MRID 49090501 CDPR IMI Melon

1 Reference
Beedle, E. (2012) Determination of Residues of Imidacloprid and its Metabolites 5

- hydroxy
Imidacloprid and Imidacloprid Olefin in Bee Relevant Matrices Collected from Melons Grown at
Locations Treated with Imidacloprid at Least Once per Year During Two suc cessive Years: Amended. Project Number: EBNTL056/02, EBNTL056/02/1/AMENDED. Unpublished study prepared by Bayer

Cropscience LP and Bee Alert Technology, Inc. 237p. MRID 49090501 , CDPR Study ID 268741 , Data

Volume 51950

- 0811 , Tracking ID# 256591

1. STUDY INFORMATION

Chemical: Imidacloprid PC Code 129099

Test Material: Multiple formulations were
used during this study and

some materials are

unknown

. Percent Active
Ingredient: Various and unkown

Study Type: Non

- Guideline field residue study on melons to establish imidacloprid and

metabolite levels in bee collected nectar, bee collected pollen and leaves in site
locations that have been previously treated with imidacloprid at least once for

two successive years

.

Sponsor: Bayer CropScience

2T.W. Alexander Drive
Research Triangle Park, NC

USA 27709 Experiment Start and

End Date: April 4, 2011

– December 12, 2012

Sponsor Study

ID: EBNTL056

- 02

Study Locations: Ten trial sites located in
Imperial County of

California. Study
Completion

Date: December 27, 2012

GLP Status: Non

- GLP; protocol reviewed by CDPR

. [CDPR study ID 259804 , Data Volume 51950

- 0789 , Tracking ID# 246838 ]

2. REVIEWER INFORMATION

Study Reviewed by:
California Department
of Pesticide Regulation Richard Bireley, Sr. Environmental Scientist (Specialist)

John Troiano, Ph.D., Research Scientist III

Alexander Kolosovich, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)

Russel l Darling, Environmental Scientist

3. EXECUTIVE SUMMARY

Ten trials were conducted in California in soils classified as either “ heavy ” (fin e

- textured), or "medium ”
(medium

- textured) to determine the residu es of imidacloprid and its metabolites (5

- hydroxy
imidacloprid and imidacloprid olefin) in bee collected nectar (hive deposited ), b ee collected pollen
(pollen traps), and leaves (hand coll ected) from melon plants (cucurbits) g rown at locations treated
previously with imidacloprid.MRID 49090501 CDPR IMI Melon

2 Imidaclopri d application rates and application methods for the trial locations during the ye ars of 2008
through 2011 were collected from grower communications

. Individual application rates ranged from

0.23 to 0.38 lb. imidacloprid/acre /application . Applications in 20 11 were made at or near the melon
transplant s

. Within each field site, two plots were established, each with a bee

- tight, ventilated m esh tent (24 ft. x
100 ft. x 10 ft. tall) for sample collection. One normally developed, apparently healthy and queen

- righ t honey bee colony was placed in each tented area shortly after the beginning of flowering when enough
blossoms were open to allow orientation and foraging of the bee s.

Leaves (hand collected), b ee co llected nectar (hive deposited) and pollen (pollen trap s ) were collected

from each plot. Composite n ectar samples were collected by syringe at several sampling intervals over
several days. Sampling was from several cells of a brood frames, as no nectar was stored in an empty
frame placed next to the brood frame. Composite pollen sam p l es were collected from plastic pollen
traps that were mounted at the entrance of each hive for over a period of two to three weeks.
Composite leaf samples were collec ted from each plot near the middle of the nectar and pollen
sampling period.

The residues of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin were quantitated by high performance liquid chromatography/triple stage quadrupole mass spectrometry (L C/MS/MS ) using
stable isotopically labeled internal standards. The individual analyte residues were summed to give a
total imidacloprid residue. The limits of quant itation (LOQs) and limits of detection (LODs) are shown

below in section 5F.

4. STUDY VALIDITY

Guideline Followed: Non

- guideline study (protocol was reviewed by U.S. EPA/PMRA/CDPR)

Guideline Deviations: N/A

Other Deviations: N/A

Classification: Supplemental

Rationale: N/A

Reparability: N/A

5. MATERIALS AND METHODS

Test Material Characterization

Test item: Admire Pro, Alias and

Unknown Formulations pH (24°C) 17.4% A.I.

Description: U nknown Density (20°C) : 1.54 g/mL

CAS #: 138261

- 41

- 3 Solubility: 0.51 to 0.61 g/L

5A. S TUDY D ESIGN

The non

- GLP sampling phase of this study was conducted in April and May, 2011. The GLP experimental
start date (first analysis) was on November 30, 2012, and the experimental end date (last analysis) was

December 12, 2012.

