1 

# Data Evaluation Report

Study Titles:

Bocksch, S. (2014): Honey bee brood and colony level effects

following imidacloprid intake via treated artificial diet in a field

study in North Carolina

Final Report Source:

Eurofins Agroscience Services Inc., unpublished report No: S13

- 03176, 06 Nov 2014

Year of study: 2013

- 2014

PMRA#: 2474495

PMRA DACO#: 9.2.4.9

MRID : 49510001

Study Type:

Tier II colony feeding study conducted in an open field

Review Date (final) : January 28, 2016

PMRA reviewer:

Primary Evaluator: Wayne Hou

Data Statistical Analysis : Keith O'Rourke

E PA reviewer:

Primary Evaluators: Justin Housenger , Biologist

Keith G. Sappington , Senior Science Advisor

Data Statistical Analysis: Christine Hartless , Wildlife Biologist

CDPR reviewer:

Primary Evaluators: Richard Bireley, Sr. Environmental Scientist (Specialist)

Alexander Kolosovich, Environmental Scientist

Brigitte Tafarella, Environmental Scientist

Data Statistical Analysis: John Troiano, Ph.D., Research Scientist III2 

## Table of Contents

Executive Summary

................................ ................................ ................................ ................................ ....... 7

Consideration of Study Strengths, Limitations and Interpretation

................................ .......................... 9

1. Study Objective

................................ ................................ ................................ ................................ ... 12

2. Study Methods

................................ ................................ ................................ ................................ .... 12

2.1. Test crop

................................ ................................ ................................ ................................ ..... 12

2.2. Test chemical

................................ ................................ ................................ .............................. 12

2.3. Test sites

................................ ................................ ................................ ................................ ..... 12

2.4. Test organisms

................................ ................................ ................................ ............................ 14

2.5. Tre atments

................................ ................................ ................................ ................................ .. 15

2.5.1. Preparation of stock solution

................................ ................................ .............................. 16

2.5.2. Preparation of sugar solution

................................ ................................ ............................. 16

2.5.3. Preparation of feeding solution

................................ ................................ .......................... 16

2.5.4. Artificial Feeding

................................ ................................ ................................ ................. 17

2.6. Meteorological Data

................................ ................................ ................................ ................... 17

2.7. Observations

................................ ................................ ................................ ............................... 17

Important activity and dates

................................ ................................ ................................ ............... 17

2.7.1. Colony mortality

................................ ................................ ................................ .................. 19

2.7.2. Colony Condition Assessments (CCA)

................................ ................................ ................. 19

2.7.3. Evaluation of Disease or Pests in the Hive

................................ ................................ ......... 20

2.7.4. Hive weights

................................ ................................ ................................ ........................ 20

2.8. Residue analysis

................................ ................................ ................................ .......................... 20

2.8.1. Pollen from outside sources

................................ ................................ ............................... 21

2.8.2. Stored pollen and nectar in test hives

................................ ................................ ................ 2 1

2.8.3. Feeding solution and stability of test item

................................ ................................ ......... 21

3. Results

................................ ................................ ................................ ................................ ................. 22

3.1. Land use near test hives

................................ ................................ ................................ .............. 22

3.2. Pollen sources of test hives

................................ ................................ ................................ ......... 23

3.3. Consumption of spiked sucrose

................................ ................................ ................................ .. 23

3.4. Examination of pesticides from other sources

................................ ................................ ........... 24

3.5. Confirmation of test concentrations

................................ ................................ ........................... 25

3.6. Stability of the test item in feeding solution

................................ ................................ .............. 263 

### 3.7. Residues in hive matrices

................................ ................................ ................................ ............ 27

3.7.1. Background imidacloprid contamination in hives prior to the feeding exposure

.............. 27

3.7.2. Residues in hive matrices during and after feeding exposure

................................ ............ 27

3.8. Pathogens

................................ ................................ ................................ ................................ ... 36

3.8.1. Varroa Presence

................................ ................................ ................................ .................. 36

3.9. Statistical Analysis

................................ ................................ ................................ ....................... 38

3.9.1. Study Authors Analysis

................................ ................................ ................................ ........ 38

3.9.2. Study Reviewer Analysis

................................ ................................ ................................ ..... 38

3.9.3. EPA Analysis

................................ ................................ ................................ ........................ 38

3.9.4. PMRA Analysis

................................ ................................ ................................ ..................... 39

3.9.5. C DPR Analysis

................................ ................................ ................................ ...................... 39

3.10. Hive mortality

................................ ................................ ................................ .......................... 39

3.11. Colony Condition Assessment Response Variables

................................ ................................ 41

Life Stages

................................ ................................ ................................ ................................ ............... 42

3.11.1. Adults

................................ ................................ ................................ ................................ .. 42

3.11.2. Eggs

................................ ................................ ................................ ................................ ..... 45

3.11.3. Larvae (Open/Uncapped brood)

................................ ................................ ......................... 50

3.11.4. Pupae (Capped brood)

................................ ................................ ................................ ........ 54

3.11.5. Total individuals in hives

................................ ................................ ................................ ..... 58

Colony Condition Assessments

– Food Store Response Variables

................................ ......................... 65

3.11.6. Pollen

................................ ................................ ................................ ................................ .. 65

3.11.7. Nectar / Honey

................................ ................................ ................................ .................... 68

3.11.8

. Hive Weight

................................ ................................ ................................ ......................... 77

4. Reviewer comments:

................................ ................................ ................................ .......................... 79

General Considerations for Biological Interpretation

................................ ................................ ............. 79

Control Performance

................................ ................................ ................................ .............................. 80

Consider ation of CCA8

................................ ................................ ................................ ............................ 81

Control overwintering mortality

................................ ................................ ................................ ......... 81

Response Variables from Surviving Hives

................................ ................................ ........................... 81

Consideration of Study Strengths, Limitations and Interpretation

................................ ........................ 82

5. Conclusions

................................ ................................ ................................ ................................ ......... 854 

## List of Table s

Table 1. Details about the test substance

................................ ................................ ................................ .. 12

Table 2: GPS

- coordinates of the test apiary sites

................................ ................................ ....................... 13

Table 3. Treatment groups, feeding rates and feeding volume

................................ ................................ . 16

Table 4. Hive assignment to test ap iaries

................................ ................................ ................................ .. 16

Table 5. Chronological list of key dates and activities

................................ ................................ ................ 18

Table 6. Schedule for colony assessment and beekeeper checks

................................ .............................. 19

Table 7. LOD for imidacloprid and its metabolites

................................ ................................ ..................... 20

Table 8. Sampling schedule for feeding solution and stability of test chemic al.

................................ ........ 21

Table 9: Percent (%) land use pattern

................................ ................................ ................................ ......... 22

Table 10: Cultivated cropping area near each test apiary

................................ ................................ .......... 23

Table 11. Residues from outside sources in pollen samples from pollen trap (non

- GLP)

.......................... 25

Table 12. Dosing solution residue data from 2 July 2013 (Week 1), 12 July 2013 (Week 2) and 2 August

2013 (Week 5)

................................ ................................ ................................ ................................ ............. 26

Table 13. The stability of imidacloprid in feeding solution on 3 Jul, 12 Jul, and 2 Aug, 2013.

.................. 26

Table 14. Detailed hive pollen residue data, pre

- study collection (30 May 2013)

................................ .... 27

Table 15. Detailed hive nectar residue data, pre

- study sample collection (30 May 2013)

....................... 27

Table 16. Imidacloprid concentrations (ppb) in hive pollen sampled three weeks after the start of

artificial feeding on 18 July 2013 (CCA4).

................................ ................................ ................................ ... 29

Table 17. Imidacloprid concentrations (ppb) in uncapped hive nectar sampled three weeks after the start

of artificial feeding on 18 Jul, 2013 (CCA4).

................................ ................................ ................................ 30

Table 18. Imidacloprid concentrations (ppb) in beebread and uncapped hive nectar sampled one week

after the end of art ificial feeding on 14 Aug, 2013 (CCA5).

................................ ................................ ........ 31

Table 19: Imidacloprid concentrations (ppb) in bee bread and honey sampled after overw intering on

March 24, 2014 (CCA8).

................................ ................................ ................................ .............................. 32

Table 20. Imidacloprid (ppb) detected in test hives before the exposure (CCA2), and in control hives

during the exposure (CCA4) and after the exposure (CCA5 and CCA8).

................................ ..................... 34

2

................................ ................................ ................................ ................................ ................................ .. 34

Table 22. Imidacloprid concentration measured in hive uncapped nectar and hive bee bread

................ 36

Table 23a. Hive survival at CCA8 (after overwintering)

................................ ................................ .............. 40

Table 23b. Hive mortality after overwintering measure at CCA8

................................ .............................. 40

Table 24. Estimated percent reduction from control for number of adults

................................ ............... 43

Table 25. Estimated percent reduction from control for number of eggs

................................ ................. 46

Table 26. Es timated percent reduction from control for number of larvae (open/uncapped brood)

....... 50

Table 27. Estimated percent reduction from control for number of pupae

................................ .............. 54

Table 28. Estimated percent reduction from control for total number of individuals

............................... 58

Table 29. Estimated percent reduction from control for pollen stores

................................ ...................... 65

Table 30. Estimated percent reduction from control for nectar/honey stores

................................ .......... 69

Table 31. Estimated percent reduction from control for hive weights

................................ ...................... 785 

## List of Figures

Figure 1: Location of test apiary sites

................................ ................................ ................................ ......... 13

Figure 2. Layout of test hives in a test site

................................ ................................ ................................ . 15

Figure 3. Mean total food consumption (mL) per colony during the 6

- week exposure period

................ 24

Figure 4. Mean number of Varroa mites per 100 bees

................................ ................................ ............. 37

Figure 5. Mean number of Nosema spores per bee

................................ ................................ ................... 37

Figure 6. Hive mortality after overwintering.

................................ ................................ ............................. 41

Figure 7. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8.

................................ ................................ ................................ ................................ ... 43

Figure 8. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg /L groups only.

................................ ..................... 44

Figure 9. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

acros s CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

................................ ............................. 45

Figure 10. Proportion of eggs following exposure to varying conce ntrations of imidacloprid in the diet

across CCA3

– CCA8.

................................ ................................ ................................ ................................ ... 47

Figure 11. Proportion of eggs following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

................................ ..................... 48

Figure 12. Pr oportion of eggs following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

................................ ............................. 49

Figure 13. Proportion of larval cells following exposure to varying concentrations of imidacloprid in the

diet across CCA3

– CCA8.

................................ ................................ ................................ ............................ 51

Figure 14. Proportion of larval cells following exposure to varying concentrations of imidacloprid in the

diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

................................ .............. 52

Figure 15. Proportion of larval cells following exposure to varying concentrations of imidacloprid in the

diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

................................ ..................... 53

Figure 16. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8.

................................ ................................ ............................. 55

Figure 17. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

.............. 56

Figure 18. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

...................... 57

Figure 19. Proportion of total individuals (adult, eggs, larvae, pupae) following exposure to varying

concentrations of imidacloprid in the diet across CCA3

– CCA8.

................................ ............................... 59

Figure 20. Summary of living organism parameters at the 12.5 μg/L treatment group

............................ 60

Figure 21. Summary of living organism parameters at the 25 μg/L treatment group

............................... 61

Figure 22. Summary o f living organism parameters at the 50 μg/L treatment group

............................... 61

Figure 23. Trends of life stages across treatment groups within CCA4

................................ ...................... 62

Figure 24. Trends of life stages across treatment groups within CCA5

................................ ...................... 63

Figure 25. Trends of life stages across treatment groups within CCA

................................ ........................ 63

Figure 26. Trends of life stages across treatment groups within CCA7

................................ ...................... 64

Figure 27. Trends of life stages across treatment groups within CCA8

................................ ..................... 656 

### Figure 28. Proportion of pollen stores following exposure of honey bees to varying concentratio ns of

imidacloprid in the diet across CCA3

– CCA8.

................................ ................................ ............................. 66

Figure 29. Proportion of pollen stores following exposure to varying concen trations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

................................ ....... 67

Figure 30. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

................................ ............... 68

Figure 31. Proportion of honey stores following exposure of honey bees to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8.

................................ ................................ ............................. 70

Figure 32. Proportion of honey stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

................................ ....... 70

Figure 33. Proportion of honey stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CC A8 in the control, 12.5, 25, and 50 μg/L groups.

................................ ............... 71

Figure 34. Summary of hive food supply parameters at the 12.5 μg/L treatment group

.......................... 73

Figure 35. Summary of hive food supply parameters at the 25 μg/L treatment group

............................. 73

Figure 36. Summary of hive food supply parameters at the 50 μg/L treatment group

............................. 74

Figure 37. Trends of food store parameters across treatment groups within CCA4

................................ .. 75

Figure 38. Trends of food store parameters across treatment groups at CCA5

................................ ......... 75

Figure 39. Trends of food store parameters across tr eatment groups at CCA6

................................ ......... 76

Figure 40. Trends of food store parameters across treatment groups at CCA7

................................ ......... 76

Figure 41. Trends of food store parameters across treatment groups at CCA8

................................ ......... 77

Figure 42. Proportion of hive weight following exposure of honey bees to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8

................................ ................................ .............................. 78

Figure 43. Fate of control hives as compared to number of adults across CCAs

................................ ....... 81

Figure 44. Fate of control hives as compared to the honey stores across CCAs

................................ ....... 82



## Table of Appendices

Appendix A: Details of EPA Statistical Analysis

................................ ................................ ........................... 91

Appendix B: Details of PMRA Statistical Analysis

................................ ................................ ..................... 107

Appendix C: Details of CDPR Statistical Analysis

................................ ................................ ....................... 1467 

## Executive **Summary**

A colony feeding study was conducted with honey bees to assess t he potential for long

- term effects,

including overwintering survival

, resulting from exposure to imidacloprid . The study was

conducted in twelve test areas of low agricultural cultivation (Apiaries A

– L) in North Carolina

from June 21, 2013 to March 24, 2014. Eighty

- four hives were divided according to hive strength

(number of brood frames) with the strongest 7 hives ssigned to Apiary A and the weakest 7 hives

assigned to Apiary L. Within each apiary, the 7 hives were randomly assigned to treatment groups.

At each apiary, five test hives were artificially fed with 50% sugar solution spiked with

imidacloprid at 12.5, 25, 50, 100 or 200 μg ai/L for six weeks continuously in the field , with two

hives at each apiary serving as controls . Assuming the density of a 50% sugar solution is 1.2296

g/ml, the reviewer calculated that the test concentrations at 12.5, 25, 50, 100, and 200 μg/L are

equivalent to 10.2, 20.3, 40.7, 81.3, and 162.7 ppb (μg/kg), respectively. The average measured

ppb (μg/kg) concentrations in dosing solutions was confirmed to be 11.0, 23.3, 46.7, 96.3, 189.6

ppb (μg/kg), respectively.

Eight Colony Condition Assessments (CCAs) were conducted during the study. Three CCAs

(CCA1

- 3) were conducted prior to feeding to determine hive strength and initial hive conditions.

A CCA was conducted during exposure with another one conducted one week afte r termination of

exposure (CCA4 and CCA5, respectively ) which characterize hive conditions during exposure.

Two more CCAs were conducted at 5 and 10 weeks after exposure (CCA6 and CCA7, respectively )

to assess the chronic effect following exposure to imid acloprid and to characterize pre

overwintering hive conditions. A final CCA was conducted after overwintering in March 2014

(CCA8) to assess potential exposure impact on survival and chronic colony level effects. Multiple

parameters, such as hive weight, number of individuals at different life stages in the hive, hive

honey and pollen stores, and hive overwintering survival, were measured during the course of the

study.

Levels of imidacloprid residues in hives were measured before, during and after the f eeding

exposure. Potential contamination of pesticides from other food sources was monitored using

pollen collected in additional hives at each apiary that served as monitoring hives

. The results

showed that while there were a few instances of imidacloprid detected in the pollen and nectar of

the control hives, the frequency and magnitude of these detections is not expected to confound the

results of this study. Residues measured in hive matrices demonstrated that higher treatment

exposures corresponded wel l to higher residues in hive matrices. There were individual hive

variations in measured residues, with some overlap in measured hive concentrations, particularly

at the lower doses. This variability likely originates from the limited spatial and temporal sampling

methodology ( i.e. one sample from one side of the comb on one frame to represent a hive, and

only at 3 CCAs) employed for this study. Overall during the exposure period, imidacloprid

concentration in hive nectar averaged 63.7% of the nominal con centration in the feeding solution,

whereas imidacloprid concentration in hive pollen averaged 30.2% of the nominal concentration

in feeding solution. This dilution is expected since bees could forage on outside pollen and nectar

sources, and hive pollen ( bee bread) includes only some nectar

. See Section 3 .7 for more details

regarding the residues of imidacloprid in the dosing solutions and hive matrices.8 

### During the review of the study, a joint review effort was conducted by the United States

Environmental Protection Agency (EPA), Health Canada’s Pest Management Regulatory Agency

(PMRA), and the California Department of Pesticide Regulation (CDPR). As part of that effort,

a separate stat istical analysis was conducted by each regulatory entity as an independent

verification of the results from the analysis provided by the registrant. These analyses (described

in detail in Appendices A, B, and C for EPA’s, PMRA’s and CDPR’s approaches, res pectively)

were distinct in approach but generally yielded similar statistical results. When weighing these

results as well as biological concerns, particularly as they relate to honey bee biology at the colony

level, EPA, PMRA, and CDPR arrived at the sa me conclusions and are therefore harmonized in

terms of the determination of an overall No Observed Adverse Effect Concentration (NOAEC)

and Lowest Observed Adverse E ffect Concentration (LOAEC) for this study.

As will be discussed in Section 3 (Results) the PMRA, EPA, and CDPR analyses determined

statistically significant imidacloprid dose

- related effects in the 50, 100, and 200 μg/L treatment

groups across multiple CCAs for the majority of response variables. Indeed, for the 100 and 200

μg/L treatment g roups, significant effects (p<0.05) were determined for every response variable

, persisted across multiple CCAs and eventually resulted in loss of nearly all hives in those treatment

groups after the overwintering period. The 50 μg/L treatment group also s howed significant effects

for multiple response variables across multiple CCAs, and poorer colony condition in surviving

hives after overwintering in comparison to controls.

Conversely, there was not a strong indication from the PMRA, EPA, and CDPR analyse s of an

impact at the colony level for the 12.5 and 25 μg/L treatment group s for individual life stages . This

is evidenced not only by a general lack o f statistically significant effects (p>0.1) at these treatment

levels but in cases where significant effects were determined, they either did not show strong dose

responsive ness, did not persist across multiple CCAs, or were considered potential transient effects

which did not persist after overwintering . This latter point was the case for the statistica lly

significant effects noted at CCA6, which included pupal cell and total individual effects for which

the PMRA analyses determined significant effects at all treatment levels , and EPA determined

significant effects for pupae at 12.5 , 100 and 200 μg/L (but not at 25 or 50 μ g a.i/L)

. As well at

CCA6, PMRA determined significant effects with eggs and larvae at 25 μg/L treatment (but not

at the 50 μg/L). For the two lowest treatment groups ( 12.5 and 25 μg/L), the colony condition of

surviving hives at CCA8 following overwintering was similar to controls, indicating the effects

observed at CCA6 were likely transient and the colony was able to compensate for these effects.

When examining the effects on food stores (pollen and nectar), the PMRA

, EPA , and CDPR

analyses did not determine any consistent and significant reductions in pollen and nectar stores at

the 12.5 and 25 μg/L treatment groups. This is distinguished from the 50 μg/L group where effects

on nectar in particular were very apparent

, when compared alongside the response of the control

, in terms of the le vel of nectar buildup before hive preparation for overwintering at CCA7. This

finding was also confirmed statistically in all three analyses with significant reductions in honey

stores at CCAs 6, 7, and 8 (CCA8 data excluded from the EPA analysis for the 100 and 200 μg/L

groups ). Significant reducti ons in pollen stores were also confirmed at CCAs 4 and 5 ( i.e. during

the exposure period) at the 50 μg/L treatment group.

Specifically, when considering the proportion of adults as well as honey and pollen stores response

variables, the differences from control were apparent both visually and statistically, particularly in9 

### the three highest treatment groups. For the proportion of adults, the onset of a decline in numbers

occurred one CCA earlier in these groups than in the control, 12.5 and 25 μg/L treat ment groups. For honey stores, the buildup that occurred starting at CCA5 in the 50 μg/L treatment group, reached only half the level reached in the control, 12.5, and 25 μg/L treatment groups by CCA7.

Pollen stores were also reduced at CCA4 and CCA 5 comp ared to controls for the three highest

treatment groups, as well as at CCA6 and CCA7 at the highest treatment group. These effects were

statist ically significant (p<0.05) and indicate that the 50 μg/L treatment group was associated with

trends and proportions of abundance for life stages and food stores not observed in the control,

12.5, and 25 μg/L treatment groups.

The study is considered to be informative and will be used as a line of evidence in the pollinator

risk assessment . While there were uncertainties that were generally related to inherent aspects of

any semi

- field or full field study design (described in the section below) this study still provides

information on a number of colony health parameters about the long term (including overwintering)

exposure to imidacloprid at the colony level. When weighing biological significance and the

natural seasonal changes of honey bee colonies, as well as supporting conclusions from the

statistical ap proach es used by PMRA, EPA, and CDPR, the NOAEC and LOAEC for this

study are determined to be 25 and 50 μg/L , respectively.



## Consideration of Study Strengths, Limitations and Interpretation

It is important to recognize the inherent strengths and limitations of this study as results are

interpreted and potentially considered in risk assessment.

In the context of available field studies involving honey bees and imidacloprid, this study contains

a number of strengths including:

• Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power

• Demonstration of a general ized concentration

- response relationship with respect to the

concentration of imidacloprid in sucrose solution and the magnitude and duration of

adverse effects

• Quantification of exposure to parent (imidacloprid) and toxicologically

- relevant

metabolites in diet and in hive matrices ( uncapped nectar, pollen, honey , bee bread )

• Use of a 6

- week exposure duration to represent a “high end” exposure scenario

• Inclusion of multiple colony

- level endpoints reflecting hive strength, brood development

and food stores

• De tailed QA/QC results regarding quantification of chemical residues in various matrices

• Availability of raw data for conducting statistical analysis

.

A number of limitations are also noted with this study, including:

• Exposure of bees to imidacloprid occurr ed through nectar (sucrose) alone, whereas bees in

the field are likely exposed through both pollen and nectar routes. Therefore, the design

of this study may not reflect a “worst case” exposure scenario in which bees are

experiencing prolonged exposure t o both contaminated nectar and pollen. While exclusion10 

### of the pollen route is expected to reduce overall exposure, the impact of this exclusion on the study results is uncertain and will likely depend on the life stage/caste of bee

.

• It is noted that imidacloprid was found in both hive nectar and hive pollen (beebread), at

concentrations lower than the feeding solutions. Dilution compared to the treatment

feeding solution is expected since bees could also forage on outside nectar and pollen

sources. A dditionally , hive pollen contains only some hive nectar, thus would not be

expected to have a concentration equivalent to nectar alone, and it is mixed with pollen

which will come from outside sources. Therefore exposure through both hive pollen and

nectar occurred via exposure to the sucrose feeding solution, but how this compares to

exposure through contaminated pollen directly is not known. A recent paper by Dively

(2015) 1 

### showed that higher residues throughout the hive resulted from feeding pollen

treat ments compared to feeding sucrose solution treatments. It is also noted that while

nectar is considered the dominant exposure route for forager bees; other hive bees and

larvae consume both nectar and pollen. In addition, since bees were forced to forage f or pollen in this study, the potential impact of imidacloprid exposure on reducing pollen

foraging efficiency of bees could be incorporated into the overall expression of adverse

effects, as suggested by published literature. Had contaminated pollen been provided to

bees, it is not known if the potential impact on pollen foraging efficiency would have been

masked.

• The quantity of nectar provided to hives (2 L per week per hive) likely did not fulfill the

complete carbohydrate needs of the colony, as ind icated by colony bioenergetics and the

lack of remaining sucrose solution upon their renewal. This suggests that bees could be

exposed to a greater dose of imidacloprid in nectar had a greater volume of spiked sucrose

been provided. Although one can infe r that the dosing regimen may have underestimated

exposure through sucrose relative to 100% contaminated diet, it is also noted that bees had

to supplement their spiked sucrose by foraging on their own for other sources of nectar.

As with the previous dis cussion of pollen, it is noted that had 100% of the carbohydrate

needs of the colony been provided via feeders, the potential impact of purported reductions

in nectar foraging efficiency may have been masked to some degree.

• Overwintering success of contro ls was impacted (36% hive mortality)

. This may have

reduced the ability to detect adverse effects related to hive loss following overwintering.

Although comparable to overwintering losses of commercial beekeepers, it is possible that

elements of the stud y design may have contributed to this loss (e.g., lack of supers to allow

for colony growth, delayed supplemental feeding during fall).

• Hive detections with pesticides from food sources other than the artificial feeding was

detected d uring the exposure period and post

- exposure periods through collection of pollen

from pollen traps . Although the study was deliberately conducted in a low agricultural area

in order to minimize the potential for pesticide contamination from other sources, the bees

still appe ared to be foraging on contaminated pollen and possibly nectar. During both

1 Dively GP, Embrey MS, Kamel A, Hawthorne DJ, Pettis JS (2015) Assessment of Chronic Sublethal Effects of

Imidacloprid on Honey Bee Colony Health. PLoS ONE 10(4): e0126043. doi: 10.1371/journal.pone.012604311 

### exposure and post

- exposure periods, a high level of multiple pesticides that may cause

concern for bees were detected in most monitoring hives , such as spiromesifen (maximum

at 961 ppb) and piperonyl butoxide (maximum at 591 ppb). Trace amount s of other bee

toxic pesticides, such as chlorpyifos (LOD = 1.0 ppb) and malathion (LOD = 4.0 ppb) were

also detected. The test chemical imidacloprid was found at 12.1 ppb in pollen from one (a piary L) of the total of six sites analysed. This level is similar to one of the test

concentrations.

• R esidues of imidacloprid in uncapped nectar and bee bread with in the hives at CCAs 4, 5,

and 8 represent a single sample per hive on a single frame rather than a composite sample

from multiple portions of the comb within a hive. This means that residue results may

reflect “hit or miss” scenario with respect to detecting residues in nectar laid down from

con taminated (fed) vs. outside sources.

• The exposure, based on residues measured in the hive (hive nectar and hive pollen)

indicated that, overall, higher measured hive residues correlated with higher nominal

residues in feeding solutions. However, individ ual hive residue values varied, and there

was some overlap in measured values, particularly among the three lowest doses. Given

the limited spatial and temporal sampling methodology (as mentiond above), there is

uncertainy in whether these residues represe nt actual in hive residues across all portions

of the frame. Specifically, one sample of one area of the comb on one side of the frame

to represent the nectar or pollen residues of an entire hive may not reflect the true nature

of the residues across all portions of a given hive

.

• E xposure dilution during the study was evident . P ollen stor age was observed consistently

in the control hives and hives exposed to low er test concentrations during the exposure

period , indicating that test bees were foraging on food sources other than the spiked sugar

solution . Remarkably lower residue concentrations detected in bee bread and hive nectar

in some test hives compared to the feeding concentrations may also indicate foraging on

other food sources. This uncertainty is inherent in any semi

- field or full

- field study design.12 

## 1. **Study Objective**

To determine the potential long term effects on the honey bee ( Apis mellifera L.) colony health

during and after dietary intake of imidacloprid, including the potential effect s on overwintering.

The long term exposure allows for the characterization and distinction of short

- term versus a persistent nature of effects.



## 2. **S tudy Methods** 

**2.1. Test crop** 

Not appl ied . The study was conducted in an open field where multiple field flowers were available

and may serve as food sources for the test bees, in addition to the artificial feeding of spiked sugar

solution

.



## 2.2. **Test chemical**

The test substance was technical imidacloprid. Further details are provided in Table 1 below.

1 Table 1

. Details about the test substance



## 2.3. **Test s ites** 

The field and sampling phases of this study were conducted by Eurofins Agroscience Services Inc.,

Cedar Grove Research Station Mebane, NC, USA; the analytical phase was conducted by Bayer

CropScience in Durham, NC, USA. The apiary sites were located in the vicinity of the EASI Cedar

Grove Research Station in Orange, Caswell, Person and Alamance counties, North Carolina.

There were 12 apiaries separated by more than 1 mile. L and use s urveys in 1

- mile radius and 3

- mile radius were conducted. Pollen species identification and multiple pesticide analysis were

conducted using pollen samples collected from the monitoring hives to characterize outside food

sources of the test bees and contamination. Pollen samples were collected for a period of 24

- 48

hours usin g pollen traps at 5 times during the feeding exposure period (Jun 28, July 3, 12, and 19,

and Aug 2 , 2013 ) and 1 time after the exposure (Oct 17 , 2013 ). Test Item

Name Imidacloprid TC Batch number : EDE0015669

Test item code : NC

- 0116 Appearance / colour : Solid / beige, light

Formulation type : Technical compound Intended Usage : Insecticide

Active ingredient : imidacloprid Content of a.i. analysed : 98.7 %

CAS number : 138261

- 41

- 3

Density (20 °C)

analysed : Not applicable Risk symbol(s) : Not available

Date of analysis : 17 July 2014 Expiry date : 17 July 2016

Stability in solution : sufficient for the test purpose

(at least 1h) Storage conditions : ambient13 

### 1 Figure 1: Location of test apiary sites

2 Table 2 : GPS

- coordinates of the test apiary sites

Apiary GPS

- coordinates

A

p i a r y A 36°12’01.33” N, 79°06’33.76” W

Apiary B 36°13’55.12” N, 79°08’58.15” W

Apiary C 36°15’12.77” N, 79°06’11.58” W

Apiary D 36°13’20.79” N, 79°10’51.85” W

Apiary E 36°14’55.69” N, 79°14’13.95” W

A

p i ar y F 36°9’59.15” N, 79°10’18.26” W

Apiary G 36°11’22.53” N, 79°15’59.81” W

Apiary H 36°15’41.51” N, 79°11’47.16” W

Apiary I 36°16’50.40” N, 79°11’00.11” W

Apiary J 36°13’22.39” N, 79°12’29.23” W

Apiary K 36°13’55.09” N, 79°14’21.00” W

Apiary L 36°11’22.12” N, 79°10’10.64” W

From Table 2, page 19 of the study report.14 

## 2.4. **Test organisms** 

The test species was the h oney bee ( Apis mellifera ), Italian race ( Apis mellifera ligustica ). Hives

were established from package bees bought from the commercial bee supplier (J J’s Honey, 5748

Chancey Road, Patterson, GA 31557, USA), typical of the bee stock used in commercial

beekeeping operations. A new queen was introduced into each colony. Al l queens were purch ased

from the package supplier. The colonies were maintained in 10

- frame Langstroth boxes with an

empty deep super on top as a feeder box. In the test field, hives were raised above ground level.

Eighty

- four hives that met the conditions provided below at the third Colony Condition

Assessments (CCA3) were selected for the study. More than 100 inspected hives were screened

based on the outcome of CCA2. Hives were checked for the “appearance” of a healthy colony

with no visible symptoms of Varroa or Nosema , as well as having all stages of brood, a queen, and

some food stores.

• 4

- 8 brood combs containing eggs, larvae or capped cells (except one colony in

the control which contained only 2 brood combs at test start);

• 6

- 10 food combs containing honey and pollen;

Reviewer note: Although the number of adult bees was not considered here as a criteria, it was

estimated to be 7000

- 8000 bee s per hive according to the study report.

Eight y

- four h ives were blocked into 12 apiary sites by brood strength of the colony, starting with

Apiary A as the strongest group of hives, and Apiary L as the weakest group of hives. Assignment

of apiaries to the geographic locations was done randomly.

Hives were moved on 18/19 Jun 2013 to the Cedar Grove site temporarily from their original

apiary locations. On the night of 19 Jun 2013, hives for Apiary sites A

- F were moved to their

study locations and had their CCA 3 on 21 Jun 2013. Hives for Apiary sites G

- L were moved on

21 Jun 2013 and had their CCA 3 on 23 Jun 2013. After evaluating the assessments, 6 hives were

deemed unsuitable and were replaced the morning of 26 Jun 2013, just before exposure began.

CCA3 on the 6 replaced hives were conducted. The replaced hives were A1, B8, G1, F2, I4, and

J1.

There were e ight hives at each site (7 hives for biological assessments and one as the monitoring

hive for pollen sample collection). Each hive was spatially isolated from other treatment rates by

30 feet (9 m) spacing at each apiary site ( Figure 2 ) . Hives were arranged in a semi

- circular pattern,

facing east to west, with 125 feet (38 m) spacing between the two end hives.15 

### 2 Figure 2

. Layout of test hives in a test site

During the study, all hives were treated with one application of Apiguard® (active ingredient:

thymol) following typical apicultural practice for the region. The initial application occurred

immediately after the CCA6 ( 17 Sep , 2013) to prevent high mite lo ads. No treatments for any other

hive pests, predators or diseases were administered to any hives.

To minimize the potential for robbing amongst test hives, hives at 100 and 200 ppb treatment s

w ere removed from all test apiaries in week 10 ( 5

- 6 Sep, 2013 ) immediately before CCA6. The

hives were placed at a separate apiary. Information on the separate apiary was not provided

. For

over wintering, the surviving colonies were fed with 1 L of 2:1 sugar syrup on 13 Dec 2013, 19

Dec 2013, 13 Jan 2014, 20 Jan 2014, 27 Jan 2014, 07 Feb 2014, 18 Feb 2014, 02 Mar 2014 and

11 Mar 2014.

The monitoring hives were used for outsource pollen sample collection. In addition, test solutions

were sealed and placed in monitoring hives in order to assess imidacloprid stabil ity under field test

conditions. These stability solutions were not available as a food source to the monitoring hives.



## 2.5. **Treatment s** 

The re were:

o 6 treatment groups (5 test concentrations and control ): 0, 12.5, 25, 50, 100, or 200

μg/L . At each site, t here were 2 control hives, and one hive for each test concentration.

o 12 replicates per treatment group (apiaries)

,

The individual treatment groups , the respective feeding rates and the respective feeding volumes

are summarized in 3 Table 3

.16 

### 3 Table 3

. Treatment groups, feeding rates and feeding volume

Tr e at m e n t G r o up Code Feeding T i m ing Concentration

a.i. Feeding

Volume

1 : UTC UTC

(T1+ T 2) Twice a week

--- 1000 mL

2 : Lowest Rate T 3 Twice a week 12.5 ppb 1000 mL

3 : L o w rat e T 4 Twice a week 25 ppb 1000 mL

4 : Moderate rate T 5 Twice a week 50 ppb 1000 mL

5: High rate T 6 Twice a week 100 ppb 1000 mL

6: Highest rate T 7 Twice a week 200 ppb 1000 mL

From Table 3, page 21 of the study report.



### T he assignment of each test hive at 12 apiaries is summarized in Table 4.

4 Table 4

. Hive assignment to test apiaries

Treatment

group Apiary

A B C D E F G H I J K L

UTC A1 B2 C7 D4 E1 F4 G6 H5 I5 J1 K3 L7

UTC A2 B8 C6 D6 E8 F7 G5 H3 I8 J7 K4 L5

12.5 ppb A4 B3 C3 D8 E5 F5 G4 H7 I7 J3 K2 L6

25 ppb A6 B4 C1 D1 E4 F3 G8 H6 I6 J4 K6 L2

50 ppb A8 B7 C5 D5 E7 F2 G1 H2 I3 J2 K5 L3

100 ppb A5 B6 C2 D7 E2 F8 G3 H8 I4 J5 K1 L4

200 ppb A7 B1 C8 D3 E6 F1 G7 H1 I2 J6 K8 L8

Monitoring A3 B5 C4 D2 E3 F6 G2 H4 I1 J8 K7 L1

From Table 4, page 22 of the study report.



## 2.5.1. **Preparation of stock solution**



### Stock solution was created by combining 0.051 g of Imidacloprid Technical Compound, dissolved

in approx. 20 mL of acetone, in 1000 mL of distilled water. After preparation, the stock solution

was re

- stored in a refrigerator until use or replacement. Stock solution was replaced once during

feeding on 16 Jul 2013.



## 2.5.2. **Prep aration of sugar solution** 



### Sugar syrup was created by combining 10, 100 mL tap water with 10,100 g of sugar in a 5

- gallon

(19 L) container to make approximately 17 L of sugar syrup.



## 2.5.3. **Preparation of feeding solution** 



### o 12.5 μg/ L : mixing 4.25 mL of stock solution into the 17 L of sugar solution.

o 25 μg/L : mixing 8.5 mL of stock solution into the 17 L of sugar solution17 

### o 50 μg/L : mixing 17 mL of stock solution into the 17 L of sugar solution

o 100 μg/L : mixing 34 mL of stock solution into the 17 L of sugar solution

o 200 μg/L : mixing 68 mL of stock solution into the 17 L of sugar solution.

The test concentration s were reported as “ppb” in the study report. However, the value s are in fact

in the unit o f μg/L , not ppb (ug/kg)

. For example, 12.5 μg/ L: can be calculated by 4.25 ml * 0.051

g /1020 ml)/17 L.

The test solution density was not provided. A ssum ing the density of a 50% sugar solution is 1.2296

g/ml 2 

### , the reviewer calculated that the test concentrations at 12.5, 25, 50, 100, and 200 μg/L are equivalent to 10.2, 20.3, 40.7, 81.3, and 162.7 μg/L (ug/kg) , respectively

.



## 2.5.4. **Artificial Feeding** 



### Each h ive had an empty deep super on top, between the lid and the inner cover to allow dark space

t o place the feeder inside the hive. This allowed the feeder to be placed on the inner cover so that

the bees had easy access without allowing the feeder to come into constant contact with light.

The treated sugar syrup was prepared one day in advance for each feeding event and stored

overnight at room temperature. The feeding started on 26 Jun , 2013 and continued for 6 weeks.

All of the h ives were artificially fed with 1 liter of 50% sugar soluti on, two times per week. The

remaining feeding syrup was removed from the feeder and weighed to determine the consumed

amount. The study observation period was 21 Jun , 2013

– 24 Mar , 2014, which includes the

overwintering period.



