# Methods fo r Eva lua ting Toxicity of Pe st icides toB ees **1** U.S. Environmental Pr otection Agency



### Joseph DeCan t, M.S. Environmental F ate and E ffects Division Office of P es ticide Pr ogr ams

# Overview of the Presen tat ion

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### Introduction to the tier ed appr oach to to xicity te st ing • 

### Tier I: labor ator y • 

### Tier II: semi - field whole hiv e • 

### Tier III: full field whole hiv e Char ge q ues tions 9, 10, 11, 12 U.S. Environmental Pr otection Agency **2**

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### Char ge q ues tions 9, 10, 11, 12

# Assessmen t and Measuremen t Endpoin ts U.S. Environmental Pr otection Agency **3**

**Pr ot**  **ection Goal**  **Assessment**

**Endpoints Measur**  **ement**

**Endpoints (P**  **opulation Level**  **and Higher**  **) Measur**  **ement Endpoints**  **(Individual Level)** 1) Provision of Po pulation

siz e and Colon y strength and Individual work er surviv al Queen fecundity **Ta ble**  **1. Pr oposed pr**  **otection goals and examples**  **of associat ed**  **assessment and**  **measur ement**  **(population and individual)**  **endpoints f or**  **bees.** 1) Provision of Po llination Services siz e and

sta bility of managed bees Colon y strength and

survival Queen fecundity Br ood siz e Wo rker bee longevity 2) Production of Hive Products Quantity and

quality of hive pr oducts Quantity and quality of

hive pr oducts; r esidue levels on honey/w ax Individual work er surviv al Queen fecundity Br ood success 3) Contribution to P ollinator Biodiver sity Species

richness and abundance Colon y strength and

survival Species richness and abundance Individual work er surviv al Br ood success

# Tiered Approach toT

ox icity Te st ing Eco logical effects assessmen t describes the effects elicited by a pes ticide U.S. Environmental Pr otection Agency **4** 

### Scr eening Lev el Tier I: • Individual based • Labor ator y assa yIncreasingLevelofRealism 

## a pes ticide Tier ed appr oach to allow for incr easing levels of r ealism and ref inemen t for both to xicity and e xposur e Tier II: • Whole hiv e • Semi - Field (tunnel/feeding) Tier III: • Whole hiv e • Full FieldIncreasingLevelofRealism

# Tier I T ox icity

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### Curr en t U. S. EP

A 40 CFR § 158 r equir emen t includes a 48hr acut e contact assa y with young adults • 

### Expansion to include acute or al studies with both adults and lar va e • 

### Appr oach similar to U .S. EP A Interim Guidance on Honey Bee U.S. Environmental Pr otection Agency **5**

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### Appr oach similar to U .S. EP A Interim Guidance on Honey Bee Data Requirements • Individual based • Focus on mortality • Scr eening lev el evaluation of dietary and contact e xposur e r out es

# Tier 1: Adult Acut e Toxicity Assa ys - Con ta ct EP A 850.3020/OE CD 214: Labor atory acute con ta ct to xicity with y oung adult bees • 

### 48 hr acute contact to xicity – LD 50 U.S. Environmental Pr otection Agency **6**

• LD 50 > 11 μg a.i ./bee = pr actically non - tox ic • 2 < LD 50 < 10.99 μg a.i ./bee = moder ately to xic • LD 50 < 2 μg a.i ./bee = highly tox ic 

## EP A 850.3030: To xicity of r esidues on foliage • 

### RT 25 

### : Measur e of r esidual t ox icity

# Tier 1: Adult Acut e Toxicity Assa ys - Die ta ry

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# OE CD 213: Labor ato ry acut e or al t ox icity with yo ung adults • 

## 48 hr acut e or al t ox icity – LD U.S. Environmental Pr otection Agency **7**



### • 

## 48 hr acut e or al t ox icity – LD 50

# Tier 1: Larva l Acut e Toxicity Assa y

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## Br ood Study – consider ed a semi - field st udy • 

## In Vitro Assa y • 

### 1 st 

### ins ta r bee lar va e ar e tr ans fe rr ed in to 48 - we ll plates • 

### Lar va e giv en s yn thetic food sour ce containing known U.S. Environmental Pr otection Agency **8**

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### Lar va e giv en s yn thetic food sour ce containing known quan tities of the t es t chemical ove r the 48hr s tudy period • 