Ten treated field sites in Cali fornia representing two soil categories classified a s either “heavy” (fine

textured) , or “medium ” (medium

- textured) were selected based on previous applications of
imidacloprid according to grower communications. Classification of the soils was obtained fr om the SoilMRID 49090501 CDPR IMI Melon

3 Survey Geographic (SSURGO) Database provided by the Natural Resources Conservation Service. Each
site contained a treated area of at least one acre (from placement of a pair of tents and sample
collection) that was located in a larger field ran ging from approximately 30 acres to more than 100
acres. Individual application rates ranged from 0.23 to 0.38 lb. imidacloprid/a cre/application.

Applications to the sites in 2011 were made at or near the me lon (cucurbits) transplants

.

Shortly after the b eginning of flowering within each melon site, two b ee

- tight, ventilated mesh tents
( each approximately 24 ft. x 100 ft. x 10 ft. tall) were placed in each of the ten selected field sites
covering three rows of established melon plants. Tents were placed be tween April 4, 2011 and April 20,

2011.

One queen

- right honey bee colony, Apis mellifera L. (Hymenoptera, Apidae) was placed in each tent
when enough blossoms were present to allow orientation and foraging of the bees. Colonies were
chosen from nearby colonies on pallets surrounding each field provided by commercial beekeepers to
pollinate th e imidacloprid treated fields. Each colony was elevated on plastic stands to reduce dust
infiltration and placed about 6 feet from the end of the tent. Each colony consisted of at least six frames

of bees housed in a two

- story hive, consisting of a stand ard Langstroth Hive body on the bottom, and
either a shallow or standard hive body as a second story.

The collection of the pollen (pollen traps), nectar (hive deposited), and leaves (hand collected) was
performed at the test site. Sample analysis and re port preparation was performed at Bayer CropScience
located in Research Triangle Park, North Carolina.

All raw data associated with this study are retained along with the protocol, protocol amendments and
the final report under Notebook Number EBNTL056

- 0 2 at locations s pecified by Bayer CropScience.

5B. A PPLICATION T IMING AND R ATES

Table 1

. Application History aField NumberFieldIdentificationLocation(City, State,NAFTA Region)Soil TypebApplicationMethodbYearDateRate (lb. a.i./A)c

1 NT209 Imperial

County,

CA, NAFTA

Region 10 Holtville Silty
Clay (Heavy) Injected 2011 Jan 10 0.36

None 2010 NA d NA
Injected 2009 Oct 30 0.36
Injected 2008 Oct 5 0.31

2 NT210 Imperial

County,

CA, NAFTA

Region 10 Holtville Silty
Clay (Heavy) Injected 2011 Jan 10 0.36

None 2010 NA NA
Injected 2009 Oct 30 0.36
Injected 2008 Oct 5 0.31

3 NT201 Imperial

County,

CA, NAFTA

Region 10 Holtville Silty
Clay (Heavy) Injected 2011 Jan 7 0.29

Injected 2010 Jan 25 0.29

None 2009 NA NA

None 2008 NA NAMRID 49090501 CDPR IMI Melon

4Field NumberFieldIdentificationLocation(City, State,NAFTA Region)Soil TypebApplicationMethodbYearDateRate (lb. a.i./A)c

4 NT202 Imperial

County,

CA, NAFTA

Region 10 Meloland

Very Fine
Sandy Loam
(Medium) Injected 2011 Jan 10 0.29

None 2010 NA NA
Injected 2009 Jan 24 0.29

None 2008 NA NA

5 NT203 Imperial

County,

CA, NAFTA

Region 10 Holtville Silty
Clay (Heavy) e Injected 2011 Jan 10 0.29

None 2010 NA NA
Injected 2009 Jan 24 0.29

None 2008 NA NA

6 NT208 Imperial

County,

CA, NAFTA

Region 10 Imperial

Glenbar Silty
Clay Loam
(Medium) Seed Line 2011 Jan 3 0.36

None 2010 NA NA

Seed Line 2009 Oct 4 0.30

None 2008 NA NA

7 NT206 Imperial

County,

CA, NAFTA

Region 10 Imperial

Glenbar Silty
Clay Loam
(Medium) None 2011 NA NA

Seed Line 2010 Oct 21 0.28

Seed Line

Seed Line 2009

2009 Nov 21

March 2 0.23

0.28

None 2008 NA NA

8 NT205 Imperial

County,

CA, NAFTA

Region 10 Meloland

Very Fine
Sandy Loam
(Medium) Seed Line 2011 Jan 9 0.38

None 2010 NA NA

None 2009 NA NA

None 2008 NA NA

9 NT204 Imperial

County,

CA, NAFTA

Region 10 Holtville Silty
Clay (Heavy) Seed Line 2011 Jan 3 0.36

None 2010 NA NA

None 2009 NA NA

None 2008 NA NA

10 NT207 Imperial

County,

CA, NAFTA

Region 10 Meloland

Very Fine
Sandy Loam
(Medium) Injected 2011 Jan 20 0.36

None 2010 NA NA
Injected 2009 Jan 20 0.36

None 2008 NA NA

a All rates, methods and dateswere collected from verbal communications with the growers and could not be

confirmed.

