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Reference

Murphy, I., Bowers, L., Dyer, D., and Jerkins, E. (2014). ADMIRE PRO - Magnitude of the Residues of 

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

Matrices Collected from Citrus Trees following Foliar Applications of Imidacloprid Over Two 

Successive Years. Bayer Crop Science, Research Triangle Park, NC. MRID 49521301

1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material Gaucho 600 FL Admire Pro 

Systemic Protectant (SC)

Purity 42.9% a.i. w.w.

Study Type: Non-Guideline field residue study on citrus (orange) to establish imidacloprid and 

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

annual foliar 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 66085

(Trial NT004-12ZA): Florida Pesticide Research, 

1810 Deleon St, Ovliedo, FL 32765 

(Trial NT005-12ZA): Eurofins Agroscience 

Services, Inc. 940 W. Oakland Ave, Oakland, FL 

34787 (ID: S12-00586-01)

(Trial NT006-12ZA): Eurofins Agroscience 

Services, Inc. 940 W. Oakland Ave, Oakland, FL 

34787 (ID: S12-00586-02)

Sponsor Study 

ID:

EBNTY007

Study 

Completion 

Date:

21 November 2014

Experiment 

Start/End Date:

18 February 2012 – 18 

August 2014

Study Location: 3 Field Trials:

Bithio, FL (NT004)

Umatilla, FL (NT005)

Umatilla, FL (NT006)

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

2. REVIEWER INFORMATION

Primary Reviewer:

Signature: 

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

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

Date: 1/28/2016

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 citrus trees following two 

pre-bloom foliar applications of 0.25 lb a.i./A per year (current U.S. label rate). Three separate 

trials were conducted in Florida citrus groves (NT004, NT005 and NT006), each with a treated 

(TRTD) and untreated control (UTC) plot. Each year, two foliar applications (0.25 lb a.i./A x 2) 

were made, targeting a pre-bloom interval (PBI) of 10 days (after the second application). The 

retreatment interval ranged from 8-10 days. Trial NT004 experienced multiple additional 

pesticide applications containing imidacloprid prior to and during the experimental trial. 
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Therefore, results from this trial may not accurately reflect residues associated with the 

experimental applications. Trials NT005 and NT006 appear to be located on adjacent fields. 

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 pre-bloom, foliar application of approximately 0.25 lb a.i./A x 2 results in 

residues of total IMI (sum of IMI, IMI-olefin and 5-OH-IMI) in individual composite 

nectar samples of up to 430 ppb and 4,100 ppb in pollen (excluding Trial NT004 because 

of inadvertent IMI applications). Mean residues of total IMI in nectar varied from 76 to 

125 ppb and that for pollen varied from 1,800 to 2,200 ppb (excluding trial NT004). 

Total IMI residues in nectar decline substantially over time, such that mean residues at 

or near 10 days post application are typically below 100 ppb (excluding Trial NT004). 

Total IMI residues in pollen were approximately 10X those observed in nectar, and 

generally did not decline at the rate observed with nectar. This may reflect differences 

in the timing and duration of nectar vs. pollen production by citrus (i.e., production of 

nectar over a longer time period vs. pollen). Three DT50 values of total IMI in nectar from 

Trials NT005 and NT006 ranged from 9-14 days. Only 1 DT50 could be calculated for 

pollen (16 days) because other trials showed little or no dissipation in pollen or had too 

few data points.

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

IMI residues in pollen and nectar. Total IMI residues in flowers appear to be most 

predictive of nectar concentrations, although the extent to which this relationship holds 

for other weather/soil conditions is uncertain. Variability in sampling times and 

inadvertent pesticide application in Trial NT004 greatly limits the ability to assess yearto-year accumulation of IMI residues in plant tissues.

4. STUDY VALIDITY

Guideline Followed: Non-guideline study (protocol was reviewed by EPA/PMRA/CDPR)

Guideline 

Deviations: N/A

Other Deviations: Multiple, inadvertent applications of imidacloprid prior to and during 

the NT004 trial renders the results of that trial of questionable utility 

for relating foliar IMI applications to residues in pollen and nectar

Classification: SUPPLEMENTAL

Rationale: Data from Trial NT004 are not considered suitable for risk assessment 

purposes due to inadvertent imidacloprid applications. Data from the 

other two trials (NT005 and NT006) are considered scientifically 

sound and useful for quantitative use in risk assessment, although 

these trials are highly similar in many respects do to their close 

(adjacent) proximity.