## 2.6. **Met eo**  **rological Data**

Tempe rature, humidity and rainfall data were obtained from two apiary sites (from the EASI

weather stations located at Apiaries K and J; distance to the other apiaries between 0.1 to 7.5 miles).

A total of 11.93 inches (303 mm) of rainfall accumulated throughout the exposure period

– including CCA3 (from 21 Jun 2013 until 08 Aug 2013), with 2.63 inches (67 mm) in June, 7.96

inches (202 mm) in July and 1.34 inches (34 mm) in August. For this period the on

- site temperature

minimum was 13 °C (55.4 °F) and the temperature maximum was 34 °C (93.2 ° F). The humidity

ranged from 38

- 100 %.



## 2.7. **Observation s** 

Important activity and dates are summarized in T able 5

.

2 Cell Biology Laboratory Manual, http://homepages.gac.edu/~cellab/chpts/chpt3/table3

- 2.html , accessed on Dec 12,

201418 

### 5 Table 5

. Chronological list of key dates and activities

Week Date Activity Week Date Activity

- 7 12 May 2013 CC A 1 6 07 Aug 2013 1 st sample shipment

- 4 30 May 2013 Hive samples (uncapped nectar, bee bread) 6 08 Aug 2013 Measurement of remaining food

- 4 30 May

– 13 Jun 2013 CCA2 7 12 Aug 2013 Recording of hive weights

- 1 21 Jun 2013 Recording of hive weights 7 14 Aug 2013 Hive samples (uncapped nectar, bee bread)

- 1 21

– 25 Jun 2013 CC A 3 7 14/15 Aug 2013 CC A 5

- 1 21

– 23 Jun 2013 Hive bee sampling for Varroa and Nosema

assessment 7 14/15 Aug 2013 Hive bee sampling for Varroa and Nosema

assessment

0 26 Jun 2013 Feeding 8 21 Aug 2013 Varroa counts CCA5

0 28 Jun 2013 Feeding; Measurement of remaining food 10 05/06 Sep 2013 Removal of 100 and 200 ppb hives to separate apiary

0 28 Jun 2013 Pollen samples from pollen trap 11 10/11 Sep 2013 CCA6 (UTC, 12.5 ppb, 25 ppb, 50 ppb)

1 01 Jul 2013 Feeding; Measurement of remaining food 12 17 Sep 2013 Recording of hive weights

1 03 Jul 2013 Stability samples 12 18/20 Sep 2013 CCA6 (100 ppb, 200 ppb)

1 03 Jul 2013 Feeding; Measurement of remaining food 15 08 Oct 2013 Recording of hive weights

1 03 Jul 2013 Pollen samples from pollen trap 16 16/17 Oct 2013 CCA7 (UTC, 12.5 ppb, 25 ppb, 50 ppb)

2 09 Jul 2013 Feeding; Measurement of remaining food 16 17 Oct 2013 Pollen samples from pollen trap

2 12 Jul 2013 Feeding; Measurement of remaining food 16/17 18/23 Oct 2013 CCA7 (100 ppb, 200 ppb)

2 12 Jul 2013 Stability samples

-- 13 Dec 2013 Feeding 1 L 2:1 sugar syrup per hive

2 12 Jul 2013 Pollen samples from pollen trap

-- 19 Dec 2013 Feeding 1 L 2:1 sugar syrup per hive

3 16 Jul 2013 New stock solution

-- 13 Jan 2014 Feeding 1 L 2:1 sugar syrup per hive

3 17 Jul 2013 Feeding; Measurement of remaining food

-- 20 Jan 2014 Feeding 1 L 2:1 sugar syrup per hive

3 17/18 Jul 2013 CC A 4

-- 27 Jan 2014 Feeding 1 L 2:1 sugar syrup per hive

3 18 Jul 2013 Hive samples (uncapped nectar, bee bread)

-- 07 Feb 2014 Feeding 1 L 2:1 sugar syrup per hive

3 19 Jul 2013 Feeding; Measurement of remaining food

-- 18 Feb 2014 Feeding 1 L 2:1 sugar syrup per hive

3 19 Jul 2013 Pollen samples from pollen trap

-- 02 Mar 2014 Feeding 1 L 2:1 sugar syrup per hive

4 22 Jul 2013 Recording of hive weights

-- 11 Mar 2014 Feeding 1 L 2:1 sugar syrup per hive

4 24 Jul 2013 Feeding; Measurement of remaining food After over

- wintering

4 26 Jul 2013 Feeding; Measurement of remaining food

-- 22 Mar 2014 CC A 8

5 27/28 Jul 2013 Apiary C vandalized

-- 22 Mar 2014 Hive bee sampling for Varroa and Nosema

assessment

5 31 Jul 2013 Feeding; Measurement of remaining food

-- 22 Mar 2014 Hive samples (capped honey, bee bread)

5 02 Aug 2013 Feeding; Measurement of remaining food

-- 24 Mar 2014 Recording of hive weights

5 02 Aug 2013 Stability samples

-- 15 Apr 2014 2 nd sample shipment

5 02 Aug 2013 Pollen samples from pollen trap19 

## 2.7.1. **Colony mortality**



### Any colony (hive) that did not show the presence of a queen and had no open brood or eggs, or was

devoid of worker (female) bees was considered “dead”. If a hive was considered “dead” at the time of

assessment, it was no longer used in the analysis of endpoints (e.g., adult bee numbers, hive weight). The

number of individual dead bees was not recorded.



## 2.7.2. **Colony Condition Assessments (CCA)**



### Observations wer e blocked by the observer, with the same person always observing the same set of hives

to avoid viewer discrepancies in the data, Apiary A, B, C, G, J, and L were inspected by one inspector and

apiaries D, E, F, H, I, and K by another inspector.

Eight CCAs were conducted during the entire study. CCA1 (day

- 45), and CCA2 (day

- 13 to

- 27) were

conducted during the hive establishment. CCA3 (day

- 1 to

- 5 days) was conducted 1 week prior to the

feeding exposure which served as initial hive conditions prior to the feeding exposure. CCA4 (17/18 Jul

2013) was conducted 3 weeks after the start of feeding exposure. After the end of feeding exposure (Week

6), the following additional CCAs were conducted: CCA5 (week 7), CCA6 (week 11), CCA7 (week 16)

and after ove rwintering CCA8 on 22

- 24 Mar 2014. Each CCA period in 2013 took two or more days to

complete. F or summary statistics, the first day is used to characterize any given CCA.

The t ime schedule of CCAs is summarized in T able 6

.

6 Table 6

. S chedule for colony assessment and beekeeper checks

Date Timing Evaluation/Activity

12 May 2013 45 DBE CCA1 (non

- GLP)

30, 31 May, and 3, 4, 5, 9,

10, 12 and 13 Jun 2013 27 to 13 DBE CCA2 (non

- GLP)

21, 23 and 25 Jun 2013 5 to 1 DBE CCA3 (before start of feeding ; feeding began

26 June )

17 and 18 Jul 2013 21/22 DAE CCA4 (during exposure , 3 weeks after start of

exposure )

14 and 15 Aug 2013 49/50 DAE CCA5 ( 1 week after exposure ended )

10, 11, 18 and 20 Sep 2013 76/77/86 DAE CCA6 (5 weeks after exposure ended)*

16, 17, 18 and 23 Oct 2013 112 to 119 DAE CCA7 (prior to over

- wintering)

22 Mar 2014 269 DAE CCA8 (after over

- wintering)

DBE: Days be f ore start o f exposure; DAE: Days a fter start o f exposure

*CCA6 timing allows all bee individuals (eggs, larvae, pupae) present during the exposure period to complete their development cycle

to adults.



### During the colony condition assessment s , each frame was removed and inspected one at a time, with

measurements for endpoints taken as percent of total frame area covered by honey / nectar, bee bread /

pollen, eggs, open brood ( larvae ) , capped brood (pupae) , and adult bees.20 

### The estimation wa s made by:

o Each hive consisted of 20 observed panels (10 frames with two sides of each frame), with an area

of 860 cm 2 

### per side, or a total area of 17,200 cm 2 

### for all 10 frames.

o There were 130 bees per 100 cm 2



### .

o The total number of cells per frame is 3440. Estimated to be 4 cells/cm 2



### .



## 2.7.3. **Evaluation of Disease or Pests in the Hive** 



### Colonies were also checked for visible symptoms of disease or pests, such as Nosema , foulbrood, Varroa

mites or small hive beetle.

To assess the presence of Varroa in the hive, bee samples were taken at the CCA 3, CCA5 and CCA8 . Bees

were washed in alcohol to remove mites. The number of mites per 100 bees was calculated.



## 2.7.4. **Hive weights**



### Hive weights were recorded after 10 a.m. once a month from June to October, as well as after over

- wintering,

on 21 Jun 2013 (week

- 1), 22 Jul 2013 (week 4), 12 Aug 2013 (week 7), 17 Sep 2013 (week 12), 08 Oct

2013 (week 15) and 24 Mar 2014 (week 39).



## 2.8. **R esidue analysis** 

All residue and stability samples collected from feeding solution , pollen traps, and test hives were analysed

for imidacloprid, olefin

- and 5

- hydroxy imidacloprid. Samples from pollen trap s in the monitoring hives

were also analysed for residues of multiple pesticides from outside sources at the National Science

Labora tories of USDA in Gastonia (non

- GLP). T he residue results were reported as ng per g of sample

matrix (ppb) , which is d ifferent from the test solution that was reported in μg/L

,

The LOQ was 5 ppb for imidacloprid, olefin

- and 5

- hydroxy imidacloprid in feeding solution and hive

nectar samples

, and the LOQ was 2 ppb for pollen samples

. The LODs are listed in Table 7

.

7 Table 7

. LOD for imidacloprid and its metabolites

Matrix Imidacloprid

Olefin Imidacloprid 5

- hydroxy Imidacloprid

Dosing/Stability Solutions 2.07 ppb 2.22 ppb 0.38 ppb

Hive Collected Nectar 1.38 ppb 1.43 ppb 1.43 ppb

Pollen 0.74 ppb 0.18 ppb 0.36 ppb

Taken from page 175 of the study report



### Olefin

- and 5

- hydroxy imidacloprid were not detected in any samples except for two samples. The reported

average residue results included only imidacloprid. For the values <LOD, a half of the LOD value was used

in order to calculate the means. Multiple pesticide analysis was conducted in order to monitor pesticide

contamination from outside food sources using pollen collected from pollen traps on the monitoring hives.21 

### All samples for residue analysis were protected from sunlight by using amber vials and transported to freezer

storage after field collection. All samples were placed in frozen storage upon receipt at the test facility.

Samples were maintained frozen (≤

- 15° C) (up to

- 13.9° C for a short period of time ) at the test facility

until shipment under frozen conditions to the test site for residue analysis. Daily mini mum/maximum

temperatures were recorded for the duration of the storage period at the test facility.



## 2.8.1. **Pollen from outside sources** 



### Pollen samples were collected from pollen traps attached for 24

- 48 hours to the monitor ing hives at each

site to assess the potential contaminant exposure from outside sources. Pollen amounts collected from each

hive were variable and samples were not available from every site each time. Pollen samples from the

monitoring hives were taken at w eek s 0 (CCA3)

, 1

, 2, 3 (CCA4), 5, and 16 (17 Oct 2013).



## 2.8.2. **Stored pollen and nectar in test**  **hive s** 



### Stored bee bread and bee

- collected nectar were collected within the study hives for imidacloprid residue

analysis. Samples weigh ed at least 500 mg each. Bee bread and uncapped nectar were collected at week s

- 3 (CCA4), 4, and 7 ( CCA5 ). Bee bread and capped honey were collected at CCA8 (after overwintering).

All test hives were sampled at CCA4, but only part of them were sampled at the other sampling time s

. However, bee bread and honey were not available from every colony each time

.



## 2.8.3. **Feeding s**  **olution and**  **s tability of**  **t est**  **i tem** 



### The monitoring hives were used for dose verification and to evaluate stability of the test item in a hive

environment. Monitoring hives were set up in the same manner as test hives except the colony was denied

access to the spiked or unspiked sucrose. Residue samples comprising approx. 5 g each from the sugar syrup

were taken on week 1 (3 July 2013) , week 2 (12 July 2013) and week 5 (2 August 2013)

.

8 Table 8

. Sampling schedule for feeding solution and stability of test chemical.

Timing Week 1 Week 2 Week 5

Apiary / replicate 03 Jul 2013 12 Jul 2013 02 Aug 2013

UTC X X X

12.5 ppb X X X

25 ppb X X X

50 ppb X X X

100 ppb X X X

200 ppb X X X

X = samples taken but no sample ID available22 

## 3. **Results**

**3.1. Land use near test hives** 

Land use pattern within a 1

- mile and 3

- mile radius around the 12 apiaries are summarized in Table 9

. The cultivated crop area occupied 0.2

- 5.5% of the total land within 1 mile radius, and 1.1

- 2.7% within a 3

mile radius range from the test apiaries. Using the raw data provided, the reviewer calculated the area of

cultivated crops as summarised in Tabl e 10 . The mean area of cultivated cropping land was 19 and 168

ha within 1 mile and 3 miles, respectively, of the radius from each apiary.

9 Table 9 : Percent (%) land use pattern

1 Mile Radius Apiary

Land Use Category A B C D E F G H I J K L

Open Water 0.7 0.8 0.2 1.0 0.7 6.1 0.5 0.6 0.9 0.7 0.8 1.3

Developed, Open Space 5.7 6.4 2.6 6.9 1.6 5.0 3.5 1.9 1.4 6.5 4.3 7.1

Developed, Low Intensity 1.1 1.5 0.6 1.8 0.8 0.1 1.0 2.9 1.9 2.9 0.8 1.7

Developed, Medium Intensity 0.0 0.1 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.2 0.0 0.0

Developed, high Intensity 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0

Barren Land

(Rock/Sand/Clay) 0.0 0.1 2.4 0.2 0.0 0.2 0.0 0.0 0.1 0.0 0.0 0.1

Deciduous Forest 44.5 38.7 52.2 33.7 49.9 48.3 39.1 46.5 35.4 32.6 40.1 28.8

Evergreen Forest 5.9 4.2 2.5 8.5 9.6 3.3 7.7 5.8 5.5 4.4 6.8 4.7

Mixed Forest 2.3 2.3 3.6 4.0 4.2 2.1 3.5 3.8 4.6 2.7 4.2 2.4

Shrub/Scrub 1.2 1.8 2.1 0.1 1.1 3.7 2.6 0.9 2.7 0.7 2.9 0.8

Grassland/Herbaceous 4.5 3.2 4.7 3.4 3.6 2.0 5.1 4.0 7.3 3.0 3.2 2.1

Pasture/Hay 31.6 39.1 27.1 36.7 25.7 29.0 35.7 31.1 34.7 41.1 33.9 48.3

Cultivated Crops 0.7 1.9 1.8 2.9 1.4 0.2 0.6 2.4 5.5 5.0 2.8 2.4

Woody Wetlands 1.8 0.1 0.3 0.7 1.3 0.0 0.4 0.1 0.0 0.0 0.2 0.0

Emergent Herbaceous

Wetland 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0

3 Mile Radius Apiary

Land Use Category A B C D E F G H I J K L

Open Water 0.6 0.6 0.5 0.8 0.5 2.3 0.6 0.4 0.6 0.8 0.5 2.5

Developed, Open Space 5.6 4.8 3.5 5.6 3.3 5.1 4.9 2.6 2.1 5.2 4.2 6.1

Developed, Low Intensity 1.0 1.6 1.2 2.0 1.6 0.8 1.2 1.7 1.6 1.8 1.4 1.4

Developed, Medium Intensity 0.1 0.0 0.0 0.1 0.1 0.0 0.1 0.0 0.0 0.1 0.1 0.1

Developed, High Intensity 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Barren Land

(Rock/Sand/Clay) 0.1 0.4 0.3 0.1 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.1

Deciduous Forest 41.2 41.2 44.4 39.4 44.0 45.8 40.2 43.4 44.0 39.2 42.6 38.3

Evergreen Forest 6.3 5.6 4.8 5.5 6.5 5.0 8.1 6.7 7.2 5.9 6.3 4.9

Mixed Forest 2.2 2.7 2.7 2.7 3.1 2.2 2.8 3.8 3.8 3.1 3.1 2.2

Shrub/Scrub 1.6 1.6 2.0 1.4 2.6 2.2 1.9 2.6 3.1 1.5 1.8 1.5

Grassland/Herbaceous 3.5 4.6 4.4 4.7 7.1 3.0 4.2 7.9 9.8 5.2 5.3 3.3

Pasture/Hay 34.9 34.1 33.2 34.7 28.4 31.7 33.9 27.8 24.7 33.9 31.5 36.823 

### 10 Table 1 0 : Cultivated cropping area near each test apiary

Apiary

Mean A B C D E F G H I J K L

1 mile radius (813 ha)

Cultivated

Crops (%) 0.7 1.9 1.8 2.9 1.4 0.2 0.6 2.4 5.5 5.0 2.8 2.4 2.3

Area of

cultivated

crop (ha) 5.7 15.4 14.6 23.6 11.4 1.6 4.9 19.5 44.7 40.7 22.8 19.5 18.7

3 mile radius (7323 ha)

Cultivated

Crops (%) 1.7 2.4 2.5 2.5 2.6 1.1 1.7 2.7 2.7 2.6 2.7 2.0 2.3

Area of

cultivated

crop (ha) 124.5 175.8 183.1 183.1 190.4 80.6 124.5 197.7 197.7 190.4 197.7 175.8 168.4



## 3.2. **Pollen sources of test hives** 

Monitoring hives were used at each test apiary to collect pollen for assessment of the local pollen flora (non

GLP). Pollen trap samples from the monitoring hives were taken at CCA3 (28 Jun 2013; week 0) and CCA4

(03 Jul 2013; week 1), as well as on 12 Jul 2013 (week 2), 19 Jul 2013 (week 3) and 02 Aug 2013 (week 5),

and at CCA5 (week 7) and after CCA7 (week 16).

The major pollen was from non

- cultivated crops, such as Parthenocissus, Melilotus, Plantago, Rhus, and

Asteraceae. Cultivated crops such as Zea mays (maize) and Fagopyrum esculentum (Buckwheat) were

identified occasionally, and took up the maximum of 13% and 21% of the total pollen particles, respectively.

Full results can be found in Table 49 of the study report (pages 128

- 129).



## 3.3. **C onsumption**  **of spiked sucrose** 

Hive consumption rates for the feeding solution (sugar syrup) ranged from 10,290 mL to 12,000 mL of the

total 12,000 mL per hive provided during a 6

- week period ( i.e 1 litre per colony 2 times a week for a total

of 12,000 mL per colony during the exposure period) . All colonies consumed most or all of the sugar

solution ( see 3 Figure 3 ) with a slightly low er consumption in 100 μg/L and 200 μg/L treatment.24 

### 3 Figur e 3

. Mean total food consumption (mL ) per colony during the 6

- week exposure period



## 3.4. **Examination of pesticides from other sources** 

Monitoring hives were used to assess the potential contaminant exposure from outside sources (non

- GLP)

at each site. Pollen trap samples from the monitoring hives were taken after CCA3 (28 Jun 2013; week 0)

and CCA4 (03 Jul 2013; week 1), as well as on 12 Jul 2013 (week 2), 19 Jul 2013 (week 3) and 02 Aug

2013 (week 5 ), and at CCA5 (week 7) and after CCA7 (week 16). The amount of pollen collected from

traps on the monitoring hives varied. A large portion of the pollen samples collected on Aug 2, and Oct 17

w ere reported as either “No sample” or “no sample sent to USDA ”. For these

, samples with no pollen

collected were indicated as “No sample”

, while those samples without enough to meet the mass requirement

for pesticide analysis were indicated as ‘No sample sent to USDA’. It is noted that out of 16 weeks from

the begin ning of feeding exposure to the last CCA before overwintering, pollen samples were collected 6

times with each collection over a period of 1

- 2 days and a few of them were no t ana lyzed for residue analysis

due an insufficient amount of pollen for analysis

. Pesticide contamination was unknown for those intervals

when pollen samples were not collected.

Dimethenamid (maximum at 87 ppb), Fenamidone (maximum at 345 ppb), Spiromesifen (maximum at 961

ppb) were the major pesticides detected in the pollen sampl es originat ing from outside food sources ( Table

11 ). High level s of piperonyl butoxide (maximum at 591 ppb) were detected in th e monitoring hive at

A piary A along with several other pesticides. Out of 6 test sites where the pollen samples were collected

on 02 Aug 2013, imidacloprid was detected in one pollen sample at A p iary L at 12.1 ppb . The detection

frequency by the test sites was 1/6. It was found that the contaminated pollen sample at A pi ary L consisted

of 92.4% of Rhus , 5.2% plantago , 2.3% of Lagerstroemia indica, none of which a re major cultivated crops.25 

### 11 Table 11. Residues from outside sources in pollen samples from pollen trap (non

- GLP)

Apiary Sampling Date

6/28/13 7/3/13 7/12/13 7/19/13 8/2/13 10/17/13

A No detects Chlorpyrifos

(trace) No detects DDD p,p’ (14.4)

Dimethenamid (87.0)

Fenamidone (345)

Fenhexamid (trace)

Metalaxyl (trace)

Piperonyl butoxide

(591)

Quinoxyfen (474)

Spiromesifen (961) No detects No sample sent

to USDA

B No detects No detects No sample sent to

USDA No sample sent to

USDA THPI (299) No sample sent

to USDA

C No detects No detects No detects Metalaxyl (6.0)

Methamidophos (62.3) No sample No sample

D Hydroprene

(trace) No detects No detects Dimethenamid (71.7)

Fenamidone (215)

Spiromesifen (584) No detects No detects

E No sample No sample

sent to USDA No sample No sample No sample No sample

F No detects No detects No sample sent to

USDA Dimethenamid (47.0)

Fenamidone (90.5)

Spiromesifen (362) No sample sent to

USDA No sample

G No detects No detects No detects Dimethenamid (14.6)

Spiromesifen (148) No detects No sample sent

to USDA

H No detects No detects Dimethenamid (44.9)

Fenamidone (134) No sample sent to

USDA No sample sent to

USDA No sample

I No detects No detects Fenhexamid (trace) No detects No sample sent to

USDA No sample sent

to USDA

J No detects No detects Dimethenamid (26.5) No sample sent to

USDA No sample sent to

USDA Thymol (193)

K No detects No detects Dimethenamid (10.3)

Spiromesifen (80.1) Trifluralin (trace) No detects No sample

L No detects No sample MGK

- 326 (trace) No detects Imidacloprid (12.1)

Malathion (trace) No detects

THPI = tetrahydrophthalimide

Residue values in parentheses are ppb.

From Table 64, page 142 of study report.



## 3.5. **Confirmation of test concentrations**

Imidacloprid and its major transformation products were analyzed from feeding solutions sampled after they

were prepared before start of feeding, three times on 2 July (week1), 12 July (week 2) and 2 Aug ust 2013

( week 5). The averages of measured concentrations were <LOD, 11.0, 23.3, 46.7, 96.3, and 189.6 ppb for

the nominal concentrations of control, 12.5, 25, 50, 100, and 200 μg/ L , respectively. It is noted that

imidacloprid and 5

- OH

- imidacloprid were detected in one control feeding solution at 0.45 and 4.22 ppb

respectively, sampled at week 2 on 12 Jul y 2013. It is unknown which control hives were fed with the

contaminated feedi ng solution. The data are tabulated below in Table 12

.26 

### 12 Table 12

. Dosing solution residue data from 2 July 2013 (Week 1), 12 July 2013 (Week 2) and 2

Aug ust 2013 (Week 5)

Nominal concentrations Average of

measured

concentrations

(ppb) Measured imidacloprid concentrations

(ppb) (n=6) (μg/ L ) (ppb)

0 (Control) 0 <LOD † <LOD <LOD <LOD 0.45 * <LOD <LOD

12.5 10.2 11.0 11.3 11.96 10.62 10.2 10.73 11.22

25 20.3 23.3 24.24 23.45 22.91 22.14 23.45 23.75

50 40.7 46.7 44.34 46.71 48.99 43.5 48.66 48.11

100 81.3 96.3 99.15 99.23 91.04 96.1 96.87 95.12

200 162.7 189.6 193.51 195.06 186.9 190.07 189.04 182.94

- Regenerated from Table section 5.5, on page 189

- 190 in the study report

† : LOD=0.38 ppb for imidacloprid;

* : In the same control sample, 5

- OH

- imidaclopri d was also detected at 4.22 ppb



## 3.6. **Stability of the test item in feeding solution**

Stability of imidacloprid in the sugar solution during the feeding period was examined from diet collected

from closed

- off feed ing solutions placed in the monitoring hives, sampled three times on 3 Jul y 12 Jul y , and

2 Aug ust 2013 . No reduction of test concentrations in the feeding solution was noticed during the feeding

period. The stability of imidacloprid at 200 μg/ L in the feeding solution was not provided, but a significant

reduction is not expected based on the reported data for all other concentrations. It is noted that imidacloprid

was detected at 0.56 ppb in one of the control solution for the control hive H4 s ampled on 12 Jul y 2014

. No

imidacloprid olefin or imidacloprid 5

- hyrdoxy was detected in any of the samples (LOD of 2.07 ppb and

2.22 ppb, respectively). Average imidacloprid residue data for the stability solution are presented in Table

13

.

13 Table 13

. The stability of imidacloprid in feeding solution on 3 Jul

, 12 Jul , and 2 Aug , 2013

.

Nominal

concentration

(μg/ L ) Average of measured

concentrations (ppb) Number of

samples

measured Measured im idacloprid

concentrations (ppb)

03 Jul

,

2013 12 Jul

, 2013 02 Aug

, 2013

Control <LOD † 20 0.56 ppb in one out of 20 samples.

<LOD in 19 samples;

12.5 11.4 12 11.74 11.86 10.65

25 23.2 10 23.65 23.40 22.89

50 47.4 10 46.62 46.09 51.78

100 93.6 12 95.77 92.09 92.98

200 N/A * N/A N/A

- Regenerated from Section 5.6, on page 191

- 193 in the study report

†: LOD=0.38 ppb for imidacloprid;

*: N/A: data not available.27 

## 3.7. **Residues in hive matrices**

It is noted here as it was in the uncertainties section that the residue samples from the different hive matrices

represent a single sample from a single hive. Therefore there is variation in the residues t hat likely stems

from the sampling procedure empl oyed for this study (single sample, one side of the comb).



## 3.7.1. **Background imidacloprid contamination in hives prior to the feeding exposure**



### The background imidacloprid contamination in test hives was examined using hive bee bread (hive pollen)

and nectar c ollected about a month ( 30 May 2013) prior to the beginning of feeding exposure

. Imidacloprid

was detected in two out of a total of six hive pollen samples at 0.43 ppb and 1.19 ppb, respectively ( Table

14 ), with a mean of 0.81 ppb and a detection frequency of 33% (2 hives out of total 6 hives). It is noted that

the limit of detection for imidacloprid i n pollen for this study was 0.36 ppb. Imidacloprid was not detected

in any hive nectar samples collected prior to the feeding exposure ( Table 15 ) . Residue a nalysis for other

pesticides was not conducted prior to exposure

.

14 Table 1 4

. Detailed hive pollen residue data , pre

- study collection ( 30 May 2013)

Location Imidacloprid Olefin (ppb) Imidacloprid 5 Hydroxy

(ppb) Imidacloprid (ppb)

LODs 0.74 0.18 0.36

Pope <LOD <LOD <LOD

Maple <LOD <LOD <LOD

Greenhouse <LOD <LOD 0.43

Corbett Ridge <LOD <LOD <LOD

Cedar Grove <LOD <LOD <LOD

Prospect Hill <LOD <LOD 1.19

Residue values were not corrected for recovery or moisture content of the sample.

Residues below are LOD are reported as <LOD.

From page 183 of the study report.



### 15 Table 15

. Detailed hive nectar residue data, pre

- study sample collection ( 30 May 2013)

Location Imidacloprid Olefin

(ppb) Imidacloprid 5 Hydroxy

(ppb) Imidacloprid (ppb) Brix (%) *

LODs 1.38 1.43 1.43

Pope <LOD <LOD <LOD >80

Maple <LOD <LOD <LOD 79

Greenhouse <LOD <LOD <LOD 78

Corbett Ridge <LOD <LOD <LOD 80

Cedar Grove <LOD <LOD <LOD 80

Prospect Hill <LOD <LOD <LOD 80

Taken from page 194 of the study report. *Brix % is the percentage of sugar content in honey by mass, measured by a refractometer.



## 3.7.2. **Residues in hive matrices during and after feeding exposure**



### Imidacloprid and its two major transformation products in hives (imidacloprid olefin and 5

- hydroxy

imidacloprid) were examined three times after the feeding started using hive bee bread and hive nectar. All

test hives were sampled at 1 st 

### batch of sampling (CCA4, 18 Jul y 2013) during the exposure phase, but only

part o f test hives were sampled at the other two sampling times, CCA5 which was one week after the feeding

exposure, and CCA8 after the overwintering).28 

## 3.7.2.1. Residues in hive matrices at CCA4 (after 3 weeks of exposure)



### The level of imidacloprid in hive bee bread and uncapped nectar after 3 weeks of feeding (CCA4) was

summarized in Table 16 and 17 . All test hives were sampled at CCA4 ( 18 Jul y 2013). A dose

- response correlation was observed between the imidacloprid concentrations in the feeding solution and measur ed

concentrations in both bee bread and uncapped hive nectar. However, the imidacloprid concentration in

hive uncapped nectar and pollen was 64% and 26% of the mean concentration s in feeding solution

, respectively. It is possible that dilution of stored pollen and nectar from other food sources occur red during

the exposure period since, as indicated in the study, a significant degradation of imidacloprid in test solution

was not detected in the study.

Imidacloprid in bee bread at CCA4: The level of imidacloprid in hive bee bread after 3 weeks of feeding

(CCA4) was summarized in the Table 16

. Imidacloprid was detected in all measured treatment samples.

No imidacloprid metabolites were detected. It was noted that not all residue information in pollen was available. No residue information for treatment at 200 ug/ L in bee bread was provided. Out of 12 hives, four

hives at 100 ug/ L and eight hives at 50 ug/ L were measured

, respectively.

The result s showed a dose

- response correlation between the a verage concentrations measured in hive bee

bread and the concentrations in the feeding solution. However, the concentrations varied within each

treatment group (see Table 16 ). The mean of the measured concentrations in bee bread within each

treatment gr oup of 12.5, 25, 50 and 100 ug/L was 2.86 (range: 0.77

- 5.34), 5.37 (range: 1.45

- 9.41), 10.84

(range: 4.2

- 19.41), and 17.89 ppb (range: 2.66

- 35.1), respectively. By average, the measured concentration

was 25.8% (range 22

- 28.1%) of the concentration in feeding solution, and 27.8% (range 24.9

- 31.8 %) of

the measured concentrations in uncapped hive nectar (data not shown in the table). The result s showed that

after 3 weeks of feeding, imidacloprid concentrations in hive bee bread appeared remarkably lower than

that in the feeding solutions and in hive nectar. The lower concentration in bee bread is expected due to the

dilution since bee bread is a mixture of nectar and pollen from various sources.29 

### 16 Table 16. Imidacloprid concentrations (ppb) in hive pollen sampled three weeks after the start of

artificial feeding on 18 Jul y 2013 (CCA4).

Apiary Measured imidacloprid concentrations (ppb) (LOD = 0.38 ppb)*

Nominal concentration (ug/L)

Control 1 Control 2 12.5 25 50 100 200

Nominal concentration (ppb) ‡

0 0 10.2 20.3 40.7 81.3 162.7

A <LOD <LOD 3.02 4.01 7.17 9.84

-
B <LOD 1.05 3.33 2.34 4.2

- - C <LOD <LOD 3.47 7.17 9.56 23.97

-
D <LOD <LOD 0.77 2.32 5.79 2.66

-
E <LOD <LOD 1.59 3.03 10.1

- -
F <LOD <LOD 2.09 5.19

- - - G <LOD <LOD 1.54 6.38 11.19 35.1

-
H 1.24

- 5.34 1.45

- - -
I <LOD <LOD 4.15 9.41 19.31

- -
J <LOD <LOD 3.51 7.81

- - -
K <LOD <LOD 2.4 8.05

- - -
L <LOD <LOD 3.17 7.31 19.41

- -

Number of samples

measured 12 12 12 12 8 4 0

Average

concentration <LOD 2.86 5.37 10.84 17.89

-

% of the feeding

concentration †† Not applicable 28.1 26.4 26.7 22.0

-

% of the average

detection in hive

Nectar ††† Not applicable 31.8 27.3 27.4 24.9

-

* regenerated from the additional residue information (email forwarded by Keith S appington ( EPA ) to Tina Singal ( PMRA ) on March 10,

2015) ; “

- “ indicates that data are not available

‡ Nominal concentration in ppb is estimate d from the concentration in μg/L by assuming the volume density of the test solution to be

1.2296 g/ml.

†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentrations in ppb.

††† % of the average detection in hive Nectar : the average of measured concentration in pollen compared with the average measured

concentration in nectar ppb without corrections for sugar.



### Imidacloprid in hive uncapped nectar at CCA4: The level of imidacloprid in hive uncapped nectar during

the feeding exposure (CCA4) was summarized in Table 17 . All twelve test hives were measured.

Imidacloprid was detected in the majority of the measured treatment samples. Out of 12 hives measured

for each concentration, <LOD was reported in tw o hives at 12.5 ug/L ( A piary E and H) and two hives at

200 ug/ L ( A piary D and F). No imidacloprid metabolites were detected.

The result s showed a dose

- response correlation between the average concentrations measured in uncapped

hive nectar and the concentrations in the feeding solution. However, the concentrations varied remarkably

within each treatment group (see Table 17 ). Remarkably low er concentrations were detected in nine test

hives, including all 5 treatment hives at apiary D, E5, E7, F1, and H7. <LOD was re ported for treatment at

200 μg/L in hive D3 and F1. After correction with Brix values to 50% sugar concentration, the mean of t he

measured concentrations in uncapped hive nectar within each treatment group of 12.5, 25, 50, 100, and 200

ug/L was 6.31 (range: 0.88

- 9.42), 13.24 (range: 1.19

- 20.53), 27.66 (range: 2.31

- 40.59), 46.87 (range: 2.1

-30 

### 80.15), and 109.14 ppb (range: 0.89

- 152.9 4) respectively. By average, the measured concentration in hive

nectar was 64% (range 62.0

- 68.0%) of the concentration in feeding solution. The result s showed that after

3 weeks of feeding, imidacloprid concentrations in hive nectar appeared lower than that in the feeding

solutions, which indicated that the foraging bees also foraged on nectar sources other than the provided

sugar sources which diluted t he level of treatment. It is noted that this result is expected, as bees were

allowed to freely forage, and also, under natural conditions bees typically forage on multiple plant pollen

and nectar sources.

17 Table 17

. Imidacloprid concentrations (ppb) in uncapped hive nectar sampled three weeks after the

start of artificial feeding on 18 Jul , 2013 (CCA4).

Apiary Measured imidacloprid concentrations (ppb) (LOD=1.43 ppb) *

Nominal concentration (ug/ L )

Control 1 † Control 2 † 12.5 25 50 100 200

Nominal concentration (ppb) ‡

0 0 10.2 20.3 40.7 81.3 162.7

A <LOD <LOD 9.42 14.06 40.36 48.85 96.98

B <LOD 2.98 7.91 20.53 31.12 57.45 124.53

C <LOD <LOD 6.29 7.97 38.61 66.88 134.77

D <LOD <LOD 3.37 1.19 2.91 22.58 0.89

E <LOD <LOD 0.89 7.12 2.31 80.15 90.19

F <LOD <LOD 8.04 13.89 34.71 62.15 0.89

G <LOD <LOD 7.6 14.82 16.71 58.9 150.79

H <LOD <LOD 0.88 10.47 32.32 26.17 137.27

I <LOD <LOD 8.02 13.69 18.15 23.55 136.91

J <LOD <LOD 7.66 15.51 40.59 47.7 139.32

K <LOD <LOD 7.01 19.67 38.83 2.1 144.27

L <LOD <LOD 8.57 20 35.25 65.95 152.94

Number of samples 12 12 12 12 12 12 12

Average concentration < LOD 6.31 13.24 27.66 46.87 109.14

% Feeding

concentration †† 62.0 65.1 68.0 57.6 67.1

* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in the table, but

were in the table section 5.9 on page 195

- 197 of the study report

† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations.

‡ Nominal concentration in ppb is estimated from the concentration in μg/ L by assuming the volume density of the test solution to be

1 .2296 g/ml.

†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb

.



## 3.7.2.2. Residues in Hive Matrices at CCA5 (1 week after end of exposure)



### The level of imidacloprid in hive bee bread and uncapped nectar one week after the end of feeding exposure

(CCA5, 14

- 15 Aug 2013 ) was summarized in Table 18

. Only three apiaries were sampled (Apiaries A, B,

and L). Again, the level of imidacloprid residues in hive nectar was reported for all treatment concentrations

but not for all the bee bread, especially for 100 and 200 μg/ L . In summary, similar to CCA4, a dose

- response correlation was observed between the average concentrations of imidacloprid measured in both bee bread

and uncapped hive nectar and the concentrations in the feeding solution. However, the imidacloprid

concentration in hive uncapped nectar and beebread was lower than what was in the feeding solutions,

indicating dilution of stored bee bread and nectar from other food sources.31 

### Shown in Table 18

, a dose

- response correlation was shown between the average concentrations of

imidacloprid measured in both bee bread and uncapped hive nectar and the concentrations in the feeding

solu tion. However, the concentrations varied remarkably within some treatment group s despite the low

number of samples measured. In bee bread, the mean of the measured concentrations for 12.5, 25, 50 and

100 ug/ L was 4.22 (range: 3.26

- 5.25), 5.74 (range: 4.89

- 6.4), 16.44 (range: 14.37

- 18.00), and 22.89 ppb ( no

range, only one measurement), respectively. By average, the measured concentration was 28.1% (range

22.9

- 33.8%) of the concentration in feeding solution, and 40.3% (range 26.0

- 51.5 %) of the measured

con centrations in uncapped hive nectar without correction for sugar content (data not shown in the table).