b Classification of the soils was obtained from the Soil Survey Geographic (SSURGO) Database provided by the
Natural Resources Conservation Service. “Heavy” class represents soil with slow drainage capacity.

c Applications were made either by injection to a drip issigation system or with a drench application over the seed

line.

d NA

- Per grower i nformation, an imidacloprid application was not made during the year.

e One tent was in Holtville Silty Clay. The other tent in this site was divided between Holtville Silty Clay and
Imperial

- Glenbar Silty Clay Loam. The trial was designated “heavy” since the majority of the sampling was from

Holtville Silty Clay.MRID 49090501 CDPR IMI Melon

5 5C. S TUDY S ITE L OCATION AND C HARACTERISTICS

Table 2

. Trial Site Conditions for Melon Grown at Sites Previously Treated with Imidacloprid

Trial (Field)

Identification Trial Location (County,
State) Soil Types a Rainfall
(in) b Temperature Range (°F) b

1 (NT209) Imperial County, CA Holtville Silty Clay
(Heavy) 0.00 54

- 101

2 (NT210 Imperial County, CA Holtville Silty Clay
(Heavy) 0.00 54

- 101

3 (NT201) Imperial County, CA Holtville Silty Clay
(Heavy) 0.00 54

- 101

4 (NT202) Imperial County, CA Meloland Very Fine
Sandy Loam

(Medium) 0.00 54

- 101

5 (NT203) Imperial County, CA Holtville Silty Clay
(Heavy) c 0.00 54

- 101

6 (NT208) Imperial County, CA Glenbar Silty Clay
Loam (Medium) 0.00 54

- 101

7 (NT206) Imperial County, CA Glenbar Silty Clay
Loam (Medium) 0.00 54

- 101

8 (NT205) Imperial County, CA Meloland Very Fine
Sandy Loam

(Medium) 0.00 54

- 101

9 (NT204) Imperial County, CA Holtville Silty Clay
(Heavy) 0.00 54

- 101

10 (NT207) Imperial County, CA Meloland Very Fine
Sandy Loam

(Medium) 0.00 54

- 101

a Classification of the soils was obtained from the Soil Survey Geographic (SSURGO) Database provided by the

Natural Resources Conservation Service. “Heavy” class re presents soil with slow drainage capacity.

b Meteorological data is from a NOAA station in El Centro, CA, a location in the same vicinity of all trials and covers
the sampling period from April 23, 2011 through May 8, 2011.

c From this site, the location of one tent was in Holtville Silty Clay soil, and the other tent was divided between

Holtville Silty Clay and Imperial

- Glenbar Silty Clay Loam. The trial was designated “heavy” since the majority of the
sampling was from Holtvi lle Silty Clay.

5D. S AMPLE C OLLECTION
, H ANDLING
, P ROCESSING

When each colony was placed in a tent, a frame closest to the edge of the brood frame was replaced

w ith either an empty, fully drawn, new plastic frame or an empty, drawn wax comb; however, no nectar
was stored in these frames. Therefore, hive

- deposited nectar samples were collected from several cells

of a brood frame nearest the brood, taking care not to sample nectar from cells near parts of a comb
containing capped stored honey. Nectar collec tion began one to two days after the hives were placed in
the tents. Sampling occurred between April 23 and May 8, 2011, although the majority of the nectar
samples were collected between May 2 and May 4, 2011.

Pollen collected by the bees was collected in plastic pollen traps that were mounted at the entrance of

the hive. Pollen traps were affixed to the hive and left in the open position at least one day prior toMRID 49090501 CDPR IMI Melon

6 sampling. To collect pollen, the screens were closed. A few, small pollen pellets were avai lable after two
to three days; therefore, a composite sample was collected for each hive which consisted of repeated
visits over a period of two to three weeks.

Composite leaf samples were collected from a minimum of 12 plants distributed throughout the four
quadrants inside the tent. From each plant, a basal, a terminal, and a middle leaf were collected,
yielding a sample size of 36 leaves. Leaf samples were generally collected near the middle of the nectar
and pollen sampling period. Leaves were collect ed on April 26 and 27, 2011 except for two fields which
were collected on May 2 and May 8, 2011.

