1 

## Appendix D

.

Supporting Information for Honey Bee Risk Assessment

I. Individual Level Toxicity Studies

Acute Contact Toxicity of TGAI to Adult Bees

Bergfield A (2007; MRID 47832102 ). The acute toxicity of sulfoxaflor (purity 96.6 % w/w) to the honeybee ( Apis mellifera ) was determined after contact exposure. Adult worker bees were topically exposed to nominal doses of 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 μg a.i./bee and observed for 72 hours

. In addition, a dilution water control was tested. T here was 7% mortality in the control group with dose

response mortality ranging 3

- 97% in the treatment groups. No sublethal effects were noted at the end
of the test. The 72

- hour LD 50 was 0.379 μg a.i./bee. This study is classified as a cceptable

.

Acute Contact Toxicity of Formulated Product to Adult Bees

Vinall (2010; MRID 47832511 ). The acute toxicity of GF

- 2372 (water

- dispersible granule, 50% w/w sulfoxaflor) to the honeybee ( Apis mellifera ) was determined after contact exposure. Adult worker bees

wer e topically exposed to nominal doses of 0.013, 0.032, 0.08, 0.2 and 0.5 μg a.i./bee and observed for
48 hours . In addition, a wetting agent control and a water control were tested. There was 2% mortality in the wetting agent control, no mortality in the water only control, and dose

- response mortality was observed in the treatment groups ranging 0

- 90% in the treatment groups. No sublethal effects were
noted at the end of the test. The 48

- hour LD 50 was 0.224 μg a.i./bee. This study is classified as

a cceptab le

.

Vinall (2009; MRID 47832419 ). The acute toxicity of GF

- 2032 (suspension concentrate, 22% w/w sulfoxaflor) to the honeybee ( Apis mellifera ) was determined after contact exposure. Adult worker bees were topically exposed to nominal doses of 0.021, 0.047 , 0.103, 0.227 and 0.5 μg a.i./bee and observed
for 48 hours . In addition, a wetting agent control and a water control were tested. There was 6% mortality in both controls, and dose

- response mortality was observed in the treatment groups ranging
8

- 98% in t he treatment groups. No sublethal effects were noted at the end of the test. The 48

- hour LD 50 was 0.130 μg a.i./bee. This study is classified as a cceptable

.

Vinall (2009; MRID 47832418 ). The acute toxicity of GF

- 2032 (suspension concentrate, 22% w/w sulfoxaflor) to the bumblebee ( Bombus terrestris ) was determined after contact exposure. Adult worker bees were topically exposed to nominal doses of 0.01, 0.1, 1, 10 and 100 μg a.i./bee and observed for 72
hours . In addition, a wetting agent control and a water control were tested. There was 6.7% mortality in the water control, 3.3% mortality in the wetting agent control, and dose

- response mortality was observed in the treatment groups ranging 3.3

- 100.0% in the treatment groups. No sublethal effects in

sur viving bees were noted. The 72

- hour LD 50 was 7.554 μg a.i./bee.

Acute Oral Toxicity of TGAI to Adult Bees

Bergfield A (2007; MRID 47832103 ). The acute toxicity of sulfoxaflor (purity 96.6 % w/w) to the honeybee ( Apis mellifera ) was determined after oral exposure. Adult worker bees were exposed to2 nominal doses of 0.0625, 0.125, 0.25, 0.50 and 1.0 μg a.i./bee dispersed in sucrose solution and
observed for 48 hours . In addition, a sucrose solution control was tested. A dose

- respo nse effect on diet consumption was observed beginning at the lowest dose indicating sulfoxaflor was not palatable to the bees. There was 7% mortality in the control group with dose

- response mortality ranging 27

- 100% in the treatment groups. One bee (3%) in each of the 0.125, 0.25 and 0.50 μg a.i./bee treatments exhibited sublethal effects (lying on back, lethargy) at the end of the test, for which death may have been
imminent. The 48

- hour LD 50 was 0.146 μg a.i./bee. This study is classified as a cceptable

.

Vinall S (2009; MRID 47832107). The acute toxicity of the sulfoxaflor degradation product, X11719474 (purity 99.9 % w/w) to the honeybee ( Apis mellifera ) was determined after oral exposure. Adult worker bees were exposed in two separate tests to a nominal dose of 100 μg/bee (48

- hour limit test) and nominal doses of 20, 40, 60, 80 and 100 μg/bee (96

- hour dose

- response test) dispersed in sucrose
solution . In addition, a sucrose solution controls were tested. There was no effect on diet consumption
in either test indicating X11719474 was palatable to the bees. In the 48

- hour limit test, there was 4% mortality in the controls and 28% mortality in the 100 μg/bee treatment . In the 96

- hour dose

- response test, there was no treatment

- related mortality. No sublethal effects were noted at the end of either
test. Both 48

- hour and 96

- hour LD 50 values were >100 μg/bee

. This study is classified as a cceptable

.

Vinall (2010; MRID 48445809 ). The acute toxicity of the sulfoxaflor metabolite X11721061 (purity 99 % w/w) to the honeybee ( Apis mellifera ) was determined after oral exposure. Adult worker bees (approximately 2 weeks of age) were exposed to in a 48

- h limit test to a nominal dose of 100 μg a.i./bee in a 50% sucrose solution distributed among 5 replicate cages containin g 10 bees each. Results from a previous range finding test indicated the acute oral LD50 was > 100 μg a.i./bee . In addition, a sucrose solution controls were tested. No mortality occurred after 48 hours in both the control and treatment groups. Based on the amount of material consumed, the 48

- hour LD 50 value is >103.5 μg a.i./bee . No sublethal signs of toxicity were observed (e.g., lethargy, bees on back).

To confirm the sensitivity of the test insects, bees from the same hive were also tested with technical

grade dimethoate (dissolved in acetone and diluted with 50% w/v sugar solution) in a separate bioassay. The dimethoate was applied at a series of doses (nominally 0.20, 0.175, 0.15, 0.125 and 0.10 μ g a.i./bee). Three replicate cages of 10 bees each (i.e. 30 bees per treatment in total) were used for each treatment and acetone diluted in 50% w/v sugar solution was used as a control. The acute oral LD 50 fo r dimethoate is 0.16 (0.138

- 0.177) μ g a.i./bee, which is within the historical range for this chemical ( 0.10
and 0.35 μ g a.i./bee). This study is classified as a cceptable

.

Acute Oral Toxicity of Formulated Product to Adult Bees

Vinall (2009; MRID 47832417). The acute toxicity of GF

- 2032 (suspension concentrate, 22.0% w/w sulfoxaflor) to the honeybee ( Apis mellifera ) was determined after oral exposure. Adult worker bees were exposed to nominal doses of 0.0063, 0.0125, 0.025, 0.05, 0.1 and 0.2 μg a.i ./bee dispersed in
sucrose solution and observed for 48 hours . In addition, a sucrose solution control was tested. A slight effect on diet consumption beginning at 0.1 μg a.i./bee was observed indicating sulfoxaflor was not palatable to the bees. After 48 hours, there was no mortality in the control group and dose

- response mortality was observed in the treatment groups ranging 2

- 96%. One bee (3%) in the 0.05 μg a.i./bee treatment exhibited sublethal effects (such as uncoordinated attempts to move, increased amounts of grooming, lethargy or diarrhea ) at the end of the test. The 48

- hour LD 50 was 0.0515 μg a.i./bee. This study is classified as a cceptable

.3 Vinall (2009; MRID 47832418). The acute toxicity of GF

- 2032 (suspension concentrate, 22% w/w sulfoxaflor) to the bumblebee ( Bombus terrestris ) was determined after oral exposure. Adult worker bees were exposed to nominal doses of 0.010, 0.019, 0.035, 0.065 and 0.120 μg a.i./bee disper sed in
sucrose solution and observed for 72 hours . In addition, a sucrose solution control was tested. After 72 hours, there was 6.7% mortality in the control group and dose

- response mortality was observed in the treatment groups ranging 10.0

- 96.7%. No sub lethal effects were observed throughout the duration of
the test. The 72

- hour LD 50 was 0.027 μg a.i./bee. This study is classified as supplemental (but acceptable for quantitative use in risk assessment) because b umblebee studies do not currently have an internationally accepted guideline; however, the 72

- hour LD 50 was determined to be reliable for regulatory purposes.

