERIALS AND METHODS

Test Material Characterization

Test item: Gaucho 600 Flowable seed

treatment ( ST ), 600 g a.i./L CAS #: 138261

- 41

- 3

Description: N/A Purity: 4 8.59 %

Lot No./Batch No. Batch No. NK41BX0356 Density: 1.41

– 1.54 g/mL

Material Source: Bayer Crop Science Cert. # DEH028: 128

Material Receipt

Date: Not reported Analysis

Date: 03/13 /2012

Expiration Date: 3 / 1 3/2014 Solubility: 0.51

- 0.61 g/L

Storage of Test Mat’l : Ambient ( 54

- 8 0 o F) Sample

Storage:

- 24 o to 26 o F

5A. S TUDY D ESIGN

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

, tassel, leaves, and

soil associated with corn plants grown from imidacloprid

- treated seeds (Gaucho 600 Flowable)

. In

addition, a second 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 seed treated corn pla nts were grown the previous year

. Besides quantifying the

magnitude of residues in bee

- relevant matrices of co rn and white clover , this study was also intended to evaluate the following attributes:
1) The extent of year

- to

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

matrices
2) The dissipation rates 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, nectar, leaf,

flower).

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

consisted of seed treatment at a rate of 1.34 mg a.i./ seed (equivalent to a soil loading rate of 0.0 11

– 0.12

lb a.i./ A) over two successive years. The TRTDB trials consisted of seed treatment of 1.34 mg a.i./ seed

(equivalent to a soil loading rate of 0.12 lb a.i./ A) during year 1 followed by planting of wh ite clover and

residue sampling of clover during year 2. All plots were tilled or disked at least once prior to the year 2

planting of corn or clover. I n addition to the treated crop study sites, two control sites were included for

each trial (UTCA and UT CB).

Five composite treated samples (separate runs through the plot) of field corn leaves, tassels, and pollen

were collected by hand per sample period from treated plot TRTSTA in years 1 and 2 and from treated

plot TRTSTB in year 1. For TRTDB sites i n y ear 2, clover seeds were planted on plots UTCB and TRTDB

p reviously treated in year 1

. Five composite samples (separate runs through the plot) of white clover

flowers and leaves were collected by hand and five composite samples of nectar and pollen were

c ollected from the hives of tented bees in treated plot TRTSTB in year 2. For collection of clover samples

, one tent was placed per plot, and inside each tent one honey bee hive was placed 1

- 2 prior to clover

sampling

. Composite samples of white clo ver leaves and blossoms for direct analysis were collected byDP Barcode: D424267 MRID : 495 11701

4 hand, and composite sa mples of nectar and pollen were collected from the bee hives. Nine soil samples

were collected using a soil sampling device prior to seed planting and at the end of the gr owing season

per year from treated plots TRTSTA and TRTSTB.

Two composite samples of all field corn and clover matrices and nine composite samples of soil were

collected from the control plots UTCA and UTCB, at the same sampling periods as used for the tr eated

samples of that sample type. QA/QC included field and lab storage/stability samples, blanks, and matrix

spikes to validate analyte recovery

.

5 B

. A PPLICATION T IMING AND R ATES

Plot TRTSTA received field corn seed treated with Gaucho 600 Flowable in years 1 and 2 of the study

(2012 and 2013, respectively). Plot TRTSTB received field corn seed treated with Gaucho 600 Flowable in

year 1 and untreated forage crop (white clover) seed in year 2. All plots were tilled or disked at least once

prior to the year 2 planting of corn or clover. For plot TRTSTA, the seed planting rate ranged from 36,440

to 41,480 seeds/A across both years. Soil application rates due to seed treatment for TRTSTA ranged from

0.115 to 0.122 Ib imidacloprid/A (0.129 to 0.137 kg imida cloprid/ha) in year 1 and from 0.108 to 0.121 Ib
imidacloprid/A (0.120 to 0.135 kg imidacloprid/ha) in year 2. For plot TRTSTB, the treated seed planting
rate ranged from 38,820 to 41,330 seeds/A in year 1. Soil application rates due to seed treatment for
TRTSTB ranged from 0.115 to 0.122 Ib imidacloprid/A (0.129 to 0.137 kg imidacloprid/ha) in year 1.