### Te st pr ovides LD 50 

### and with sufficient r eplication it also pr ovides a NO AEC • 

### Uncert ain ties associated with this design • Hiv e v er sus labor ator y conditions • Artificial food source

# Tier 1: Approaches toEva lua teC hronic Toxicity **Proposed approach**  : • 

### To date ther e ar e no formal guidelines fo r conducting chr onic te st s with adults or lar va e • 

### Adult chr onic or al to xicity assa y (10 da y) • 

### Lar va l chr onic to xicity assa y (7 da y) U.S. Environmental Pr otection Agency **9**

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### Lar va l chr onic to xicity assa y (7 da y) **Po ssible approach:**  Chr onic to xicity es timated fr om acute labor ator y st udies • NOAEC fr om acut e surviv al s tudies • Re gr ession v er sus h ypothesis based tes ting

# Tier 1: Other T

ox icity Da ta S ources

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### Scien tific Open Lit er atur e • Mor e sensitiv e endpoints than r egis tr ant submitted s tudies • Ma y be used to deriv e risk quotients (quantitativ e) • 

### Non - ta rget Arthr opods • Re gis tr ant - submitted studies, e.g. , gr een lacewing , par asitoid w asp U.S. Environmental Pr otection Agency **10**

• Re gis tr ant - submitted studies, e.g. , gr een lacewing , par asitoid w asp • Qualitative use in risk char act eriz at ion • 

### Inciden ts • Re ports of honey bee mortality ev ents fo llowing pesticide e xposur e • Po tentially useful in ev aluating the risk conclusions

# Tier 1: Sublethal eff

ects

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### Ty pes of sublethal effects include impairmen ts to: • Mov ement and communication • For aging and homing beha vior • Re cruitment of fellow for ager s • Health of queen • Deto xification pathwa ys U.S. Environmental Pr otection Agency **11**

• Deto xification pathwa ys • Hiv e and individual immune r esponse • Colon y maintenance activity • Pr oboscis e xtension r efle x • 

### EF SA: sublethal endpoin ts inadequately cov er ed by e xis ting tests • 

### Lack of dir ect connection between measur emen t endpoints and assessmen t endpoin ts • 

### Qualit ative use in the risk assessmen t

# Tier 1: Summary of Measuremen t Endpoin ts U.S. Environmental Pr otection Agency **12** 

## Measur emen t Endpoin ts (RQ Calculation): **1. Individual**  **Wo rk** **er Surviv al:**  • Acut e LD 50 (Contact and Dietary) • Adult 10 - da y Dietary Chr onic NOAEL or EDx **2. Br**  **ood Success:**  • Larv al Acute and Chr onic NOAEL or LD 

## Qualit ative (Risk Description): • Larv al Acute and Chr onic NOAEL or LD 50 

## Assessmen t Endpoin ts: • Po pulation Siz e and Stability of Manag ed Bees • Quan tity of Hiv e Pr oducts • Species Richness and Abundance 

## Description): • RT 25 

### • Sublethal effects • Other data sour ces 

## Risk Managemen t

# Transition From Tier I t o Tier II U.S. Environmental Pr otection Agency **13**

• Mortality - based • Labor ator y • Individual • No fo rmal guidelines to assess chr onic to xicity 

### Limit ations of Tier I T

es ting • Incr easing lev el of realism

• Eva luate potential effects identified at Tier I for impact to the entire colon y • Integr at ion of sub - lethal effects, 

### Str eng ths of Tier II T

es ting



## Eva lua te the need for further ref inement • 

### Da ta needs iden tified by risk manag er • Decision based on to xicity and exposure data • Other lines of evidence • Uncertainties ( e.g. , mode of action, chemistry) • Mitig ation • Of f- label specific uses chr onic to xicity • Integr at ion of sub - lethal effects, chr onic e xposur e, and degr ada te toxicity

# Tier II: Semi - Field Study

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## Purpose • 

### Ref inemen t of Tier I assessmen t • 

### Shift of focus fr om the individual to the colon y • 

### Addr ess specific issues r aised at the initial tier s of the risk assessmen t U.S. Environmental Pr otection Agency **14**



### assessmen t

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

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### 40 CFR part 158 r equir emen t • 

### Re quir ed when the following conditions ar e met: • Ev idence of e xtended r esidual t ox icity (RT 25 > 8hr s) to honey bees as seen in the 850.3030 test fo r the to xicity of r esidues on foliage • Da ta fr om other sour ces for honey bees indicate that there is the U.S. Environmental Pr otection Agency **15**

• Da ta fr om other sour ces for honey bees indicate that there is the pot ential for effects other than mortality on the colon y • Da ta fr om s tudies with terr es trial arthr opods other than bees indicate pot ential chr onic, r epr oductiv e, or beha vior al effects

# Tier II: Design Options fo r the Semi -field Study

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### Tu nnel Design: pes ticide ex posur e facilit ate d by te st c ro p • Adult/Br ood U.S. Environmental Pr otection Agency **16**

