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Eurofins Agroscience Services Inc., unpublished report No: S13-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 

EPA 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 III
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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. Treatments..................................................................................................................................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................................................................................21

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 ..............................................................................26
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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. CDPR 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

Consideration of CCA8 ............................................................................................................................81

Control overwintering mortality .........................................................................................................81

Response Variables from Surviving Hives...........................................................................................81

Consideration of Study Strengths, Limitations and Interpretation ........................................................82

5. Conclusions.........................................................................................................................................85
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List of Tables

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 apiaries..................................................................................................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 chemical.........................................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 artificial feeding on 14 Aug, 2013 (CCA5).........................................................................31

Table 19: Imidacloprid concentrations (ppb) in bee bread and honey sampled after overwintering 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. Estimated 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......................................................78
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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 

across CCA3 – CCA8 in the control, 12.5, 25, and 50 μg/L groups..............................................................45

Figure 10. Proportion of eggs following exposure to varying concentrations 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. 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..............................................................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 of 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 .....................................................65
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Figure 28. Proportion of pollen stores following exposure of honey bees to varying concentrations of 

imidacloprid in the diet across CCA3 – CCA8..............................................................................................66

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. .......................................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 – CCA8 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 treatment 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.......................................................................................146
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Executive Summary 

A colony feeding study was conducted with honey bees to assess the 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 after 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 imidacloprid and to characterize preoverwintering 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 feeding 

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 well 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 concentration 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.
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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 statistical 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, respectively) 

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 same conclusions and are therefore harmonized in 

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

and Lowest Observed Adverse Effect 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 groups, 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 showed 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 analyses of an 

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

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

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

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 μg 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 level 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 reductions 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 in 
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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 treatment 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 CCA5 compared to controls for the three highest 

treatment groups, as well as at CCA6 and CCA7 at the highest 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. 

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 approaches 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 generalized 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

• 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 to imidacloprid occurred 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 
10

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. Additionally, 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 

treatments 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 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 

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 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 efficiency 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 overwintering 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 detections with pesticides from food sources other than the artificial feeding was 

detected during 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 appeared 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.0126043
11

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 amounts of other beetoxic 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. 

• Residues of imidacloprid in uncapped nectar and bee bread within 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 

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 solutions. However, individual 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 represent 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. 

• Exposure dilution during the study was evident. Pollen storage was observed consistently 

in the control hives and hives exposed to lower 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 effects on overwintering. 

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

persistent nature of effects.

2. Study Methods

2.1. Test crop

Not applied. 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. 

1Table 1. Details about the test substance

2.3. Test sites

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. Land use surveys 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 using 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: ambient
13

1Figure 1: Location of test apiary sites

2Table 2: GPS-coordinates of the test apiary sites

Apiary GPS-coordinates

Apiary 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

Apiary 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 honey 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. All queens were purchased 

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 bees per hive according to the study report. 

Eighty-four hives 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 CCA3 on 21 Jun 2013. Hives for Apiary sites G-L were moved on 

21 Jun 2013 and had their CCA3 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 eight 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

2Figure 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 loads. 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 treatments

were 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 stability under field test 

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

2.5. Treatments

There were:

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

μg/L. At each site, there 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 in3Table 3.
16

3Table 3. Treatment groups, feeding rates and feeding volume

Treatment Group Code Feeding Timing Concentration

a.i.

Feeding

Volume

1 : UTC UTC

(T1+T2)

Twice a week --- 1000 mL

2 : Lowest Rate T3 Twice a week 12.5 ppb 1000 mL

3 : Low rate T4 Twice a week 25 ppb 1000 mL

4 : Moderate rate T5 Twice a week 50 ppb 1000 mL

5: High rate T6 Twice a week 100 ppb 1000 mL

6: Highest rate T7 Twice a week 200 ppb 1000 mL

From Table 3, page 21 of the study report.

The assignment of each test hive at 12 apiaries is summarized in Table 4.