Reparability: None.
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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: 42.9 %. w/w

Lot No./Batch No. Batch No. NK41BX0170 Density: 1.41 – 1.54 g/mL

Material Source: Bayer Crop Science Cert. # 212PG1962

Material Receipt 

Date:

Not reported Analysis 

Date:

03/30/2012

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

Storage of Test 

Mat’l:

Ambient (35-88oF) Sample 

Storage: -25oF to 35oF

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 citrus trees following two 

pre-bloom foliar applications per year of ADMIRE PRO Systemic Protectant containing 550 g/L 

imidacloprid (IMI). 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 year, two foliar applications (0.25 lb a.i./A x 2) were made, targeting a pre-bloom 

interval (PBI) of 10 days (after the second application). The retreatment interval ranged from 8-

10 days. Five composite samples of flowers, pollen, nectar and leaves were made at various 

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

samples were collected prior to treatment and at the end of the growing season from each TRTD 

plot/year. Control plots were sampled for all citrus matrices (two composites) and soil (9 

composites) at the same time the TRTD plots were sampled. QA/QC included field and lab 

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

5B. APPLICATION TIMING AND RATES

Two pre-bloom foliar applications of approximately 0.25 lb a.i./A were applied at least 10 days 

prior to bloom in successive years at each of the three treatment sites (total annual application 

rate = 0.5 lb a.i./A). All applications were made with adjuvant (Dyne-Amic, 0.25% to 0.50%) using 

a ground-based Skid Mount Radiarc Sprayer. The volume of application was similar across trials 

and years (data not shown). The interval between the 1st and 2nd applications was 8-10 days. In 

addition, a reference site (untreated) was also included for each of the three trials. All 

applications were made between BBCH growth stages 51 and 59 (BBCH 51: inflorescence buds 
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swelling: buds closed, light green scales visible; BBCH 59: most flowers with petals forming a 

hollow ball), except for year 2 in trial NT004-12ZA. In that year/trial, applications were made 

between BBCH growth stages 31 and 61 (BBCH 31: beginning of shoot growth, axes of shoots 

visible; BBCH 61: beginning of flowering, about 10% of flowers open) in year 2.

Due to an oversight by the Principle Investigator, the plots in trial NT004-12ZA were sprayed 

with PROVADO, an insecticide containing imidacloprid (1.6 lb a.i./gal, corresponding to 0.1 lb 

a.i./acre application), in both 2010 and 2011, which was not known at the time of the 

experiment. Additionally, PREY, another insecticide containing imidacloprid, was used as a 

maintenance pesticide on both the UTRD and UTC plots of this trial in September 2012 and 

September 2013 (single application of 12 fl oz/ac or 0.15 Ib/ac each year). Because of the 

additional imidacloprid added to the plots prior to the study, the residue values are 

presumably higher in the NT004-12ZA than what would be expected from the study 

applications alone.

Information on application timing is shown in the Table 1 below.

Table 1. Summary of foliar application rates and timing

Location Orange 

Variety

Year Appl. 

No.

Appl. Date 

(DAFA/Stage)1

DAFYA2Individual 

Rate 

(lb a.i./A)

Total Rate 

(lb a.i./A)

Trial NT004-12ZA

Bithio, FL Valencia 2012

1 2/18/2012

(0/BBCH 51) NA 0.252

0.50 2 2/26/2012

(8/BBCH 56) 8 0.251

Bithio, FL Valencia 2013

1 1/16/2013

(333 / BBCH 31-61) NA 0.254

0.51 2 1/26/2013

(343 / BBCH 31-61) 10 0.254

Trial NT005-12ZA

Umatilla, FL Valencia 2012

1

2/23/2012

(0 / BBCH 53) NA 0.260

0.52

2

3/2/2012

(8 / BBCH 59) 8 0.255

Umatilla, FL Valencia 2013

1

1/29/2013

(341 /BBCH 55) NA 0.257

0.51

2

2/8/2013

(351 / BBCH 57) 10 0.257

Trial NT006-12ZA

Umatilla, FL Hamlin 2012

1

2/23/2012

(0 / BBCH 53) NA 0.257

0.51

2

3/2/2012

(8 / BBCH 56) 8 0.257

Umatilla, FL Hamlin 2013

1

2/8/2013

(351 / BBCH 51) NA 0.258

0.52

2

2/18/2013

(361 / BBCH 56) 10 0.259

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

2 DAFYA = days after 1st yearly application
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5C. STUDY SITE LOCATION AND CHARACTERISTICS 

Maps showing the locations of the three TRTD study trials are provided in Appendix A. It is 

evident that Trials NT005 and NT006 were located in neighbouring fields near Lake Peal in

Umatilla, FL. Trial NT004 was approximately 60 miles to the southeast near Bithio, FL. Various 

aspects of the study sites are summarized in Table 2. In general, soil type, plot size, row/plant 

spacing were similar among the three study sites/trials. The variety of orange differed between 

NT004/NT005 and NT006 (Valencia vs. Hamlin, respectively). Also, orange trees were 

considerably younger in the NT004 trial (8 years old) vs. NT005/006 (23-28 years). 

