Guidance on Exposure and Effects
Testing for Assessing Risks to Bees

Office of Pesticide Programs
U.S. Environmental Protection Agency
July 5, 2016

1

Disclaimer
This guidance is not a regulation and, therefore, does not add, eliminate or change any existing regulatory
requirements. The statements in this document are intended solely as guidance. This document is not intended,
nor can it be relied on, to create any rights enforceable by any party in litigation with the United States. EPA
staff may decide to follow the guidance provided in this document, or to act at variance with the guidance, based
on analysis of pesticide-specific risks and benefits. Deviations from this guidance shall not constitute grounds
for challenging pesticide registration decisions made by EPA. This guidance may be revised without public
notice to reflect changes in EPA’s policy.
3

1. Purpose
The  intent  of  this  document  is  to  provide  guidance  to  risk  assessors  within  the  U.S.  Environmental
Protection Agency (EPA) Office of Pesticide Programs (OPP) for determining which exposure and effects
(toxicity) studies should be considered when characterizing the potential risk of pesticides to bees. This
guidance supersedes the interim guidance1 to risk assessors issued by the Environmental Fate and Effects
Division  (EFED)  in  2011  and  complements  the  broader  risk  assessment  process  outlined  in  the  2014
Guidance for Assessing Pesticide Risks to Bees2, which was developed as a collaborative effort between
the EPA, Health Canada Pest Management Regulatory Agency (PMRA) and the California Department of
Pesticide  Regulation  (CDPR).  Further information for  risk  managers  within  OPP  to  determine  whether
honey  bee  exposure  and  effects  data  are  necessary  for  regulatory  actions  is  provided  in  a  companion
guidance entitled, Process for Requiring Exposure and Effects Testing for Assessing Risks to Bees during
Registration and Registration Review (also referred to as the implementation guidance). To provide the
public  with  more  insight  into  OPP's  regulatory  program,  this  document,  along  with  the  companion
implementation guidance, are being made available on our website. By doing so, EPA expects the regulated
community to become more familiar with how OPP plans to move forward with assessing risks to bees. It
is important to note that the guidance documents EPA has developed focus primarily on studies that may
be  useful  in  evaluating  the  exposure  and  effects  to  honey  bees  of  conventional  pesticides.  While  they
provide some guidance to risk assessors evaluating other types of pesticide products, further consideration
is needed to determine whether the studies are appropriate for evaluating other types of pesticides or if
different types of studies may be more useful. Therefore, this guidance document is focused on conventional
pesticides; EPA discusses its current approach for other types of pesticides in section 4.
2. Background
Based on survey data collected through the U.S. Department of Agriculture National Agricultural Statistics
Service (NASS3) on the number of managed honey producing colonies in the U.S., the numbers of such
colonies have been in decline since the mid-1940s. The NASS survey reported in 1947 that there were
approximately 5.8 million colonies used to produce honey4; however, as of 2016, there were roughly 2.59
million5. Additional information on declines in pollinator species such as the honey bee in North America
was reported by the National Research Council6 in 2007. In 2006, the magnitude of honey bee colony losses
increased as beekeepers reported the sudden disappearance of adult bees from colonies in a phenomenon

1 USEPA. 2011. Interim Guidance on Honey Bee Data Requirements.
2 USEPA,  PMRA,  CDPR.  2014.  Guidance  for  Assessing  Pesticide  Risks  to  Bees.  Office  of  Pesticide  Programs  United  States  Environmental
Protection Agency, Health Canada Pest Management Regulatory Agency (PMRA), California Department of Pesticide Regulation (CDPR). June
19,  2014.  http://www2.epa.gov/sites/production/files/2014-06/documents/pollinator_risk_assessment_guidance_06_19_14.pdf  (last  accessed
06/28/2016).
3
USDA.
2014.
National
Agricultural
Statistics
Service
Bee
and
Honey
Inquiry.
https://www.nass.usda.gov/Surveys/Guide_to_NASS_Surveys/Bee_and_Honey/#skipnav  (last accessed 06/27/2016).
4 USDA. 1947. Bureau of Agricultural Economics, Washington DC. http://usda.mannlib.cornell.edu/usda/nass/HoneProd//1940s/1947/HoneProd-
01-24-1947.pdf (last accessed 12/19/2015).
5
USDA.
2016.
National
Agricultural
Statistics
Survey
(NASS)
Honey
Bee
Colonies.
http://usda.mannlib.cornell.edu/usda/current/BeeColonies/BeeColonies-05-12-2016.pdf  (last accessed 06/27/2016).
6 National Research Council. 2007. Status of Pollinators in North America. Committee on the Status of Pollinators in North America Board of Life
Sciences
National
Research
Council
of
the
National
Academies.
National
Academies
Press,
Washington
DC.
http://www.nap.edu/openbook.php?record_id=11761 (last accessed 06/27/2016).
4

termed “Colony Collapse Disorder (CCD7)” where colonies were left with insufficient numbers of bees to
survive.
In 2007, Congress charged the USDA as the lead federal agency  to determine the causes and potential
mitigation measures for losses associated with CCD and more recently with general declines in honey bee
health. Based on six years of research, the USDA has attributed losses to a number of factors including
pesticides, pathogens (e.g., fungal, bacterial, viral disease[s]), pests (Varroa mite; Varroa destructor), poor
nutrition, and bee management practices)8. While no single factor has been identified as a specific cause of
honey bee declines, available data suggest that CCD and declines in honey bee health are associated with
combinations of these factors; however, the exact combination remains uncertain9.  Pesticides have been
identified as a factor, and EPA is responsible under Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) for evaluating the potential for pesticides to adversely affect non-target organisms. Therefore,
regulatory  authorities  in  North  America  and  elsewhere  are  implementing  improved  procedures  for
evaluating the potential risks of pesticides to bees.
In response to the uncertainties regarding the potential role that pesticides may play in pollinator declines,
OPP established the Pollinator Protection Team in 2007 with representatives from each of the Divisions
within OPP. This team was charged with:
  Advancing the Agency’s scientific knowledge and assessment of pesticide risks to pollinators;
  Improving risk management tools for mitigating potential risks to pollinators; and,
  Increasing  and  broadening  collaboration  and  communication  with  governmental  and  non-
governmental organizations and the public in addressing pollinator issues.
Members of the Pollinator Protection Team  have been and continue to be engaged in a broad range of
efforts to advance the science, management and understanding of the extent to which pesticides play a role
in any of the adverse effects being seen in pollinator populations. In 2014, EPA issued guidance to risk
assessors in OPP for evaluating the potential risks of pesticides to bees10. The guidance identifies a tiered
risk assessment process and the underlying data necessary to implement that process. The guidance is based
on a White Paper11 submitted to the FIFRA Scientific Advisory Panel (SAP) for review and comment in
September  2012.  The  White  Paper  was  in  turn  informed  by  efforts  underway  in  Europe  through  the
European  and  Mediterranean  Organization  for  Plant  Protection  (EPPO12),  the  European  Food  Safety
Authority  (EFSA13)  as  well as  the  Organization for  Economic  Cooperation  and Development  (OECD).
Non-governmental organizations such as the International Commission for Plant-Pollinator Relationships