4Table 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. Preparation 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 solution 
17

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 concentrations were reported as “ppb” in the study report. However, the values are in fact 

in the unit of 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. Assuming the density of a 50% sugar solution is 1.2296 

g/ml2, 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 hive had an empty deep super on top, between the lid and the inner cover to allow dark space 

to 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 hives were artificially fed with 1 liter of 50% sugar solution, 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. Meteorological Data

Temperature, 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. Observations

Important activity and dates are summarized in Table 5.

 

2 Cell Biology Laboratory Manual, http://homepages.gac.edu/~cellab/chpts/chpt3/table3-2.html, accessed on Dec 12, 

2014
18

5Table 5. Chronological list of key dates and activities

Week Date Activity Week Date Activity

-7 12 May 2013 CCA1 6 07 Aug 2013 1st 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 CCA3 7 14/15 Aug 2013 CCA5

-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 CCA4 -- 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 CCA8

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 2nd sample shipment

5 02 Aug 2013 Pollen samples from pollen trap
19

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 were 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 overwintering CCA8 on 22-24 Mar 2014. Each CCA period in 2013 took two or more days to 

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

The time schedule of CCAs is summarized in Table 6 .

6Table 6. Schedule 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 before start of exposure; DAE: Days after start of 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 assessments, 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 was made by: 

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

of 860 cm2 per side, or a total area of 17,200 cm2 for all 10 frames. 

o There were 130 bees per 100 cm2.

 

o The total number of cells per frame is 3440. Estimated to be 4 cells/cm2.

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 CCA3, 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. Residue 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 traps in the monitoring hives 

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

Laboratories of USDA in Gastonia (non-GLP). The residue results were reported as ng per g of sample 

matrix (ppb), which is different 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.

7Table 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 minimum/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 monitoring 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 weeks 0 (CCA3), 1, 2, 3 (CCA4), 5, and 16 (17 Oct 2013).

2.8.2. Stored pollen and nectar in test hives

Stored bee bread and bee-collected nectar were collected within the study hives for imidacloprid residue 

analysis. Samples weighed at least 500 mg each. Bee bread and uncapped nectar were collected at weeks -

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 times.

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

2.8.3. Feeding solution and stability of test item

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).

8Table 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 available
22

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 Table 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.

9Table 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 36.8
23

10Table 10: Cultivated cropping area near each test apiary

Apiary

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

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 (nonGLP). 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. Consumption 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 3Figure 3) with a slightly lower consumption in 100 μg/L and 200 μg/L treatment.
24

3Figure 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 

were 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 beginning 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 not analyzed 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 samples originating from outside food sources (Table 

11). High levels of piperonyl butoxide (maximum at 591 ppb) were detected in the monitoring hive at 

Apiary 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 Apiary L at 12.1 ppb. The detection 

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

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

11Table 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 August 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 July 2013. It is unknown which control hives were fed with the

contaminated feeding solution. The data are tabulated below in Table 12.
26

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

August 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-imidacloprid 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 feeding solutions placed in the monitoring hives, sampled three times on 3 July 12 July, and 

2 August 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 sampled on 12 July 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.

13Table 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 imidacloprid 

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 that likely stems 

from the sampling procedure employed 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 collected 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 in 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 analysis for other 

pesticides was not conducted prior to exposure.

14Table 14. 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.

15Table 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 1st batch of sampling (CCA4, 18 July 2013) during the exposure phase, but only 

part of 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 July 2013). A dose-response 

correlation was observed between the imidacloprid concentrations in the feeding solution and measured 

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 concentrations in feeding solution,

respectively. It is possible that dilution of stored pollen and nectar from other food sources occurred 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 results showed a dose-response correlation between the average 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 group 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 results 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

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

artificial feeding on 18 July 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 Sappington (EPA) to Tina Singal (PMRA) on March 10, 

2015) ; “-“ 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 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 two hives at 12.5 ug/L (Apiary E and H) and two hives at 

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

The results 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 lower concentrations were detected in nine test 

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

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

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: 0.89-152.94) respectively. By average, the measured concentration in hive 

nectar was 64% (range 62.0-68.0%) of the concentration in feeding solution. The results 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 the 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.