Table 2. Summary of study site characteristics.

Attribute Trial NT004-12ZA Trial NT005-12ZA Trial NT006-12ZA

Plot ID UTC TRTD1 UTC TRTD1 UTC TRTD1

Crop Orange Orange Orange Orange Orange Orange

Variety Valencia Valencia Valencia Valencia Hamlin Hamlin

Crop Age 8 8 23 23 28 28

Soil Type Sand Sand Sand Sand Sand Sand

Plot Length 250 ft 237.5 ft 264 ft 324 ft 240 ft 324 ft

Plot Width 75 ft 125 ft 24 ft 72 ft 24 ft 72 ft

Plot Area 18750ft2 29687.5 ft2 6336 ft2 23328 ft2 5760 ft' 23328 ft2

Row Spacing 25 ft 25 ft 24 ft 24 ft 24 ft 24 ft

Plant Spacing 

within Row

12.5 ft 12.5 ft 12ft 12ft 12ft 12ft

GPS Coordinates 

(Latitude, 

Longitude)

(28,5913638, 

-81.11696)

(28.5913638, 

-81.11696)

(28.939596, 

-81.648202)

(28.94102, 

-81.64804)

(28.936156, 

-81.653054)

(28.93859, 

-81.65357)

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

Not surprisingly given its different location, soil and weather conditions at Trial NT004 were

most different from the other two trials (NT005 and NT006). Soil at trial NT004 had about 5X 

more organic carbon (1.9%) than trials NT005 and 006 (0.0.35% each), although overall, all three 

plots can be considered to contain relatively low amounts of organic carbon. The pH was lowest 

in soils at NT004 (6.7) vs. pH 7.2 and 7.0 for soils in trials NT005/006, respectively. 

Total rainfall was slightly higher in trial NT004 (44.7 inches) vs. 37.7 and 39.9 inches in trial 

NT005 and 006, respectively. Temperature and precipitation records were identical between 

Trials NT005 and NT006 due to their adjacent location (Figure 1). All three sites were irrigated 

during the study, with NT004 receiving “1-2 inches per week when no rain occurred” (no further 

data were provided in the study report). Monthly irrigation records for Trials NT005 and NT006 

were similar, ranging from 1 – 3 inches per month. 
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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

5D. SAMPLE COLLECTION, HANDLING, PROCESSING

Plant Matrices. In all trials, five composite samples (separate runs through the plot) of citrus 

flowers for direct analysis, flowers to be processed for nectar and pollen, and leaves were 

collected by hand at each sampling period from plot TRTD in years 1 and 2 (Table 4). Each 

composite sample of flowers contained a minimum of 125 g collected from at least 5 individual 

trees. Each composite sample of leaves contained a minimum of 100 g collected from at least at 

least 5 individual trees. Citrus flowers were collected at four sampling periods, when the citrus 

trees were at growth stages of BBCH 61, 64, 65, and 67 (BBCH 61: beginning of flowering, about 

10% of flowers open; BBCH 67: flower fading, majority of petals fallen), corresponding to 4 to 38 

days after the last application (DALA). Exceptions are trial NT006 year 2, when only BBCH 64 and 

65 flowers were collected, and trial NT004-12ZA year 2, when samples were collected at BBCH 

60 and not at BBCH 64.
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Citrus leaves were collected at six sampling periods, when the citrus trees were at growth stages 

of BBCH 59, 61, 64, 65, 67, and 69 (BBCH 59: most flowers with petals forming a hollow ball; 

BBCH 69: end of flowering, all petals fallen), corresponding to 3 to 49 DAA. The exceptions are 

trial NT004-12ZA, when leaves were collected at BBCHs of 55, 60, 61, 65, 67, and 83 (BBCH 55: 

flowers visible, still closed (green bud), borne on single or multi-flowered leafy or leafless 

inflorescences; BBCH 83: fruit ripe for picking; fruit has not yet developed variety-specific color), 

and trial NT006-12ZA, when no BBCH 61 samples were collected in year 2. Nine soil samples 

were collected prior to treatment and at the end of the growing season per plot per year, except 

in trial NT006-12ZA, when only seven samples were collected prior to the year 2 applications.

It is evident from Table 4 that the citrus developed at different rates across different years and 

trials, such that the days after last application (DALA) varied somewhat for the same BBCH 

stage.

