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Reference

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

Imidacloprid and its Metabolites 5-Hydroxy Imidacloprid and Imidacloprid Olefin in Bee

Relevant Matrices Collected from Blueberries following Soil Application of Imidacloprid over

Two Successive Years. Bayer Crop Science, Research Triangle Park, NC. MRID 49536502

1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material Gaucho 600 FL Admire Pro 

Systemic Protectant (SC)

Purity 41.8 - 42.9% a.i. w.w.

Study Type: Non-Guideline field residue study on blueberry to establish imidacloprid and 

metabolite levels in pollen, nectar, flowers, leaves and soil after two successive 

annual soil applications. 

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, Flower, Soil): SynTech Research 

Laboratory Services, 17745 S. Metcalf Ave, 

Stillwell, KS USA 66085

(Trial NT001-12ZA): A.C.D.S. Research Inc., 100

Willow Lane, Dundee, NY USA 14837 (ID: 

AR12238)

(Trial NT002-12ZA): Great Lakes Agricultural 

Research Service, N6084 Johnson Road, Delavan, 

WI USA 53115 (ID: 12-GLARS-1606)

(Trial NT003-12ZA): Eurofins Agroscience 

Services, Inc. 1035 Country Road 300 East,

Seymour, IL USA 61875 (ID: S12-04201-01)

Sponsor Study 

ID:

EBNTY00X

Study 

Completion 

Date:

17 December 2014

Experiment 

Start/End Date:

18 February 2012 – 18 

August 2014

Study Location: 3 Field Trials:

Penn Yan, NY (NT001)

Marengo, IL (NT002)

Fennville, MI (NT003)

GLP Status: GLP-compliant; protocol reviewed by EPA, PMRA, CDPR

2. REVIEWER INFORMATION

Primary Reviewer:

Secondary Reviewer:

Christina Wendel, M.S., Biologist, ERB2, Environmental Fate and Effects 

Division, Office of Pesticide Programs, USEPA

Keith Sappington, M.S., Science Advisor, ERB5, Environmental Fate and 

Effects Division, Office of Pesticide Programs, USEPA

Signature: Date: 01/28/2016

3. EXECUTIVE SUMMARY

This study was designed to measure the magnitude of imidacloprid residues in bee-relevant pollen and 

nectar samples and in/on flowers, leaves, and soil from blueberry plants following one post-harvest 

banded soil application of 0.5 lb a.i./A per year (current U.S. label rate) for two successive years. Three 

separate trials were conducted for this study, one in NY (NT001), IL (NT002) and MI (NT003) blueberry 

fields, each with a treated (TRTD) and untreated control (UTC) plot. Each year, one post-harvest soil 

application (0.5 lb a.i./A) was made as an 18-inch band on each side of the row, 3 days post-harvest (PHI). 
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The first year applications (2012) and second year applications (2013) were both made after blueberry 

harvest early fall (between September 26 and October 4), when the plants were between BBCH growth 

stages 92 and 97 (BBCH 92: leaves begin to change color or fall; and BBCH 97: plant resting or dormant). 

Crop varieties representing those commonly grown in the area of the trials and agronomic practices 

typical for commercial production of blueberries were used at all trial locations. Samples of flowers, 

pollen, nectar and leaves were made at various sampling times during the flowering period from each 

TRTD plot/year. Soil samples were also collected. QA/QC included field and lab storage/stability samples, 

blanks, and matrix spikes to validate analyte recovery. 

Results indicate that post-harvest, soil application of approximately 0.5 lb a.i./A results in residues of total 

IMI (sum of IMI, IMI-olefin and 5-OH-IMI) in individual composite nectar samples of up to 16 ppb and 42 

ppb in pollen. Within each trial year, mean residues of total IMI in nectar varied from 0.81-7.25 ppb and 

that for pollen varied from 0.6-14.0 ppb. 

The residues in pollen and nectar for trial sites NT001 and NT002 were below the level of detection (LOD) 

and/or level of quantification (LOQ) for a majority of the time points of collection for all measured

matrices. The confidence in the endpoints that were extrapolated for these were low; therefore the 

results for further analyses were completed on the NT003 trial data only, as that trial location results 

obtained measured values for both pollen and nectar matrices. The mean daily residue values of total IMI 

in nectar ranged from 0.9-8.8 ppb and that for pollen ranged from 8.9-16.5 ppb for trial NT003. The 

overall higher concentrations measured in trial NT003 may be partly a function of the high sand content 

of its soil (90%) compared to the other two trial sites (31%-39%).

Total IMI residues in nectar for trial NT003 increase over time, such that in trial year 1 mean daily residues 

at 236 days post application were 8.67 ppb, compared to 2.03 ppb at 228 days post application. In NT003 

trial year 2 mean daily residues also increase over time; at 235 days post application they were 0.9 ppb, 

compared to 3.24 ppb 240 days post application. Unlike the intra-annual increase in total IMI 

concentrations in nectar in trial NT003, no consistent intra-annual increase is seen with pollen in either of 

the trial years. 

Limited sampling of plant matrices over each growing season prevented determination of reliable 

estimates of dissipation kinetics (DT50) in plant matrices for all trials except NT003. DT50 values of total 

IMI in nectar from trial NT003 could not be calculated as the concentrations in nectar showed little or no 

dissipation or were increasing over time. Only 1 DT50 could be calculated for pollen (35.6 days) because, 

like the nectar the other year in trial NT003 showed little or no dissipation or were increasing over time.

Two metabolites of IMI (IMI-olefin and 5-OH-IMI) typically represented <15% of total IMI residues in 

pollen and nectar for trial NT003. The predictability of total IMI residues in nectar and pollen based on 

measured concentrations in other plant matrices (flowers, leaves) was generally poor for all trials, 

including NT003, in which analyses could be completed. 

