Methods for Evaluating Toxicity of 

Pesticides to Bees

U.S. Environmental Protection Agency 1

Joseph DeCant, M.S.

Environmental Fate and Effects Division

Office of Pesticide Programs
Overview of the Presentation

• Introduction to the tiered approach to toxicity testing

• Tier I: laboratory

• Tier II: semi-field whole hive

• Tier III: full field whole hive

Charge questions 9, 10, 11, 12

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• Charge questions 9, 10, 11, 12
Assessment and Measurement Endpoints

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Protection 

Goal

Assessment 

Endpoints

Measurement 

Endpoints (Population 

Level and Higher)

Measurement Endpoints

(Individual Level)

1) Provision of 

Population 

size and 

Colony strength and 

Individual worker survival

Queen fecundity

Table 1. Proposed protection goals and examples of associated assessment and 

measurement (population and individual) endpoints for bees. 

1) Provision of 

Pollination 

Services

size and 

stability of 

managed 

bees

Colony strength and 

survival

Queen fecundity

Brood size

Worker bee longevity

2) Production 

of Hive 

Products

Quantity and 

quality of hive 

products

Quantity and quality of 

hive products; residue 

levels on honey/wax

Individual worker survival

Queen fecundity

Brood success

3) Contribution 

to Pollinator 

Biodiversity

Species 

richness and 

abundance

Colony strength and 

survival

Species richness and 

abundance

Individual worker survival

Brood success
Tiered Approach to Toxicity Testing

Ecological effects 

assessment describes 

the effects elicited by 

a pesticide

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Screening Level Tier I: 

•Individual based

•Laboratory assay

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a pesticide

Tiered approach to

allow for increasing 

levels of realism and 

refinement for both 

toxicity and exposure

Tier II: 

•Whole hive

•Semi-Field (tunnel/feeding)

Tier III: 

•Whole hive

•Full Field

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Tier I Toxicity

• Current U.S. EPA 40 CFR § 158 requirement includes a 48hr 

acute contact assay with young adults

• Expansion to include acute oral studies with both adults and 

larvae

• Approach similar to U.S. EPA Interim Guidance on Honey Bee 

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• Approach similar to U.S. EPA Interim Guidance on Honey Bee 

Data Requirements

• Individual based

• Focus on mortality

• Screening level evaluation of dietary and contact exposure routes
Tier 1: Adult Acute Toxicity Assays- Contact

EPA 850.3020/OECD 214: Laboratory acute contact 

toxicity with young adult bees

• 48 hr acute contact toxicity – LD50

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• LD50 > 11 μg a.i./bee = practically non-toxic

• 2 < LD50 < 10.99 μg a.i./bee = moderately toxic

• LD50 < 2 μg a.i./bee = highly toxic

EPA 850.3030: Toxicity of residues on foliage

• RT25: Measure of residual toxicity
Tier 1: Adult Acute Toxicity Assays- Dietary

•OECD 213: Laboratory acute oral toxicity with 

young adults

• 48 hr acute oral toxicity – LD

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• 48 hr acute oral toxicity – LD50
Tier 1: Larval Acute Toxicity Assay

• Brood Study – considered a semi-field study

• In Vitro Assay

• 1

st instar bee larvae are transferred into 48-well plates

• Larvae given synthetic food source containing known 

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• Larvae given synthetic food source containing known 

quantities of the test chemical over the 48hr study period

• Test provides LD50 and with sufficient replication it also 

provides a NOAEC

• Uncertainties associated with this design

• Hive versus laboratory conditions

• Artificial food source
Tier 1: Approaches to Evaluate Chronic Toxicity

Proposed approach: 

• To date there are no formal guidelines for conducting chronic 

tests with adults or larvae

• Adult chronic oral toxicity assay (10 day)

• Larval chronic toxicity assay (7 day)

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• Larval chronic toxicity assay (7 day)

Possible approach: Chronic toxicity estimated from acute 

laboratory studies

• NOAEC from acute survival studies

• Regression versus hypothesis based testing
Tier 1: Other Toxicity Data Sources

• Scientific Open Literature

• More sensitive endpoints than registrant submitted studies

• May be used to derive risk quotients (quantitative)

• Non-target Arthropods

• Registrant-submitted studies, e.g., green lacewing, parasitoid wasp

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• Registrant-submitted studies, e.g., green lacewing, parasitoid wasp