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

NT004

Pollen, Nectar and Flower Sampling Leaf Sampling

Year 1 Year 2 Year 1 Year 2

BBCH* DALA* BBCH DALA BBCH DALA BBCH DALA

-- -- -- -- 55 8 55 3

61 13** 60 10 60 13 60 10

64 16 61 16 61 16 61 16

65 18 65 18 65 18 65 18

67 23 67 23 67 23 67 23

-- -- -- -- 83 31 83 46

NT005

-- -- -- -- 59 3 59 3

61 4 61 12 61 4 61 12

64 6 64 14 64 6 64 14

65 9 65 17 65 9 65 17

67 12 67 20 67 12 67 20

-- -- -- -- 69 17 69 25

NT006

-- -- -- -- 59 3 59 10

61 4 -- -- 61 4 0 0

64 7 64 30 64 7 64 30

65 11 65 38 65 11 65 38

67 14 0 44*** 67 14 67 44

-- -- -- -- 69 17 69 49

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

citrus; DALA = days after last application

** nectar only

*** pollen only

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

per plot per year, except in trial NT006-12ZA, when only seven samples were collected prior to 

the year 2 applications. Soil samples were collected from the surface (0-6 inches [15 cm]) of the 
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plots using a soil auger or probe 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 ft x 200 ft 

[30 x 60 m] area of a 200 ft x 400 [60 x 120 m] ft plot), and locations were separated by 10 feet 

(3 m).

Control Plots. Two composite samples of all citrus matrices and nine composite samples of soil 

were collected from the control plot of each trial at the same sampling periods as for the 

treated samples of their sample type.

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. The flowers to 

be processed for nectar and pollen were processed the same day they were collected. Nectar 

from the floral nectary was removed using a micropipette and placed in a 2-mL glass collection 

vial. Then, the flowers were allowed to dry at room temperature to improve pollen collection. 

Pollen was removed from the anthers of the flower sample by either vacuum aspiration with 

filter tips or by tapping the pollen from the blossom. 

All resulting nectar and pollen samples were labeled and placed in the freezer immediately after 

they were generated. After the pollen and nectar were harvested, the processed flowers were 

discarded. Composite samples were placed into labeled (study number and sample number)

containers for shipment. All soil, flower, and leaf samples were frozen the day of their collection 

from the field, and all nectar and pollen samples were frozen the day of their processing.

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 (LCDs) 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 1080 days (36 months) in 

citrus 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, transit stability samples were evaluated 

using sets of two pollen unfortified controls, four fortified pollen samples, two unfortified nectar 

surrogate controls, and four fortified nectar surrogate samples. 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, except the transit nectar spike set 

from trial NT005-12ZA which was analyzed earlier in August 2013 by oversight, and they 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 citrus 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.
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6. RESULTS:

6.A. QUALITY ASSURANCE RESULTS

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

was 790 days for citrus flowers, 787 days for citrus leaves, 532 days for citrus nectar, 770 days 

for citrus pollen, and 910 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 91%-96% in nectar 

and 86%-98% in pollen.

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

(pollen, nectar, flower, leaf, soil), 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%. 

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

Nectar. Summary statistics of the overall magnitude of IMI and related metabolites in citrus 

nectar is shown in Table 6. These statistics reflect analysis of individual composite samples 

(usually 5/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, IMI-olefin and 5-OH-IMI). Mean residues of total IMI in citrus nectar range 

from approximately 77 – 125 ppb while maxima range from 280-450 ppb. Median values of 

total IMI range from 54-85 ppb while the upper 90th percentiles range from 170-278 ppb. As 

noted earlier in Section 5B, multiple inadvertent applications of pesticide containing IMI 

occurred prior to and during trial NT004. Therefore these residues may be higher than expected 

based on the four foliar applications which occurred during the trial. Notably, however, mean, 

median and maximum concentrations of total IMI in nectar are quite similar between trials 

NT004 and NT006. This suggests that the contribution of inadvertent IMI applications in trial 

NT004 did not result in residues which were elevated beyond the realm of environmental 

realism.

The mean proportion of total IMI as olefin-IMI and 5-OH-IMI in nectar ranged from 4-10% 

across the three trials.

Table 6. Magnitude of IMI and related metabolites in citrus nectar (ppb).