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### Fe eding Design: pes ticide ex posur e facilit ate d by pes ticide spik ed diet • Br ood • Adult/Br ood

# Tier II: Semi -field Tu nnel Design

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### Design: U. S. EP

A 850.3040/EPPO 170 (4)/OECD 75 • Cr op gr own outdoor s in an enclosed s ystem ( i.e. , tunnel) with surr og ate species ( e.g. , Phacelia ta nacetifolia ) • Ma ximum application r ate, minimum application inter va l • Small nucleus hiv e of appr ox imately 5,000 bees U.S. Environmental Pr otection Agency **17**

• Hiv es ar e k ept in tunnel for 7 – 10 da ys • Hiv es r emoved fr om tunnel after 10 da ys and allowed to freely for age • Monitoring of hiv e conditions pr oceed for up to 28 da ys • Refer ence to xicant frequently used ( e.g. , fe no xycarb or dimethoate) • Measur e residues in flower or bee - collected pollen and nectar • 

### Applicable scenarios include seed tr ea tmen t, soil application, trunk injection, and foliar spr ay

# Tier II: Semi -field Fe eding Design - Brood

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## Ty pically used to assess br ood to xicity • 

### Based on Oomen et al. 1992 • 

### Hiv es allowed to fr eely for ag e • 

### 1 liter of spik ed/ control solution placed in feeder near hiv e U.S. Environmental Pr otection Agency **18**

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### We ekly measur emen ts on br ood dev elopmen t

# Tier II: Semi -field Fe eding Design - Brood and/or Adults • 

## Adult open field feeding s tudy • 

### Spik ed pollen pat ties/sucr ose solution fed t o hiv es r anging in concentr ation to establish NOAEC/L OA EC • 

### Location in ar ea of low pes ticide use Bees allowed to fr eely for ag e (use of pollen tr aps limit U.S. Environmental Pr otection Agency **19**

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### Bees allowed to fr eely for ag e (use of pollen tr aps limit incoming pollen) • 

### Use of full siz e hiv es (minimum 10k bees) • 

### Dur ation of study can include overwintering • 

### Measur emen t of endpoin ts at specified time periods

# Tier II: Semi -field Measuremen t Endpoin ts: Adults and/or Brood U.S. Environmental Pr otection Agency **20**


### 1. Adult Bee Mortality Ref inemen t of Tier I: colon y lev el 1. Br ood condition assessment Additional endpoin ts beyond Tier I 1. Br ood condition assessment • Egg abundance/larv al/ pupal abundance, br ood pa ttern, emergence success 

### 2. Hiv e strength • Pe rc ent of comb area covered with bees 

### 3. Queen health 4. Hiv e R esour ces • Hiv e weight • Pe rc ent of fr ame cov er ed by resources • Quality of hiv e pr oducts

# Tier II: Semi -field Uncert ain ties **Both Designs** • Adv er se weather conditions • Va riability in measur ement endpoints 

### Tu **nnel Design** • Quantity and quality of food ma y be compr omised • Confinement stress on for aging bees U.S. Environmental Pr otection Agency **21**

• Confinement stress on for aging bees • Short period of e xposur e 

### Fe **eding Design** • For aging not limited to the pr ovided food • Artificial aspect of feeding in r elation to natur al for aging • Ma y not integr ate impact t o for aging beha vior in the field • Uncertain performance of colonies to long - term supplemental feeding • Inter ve ntions required for overwintering

# Re conciling St at istical and Biological Signific ance • 

### St atistical Analysis • Comparison of treatment means • Centr al tendency (mean, median), disper sion (s tandard err or , s tandard deviation) • Time tr ends not r outinely r eported but can be qualitativ ely evaluated U.S. Environmental Pr otection Agency **22**

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### Biological significance as it r ela te s to assessment endpoints • High v ariability in measur ement endpoints between hiv es and ov er time • Uncertainty reg ar ding effects tha t ar e not s tat istically significant • Compensatory mechanisms of the hiv e • Ef fe cts that ar e tr ansient

# Tier II: Use of Semi -field st udies

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### Addr ess concerns r aised at the fir st Tier • Pr ovide clear er lines of evidence for linking multiple r out es of e xposur e to adver se ecological effects • Assess both e xposur e and effects tha t ar e less conserv at iv e and mor e re alis tic than labor ator y methods Determine e xtent to which effects on individuals tr anslates into effects a t U.S. Environmental Pr otection Agency **23**

