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Reference

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

Clover Planted after Seed Treated Field Corn. Study ID EBNTY009. Bayer Crop Science, Research 

Triangle Park, NC. MRID 49511701.

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 successive 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 NT010) Heartland Ag Research, LLC, 1 

5635 W. 83rd Terrace, Lenexa, KS 66219

(Field Trial NT011): 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: EBNTY009

Study 

Completion 

Date: 3 November 2014

Experiment 

Start/End Date: 1 May 2012 – 9 July 2014

Study Location: 3 Field Trials:

Edgerton, KS,(NT010); 

Springfield, NE (NT011); 

York, NE (NT012) 

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 corn 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 corn the previous year. Six separate trials were 

conducted in three study sites in KS and NE (NT010, NT011, and NT012); three of the trials focused on 

residues in corn over two years (TRTSTA) and the other three trials focused on residues in corn after 1 

year and rotationally-planted white clover during the second year (TRTSTB). Each trial included an
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untreated control (UTC) plot. Each year, seed treated corn (1.34 mg a.i./seed; 0.12 a.i./A) was planted at 

the beginning of the growing season. Five composite samples of corn pollen, tassels, and leaves were 

made at various sampling times during the 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 findings is as follows:

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

maximum of 39.7 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 corn pollen is 22.3 ppb and the maximum yearly average

concentration is 7.5 ppb.

2. Yearly average concentrations of total IMI in corn tassels are about 2X – 3X the mean residues 

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

maxima range 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. The 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 

increasing 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 increasing trends seen in concentrations of total IMI in corn pollen, 

DT50 values were not determined. 

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

is fair, with most of the predicted values being within a factor 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/A
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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: 48.59% 

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: 3/13/2014 Solubility: 0.51-0.61 g/L

Storage of Test Mat’l: Ambient (54-80

o

F) Sample 

Storage: -24oto 26oF

5A. STUDY DESIGN

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 plants were grown the previous year. Besides quantifying the 

magnitude of residues in bee-relevant matrices of corn 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 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 0.011 – 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 white 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. In addition to the treated crop study sites, two control sites were included for 

each trial (UTCA and UTCB). 

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 in year 2, clover seeds were planted on plots UTCB and TRTDB 

previously 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 

collected 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 clover leaves and blossoms for direct analysis were collected by 
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hand, and composite samples 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 growing 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 treated 

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

spikes to validate analyte recovery.

5B. APPLICATION TIMING AND RATES

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 imidacloprid/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.
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Table 1. Imidacloprid application and corn seeding rates for the three study trials* 

* from Table 4 of the study report)

5C. STUDY SITE LOCATION AND CHARACTERISTICS 

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 Corn study site characteristics (treated sites only).

Attribute NT010-12ZA

Edgerton, KS

Trial NT011-12ZA

Springfield, NE

Trial NT012-12ZA

York, NE

Plot ID TRTSTA / (TRTSTB) TRTSTA / (TRTSTB) TRTSTA / (TRTSTB)

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. 2012)

8-May-2012,

7-May 2013 

(6 Sept. 2012)

11-May-2012, 

29-April 2013 

(16 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 ft2 44,400 ft2 44,000 ft2

Row Spacing 30 in 30 in 30 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, -97.600888
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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, 

soils from NT012 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 site 

NT010, monthly 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 Corn study sites* 

* (from Table 3 of study report)

Figure 1. Monthly precipitation for the 3 study trial sites (NT010, NT011, NT012).
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5D. SAMPLE COLLECTION, HANDLING, PROCESSING

Corn 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 flowering, stigmata completely dry; Table 4). 

Corn leaves were sampled at six times from BBCH 59-71. Five composite samples of corn 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 corn 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

NT0110

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 = Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie growth stage scale for corn; 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 targeted 
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growth stages of BBCH 63, 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 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 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 sampled 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 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] 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 nectar and pollen, which were 

processed for pollen and nectar by the field investigators. 

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 LOD and LOQ values for each matrix are shown below in 

Table 5.
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Table 5. 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 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 imidacloprid 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 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 analysed 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.
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Spike Recovery. The method for determining imidacloprid, 5-hydroxy imidacloprid, and imidacloprid 

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

from control matrices fortified at their respective matrix 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 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 Recoveries. As indicated in Table 5 of the study report, all matrix spike recoveries (pollen, 

nectar, tassel, blossom, leaf, soil), were within the acceptable range of 70 to 120%, and the standard 

deviation values were below 20%. 

