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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 Treated Cotton During Two Successive Years and in White Clover Planted after Treated Cotton. 

Study ID EBNTY010. Bayer Crop Science, Research Triangle Park, NC. MRID 49511702.

1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material #1 Gaucho 600 FL Admire Pro 

Systemic Protectant (SC)

Purity 48.59

Test Material #2 Admire Pro Systemic 

Protectant

Purity 42.9%

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

metabolite levels in pollen, nectar, flowers, 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, Flower, Soil): SynTech Research 

Laboratory Services, 17745 S. Metcalf Ave, 

Stillwell, KS 66085

(Trial NT013, NT014, NT015): Shoffner Farm

Research, Inc, 4809 Highway FF, Fisk, MO

Sponsor Study 

ID: EBNTY010

Study 

Completion 

Date: 6 November 2014

Experiment 

Start/End Date: 2 May 2012 – 3 Sept. 2014

Study Location: 3 Field Trials:

Maiden, MO (NT013); 

Glennonville, MO (NT014); 

Fisk, MO (NT015) 

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 and 

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

imidacloprid-treated seeds (Gaucho) and pre-bloom, foliar applications of imidacloprid (Admire Pro) over 

two successive years. In addition, this study examined the occurrence of imidacloprid residues in bee 

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

previous year. Six separate trials were conducted in three study sites in MO (NT013, NT014, NT015); three 
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of the trials focused on residues in cotton over two years (TRTDA) and the other half focused on residues 

in cotton after 1 year and rotationally-planted white clover during the second year (TRTDB). Each trial 

included an untreated control (UTC) plot. Each year, five foliar applications (0.06 lb a.i./A x 5) were made

5-8 days apart to seed treated cotton (0.375 mg a.i./seed; 0.05 lb a.i./A) for a total application rate of 0.35 

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

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

various sampling times during the flowering period of the growing season between 12-55 days after the 

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

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

A summary of the key findings is as follows:

1. Results indicate that pre-bloom, foliar applications of approximately 0.06 lb a.i./A x 5 (5-8 day 

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

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

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

average concentration of total IMI in cotton floral and extrafloral nectar is 29.0 and 15.7 ppb. 

2. These same pre-bloom, foliar applications of imidacloprid resulted in a maximum of 56.7 ppb of 

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

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

ppb. 

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

measured after the second year of foliar applications to seed treated cotton were 1.2X-2.9X those 

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

occurred within 13-20 days following the last foliar application. It is not clear if the increase in 

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

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

4. Total IMI residues in pollen were approximately 1-2 orders of magnitude greater than those 

observed in nectar. 

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

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

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

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

6. Two metabolites of IMI (IMI-olefin and 5-OH-IMI) were present below the limits of quantification 

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

7. DT50 values for total IMI in floral and extrafloral nectar varied from 19-68 days, except for one trial 

year where residues were relatively constant over the 40-d sampling period. Three DT50 values for 

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

relatively constant concentrations of total IMI over time.

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

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

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

about 2X those measured in nectar (floral and extrafloral) while those in leaves were about 4-5X 

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

measured concentrations in flowers and leaves is poor.

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

application to seed treated cotton harvested the previous year. In most samples, total IMI 
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residues were at or below the level of detection (0.7 ppb). The maximum concentrations of total 

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

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

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

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

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

conditions (all three located in MO). 

4. STUDY VALIDITY

Guideline Followed: Non-guideline study (protocol was 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

5. MATERIALS AND METHODS

Test Material Characterization

Test item #1 (foliar) Admire Pro Systemic 

Protectant SC (Imidacloprid) 

550 g a.i./L

CAS #: 138261-41-3

Test item #2 (seed 

treatment)

Gaucho 600 FL Flowable 

concentrate for seed

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

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

48.59% (Gaucho)

Lot No./Batch No. Batch No. NK41BX0170

(Admire Pro)

NK41BX0356 (Gaucho 600)

Density: 1.41 – 1.54 g/mL

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

DEH028: 128 (Gaucho)

Material Receipt 

Date:

Not reported Analysis 

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

03/13/2012 (Gaucho)

Expiration Date: 03/31/2014 (Admire Pro); 

3/13/2014 (Gaucho)