Oral Toxicity of TGAI To Larval Bees

Stempniewicz A (2012 ; MRID 48755602)

. This laboratory study was conducted based on the publication of Aupinel et al. 2009 1 to determine the effects of XDE

- 208 (active ingredient sulfoxaflor on the larval, pupal and adult emergence of the honeybee, Apis mellifera carnica L. This study included five treatment groups of the test item applied at concentrations of 0.0002, 0.002, 0.02, 0.2 and 2.0 μg a.i. in 30 μl diet/bee larvae. The control group was treated with 30 μl diet in purified water and the reference item (dimethoate) was applied at 5 μg a.i. in 30 μl diet/bee larvae. All treatment groups were treated once on day four of the experiment using a micropipette. Two test units (replicates) containing 30 first instar honeybee larvae were set up for each treatment. Larvae were fed once a day. O n day four, treatments were applied to the culture plates that contained one larvae plus diet per cell. The culture plates were placed into a hermetic container that kept relative humidity at a mean of 90%. The container was then kept in a climate chambe r at 33

- 35°C. On day 7, when pupation normally occurs, larvae mortality was assessed and the dead larvae was removed. On day 15, pupae mortality was assessed and dead pupae were removed. The remaining living pupae were then transferred to an emergence b ox that was again, kept in the hermetic container that was kept in a climate chamber. Adult bee emergence occurred from day 15 to 18. Mortality and rate of emergence were assessed on day 18. Mortality values were corrected using Abbott’s formula (1925). LC 10 and LC 50 values were calculated with logistic regression using corrected mortality data from days 7 and 18.

In Aupinel et al. 2009, two validity criterions were set: control mortality must be lower than 15% at day 6; and successful hatch of adults in at least the control group. The validity criteria expressed in this trial included: larval mean control mortality should not be > 15% by day 7; and mean mortality of the reference item should be ≥ 50%. The mean mortality of larvae in the control treatments on day 7 was 8.3%. The mean mortality of the reference item (dimethoate) on day 18 was 100%. Both validity
criterions were met.

A rate effect was evident across the range of doses tested. The LD 50 for larvae by day 7 was greater than 2.0 μg a.i./bee larvae and the LD 50 for total mortality (larvae + pupae) by day 18 was 0.22 μg a.i./bee larvae (0.08

– 0.35 CI). Because of the very high mortality observed in the contro l groups at 18 days, the

7

- d LD 50 is thought to be more reliable. The emergence rate on D18 was 50.0 %, 68.3 %, 70.0 %, 46.7 %, 35.0 %, 0.0 % in the control and the 0.0002, 0.002, 0.02, 0.2, 2.0 μg a.i. / bee larvae treatment groups, respectively. There we re no morphological parameter differences recorded at any time, across any of

1 Aupinel et al . 2009. Honey bee brood ring

- test: method for testing pesticide toxicity on honeybee brood in

laboratory conditions. In: Julius

- Kühn

- Archiv 423 (2009), Hazards of pesticides to bees

- 10th Int. Symp. of the ICP

- BR Bee Protection Group, Bucharest (Romania), Oct 8

- 1 0, 2008.4 the different honeybee lifestages. This study is classified as supplemental (the 7

- D LD50 is considered acceptable for quantitative use in risk assessment).

Stempniewicz A (2012; MRID 48755603

. This laboratory study was conducted based on the publication of Aupinel et al. 2009 to determine the effects of repeated doses of XDE

- 208 (active ingredient sulfoxaflor) on the larval, pupal and adult emergence of the honeybee, Ap is mellifera carnica L. This study included five treatment groups of the test item applied at cumulative doses of 0.0001, 0.0002, 0.002, 0.02 and 0.2 μg a.i. XDE

- 208 / bee larvae in 20

- 50 μl diet/bee larvae. The control group was
treated with 20

- 50 μl die t in purified water and the reference item (dimethoate) was applied at a cumulative dose of 5 μg a.i. in 20

- 50 μl diet/bee larvae. All treatment groups were treated once/day (except day 2) on days 1 through 6, consistent w/ the protocol of Aupinel et al. 2009.

Two test units (replicates) containing 30 first instar honeybee larvae were set up for each treatment. Larvae were fed once a day. On days 1, 3, 4, 5, and 6, treatments were applied to the culture plates that
containe d one larvae plus diet per ce ll. The culture plates were placed into a hermetic container that kept relative humidity at a mean of 93%. The container was then kept in a climate chamber at 33

- 35°C. On day 7, when pupation normally occurs, larvae mortality was assessed and the dead la rvae was removed. On day 15, pupae mortality was assessed and dead pupae were removed. The remaining living pupae were then transferred to an emergence box that was again, kept in the hermetic container that was kept in a climate chamber

. Adult bee emerg ence occurred from day 15 to 18. Mortality and rate of emergence were assessed on day 18. Mortality values were corrected using Abbott’s formula (1925). LD 10 and LD 50 values were calculated with logistic regression using corrected mortality from days

7 a nd 18.

In Aupinel et al. 2009, two validity criterions were set: control mortality must be lower than 15% at day 6; and successful hatch of adults in at least the control group. The validity criteria expressed in this trial included: larval mean control mortality should not be > 15% by day 7; and mean mortality of the reference item should be ≥ 50%. The mean mortality of larvae in the control treatments on day 7 was 5%. The mean mortality of the reference item (dimethoate) on day 18 was 100%. Both validity criteria

were met.

Mean control mortality by day 7 was low ( 5%) while that by day 15 and 18 was high (38.3% and 58.3%). Furthermore, mean emergence in controls by day 18 was relatively low (42%). Results from Aupinel et al. 2007 2 indicate mean adult emergence in controls of 70% or higher. Therefore, results from day 7 are considered most applicable to risk assessment purposes. At and below a cumulative 6 day dose of 0.02 μg a.i./bee, larval mortality ranged from 8.3 to 15% with no obvious dose

- response relationship by day 7. At 0.2 μg a.i./bee, 45% mortality oc curred on day 7. The 7 day LD 10 = 0.085 μg a.i./bee larvae (CI =

0.005

– 1.49) and the 7 day LD 50 was greater than 0.2 μg a.i./bee larvae. The emergence rate was 41.7 %, 38.3 %, 41.7 %, 20.0 %, 38.3 %, 0.0 % and 0.0 % for the control and the 0.0001, 0.00 02, 0.002, 0.02, 0.2 μg a.i. / bee larvae treatment groups, respectively. There were no morphological parameter differences recorded at any time, across any of the different honeybee lifestages. This study is classified as supplemental (the 7

- D LD50 is co nsidered acceptable for quantitative use in risk assessment).