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 
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scientific integrity of this study.

Reparability: N/A

5. MATERIALS AND METHODS

Test Material Characterization

Test item: Gaucho 600 FL Admire Pro 

Systemic Protectant SC 

(Imidacloprid) 550 g a.i./L

CAS #: 138261-41-3

Description: N/A Purity: 41.8 - 42.9% a.i. w.w.

Lot No./Batch No. Batch No. NK41BX0170;

NK41BX0190

Density: 1.41 – 1.54 g/mL

Material Source: Bayer Crop Science Cert. # 212PG1962 & 212PG2246

Material Receipt 

Date:

Not reported Analysis 

Date: 03/30/2012 & 09/27/2012

Expiration Date: 03/31/2014 & 09/27/2014 Solubility: 0.51-0.61 g/L

Storage of Test Mat’l: Ambient (35-86oF) once at 

field locations

Sample 

Storage: -32o

F to 30oF

5A. STUDY DESIGN

This study was designed to measure the magnitude of imidacloprid residues in bee-relevant pollen and 

nectar samples and in/on flowers, leaves, and soil from blueberry plants following one post-harvest 

banded soil application per year of ADMIRE PRO Systemic Protectant containing 550 g/L imidacloprid (IMI)

for two successive years. In addition to quantifying the magnitude of residues, this study was designed to 

evaluate the following additional attributes:

1) The extent of year-to-year ‘carry over’ of pesticide residues 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). 

Three separate trials were conducted, each with a treated (TRTD) and untreated control (UTC) plot. Each 

treated plot was divided into 5 sampling subplots, for the construction of bee-tight, ventilated meshcovered tents (tunnels). Each year, a single post-harvest banded soil application (0.5 lb a.i./A) was made 3 

days post-harvest (PHI). The first year application (2012) occurred in early fall (between BBCH growth 

stages 92 and 97 (BBCH 92: leaves begin to change color or fall; BBCH 97: plant resting or dormant)), and 

the second year application (2013) occurred between 360 to 366 days after the first year’s application.

Five composite samples (one sample per bee tunnel) of flowers, pollen, nectar and leaves were made at 

various sampling times during the flowering period from TRTD plot/year. Nine composite soil samples 

were collected prior to treatment and after sampling from each TRTD plot/year, exceptions were trial 

NT001 year 2, when the soil samples were collected before the spray application and just before 

sampling, and in trial NT003, when the only soil sample collected were after sampling in year 2 (no year 1 

soil samples). Control (UTC) plots were sampled for all blueberry matrices (two composites) and soil (9 

composites) at the same time the TRTD plots were sampled. Except in trial NT002, in which no year 2 UTC 

samples could be collected because the UTC blueberry plants died during the previous winter. In all trials, 

not enough plant matrix material was present at every interval to allow for the full number of target 
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samples or sampling intervals to be collected. QA/QC included field and lab storage/stability samples, 

blanks, and matrix spikes to validate analyte recovery. 

5B. APPLICATION TIMING AND RATES

One post-harvest banded soil application of approximately 0.5 lb a.i./A was applied 3 days post-harvest as 

an 18-inch band on each side of the row in successive years at each of the three treatment sites (total 

annual application rate is equivalent to the individual rates; 0.5 lb a.i./A) using ground-based equipment.

The volume of application was similar across trials and years (data not shown). The interval between the 

1

st and 2nd applications was approximately one year (360-366 days). In addition, a reference site 

(untreated; UTC) was also included for each of the three trials. All applications were made after blueberry 

harvest between September 26 and October 4 of each year, when the plants were between BBCH growth 

stages 92 and 97 (BBCH 92: leaves begin to change color or fall; BBCH 97: plant resting or dormant). 

Maintenance pesticides were used at some point during all three trials. Information on application timing 

is shown in the Table 1 below.

Table 1. Summary of banded soil application rates and timing

Location Blueberry 

Variety Year Appl. No.

Appl. Date 

(DAFA/Stage)1 DAFYA2

Individual 

Rate

(lb a.i./A)

Total Rate

(lb a.i./A)

Trial NT001-12ZA

Penn Yan, 

NY Blue Ray 2012 1

09/29/2012

(0/BBCH 92)

NA 0.50 0.50

Penn Yan, 

NY Blue Ray 2013 2

09/30/2013

(366/BBCH 92)

366 0.50 0.50

Trial NT002-12ZA

Marengo, 

IL Duke 2012 1

10/01/2012

(0/BBCH 97)

NA 0.50 0.50

Marengo, 

IL Duke 2013 2

09/26/2013

(360/BBCH 97)

360 0.50 0.50

Trial NT003-12ZA

Fennville, 

MI Bluecrop 2012 1

10/04/2012

(0 /BBCH 92)

NA 0.50 0.50

Fennville, 

MI Bluecrop 2013 2

10/01/2013

(362/BBCH 92)

362 0.50 0.50

1 DAFA = days after 1st application; BBCH = plant growth stage.

2 DAFYA = days after 1st yearly application

5C. STUDY SITE LOCATION AND CHARACTERISTICS 

Various aspects of the study sites are summarized in Table 2. In general, plot size, row/plant spacing were 

similar among the three study sites/trials. However, the soil type (loam, silt loam and sand), and the 

variety of blueberry (Blue Ray, Duke, and Bluecrop) differed between the three trials, respectively (Table 

3). Maps showing the locations of the three TRTD study trials and the UTC for NT002 are provided in 

Appendix A. 
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Table 2. Summary of study site characteristics.