Solubility: 0.51-0.61 g/L

Storage of Test Mat’l: Ambient (56-87oF) Sample 

Storage: -39oto 3

o

F

5A. STUDY DESIGN

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

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

imidacloprid-treated seeds (Gaucho 600 Flowable) followed by five foliar applications per year of ADMIRE 

PRO Systemic Protectant containing 550 g/L imidacloprid for two consecutive years. In addition, a second 
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series of trials was conducted to measure the same residues in/on bee-relevant white clover pollen and 

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

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

year. Besides quantifying the magnitude of residues in bee-relevant matrices of cotton and white clover, 

this study was also intended to evaluate the following attributes:

1) The extent of year-to-year ‘carry over’ of pesticide residues measured in plant and soil 

matrices

2) The dissipation 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 (0.375 mg a.i./seed or 0.047 lb a.i./A) and 5 x 0.06 lb a.i./A foliar applications 

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

x 0.06 lb a.i./A foliar applications (5-8 days apart) during year 1 followed by planting of white clover and 

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

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

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

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

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

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

various sampling times during the flowering period from each TRTD plot/year. For TRTDB sites in year 2, 

clover seeds were planted on plots UTCB and TRTDB previously treated in year 1. However, no clover 

grew in trial NT013-12ZA, so no clover samples could be collected and no bees were used. In trials NT014-

12HA and NT015-12ZA, clover samples were collected from within erected bee tents; one tent was placed 

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

Composite samples of white clover leaves and blossoms for direct analysis were collected by hand, and 

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

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

1, before cotton planting in year 2, and after all sampling was complete in year 2. QA/QC included field 

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

showing the application and residue sampling design is shown in Figure 1. 
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Figure 1. Timeline of residue sampling of nectar, pollen, flower, leaf relative to foliar pesticide 

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

(panel B).

5B. APPLICATION TIMING AND RATES

During each of the two successive years, five foliar applications of approximately 0.06 lb a.i./A were 

applied at intervals from 5-8 days apart at each of the three treatment sites for a total annual application 

rate of 0.35 lb a.i./A (annual foliar application rate = 0.3 lb a.i./A + 0.05 lb a.i./A from seed treatment). All 

applications were made with adjuvant (Dyne-Amic, 0.25%) using a ground-based equipment. Plot 

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

applications is provided in Appendix A.

5C. 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 type of cotton were similar.
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Table 1. Summary of Cotton study site characteristics (treated sites only).

Attribute NT013-12ZA

Malden, MO

Trial NT014-12ZA

Glennonville, MO

Trial NT015-12ZA

Fisk, MO

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

Variety Pima cotton 

(Landino clover)

Pima cotton 

(Landino clover)

Pima cotton 

(Landino clover)

Planting Date 9-May-2012, 

8-May 2013 

(15 Apr. 2013)

24-May-2012,

8-May 2013 

(15 Apr. 2013)

22-May-2012, 

26-May 2013 

(15 Apr. 2013)

Soil Type Sandy loam Silty loam Sand

Plot Length 1150 ft 600 ft 625 ft

Plot Width 90 ft 180 ft 173 ft

Plot Area 103,500 ft2 108,000 ft2 108,331 ft2

Row Spacing 40 in 40 in 40 in

Plant Spacing 

within Row

2.7 in 2.7 in 2.7 in

GPS Lat. Long. 36.60981, -90.00092 36.56850, -90.13092 36.70934, -90.19482

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

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

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

(66%). Temperatures and total precipitation were similar among sites. Monthly precipitation patterns 

follow similar temporal patterns which is expected given the relatively close proximity of study sites 

(Figure 2). All three sites were irrigated to some extent during the summer months (0.5 – 3 in/month). 

Maps showing the locations of the trials are found in Appendix B.

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

* (from Table 3 of study report)
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Figure 2. Monthly precipitation for the 3 study trial sites (NT013, NT014, NT015).