In uncapped hive nectar, after correction with Brix values (amount of sugar dissolved in solution) to 50%

sugar concentration, the mean of the measured concentrations within each treatment group of 12.5, 25, 50,

100, and 200 ug/ L was 5.88 (range: 3.36

- 7.28 ) , 7.18 (range: 0.89

- 10.68), 27.46 (range: 22.93

- 33.39), 54.98

(range:5.79

- 79.79), and 127.93 ppb (range:103.32

- 144.27) respectively. By average, the measured

concentration in hive n ectar was 61.4% (range 35.3

- 78.7 %) of the concentration in feeding solution.

18 Ta ble 18

. Imidacloprid concentrations (ppb) in beebread and uncapped hive nectar sampled one week

after the end of artificial feeding on 14 Aug , 2013 (CCA5).

Matrix Apiary Measured imidacloprid conce ntrations (ppb) (LOD=0.38 ppb pollen;

1.43 ppb nectar )*

Nominal concentration (ug/L )

Control 1 Control 2 12.5 25 50 100 200

Nominal concentration (ppb) ‡

0 0 10.2 20.3 40.7 81.3 162.7

Residues in

bee bread A <LOD <LOD 5.25 4.89 14.37 22.89

-
B <LOD <LOD 4.16 5.94 18 .00

- -
L <LOD 0.55 3.26 6.4 16.96

- - Number of samples measured 3 3 3 3 3 1 0

Average <LOD 4.22 5.74 16.44 22.89

- % Feeding Solution (ppb) †† No t applicable 41.5 28.2 40.4 28.1

% Nectar ††† No t applicable 45.6 51.5 38.1 26.0

-

Residues in

uncapped

nectar after

correction to
50% sugar A <LOD <LOD 7.28 0.89 22.93 5.79 136.19

B <LOD <LOD 7.01 10.68 33.39 79.79 144.27

L <LOD <LOD 3.36 9.97 26.06 79.36 103.32

Number of samples measured 3 3 3 3 3 3 3

Average <LOD 5.88 7.18 27.46 54.98 127.93

% of the Feeding Solution †† No t applicable 57.8 35.3 67.5 67.6 78.7

* Concentration in all treatments except for the controls are corrected to 50% sugar using Brix values. “

- “ indicates that data are not

available

‡ Nominal concentration in ppb is estimated from the concentration in μg/L by assuming the volume density of the test solution to be

1.2296 g/ml.

†† % Feeding solution : the average of measured concentration compared with the nominal feeding concentrations in ppb.

††† % Nectar: percent of the average of measured concentration in bee bread (hive pollen) compared with the average measured

concentration in nectar ( ppb ) without corrections for sugar.



## 3.7.2.3. Residues in hive matrices at CCA8 (after overwintering)



### The level of imidacloprid in hive bee bread and capped hone y after overwintering (CCA8, 22 March 2014)

was summarized in Table 19

. Only surviving hives in four apiaries were sampled (Apiaries E, I, J, and L).

Again, imidacloprid residue was not reported for all hives sampled. I n bee bread, imidacloprid was not

detected in treatments of control, 12.5, 2 5 and 100 ug/L , but was detected at 0.52 ppb (E7), 0.52 ppb (I3),

and 0.40 ppb (J2 in three 50 ug/L treatment hives. No measurement was pr ovided for treatment at 200 ug/L

.32 

### In honey, no imidacloprid residues were detected in all measured hives except for one at 13.53 ppb (L4) in

the 100 ug/ L treatment group

.

The average concentration of imidacloprid in hives after overwintering (CCA8) is considered to be

uncertain, especially for hives at 100 and 200 ug/ L . After overwintering, residues were analyzed only from

surviving hives, not from the dead hives. In the study, a high number of hives was reported dead after

overwintering. At 100 and 200 ug/ L treatments, only one and two hives survived, respectively . T he

unmeasured level of residues in dead hives presents an uncertainty as to the average of residues that might

represent the level of treatments at CCA8.

19 Table 19 : Imidacloprid concentrations (ppb) in bee bread and honey sampled after overwintering on

March 24, 2014 (CCA8).

Matrix Apiary Measured imidacloprid concentrations (ppb) (LOD=0.38 ppb pollen; 1.43 ppb

nectar )*

Nominal concentration (ug /L )

Control 1 Control 2 12.5 25 50 100 200

Nominal concentration (ppb) ‡

0 0 10.2 20.3 40.7 81.3 162.7

Residues in bee

bread E <LOD <LOD <LOD <LOD 0.52

- -

I <LOD <LOD <LOD <LOD 0.52

- -

J <LOD <LOD

- <LOD 0.4

- -

L <LOD <LOD <LOD <LOD <LOD <LOD

-

Number of samples

measured 4 4 3 4 4 1 0

Average <LOD <LOD <LOD 0.48 <LOD

- % Feeding Solution †† Not applicable 1.2 Not applicable

Residues in

uncapped

nectar after

correction to

50% sugar E <LOD <LOD <LOD <LOD <LOD

- -

I <LOD <LOD <LOD <LOD <LOD

- -

J <LOD <LOD

- <LOD <LOD

- -

L <LOD <LOD <LOD <LOD <LOD 13.53

-

Number of samples

measured 4 4 3 4 4 1 0

Average <LOD <LOD <LOD <LOD 54.98

- % Feeding Solution †† Not applicable 16.7 Not applicable

* “

- “ indicates that data are not available

‡ Nominal concentration in ppb is estimate d from the concentration in μg/L by assuming the volume density of the test solution to be

1.2296 g/ml.

†† % Feeding concentration: the average of measured concentration compared with the nominal f eeding concentrations in ppb.



## 3.7.2.4. Detection of imidacloprid in test hives at CCA2, CCA4, CCA5, and CCA833 

### Imidacloprid was detected during the entire course of the study in test hives before the feeding exposure

(CCA2) and in the control hives during the exposure (CCA4) and post exposure (CCA5). However, it was

not detected in the live control hives after over wintering (CCA8). The magnitude and the frequency of

detection of imidacloprid in control hives are summarized in Table 20 . The detection in control hives was

relatively low and was <LOD in the majority of control hives. In a total of 35 bee bread sampl es from

control hives, imidacloprid was detected in 6 samples with a maximum of 1.24 ppb. In a total of 36 uncapped

nectar samples, imidacloprid was detected in only one control hive at 2.98 ppb. 33% of the hives had been

exposed to the test chemicals bef ore the start of the test (CCA2), and <20% of control hives were

contaminated with the test chemical during (CCA4), and one week after, the exposure (CCA5).

Before the start of the artificial feeding exposure (CCA2), test hives had been contaminated with a low level

of imidacloprid. Out of the total six hives measured, imidacloprid was detected in bee bread of two hives

(33% of the measured hives) at 0.43 and 1.19 ppb respectively (LOD= 0.36 ppb). However, it was not

detected in uncapped nectar (LOD= 1.43 ppb) in all six measured hives.

During the exposure period (CCA4), the control hives were contaminated with a low level of imidacloprid.

Out of 23 control hives measured, imidacloprid was detected in hive bee bread in three control hives at 1.05,

1.24, and 0.68 ppb (LOD= 0.36 ppb), respectively. Out of 24 control hives measured, imidacloprid was

detected in uncapped nectar in one control hive at 2.98 ppb (LOD=1.43 ppb). The frequency of detections

in the control hives was 3/21 in bee bread and 1/24 in n ectar.

One week after the end of the feeding exposure (CCA5), a low level of imidacloprid was also detected in

the control hives. Out of five control hives measured, imidacloprid was detected in one control hives in bee

bread at 0.55 ppb (LOD=0.36 ppb). Imidacloprid was not detected in any of the six control hives in nectar

(LOD = 1.43 ppb). Out of 8 control hives, no imidacloprid were detected in hive bee bread and honey

samples after overwintering (CCA8). It was noted that the frequency of detection of imidacloprid was lower

in hive nectar than in the bee bread during CCA2, CCA4 and CCA5. This likely resulted from the less

sensitive LOD in nectar as the maximum detection in bee bread was even lower than the LOD for nectar.

In order to consider the p otential impact of the detected imidacloprid contamination, the maximum

detections in the control hives were compared with the average residues detected in the treatment hives and

expressed as a percentage in Table 21 . The maximum residues in the controls counted for 1.8

- 41.6% of the

average detections in treatment hives. The percentage varied by the test concentrations, low in treatments

with high concentrations but high in treatments with low concentrations. It appeared that the level of

imidacloprid c ontamination might have a greater impact to the treatments with low concentrations than the

higher ones. Therefore impact of the contamination should not be ignored especially for treatment at low

concentrations. However, due to the low detection frequen cy in control hives, this impact is likely to be

only on a few individual hives.

Imidacloprid detected in uncapped nectar in the control hives indicated that a slight level of cross foraging

among test hives might have occurred during the exposure perio d. Overall: (1) imidacloprid (2.98 ppb) was

detected in one control hive nectar during the exposure period at CCA4, but no detection (<LOD =1.43

ppb) in all hives prior to the feeding exposure ( Table 20 ); (2) Although a low level of imidacloprid was

de tected in one of the control feeding solutions at 0.45 ppb (Table 14), this level was so low it would unlikely

result in such a high level of the detection in hive nectar (about 6X increase); (3) No other sources of

imidacloprid were detected at CCA4 and earlier. Imidacloprid was not detected (LOD=0.36 ppb) in any34 

### pollen samples trapped in the monitoring hives at CCA4 and earlier ( Table 21 ). Considering the dose

response relationship of the residues detected in the treatment hives, the imidacloprid contam ination in

control hives is expected to have minimal impact to the colony level effect, especially for the treatments

with high concentrations.

Some individual hives with measured residues had exposure levels more similar to exposure levels measured

in higher or lower doses. While the residue data provides a good indication of exposure, there is some

uncertainty regarding the extent of the variab ility in exposure since r esidues in all hives were not measured,

residues were measured only at certain timepoints , and as discussed earlier, residue samples were taken only

from one location in the hive. Therefore, there is some uncertainty regarding the true extent of variability in

measured hive residues and exposure . The residue data does clearly i ndicate that a dose response

relationship is expected since higher treatment level s had higher mean measured residues in the hive pollen

and nectar

.

20 Table 20

. Imidacloprid (ppb) detected in test hives before the exposure (CCA2), and in control hives

during the exposure (CCA4) and after the exposure (CCA5 and CCA8).

Pre

- exposure (background) During exposure

( 26 Jun

– 8 Aug

, 2013 ) Post exposure

( 8 Aug 2013

– 22 Mar , 2014)

CCA2 CCA4 CCA5 CCA8

Sampling dates 30 May , 2013 18 Jul , 2013 14 Aug , 2013 22 Mar, 2014

Sample matrix Bee bread Uncapped

nectar Bee bread Uncapped

nectar Bee

bread Uncapped

nectar Bee bread Honey

LOD (ppb) 0.36 1.43 0.36 1.43 0.36 1.43 0.36 1.43

Total number of

samples measured 6 6 23 24 6 6 8 8

Number of samples

with quantifiable

level of residues

(Residues in ppb) 2

(1.19,

0.43) 0 3

(1.05, 1.24,

0.68) 1

(2.98) 1

(0.55) 0 0 0

% of samples with

detected residue

(95% confidence

limit, low

- upper) 33.3

(4.3

- 77.7) 0.0

(0.0

- 45.9) 13.0

(2.8

- 33.6) 4.2

(0.1

- 21.1) 16.7

(0.4

- 64.1) 0.0

(0.0

- 45.9) 0.0

(0.0

- 36.9) 0.0

(0.0

- 36.9)



### 21 Table 21

. Comparison between the maximum detections of imidacloprid in control hives and the

average residues detected in the same hive matrices in the treatment hives fed at different concentrations

of imidacloprid for three weeks (CCA4)

.

Test concentration

in feeding solution Average

residue

measured in

bee bread) at

CCA4 (ppb Average residue

in hive nectar at

CCA4 (ppb) % of the maximum detection in control hives in comparison

to the average of measured concentrations in treatment

hives at CCA4

Bee bread Hive

nectar

ug/L ppb Pre

exposure (CCA2)

(Max=1.19

ppb) During

exposure (CCA4)

(max=1.25

ppb) Post

exposure (CCA5)

(max=0.55

ppb) During

exposure (CCA4)

(max=2.98

ppb)

12.5 10.2 2.86 9 41.6 43.7 19.2 33.1

25 20.3 5.37 19.7 22.2 23.3 10.2 15.135 Test concentration

in feeding solution Average

residue

measured in

bee bread) at

CCA4 (ppb Average residue

in hive nectar at

CCA4 (ppb) % of the maximum detection in control hives in comparison

to the average of measured concentrations in treatment

hives at CCA4

Bee bread Hive

nectar

ug/L ppb Pre

exposure (CCA2)

(Max=1.19

ppb) During

exposure (CCA4)

(max=1.25

ppb) Post

exposure (CCA5)

(max=0.55

ppb) During

exposure (CCA4)

(max=2.98

ppb)

50 40.7 10.84 39.5 11 11.5 5.1 7.5

100 81.3 17.89 71.8 6.7 7 3.1 4.2

200 162.7 NA 162.4 Not

applicable Not

applicable Not

applicable 1.8

- Imidacloprid was not detected in hive nectar (LOD=1.43 ppb) in any control hives in CCA2 and CCA5

- NA: data was not available.

- Measured concentration in hive nectar was not corrected for sugar content.



## 3.7.2.5. Comparison of concentration in feeding solution and hive matrices



### A correlation between the imidacloprid concentrations in the feeding solution and the concentrations

measured in hive beebread and uncapped nectar was observed during the exposure period (CCA4) and one

week after the end of exposure (CCA5). However, imidacloprid measured concentrations in hive matrices

were lower than that in the feeding solution. The av erage concentrations in hive uncapped nectar and hive

bee bread were 62.7% and 30.2%, respectively, of the concentration in the feeding solution ( Table 22 ).

22 Table 22. Imidacloprid concentration measured in hive uncapped nectar and hive bee bread

Nominal concentration in

test feeding solution μg/L 12.5 25 50 100 200 Average

ppb 10.2 20.3 40.7 81.3 162.7

Imidacloprid concentration

in hive uncapped nectar in

% of the concentration of CCA 4

(n=12) 62.0

(6.31) 65.1

(13.24) 68.0

(27.66) 57.6

(46.87) 67.1

(109.14) 64.036 Nominal concentration in

test feeding solution μg/L 12.5 25 50 100 200 Average

ppb 10.2 20.3 40.7 81.3 162.7

nominal feeding solution

(average measured

concentration in ppb) ¥ CCA 5 (n=3) 57.8

(5.88) 35.3

(7.18) 67.5

(27.46) 67.6

(54.98) 78.7

(127.93) 61.4

Imidacloprid concentration

in hive beebread in % of the

concentration of nominal

feeding solution (average

measured concentration in

ppb) CCA 4

(n=12) 28.1

(2.86) 26.4

(5.37) 26.7*

(10.84) 22.0**

(17.89) NA 25.8

CCA 5 (n=3) 41.5

(4.22) 28.2

(5.74) 40.4

(16.44) 28.1***

(22.89) NA 34.6

¥ Measured concentrations in uncapped nectar were corrected for sugar concentration equivalence.

* n=8; **n=4. *** n=1;



### The study did not find a significant degradation of imidacloprid in the test solution. No imidacloprid

transformation products (olefin imidacloprid and 5

- hydroxy imidacloprid) were detected in almost all the

samples of test solution and hives matrices.

Considering the stability of imidacloprid in the test solution, the reduced concentrations of imidacloprid in

hive matrices likely indicates that test bees were also foraging for pollen and nectar from sources other than

the feeding solution.



## 3.8. **Pathogen s** 

Besides a standard treatment for Varroa mites, n o treatments for any other hive pests, predators or

diseases were administered to any hives.



## 3.8.1. **Varroa Presence** 



### Varroa mite occurrence in the colonies was assessed the week before and after the feeding period , as well

as after over

- wintering ( CCA3, CCA5 and CCA8 ) . The number of mites per 100 bees was calculated

( 4 Figure 4 ). Hives were treated with one application of Apiguard® (active ingredient: thymol) following

typical apicultural practice for the region immediately after the September CCA’s to prevent high mite loads.

After over

- wintering, the colonies of all treatment groups, except the 100 μg/L group, had similar Varroa

infestation level s

.

The study showed no correlation between the treatments and the level of Varroa infestation.37 

### 4 Figure 4

. Mean number of Varroa mites per 100 bees



## 3.8.2. Nosema **presence**

The number of Nosema spores per bee was determined at three time points at CCA3, CCA5 and CCA8. The

study showed no correlation between the treatments and the level of Nosema infestation.

5 Figure 5

. Mean number of Nosema spores per bee38 

## 3.9. **Statistical Analysis** 

What follows are brief summaries of each of the statistical analyses employed for the review of this study.

It is noted here, and later, when discussing the results that the PMRA, EPA, and CDPR statistical approaches,

and when weighing statistical and bio logical lines of evidence, that the three Agencies are harmonized in

the determination of the overall NOAEC and LOAEC.



## 3.9.1. **Study Authors Analysis**



### The study author conducted statistical analysis using SAS (version 9.3). The analysis included colony

strength (as indicated by mean number of adults), brood stages (as indicated by the mean number of eggs,

larval cells, and pupal cells) and food stores (as indicated by the mean number of pollen and nectar/honey

cells). For the pre

- test data, all tests were done i n a two tailed approach, whereas for the data assessed after

application, one tailed (lower) tests were conducted. According to the study author, procedure GLM was

used for ANOVA analysis. Williams’ Trend Test was used to test data that passed statistica l tests that

assessed the assumptions of normality, variance homogeneity, and monotonicity. Dunnett’s t

- Test was used

to test data that are non

- monotonic, but pass tests of normality and variance homogeneity. Dunnett’s T3

Test was used to test data that sa tisfy the criteria for normality, but fail the criteria for homogeneity of

variance. For hive mortality, Cochran Armitage Exact Trend Test was used.



## 3.9.2. **Study Reviewer Analysis**



### During the review of the study, a separate statistical analysis was conducted using the raw data submitted

by the s tudy author. As part of the collaborative review effort of the st udy by EPA, PMRA, and CDPR , a variety of statistical analyses were conducted for the evaluation of the data. The detailed methods of these

analyses incl uding statistical model selection and parameterization are presented in Appendices A, B, and

C for the EPA, PMRA, and CDPR analyses, respectively. What follows is a brief summarization of each

method. It is noted that while each method was distinct in th e manner in which the data were analyzed, all

three methods produced similar statistical results, that is, similar findings of significance for a given

response variable at a given treatment level and CCA at a specified alpha level.



## 3.9.3. **EPA Analysis**



### The general experimental design was a randomized complete block (apiary) with repeated measures (CCA)

. Since hives were not randomly assigned and placed in the study apiaries un til shortly before CCA3, the data

for the statistical analysis only included CCA3 t hrough CCA8. For the two highest treatment levels (100

and 200 μg/L ), data obtained from CCA8 was deleted from the analysis as only one and two hives

(respectively) were surviving at the CCA8 measurement time. Temporal correlations were evaluated for

each response variable; compound symmetry with heterogeneous variance was selected as the best fitting

covariance structure. PROC MIXED in SAS was used for the data analysis, and the TREATMENT*CCA

interaction was statistically significant for all evaluated response variables

. This interaction was explored

by 1) a t each CCA, treatment means were compared to the control using a one

- sided Dunnett’ s test; and 2)

for each treatment level, CCAs 4 through 8 were comp ared to CCA3 using a two

- sided Dunnett’s test.

Further details of the EPA statistical analysis can be found in Appendix A.39 

### The EPA approach controlled for multiplicities by using a Dunnett’s test which holds the family wise error

rate ( i.e the probability of make Type I errors or false discoveries of significance) at or below the level of

α.



## 3.9.4. **PMRA Analysis**



### The differences of each measurement parameter between the treatment and control at the same apiary were

calculated for each apiary site. The means of the differences among 11 sites (Apiary C excluded due to

vandalism) were plotted. A formal comparison from the highest to lowest concentration with the control

was carried out using an often used conventional analysis of the block randomised experiments using the

raw data with adjustment for baseline measurements: linear modeling (or ANOVA) stratified on Apiary

(block) and adjusted for baseline measurements at CCA3 with one

- side testing for effect. In the remainder

of the document where PMRA analysis res ults are referred to as “raw data” it is noted that it is actually the

model estimates using the raw data with adjustment for baseline measurements. Tak ing into consideration

a limited detection power in a typical field level study, alpha levels of both 0 .1 and 0.05 were considered as

statistically significant. A list of statistical P values for each measuring parameter is summarized and

included in this report.

Prior to the data analysis, for the purposes of controlling for multiplicity in the statistic al analysis, a primary

parameter for detection of treatment effects was defined as the total individuals in hives (sum of eggs, larvae,

pupa and adults) at CCA6. However, when determining an overall NOAEC all response variables were

examined with equal we ight in considering treatment related effects , but were considered against statistical

results for the primary parameter

. Further details of the PMRA statistical approach can be found in

Appendix B.



## 3.9.5. **CDPR Analysis**



### A multivariate mixed repeated measures model approach was employed and is distinguished from the

univariate approach es above in that all bee life stages or hive food storage variables are simultaneously

analyzed as a single model. Multivariate analyses of v ariance for fixed effects models are conducted, using

Statistical Analysis System (SAS) software, through implementing the MANOVA option in the PROC

GLM procedure. Recently, multivariate analyses have been extended to mixed models using the PROC

MIXED proc edure. The MIXED procedure is designed to conduct a mixed model analysis of variance where

fixed and random effects can be specified. Inclusion of random effects in a model provides a broader

application of results. For this study, locations were denoted as apiaries with individual hives as test

subjects. Use of a mixed model with apiaries identified as a random variable provides some assurance that

the results can be generalized to other locations and hives. Further details of the CDPR analysis can be

fou nd in Appendix C.

The CDPR approach controlled for multiplicities by way of a Bonferroni adjustment to fix the family wise

error rate at α.



## 3.10. **Hive mortality**

The study author reported that 72 out of 84 colonies for biological observations were maintained over the

6

- week exposure period. Apiary C was vandalized after 8 feedings during the weekend of 27/28th Jul 201340 

### during which hives were left open. This allowed all colonies in this apiary access to treated sugar solution

and treated food stores in the hives. Since all hives were compromised, they were no longer used to collect

data for the study and this apiary was thereafter removed from the study and subsequent data analysis

.

From the 11 remaining sites with a combined 77 hives, 34 hives were consid ered dead by 22 Mar ch 2014

(end of test), as summarized by Table 23 and Figure 6

. The author reported that b ased on an one

- sided

Cochran

- Armitage exact trend test, mortality in the 100 ppb and 200 μg/L treatments are significantly

different from the cont r ol mortality (p = 0.01 and p <0.01 respectively).

After overwintering, only one hive survived (A pi ary L) at the treatment 100 μg/L , and two hives survived

( A piary D and F) at the treatment 200 μg/L . The hive mortality showed a U

- shaped response to the

treatments ( Figure 6 ). The percent mortality decreas ed from control (36%) to 25 μg/L (9%), and the n

i ncreased from 25 μg/L to 50 ug/L (36%) to 100 μg/L (91%) and 200 μg/ L (82%). It is noted from the

results presented in Table 23 a and b and Figure 6 below show that control mortality after overwintering

was higher than it was for the 12.5 and 25 μg/L groups. For this reason, the ability to detect treatment

related decreases in overwintering colony survival may be masked by the magnitude of control colony l oss.

23 Table 23a

. Hive survival at CCA8 (after overwintering)

Treatment

group Apiary

A B C D E F G H I J K L

UTC A1

- - - E1

- G6

- I5 J1

- L7

UTC A2 B8

- - E8 F7

- H3 I8 J7

- L5

12.5 ppb A4 B3

- D8 E5 F5 G4 H7 I7 J3 K2 L6

25 ppb

- B4

- D1 E4 F3 G8 H6 I6 J4 K6 L2

50 ppb A8 B7

- - E7

- - H2 I3 J2 K5 L3

100 ppb

- - - - - - - - - - - L4

200 ppb

- - - D3

- F1

- - - - - -



### 24 Table 23 b

. Hive mortality after overwintering measure at CCA8

Treatment (μg/L ) Control 12.5 25 50 100 200

Number of deceased colonies

/total colonies 8/22 2/11 1/11 4/11 10/11 9/11

Colony mortality (%) 36 18 9 36 91 82

Colony survival (%) 64 82 91 64 9 1841 

### 6 Figure 6

. Hive mortality after overwintering

.



## 3.11. **Colony Condition Assessment Response Variables** 

What follows is a breakdown of each response variable assessed and the significant effects that were

determined at each CCA. A couple of general points are made below when examining the results of the

response variables:

• Unless explicitly stated otherwise, all discussion of statistical findings refer to shared determinations

from the PMRA, EPA, and CDPR analyses.

• All analyses considered effects at both the 0.05 and 0.1 alpha levels when weighing statistically

significant effects with biological considerations.

• The tables are the percent differences from control based on raw counts of the data ( model

estimations using the raw count data with adjustment for baseline measu rements at CCA3) and

generated by PMRA. The figures with significance “dots” were based off of the proportions of

frame coverage for each hive for each response variable (with the exception of hive weight) and

were generated by EPA.

• In the EPA analysis, t he data in the 100 and 200 μg/L treatment groups were excluded from the

analysis at CCA8. This was done primarily to facilitate the statistical model converging . Excluding

these two treatment groups from the analysis at CCA8 is not expected to have an im pact on the

interpretation of results as there was a clear effect at these two treatment groups by the time of CCA8

indicated primarily by hive mortality.

• Even though data from CCA8 at the 100 and 200 μg/L groups were included in the PMRA analysis,

the lac k of statistical difference from control in these two groups is considered unreliable as there

were only one and two remaining hives in these groups at CCA8 , respectively.

• The PMRA approach used raw counts of the each response variable while the EPA and CD PR

approach es converted these data into proportion of frame coverage (using methods described in

Appendix A ) to facilitate convergence of the statistical model. The tables of percent differences

from control are based off of the raw counts of the data (model estimates using raw counts adjusted

for baseline measurements ) while the figures present the trends of the proportions of life stages and

food stores with significant findings indicated. When differences in statistical findings are

discussed, the findings of PMRA were based off of the raw counts (model estimates using raw counts 36

18

9 36 91

82

0 20 40 60 80 100

Control 12.5 25 50 100 200 M

o

r

t

a

l

i

t

y( %)

Treatments ( μ g/l) 

### Hive Mortality (%) After Overwintering42 

### adjusted for baseline measurements) while the findings of the EPA and CDPR approaches were based off proportions.

• CCA3 was the baseline covariate and therefore is not prese nted in the tables for each response

variable with percent reductions.

• CDPR did not include the “total individuals” endpoint in its analysis so those results will only pertain

to EPA and PMRA findings.

• For its simplicity in visualizing the trends and findi ngs of statistical significance simultaneously, the

EPA

- generated figures are presented below. The figures generated by PMRA can be found in

Appendix B and those generated by CDPR can be found in Appendix C

. It is noted here as well as

above that in the d iscussion of each response variable, the results of all approaches will be discussed

and noted where divergent from each other.

• The figures below indicate significance with black and red “dots” denoting a significant finding at

the 0.1 and 0 .05 alpha levels, respectively. Although these figures refer to EPA’s analysis, as

mentioned previously, the PMRA, EPA, and CDPR statistical results were generally in agreement

and it is noted below where there were differences. As also mentioned previou sly, although there

were different statistical findings in a few cases depending on the method employed, the

interpretation of the results leads to a shared overall NOAEC /LOAEC of the study.

• While the EPA and PMRA analyses looked at each response variable across CCAs, the CDPR is

distinguished from this approach by looking at all life stage s or food store variable s simultaneously

within a single CCA across treatments

.

• While it is not depicted in the figures below, it is acknowledged (and addressed in a var iety of ways

through the various statistical approaches) that there was considerable variability for some response

variables at certain treatment groups and CCAs. Please refer to Appendix A for summary statistics

tables ( i.e. max, min, standard deviation values) of the proportions of each response variable for

further information.



## Life Stages

**3.11.1. Adults** 



### Figure 7 below shows the effects on adult honey bees across CCAs and treatment groups

. Compared with

the control, no differences in the number of adults in hives (p >0.1) during the CCA4 exposure period were

apparent in any of the treatments with the exception of the CDPR analysis where a marginally significant

(0.05 < p < 0.1) reduction was determined at the 200 μg/L group. Additionally, t he total number o f a dults

in the 12.5 and 25 μg/L treatments was not reduced in any of the CCAs. However, the numbers of the adults

in the 100 and 200 μg/ L treatments were consistently reduced (p <0.05) at CCA5, CCA6, and CCA7 with

reductions ranging from 24.4

– 59.4% (data from CCA8 excluded from EPA analysis in the 100 and 200

μg/L group due to clear effects on hive mortality)

. An exception is that the EPA and CDPR analyses did

not determine a significant reduction s (p>0.05) at the 200 μg/L treatment group at CCA5 w hile the PMRA

analysis did. However, it is apparent from all analyses that there were impacts to adults at the 200 μg/L

group during the course of the study. The number of adul ts in the 50 μg/L treatment was also reduced with

marginal statistical signifi cance at CCA5 (0.05 < p <0.1 ) but at p<0.05 at CCA6 and CCA 8 with percent

reductions from control of 21.7 %, 19.8 % and 78 %, respectively based off raw counts ( Table 24)

. The

PMRA analysis also determined a significant reduction from control at CCA 7 while the EPA and CDPR43 

### analyses did not, but like the 100 and 200 μg/L treatment group s , the persistent nature of significant

reductions to adults at the 50 μg/L group is evident.

25 Table 24

. Estimated percent reduction from control for number of adults

Test

concentration

(μg/L ) Estimated reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 2.2

- 5.4 2.3

- 12.6

- 11

25 2.7 8.7 7.7

- 4.1

- 4.3

50

- 23.3 21.7 * 19.8 * 18.6 * 78 **

100 5.2 34 ** 28.7 ** 51 ** 172.5 **

200

- 29.2 24.4 ** 52.8 ** 59.4 ** 7.3

Note: Negative value indicates increased number of adults in comparison to control.

*0.05<p<0.1

**p<0.05 1 Percent differences from control are based on the raw counts of adults, not proportions of the adults as Figure 7 below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups,

re spectively. These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis

.



### 7 Figure 7

. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8.

Figure 8 below shows the trends proportions of adults across the CCAs for the control and three lowest

treatment groups only as the impact at the two highest groups was evident, especially when considering

overwintering mortality. Removing the two highest treatment concentrations adjusts the scale of the figures

to see the trends more clearly at the lower treatment groups

. There is a clear divergence in the trends at the44 

### 50 μg/L treatment group not only in the decline in numbers beginning one CCA earlier (CCA4 as compared

to CCA5 in the control, 12.5, and 25 μg/L groups) but also the average proportion of adults after

overwintering at CCA8 (approximately 20% frame coverage as opposed to 33

- 35% for control and the lower

groups).

8 Figure 8

. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

I n an examination of the trends of adults in the control group, b y CCA6, the average number of adults began

to decline to 39% based off the proportions of adults covering the hive frames ( Figure 9 below)

. CCA6

represents the time when the colony as a whole starts to prepare for overwintering and therefor e starts to begin a “shut

- down” phase where the numbers of adults and other life stages are clearly decreased by the

time of CCA7. During this pre

- overwintering phase, adult proportions decline due to natural die off of

worker bees and reduced rate s of replenishment from reduced egg laying by the queen. It is noted that the

average proportion of comb area as adults is similar after overwintering at CCA8 (35%) as compared to

before exposure at CCA3 ( 33% ) when the hives were initially placed in the t est sites.

Also n otably, as distinguished from the control and 12.5 and 25 μg/L groups, w hile the proportions of adults

for those groups generally increased through CCA5 before beginning to decline, the numbers of adults at

the 50 μg/L began to decline a s early as CCA4, where these numbers were being built up in the control and

lower treatment groups to support the foraging worker bee force for nectar and pollen collection. This again

is evidenced by the average proportion of adults at CCA5 in the 50 μg/ L group which was 33% as compared

to 45, 48, and 42% for the control, 12.5 and 25 μg/L groups, respectively (percent reductions ranging from

24

– 37% based off the proportions of frame coverage ). 0 0.1 0.2 0.3 0.4 0.5 0.6

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Adults



### Colony Condition Assessment 

## Adults

Control

12.5

25

5045 

### 9 Figure 9

. Proportion of adults following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the contr ol, 12.5, 25, and 50 μg/L groups

.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for adults is

determined to be 25 and 50 μg/L, respectively.



## 3.11.2. **Eggs**



### T here were consistently lower numbers of eggs in treatments at 100 and 200 μg/ L (p<0.05) during the course of the CCAs following exposure. There were minor differences in the statistical findings of the three 0 0.1 0.2 0.3 0.4 0.5Proportion of Adults



### Colony Condition Assessment 

## Control

Adults 0 0.1 0.2 0.3 0.4 0.5Proportion of Adults



### Colony Condition Assessment 

## 12.5 μg/L

Adults

0 0.1 0.2 0.3 0.4 0.5Proportion of Adults



### Colony Condition Assessment 

## 25 μg/L

Adults 0 0.1 0.2 0.3 0.4 0.5Proportion of Adults



### Colony Condition Assessment 

## 50 μg/L

Adults46 

### anal yses not only at a given CCA and treatment group but also at what alpha level an effect was significant

at, but these differences do not have an impact on the determination that there was not only an early onset

but a persistent nature of a reduction in the numbers of eggs that was not confined to one CCA. At the 100

and 200 μu/L treatment groups, all analyses determined significant reductions (p<0.1) in eggs for the 100

and 200 μg/L treatment groups for at least two CCAs . The sole finding of signif icance at the 50 μg/L group

was at CCA8 at the 0.05 alpha level

.

For the 12.5 μg/L the PMRA and EPA analyses only determined a significant reducti on in the number of

eggs at CCA 8 (p<0.1). A similar finding was made at CCA4 in the PMRA analysis only. Ho wever, there

were no significant reductions (p>0.1) at both CCA4 and CCA8 in the 25 μg/L group, indicating a lack of

dose responsiveness within these time points . The biological significance of this finding at 12.5 μg/L is

therefore considered to be low. Similarly, there was a significant reduction in eggs determined at CCA6 in

the 25 μg/L group (PMRA analysis only). However, similar findings of statistical significance at this CCA

was not determined for the 50 μg/L group. Finally, this effect was not o bserved before CCA6 or in the

subsequent CCAs (CCAs 7 and 8) indicating this effect was isolated to this time point

.

At the 50 μg/L treatment level, all analyses determined a significant reduction (p< 0.05) at CCA8. While

this effect was isolated to just CCA8 for this treatment group, there is uncertainty as to whether hives could

have compensated for this reduction as could or may not have been shown by an additional CCA.

26 Table 25

. Estimated percent reduction from control for number of eggs

Test

concentration

(μg/L ) Estim ated reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 22.8 * 11.9 5.9 0.1 37.7 *

25

- 1.1 8.3 26.3 * 31.3 5.8

50

- 9.6 1.8 11.7 2.6 78.2 **

100 37.9 ** 39.4 ** 70.8 ** 46.6 ** 138 **

200 14.5 32.2 ** 60.1 ** 77.8 ** 153.2 **

Note: Negative value indicates increased number of eggs in comparison to control.

*0.05<p<0.1

**p<0.05

1 Percent differences from control are based on the raw counts of eggs , not proportions of the eggs as Figure 10 below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.

These data are included in the PMRA and CDPR analyses anal ysis but excluded from the EPA analysis

.47 

### 10 Figure 10

. Proportion of eggs following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8.

Figure 11 below shows the responses for the control, 12.5, 25, and 50 μg/L treatment groups. Removing

the two highest treatment concentrations adjusts the scale of the figures to see the trends more clearly. It is

noted from this graph the variability present in the groups at CC3 before exposure had started. Particularly,

the variation in egg coverage of the frame at the early CCAs is noted which may have contributed to some

of these findings at CCA4.48 

### 11 Figure 11

. Proportion of eggs following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

By CCA8

, the average of eggs in the control group increased relative to CCA7, but the average proportion

at CCA8 (4.8%) was approximately half the proportion initially recorded at CCA3 (8.4%). It is noted that

the 50 μg/L group was the only group of t he control and three lowest treatment levels that und erwent a

downward trend from CCA7 to CCA8

. Additionally, the average proportions of egg cells at CCA8 for the

50 μg/L group are approximately half of the proportion for the control, 12.5, and 25 μg/L groups ( Figure

12 ). 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Eggs



### Colony Condition Assessment 

## Eggs

Control

12.5

25

504 9 

### 12 Figure 12

. Proportion of eggs following exposure to varying concentrations of imidacloprid in the diet

across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for eggs is

determined to be 25 and 50 μg/L, respectively, based on a significant reduction in eggs at CCA8 that

represented a 78.2 % reduction based on raw counts as well as being clearly divergent in its response

at CCA8 (based off the proportions) from the control and 12.5 and 25 μg/L groups. 0 0.02 0.04 0.06 0.08

0.1Proportion of Eggs



### Colony Condition Assessment 

## Control

Eggs 0 0.02 0.04 0.06 0.08

0.1Proportion of Eggs



### Colony Condition Assessment 

## 12.5 μg/L

Eggs

0 0.02 0.04 0.06 0.08

0.1Proportion of Eggs



### Colony Condition Assessment 

## 25 μg/L

Eggs 0 0.02 0.04 0.06 0.08

0.1Proportion of Eggs



### Colony Condition Assessment 

## 50 μg/L

Eggs50 

## 3.11.3. **Larvae (Open/Uncapped brood)** 



### T here were consistently and significantly lower number s of larvae in the 100 and 200 μg/L groups as

compared to control (p < 0.05 ) beginning at CCA4 and persisting throughout the entirety of the study up to and including after overwintering at CCA8 (data from CCA8 in these groups excluded from EPA analysis)

. There were no significant reductions from contr ol at any CCA for the 12.5, 25, and 50 μg/L treatment groups in the EPA analysis.