Attribute Trial NT001-12ZA Trial NT002-12ZA Trial NT003-12ZA

Plot ID UTC TRTD UTC TRTD UTC TRTD

Crop Blueberry Blueberry Blueberry Blueberry Blueberry Blueberry

Variety Blue Ray Blue Ray Duke Duke Bluecrop Bluecrop

Crop Age 35 35 4 14 4 4

Soil Type Loam Loam Silt Loam Silt Loam Sand Sand

Plot Length 315 ft 300 ft 220 ft 220 ft 252 ft 252 ft

Plot Width 56 ft 210 ft 40 ft 140 ft 84 ft 192 ft

Plot Area 17640 ft2 63000 ft2 8800 ft2 30800 ft2 21168 ft2 48384 ft2

Row Spacing 14 ft 14 ft 10 ft 10 ft 12 ft 12 ft

Plant Spacing 

within Row 5.5 ft 5.5 ft 4 ft 4 ft 4 ft 4 ft

Latitude, 

Longitude Unknown (42.570667, -77.037833)

(42.27780, 

-88.6807)

(42.29122, 

-88.62452)

Unknown (42.60427, 

-86.15298)

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

Irrigation supplemented normal rainfall in some trials, and irrigation or rainfall occurred within 24 hours 

following each application except in trial NT002 year 1, when rainfall occurred 2 days after the

application. Overall, the total rainfall was similar between all trials, despite the difference in locations 

(New York, Illinois and Michigan). The total rainfall over the study period was 58.1, 58.5 and 60.2 in for 

trial NT001 (NY), NT002 (IL), and NT003 (MI), respectively (Table 3). A summary of the monthly 

precipitation data for all three study trials is presented in Figure 1 below. 

Irrigation type and source differed between trial locations. Trial NT001 irrigation occurred in September 

2012 and 2013 (0.9 and 1.13 in, respectively) via drip irrigation from a pond. Trial NT002 used drip 

irrigation from a well in October 2012 (0.5 in, treatment site only), and “periodically during growing 

season.” NT003 received irrigation water from a sprinkler solid set throughout the year (10 in. in 2012; 3 

in. in 2013, and 0.5 in through August 2014). 

Fertilizer was used in trials NT002 and NT003, but not NT001. Fertilizer was applied in May 2013 and 

2014 in trial NT002. At trial NT003 a slow release fertilizer was applied in September 2012 and March 

2013.

The temperature range was the largest at trial NT002 (IL), ranging from -1 to 82F; and the study authors 

postulated that the cold period in trial NT002; which perhaps contributed to the death of the blueberry 

bushes in the UTC site prior to year 2 of the study. The organic carbon was the greatest in trial NT002 at

the TRTD site, at 3.78%. Whereas the organic carbon at both trials NT001 and NT003 was less at 2.79% 

and 2.38%, respectively. Although the soil types of Trials NT001 and NT002 were different, loam and silt 

loam, the soil consistency, was similar: trial NT001 consisted of 39% sand, 46% silt and 15% clay; whereas 

trial NT002’s soil consisted of 31% sand, 53% silt and 16% clay. However, trial NT003 soil type, sand, was 

very different than the other two trials; and it consisted of 90% sand, 10% silt and 0% clay. The pH was 

lowest in soils at NT001 and NT003 (4.8 and 4.4, respectively) vs. pH 6.4 for the soil in the treatment site 

in trial NT002. 
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Table 3. Soil and meteorological characteristics of the study sites* 

* (from Table 3 of study report)

Figure 1. Monthly precipitation for the 3 study trials

0

1

2

3

4

5

6

7

8

9

10

Rainfall (inches)

Month

NT001

NT002

NT003
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5D. SAMPLE COLLECTION, HANDLING, PROCESSING

Plant Matrices. Samples that were treated in 2012 were collected in 2013 (year 1 of the study), and 

samples treated in 2013 were collected in 2014 (year 2 of the study). Each trial location was divided into 

sampling subplots, for the construction of bee-tight, ventilated mesh-covered tents (tunnels), which were 

used for collection of pollen and nectar. Two tunnels were erected on control plot UTC, and five tunnels 

were erected on each TRTD plot. Each tunnel enclosed one or two rows of blueberry bushes. The bee 

tunnels were erected over the same blueberry bushes in 2013 and 2014, except in trial NT002-12ZA, when 

the tunnel size was expanded in 2014 to include bushes that were treated but not sampled in 2013. One 

honey bee hive was placed in each bee tunnel 1 to 5 days before the first nectar/pollen samples were 

collected. Each honey bee colony was contained in a standard 10 frame Langstroth hive body or in a 2 

story hive with a standard Langstroth body on bottom and a standard hive body as the second story (ten 

total frames). Honey bee hives were equipped with pollen traps, and the honey bees were provided no 

supplemental feed. In year 2 apparently healthy bumble bees (Bombus impatiens, purchased) were used

alongside the honey bees to aid in pollen collection. One honey bee hive and one to four bumble bee 

colonies were placed in each bee tunnel 3 to 6 days before the first nectar/pollen samples were collected. 

In trials NT001-12ZA and NT003-12ZA, the bumble bee boxes contained sugar water feeders inside the 

box. 

Composite samples (separate runs through the plot) of blueberry leaves, and flowers, were collected from 

plots UTC and TRTD in years 1 and 2 of the study, except in trial NT002, in which no year 2 UTC samples 

could be collected because the UTC blueberry plants died during the previous winter. All samples were 

collected from within the erected bee tunnels on both plots except in trial NT002, when leaf samples 

were taken prior to and after pollen and nectar sampling and were collected from the bushes on which 

the tunnels would be/were erected because the tunnels were not yet present. Five samples were 

targeted for collection from TRTD plots and two samples from an UTC untreated control plots at each 

sampling period, corresponding to one sample per erected bee tunnel (subplot). UTC samples were 

collected at the same sampling intervals and via the same methods as the TRTD samples. However, in all 

trials, not enough plant matrix material was present at every interval to allow for the full number of target 

samples or sampling intervals to be collected.