2 Aupinel et al. 2007. Toxicity of dimethoate and fenoxycarb to honey bee brood ( Apis mellifera ), using a new in
vitro standardized feeding method. Pest. Manag. Sci . 63:1090

- 1094.5 Acute Foliar Residue Toxicity

Bergfield (2009 ; MRID 47832512 ). The effects of foliar residues of GF

- 2372 (water

- dispersible granule, 50% w/w sulfoxaflor) on the honeybee ( Apis mellifera ) were determined after 24 hours of contact exposure. Alfalfa foliage was sprayed at nominal rates of 100 and 200 g a.i./ha. Residues were allowed to weather in the field for 3, 6 and 24 hours of application. In addition, untreated alfalfa foliage was
maintained for the controls. The alfalfa was harvested and placed into cages containing the bees. After 24 hours, mean m ortality in the controls was 5%. In the 3

- , 6

- , and 24

- hour weathered treatments, the corrected mean mortalities were 0.7, 2.8 and 14% at 100 g a. i./ha and 9.9, 9.9 and 15% at 200 g a.i./ha. Sublethal symptoms were not observed in the surviving bees. This study is classified as a cceptable

.

Lee (2008 MRID 47832420 ). The effects of foliar residues of GF

- 2032 (suspension concentrate, 22% w/w sulfoxaflor) on the honeybee ( Apis mellifera ) were determined after 24 hours of contact exposure. Alfalfa foliage was sprayed at a nominal rate of 200 g a.i./ha. Residues were allowed to weather in the field for 3, 6 and 24 hours of application. In additio n, untreated alfalfa foliage was maintained for the controls. The alfalfa was harvested and placed into cages containing the bees. After 24 hours, mean

m ortality in the controls was 0.7%. In the 200 g a.i./ha treatments weathered for 3, 6 and 24 hours, the corrected mean mortalities were 4.7, 2.0 and 1.3%, respectively. Sublethal symptoms in the surviving bees included bees lying on their back and lethargy. This study is classified as a cceptable

.



### II. Semi

- Field Tunnel Studies

1. Hecht

- Rost (2009; MRID 48445806 ). This semi

- field tunnel study was conducted to determine the
effects of GF

- 2032 (nominally a 240 g a.i./L SC formulation containing the insecticide sulfoxaflor on the honeybee, Apis mellifera carnica L. This study included five treatment groups o f the test item GF2032 applied at calculated rates of 99, 50, 24, 13.6 and 6.5 g a.i./ha in separated tunnels. A sixth group (tunnel) treated with tap water served as control. As reference item "Perfekthion" BAS 152 11 I (dimethoate) was applied at a rate of 400 g ai/ha (nominal). All applications were conducted when bees were actively foraging (> 5 bees/m 2 ) during daily bee

- flight and with a spay volume of 300 L water/ha. The effect of the test item was examined on small bee colonies in tunnels (approx. 10 0 m 2 ) placed on plots with Phacelia tanacetifolia.

Mortality, behavior, flight intensity, condition of the colonies and the development of the bee brood (% comb area with eggs, larvae, and capped brood) w ere assessed prior to application and up to 7 days after the application. In order to evaluate the magnitude of residues of the test item GF

- 2032 pollen samples from inside the hives and flowers of P. tanacetifolia were taken for analysis.

Adult Mortality and Flight Intensity

. Application of sulfoxaflor up to 99 g ai/ha appeared to increase bee mortality by up to 7X compared to controls during the first 3 days after application. After this time, bee mortality returned to levels observed in the controls. An approximate dose

- dep endent decline in flight intensity was also observed from 0DAA through 3DAA in the sulfoxaflor treatments. Flight intensity was reduced in sulfoxaflor treatments relative to controls from 4DAA through 7DAA, although dose

dependency was not observed. Mort ality in the reference toxicant (dimethoate) treatment was elevated by up to 10X of controls from 0DAA to 3DAA, indicating that the application procedures resulted in significant exposure to bees. Similarly, flight intensity was extremely impacted (reduce d) in
the dimethoate treatment

.6 Colony Strength. The effect of sulfoxaflor on colony strength is difficult to interpret due to the large difference among control and treated hives prior to pesticide application (# bees/hive in controls was 2X
that of trea ted hives on

- 2DAA). On 7DAA, no obvious dose

- dependent trend in colony strength was apparent among hives from plots treated with 6.5 to 99 g ai/ha sulfoxaflor. The effects of sulfoxaflor on bee brood are considered inconclusive due to the presence of Va rroa mites in control hives and the short observation period (7 days).

Residues. Residues of sulfoxaflor up to 1 ppm were detected in Phacelia pollen 7 days after application and showed a general decline with application rate. Residues in flowers (max of 1.7 ppm on 0DAA) declined steadily in the T3/24 g ai/ha treated plots to about 0.1 ppm by 7DAA. Based on this decline, one can infer higher residues in pollen on 0DAA.

Conclusions. Although this study had several strengths (multiple dosing levels, mea surement of residues, documented exposure and effects with reference toxicant (dimethoate), it also had several limitations that either confounded the interpretation of results and/or limits there use in pollinator risk assessment. Specifically, the maxim um application rate tested (99 g ai/ha) was ont third the proposed seasonal maximum on the US label (300 g ai/ha). The presence of Varroa mites in control hives may have compromised control brood performance measures (no Varroa were reported in other

trea tments). Furthermore, the post treatment observation was only 7 days which may be insufficient to detect effects on developing brood. For brood development, OECD Guideline 75 suggests 7 d exposure + 19 d observation period.

2. Schmitzer (2010 ; MRID 4844 5807 )

. Tunnels (14 m length x 5.5 m width x 2.5 m height) were set up

on a ca. 40 m 2 plot of Phacelia tanacetifolia (4m x 10m) and small bee colonies were introduced eight days before the daytime application. A water control and a toxic reference and Perfection EC [400g/L dimethoate]) were included in the study. Three application scenarios were conducted:

• Scenario 1: 48 g a.i./ha of GF

- 2032 was applied to the crop in the evenin g after bee flight to evaluate the impact of dried residues on foliage. The day after following this application, the bees were introduced to the tunnels and were exposed to the residues of the test item for 9 days.

• Scenario 2: 24 g a.i./ha of GF

- 2032 was applied during the day with bees actively foraging.

• Scenario 3: 48 g a.i./ha of GF

- 2032 was applied in the middle of the day with bees actively foraging.

The water

- treated control and reference item (600 g dimethoate/ha) were applied to the whole plot in two operations, in the middle of the day with foraging bees present (daytime applications). The trial was carried out using three tunnels (i.e. replicates) for each treatment group, with one bee hive per tunnel. Mortality, foraging activity and behavior of adult bees were recorded daily over the course of the study. Brood condition was assessed 4 days prior to application and 9 days following application.

Adult Mortality. Adult foraging bees exposed to GF

- 2032 at rates of 24 and 48 g a.i./ha (during flight) exhibited a statistically

- significant increases in mortality of up to 20X the rate observed in controls on the day of application. This increase in mea n daily worker bee mortality was short lived, however, having returned to a factor of 1.5X of controls by 1DAA (for the 24 g a.i./ha during flight; 48 g a.i./ha after flight treatments) and 3DAA(for the 48 g a.i./ha during flight treatment). No statistica lly significant effects on daily mortality rates were detected after 0DAA or when data were combined from 0DAA and 7DAA. The lack of statistical significance should be interpreted with caution because of the apparent low statistical power of the test for this endpoint.7 Foraging Intensity. Application of GF

- 2032 led to a reduction of foraging activity of bees on the day of application. Relative to control bees, mean foraging intensity on 0DAA was reduced by 25% in the 24 g a.i./ha (during flight) and 48 g a.i./ha (after flight) treatments and was reduced by 50% in the 48 g a.i./ha treatment. No statistical analysis was conducted on the 0DAA results. For the remainder of the test, mean forage intensity of bees was comparable between the controls and GF

- 20 32 treatments, indicating the reduction in foraging intensity was a short

- term effect. When forage intensity was evaluated from 0DAA through 7DAA, no statistically significant differences were detected according to the study author. Foraging activity in the dimethoate

- treated tunnels (reference item) was severely reduced from 0DAA through 7DAA, which indicates the methods used to quantify foraging activity were appropriately
sensitive.