Information on the application rates and timing of applications is provided in Table 1

.DP Barcode: D424267 MRID : 495 11701

5 Table 1 . Imidacloprid application and corn se eding rates for the three study trials *

* from Table 4 of the study report)

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 variety of corn were similar

.

Table 2 . Summary of C orn study site characteristics (treated sites only)

. Attribute NT01 0

- 12ZA

Edgerton, KS Trial NT01 1

- 12ZA Springfield, NE Trial NT01 2

- 12ZA
York, NE

Plot ID TRTST A / (TRT ST B) TRTST A / (TRT ST B) TRTST A / (TRT ST B)

Variety Corn 56N67

, 56N667 VTP3

( Dutch White clover) Corn 56N67

, 56N667 VTP3
(Dutch White clover) Corn 56N67, 56N667 VTP3
(Dutch White clover)

Planting Date 5

- May

- 2012,

25

- May 2013
( 24 Sept . 201 2 ) 8

- May

- 2012,

7

- May 2013
( 6 Sept. 201 2 ) 11

- May

- 2012,

2 9

- April 2013
(1 6 May 2013)

Soil Type Silty clay Silt loam Loam

Plot Length 300 ft 370 ft 200 ft

Plot Width 150 ft 120 ft 220 ft

Plot Area 45,000 ft 2 44, 4 00 ft 2 44,000 ft 2

Row Spacing 30 in 30 in 3 0 in

Plant Spacing

within Row 5.06 in 5 in 8 in

GPS Lat . Long. (38.69305,

- 94.986267 41.03567,

- 96.14668 (40.945793,

- 97.600888DP Barcode: D424267 MRID : 495 11701

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

carbon varied from 1.3% to 2.4% across all three trials (control sites included)

. Among the treated sites,

s oils from NT01 2 contained the most sand ( 38%), followed by NT011 (28%) and NT010 (16%).

Temperatures were similar among sites , but considerably more rainfall occurred at site NT010 (55.7 in) vs

the other two treated sites (33 and 37 in). With the exception of the spring 2012 and winter 2013 at s ite

NT010, m onthly precipitation patterns follow similar temporal patterns among the sites ( Figure 2 ). Plots

at sites NT010 and NT011 were irrigated during summer (2

- 3 months; 1.5

- 2 in/month) while plots at

NT012 were irrigated 4 months during late spring /summer (0.75

- 3.75 in/month). Maps showing the

locations of the trials are found in Appendix A

.

Table 3 . Soil and meteorological characteristics of the Co rn study sites *

* (from Table 3 of study report)

Figure 1

. Monthly precipitation for the 3 study trial sites (NT01 0 , NT01 1 , NT01 2 ).DP Barcode: D424267 MRID : 495 11701

7 5D. S AMPLE C OLLECTION
, H ANDLING
, P ROCESSING

Co rn Plant Matrices. Corn sample collection began 58

- 78 days after planting seed treated corn (DAA)

each year. Samples of corn pollen and tassels were collected at four sampling intervals per year of

collection. Sampling of corn pollen and tassels targeted growth stages BBCH 63, 65, 67, and 69 . For corn,

BBCH 63 ( male ) represents the beginning of pollen shedding ; BBCH 63 ( female ) represents tips of stigmata

visible; and BBCH 69 represents the end of flo wering, stigmata completely dry; Table 4 ).

Corn leaves were sampled at six times from BBCH 59

- 71. F ive composite samples of co rn leaves (and

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

each six sampling intervals . Two composite samples were taken from the control plots in the same

manner.

Table 4 . Sampling dates and co rn developmental stages for plant residue analysis

Pollen and Tassel 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 110

63 59

- 60 63 59 59 56 59 58

65 62 65 61

- 62 63 59 63 59

67 64 67 63

- 64 65 62 65 61

69 66 69 66

- 67 67 64 67 63

--- --- --- --- 69 66 69 67

--- --- --- --- 71 75 71 73

NT011

63 58 63 65 59 56 59 64

65 60 65 67 63 58 63 65

67 62 67 69 65 60 65 67

69 65 69 72 67 62 67 69

--- --- --- --- 69 65 69 72

71 70 71 76

NT012

63 62 63 78 59 61 59 77

65 63 65 80 63 62 63 78

67 66 67 82 65 63 65 80

69 68 69 84 67 66 67 82

--- --- --- --- 69 68 69 84

71 75 71 87

*BBCH = B iologische Bundesanstalt, B undessortenamt und CH emische Industrie growth stage scale for c o rn ; 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 of clover flowers targetedDP Barcode: D424267 MRID : 495 11701

