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 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

 WASHINGTON D.C., 20460

OFFICE OF CHEMICAL SAFETY

 AND POLLUTION PREVENTION

 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 (EFED) 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 Application 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 technical registrant, Nippon Soda Co., Ltd., in response 

to a generic data call-in1

 for a field pollinator study (OCSPP Guideline 850-30402). The 

preliminary problem formulation written in support of the Registration Review of tebufenozide3

identified that a field pollinator toxicity test using the honeybee (Apis mellifera) is conditionally 

required under 40CFR 1584

 data requirements for conventional pesticides. 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 sufficient 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 and 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 Species, 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 Effects) §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, 

Thomas

Digitally signed by Steeger, 

Thomas 

DN: cn=Steeger, Thomas, 

email=steeger.thomas@epa.gov 

Date: 2014.07.08 05:06:14 -04'00'
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with bees. While the acute toxicity studies of honeybees indicate that the technical grade 

tebufenozide is practically non-toxic to young adult honey bees on an acute contact exposure basis 

(LD50>234 μg/bee; MRID 424362-445

), 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 756

. The study is classified as supplemental due to 

uncertainties related to the extent to which bees foraged on the treated crop as well as the statistical 

power of the study to discriminate treatment effects. In spite of these uncertainties, the study 

provides useful information for qualitatively assessing 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 questions/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 of 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)22
Honeybee (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 EcoChem 

GmbH, Eutinger Str. 24, D-75223 NiefernOschelbronn, Germany 

 

Sponsor: Nippon Soda Co., Ltd. Shin-Ohtemachi 

Bldg., 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 751

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)22 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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 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'
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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 for 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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. 
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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. 
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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 diseasefree 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 - 33.2oC 

Relative humidity 

during exposure

Range: 33.0 - 100% 

Lighting Ambient lighting conditions. 
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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 (5th) 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 were at least 

5 bees/m2 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 30oC or drop below 15oC. 

Tunnels were covered with a fine 

plastic mesh. Colonies were 

acclimated to the tunnels for 6 days 

before application (DBA) of test 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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Guideline Criteria Reported Information

material. For 5 DBA, data were 

collected on bee behavior and 

mortality. Foraging activity in a 1 

-m2 area of phacelia for 10 15 seconds 

were monitored the day of application 

and for 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 fixing day 0; 

BFD 0) and 4, 8, 14, 22 and 27 DAA 

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 study 
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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?

Yes 

 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 m2

(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 m2

) 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 

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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 5th 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 

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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. 
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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 

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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 <LOQT <LOQT

<LOQF nd 0.984T

2.58T 

<LOQM 

<LOQF

4 DAA nd nd <LOQT nd 1.48T 

<LOQM 

4.62T 

0.01M

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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Table 3. Mean mortality by treatment group (Controls Ca-d; 

Tebufenozide T1a-d; Tebufenozide T2a-d; fenoxycarb Ra-d) across preapplication )5DBA - 0DBA), day of application (DAA0) and postapplication (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 

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Figure 3. Total mortality of worker bees in control (C), tebufenozidetreated (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. 
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The mortality index, used to illustrate the incidences of 

treatment effect on mortality relative to controls, 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 incidence 

of 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/m2

 in control, T1, T2 and fenoxycarb-treated tunnels, 

respectively. Following application, foraging activity averaged 

11.6, 25.1, 17.6 and 15.8 bees/m2

 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/m2

 in the control, T1, T2 and 

fenoxycarb-treated tunnels, respectively, and this reduced 

activity was attributed to rainfall. 
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Figure 4. Mean foraging activity (mean number of forager bees/m2) 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 to 
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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 the 
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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 for 
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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 that 
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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. 
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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. 
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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. 
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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 (T2e) is 
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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 was 

significantly (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 was 
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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 marked 

as 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 0.38 
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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. 
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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 similar 
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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. 
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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 R 0 0 4 1.0000 1.0000 . 0.00000 0.00000 . 2 R 5 0 4 0.2825 0.3100 90.4200 0.41412 0.40008 13.5655 3 R 9 0 4 0.3150 0.3425 92.1600 0.52874 0.52079 13.2205 4 R 15 0 4 0.3050 0.3750 92.3850 0.50895 0.47843 12.7749 5 R 23 0 4 0.3800 0.4925 92.3850 0.63964 0.70802 12.7749 6 T1 0 0 4 1.0000 1.0000 . 0.00000 0.00000 . 7 T1 5 0 4 1.4475 1.4675 50.3975 0.68075 0.65774 22.7920 8 T1 9 0 4 1.7100 1.7825 57.2075 1.12389 1.01329 28.1397 9 T1 15 0 4 1.6925 1.8700 57.7025 1.15448 0.92329 28.8365 

10 T1 23 0 4 2.1150 2.7625 60.1650 1.44477 0.81619 27.4196 11 T2 0 0 4 1.0000 1.0000 . 0.00000 0.00000 . 12 T2 5 0 4 1.9200 1.9500 35.8700 0.67897 0.69517 22.8245 13 T2 9 0 4 2.3950 2.4625 40.1300 0.84089 0.89343 20.9631 14 T2 15 0 4 2.3900 2.4950 40.2500 0.83893 0.91541 20.9162 
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Obs TREAT DAY _TYPE_ _FREQ_ BROOD COMP TERM BR CP TM 

15 T2 23 0 4 2.9875 3.6325 42.2500 1.04650 0.51110 22.4170 16 c 0 0 4 1.0000 1.0000 . 0.00000 0.00000 . 17 c 5 0 4 2.1900 2.1900 24.2950 0.61984 0.61984 20.4826 18 c 9 0 4 2.6950 2.6950 32.7250 0.86781 0.86781 21.8915 19 c 15 0 4 2.6750 2.7350 33.1650 0.86631 0.78134 21.7193 20 c 23 0 4 3.3400 3.7550 34.4275 1.08683 0.77004 23.6536 
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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 0.0106 
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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 R 

 
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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 0.0352 
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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 <.0001 
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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 R 

 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.0174 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.0142 
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Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 c 

 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.0465 
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Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.71880 
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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 0.0016 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 T1 

 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.0246 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.4186 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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Honeybee (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 T1 

 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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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 0.4885 
Honeybee (Apis mellifera) Semi-Field (Tunnel) Test 

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