Behavioral Effects. As seen with bee mortality and flight intensity results, the behavioral abnormalities reported for adult worker bees were short lived, having occurred only on 0DAA for the 48 g a.i./ha (after flight) and 24 g a.i./ha (during flight) treatments and 0DAA thr ough 1DAA for the 48 g a.i./ha (during flight) treatment. Behavioral abnormalities included uncoordinated movement, cramps, intensive cleaning and aggressiveness.

Brood Condition. The condition of brood among the hives was similar 4 days prior to pestic ide application, which indicates differences in brood condition among hives would not likely confound interpretation of the study results. Nine days following the applications, all brood stages could be
found at the end of the test in each of the colonies . The presence of nectar and pollen in the combs on 9DAA indicates that bees were able to forage successfully on the crop. The mean % comb area with nectar, pollen, eggs and larvae were comparable among treatments and controls, although no
statistical an alysis was conducted of these data. The only noticeable difference among brood condition was a slight increase in the percent capped brood in treatments (20

- 40%) compared to controls (20

- 25%). Given the small magnitude of increase and the high variabilit y within treatments, this difference is not expected to be statistically significant and its biological significance is
uncertain.

Conclusions . This study had a number of strengths including:

• A number of exposure scenarios were used ( e.g. , exposure to ag ed residues and direct exposure to daytime applications which simulate actual use conditions of the product )

.

• Replicated treatments were used which permitted statistical analysis of the data.

• Colony attributes were similar across treatments prior to test initiation

• The reference toxicant treatment demonstrated that the application methods used
documented sufficient exposure to foraging bees.

However, this study also has some significant limitations including:

• The application rates tested (24 and 48 g a.i ./ha) were below the maximum single and annual application rates proposed for registration in the US ( e.g. , 2 x 150 g a.i./ha for cotton).

• Colonies were not evaluated subsequent to the 9

- day tunnel exposures; therefore, longer

term effects of the treatme nts on colony and brood condition could not be evaluated.

• Colonies were introduced into tunnels 8 days prior to pesticide application, which is greater
than the 2

- 3 days recommended in the test guideline. Confinement in tunnels is known to adversely affe ct honeybees and should be minimized.8 • No assessment of overall colony strength (numbers of bees) was reported at test initiation or termination. Such information would have provided context to the number of worker bees killed by the test product and refe rence toxicant.

• Statistical analysis was used to analyse the data however large differences were required in the means between the treatment and control groups in order to detect a statistically significant difference. For example, for the 48 g a.i./ha application after bee flight, the mean number of dead bees per day on 0DAA 164.3 (SD=28.0) and was found not to be significantly different from the control dead bee rate of 26.7 (SD=12.1). This reflects the high variability in this measurement endpoint wi thin treatments and the low statistical power. Additional replicates would be needed to improve statistical power of this study.

3. Schmitzer (2011a; 48755604 ). The effects of the test item on small bee colonies were examined in tunnels (14 m length x 5. 5 m width x 2.5 m height) placed on plots of Phacelia tanacetifolia . The study
included a water control and a toxic reference (Insegar [250 g/kg fenoxycarb]). Three application
scenarios were conducted:

• Scenario 1: 48 g a.i./ ha of GF

- 2626 was applied to the crop before flowering. Nine days following this application, the bees were introduced to the tunnels when the Phacelia was now in full flower and were exposed to the residues of the test item. This application was conducted 13 day before the daytime applications.

• Scenario 2: In the evening before the daytime application two test item rates of 24 and 48 g a.i./ha were applied after the bees were active in order to expose the bees to dried residues of
the test item the next da y.

• Scenario 3: 24 g a.i./ha of GF

- 2626 was applied in the middle of the day with foraging bees present (daytime applications).

The water

- treated control and the reference item (300 g fenoxycarb/ha) were applied also during daytime with foraging bees pres ent. The trial was carried out using three tunnels (i.e. replicates) for each treatment group, with one bee hive per tunnel. Following the daytime applications, ontogenesis of a defined number of honey bee eggs was observed for each treatment group and col ony. Mortality of adult bees and pupae as well as foraging activity of the adult bees was also assessed. The condition of the colonies was assessed in regular intervals until the end of the trial.

The exposure period of the bees to the water, test item an d reference item treated crops in the tunnels was 7 days (10 days for the pre

- flower treatment). Afterwards the bee hives were removed from the tunnels to an area with no main flowering, bee attractive crops. Ontogenesis of the bees from egg to
adult worke rs was observed for a period of 22 days. This was done by marking 120 eggs at the first brood area fixing day BFDO (BFD = Brood Area Fixing Day) and investigating the further progress of their development in regular intervals until day 21 following the day time application (BFD 22 following BFDO).

Adult and Pupae Mortality. Adult foraging bees exposed to pre

- flower treatment with 48 g a.i./ha sulfoxaflor, to dried residues applied at 24 and 48 g a.i./ha after bee flight and to direct exposure to 24 g a.i./ha had no statistically significant mortality compared to the control over the 7 day exposure period and up to 27 days following application. The results should be interpreted with caution because of the statistical power of the test. The mean mortality wa s higher in all of the treatment groups compared to the control for the entire 27 day observation period. Pupae exposed to pre

flower treatment with 48 g a.i./ha sulfoxaflor, to dried residues applied at 24 and 48 g a.i./ha after bee flight and to direct exposure to 24 g a.i./ha had no statistically significant effects compared to9 the control over the 7 day exposure period and up to 27 days following application. Very few dead pupae were found in the control or in any of the treatments over the period of the test.

Foraging Activity. Foraging activity of bees exposed to pre

- flower treatment with 48 g a.i./ha sulfoxaflor, to dried residues applied at 24 g a.i./ha after bee flight and to direct exposure to 24 g a.i./ha had no statistically significant effects compared to the control over the 7 day exposure period. Foraging activity was, however statistically significantly reduced in the 48 g a.i./ha application after bee flight compared to the control over the 7 day exposure period. The results should be interpreted
with caution because of the statistical power of the test. The mean foraging activity was lower in all of the treatment groups compared to the control for the 7 day exposure period.

Brood and Colony Development. There was a similar pattern of colony development in the control and the test item treatment groups 24 and 48 g a.i./ha after bee flight and 24 g a.i./ha during bee flight. Colony sizes remain on a comparable level until day +27 ranging around 57 to 129 % compared to the initial values. Colony sizes in the 48 g a.i./ha pre

- flowering treatment group was somewhat lower compared to the other colonies (with and without the exclusion of the colony which was temporarily queenless).