7 vanEngelsdorp,  D.,  J.  D.  Evans,  C.  Saegerman,  C.  Mullin,  E.  Haubruge,  B.  K,  Nguyen,  M.  Frazier,  J.  Frazier,  D.  Cox-Foster,  Y.  Chen,  R.
Underwood,  D.  R.  Tarpy,  J.  S.  Pettis.  2009.  Colony  Collapse  Disorder:    A  Descriptive  Study.  PLoSONE  4(8):  e6481.
Doi:10.1371/journal.pone.0006481 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006481 (last accessed 06/27/2016).
8 USDA. 2013. Report on the National Stakeholders Conference on Honey Bee Health. National Honey Bee Health Stakeholder Conference Steering
Committee. October 15 – 17, 2012. http://www.usda.gov/documents/ReportHoneyBeeHealth.pdf  (last accessed 06.27/2016)
9 Pettis,  J.  S.  and  K.  S.  Delaplane.  2010.  Coordinated  responses  to  honey  bee  decline  in  the  USA.  Apidologie  41:    256-  263.
http://www.apidologie.org/articles/apido/pdf/2010/03/m09140.pdf (last accessed 06/27/2106).
10 Ibid USEPA, PMRA, CDPR. 2014.
11USEPA. 2012. White Paper in Support of the Proposed Risk Assessment Process for Bees. Submitted to the FIFRA Scientific Advisory Panel for
Review and Comment September 11 – 14, 2012. Office of Chemical Safety and Pollution Prevention Office of Pesticide Programs Environmental
Fate  and  Effects  Division,  Environmental  Protection  Agency,  Washington  DC;  Environmental  Assessment  Directorate,  Pest  Management
Regulatory Agency, Health Canada, Ottawa, CN; California Department  of Pesticide Regulation  http://cues.cfans.umn.edu/old/pollinators/pdf-
EPA/EAP-SAP-whitepaper.pdf (last accessed 06/27/2016).
12 EPPO. 2010. Efficacy Evaluation of Plant Protection Products: Side-effects on Honey bees. PP 1/170 (4). OEPP/EPPO Bulletin 40: 313–319
13 EFSA. 2013. Guidance on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees). European
Food Safety Authority. EFSA Journal 11(7): 3295. https://www.efsa.europa.eu/en/efsajournal/pub/3295 (last accessed 06/27/2016).
5

(ICP-PR)  and  the  Society  of  Environmental  Toxicology  and  Chemistry  (SETAC)  also  have  sponsored
efforts to advance the science of assessing risks to bees from pesticides.
In June 2014, President Obama issued a memorandum14 establishing the Pollinator Health Task Force and
requiring  the  development of  a  federal  strategy  to  protect the health  of  bees and  other pollinators. The
Pollinator Health Task Force identified in the strategy is co-chaired by EPA and USDA and is tasked with
collecting, collating and disseminating information on stressors (e.g., pesticides) that may affect bees and
other pollinators.  In May 2015, the White House published the National Strategy to Promote the Health of
Honey Bees and other Pollinators15 document.
Assessing risks to bees is a complex matter. There are many factors that contribute to potential adverse
effects on bees. Consistent with EPA’s process for evaluating risk to other taxa, it relies on multiple studies
identified in Title 40 (Protection of the Environment) of the Code of Federal Regulations, Part 158 (Data
Requirements for Pesticides; abbreviated as 40 CFR Part 15816). OPP's assessments of risk must adjust as
science evolves. As an Agency, EPA is committed to using the best available and sound science in its
decision making process. As such, data requirements will change over time. The number and nature of
studies  needed  to  support  regulatory  decisions  has  continued  to  evolve  as  the  science  evolves  and  the
process for implementing those studies through rule making (e.g., codifying in the 40CFR158) can take
considerable  time.  EPA  also  recognizes  that  the  capacity  of  contract  research  organizations  (CROs)  to
conduct new studies on behalf of the regulated community depends on the nature of the studies and the
laboratory’s familiarity with the test species and/or study conditions.
The honey bee exposure and effect studies discussed in this guidance reflect the evolving science and EPA’s
effort to address uncertainties regarding the extent to which bees may be exposed and the nature of potential
effects on bees at differing stages of development (e.g., larval vs. adult bees) and social organization (e.g.,
individual bee vs. bee colony). In the absence of data, there will be uncertainties regarding the potential for
exposure  and  effects  to  bees.    For  pesticides  where bees  are  not  considered  likely  to  be  exposed  or  in
situations where acute and/ or chronic toxicity is not expected based on other lines of evidence (e.g., mode
of action, toxicity data for other related taxa), additional data may not be warranted to support regulatory
decisions.    Decisions  to  proceed  with  a  particular  regulatory  action  will  consider  the  nature  of  the
uncertainties (e.g., which data may not be available), the benefits associated with the use, whether there are
alternatives and the potential risks associated with those alternatives, and the extent to which mitigation
measures  can  reduce  exposure/effects  from  the  pesticide  undergoing  the  registration  action.    Further
information for determining whether the honey bee exposure and effects studies described in this document
are required for various regulatory actions is provided in the companion guidance document, Guidance for
Implementation of Exposure and Effects Testing for Assessing Risks to Bees.