17Table 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 

solution. However, the concentrations varied remarkably within some treatment groups 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 

concentrations 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 nectar was 61.4% (range 35.3-78.7%) of the concentration in feeding solution.

18Table 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 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

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) †† Not applicable 41.5 28.2 40.4 28.1

% Nectar ††† Not 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†† Not 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 honey 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. In bee bread, imidacloprid was not 

detected in treatments of control, 12.5, 25 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 provided 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. The 

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.

19Table 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 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 concentrations in ppb. 

3.7.2.4. Detection of imidacloprid in test hives at CCA2, CCA4, CCA5, and CCA8
33

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 overwintering (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 samples 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 before 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 nectar. 

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 potential 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 contamination 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 frequency 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 period. 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 

detected 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 any 
34

pollen samples trapped in the monitoring hives at CCA4 and earlier (Table 21). Considering the doseresponse relationship of the residues detected in the treatment hives, the imidacloprid contamination 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 variability in exposure since residues 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 indicate that a dose responserelationship is expected since higher treatment levels had higher mean measured residues in the hive pollen 

and nectar. 

20Table 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)

21Table 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

Preexposure

(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 15.1
35

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

Preexposure

(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 average 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). 

22Table 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) 64.0
36

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. Pathogens

Besides a standard treatment for Varroa mites, no 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 

(4Figure4). 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 levels.

The study showed no correlation between the treatments and the level of Varroa infestation.
37

4Figure 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.

5Figure 5. Mean number of Nosema spores per bee 
38

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 biological 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 in 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 statistical 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 satisfy 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 study author. As part of the collaborative review effort of the study by EPA, PMRA, and CDPR, a 

variety of statistical analyses were conducted for the evaluation of the data. The detailed methods of these 

analyses including 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 the 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 until shortly before CCA3, the data 

for the statistical analysis only included CCA3 through 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) at 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 compared 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 results 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. Taking 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 statistical 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 weight 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 approaches above 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, 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 

found 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 2013 
40

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 considered dead by 22 March 2014 

(end of test), as summarized by Table 23 and Figure 6. The author reported that based on an one-sided 

Cochran-Armitage exact trend test, mortality in the 100 ppb and 200 μg/L treatments are significantly 

different from the control mortality (p = 0.01 and p<0.01 respectively).

After overwintering, only one hive survived (Apiary L) at the treatment 100 μg/L, and two hives survived 

(Apiary 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 decreased from control (36%) to 25 μg/L (9%), and then 

increased 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 23a 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 loss.

23Table 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 - - - - - -

24Table 23b. 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 18
41

6Figure 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 measurements 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, the 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 impact 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 lack 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 CDPR 

approaches 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 (μg/l) 

Hive Mortality (%) After Overwintering 
42

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 presented 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 findings 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 discussion 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 previously, 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 stages or food store variables simultaneously 

within a single CCA across treatments. 

• While it is not depicted in the figures below, it is acknowledged (and addressed in a variety 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, the total number of adults 

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 reductions (p>0.05) at the 200 μg/L treatment group at CCA5 while 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 adults in the 50 μg/L treatment was also reduced with 

marginal statistical significance at CCA5 (0.05< p<0.1) but at p<0.05 at CCA6 and CCA8 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 CCA7 while the EPA and CDPR
43

analyses did not, but like the 100 and 200 μg/L treatment groups, the persistent nature of significant 

reductions to adults at the 50 μg/L group is evident. 

25Table 24. Estimated percent reduction from control for number of adults

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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<0.051

Percent differences from control are based on the raw counts of adults, not proportions of the adults as Figure 7 below shows. 2At 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. 

7Figure 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 the 
44

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). 

8Figure 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. 

In an examination of the trends of adults in the control group, by 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 therefore 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 rates 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 test sites. 

Also notably, as distinguished from the control and 12.5 and 25 μg/L groups, while 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 as 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 CCA8

Proportion of Adults

Colony Condition Assessment

Adults 

Control

12.5

25

50
45

9Figure 9. 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. 

