Page 1 of 2 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 20460

O FFICE OF C HEMICAL S AFETY
AND P OLLUTION P REVENTION

PC Code : 129026 DP Code: D413482 Date : July 7, 2014 MEMORANDUM

Subject: Review of Field Pollinator Study with Tebufenozide

TO: Kaitlin Keller, Chemical Review Manager Pesticide Re-evaluation Division, 7508P

FROM: Katrina White, Ph.D., Biologist Thomas Steeger, Ph.D., Senior Advisor Environmental Risk Branch IV Environmental Fate and Effects Division (7507P)

THRU: Sarah Winfield, Acting Branch Chief Environmental Risk Branch IV Environmental Fate and Effects Division (7507P)

The Environmental Fate and Effects Division (EFE D) has completed its review (DP Barcode D413482 ) of a study entitled “ Assessment of Side Effects of Tebufenozide on the Honeybee (Apis mellifera) in the Semi-Field after One Applicat ion in Phacelia tanacetifolia in Germany 2012 ” (MRID 491587-01) examining the effects of tebufenozide (PC Code 129026) on honeybee colonies. The study was submitted by the technica l registrant, Nippon Soda Co., Ltd., in response to a generic data call-in 1 for a field pollinator study (OCSPP Guideline 850-3040 2 ). The preliminary problem formulation written in support of the Registration Review of tebufenozide 3 identified that a field pollinator toxicity test using the honeybee ( Apis mellifera ) is conditionally required under 40CFR 158 4 data requirements for conventional pe sticides. This study is required when there are data for terrestrial arthropods other than bees indicating potential chronic, reproductive or behavioral effects.

Given the mechanism of action of tebufenozide as an ecdysone agonist that can induce a premature molt in susceptible invertebrates, there was suff icient uncertainty to warrant more refined testing

1 USEPA. 2010. Generic Data Call-in. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP- 2008-0824-0016

2 USEPA. 2012. Ecological Effects Test Guidelines OCSPP 850.3040: Field Testing for Pollinators. Office of Chemical Safety an d Pollution Prevention (7101). EPA 712-C0017. January 2012. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0154-0018

3 USEPA. 2008. Registration Review- Preliminary Problem Formulation for Ecological Risk and Environmental Fate, Endangered Spec ies, and Drinking Water Assessments for Tebufenozide (PC Code 129026; DP Barcodes D352989, D352987, D352988). http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP- 2008-0824-0004

4 eCFR. 2014. Title 40 (Protection of the Environment), Part 158 (Data Requirements for Pesticides) Subpart G (Ecological Effec ts) §158.630 (Terrestrial and aquatic non-target organisms data requirements table. http://www.ecfr.gov/cgi-bin/textidx?SID=40eeaf61a19d1c8255cbb1c8a849b100&node=40:24.0.1.1.9.7.1.1&rgn=div8 

### Steeger,
T homas Digitally signed by Steeger,
T homas DN: cn=Steeger, Thomas, email=steeger.thomas@epa.gov Date: 2014.07.08 05:06:14 -04'00'Page 2 of 2 with bees. While the acute toxicity studies of honeybees indicate that the technical grade tebufenozide is practically non-toxic to young a dult honey bees on an acute contact exposure basis (LD 50 >234 μ g/bee; MRID 424362-44 5 ), the acute toxicity study did not provide information on the potential effects of tebufenozide exposure on the development of honeybee eggs/larvae/pupae (collectively referred as brood).

The semi-field study was conducted following the Organization for Economic Cooperation and Development (OECD) Test Guidance 75 6 . The study is classified as supplemental due to uncertainties related to the extent to which bees for aged on the treated crop as well as the statistical power of the study to discriminate treatment effe cts. In spite of these uncertainties, the study provides useful information for qualitatively as sessing the effects of tebufenozide on bees following prolonged exposure from a single application of tebufenozide during full bloom of phacelia

. This information is summarized in Table 1. The final Data Evaluation Record (DER) for this study is attached to this memo.

Please let me know if you have any questi ons/concerns regarding the study review.

Table 1. Reference Information for Attached Ecotoxicity Study

Guidance Data Requirement Test Substance MRID Study Classification

OECD 75 Field Pollinator Study Tebufenozide 491587- 01 Supplemental

5 Atkins, E. L. 1990. RH-5992 Technical: Bee Adult Toxicity Dusting Test. Conducted by Department of Entomology, University o f California, Riverside, CA. Laboratory Project No.l BATDT #445 and #446. Submitted by Rohm and Haas Company, Spring House, PA MRID 424362- 44.
6 OECD. 2007. Guidance Document No. 75 on the honeybee ( Apis mellifera L.) brood test under semi-field conditions. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 75, ENV/JM/MONO(2007)22Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

1 **DATA EVALUATION RECORD**

**1. CHEMICAL:** Tebufenozide PC Code No.: 129026

**2. TEST MATERIAL:** Confirm 2F (tebufenozide; 240 g/L) Purity: 22.84%

**3. CITATION**
Authors: S. Bocksch
Title: Assessment of Side Effects of Tebufenozide on the Honeybee ( Apis
mellifera ) in the Semi-Field after One Application in Phacelia tanacetifolia in Germany 2012 Study Completion Date: May 16, 2013 Laboratory: Eurofins Agroscience Services EcoChemGmbH, Eutinger Str. 24, D-75223 NiefernOschelbronn, Germany

Sponsor: Nippon Soda Co., Ltd. Shin-OhtemachiBldg., 2-1, Ohtermachi 2-Chome, Chiyodaku, Tokyo 100-8165 Japan Laboratory Report ID: S12-02218
MRID No.: 491587-01
DP Barcode: D413482 Guideline: 850.3040 (modified); OECD 75 1

**4. REVIEWED BY:** Thomas Steeger, Ph.D., Senior Science Advisor
Environmental Fate and Effects Division

**Signature: Date: 07/07/14** 

**SECONDARY REVIEW BY:** Katrina White, Ph.D., Biologist
Environmental Fate and Effects Division

OECD. 2007. Guidance Document No. 75 on the honeybee ( Apis mellifera L.) brood test under semi-field conditions. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 75, ENV/JM/MONO(2007)22Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

2 **Signature: Date: 07/07/14** 

**5. STUDY PARAMETERS**

**Age of Test Organisms at Test Initiation:** nucleus colonies containing 6-10 frames with brood and food combs **Exposure Duration:** Colonies monitored 5 days prior to application and for 28 days post-treatment.

**6. Executive Summary:**

The effect of formulated tebufenozide (Confirm 2 F; 22.84% ai) was tested under semi-field conditions using phacilia, Phacelia tanacetifolis, in tunnels (16 m L x 5 m W x 3.5 m H) containing a single nucleus honey bee colony per tunnel. Test groups consisted of phacelia treated at full bloom with either tebufenozide at 280 g ai/ha (0.250 lbs/A) (low), tebufenozide at 530 g ai/ha (0.473 lbs/A)(high), water (negative control), or a reference toxicant (Insegar 25 WG; fenoxycarb) at a rate of 150 g a.i./ha (0.134 lbs/A) while bees were actively foraging. Measured levels of tebufenozide applied in the low and high group were 287 g (0.256 lbs ai/A) and 545 g a.i./ha (0.486 lbs ai/A), respectively; the measured level of fenoxycarb applied was 151 g a.i./ha (0.135 lbs ai/A). Treatments were made while bees were actively foraging. Colonies were removed from tunnels 7 days after application (DAA) and moved to a monitoring site; then 5 days later, the colonies were moved to another monitoring site.