A chronological listing of significant study dates are given in Appendix 1 of the study report

. Appendix 2
of the study report contains a field report summary for each trial location, the history of imidacloprid
applications and a field report describing all field activities including placement of tents, placement of
hives, sampling, etc.

Composite samples of nectar and pollen were collected in labeled amber glass vials. The vials were
transported to each tent for each sampling event in a cooler with dry ice. New, disposable gloves were
used for each sampling event in each tent to prevent sampling contamination. A new plastic pipette tip

was used for ea ch nectar sample. Pollen samples were transferred from the pollen trap to vials using
forceps. Leaves were placed in plastic

- lined cloth bags. Care was taken to protect all samples from
sunlight before placement in the ice chest.

All samples were placed in an iced chest containing dry ice and the transit (spiked) stability samples. At
the end of each day, all samples, along with the transit stability samples, were transferred to local frozen
storage. All samples, including the transit stabili ty samples, w ere shipped frozen via freezer truck to
Bayer Research Park (BRP) in Stilwell, Kansas, where they were placed together in frozen storage. During
storage at BRP, all samples remained frozen.

Sample Storage

.

The nectar and pollen samples, along with the transit stability samples, were shipped frozen to Bayer
CropScience, Research Triangle Park (RTP), North Carolina, via freezer truck. Homogenization of the leaf
samples was conducted at BRP and shipped frozen via FedEx overnight delivery to RTP.

Freezer s torage stability studies have indicated the imidacloprid residues are stable (<30%
decomposition) for 24 months (728 to 769 days) or freezer storage in the following representative crops:
an oilseed (cottonseed), a non

- oily grain (wheat), a leafy vegetable (lettuce), a root crop (potato), a tree
fruit (apple), and a fruiting vegetable (tomato). 14

- 19 The nectar, pollen, and leaf samples analysed in this
study were held in frozen storage for a maximum of 598 days (20 months) prior to extraction.

Samples re mained frozen at all times at RTP, except during preparation for analysis and the following
exception. During storage at RTP, the pollen and nectar samples and the transit stability samples

reache d room temperature for approximately three days due to freez er failure.

5E. A NALYTICAL M ETHODSMRID 49090501 CDPR IMI Melon

7 The analytical method 11 used in this study measured the resi dues of imidacloprid, 5

- hydroxy
imidacloprid, and imidacloprid olefin in nectar, pollen, and leaves collected from melon plants

. These
data are reported in Appendix 3 of the study report

.

All neat analytical reference standards were stored froz en prior to dilution. All reference s tandard
solutions were prepared in parent equivalents and corrected for purity during initial preparation

. The

reference standard solutions were stored refrigerated (average temperature <5 °C ) an d have been
shown to be stable for the length of storage time required for this study. 12 All reference standard
solutions were prepared in parent imidacloprid equivalents and corrected for purity during initial

preparation

.

Leaf samples were extracted by blending them with 2:1 acetonitrile/water (ACN/H20). The mixture was
amended with magnesium sulfate (MgS0 4 ), sodium chloride (NaCI), and isotopically labeled internal

standards, mixed well, and centrifuged. An aliquot of the supernatant was transferred to a centrifuge
tube containing MgS0 4 and Bondesil

- PSA. The contents of the tube were mixed well and centrifuged.
About aliquot of the supernatant was percolated through a C18 s olid phase extr action (SPE) cartridge
(Bond Elut) into a culture tube. The eluate was evaporated to dryness and the residue was reconstituted
with 1:9 methanol/water (MeOH/H 2 0) for analysis by high performance liquid chromatography/tandem
mass spectrometry (LC/MS/MS).

For nectar, a sample was weighed into a culture tube and dissolved in water. The mixture was amended
with isotopically labeled internals standards, mixed well and applied to a C18 Bond Elute cartridge. The
analytes were extracted from the cartridge with me thanol/water (2:1 v/v). The eluate was evaporated
to dryness and the residue was reconstituted with 1:9 methanol/water (MeOH/H 2 O) for analysis by
LC/MS/MS.

For pollen, a sample was extracted by shaking with water for 10 minutes, adding CAN and shaking fo r an additional 55 minutes. The mixture was amended with isotopically labeled inter nal standards, mixed
well and centrifuged. The supernatant was drawn off, evaporated to an aqueous remainder, amended
with water and applied to a Chem Elute cartridge. The a nalytes were extracted from the cartridge with hexane/ethyl acetate (1:1 v/v). The eluate was evaporated to dryness and the residue was reconstituted
with 1:9 methanol/water (MeOH/H 2 O) for analysis by LC/MS/MS.