8 growth stages of BBCH 6 3 , 65, 67 and 69 ( clover BBCH 63 corresponds to ~ 30% flowering and BBCH 69 is

the end of flowering ). For pollen and nectar collection , two bee

- tight, ventilated mesh

- covered tents

(tunnel) were constructed on plot UTCB. Five tunnels were constructed on plot TRTDB at the beginning of

clover blooming. Normally developed, apparently healthy and queen

- right honey bees, Apis mellifera

( purchased new from Heartland Honey in Orland, GA ), 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 1

- 2 days

prior to sample collection. The colonies were equipped with pollen traps. Colonies and tents were

removed after the end of polle n 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 samples 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 1

- 2

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

a disposable spoon and sample vial. Pollen from the blossoms in the tent was sampl ed from the pollen

traps affixed to the colonies. All available pollen and nectar was collected, and the samples were

individually placed in pre

- weighed amber glass containers.

Soil. Nine soil samples were collected from all plots using a soil sampling device prior to corn planting and

at the end of the growing season each year. Soil samples were collected from the surface of the plots (6

inches [15 cm] deep) by bulb planter or soil probe and placed in a plastic bag. In each sampling period,

sample locat ions 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] plot), 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 facility, all composite samples were

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

processed for pollen and nectar by the field investigators

.

5E. A NALYTICAL M ETHODS

The residues of 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. 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 residue 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

.DP Barcode: D424267 MRID : 495 11701

9 Table 5 . Limits of quantification and detection for imidacloprid and metabolites

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 741 days ( 24 months) in representative crops and 793

-

1281 days ( 26

- 42 months) in soil matrices prior to analysis

.

Transit Stability. To ensure that imidaclop rid and its metabolites 5

- hydroxy imidacloprid and imidacloprid

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

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

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

with approximately 0.10 ppm each of imidacloprid and its metabolites 5

- hydroxy imidacloprid and

imidacloprid olefin, acco mpanied the respective study sample types throughout the sampling, shipping,

and storage events of the field phase of the study. They were analysed after all sample analyses of a given

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

transport conditions encountered by the samples.DP Barcode: D424267 MRID : 495 11701

10 Spike Recovery. The method for determining imidacloprid, 5

- hydrox y imidacl oprid, and i midacloprid

olefin residues in/on co rn 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 validated the method for the highest residues observed in ind ividual 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 in corn and

clover matrices was 734 days and that for soil was 728 days. These storage times are within the bounds

of the storage stability results described earlier and indicate that storage times are acceptable.

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

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

Matrix Spike Recov eries. As indicated in Table 5 of the study report, all matrix spike recoveries (poll en,

nectar, tassel, blossom , 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

Co rn Pollen and Tassel

. Summary statistics of t he overall magnitude of total IMI residues in corn pollen

and tassels are shown in Table 6

. These statistics reflect analysis of individual composite samples (usually

5 replicates per sampling date) among the sampling times within each year of each trial. Plots of the raw

data for each trial are found in Appendix B

. Since IMI

- olefin and 5

- OH IMI are considered of similar

toxicity as parent IMI, this discussio n focuses on total IMI residues (sum of IMI, IMI

- olefin and 5

- 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 b y 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 le ast one analyte was >LOQ. As a result, residue values of total IMI at or below 1.2

ppb essentially reflect these assumptions regarding <LOD and <LOQ.

Across all three trials, yearly m ean residues of total IMI in corn pollen r ange from approximately 2.1

– 7.5

ppb while yearly maxima range from 4.8

– 39.7 pp b

. Based on yearly means, r esults for corn tassels are

about 2X

– 3X the mean residues reported for corn pollen , with yearly average total IMI residues ranging

from 4. 8 to 1 3.7 ppb and yearly maxima ranging from 9.4 to 26.1 ppb

. Not surprisingly, overall trends in

mean residues of total IMI in tassels across trials follow those seen in pollen. Means from years 1 and 2 of

TRTSTA are comparable, suggesting no obvious year

- to

- year carry over in total I MI residues in corn pollen

and tassels. Notably, the highest mean (and maximum) values of total IMI in corn pollen and tassels

occurred in trial NT01 2

. Precipitation patterns are comparable among the three trials (especially duringDP Barcode: D424267 MRID : 495 11701

11 year 2). The percent s and in soils from trial NT01 2 (36%) is about 2X that of trial NT010 (16%) and 30%

greater than trial NT011 (28%)

. This suggests that the greater accumulation in pollen and tassels n trials

NT01 2 may be due its greater fraction of sand in soil.