• Qualitative use in risk characterization

• Incidents

• Reports of honey bee mortality events following pesticide exposure

• Potentially useful in evaluating the risk conclusions
Tier 1: Sublethal effects

• Types of sublethal effects include impairments to:

• Movement and communication

• Foraging and homing behavior

• Recruitment of fellow foragers

• Health of queen

• Detoxification pathways

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• Detoxification pathways

• Hive and individual immune response

• Colony maintenance activity

• Proboscis extension reflex

• EFSA: sublethal endpoints inadequately covered by existing tests

• Lack of direct connection between measurement endpoints and 

assessment endpoints

• Qualitative use in the risk assessment
Tier 1: Summary of Measurement Endpoints

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Measurement Endpoints (RQ 

Calculation):

1. Individual Worker Survival:

•Acute LD50 (Contact and Dietary)

•Adult 10-day Dietary Chronic NOAEL or EDx

2. Brood Success:

•Larval Acute and Chronic NOAEL or LD

Qualitative (Risk 

Description): •Larval Acute and Chronic NOAEL or LD50

Assessment Endpoints:

•Population Size and Stability of 

Managed Bees

•Quantity of Hive Products

•Species Richness and Abundance

Description):

•RT25

•Sublethal effects

•Other data sources

Risk Management
Transition From Tier I to Tier II

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•Mortality-based

• Laboratory

•Individual

•No formal guidelines to assess 

chronic toxicity

Limitations of Tier I Testing

•Increasing level of realism

•Evaluate potential effects 

identified at Tier I for impact to 

the entire colony

•Integration of sub-lethal effects, 

Strengths of Tier II Testing

Evaluate the need for further refinement

• Data needs identified by risk manager

• Decision based on toxicity and exposure data

• Other lines of evidence

• Uncertainties (e.g., mode of action, chemistry)

• Mitigation

• Off-label specific uses

chronic toxicity •Integration of sub-lethal effects, 

chronic exposure, and 

degradate toxicity
Tier II: Semi-Field Study

• Purpose

• Refinement of Tier I assessment 

• Shift of focus from the individual to the colony

• Address specific issues raised at the initial tiers of the risk 

assessment

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assessment
Tiers II and III: Field/Semi-field Study

• 40 CFR part 158 requirement

• Required when the following conditions are met:

• Evidence of extended residual toxicity (RT25 > 8hrs) to honey bees as 

seen in the 850.3030 test for the toxicity of residues on foliage

• Data from other sources for honey bees indicate that there is the 

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• Data from other sources for honey bees indicate that there is the 

potential for effects other than mortality on the colony

• Data from studies with terrestrial arthropods other than bees indicate 

potential chronic, reproductive, or behavioral effects
Tier II: Design Options for the Semi-field Study

• Tunnel Design: pesticide 

exposure facilitated by 

test crop

• Adult/Brood

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• Feeding Design: pesticide 

exposure facilitated by 

pesticide spiked diet

• Brood

• Adult/Brood
Tier II: Semi-field Tunnel Design 

• Design: U.S. EPA 850.3040/EPPO 170 (4)/OECD 75

• Crop grown outdoors in an enclosed system (i.e., tunnel) with surrogate 

species (e.g., Phacelia tanacetifolia ) 

• Maximum application rate, minimum application interval

• Small nucleus hive of approximately 5,000 bees

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• Hives are kept in tunnel for 7 – 10 days

• Hives removed from tunnel after 10 days and allowed to freely forage

• Monitoring of hive conditions proceed for up to 28 days

• Reference toxicant frequently used (e.g., fenoxycarb or dimethoate)

• Measure residues in flower or bee-collected pollen and nectar 

• Applicable scenarios include seed treatment, soil application, 

trunk injection, and foliar spray
Tier II: Semi-field Feeding Design - Brood

• Typically used to assess brood toxicity

• Based on Oomen et al. 1992

• Hives allowed to freely forage

• 1 liter of spiked/control solution placed in feeder near hive

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• Weekly measurements on brood development
Tier II: Semi-field Feeding Design - Brood 

and/or Adults

• Adult open field feeding study

• Spiked pollen patties/sucrose solution fed to hives ranging in 

concentration to establish NOAEC/LOAEC

• Location in area of low pesticide use

Bees allowed to freely forage (use of pollen traps limit 

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• Bees allowed to freely forage (use of pollen traps limit 

incoming pollen)