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

There 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

0.5

Proportion of Adults

Colony Condition Assessment

Control

Adults

0

0.1

0.2

0.3

0.4

0.5

Proportion of Adults

Colony Condition Assessment

12.5 μg/L

Adults

0

0.1

0.2

0.3

0.4

0.5

Proportion of Adults

Colony Condition Assessment

25 μg/L

Adults

0

0.1

0.2

0.3

0.4

0.5

Proportion of Adults

Colony Condition Assessment

50 μg/L

Adults
46

analyses 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 significance 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 reduction in the number of 

eggs at CCA8 (p<0.1). A similar finding was made at CCA4 in the PMRA analysis only. However, 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 observed 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. 

26Table 25. Estimated percent reduction from control for number of eggs

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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

1Percent differences from control are based on the raw counts of eggs, not proportions of the eggs as Figure 10 below shows. 2At 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 analysis but excluded from the EPA analysis. 
47

10Figure 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

11Figure 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 the control and three lowest treatment levels that underwent 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 CCA8

Proportion of Eggs

Colony Condition Assessment

Eggs 

Control

12.5

25

50
49

12Figure 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

0.1

Proportion of Eggs

Colony Condition Assessment

Control

Eggs

0

0.02

0.04

0.06

0.08

0.1

Proportion of Eggs

Colony Condition Assessment

12.5 μg/L

Eggs

0

0.02

0.04

0.06

0.08

0.1

Proportion of Eggs

Colony Condition Assessment

25 μg/L

Eggs

0

0.02

0.04

0.06

0.08

0.1

Proportion of Eggs

Colony Condition Assessment

50 μg/L

Eggs
50

3.11.3. Larvae (Open/Uncapped brood)

There were consistently and significantly lower numbers 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 control 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, at 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). 

27Table 26. Estimated percent reduction from control for number of larvae (open/uncapped brood)

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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

1Percent 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.

2At 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 decline while the other treatment groups generally 

trend with control.
51

13Figure 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

14Figure 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, the 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 time 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 CCA8

Proportion of Larvae

Colony Condition Assessment

Larvae (Open) Cells 

Control

12.5

25

50
53

15Figure 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 the 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 

approximately 6% of frame coverage. The 25 ppb group also look to undergo a more marked decline 

beginning at CCA4 as compared to the 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

0.12

Proportion of Larvae

Colony Condition Assessment

Control

Open

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Larvae

Colony Condition Assessment

12.5 μg/L

Open

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Larvae

Colony Condition Assessment

25 μg/L

Open

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Larvae

Colony Condition Assessment

50 μg/L

Open
54

When weighing 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 

effect). 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. Pupae (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 any 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, respectively. However, responses at the lower three doses were all similarly 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 treatment 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%.

28Table 27. Estimated percent reduction from control for number of pupae

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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

1Percent differences from control are based on the raw counts of pupae (capped) brood, not proportions of the pupae (capped) brood 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

16Figure 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, same 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 while 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 selections 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

17Figure 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 imidacloprid 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 12.5 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 proportions 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 CCA8

Proportion of Pupae

Colony Condition Assessment

Capped (Pupal) Cells

Control

12.5

25

50
57

18Figure 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 pupal 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 group 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 proportions 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

0.3

Proportion of Pupae

Colony Condition Assessment

Control

Capped

0

0.05

0.1

0.15

0.2

0.25

0.3

Proportion of Pupae

Colony Condition Assessment

12.5 μg/L 

Capped

0

0.05

0.1

0.15

0.2

0.25

0.3

Proportion of Pupae

Colony Condition Assessment

25 μg/L 

Capped

0

0.05

0.1

0.15

0.2

0.25

0.3

Proportion of Pupae

Colony Condition Assessment

50 μg/L 

Capped
58

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 similar 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, 

respectively). 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 after 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 coverage 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 from 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.

29Table 28. Estimated percent reduction from control for total number of individuals

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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

1Percent differences from control are based on the raw counts of total individuals, not proportions of the total individuals as Figure 19 below 

shows.