In the PMRA analysis at CCA6, a significant reduction from control was determined at 25 μg/ L ( p <0.1 ),

but not at 12.5 μg/L and 50 μg/ L (p>0.1 for both treatments). Additionally, the effect was not determined

at CCA4, CCA5, and CCA7

. Similarly, the CDPR analysis determined a significant difference (p<0. 05 ) at

CCA7 at 12.5 μg/L , but this finding was not determined at the 25 and 50 μg/L treatment groups (p>0.1).

Also in the PMRA analysis only , a t CCA8, a statistical reduction was determined at 50 μg/ L treatment group

( p < 0. 05 )

. This effect was not determined to be significantly reduced from control from CCA4 to CCA7

(p >0.1). Although this difference was not detected in the EPA and CDPR analyses , the percent reduction in

larval cells at CCA8 for this group was 43% (based on raw counts).

27 Table 26

. Estimated percent reduction from control for number of larvae (open/uncapped brood)

Test

concentration

(μg/ L ) Estim ated reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 12.3 10.7

- 15.5

- 62.6 0.8

25 8.1 16.2 23 *

- 25.5

- 9.9

50 12.1 15.3

- 2.1

- 32.4 42.6 **

100 37.1 ** 30.9 ** 52.3 ** 64.4 ** 159.9 **

200 64 ** 65 ** 57.2 ** 78.3 ** 54.1

Note: Negative value indicates increased number of larvae in comparison to control.

*0.05<p<0.1

**p<0.05

1 Percent differences from control are based on the raw counts of larvae (open) brood , not proportions of the larvae (open) brood as Figure 13

below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.

These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis

.



### Figure 13 below shows the trends of the control and all treatment groups for larval cells across all CCAs

assessed. A clear divergence in the 100 and 200 μg/L groups is evident beginning at CCA4 where the

numbers of larvae in these groups undergo a marked declin e while the other treatment groups generally

trend with control.51 

### 13 Figure 13

. Proportion of larval cells following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8.

When examining the trends with the control and three lowest treatment groups alone, differences are less

apparent than when the 100 and 200 μg/L groups depicted alongside with the exception of the separation of

the 50 μg/L treatment group at CCA8 ( Figure 14 )52 

### 14 Figure 14

. Proportion of larval cells following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

When examining the trends in the control group, t he average proportion of larval cells increased from CCA7

to CCA8, and at CCA8 had a similar level (8.2%) that was recorded for CCA3 (10%) ( Figure 15 )

. With

the 12.5 μg/L group, the starting average proportions of life stages at CCA3, prior to exposure, were similar

to those in the control group with respect to the proportions of larval cells being approximately 5

- 8% of the

comb area. The average proportion of larval cells trended down beginning as early as CCA4 (7%) before

experiencing a more marked decline ahead of CCA7 (2%) as with the other life stages, which is anticipated

ahead of overwintering

. The proportions of larval cells were again relatively stable from the ti me of CCA3

(10%) to CCA6 (10%). This was preceded by a marked decline at CCA7 to an average proportions of 4%

. 0 0.02 0.04 0.06 0.08

0.1 0.12

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Larvae



### Colony Condition Assessment 

## Larvae (Open) Cells

Control

12.5

25

5053 

### 15 Figure 15 . Proportion of larval cells following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

It is also noted from the treatment by treatment comparisons below for larval cells across CCAs that

although the trends for the control, 12.5, 25, and 50 μg/L groups are similar, that t he 50 μg/L group was the

only group of the 4 that did not end up at approximately 8% coverage of the frame, but instead was

approxi mately 6% of frame coverage . The 25 ppb group also look to undergo a more marked decline

beginning at CCA4 as compared to th e control, 12.5, and 50 μg/L group but as stated earlier, the difference

was only marginally significant in the PMRA analysis, similar findings were not determined in the 50 μg/L

group, and the effect at 25 μg/L was not significant at CCA7 and CCA8 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Larvae



### Colony Condition Assessment 

## Control

Open 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Larvae



### Colony Condition Assessment 

## 12.5 μg/L

Open

0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Larvae



### Colony Condition Assessment 

## 25 μg/L

Open 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Larvae



### Colony Condition Assessment 

## 50 μg/L

Open54 

### When w eighing statistical and biological significance, the overall NOAEC and LOAEC for larval cells

is determined to be 25 and 50 μg/L, respectively. This is based on a significant reduction at the 50

μg/L at CCA8. While this finding is isolated to CCA8, it is an uncertainty what the response would

have been at an additional CCA ( i.e. , whether the hives could have compensated for this potential

effec t ). It is also noted, as has been above, that the 50 μg/L group showed a reduced percentage of

frame coverage of larval cells at CCA8 as compared to the control and two lower treatment groups.



## 3.11.4. **Pupa e (Capped brood)** 



### In the 50, 100 and 200 μg/L treatment groups, there were significant reductions from control ( p <0.05) that

persisted through most of the study (EPA findings at the 50 μg/L were significant at two CCAs , CDPR at 3

CCAs and the PMRA analysis determined significant reductions from control at 5 CCAs)

. The percent

reductions from control based on the raw counts of pupal cells in the 100 and 200 μg/L groups ranged from

49.7

– 93.5% during CCA4

– CCA7.

At the 12.5 μg/L treatment group, there were significant reductions determined at CCA6 for the EPA and

PMRA analyses . While the findings were not determined at the 25 μg/L treatment group at CCA6 for the

EPA and CDPR analyses , they were for the approach used by PMRA

. It is noted that significant reductions

in pupal cells were not determined by an y analysis at 12.5 and 25 μg/L in any CCA preceding or subsequent

to CCA6 , thus the significant effect for the 12.5 and 25 μg/L treatments was isolated to the CCA6 timepoint

. Additionally, although PMRA determined significant reductions at CCA6 for all treatment groups, the

effects did not demonstrate a dose response at the lowest three doses with the percent reductions from

control (based on raw counts of the data) at 22.3, 18.3, 12.5, 49.7 and 75.5 % for the 12.5, 25, 50, 100 and

200 μg/L groups, respec tively. However, responses at the lower three doses were all similar ly reduced

compared to controls (12.5

- 22.3%) , and some overlap in dose

- response might be expected at the lower

doses, given the variability and overlap in exposure among individual hives

. It is also noted that the

confidence intervals among the three lowest doses are similar and overlapping (see Bees 8, PMRA analysis

Appendix B). Finally, after overwintering at CCA8, the levels of pupae in the surviving hives at the 12.5

and 25 μg/L treat ment groups were actually above the level of control (based on raw counts of the data) by

1.3 and 10.8%, respectively. The percent reduction from control at CCA8 in the 50 μg/L group was 70.6%.

28 Table 27

. Estimated percent reduction from control for number of pupae

Test

concentration

(μg/L ) Estim ated reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 2.8

- 3.7 22.2 **

- 8.4

- 1.3

25 17.7 5.8 18.3 ** 18.1

- 10.8

50 28.1 * 34.6 ** 12.5 * 9.7 70.6 **

100 51.7 ** 56.6 ** 49.7 ** 75.6 ** 150.9 *

200 83.3 ** 79.5 ** 75.5 ** 93.5 ** 42

Note: Negative value indicates increased number of pupae in comparison to control.

*0.05<p<0.1

**p<0.05

1 Percent differences from control are based on the raw counts of pupae (capped) brood, not proportions of the pupae (capped) b rood as

Figure 16 below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups,

respectively. These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis

.55 

### 16 Figure 16

. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8.

In summarizing the information provided by the different analyses for this response variable, a few points

can be made:

• All analyses find significant differences at the 100 and 200 μg/L levels starting at CCA4 and

persisting until CCA7 (data from CCA8 excluded from EPA analysis at these two treatment groups )

• PMRA analysis determined significant difference at the 50 μg/L group (at either 0.1 or 0.05 alpha

level) for all CCAs assessed except CCA7 (for CDPR, s ame findings except no significant findings

at CCA6 and CCA7)

• EPA analysis determined significant differences at 50 μg/L group (at either 0.1 or 0.05 alpha level)

at CCA5 and CCA8

• PMRA determined significant differences at all treatment groups at CCA6 whil e EPA determined

significant effects for the 12.5, 100 and 200 μg/L groups , and CDPR only at 100 and 200 μg/L

.

In further exploring this last point, the difference in findings can potentially be explained by the statistical

model selection s employed for each analyses . The discussion below focuses on additional lines of evidence

to further characterize the findings

.

Figure 17 shows the trends in pupal cells over the course of the study in the control and three lowest

treatment groups only. As indicated in both the PMRA and EPA analyses, the impacts to pupal cells occurs

early as there is divergence from the control as early as CCA4.56 

### 17 Figure 17

. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

It is noted that the average proportion of comb area as pupal cells in controls is similar after overwintering

at CCA8 ( 17 %) as compared to CCA3 ( 16% ) when the hives were initially placed in the test sites. Also

notably , the average proportion of pupal cells in the 12.5 and 25 μg/L groups (22%) is higher than it was

for the control group (16%) but this is obviously not a finding related to imi dacloprid treatment, given that

exposure had not occurred yet.

In the 12.5 μg/L group, unlike the control group, where the average proportion of pupal cells remained

stable between the time of CCA5 and CCA6 (26%), there was an apparent decrease in the 1 2.5 μg/L group from CCA5 (26%) to CCA6 (20%) based on average proportions . This decrease continued for the CCA6

to CCA7 interval to an equivalent level as controls (approximately 8%). After the overwintering period in

the 12.5 μg/L group, the proportions of life stages were similar to CCA8 for the control group in that the

proportion of pupal cells 17% at CCA8 (as compared to 17% in the control)

.

In the 25 μg/L treatment group, there were again no significant differences in the proportions of all life

stages at CCA3 before the start of exposure (p>0.05) . As opposed to the steady buildup that was observed

in the control and 12.5 μg/L treatment groups from CCA3 to CCA5, the numbers of pupal cells remained

similar from CCA3 to CCA5; they were decreased slightly at CCA4 (21%) as compared to CCA3 (23%)

but at CCA5 (24%) were again to the level of CCA3. As with the other life stages, a decline in numbers

was observed between CCA6 and CCA7 as the hives prepared for overwintering . The average proportion

of pupal cells at CCA8 for the 25 μg/L treatment group were similar to the proportion s in CCA8 of the

control group, that is, 16% and 17% frame coverage at CCA8 for the 25 μg/L group and control group,

respectively. 0 0.05

0.1 0.15

0.2 0.25

0.3

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Pupae



### Colony Condition Assessment 

## Capped (Pupal) Cells

Control

12.5

25

5057 

### 18 Figure 18

. Proportion of pupal (capped) cells following exposure to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

As distinguished from the control and 12.5 and 25 μg/L groups, the number of p upal cells at the 50 μg/L

group underwent a steady decline beginning at CCA4 and continuing through CCA5 (average proportion at

CCA3 was 19% compared to 16% at CCA5). This is also evidenced by the average proportion of pupal

cells at CCA5 at the 50 μg/L g roup which was 16% of the comb areas as compared to 26, 26, and 24% at

CCA5 for the control, 12.5, and 25 μg/L groups, respectively. This finding is also statistically significant

for all analyses conducted as indicated above. An examination of the propo rtions at CCA8 also suggest the

persistent nature of these effects and their lasting impact at this treatment group. The average proportion of

pupal cells at CCA8 for the 50 μg/L group was 9% as compared to 17, 17, and 16% for the control, 12.5,

and 25 μg /L groups, respectively. This finding was significant at the α = 0.1 in the EPA analysis and a α =

0.05 for the PMRA analysis (with 70.6 % reduction compared to control). 0 0.05

0.1 0.15

0.2 0.25

0.3Proportion of Pupae



### Colony Condition Assessment 

## Control

Capped 0 0.05

0.1 0.15

0.2 0.25

0.3Proportion of Pupae



### Colony Condition Assessment 

## 12.5 μg/L

Capped

0 0.05

0.1 0.15

0.2 0.25

0.3Proportion of Pupae



### Colony Condition Assessment 

## 25 μg/L

Capped 0 0.05

0.1 0.15

0.2 0.25

0.3Proportion of Pupae



### Colony Condition Assessment 

## 50 μg/L

Capped58 

### Regarding the statistical analyses, all methods found significant differences at 100 and 200 μg/L that were

apparent at early CCAs and persisted throughout the study. Additionally, effects were noted at multiple

CCAs for 50 μg/L, and the effects continued following overwintering. While all analyses found a significant

effect at the 12.5 μg/L treatment level at CCA6, and the PMRA analysis also found significant effect at the

25 μg/L treatment level, these effects were considered transient. This is because effects at 12.5 and 25μg/L

were isolated to CCA6 with levels returning to those simil ar to control after overwintering, and at CCA6

the effects lacked a clear dose

- response relationship and were similar among all three lower treatment levels

(12.5, 25, and 50 μg/L; 22.2, 18.3, and 12.5 % reductions compared to control based on raw data,

re spectively). Additionally, the discussion presented above indicates that the average proportions of pupal

cells in the 12.5 and 25 μg/L group at different CCAs resemble the responses found in the control group in

terms of their level before, during, and af ter exposure and overwintering. The effects at the 50 μg/L however,

appear earlier, persist longer, and have a clear impact, especially after overwintering, when compared to the

control.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for pupal cells

is determined to be 25 and 50 μg/L, respectively.



## 3.11.5. **Total individuals in hives**



### When evaluating the proportion of frame coverage of total individuals

, the pattern of effects has some

similarity to the proportion of frame cove rage of adults and pupae , as these two life stages make up the

largest components of the hive population throughout the course of the study. In the 100 and 200 μg/L

treatment groups, total individuals were significantly reduced f rom the level of control from CCA4 to CCA7

( p <0.05). The EPA analysis did not find a significant difference (p>0.1) at the 200 μg/L group at CCA4

but the impact at this treatment level is evident at other CCAs. The CDPR analysis did not assess this

response variable.

29 Table 28

. Estimated percent reduction from control for total number of individuals

Test

concentration

(μg/ L ) Estima ted reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 4 1 8.7 *

- 16.9

- 2.6

25 10.8 10.9 17.4 ** 2.2

- 11.4

50 10.3 25.4 * 12.1 ** 8.1 49.1 **

100 35.3 ** 46 ** 48.2 ** 60.9 ** 145.1 **

200 48.6 ** 60.5 ** 65.9 ** 74.6 ** 54.1

Note: Negative value indicates increased number of total individuals in comparison to control.

*0.05<p<0.1

**p<0.05

1 Percent differences from control are based on the raw counts of total individuals, not proportions of the total individuals a s Figure 1 9 below

shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.

These data are included in the PMRA analysis but excluded from the EPA analysis59 

### 19 Figure 19

. Proportion of total individuals (adult, eggs, larvae, pupae) following exposure to varying

concentrations of imidacloprid in the diet across CCA3

– CCA8.

For the 50 ppb treatment group, reductions relative to the control at CCA5 and CCA6 were significant at
α=0.10 and reductions relative to the control at CCA8 were significant for α=0.05 (CCA6 result was

significant at 0.05 in the PMRA analysis). For the EPA analysis, there were no further findings of statistical

significance which includes all CCAs at the 12.5 and 25 μg/L treatment groups. In the PMRA analysis,

total individuals were signific antly reduced at 12.5 (p<0.1) and at the 25 μg/L treatment groups (p<0.05).

It is noted here, as it has been previously for other response variables, that the effects determined at 12.5

and 25 μg/L are isolated to CCA6, with no determinations of significa nce before and after this CCA,

indicating this may be a transient effect. After overwintering at CCA8, the surviving hives in the 12.5 and

25 μg/L groups were actually above the level of control by 2.6 and 11.4% respectively (based on the raw

counts of to tal individuals) while the 49% reduction from control at CCA8 in the 50 μg/L group was

significantly reduced at a 0.05 alpha level.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for total

individuals is determined to be 25 and 50 μg/L, respectively.

Figures 20

- 22 below provide another visual representation of the effects across CCAs variables within a

response variable for the various life stages of bees during the course of the study. The bar charts represent

the percent differences from control with negative percent differences from control indicating an increase

in a given response variable above the level of contro l. These figures provide further evidence of the general

lack of dose responsiveness in effects at the three lowest treatment groups. Furthermore these charts are

effective in indicating how the percent differences with a given response variable, changed over the course

of the study within a treatment group. The 50 μg/L group chart ( Figure 22 ) in particular shows a progression60 

### of a continuous impact throughout the study for certain response variables that is still present to a higher

degree after overwin tering at CCA8. It is also noted here that the scale for percent difference from control

(y

- axis) has been standardized across all charts

. It is noted here also that negative (“

- “) responses refer to a

percent increase above the level of control. Charts are not shown for the 100 and 200μg/L group s given the

clear impacts on those hives across multiple response variables. Additional charts of the data represented

in a slightly different way, across response variables within a CCA, are provided in Appendix B

.

20 Figure 20

. Summary of living organism parameters at the 12.5 μg/L treatment group 4.0 2.2 2.8 12.3 22.8 1.0

- 5.4

- 3.7 10.7 11.9 8.7

2.3 22.2

- 15.5 5.9

- 16.9

- 12.6 - 8.4

- 62.6 0.1

- 2.6

- 11.0 - 1.3 0.8 37.7

-80.0 -60.0 -40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

TotalLife Adult Pupae Larvae EggsDifference from control (%)



### CCA4 CCA5 CCA6 CCA7 CCA861 

### 21 Figure 21

. Summary of living organism parameters at the 25 μg/L treatment group

22 Figure 22

. Summary of living organism parameters at the 50 μg/L treatment group 10.8 2.7 17.7

8.1

- 1.1 10.9

8.7 5.8 16.2

8.3 17.4

7.7 18.3 23.0 26.3

2.2

- 4.1 18.1

- 25.5 31.3

- 11.4 - 4.3

- 10.8

- 9.9 5.8

-40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

TotalLife Adult Pupae Larvae EggsDifference from the control (%)



### CCA4 CCA5 CCA6 CCA7 CCA8

10.3

- 23.3 28.1

12.1

- 9.6 25.4 21.7 34.6

15.3

1.8 12.1 19.8

12.5

- 2.1 11.7

8.1 18.6

9.7

- 32.4 2.6

49.1 78.0

70.6

42.6 78.2

-40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

TotalLife Adult Pupae Larvae EggsDifference from the control (%)



### CCA4 CCA5 CCA6 CCA7 CCA862 

### Figures 23

- 27 provide an additional visual representation of the effects on life stages during the course of

the study. This representation is distinguished from the figures p reviously presented for the life stage

response variables in that the trends for each variab le are presented within a CCA

. It is noted for these

figures that the scale has been adjusted to match for the y

- axis for all CCAs. This helps visualize the trends

of the response variables particularly in the later CCAs as compared to the earlier ones as well as to highlight

the fact that the control hives themselves experience seasonal reductions and inc reases in certain life stages

through time.

Although the assessment of hive health at CCA4 was taken only 3 weeks after the exposure period began

, decreasing numbers of pupal and larval cells are indicated with increase in imidacloprid dose , particularly

at the 100 and 200 μg/L groups ( Figure 23)

. These effects were also confirmed statistically by the three

analyses. Effects on pupal and larval cells numbers were persistent throughout the subsequent CCAs as was

discussed above.

23 Figure 23

. Trends of life stages across treatment groups within CCA4

At CCA5 ( Figure 24 )

, the effects become more readily apparent in the 50 μg/L group for adults and pupal

cells especially. In the 100 and 200 μg/L groups, adults, pupal cells, and egg cells continue to be repressed

as they were from CCA4. This is also visualized by the sharp dip in the trend lines for adult and pupal cells

in particular at the 50 μg/L groups and above in comparison to the level of control while the responses in

the 12.5 and 25 μg/L groups remain generally at the level of the control. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA4

Adult

Egg

Larval

Pupal63 

### 24 Figure 24

. Trends of life stages across treatment groups within CCA5

At CCA 6 ( Figure 25 ) , reductions that were determined in the three analyses for all life stage response

variables at the two highest treatment groups continue to be repressed from the level of control. At the 50

μg/L group, the reductions in adults and pupal cells are also e vident. Most noteworthy is the reduction in

pupal cells that was confirmed as statistically significant (p<0.05) by the EPA and PMRA analyses at the

12.5 μg/L (the PMRA analysis also determined a significant reduction at the 25 μg/L treatment group) . Thi s is also evidenced visually by the dip in the proportion of pupal cells of frame coverage at these two groups

(12.5 and 25 μg/L) while other response variables in this group (12.5 μg/L) at this CCA are in line with the

level of the control.

25 Figure 25

. Trends of life stages acr oss treatment groups within CCA 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA5

Adult

Egg

Larval

Pupal

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA6

Adult

Egg

Larval

Pupal64 

### As mentioned previously, CCA 7 represented the time during the study when hives are in a “shut down”

mode and preparing for overwintering. As a result, the proportions of all life stages are in a natural decline,

independent of imidacloprid exposure. The significant effects for adults, eggs, larvae, and pupal cells at the

100 and 20 0 μg/L treatment groups that were identified in previous CCAs were again determined by all

three analyses which are also visually evident in Figure 26 with the proportions of life stages in these two

groups clearly being below the level of that in the control. Effects at the 50 μg/L for adults (no other

significant effects at this group for other life stages) were determined as statistically significant by the

PMRA analysis but the level at CCA7 is reduced from control as well as in previous CCAs.

26 Figure 26

. Trends of life stages across treatment groups within CCA7

After overwintering at CCA8 ( Figure 27 a and 27 b ) , there were a number of hives that were lost due to

hive mortality. Despite this, colony health response variables of surviving hives are still considered

informative in examining the success of certain treatment groups over others (see Section 5

– Reviewer’s

Comment s for more details regarding this) . As evidenced by the hive mortality data previously discussed

as well as the trends shown below at the 100 and 200 μg/L group, there was a clear impact at these groups.

It is worth noting that significant reductions in these life stage response variables were evident well before

the time of CCA8 , with reductions being determined as early as CCA4 . What is noteworthy is the levels of

proportions at the 50 μg/L group as compared to the control and 12.5 and 25 μg/L groups. In addition to

the statistically significant findings of all analyses, the levels of proportions in all life stages is shown b elow

to be reduced from the level of control (EPA and CDPR analyses did not determine significance for larval

cells). This is distinguished from the 12.5 and 25 μg/L groups whose levels are generally in line or above

the level of control. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA7

Adult

Egg

Larval

Pupal65 

### 27 Figure 27

. Trends of life stages across treatment groups within CCA8



## Colony Condition Assessment **s**

**– Food Store Response Variables** 

**3.11.6. Pollen** 



### Pollen store s were significantly reduced in the 200 μg/L treatment group during from CCA4 to CCA7

( p <0.05) , with the CDPR analysis not finding a significant difference at CCA7 only. Pollen stores were

reduced at 100 μg/L treatment group at CCA4 andCCA5 (p<0.05). Similarly, pollen stores were

significantly reduced at the 50 μg/L treatment grou p at the 0.05 alpha level at CCA4 and CCA5, but not

CCA6 and CCA7 (p >0.1). A marginal reduction at CCA7 (0.05 < p <0.1) for the 50 μg/L was determined

in the PMRA analysis but not in the EPA or CDPR analyses

. The reduction of pollen store s was not

determi ned in the 12.5 and 25 μg/L treatment groups for any CCA assessed. The reduced pollen store was

most prevalent during and just after the exposure phase (CCA4 and CCA5) of the treatment levels exhibiting

effects.

30 Table 29

. Estimated percent reduction from control for pollen store s

Test

concentration

(μg/L ) Estima ted reduction from control (%) 1



### CCA4 CCA5 CCA6 CCA7 CCA8 2



### 12.5 11.8

- 6.8

- 53.3

- 26.1

- 12.2

25 5.2 2.1

- 25.2

- 20.7 0.7

50 56.1 ** 62.5 ** 15.5 29.2 * 63.8 **

100 83.7 ** 83.6 ** 15.3 12.9 100.6 **

200 94.5 ** 90.4 ** 54.7 ** 50.6 ** 12.3

Note: Negative value indicates increased pollen store s in comparison to control.

*0.05<p<0.1 

### 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA8

Adult

Egg

Larval

Pupal66 **p<0.05

1 Percent differences from control are based on the raw counts of pollen stores , not proportions of the pollen stores as Figure 28 below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.

These data are included in the PMRA and CDPR anal yses but excluded from the EPA analysis

.



### At CCA8, in the hives that survived overwintering, the total amount of pollen store was reduced in in the

50 μg/L group (↓64% based off raw counts ) . The PMRA analysis also determined a significant reduction

in the 100 μg/L group (p<0.05) but not in the 200 μg/L group (p>0.1) (this data excluded from EPA’s

analysis). It is noted however, that the lack of statistical difference of pollen store s in th e 200 μg/L group

is considered to be uncertain as there were only two hives surviving overwinter and a large confidence

interval.

28 Figure 28

. Proportion of pollen stores following exposure of honey bees to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8.

Figure 29 below shows the clear divergence of pollen stores in the 50 μg/L group as compared to the

control, 12.5, and 25 μg/L groups where stores begin to decline immediately following exposu re and

continue to be repressed throughout the study including after overwintering.67 

### 29 Figure 29

. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only.

In examining the trends of pollen stores in the control ( Figure 30 below), there was a buildup that occurred

from CCA3 (7% of frame coverage area ) to CCA5 (10%). This increase supports the queen in her effort to

build up brood during the late spring and early summer months. P ollen stores experienced a decline in

numbers from CCA5 (10%) to CCA7 (4%) before showing an upward trend from CCA7 to CCA8 (8%).

This downward trend reflect s that the fact that up to overwintering, brood production will slow as the hive

prepares for winter and therefore there is a reduced need for pollen within the hive.

As depicted in the Figure 30 below , the trends of t he proportions in the 12.5 and 25 μg/L groups tracks very

similarly with the control. Pollen stores at the 50 μg/L group , in contrast to the control, 12.5 and 25 μg/L

groups, began a decline in stores earlier than the other groups as well as having an ave rage proportion of

approximately 50% of the stores after wintering in CCA8 among the surviving hives. 0 0.02 0.04 0.06 0.08

0.1 0.12

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Polen Stores



### Colony Condtion Assessment 

## Pollen

Control

12.5

25

5068 

### 30 Figure 30

. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for pollen

stores is determined to be 25 and 50 μg/L, respectively.



## 3.11.7. **Nectar / Honey**



### There was c onsistently and significantly (p <0.1) a lower amount of honey stored in treatment hives at 50,

100 and 200 μg/L than in the control at CCA6 and thereafter ( Figure 31 below ) . One exception is the

absence of a determination of significance at CCA6 for the 100 μg/L group which PMRA determined as significant but EPA did not. All other findings after CCA6 at the 50, 100 and 200 μg/L (EPA excluded data 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of pollen stores



### Colony Condition Assessment 

## Control

Pollen 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Pollen Stores



### Colony Condition Assessment 

## 12.5 μg/L

Pollen

0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Pollen Stores



### Colony Condition Assessment 

## 25 μg/L

Pollen 0 0.02 0.04 0.06 0.08

0.1 0.12Proportion of Pollen Stores



### Colony Condition Assessment 

## 50 μg/L

Pollen69 

### at CCA8 for the 100 and 200 μg/L groups) were in general agreement with slight variations of the alpha

level that the effect was significant. N o reduction of the honey store s was determined at the 12.5 and 25

μg/L during the study, with the exception of the PMRA finding of a reduction at CCA6 at the 12.5 μg/L

treatment group ( p < 0 .1 ) at CCA6. This statistical difference at 12.5 μg/ L was unlikely to be treatment

related, as there were no reductions before or after the CCA6 at the same concentration, nor at the higher

concentration of 25 μg/L at CCA6. While there were no significant findings of impact at CCA8 in the 200

μg/L group (EPA excluded this data) it is noted that this lack of finding is considered to be uncertain as

there were only 2 surviving hives at this treatment group.

31 Table 3 0

. Estimated percent reduction from control for nectar/ honey stores

Test

concentration

(μg/L ) Estimated reduction from control (% )

CCA4 CCA5 CCA6 CCA7 CCA8

12.5

- 7.5 1.3 15.8 * 12.5

- 10.9

25

- 10.4

- 15.3

- 2.4 10.6 13.3

50

- 6.2 21.6 * 36 ** 41.2 ** 60.4 **

100

- 8 7.1 21.8 * 52.9 ** 156.6 **

200

- 21.1

- 84.1 70.5 ** 80 ** 5.1

Note: Negative value indicates increased nectar/ honey store s in comparison to control.

*0.05<p<0.1

**p<0.05

1 Percent differences from control are based on the raw counts of nectar/honey stores, not proportions of the nectar/honey stores as Figure 31

below shows.

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives s urvived in these groups, respectively.

These data are included in the PMRA analysis but excluded from the EPA analysis



### Figure 32 below for the honey store trends in the control, 12.5, 25, and 50 μg/L groups only show a marked

divergence at the 50 μg/L treatment group beginning at CCA6 and persisting up to and after overwintering

at CCA8.70 

### 31 Figure 31

. Proportion of honey stores following exposure of honey bees to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8.

32 Figure 32

. Proportion of honey stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups only. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Nectar (honey) stores



### Colony Condition Assessment 

## Nectar (Honey) Stores

Control

12.5

25

5071 

### For the control group, honey stores underwent an upward trend from CCA3 (16% of the frame coverage ) to

CCA4 (20%), before a subsequent decline in average proportion from CCA4 to CCA5 (13%). This was

followed by an approximately 140% increase in honey cells from CCA5 to CCA6 (average proport ion of

30%) that remained stable until CCA7 (30%). This buildup of honey stores took place ahead of CCA7 that

represented the last time point before overwintering. The honey stores declined markedly from CCA7 to

CCA8 (16%) which is expected given lack of foraging and utilization of these reserves during the

overwintering period. It is noted that the proportion of comb cells containing honey stores at CCA3 and at

CCA8 were approximately the same at 16%.

33 Figure 33

. Proportion of honey stores following exposure to varying concentrations of imidacloprid in

the diet across CCA3

– CCA8 in the control, 12.5, 25, and 50 μg/L groups. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35Proportion of nectar/honey stores



### Colony Condition Assessment 

## Control

Honey 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35Proportion of nectar/honey stores



### Colony Condition Assessment 

## 12.5 μg/L

Honey

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35Proportion of honey/nectar stores



### Colony Condition Assessment 

## 25 μg/L

Honey 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35Proportion of honey/nectar stores



### Colony Condition Assesment 

## 50 μg/L

Honey72 

### In the 12.5 μg/L treatment group, honey stores also underwent an initial build up from CCA3 (16%) to

CCA4 (21%) before a subsequent decline from CCA4 to CCA5 (12%), similar to that of the control. This

was followed by a large buildup of honey stores from CCA5 to CCA7 (27%). Honey stores declined during

ov erwintering and represented 15% of the brood area at CCA8, as compared to 16% at CCA3, which a

similar finding to that in the control group. The proportion of honey stores at CCA8 was comparable to that

of the control group (15% for both 12.5 μg/L and con trol). A similar picture was found for the 25 μg/L

treatment group

, in that honey stores underwent an initial build from CCA3 (15%) to CCA4 (21%) before

a decline at CCA5 (14%). The subsequent build up reached 29% at CCA7 before a decline through

overwin tering to CCA8 (14%). CCA8 concentrations of nectar and pollen were comparable to that of the

control at CCA8 (honey: 14% and 15%; 25 μg/L and control groups, respectively).

These trends in the control and two lower treatment groups are distinguished from the response at the 50

μg/L group. While honey stores underwent an initial buildup and then decline from CCA 3 (17%) to CCA4

(21%), the subsequent larger buildup leading up to CCA7 that took place in the control and lower treatment

groups was much less pronounced with the 50 μg/L group. Specifically, the proportion of honey stores

from CCA3 to CCA7 roughly double in numbers from 15 to 30% of the brood comb in the control, 12 .5

and 25 μg/L treatment groups. This is distinguished from the 50 μg/L group were the buildup that occurred

from CCA5 to CCA7 reached a marginally higher level that the starting proportion at CCA3 (18% and 17%

respectively). That is to say, that the amo unt of honey stores at CCA7 (before the overwintering period) in

the 50 μg/L group was approximately half of that in the control, 12.5, and 25 μg/L treatment groups (18%

for 50 μg/L as compared to 30, 27, and 29% at CCA7 for the control, 12.5 and 25 μg/L t reatment groups, respectively). Notably, it is also the only group out of these 4 in which the proportion of honey stores at

CCA8 was markedly lower than that of CCA3 (8% at CCA8 and 17% at CCA3).

It is noted that the feeding solutions (sugar solutions ) provided during the exposure period might have

affected natural honey storage patterns; however, effects on honey storage are still able to be considered as

all treatments were compared to control hives (which also received feeding solutions).

When weig hing statistical and biological significance, the overall NOAEC and LOAEC for honey

stores is determined to be 25 and 50 μg/L, respectively.

Figures 34

- 36 below show an additional visual representation of the impacts on food stores across the CCAs

for each treatment group (Figures not shown for the 100 and 200 μg/L treatment groups due to the clear

impacts on the hives at that level, primarily on hive morta lity). It is noted that the scale for percent difference

from the control (y

- axis) was standardized to the level of the 50 μg/L chart. When visualized this way, the

impact of the early on and persistent nature of effects (particularly with pollen) at the 50 μg/L is clearly

divergent from the responses at the 12.5 and 25 μg/L, which generally show a buildup in food stores that

was also observed in the control group. It is noted here that negative (“

- “) responses refer to a percent

increase above the level of control.73 

### 34 Figure 34 . Summary of hive food supply parameters at the 12.5 μg/L treatment group

35 Figure 35

. Summary of hive food supply parameters at the 25 μg/L treatment group - 7.5 1.3 15.8 12.5

- 10.9 11.8

- 6.8

- 53.3 - 26.1 - 12.2

-60.0 -50.0 -40.0 -30.0 -20.0 -10.0

0.0 10.0 20.0



### CCA4 CCA5 CCA6 CCA7 CCA8Difference from the control (%) 

### Honey Pollen



### - 10.4

- 15.3 - 2.4 10.6 13.3

5.2 2.1

- 25.2 - 20.7 0.7

-40.0 -20.0

0.0 20.0 40.0 60.0 80.0

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Honey Pollen74 

### 36 Figure 36

. Summary of hive food supply parameters at the 50 μg/L treatment group

Figures 37

- 41 provide an additional visual representation of the effects on food store parameters during the

course of the study. This representation is distinguished from the figures previously presented for the food

store response variables in that the trends for each varia ble are presented within a CCA8 across treatment

groups. It is noted for these figures that the scale has been adjusted to match for the y

- axis for all CCAs.

This helps visualize the trends of the response variables particularly when examining the level o f control at

each CCA as honey stores for example initially fall then build up across multiple CCAs in preparation for

overwintering heading into CCA7. - 6.2 21.6 36.0 41.2 60.4 56.1 62.5

15.5 29.2 63.8

-20.0 -10.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Honey Pollen75 

### At CCA4, 3 weeks into the exposure period, honey stores across all treatment groups remained at the level

of the control, as shown below, and this was also confirmed statistically by all analyses. Pollen stores

however, began to undergo an immediate reduction at the 50, 100, and 200 μg/L groups while the 12.5 and

25 μg/L remain ge nerally at the level of control.

37 Figure 37

. Trends of food store parameters across treatment groups within CCA4

The responses at CCA5 are the same as those at CCA4 in terms of honey responses although the trend at

the 200 μg/L group rises above the level of the control. Pollen stores continue to be suppressed at the 50,

100 and 200 μg/L, while the responses at 12.5 and 25 μg/L are generally at the level of the negative

control.

38 Figure 38

. Trends of food store parameters across treatment groups at CCA5 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA4

Honey

Pollen

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA5

Honey

Pollen76 

### At CCA6, significant reductions in honey stores began to become apparent and were confirmed statistically

at the 50 and 200 μg/L levels. These groups also appear visually reduced from the level of control. Of note

is that the proportions of honey in the control are above that of the level of CCA5 by approximately 2.5

fold, indicating a buildup in stores in the weeks before overwintering.

39 Figure 39

. Trends of food store parameters across treatment groups at CCA6

At CCA7, just before the overwintering phase begins, there is a marked decline in honey stores in the 50,

100 and 200 μg/L groups below that of the level of control. This is noteworthy as the reduced stores provide

an indication that these hives will have reduced success after overwintering. In the case of the 100 and 200

μg/L groups, these hives largely did not survive overwintering.

40 Figure 40

. Trends of food store parameters across treatment groups at CCA7 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA6

Honey

Pollen

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA7

Honey

Pollen77 

### Finally, at CCA 8 after overwintering, honey stores were markedly decreased in the control group from their

level heading into overwintering at CCAs 6 and 7. The levels at the 100 and 200 μg/L groups are clearly

suppressed as these groups had one and two hives surviv ing, respectively. The response at the 50 μg/L

level was also reduced from control while the response at the 12.5 and 50 groups again are in line with or

above the level of control.

41 Figure 41

. Trends of food store parameters across treatment groups at CCA8



## 3.11.8. **Hive Weight**



### There were significant reductions from control observed at the two highest dose levels (100 and 200 μg/L)

beginning at CCA4 and persisting until CCA 8 (p< 0.05, data from CCA8 excluded from EPA analysis).

Additionally, there were no significant reductions from control in the 12.5 and 25 μg/L treatment groups

determined for all CCAs assessed (p>0.1). For the 50 μg/L group, there were significant reductions at both

the 0.05 and 0.1 alpha level starting as early as CCA4 (CDPR and PMRA analyses) . The PMRA analysis

determined significant reductions at the 0.1 alpha level for CCA 4 and CCA6 and at the 0.05 alpha level for

CCAs 5, 7, and 8. The EPA analysis deter mined significant reductions from the control at the 0.1 alpha

level at CCAs 7 and 8 only. Despite these differences in statistical findings, there is an apparent effect on

hive weight at the 50 μg/L level that is supported by both analyses indicating sig nificant reductions at

multiple CCAs

.