14 White  House.  2014.  Presidential  Memorandum  Creating  a  Federal  Strategy  to  Promote  the  Health  of  Honey  Bees  and  Other  Pollinators.
Memorandum  for  Heads  of  Executive  Departments  and  Agencies.  June  20,  2014.  http://www.whitehouse.gov/the-press-
office/2014/06/20/presidential-memorandum-creating-federal-strategy-promote-health-honey-b (last accessed 06/27/2016).
15 White House.  2015.  National Strategy to Promote the Health of Honey Bees and other Pollinators.  Pollinator Health Task Force, May 19, 2015.
https://www.whitehouse.gov/sites/default/files/microsites/ostp/Pollinator%20Health%20Strategy%202015.pdf  (last accessed 06/27/2016).
16 CFR. 2016.  Code of Federal Regulations Title 40 (Protection of the Environment) Chapeter I (Environmental Protection Agency) Subchapter E
(Pesticide Programs) Part 158 (Data Requirements for Pesticides) http://www.ecfr.gov/cgi-bin/text-
idx?tpl=/ecfrbrowse/Title40/40cfr158_main_02.tpl (last accessed 06/27/2016).
6

3. USEPA Toxicity Testing Requirements for Bees
3.1.  Statutory/Regulatory Provisions
In general, pesticides can only be sold and distributed in the United States if they have been registered by
EPA. Prior to EPA granting a registration, each applicant must establish that its product meets the standards
set forth in FIFRA section 3(c)(5) and/or 3(c)(7). These standards include finding that when a pesticide is
used  in  accordance  with  widespread  and  commonly  recognized  practice,  it  will  not  generally  cause
unreasonable  adverse  effects  on  the  environment.  FIFRA  also  provides  for  regular  review  of  existing
pesticide registrations. FIFRA section 3(g) and its implementing regulations at 40 CFR Part 155 set forth
the process for the reevaluation of currently-registered pesticides (i.e., Registration Review).
FIFRA’s  implementing  regulations  at  40  CFR  Part  158  set  forth  the  data  requirements  for  pesticide
registration. Additionally, these regulations discuss the flexibility EPA has in evaluating when data may be
required for pesticide registrations. Under 40 CFR Part 158.30, EPA may determine to modify the data
requirements on an individual or case-by-case basis to fully characterize the effects of a pesticide product.
Additionally, these regulations make clear the data routinely required under Part 158 may not always be
sufficient to assess whether there are unreasonable adverse effects on the environment. Under 40 CFR Part
158.30(b)  and  40  CFR  Part  158.75,  EPA  may  require  additional  information  to  better  characterize  the
potential risks.
As noted earlier, EPA has developed guidance documents17 18 for risk assessors that identify additional data
that may be useful in evaluating effects of pesticides on honey bees. These guidance documents identify
three tiers of data, and currently provide the most up-to-date information on the data that might be needed
by EPA. Given the advancement of the science, EPA believes that there are benefits associated with revising
the existing insect pollinator data requirements in part 158. The enhanced clarity and transparency of the
information  presented  in  part  158  should  enhance  the  ability  of  industry  to  efficiently  manage  their
registration  submissions.  Applicants  for  registration  may  save  time  and  money  by  understanding  when
higher-tiered studies are needed. Having all required studies available to EPA at the time  of application
should halt potential delays in the registration process. This should enable the registration of products that
could decrease risks to pollinators and therefore allow such products to enter the market earlier.
EPA intends to codify all of the data required to support each tier of the risk assessment process for bees in
40 CFR Part 158. EPA initiated the rulemaking process in 2015 with the understanding that the process can
take time to complete. EPA’s process for developing a rulemaking is intended to assure that the action (1)
is supported by strong analysis, (2) is developed via an open process, and (3) meets the requirements of the
Administrative Procedures Act. To support the science analysis for this rulemaking, EPA’s research on
insect pollinator issues has included a presentation to a peer review panel (i.e., the FIFRA SAP). For a
rulemaking, EPA also prepares an economic analysis (EA) to describe the costs and benefits of the action.

17 Ibid USEPA 2011
18 Ibid USEPA 2014.
7

The draft notice of proposed rulemaking and the EA are reviewed via an EPA-internal review process and
via an external interagency review process before publication in the Federal Register. EPA will publish the
proposed rule and proposed EA so that members of the public can consider the proposal and send their
comments  to  us.  EPA  accepts  comments  via  the  official  docket  at  http://www.regulations.gov/.  EPA,
considers, reviews and evaluates all comments submitted on the proposed rule and EA, and determines
whether or not any changes are needed. Then, drafts of the final documents are prepared and these draft
final  documents  also  undergo  the  internal-EPA  and  external  interagency  review  processes  before
publication in the Federal Register. The final rule is not effective until the new regulatory text becomes part
of CFR, i.e., the new data requirements are codified in the 40 CFR Part 158. This happens 60 days after
publication of the final rule. EPA expects to publish the proposed new data requirements in 2016, which
would  be  followed  by  the  public  comment  period.  The  timing  of  the  codification  of  the  new  data
requirements depends somewhat on the number and complexity of the comments submitted, as well as other
external factors. EPA projects the new rule to be effective by mid-to-late 2017.
While the Agency’s data requirements are established to provide the information needed by EPA to make
decisions about whether new pesticide products and new uses of existing products should be registered,
EPA may determine that additional data (e.g., bee studies) are required to support an existing registration
of a pesticide. In such cases, the EPA notifies registrants of the pesticide through issuance of a Data Call-
In Notice or DCI under FIFRA section 3(c)(2)(B). The DCI requires each affected registrant to provide
evidence  within  90  days  that  the  affected  registrant  is  taking  appropriate  steps  to  respond  to  the  DCI.
Additionally,  the  Notice  sets  deadlines  for  data  submission  and  may  specify  interim  deadlines.  Before
issuing  a  DCI,  OPP  must  submit  to  the  Office  of  Management  and  Budget  (OMB)  its  justification  for
requiring the additional information. Once OMB has approved the DCI, OPP may issue the order.
The next sections discuss the current data requirements and also explain what and why additional data may
be needed on a case-by-case basis.
3.2.  Conventional Pesticides: Current Data Requirements (40 CFR Part 158,
Subpart G)
The current EPA data requirements for insect pollinator testing, for conventional pesticides, are specified
in  the  40  CFR  Part  158  Subpart  G  (Ecological  Effects)  §158.630  (Terrestrial  and  Aquatic  Non-target
Organism Data Requirements Table).19 Data specified in the 40 CFR Part 158 are used to inform regulatory
decisions  under  FIFRA  about  the risks  and  benefits  of  pesticide  products.  Current  toxicity  testing  data
requirements specified in 40 CFR Part 158 for insect pollinators are shown in Table 1.