Each test group had 4 replicates for collecting biological measurement endpoints; tebufenozide-treated tunnels and negative controls had an additional ‘‘replicate’’ used to collect samples for residue analyses. Colonies were acclimated to the tunnels for 6 days before application (DBA) of test material. For 5 DBA, data were collected on bee behavior and mortality. Foraging activity was monitored the day of application and for 7 DAA. Colony condition (strength, food stores, and brood) was evaluated Digitally signed by Katrina White
Date: 2014.07.07 12:50:59 -04'00'Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

3 1 DBA and at 4, 8, 14, 22 and 27 DAA. Detailed brood assessments were conducted 1 DBA (brood fixing day 0; BFD 0) and 4, 8, 14 and 22 DAA representing BFD 5, 9, 15 and 23.

Residues of tebufenozide were measured in pollen collected by worker bees at 2 and 4 DAA while only trace amounts were detected in nectar collected from forager bees. Residues were not detected in comb pollen or nectar/honey.

On the day of application and the day following, there was no statistical difference between control and T1; however, T2 exhibited statistically higher (p<0.05) mortality both days. Overall daily mean mortality (adults + pupae) 0 - 28 DAA was similar with 14.6, 18.2, 22.4 and 22.6 dead bees/colony/day in the control, T1, T2 and reference toxicant groups, respectively. Statistically different mortality between controls and the T1 treatment was detected 21DAA and in T2 on 0, 1, 4, and 6 DAA. Although mean mortality was not substantially different across the different groups over the 0DAA - 28 DAA, mortality in T2 was 2.2- fold higher than controls at DAA0 and 1.5-fold higher 0DAA - 28DAA while in the fenoxycarb-treated colonies, the mortality was 1.9- fold and 1.6-fold higher than controls in the DAA0 and the 0DAA - 28DAA periods, respectively. Mortality quotients, intended to quantify post-application mortality relative to pre-treatment, were 0.5, 0.7, 1.3 and 0.9 for the C, T1, T2 and fenoxycarb groups, respectively.

Over the entire study period, the sum of dead pupae per colony was 7, 29.8, 9.5 and 140.4 in the control, T1, T2 and fenoxycarb groups, respectively; elevated overall mortality in T1 was associated with one of the replicates within that treatment.

There were no statistical differences in brood termination rate, brood index nor brood compensation index for colonies in tunnels
treated with tebufenozide relative to controls. All of the colonies in the study decreased in the percentage of comb covered by brood as well as in the number of cells containing pollen and nectar and overall colony weights declined as well over the duration of the study. Although the brood terminations rate forHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

4 cells marked as egg stage on brood fixation day 0 (BFD0) were higher than controls in tebufenozide-treated groups, and for T1, the brood termination rate was roughly double (60.17 ± 27.42%) compared to controls (33.17 ± 21.72%); however, these differences were not statistically significant given the high level of variability associated with these estimates.

While bee foraging behavior was not substantially different between study groups over the course of the study, bees in both tebufenozide treatments exhibited increased grooming behavior and two colonies in T2 discarded drones and in one colony in T2 at 2 DAA. Clustering of bees was reported in T1 1 DAA.

The reviewer concurs with the study authors’ conclusion that there was a statistically significant (p<0.05) effect on bee mortality associated with the application of tebufenozide at 545 g a.i./ha (0.486 lbs ai/A) and that this effect appeared to be transitory. Also, although colony weights in all of the tebufenozide- and fenoxycarb-treated colonies were statistically different (p<0.05) at Day 23 after application of tebufenozide where the treated colonies weighed less than controls, by Day 29 of the study there was not difference in weights across controls and treated colonies. In general, colonies in tunnels treated with tebufenozide did not appear to perform substantially different from controls by the end of the study.

The study is classified as supplemental as it is a nonguideline study and there is uncertainty regarding the extent to which bees may have foraged in treatment areas following their removal from tunnels and subsequent relocation to a monitoring site that was within 2 km of the treatments. Although there were four replicates per treatment, the variability associated with various measurement endpoints limits the ability of the study to detect treatment effects. Residues of the reference toxicant, fenoxycarb, were sporadically detected in samples collected from the tebufenozide treatment groups and may indicate colony robbing activity by the bees. In spite of these uncertainties, the study provides useful qualitative information for assessing the effects of tebufenozide on honey bee colonies.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

5 **7. ADEQUACY OF THE STUDY**

**A. Classification:** Supplemental

**B. Rationale:** Non-guideline study

**C. Repairability:** not applicable

**8. GUIDELINE DEVIATIONS**

x The first monitoring site was less than 2-km away from the field site; hives were then moved 15.8 km from the field
site.

x The first samples of pollen/nectar from comb were collected
8 DBA instead of 5 - 7 DBA.

x No nectar samples for the fenoxycarb Rb (Reference toxicant subsample) and the T2c (A- and R-subsample) could be taken
on 28 DAA. According to the study authors, these deviations had no impact on the study.

**9. SUBMISSION PURPOSE:** To determine the potential effect of tebufenozide on brood development, mortality, foraging activity and behavior of the honey bee under semi-field
conditions in P. tanacetafolia following OECD Guidance 75. Residue analyses were also performed to assess the magnitude of potential residues in nectar and pollen.

**10. MATERIALS AND METHODS**

A. **Test Organisms:**

**Guideline Criteria Reported Information** 

**Species** Honey Bee ( Apis mellifera L. )

**Age at beginning of** Adult bees and developing brood at various ages/stages of development.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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6 **Guideline Criteria Reported Information** 

**test**
Worker bees of uniform

age. Queen bees were 1-yr old.

**Source** Colonies obtained from a beekeeper in Rheinland Pfalz, Germany (Mr. Berthold Nengel **)**

**Were bees from disease**
**free colonies?** Bees were free of Nosema and Varroa disease symptoms and other bee diseases ( e.g ., amoebiosis, chalkbrood, sacbrood, and American or European foulbrood) at test start.

**Were bees kept in conditions conforming**  **to proper cultural practices?**  Bees were from commercial beekeeper and are presumed to have been maintained under proper cultural practices.

**B. Test System**

**Guideline Criteria Reported Information** 

**Test Colonies** Healthy nucleus colonies containing 6
- 10 brood frames and 6 - 10 honey/pollen (food) combs. Colonies
consisted for 6125 - 12563 bees per hive at test start. Corresponding queens were 1-yr old and originated from a single breeding line.