Trial NT004 (n=40)* Trial NT005 (n=40) Trial NT006 (n=27)

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

Mean 9.2 13 102.4 124.3 3.8 6.2 65.7 76.6 5.4 8.5 110.8 125.0

SD 2.8 6.5 88.6 96.7 2.0 3.3 60.7 67.2 2.7 4.5 104.4 109.7

CV 31% 52% 87% 78% 53% 52% 92% 88% 50% 52% 94% 88%

Min. 5.0 5.2 24 36 1.1 2.2 13 17 1.1 1.4 7.6 10

Median 8.1 11 64 85 3.2 5.7 46.0 54 5.9 8.1 71 82
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Trial NT004 (n=40)* Trial NT005 (n=40) Trial NT006 (n=27)

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

75th 11 15 150 170 4.1 6.6 66 76 7.3 11 130 150

90th 14 23 220 250 6.0 9.4 150 170 8.9 14 258 278

Max. 15 29 400 450 10 17 250 280 10 20 410 430

% of 

Total

7.4% 10% 82% --- 4.9% 8.1% 86% --- 4.4% 6.8% 89% ---

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.

*Prey, a pesticide containing imidacloprid, was inadvertently applied for maintenance by the grower in 

trial NT004-12ZA (0.15 Ib/ac in Sept. 2012 and 2013. Provado, containing 0.1 lb a.i./A of IMI, was also 

used on the area of both plots prior to the study, in 2010 and 2011).

Pollen. Mean residues of total IMI in citrus pollen are about 10X those measured in nectar 

(Table 7). Mean residues of total IMI range in pollen from approximately 1,800 – 3,500 ppb

while maxima range from 3,400-7,400 ppb. Median values of total IMI in pollen range from 

2,000-3,200 54-85 ppb while the upper 90th percentiles range from 170-278 ppb. As noted 

earlier in Section 5B, multiple inadvertent applications of pesticide containing IMI occurred prior 

to (and during) trial NT004. Therefore, these residues may be higher than expected based on 

the four foliar applications which occurred during the trial. Notably, however, mean, median 

and maximum concentrations of total IMI in nectar are quite similar between trials NT004 and 

NT006. This suggests that the contribution of inadvertent IMI applications in trial NT004 did not 

result in residues which were elevated beyond the realm of environmental variability.

The mean proportion of total IMI as olefin-IMI and 5-OH-IMI in pollen ranged from 5-8% across 

the three trials.

Table 7. Magnitude of IMI and related metabolites in citrus pollen (ppb).

NT004 (n=13)* NT005 (n=29) NT006 (n=24)

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

Mean 218 191 3055 3455 172 128 1869 2175 126 103 1614 1834

SD 58 78 1944 2056 46 35 585 654 82 68 1250 1386

CV 27% 41% 64% 60% 27% 27% 31% 30% 66% 66% 77% 76%

Min. 110 58 360 520 84 70 800 980 10 7.4 67 87

Median 220 190 2800 3200 170 120 1700 2000 170 140 2000 2250

75th 240 230 4300 4700 210 150 2200 2600 180 150 2300 2600

90th 288 306 5740 6320 232 180 2800 3220 221 181 3330 3700

Max. 320 320 6800 7400 250 210 3000 3400 250 210 3700 4100

% of 

Total

6.3% 5.5% 88% 7.9% 5.9% 86% 6.9% 5.6% 88.0%

*Prey, a pesticide containing imidacloprid, was inadvertently applied for maintenance by the grower in 

trial NT004-12ZA (0.15 Ib/ac in Sept. 2012 and 2013. Provado, containing 0.1 lb a.i./A of IMI, was also 

used on the area of both plots prior to the study, in 2010 and 2011.

Flowers. Mean residues of total IMI in flowers (Table 8) range from 670-740 ppb and are about 

1/5th (trial NT004) to 1/3rd (trial NT005 & 006) those found in pollen but are substantially 

greater than residues measured in nectar. The maximum reported residues of total IMI in 

flowers range from approximately 2,000-3,000 ppb. About 90% of total IMI residues are 
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represented by IMI on average. Although additional IMI applications occurred in trial NT004, 

mean residue levels of total IMI are comparable across all 3 trials and median levels in trial 

NT004 (349 ppb) are about 60% of those recorded in trials NT005 and NT006 (539 and 601, 

respectively).

Table 8. Magnitude of IMI and related metabolites in citrus flowers (ppb).