5D. SAMPLE COLLECTION, HANDLING, PROCESSING

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

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

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

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

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

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

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

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

Pollen, Nectar and Flower Sampling Leaf Sampling

Year 1

(TRTDA & B)

Year 2

(TRTDA)

Year 1

(TRTDA & B)

Year 2

(TRTDA)

BBCH* DAA* BBCH DAA BBCH DAA BBCH DAA

NT013

60 14 60 15 60 15 60 15

61 21 61 22 61 22 61 22

65 28 65 29 65 29 65 29

67 36 67 35 67 35 67 35

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

NT014

60 13 60 14 60 13 60 14

61 20 61 21 61 20 61 21

65 26 65 33 65 26 65 33

67 33 67 40 67 33 67 40

69 41 69 50 69 41 69 50
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Pollen, Nectar and Flower Sampling Leaf Sampling

Year 1

(TRTDA & B)

Year 2

(TRTDA)

Year 1

(TRTDA & B)

Year 2

(TRTDA)

BBCH* DAA* BBCH DAA BBCH DAA BBCH DAA

NT015

60 13 60 14 60 13 60 14

61 20 61 21 61 20 61 21

65 26 65 28 65 26 65 28

67 33 67 35 67 33 67 35

69 41 69 43 69 41 69 43

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

= days after last application

Clover Plant Matrices. Samples of white clover leaves, blossoms, nectar (hive-collected), and pollen (hive

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

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

flowering declining). For trials NT014-12HA and NT015-12ZA, two bee-tight, ventilated mesh-covered 

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

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

of clover blooming. Normally developed, apparently healthy and queen-right honey bees, Apis mellifera

(purchased from local sources), were used to collect the clover pollen and nectar samples. One bee colony 

housed in a standard 10 frame Langstroth hive was placed in each tent 3-4 days prior to sample collection. 

The colonies were equipped with pollen 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 3-4 

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 (up to 40-mL), and the samples 

were individually placed in 40-mL amber glass containers.

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

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

collected from the surface of the plots (6 inches [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. 
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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.

Table 4. Limits of quantification and detection for imidacloprid and metabolites
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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 cotton flower and leaf 

matrices and for at least 1281 days (42 months) in soil matrices prior to analysis.

Transit Stability. To ensure that imidacloprid and its metabolites 5-hydroxy imidacloprid and imidacloprid

olefin were stable in nectar and pollen, sample sets of two pollen unfortified 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 cotton 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 was 621

days for cotton flowers, 595 days for cotton leaves, 413 for cotton nectar, and 544 days for cotton pollen. 

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

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

storage times are acceptable.

Transit Stability. 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, flower, 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 

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

floral nectar are shown in Table 6. These statistics in Table 6 reflect analysis of individual composite 

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

Plots of the raw data for each trial are found in Appendix C. Since IMI-olefin and 5-OH IMI are considered 

of similar toxicity as parent IMI, this discussion focuses on total IMI residues (sum of IMI, IMI-olefin and 5-
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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 cotton extrafloral nectar range from 

approximately 3.4 – 11 ppb while yearly maxima range from 21– 30 ppb. Results for floral nectar are 

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

4.3 to 16.2 ppb and yearly maxima ranging from 10.7 to 39.5 ppb. Notably, the highest mean (and 

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

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

trial NT014 (19%). This suggests that the greater accumulation in nectar in trials NT013 and NT015 may 

be due to their coarser soils. 

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

Extrafloral Nectar (ppb)

Trial NT013* Trial NT014* Trial NT015*

TRTDA TRTDB TRTDA TRTDB TRTDA TRTDB

2012 2013 2012 2012 2013 2012 2012 2013 2012

Mean 4.1 11 3.4 4.2 5.1 4.2 4.2 7.1 2.5

Min. 1 2.8 1.8 1.2 1.2 1.2 1.2 2.2 1.2

Med. 3.2 12 3.1 2.4 2.6 1.8 4.2 5.1 2.5

90th 7.5 19 7.2 12 14 11 9.3 11 4.9

Max. 9.1 24 8.9 16 21 16 14 30 6.2

Floral Nectar (ppb)

Mean 5.2 14.4 9.0 5.2 7.4 4.3 9.8 16.2 6.6

Min. 2.4 5.9 2.3 2.0 1.9 1.8 2.7 4.2 3.3

Med. 4.6 11.0 5.0 3.3 2.6 2.6 9.6 11.7 6.0

90th 8.8 26.3 19.9 12.9 18.0 10.1 14.3 29.8 9.9

Max. 11.6 28.9 38.4 15.7 21.9 17.7 19.8 39.5 10.7

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 cotton floral nectar are shown in 