**Temperature during**

**exposure** Range: 4.6 - 33.2 o C

**Relative humidity during exposure**  Range: 33.0 - 100%

**Lighting** Ambient lighting conditions.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

7 **Guideline Criteria Reported Information** 

**Feeding** Bees were allowed to forage freely on phacelia while confined to the
tunnels.

**C. Test Design**

**Guideline Criteria Reported Information** 

**Nominal dosage levels**
**tested** 280 g a.i./ha (0.250 lbs ai/Acre) 530 g a.i./ha (0.473 lbs ai/Acre)

**Number of bees exposed per dosage level**  Each treatment consisted of 4 replicates with each replicate containing a single nucleus colony

**Other experimental design information**  An additional (5 th ) replicate was
included in the tebufenozide and negative control treatments. This replicate served to support residue analyses. Reference toxicant ( Insegar 25 WG; fenoxycarb; applied at 150 g a.i./ha.). All applications were made while bees were actively foraging. Applications were carried out with a portable boom sprayer when phacelia was in full bloom, there we re at least 5 bees/m 2 foraging in each treatment tent, the wind speed was <2 m/sec, no
rainfall occurred within 2 hours of application and air temperatures did
not exceed 30 o C or drop below 15 o C

. Tunnels were covered with a fine plastic mesh. Colonies were acclimated to the tunnels for 6 days before application (DBA) of testHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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8 **Guideline Criteria Reported Information** 
material. For 5 DBA, data were
collected on bee behavior and mortality. Foraging activity in a 1

-m 2 area of phaceli a for 10 15 seconds

were monitored the day of application
and f or 7 days after application (DAA). Colony condition (strength, food stores, and brood) was evaluated 1 DBA and at 4, 8, 14, 22 and 27 DAA.
Detailed brood assessments were conducted 1 DBA (brood fixin g day 0; BFD 0) and 4, 8, 14, 22 and 27 D AA representing BFD 5, 9, 15, 23 and 28.

**Bees randomly or impartially assigned to**  **test groups** Yes

**Control** Yes

Negative (water control), 5 replicates with one replicate used for residue analysis

Positive control (fenoxycarb 150 g a.i./ha), 4 replicates

**Solvent control** No

**Total observation period and frequency of interim** 
**observations** 28-day studyHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

9 **11. REPORTED RESULTS**

**Guideline Criteria Reported Information** 

**Quality assurance and**
**GLP compliance statements** 

**were included in the report?**  Yes; study was conducted in compliance with Principles of good Laboratory Practice (GLP), Chemikaliengestz, and the OECD Principles of Good Laboratory
Practice.

**Observed adverse effects on bees at respective**  **dosages** Yes, effects on mortality and sublethal effects (foraging activity, brood development) were reported for each test group.

**Control and Solvent Control Mortality**  No solvent control.

**Were raw data included?** YesHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

10 **Study Design Elements**

Study was conducted in southern Germany (Bodelshausen) near Tübinger; **Figure 1** depicts the layout of tunnels in the field of phacilia . The minimum distance between tunnels was 3 m and the minimum distance to the edge of the field was 2.5 m.

**Figure 1. Layout of each tunnel (experimental unit).** 

**Figure 2** depicts tunnels used in the study; each 5-m W x 16-m L x 3.5-m H tunnel was covered with fine white plastic mesh screening. Total area of phacelia treated (plant area) consisted of 65.12 m 2 (two strips measuring 2.2 x 14.8 m separated by a 0.6-m path. The path as well as a 5 x 0.6 m area on either side of the colony was covered with linen sheet (representing an area of 14.88 m 2 ) was used to assess dead bees. Wooden dead bee traps were at the **Figure 2. Study design layout for tebufenozide, fenoxycarb and control colonies in phacilia field.** Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

11 entrance to each colony and were also used to assess bee mortality. Each tunnel contained a 4-L water source; water containers were removed during application.

Colonies were introduced into tunnels 6 DBA and were removed from tunnels 7 DAA at which time the colonies were moved to a monitoring site; then 5 days later, they were moved to another monitoring
site.

Mortality was evaluated at 5 - 0 DBA, and at 1, 2, 4, and 6 hrs the day of application as well as daily 1 - 7 DAA (during bee flight activity) and from 8 - 28 DAA (dead bee trap only).

Brood development was assessed to overlap the pre-imaginal development period of worker bees which typically averages 21 days.

Colony condition was assessed based on:

x Colony strength (number of bees);

x Presence of a healthy queen (evidenced by presence of eggs);

x Pollen and nectar/honey storage area;

x Area containing cells with brood stages; and

x Presence of disease.

The comb area containing bee and cells with nectar, pollen, eggs, larvae and capped cells was estimated for each side of the comb.
Totals for each were then calculated for each hive and means were determined for each index by treatment and assessment date.

Samples for residue analyses were collected from the 5 th replicate of the tebufenozide-treated and negative control colonies. Pollen (1-g samples) as well as nectar (1-g samples) was collected directly from the legs and honey stomach, respectively, of forage bees. Bee samples consisted of 250 bees. Plant samples (50 g) were collected after application and just prior to the removal of the colonies from the tunnels (4DAA). Residue analyses were conducted at 8 DBA and 2, 4, 5 and 28 DAA. Analyses included tebufenozide, its metabolite RH-1788 and fenoxycarb.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

12 The following brood indices were calculated:

Termination rate : the percentage of brood which did not successfully complete development to adult bee (number terminated by total number of cells monitored.)

Brood index : if a cell did not contain the expected brood stage or food was stored in the cell during BFD 5 - 15, the cell was assigned a value of 0 (index=sum/total number of cells monitored)

Brood compensation index : if the cell was filled again with brood, the cell was assigned a value associated with the particular development stage summarized in **Table 1** . (index=sum of values in each replicate assessed on a particular date and divided by the
number of observed cells.

**Table 1. Codes used to evaluate developmental stage of**
**cells in comb.**Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

13 **Results**

According to the study authors temperatures and weather conditions were mostly favorable for bees on almost all days except for 4 -
2 DBA when there was rain.

According to the study authors, recoveries of tebufenozide, its metabolite (RH-1788), and fenoxycarb from spike matrices using tandem mass spectroscopy ranged between 70 - 110% and were within ‘‘acceptable limits’’.

No residues of tebufenozide, its metabolite or fenoxycarb were detected in samples of pollen or nectar/honey collected from comb at 8 DBA and 2, 4, 5 and 28 DAA except for a residue of tebufenozide at less than the limit of quantification (LOQ=0.01 mg/kg) but greater than the limit of detection (LOD=0.003 mg/kg)in comb nectar from the high tebufenozide treatment, 2.29 mg/kg detected in comb pollen in the low tebufenozide colonies, and residues of the tebufenozide metabolite at less than the LOQ at 5DAA.

In nectar sampled directly from bees, residue levels were less than the LOQ but greater than the LOD in both the low and high
tebufenozide-treated tunnels at 2DAA for tebufenozide and in the high treatment for residues of fenoxycarb ( **Table 2** ). At 4 DAA, residues of tebufenozide in the high treatment were also between the LOD and LOQ.