While there was no difference (p >0.1) in the hive weight at the 200 μg/L treatment groups (data not included

in EPA analysis), the lack of statistical difference is considered to be uncertain as there were only two hives

surviving overwintering

. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA8

Honey

Pollen78 

### 32 Table 3 1. Estimated percent reduction from control for hive weights

Test

concentration

(μg/L ) Estima ted reduction from control (%)

CCA4 CCA5 CCA6 CCA7 CCA8 1



### 12.5

- 0.5 2.4 0.2

- 1.6

- 1.1

25 2.7

- 0.4

- 1.5 0.6 1.2

50 4.1 * 11 ** 12.3 * 15 ** 20.9 **

100 7.1 ** 14.9 ** 15 ** 18.9 ** 67.7 **

200 10.1 ** 10.4 ** 30.4 ** 33.3 **

- 25.5

Note: Negative value indicates increased hive weight in comparison to control.

*0.05<p<0.1

**p<0.05

2 At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.

These data are included in the PMRA analysis but excluded from the EPA analysis



### 42 Fi gure 42

. Proportion of hive weight following exposure of honey bees to varying concentrations of

imidacloprid in the diet across CCA3

– CCA8

When weighing statistical and biological significance, the overall NOAEC and LOAEC for hive

weight is determined to be 25 and 50 μg/L, res pectively.79 

## 4. **Review er**  **comments:**

What follows is brief discussion of some of the elements taken into consideration when evaluating the results

of this study.



## General Considerations for Biological Interpretation

While the hive mortality is considered as the most relevant measurement of survival at the colony level,

sublethal effects at the colony level were estimated by measuring multiple parameters during the course of

study. Each measured parameter is expected to reflect only part of the colony condi tions, and all

measurements have to be integrated for a better understanding of the hive status at the colony level. A

honey bee colony is a super

- organism in which live individuals and food supply are the two major

components in maintaining the proper fu nction of the colony. There are interactions between the two

components and even within each component.

Bee individuals are present in the colony as eggs, larvae, pupa and adults and they develop from one stage

to another and interact with each other to perform a variety of tasks to maintain the integrity of the colony.

The measurement of each stage of the bees is expected to provide information on the potential treatment

effect on a specific life stage of bees during their development.

Hive food supplies including hive pollen and nectar are collected and processed by adults and are expected

to have a large impact on the development of all stages of bees in hives. However, the amount of hive food

storage is dependent on not only the power/number of foragers available for food collection, but also the

number of individuals that consume the food. In addition, the seasonal availability of outside pollen and

nectar sources also affects the amount of storage, thus impacting hive development. As well, sucrose

feeding solutions were provided to the hives as a means of treatment and as a supplement for hive

overwintering, which may have affected foraging and food storage during those time periods.

Hive weight was measured during the study. However, it is largely affected by the honey storage and number

of bees that consume the food. A strong colony with a high number of bees likely consumes a high amount

of stored honey and may result in a reduced hive weight . In this study, additional sugar solution was

provided as the means of treatment and a supplement for hive overwintering in the study. Such feeding

likely further confounds the relevance of the hive weight to the treatment effect. In addition, hive weigh ts

were taken after 10 a.m. However, weighing hives at different time periods of the day may result in an

increased variation of the measurement due to the fact that foragers may not be present in the hive when the

weigh t is measured . Hive weights may be a rtificially lower in hives which contain a high number of forager

bees that may be out collecting food during a different time of the day.

Considerations regarding the measurement time points :

• CC A3 represents the background hive conditions as the first colony assessment after the hives

were placed in the test fields prior to the exposure.

• CCA4 and CCA5 represent the hive conditions during the exposure phase. It was noted that the

CCA5 was conducted a week after the end of the 6

- week exposure period , but is expected to

represent effects during the exposure period

.80 

### • CCA6 was measured at 5 weeks after the end of exposure. It allows all bee individuals, including

eggs, larvae and pupa that were exposed to treatment to finish their development cycle and become

adults.

• CCA7 represents the hive conditions prior to overwintering. It is considered that hives were

physiologically preparing for overwintering by reducing the production of immature bee

individuals. Treatment effect s may be masked by the natural dec line of hive individuals.

• CCA8 represents hive conditions of surviving hives after overwintering. Additionally, hive

overwintering mortality at CCA8 is expected to be directly relevant to the treatment effect at the

colony level.



## Control Performance

Control mortality:

The leve l of colony loss after overwintering in controls (36 %), though not desirable, is consistent with that

historically experienced by beekeepers on average across the United States 3 

### ; the 2014

- 2015 preliminary

results estimate US overwintering loss on average to be 23.1%

. There are , however, differences in bee

management practices associated with the test hives compared to commercial hives, making a direct

comparison of expected overwintering success challenging

. C ommercial hives c ould experience very

different beekeeping practices than test hives , including being transported for pollination services, being

harvested for honey production, given additional feedings, receiving different pest and disease control

treatments, having diff erent sized hives and different preparation for overwintering. The test hives, while

closely monitored, may also experience invasive disruptions during the col ony condition assessments, have

different/less supplemental feeding or mite treatments than comm ercial hives , have a lower colony size or

be prepared differently going into overwintering as was the case with hives in this study which were not

given supers to allow for growth as well as having supplemental feeding delayed

.

Sublethal effect s on life stages and food stores

As described with discussion of the response variables in Section 3 above, the parameters measured indicate

that control hives behaved as would be expected through the seasonal changes that a honey bee colony

undergoes . Althoug h a large variation among apiaries was detected for each parameter in all control hives,

the average of the total number of individuals in hives, as well as eggs, larvae, pupae and adults increased

or remained at similar levels during the exposure period f rom CCA3 to CCA5. After the end of the exposure,

all these biological parameters also increased or remained at a similar level at CCA5 and CC A 6, and then

all decreased sharply from CCA6 to CCA7.

The similarity in the dynamics of all parameters for the i ndividual living organisms at various stages

indicates that control hives were well developed during CCA3 to CCA6, and the hives were preparing for

overwintering at CCA7 as expected in the late fall in October. The increase of hive food supply (pollen and

honey store) and hive weight also confirms that hives were actively developing during the exposure period

from CCA3 to CCA5. The increased level of honey store from CCA5 to CCA6 and CCA7 indicates that

there were plenty of outside nectar sources in the t est area after the end of feeding exposure period.

3 http://beeinformed .org/2015/05/colony

- loss

- 2014

- 2015

- preliminary

- results/81 

### However, the pollen store reduction observed during CCA5 to CCA7 might have resulted from increased

pollen consumption due to the increase of live individuals and/or limited availability of outside polle n sources. If the outside pollen source were limited, it may have impacted the further development of control

hives after CCA5. However, the decline of pollen stores in the control hives after CCA5 is likely normal

for the local region of the study, and c ontrol hives did not appear to be impacted, therefore, this is not

expected to have biased the study.



## Consideration of CCA8

**Control overwintering mortality** 



### While the level of control mortality after overwintering is generally in line with historical findings from

North American beekeepers as described above, it is noted that the use of data from CCA8 to distinguish

treatment

- related effects on colony survival ( i.e. dead or alive) is compromised due to the level of control

mortality observed

. That is to say , the ability to detect treatment

- related decreases in overwintering colony

survival may be masked by the magnitude of control hive loss.

**Response Variab les**  **from Surviving Hives**



### While the measure of overwintering success may be compromised by the control hive loss, the evaluation

of life stage and food store metrics at CCA8 for surviving hives is considered to be useful and important to

interpreting the s tudy results. Specifically, the data suggest that the weaker control hives were not able to

survive overwintering because they had disproportionately fewer numbers of adults and honey stores

compared to control hives that survived overwintering

. ( Figure 4 3 ).

43 Figure 43

. Fate of control hives as compared to number of adults across CCAs

Control hives that survived overwintering had 2.5X more honey on average at CCA7 than those that did not

survive overwintering ( Figure 44 )82 

### 44 Figure 4 4. Fate of control hives as compared to the honey stores a cross CCAs

This, coupled with the experimental design which limited colony development due to lack of supers,

provides a plausible explanation for the loss of control hives. Assuming this hypothesis, the bias introduced

by this “culling” of weaker hives would theoretically render the remaining control hives at CCA 8 stronger

(on average) than the initial population of control hives. This could conceivably improve the ability to

detect treatment

- related colony condition effects at CCA8 , since the actual values for controls would be

weaker (on average) if the data fr om the dead hives were available for inclusion , and b ecause there were

more non

- survived hives among controls than the two lowest treatments

.



## Consideration of Study Strengths, Limitations and Interpretation

It is important to recognize the inherent st rengths and limitations of this study as results are interpreted and

potentially considered in risk assessment.

In the context of available field studies involving honey bees and imidacloprid, this study contains a number

of strengths including:

• Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power

• Demonstration of a generalized concentration

- response relationship with respect to the

concentration of imidacloprid in sucrose solution and the magnitude and duratio n of adverse effects

• Quantification of exposure to parent (imidacloprid) and toxicologically

- relevant metabolites in diet

and in hive matrices (uncapped nectar, pollen, honey, bee bread)

• Use of a 6

- week exposure duration to represent a “high end” exposure scenario

• Inclusion of multiple colony

- level endpoints reflecting hive strength, brood development and food

stores83 

### • Detailed QA/QC results regarding quantification of chemical residues in various matrices

• Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:

• Exposure of bees through nectar (sucrose) alone, whereas bees in the field are likely exposed through

both pollen and nectar routes. Therefore, the design of this study may not reflect a “worst case”

exposure scenario in which bees are experiencing prolonged exposure to both contaminated nectar

and pollen. While exclusion of the pollen route is expected to reduce overall exposure, the impact

of this exclusion on the study results is uncertain and will likely depend on the life stage/caste of

bee

.

• It is noted that imidacloprid was found in both hive nectar and hive pollen (beebread), at

concentrations lower than the feeding solutions. Dilution compared to the treatment feeding s olution

is expected since bees could also forage on outside nectar and pollen sources. As well, hive pollen

contains only some hive nectar, thus would not be expected to have a concentration equivalent to

nectar alone, and it is mixed with pollen which wil l come from outside sources. Therefore exposure

through both hive pollen and nectar occurred via exposure to the sucrose feeding solution, but how

this compares to exposure through contaminated pollen directly is not known. A recent paper by

Dively (2014) showed that higher residues throughout the hive resulted from feeding pollen

treatments compared to feeding sucrose solution treatments. It is also noted that n ectar is considered

the dominant exposure route for forager bees; other hive bees and larvae c on sume both nectar and

pollen. In addition, s ince bees were forced to forage for pollen in this study, the potential impact of

imidacloprid exposure on reducing pollen foraging efficiency of bees could be incorporated into the

overall expression of adverse e ffects, as suggested by published literature. Had contaminated pollen

been provided to bees, it is not known if the potential impact on pollen foraging efficiency would

have been masked.

• The quantity of nectar provided to hives (2 L per week per hive) likely did not fulfill the complete

carbohydrate needs of the colony, as indicated by colony bioenergetics and the lack of remaining

sucrose solution upon their renewal. This suggests that bees could be exposed to a greater dose of

imidacloprid in nectar had a greater volume of spiked sucrose been provided. Although one can

infer that the dosing regimen may have underestimated exposure through sucrose relative to 100%

contaminated diet, it is also noted that bees had to supplement their spiked sucrose by foraging on

their own for other sources of nectar. As with the previous discussion of pollen, it is noted that had

100% of the carbohydrate needs of the colony been provided via feeders, the potential impact of

purported reductions in nectar foraging effi ciency may have been masked to some degree.

• Overwintering success of controls was impacted (36% hive mortality)

. This may have reduced the

ability to detect adverse effects related to hive loss following overwintering. Although comparable

to overwinteri ng losses of commercial beekeepers, it is possible that elements of the study design

may have contributed to this loss (e.g., lack of supers to allow for colony growth, delayed

supplemental feeding during fall).

• Hive c ontamination with pesticides from fo od sources other than the artificial feeding was detected

d uring the exposure period and post

- exposure periods through collection of pollen from pollen traps

.84 

### Although the study was deliberately conducted in a low agricultural area in order to minimize the

potential for pesticide contamination from other sources, the bees still appeared to be foraging on

contaminated pollen and possibly nectar. During both exposure and post

- exposure periods, high

level s of multiple pesticides that may cause concern for bees were detected in most monitoring hives

, such as spiromesifen (maximum at 961 ppb) and piperonyl butoxide (maximum at 591 ppb). Trace

amount s of other bee

- toxic pesticides, such as chlorpyifos (LOD = 1.0 ppb) and malathion (LOD = 4.0 ppb) were also detected. The test chemical imidacloprid was found at 12.1 ppb in pollen from

one (apiary L) of the total of six sites analysed. This level is similar to one of the test concentrations.

• R esidues of imidacloprid in uncapped nectar and bee bread with in the hives at CCAs 4, 5, and 8

represent a single sample per hive on a single frame rather than a composite sample from multiple

portions of the comb within a hive. This means that residue results may reflect “hit or miss” scenario

with r espect to detecting residues in nectar laid down from contaminated (fed) vs. outside sources.

• The exposure, based on residues measured in the hive (hive nectar and hive pollen) indicated that

, overall, higher measured hive residues correlated with higher nominal residues in feeding solution s

. However, individual hiv e residue values varied , and there was some overlap in measured values,

particularly among the three lowest doses.

• E xposure dilution during the study was evident . P ollen stor age was observed consistently in the

control hives and hives exposed to low er test concentrations during the exposure period , indicating

that test bees were foraging on food sources other than the spiked sugar solution . Remarkably lower

residue concentrations detected in bee bread and hive nectar in some test hives compared to the

feeding concentrations may also indicate foraging on other food sources. This uncertainty is inherent

in an y semi

- field or full

- field study design.85 

## 5. **Conclusions**

The study is considered to be informative and will be used as a line of evidence in the pollinator risk

assessment . While there were uncertainties that were generally related to inherent aspects of any semi

- field

or full field study design (such as dilution of the tes t chemical through alternative sources of forage,

d etection of other chemicals in the monitoring hives ), this study still provides information on a number of

colony health parameters about the long term (including overwintering) exposure to imidacloprid at the

colony level.

As indicated in the results section above, the PMRA, EPA, and CDPR analyses determined significant

effects ( at both the 0.05 and 0.1 alpha level s) in the 50, 100, and 200 μg/L groups across multiple CCAs for

the majority of response variables

. Specifically, for the 100 and 200 μg/L treatment groups, significant

effects (p<0.05) were determined for every response variable and persisted across at least 2 CCAs , along

with very high overwintering mortality. W hile the 50 μg/L group had overwintering mortality similar to the

controls, colony condition effects were different from controls with an early onset of effects which tended

to persist, and notably poorer colony condition in surviving hives after overwinte ring in comparison to

controls.

Conversely, there was not a strong indication from the PMRA, EPA, and CDPR analyses of an impact at

the colony level at the 12.5 and 25 μg/L treatment group s

. This is evidenced not only by a general lack of

statistical findings (p>0.1) at these treatment levels but in cases where significant effects were determined,

they either did not show strong dose

- responsiveness, did not persist across multiple CCAs, or were

considered potential transient effects (e.g. at CCA6) which did not persist after overwintering . This latter

point was the case for the total life and pupal cell findings in which the PMRA analysi s determined

significant effects at all treatment levels at CCA 6 (EPA also determined a significant reduction in pupal

cells at the lowest treatment group of 12.5 μg/L at CCA6)

. As well at CCA6, PMRA determined significant

effects with eggs and larvae at 25 μg/L treatment (but not at the 50 μg/L). For these two lowest treatment

groups ( 12.5 and 25 μg/L), t he colony condition of surviving hives at CCA8 following overwintering was

similar to controls

, indicating the effects observed at CCA6 were likely transient and the colony was able

to compensate for these effects

.

The figures below present the trends o f life stages across all CC As in the 12.5, 25, and 50 μg/L treatment

groups compared alongside the response of that in the control.86 

### 0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## Control

Adults Eggs Open Capped

0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## 12.5 μg/L

Adults Eggs Open Capped

Significantly reduced from control in the

PMRA, EPA, and CDPR analyses (either

0.1 or 0.05 level depending on the

analysis) Proportions at CCA7 and CCA8 are

approximately the same as control (8%

and 17% of frame coverage,

respectively)87 

### 0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## Control

Adults Eggs Open Capped

0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## 25 μg/L

Adults Eggs Open Capped

Significantly reduced from control in the

PMRA, EPA, and CDPR analyses (either

0.1 or 0.05 level depending on the

analysis) Proportions at CCA7 and CCA8 are

approximately the same as control (7% in

treatment vs 8% in control and 16% in

treatment vs 18% in control, respectively88 

### 0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## Control

Adults Eggs Open Capped

0 0.1 0.2 0.3 0.4 0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8Proportion of Abundance



### Colony Condition Assessment 

## 50 μg/L

Adults Eggs Open Capped

Significantly reduced from control in the PMRA,

EPA, and CDPR analyses (either 0.1 or 0.05 level

depending on the analysis). Effects begin at a time

when buildup of adults is still taking place in

controls and two lower treatment groups and

repression continues up to and including after

overwintering. Significantly reduced from control in the PMRA and EPA (at 0.05

level). The 50 μg/L is the only group of the three lower treatment

groups that does not show an upward trend from CCA7 to CCA8.

Significantly reduced from control in EPA, CDPR and PMRA analyses

(p<0.05 or p<0.01, depending on the analysis, CCA6 not determined in EPA

analysis). Effects are clearly divergent from control in which a buildup in

pup al cells occurs in the control from CCA3 to CCA6 whereas the 50 μg/L

group underg oes an initial decline from CCA4 to CCA589 

### 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8 

## Control

Pollen Honey

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8 

## 50 ppb

Pollen Honey

Level of honey stores at their peak in

control group heading into overwintering

at CCA7. The control group had

approximately 30% of frame coverage of

honey stores at CCA7 while the 50μg/L

group had 19% Significantly reduced from control in the PMRA,

EPA, and CDPR analyses (either 0.1 or 0.05 level

depending on the analysis). The level of honey

stores at CCA8 in the 50 μg/L group is

approximately half of that of the control at CCA8.90 

### When examining the effects on food stores (pollen and nectar), the PMRA, EPA, and CDPR analyses did

not determine any consistent and significant reductions in pollen and nectar stores at the 12.5 and 25 μg/L

treatment groups. This is distinguished from the 50 μg/L group where effects on nectar in particular were

very apparent when compared alongside the response of the control in terms of the level of nectar buildup

before the hive preparation for overwintering at CCA7. This finding was also confirmed s tatistically in all

three analyses with significant reductions in honey stores at CCAs 6, 7, and 8 (CCA8 data excluded from

the EPA analysis for the 100 and 200 μg/L groups). Significant reductions in pollen stores were also

confirmed at CCAs 4 and 5 at th e 50 μg/L treatment during the exposure period.

Specifically, when considering the adult and honey and pollen stores response variables, the differences

from control were apparent both visually and statistically, particularly in the three highest treatmen t groups. For the proportion of adults, the onset of a decline in numbers occurred one CCA earlier in these groups

than in the control, 12.5 and 25 μg/L treatment groups. For honey stores, the buildup that occurred starting

at CCA5 in the 50 μg/L treatme nt group, reached only half the level reached in the control, 12.5, and 25

μg/L treatment groups by CCA7. Pollen stores were also reduced at CCA4 and CCA5 compared to controls

for the three highest treatment groups, as well as at CCA6 and CCA7 at the highe st treatment group. These

effects were statistically significant (p<0.05) and indicate that the 50 μg/L treatment group was associated

with trends and proportions of abundance for life stages and food stores not observed in the control, 12.5,

and 25 μg/L treatment groups.

Therefore, when weighing biological significance and the natural seasonal changes of honey bees

colonies, as well as supporting conclusions from the statistical approach es used in PMRA, EPA, and

CDPR , the NOAEC and LOAEC for this stud y is determined to be 25 and 50 μg/L , respectively.91 

## 1

. A ppendix A : Details of EPA Statistical Analysis

In the statistical analysis of the Tier II colony feeding study for imidacloprid and honey bees, there were

three main questions that were investigated:

1. For given CCA (colony condition assessment) and treatment level, what treatments were having an

effect on the measures of hive health, as determined relative to control?

2. Were the observed treatment effects consistent over time?

3. What was nature of the onset of effects over time for various treatments?

To answer these questions, a variety of statistical approaches were considered and a repeated measure mixed

model analysis was deemed best to address the above questions.

For this analysis the time by treatment interaction was evaluated across all CCAs and treatment groups for

each response variable. In this way, the trends for each response variable ( i.e. adult, eggs, etc.) across all

CCAs for a given treatment group c ould be examined. The details of the parameterization of the repeated

measure mixed model along with the statistical results (p

- values) are provided below.

Background on data manipulation

• Data utilized for the statistical analysis were provided in an Excel file by the study author. This file

was made available to the Agency on May 15, 2015. Additional QA of the entire data file was

completed by the study author, as some transcription errors were found in an earlier electronic file

submitted by the study author.

• Data to be included in the data analysis were data that were collected on or after CCA3. CCA3

represents the first time hive parameters were measured after the hives were placed in t heir treatment

apiaries/locations.

• Zeros in the data set represent instances when no frame contained >5% coverage of a given matrix.

Missing values for all matrices during a given timepoint indicate the hive was dead and no

measurements were taken. Defin ition of a dead hive is provided in the study report. For these data

analyses, all entered zeros were maintained in the data set. Missing values were kept as ‘missing’.

These parameters were not replaced with zeros.

• Time (days) between measurements was ro ughly even for CCA3 through CCA7, while CCA8 was

measured during the following spring (to evaluate any impacts on hive overwintering). Number of

days was estimated using the median number of days between CCAs. Assuming that CCA3 was on

day 0, CCA4 through CCA8 occurred on the following days: 25, 53, 84, 119, and 272.

• In the initial analyses, there were difficulties with convergence of the PROC MIXED algorithm for

many of the more complicated temporal covariance structures. John Troiano (California Departme nt of Pesticide Regulation) provided an article from SAS suggesting that by re

- scaling the response

variable, the likelihood of convergence would be improved. Given the nature of the response variable

(most were count cells of a given matrix within in a hi ve), a re

- scaling was straight

- forward. For

eggs, open, capped, pollen, and honey cells, the count (the variable recorded in the excel spreadsheet)

was divided by 68800 (total number of cells in the hive). This resulted in the proportion of cells

occupied by a given matrix (values between zero and one). Number of adult bees, as recorded in the

spreadsheet, was derived from the percent coverage, the density estimate of 1.3 bees/cell, and the

density estimate of 4 cells/cm 2 

### . Therefore, the conversion from th e data stored in the spreadsheet92 

### was to divide by 68800, divide by 1.3, and multiply by 4. The resulting number is the proportion of

cells occupied by adult bees. Following the re

- scaling of the response variables, the model

convergence improved dramatica lly.

• The variables to be analyzed included:

o Proportion of hive covered in adults

o proportion of hive covered in eggs

o proportion of hive covered in open cells (larvae)

o proportion of hive covered in capped cells (pupae)

o Proportion of hive covered in live be es (at any life stage). This was obtained by summing the

proportions of the four life stages (described above). Note that it is possible for the proportion

of a hive covered in live bees to be greater than 1.0, as adult bees will be observed on top of

cell s containing eggs, larvae, or pupae.

o Proportion of hive covered in pollen cells

o proportion of hive covered in honey cells, and

o Hive weight.

Examination of the associations among the variables

• Based on physical hive constructs and the nature of the honey bees, it is generally accepted that the

colony condition assessment (CCA) variables may be correlated over time and may also be

correlated within a time point (sampling time). Given this backgro und, a series of scatterplots,

correlation matrices, and principal component analyses for this bee study was prepared (Further

details are provided in the section entitled “ Scatterplot and Principal Component Discussion ”

and the full SAS output is included as Attachment 3 ).

• Some of the general summary points are:

o Variables tended to have stronger correlations at adjacent time points than at farther time

points (i.e., correlations for CCA3 and CCA4 tended to be stronger than correlations for

CCA3 and CCA7 or for CCA4 and CCA7).

o CCA8 tended to have weaker pairwise correlations with all the other time points than CCA3

through CCA7 had with each other.

o The first principal component for each of the CCAs explained 20

- 50% of the total

variation. The lowest was cap ped, and the highest were adults, honey, and pollen.

o The general interpretation of the first principal component was a weighted average over all

time points (weights varied depending on variable).

Analysis Approach and Model Setup

The general experimental design was a randomized complete block with repeated measures. Apiary was the

block effect and the repeated measures were the CCAs. Within each block, the control treatment was

replicated 2x and each treatment occurred one time. Si nce hives were not placed till shortly before CCA3,

the data for the statistical analysis only included CCA3 through CCA8. Exploring the interaction between

treatment and CCA will address the first two questions above.

Once the design component of the an alysis was established, the next part of the analysis was to determine

which correlation structure (across time) was the best fitting for these data. One of the challenges was that

many hives died before the end of the study (especially the 100 and 200 ppb treatments in CCA8), creating93 

### censored data or an imbalanced design. PROC MIXED in SAS can handle an unbalanced design; however,

convergence may not be attained for some correlation structures. In this case the imbalance was due to

treatment (the higher t reatments had a higher rate of hive death) as well as random mortality (control hive

mortality rate during overwintering was similar to background mortalities of commercial hives). After

exploring several options ( which are detailed in the section entitled “Options for Addressing the Data

Imbalance” ), the data were analyzed after removing data from the few remaining hives in treatment groups

100 and 200 ppb from CCA8. Data from hives treated with 100 or 200 ppb imidacloprid remain in the data

set and analys is for CCA3 through CCA7 permitting utilization of a majority of the data. These deletions

create a 5x6 factorial design matrix with two of the cells (CCA8, treatment=100) and (CCA8,

treatment=200) missing all data. CCA*treatment interaction means can stil l be calculated. Least square

means on the main effects (treatment and CCA) cannot be estimated for Concentration=100,

Concentration=200, and CCA8. Since the expectation was that the interaction would be significant and all

further statistical tests would be exploring the interaction, this was not considered a significant hindrance.

To address the primary research questions above, the “treatment * CCA” interaction was evaluated in two

ways:

• At each CCA, was there a reduction in the response relative to t he control? This was evaluated using

a one

- sided Dunnett’s test.

• At each treatment level, was there a difference in the response relative to the baseline? This was

evaluated using a two

- sided Dunnett’s test comparing CCA4 through CCA8 against CCA3.



## Scatterplot and Principal Component Discussion

Based on physical hive constructs and the nature of the honey bees, it is generally accepted that the colony

condition assessment (CCA) variables may be correlated over time and may also be correlated within a time

point (sampling time). Given this backgro und, a series of scatterplots, correlation matrices, and principal

component analyses (PCA) for this bee study were prepared. The full printout is included as Attachment

3 . Some of the general summary points are:

The first series looked at: for a given re sponse variable, what were the pairwise correlations over time, and

how would a principal components analysis best explain the observed variation. Data were plotted and

subjected to a PCA without accounting for treatment (i.e., all data were included in a single series of plots

and PCAs; separate assessments were not done for each treatment). Some general interpretations are:

• Scatterplot and correlation matrices indicated that variables tended to have stronger correlations at

adjacent time points than at fa rther time points.

• CCA8 tended to have weaker pairwise correlations with all the other time points than CCA3 through

CCA7 had with each other.

• The first principal component for each of the variables explained 26

- 53% of the total variation. The

lowest was proportion frame coverage of eggs, and the highest were adults, honey, and hive weight.

The general interpretation of the first principal compon ent was a weighted average over all time

points (weights varied depending on variable). For most variables, CCA3 tended to carry the least

weight in the weighted average.

• The second principle component explained an additional 16 to 27 percent of the total variation. For

most endpoints, a general interpretation of the principle component eigenvector was a difference

between measured taken early in the study and measure recorded later in the study ( e.g ., average of

CCA3 and CCA4 minus the average of CCA7 and CCA8).94 

### These scatterplots, correlation matrices, and principle component analyses were used to inform the choice

of covariance structure used in the repeated measure analysis. Some summary points are:

• Variance for a given response variable was not consis tent across all CCAs. This may indicate that

the correlation structures with a constant variance for all CCAs ( e.g ., CS, SP(pow)(1), and AR(1))

may not fit as well as those that allow for heterogenous variance ( e.g ., CSH, ToepH(1), and

ToepH(2)).

• There d id not appear to be a consistent decrease in correlation if the paired CCAs were farther apart

(i.e., correlation between CCA4 and CCA8 (three time steps apart) was not consistently less than

correlation between measurements 2 time steps apart, e.g., (CCA4 , CCA6), (CCA5, CCA7), and

(CCA6, CCA8). This may indicate that the AR(1) and SP(pow)(1) covariance structures may not fit

the data as well as other structures.

The second series of scatterplots and PCAs looked at: for a given CCA, what were the pairwise correlations

across matrices, and how would a principal components analysis best explain the observed variation. Data

were plotted and subjected to a PCA without accounting for treatment ( i.e ., all data were included in a single

series of plots and PCAs; separate assessments were not done for each treatment). Some of the general

summary points are:

o Honey had the weakest correlations (honey and any of the other measured matrices) amongst

all the pairwise correlations.

o The first principal component explaine d 36

- 66% of the total variation. The CCAs with the

lowest percent of variation explained were CCA3 and CCA4. The percent variation

explained tended to increase over time. At each time point the first principle component

tended to be interpreted as a weight ed average, with honey receiving the least weight.



# o The second principal component explained an additional 15 to 24% of the total variation.

The interpretations of the eigenvectors from the second principal component were less clear

and consistent. They tended to be an average of a measure of hive food stor es (pollen, honey,

and/or hive weight) or an average of the hive food stores contrasted with a subset of the

population matrices (e.g., eggs, open, capped, adult, and/or total).



## Options for Addressing the Data Imbalance

Once the design component of the analysis was established, the next part of the analysis was to determine

which correlation structure (across time) was the best fitting for these data. One of the challenges was that

many hives died before the end of the st udy (especially the 100 and 200 ppb treatments in CCA8), creating

censored data or an imbalanced design. PROC MIXED in SAS can handle an unbalanced design; however,

convergence may not be attained for some correlation structures. In this case the imbalance was due to

treatment (the higher treatments had a higher rate of hive death) as well as random mortality (control hive

mortality rate during overwintering was similar to background mortalities of commercial hives). Several

options were explored for addres sing the data imbalance and convergence issues: 4

4 These analyses were conducted utilizing an earlier version of the data set that was provided by the study author. It was late r
determined that there were some data entry errors. The errors were cor rected and the main analyses were re

- run. These

exploratory analyses discussed in Appendix B were not re

- run utilizing the corrected data set; however, the scope of the data

errors was such that it was unlikely that the analysis choices would have changed.95 

### • Analyze the data with treatment groups 100 and 200 deleted for all CCAs. The reasoning behind this

path forward are:

o There was consensus that treatments 100 and 200 impacted hive health with no recovery

amo ngst all evaluators.

o Removing these treatment groups greatly improves the percentage of covariance structures

that were able to be fitted in PROC MIXED.

o During the study, hives treated with 100 and 200 concentrations of test material were

physically moved away from the initial placements in the field to minimize the potential

impact these hives may have on the other nearby hives. Thus data obtained on these hives

after moving may not be comparable to data obtained before moving.

• Analyze the data with CCA8 removed and conduct separate analysis for CCA8. The justifications

are:

o The majority of hive deaths occurred between CCA7 and CCA8; therefore, data are better

balanced from CCA3 to CCA7 and convergence success will be much higher for all

responses.

o Number of days between CCAs was very similar from CCA3 to CCA8; therefore, the AR(1)

covariance structure which requires equal spacing between time points could be evaluated.

• Analyze the data analyzed after removing data for the few remaining hives in treatment groups 100

and 200 ppb from CCA8. The justifications are:

o The vast majority of the data can still be included in the analysis.

o Data on some hives after overwintering can be included in the full analysis.

o Pulled error terms (and standard errors) will utili ze the majority of the data set, thus

increasing confidence in the estimates.

After exploring these options, the data were analyzed after removing data from the few remaining hives in

treatment groups 100 and 200 ppb from CCA8 (third option above). Data from hives treated with 100 or

200 ppb imidacloprid remain in the data set and analysis for CCA3 through CCA7 permitting utilization of

a majority of the data. These deletions create a 5x6 factorial design matrix with two of the cells (CCA8,

treatment=100) and (CCA8, treatment=200) missing all data. CCA*treatment interaction means can still be

calculated. Least square means on the main effects (treatment and CCA) cannot be estimated for

Concentration=100, Concentration=200, and CCA8. Since the expectation w as that the interaction would

be significant and all further statistical tests would be exploring the interaction, this was not considered a

significant hindrance.



## Determining the temporal covariance structure

Before conducting Dunnett’s test, several different correlation structures to best fit the temporal correlation

were evaluated. The structures that were fitted included:

• Compound symmetry (CS) : assumes equal correlation for all pairwise correlations (regardless of

distance of timepoint).

• Compound symmetry with heterogeneous variance (CSH): Estimates a unique variance at each

time point, but assumes equal correlation for all pairwise correlations (regardless of distance of time

point).

• Sp(pow)(1): this is a correlation structure that fits an AR(1) but it adjusts for unequal spacing

between time points.96 

### • AR(1): autoregressive correlation . Assumes equal correlation between adjacent timepoints. Time

points further apart have a lesser correlation.

• Heterogeneous Toeplitz TOEPH(1): models a unique varianc e for each timepoint. Correlation

between timepoints was zero.

• Heterogeneous Toeplitz TOEPH(2): models a unique variance for each timepoint and a common

correlation for adjacent timepoints. Correlation for timepoints not immediately adjacent was

assumed to be zero.

More information about each of the correlation structures can be found here:

http://support.sas.com/documentation/cdl/ en/statug/63033/HTML/default/viewer.htm#statug_mixed_sect0

19.htm

. The full SAS output is provided in Attachment 4

.

To compare structure fit, Bayesian Information Criterion (BIC) was utilized. The BIC is similar to the AIC

and both are functions of the log likelihood with a penalty for an increase in the number of covariance

parameters to be fitted. The BIC value for ea ch fitted model for all eight response variables is reported in

Table A

- 1 ; s maller values of the BIC indicate a better fit (bolded). For many of the endpoints, heterogeneity

of variance at different time points was indicated as CSH, ToepH(1), or ToepH(2) w ere the covariance

structures providing the best fits. This is not surprising as unequal variances were observed in the

exploratory multivariate/principle component analysis. Although the AR(1) model was fit here, it may not

be appropriate as the spacing b etween measurement times is not consistent (minor deviations as between

CCA3, CCA4, CCA5, CCA6, and CCA7 are acceptable, but CCA8 is clearly not equidistant from CCA7).

For the variables that represent the individual life stages (adults, eggs, open cell s, and capped cells),

ToepH(2) is the one covariance structure that provides a good fit for all four matrices of the evaluated

structures. In addition, the BIC statistics suggests that CSH, AR(1) and ToepH(1) provide adequate fits for

at least one of the life stages. For proportion frame coverage of total individuals, ToepH(2) provides the

best fit to the covariance structure of all the evaluated models. For pollen, CSH, ToepH(1) and ToepH(2)

provided the best fits to the data. For honey, SP(pow)(1) and AR (1) provided the best fits to the covariance

structure; ToepH(2) did not converge (infinite likelihood). For hive weight, SP(pow)(1), AR(1), and

ToepH(1) were the best fitting covariance structures. Compound symmetry (CS) was not identified as

quality fit to the data for any of the eight evaluated response variables.

Residual plots were also evaluated for each of the response variables and covariance structures.

Patterns indicative of heterogeneous variance of the residuals were evident for many of the r esponse variables and models where an assumption of equal variance at each time point was made. It was recognized

that many of the response variables were proportions, hence the distribution of the response variable and

the residuals may not meet assumpti ons of normality. Review of the residual plots indicated that estimating

unique variances at each CCA (e.g., CSH, ToepH(1), or ToepH(2) covariance structures) appears to address

the concern of unequal variance.

The varying strength and pattern of correla tion seen in the pairwise scatterplots (CCA x vs. CCA y for any

given response variable for x and y equal to 3, 4, 5, 6, 7, and 8), the BIC comparisons, and the residual plot

review indicate that there is not a single covariance structure that is clearly best for all eight response

variables. General conclusions that can be made from the scatter plots a nd covariance analysis are that

within a response variable, variance varies over time and that there is correlation in response variable over

time; however, the pattern of correlation is not particularly strong nor is it consistent. Given these97 

### interpretat ions along with the BIC analysis and residual plots, the CSH covariance was chosen for the mixed

model structure.

Table A

- 1. BIC values for fitted models. CCA3

- CCA8; concentrations 100 and 200 ppb deleted from

CCA8

Variable →

Model ↓ Adults Eggs Open Capped Total

indiv. Pollen Honey Hive

weight

SP(pow)(1)

- 389

- 1457

- 1268

- 735 1.95

- 1518

- 724 2502

CS

- 374

- 1459

- 1265

- 724 33.3

- 1518

- 679 2588

CSH

- 385

- 1549

- 1321

- 816

- 5.85

- 1534

- 709 2533

AR(1)

- 401*@

- 1456

- 1268@

- 738@

- 2.27@

- 1522

- 729 2492@

ToepH(1)

- 381

- 1552

- 1320

- 815 1.99

- 1532

- 711 2547

ToepH(2)

- 406

- 1550

- 1319

- 824

- 35.5

- 1535 Inf. lklhd 2484

*Within a response variable, bolded BIC values indicate better covariance model fit. Kass and Raferty (1995) suggested that

differences of greater than 10 in BIC values provides very strong evidence that model fits are not equivalent.

@Convergence was att ained, but estimated G matrix was not positive definite.



## Treatment by time interaction and follow

- up contrasts

The text box provides the SAS code for the mixed model that was used for the Dunnett’s comparisons.

Table A

- 2 provides the results from the Dunnett’s comparisons in which treatment means were tested to

see if significantly less than control at each CCA. For these analyses, the CSH covariance matrix was used

for each of the variables.