The effects of sulfoxaflor on bee brood are considered inconclusive due to the fact tha t the mean
brood termination rate was 56.4% in the control which appears unusually high

. Notably, Schur et al. (2003) 3 reported that brood termination rate in control hives varied from 8% to 43% in a ring

- test of five trials of the OECD 75 tunnel study des ign. Importantly, they attributed the high brood termination rates (32

- 43%) in three trials to poor weather conditions that occurred during the studies. In a recent review of historical control data for brood termination rate, Pistorius et al 4

. correlate d increases in control brood termination rate with season of test initiation and available forage area. Regardless of the source of the high brood termination rate in the control treatments of this study, it likely reflects some stress on the bees caused by the study design and creates substantial uncertainty as to the ability of the study to detect the effects of sulfoxaflor on developing
brood

. Application of the reference item Insegar (300 g fenoxycarb/ha) resulted in high numbers of dead pupae after ap plication, which was statistically significant different from the control for the period day 8 after application to day 27 (529 dead pupae) and from day 0 to day 27 (531 dead pupae). Colonies in the reference item treatment group developed normal until day +15 but thereafter decreased down to < 50 % at the last assessment, compared to the initial value.

Conclusions. This study had several strengths including the use of a number of exposure scenarios e.g., exposure to aged residues and direct exposure to daytime applications which simulate actual use conditions of the product, replicated treatments which permit statist ical analysis, and exposure to bee brood was confirmed because of recorded mortality of pupae with the reference toxicant (fenoxycarb) which is an insect growth regulator.

The study also had several limitations that confounded the interpretation of resul t s and/or limits their use in a pollinator risk assessment including:

3 Schur, A., et al. 2003. Honey bee brood ring

- test in 2002: method for the assessment of side effects of plant protection products on the honey bee brood under semi

- field conditions

. Bull. Insectol. 56(1):91

- 96.
4 Pistorius, J. et al. 2011. Effectiveness of method improvements to reduce variability of brood termina tion rate in honeybee brood studies under semi

- field conditions

. International Commission For Plant

- Bee Relationships Bee Protection Group 11th International Symposium Hazards Of Pesticides To Bees

. Wageningen, The Netherlands

, November 2

- 4, 2011

.10 • The application rates tested ( 24 and 48 g a.i./ha) were below the maximum single airblast application rates proposed for registration in the US (e.g., 2 x 150 g ai/ha for stone fruit )

.

• The mean brood termination rate was 56.4% in the control which seems unusually high, which makes any conclusions regarding the development of the brood in the treatment groups difficult to interpret. Although no specific guidance is available regarding b rood termination rate in controls, it is generally thought that more than 25% brood termination rate in controls in unacceptable.

• Statistical analysis was used to analyse the data however large differences were required in
the means between the treatment a nd control groups in order to detect a statistically significant difference. For example, for the evening application of 24 and 48 g a.i./ha the day following application a mean of 24.3 and 24.7 dead bees were found in both treatment groups (9.7 in the co ntrol group), which was not statistically significant for both treatment groups. This due to the statistical power of the analysis used.

4. Schmitzer (2011b ; 48755605)

. Tunnels (14 m length x 5.5 m width x 2.5 m height) were set up on a

ca. 50 m 2 plot o f Phacelia tanacetifolia (2 x 24 m 2 ) and small bee colonies were introduced ten days before daytime applications. A water control and two toxic references (Insegar [250 g/kg fenoxycarb] and Perfection EC [400g/L dimethoate]) were included in the study. Thr ee application scenarios were
conducted:

• Scenario 1: 48 g a.i./ha of GF

- 2626 was applied to the crop before flowering. Five days following this application, the bees were introduced to the tunnels and were exposed to the residues of
the test item. This application was conducted 15 days before the daytime applications.

• Scenario 2 : In the evening before the daytime application 24 g a.i./ha was applied after the bees were active in order to expose the bees to dried residues of the test item the next day.

• Sc enario 3: 24 g a.i./ha of GF

- 2626 was applied in the middle of the day with foraging bees present (daytime applications). The water

- treated control and both reference items (300 g fenoxycarb/ha and 600 g dimethoate/ha) were applied to the whole plot in two operations, in the middle of the day with foraging bees present (daytime applications).

The trial was carried out using three tunnels (i.e. replicates) for each treatment group, with one bee hive per tunnel. Following the daytime applications, ontogenes is of a defined number of honey bee eggs was observed for each treatment group and colony. Mortality of adult bees and pupae as well as foraging activity of the adult bees was also assessed. The condition of the colonies was assessed in regular
intervals u ntil the end of the trial.

The exposure period of the bees to the water, test item and reference item treated crops in the tunnels was 7 days (17 days for the pre

- flower treatment). Afterwards the bee hives were removed from the
tunnels to an area with no main flowering, bee attractive crops. Ontogenesis of the bees from egg to adult workers was observed for a period of 21 days. This was done by marking 120 eggs at the first brood area fixing day BFDO (BFD = Brood Area Fixing Day) and investigating the fur ther progress of their development in regular intervals until day 20 following the daytime application (BFD 21 following BFDO).

Adult and Pupae Mortality. Adult foraging bees exposed to pre

- flower treatment with 48 g a.i./ha sulfoxaflor, to dried residues applied at 24 g a.i./ha after bee flight and to direct exposure to 24 g a.i./ha had no statistically significant effects compared to the control over the 7 day exposure period and up to 27 days following application. The results should be interpreted wit h caution because of the statistical11 power of the test. Specifically, on day 0 in both 24 g a.i./ha treatments, a 3

- fold increase in mortality occurred but this was not statistically significant. By day 1 after application, mortality returned to levels

co mparable to controls. The mean mortality was slightly higher in all of the treatment groups compared to the control for the entire 27 day observation period, although differences were not statistically significant. Pupae exposed to pre

- flower treatment wi th 48 g a.i./ha sulfoxaflor and to direct exposure to 24 g a.i./ha had no statistically significant effects compared to the control over the 7 day exposure period and up to 27 days following application. A high number of dead pupae (71) were observed in t he 24 g a.i./ha treatment group after bee flight which was statistically significant compared to the control. This pupae mortality, however, was limited almost entirely to one hive and it is therefore unclear if this
is treatment related.

Application of the reference item Insegar (300 g fenoxycarb/ha) resulted in high numbers of dead pupae after application, which was statistically significant different from the control for the period day 8 after application to day 27 (97 dead pupae) and from day 0 to da y 27 (117 dead pupae). Only 1 dead pupa was found following the application with the reference item Perfekthion (600 g dimethoate/ha).

Foraging Activity

. Foraging activity of bees exposed to pre

- flower treatment with 48 g a.i./ha sulfoxaflor, to dried residues applied at 24 g a.i./ha after bee flight and to direct exposure to 24 g a.i./ha had no statistically significant effects compared to the control over the 7 day exposure period. The results should be interpreted with caution because of the statistical power of the test. The mean foraging activity was lower in all of the treatment groups compared to the control for the 7 day exposure period.