Quantitation of each analyte was based on th e daughter ion transitions of the analytes and the respective internal standard analogs. The response of the LC/MS/MS system to each analyte and its

internal standard were measured in samples and in standards and a relative response was calculated as

the r ation of the analyte and the stable isotopically labeled internal standard responses. The relative
response of the analyte in each sample was compared to the relative response of the analyte in the
standards (Appendix 3 of the report study ).

The relative responses of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin in solvent were
measured over the range of 1 ppb to 2000 ppb for each analyte. The coefficients of determination (r 2 )
were calculated using linear regression analysis (XCalibur 2.0 data handling software).

The total imidacloprid residue is the sum of the individual measured residue values of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin in parent equivalents. For the purpose of calculating total
imidacloprid residue s where individual analyte residue values were less than the limit of detectionMRID 49090501 CDPR IMI Melon

8 (LOD), the residues were assigned a finite value of 1⁄2 the value of the respective LOD. This value is only
an estimate of potential residue in a sample, not a measured value.

To conduct method validation, LOD determination and concurrent recoveries, commercial honey was
diluted to approximately 25% sugar content to produce control nectar surrogate, and commercially
obtained bee pollen was used as a surrogate for control pollen.

The analytical method for determining imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin
residue was validated by measuring the recoveries of these analytes from the nectar surrogate, the
control bee pollen, and leaves (control strawberry use d as a surrogate). Nectar and pollen were fortified

at 1 ppb with each analyte and leaves were fortified at 10 ppb . Recoveries from the fortified control
samples were corrected for any apparent residue in the associated controls before the method

efficienc y was conducted. 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 were measured with e ach set of samples to verify method
performance.

5F. Q UALITY A SSURANCE R ESULTS

The responses of the LC/MS/MS system to imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin
were linear in solvent over the range of .5 ppb to 30 ppb for imidacloprid, 5

- hydroxy imidacloprid, and
imidacloprid olefin. The coefficients of determination (R 2 ) were >0.99

.

Average control interferences in the nectar, pollen, and leaves were less than their respective
determined Limits of Quantitation (LOQs) for each analyte

. All recoveries were corrected for any

interferences in corresponding controls. The overall mean of the recoveries of each analyte from each

matrix was within the acceptable range of 70% to 110%. All percent standard deviation (%SD ) value s

we re below 20%.

The limit of quantitation LOQ is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values
assigned to the individual analytes for a p articular matrix.

The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be determined
to be statistically different from a blank. The LODs were determined from method validation data
obtained from control samples fortified at the respective analyte LOQs. The LODs were calculated by
multiplying the standard deviation of recovery measurements at the LOQ by t 0.99 [where t 0.99 is the one

tailed t

- statistic at the 99% confidence level for the number of replicates (n)]. 1 3 The LOD for the total
imidacloprid residue in each matrix is the highest LOD value of any one individual analyte for that

particular matrix.

Summary of LOQs and LODs

Matrix Analyte LOQ (ppb , parent equivalents) LOD (pp b , parent equivalents)

Melon Nectar Imidacloprid 1.0 0. 03

5

- hydroxy Imidacloprid 1.0 0. 79
Imidacloprid olefin 1.0 0. 57
Total Imidacloprid 1.0 0. 79

Melon Pollen Imidacloprid 10 1. 7MRID 49090501 CDPR IMI Melon

9 5

- hydroxy Imidacloprid 10 2.2
Imidacloprid olefin 10 5.0
Total Imidacloprid 10 4.6

Melon Leaves Imidacloprid 10 2.2

5

- hydroxy Imidacloprid 10 3.0
Imidacloprid olefin 10 4.6

Total Imidacloprid 10 4.6

Any analyte (or total) residue measured to be less than the LOQ was reported as less than the LOQ

value. Measured residue values between the LOQ and LOD are provided in Appendix 3 of the study

report ; any residue value that was below the LOD was reported as less than the LOD. The total
imidacloprid residue is a sum of the analyte residue values that are greater than the respective analyte

LODs. If the analyte value was less than the LOD, a default value equal to 1⁄2 the LOD was added into the

sum.

6. RESULTS:

In all matrices (nectar, pollen, and leaves) concentrations of total imidacloprid were higher in melons
grown in medium soil than in fine soil. Melons grown in coarse soil were not included in this study. The
maximum total residues in melon pollen were mea sured to be 12 ppb (mean ± SD, <10 ± 2.8 ) and 32 ppb
(mean ± SD, 13 ± 8.6 ) grown in fine and medium

- textured soils, respectively. The maximum total
residues in melon nectar were measured to be 5.3 ppb (mean ± SD, 3.0 ± 1.5 ) and 8.0 ppb (mean ± SD,

3.9 ± 2. 5) grown in fine and medium

- textured soils, respectively. The maximum total residues in melon

leaves were measured to be 28 ppb (mean ± SD, 16 ± 6.7 ) and 71 ppb (mean ± SD, 27 ± 25 ) grown in fine

and medium

- textured soils, respectively. Summary statistics for concentration of total imidacloprid in
melon nectar, pollen and leaves are contained in Table 3.

The imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin residue data for bee collected nectar
(hive deposited), bee collected pollen (pollen tra ps), and leaf samples are provided in Table 4 through

Table 6 . The total imidacloprid residue data are summarized below.

Table 3

. Magnitude of total imidacloprid residues in melon pollen, nectar and leaves.

Commodity Soil Texture

Category a Total Imidacloprid Residue Levels (ppb) b

N Min Max Highest

Avg

. Site

Residue Median Mean Standard

Deviation

Bee collected
(hive deposited)

melon nectar Fine 10 1.2 5.3 3.9 2.4 3.0 1.5

Bee collected
(hive deposited)

melon nectar Medium 10 1.6 8.0 4.9 3.0 3.9 2.5

Bee collected
(pollen traps)

melon pollen Fine 10 <10 12 11 <10 <10 2.8MRID 49090501 CDPR IMI Melon

10 Bee collected
(pollen traps)

melon pollen Medium 10 <10 32 19 <10 13 8.6

Melon leaves Fine 10 <10 28 27 13 16 6.7

Melon leaves Medium 10 <10 71 55 10 27 25

a Classification of the soils was obtained from the Soil Survey Geographic (SSURGO) Database provided by the

Natural Resources Conservation Service and converted to soil texture categories.

b Abbreviations used are as follows: Min is the lowest treated resid ue value; Max is the highest treated residue

value; Median is the geometric median of the treated residue values; Mean is the mathematical average of the
treated residue values; Standard Deviation is the standard deviation for a small population of “N” sam ples.

Table 4. Total Imidacloprid Residue in/on Nectar (Hive Deposited) from Melon Plants Grown in Soil

Previously treated with Imidacloprid. a *Trial IdentificationLocation (County)CropBee Relevant MatricSoil TypeImidacloprid OlefinResidue (ppb)5-HydroxyImidacloprid Residue(ppb)Imidacloprid Residue(ppb)Total ImidaclopridResidue (ppb)

NT209 Imperial,

CA Melon Nectar Heavy <1.0

<1.0 <1.0

2.8 1.5

2.2 2.5

5.3

Average

3.9

NT210 Imperial,

CA Melon Nectar Heavy <1.0

<1.0 <1.0

1.0 1.2

1.0 1.9

2.3

Average

2.1

NT201 Imperial,

CA Melon Nectar Heavy <1.0

<1.0 1.3

<1.0 <1.0

<1.0 2.2

1.2

Average

1.7

NT202 Imperial,

CA Melon Nectar Medium 1.6

1.2 2.7

1.4 1.9

<1.0 6.3

3.5

Average

4.9

NT203 Imperial,

CA Melon Nectar Heavy <1.0

1.2 2.1

<1.0 2.7

<1.0 5.1

2.5

Average

3.8

NT208 Imperial,

CA Melon Nectar Medium <1.0

<1.0 <1.0

<1.0 6.4

<1.0 8.0

1.6

Average

4.8

NT206 Imperial,

CA Melon Nectar Medium <1.0

<1.0 2.0

1.1 <1.0

<1.0 2.6

1.9

AverageMRID 49090501 CDPR IMI Melon

11Trial IdentificationLocation (County)CropBee Relevant MatricSoil TypeImidacloprid OlefinResidue (ppb)5-HydroxyImidacloprid Residue(ppb)Imidacloprid Residue(ppb)Total ImidaclopridResidue (ppb)

2.3

NT205 Imperial,

CA Melon Nectar Medium <1.0

<1.0 <1.0

1.6 1.1

1.5 1.8

3.4

Average

2.6

NT204 Imperial,

CA Melon Nectar Heavy <1.0

1.1 <1.0

1.4 1.2

1.7 1.9

4.2

Average

3.0

NT207 Imperial,

CA Melon Nectar Medium <1.0

<1.0 <1.0

<1.0 1.3

5.7 2.0

7.5

Average

4.7

a See Appendix 3 of the study report for the analytical data summaries used to prepare this summary. Total
imidacloprid residue is the sum of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin residues, in parent

equivalents. Any analyte residue measured to be less than the LOQ wa s reported in this table as less than the LOQ
value (1.0 ppb). Measured residue values between the LOQ and LOD are provided in Appendix 3 of the study

report . The total Imidacloprid residue is a sum of the analyte residue values that are greater than the r espective
analyte LODs. If the analyte value was less than the LOD, a default value equal to 1⁄2 the LOD was added into the

sum. *Table 6 in study report.