• Use of full size hives (minimum 10k bees)

• Duration of study can include overwintering

• Measurement of endpoints at specified time periods
Tier II: Semi-field Measurement Endpoints: 

Adults and/or Brood 

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Ref 1. Adult Bee Mortality inement of Tier I: 

colony level

1. Brood condition assessment

Additional endpoints 

beyond Tier I

1. Brood condition assessment

•Egg abundance/larval/pupal abundance, 

brood pattern, emergence success

2. Hive strength

•Percent of comb area covered with bees

3. Queen health

4. Hive Resources

•Hive weight

•Percent of frame covered by resources

•Quality of hive products
Tier II: Semi-field Uncertainties

Both Designs

• Adverse weather conditions

• Variability in measurement endpoints

Tunnel Design

• Quantity and quality of food may be compromised

• Confinement stress on foraging bees

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• Confinement stress on foraging bees

• Short period of exposure

Feeding Design

• Foraging not limited to the provided food

• Artificial aspect of feeding in relation to natural foraging

• May not integrate impact to foraging behavior in the field

• Uncertain performance of colonies to long-term supplemental feeding

• Interventions required for overwintering
Reconciling Statistical and Biological 

Significance

• Statistical Analysis

• Comparison of treatment means

• Central tendency (mean, median), dispersion (standard error, standard 

deviation)

• Time trends not routinely reported but can be qualitatively evaluated

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• Biological significance as it relates to assessment endpoints

• High variability in measurement endpoints between hives and over time

• Uncertainty regarding effects that are not statistically significant 

• Compensatory mechanisms of the hive

• Effects that are transient
Tier II: Use of Semi-field studies

• Address concerns raised at the first Tier

• Provide clearer lines of evidence for linking multiple routes of exposure 

to adverse ecological effects

• Assess both exposure and effects that are less conservative and more 

realistic than laboratory methods

Determine extent to which effects on individuals translates into effects at 

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• Determine extent to which effects on individuals translates into effects at 

the colony level

• Integrate toxicity of degradates, sub-lethal and chronic effects into the 

performance of the hives as they relate to assessment endpoints

• The modified feeding design NOAEC can be compared with 

residue levels from targeted monitoring data

• Qualitative use to inform risk management regarding potential 

mitigation or further refinement
Transition From Tier II to Tier III

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Tier II 

Limitations

Tunnel:

•Stress of tunnel

•Short duration

•Compromised 

quantity and quality 

of food

Feeding: Brood

•Artificial food 

source: uncertain 

exposure

•May not integrate 

effects on foraging 

behavior

Feeding: Adult/Brood

•Artificial food source: 

uncertain exposure

•May not integrate 

effects on foraging 

behavior

Tier III 

Advantages

•Eliminate the 

stress of the tunnel 

and limited food

•Allows for larger 

size hives

•More flexibility in 

duration of the 

study

behavior

•Natural feeding 

mechanism

•Integration of 

foraging activity

•Natural feeding 

mechanism

•Integration of foraging 

activity

•Refine the effects 

assessment when 

residue levels are 

above the NOAEC
Basis for Transition from Tier II to Tier III

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Evaluate the need for further refinement

• Data needs identified by risk manager

• Decision based on toxicity and exposure data

• Other lines of evidence

• Uncertainties ( Uncertainties (e.g., mode of action, chemis mode of action, chemistry, use pa , use pattern)

• Mitigation
Tier III: Purpose

• Final level of refinement to Tiers I and II

• Evaluate the consequences of exposure to real-world 

application of pesticide

• Test for the potential of an effect, not the mechanistic cause of 

an effect

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an effect

• Focused risk hypothesis
Tier III: Guidance Pertaining to the Field Study

• EPA 850.3040

• Flexibility to design study based upon needs of the risk assessment

• EPA 850.2500

• Screening versus definitive level study

• Geographic area

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• Geographic area

• Size and distance between study sites

• Sample size depends on the required level of confidence

• Test conditions should resemble those encountered under actual use 

conditions

• EPPO 1/170 (4)

• Provides guidance on the design of a field study
Tier III: Design Elements of a Field Study

• Flexible design

• Actual use conditions

• Honey bees are free foraging and unrestricted by a tunnel

• Crop grown according to good agricultural practices

Hives of at least 10k bees

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• Hives of at least 10k bees

• Duration of exposure

• Length of field study monitoring

• Allows short term through overwintering
Tier III: Design Elements of a Field Study, cont.