## Treatment effects within a CCA

The table of p

- values resulting from the Dunnett’s tests for evaluating whether within a CCA, the treatment

mean are significantly less than control means) are summarized in Table A

- 2. Figures 1

- 7 below show the

results for each response variable across al l CCAs analyzed (CCA3

- CCA8) and all treatment levels. It is

noted as discussed previously that the 100 and 200 ppb treatment groups were excluded from CCA8 due to

high hive mortality. For all figures presented below, significant reductions from the negat ive control with

p

- values below the 0.05 alpha level are denoted by a red dot at a given treatment level and CCA and those

reductions with p

- values between 0.05 and 0.1 are denoted by a black dot. Statistical NOAECs and

LOAECs will be determined using an alpha

- level of 0.05. Additional comparisons using and alpha

- level of

0.10 are included for additional characterization. The tables of p

- values resulting from the Dunnett’s test

title 'concentration 100 and 200 deleted for CCA=8, covariance=csh' ;

**proc mixed**  data =cca3_8 ;

title2 "Dunnett's tests

- adult" ;

class apiary cca concval replicate;

model adult_p = concval|cca / DDFM =SATTERTHWAITE;

random apiary ;

repeated cca/ subject =replicate*concval(a piary) type =csh ;

lsmeans concval*cca;

slice concval*cca /sliceby=cca diff=controll adjust=dunnett;

slice concval*cca /sliceby=concval diff=control adjust=dunnett;

**run** ;98 

### are summarized in Table A

- 2 . The associated SAS output containing the full res ults of the Dunnett’s

comparisons can be found in Attachment 5

.

Table A

- 2

. Results of one

- sided Dunnett’s test (comparing control to each treatment group) with 100 and

200 concentrations deleted from CCA8, correlations modeled using CSH.*

Adults Eggs Open Capped Total

indiv. Pollen Honey Hive weight

CCA3 NS NS NS NS NS NS NS NS

CCA4 NS 100 100

200 100

200 100 50

100

200 NS 100

200

CCA5 50

100 100 100

200 50

100

200 50

100

200 50

100

200 NS 50

100

200

CCA6 50

100

200 100

200 100

200 12.5

100

200 50

100

200 200 50

200 100

200

CCA7 100

200 200 100

200 100

200 100

200 200 50

100

200 50

100

200

CCA8 50 12.5

50 NS 50 50 50 50 50

*Listed concentrations are those that were significantly less than the control following the results of Dunnett’s

test. NS indicates that there were no test concentrations with means significantly less than the control (p>0.10).

Bolded concentration = sig nificantly less than control (p< 0.05)

Italicized concentration = less than control (0.05< P < 0.10)



## Temporal trends within a treatment level

A second component to evaluating the “treatment x CCA” interaction is to look at the temporal changes

within a treatment group. This was accomplished by comparing each CCA (CCA4 through CCA8) to CCA3

by use of a two

- sided Dunnett’s test ( Table A

- 3 and Table A

- 4 ). This suite of comparisons is not as

informative as the contrasts of control against the treatment group within a CCA for establishing a NOAEC

and LOAEC. However, it may aid in interpretations and further biological understanding of temporal shifts

in th e life stages and food components present in the hive. Differences in patterns of temporal shifts between

the control and various treatment groups can provide further understanding of the potential impacts of

imidacloprid on beehive population dynamics.

Table A

- 3

. Results of two

- sided Dunnett’s test (comparing CCA3 to each following CCA) with 100 and

200 concentrations deleted from CCA8, correlations modeled using CSH.*

Trt

Group Response Variable

Adults Eggs Open Capped Total indiv

Control CCA5> CCA3 CCA7 and CCA8

< CCA3 CCA7<CCA3 CCA4

- 6 > CCA3

CCA7 < CCA3 CCA7 < CCA3

12.5 NS NS NS CCA7 < CCA3 CCA7 < CCA3

25 NS CCA7 < CCA3 CCA7<CCA3 CCA7 < CCA3 CCA7 < CCA399 50 NS CCA7 and CCA8

< CCA3 CCA7<CCA3 CCA7 < CCA3 CCA7

- 8 < CCA3

100* CCA7<CCA3 CCA6 and CCA7

<CCA3 CCA7<CCA3 CCA5

- 7 < CCA3 CCA6

- 7<CCA3

200* CCA6 and

CCA7<CCA3 CCA6 and CCA7

<CCA3 CCA4

- 7 < CCA3 CCA4

- 7 < CCA3 CCA4

- 7<CCA3

* CCA8 not included for test concentrations 100 and 200

NS

– No significant differences from control (p>0.05)



### Table A

- 4

. Results of two

- sided Dunnett’s test (comparing CCA3 to each following CCA) with 100 and

200 concentrations deleted from CCA8, correlations modeled using CSH.*

Trt

Group Response Variable

Pollen Honey Hive weight

Control CCA5>CCA3

CCA7 < CCA3 CCA6

- 7 > CCA3 CCA4

- 7> CCA3

12.5 CCA5 and CCA8>CCA3 CCA6

- 7 > CCA3 CCA4, 6

- 7 >CCA3

25 CCA5>CCA3 CCA6

- 7 > CCA3 CCA4

- 7> CCA3

50 CCA4 and CCA7<CCA3 CCA5 and CCA8< CCA3 CCA4>CCA3

CCA8<CCA3

100* CCA4

- 5<CCA3 NS CCA4>CCA3

200* CCA4

- 5 and CCA7<CCA3 CCA4

- 5> CCA3 CCA4> CCA3

* CCA8 not included for test concentrations 100 and 200

NS

– No significant differences from control (p>0.05)



### Tables A

- 5

– A

- 12 tabulate the summary statistics (including mean and standard deviation) of each

response variable for all treatment levels across CCAs 3

- 8.100 

### Table A

- 5. Summary statistics for adults

Table A

- 6. Summary statistics for eggs

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0 0.025 0.0075 0.03 0 0.0175

0 MAX 0.165 0.1525 0.12 0.1225 0.0675 0.08

0 MEAN 0.083929 0.069205 0.070909 0.07369 0.025 0.04875

0 STD 0.052331 0.026574 0.028301 0.027552 0.015749 0.019728 Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0.157513 0.127504 0.077504 0.155009 0.130009 0.047496

0 MAX 0.489982 0.632513 0.742487 0.875 0.532513 0.605009

0 MEAN 0.328452 0.34023 0.451138 0.394288 0.29725 0.36411

0 STD 0.099238 0.15064 0.170406 0.170447 0.117737 0.169285

12 N 11 11 11 11 11 9

12 MIN 0.15 0.130009 0.212522 0.194991 0.130009 0.180009

12 MAX 0.727504 0.680009 0.687522 0.555009 0.485018 0.577504

12 MEAN 0.374093 0.36932 0.484327 0.375224 0.342958 0.369728

12 STD 0.177567 0.183432 0.157566 0.124439 0.09466 0.13788

25 N 11 11 11 11 11 10

25 MIN 0.155009 0.219991 0.175 0.139982 0.117487 0.077504

25 MAX 0.705009 0.614982 0.672496 0.597496 0.6 0.632513

25 MEAN 0.379094 0.357501 0.422731 0.361364 0.328639 0.346498

25 STD 0.16288 0.141233 0.173179 0.169438 0.13711 0.191584

50 N 11 11 11 10 9 7

50 MIN 0.175 0.175 0.155009 0.147496 0.172496 0.039982

50 MAX 0.535018 0.707513 0.562522 0.407513 0.432513 0.305501

50 MEAN 0.302053 0.396361 0.334323 0.264244 0.255282 0.184718

50 STD 0.098915 0.174624 0.148201 0.086622 0.077175 0.10027

100 N 11 11 11 10 10 1

100 MIN 0.155009 0.117487 0.027504 0.037522 0.042487 0.010018

100 MAX 0.560018 0.664982 0.647496 0.597496 0.397496 0.010018

100 MEAN 0.301374 0.312047 0.281591 0.253001 0.144253 0.010018

100 STD 0.132728 0.157151 0.21117 0.21868 0.137779

200 N 11 11 11 11 10 2

200 MIN 0.214982 0.202504 0.225 0.112522 0.037522 0.035018

200 MAX 0.589982 0.787522 0.632513 0.302504 0.260018 0.077504

200 MEAN 0.378175 0.479541 0.351374 0.172508 0.143256 0.056261

200 STD 0.120328 0.180595 0.133209 0.059981 0.069188 0.030043101 12 N 11 11 11 11 11 9

12 MIN 0 0 0 0.03 0.0125 0

12 MAX 0.15 0.0875 0.1125 0.175 0.05 0.0925

12 MEAN 0.058636 0.055909 0.065227 0.0675 0.0275 0.031389

12 STD 0.041418 0.026369 0.030485 0.039655 0.01199 0.027475

25 N 11 11 11 11 11 10

25 MIN 0 0 0 0 0 0.0275

25 MAX 0.105 0.12 0.1175 0.1175 0.035 0.1

25 MEAN 0.0675 0.071364 0.065227 0.055909 0.017727 0.04575

25 STD 0.034605 0.030951 0.034414 0.034229 0.010214 0.021572

50 N 11 11 11 10 9 7

50 MIN 0 0.0125 0.0125 0.0375 0.0125 0.0075

50 MAX 0.1425 0.1475 0.14 0.1125 0.0475 0.0375

50 MEAN 0.080227 0.077045 0.069318 0.068 0.024444 0.017857

50 STD 0.039851 0.037213 0.038342 0.023682 0.01191 0.011586

100 N 11 11 11 10 10 1

100 MIN 0 0 0 0 0 0.0025

100 MAX 0.15 0.105 0.1125 0.0675 0.0425 0.0025

100 MEAN 0.066818 0.043864 0.044091 0.0255 0.01625 0.0025

100 STD 0.053445 0.039376 0.046088 0.026557 0.014589

200 N 11 11 11 11 10 2

200 MIN 0 0 0 0 0 0.015

200 MAX 0.1225 0.1175 0.1 0.065 0.015 0.0175

200 MEAN 0.071136 0.060682 0.045682 0.029545 0.007 0.01625

200 STD 0.036372 0.037167 0.035019 0.021961 0.006433 0.001768



### Table A

- 7. Summary statistics for larval (open) cells

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0 0.015 0 0.0425 0 0

0 MAX 0.215 0.185 0.1725 0.18 0.0675 0.1425

0 MEAN 0.101905 0.110795 0.100682 0.099048 0.03425 0.083036

0 STD 0.055778 0.037736 0.044207 0.033001 0.017698 0.042508

12 N 11 11 11 11 11 9

12 MIN 0 0.005 0 0.0425 0.0275 0

12 MAX 0.15 0.1525 0.1425 0.2275 0.085 0.15

12 MEAN 0.087045 0.094773 0.09 0.113864 0.053636 0.078333

12 STD 0.05073 0.041132 0.040481 0.055389 0.017189 0.049418

25 N 11 11 11 11 11 10

25 MIN 0 0.075 0 0 0 0.0125

25 MAX 0.1775 0.16 0.1575 0.1125 0.1075 0.1475102 25 MEAN 0.11 0.102955 0.084318 0.072727 0.039773 0.07925

25 STD 0.049422 0.025637 0.047593 0.036408 0.027075 0.04026

50 N 11 11 11 10 9 7

50 MIN 0 0.0225 0 0.0575 0.02 0.01

50 MAX 0.2075 0.155 0.1775 0.1475 0.0725 0.125

50 MEAN 0.1 0.096591 0.085455 0.0955 0.041667 0.058929

50 STD 0.054118 0.040316 0.058372 0.029411 0.018028 0.042127

100 N 11 11 11 10 10 1

100 MIN 0 0 0 0 0 0.0025

100 MAX 0.12 0.1325 0.15 0.09 0.045 0.0025

100 MEAN 0.071818 0.056591 0.063409 0.0445 0.01425 0.0025

100 STD 0.041399 0.049816 0.063946 0.03704 0.014484

200 N 11 11 11 11 10 2

200 MIN 0.0425 0 0 0 0 0.005

200 MAX 0.1375 0.1125 0.15 0.085 0.0225 0.0275

200 MEAN 0.099318 0.039545 0.034318 0.039091 0.007 0.01625

200 STD 0.03406 0.042849 0.043288 0.030522 0.0084 0.01591



### Table A

- 8. Summary statistics for pupal (capped) cells

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0 0 0 0.19 0.04 0

0 MAX 0.345 0.36 0.4075 0.3675 0.1525 0.31

0 MEAN 0.16381 0.228523 0.259318 0.260595 0.080875 0.17625

0 STD 0.123803 0.093985 0.119229 0.053003 0.031843 0.093627

12 N 11 11 11 11 11 9

12 MIN 0.0625 0.195 0.13 0 0.0325 0

12 MAX 0.285 0.3025 0.3575 0.315 0.16 0.2875

12 MEAN 0.224545 0.246818 0.257045 0.197955 0.084773 0.178611

12 STD 0.068628 0.037518 0.062897 0.104228 0.038705 0.085961

25 N 11 11 11 11 11 10

25 MIN 0.18 0.04 0 0 0.0175 0.0325

25 MAX 0.3375 0.3275 0.35 0.3275 0.115 0.2725

25 MEAN 0.233409 0.214091 0.235227 0.208409 0.065 0.1655

25 STD 0.042519 0.086373 0.120931 0.084656 0.034893 0.085965

50 N 11 11 11 10 9 7

50 MIN 0 0 0 0.145 0.05 0.02

50 MAX 0.3325 0.3475 0.325 0.2975 0.115 0.1625

50 MEAN 0.189773 0.170909 0.162955 0.2185 0.077222 0.092143

50 STD 0.108975 0.114511 0.112704 0.051763 0.024253 0.058531

100 N 11 11 11 10 10 1103 100 MIN 0 0 0 0 0 0.005

100 MAX 0.355 0.2425 0.3175 0.255 0.0825 0.005

100 MEAN 0.211136 0.128864 0.105 0.1215 0.025 0.005

100 STD 0.097477 0.082016 0.128826 0.110001 0.024438

200 N 11 11 11 11 10 2

200 MIN 0.1425 0 0 0 0 0.01

200 MAX 0.33 0.3025 0.195 0.165 0.03 0.045

200 MEAN 0.250682 0.065227 0.041136 0.057955 0.0115 0.0275

200 STD 0.053934 0.095796 0.059091 0.062077 0.012315 0.024749



### Table A

- 9. Summary statistics for total individuals

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0.242513 0.210013 0.102504 0.515009 0.212509 0.064996

0 MAX 1.012491 1.142513 1.242487 1.4775 0.664996 1.107509

0 MEAN 0.678095 0.748753 0.882047 0.827622 0.437375 0.672146

0 STD 0.237041 0.254076 0.312739 0.233682 0.147805 0.30578

12 N 11 11 11 11 11 9

12 MIN 0.2125 0.450004 0.617522 0.5175 0.205009 0.180009

12 MAX 1.267504 1.132504 1.160013 0.972487 0.714991 1.067496

12 MEAN 0.744321 0.76682 0.896599 0.754542 0.508867 0.658061

12 STD 0.264013 0.236201 0.212789 0.132926 0.140911 0.277451

25 N 11 11 11 11 11 10

25 MIN 0.477509 0.479991 0.175 0.15 0.149987 0.152504

25 MAX 1.112509 1.087482 1.184996 1.049996 0.735 1.107513

25 MEAN 0.790003 0.74591 0.807504 0.698409 0.451139 0.636998

25 STD 0.197306 0.219977 0.332487 0.246998 0.151117 0.325348

50 N 11 11 11 10 9 7

50 MIN 0.47 0.45 0.219996 0.462491 0.270004 0.092482

50 MAX 1.062518 1.150013 1.080022 0.787496 0.580013 0.575018

50 MEAN 0.672053 0.740907 0.65205 0.646244 0.398616 0.353646

50 STD 0.187031 0.261104 0.317216 0.122113 0.094003 0.204817

100 N 11 11 11 10 10 1

100 MIN 0.347509 0.124991 0.027504 0.064982 0.059996 0.020018

100 MAX 1.152518 0.860018 1.199996 0.9525 0.492496 0.020018

100 MEAN 0.651147 0.541365 0.494091 0.444501 0.199753 0.020018

100 STD 0.235144 0.251096 0.431212 0.370248 0.179209

200 N 11 11 11 11 10 2

200 MIN 0.519996 0.212504 0.3 0.117487 0.050022 0.065018

200 MAX 1.042522 1.262522 0.952513 0.610004 0.260018 0.167504

200 MEAN 0.799312 0.644996 0.472511 0.299099 0.168756 0.116261104 200 STD 0.175532 0.29121 0.196226 0.155814 0.070029 0.072469



### Table A

- 10. Summary statistics for nectar (honey) cells

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0.0375 0.0625 0.0075 0.0975 0.0325 0.055

0 MAX 0.305 0.345 0.25 0.4875 0.5325 0.2925

0 MEAN 0.156667 0.202045 0.125341 0.295476 0.293625 0.168214

0 STD 0.069211 0.07982 0.059833 0.12647 0.156801 0.086937

12 N 11 11 11 11 11 9

12 MIN 0.0625 0.06 0.0125 0.115 0.105 0.0325

12 MAX 0.4275 0.415 0.285 0.4475 0.55 0.3375

12 MEAN 0.160909 0.211591 0.121818 0.259773 0.270227 0.168056

12 STD 0.098503 0.110478 0.09819 0.121431 0.135906 0.080833

25 N 11 11 11 11 11 10

25 MIN 0.0125 0.0125 0 0.1075 0.075 0.065

25 MAX 0.2725 0.3575 0.2675 0.55 0.49 0.29

25 MEAN 0.152045 0.213636 0.137045 0.311591 0.288864 0.139

25 STD 0.09124 0.120781 0.093252 0.138912 0.153913 0.067063

50 N 11 11 11 10 9 7

50 MIN 0.08 0.1 0.02 0.0475 0.06 0

50 MAX 0.2775 0.405 0.195 0.3275 0.445 0.2325

50 MEAN 0.168636 0.213409 0.101136 0.18975 0.193889 0.085

50 STD 0.066533 0.08095 0.05968 0.099467 0.122226 0.072414

100 N 11 11 11 10 10 1

100 MIN 0.055 0.085 0.0175 0.03 0.0175 0.0325

100 MAX 0.2775 0.3225 0.3 0.4675 0.355 0.0325

100 MEAN 0.173864 0.219773 0.125682 0.22725 0.13975 0.0325

100 STD 0.080602 0.084915 0.085089 0.162235 0.112023

200 N 11 11 11 11 10 2

200 MIN 0.035 0.0975 0.055 0.0175 0.0025 0.08

200 MAX 0.25 0.33 0.3575 0.17 0.16 0.09

200 MEAN 0.135227 0.2225 0.207273 0.092727 0.07375 0.085

200 STD 0.06755 0.076722 0.10878 0.053074 0.048265 0.007071105 

### Table A

- 11. Summary statistics for pollen cells

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 20 14

0 MIN 0.0175 0 0.0025 0.02 0.0025 0.0225

0 MAX 0.18 0.1375 0.205 0.1 0.09 0.12

0 MEAN 0.069286 0.066136 0.103182 0.0525 0.043125 0.076607

0 STD 0.043805 0.031156 0.048936 0.022389 0.02484 0.027185

12 N 11 11 11 11 11 9

12 MIN 0.0225 0.005 0.05 0.0225 0.0275 0.03

12 MAX 0.105 0.1025 0.205 0.1475 0.1125 0.13

12 MEAN 0.054091 0.054773 0.104318 0.075455 0.051136 0.0875

12 STD 0.024425 0.035151 0.045772 0.039351 0.024555 0.032089

25 N 11 11 11 11 11 10

25 MIN 0.01 0.0325 0.0275 0.0075 0.0125 0.035

25 MAX 0.115 0.095 0.17 0.1325 0.1025 0.1475

25 MEAN 0.045682 0.055455 0.086591 0.059545 0.047273 0.0755

25 STD 0.026671 0.023044 0.053083 0.039605 0.02425 0.032783

50 N 11 11 11 10 9 7

50 MIN 0.0125 0 0 0.0075 0.0025 0.015

50 MAX 0.16 0.0625 0.11 0.09 0.0825 0.08

50 MEAN 0.067727 0.0275 0.037045 0.04225 0.0325 0.039643

50 STD 0.040488 0.025471 0.035686 0.026574 0.026071 0.026826

100 N 11 11 11 10 10 1

100 MIN 0.0275 0 0 0.0175 0.01 0.015

100 MAX 0.125 0.07 0.0775 0.0725 0.0925 0.015

100 MEAN 0.062727 0.008636 0.013409 0.0435 0.0395 0.015

100 STD 0.024886 0.021429 0.023218 0.018379 0.024743

200 N 11 11 11 11 10 2

200 MIN 0.025 0 0 0 0 0.0125

200 MAX 0.085 0 0.0125 0.08 0.0875 0.015

200 MEAN 0.046818 0 0.001136 0.020227 0.0165 0.01375

200 STD 0.019464 0 0.003769 0.027916 0.026358 0.001768106 

### Table A

- 11. Summary statistics for hive weight

Treatment

Group μg/L Parameter CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

0 N 21 22 22 21 21 14

0 MIN 32 49 31 30 31.5 25.5

0 MAX 50 66 56.5 68 66.5 53.5

0 MEAN 40.19524 58.06818 47.43182 52.59524 52.14286 41.71429

0 STD 4.284913 5.005462 5.910635 10.71286 10.99675 7.392081

12 N 11 11 11 11 11 9

12 MIN 33.5 50 37 39 41 27.5

12 MAX 49 71.5 56.5 68 74 49

12 MEAN 42.13636 58.5 45.86364 52.27273 53 40.11111

12 STD 5.822761 6.492303 5.263511 8.866689 9.721111 6.436118

25 N 11 11 11 11 11 10

25 MIN 33.5 48.5 42 37.5 41 34

25 MAX 51 64 54.5 69.5 64.5 48.5

25 MEAN 42.36364 57.31818 47.59091 54.09091 53.18182 42.1

25 STD 5.186959 5.891828 4.597924 9.194613 9.453234 4.629615

50 N 11 11 11 10 10 7

50 MIN 33.5 47.5 36 30 22.5 30

50 MAX 55.5 65 47 55.5 57.5 43

50 MEAN 42.95455 55.31818 41.54545 45.85 44 35

50 STD 6.254816 6.108489 4.143999 8.131045 9.436925 4.481443

100 N 11 11 11 10 10 1

100 MIN 35.5 47 32.5 33 32.5 26.5

100 MAX 50 65 50.5 67 62.5 26.5

100 MEAN 39.90909 53.31818 40 44.05 42.15 26.5

100 STD 4.559705 6.569904 6.160357 11.48296 10.78592

200 N 11 11 11 11 11 2

200 MIN 35.5 47 38 34.5 33.5 32

200 MAX 52 60 49.5 43.5 42 32

200 MEAN 41.90909 52.63636 42.54545 38.18182 36.5 32

200 STD 5.337688 4.080998 3.173756 3.356134 3.138471 0107 

## 2 Appendix B : Details of PMRA Statistical Analysis

During the review of the study, a separate statistical analysis was conducted with the program R (version

3.1.2 ) 5 

### using the raw data submitted by the study author.



## S tat istical analysis

Analysis Strategy

Hive condition data:

To analyze colony condition data which contains many components over many assessments at different

times , a primary analysis was set out to effectively prevent multiplicities from interfering with the

interpretation of p_val ues and confidence intervals. These multiplicities arise from having multiple dose

levels, multiple outcomes and multiple time points , and are dealt with as follows:

• The multiplicities from having multiple dose levels was dealt with by using step down testing, the

highest dose group’s data was compared directly to the control group’s data, if statistically

significant at a chosen alpha level the next lowest dose group’ s data was compared to the control

group’s data and this was continued down to the dose where statistical significance was no longer

achieved. A technical reference for this step down testing would be Multiple Comparison

Procedures in Dose Response Studie s. Tamhane, Ajit C. and Logan, Brent R., in Dose Finding in

Drug Development edited by Ting, Naitee. Springer New York 2006. This step down procedure

(referred to as the SD2PC procedure in the technical reference) was chosen as it provides good power

for d etecting the minimum effective dose (lowest does where effect is present) when monotonic dose

effects are expected while providing stringent control of type one error, regardless of the true pattern

of dose effects . That is, with minimal assumptions, the p rocedure strongly controls family wise type

one error rate while maintaining good power for effect patterns that are expected.

The applicant’s choice of multiplicity adjustment procedure, which was Willi am’s trend test

(Williams 1972), presumably chosen to be in accord with OECD. 2003. Draft guidance document

for the statistical analysis of ecotoxicity data . They are both step down procedures but ours differs

from William’s in that it uses only within dose group data based estimates of means rather than

m aximum likelihood estimates of dose group means using all group’s data simultaneously

- under

monotonicity assumption ( i.e. order restricted or isotonic means) additionally assuming

homogeneous variances . Although these additional assumptions may not be problematic and are

within the OECD guidelines, we simply chose not to rely on them (and by doing so, exceed the

OECD guidelines.)

• The multiplicities from having multiple outcomes, was dealt with by choosing to focus on the

assessment of total life in th e hive

– simply the number of viable life forms at any stage in the hive.

It is considered that the total number of individuals includes all live individuals in hives and is

expected to be a better indicator of the hive status at the colony level than any single stage of bees

alone. This outcome would provide good power when background knowledge is lacking on the

stage most likely to be affected (i.e. it cannot be well anticipated) and it is not expected that there

5 R Core Team (2014). R: A language and environment for statistical computing. R Foundation for Statistical

Computing, Vienna, Austria. URL http://www.R

- project.org/.108 

### will be simultaneous trade

- offs effects b etween the stages. That is, when it is not expected that a

toxic effect on one stage would have a beneficial effect for another stage at the same point in time.

• The multiplicities from having multiple time points was dealt with by choosing to focus on the time

when the effects were believed most pronounced both in terms of having an impact on total life and

having a high powered assessment of that. In this case CCA6 was selected for the following reasons

.

o CCA6 (five weeks after the end of feeding exposure ) was selected as it maximise s the time

period for detecting a potential latent effect from exposure and occurs before the start of hive

decline prior to overwintering.

o CCA7 was not chosen simply due to the natural decline of hive size in the late fall tha t may

mask the effect of treatment.

o CCA8 was not selected because of the higher hive mortality observed in the controls in

comparison to lower dose groups, and because data was available only for surviving hives

. T he hive mortality in the control was higher than the two lowest test concentrations (12.5

and 25 ug/l) and equal to the 50 ug/l treatment. The uncertainty regarding the cause of dead

hives, (i.e. whether mortality resulted from the treatment effects or random background effect

or both ) , is co nsidered to reduce the confidence of using CCA 8 data as the primary time

point

. Additionally, n o hive condition data were available on hives that died during the

overwintering , meaning only the condition of hives that survived overwintering

(informatively selected on ability to survive) were able to be compared

. The lack of data on

dead hives poses difficulties in the statistical analysis for hive condition at CCA8.

While the total individuals at CCA6 is considered as a primary parameter to control mult iplicity for

statistical analysis, all parameters including eggs, open brood and capped brood, adults, hive weight, pollen

and nectar store, that were observed during the entire study including CCA4, CCA5, CCA6, CCA7 and

CCA8 were also considered in the re view. Given that the primary analysis has prevented multiplicities from

interfering with the interpretation of p_values and confidence intervals, if statistical significance has been

achieved (at given dose levels), further analysis with all other outcome s is undertaken “with prejudice” for

the assessment of similar effects as being significant. More formally, re

- allowance for multiplicities is not

required and less stringent alpha levels are allowed. Essentially the price has been paid for searching for t he

pattern in the primary analysis (measures taken to prevent multiplicities) and it need not be re

- paid

evaluating the same pattern elsewhere. On the other hand, if statistical significance has not been achieved

(at given dose levels), further analysis wi th all other outcomes is undertaken “with prejudice” for assessment

of other effects as likely being just noise. Here though dramatic effects should not be ignored but carefully

considered and noted.

With the primary focus on CCA6 to discern treatment e ffects, the later assessment of recovery from detected

effects at CCA7 and CCA8 was subsequently addressed. Here the use of confidence intervals was chosen

to provide assurance that important underlying differences at the later period had been reliably rul ed out

– that is the upper confidence limit did not include worrisome differences. Given the arguable need to make

important bias adjustment for confidence intervals in CCA8 but little to no background information to

accurately make these bias adjustments, the assessment of recovery was limited to CCA7. There was no

formal analysis of whether and when treatment effects detected at CCA6 were present before CCA6. The

consideration of recovery at CCA7, while considered, was not formally presented because of c oncern regarding relying on parameters at CCA7 which are all decreasing as colonies prepare for overwintering.

Hive mortality data:109 

### The analysis of Mortality at CCA8 had been anticipated as the primary or key assessment for this study

design and the only multiplicities to be dealt with there were from the multiple dose levels (for which the

step down method was used). Unfortunately, t he observed hive mortality in the control was higher than the

two lowest test concentrations (12.5 and 25 ug/l) and equal to the 50 ug/l treatment which is at odds with

underlying biological understanding and thus greatly reduce d the confidence of using CCA 8 mortality as

the primary assessment.

Analysis methods for hive conditions

For all hive conditions total life, eggs, open brood and capped brood, adults, hive weight, pollen and nectar

store at CCA4, CCA5, CCA6, CCA7 and CCA8 a conventional analysis of block randomised experiments

with a baseline measurements was undertaken. In line with the statistical strategy discussed above, the focus

was on total life at CCA6 (with step down adjustment for multiplicities applied) but identical analysis was

carried out (less the step down adjustment) on all other hive conditions assessed at the given assessment

points (with CC A8 considered biased and problematic). This analysis comprised of li near modeling (or

ANOVA) stratified on Apiary (block) and adjusted for baseline measurements at CCA3 with one

- side

testing for harm using only the control group data and the data from a si ngle dose group at a time, starting

with the highest and then through lowest dose groups. It is a series of robust “t.test like” analyses that

conservatively implement the step down testing procedure. Under the assumption of no effect in the single

dose gr oup being tested (relevant to type one error control), the means and variances and covariate effects

should be identical in both the control group and the single dose group being tested. (In an analysis that

includes all dose group data together e.g. Willi am’s procedure, an impact of a treatment effect on the

variance and covariate effects at a higher dose, in addition to an effect on the mean, would invalidate the

assumptions needed to control type one error rate in the lower doses.) The results of these a nalyses are

presented in tables of unadjusted p_values (adjusted p_values can be simply read off as the maximum of all

p_values in higher doses), effect estimates and upper and lower confidence intervals (currently labelled as

Table A

- 2) as well as plots o f the confidence intervals (pdf file Bees8.pdf).

The code snippet to implement these analyses in R was:

glm(out come ~Apiary + base line + exposed, data= x[x$exposed == " control " | x$exposed == dose,])

Sensitivity analysis was undertaken on the primary analysis (total life) by taking logs. The resulting p_values

were 0.089

, 0.043

, 0.042

, 0.000 and 0.000 for dose of 13, 25, 50, 100 and 200 the largest difference for the

original scale being .013.

Analysis methods for hive mortality

The analysis of hive mortality at CCA8, like all other outcomes was also blocked by Apiary, but unlike

other outcomes, it was not adjusted for a baseline measurement at CCA3. Additionally, given the sparsity

of the outcome when blocked, common methods of a nalysis for binary data like Cochran

– Armitage test for

trend, Pearson's chi

- square test or logistic regression should not be used as they are well known to be biased.

Instead, conditional logistic regression or exact tests are required. The code snippet to implement conditional

logistic regression in R was:

clogit(Mortality ~ Conc + strata(Apiary), data=filter(mm,Conc =="CON" | Conc== dose)).110 

### As with other outcomes, step down testing from highest dose, and then in turn lower doses was us ed to

control for multiplicity.

Transcript/program of analyses carried out

The file RunJune25.2015 contains the transcript of the final run of the R program used to carry out the

analysis and generate the tables and plots.

Supporting graphs

The following graphs were p roduced as part of the analysis.

Bees1.pdf

– Plots of individual hive condition assessments over

- time group by Apiary.

Bees2.pdf

– Plots of control versus exposed condition assessments over

- time group by Apiary.

Bees3a.pdf

– Plots of mean and Apiary mea n control condition assessments over time.

Bees5.pdf

– Plots of m odel estimated difference s and confidence interval s (title revised).

Bees6.pdf

– Plots of m odel estimated difference versus observed mean for total life.

Bees7.pdf

– Plots of individual exposed hive versus control condition assessments fo r all parameters

.

Bees8.pdf

– Plots of effect estimates and confidence intervals for all parameters.111 

### Table B

- 1 : Summary of the differences between treatment and controls on the basis of observations and model estimations, and P values

Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

TotalLife 3 200

- 5284 3194 11 0 32147

- 1.729

TotalLife 3 100 1344 3438 11 0 32147

- 1.729

TotalLife 3 50

- 63 4496 11 0 32147

- 1.729

TotalLife 3 25

- 4600 3028 11 0 32147

- 1.729

TotalLife 3 13

- 1690 5092 11 0 32147

- 1.729

TotalLife 4 200 13608 3399 11 17344.8 3970.078 0 0.678 0.293 0.486 24210 35714

- 1.729

TotalLife 4 100 12959 3492 11 12608 3851.589 0.002 0.54 0.167 0.353 19268 35714

- 1.729

TotalLife 4 50 3147 3246 11 3667.244 3939.317 0.182 0.293

- 0.088 0.103 10479 35714

- 1.729

TotalLife 4 25 998 2684 11 3863.666 3887.011 0.166 0.296

- 0.08 0.108 10585 35714

- 1.729

TotalLife 4 13 108 2566 11 1412.154 3701.529 0.354 0.219

- 0.14 0.04 7813 35714

- 1.729

TotalLife 5 200 23543 3240 11 24026.8 4838.476 0 0.815 0.394 0.605 32393 39734

- 1.729

TotalLife 5 100 18818 4838 11 18258.98 5313.62 0.001 0.691 0.228 0.46 27447 39734

- 1.729

TotalLife 5 50 10399 6533 11 10080.39 6256.939 0.062 0.526

- 0.019 0.254 20899 39734

- 1.729

TotalLife 5 25 3809 6432 11 4333.576 6380.06 0.253 0.387

- 0.169 0.109 15366 39734

- 1.729

TotalLife 5 13 540 3894 11 385.681 4958.292 0.469 0.225

- 0.206 0.01 8959 39734

- 1.729

TotalLife 6 200 25992 1870 11 25425.35 2545.607 0 0.774 0.545 0.659 29840 38559

- 1.734

TotalLife 6 100 19875 4884 10 18601.92 3977.831 0 0.662 0.303 0.482 25522 38559

- 1.74

TotalLife 6 50 6104 2836 10 4674.351 2657.457 0.048 0.241 0.001 0.121 9297 38559

- 1.74

TotalLife 6 25 7290 3261 11 6715.559 3326.913 0.029 0.324 0.025 0.174 12485 38559

- 1.734

TotalLife 6 13 4072 1865 11 3357.78 2424.369 0.091 0.196

- 0.022 0.087 7562 38559

- 1.734

TotalLife 7 200 11178 1781 10 11783.5 1864.738 0 0.952 0.54 0.746 15039 15794

- 1.746

TotalLife 7 100 9015 2385 10 9612.191 2061.392 0 0.836 0.381 0.609 13211 15794

- 1.746

TotalLife 7 50 1212 779 9 1275.1 1411.714 0.19 0.237

- 0.076 0.081 3750 15794

- 1.753

TotalLife 7 25 18 2073 11 353.425 1937.944 0.429 0.236

- 0.191 0.022 3725 15794

- 1.74112 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

TotalLife 7 13

- 3289 2471 11

- 2666.66 1985.142 0.902 0.05

- 0.387

- 0.169 787 15794

- 1.74

TotalLife 8 200 14495 NA 1 15094.09 19208.88 0.238 2.009

- 0.927 0.541 56044 27899

- 2.132

TotalLife 8 100 42178 NA 1 40478.37 16670.41 0.036 2.725 0.177 1.451 76017 27899

- 2.132

TotalLife 8 50 15852 5456 7 13690.55 6342.992 0.028 0.903 0.079 0.491 25187 27899

- 1.812

TotalLife 8 25

- 2246 6876 8

- 3173.88 7643.389 0.657 0.378

- 0.606

- 0.114 10553 27899

- 1.796

TotalLife 8 13 936 2165 8

- 724.634 4642.539 0.561 0.273

- 0.325

- 0.026 7613 27899

- 1.796

Eggs 3 200 1134 976 11 0 6028

- 1.729

Eggs 3 100 1431 1163 11 0 6028

- 1.729

Eggs 3 50 508 1151 11 0 6028

- 1.729

Eggs 3 25 1384 1043 11 0 6028

- 1.729

Eggs 3 13 1994 1280 11 0 6028

- 1.729

Eggs 4 200 586 907 11 691.939 847.57 0.212 0.453

- 0.162 0.145 2157 4761

- 1.729

Eggs 4 100 1743 628 11 1805.239 706.582 0.01 0.636 0.123 0.379 3027 4761

- 1.729

Eggs 4 50

- 539 1014 11

- 457.571 898.501 0.692 0.23

- 0.422

- 0.096 1096 4761

- 1.729

Eggs 4 25

- 149 737 11

- 54.052 763.511 0.528 0.266

- 0.289

- 0.011 1266 4761

- 1.729

Eggs 4 13 915 573 11 1087.188 680.722 0.063 0.476

- 0.019 0.228 2264 4761

- 1.729

Eggs 5 200 1736 1002 11 1571.261 864.911 0.043 0.629 0.016 0.322 3067 4879

- 1.729

Eggs 5 100 1845 1193 11 1920.253 1006.424 0.036 0.75 0.037 0.394 3660 4879

- 1.729

Eggs 5 50 109 794 11 88.23 736.676 0.453 0.279

- 0.243 0.018 1362 4879

- 1.729

Eggs 5 25 391 1094 11 405.731 941.939 0.336 0.417

- 0.251 0.083 2034 4879

- 1.729

Eggs 5 13 391 840 11 578.592 788.155 0.236 0.398

- 0.161 0.119 1941 4879

- 1.729

Eggs 6 200 2916 744 11 2973.088 707.187 0 0.849 0.353 0.601 4199 4949

- 1.734

Eggs 6 100 3449 721 10 3505.81 669.65 0 0.944 0.473 0.708 4671 4949

- 1.74

Eggs 6 50 396 497 10 578.756 531.111 0.146 0.304

- 0.07 0.117 1503 4949

- 1.74

Eggs 6 25 1102 860 11 1302.118 787.695 0.058 0.539

- 0.013 0.263 2668 4949

- 1.734

Eggs 6 13 305 1045 11 291.007 940.826 0.38 0.388

- 0.271 0.059 1922 4949

- 1.734113 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Eggs 7 200 1170 322 10 1259.78 371.642 0.002 1.179 0.378 0.778 1909 1618