2At 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
59

19Figure 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 significantly 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 significance 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 total 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 control. 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 progression 
60

of a continuous impact throughout the study for certain response variables that is still present to a higher 

degree after overwintering 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 groups 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.

20Figure 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 Eggs

Difference from control (%)

CCA4 CCA5 CCA6 CCA7 CCA8
61

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

22Figure 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 Eggs

Difference 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 Eggs

Difference from the control (%)

CCA4 CCA5 CCA6 CCA7 CCA8
62

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 previously presented for the life stage 

response variables in that the trends for each variable 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 increases 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. 

 

23Figure 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA4

Adult

Egg

Larval

Pupal
63

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

At CCA6 (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 evident. 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). This 

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. 

25Figure 25. Trends of life stages across 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA6

Adult

Egg

Larval

Pupal
64

As mentioned previously, CCA7 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 200 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. 

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

After overwintering at CCA8 (Figure 27a 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 

Comments 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 below 

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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA7

Adult

Egg

Larval

Pupal
65

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

Colony Condition Assessments – Food Store Response Variables

3.11.6. Pollen

Pollen stores 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 group 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 stores was not 

determined 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.

30Table 29. Estimated percent reduction from control for pollen stores

Test 

concentration 

(μg/L)

Estimated reduction from control (%)1

CCA4 CCA5 CCA6 CCA7 CCA82

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 stores 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA8

Adult

Egg

Larval

Pupal
66

**p<0.05

1Percent differences from control are based on the raw counts of pollen stores, not proportions of the pollen stores as Figure 28 below shows. 2At 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. 

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 stores in the 200 μg/L group 

is considered to be uncertain as there were only two hives surviving overwinter and a large confidence 

interval. 

28Figure 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 exposure and 

continue to be repressed throughout the study including after overwintering.
67

29Figure 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. Pollen 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 reflects 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 the 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 average 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 CCA8

Proportion of Polen Stores

Colony Condtion Assessment

Pollen 

Control

12.5

25

50
68

30Figure 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 consistently 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

0.12

Proportion of pollen stores

Colony Condition Assessment

Control

Pollen

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Pollen Stores

Colony Condition Assessment

12.5 μg/L

Pollen

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Pollen Stores

Colony Condition Assessment

25 μg/L

Pollen

0

0.02

0.04

0.06

0.08

0.1

0.12

Proportion of Pollen Stores

Colony Condition Assessment

50 μg/L

Pollen
69

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. No reduction of the honey stores 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. 

31Table 30. 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 stores in comparison to control.

*0.05<p<0.1

**p<0.05

1Percent 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.

2At 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

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

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

imidacloprid in the diet across CCA3 – CCA8.

32Figure 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

Proportion of Nectar (honey) stores

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

Colony Condition Assessment

Nectar (Honey) Stores

Control

12.5

25

50
71

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 proportion 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%. 

33Figure 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

0.35

Proportion of nectar/honey stores

Colony Condition Assessment

Control

Honey

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Proportion 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

0.35

Proportion of honey/nectar stores

Colony Condition Assessment

25 μg/L

Honey

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Proportion of honey/nectar stores

Colony Condition Assesment

50 μg/L

Honey
72

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 

overwintering 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 control). 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 

overwintering 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 amount 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 treatment 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 weighing 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 mortality). 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

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

35Figure 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 CCA8

Difference 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 CCA8

Difference from control (%)

Honey Pollen
74

36Figure 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 variable 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 of 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 CCA8

Difference from control (%)

Honey Pollen
75

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 generally at the level of control.

37Figure 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.

38Figure 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

Proportion of Frame Coverage

Control 12.5 25 50 100 200

Treatment Group

CCA4

Honey

Pollen

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control 12.5 25 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA5

Honey

Pollen
76

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.

 

39Figure 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. 

 

40Figure 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA7

Honey

Pollen
77

Finally, at CCA8 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 surviving, 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.