Table 5. Total Imidacloprid Residue in/on Pollen (Pollen Traps) from Melon Plants Grown in Soil

Previ ously treated with Imidacloprid. a *TrialIdentificationLocation(County)CropBee RelevantMatricSoil TypeImidaclopridOlefin Residue(ppb)5-HydroxyImidaclopridResidue (ppb)ImidaclopridResidue (ppb)TotalImidaclopridResidue (ppb)

NT209 Imperial,

CA Melon Pollen Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

<1.0

Average

<1

NT210 Imperial,

CA Melon Pollen Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.0

1.2

Average

1.1MRID 49090501 CDPR IMI Melon

12TrialIdentificationLocation(County)CropBee RelevantMatricSoil TypeImidaclopridOlefin Residue(ppb)5-HydroxyImidaclopridResidue (ppb)ImidaclopridResidue (ppb)TotalImidaclopridResidue (ppb)

NT201 Imperial,

CA Melon Pollen Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

<1.0

Average

<1.0

NT202 Imperial,

CA Melon Pollen Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

<1.0

Average

<1.0

NT203 Imperial,

CA Melon Pollen Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

1.1

Average

<1.0

NT208 Imperial,

CA Melon Pollen Medium <1.0

<1.0 <1.0

<1.0 <1.0

2.9 <1.0

3.2

Average

1.8

NT206 Imperial,

CA Melon Pollen Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.3

1.2

Average

1.3

NT205 Imperial,

CA Melon Pollen Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

<1.0

Average

<1

NT204 Imperial,

CA Melon Pollen Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1

1.0

Average

<1

NT207 Imperial,

CA Melon Pollen Medium <1.0

<1.0 <1.0

<1.0 1.5

1.5 1.9

1.8

Average

1.9

a See Appendix 3 of the study report f or the analytical data summaries used to prepare this summary. Total
imidacloprid residue is the sum of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin residues, in parent

equivalents. Any analyte residue measured to be less than the LOQ was reported in this table as less than the LOQ
value (1.0 ppb). Measured residue valu es between the LOQ and LOD are provided in Appendix 3 of the study

report . The total Imidacloprid residue is a sum of the analyte residue values that are greater than the respective
analyte LODs. If the analyte value was less than the LOD, a default value equal to 1⁄2 the LOD was added into the

sum. *Table 7 in study reportMRID 49090501 CDPR IMI Melon

13 Table 6. Total Imidacloprid Residue in/on leaves from Melon Plants Grown in Soil Previously treated

with Imidacloprid. a *TrialIdentificationLocation(County)CropBee RelevantMatricSoil TypeImidaclopridOlefin Residue(ppb)5-HydroxyImidaclopridResidue (ppb)ImidaclopridResidue (ppb)TotalImidaclopridResidue (ppb)

NT209 Imperial,

CA Melon Leaves Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.1

1.2

Average

1.1

NT210 Imperial,

CA Melon Leaves Heavy <1.0

<1.0 <1.0

<1.0 2.3

2.2 2.8

2.6

Average

2.7

NT201 Imperial,

CA Melon Leaves Heavy <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.2

<1.0

Average

1.1

NT202 Imperial,

CA Melon Leaves Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.0

1.1

Average

1.0

NT203 Imperial,

CA Melon Leaves Heavy <1.0

<1.0 <1.0

<1.0 <1.0

1.5 1.2

1.9

Average

1.5

NT208 Imperial,

CA Melon Leaves Medium <1.0

<1.0 <1.0

<1.0 4.4

5.3 5.2

5.8

Average

5.5

NT206 Imperial,

CA Melon Leaves Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 1.0

<1.0

Average

<1.0

NT205 Imperial,

CA Melon Leaves Medium <1.0

<1.0 <1.0

<1.0 <1.0

<1.0 <1.0

<1.0

Average

<1.0

NT204 Imperial,

CA Melon Leaves Heavy <1.0

<1.0 <1.0

<1.0 <1.0

1.7 1.4

2.0MRID 49090501 CDPR IMI Melon

14TrialIdentificationLocation(County)CropBee RelevantMatricSoil TypeImidaclopridOlefin Residue(ppb)5-HydroxyImidaclopridResidue (ppb)ImidaclopridResidue (ppb)TotalImidaclopridResidue (ppb)

Average

1.7

NT207 Imperial,

CA Melon Leaves Medium <1.0

<1.0 <1.0

<1.0 6.0

3.0 7.1

3.6

Average

5.3

a See Appendix 3 of the study report for the analytical data summaries used to prepare this summary. Total
imidacloprid residue is the sum of imidacloprid, 5

- hydroxy imidacloprid, and imidacloprid olefin residues, in parent

equivalents. Any analyte residue measured to be less than the LOQ was reported in this table as less than the LOQ
value (1.0 ppb). Measured residue values between the LOQ and LOD are provided in Appendix 3 of the study

report . The total Imidacloprid residu e is a sum of the analyte residue values that are greater than the respective
analyte LODs. If the analyte value was less than the LOD, a default value equal to 1⁄2 the LOD was added into the

sum. *Table 8 in study report.