In pollen collected from forager bees, residues of tebufenozide at 2 DAA were 0.984 and 2.58 mg/kg in the low and high tebufenozide-treated tunnels, respectively, and residues of the tebufenozide metabolite (RH-1788) as well as fenoxycarb were between the LOD and LOQ in the high tebufenozide-treated tunnel ( **Table 2** ). At 4 DAA, residues of tebufenozide in pollen from the low and high-treated tunnels were 1.48 and 4.62 mg/kg, respectively, while residues of the metabolite were between the LOD and LOQ in the low treatment group and at 0.01 mg/kg in the high-treatment group.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

14 **Table 2. Summary of residues (tebufenozide, metabolite RH-1788 and fenoxycarb) in nectar and pollen collected directly from forage bees in**  **control (Cntr) and tebufenozide-treated (280 g/ha and 530 g/ha) tunnels at 2 and 4 days after application (DAA).** 

**Sample Day**  **Forager Nectar Forager Pollen** 

**Cntr Tebufenozide**  **Cntr Tebufenozide** 
**280 530**  **280 530** 

2 DAA nd <LOQ T <LOQ T
<LOQ F nd 0.984 T 2.58 T
<LOQ M
<LOQ F

4 DAA nd nd <LOQ T nd 1.48 T
<LOQ M 4.62 T

0.01 M

nd not detected; < limit of detection LOD (0.003 mg/kg) LOQ limit of quantification (LOQ; 0.01 mg/kg)
tebufenozide residue fenoxycarb residue
tebufenozide metabolite RH-1788 residue

**Figure 3** depicts the total mortality of worker bees in control (C), tebufenozide treatments T1 and T2 and the reference toxicant (R). According to the study authors, mean daily bee mortality prior to application was 45.8, 29.2, 36.6 and 46.6 in the control, T1, T2 and reference toxicant groups, respectively ( **Table 3** ). On the day of application and the day following, there was no statistical difference between control and T1; however, T2 exhibited statistically higher (p<0.05) mortality both days. Overall daily mean mortality (adults + pupae) 0 - 28 DAA was similar with 14.6, 18.2, 22.4 and 22.6 dead bees/colony/day in the control, T1, T2 and reference toxicant groups, respectively. Statistically different mortality between controls and the T1 treatment was detected 21DAA and in T2 on 0, 1, 4, and 6 DAA. The study authors believed that the mortality reported in the control and tebufenozide-treated tunnels was low and was not of biological significance for colony strength.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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15 **Table 3. Mean mortality by treatment group (Controls C a-d;**  **Tebufenozide T1 a-d**  **; Tebufenozide T2 a-d**  **; fenoxycarb R a-d**  **) across pre application )5DBA -** **0DBA), day of application (DAA0) and post application (0DAA -**  **28DA). Reproduced from Table 28 of MRID 491587-**  **01).**

In the reference group, mortality was significantly (p<0.05) higher than controls at 11, 13, 14 17 and 19 DAA. According to the study authors, the delayed pattern of mortality in the fenoxycarb-treated colonies was consistent with historical data where mortality is increased between 10 - 20 DAA.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

16**Figure 3. Total mortality of worker bees in control (C), tebufenozide treated (low T1; high T2) and the reference toxicant (R) across the**  **sampling days after application (DAA). The * indicates significantly** **(p<0.05) higher than control using one-side Dunnett’s t-test;   ** indicates significantly higher (p<0.05) than control using one-sided** **Bonferroni U-test. Reproduced from MRID 491587-01.**

Over the entire study period, the sum of dead pupae per colony was 7, 29.8, 9.5 and 140.4 in the control, T1, T2 and fenoxycarb groups, respectively, and according to the study authors, the elevated overall mortality in T1 as associated with one of the replicates within that treatment. Mortality quotients, intended to quantify post-application mortality relative to pre-treatment, were 0.5, 0.7, 1.3 and 0.9 for the C, T1, T2 and fenoxycarb groups, respectively. According to the study authors, higher mortality was observed the day of application in T2 and in the reference group; however, the mean post-application mortality was in the same range for all treatment groups and in all treatments, the post-application mortality was lower than pre-application mortality.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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17 The mortality index, used to illustrate the incidences of

treatment e ffect on mort ality relative to cont rols, were 1.0, 1.4, 1.4, and 1.6 on 0DAA, 1.0, 1.2, 1.0 and 1.1 for 0 - 7DAA and 1.0, 1.0, 1.0 and 1.3 for 0 - 28 DAA in the C, T1, T2 and fenoxycarb treatments, respectively. These ratios indicate that incidenceof mortality after application was roughly similar to preapplication mortality.

A similar index was developed for forager bees, i.e ., ratios for pre- and post-treatment numbers of forager bees. The ratios were

- 0.4, 0.1, 0.5 and 0.8 at 0 DAA, and 0.2, 0.2, 0.4 and - 0.6 from 0

- 7 DAA in the C, T1, T2 and fenoxycarb treatments, respectively. For bees present in the treated phacelia, the ratios were 1.0,

- 0.2, - 1.2 and - 1.8 on 0 DAA and 1.0, 0.8, 1.8 and 2.5 from 0 - 7 DAA in the C, T1, T2 and fenoxycarb-treated tunnels, respectively. According to the study authors, negative numbers indicate a lower mortality after application as well as fewer bees present in the crop relative to controls.

Mean foraging activity is depicted in **Figure 4** ; according to the study authors, there were no statistical differences between study groups during the pre- and post-exposure period. On 0 DAA prior to application mean foraging activity was 12.2, 25.8, 14.2, and 12.3 bees/m 2 in control, T1, T2 and fenoxycarb-treated tunnels, respectively. Following application, foraging activity averaged 11.6, 25.1, 17.6 and 15.8 bees/m 2 in the control, T1, T2 and fenoxycarb-treated tunnels, respectively. At 1 DAA, activity averaged 1.2, 0.8, 0.7 and 0.8 bees/m 2 in the control, T1, T2 and fenoxycarb-treated tunnels, respectively, and this reduced activity was attributed to rainfall.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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18 **Figure 4. Mean foraging activity (mean number of forager bees/m 2**  **) in control (C), low tebufenozide (T1), high tebufenozide (T2) and toxic**  **reference (R) across days after application (DAA). Reproduced from**
**MRID 491587-01.**

According to the study authors bees in the tebufenozide treatments showed simple and intensive grooming behavior on the day of application and two colonies in T2 discarded drones and in one colony in T2 at 2 DAA. Clustering of bees was reported in T1 1 DAA. In the fenoxycarb-treated tunnels, cramping, grooming, clustering and immobile bees were observed on the day of application and in one of the replicates, drones were discarded. At 12 DAA, pupae from the reference toxicant colonies exhibited malformed eyes which the study author’s claim is typical for fenoxycarb.

Estimates of colony strength across treatments and sampling dates are depicted in **Figure 5** . At the assessment just prior toHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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19 application, the mean number of bees was 9313, 9328, 9797 and 9360 in control, T1, T2 and fenoxycarb treatment groups, respectively. The number of bees in each group increased until 14DAA when the mean number of bees was 14875(C), 15453 (T1), 15485 (T2), 13282 (fenoxycarb). By the end of the study (28 DAA; BFD28), the number of bees had declined to 10625 (C), 10094 (T1), 9954 (T2), and 9407 (fenoxycarb) representing percent changes of 14.1%, 8.2%, 1.6% and 0.5% from pre-application numbers in the control, T1, T2 and fenoxycarb groups, respectively. No statistical differences were noted for colony strength between the different study groups at any of the sampling dates.

**Figure 5. Mean number of bees per hive in control (C), low tebufenozide (T1), high tebufenozide (T2) and reference toxicant (fenoxycarb; R)**  **groups after brood fixing day (BFD). Vertical arrow depicts the day of application. Reproduced from MRID 491587-01.** 

The authors reported that brood nest size decreased in all of theHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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20 study groups across the course of the study from a high of 31100 (C), 29900 (T1), 32150 (T2), 31750 (fenoxycarb) at BFD0 to 22350 (C), 19100 (T1), 24250 (T2), 11250 (fenoxycarb) by BFD28 ( **Figure**
**6** ). This represented a decrease of -28.1%, -56.5%, -24.6% and - 64.6% in the control, T1, T2, and fenoxycarb groups, respectively.

