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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 4 2.9 % 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, Oak land, 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)

Umatil l a, FL (NT005)

Umatil l a, 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 fol iar 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

. S amples 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 v alidate analyte recovery.

Results indicate p 

### re

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

- olefin and 5

- OH

- IMI) in individual composite nectar samples of up to 4 3 0 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 subst antially over time, such that mean residues at or near 10 days post application are typically below 100 ppb (excluding Trial NT004).

T otal IMI residues in pollen were approximately 10X those observed in nectar, and generally did not decline at the rate o bserved 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 DT 50 

### values of total IMI in nectar from Trials NT005 and NT006 ranged from 9

- 14 days. Only 1 DT 50 

### 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

- o lefin 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 co nditions is uncertain. Variability in sampling times and inadvertent pesticide application in Trial NT004 greatly limits the ability to assess year

to

- 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: D ata 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

- 88 o 

### F) Sample
Storage:

- 25 o 

### F to 35 o 

### F

5A. S TUDY 

### D ESIGN



### 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 Protectan t 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

soi l 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 differen t 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 composit e 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 cit r us 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.

5 B

. A PPLICATION 

### T IMING AND 

### R ATES



### 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 1 st 

### and 2 nd 

### applications was 8

- 10 days. In addition, a reference site (untreated) was also included for each of the three trials. A ll applications we re 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 P ROVADO , 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, P REY , 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 imid acloprid 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

.

Inf ormation on application timing is shown in the T able 1 below.

Table 1 . Summary of foliar application rates and timing

Location Orange

Variety Year Appl.

No. Appl. Date

(DAFA/Stage) 1 DAFYA 2 Individual

Rate (lb a.i./A) Total Rate

(lb a.i./A)

Trial NT004

- 12ZA

Bithio, FL Valencia 2012 1 2/18/ 20 12 ( 0/ BBCH 51) NA 0.252

0.50 2 2/26/ 20 12

( 8/ BBCH 56) 8 0.251

Bithio, FL Valencia 2013 1 1/16/ 20 13 (333 / BBCH 31

- 61) NA 0.254

0.51 2 1/26/ 20 13 (343 / BBCH 31

- 61) 10 0.254

Trial NT005

- 12ZA

Umatilla, FL Valencia 2012 1 2/23/ 20 12 (0 / BBCH 53) NA 0.260

0.52

2 3/2/ 20 12 (8 / BBCH 59) 8 0.255

Umatilla, FL Valencia 2013 1 1/29/ 20 13 (341 /BBCH 55) NA 0.257

0.51

2 2/8/ 20 13 (351 / BBCH 57) 10 0.257

Trial NT006

- 12ZA

Umatilla, FL Hamlin 2012 1 2/23/ 20 12 (0 / BBCH 53) NA 0.257

0.51

2 3/2/ 20 12 (8 / BBCH 56) 8 0.257

Umatilla, FL Hamlin 2013 1 2/8/ 20 13 (351 / BBCH 51) NA 0.258

0.52

2 2/18/ 20 13 (361 / BBCH 56) 10 0.259

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

. 2 DAFYA = days after 1 st yearly application

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### 5C. S TUDY 

### S ITE 

### L OCATION AND 

### C HARACTERISTICS



### 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 wer e similar among the three study sites/trials. T he variety of orange differed between NT004/ NT 005 and N T006 (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 stud y 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 18750ft 2 29687.5 ft 2 6336 ft 2 23328 ft 2 5760 ft' 23328 ft 2
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 NT 004 were most different from the other two trials ( NT 005 and NT 006). 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. 3 7.7 and 39.9 inches in trial NT005 and 006, respectively

. Temperature and precipitation records were identical betwee n 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. S AMPLE 

### C OLLECTION 

### , H ANDLING 

### , P ROCESSING



### 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 containe d 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 (DA L A). Exceptions are trial N T006 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 floweri ng, 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 lea fless 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 gr owing 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 d ays 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 = B iologische Bundesanstalt, B undessortenamt und CH emische 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).

Contr ol 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 pro tected 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 th e 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. A NALYTICAL 

### M ETHODS



### The res idues 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 isotopic ally

labeled internal standards. Details of the analytical methods are provided in the study report.

The limit of detection (LO D) for the total residue is the highest LO D value for an individual analyte in a particular matrix. The limit of quantification (LOQ) for the total residue is the highest LOQ value for an individual analyte in a particular matrix. The 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. Q UALITY 

### A SSURANCE



### Storage Stability

. Storage stability studies and transit spikes indicate that the imidacloprid residues would have been stable during frozen storage for at least 1080 days (36 months) in citrus matrices and for at least 1281 days (42 months) in soil matrices prior to analysi s.