19 CFR40. 2016. Part 158, subpart G, §158.630  http://www.ecfr.gov/cgi-bin/text-
idx?SID=3da251be263b16deffd269aa64e0098c&mc=true&node=sp40.24.158.g&rgn=div6 (last accessed 06/27/16).
8

Table 1. Toxicity Testing Requirements for Insect Pollinators as Specified in 40 CFR Part 158, Subpart G.
Use Pattern
Test
Guideline
Data
Residential
Green-
Test
Note
Indoor
Number
Requirement  Terrestrial  Aquatic Forestry  Outdoor
house 5
substance  No.
Insect Pollinator Testing
Honey bee adult
850.3020
acute contact
R
CR
R
R
NR
NR
TGAI
1
toxicity
Honey bee toxicity
850.3030
of residues on
CR
CR
CR
CR
NR
NR
TEP
2
foliage
Field testing for
850.3040
CR
CR
CR
CR
NR
NR
TEP
3
pollinators
Definitions: R = Required; CR = Conditionally Required; NR = Not Required; TGAI = Technical Grade of the Active Ingredient; TEP = Typical
End-Use Product
Test Notes:
1.  Data using the TGAI are required to support all outdoor end-use product uses.  Data are generally not required to support end-use products in
the form of a gas, a highly volatile liquid, a highly reactive solid, or a highly corrosive material.
2.  Data are required only when the formulation contains one or more active ingredients having an acute LD50 of <11 micrograms per bee as
determined in the honey bee acute contact study and the use pattern(s) indicate(s) that honey bees may be exposed to the pesticide.  (Note that
in the regulatory text this is actually Test Note 24.)
3.  Required if any of the following conditions are met:  (Note that in the regulatory text this is actually Test Note 25.)
i.  Data from other sources (Experimental Use Permit program, university research, registrant submittals, etc.) indicate potential adverse effects
on colonies, especially effects other than acute mortality (reproductive, behavioral, etc.);
ii.  Data from residual toxicity studies indicate extended residual toxicity.
iii.  Data derived from studies with terrestrial arthropods other than bees indicate potential chronic, reproductive or behavioral effects

As indicated in Table 1, current data requirements specified under Part 158 include the honey bee acute
contact  toxicity  test  (OCSPP  Guideline  850.3020)20,  the  honey  bee  toxicity  of  residues  on  foliage  test
(OCSPP  Guideline  850.3030)21 and  field  testing  for  pollinators  (OCSPP  Guideline  850.3040)22 when
certain pesticide use patterns or triggers are met. These data are used to provide risk assessors with an
understanding of the effects of pesticides to which non-target insects are exposed through contact with
residues on various surfaces or through direct contact via spray and/or dust. The honey bee acute contact
toxicity test is required for pesticide technical grade active ingredients (TGAI) with terrestrial, forestry and
residential outdoor uses and is conditionally required for pesticides with aquatic uses as a Tier  1 screen
conducted under laboratory conditions. EPA will consider limit tests (100 µg a.i./bee) in the acute toxicity
tests; however, the registrant should provide a rationale for conducting a limit test. The rationale should
demonstrate  that  the  limit  test  is  protective  for  the  highest  estimated  environmental  exposure  level  for
individual bees. If the results of the honey bee acute contact toxicity test indicate that a pesticide has a
median acute lethal dose to 50% of the animals tested, i.e., the LD50 value, of less than (<) 11 micrograms
(µg) per bee, and the use pattern indicates that honey bees may be exposed, then the toxicity of residues on
foliage test is conditionally required as a laboratory-based test using the technical end-use product (TEP).
Toxicity of residues on foliage studies are designed to determine the time required for fewer than 25% of
bees exposed via contact with aged residues on foliage to die (RT25). Notably, information indicates that

20  USEPA.  2012a.  “Honey  Bee  Acute  Contact  Toxicity”  Ecological  Effects  Test  Guidelines  OCSPP  850.3020.  EPA  712-C-019  Web:
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines (last accessed 06/27/2016).
21 USEPA. 2012b. “Honey Bee Toxicity of Residues on Foliage.” Ecological Effects Test Guidelines  OCSPP 850.3030. EPA 712-C-018. Web.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines (last accessed 06/27/2016).
22  USEPA.  2012c.  “Field  Testing  for  Pollinators.”  Ecological  Effects  Test  Guidelines  OCSPP  850.3040.  EPA  712-C-017.  Web.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines (last accessed 06/27/2016).
9

the RT25 value may be strongly dependent on the formulation and environmental conditions. The Interim
Guidance on Honey Bee Data Requirements23 identified several other types of studies that can be used to
assess  the  persistence  of  pesticide  residues  on  foliage,  including  plant  metabolism  studies  (OCSPP
Guideline  860.130024,  rotational  crop  studies  (OCSPP  Guideline  860.190025)  and  magnitude  of  residue
studies (OCSPP Guideline 860.150026). This information can be coupled with acute contact toxicity data to
obtain estimates of the period over which residues may be toxic to bees and thus, may provide an alternative
to RT25 estimates. Notably, however, obtaining and analyzing these crop residue studies for determination
of foliar dissipation half-life values can be challenging and resource intensive.

As specified in 40 CFR § 158.63027, field testing of pollinators is required if any of the following conditions
are met:
  Data  from  other sources  (Experimental  Use  Permit program,  university  research,  registrant
submittals,  etc.)  indicate  potential  adverse  effects  on  colonies,  especially  effects  other  than
acute mortality (reproductive, behavioral, etc.);
  Data from residual toxicity studies indicate extended residual toxicity; or,
  Data derived from studies with terrestrial arthropods other than bees indicate potential chronic,
reproductive or behavioral effects.

Field testing of pollinators may include semi-field/feeding (Tier 2) or full-field studies (Tier 3) although
historically, studies conducted under OCSPP Guideline 850.3040 have focused on full-field testing. Full-
field  studies  are  intended  to  represent  real  world  conditions  and  are  considered  the  highest  level  of
refinement  (Tier  3)  for  bee  toxicity  testing  according  to  the  White  Paper 28  and  the  2014  Bee  Risk
Assessment Guidance29 document. As such, full-field studies should ideally be designed to address specific
uncertainties that have been identified in lower-tier tests. Pollinator full-field study designs received by
OPP  to  date  have  varied  considerably;  therefore,  rather  than  a  rigid  study  methodology,  study  design
elements that should be considered for these studies are provided to risk assessors (Appendix 1). Variability
has on occasion resulted from study designs attempting to collect too much information where colonies are
stressed by the collection of repeated measures. At a full-field level, study colonies are also vulnerable to
the same factors (e.g., disease, pests, poor nutrition) that have been associated with declines and honey bee
health and these can confound efforts to conduct such studies. Therefore, study protocols for Tier 3 tests
should be developed by pesticide applicants/registrants to address the specific hypothesis being tested. Such
protocols should ideally be reviewed by EPA staff prior to study initiation.