- 1.746

Eggs 7 100 662 405 10 754.486 410.884 0.042 0.909 0.023 0.466 1472 1618

- 1.746

Eggs 7 50

- 38 242 9 41.97 335.082 0.451 0.389

- 0.337 0.026 629 1618

- 1.753

Eggs 7 25 399 376 11 506.208 404.398 0.114 0.747

- 0.122 0.313 1210 1618

- 1.74

Eggs 7 13

- 274 437 11 2.106 429.799 0.498 0.463

- 0.461 0.001 750 1618

- 1.74

Eggs 8 200 3956 NA 1 4993.274 916.963 0.003 2.132 0.932 1.532 6948 3258

- 2.132

Eggs 8 100 3612 NA 1 4496.733 793.551 0.002 1.899 0.861 1.38 6188 3258

- 2.132

Eggs 8 50 2027 387 7 2548.007 434.937 0 1.024 0.54 0.782 3336 3258

- 1.812

Eggs 8 25

- 54 428 8 189.139 542.807 0.367 0.357

- 0.241 0.058 1164 3258

- 1.796

Eggs 8 13 1032 723 8 1227.919 719.885 0.058 0.774

- 0.02 0.377 2521 3258

- 1.796

Open

brood 3 200 235 1230 11 0 7068

- 1.729

Open

brood 3 100 2127 990 11 0 7068

- 1.729

Open

brood 3 50 188 1511 11 0 7068

- 1.729

Open

brood 3 25

- 500 1105 11 0 7068

- 1.729

Open

brood 3 13 1079 1612 11 0 7068

- 1.729

Open

brood 4 200 4902 1036 11 4876.767 1033.897 0 0.874 0.405 0.64 6665 7623

- 1.729

Open

brood 4 100 3729 1093 11 2825.234 1049.719 0.007 0.609 0.133 0.371 4640 7623

- 1.729

Open

brood 4 50 977 1014 11 918.781 1063.42 0.199 0.362

- 0.121 0.121 2758 7623

- 1.729

Open

brood 4 25 539 663 11 618.534 861.531 0.241 0.277

- 0.114 0.081 2108 7623

- 1.729

Open

brood 4 13 1102 717 11 935.28 920.463 0.161 0.331

- 0.086 0.123 2527 7623

- 1.729

Open

brood 5 200 4566 1188 11 4505.546 1123.44 0 0.931 0.37 0.65 6448 6927

- 1.729114 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Open

brood 5 100 2564 931 11 2137.769 1035.374 0.026 0.567 0.05 0.309 3928 6927

- 1.729

Open

brood 5 50 1048 1563 11 1060.273 1359.971 0.223 0.493

- 0.186 0.153 3412 6927

- 1.729

Open

brood 5 25 1126 1360 11 1125.489 1237.741 0.187 0.471

- 0.146 0.162 3266 6927

- 1.729

Open

brood 5 13 735 991 11 743.713 1041.675 0.242 0.367

- 0.153 0.107 2545 6927

- 1.729

Open

brood 6 200 4042 491 11 3848.744 692.846 0 0.75 0.393 0.572 5050 6731

- 1.734

Open

brood 6 100 3844 797 10 3519.266 851.435 0 0.743 0.303 0.523 5000 6731

- 1.74

Open

brood 6 50 189 652 10

- 144.032 726.133 0.577 0.166

- 0.209

- 0.021 1119 6731

- 1.74

Open

brood 6 25 1728 989 11 1548.347 905.214 0.052 0.463

- 0.003 0.23 3118 6731

- 1.734

Open

brood 6 13

- 1102 1420 11

- 1046.67 1215.331 0.8 0.158

- 0.469

- 0.155 1061 6731

- 1.734

Open

brood 7 200 1737 385 10 1738.782 405.588 0 1.102 0.464 0.783 2447 2220

- 1.746

Open

brood 7 100 1462 421 10 1431.022 433.195 0.002 0.985 0.304 0.644 2187 2220

- 1.746

Open

brood 7 50

- 698 370 9

- 719.181 396.111 0.955

- 0.011

- 0.637

- 0.324

- 25 2220

- 1.753

Open

brood 7 25

- 516 681 11

- 565.735 573.919 0.831 0.195

- 0.704

- 0.255 433 2220

- 1.74

Open

brood 7 13

- 1470 476 11

- 1389.37 456.941 0.996

- 0.268

- 0.984

- 0.626

- 594 2220

- 1.74

Open

brood 8 200 3096 NA 1 2958.927 3590.736 0.228 1.942

- 0.859 0.541 10614 5466

- 2.132

Open

brood 8 100 8944 NA 1 8738.39 3111.431 0.024 2.812 0.385 1.599 15371 5466

- 2.132

Open

brood 8 50 2138 1418 7 2328.118 1194.927 0.04 0.822 0.03 0.426 4494 5466

- 1.812

Open

brood 8 25

- 677 1406 8

- 543.73 1466.604 0.641 0.382

- 0.581

- 0.099 2090 5466

- 1.796115 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Open

brood 8 13 43 585 8 45.576 856.197 0.479 0.29

- 0.273 0.008 1583 5466

- 1.796

Capped

brood 3 200

- 5684 2040 11 0 11563

- 1.729

Capped

brood 3 100

- 2963 2161 11 0 11563

- 1.729

Capped

brood 3 50

- 1493 3468 11 0 11563

- 1.729

Capped

brood 3 25

- 4495 1327 11 0 11563

- 1.729

Capped

brood 3 13

- 3886 2448 11 0 11563

- 1.729

Capped

brood 4 200 11235 1859 11 13101.38 2806.568 0 1.142 0.525 0.833 17954 15722

- 1.729

Capped

brood 4 100 6857 1666 11 8125.651 2490.725 0.002 0.791 0.243 0.517 12432 15722

- 1.729

Capped

brood 4 50 3964 2316 11 4411.824 2826.411 0.068 0.591

- 0.03 0.281 9299 15722

- 1.729

Capped

brood 4 25 993 1631 11 2778.589 2653.562 0.154 0.469

- 0.115 0.177 7367 15722

- 1.729

Capped

brood 4 13

- 1259 1275 11 433.306 2337.261 0.427 0.285

- 0.229 0.028 4475 15722

- 1.729

Capped

brood 5 200 15011 1722 11 14191.99 2739.248 0 1.061 0.53 0.795 18929 17841

- 1.729

Capped

brood 5 100 10617 2093 11 10099.86 2683.038 0.001 0.826 0.306 0.566 14739 17841

- 1.729

Capped

brood 5 50 6630 3684 11 6173.291 3431.445 0.044 0.679 0.013 0.346 12107 17841

- 1.729

Capped

brood 5 25 1657 3361 11 1028.506 3529.735 0.387 0.4

- 0.284 0.058 7132 17841

- 1.729

Capped

brood 5 13 156 2183 11

- 665.442 2729.606 0.595 0.227

- 0.302

- 0.037 4054 17841

- 1.729

Capped

brood 6 200 14026 1220 11 13595.89 1412.206 0 0.891 0.619 0.755 16045 18013

- 1.734

Capped

brood 6 100 9477 2550 10 8945.157 2165.524 0 0.706 0.287 0.497 12712 18013

- 1.74116 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Capped

brood 6 50 2761 1515 10 2244.065 1367.303 0.06 0.257

- 0.007 0.125 4623 18013

- 1.74

Capped

brood 6 25 3675 1774 11 3304.405 1729.37 0.036 0.35 0.017 0.183 6303 18013

- 1.734

Capped

brood 6 13 4394 2297 11 3994.561 2028.775 0.032 0.417 0.026 0.222 7513 18013

- 1.734

Capped

brood 7 200 4730 610 10 5046.549 808.018 0 1.197 0.674 0.935 6457 5395

- 1.746

Capped

brood 7 100 3750 848 10 4078.307 875.563 0 1.039 0.473 0.756 5607 5395

- 1.746

Capped

brood 7 50 506 353 9 523.439 697.699 0.232 0.324

- 0.13 0.097 1747 5395

- 1.753

Capped

brood 7 25 923 755 11 974.261 846.077 0.133 0.453

- 0.092 0.181 2446 5395

- 1.74

Capped

brood 7 13

- 438 1038 11

- 454.128 917.895 0.686 0.212

- 0.38

- 0.084 1143 5395

- 1.74

Capped

brood 8 200 4816 NA 1 4816 12138.26 0.356 2.674

- 1.835 0.42 30693 11476

- 2.132

Capped

brood 8 100 17716 NA 1 17320.16 10822.64 0.092 3.52

- 0.501 1.509 40392 11476

- 2.132

Capped

brood 8 50 6917 2328 7 8097.621 4134.314 0.039 1.359 0.053 0.706 15591 11476

- 1.812

Capped

brood 8 25

- 785 2926 8

- 1239.48 3942.057 0.62 0.509

- 0.725

- 0.108 5840 11476

- 1.796

Capped

brood 8 13 172 1273 8

- 148.408 2879.268 0.52 0.438

- 0.464

- 0.013 5022 11476

- 1.796

Adults 3 200

- 968 577 11 0 7488

- 1.729

Adults 3 100 749 896 11 0 7488

- 1.729

Adults 3 50 734 638 11 0 7488

- 1.729

Adults 3 25

- 988 904 11 0 7488

- 1.729

Adults 3 13

- 877 926 11 0 7488

- 1.729

Adults 4 200

- 3115 980 11

- 2220.6 1189.671 0.961

- 0.021

- 0.562

- 0.292

- 164 7608

- 1.729

Adults 4 100 630 1345 11 391.889 1414.562 0.392 0.373

- 0.27 0.052 2838 7608

- 1.729117 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Adults 4 50

- 1255 1159 11

- 1771.83 1144.327 0.931 0.027

- 0.493

- 0.233 207 7608

- 1.729

Adults 4 25

- 386 741 11 205.329 1175.032 0.432 0.294

- 0.24 0.027 2237 7608

- 1.729

Adults 4 13

- 650 1085 11 168.16 1163.063 0.443 0.286

- 0.242 0.022 2179 7608

- 1.729

Adults 5 200 2231 598 11 2466.328 1208.532 0.028 0.452 0.037 0.244 4556 10087

- 1.729

Adults 5 100 3791 1342 11 3425.479 1451.632 0.015 0.588 0.091 0.34 5936 10087

- 1.729

Adults 5 50 2612 1430 11 2191.531 1483.044 0.078 0.471

- 0.037 0.217 4756 10087

- 1.729

Adults 5 25 635 1120 11 874.801 1378.486 0.267 0.323

- 0.15 0.087 3258 10087

- 1.729

Adults 5 13

- 742 1148 11

- 544.515 1369.016 0.652 0.181

- 0.289

- 0.054 1823 10087

- 1.729

Adults 6 200 5008 728 11 4677.835 932.194 0 0.71 0.345 0.528 6294 8865

- 1.734

Adults 6 100 3105 1397 10 2544.891 1196.879 0.024 0.522 0.052 0.287 4627 8865

- 1.74

Adults 6 50 2759 1078 10 1754.005 1053.102 0.057 0.404

- 0.009 0.198 3586 8865

- 1.74

Adults 6 25 785 642 11 686.452 950.173 0.24 0.263

- 0.108 0.077 2334 8865

- 1.734

Adults 6 13 475 953 11 199.536 910.841 0.415 0.201

- 0.156 0.023 1779 8865

- 1.734

Adults 7 200 3541 1095 10 3895.534 993.559 0.001 0.858 0.329 0.594 5630 6561

- 1.746

Adults 7 100 3142 965 10 3347.475 917.098 0.001 0.754 0.266 0.51 4949 6561

- 1.746

Adults 7 50 1441 650 9 1222.715 799.636 0.074 0.4

- 0.027 0.186 2625 6561

- 1.753

Adults 7 25

- 788 1243 11

- 272.138 1019.519 0.604 0.229

- 0.312

- 0.041 1501 6561

- 1.74

Adults 7 13

- 1108 959 11

- 826.715 874.128 0.821 0.106

- 0.358

- 0.126 694 6561

- 1.74

Adults 8 200 2627 NA 1 564.155 2791.126 0.425 0.846

- 0.7 0.073 6514 7699

- 2.132

Adults 8 100 11906 NA 1 13280.14 2402.712 0.003 2.39 1.06 1.725 18402 7699

- 2.132

Adults 8 50 4770 1434 7 6008.543 1473.09 0.001 1.127 0.434 0.78 8678 7699

- 1.812

Adults 8 25

- 730 2337 8

- 329.695 2346.527 0.555 0.505

- 0.59

- 0.043 3884 7699

- 1.796

Adults 8 13

- 311 695 8

- 845.547 1275.142 0.74 0.188

- 0.407

- 0.11 1444 7699

- 1.796

Honey 3 200 1407 1690 11 0 10711

- 1.729

Honey 3 100

- 1251 1594 11 0 10711

- 1.729

Honey 3 50

- 891 1300 11 0 10711

- 1.729118 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Honey 3 25 250 1801 11 0 10711

- 1.729

Honey 3 13

- 360 2276 11 0 10711

- 1.729

Honey 4 200

- 1407 2205 11

- 2934.5 1555.065 0.963

- 0.018

- 0.405

- 0.211

- 246 13901

- 1.729

Honey 4 100

- 1220 1573 11

- 1111.34 1055.672 0.847 0.051

- 0.211

- 0.08 714 13901

- 1.729

Honey 4 50

- 782 2012 11

- 864.527 1431.841 0.723 0.116

- 0.24

- 0.062 1611 13901

- 1.729

Honey 4 25

- 797 1894 11

- 1438.89 1134.467 0.89 0.038

- 0.245

- 0.104 523 13901

- 1.729

Honey 4 13

- 657 1710 11

- 1036.17 1160.622 0.808 0.07

- 0.219

- 0.075 971 13901

- 1.729

Honey 5 200

- 5637 2476 11

- 7251.97 1690.564 1

- 0.502

- 1.18

- 0.841

- 4329 8623

- 1.729

Honey 5 100

- 23 1814 11 609.048 1392.03 0.333 0.35

- 0.208 0.071 3016 8623

- 1.729

Honey 5 50 1665 1294 11 1862.689 1342.59 0.091 0.485

- 0.053 0.216 4184 8623

- 1.729

Honey 5 25

- 805 2119 11

- 1323.61 1297.1 0.84 0.107

- 0.414

- 0.153 919 8623

- 1.729

Honey 5 13 242 2130 11 112.248 1320.202 0.467 0.278

- 0.252 0.013 2395 8623

- 1.729

Honey 6 200 14526 2808 11 14734.61 2475.396 0 0.91 0.499 0.705 19027 20906

- 1.734

Honey 6 100 4059 3930 10 4547.091 3224.44 0.088 0.486

- 0.051 0.218 10156 20906

- 1.74

Honey 6 50 7327 3279 10 7524.125 2905.267 0.01 0.602 0.118 0.36 12578 20906

- 1.74

Honey 6 25

- 532 2597 11

- 507.008 2427.394 0.582 0.177

- 0.226

- 0.024 3702 20906

- 1.734

Honey 6 13 3033 2428 11 3297.32 2397.491 0.093 0.357

- 0.041 0.158 7455 20906

- 1.734

Honey 7 200 16194 3296 10 16768.37 3080.791 0 1.057 0.543 0.8 22147 20961

- 1.746

Honey 7 100 10243 3273 10 11091.16 3101.539 0.001 0.787 0.271 0.529 16506 20961

- 1.746

Honey 7 50 8485 4632 9 8636.163 3907.923 0.022 0.739 0.085 0.412 15487 20961

- 1.753

Honey 7 25 1087 3820 11 2226.627 3127.457 0.243 0.366

- 0.153 0.106 7667 20961

- 1.74

Honey 7 13 2369 3644 11 2614.224 3427.576 0.228 0.409

- 0.16 0.125 8577 20961

- 1.74

Honey 8 200 516 NA 1 559.044 7145.141 0.471 1.447

- 1.345 0.051 15791 10912

- 2.132

Honey 8 100 17114 NA 1 17084.95 6067.538 0.024 2.751 0.38 1.566 30020 10912

- 2.132

Honey 8 50 6217 2261 7 6589.059 2225.801 0.007 0.973 0.234 0.604 10623 10912

- 1.812

Honey 8 25 1333 3139 8 1450.279 2523.066 0.288 0.548

- 0.282 0.133 5981 10912

- 1.796119 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Honey 8 13

- 1161 1972 8

- 1185.77 2059.405 0.712 0.23

- 0.448

- 0.109 2513 10912

- 1.796

Pollen 3 200 1392 645 11 0 4613

- 1.729

Pollen 3 100 297 717 11 0 4613

- 1.729

Pollen 3 50

- 47 1057 11 0 4613

- 1.729

Pollen 3 25 1470 764 11 0 4613

- 1.729

Pollen 3 13 891 833 11 0 4613

- 1.729

Pollen 4 200 4550 478 11 4297.751 690.417 0 1.207 0.682 0.945 5492 4550

- 1.729

Pollen 4 100 3956 651 11 3806.351 713.408 0 1.108 0.565 0.837 5040 4550

- 1.729

Pollen 4 50 2658 385 11 2551.377 594.965 0 0.787 0.335 0.561 3580 4550

- 1.729

Pollen 4 25 735 615 11 236.8 695.034 0.369 0.316

- 0.212 0.052 1439 4550

- 1.729

Pollen 4 13 782 541 11 536.343 685.609 0.222 0.378

- 0.143 0.118 1722 4550

- 1.729

Pollen 5 200 7021 642 11 6419.711 1061.603 0 1.163 0.646 0.904 8255 7099

- 1.729

Pollen 5 100 6176 899 11 5935.685 1078.422 0 1.099 0.573 0.836 7800 7099

- 1.729

Pollen 5 50 4550 704 11 4438.326 1047.955 0 0.88 0.37 0.625 6250 7099

- 1.729

Pollen 5 25 1141 1327 11 145.606 1282.746 0.455 0.333

- 0.292 0.021 2364 7099

- 1.729

Pollen 5 13

- 78 1005 11

- 485.925 1177.574 0.658 0.218

- 0.355

- 0.068 1550 7099

- 1.729

Pollen 6 200 2119 493 11 1921.621 506.636 0.001 0.798 0.297 0.547 2800 3510

- 1.734

Pollen 6 100 731 610 10 537.423 519.822 0.158 0.411

- 0.105 0.153 1442 3510

- 1.74

Pollen 6 50 628 595 10 544.13 502.694 0.147 0.404

- 0.094 0.155 1419 3510

- 1.74

Pollen 6 25

- 586 699 11

- 885.905 606.289 0.919 0.047

- 0.552

- 0.252 165 3510

- 1.734

Pollen 6 13

- 1681 584 11

- 1872.63 550.502 0.998

- 0.262

- 0.805

- 0.533

- 918 3510

- 1.734

Pollen 7 200 1531 748 10 1400.095 688.615 0.029 0.94 0.071 0.506 2602 2768

- 1.746

Pollen 7 100 292 660 10 357.835 605.124 0.281 0.511

- 0.252 0.129 1414 2768

- 1.746

Pollen 7 50 860 506 9 807.288 538.712 0.077 0.633

- 0.05 0.292 1752 2768

- 1.753

Pollen 7 25

- 485 568 11

- 573.884 549.941 0.844 0.138

- 0.553

- 0.207 383 2768

- 1.74

Pollen 7 13

- 751 776 11

- 723.548 633.978 0.865 0.137

- 0.66

- 0.261 379 2768

- 1.74120 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Pollen 8 200 688 NA 1 611.922 2401.029 0.406 1.153

- 0.907 0.123 5731 4969

- 2.132

Pollen 8 100 4902 NA 1 4997.097 2082.223 0.037 1.899 0.112 1.006 9436 4969

- 2.132

Pollen 8 50 2949 672 7 3170.529 780.622 0.001 0.923 0.353 0.638 4585 4969

- 1.812

Pollen 8 25

- 172 1125 8 34.473 1041.741 0.487 0.383

- 0.37 0.007 1905 4969

- 1.796

Pollen 8 13

- 580 787 8

- 605.432 809.164 0.765 0.171

- 0.414

- 0.122 848 4969

- 1.796

Weight 3 200

- 2 2 11 0 40

- 1.729

Weight 3 100 0 2 11 0 40

- 1.729

Weight 3 50

- 3 2 11 0 40

- 1.729

Weight 3 25

- 2 2 11 0 40

- 1.729

Weight 3 13

- 2 2 11 0 40

- 1.729

Weight 4 200 5 1 11 5.863 1.472 0 0.145 0.057 0.101 8 58

- 1.729

Weight 4 100 5 2 11 4.147 1.436 0.005 0.114 0.029 0.071 7 58

- 1.729

Weight 4 50 3 2 11 2.382 1.532 0.068 0.087

- 0.005 0.041 5 58

- 1.729

Weight 4 25 1 2 11 1.569 1.495 0.154 0.072

- 0.017 0.027 4 58

- 1.729

Weight 4 13 0 2 11

- 0.271 1.798 0.559 0.049

- 0.058

- 0.005 3 58

- 1.729

Weight 5 200 5 2 11 4.929 1.972 0.011 0.176 0.032 0.104 8 47

- 1.729

Weight 5 100 7 2 11 7.088 2.034 0.001 0.224 0.075 0.149 11 47

- 1.729

Weight 5 50 6 2 11 5.241 2.189 0.014 0.19 0.031 0.11 9 47

- 1.729

Weight 5 25 0 2 11

- 0.188 2.015 0.537 0.069

- 0.077

- 0.004 3 47

- 1.729

Weight 5 13 2 2 11 1.146 2.222 0.306 0.105

- 0.057 0.024 5 47

- 1.729

Weight 6 200 15 3 11 16.153 3.421 0 0.415 0.192 0.304 22 53

- 1.734

Weight 6 100 8 4 10 7.991 3.955 0.03 0.28 0.021 0.15 15 53

- 1.74

Weight 6 50 8 3 10 6.564 3.966 0.058 0.253

- 0.006 0.123 13 53

- 1.74

Weight 6 25

- 1 3 11

- 0.802 3.677 0.585 0.105

- 0.135

- 0.015 6 53

- 1.734

Weight 6 13 1 3 11 0.088 3.5 0.49 0.116

- 0.112 0.002 6 53

- 1.734

Weight 7 200 16 3 11 17.512 3.434 0 0.446 0.22 0.333 23 53

- 1.734121 Parameter Time

(CCA)

1 Test

conc. (μg/l) 2 Observed

mean

difference

from

control 3 Standard

error

observed

mean n Model

estimate

mean

difference

from

control 4 Standard

error of

estimated

mean P value for

comparison

with the

control 90%

confidence

upper limit 90%

confidence

low er limit Estimated

reduction

from

control (%) 5 Estimated

reduction

from control

(number) Obeserve

d means

in control Ttest

confidence

limit

Weight 7 100 9 4 10 9.946 3.841 0.01 0.316 0.062 0.189 17 53

- 1.74

Weight 7 50 9 4 10 7.903 4.104 0.036 0.286 0.015 0.15 15 53

- 1.74

Weight 7 25

- 1 4 11 0.306 3.853 0.469 0.133

- 0.121 0.006 7 53

- 1.734

Weight 7 13 0 3 11

- 0.825 3.756 0.586 0.108

- 0.139

- 0.016 6 53

- 1.734

Weight 8 200

- 6 NA 1

- 10.331 5.62 0.93 0.041

- 0.551

- 0.255 2 41

- 2.132

Weight 8 100 26 NA 1 27.447 3.867 0.001 0.881 0.474 0.677 36 41

- 2.132

Weight 8 50 8 2 7 8.475 2.777 0.006 0.333 0.085 0.209 14 41

- 1.812

Weight 8 25

- 1 4 8 0.503 3.511 0.444 0.168

- 0.143 0.012 7 41

- 1.796

Weight 8 13

- 1 1 8

- 0.454 1.484 0.617 0.055

- 0.077

- 0.011 2 41

- 1.796

Note s :

1, Observation d ates for h ive weight measurement were slight different from the dates when colony assessments were conducted.

2, T he t est concentration labelled as “13” in the table was originated from the raw data submitted by the study author. The actual concentration is expected to be 12.5 μg/l .

3, Mean of observations in controls minus the observation in the treatment.

4, Difference between the mean of observation in controls and estimated number in treatment after adjustment for covariance f or CCA3 to be a 0 baseline.

5

, The percentage of the estimated difference between the treatment and control divided by the number in the control.[Value in c olumn must be multiplied by 100 t o be a %]122 

## Results

The following tables and graphs present results for individual measurement endpoints (total

individuals, eggs, larvae, pupae, pollen stores, nectar stores, hive weight). The percent reductions

are the means of the differences between each treatment and con trol at the same apiary, based on

observations and expected values estimated by the statistical model that adjusted baseline

measurement for CCA3, using raw count data.

Table B

- 2

. Estimated percent reduction from control for total number of individuals

Test

concentration

(μg/L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 4

(0.354) 1

(0.469) 8.7

(0.091)* - 16.9

(0.902) - 2.6

(0.561)

25 10.8

(0.166) 10.9

(0.253) 17.4

(0.029)** 2.2

(0.429) - 11.4

(0.657)

50 10.3

(0.182) 25.4

(0.062)* 12.1

(0.048)** 8.1

(0.19) 49.1

(0.028)**

100 35.3

(0.002)** 46

(0.001)** 48.2

(<0.001)** 60.9

(<0.001)** 145.1

(0.036)**

200 48.6

(<0.001)** 60.5

(<0.001)** 65.9

(<0.001)** 74.6

(<0.001)** 54.1

(0.238)

Note: Negative value indicates increased number of individuals in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.123 Figure B

- 1 : Estimates and 90% C I s for Total Life with thresholds of statistical (red) significance shown. The green

line shows 10% difference from control.

Figure B

- 2 : Mean of the differences of the total individuals in hives between treatments and control at the

same apiary.



### A B124 Figure A: Total number of individuals in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the total individuals in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: Mean of the differences of the total individuals in hives between the treatments and control at the same apiary

(n=11) with 90% upper and lower confidence interval.

X

- axis represents the num ber of CCA; Y

- axis represents the mean of the differences of the total individuals in hives between

the control and treatment at the same apiary (control minus treatment).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more live individuals in hives in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 3 : Estimated percent reduction from control for number of adults

Test

concentration

(μg/L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 2.2

(0.443) - 5.4

(0.652) 2.3

(0.415) - 12.6

(0.821) - 11

(0.74)

25 2.7

(0.432) 8.7

(0.267) 7.7

(0.24) - 4.1

(0.604) - 4.3

(0.555)

50 - 23.3

(0.931) 21.7

(0.078)* 19.8

(0.057)* 18.6

(0.074)* 78**

(0.001)

100 5.2

(0.392) 34

(0.015)** 28.7

(0.024)** 51

(0.001)** 172.5

(0.003)**

200 - 29.2

(0.961) 24.4**

(0.028) 52.8**

(<0.001) 59.4**

(0.001) 7.3

(0.425)

Note: Negative value indicates increased number of adults in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the s tep

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.125 Figure B

- 3 : Estimates and 90% C I s for adult bees with thresholds of statistical (red) significance shown. The

green line shows 10% difference from control.

Figure B

- 4 : Mean of the differences of adult bees between treatments and control at the same apiary.



### A B126 Figure A: Total member of adults in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the number of adults in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: Mean of the differences of the number of adults in hive s between the treatments and control at the same apiary

(n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the number of adults in hives

between the control and treatment at the same apiary (control minus treatme nt).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA 3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more adult bee in hives in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 3: Estimated percent reduction from control for number of eggs

Test

concentration

(μg/ L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 22.8

(0.063*/0.69) 11.9

(0.236) 5.9

(0.38) 0.1

(0.498) 37.7

(0.058*/0.36)

25 - 1.1

(0.528) 8.3

(0.336) 26.3

(0.058*/0.14) 31.3

(0.114) 5.8

(0.367)

50 - 9.6

(0.692) 1.8

(0.453) 11.7

(0.146) 2.6

(0.451) 78.2

(<0.001)**

100 37.9

(0.01**/0.21) 39.4

(0.036)** 70.8

(<0.001)** 46.6

(0.042)** 138

(0.002)**

200 14.5

(0.212) 32.2

(0.043)** 60.1

(<0.001)** 77.8

(0.002)** 153.2

(0.003)**

Note: Negative value indicates increased number of eggs in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.127 Figure B

- 5 : Estimates and 90% CIs for eggs with thresholds of statistical (red) significance shown. The green line

shows 10% difference from control.

Figure B

- 6 : Mean of the differences of number of eggs in hives between treatments and control at the same

apiary.



### A B128 Figure A: Total number of eggs in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the number of eggs in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: : Mean of the differences of the number of eggs in hives between the treatments and control at the same apiary

(n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the number of eggs in hives between

the control and treatment at the same apiary (control minus treatment).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA 3

for e ach CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more eggs in hives in the control than in the treatment, and a negative value indicate s
reversely.

A dash line indicates the 90% c onfidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 4: Estimated percent reduction from control for number of larvae

Test

concentration

(μg/L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 12.3

(0.161) 10.7

(0.242) - 15.5

(0.8) - 62.6

(0.996) 0.8

(0.479)

25 8.1

(0.241) 16.2

(0.187) 23

(0.052*/0.57) - 25.5

(0.831) - 9.9

(0.641)

50 12.1

(0.199) 15.3

(0.223) - 2.1

(0.577) - 32.4

(0.955) 42.6

(0.04)**

100 37.1

(0.007)** 30.9

(0.026)** 52.3

(<0.001)** 64.4

(0.002)** 159.9

(0.024)**

200 64

(<0.001)** 65

(<0.001)** 57.2

(<0.001)** 78.3

(<0.001)** 54.1

(0.228)

Note: Negative value indicates increased number of larvae in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 t reatment levels where very few

hives survived.129 Figure B

- 7 Estimates and 90% CIs for larvae with thresholds of statistical (red) significance shown. The green

line shows 10% difference from control.

Figure B

- 8 : Mean of the differences of number of larvae in hives between treatments and control at the same

apiary.



### A B130 Figure A: Total number of larvae in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the number of larvae in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: : Mean of the differences of the number of larvae in hives between the treatments and control at the same apiary

(n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the number of larvae in hives

between the control and treatment at the same apiary (control minus treatment).

The line of “0” indicates no effect from the control with in the same Apiary and it was adjusted for baseline covariate at CCA3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more larvae in hives in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 5: Estimated percent reduction from control for number of pupae

Test

concentration

(μg/ L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 2.8

(0.427) - 3.7

(0.595) 22.2

(0.032**/

0.06*) - 8.4

(0.686) - 1.3

(0.52)

25 17.7

(0.154) 5.8

(0.387) 18.3

(0.036**/

0.06*) 18.1

(0.133) - 10.8

(0.62)

50 28.1

(0.068)* 34.6

(0.044)** 12.5

(0.06)* 9.7

(0.232) 70.6

(0.039)**

100 51.7

(0.002)** 56.6

(0.001)** 49.7

(<0.001)** 75.6

(<0.001)** 150.9

(0.092)*

200 83.3

(<0.001)** 79.5

(<0.001)** 75.5

(<0.001)** 93.5

(<0.001)** 42

(0.356)

Note: Negative value indicates increased number of pupae in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the s ignificance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.131 Figure B

- 9 : Estimates and 90% CIs for pupae with thresholds of statistical (red) significance shown. The green

line shows 10% difference from control.

Figure B

- 10 : Mean of the differences of number of pupae in hives between treatments and control at the same

apiary



### A B132 Figure A: Total number of capped brood (pupae) in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the number of pupae in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: Mean of the differences of the number of capped brood (pupae) in hives between the treatments and control at

the same apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the number of pupae in hiv es between the control and treatment at the same apiary (control minus treatment).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA 3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more pupae in hives in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM m odel for a solid line with the same color.



### Table B

- 6: Estimated percent reduction from control for pollen store

Test

concentration

(μg/L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 11.8

(0.222) - 6.8

(0.658) - 53.3

(0.998) - 26.1

(0.865) - 12.2

(0.765)

25 5.2

(0.369) 2.1

(0.455) - 25.2

(0.919) - 20.7

(0.844) 0.7

(0.487)

50 56.1

(<0.001)** 62.5

(<0.001)** 15.5

(0.147) 29.2

(0.077*/0.28) 63.8

(0.001)**

100 83.7

(<0.001)** 83.6

(<0.001)** 15.3

(0.158) 12.9

(0.281) 100.6

(0.037)**

200 94.5

(<0.001)** 90.4

(<0.001)** 54.7

(0.001)** 50.6

(0.029)** 12.3

(0.406)

Note: Negative value indicates increased pollen store in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives sur vived.133 Figure B

- 11 : Estimates and 90% CIs for pollen store with thresholds of statistical (red) significance shown. The

green line shows 10% difference from control.

Figure B

- 12 : Mean of the differences of the amount of pollen store in hives between treatments and control at

the same apiary.



### A B134 Figure A: Total amount of pollen store in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the amount of pollen store in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: Mean of the differences of the amount of pollen store in hives between the treatments and control at the same

apiary (n=11).

X

- axis represents the nu mber of CCA; Y

- axis represents the mean of the differences of the amount of pollen store in hives

between the control and treatment at the same apiary (control minus treatment).

The line of “0” indicates no effect from the control within the same Apiary a nd it was adjusted for baseline covariate at CCA3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates there are more pollen store in hives in the control than in the treatment, and a negativ e value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 7: Estimated percent reduction from control for honey store

Test

concentration

(μg/ L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 - 7.5

(0.808) 1.3

(0.467) 15.8

(0.093*/0.58) 12.5

(0.228) - 10.9

(0.712)

25 - 10.4

(0.89) - 15.3

(0.84) - 2.4

(0.582) 10.6

(0.243) 13.3

(0.288)

50 - 6.2

(0.723) 21.6

(0.091*/0.33) 36

(0.01)** 41.2

(0.022)** 60.4

(0.007)**

100 - 8

(0.847) 7.1

(0.333) 21.8

(0.088)* 52.9

(0.001)** 156.6

(0.024)**

200 - 21.1

(0.963) - 84.1

(1) 70.5

(<0.001)** 80

(<0.001)** 5.1

(0.471)

Note: Negative value indicates increased honey store in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, th e step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.135 Figure B

- 13 : Estimates and 90% CIs for honey store with thresholds of statistical (red) significance shown. The

green line shows 10% difference from control.

Figure B

- 14 : Mean of the differences of the amount of honey store in hives between the treatments and control

at the same apiary.136 

### A B

Figure A: Total amount of honey store in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the amount of honey store in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: : Mean of the differences of the amount of honey s tore in hives between the treatments and control at the same

apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the amount of honey store in hives

between the control and treatment at the same apiary (cont rol minus treatment).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA 3

for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y valu e indicates there is more honey store in hives in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.



### Table B

- 8: Estimated percent reduction from control for hive weights

Test

concentration

(μg/L ) Estimated reduction from control (%)

(P value)

CCA4 CCA5 CCA6 CCA7 CCA8

12.5 - 0.5

(0.559) 2.4

(0.306) 0.2

(0.49) - 1.6

(0.586) - 1.1

(0.617)

25 2.7

(0.154) - 0.4

(0.537) - 1.5

(0.585) 0.6

(0.469) 1.2

(0.444)

50 4.1

(0.068)* 11

(0.014)** 12.3

(0.058)* 15

(0.036)** 20.9

(0.006)**

100 7.1

(0.005)** 14.9

(0.001)** 15

(0.03)** 18.9

(0.01)** 67.7

(0.001)**

200 10.1

(<0.001)** 10.4

(0.011)** 30.4

(<0.001)** 33.3

(<0.001)** - 25.5

(0.93)

Note: Negative value indicates increased hive weight in comparison to control.

*0.05<P<0.1

**P<0.05

Where two p values are listed, the first is the non

- adjusted p value, the second is the p value adjusted for

the step

- down approach. The step

- down adjustment was shown only if it changed the significance level.

At CCA8, the step

- down approach was not applied to the 200 or 100 treatment levels where very few

hives survived.137 Figure B

- 15 : Estimates and 90% CIs for hive weight with thresholds of statistical (red) significance shown. The

green line shows 10% difference from control.

Figure B

- 16 Mean of the differences of the hive weight between the treatments and control at the

same apiary.



### A B138 Figure A: Total hive weight in control hives at each apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the hive weight in control hives.

Apiaries are coded with various colors.

Black dash line represents the mean of all apiaries.

Figure B: Mean of the differences of the hive weight between the treatments and control at the same apiary (n=11).

X

- axis represents the number of CCA; Y

- axis represents the mean of the differences of the hive weight between the

control and treatment at t he same apiary (control minus treatment).

The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate

at CCA3 for each CCAs.

Labels at the end of each line are the treatment concentrations (μg/L).

A positive Y value indicates that the hive weight was more in the control than in the treatment, and a negative value

indicates reversely.

A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color

.