Blueberry flowers were collected during four target sampling periods 228 to 257 days after the last

application (DAA) at growth stages of BBCH 60 to 69 (BBCH 60: first flowers open; and BBCH 69: end of 

flowering, fruit set visible) in years 1 and 2. Blueberry leaves were collected at six target sampling 

periods, 228 to 264 DAA when the blueberry plants were at growth stages BBCH 59 to 74 (BBCH 59: first 

flower petals visible in petalled forms) in years 1 and 2 (Table 4).
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Table 4. Sampling dates and blueberry developmental stages for plant residue analysis

NT001

Flower & Nectar Sampling Leaf Sampling

Year 1 Year 2 Year 1 Year 2

BBCH DALA BBCH DALA BBCH DALA BBCH DALA

-- -- -- -- 59 227 -- --

61 230 -- -- 61 230 59 230

65 236 60 236 65 236 60 236

67 238 61 238 67 238 61 238

-- -- 65 243 -- -- 65 243

-- -- -- -- 69 252 -- --

-- -- 67 254 -- -- 67 254

-- -- -- -- -- -- 69 262

Pollen Sampling (Honey Bees) Pollen Sampling (Bumble Bees)

Year 1 Year 2 Year 1 Year 2

BBCH DALA BBCH DALA BBCH DALA BBCH DALA

-- -- -- -- -- -- -- --

-- -- -- -- -- -- -- --

65 236 -- -- -- -- 60 236-37

67 238 61 238 -- -- 61 238

-- -- 65 243 -- -- 65 243

-- -- 67 257 -- -- 67 257

NT002

Flower, Nectar & Pollen (YR2) Sampling** Leaf Sampling

Year 1 Year 2 Year 1 Year 2

BBCH DALA BBCH DALA BBCH DALA BBCH DALA

-- -- -- -- 59 228 -- --

60 232 -- -- 60 232 59 232

61 233 -- -- 61 233 -- --

65 234 -- -- 65 234 -- --

67 235 60 235 67 235 60 235

-- -- 61 237 -- -- 61 237

-- -- 65 238 -- -- 65 238

-- -- 67 239 -- -- 67 239

-- -- -- -- 69 241 -- --

-- -- -- -- -- -- 69 243

NT003

Nectar, Pollen & Flower Sampling Leaf Sampling

Year 1 Year 2 Year 1 Year 2

BBCH DALA BBCH DALA BBCH DALA BBCH DALA

-- -- -- -- 59 223 -- --

65 228 -- -- 65 228 -- --

-- -- 60 232*** -- -- 60 232

66 233 -- -- 66 233 -- --

-- -- -- -- -- -- 62 234

-- -- 65 235 -- -- 65 235

68 236 65 236 68 236 65 236

-- -- 67 238 -- -- 67 238

-- -- 69 240 -- -- 69 240

-- -- -- -- -- -- 69 254

-- -- -- -- 74 264 -- --

*BBCH = Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie growth stage scale for blueberry; 

DALA = days after the last yearly application; ** Pollen was collected for trial NT002 in year 2 only; *** Flower only.
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Honey bees were used to collect pollen and nectar samples, and bumble bees were used to aid in the 

collection of pollen samples in year 2. Table 5 below provides the date of hive introduction for both 

years, for all three trials. Introduction of the hives occurred approx. 8 months after application in each 

year for each trial (Table 5). After 1 to 5 days of bee foraging, nectar was collected from samples 

deposited in uncapped cells within a newly-placed bee hive using a disposable spoon and sample vial. For 

honey bees pollen samples were collected via pollen traps affixed to the hives. In year 2 when bumble 

bees were used to aid in the collection of pollen, pollen was collected from the pollen basket on the

bumble bee’s leg. The bumble bees with pollen had the pollen collected from the baskets and placed into

vials. Blueberry nectar and pollen were collected at four target sampling periods 228 to 257 days after 

the last application (DAA) at growth stages of BBCH 60 to 69 (BBCH 60: first flowers open; BBCH 69: end of 

flowering, fruit set visible) in years 1 and 2. In trial NT001, pollen collected from bumble bees was 

shipped as separate samples from the honey bee hive-collected pollen; in trials NT003, pollen collected 

from both bee sources was combined into one pollen sample for the subplot and interval, while in trial 

NT002, no pollen was collected using honey bees. Honey bee hives and bumble bee colonies were 

removed from the tents after the end of sampling. All bees remained in good condition, with no visible 

signs of infection or infestation, throughout the course of the study. 

Table 5. Be Summary Information for the three trials NT001, NT002 and NT003

Soil. Nine soil samples were collected prior to treatment and at the end of growing season in each plot, 

each year. The exceptions were trial NT001 year 2 (2014), when the soil samples were collected before 

the spray application and just before the start of the blueberry collection in year 2, (2014) and trial NT003, 

when the only soil samples collected were after the growing season in year 2 2014 (no year 1 2013 soil 

samples were collected). Soil samples were collected from the surface (6 inches or 15 cm deep) of the 

plots using a soil core sampler or shovel and placed in a plastic bag. In each sampling period, sample 

locations were randomly selected from the inner half of the plot (e.g., the inner 100 x 200 ft [30 x 60 m] 

area of a 200 x 400 ft [60 x 120 m] plot), and locations were separated by at least 10 feet (3 m).