23 USEPA 2011. Interim Guidance on Honey bee Data Requirements. Memorandum from Donald Brady, Director, Environmental Fate and Effects
Division, dated October 19, 2011.
24 USEPA. 1996. Residue Chemistry Test Guidelines. OPPTS 860.1300 Nature of the Residue—Plants, Livestock. Office of Chemical Safety and
Pollution  Prevention  formerly  the  Office  of  Prevention,  Pesticides  and  Toxic  Substances  (7101)  EPA  712-C-96-172.  August  1996.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-860-residue-chemistry-test-guidelines (last accessed 06/27/2016).
25 USEPA. 1996. Residue Chemistry Test Guidelines OPPTS 860.1900 Field Accumulation in Rotational Crops. Office of Chemical Safety and
Pollution  Prevention  formerly  the  Office  of  Prevention,  Pesticides  and  Toxic  Substances  (7101)  EPA  712-C-96-189  August  1996.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-860-residue-chemistry-test-guidelines (last accessed 06/27/2016).
26 USEPA. 1996. Residue Chemistry Test Guidelines OPPTS 860.1500. Crop Field Trials. Office of Chemical Safety and Pollution Prevention
formerly the Office of Prevention, Pesticides and Toxic Substances (7101) EPA 712-C-96-183. August 1996. http://www.epa.gov/test-guidelines-
pesticides-and-toxic-substances/series-860-residue-chemistry-test-guidelines (last accessed 06/27/2016).
27 Ibid CFR40 2016.
28 Ibid USEPA. 2012
29 Ibid USEPA et al. 2014.
10

3.3.   Conventional Pesticides: Additional Bee Testing Guidance
After considering input from the  SETAC Pellston, EPPO and EFSA (discussed above), in 2011, EFED
issued interim guidance30 for ecological risk assessors  to determine whether and what additional honey bee
studies beyond the existing data requirements may be needed to better characterize the potential hazard
(i.e.,  adverse  effects)  of  chemicals  to  honey  bees.  That  guidance  also  more  clearly  establishes  a  tiered
structure for assessing effects of pesticides on bees. Relevant studies for Tier 1 include the acute and chronic
oral toxicity studies with adult bees and acute and chronic toxicity studies with larval bees, in addition to
the currently required honeybee acute contact toxicity study. For Tier 2, relevant data may include semi-
field effects studies conducted with full colonies (e.g., field feeding and tunnel studies) as well as studies
quantifying pesticide concentrations in pollen and nectar from treated plants. Finally, Tier 3 may involve
full-field testing with honeybee colonies, similar to the existing field testing for pollinator data requirement.
As EPA gains experience evaluating these data, it may be possible to consider data on similarly structured
chemicals with a common mode of action to that of the chemical under review depending on the quality of
those data.
The following subsections discuss the types of studies that are required and the additional studies that may
be needed on a case-by-case basis to fully characterize risk in various tiers of the risk assessment process
for  bees.  The  decision  to  recommend  additional  data  should  be  based  on  the  extent  to  which  bees  are
considered likely to be exposed and if so, whether there are any existing data on the chemical or similarly
structured chemical with respect to exposure and effects. Additional considerations may include whether
mitigation has been imposed/proposed that would reduce the likelihood of exposure/effects.
Bees can be exposed to pesticides through multiple pathways including contact with sprays and dusts and
through  ingestion  of  residues  in  food/water  (e.g.,  pollen/nectar  and  water  used  to  maintain  colony
temperature). Worker bees foraging on flowers for pollen and nectar can be exposed to residues in pollen
and  nectar  either  through  direct  contamination  of  these  matrices  by  foliar  sprays  and/or  dusts  through
translocation of residues via systemic transport of the active ingredient. Residues can in turn be brought
back to bee colonies where in-hive bees including young adults and developing brood (i.e., eggs, larvae and
pupae) may be exposed. EPA guidance31 on assessing the risk of pesticides to bees identifies a suite of
laboratory-based studies intended to serve as the foundation for screening chemicals for potential acute and
chronic effects to individual adult and larval bees.

With respect to the acute oral and chronic toxicity tests with adult bees, terrestrial invertebrates are likely
to be impacted if exposed to pesticides in various use settings. Pesticide residues may be transferred to or
come in contact with pollen and/or nectar of treated plants and ingested directly by individual bees or for
social species brought back to the colony. Therefore, potential acute effects to adult honey bees and other
insect pollinators from oral exposure to some pesticides could exist. The acute contact toxicity test does not
fully address possible effects of oral exposure on adult terrestrial insect survival. Because of the potential
for pollen and nectar to be contaminated with pesticide residues, and subsequently brought back to the hive
and  ingested,  it  is  important  to  determine  the  acute  oral  toxicity  to  adult  honey  bees  and  other  insect
pollinators for chemicals where exposure is considered possible.


30 Ibid USEPA. 2011.
31 Ibid USEPA et al. 2014.
11

With respect to the acute and chronic toxicity tests with larval bees, these studies evaluate both oral and
contact toxicity since larvae both ingest residues and are in contact with the treated diet during the study.
Similar to the potential route of exposure described for adult bees, pesticide residues may be transferred to
or come in contact with pollen and/or nectar of treated plants and these contaminated food items may in
turn be ingested by developing larvae during certain stages of their development. The acute contact toxicity
test with adult bees may not adequately address uncertainties related to differential sensitivity of different
life stages of bees. The chronic toxicity test provides a means of evaluating other endpoints (e.g., growth,
development  and  survival)  of  individual  bees  as  they  transition  from  larvae,  through  pupation  and
emergence as adults.

Although  EPA  guidelines  have  not  been  developed  for  these  studies,  the  Organization  for  Economic
Cooperation and Development (OECD) has developed a formal test guideline for an acute oral toxicity
study with adult bees (OECD 21332) as well as a test guideline for an acute oral toxicity study with honey
bee larvae (OECD 237)33. OECD also has a test guideline for assessing acute contact toxicity with young
adult bees (OECD 21434), that may provide sufficient data to fulfill the 40 CFR Part 158 adult contact
toxicity  test  requirement  (OCSPP  Guideline  850.3020).  Although  the  OECD  has  not  yet  finalized  test
guidelines  for  chronic  studies,  efforts  are  currently  underway  to  develop  standardized  guidelines  for
assessing the effects from chronic exposures to adult and larval honey bees in the laboratory. For Tier 2,
EPA  does  not  have  a  formal  guideline;  however,  OECD  7535 and  Oomen  et  al.  199236 represent  useful
guidance that can help shape the conduct of a tunnel or feeding study. As stated in 40 CFR Part 158.70(d)(2),
registrants/applicants can satisfy EPA data requirements by conducting studies in accordance with OECD
requirements  and  recommendations.  Importantly,  non-guideline  studies  should  not  automatically  be
classified as supplemental by data reviewers. Scientifically sound, non-guideline studies may be considered
acceptable  if  tests  were  conducted  according  to  OECD  guidelines  or  test  protocols  were  previously
reviewed by EPA.