**Figure 6. Mean comb area covered with brood in control (C), low tebufenozide (T1), high tebufenozide (T2) and reference toxicant**  **(fenoxycarb; R) hives after the brood fixing day (BFD). Reproduced**
**from MRID 491587-01.**

According to the study authors, there was no statistical difference in mean brood termination rate at BFD23 for the egg stage between control (33.17 ± 21.72) and T1 (60.17 ± 27.42) and T2 (42.25 ± 22.42); however, the fenoxycarb-treated group was significantly different (92.39 ± 12.77; p<0.5) from controls and demonstrates a significant increase in brood termination rate forHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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21 fenoxycarb. Across the multiple assessment dates for the initial egg stage, the brood termination rate was significantly higher (p<0.5) for hives in fenoxycarb-treated tunnels ( **Figure 7** ).

**Figure 7. Average brood termination rate per colony for control, low tebufenozide (Test item 1), high tebufenozide (Test item 2) and**  **fenoxycarb (Reference item) treated groups by days after brood fixing day (BFD). Reproduced from MRID 491587-01.** 

According to the study authors, at the end of the assessment period, the brood index ( **Table 3** ) for eggs in control, T1, T2 and fenoxycarb treatments was 3.34, 2.12, 2.99 and 0.38, respectively. The fenoxycarb treatment was the only index that was significantly lower (p<0.5) than controls.

The mean brood compensation indices ( **Table 4** ) for in control, T1, T2 and fenoxycarb treatments were 3.76, 2.76, 3.63 and 0.49, respectively, which according to the study authors indicated thatHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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22 for the most part, cells with terminated brood were refilled with new eggs except for a ‘‘slight’’ effect in T1 and a statistically significant (p<0.5) effect in the fenoxycarb-treated group.

**Table 4. Summary of brood and compensation indices based on individual brood assessments of single cells by days after brood**  **fixation day 0 (BFD0). Reproduced from MRID 491587-01.**Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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23 According to the study authors, food stores in control and the low tebufenozide-treated groups were roughly similar, but decreased slightly in the high tebufenozide and reference toxicant treatments ( **Figure 8** ). At the start of the study there were 11700 8600, 9850 and 8300 nectar cells and 1350, 1050, 1600, and 1550 pollen cells in the control, T1, T2 and reference treatments, respectively. In one of the T1 replicates though, there were no pollen or nectar cells at BFD15.

**Figure 8. Mean number of cells per hive covered with brood (eggs, larvae, pupae) and food (nectar, pollen) in control, low**  **tebufenozide (Test item 1), high tebufenozide (Test item 2) and fenoxycarb (Reference item) by day after brood fixing day 0 (BFD0).**  **Reproduced from MRID 491587-01.**

**Table 5** summarizes mean (± std. deviation) hive weight by treatment and brood fixation day; there were no significant differences across treatments and sampling days.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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24 **Table 5. Mean (± std deviation) hive weights in control, tebufenozide low (T1), tebufenozide high (T2) and reference (fenoxycarb) groups over**  **brood fixation days. Last row represents grand mean (± std. deviation) across all BFD by treatment.** 
**BFD Control**  **T1 T2**  **Fenoxycarb**

0 25.7 ± 0.5 24.0 ± 2.0 25.8 ± 1.7 23.6 ± 0.6
5 24.4 ± 0.4 2304 ± 1.1 24.4 ± 1.3 22.4 ± 0.4
9 24.2 ± 0.8 22.1 ± 2.1 22.9 ± 1.1 21.9 ± 0.6
15 23.0 ± 0.6 21.7 ± 2.5 22.6 ± 1.5 21.2 ± 1.4
23 23.4 ± 0.7 19.7 ± 1.2 20.5 ± 1.2 20.8 ± 0.8
22 22.2 ± 0.8 20.9 ± 1.4 21.8 ± 1.4 20.9 ± 1.8

**Mean 23.8 ± 1.2**  **22.0 ± 1.6 23.0 ± 1.9**  **21.8 ± 1.1**

Overall, the study authors conclude that although statistically significant mortality was observed for T1 at 21DAA and for T2 on 1, 1, 4 and 6 DAA, these differences were not biologically relevant. Comparisons of mortality following application relative to mortality prior to application did not reveal any significant differences. Also, although pupal mortality was slightly elevated in T1 compared to controls, the authors considered this an artifact of a single replicate. However, the increased pupal mortality in T2 during the early post-application period was considered treatment-related, the level was considered ‘‘slight’’. The authors also concluded that there were no effects of tebufenozide treatment on foraging activity or colony strength over the 27-day study period although there was an effect on foraging bee behavior on the day of application. While the amount of brood was temporarily reduced in T1, the colonies recovered within the 27-day study period. Mean brood termination rate of the marked egg stage were slightly elevated, they were not statistically different nor were they dose-dependent. Also, the brood and compensation indices increased over the study period, with slightly lower levels for the tebufenozide treatments; however, these were not statistically significant and did not exhibit a dose-response. The authors state that application of Confirm 2F to the flowering phacelia where honey bees are actively foraging poses ‘‘a slight and temporary’’ effect on adult bee mortality at an application rate of 530 g/ha but no effect at 280 g/ha.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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25 **Reviewer Comments**

According to the study authors, weather conditions for the most part were favorable to bees except for 4 - 2 DBA. During this period it rained, 25.2, 15, and 4 mm. While it did not rain the day of application, it did rain 7, 3 and 10 mm on 1, 2 and 3 DAA, respectively, and 11 mm on 6 DAA. The rain for three consecutive days following application may have affected the availability of residues as well as bee foraging activity.

At 7 DAA, colonies were moved from the tunnels and transferred to a monitoring site that was 2-km away from the treatment site; 5 days later, the colonies were moved to a second monitoring site that was 15.8 km away from the treatment site. It is unclear from the study report whether bees from the different treatment groups may have continued to forage in the treatment areas given that the initial monitoring site was only 2 km away. Also, the nature of the monitoring sites is not described in terms of available forage and/or proximity to agricultural crops. Residues of chlorpyrifos, ethion, fenpropathrin, flutriafol, and spinosad were also detected in pollen and nectar samples, and the study author speculated that this was due to forager bees returning from surrounding agricultural crops.

The study reports measuring residues of the metabolite RH-1788; however, the nature (structure/chemical name) of this metabolite is not specified. Residue analyses indicate that although detectable residues of test material were infrequently detected in pollen and nectar/honey samples collected directly from comb, trace residues ( i.e ., LOD<residues<LOQ) of tebufenozide were detected in nectar and 2.29 mg/kg were detected in comb pollen 5
DAA in the low tebufenozide-treated tunnel. Trace residues of tebufenozide were also detected in nectar collected directly from forager bees; however, quantifiable levels were detected in pollen in both tebufenozide treatment group at 2 and 4 DAA. It’s not clear what to make of the fenoxycarb residues that were detected at trace levels in forager bee pollen and nectar from the high tebufenozide-treated tunnels at 2 DAA; however, both samples were taken from the same tunnel. The tebufenozide treatment (T2

e ) isHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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26 depicted in Figure 1 as being positioned diagonally between two fenoxycarb treatments.