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

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

during 13-20 DAA for year 2 in trials NT014 and NT015. Furthermore, mean daily concentrations of total 

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

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

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

maximum daily average concentration of total IMI in cotton floral nectar is 29.0 ppb. 
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Plots of the daily average residue values of total IMI (and daily ranges) in cotton extrafloral nectar are 

shown in Figure 4. With the exception of the latter sampling dates in Trial NT013, average daily residues 

of total IMI from year 2 are similar to those from year 1. As observed with floral nectar, the mean daily 

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

trail. The maximum daily average concentration of total IMI in extrafloral nectar is 15.7 ppb. 
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Figure 3. Total imidacloprid residues measured in cotton floral nectar for trials NT013, NT014 and NT015

(symbols = mean, error bars = range).
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Figure 4. Total imidacloprid residues measured in cotton extrafloral nectar for trials NT013, NT014 and 

NT015 (symbols = mean, error bars = range).

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

Yearly mean residues of total IMI pollen vary from 1.5 – 6.9 ppb while maxima range from 3.9 – 56.7 ppb. 

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

pollen occurred in year 2 of the TRTDA trials, which were 1.5 – 3X the respective yearly means from year 

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

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

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

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

respectively).

Table 6. Magnitude of total IMI residues in cotton pollen (ppb).

Concentration in Pollen (ppb)

Trial NT013 Trial NT014 Trial NT015

TRTDA TRTDB TRTDA TRTDB TRTDA TRTDB

2012 2013 2012 2012 2013 2012 2012 2013 2012

Mean 2.4 6.9 2.7 4.0 6.1 5.1 1.5 3.0 1.5

Min. 0.9 0.6 0.9 0.6 0.6 0.6 0.6 0.6 0.6

Med. 2.1 2.8 1.7 1.7 0.6 1.9 0.9 0.9 0.9

90th 3.9 16.8 6.3 12.2 19.4 5.2 3.2 6.2 3.5

Max. 5.3 36.0 11.7 17.4 47.4 56.7 3.9 19.4 4.4

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

concentration shown is in bold. 

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

assumed equal to 1⁄2 the LOQ.

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

Figure 5. Generally, average daily residues of total IMI in pollen between 13-20 DAA from year 2 are 

greater than those from year 1 across all three trials. As observed with floral and extrafloral nectar, the 
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mean daily concentrations of total IMI in pollen from year 1 of TRTDA and TRTDB are similar within each 

trail. The maximum daily average concentration of total IMI in pollen is 25.2 ppb. 

Figure 5. Total imidacloprid residues measured in cotton pollen for trials NT013, NT014 and NT015

(symbols = mean, error bars = range).
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Flowers. Across all trials and years, mean residues of total IMI in flowers range from 6.0-13.6 ppb in 2012

and 6.0-10.2 ppb in 2014 (Table 7). No consistent trend is observed between mean residues measured in 

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

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

Table 7. Mean concentrations of total IMI in cotton flowers (ppb).

Treatment (Year)

Residue Trial

NT013 NT014 NT015

TRTDA (2012) 8.8 13.6 8.8

TRTDA (2013) 10.2 6.0 8.4

TRTDB (2012) 9.6 12.0 6.0

6.C. MAGNITUDE OF RESIDUES IN LEAVES AND SOIL 

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

Interestingly, mean residues in year 2 (2013) are substantially lower (by 2X-3X) than those measured 

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

concentrations of total IMI in leaves are higher by up to 2X-20X those measured in pollen (Table 8 vs. 

Table 6).

Table 8. Mean concentrations of total IMI in cotton leaves (ppb).

Treatment (Year)

Residue Trial

NT013 NT014 NT015

TRTDA (2012) 29.3 54.0 27.8

TRTDA (2013) 16.4 14.6 12.0

TRTDB (2012) 28.2 41.4 17.8

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

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

during years 1 and 2, the concentration of IMI in soil increases from 0.3-12 ppb (year 1) to 24-45 ppb prior 

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

pesticide carryover could not be determined immediately after applications.

Table 9. Magnitude of IMI residues in soil (ppb).