Tier 1 of the risk assessment process consists of acute and chronic laboratory toxicity studies of individual
bees (adults and larvae) and is used as a basic screen. Depending on whether screening-level risk estimates
exceed EPA levels of concern (LOCs) and the extent to which additional information is needed to inform risk
management decisions, more refined (or higher-tier) studies, may be required. Higher-tier studies examine
the  whole  colony  rather  than  individual  bees  and  may  be  conducted  under  relatively  controlled
environmental conditions where colonies are confined to tunnels/enclosures (Tier 2), are left unconfined
and fed pesticide-spiked diets (Tier 2) or are allowed to freely forage in unconfined areas where pesticide
applications may be  made  (Tier 3). Appendix 2 contains example justification tables for exposure and
effects  studies  with  bees  which  are  intended  for  use  in  Registration  Review  Problem  Formulation
documents.

32  OECD.  1998a.  OECD  Guidelines  for  the  Testing  of  Chemicals.  Honeybees,  Acute  Oral  Toxicity  Test.  213.  http://www.oecd-
ilibrary.org/environment/test-no-213-honeybees-acute-oral-toxicity-test_9789264070165-en (last accessed 06/27/2016).
33 OECD.  2013.  OECD  Guidelines  for  Testing  Chemicals.  Honey  bee  (Apis  mellifera)  larval  toxicity  test,  single  exposure.  http://www.oecd-
ilibrary.org/environment/test-no-237-honey-bee-apis-mellifera-larval-toxicity-test-single-exposure_9789264203723-en
(last
accessed
06/27/2016).
34  OECD.1998b.  OECD  Guidelines  for  the  Testing  of  Chemicals.  Test  Number  214,  Acute  Contact  Toxicity  Test.  http://www.oecd-
ilibrary.org/environment/test-no-214-honey  bees-acute-contact-toxicity-test_9789264070189-en;jsessionid=43gvto47wnue9.delta    (last  accessed
06/27/2016).
35 OECD. 2007. Guidance document on the honey bee (Apis mellifera L.) brood test under semi-field conditions. Series on Testing and Assessment
No. 75. ENV/JM/MONO(2007)22.
36 Oomen, P. A. A. DeRuijter and J. Van der Steen. 1992. Method for honey bee brood feeding tests with insect growth-regulating insecticides. Bul
OEPP/EPPO Bulletin 22:  613 – 616.
12

Screening-level Toxicity Studies (Tier 1)

The additional toxicity data for honey bees at Tier 1 may apply to situations where exposure of bees to the
pesticide is considered likely. As noted earlier, the decision to recommend specific studies should be based
on  the  extent  to  which  bees  are  considered  likely  to  be  exposed  and  if  so,  the  extent  to  which  other
scientifically relevant information may not be available to address uncertainties regarding exposure and
effects  to  bees.  For  pesticides  where  acute  or  chronic  toxicity  is  not  expected,  based  on  other  lines  of
evidence (e.g., mode of action, toxicity data for other related taxa), the assessor may determine that a limit
test37 according to OCSPP or OECD guidelines is appropriate prior to performing a definitive test. If the
limit dose causes increased mortality to bees, then a definitive test would be triggered. Use of a limit test
for the chronic larval toxicity test may be less labor intensive studies compared to the other Tier 1 bee
toxicity tests.  If there are data to indicate that a TEP is potentially more toxic than the TGAI and there is
reason to believe that bees may come directly in contact with the intact TEP, then testing of such formulated
products should be considered.

Acute Oral Adult Toxicity

The acute oral toxicity study with young (newly emerged) adult bees provides median lethal dose (LD50)
value for honey bees (A. mellifera) based on acute oral exposure following OECD test guideline 21338.
These data are used in conjunction with acute contact LD50 data obtained through OCSPP 850.302039 to
estimate the acute toxicity of the technical grade active ingredient (TGAI) to individual young adult honey
bees. The studies also provide slopes for the dose-response curves that can in turn be used for estimating
the  likelihood  of  individual  effects.  In  addition,  sublethal  effects  observed  in  the  study  (e.g.,  abnormal
behavior or movement) are used to further characterize effects following a single exposure to the technical
grade material. Data obtained from this study are used in estimating acute risk to individual adult bees based
on  ingestion  of  residues.  Risk  estimates  based  on  these  data  are  considered  along  with  other  lines  of
evidence (e.g., the likelihood of colony exposure and the potential magnitude of effect based on toxicity
data collected on individual bees) to determine whether higher-tier studies are needed at the whole colony
level.


37 Limit testing should ensure that the highest level tested accounts for high-end exposure levels that be encountered at the maximum application
rate of the compound. Tier 1 exposure modeling estimates based on the 2014 Bee Risk Assessment Guidance should be used in determining the
limit test.
38 Ibid OECD 1998a
39Ibid USEPA 2012a.
13

Acute Contact Adult Toxicity

This acute contact toxicity study with young (newly emerged) adult bees is currently an EPA guideline
(OCSPP 850.302040) as well as an OECD test guideline (OECD 21441). Data from the acute contact toxicity
test are used in conjunction with acute oral LD50 data obtained through OECD 21342 to estimate the acute
toxicity of the TGAI to individual young adult honey bees. The studies also provide slopes for the dose-
response curves that can be used for estimating the likelihood of individual effects. In addition, sublethal
effects observed in the study (e.g., abnormal behavior or movement) are used to further characterize effects
following  a  single  exposure  to  the  technical  grade  material.  Data  obtained  from  this  study  are  used  in
estimating acute risk to individual adult bees based on contact exposure. Risk estimates based on these data
are considered along with other lines of evidence to determine whether higher-tier studies are needed at the
whole colony level. For highly volatile chemicals used as fumigants, this test can be adapted to address
exposure through the vapor phase.

Acute Larval Toxicity

The 7-day single dose study with larval bees provides a 96-hr LD50 for larval bees following OECD test
guideline 23743. Data obtained from this study are used in estimating acute risk to individual larval bees
based on ingestion of residues. Risk estimates based on these data are considered along with other lines of
evidence to determine whether higher-tier studies are needed at the whole colony level.

10-day Adult Chronic Toxicity Study

The 10-day toxicity study with young adult bees (guideline under development by OECD) provides a no-
observed adverse effect level (NOAEL) and lowest-observed adverse effect level (LOAEL) for assessing
chronic effects.  Although the study focuses primarily on survival and growth (weight of bees), sublethal
effects on behavior and food consumption can be obtained as well. Data obtained from this study are used
in estimating chronic risk to individual adult bees. Risk estimates based on these data are considered along
with other lines of evidence to determine whether higher-tier studies are needed at the whole colony level.