 

41Figure 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 CCA8 (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 CCA4 and CCA6 and at the 0.05 alpha level for 

CCAs 5, 7, and 8. The EPA analysis determined 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 significant 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA8

Honey

Pollen
78

32Table 31. Estimated percent reduction from control for hive weights

Test 

concentration 

(μg/L)

Estimated reduction from control (%) 

CCA4 CCA5 CCA6 CCA7 CCA81

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

2At 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

42Figure 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, respectively.
79

4. Reviewer 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 conditions, 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 function 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 weights 

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

weight is measured. Hive weights may be artificially 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: 

• CCA3 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 effects may be masked by the natural decline 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 level of colony loss after overwintering in controls (36%), though not desirable, is consistent with that 

historically experienced by beekeepers on average across the United States3

; 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. Commercial hives could 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 different sized hives and different preparation for overwintering. The test hives, while 

closely monitored, may also experience invasive disruptions during the colony condition assessments, have 

different/less supplemental feeding or mite treatments than commercial 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 effects 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. Although 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 from CCA3 to CCA5. After the end of the exposure, 

all these biological parameters also increased or remained at a similar level at CCA5 and CCA6, and then 

all decreased sharply from CCA6 to CCA7. 

The similarity in the dynamics of all parameters for the individual 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 test 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 pollen 

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 control 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 Variables 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 study 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 43).

43Figure 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

44Figure 44. Fate of control hives as compared to the honey stores across 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 CCA8 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 from the dead hives were available for inclusion, and because 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 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 generalized 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
83

• 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 solution 

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 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 (2014) showed that higher residues throughout the hive resulted from feeding pollen 

treatments compared to feeding sucrose solution treatments. It is also noted that 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 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 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 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 efficiency 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 overwintering 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 contamination with pesticides from food sources other than the artificial feeding was detected 

during 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 

levels 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 

amounts 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. 

• Residues of imidacloprid in uncapped nectar and bee bread within 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 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 solutions. 

However, individual hive residue values varied, and there was some overlap in measured values, 

particularly among the three lowest doses. 

• Exposure dilution during the study was evident. Pollen storage was observed consistently in the 

control hives and hives exposed to lower 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.
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 test chemical through alternative sources of forage, 

detection 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 levels) 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. While 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 overwintering 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 groups. 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 analysis determined 

significant effects at all treatment levels at CCA6 (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), 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. 

The figures below present the trends of life stages across all CCAs 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 CCA8

Proportion 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 CCA8

Proportion 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 CCA8

Proportion 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 CCA8

Proportion 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, respectively
88

0

0.1

0.2

0.3

0.4

0.5

CCA3 CCA4 CCA5 CCA6 CCA7 CCA8

Proportion 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 CCA8

Proportion 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 

pupal cells occurs in the control from CCA3 to CCA6 whereas the 50 μg/L 

group undergoes an initial decline from CCA4 to CCA5
89

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 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 reductions in pollen stores were also 

confirmed at CCAs 4 and 5 at the 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 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 treatment 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 CCA5 compared to controls 

for the three highest treatment groups, as well as at CCA6 and CCA7 at the highest 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 approaches used in PMRA, EPA, and 

CDPR, the NOAEC and LOAEC for this study is determined to be 25 and 50 μg/L, respectively. 
91

1.Appendix 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 could 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 their 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. Definition 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 roughly 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 Department 

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 hive), 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/cm2

. Therefore, the conversion from the data stored in the spreadsheet 
92

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 dramatically. 

• 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 bees (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 

cells 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 background, 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 capped, 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. Since 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 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 study (especially the 100 and 200 ppb treatments in CCA8), creating 
93

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). 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 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 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 the 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 background, 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 response 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 farther 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 component 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 consistent 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 did 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 explained 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 weighted 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 stores (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 study (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 addressing 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 later 

determined that there were some data entry errors. The errors were corrected 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 

amongst 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 utilize 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 was 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 variance 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 each fitted model for all eight response variables is reported in 

Table A-1; smaller 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) were 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 between 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 cells, 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 response 

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 assumptions 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 correlation seen in the pairwise scatterplots (CCAx vs. CCAy 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 and 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 these 
97

interpretations 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 attained, 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 all 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 negative 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(apiary) 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 results 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 = significantly 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 the 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 < CCA3
99

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 0.030043
101

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 0.1475
102

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 1
103

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 0.116261
104

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 0.007071
105

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 0.001768
106

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 0
107

2Appendix 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 )5using the raw data submitted by the study author. 