Although the study authors attribute the reduced foraging activity at 1 DAA to rain ( **Figure 4** ) that day, the environmental data indicate that larger quantities of rain fell 3 DAA and again 6 DAA; however, the declines in foraging activity on those dates were not as accentuated as at 1 DAA. It should be noted though that at 1 DAA there was 100% cloud cover compared to 0 - 60% at 3
DAA and 15 - 100% at 6 DAA.

Colony strength depicted as mean number of bees per hive in **Figure**
**5** shows an increase in the number of bees beginning at 8 DAA (BFD9) and continuing to 14 DAA (BFD 15) and may reflect the effects from the colonies having been moved out of the tunnels at 7 DAA (BFD8). The peak in colony strength at 14 DAA occurred two days after the colonies were again moved to a different monitoring site.

Mean mortality rates reported in **Table 3** indicate no significant differences between pre-treatment (5DBA - 0DBA), the day of treatment (DAA0), and post-treatment (0DAA - 28DAA) values for T1 relative to controls. In T2 though, mean mortality wassignificantly (p<0.05) different than controls at DAA0 and posttreatment where mortality was 2.2-fold and 1.5-fold higher than controls. For the fenoxycarb-treated colonies, mean mortality at DAA0 and post-treatment was also significantly (p<0.05) different than controls with 1.9-fold and 1.60-fold higher mean mortality levels, respectively. While overall mortality was significantly higher than controls in the highest tebufenozide treatment, **Figure**
**3** indicates that mortality levels continued to decline following application at 0DAA and by 28DAA, was not significantly different
from controls.

Estimates of brood nest size declined until BFD9, but started to increase afterwards. The increase appears to correspond to when the colonies were removed from the tunnels. Although declines by as much as 56.5% were reported in the tebufenozide-treated (T1) group, there did not appear to be a dose response given that the decline in the higher tebufenozide group, i.e ., 24.6% for T2 wasHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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27 roughly similar to that reported for controls, i.e ., 28.1%. The highest percent decrease (64.6%) occurred in the toxic reference hives by the end of the study on BFD28; however, even these hives were beginning to increase from the lowest levels depicted for
BFD15.

The reviewer conducted separate statistical analyses using both parametric (ANOVA Bonferroni) and nonparametric (Wilcoxon/Chi Square) of brood index, brood compensation and brood termination rates ( **Appendix 1** ). Mean brood index, mean brood compensation index and mean brood termination rates are reported in **Tables 6** through **8** , respectively. The only effects that were statistically (p<0.05) from controls were those reported for colonies treated
with the toxic reference.

Although the brood terminations rate ( **Table 8** ) for cells markedas egg stage on BFD0 were higher than controls in tebufenozidetreated groups, and for T1, the brood termination rate ranged from 50.40 to 60.17 compared to controls which ranged from 20.48 to 23.65, these differences were not statistically significant given the high level of variability associated with these estimates. Average brood termination rate across each of the brood assessment dates ( **Figure 7** ) shows similar levels of variability. It is noteworthy that the lowest brood termination rate was consistently in controls which averaged 31.15 ± 4.62% (mean ± std dev); however, the T2 group (39.6 3± 2.69%) was not substantially different from controls when averaged across all of the sampling periods. The fenoxycarb-treated group did exhibit a consistently high brood termination rate around 90% across the study periods and were significantly higher (p<0.05) than controls.

**Table 6. Mean brood index across control, low tebufenozide (T1), high tebufenozide (T2) and fenoxycarb treatments after brood fixation day**  **(BFD).**
**BFD Control**  **T1 T2**  **Fenoxycarb**
0 1 1 1 1
5 2.19 1.45 1.92 0.28
9 2.70 1.71 2.40 0.32
15 2.68 1.69 2.39 0.30
23 3.34 2.12 2.99 0.38Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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28 **Table 7. Mean brood compensation index across control, low tebufenozide**  **(T1), high tebufenozide (T2) and fenoxycarb treatments after brood fixation day (BFD).** 
**BFD Control**  **T1 T2**  **Fenoxycarb**
0 1 1 1 1
5 2.19 1.47 1.95 0.31
9 2.70 1.78 2.46 0.34
15 2.74 1.87 2.50 0.38
23 3.76 2.76 3.63 0.49

**Table 8. Mean ( ± std. deviation)**  **brood termination rate (%) across control, low tebufenozide (T1), high tebufenozide (T2) and fenoxycarb**  **treatments after brood fixation day (BFD).**

**BFD Control**  **T1 T2**  **Fenoxycarb**
0

. . . . 5 24.30 ± 20.48 50.40 ± 22.79 35.87 ± 22.82 90.42 ± 13.57
9 32.72 ± 21.89 57.21 ± 28.14 40.13 ± 20.96 92.16 ± 13.22
15 33.16 ± 21.72 57.70 ± 28.84 40.25 ± 20.92 92.38 ± 12.77
23 34.43 ± 23.65 60.17 ± 27.42 42.25 ± 22.42 92.38 ± 12.77

The bar graph **Figure 8** depicting food reserves in each treatment group across the study period is difficult to interpret since the individual components on each bar are varying by sampling date and the number of cells with pupae appear to be a major determinant in the height of the bar. For each treatment, BFD15 appears to be
associated with marked declines in both brood and food reserves (particularly pollen) and would represents two days after the colonies were moved to a second monitoring location. By BFD23, there appear to be increased reserves of nectar while pollen cells continued to decline in all of the treatment groups.

Mean weight per colony summarized in **Table 5** shows a general pattern of decreased colony weights across sampling dates until BFD23; colony weights appear to have increased slightly by BFD29. Appendix 1 contains the results of both parametric and nonparametric analysis of colony weights at Day 23 and Day 29.Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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29 Both tebufenozide and fenoxycarb-treated colonies had statistically significant (p<0.05) colony weights at Day 23; however, by Day 29, there was not difference between control and treated colonies. In general, although not statistically significant, control colonies tended to weigh more than the other treatment groups. Although not statistically significant, hives from fenoxycarb-treated tunnels had lower mean weights (except for BFD23 where hives from tebufenozide-treated tunnels had slightly lower weights) than other treatment groups across each of the sampling periods.

Given the amount of variability associated with the measurement endpoints evaluated in this study, it is difficult to discern marked effects that can be readily attributed to exposure to tebufenozide. The authors’ conclusion that application of Confirm 2F to the flowering phacelia where honey bees are actively foraging poses ‘‘a slight and temporary’’ effect on adult bee mortality at an application rate of 530 g/ha but no effect at 280 g/ha appears to be consistent with the data. Although tebufenozide is an ecdysone agonist that is intended to target lepidopterans, significant effects were observed on honey bees following application while bees were actively foraging. Residue data confirm that tebufenozide was carried back to colonies primarily associated with corbicular pollen and appear to have been consumed given that similar residues were not detected in comb pollen. Rain events within a day of application however may have reduced available residues, i.e ., limited prolonged exposure. Significant effects associated with the reference toxicant fenoxycarb indicate that sufficient amounts of the compound were brought back to the colony to result in statistically significant effects that were consistent with this insect growth regulator.

There is uncertainty regarding the extent to which bees may have foraged in treatment areas following their removal from tunnels and subsequent relocation to a monitoring site that was within 2 km of the treatments. Trace residues of fenoxycarb were detected in corbicular pollen 2 DAA in the tebufenozide treatments; therefore, it is uncertain whether residues may have drifted between treatments during application even though similarHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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30 contamination was not reported in any of the other treatment

groups.