21-day Larval Toxicity Study

Developing bee brood (i.e., larvae and pupae) can be exposed to the active ingredient through residues
brought back to the colony by worker bees foraging in areas where the pesticide has been applied. While
larvae are typically fed royal or brood jelly during their early stages of development, worker bee (females)
and drone (males) larvae are also fed pollen/honey (bee bread) directly by in-hive nurse bees. The 21-day
larval toxicity study (guidance under development by OECD) provides chronic toxicity data on developing
bee brood, expressed in terms of a 21-day no-observed adverse effect level (NOAEL) and lowest-observed
adverse effect level (LOAEL), for assessing chronic effects. Effects on survival and development (adult
bee  emergence  and  body weight) from  repeat  exposures  to  the  active  ingredient  are used  in  estimating

40Ibid USEPA 2012a.
41 Ibid OECD 1998b.
42 Ibid OECD 1998a
43 Ibid OECD 2013.
14

chronic risk to individual brood. In some cases, it may be possible to document food consumption by larvae
during the feeding component of the study. Risk estimates based on data from this study  are considered
along with other lines of evidence to determine whether higher-tier studies are needed at the whole colony
level. Special considerations for the design of the 21-day larval feeding study are provided in Appendix 3.
When all of the Tier 1 data are not available to evaluate potential exposure and effects to bees, it may be
difficult  to  develop  suitable  mitigation  measures  for  some  compounds  (e.g.,  systemic  insecticides)
especially when the use is on an indeterminate blooming plant (e.g., cotton, cucurbits) which is attractive
to pollinators. If the EPA cannot evaluate the potential exposure and effects to bees, EPA may not be able
to make the necessary determination under FIFRA to register the pesticide or the new use.

Tier 2 Toxicity Testing

As  is  the  case  with  current  data  requirements  for  pollinators,  the  need  for  Tier 2  studies to  more  fully
characterize risk is based on the outcome of the screening-level assessment where acute and/or chronic risk
LOCs have been exceeded for bees. Bees are likely to be impacted if exposed to pesticides in various use
settings. For social bees, pesticide residues may be transferred to pollen and/or nectar of treated plants and
subsequently brought back to the hive and may adversely affect developing brood (egg, larvae, and pupae)
and adult bees. Screening-level (Tier 1) studies of individual bees are not meant to fully address possible
effects and/or exposure to pesticide residues at the colony-level, and for many pesticides, assessing effects
at the colony-level may not be necessary (e.g., when RQs do not exceed LOCs or when the potential for
exposure can be mitigated). Because of the potential for pollen and nectar to be contaminated with pesticide
residues,  and  subsequently  brought  back  to  the  hive,  it  may  be  important  on  a  case-by  case  basis  to
determine whether bee colonies may be negatively affected under relatively controlled exposure conditions
of  a  semi-field  study.  In  addition to  providing  effects  data,  these  studies  can  provide  data  on  pesticide
residues in pollen/nectar of treated plants.

Semi-field Testing with Honey Bee Colonies

If screening-level RQ values exceed the acute risk LOC (RQ ≥0.4) and/or chronic risk LOC (RQ≥1.0) and
depending on the need for additional information to characterize risk, higher-tier studies may be required
to examine potential effects at the colony level. At Tier 2, semi-field studies are conducted under relatively
controlled conditions (i.e., through use of enclosures/tunnels or outdoor feeding studies) to better ensure
that  bees  are  confined  to  the  treatment  area  and  that  exposure  has  taken  place.  Depending  on  the  risk
management question, semi-field studies can be conducted with TEP at the maximum application rate on a
pollinator-attractive  crop  or  multiple  exposure  levels  can  be  tested  to  enable  the  development  of
concentration-response data.

15

Honey Bee Brood Study (OECD 75)

Although a general study guidance for conducting a semi-field study is still under development, the OECD
75 guidance document on honey bee brood testing44, and the European and Mediterranean Plant Protection
Organization (EPPO) 170 describe basic semi-field study elements that should be considered.

In a tunnel study, there is typically a pesticide exposure period in the tunnel and an extended observation
period when test bees are allowed to freely forage from the landscape. While typically honey bee colonies
can only be maintained within enclosures for a limited exposure time (~10 days), these colonies may be
monitored  following  their  removal  from  the  enclosure  to  evaluate  chronic  effects  resulting  from  the
exposure period or delayed exposure from ingestion of stored pollen/nectar. However, the tunnel studies
are conducted with smaller colonies (referred to as nucleus “nuc” colonies)  and can only accommodate
relatively short exposure periods in the tunnel (e.g., up to 10 days or so) due to confinement-related stress
on the bees. If overwintering is an additional measurement endpoint (not identified in OECD 7545), the
colonies  must  be  provided  appropriate  time  and  forage  to  buildup  sufficiently  to  test  their  ability  to
overwinter  successfully.  Typical  endpoints  measured  in  tunnel  studies  include  adult  mortality,  flight
activity, brood development, hive strength (numbers of adult bees and brood; food reserves), and abnormal
behavior. These  endpoints are  typically  expressed in terms  of the  pesticide application rate  used  in  the
study, although measurement and expression of results in terms of measured pesticide residues may also be
conducted.

Feeding Studies

The feeding study methodology described by Oomen et al. 199246 and the extended-feeding field study
design proposed in the SAP White Paper47 may also be considered useful for assessing the potential effects
of pesticides on bees at the colony level. Rather than restricting bees to tunnel enclosures with a treated
crop, colonies are unrestricted and fed food sources spiked with known concentration of pesticides. The
amount of pesticide in the diet and the quantity of diet consumed by the bees can be monitored to provide
an estimate of an overall amount of pesticide  “dose” consumed in hives. These studies  are intended to
provide a NOAEC and LOAEC based on a range of measurement endpoints including colony strength, i.e.,
numbers of adult bees and brood (eggs, larvae, and pupae) covering each frame of the colony, as well as
sublethal endpoints (e.g., foraging behavior). Unlike the typical tunnel study designs, feeding studies are
designed  to  provide  a  dose-response  relationship  between  pesticide  residues  in  diet  and  effects  on  the
colony. The NOAEC and LOAEC values from feeding studies can then be compared to pesticide residues
measured  in  pollen  and/or nectar  of  crops  to qualitatively  characterize  risk  and identify  risk  mitigation
options. Furthermore, feeding studies can provide information on the effects to honey bees over longer
durations of exposure compared to tunnel studies. However, it is worth noting that the feeding study design
also has some uncertainty with respect to how well it mimics actual forage activity and pesticide exposure
experienced by colonies with actual pesticide-treated crops. Considerations for the design of the colony
feeding study are provided in Appendix 4.