Statistical 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_values 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 Studies. 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 detecting 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 procedure 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 William’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 

maximum 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 the 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 between 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 maximises 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 that 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. 

The 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 considered to reduce the confidence of using CCA8 data as the primary time 

point. Additionally, no 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 multiplicity 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 review. 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 outcomes 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 the 

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 with 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 effects, 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 ruled 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 concern 

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, the 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 reduced the confidence of using CCA8 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 CCA8 considered biased and problematic). This analysis comprised of linear 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 single 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 group 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. William’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 analyses 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 of the confidence intervals (pdf file Bees8.pdf). 

The code snippet to implement these analyses in R was:

glm(outcome~Apiary + baseline + 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 analysis 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 used 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 produced 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 mean control condition assessments over time.

Bees5.pdf – Plots of model estimated differences and confidence intervals (title revised).

Bees6.pdf – Plots of model estimated difference versus observed mean for total life.

Bees7.pdf – Plots of individual exposed hive versus control condition assessments for 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 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.74
112

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.734
113

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.729
114

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.796
115

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.74
116

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.729
117

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.729
118

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.796
119

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.74
120

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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 -1.734
121

Parameter

Time 

(CCA)

1

Test 

conc.

(μg/l)2

Observed 

mean 

difference 

from 

control3

Standard 

error 

observed 

mean

n

Model 

estimate 

mean 

difference 

from 

control4

Standard 

error of 

estimated 

mean

P value for 

comparison 

with the 

control

90% 

confidence 

upper limit

90% 

confidence 

lower 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

Notes:

1, Observation dates for hive weight measurement were slight different from the dates when colony assessments were conducted.

2, The test 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 for 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 column must be multiplied by 100 to 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 control 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% CIs 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 B
124

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 number 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 step-down approach was not applied to the 200 or 100 treatment levels where very few 

hives survived.
125

Figure B-3: Estimates and 90% CIs 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 B
126

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 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 adults 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 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 B
128

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 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 eggs 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-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 treatment 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 B
130

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 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 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 significance 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 B
132

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 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 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 model 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 survived.
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 B
134

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 number 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 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 pollen 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-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, the 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 store 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 (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 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 B
138

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 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 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 CCA8

Difference from control (%)

Total individuals

12.5 25 50 100 200

-50

0

50

100

150

200

CCA4 CCA5 CCA6 CCA7 CCA8

Difference from control (%)

Adults

12.5 25 50 100 200
139

Figure B-19. Eggs (percent difference from control)

Figure B-20. Larvae (percent difference from control)

Figure B-21. Pupae (percent difference from control)

-500

50

100

150

200

CCA4 CCA5 CCA6 CCA7 CCA8

Difference from control (%)

Eggs

12.5 25 50 100 200

-100

-500

50

100

150

200

CCA4 CCA5 CCA6 CCA7 CCA8

Difference from control (%)

Larvae

12.5 25 50 100 200

-500

50

100

150

200

CCA4 CCA5 CCA6 CCA7 CCA8

Difference from control (%)

Pupae

12.5 25 50 100 200
14

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

-500

50

100

150

200

CCA4 CCA5 CCA6 CCA7 CCA8

Difference from control (%)

Honey

12.5 25 50 100 200

-100

-500

50

100

150

Difference from control (%)

CCA4 CCA5 CCA6 CCA7 CCA8

Pollen

12.5 25 50 100 200

-40

-200

20

40

60

80

Difference from control (%)

CCA4 CCA5 CCA6 CCA7 CCA8

Weight

12.5 25 50 100 200
141

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 CCA8

Difference 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 CCA8

Difference from the control (%)

TotalLife Adult Pupae Larvae Eggs
142

Figure B-27. Summary of living organism parameters at 50 μg/L

Figure 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 CCA8

Difference 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 CCA8

Difference from the control (%)