Year Date Event DAFA1 Mean IMI in Soil 

TRTDA (ppb)2

Mean IMI in Soil 

TRTDB (ppb)2

NT013

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

1 7/9/12 – 7/30/12 Foliar applications 61-82

1 NA End of 1

st growing season NA NA NA

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

2 6/27/13 – 7/18/13 Foliar applications 414-435

2 11/21/13 End of 2

nd growing season 561 79 23

NT014

1 05/10/12 Prior to planting -14 5 2

1 7/5/12 – 7/27/12 Foliar applications 42-64
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Year Date Event DAFA1 Mean IMI in Soil 

TRTDA (ppb)2

Mean IMI in Soil 

TRTDB (ppb)2

1 NA End of 1st growing season NA NA NA

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

2 6/27/13 – 7/18/13 Foliar applications 399-420

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

NT015

1 5/21/12 Prior to planting -1 0.3 0.3

1 7/3/13 – 7/26/13 Foliar applications 42-65

1 NA End of 1st growing season NA NA NA

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

2 7/3/13 – 7/24/13 Foliar applications 407-428

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

1 DAFA = Days After First Application in Year 1

2 Mean of 3 soil composite samples for IMI only

6.D. YEAR TO YEAR ACCUMULATION IN POLLEN, NECTAR, AND SOIL

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

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

and 6 and Figures 4-6. Based on yearly mean values of total IMI in floral and extrafloral nectar, year 2 

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

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

residues of total IMI occurred sooner after application (13-20 days) across the trials. Interestingly, the two

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

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

contained mostly silt (1.2-1.5X). It is not certain whether this differential increase is related to differences 

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

42 ppb). 

It is clear from Table 9 that IMI residues in soil measured prior to planting in year 2 (24-45 ppb) are 

elevated compared to those measured prior to planting in year 1 (0.3-12 ppb). This could explain some of 

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

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

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

may also contribute to these observed differences. 

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

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

determined via linear regression of total IMI residues (log transformed) vs. time (days after last pesticide 

application). The resulting DT50 values were determined for those trial years with 4 or more sampling 

days with residues measured above the LOD. Results are shown in Table 10. Plots of the residue decline 

determinations are shown in Appendix D. For residues of total IMI in cotton floral nectar, DT50 values 

vary from 19-68 days while those for extrafloral nectar vary from 27 – 373 days. With floral and 

extrafloral nectar, 14/16 of the DT50 values of total IMI are between 19 and 51 days. The large DT50 value 
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of 373 days for year 2 of trial NT013 is unusual among the cotton trials in that concentrations of total IMI 

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

With pollen, most of the trials contained insufficient data for reliable determination of DT50 values (<4 

sampling points with detectable residues of total IMI). Three DT50 values for total IMI in pollen varied from 

14 to 58 days. The negative DT50 values of -450 represents slightly increasing concentrations of total IMI 

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

reliable than other DT50 values.

Trial Trial Year DT50 (days) r

2 P-Value

Floral Nectar

NT013 2012A 32 0.75 <0.001

2013A 39 0.65 <0.001

2012B 19 0.67 <0.001

NT014 2012A 44 0.32 0.003

2013A 24 0.86 <0.001

2012B 41 0.32 0.004

NT015 2012A 50 0.43 <0.001

2013A 33 0.50 <0.001

2012B 68 0.37 0.001

Extrafloral Nectar

NT013 2012A 28 0.63 <0.001

2013A 373 0.00 0.75

2012B ND

NT014 2012A 27 0.52 <0.001

2013A 27 0.71 <0.001

2012B ND

NT015 2012A 27 0.65 <0.001

2013A 33 0.61 <0.001

2012B 51 0.31 0.01

Floral Pollen

NT013 2012A -450 0.00 0.83

2013A 14 0.76 <0.001

2012B ND

NT014 2012A 25 0.33 0.003

2013A ND

2012B 58 0.06 0.25

NT015 2012A ND

2013A ND

2012B ND

ND = not determined because <4 sampling events contained detectable residues.
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6.F. OCCURRENCE OF RELEVANT IMI METABOLITES 

Very few samples contained concentrations of IMI-olefin or 5-OH-IMI above the limits of quantitation in 

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

these two metabolites was not conducted.