7. STUDY VALIDITY/ CLASSIFICATION AND STUDY LIMITATIONS

Classification/Utility for Bee Risk Assessment

. This study is classified as SUPPLEMENTAL primarily due
to the many uncertainties which may have confounded the results and could have caused the reported
residues to be not representa tive of actual field conditions. The study design outlined by the
Department of Pesticide Regulation (DPR ) requested that sites be selected with a preference to sites
receiving soil applications of imidacloprid during two successive years. However, half of the field sites
used in this study did not have sequential applications. In two sites there was only one application in
2011, with no data indicating any previous applications had been made. Additionally, in one site no
pesticide applications were made du ring the study year. If the data is to be used as part of a risk

assessment, then the data should be re

- analyzed taking these variations into consideration: (1) It is
unclear how much of the composite nectar samples was collected before the hives were move d to the
treated field; (2) It is not known how much of the nectar was collected before the bees were exposed to
imidacloprid; and (3) Additional uncertainties exist for crops grown in coarse soil.

Temporal Variability in Residues . Applications were made from January 3 to January 20, 2011 (except
the one site that did not receive any application in 2011), and sampling was conducted from April 23 to
May 8, 2011. This study was not designed for temporal analysis of declining concentrations, but rather,

to pr ovide a snapshot of residue concentrations during flowering.

Spatial Variability in Residues. All ten sites in this study were in Imperial County in Southern California.

All of the sites had similar climatic conditions. Five of the sites had soil with fin e texture and five sites
had soil that was described as medium texture. Coarse soil was not included in this study.

C oncentrations of total imidacloprid were higher in melons grown in medium soil than in fine soil.MRID 49090501 CDPR IMI Melon

15 Pesticide Carryover. The extent to which prior year applications of imidacloprid contributed to year

- to

year carryover was not analyzed by the study authors. There was substantial variability in the
application history but the study authors did not consider application history in their data analysis.
Therefore, the effects of pesticide carryover in melons are unknown.

Additional Limitations. Using bee hives to collect pollen and nectar samples appeared to be
inappropriate in this study. Both pollen and nectar samples may not reflect the re al concentrations of
imidacloprid and its transformation products in plant nectar and pollen. Nectar was collected from
brood cells where the source of nectar cannot be confirmed (although care was taken to not sample
nectar from cells near any parts of a comb containing capped, stored honey). Pollen was collected in
pollen traps for a long period of time when degradation of imidacloprid may occur.

Melon flowers are considered to be attractive to bees. However, there appeared to have insufficient

amount nutritional sources to support the hive development in this study. All 20 colonies in this study
were under severe stress and continually shrank while colonies were in the tents. No nectar storage was

observed in new frame inserted in the hives that was d esigned to collect nectar from. The small
amounts of nectar observed in bee stomachs confirm very low level of foraging on the test crops and
low nectar collection activity. In addition only a few pollen loads were found in pollen traps even during
couple of days. Degradation of imidacloprid could not be excluded in pollen samples that were
combined from collection during multiple days, over a period of two to three weeks.

8. CONCLUSION

Ten trials were conducted in California in soils classified as either “heavy (fine

- textured)”, or “medium
(medium

- textured)” to determine the residues of imidacloprid and its metabolites (5

- hydroxy
imidacloprid and imidacloprid olefin) in bee collected nectar (hive deposited), bee collected pollen
(pollen traps), and leaves (hand collected) from melon plants (cucurbits) grown at locations treated

previously with imidacloprid.

In all matrices (nectar, pollen, and leaves) concentrations of total imidacloprid were higher in melons
grown in medium soil than in fine soil. The maximum total residues in melon pollen were measured to
be 12 ppb (mean ± SD, <10 ± 2.8 ) and 32 ppb (mean ± SD, 13 ± 8.6 ) grown in fine and medium

- textured
soils, respectively. The maximum total residues in melon nectar were measured to be 5.3 ppb (me an ±

SD, 3.0 ± 1.5 ) and 8.0 ppb (mean ± SD, 3.9 ± 2.5) grown in fine and medium

- textured soils, respectively.

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