Colony a nd Brood Development. Colony sizes among all treatment groups did not differ very much in size over the course of the study. Only at the last assessment on day 60, was there a clear decrease. There was a similar pattern of development in the control and al l test item treatment groups. Colony sizes remain on a comparable level until day +27 ranging around 81 to 131 % compared to the initial values. At the last assessment period (day +60) all test items and control colonies ranged from 69 % to 81 % compared t o their initial values. Colonies in the reference item treatment group with Insegar showed a progress until day 16 and thereafter decreased. Reference item Perfekthion EC led to a
continuous decrease of number of bees in the treated colonies until test en d on day 60

The effects of sulfoxaflor on bee brood are considered inconclusive due to the fact that the mean
brood termination rate was 65.3% in the control which appears unusually high. Notably, Schur et al. (2003) 5 reported that brood termination rate in control hives varied from 8% to 43% in a ring

- test of five trials of the OECD 75 tunnel study design. Importantly, they attributed the high brood termination rates (32

- 43%) in three trials to poor weather condition s that occurred during the studies. In a recent review of historical control data for brood termination rate, Pistorius et al 6

. correlated increases in control brood termination rate with season of test initiation and available forage area. Regardless of the source of the high brood termination rate in the control treatments of this study, it likely reflects some stress on the bees caused by the study design and creates

5 Schur, A., et al. 2003. Honey bee brood ring

- test in 2002: method for the assessment of side effects of plant protection products on the honey bee brood under semi

- field conditions

. Bull. Insectol. 56(1):91

- 96.
6 Pistorius, J. et al. 2011. Effectiveness of metho d improvements to reduce variability of brood termination rate in honeybee brood studies under semi

- field conditions

. International Commission For Plant

- Bee Relationships Bee Protection Group 11th International Symposium Hazards Of Pesticides To Bees

. Wage ningen, The Netherlands

, November 2

- 4, 2011

.12 substantial uncertainty as to the ability of the study to detect the effects of sulfox aflor on developing
brood

Conclusions. This study had several strengths including 1) the use of a number of exposure scenarios

e.g. , exposure to aged residues and direct exposure to daytime applications which simulate actual use conditions of the product , 2) replicated treatments which permit statistical analysis, and 3) exposure to bee brood and adults was confirmed because of recorded mortality of pupae and adults with the reference toxicants fenoxycarb and dimethoate, respectively.

The study also had several limitations that confounded the interpretation of results and/or limits their use in a pollinator risk assessment including:

• The application rates tested ( 24 and 48 g a.i./ha) were below the maximum single airblast application rates registered i n Canada for pome fruits, grapes, stone fruits and tree nuts and below the maximum seasonal application rates for all registered uses in Canada with the exception of succulent and edible podded beans and dry beans. The rates used in this study are
also be low maximum single rates proposed for registration in the United States ( e.g. , 100 g ai/ha for cotton).

• The mean brood termination rate was 65.3% in the control which seems unusually high, which makes any conclusions regarding the development of the brood in the treatment groups difficult to interpret.

• Statistical analysis was used to analyse the data however large differences were required in the means between the treatment and control groups in order to detect a statistically significant
difference. For example, for the evening application of 24 g a.i./ha on day 0 following the evening application a mean of 81.7 dead bees was found in the treatment group compared to 26.7 mean dead bees in the water control group. This was not statistically significant. T his is due to the lack of statistical power of the analysis used. Additional replicates for the treatment groups would be required to increase the statistical power of the test.

5. Schmitzer (2011c)

. This study was conducted to determine the effects on foraging bees due to direct exposure (application during bee activity) on flowering crops at 4, 8 and 24 g a.i./ha of GF

- 2626. The control group (water) and two reference item groups (thiamethoxam or dimethoate, respectively) were treated also during bee f light. Tunnels (14 m length x 5.5 m width x 2.5 m height) were set up on a 40

m 2 plot of flowering Phacelia tanacetifolia (2 x 20 m 2 ) and small bee colonies were introduced 8 days before the applications. One bee hive was used per tunnel.

Small colonies of honey bees (Apis mellifera carnica L.) were maintained according to normal beekeeping p ractice, containing 4 honeycombs each. The preliminary brood check indicated healthy colonies with a present queen. Due to the late season in which the study was cond ucted ( October in Germany ), not all brood stages could be found. The study authors indicated that either a queen or a sufficient amount of eggs were found indicating that the colonies were queen

- right. The mean number of bees per colony in the test groups three days before the application was similar.

The trial was performed using three tunnels for the different test item treatments (3 replicates/treatment), the control and both reference item treatments, respectively (total = 15 tunnels). The exposure p hase of the bees to the treated crop was 7 days following the daytime applications. The conditions of the colonies were examined until 3 months following the initial applications. Mortality and foraging activity of the bees were assessed before and after application. Sublethal effects, such as changes in behavior, were also monitored. Condition of the colonies (food stores, brood status and13 colony strength) was assessed 3 days before, 7 and 14 day after the application. 32 day and 3 months following the ap plication the condition of the colonies was examined concerning the overall survival.

Mortality. Application of GF

- 2626 at rates of 4, 8 and 24 g ai/ha resulted in an increase in mean number of dead bees/colony relative to controls on the day of applicati on (1.5X to 4X higher), although these increases were not statistically significant. From 1DAA through 7DAA, results
between the control and GF

- 2626 treated hives were similar, indicating a transient effect of sulfoxaflor on bee mortality. Application of dimethoate and thiamethoxam (reference toxicants) resulted in much greater mortality compared to controls (40X) and for a longer period of time.

Foraging Activity. Similar to mortality, application of GF

- 2626 at rates of 4, 8 and 24 g ai/ha resulted

in a reduction in foraging activity of bees on the day of application by 30

- 40% relative to controls. However, foraging activity in subsequent days recovered to levels that were similar or exceeded slightly those of controls. Application of the dimethoate and thiamethoxam reference toxicants resulted in a sustained (and statistically significant) depression of foraging activity which indicates the study design is able to detect effects on this endpoint.

Behavioral Abnormalities

. As seen with bee mortality and flight intensity results, the behavioral abnormalities reported for adult worker bees were short lived, having occurred only on 0DAA for the 24 g a.i./ha treatment and up to 2DAA in the reference toxicant treatments.

Brood D evelopment and Colony Strength

. Due to the confounding influence of the late season and declining colony condition, the effect of GF

- 2626 or the reference toxicants on brood condition and colony strength are not considered valid as measured in this study. The late season at which the study was conducted resulted in severely depleted brood stock which likely confounded the ability
to detect treatment related effects on brood condition.

6. Ythier 2012 MRID 48755606. GF

- 2372 (Batch No. E3461

- 80, content of s ulfoxaflor 49.1 % w/w) was
diluted in water and applied with a spray application to protected cotton at full bloom, at the following rates: 0.045 lb a.i./acre (1 or 2 applications at 5

- day interval), 0.089 lb a.i./acre (2 applications at 5 day

interval) an d 0.134 lb a.i./acre (2 applications at 5

- day interval). When spray residues were dry, honey bee colonies were exposed to the treated crop in tunnels (two bee hives per tunnel) for 10 days. Samples of pollen (extracted from flowers), forager honeybees (for subsequent extraction of pollen loads and nectar) and pollen and larvae from the combs were taken and analyzed for sulfoxaflor (XDE

208) and its major metabolite X11719474. General conditions and weight of the colonies were recorded before and after expos ure during the experimental phase, to ensure the colonies were in a suitable
condition to fulfil l the purpose of the experiment.

Assessment of the colonies (three days before first exposure to the test item = 3DBE) indicated that the
test colonies were in a suitable condition to fulfill the purpose of the experiment (adequately fed, healthy and queen

- right colonies, with at least 10

, 000 young honey bees, 4

- 5 combs of brood with all brood instars present and 2

- 3 combs of nectar and pollen).

• In pollen fro m plants, results ranged from below the LOD (0.01 ug/g) to 6.6560 μg/g for XDE

- 208 and from below the LOD (0.01 ug/g) to 0.0176 μg/g for X11719474.

• In pollen from bees, results ranged from 0.0126 to 2.780 μg/g for XDE

- 208 and from below the
LOD to 0.0650 μg/g for X11719474.14 • In nectar from bees, results ranged from below the LOD to 1.010 μg/g for XDE

- 208 and from below the LOD to 0.0210 μg/g for X11719474.

• In pollen from combs, results ranged from below the LOD to 1.190 μg/g for XDE

- 208 and from
below the L OD to 0.0637 μg/g for X11719474.