Control Plots. Two composite samples (one sample per bee tunnel) of all blueberry matrices and nine 

composite samples of soil were collected at the same sampling periods as for the treated samples of their 

sample type. Except in trial NT002, in which no year 2 control samples could be collected because the

control blueberry plants died during the previous winter. However, in all trials (including the controls), 
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not enough plant matrix material was present at every interval to allow for the full number of target 

samples or sampling intervals to be collected. 

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. Composite samples were placed into labeled (study number and 

sample number) containers for shipment. All samples were frozen the day of their collection, and if 

analyses were to be completed at a different facility (Bayer CropScience facility (Stilwell, KS or RTP, NC)), 

all samples were shipped frozen.

5E. ANALYTICAL METHODS

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 LOD 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 limits of detection (LODs) are shown below in Table 6.

Table 6. Limits of quantification and detection for imidacloprid and metabolites

5F. QUALITY ASSURANCE 

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 blueberry matrices and for at 

least 1281 days (42 months) in soil matrices prior to analysis. 
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Transit Stability. To ensure that imidacloprid, 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 samples, 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, accompanied the respective study sample types throughout the sampling, shipping, and storage 

events of the field phase of the study. They were analyzed after 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-hydroxy imidacloprid, and imidacloprid 

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

analytes from control matrices fortified at their respective LOQs. Additional recoveries at higher 

fortification levels validated 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. QUALITY ASSURANCE RESULTS

Sample Storage. The maximum storage period of frozen samples in this study for imidacloprid was 273 

days for blueberry flowers, 489 days for blueberry leaves, 443 days for blueberry nectar, 429 days for 

blueberry pollen, and 679 days for soil prior to extraction. 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. Mean transit 

recoveries for IMI, IMI-Olefin, and 5-hydroxy IMI range from 85%-96% in nectar and 86%-186% in pollen.

Matrix Spike Recoveries. As indicated in Table 5 of the study report, all matrix spike recoveries (pollen, 

nectar, flower, leaf, soil), were corrected for any interferences in corresponding controls. The overall 

mean of the recoveries for each matrix was within the acceptable range of 70 to 120%, and the 

standard deviation values were below 20%, with the exception of the 0.005 ppm imidacloprid pollen 

group, which had a standard deviation of 22%.

6.B. MAGNITUDE OF RESIDUES IN BEE-RELEVANT MATRICES 

Nectar. Summary statistics of the overall magnitude of IMI and related metabolites in blueberry nectar is 

shown in Table 7. These statistics reflect analysis of individual composite samples (usually 5 replicates per 

sampling date) among all of the sampling times within each trial. 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, IMIolefin and 5-OH-IMI). IMI residues in blueberry nectar from trials NT001 (year 1) and all but one sample in 

NT002 (both years) were <LOD. Total IMI residues in trial NT003 (both years) had two samples containing 

residues of >10 ppb. 
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Table 7. Magnitude of IMI and related metabolites in blueberry nectar (ppb).

Trial NT001 (n=23)* Trial NT002 (n=35)* Trial NT003 (n=29)*

Olefin 5-OH IMI Total Olefin 5-OH IMI Total Olefin 5-OH IMI Total

Mean 0.43 0.45 0.72 1.58 0.32 0.36 0.17 0.84 0.42 0.49 2.62 3.49

SD 0.33 0.40 0.82 1.30 0.00 0.16 0.00 0.17 0.22 0.34 3.41 3.72

CV 75% 88% 114% 82% 0% 46% 0% 20% 52% 70% 130% 106%

Min. 0.32 0.33 0.17 0.81 0.32 0.33 0.17 0.81 0.32 0.33 0.17 0.81

Median 0.32 0.33 0.17 0.81 0.32 0.33 0.17 0.81 0.32 0.33 1.10 2.00

90th 0.50 0.47 1.90 3.42 0.32 0.33 0.17 0.81 0.50 1.06 7.22 8.82

Max. 1.50 2.10 3.20 5.80 0.32 1.30 0.17 1.80 1.20 1.50 14.00 16.00

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.

* 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 individual measured residues of total IMI in nectar for each trial year are shown in Figure 2AC. Mean values by residue trial and year are shown in Table 8 below. Mean residues measured during 

year 1 from the MI site (2.0-8.8 ppb) is about 2X higher than those measured during year 1 of the NY site 

and that from the year 1 and 2 of the IL site (1.8 ppb for NT003) is 2X the mean value measured during 

year 2 (0.81 and/or 0.87 ppb, year 1 and 2, respectively). 

Table 8. Mean total IMI residue in blueberry nectar (ppb) by trial and study year.

Study Year Residue Trial

NY (NT001) IL (NT002) MI (NT003)

Year 1 (2013) 0.81 0.81 7.25

Year 2 (2014) 1.8 0.87 1.8
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Figure 2. Total imidacloprid residues measured in blueberry nectar for Trials NT001, NT002 (panel A), 

NT003 (panel B), and all Trials combined (panel C).

Mean daily residues of total IMI in blueberry nectar range from approximately 0.81-8.8 ppb across all 

three trials, while maxima range from 0.81–16 ppb over year 1 (2013) and 2 (2014). Plots showing the 

mean daily residues of total IMI in blueberry nectar for each trial are found in Figure 3A-C below.
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Figure 3. Mean daily total imidacloprid residues measured in blueberry nectar for trials NT001 (panel A), 

NT002 (panel B), NT003 (panel C); error bars = range.