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 ol efin, accompanied the respective study sample types throughout the sampling, shipping, and storage events of the field phase of the study. They

were analy z ed 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 im idacloprid, 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 fortifica tion 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. Q UALITY 

### A SSURANCE 

### R ESULTS



### 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 c itrus 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 indica te 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 (poll en, nectar, flower, leaf, soil), t he overall mean of the recoveries for each matrix was within the acceptable range of 70 to 120%, and the standard devia tion values were below 20%.

6. B

. M AGNITUDE OF 

### R ESIDUES IN 

### B EE



### - R ELEVANT 

### M ATRICES



### Nectar. Summary statistics of t he overall magnitude of IMI and related metabolites in citrus

nectar is shown in Table 6

. These statistics reflect analysis of individual c omposite 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

- IM I). M ean 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 90 th 

### 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 app lications 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

NT0 04 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 I MI 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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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%

---

N OTES : 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 va lues of total IMI in pollen range from
2,000

- 3,200 54

- 85 ppb while the upper 90 th 

### 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 sug gests 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 1/5 th 

### (trial NT004) to 1/3 rd 

### (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 leve ls 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% 9 1 % 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

. M AGNITUDE OF 

### R ESIDUES IN 

### L EAVES AND 

### S OIL



### 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 40 1 5110 5594

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

Min. 22.4 26.4 21 3 262 67.9 14 3 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 DAFA 1 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

. Y EAR TO 

### Y EAR 

### A CCUMULATION IN 

### P OLLEN AND 

### N ECTAR



### 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 ye ars).

T his 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). F irst, data from Trial NT004 may be compromised by the inadvertent application of I MI 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, i t is also possible that the wide range of citrus growth stages upon application in 2013 (BBCH3 1

- 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. T his difference in sampling times likely reflects slower citrus flower

d evelopment during 2013 vs. 2012. While pollen and nectar sampling times were similar in terms of the citrus growth stages between 2012 and 2013, the differe nces 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

. R ESIDUE 

### D ECLINE 

### (DT 50 

### ) IN 

### P OLLEN AND 

### N ECTAR



### Nectar. The decline in total IMI residues in pollen and nectar can be described in terms of a dissipation half life (DT 50 

### ). The DT 50 

### values were calculated from individual composite samples of nectar for Trials NT005 and NT006 (Trial NT004 was excluded from analy sis due to the potential influence of additional pesticide applications on residue kinetics; Figure 3 A&B ). The three nectar DT 50 

### values range from approximately 9

- 14 days. Correlation coefficients (r 2 

### ) 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 4 A and 4B for Trials NT005 and

NT006 , respectively . Only one data set was considered marginally sui table for DT 50 

### calculation (Trial NT006, 2013 with a DT 50 

### of approximately 16 days.) DT 50 

### 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 fi t 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 flowerin g 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 b e 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 DT 50 

### 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

. O CCURRENCE OF 

### R ELEVANT 

### I MI M ETABOLITES



### 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 resi dues 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 additi on to any degradation of

these two metabolites . The fraction of total IMI residues in nectar represented by olefin

- IMI and 5

- OH

IMI 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 olefin

IMI 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

. R ELATIONSHIP OF 

### R ESIDUES 

### A MONG 

### P LANT 

### T ISSUE S



### 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 (r 2 

### = 0.83

, Panel A ). Nectar concentratio ns tended to be under

predicted 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 (r 2 

### =0.54, Panel

B ), particularly at the hi gh 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 8 A and 8 B

, 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 (r 2 

### = 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 (r 2 

### =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 an d 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. Sa mpling 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. Inadve rtent 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 DT 50 

### calculations (generally 4 time points or less).

4. The DT 50 

### values reflect dissipation kinetics associated with a pre

- bloom interval of 10 days. Therefore, application of these DT 50 

### values to other application patterns (e.g., shorter or longer

pre

- bloom intervals) introduces uncertainty.

5. Variability in sampling t imes , 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 fol lowing 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 p pb 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 nec tar 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 res pects 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 i n 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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23 Appendix A. Location of Citrus Residue Trials.

Figure A1. Trial NT004

- 12ZA: Latitude: 28.591364 | Longitude:

- 81.11696 (Bithio, FL)

Figure A 2

. Trial NT005

- 12ZA Latitude: 28.94102 | Longitude:

- 81.64804 (Umatilla, FL)

Figure A 3

. Trial NT006

- 12ZA: Latitude: 28.93859 | Longitude:

- 81.65357 (Umatilla, FL)

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24 Figure A2. Relative location of Study Trials NT004 (Bithio), NT005, NT006 (Umatilla)