Table 6 . Magnitude of t otal IMI in corn pollen and tassels

Corn Pollen (ppb)

Trial NT01 0 * Trial NT01 1 * Trial NT01 2 *

TRTDA TRTDB TRTDA TRTDB TRTDA TRTDB

2012 2013 2012 2012 2013 2012 2012 2013 2012

Mean 2.9 2.6 3.2 3.2 2.1 2.7 7.5 7.4 7.0

Min. 0.9 0.60 0.9 0.9 0.9 0.9 0.6 0.6 3.30

Med. 2.7 2.5 2.7 2.8 1.8 3.0 7.2 2.8 6.9

90th 4.9 4.7 5.7 5.1 4.6 4.2 12.1 16.4 9.5

Max. 6.5 4.8 8.6 9.3 5.3 6.1 16.4 39.7 15.4

Corn Tassel (ppb)

Mean 8.8 10.3 10.8 9.6 4.8 7.9 13.7 10.5 13.3

Min. 3.2 4.5 5.3 4.3 2.6 4.4 4.9 3.2 4.3

Med. 8.1 11.0 9.9 8.7 3.8 7.1 10.7 9.0 14.0

90th 13.6 14.5 17.6 14.3 8.5 11.0 25.2 18.4 25.2

Max. 20.1 17.2 23.0 22.3 9.4 15.2 26.1 19.8 25.7

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 corn pollen are shown in Figure

2

. Temporal trends in daily average total IMI in pollen were variable or declining with time in 2012 while

interestingly in 2013, concentrations increased with time in each of the three trials. The maximum daily

average concentration of total IMI ( 22.3 ppb ) from DAA 84 in trial NT012 is much greater than the

previous sampling event for that trial (4.0 ppb) and is also greater than daily means from all other trials.

Inspection of the raw data indicates this high value is not a result of a single outlier among the 5 replicates

( Appendix B ). The reason for the increasing concentrations of total IMI during the second year of

sampling is not clear but may reflect greater desiccation of pollen over time. This explanation , however,

does not explain why such increasing trends were not consistently observed during year 1 (2012)

.

With corn tassels, the daily average of total IMI residues generally followed a similar trend as that

described previously with pollen ( Figure 3 ). This makes sense given that tassels are the pollen bearing

portion of the corn plant. This also suggests that the increase in concentration over time consistently

observed in year 2 (and in year 1 of tri al NT012) is not limited to the pollen matrix alone, but rather

reflects the pattern in the entire corn tassel.DP Barcode: D424267 MRID : 495 11701

12 Figure 2

. Total imidacloprid residues measured in corn pollen f rom trials NT010, NT011 and NT012 (dots
= mean; error bars = range)

.DP Barcode: D424267 MRID : 495 11701

13 Figure 3

. Total imidacloprid residues measured in c orn tassels from trials NT010, NT011 and NT012 (dots
= mean; error bars = range)

.DP Barcode: D424267 MRID : 495 11701

14 6. C

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

Mean concentrations of total IMI in co rn leaves within each trial /study year are shown in Table 7

. Residues of total IMI in leaves were often 10X or greater than those observed in corn pollen ( Table 6 ).

Unlike the results for corn pollen and tassels, mean yearly residues of total IMI in leaves from trial NT012

are consistently greater than those from trials NT010 and NT011. To explore whether the trend of

increasing total IMI concentrations observed in pollen and tassel are also seen with leaves, plots of the

daily average concentrations in leaves are shown in Figure 4

. With the exception of 2013 in trial NT012,

temporal trends in the daily mean residues of total IMI in corn leaves were either variable or remained

relatively constant with time. This sugges ts that leaves are not a reliable surrogate for corn pollen (and

tassel) at least under the conditions of this study.

Table 7

. Mean concentrations of total IMI in co rn leaves (ppb).