This is a non-guideline study and is classified as supplemental. Although there are multiple uncertainties associated with the study, it provides qualitative information regarding the effects of tebufenozide on honey bee colonies. Colonies treated with tebufenozide at 530 g ai/ha at full bloom exhibited increased mean mortality relative to controls; however, the statistically significant (p<0.05) mortality relative to controls appeared be sporadic and decreased over the study period.Honeybee (Apis mellifera) Semi-Field (Tunnel) TestDP Barcode: D413482 MRID No.: 491587-0131**Appendix--- -Statistical Analysis** MEAN BROOD INDEX, COMPENSATION, TERMINATION RATE BY TREATMENT AND DAY(STANDARD DEVIATION FOR EACH REPORTED AS BR CP TM)**Obs TREAT** **DAY _TYPE_** **_FREQ_ BROOD** **COMP TERM** **BR CP** **TM 1** R0041.00001.0000.0.000000.00000.**2**R5040.28250.310090.42000.414120.4000813.5655**3**R9040.31500.342592.16000.528740.5207913.2205**4**R15040.30500.375092.38500.508950.4784312.7749**5**R23040.38000.492592.38500.639640.7080212.7749**6**T10041.00001.0000.0.000000.00000.**7**T15041.44751.467550.39750.680750.6577422.7920**8**T19041.71001.782557.20751.123891.0132928.1397**9**T115041.69251.870057.70251.154480.9232928.8365**10**T123042.11502.762560.16501.444770.8161927.4196**11**T20041.00001.0000.0.000000.00000.**12**T25041.92001.950035.87000.678970.6951722.8245**13**T29042.39502.462540.13000.840890.8934320.9631**14**T215042.39002.495040.25000.838930.9154120.9162Honeybee (Apis mellifera) Semi-Field (Tunnel) TestDP Barcode: D413482 MRID No.: 491587-0132**Obs TREAT** **DAY _TYPE_** **_FREQ_ BROOD** **COMP TERM** **BR CP** **TM 15** T223042.98753.632542.25001.046500.5111022.4170**16**c0041.00001.0000.0.000000.00000.**17**c5042.19002.190024.29500.619840.6198420.4826**18**c9042.69502.695032.72500.867810.8678121.8915**19**c15042.67502.735033.16500.866310.7813421.7193**20**c23043.34003.755034.42751.086830.7700423.6536Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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33 ANOVA FOR BROOD INDEX ON DAY 23

The ANOVA Procedure
**Class Level Information**

**Class Levels**  **Values**

**TREAT** 4 R T1 T2 c

**Number of Observations Read** 16

**Number of Observations Used** 16

Dependent Variable: BROOD

**Source DF**  **Sum of Squares Mean Square**  **F Value Pr > F** 

**Model** 3 20.95701875 6.98567292 5.85 0.0106

**Error** 12 14.31857500 1.19321458

**Corrected Total** 15 35.27559375

**R-Square Coeff Var**  **Root MSE BROOD Mean** 

0.594094 49.52536 1.092344 2.205625

**Source DF**  **Anova SS Mean Square**  **F Value Pr > F** 

**TREAT** 3 20.95701875 6.98567292 5.85 0.0106Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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34Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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35 ANOVA FOR BROOD INDEX ON DAY 23

The ANOVA Procedure

Bonferroni (Dunn) t Tests for BROOD

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. **Alpha** 0.05

**Error Degrees of Freedom** 12

**Error Mean Square** 1.193215

**Critical Value of t** 3.15268

**Minimum Significant Difference** 2.4351

**Means with the same letter are not significantly different.** 

**Bon Grouping Mean**  **N TREAT** 

A 3.3400 4 c

A

A 2.9875 4 T2

A

B A 2.1150 4 T1

B

B 0.3800 4 RHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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36 NONPARAMETRIC COMPARISON OF BROOD INDEX ON DAY 23

The NPAR1WAY Procedure **Wilcoxon Scores (Rank Sums) for Variable BROOD Classified by Variable TREAT** 

**TREAT N**  **Sum of**
**Scores Expected** 
**Under H0 Std Dev** 
**Under H0 Mean** 
**Score**

**R** 4 12.0 34.0 8.240146 3.000

**T1** 4 32.0 34.0 8.240146 8.000

**T2** 4 45.0 34.0 8.240146 11.250

**c** 4 47.0 34.0 8.240146 11.750

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 8.5935

**DF** 3

**Pr > Chi-Square** 0.0352Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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38 ANOVA FOR COMPENSATION ON DAY 23

The ANOVA Procedure
**Class Level Information**

**Class Levels**  **Values**

**TREAT** 4 R T1 T2 c

**Number of Observations Read** 16

**Number of Observations Used** 16

Dependent Variable: COMP

**Source DF**  **Sum of Squares Mean Square**  **F Value Pr > F** 

**Model** 3 27.41336875 9.13778958 18.08 <.0001

**Error** 12 6.06492500 0.50541042

**Corrected Total** 15 33.47829375

**R-Square Coeff Var**  **Root MSE COMP Mean** 

0.818840 26.72012 0.710922 2.660625

**Source DF**  **Anova SS Mean Square**  **F Value Pr > F** 

**TREAT** 3 27.41336875 9.13778958 18.08 <.0001Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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39Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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40 ANOVA FOR COMPENSATION ON DAY 23

The ANOVA Procedure

Bonferroni (Dunn) t Tests for COMP

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. **Alpha** 0.05

**Error Degrees of Freedom** 12

**Error Mean Square** 0.50541

**Critical Value of t** 3.15268

**Minimum Significant Difference** 1.5848

**Means with the same letter are not significantly different.** 

**Bon Grouping Mean**  **N TREAT** 

A 3.7550 4 c

A

A 3.6325 4 T2

A

A 2.7625 4 T1

B 0.4925 4 RHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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41 NONPARAMETRIC COMPARISON OF COMPENSATION INDEX ON DAY 23

The NPAR1WAY Procedure **Wilcoxon Scores (Rank Sums) for Variable COMP Classified by Variable TREAT** 

**TREAT N**  **Sum of**
**Scores Expected** 
**Under H0 Std Dev** 
**Under H0 Mean** 
**Score**

**R** 4 10.0 34.0 8.240146 2.500

**T1** 4 32.0 34.0 8.240146 8.000

**T2** 4 47.0 34.0 8.240146 11.750

**c** 4 47.0 34.0 8.240146 11.750

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 10.1399

**DF** 3

**Pr > Chi-Square** 0.0174Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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43 ANOVA FOR TERMINATION RATE ON DAY 23

The ANOVA Procedure
**Class Level Information**

**Class Levels**  **Values**

**TREAT** 4 R T1 T2 c

**Number of Observations Read** 16

**Number of Observations Used** 16

Dependent Variable: TERM

**Source DF**  **Sum of Squares Mean Square**  **F Value Pr > F** 

**Model** 3 7955.27607 2651.75869 5.37 0.0142

**Error** 12 5931.14608 494.26217

**Corrected Total** 15 13886.42214

**R-Square Coeff Var**  **Root MSE TERM Mean** 

0.572882 38.79466 22.23201 57.30688

**Source DF**  **Anova SS Mean Square**  **F Value Pr > F** 

**TREAT** 3 7955.276069 2651.758690 5.37 0.0142Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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45 ANOVA FOR TERMINATION RATE ON DAY 23