• Colonies equally distributed among plots 

• Hives are queen-right, use sister queens, and be of similar 

strength at test initiation

• Equipment free of other chemicals

• Hives with low occurrence of disease and parasites

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• Hives with low occurrence of disease and parasites

• No positive control at the full field level
Tier III: Measurement Endpoints 

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1. Adult Bee Mortality

1. Brood condition assessment

Refinement of Tier I: 

colony level

1. Brood condition assessment

•Egg abundance/larval/pupal abundance, 

brood pattern, emergence success

2. Hive strength

•Percent of comb area covered with bees

3. Queen health

4. Hive Resources

•Hive weight

•Percent of frame covered by resources

•Quality of hive products

Additional endpoints 

similar to Tier II
Tier III: Analysis and Interpretation of Effects

• Exposure

• Potential for bees to forage minimally on test plots

• Residues in relevant bee matrices

• Outside hive: pollen traps

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Outside hive: pollen traps

• Inside hive: stored pollen/nectar, honey/bee bread

• Pollen and nectar identification analysis

• Measures of foraging and crop attractiveness
Tier III: Experimental Design Considerations

• Exposure to test chemical

• Lack of control over foraging

• Potentially relatively lower level of foraging on treatment plot 

compared to control plot

• Uncontaminated reserves of pollen and honey collected prior 

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• Uncontaminated reserves of pollen and honey collected prior 

to study initiation

• Weather

• Plot sizes
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5 mile maximum 

foraging radius = 

78.5 mi2

Tier III: Typical Crop Specific Field Study

2.5 – 13 acres plot 

sizes = 0.004 – 0.02 

mi2

0.0051 - 0.025% 

of maximum 

foraging area
Tier III: Experimental Design Considerations, 

cont.

• Colony size

• 10k adult bees minimum size

• Larger hives are more efficient, smaller hives may be more 

sensitive

• Exposure to other pesticides

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• Exposure to other pesticides

• Standard grower practice on-field and off-site

• Separation of treatment from controls

• Cross-contamination or dilution from control plots

• Parasites and disease

• Nosema spp., Varroa mites, small hive beetle, viruses, bacteria, 

wax moth, and fungal pathogens

• Duration of the study

• Replication and variability 
Tier III: Suggested Modifications to Design

1. Test plots

• Increase size

• Ensure adequate separation

2. Statistical analysis should be performed for all field studies

• Power analysis before and after conduct of the study

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• Power analysis before and after conduct of the study

3. Exposure to be evaluated

• Residues in floral pollen/nectar

• Residues in hive products

• Identify sources of incoming pollen/nectar

• Measure bloom intensity and duration of that intensity

4. Limit disease/pests using new hive equipment or obtain bees 

from sources with low pest incidence
Tier III: Suggested Modifications to Design, 

cont.

5. Duration of the study should match the concern of the chemical

• Monitoring to span flowering period and at least two brood cycles

• For persistent residues, monitoring should continue to after the 

overwintering period

6. Use of a highly attractive crop

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6. Use of a highly attractive crop

7. Assessment of hive brood following OECD 75 guidance 

document

8. Residue analysis of in-hive bees (in particular nurse bees)/dead 

bees in bee trap

9. Evaluation of the storage of food and the residues in stored food

10. Potential removal of food stocks
Tier III: Intensity of Resource Use

• Additional value to the 

risk assessment process

• Size of study and 

complexity

• Financial cost to registrant

• Agency resources to 

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Tier III • Agency resources to 

design and review

Increasing 

realism, 

variability, 

complexity, 

and cost

Tier I

Tier II

Tier III
Tier III: Use of the Field Study

• Screening Field Study

• Determine the presence/absence of an effect

• Definitive Study

• Define magnitude and duration

• Use qualitatively to describe risk

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• Use qualitatively to describe risk

• Inform risk management regarding the need for potential 

mitigation 
Weight of Evidence

• Interpretation of field study within risk assessment will be 

made in the context of data drawn from lower Tiers

• Available data taken into account for consistency, coherence, 

and biological plausibility

• Available data may include:

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Available data may include:

• Tier I laboratory acute/chronic toxicity

• Open literature

• Non-target arthropod data

• Sublethal effects

• Incident reports

• Tier II semi-field studies

• Tier III full field studies

• Exposure data
Questions

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Questions