Treatment ( Year ) Residue Trial

NT01 0 NT01 1 NT01 2

TRTDA (2012 ) 77.2 22.7 34.0

TRTDA (2013 ) 14.7 8.3 18.6

TRTDB (2012) 66.7 19.4 28.4DP Barcode: D424267 MRID : 495 11701

15 Figure 4 . Concentration of total IMI in corn leaves (ppb) for trials NT010, NT011, and NT012

Imidacloprid concentrations (parent only) measured in soils of all three study trials are shown in Table 8

. Concentrations are 103 ppb or less at all sampling times. Based on samples taken prior to planting during

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

- 4 ppb (year 1) to 9

- 80 ppb prior to planting

in year 2. Based on samples taken at the end of each growing season for TRTSTA, the concentration of

IMI in soil increases from 9

- 19 ppb (year 1) to 37

- 103 at the end of the growing season in year 2.

Table 8

. Magnitude of IMI residues in soil (ppb).

Year Date Event DAFA 1 Mean IMI in Soil

TRT ST A (ppb) 2 Mean IMI in Soil

TRT ST B (ppb) 2

NT 01 0

1 5/ 4 /12 Prior to planting

- 1 2 2

1 5/5/12 Seed planting 0

--- ---

1 10/17/12 End of 1 st growing season 165 17 60

2 5/21 /13 Prior to planting 381 18 26

2 5/25 /13 Seed planting 385

--- ---DP Barcode: D424267 MRID : 495 11701

16 Year Date Event DAFA 1 Mean IMI in Soil

TRT ST A (ppb) 2 Mean IMI in Soil

TRT ST B (ppb) 2

2 10/10 /13 End of 2 nd growing season 523 103 28

NT0 1 1

1 05/ 8 /12 Prior to planting 0 4 4

1 05/ 8 /12 Seed planting 0

--- ---

1 9/6/12 End of 1 st growing season 121 19 11

2 4/25/13 Prior to planting 352 80 9

2 5/7/13 Seed planting 364

--- ---

2 9/30/13 End of 2 nd growing season 5 10 71 6

NT01 2

1 5/1 1/12 Prior to planting 0 3 2

1 5/1 1/12 Seed planting 0

--- ---

1 10/24/12 End of 1 st growing season 166 9 2

2 4/ 4 /13 Prior to planting 3 2 8 22 12

2 4/29/13 Seed planting 353

--- ---

2 11/ 13 /13 End of 2 nd growing season 551 37 17

1 DAFA = Days After First Application (seed planting) 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
, TASSEL AND S OIL

A qualitative evaluation of the potential for ‘year

- to

- year’ carryover in the accumulation of total IMI

residues in corn pollen and tassels is conducted here for the three trials based on results presented in

Table 6 and Figures 2 and 3 . Based on yearly mean values of total IMI in corn pollen and tassels from

TRTSTA

, there appears to be no consistent increase or decrease year 2 mean values relative to year 1.

It is clear from Table 8 that IMI residues in soil measured prior to planting in year 2 ( 9

- 80 ppb ) are

elevated compared to those measured prior to planting in year 1 ( 2

- 4 ppb ). Within each trial/plot,

concentrations of IMI increased in soil by 2

- 13X prior to planting in year 1 vs. year 2. Comparison of soil

concentrations measured at the end of each growing season in plot TRTSTA also suggest year

- to

- year

carry over in soil, with year 2 IMI concentrations increasing by 4 X to 5X relative to year 1. It is important

to recognize that these trends reflect parent IMI only and do not include possible contribution of toxic
degradates (IMI

- olefin, 5

- OH IMI).

6. E

. R ESIDUE D ECLINE (DT 50 ) IN C ORN P OLLEN

The temporal patterns associated with the concentrations of total IMI in corn pollen are not considered

suitable for DT 50 analysis because with two exceptions, concentrations were either highly variable with

time or increased with time ( Figure 2 ). The two exceptions are TRTSTA and TRTSTB for 2012 in trial

NT010, which showed declining total IMI in pollen with time. However, it is evident from the

preponderance of pollen and tassel residue data that declines of total IMI over the course of the

b looming period are the exception rather than the rule for seed treated corn.DP Barcode: D424267 MRID : 495 11701

17 6. F

. O CCURRENCE OF R ELEVANT IMI M ETABOLITES

Very few samples of corn pollen contained concentrations of IMI

- olefin or 5

- OH

- IMI above the limits of

detection in pollen (0. 3 and 0. 5 ppb, respectively ). 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 )

The concentrations of total IMI measured in whi te clover nectar and pollen planted following planting and

harvesting of seed treated corn the previous year were near or below the combined limits of detection for

total IMI (1. 24 ppb for pollen; 1.33 ppb for nectar ) in the majority of samples analyzed (d etection

frequency = 28 % for clover pollen and 0 % for clover nectar )

. The maximum concentrations of total IMI

measured in clover pollen in three trials is 3.8 ppb

.