NT004 (n=40)* NT005 (n=40) NT006 (n=27)

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

Mean 21.4 39.9 606 668 17.9 37.2 629 684 20.2 36.2 682 739

SD 10.8 30.3 642 681 8.7 19.3 461 487 15.4 27.7 677 717

CV 50% 76% 106% 102% 48% 52% 73% 71% 77% 76% 99% 97%

Min. 5.0 10.1 92.0 116 <3.3 6.3 116 135 <3.3 <1.1 36.3 38.5

Median 18.5 30.3 295 349 18.6 34.0 487 539 19.2 35.9 556 601

Max. 56.5 135.8 2789 2981 39.1 90.8 1860 1975 56.9 103 2608 2767

% of 

Total

3.2% 6.0% 91% 2.6% 5.4% 92% 2.7% 4.9% 92%

*Prey, a pesticide containing imidacloprid, was inadvertently applied for maintenance by the grower in 

trial NT004-12ZA (0.15 Ib/ac in Sept. 2012 and 2013. Provado, containing 0.1 lb a.i./A of IMI, was also 

used on the area of both plots prior to the study, in 2010 and 2011.

6.C. MAGNITUDE OF RESIDUES IN LEAVES AND SOIL 

As expected, residues of IMI and related metabolites measured in leaves are highest among the 

plant tissues sampled (Table 9). Mean residues of total IMI measured in leaves varied from 

approximately 6,000 to 8,600 ppb with maxima varying from 18,000 to 34,700 ppb among the 

three trials. Although additional IMI applications occurred in trial NT004, median levels of total 

IMI (3,600 ppb) are about 1⁄2 those recorded in trials NT005 and NT006 (6,600 and 6,200, 

respectively).

Table 9. Magnitude of IMI and related metabolites in citrus leaves (ppb).

NT004 (n=40-59)* NT005 (n=40) NT006 (n=23)

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

Mean 109 358 5526 5994 236 941 7375 8551 249 665 6324 7238

SD 88.3 296 6623 6930 95.4 445 5128 5545 149 401 5110 5594

CV 81% 83% 120% 116% 40% 47% 70% 65% 60% 60% 81% 77%

Min. 22.4 26.4 213 262 67.9 143 1006 1281 45.2 108 465 637

Med. 102 260 3078 3642 221 989 5254 6676 276 671 5187 6208

Max. 670 1530 33300 34720 480 1913 17480 18830 548 1434 16050 18020

% of 

Total

1.8% 6.0% 92% 2.8% 11% 86% 3.4% 9.2% 87%

*Prey, a pesticide containing imidacloprid, was inadvertently applied for maintenance by the grower in 

trial NT004-12ZA (0.15 Ib/ac in Sept. 2012 and 2013. Provado, containing 0.1 lb a.i./A of IMI, was also 

used on the area of both plots prior to the study, in 2010 and 2011.

Residues of IMI measured in soil are low relative to most plant matrices, which is expected given 

the foliar method of pesticide application (Table 10). Levels were highest in Trial NT004, 

although concentrations may reflect the additional inadvertent IMI application which occurred 

only in this trial. Carry over from year to year was not evident, although lack of pre-treatment 
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measurements in two trials (NT005 and NT006) limit the ability to make firm conclusions 

regarding year-to-year accumulation.

Table 10. Magnitude of IMI residues in soil (ppb).

Trial Year Event DAFA1 Mean IMI in Soil (ppb)2

NT004

2012 Prior to treatment -1 92

End of growing season 292 320 *

2013

Prior to treatment 332 110

End of growing season 767 420 *

NT005

2012

Prior to treatment -2 35

End of growing season 312 120

2013

Prior to treatment 340 No data

End of growing season 657 57

NT006

2012

Prior to treatment -1 110

End of growing season 312 54

2013

Prior to treatment 340 No data

End of growing season 657 44

1 DAFA = Days After First Application

2 Mean of 3 soil composite samples for IMI only

*Prey, a pesticide containing imidacloprid, was inadvertently applied for maintenance by the grower in 

trial NT004-12ZA (0.15 Ib/ac in Sept. 2012 and 2013. Provado, containing 0.1 lb a.i./A of IMI, was also 

used on the area of both plots prior to the study, in 2010 and 2011.

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

The temporal pattern of total IMI residues measured in nectar and pollen is shown in Figure 2

for each trial and application year. The mean and range of total IMI concentrations are plotted 

for each sampling day following the second yearly application of ~ 0.25 lb a.i./A IMI. Within an 

application year, total IMI residues in nectar declined with time while residues in pollen 

remained stable (3 trial-years) or declined (3 trial years). 