• In larvae from combs, results ranged from below the LOD to 0.0815 μg/g for XDE

- 208 and from below the LOD to 0.0752 μg/g for X11719474.

Biological measurements of hive attributes indicated that foragers were able to obtai n sufficient nectar to maintain the number of foragers in hives following 10 days exposure in tunnels treated with sulfoxaflor. However, pollen reserves were exhausted in all treatments by 10DAE, indicating the quantity and/or quality of cotton pollen was insufficient to maintain pollen stores. This apparent lack of pollen likely influenced the reduction in brood condition on 10DAE and 17DAE relative to 3DBE. Lack of a concurrent control precluded evaluation of sulfoxaflor effects separate from those whic h may have been caused by the tunnel enclosure environment.

Colony strength (total numbers of bees) was similar between the pre

- and post application measurements within and among all treatments . This implies that conditions of the sulfoxaflor

treatment s did not result in an obvious decline in mean colony strength by 17DAFA, even at the maximum US application rate of 2 x 0.134 lb ai/A. Although lack of a current control and limited observation period precludes definitive conclusions regarding the effect of sulfoxaflor on colony strength in this study, these results suggest that major impacts on honey bee colony strength are not apparent with sulfoxaflor applications at the maximum US application rate, at least over the short term ( e.g., 17DAFA).



### I II

. Sulfoxaflor Residue Studies

Cotton Residue Study (MRID 48755606). GF

- 2372 (sulfoxaflor 49.1 % w/w) was diluted in water and applied with a spray application to protected cotton at full bloom in a series of tunnel enclosures. Application rates include : 0.045 lb a.i./acre (1 or 2 applications at 5

- day interval), 0.089 lb a.i./acre (2 applications at a 5

- day interval) and 0.134 lb a.i./acre (2 applications at 5

- day interval). When spray residues were dry, honey bee colonies were exposed to the treated cr op in tunnels (two bee hives per tunnel) for 10 days. Samples of pollen (extracted from flowers), forager honeybees (for subsequent extraction of pollen loads and nectar) and pollen and larvae from the combs were taken and analyzed
for sulfoxaflor and its major metabolite X474 on Day 0 after application (0DAA) through 10DAA . General conditions and weight of the colonies were recorded before and after exposure during the experimental phase, to ensure the colonies were in a suitable condition to fulfill the p urpose of the experiment.

Assessment of the colonies (three days before first exposure to the test item = 3DBE) indicated that the test colonies were in a suitable condition to fulfill the purpose of the experiment (adequately fed, healthy and queen

- right colonies, with at least 10

, 000 young honey bees, 4

- 5 combs of brood with all brood instars present and 2

- 3 combs of nectar and pollen).

A summary of reported residue values in various matrices from the cotton study is shown in Table D

- 1. One notable a spect of this study is how the biology of the cotton plant affects interpretation of the residue measurements made over time. Specifically, cotton flowers are known to remain open for approximately one day or less. Furthermore, at the relatively at high temperatures associated with the conduct of this study, pollen was noted to degrade relatively quickly in the flower (generally by mid

- day to early afternoon). Thus, on days following sulfoxaflor applications, bees would not be able to forage15 on flowers w hich were previously open during pesticide applications because flowers would not remain open beyond the day of pesticide application. It is therefore concluded that residues measured on the first application day reflect sulfoxaflor that has deposited ont o pollen and nectar immediately after application. On days 1

- 4 and 6

- 10, residues in pollen and nectar are considered to reflect systemically transported chemical, since these sampled flowers were not open during the day of application. Pollen
and nectar residues measured on DAA5 reflect both deposited and systemically transported chemical. An illustration of this interpretation of the sulfoxaflor residues in forager bee

- collected pollen and nectar
is shown in Figure D

- 1 and D

- 2

.



### Table D

- 1. Sulfoxaflor residues in pollen collected from cotton plants, pollen collected from foragers, and nectar collected from foragers (MRID 48755606) Application Rate (lb ai/A) Application Days Non

- Application Days (D1

- D4 & D6

- D10)
D0 D5 Min Max
Pollen & Stamen from Plants (ppb)

0.045 x 1 1,263 n/a <10 <10
0.045 x 2 1,077 2,540 <10 61.3
0.089 x 2 6,656 691 <10 56.7
0.134 x 2 2,612 74.8 12.9 69.5 Pollen from Foragers (ppb)
0.045 x 1 127

- 187 n/a 12.6 222
0.045 x 2 173

- 226 192

– 830 32.3 296
0.089 x 2 512

- 2,782 787

- 1,146 99.5 2,262
0.134 x 2 1,209

- 2,218 1,150

– 1,420 129 2,226 Nectar from Foragers (ppb)

0.045 x 1 21.7

– 32.7 n/a <10 126
0.045 x 2 <10 45.0

– 49.0 <10 42.0
0.089 x 2 <10

– 73.8 21.9

– 22.9 <10 35.5
0.134 x 2 51.7

– 109 21.9

– 43.7 < 10 1,00616 

### Figure D

- 1. Sulfoxaflor residues reported in pollen collected by forager bees from treated
cotton

Figure D

- 2. Sulfoxaflor residues reported in nectar collected by forager bees from treated
cotton

Biological measurements of hive attributes indicated that foragers were able to obtain sufficient nectar to maintain the number of foragers in hives following 10 days exposure in tunnels treated with sulfoxaflor. However, pollen reserves were exhausted in all treatments by 10DAE, indicating the quantity and/or quality of cotton pollen was insufficient to maintain pollen stores.17 

### This apparent lack of pollen due to the artificial confinement likely influenced the reduction in brood condition on 10DAE and 17D AE relative to 3DBE. The l ack of a concurrent control precluded evaluation of sulfoxaflor effects separate from those likely caused by the tunnel enclosure itself.

Phacelia Residue Stud y (MRID 48446601 ). A semi

- field study was conducted to investigate the residues of sulfoxaflor (GF

- 2626) in nectar, pollen and whole plant tissue after four types of applications. Specifically, the test substance was applied at rates of 24 g a.i./ha (treatment groups Tl and T3) and 48 g a.i./ha (treatment groups T2 and T4) in separated tunnels. A fifth group (tunnel) left untreated served as control. Applications in treatment group T I and T2 were conducted before flowering, applications in treatment group T3 and T4 were mad e during flowering and during daily bee fl ight. All applications were made with a rate of 400 L water/ha. Commercial bee colonies were placed in tunnels (approx. 200 rn 2 ) on plots with Phacelia tanacetifolia. Condition of the colonies and the development o f the bee brood

were assessed once before the start of exposure of the honeybees in the tunnels.

In order to evaluate the magnitude of residues of the test item GF

- 2626, nectar stomachs from forager bees, pollen samples from pollen traps, and the treated phacelia plants were taken for analysis o n days 0, 5, and 6 after application in T3 and T4 treatments (10, 15 and 16 days after application in T1 and T2

treatments ; Table D

- 2 ).



### Table D

- 2

. Residues of sulfoxaflor detected in nectar collected from forager s, pollen collected from hive traps, and whole Phacelia (MRID MRID 48446601 )

Day Treatment Application Rate (g ai/ha) Nectar (mg/kg) Pollen (mg/kg) Whole Plant (mg/kg)

0DAA T1 24 (before flower) n.d. n.d. n.d.

T2 48 (before flower) n.d. n.d. 0.034

T3 24 (during flower) 0.04

- 0.05 0.29 0.52

T4 48 (during flower) 0.05

- 0.09 0.81 1.48

5DAA T1 24 (before flower) n.d. n.d. n.d.