X : 0.1>P>0.05,  : 0.05>P 

### Figure B

- 17

. Total Individuals (percent difference from control)

Figure B

- 18

. Adults (percent difference from control) -50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Total individuals

12.5 25 50 100 200

-50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Adults

12.5 25 50 100 200139 

### Figure B

- 19

. Eggs (percent difference from control)

Figure B

- 20

. Larvae (percent difference from control)

Figure B

- 21

. Pupae (percent difference from control) -50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Eggs

12.5 25 50 100 200

-100

-50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Larvae

12.5 25 50 100 200

-50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Pupae

12.5 25 50 100 20014 0 

### Figure B

- 22

. Honey (percent difference from control)

Figure B

- 23

. Pollen (percent difference from control)

Figure B

- 24

. Weight (percent difference from control) -100

-50

0 50 100 150 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Honey

12.5 25 50 100 200

-100

-50

0 50 100 150

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Pollen

12.5 25 50 100 200

-40 -20

0 20 40 60 80

CCA4 CCA5 CCA6 CCA7 CCA8Difference from control (%) 

### Weight

12.5 25 50 100 200141 

### Figure B

- 25 Summary of living organism parameters at 12.5 μg/L

Figure B

- 26

. Summary of living organism parameters at 25 μg/L 4.0 1.0 8.7

- 16.9 - 2.6 2.2

- 5.4 2.3

- 12.6 - 11.0 2.8

- 3.7 22.2

- 8.4 - 1.3

12.3 10.7

- 15.5

- 62.6 0.8 22.8

11.9 5.9 0.1 37.7

-80.0 -60.0 -40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

CCA4 CCA5 CCA6 CCA7 CCA8Difference from the control (%)



### TotalLife Adult Pupae Larvae Eggs

10.8 10.9 17.4

2.2

- 11.4 2.7 8.7 7.7

- 4.1

- 4.3 17.7

5.8 18.3 18.1

- 10.8 8.1 16.2 23.0

- 25.5 - 9.9 - 1.1 8.3 26.3 31.3

5.8

-40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

CCA4 CCA5 CCA6 CCA7 CCA8Difference from the control (%)



### TotalLife Adult Pupae Larvae Eggs142 

### Figure B

- 27

. Summary of living organism parameters at 50 μg/L

Figu re B

- 28

. Summary of living organism parameters at 100 μg/L 10.3 25.4

12.1 8.1 49.1

- 23.3 21.7 19.8 18.6 78.0

28.1 34.6

12.5 9.7 70.6

12.1 15.3

- 2.1

- 32.4 42.6

- 9.6 1.8 11.7

2.6 78.2

-40.0 -20.0

0.0 20.0 40.0 60.0 80.0 100.0

CCA4 CCA5 CCA6 CCA7 CCA8Difference from the control (%)



### TotalLife Adult Pupae Larvae Eggs

35.3 46 48.2 60.9 145.1

5.2 34 28.7 51 172.5

51.7 56.6 49.7 75.6 150.9

37.1

30.9 52.3 64.4 159.9

37.9 39.4 70.8

46.6 138

0 20 40 60 80 100 120 140 160 180 200

CCA4 CCA5 CCA6 CCA7 CCA8Difference from the control (%)



### TotalLife Adult Pupae Larvae Eggs143 

### Figure B

- 29

. Summary of hive weight at 12.5 μg/L

Figure B

- 30

. Summary of hive weight at 25 μg/L - 0.271 1.146

0.088

- 0.825 - 0.454

-5 -4 -3 -2 -1 0 1 2 3 4

CCA4 CCA5 CCA6 CCA7 CCA8

1.569

- 0.188

- 0.802 0.306 0.503

-5 -4 -3 -2 -1 0 1 2 3 4 5

CCA4 CCA5 CCA6 CCA7 CCA8144 

### Figure B

- 31

. Summary of hive weight at 50 μg/L

Figure B

- 32

. Summary of hive weight at 100 μg/L 2.382 5.241 6.564 7.903 8.475

0 2 4 6 8 10 12 14

CCA4 CCA5 CCA6 CCA7 CCA8

4.147 7.088 7.991 9.946 27.447

0 5 10 15 20 25 30 35

CCA4 CCA5 CCA6 CCA7 CCA8145 

### Table B

- 9

. Summary of observed effects at each treatment level ( Note: Values reported in the

table are the % reduction compared to control

, based on model estimated raw numbers corrected

for baseline measurements )

.

Treatment

(μg/l) Observations

12.5 • Decreased total number of indiv iduals in hive at CCA6 (8.7%, P<0.1 )

• Decreased number of pup ae at CCA6 (22.2%, P<0.05)

• Decreased honey store at CCA6 (15.8%, P <0.1)

• At CCA8, two out of 11 colonies did not survive overwintering (better survival than control)

• At CCA8 among surviving hives, hi ve condition similar to control

• The potential colony effects at CCA6 were considered short

- term, colony able to compensate



### 25 • Decreased total number of individuals i n hive at CCA6 (17.4%, P<0.05)

• Decreased number of p upae at CCA6 (18.3%, P<0.05)

• Decreased number of eggs at CCA6 (26.3%, P <0.1)

• Decreased number of larvae at CCA6 (23%, P <0.1)

• At CCA8, one out of 11 colonie s did not survive overwintering (better survival than control)

• At CCA8 among surviving hives, hi ve c ondition similar to control

• The potential colony effects at CCA6 were considered short

- term, colony able to compensate



### 50 • Decreased total number of individuals in hive at CCA5 (25.4%, P <0.1) and CCA6 (12.1%,

P<0.05)

• Decreased pupae at CCA4 , CCA5, CCA6 (28.1, 34.6, 12.5%, P <0.1 , <0.05, <0.1)

• Decreased number of adults at CCA5

- CCA7 (18.6

- 21.7%, P <0.1)

• No ef fect on eggs or larvae observed

• Decreased p ollen store at CCA4, CCA5, CCA7

• Decre ased honey store at CCA5

– CCA7

• Decre ased hive weight at CCA4

– CCA7

• At CCA8, four out of 11 colonies did not survive overwintering, comparable to control

• At CCA8 among surviving hives, poorer hive condition compared control



### 100 and

200 • Decreased total number of i ndividuals in hive at CCA4

- CCA7

• Decreased number of adults at CCA5

- CCA7

• Decrease d number of eggs at CCA4

– CCA7

• Decreased number of larvae at CCA4

– CCA7

• Decreased number of pupae at CCA4

– CCA7

• Decrease d pollen store at CCA4 and CCA5

• Dec reased honey store at CCA6

- CCA7

• Decre ased hive weight at CCA4

– CCA 7

• High overwintering mortality (only 1 survived at 100; only 2 survived at 200)



### OVERALL

ENDPOINT • NOAEL: 25 μg/L sucrose solution (nominal 20.3 ppb; measured 23.3 ppb)

• LOAEL: 50 μg/L sucrose solution (nominal 40.7 ppb; measured 46.7 ppb)146 

## 3

. Appendix C: Details of CDPR Statistical Analysis

Statistical Summary

A clear progression of effects on hive health in response to imidacloprid dose was indicated by

the results of the multivariate mixed model analysis.

• After only 3 weeks into the exposure period pupal and larval numbers decreased in

response to higher dose levels with effects initiated on pupal cells at the 50 ppb treatment.

Pollen food stores also exhibited decreased numbers as a dose response relations hip at the

higher treatment levels with effects initiated at the 50 ppb dose level. Overall hive weight

was decreased at 100 and 200 ppb doses.

• At 7 weeks after initiation of exposure (1 week after end of treatments), numbers of adult

and egg cells were then decreased at the higher treatments with effects initiated on adult

cells at the 50 ppb dose level. A clear dose response was also observed for hive weight at

the higher doses with effects now measured at the 50 ppb treatment.

• Later at 11 weeks afte r initiation of exposure (5 weeks after end of treatments), number of

honey cells exhibited a dose response relationship at the higher treatment levels with

effects initiated at the 50 ppb dose level.

• At the final assessment before overwintering at 16 weeks after initiation of exposure (10

weeks after end of treatments) decreases in number of honey cells and hive weight were

measured at the 50 ppb dose level. Numbers of adults and pupal and larval cells were

decreased at the 100 and 200 ppb dose levels. Note that some effects previously measured

between control and 50 ppb treatment levels were not apparent at this assessment.

• Assessment of the hives after overwintering indicated that dose related effects noted at

CCA7 were expressed in CCA8 where extr eme loss of hives was observed at the 100 and

200 ppb. Decreased vigor of hives at the 50 ppb dose level was indicated due to decreased

numbers of adults and pupal and larval cells as compared to control hives.

The general conclusion is that the data indicate a NOEC value at 25 ppb and a LOEC value

at 50 ppb.



## Background

The multivariate mixed repeated measures model approach is distinguished from the univariate

approach previously in that all bee life stages or hive food storage variables are simultaneously

analyzed as a single model. Multivariate analyses of variance for fixed effects models are

conducted, using Statistical Analysis System (SAS) software, through implementing the

MANOVA option in the PROC GLM procedure. Recently, multivariate analyses have been

extended to mixed models using the PROC MIXED procedure. The MIXED procedure is designed

to conduct a mixed model analysis of variance where fixed and random effects can be specified.

Inclusion of random effects in a model provides a broader application of results. For this study,147 

### locations were denoted as apiaries with individual hives as test subjects. Use of a mixed model

with apiaries identified as a random variable provides some assurance that the results can be

generalized to other locations and hives.



## Model Setup

• The Multivariate Analysis of Variance in PRO C MIXED is conducted by combining the data

f

or response variables to be analyzed into two columns of data . One column contains the list

of variables to be analyzed, denoted as ‘RESPONSE’ in this analysis, and the other variable

contains the measured value for that variable, denoted ‘VALUE’ in this analysis.

• Factors on the effect side of the equation for the multivariate response were dose of

imidacloprid and date sampled , which was denoted as day for each CCA assessment. .

• Technically, the RESPONSE variab le is listed as a class variable in the multivariate analysis

and then included in the effects side of the model.

The SAS code below illustrates the syntax for one of the programs used to determine the

appropriate covariate model to use:

**proc mixed**  data =a7;

title4 'Overall Multivariate analysis' ;

title5 'EPA scaling

- Apiary random

- CV = un@ar(1)' ;

class day dose response hivenum apiary;

model epavaluet=response|day|dose/ noint ;

random apiary;

repeated response day/ subject =dose*rep(apiary) type =un@ar( **1** );

**run** ;



### Features in this syntax are:

1. Class Statement: The RESPONSE variable is included in the list of effects variables and

contains the variables to be analyzed for the multivariate analysis.

2. Model Statement:

a. VALUE is the respective measure for the RESPONSE variable. For example,

VALUE contains the value for number of adult bees taken for hive number 2

located in Apiary A and taken at CCA3 at the 12.5 ppb treatment. For the statistical

analysis, the original values were scaled as in t he univariate analyses: R aw values

for pupal, larval, and egg cells were dived by 68800 w ith adult cells divided by

68800/1.3 then times by 4.

b. The RESPONSE variable is tested for interactive effects with day and dose on the

effects side of the model.

3. Rand om Statement: Apiary is treated as a random variable because effects are to be

generalized to other locations.

4. Repeated Statement:

a. Provides for a repeated measures analysis of variance.

b. The subject= indicates the hive where the repeated measures were ta ken.

c. Type=UN@AR(1) indicates the covariance model used where the symbols

represent a Compound Symmetry model applied to the response variable and a first

order autoregressive model applied to day. Various covariance models were tested148 

### to determine the mo del which provided the best fit. The best model was chosen by

comparing values of informational criteria for

- 2 Res Log Likelihood, a criteria that

provided an overall estimate of the amount of variance explained by the model, and

the BIC criteria that adj usts the previous criteria based on the number of additional

parameters added to the model for each structured model.

.

The sequence of statistical analyses conducted was:

1. Conduct a full model analysis of variance as reflected in syntax above. Owing to th e large

number of dead hives at CCA8 for the 100 and 200 ppb treatments, data from CCA3

through CCA7 were used in this overall analysis to test for interactive effects. Three

covariance models relevant to this design were tested. For bee life stages the UN *AR(1)

model provided the best fit, whereas, UN@CS provided the best fit for analysis of food

stores .

2. Upon observation of a significant interactive term between dose, day, and response , further

analyses were conducted at each CCA to determine the differen tial response s among variables over time. Data for CCA8 were included in this analysis

. Seven covariance

models were tested for each CCA. The autoregressive

- first

- order model with heterogeneity

model fit best at CCA3, the compound symmetry model with heter ogeneity fit best at

CCA4, and the unstructured model fit best for CCA5 through CCA8.

Multivariate Analysis of Variance

• Two sets of analyses were conducted. One focused on the counts for the various life stages of

bees within the hive, contrasting the numbers of adults, pupal, larval, and eggs over time.

• A second analysis explored the relationship between nectar and polle n cells. These values

indicate the level of food stores in the hive over. Analysis on hive weight was conducted

separately.



## Results

Life Stages

All effects in the multivariate full model for adult, pupal, larval, and eggs cells were highly

significant ( T able C

- 1 ). Notably, t he triple interactive effect for D ay *D ose *Response indicated

that the various bee stages responded differently over time to imidacloprid dosage

. Analyses were

then conducted by CCA to determine the sequence of effects over time.

In order to determine the pattern of response for life stages at each CCA, a regression analysis was

first conducted to measure potential linear and curvilinear effects of dose at each CCA ( Table C

2 ). Quadratic and cubic dose effects were included to indicat e potential curvature in response. The

second analysis provided a LSMEANS test for each pairwise comparison between levels of dose

for each response ( Table C

- 3 ). These contrasts provide a basis for estimating potential no observed

effect concentration (NO A EC) and lowest observed effect concentration (LO A EC) values.149 

### Table C

- 1

. Results for multivariate mixed model analysis of variance for potential interactive

effects of imidacloprid dose over time on counts of bee stages for adult, pupal, larval and egg cells

.

Comparison of the pattern of significant regression r esults between CCAs provided evidence for

the differential response s in bee life stages over time ( Table C

- 2 ). In the figures for effects, the

response for each variable over dose and is plotted. In addition, oversized do ts and colors indicate

levels of significant difference between the control value and the value at each treatment level as

indicated from Table C

- 3

.

• CCA3: Prior to dietary administration of imidacloprid at CCA3, baselines for life stages

assigned to each treatment level were essentially not significantly different (p<0.05) from

control, with the exception of pupal cells ( Table C

- 3 ). For pupal cells, the initial number of

cells tended to be lower for the control group. The reason for this finding is not known but

differences caused by imidacloprid treatments were measured in subsequent analyses. At this

time, adults were in greatest number followed by pupa l cells and then larval and egg cells

( Figure C

- 1 ).

• CCA4: Although this assessment of hive health was taken only 3 weeks into the exposure

period, significant regression indicated decreasing numbers of pupal and larval cells with

increase in imidaclopr id dose ( Figure C

- 2 ). For pupal cells the effect was first measured at the

50 ppb treatment (p<.1)) and then at progressively increasing probability levels for 100 and

200 ppb treatments. For larval cells the effects were significant for the 100 ppb treatm ent and

higher. Effects on pupal and larval cells numbers were persistent throughout the subsequent

CCAs ( Figures C

- 2 to C

- 6 ).

• CCA5: Decreases in numbers of adult and egg cells were now indicated at the higher 100 and

200 ppb dose levels ( Figure C

- 3).

• CCA6: Decreases noted in the previous CCAs at the 100 and 200 pbb levels of dose were

measured for all life stages. For adult cells, decreased numbers of adults were also measured

for the 50 ppb treatment, indicating a dose response to treatments start ing at this level ( Figure

C

- 4 ).

• CCA7: At CCA7 higher order regression coefficients were significant for counts of adults

and larval cells ( Table C

- 2 ). The curvilinear nature of the response reflected the extreme Overall Model Effect Num DF Den DF PR>F

Response 3 213 <.0001

Day 4 283 <.0001

Day*Response 12 849 <.0001

Dose 5 61 <.0001

Dose*Response 15 213 <.0001

Day*Dose 20 283 0.0001

Day*Dose*Response 60 849 <.0001150 

### effects on reductions in numbers measured for the 100 and 200 ppb levels of imidacloprid dose

(Figure C

- 5 ).

• CCA8: The pattern noted at CCA7 was reflected at the final assessment at CCA8 where

curvature in response measured for all life stages reflected a grouping of treatment levels:

Results wer e similar for 0, 12.5, and 25 ppb treatments and with 50, 100, and 200 ppb

treatments reflecting detrimental effects due to imidacloprid treatment ( Table C

- 2, Figure C

6 ). Loss of hives at 100 and 200 ppb treatments was an obvious effect resulting in essentially

loss of all life stages at these treatments. But additional decreases in numbers of adults, pupal,

and egg cells were measured at the 50 ppb treatment compared to the control, indicating lower

vigor of hives at this treatment.

Table C

- 2

. Regression for regression effect s conducted within each CCA and for each bee life

stage

.

Bee Life Stage Regression Effect Regression Results for Each CCA

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

Pr>t Pr>t Pr>t Pr>t Pr>t Pr>t

Adult Dose Linear 0.6945 0.0622 0.0341 0.0003 <.0001 <.0001

Dose Quadratic 0.1763 0.1761 0.017 0.2027 0.0938 0.034

Dose Cubic 0.3279 0.143 0.5933 0.8789 0.0501 0.0417

Pupal Dose Linear 0.0923 <.0001 < .0001 <.0001 <.0001 <.0001

Dose Quadratic 0.9986 0.4165 0.1366 0.2841 0.1758 0.0437

Dose Cubic 0.2244 0.6987 0.9101 0.7008 0.1117 0.0511

Larval Dose Linear 0.626 <.0001 0.0003 <.0001 <.0001 <.0001

Dose Quadratic 0.1992 0.3551 0.8878 0.1037 0.5789 0.0968

Dose Cubic 0.2381 0.3424 0.9101 0.2008 0.0055 0.0275

Egg Dose Linear 0.7645 0.2451 0.021 <.0001 0.0003 <.0001

Dose Quadratic 0.6786 0.2995 0.4279 0.0421 0.9673 0.0244

Dose Cubic 0.7019 0.0996 0.4022 0.1842 0.9576 0.1589151 

### Table C

- 3 . Mean comparison for the response between each level of dose at each CCA for each

bee life stage. The difference is the percent frame coverage of dose1 minus dose2 where a positive

value indicates a higher value for the lower dosage and a negative value indicates a lower value

for the lower dosage.

Life

Stage Dose1 Dose2 PR>t Difference Life

Stage Dose1 Dose2 PR>t Difference Life

Stage Dose1 Dose2 PR>t Difference

Pupal 0 12.5 0.0969 -0.06 Adult 0 200 0.0225 -0.13 Adult 0 50 0.0636 0.12

0 25 0.0577 -0.07 25 200 0.0812 -0.12 0 100 0.0079 0.17

0 200 0.0185 -0.09 100 200 0.0177 -0.17 12.5 50 0.0397 0.15

Larval 0 100 0.0972 0.03 Pupal 0 50 0.0822 0.06 12.5 100 0.006 0.21

25 100 0.0709 0.04 0 100 0.0032 0.1 12.5 200 0.0674 0.13

0 200 <0.0001 0.16 25 100 0.0525 0.14

12.5 50 0.048 0.08 Pupal 0 50 0.0172 0.1

12.5 100 0.0026 0.12 0 100 0.0002 0.15

12.5 200 <0.0001 0.18 0 200 <0.0001 0.02

25 100 0.027 0.09 12.5 50 0.0428 0.09

25 200 0.0002 0.15 12.5 100 0.0014 0.15

50 200 0.0065 0.11 12.5 200 <0.0001 0.22

100 200 0.0961 0.06 25 100 0.0056 0.13

Larval 0 100 0.0003 0.05 25 200 <0.0001 0.19

0 200 <0.0001 0.07 50 200 0.0094 0.12

12.5 200 0.024 0.04 Larval 0 100 0.045 0.04

25 100 0.0065 0.05 0 200 0.0005 0.07

25 200 0.0003 0.06 12.5 200 0.0102 0.06

50 100 0.0182 0.04 25 200 0.0205 0.05

50 200 0.001 0.06 50 200 0.0179 0.05

Egg 0 100 0.0367 0.03 Egg 0 100 0.0407 0.03

25 100 0.0491 0.03 0 200 0.0538 0.03

50 100 0.0184 0.03 Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA and Each Life Stage

CCA3 CCA4 CCA5152 

### Table C

- 3 Continued

. Mean life stage comparisons continued for CCA6 through CCA8:

Life

Stage Dose1 Dose2 PR>t Difference Life

Stage Dose1 Dose2 PR>t Difference Life

Stage Dose1 Dose2 PR>t Difference

Adult 0 50 0.0246 0.14 Adult 0 100 0.0036 0.14 Adult 0 50 0.00707 0.11

0 100 0.016 0.15 0 200 0.0034 0.14 0 100 0.0004 0.23

0 200 0.001 0.21 12.5 50 0.0142 0.13 0 200 0.0007 0.22

12.5 50 0.0525 0.14 12.5 100 0.0002 0.21 12.5 50 0.0121 0.19

12.5 100 0.0374 0.15 12.5 200 0.0002 0.21 12.5 100 <0.0001 0.3

12.5 200 0.0042 0.21 25 50 0.0278 0.12 12.5 200 0.0001 0.3

25 50 0.0811 0.12 25 100 0.0004 0.2 25 50 0.0076 0.2

25 100 0.059 0.13 25 200 0.0004 0.2 25 100 <0.0001 0.31

25 200 0.0074 0.19 Pupal 0 100 0.0001 0.05 25 200 <0.0001 0.3

Pupal 0 100 <0.0001 0.14 0 200 <0.0001 0.06 Pupal 0 50 0.0942 0.05

0 200 <0.0001 0.19 12.5 100 <0.0001 0.06 0 100 0.0007 0.11

12.5 100 0.02 0.09 12.5 200 <0.0001 0.07 0 200 0.0011 0.11

12.5 200 0.0003 0.14 25 100 0.0044 0.04 12.5 50 0.0189 0.09

25 100 0.0096 0.1 25 200 0.0003 0.05 12.5 100 0.0002 0.15

25 200 0.0001 0.15 50 100 0.0063 0.04 12.5 200 0.0002 0.14

50 100 0.0191 0.09 50 200 0.0005 0.05 25 50 0.014 0.09

50 200 0.0003 0.14 Larval 0 12.5 0.0023 -0.02 25 100 0.0001 0.15

Larval 0 100 0.0005 0.05 0 100 0.0127 0.02 25 200 0.0002 0.15

0 200 0.0003 0.06 0 200 0.0009 0.02 Larval 0 100 0.0013 0.05

12.5 25 0.0178 0.04 12.5 25 0.0958 0.01 0 200 0.0022 0.05

12.5 100 <0.0001 0.07 12.5 50 0.02 0.02 12.5 100 0.0008 0.06

12.5 200 <0.0001 0.07 12.5 100 <0.0001 0.04 12.5 200 0.0012 0.06

25 100 0.0609 0.03 12.5 200 <0.0001 0.05 25 50 0.0609 0.03

25 200 0.0511 0.03 25 100 0.0017 0.03 25 100 0.0002 0.07

50 100 0.0079 0.05 25 200 0.0001 0.03 25 200 0.0003 0.07

50 200 0.0063 0.05 50 100 0.0122 0.02 50 100 0.0435 0.04

Egg 0 100 <0.0001 0.05 Egg 50 200 0.0012 0.03 50 200 0.0609 0.03

0 200 0.0006 0.04 0 200 0.0014 0.02 Egg 0 50 0.0158 0.02

12.5 100 0.0012 0.04 12.5 100 0.0284 0.01 0 100 0.0002 0.03

12.5 200 0.005 0.04 12.5 200 0.0004 0.02 0 200 0.0007 0.03

25 100 0.0148 0.03 25 200 0.0495 0.01 12.5 25 0.0871 -0.02

25 200 0.0477 0.03 50 200 0.0192 

### 0.01 12.5 100 0.0071 0.03

50 100 0.0043 0.04 12.5 200 0.0156 0.02

50 200 0.0162 0.03 25 50 0.0016 0.03

25 100 <0.0001 0.04

25 200 <0.0001 0.04 CCA6 CCA7 CCA8 Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA and Each Life Stage153 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 1. Res ponse of each life stage measured prior to the initiation of imidacloprid treatments

at CCA3. Except for pupal cells, the baseline for each group was not significantly different (p<0.05)

from control. For pupal cells, significant differences from the control were determined for

treatment locations except for the 50 ppb group. The reason f or these differences is not known.

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 2. At CCA4, significant dose related effects were measured for larval and pupal life

stages. Sporadic effects were noted for egg and adult cells, which were most likely not related to

dosing level. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA3

Adult

Egg

Larval

Pupal

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4 0.45

0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA4

Adult

Egg

Larval

Pupal154 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 3. E ffects on larval and pupal cells measured at CCA4 were sustained in CCA5 with

significant reductions from control (p<0.05) at the 50, 100, and 200 ppb levels for pupal cells and

at the 100 and 200 level for larval cells.

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 4. At CCA 6 effects on pupal and larval cells were again sustained with onset of dose

related effects measured for egg and adult cells. Specifically, significant reductions (p<0.05) in the

number of adults and eggs were determined, along with effects to larval a nd pupal cells that were

determined in CCAs 4 and 5. 0 0.1 0.2 0.3 0.4 0.5

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA5

Adult

Egg

Larval

Pupal

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA6

Adult

Egg

Larval

Pupal155 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 5. At CCA7, decreases in all life stages from control group were measured for 100 and

200 ppb levels of imidacloprid.

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 6. Clear effects of hive death were mea sured a t CCA8 at the 100 and 200 ppb

treatments. Decreased numbers of adults, pupal, and egg cells were also measured at the 50 ppb

treatment. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA7

Adult

Egg

Larval

Pupal

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA8

Adult

Egg

Larval

Pupal156 

### Food Stores

A second multivariate analysis was conducted to determine potential effects on honey and pollen

cells. Univariate mixed model analyses were conducted for hive weight. The triple interaction of

Day*Dose*Response was again significant in the combined analysis of pollen and honey cells

( Table C

- 4 ).

Table C

- 4 . Analysis of Food Stores (Pollen and honey cells, and hive weight

In light of the significant three way interaction, the approach used for bee life stages was followed

where further multivariate analyses were conducted to determine the pattern of response at each

CCA. Results from the regression analysis for each CCA indicated th at there was a differential

response over time ( Table C

- 5 ).

• CCA3: As indicated in the results for bee life stages initial values for pollen and honey cells

and hive weight were essentially similar between all treatment levels. Some slight differences

between the controls and a few treatment levels for pollen cells were measured but these

appeared to be sporadic and not related to dose levels ( Table C

- 6; Figure C

- 6 and Figure C

12 ).

• CCA4: The number of pollen cells and hive weight were first affected at CCA4 with

decreases measured in relation to increasing imidaclopri d concentration ( Figure C

- 7 and

Figure C

- 13 ). Dose related effects on pollen cells were evident at the 50 ppb treatment with

progressive decreases in numbers in relation to increases in level of dose. The pattern for hive

weight mimicked that observed for pollen cells but significant effects were measured at the

100 ppb treatment and higher. Again, these decreases are rather significant because this

assessment was made only 3 weeks into the exposure period.

• CCA5: The pattern in response for pollen cells a nd hive weight was similar to that observed

at CCA4 ( Figure C

- 8 and Figure C

- 13 ). An additional effect was observed for hive weight

in that a significant effect was also observed at the 50 ppb level of dose. Together the effects

on honey cells and hive wei ght indicated two groupings of effects for the levels of dose: One

where the numbers were similar between 0, 12.5, and 25 ppb treatments and a second where

effects of imidacloprid dose were measured for 50, 100, and 200 ppb treatments There was one Overall Model Effect Num DF Den DF Pr > F

Response 1 116 <.0001

Day 4 294 <.0001

Day*Response 4 306 <.0001

Dose 5 118 0.0002

Dose*Response 5 116 0.8633

Day*Dose 20 294 <.0001

Day*Dose*Response 20 306 <.0001157 

### signifi cant effect indicated for honey cells but it was an isolated effect where higher numbers

were recorded for the 200 ppb treatment versus the rest of the treatments.

• CCA6: At CCA6 decreased numbers of pollen cells were only measured at the 200 ppb level

of imidacloprid dose ( Figure C

- 9 ). The response of honey cells , on the other hand, now

mimicked the initial response for pollen cells in that decreased numbers compared to the

control were measured at 50 ppb and higher dose levels. The two grouping pattern no ted at

CCA5 was now reflected in the responses for number of honey cells and hive weight ( Figure

C

- 9 and Figure C

- 14).

• CCA7: The response at CCA7 was very similar to that measured at CCA6 with the same noted

grouping of effects for honey cells and hive weight ( Figure C

- 10 and Figure C

- 15 ). Numbers

of pollen cells were not as plentiful as for honey cells throughout the study. By this assessment

all treatment means were low with values either at or below 0.05% frame coverage. The lower

coverage at this CCA is most likely the cause for diminishing effects of imidacloprid treatment

at this CCA and perhaps noted at the previous CCA6. Effects though were still measured at

the 200 ppb level when compared to the control.

• CCA8: Similar to the effects measured for life stages, the pattern noted at CCA7 was reflected

at the final assessment at CCA8 where curvature in response was measured for all life stages,

reflecting the extreme loss of hives at the 100 and 200 ppb levels of imidacloprid dose ( Table

C

- 5 ). Lower ed counts for honey and pollen cells were also measured for the 50 ppb treatment

when compared to control values, indicating lower hive vigor ( Figure C

- 11 and Figure C

16 ). These results provide evidence for the sustained influence of effects noted at CCA7 on the

over wintering heath of hives.

Table C

- 5

. Regression for linear and quadratic effect conducted within each CCA and for pollen

or nectar cells and hive weight

.

Food Storage

Variable Regression Effect Regression Results for Each CCA

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

Pr>t Pr>t Pr>t Pr>t Pr>t Pr>t

Honey Cells Dose Linear 0.5037 0.5842 0.0056 0.0001 <.0001 <.0001

Dose Quadratic 0.2801 0.7943 0.0821 0.9244 0.2545 0.0272

Dose Cubic 0.7263 0.8815 0.9365 0.6355 0.644 0.1049

Pollen Cells Dose Linear 0.2855 <0.0001 <0.0001 <0.0001 0.002 <.0001

Dose Quadratic 0.5849 0.0007 0.0006 0.9176 0.6843 0.0279

Dose Cubic 0.222 0.8135 0.7822 0.4604 0.8962 0.0215

Hive Weight Dose Linear 0.9952 0.0027 0.0011 0.0014 0.0007 <.0001

Dose Quadratic 0.9164 0.2306 0.0023 0.2494 0.1424 0.0541

Dose Cubic 0.0581 0.7024 0.5848 0.4948 0.4931 0.002158 

### Table C

- 6. Mean comparisons for the response between each level of dose for number of honey

and pollen cells and hive weight conducted at each CCA. For pollen and honey cells the

difference is the percent frame coverage of dose1 minus dose2 where a positive value in dicates a

higher value for the lower dosage and a negative value indicates a lower value for the lower

dosage. For hive cells the difference represents measurements made in pounds (lbs).

Effect Dose1 Dose2 PR>t Difference Effect Dose1 Dose2 PR>t Difference Effect Dose1 Dose2 PR>t Difference

Pollen 0 25 0.0582 0.02 Pollen 0 50 <.0001 0.04 Pollen 0 50 <0.0001 0.07

0 200 0.071 0.02 0 100 <.0001 0.06 0 100 <0.0001 0.09

0 12.5 0.0149 -4 0 200 <.0001 0.07 0 200 <0.0001 0.1

12.5 100 0.0081 5.2 12.5 50 0.0067 0.03 12.5 50 0.0001 0.07

12.5 200 0.0261 4.1 12.5 100 <.0001 0.05 12.5 100 <0.0001 0.09

12.5 200 <.0001 0.05 12.5 200 <0.0001 0.1

25 50 0.0055 0.03 25 50 0.0038 0.05

25 100 <.0001 0.05 25 100 <0.0001 0.07

25 200 <.0001 0.06 25 200 <0.0001 0.09

50 100 0.0568 0.02 50 200 0.033 0.04

50 200 0.0063 0.03 Honey 0 200 0.009 -0.08

0 100 0.0208 4.3 12.5 200 0.0177 -0.08

0 200 0.0278 4.1 25 200 0.0498 -0.07

12.5 50 0.0391 4.4 50 200 0.0036 -0.11

12.5 100 0.0056 6.1 100 200 0.0233 -0.08

12.5 200 0.0074 5.9 0 12.5 0.0305 3.6

25 100 0.0273 4.8 0 50 <0.0001 7.4

25 200 0.0348 4.5 0 100 <0.0001 10.7

0 200 0.0015 5.5

12.5 50 0.0464 3.8

12.5 100 0.0005 7.1

25 50 0.0024 6

25 100 <0.0001 9.3

25 200 0.0311 4.1

50 100 0.0843 3.3

100 200 0.0081 -5.2 Hive

Weight

Hive

Weight Hive

Weight Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA for Food Stores

CCA3 CCA4 CCA5159 

### Table C

- 6 Continued

. Mean food storage comparisons continu ed for CCA6 through CCA8:

Effect Dose1 Dose2 PR>t Difference Effect Dose1 Dose2 PR>t Difference Effect Dose1 Dose2 PR>t Difference

Pollen 0 12.5 0.0086 -0.03 Pollen 0 200 0.0087 0.02 Pollen 0 12.5 0.0677 -0.02

0 200 0.0022 0.03 12.5 50 0.0203 0.02 0 50 0.0601 0.02

12.5 50 0.0011 0.04 12.5 200 0.0009 0.04 0 100 0.0003 0.05

12.5 100 0.0015 0.04 25 50 0.0492 0.02 0 200 0.0004 0.05

12.5 200 <0.0001 0.06 25 200 0.0027 0.03 12.5 50 0.017 0.05

25 50 0.0545 0.02 100 200 0.0468 0.02 12.5 100 <0.0001 0.07

25 100 0.0683 0.04 Honey 0 50 0.0392 -0.003 12.5 200 <0.0001 0.07

25 200 0.0006 -0.001 0 100 0.0083 0.11 50 100 0.0975 0.02

50 200 0.0967 0.02 0 200 0.0002 0.14 Hoiney 0 50 0.08 0.05

100 200 0.078 0.02 12.5 50 0.0649 0.11 0 100 0.0008 0.1

Honey 0 50 0.0255 0.11 12.5 100 0.0188 0.14 0 200 0.003 0.09

0 200 0.0002 0.19 12.5 200 0.0011 0.2 12.5 50 0.018 0.08

12.5 200 0.0036 0.17 25 50 0.032 0.13 12.5 100 0.0002 0.13

25 50 0.0144 0.14 25 100 0.0082 0.16 12.5 200 0.0007 0.12

25 100 0.0623 0.11 25 200 0.0004 0.22 25 50 0.0394 0.07

25 200 0.0002 0.22 0 50 0.0471 9.8 25 100 0.0006 0.12

100 200 0.0436 0.11 0 100 0.0206 11.5 25 200 0.0019 0.11

0 50 0.0818 8.5 0 200 0.0077 13.3 0 25 0.0618 -11.7

0 100 0.0389 10.2 12.5 50 0.0228 13 0 100 0.0002 24

0 200 0.0151 12 12.5 100 0.0105 14.7 0 200 0.0013 20.7

12.5 50 0.0615 10.6 12.5 200 0.0042 16.5 12.5 100 <0.0001 30.4

12.5 100 0.0316 12.2 25 50 0.021 13.2 12.5 200 0.0003 27

12.5 200 0.0137 14.1 25 100 0.0096 14.9 25 50 0.0281 16

25 50 0.0292 12.4 25 200 0.0039 16.7 25 100 <0.0001 35.9

25 100 0.014 14 25 200 <0.0001 32.5

25 200 0.0057 15.9 50 100 0.0069 19.9

50 200 0.0241 16.5 Hive

Weight

Hive

Weight Hive

Weight Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA for Food Stores

CCA6 CCA7 CCA8160 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 6. Response of pollen and honey cells in relation to treatment group at CCA3.

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 7. At CCA4, onset of dosage related effects were measured for pupal cells and hive

weight (see Figure C

- 13). 0 0.04 0.08 0.12 0.16

0.2

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA3

Honey

Pollen

0 0.05

0.1 0.15

0.2 0.25

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA4

Honey

Pollen161 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 8. At CCA5, the effects on pollen cells and hive weight (See Figure C

- 14) measured

previously at CCA4 were sustained at the 50, 100, and 200 ppb treatment groups (p< 0.05) for

pollen cells with additional significant effects at the 50 ppb level (p<0.05) measured for hive

weight. An isolated significant effect for honey was noted at the 200 ppb treatment.

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 9. At CCA6, significant treatment related effects were also measured for the number of

honey cells with decreases at the 50, 100 and 200 ppb dose levels. Effects measured on hive weight

at CCA5 were sustained at CCA6 (See Figure C

- 15). 0 0.05

0.1 0.15

0.2 0.25

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA5

Honey

Pollen

0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA6

Honey

Pollen162 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 10. At CCA7 the effects on number of honey cells and hive weight (see Figure C

- 16)

appear to form two groups where numbers are similar between 0, 12.5 and 25 ppb treatments and

then imidacloprid dose

- related effects were measured for the 50, 100, and 200 ppb t reatments (See

Figure C

- x for hive weight).

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 11. At CCA8 effects measured at the 100 and 200 ppb treatments resulted in loss of

colonies after overw intering (see Figure C

- 17for hive weight graph). Lower vigor at 50 ppb

treatment was also indicated by the lower numbers of adults, pupal, and egg cells when

compared to the control hives. 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA7

Honey

Pollen

0 0.04 0.08 0.12 0.16

Control 12.5 25 50 100 200Proportion of Frame Coverage



### Treatment Group 

## CCA8

Honey

Pollen163 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large r ed dot p<0.05.

Figure C

- 12 . Response of hive weight in relation to treatment group at CCA3

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 13 . Response of hive weight in relation to treatment group at CCA4. 38 38.5

39 39.5

40 40.5

41 41.5

42 42.5

43 43.5

Control 12.5 25 50 100 200Hive Weight (lbs)

Treatment Group 

## CCA3

49 50 51 52 53 54 55 56 57 58 59

Control 12.5 25 50 100 200Hive Weight (lbs)

Treatment Group 

## CCA4164 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 14 . Response of hive weight in relation to treatment group at CCA5

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p< 0.05.

Figure C

- 15 . Response of hive weight in relation to treatment group at CCA6 36 38 40 42 44 46 48 50

Control 12.5 25 50 100 200Hive Weight (lbs)

Treatment Group 

## CCA5

0 10 20 30 40 50 60

Control 12.5 25 50 100 200Hive Weight (lbs)

Treatment Group 

## CCA6165 

### Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 16 . Response of hive weight in relation to treatment group at CCA7

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C

- 17 . Response of hive weight in relation to treatment group at C CA8 0 10 20 30 40 50 60

Control 12.5 25 50 100 200Hive Weight

Treatment Group 

## CCA7

0 10 20 30 40 50

Control 12.5 25 50 100 200Hive Weight

Treatment Group 

## CCA8