This study presents a number of limitations in evaluating the occurrence of year-to-year 

accumulation (i.e., an increase in concentrations from year 1 to year 2). First, data from Trial 

NT004 may be compromised by the inadvertent application of IMI on two occasions prior to the 

study and two occasions during the study (Sept 2012 and 2013. The Sept. 2012 application may 

have contributed to the higher concentrations measured in 2013 relative to 2012. Second, it is 

also possible that the wide range of citrus growth stages upon application in 2013 (BBCH31-61, 

Table 1) may have also confounded the results from this year in Trial NT004. Third, pollen and 

nectar were mostly sampled at different time periods relative days after last application (DALA) 

in Trials NT005 and NT006. This difference in sampling times likely reflects slower citrus flower 

development during 2013 vs. 2012. While pollen and nectar sampling times were similar in 

terms of the citrus growth stages between 2012 and 2013, the differences in DALA between 

2012 and 2013 may influence residue levels and confound interpretation of year-to-year 

accumulation. 
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A

B

C

D

E

F

Figure 2. Residues of total IMI in citrus nectar (A-C) and pollen (D-F). Dots = mean, bars = range.

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

Nectar. The decline in total IMI residues in pollen and nectar can be described in terms of a dissipation 

half life (DT50). The DT50 values were calculated from individual composite samples of nectar for Trials 

NT005 and NT006 (Trial NT004 was excluded from analysis due to the potential influence of additional 

pesticide applications on residue kinetics; Figure 3A&B). The three nectar DT50 values range from 

approximately 9-14 days. Correlation coefficients (r2) ranged from 0.6 to 0.85. 

Figure 3. Residue decline curves of total IMI in citrus nectar.

Pollen. The pattern of residue decline in citrus pollen is shown in Figure 4A and 4B for Trials NT005 and 

NT006, respectively. Only one data set was considered marginally suitable for DT50 calculation (Trial 

NT006, 2013 with a DT50 of approximately 16 days.) DT50 values were not determined for other 

trials/years estimates extended well beyond the sampling period due to lack of substantial decline (e.g., 

>300 days) and poor fit of regression lines. 

A

B
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Figure 4. Residue decline curves of total IMI in citrus pollen.

One possible explanation for the apparently greater decline of total IMI residues in nectar vs. pollen relates 

to the temporal dynamics of nectar and pollen formation within the flower. Nectar tends to be produced 

continuously prior to and during flowering while pollen tends to be formed once over a short time period 

(Evert, 2006). Thus, decline in nectar residues may be due (at least in part) to ‘dilution’ of translocated total 

IMI by additional nectar production in addition to whatever degradation may be occurring beyond the 

metabolites measured. Given the persistence of IMI in environmental matrices, the relative stability of total 

IMI residues in pollen (3 out of 4 data sets) may reflect its deposition into pollen over a specific time period.

When interpreting these DT50 data, it is important to consider the fact that Trials NT005 and NT006 reflect 

nearly identical environmental conditions (they were located in adjacent fields), although the citrus 

variety differed. 

A

B
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6.F. OCCURRENCE OF RELEVANT IMI METABOLITES 

Nectar. The pattern of parent IMI and metabolite residues in nectar sampled after the last annual 

application is shown in Figure 5. When considering the absolute values of metabolites/IMI (panels A, C, 

E), a general decline in nectar residues with sampling day can be observed. There appears to be an 

increasing fraction of total IMI as olefin IMI (panel B) or 5-OH-IMI (panel D), but this largely reflects the 

declining contribution of parent IMI (panel F) to the total IMI residues in addition to any degradation of 

these two metabolites. The fraction of total IMI residues in nectar represented by olefin-IMI and 5-OHIMI varied from approximately 5 to 15%. 

A

C

B

D

E F

Figure 5. Occurrence of Olefin, 5-OH, and IMI in citrus nectar (A, C, E) and % of total IMI (B, D, F).
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Pollen. The pattern of parent IMI and metabolite residues in pollen sampled after the last annual 

application is shown in Figure 6. When considering the absolute values of metabolites/IMI (panels A, C, 

E), no consistent pattern in residues in pollen is evident over time across the trials/years. Similarly, no 

consistent pattern is evident in the fraction of total IMI as olefin IMI (panel B), 5-OH-IMI (panel D), or 

parent IMI (panel F) over time. Overall, the fraction of total IMI residues in pollen represented by olefinIMI and 5-OH-IMI varied from approximately 5 to 15%. 

A

C

E

B

D

F

Figure 6. Occurrence of Olefin, 5-OH, and IMI in citrus pollen (A, C, E) and % of total IMI (B, D, F).
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6.F. 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 nectar and pollen on the same sampling day is shown in Figure 7A and 

7B, respectively. Total IMI residues in nectar are predicted within 2X for 20 out of 22 values using simple 

linear regression with flower data (r2 = 0.83, Panel A). Nectar concentrations tended to be underpredicted in Trial NT004 while over-predicted in Trial NT005. For total IMI residues in pollen, the 

relationship to total IMI residues in flowers is weaker than that observed with nectar data (r2=0.54, Panel 

B), particularly at the high and low end of the range. However, 19/22 predictions are within a factor of 2X. 