6.G. OCCURRENCE OF IMI IN ROTATIONAL CROP (WHITE CLOVER)

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

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

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

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

nectar in trials NT014 and NT015 are 1.6 and 2.7 ppb, respectively. The maximum concentrations of total 

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

6.H. RELATIONSHIP OF RESIDUES AMONG PLANT TISSUES

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

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

day is shown in Figure 6A, 6B, and 6C, respectively. Total IMI residues in floral nectar are predicted within 

2X of measured residues for 36/43 samples using simple linear regression with flower data (r2 = 0.48). 

Total IMI residues in extrafloral nectar are predicted within 2X for 34/42 paired samples (r2 = 0.59). Based 

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

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

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

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

about 1/3 of total IMI residues in pollen are predicted within a factor of 2 of measured values.
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Figure 6. Relationship between total IMI residues in cotton nectar (A), extrafloral nectar (B) and pollen 

(C) to those measured in flowers

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

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

shown in Figure 7A, 7B, and 7C. With flora and extrafloral nectar, predicted concentrations of total IMI 

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

respectively. Overall, total IMI residues in cotton leaves are about 4-5X those in floral nectar and 

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

prediction of total IMI residues in pollen (Figure 7C).
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Figure 7. Relationship between total IMI residues in cotton floral nectar (A), extrafloral nectar (B) and 

pollen (C) to those measured in leaves
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7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS

In the context of documenting the magnitude of IMI residues in bee-related matrices of cotton, the 

following strengths are observed with this study. 

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

2. Concentrations were measured for toxicologically-relevant metabolites in multiple plant matrices

3. Application methods and rates were well documented

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

variability

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

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

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

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

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

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

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

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

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

1. Pre-bloom, foliar applications of approximately 0.06 lb a.i./A x 5 (5-8 day interval) to seed-treated 

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

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

about 13 days following the last application. The maximum daily average concentration of total 

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

2. These same pre-bloom, foliar applications of imidacloprid resulted in a maximum of 56.7 ppb of 

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

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

ppb. 

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

measured after the second year of foliar applications to seed treated cotton were 1.2X-2.9X those 

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

occurred within 13-20 days following the last foliar application. It is not clear if the increase in 

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

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

4. Total IMI residues in pollen were approximately 1-2 orders of magnitude greater than those 

observed in nectar. 

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

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

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

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

6. Two metabolites of IMI (IMI-olefin and 5-OH-IMI) were present below the limits of quantification

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

7. DT50 values for total IMI in floral and extrafloral nectar varied from 19-68 days, except for one trial 

year where residues were relatively constant over the 40-d sampling period. Three DT50 values for 

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

relatively constant concentrations of total IMI over time.

8. The predictability of total IMI residues in floral nectar and extrafloral nectar based on measured 
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concentrations in other plant matrices (flowers, leaves) is fair, with most of the predicted values 

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

about 2X those measured in nectar (floral and extrafloral) while those in leaves were about 4-5X 

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

measured concentrations in flowers and leaves was poor.

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

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

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

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

8. STUDY VALIDITY/CLASSIFICATION 

Data from the three trials (NT013, NT014, NT015) are considered scientifically sound and useful for risk 

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

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

quantitative use in risk assessment.
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Appendix A. Details of Imidacloprid Application Rates and Timing
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Appendix B. Location of Cotton Residue Trials.

Figure B1. Locations of Trial NT013 (Maiden, MO), NT014 (Glennonville, MO) and NT015 (Fisk, MO)

NT013

NT014

NT015
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Appendix C. Plots Of Raw Data For Total IMI In Cotton Floral Nectar, Extrafloral Nectar And 

Pollen.

Figure C-1. Total imidacloprid concentrations in cotton floral nectar for TRTDA and TRTDB trials
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Figure C-2. Total imidacloprid concentrations in cotton extrafloral nectar for TRTDA and TRTDB trials
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Figure C-3. Total imidacloprid concentrations in cotton pollen for TRTDA and TRTDB trials
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Appendix D. Plots of Total IMI Residue Decline Curves for Cotton

Figure D-1. Residue decline curves for total IMI in cotton floral nectar
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Figure D-2. Residue decline curves for total IMI in cotton extrafloral nectar
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Figure D-3. Residue decline curves for total IMI in cotton pollen (Trials NT013 and NT014 only)