• Determine e xtent to which effects on individuals tr anslates into effects a t the colon y level • Integr ate toxicity of degr ada tes, sub - lethal and chr onic effects into the performance of the hiv es as they r elate to assessment endpoints • 

### The modified feeding design NO AEC can be compar ed with re sidue lev els fr om tar gete d monitoring data • 

### Qualit ative use to inform risk manag emen t re ga rd ing potential mitig ation or further r efinemen t

# Transition From Tier II t o Tier III U.S. Environmental Pr otection Agency **24 Tier II**  **Limit at**  **ions Tu**  **nnel:**

• Str ess of tunnel • Short dur at ion • Compr omised quantity and quality of food **Feeding: Br**  **ood**

• Artificial food sour ce: uncertain ex posur e • Ma y not integr ate effects on for aging beha vior **Feeding: Adult/Br**  **ood**

• Artificial food source: uncert ain e xposur e • Ma y not integr ate effects on for aging beha vior **Tier III Adv**  **antages** • Eliminate the

st re ss of the tunnel and limited food • Allows for larger siz e hiv es • Mor e fle xibility in dur at ion of the st udy beha vior

• Na tur al feeding mechanism • Integr at ion of fo ra ging activity • Na tur al feeding

mechanism • Integr at ion of for aging activity • Refine the effects assessment when re sidue lev els ar e abov e the NOAEC

# Basis fo r T

ra nsition from Tier II t o Tier III U.S. Environmental Pr otection Agency **25** 

## Eva luate the need for further ref inemen t • 

### Data needs identified by risk manager • Decision based on to xicity and e xposur e da ta • Other lines of evidence • Uncertainties ( e.g. , mode of action, chemis try , use pa ttern) Uncertainties ( mode of action, chemis , use pa • Mitig at ion

# Tier III: Purpose

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### Final lev el of r efinemen t to Tier s I and II • 

### Eva luate the consequences of e xposur e t o r eal - world application of pesticide • 

### Te st for the potential of an effect, not the mechanis tic cause of an effect U.S. Environmental Pr otection Agency **26**



### an effect • 

### Fo cused risk h ypothesis

# Tier III: Guidance Pe rt aining tothe Field Study

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### EP A 850.3040 • Fle xibility to design s tudy based upon needs of the risk assessment • 

### EP A 850.2500 • Scr eening v er sus definitiv e lev el st udy • Geogr aphic ar ea U.S. Environmental Pr otection Agency **27**

• Geogr aphic ar ea • Siz e and dis tance between s tudy sites • Sample siz e depends on the r equir ed lev el of confidence • Te st conditions should r esemble those encountered under actual use conditions • 

### EPPO 1/170 (4) • Pr ovides guidance on the design of a field s tudy

# Tier III: Design Elemen ts of a Field Study

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### Fle xible design • 

### Actual use conditions • 

### Honey bees ar e fr ee for aging and unr es trict ed by a tunnel • 

### Cr op gr own accor ding to good agricultur al pr actices Hiv es of at least 10k bees U.S. Environmental Pr otection Agency **28**

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### Hiv es of at least 10k bees • 

### Dur ation of e xposur e • 

### Leng th of field s tudy monitoring • 

### Allows short t erm thr ough overwin te ring

# Tier III: Design Elemen ts of a Field Study , cont.

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### Colonies equally dis tribut ed among plots • 

### Hiv es ar e queen - righ t, use sis te r queens, and be of similar st re ng th at test initiation • 

### Eq uipmen t fr ee of other chemicals • 

### Hiv es with low occurr ence of disease and par asit es U.S. Environmental Pr otection Agency **29**

• 

### Hiv es with low occurr ence of disease and par asit es • 

### No positiv e contr ol at the full field lev el

# Tier III: Measuremen t Endpoin ts U.S. Environmental Pr otection Agency **30**


### 1. Adult Bee Mortality 1. Br ood condition assessment Ref inemen t of Tier I: colon y lev el 1. Br ood condition assessment • Egg abundance/larv al/ pupal abundance, br ood pa ttern, emergence success 

### 2. Hiv e strength • Pe rc ent of comb area covered with bees 

### 3. Queen health 4. Hiv e R esour ces • Hiv e weight • Pe rc ent of fr ame cov er ed by resources • Quality of hiv e pr oducts 

### Additional endpoin ts similar to Tier II

# Tier III: Analysis and In te rpret at ion of Eff ects

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## Exposur e • 

### Po te ntial for bees t o for ag e minimally on t est plots • 

### Re sidues in relev an t bee matrices • Outside hiv e: pollen tr aps U.S. Environmental Pr otection Agency **31**

Outside hiv e: pollen tr aps • Inside hiv e: stored pollen/nectar , honey/bee br ead • 