6. H

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

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

corn tassel and total IMI measured in corn pollen measured on the same sampling day is shown in Figure

5 A

. Total IMI residues in corn pollen are predicted within 2X of measured residues for 24/ 36 samples

(66%) using simple linear regression with flower data (r 2 = 0. 35 ). Overall, concentrations of t otal IMI

residues in corn tassels are about 2X those measured in pollen. In contrast, no obvious relationship is

seen be tween total IMI residues in corn pollen and leaves ( Figure 6B ).DP Barcode: D424267 MRID : 495 11701

18 Figure 5 . Relationship between total IMI residues in corn pollen to those measured in tassels (A), and leaves ( B)

.



### 7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS

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

- related matrices of c o rn resulting

from seed treatment, the following strengths are observed with this study.

1. Analytical methods were sufficiently accurate and precise based o n 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

varia bility

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

.

A number of limitation is also noted with this study :

1. Although the three trials were distributed across two states, they represent a comparable region

of the country, share similar weather patterns and soil composition were not substantially

different. This limits the extent to which differences in environmental conditions could be

assessed on residues in co rn matrices.

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

1. Planting of seed treated corn (1.34 mg a.i./ seed; 0.12 a.i./ A) over two successive years result s in a

maximum of 3 9

. 7 ppb of total IMI in individual composite samples of corn pollen , which occurred

at the end of the blooming period during the second year of planting. The maximum daily

average concentration of total IMI in co rn pollen is 2 2.3 ppb and the maximum yearly average

concentration is 7.5 ppb

. 2. Yearly average concentrations o f total IMI in corn tassels are about 2X

– 3X the mean residues

reported for corn pollen ( yearly average total IMI residues rang e from 4.8 to 13.7 ppb and yearly

maxima rang e from 9.4 to 26.1 ppb.

3. Temporal trends in the concentration of total IMI in corn pollen are either variable or showDP Barcode: D424267 MRID : 495 11701

19 decreases with time during 2012. During 2013 (year 2), concentrations of total IMI in corn pollen

consistently increased with time for each of the three trials. T he reason for this increase in

concentration with time is not clear.

4. Year to year carryover of total IMI residues was not evident based on overall average

concentrations in corn pollen, although measurements in soil from the study plots indicates

increasi ng concentrations from year 1 to year 2.

5. Two metabolites of IMI (IMI

- olefin and 5

- OH

- IMI) were present below the limits of quantification

in corn pollen ( 0.3 and 0.5 ppb, respectively ) in the vast majority of samples.

6. Due to the highly variable or increasi ng trends seen in concentrations of total IMI in corn pollen,

DT 50 values were not determined.

7. The predictability of t otal IMI residues in corn pollen based on measured concentrations in tassels

is fair, with most of the predicted values being within a f actor of 2X of the measured values. Total

IMI residues in corn leaves provides little predictive capability for corresponding concentrations in

pollen.

8. Residues of total IMI measured in white clover pollen after planting and harvesting seed treated

corn the previous year are low, with a detection frequency of approximately 28%. Residues of

total IMI in white clover nectar were not found above limits of quantitation. The maximum

concentration of total IMI detected in clover pollen is 3.8 ppb

.



### 8. STUDY VALIDITY/CLASSIFICATION

Data from the three trials (NT0 1 0 , NT0 1 1

, and NT01 2 ) are considered scientifically sound and useful for

risk assessment purposes, although these trials were located in a similar region of the country. Overall,

this study is clas sified as ACCEPTABLE for quantitative use in risk assess ment.DP Barcode: D424267 MRID : 495 11701

20 Appendix A . Location of C o rn Residue Trials.

NT010 (Edgerton, KS)

NT011 (Springfield, NE) NT010

NT011DP Barcode: D424267 MRID : 495 11701

21 NT01 2 ( York , NE)

NT012DP Barcode: D424267 MRID : 495 11701

22 

### Appendix B . Plots Of Raw Data For Total IMI In Co rn Pollen

Figure B

- 1. Total imidacloprid concentrations in co rn pollen for NT010, NT011 and NT012