The ANOVA Procedure

Bonferroni (Dunn) t Tests for TERM

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. **Alpha** 0.05

**Error Degrees of Freedom** 12

**Error Mean Square** 494.2622

**Critical Value of t** 3.15268

**Minimum Significant Difference** 49.561

**Means with the same letter are not significantly different.** 

**Bon Grouping Mean**  **N TREAT** 

A 92.39 4 R

A

B A 60.17 4 T1

B

B 42.25 4 T2

B

B 34.43 4 cHoneybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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46 NONPARAMETRIC COMPARISON OF TERMINATION RATE ON DAY 23

The NPAR1WAY Procedure **Wilcoxon Scores (Rank Sums) for Variable TERM Classified by Variable TREAT** 

**TREAT N**  **Sum of**
**Scores Expected** 
**Under H0 Std Dev** 
**Under H0 Mean** 
**Score**

**R** 4 55.0 34.0 8.240146 13.750

**T1** 4 36.0 34.0 8.240146 9.000

**T2** 4 25.0 34.0 8.240146 6.250

**c** 4 20.0 34.0 8.240146 5.000

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 7.9750

**DF** 3

**Pr > Chi-Square** 0.0465Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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47Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

48 MEAN COLONY WEIGHT BY TREATMENT AND DAY

**Obs TREAT**  **DAY _TYPE_**  **_FREQ_ MEAN**  **STD**

**1** R 0 0 4 23.550 0.57446

**2** R 5 0 4 22.425 0.38622

**3** R 9 0 4 21.850 0.61373

**4** R 15 0 4 21.150 1.42712

**5** R 23 0 4 20.775 0.80156

**6** R 29 0 4 20.925 1.75381

**7** T1 0 0 4 24.025 2.03695

**8** T1 5 0 4 23.400 1.05515

**9** T1 9 0 4 22.100 2.09603

**10** T1 15 0 4 21.675 2.53952

**11** T1 23 0 4 19.725 1.17580

**12** T1 29 0 4 20.925 1.41980

**13** T2 0 0 4 25.800 1.73781

**14** T2 5 0 4 24.375 1.28420

**15** T2 9 0 4 22.875 1.06262

**16** T2 15 0 4 22.625 1.52834

**17** T2 23 0 4 20.475 1.24466

**18** T2 29 0 4 21.750 1.35277

**19** c 0 0 4 25.700 0.46904

**20** c 5 0 4 24.350 0.43589

**21** c 9 0 4 24.150 0.79373

**22** c 15 0 4 22.975 0.60208

**23** c 23 0 4 23.350 0.71880Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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49 **Obs TREAT**  **DAY _TYPE_**  **_FREQ_ MEAN**  **STD**

**24** c 29 0 4 22.450 1.26095

ANOVA FOR COLONY WEIGHT ON DAY 23

The ANOVA Procedure
**Class Level Information**

**Class Levels**  **Values**

**TREAT** 4 R T1 T2 c

**Number of Observations Read** 16

**Number of Observations Used** 16

ANOVA FOR COLONY WEIGHT ON DAY 23

The ANOVA Procedure

Dependent Variable: WEIGHT

**Source DF**  **Sum of Squares Mean Square**  **F Value Pr > F** 

**Model** 3 29.79187500 9.93062500 9.71 0.0016

**Error** 12 12.27250000 1.02270833

**Corrected Total** 15 42.06437500

**R-Square Coeff Var**  **Root MSE WEIGHT Mean** 

0.708245 4.797108 1.011290 21.08125

**Source DF**  **Anova SS Mean Square**  **F Value Pr > F** 

**TREAT** 3 29.79187500 9.93062500 9.71 0.0016Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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50Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

51 ANOVA FOR COLONY WEIGHT ON DAY 23

The ANOVA Procedure

Bonferroni (Dunn) t Tests for WEIGHT

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. **Alpha** 0.05

**Error Degrees of Freedom** 12

**Error Mean Square** 1.022708

**Critical Value of t** 3.15268

**Minimum Significant Difference** 2.2545

**Means with the same letter are not significantly different.** 

**Bon Grouping Mean**  **N TREAT** 

A 23.3500 4 c

B 20.7750 4 R

B

B 20.4750 4 T2

B

B 19.7250 4 T1Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
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52 NONPARAMETRIC COMPARISON OF COLONY WEIGHT

The NPAR1WAY Procedure **Wilcoxon Scores (Rank Sums) for Variable WEIGHT Classified by Variable TREAT** 

**TREAT N**  **Sum of**
**Scores Expected** 
**Under H0 Std Dev** 
**Under H0 Mean** 
**Score**

**R** 4 31.50 34.0 8.240146 7.8750

**T1** 4 19.00 34.0 8.240146 4.7500

**T2** 4 27.50 34.0 8.240146 6.8750

**c** 4 58.00 34.0 8.240146 14.5000

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 9.3833

**DF** 3

**Pr > Chi-Square** 0.0246Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

53Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

54 ANOVA FOR COLONY WEIGHT ON DAY 29

The ANOVA Procedure
**Class Level Information**

**Class Levels**  **Values**

**TREAT** 4 R T1 T2 c

**Number of Observations Read** 16

**Number of Observations Used** 16

Dependent Variable: WEIGHT

**Source DF**  **Sum of Squares Mean Square**  **F Value Pr > F** 

**Model** 3 6.50250000 2.16750000 1.02 0.4186

**Error** 12 25.53500000 2.12791667

**Corrected Total** 15 32.03750000

**R-Square Coeff Var**  **Root MSE WEIGHT Mean** 

0.202965 6.780886 1.458738 21.51250

**Source DF**  **Anova SS Mean Square**  **F Value Pr > F** 

**TREAT** 3 6.50250000 2.16750000 1.02 0.4186Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

55Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

56 ANOVA FOR COLONY WEIGHT ON DAY 29

The ANOVA Procedure

Bonferroni (Dunn) t Tests for WEIGHT

Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. **Alpha** 0.05

**Error Degrees of Freedom** 12

**Error Mean Square** 2.127917

**Critical Value of t** 3.15268

**Minimum Significant Difference** 3.2519

**Means with the same letter are not significantly different.** 

**Bon Grouping Mean**  **N TREAT** 

A 22.450 4 c

A

A 21.750 4 T2

A

A 20.925 4 R

A

A 20.925 4 T1Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

57 NONPARAMETRIC COMPARISON OF COLONY WEIGHT ON DAY 29

The NPAR1WAY Procedure **Wilcoxon Scores (Rank Sums) for Variable WEIGHT Classified by Variable TREAT** 

**TREAT N**  **Sum of**
**Scores Expected** 
**Under H0 Std Dev** 
**Under H0 Mean** 
**Score**

**R** 4 28.50 34.0 8.215838 7.1250

**T1** 4 25.50 34.0 8.215838 6.3750

**T2** 4 38.00 34.0 8.215838 9.5000

**c** 4 44.00 34.0 8.215838 11.0000

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 2.4278

**DF** 3

**Pr > Chi-Square** 0.4885Honeybee ( Apis mellifera ) Semi-Field (Tunnel) Test
DP Barcode: D413482 MRID No.: 491587-01

58