Pollen. The overall magnitude of IMI and related metabolites in blueberry pollen are about 2-8X those 

measured in nectar for year 1 (2013) and year 2 (2014), respectively (Table 9). The overall mean residues 

of total IMI range in pollen from approximately 2.69-13.9 ppb, while the maxima across all three trials 

range from 5.46-42 ppb. In year 2, bumble bees were used to help honey bees collect pollen, and as 

noted earlier in Section 5D, within each trial and year, blueberry pollen residues were generally similar. 

There were no pollen samples collected by the honey bees in year 1 of trial NT002, therefore no

comparison of year on year total residues in blueberry pollen could be made. In trial NT001, pollen 

collected from bumble bees was shipped as separate samples from the honey bee hive-collected pollen, 

and the source of collection could be distinguished. Whereas in trial NT003, pollen collected from both 

bee sources was combined into one pollen sample for the subplot and interval, while in trial NT002, no 

pollen was collected using honey bees, it was only collected by bumble bees.
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Table 9. Magnitude of IMI and related metabolites in blueberry pollen (ppb).

NT001 (n=32)* NT002 (n=11)* NT003 (n=25)*

Olefin 5-OH IMI Total Olefin 5-OH IMI Total Olefin 5-OH IMI Total

Mean 0.26 0.24 2.22 2.69 0.16 0.24 4.13 4.53 1.22 0.53 12.14 13.9

SD 0.16 0.00 3.46 3.42 0.00 0.00 1.46 1.46 0.82 0.44 7.05 7.95

CV 61% 0% 156% 127% 0% 0% 35% 32% 67% 82% 58% 57%

Min. 0.16 0.24 0.20 0.60 0.16 0.24 1.60 2.00 0.16 0.24 2.70 3.10

Median 0.16 0.24 1.40 1.8 0.16 0.24 3.90 4.30 1.10 0.24 12.0 14.0

90th 0.50 0.24 5.03 5.46 0.16 0.24 6.00 6.40 2.30 1.22 17.4 20.8

Max. 0.50 0.24 19.0 19.0 0.16 0.24 6.40 6.80 2.80 1.60 38.0 42.0

Mean % 

of Total 9.5% 8.9% 83% --- 3.5% 5.3% 91% --- 8.8% 3.8% 87% ---

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. 

* 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 individual measured residues of total IMI in nectar for each trial year are shown in Figure 4AC. Mean values by residue trial and year are shown in Table 10 below. It is clear from Table 10 that 

annual mean concentrations of total IMI in pollen from the MI study sites are about 22X greater than NY 

sites in year 1 and 3X greater than those from the IL and NY sites for year 2. Similar, but not as dramatic 

differences were also observed for annual mean nectar residues (Table 8). Even with the combined 

pollen from bumble bees in year 2, in trial NT003 there was little difference in the total IMI residues in 

pollen between the two years at this site. There were differences between the other two sites (NY and IL) 

and MI; however, it is not clear if it is due to factors related to field conditions (e.g., soil characteristics) or 

other factors as the metabolite residues were below levels of analytical detection or quantification where 

1⁄2 the LOD or LOQ was assumed in both trials NT001 and NT002. The influence of irrigation on IMI 

residues in pollen is not known and other factors may account for the differences between the NY, IL, and 

MI results. Specifically, the MI site contained much coarser soils (90% sand) compared to the NY and IL 

sites (31-39% sand). Although the basis of the 22X difference in pollen residue values between the NY 

and MI sites is not clear, such differences demonstrate the value of incorporating regional differences in 

study sites into the design of pollen/nectar residue studies. 

Table 10. Mean total IMI residue in blueberry pollen (ppb) by trial and study year.

Study Year Residue Trial

NY (NT001) IL (NT002) MI (NT003)

Year 1 (2013) 0.60 Not collected 13.7

Year 2 (2014) 2.9 4.53 14.0
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Figure 4. Total imidacloprid residues measured in blueberry pollen for Trials NT001, NT002 (panel A), 

NT003 (panel B), and all trials combined (panel C).

Mean daily residues of total IMI in blueberry pollen from approximately 0.6-16.5 ppb, while the maxima 

across all three trials range from 0.6-42 ppb over year 1 (2013) and 2 (2014). Plots showing the mean 

daily residues of total IMI in blueberry nectar for each trial are found in Figure 5A-C below.
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Figure 5. Mean daily total imidacloprid residues measured in blueberry pollen for trials NT001 (panel A), 

NT002 (panel B), NT003 (panel C); error bars = range.

Flowers. The overall mean total residues of IMI measured in flowers (Table 11) are the greater than the 

residues measured in both pollen and nectar, but less than the residues measured in leaves and soil

(Table 12 and 13, respectively). In both trials NT001 and NT002 the residues of total IMI in flowers were 
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<LOD in both years. In trial NT003, the overall mean residue of total IMI was slightly higher in 2014 (25 

ppb) compared to 2013 (17.3 ppb), with the daily mean residues of total IMI in flowers ranging from 15-22

ppb in 2013 and 21-34 ppb in 2014. For trial NT003 (MI) in both 2013 and 2014, daily mean residues in 

flowers were similar and greater (approx. 1-4X) than those found in pollen; however they were 

substantially greater (approx. 2-23X) than those found in nectar on the same DAA. A plot of the raw data 

for residues in flowers for trial NT003 is provided below in Figure 6.

Table 11. Mean concentrations of total IMI in blueberry flowers (ppb).

Study Year Residue Trial

NY (NT001) IL (NT002) MI (NT003)

Year 1 (2013) 5.4 5.4 17.3

Year 2 (2014) 5.4 5.4 25.0

Figure 6. Total imidacloprid residues measured in blueberry flowers for Trial NT003.