### Po llen and nect ar iden tific ation analysis • 

### Measures of for aging and cr op at tr activeness

# Tier III: Experimen ta l Design Considerat ions

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## Exposur e t o t es t chemical • 

### Lack of contr ol over for aging • 

### Po te ntially relatively lower level of for aging on treatment plot co mpared to c on tr ol plot • 

### Uncontaminated reser ve s of pollen and honey collected prior U.S. Environmental Pr otection Agency **32**

• 

### Uncontaminated reser ve s of pollen and honey collected prior to study initiation • 

### We ather • 

### Plot siz esU.S. Environmental Pr otection Agency **33**

5 mile maximum fo ra ging r adius = 78.5 mi 2 

# Tier III: T ypic al Crop Specific Field Study

2.5 – 13 acr es plot siz es = 0.004 – 0.02 mi 2 0.0051 - 0.025% of maximum fo ra ging ar ea

# Tier III: Experimen ta l Design Considerat ions, cont. • 

## Colon y siz e • 

### 10k adult bees minimum siz e • 

### Lar ge r hives are more efficient, smaller hives ma y be mor e sensitive 

### • 

## Exposur e to other pesticides U.S. Environmental Pr otection Agency **34**



### • 

## Exposur e to other pesticides • 

### St andard gr ower pr actice on - field and off- sit e 

### • 

## Separ at ion of tr eatment from controls • 

### Cr oss - co nta mination or dilution fr om c on tr ol plots 

### • 

## Pa ra sites and disease • 

### Nosema spp ., Va rroa mit es, small hive beetle, viruses, bact eria, wax moth, and fung al pathogens 

### • 

## Dur at ion of the study • 

## Re plication and v ariability

# Tier III: Sug geste d Modific at ions toDesign 1. 

### Te st plots • Incr ease siz e • Ensur e adequate separ at ion 2. 

### St atistical analysis should be performed for all field s tudies • Po wer analysis befor e and after conduct of the s tudy U.S. Environmental Pr otection Agency **35**

• Po wer analysis befor e and after conduct of the s tudy 3. 

### Exposur e to be ev aluated • Re sidues in flor al pollen/nectar • Re sidues in hiv e pr oducts • Identify sour ces of incoming pollen/nectar • Measur e bloom intensity and dur at ion of that intensity 4. 

### Limit disease/pes ts using new hiv e equipmen t or obtain bees fr om sour ces with low pes t incidence

# Tier III: Sug geste d Modific at ions toDesign, cont. 5. 

### Dur ation of the study should match the concern of the chemical • Monitoring to span flowering period and at leas t tw o br ood cycles • For per sistent re sidues, monitoring should continue to after the overwintering period 6. 

### Use of a highly at tr activ e cr op U.S. Environmental Pr otection Agency **36**

6. 

### Use of a highly at tr activ e cr op 7. 

### Assessmen t of hiv e br ood following OECD 75 guidance documen t 8. 

### Re sidue analysis of in - hiv e bees (in particular nur se bees)/dead bees in bee tr ap 9. 

### Eva luation of the s tor ag e of food and the r esidues in s tore d food 10. 

### Po te nt ial re mov al of food stocks

# Tier III: In te nsity of R esource Use
• 

### Additional va lue t o the risk assessmen t pr ocess • 

### Siz e of study and comple xity • 

### Financial cost to registr an t • 

### Ag ency re sour ces t o U.S. Environmental Pr otection Agency **37**



# Tier III • 

### Ag ency re sour ces t o design and r eview Incr easing re alism, va riability , comple xity

, and cost 

# Tier I Tier II Tier III

# Tier III: Use of the Field Study

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### Scr eening Field Study • Determine the presence/ absence of an effect • 

### Definitiv e Study • Define magnitude and dur at ion • 

### Use qualitatively to describe risk U.S. Environmental Pr otection Agency **38**

• 

### Use qualitatively to describe risk • 

### Inform risk manag emen t re ga rd ing the need for potential mitig ation

# We igh t of Evidence

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### Interpr etation of field study within risk assessment will be made in the contex t of data dr awn fr om lower Tier s • 

### Ava ilable data tak en in to account for consistency , coher ence, and biological plausibility • 

### Ava ilable data ma y include: U.S. Environmental Pr otection Agency **39**



### Ava ilable data ma y include: • Tier I labor ator y acute/ chr onic to xicity • Open liter at ur e • Non - target arthr opod data • Sublethal effects • Incident reports • Tier II semi - field st udies • Tier III full field s tudies • Exposur e da ta

# Questions U.S. Environmental Pr otection Agency **40** 

# Questions