TotalLife Adult Pupae Larvae Eggs
143

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 CCA8
144

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 CCA8
145

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 individuals in hive at CCA6 (8.7%, P<0.1)

• Decreased number of pupae 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, hive condition similar to control 

• The potential colony effects at CCA6 were considered short-term, colony able to compensate 

25 • Decreased total number of individuals in hive at CCA6 (17.4%, P<0.05)

• Decreased number of pupae 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 colonies did not survive overwintering (better survival than control)

• At CCA8 among surviving hives, hive condition 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 effect on eggs or larvae observed

• Decreased pollen store at CCA4, CCA5, CCA7

• Decreased honey store at CCA5 – CCA7

• Decreased 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 individuals in hive at CCA4-CCA7

• Decreased number of adults at CCA5 - CCA7

• Decreased number of eggs at CCA4 – CCA7

• Decreased number of larvae at CCA4 – CCA7

• Decreased number of pupae at CCA4 – CCA7

• Decreased pollen store at CCA4 and CCA5

• Decreased honey store at CCA6-CCA7

• Decreased hive weight at CCA4 – CCA7

• 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 relationship 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 after 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 extreme 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 PROC MIXED is conducted by combining the data 

for 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 variable 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 the univariate analyses: Raw values 

for pupal, larval, and egg cells were dived by 68800 with 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. Random 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 taken. 

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 firstorder autoregressive model applied to day. Various covariance models were tested 
148

to determine the model 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 adjusts 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 the 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 differential responses 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 heterogeneity 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 pollen 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 (Table C-1). Notably, the triple interactive effect for Day*Dose*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 C2). Quadratic and cubic dose effects were included to indicate 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 (NOAEC) and lowest observed effect concentration (LOAEC) 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 results between CCAs provided evidence for 

the differential responses 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 dots 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 pupal 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 imidacloprid 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 treatment 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 starting 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 <.0001
150

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 were 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 C6). 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 effects 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 0.1589
151

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 CCA5
152

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 Dose1Dose2PR>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 Stage
153

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C-1. Response 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 for 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA4

Adult

Egg

Larval

Pupal
154

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Figure C-3. Effects 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 and 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA6

Adult

Egg

Larval

Pupal
155

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 measured at 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA8

Adult

Egg

Larval

Pupal
156

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 that 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 C12). 

• CCA4: The number of pollen cells and hive weight were first affected at CCA4 with 

decreases measured in relation to increasing imidacloprid 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 and 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 weight 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 <.0001
157

significant 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 noted 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). Lowered 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 C16). 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 0.002
158

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 indicates 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 CCA5
159

Table C-6 Continued. Mean food storage comparisons continued 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 CCA8
160

 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA3

Honey

Pollen

0

0.05

0.1

0.15

0.2

0.25

Control 12.5 25 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA4

Honey

Pollen
161

 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 50 100 200

Proportion 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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA6

Honey

Pollen
162

 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 treatments (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 overwintering (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 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA7

Honey

Pollen

0

0.04

0.08

0.12

0.16

Control 12.5 25 50 100 200

Proportion of Frame Coverage

Treatment Group

CCA8

Honey

Pollen
163

 Comparisons to control: Large gray dot 0.05<p<0.1; Large red 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 50 100 200

Hive Weight (lbs)

Treatment Group

CCA3

49

50

51

52

53

54

55

56

57

58

59

Control 12.5 25 50 100 200

Hive Weight (lbs)

Treatment Group

CCA4
164

 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 50 100 200

Hive Weight (lbs)

Treatment Group

CCA5

0

10

20

30

40

50

60

Control 12.5 25 50 100 200

Hive Weight (lbs)

Treatment Group

CCA6
165

 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 CCA8

0

10

20

30

40

50

60

Control 12.5 25 50 100 200

Hive Weight

Treatment Group

CCA7

0

10

20

30

40

50

Control 12.5 25 50 100 200

Hive Weight

Treatment Group

CCA8