T2 48 (before flower) n.d. n.d. n.d.

T3 24 (during flower) n.d. n.d. 0.027

T4 48 (during flower) 0.01 0.019 0.052

6DAA T1 24 (before flower) n.d. n.d. n.d.

T2 48 (before flower) n.d. n.d. n.d.

T3 24 (during flower) n.d. 0.016 0.048

T4 48 (during flower) n.d. 0.033 0.051

For treatments T3 and T4 (applied during flowering), residues in nectar measured on 0DAA ranged from 0.04 to 0.09 mg a.i./kg respectively, while those in pollen were up to 10X greater (0.29 & 0.81 in T3 and T4, respectively). Sulfoxaflor residues declined sharply from 0DAA to 5DAA in treatments T3 and T4, with levels below the detection limit of 0.01 mg/kg in T3 and 0.019 mg/kg in T4. On 6DAA, residues were slightly higher in T4 (0.033 mg/kg) and were just above detection in T3 (0.016 mg/kg).

The highe st residues of sulfoxaflor were observed in whole plant on 0DAA, ranging from 0.034, 0.52 and 1.48 mg a.i./kg in T2, T3 and T4 respectively. On 5DAA and 6DAA, sulfoxaflor residues were only detected in the T3 and T4 treatments at 0.03 to 0.05 ppm. Residu es of the primary metabolite (474) were below analytical detection except in whole plant on 6DAA (T4).18 Overall, a dose

- dependent increase in residues occurred on 0DAA in nectar, pollen and whole plant
tissues . This was followed by a substantial decrease to about 3%

– 10% of 0DAA residues by 5DAA
across all matrices.

Phacelia Residue Stud y (MRID 48445806 ). This semi

- field tunnel study was conducted to determine the
effects of sulfoxaflor TEP GF

- 2032 on the honeybee, Apis mellifera carnica L. This study included seven treatment groups of the test item GF2032 applied at calculated rates of 99, 50, 24, 13.6 and 6.5 g a.i./ha in separated tunnels. All applications were conducted when bees were actively foraging (> 5 bees/m 2 ) during daily bee

- flight and with a spay volume of 300 L water/ha. The effect of the test item was examined on small bee colonies in tunnels (approx. 100 m 2 ) placed on plots with P

. tanacetifolia.

Residues in pollen collected from sulfoxaflor treated plots on 7DAA showed somewhat of a dose

dependent increase with sulfoxaflor application rate although residues in T2/50.0 g ai/ha (0.225 ppm) were lower than one might expect compared to T1/99.2 g ai/ha (1.013 ppm) or T3/24 g ai/ha (0.612

ppm ; Table D

- 3 ). Residues of the primary metaboli te (X474) were low ( approaching or below the limit of
detection of 0.01 ppm).



### Table D

- 3

. Results from pollen residue analysis of sulfoxaflor treated Phacelia plots measured 7DAA (MRID 48445806 )

T1 = 99; T2 = 50, T3 = 24, T4=13.6 and T5= 6.5 g a.i./ha applications

Samples were collected from the T3/24 g ai/ha and control plots on 0DBA for residue analysis in flowers ( Table D

- 4 ) . Samples were also collected on 0DAA, 3DAA, 5DAA and 7DAA from the T3/24 g ai/ha treated plot. Residues of sulfoxaflor and X 474 metabolite were both below detection levels (<0.01 ppm) prior to application. Just after application (0DAA), 1.8 ppm of sulfoxaflor was detected in flowers, which declined by about 10X by 3DAA and about 2X further by 5DAA and 7DAA . By 7DAA , sulfoxaflo r residues in flower from the T3/24 g ai/ha (0.096 ppm) were about 6X lower than corresponding residues in pollen sampled at the same time and treatment (0.612 ppm). The metabolite X11719474 was below the limits of analytical detection throughout the expos ure period

.19 

### Table D

- 4. Results from flower residue analysis of Phacelia plots treated with 24 g ai/ha sulfoxaflor (MRID 48445806 )

Pumpkin Residue Study (MRID 48755601). This non

- good laboratory practice (GLP) study was conducted
to measure residues of sulfoxaflor in various plant tissues of a pumpkin crop following foliar application of low and high rates of sulfoxaflor. Application rates were 25 and 100 g a.i./ha (0.0223 and 0.0892 lbs/A). Each treatment (replicated 4 times) included one treated plot i n which sulfoxaflor, formulated as a 24% solution, was applied to pumpkins as a foliar spray at a target rate of 25 g a.i./ha (low rate) and 100 g a.i./ha (high rate). Sulfoxaflor was applied starting at 7 am on 25

- July

- 2001 (Week 6) and 8

- August

- 2011 (Wee k 8) using a backpack sprayer in spray volumes of 197 L/ha.

Staminate flowers first appeared 4 weeks after planting, and tissue sampling was conducted during weeks 6, 7, and 8 post

- planting when plants reached peak flowering. Five types of plant tissues w ere collected for residue analysis: nectar, pollen, nectary, peduncle (stem), and leaf tissue. Samples were analyzed for residues of sulfoxaflor using a liquid chromatograph y mass spectrometers (LC/MS/MS). Sulfoxaflor was applied on the Monday of weeks 6 and 8 post

- planting. On Tuesday and Thursday of each week, wax

- coated paper bags were placed over staminate flower buds that had not yet bloomed to prevent pollinator visits. Bags with open flowers were removed the following day and brought to the
laborato ry to extract nectar and pollen. Therefore, residue samples reflect systemically translocated chemical from a minimum of 2 to 4 days after pesticide application.

Residues in nectar averaged over the three sampling weeks were 2.3, and 9.5 ng/g for the lo w and high rates of sulfoxaflor, respectively ( Table D

- 5 ) . Residues in pollen averaged over sampling weeks were 11.2, and 76.8 ng/g for the low and high rates of sulfoxaflor, respectively. Sulfoxaflor residues were not detectable in pollen after the firs t foliar application of the low rate but averaged 33.5 ng/g after the second application. For the low and high rates of sulfoxaflor, residues averaged 41.1 and 310.7 ng/g in
leaf tissue.



### Table D

- 5

. Mean, minimum and maximum residue levels of sulfoxaflor i n pumpkin pollen, nectar and leaf tissue ( MRID 48755601 )

Rate

- foliar application
of
sulfoxaflor Weekly sampling period after planting
Week 6 (foliar application of sulfoxaflor) Week 7 (no treatment) Week 8 (foliar application of sulfoxaflor)20 (g a.i./ha) Residues of sulfoxaflor in pollen (ng/g) a

Mean Min Max Mean Min Max Mean Min Max Average b

25 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 33.5 <LOQ 84.3 11.2

100 29.7 <LOQ 53.8 <LOQ <LOQ <LOQ 200.7 53.7 380.0 76.8
Residues of sulfoxaflor in nectar (ng/g) a

Mean Min Max Mean Min Max Mean Min Max Average b

25 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 7.0 <LOQ 28.0 2.3
100 26.1 <LOQ 26.1 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 9.5 Residues of sulfoxaflor in leaf tissue (ng/g) a

Mean Min Max Mean Min Max Mean Min Max Average b

25 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 123.2 87.2 198.0 41.1

100 124.4 70.6 263.0 <LOQ <LOQ <LOQ 807.8 261.0 1270.0 310.7

a Residues reported from pooled samples collected on Wed. and Fri.; sulfoxaflor was applied on Mon.

b Averaged over the three sampling weeks, reported in ng/g. LOQ reported to be 25 ng/g (ppb)