6.C. MAGNITUDE OF RESIDUES IN LEAVES AND SOIL 

The overall mean total residues of IMI measured in leaves are the highest among the plant tissues 

sampled for trial NT003, ranging from 81-179 ppb (Table 12). Again, in both trials NT001 and NT002, the 

majority of residues of total IMI in leaves were <LOD in both years. In trial NT003, the daily mean total 

residues of IMI in leaves range from 60-110 ppb in 2013 and 135-243 ppb in 2014. The daily mean total 

residues of IMI in leaves are significantly greater than the daily mean total IMI residues measured in 

nectar and pollen (up to 150X and 27X; based on NT003 residues) on the same DAA. They are also greater 

(approx. 7X) than the mean total IMI residues in flowers (based on NT003 residues) on the same DAA. 

Based on the data available to compare the mean total IMI residues in leaves to the residues found in soil, 

for the MI site (NT003) the residues were approximately equivalent for year 2 (179 vs. 232 ppb). A plot of 

the raw data for residues in flowers for trial NT003 is provided below in Figure 7.

Table 12. Mean concentrations of total IMI in blueberry leaves (ppb).

Study Year Residue Trial

NY (NT001) IL (NT002) MI (NT003)

Year 1 (2013) 5.1 4.7 81

Year 2 (2014) 16.8 4.7 179

Unlike the other plant matrices sampled, the leaf residue data does span 22-41 days depending on the 

year of sampling. As seen in Figure 7, concentrations of total IMI in leaves from the MI site increases 

substantially during the second year (2014) sampling period. Whereas, leaf residues measured from the 
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same MI site did not show an obvious increase, it remained fairly consistent, during the first year of the 

study during the sampling period. 

Figure 7. Total imidacloprid residues measured in blueberry leaves for Trial NT003.

Residues of IMI measured in soil are high relative to most plant matrices, with the exception of leaves

(Table 13). Levels increased over time from the end of the first growing season (2013) and the end of the 

second growing season (2014). In trial NT001 (NY) concentrations in soil measured at the end of the first 

growing season increased approx. 1.5X by the end of the second growing season and those in trial NT002 

(IL) increased approx. 2X. However, no comparison could be made for trial NT003, as soil samples were 

only collected at the last scheduled time point in trial NT003 (MI), but the residues at this sampling time 

are similar to residues found in the other trials. There was some evidence of carryover from year to year 

based on sampling at the end of each growing season, although soil residues increased < 2X.

Table 53. Magnitude of IMI residues in soil (ppb).

Trial Year Date Event DAA1 Mean IMI in Soil (ppb)2

NT001

1

09/25/12

09/29/12

06/01/13

Prior to treatment -4 <LOD (0.25)

Banded soil application 0 Application (0.5 lb ai/A)

End of 1

st growing season 245 151

2

09/25/13

09/30/13

05/10/14

Prior to treatment 361 191

Banded soil application 366 Application (0.5 lb ai/A)

End of 2

nd growing season 588 233

NT002

1

09/28/12

10/01/12

07/03/13

Prior to treatment -3 <LOD (0.456)

Banded soil application 0 Application (0.5 lb ai/A)

End of 1

st growing season 275 151

2

09/23/13

09/26/13

06/02/14

Prior to treatment 357 181

Banded soil application 360 Application (0.5 lb ai/A)

End of 2

nd growing season 609 339

NT003

1

NA3

10/04/12

NA3

Prior to treatment NA No data

Banded soil application 0 Application (0.5 lb ai/A)

End of 1

st growing season NA No data

2

NA3

10/01/13

06/12/14

Prior to treatment NA No data

Banded soil application 362 Application (0.5 lb ai/A)

End of 2

nd growing season 616 232

1 DAA = Days After Last Yearly Application; Negative numbers indicate soil sampled prior to treatment. 

2

IMI residue values are averages of 9 composite samples for IMI only taken for each plot at each sampling event. 

Residue values <LOD were averaged as 1⁄2 LOD. 

3 No samples were collected in the sampling period due to PFI oversight; NA = Not applicable.
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6.D. YEAR TO YEAR ACCUMULATION IN POLLEN AND NECTAR

A qualitative evaluation of the potential for ‘year-to-year’ accumulation of total IMI residues in nectar and 

pollen could only be conducted for 1 of the 3 trials (NT003); since a majority of the mean residue data for 

trial NT001 and NT002 were below levels of detection or quantification. Examination of Figures 3C and 5C

(and Tables 9 and 10) indicates the mean of total IMI residues in nectar from Trial NT003 decreased in 

year 2 relative to year 1 (7.25 ppb vs 1.8 ppb) while those in pollen remained essentially the same (13.7 vs 

14.0 ppb). 

The overall mean of the total IMI residues in flowers increased slightly over the two trial years (Figure 6; 

Table 11), and the overall mean residues in leaves increased about 2X from year 1 to year 2 (Figure 7; 

Table 12) remained stable in year 1, but increased in year 2. Although these data suggest that year-toyear accumulation of total IMI is potentially evident for two plant matrices (flowers, leaves) they 

represent only one trial as the other locations resulted in measurements that were below levels of 

detection of both the metabolites and parent compounds. 

6.E. RESIDUE DECLINE (DT50) IN POLLEN AND NECTAR 

Due to a majority of the residues below levels of detection or quantification (LOD and/or LOQ), a 

calculation of reliable dissipation half-life (DT50) values could not be conducted for nectar and pollen for 

trials NT001 and NT002. In trial NT003, the increase in concentrations over time with nectar (both years) 

and pollen (year 1) precluded calculation of a DT50 value (Figure 8 and Figure 9). Only one DT50 could be 

calculated for pollen in year 2 (35.6 days) but the fit of the regression line was very poor (r2 = 0.017) and 

the slope of the regression line was not significantly different from zero (p=0.6).