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

Corn Pollen and Tassel. Summary statistics of the 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 discussion 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 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

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

Across all three trials, yearly mean residues of total IMI in corn pollen range from approximately 2.1 – 7.5

ppb while yearly maxima range from 4.8– 39.7 ppb. Based on yearly means, results 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 13.7 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 IMI residues in corn pollen 

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

occurred in trial NT012. Precipitation patterns are comparable among the three trials (especially during 
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year 2). The percent sand in soils from trial NT012 (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 

NT012 may be due its greater fraction of sand in soil. 

Table 6. Magnitude of total IMI in corn pollen and tassels

Corn Pollen (ppb)

Trial NT010* Trial NT011* Trial NT012*

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

NOTES: Total IMI = sum of imidacloprid, imidacloprid olefin and hydroxy imidacloprid. The highest measured 

concentration is shown 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 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 trial NT012) is not limited to the pollen matrix alone, but rather 

reflects the pattern in the entire corn tassel.
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Figure 2. Total imidacloprid residues measured in corn pollen from trials NT010, NT011 and NT012 (dots 

= mean; error bars = range).
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Figure 3. Total imidacloprid residues measured in corn tassels from trials NT010, NT011 and NT012 (dots 

= mean; error bars = range).
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6.C. MAGNITUDE OF RESIDUES IN LEAVES AND SOIL 

Mean concentrations of total IMI in corn 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 suggests 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 corn leaves (ppb).

Treatment (Year)

Residue Trial

NT010 NT011 NT012

TRTDA (2012) 77.2 22.7 34.0

TRTDA (2013) 14.7 8.3 18.6

TRTDB (2012) 66.7 19.4 28.4
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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 DAFA1 Mean IMI in Soil 

TRTSTA (ppb)2

Mean IMI in Soil 

TRTSTB (ppb)2

NT010

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 --- ---
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Year Date Event DAFA1 Mean IMI in Soil 

TRTSTA (ppb)2

Mean IMI in Soil 

TRTSTB (ppb)2

2 10/10/13 End of 2

nd growing season 523 103 28

NT011

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 510 71 6

NT012

1 5/11/12 Prior to planting 0 3 2

1 5/11/12 Seed planting 0 --- ---

1 10/24/12 End of 1

st growing season 166 9 2

2 4/4/13 Prior to planting 328 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. YEAR TO YEAR ACCUMULATION IN POLLEN, TASSEL AND SOIL

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 4X 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. RESIDUE DECLINE (DT50) IN CORN POLLEN 

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

suitable for DT50 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 

blooming period are the exception rather than the rule for seed treated corn. 
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6.F. OCCURRENCE OF RELEVANT IMI METABOLITES 

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. OCCURRENCE OF IMI IN ROTATIONAL CROP (WHITE CLOVER)

The concentrations of total IMI measured in white 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 (detection 

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. RELATIONSHIP OF RESIDUES AMONG PLANT TISSUES

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 

5A. 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 (r2 = 0.35). Overall, concentrations of total IMI 

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

seen between total IMI residues in corn pollen and leaves (Figure 6B). 
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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 corn resulting 

from seed treatment, 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.

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 corn matrices. 

Overall, considering the strengths and 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 results in a 

maximum of 39.7 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 corn pollen is 22.3 ppb and the maximum yearly average

concentration is 7.5 ppb.

2. Yearly average concentrations of total IMI in corn tassels are about 2X – 3X the mean residues 

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

maxima range from 9.4 to 26.1 ppb. 

3. Temporal trends in the concentration of total IMI in corn pollen are either variable or show 
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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. The 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 

increasing 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 increasing trends seen in concentrations of total IMI in corn pollen, 

DT50 values were not determined. 

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

is fair, with most of the predicted values being within a factor 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 (NT010, NT011, and NT012) 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 classified as ACCEPTABLE for quantitative use in risk assessment.
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Appendix A. Location of Corn Residue Trials.

NT010 (Edgerton, KS)

NT011 (Springfield, NE)

NT010

NT011
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NT012 (York, NE)

NT012
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Appendix B. Plots Of Raw Data For Total IMI In Corn Pollen 

Figure B-1. Total imidacloprid concentrations in corn pollen for NT010, NT011 and NT012