These results suggest that total IMI residues in citrus flowers may be a useful predictor of residues nectar 

(and to a lesser extent, pollen), at least under the conditions of this study.

Figure 7. 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. The relationship between mean residues of total IMI measured in leaves 

and total IMI measured in pollen and nectar on the same sampling day is shown in Figure 8A and 8B, 

respectively. Total IMI residues in pollen are predicted within 2X for 17 out of 22 values using simple 

linear regression with leaf data, however the overall relationship is weak (r2 = 041, Panel A). For total IMI 

residues in pollen, somewhat greater scatter is seen relative to residues in leaves (Panel B). Only 14 of the 

22 predictions are within 2X of observed nectar concentrations. 

Figure 8. Relationship between total IMI residues in leaves to those in pollen (A) and nectar (B)

Pollen vs. Nectar. The relationship between total IMI residues measured in citrus pollen and nectar is 

shown in Figure 9. A relatively weak relationship is observed (r2=0.52). In two cases, total IMI residues in 

pollen were over predicted by a factor of 10 and under predicted by a factor 3. 

Caution is recommended when interpreting and applying these inter-tissue regression relationships to 

other situations since values here represent means of multiple composite samples and 2 of the 3 trials 

had nearly identical weather and soil conditions.

A

B
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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 citrus, 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 and over time within a season.

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

1. Inadvertent pesticide application for Trial NT004, thereby likely confounding the relationship 

between foliar applications and plant residues. 

2. Locating two trials adjacent to each other, thereby limiting the extent to which differences in 

weather conditions and soil properties could be assessed. 

3. Sampling times are relatively limited for DT50 calculations (generally 4 time points or less). 

4. The DT50 values reflect dissipation kinetics associated with a pre-bloom interval of 10 days. 

Therefore, application of these DT50 values to other application patterns (e.g., shorter or longer 

pre-bloom intervals) introduces uncertainty.

5. Variability in sampling times, lack of data and inadvertent pesticide application in Trial NT004 

greatly limits the ability to assess year-to-year accumulation of residues in plant tissues using this 

study.

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

1. Pre-bloom, foliar application of approximately 0.25 lb a.i./A x 2 result in residues of total IMI in 

individual composite nectar samples of up to 450 ppb (430 ppb if Trial NT004 is excluded) and 

7,400 ppb in pollen (4,100 ppb if trial NT004 is excluded). 

2. Total IMI residues in nectar decline substantially over time, such that mean residues at or near 10 

days post application are typically below 100 ppb (excluding Trial NT004). A 10-d pre-bloom 

interval is currently required for foliar applications to citrus on U.S. labels.

3. Total IMI residues in pollen were approximately 10X those observed in nectar, and generally did 

not decline at the rate observed with nectar. This may reflect differences in the timing and 

duration of nectar vs. pollen production by plants.

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

in pollen and nectar. 

5. Total IMI residues in flowers appear to be most predictive of nectar concentrations, although the

extent to which this relationship holds for other weather/soil conditions is uncertain.

8. STUDY VALIDITY/CLASSIFICATION 

Due to multiple, inadvertent pesticide applications containing IMI, data from Trial NT004 are not 

considered scientifically sound for quantifying the relationship between foliarly-applied IMI and residues 

in plant matrices. These data may be useful for other purposes (e.g., evaluating residue relationships 

among plant tissues). Data from the other two trials (NT005 and NT006) are considered scientifically 

sound and useful for risk assessment purposes, although these trials are highly similar in many respects 

do to their close proximity.
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Overall, this study is classified as SUPPLEMENTAL, as data from Trial NT004 are not considered suitable 

for risk assessment purposes while data from Trials NT005 and NT006 are considered acceptable for 

quantitative use in risk assessment. 

9. REFERENCES

Evert, R.E. 2006. Esau's Plant Anatomy: Meristems, Cells, and Tissues of the Plant Body: Their Structure, 

Function and Development. John Wiley & Sons.
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Appendix A. Location of Citrus Residue Trials.

Figure A1. Trial NT004-12ZA: Latitude: 28.591364 | Longitude: -81.11696 (Bithio, FL)

Figure A2. Trial NT005-12ZA Latitude: 28.94102 | Longitude: -81.64804 (Umatilla, FL)

Figure A3. Trial NT006-12ZA: Latitude: 28.93859 | Longitude: -81.65357 (Umatilla, FL)
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Figure A2. Relative location of Study Trials NT004 (Bithio), NT005, NT006 (Umatilla)