Figure 8. Residue “decline” curves of total IMI in blueberry nectar.
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Figure 9. Residue decline curves of total IMI in blueberry pollen.

Although reliable DT50 values could not be estimated from the current data, they can be used as a line of 

evidence in support of the showing the potential total residues of IMI in nectar and pollen as remaining 

relative stable (pollen) or even increasing over time (nectar).

6.F. OCCURRENCE OF RELEVANT IMI METABOLITES 

Nectar. For trials NT001 and NT002, due to the low levels of parent IMI and metabolite residues 

measured in nectar, reliable estimates of the relative proportion of total IMI as parent or metabolites 

could not be determined, since such estimates would be unduly influenced by assumptions of 1⁄2 the LOD 

or 1⁄2 the LOQ.

Suitable data for estimating the proportion of total IMI residues in nectar present as parent IMI or 

metabolites were available from trial NT003, only. In this trial, the mean % of total IMI as IMI-olefin in 

nectar is 21% (range 2.8-50.5%). The mean % of total IMI as 5-OH IMI 23.9% (range 3.8-65%). Parent IMI

was present at 56% of total IMI in nectar on average (range 8.5-88.6%). 

Pollen. For trials NT001 and NT002, due to the low levels of parent IMI and metabolite residues 

measured in pollen, reliable estimates of the relative proportion of total IMI as parent or metabolites 

could not be determined, since such estimates would be unduly influenced by assumptions of 1⁄2 the LOD 

or 1⁄2 the LOQ.

Suitable data for estimating the proportion of total IMI residues in pollen present as parent IMI or 

metabolites were available from trial NT003, only. In this trial, the mean % of total IMI as IMI-olefin in 

pollen is 8.7% (range 1.1-14.1%). The mean % of total IMI as 5-OH IMI is 3.9% (range 1.6-8.4%). Parent 

IMI was present at 87.5% of total IMI in pollen on average (range 76.9-100%). 
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6.G. RELATIONSHIP OF RESIDUES AMONG PLANT TISSUES

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

flowers and those measured in nectar and pollen on the same sampling day for trial NT003 is shown in 

Figure 10A and 10B, respectively. Results indicate that mean daily residues in flowers have little 

predictive ability of those in nectar or pollen, as regression equations resulted in negative slopes 

(equations not shown) and low correlation coefficients. 

Figure 10. Relationship between total IMI residues in flowers to those in nectar (A) and pollen (B)

B

A
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Predictability of Leaf Residues. Similar to the findings with flowers, daily mean concentrations of total 

IMI in leaves appear to have little predictive capacity for those in pollen or nectar (Figure 11A and 11B,

respectively). The overall slopes of the regression line is negative with relatively poor correlation 

coefficients. 

Figure 11. Relationship between total IMI residues in leaves to those in pollen (A) and nectar (B)

A

B
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Pollen vs. Nectar. No predictive relationship was observed between daily mean concentrations in nectar 

vs. pollen (Figure 12), as the slope is not significantly different from zero and correlation coefficient is 

very weak (r2=0.0014). 

Figure 12. Relationship between total IMI residues in nectar to those in pollen

7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS

In the context of documenting the magnitude of IMI residues in bee-related matrices of blueberry, 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. Sites were located in three different parts of the U.S. (NY, IL, and MI) with differing climate 

conditions

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

1. There were a large number of pollen and nectar residues for trials NT001 and NT002 that were 

below the level of detection/quantification, which that limited reliable estimates of DT50 values. 

2. Some samples (e.g., pollen for year 1 for trial NT002 and soil for year 1 for trial NT003) were not 

taken which limited the study’s utility.

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

1. A single banded soil application of approximately 0.5 lb a.i./A result in residues of total IMI in 

individual composite samples of blueberry nectar during the following year up to 16 ppb. 

2. A single banded soil application of approximately 0.5 lb a.i./A result in residues of total IMI in 

individual composite samples of blueberry pollen during the following year up to 42 ppb. 
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3. Although the total IMI residues in nectar does not continually increase over the two years, the 

mean residue each year was observed to increase. Such that mean residues 236 days post 

application were 8.7 ppb in trial NT003 (year 1) and 3.24 ppb 240 days post application in year 2. 

4. Total IMI residues in pollen were approximately 2-8X those observed in nectar in year 1 and 2,

respectively; and generally showed little or no dissipation. 

5. Total IMI residues in leaves was significantly greater (2-3 orders of magnitude) than those 

observed in pollen and nectar. 

6. Two metabolites of IMI (IMI-olefin and 5-OH-IMI) typically represented < 15% of total IMI residues 

in pollen and nectar. 

7. The predictability of total IMI residues in nectar and pollen based on measured concentrations in 

other plant matrices (flowers, leaves) was poor. 

8. STUDY VALIDITY/CLASSIFICATION 

Data from the three trials (NT001, NT002, and NT003) are considered scientifically sound and useful for 

risk assessment purposes. Overall, this study is classified as ACCEPTABLE for quantitative use in risk 

assessment.
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Appendix A. Location of Blueberry Residue Trials.

Figure A1. Trial NT001-12ZA: Latitude: 42.570667| Longitude: -77.037833 (Penn Yan, NY)

Figure A2. Trial NT002-12ZA Latitude: 42.29122 | Longitude: -88.62452 (Marengo, IL)

Figure A3. Trial NT003-12ZA: Latitude: 42.60427 | Longitude: -86.15298 (Fennville, MI)

UTC

TRTD
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Zoomed in in NT003-12ZA location

Figure A2. Relative location of Study Trials NT002 (Marengo, IL), and NT003 (Fennville, MI)

Marengo, IL

Fennville, MI