44 Ibid OECD. 2007.
45 Ibid OECD. 2007.
46  Ibid Oomen, et al., 1992.
47 Ibid USEPA 2012
16

Tier 3 Toxicity Testing

As is the case with current data requirements for pollinators, whether Tier 3 studies may be necessary to
fully characterize risk is based on the outcome of the screening-level assessment (Tier 1) where acute and/or
chronic risk LOCs have been exceeded for terrestrial invertebrates and where Tier 2 studies either under
semi-field tunnel conditions and/or feeding studies have indicated potential adverse effects at the colony
level. Available toxicity studies from lower-tier studies may not address uncertainties related to possible
effects and/or exposure to pesticide residues at the colony-level under actual pesticide use conditions and
where specific uncertainties regarding the likelihood of exposure and/or effects remain. Full-field studies
also provide an opportunity to measure residues in pollen and nectar as well as various matrices (beebread,
honey, wax) within the colony to obtain a more realistic understanding of exposure. Because EPA guideline
850.3040  is  relatively  broad,  additional  information  on  Tier  3  study  design  elements  to  consider  when
recommending/reviewing such studies are provided in Appendix 1.

Full-field Testing with Honey Bee Colonies

If Tier 2 semi-field studies indicate a likelihood of adverse effects at the colony level, then Tier 3 studies
with TEP may be needed to address specific uncertainties that are identified in the lower-tier studies. With
each progressive tier, the study design should be increasingly refined to address specific questions while
the study is increasingly realistic, i.e., representative of actual use conditions and likely exposure scenarios.
Therefore, the full-field study protocol cannot be standardized given that it is intended to address specific
uncertainties identified in lower-tier studies. In general, full-field studies offer the advantage of capturing
exposure and effects of a pesticides on honey bee colonies under real-world conditions, since bees are free
to forage for pollen and nectar without constraints or supplemental feeding. However, careful design of the
field  study  is  necessary  to  ensure  that  the  range  of  exposures  expected  in  agricultural  ecosystems  are
adequately represented. Historically, many full-field studies have used treated fields of relatively small size
which tend to underestimate honey bee exposure in larger agriculturally- dominated ecosystems where a
large percentage of the crop may be treated with the pesticide of interest. Appendix 1 of this document
provides study design elements to consider in Tier 3 studies.

Table 2 lists the additional bee testing data described above that may be required on a case-by-case basis
and their respective triggers. When determining whether data listed in Table 2 are necessary, the table can
provide useful information on study tiers and triggers. As noted previously, the decision to recommend
additional data should be based on whether exposure of bees is considered likely and/or whether other
scientifically  relevant  information  may  be  available  to  address  uncertainties.  As  discussed  in  the  risk
assessment guidance48, any proposed mitigation measures should be evaluated prior to determining whether
additional  data  are  recommended.  Additional  considerations  may  include  an  analysis  of  the  benefits
associated with the proposed chemical/use as well as the alternatives and their associated risks.


48 Ibid USEPA 2014.
17

Table 2. Additional Requirements for Bee Exposure and Effects Testing.a b

Study Type
Test substance
Table Note No.
Study
Non-Guideline Study  Honey bee adult acute oral toxicity
TGAI
1
(Tier 1) (c)
Non-Guideline Study  Honey bee larvae acute oral toxicity
TGAI
1
(Tier 1) (d)
Non-Guideline Study  Honey bee adult chronic oral toxicity
TGAI
1
(Tier 1) (e) (g)
Non-Guideline Study  Honey bee larvae chronic oral toxicity
TGAI
1
(Tier 1) (e) (g)
Non-Guideline Study  Semi-field testing for pollinators (tunnel or colony
TEP (tunnel) or
2
(Tier 2) (f) (g)
feeding studies)
TGAI (feeding)
Definitions: TGAI = Technical Grade of the Active Ingredient; TEP = Typical End-Use Product
(a) Recommendations for bee toxicity data may be modified for certain types of outdoor residential uses for which exposure is
considered extremely limited (e.g., crack and crevice treatment, spot treatment, etc). In such cases, acute toxicity data may still
be warranted but chronic toxicity data may be of limited value in the risk assessment.
(b) For greenhouse uses that involve bee pollination, Tier 1 and Tier 2 bee exposure and effects data may be required.
(c) Honey bee acute oral toxicity test protocol available through OECD TG 213.49  For aquatic uses, acute oral toxicity data are
needed to evaluate exposure of bees through drinking water and in evaporative cooling of the hive and for exposure through
systemic transport into food items (pollen/nectar).
(d) Honey bee acute larval toxicity test protocol available through OECD TG 237.50
(e) Draft test protocols are currently being finalized through the OECD..
(f) Semi-field tunnel study protocol available through OECD Guidance 75.51
(g) Study protocol should be submitted for review prior to conduct of the study.
Test Notes:
1.  Data using the TGAI are required to support all outdoor end-use product uses. Data are generally not required to support
end-use products in the form of a gas, a highly volatile liquid, a highly reactive solid, or a highly corrosive material. For
greenhouse use patterns, data are required for crops that require pollination (e.g., tomatoes); for aquatic use patterns, data
are required if bees are likely to be exposed as a result of the proposed use.
2.  Tier 2 studies may be required pending the results and evaluation of Tier 1 studies. Tier 2 studies may be required if the
ratio of the EEC and larval or adult bee acute LD50 >0.4 or the ratio of the EEC and chronic NOAEC >1. Tier 2 may be
required  if  data  from  other  sources  (Experimental  Use  Permit  program,  university  research,  open  literature,  registrant
submittals, adverse effect incident reports, etc.) indicate the potential to adversely affect bee colonies, especially effects
other than acute mortality (e.g., reproductive, behavioral, etc.). Tier 2 studies may also be required if data derived from
studies with terrestrial arthropods other than honeybees indicate potential chronic, reproductive, or behavioral effects.

When determining whether additional data are necessary for risk characterization, it is important that the
assessor consider the nature of any uncertainties from existing data for the chemical or similarly structured
chemicals. As noted earlier, if there are data to indicate that a TEP is potentially more toxic than the TGAI
and there is reason to believe that bees may come directly in contact with the intact TEP, then testing of
such  formulated  products  should  be  considered.  Also,  as  is  the  current  practice,  the  additional  data,  if
required, would be tiered. At Tier 1 (screening-level), the focus is on laboratory-based studies of acute and
chronic exposure with individual bees (adults and larvae). Conditioned on the outcome of these laboratory
studies and the likelihood of exposure, semi-field and full-field studies may be required where the focus is
on whole honey bee colonies. As the assessment process is refined (i.e., moving to higher tier studies), tests
are intended to reflect increasingly realistic exposure conditions and to address specific risks/uncertainties

49 Ibid OECD 1998a.
50 Ibid OECD 2013.
51 Ibid OECD 2007.
18

(e.g., decreased brood production) identified in the lower-tier studies. As noted in Tables 1 and 2, higher-
tier testing at the semi-field and full-field level are typically conducted with TEP; however, feeding studies
(Tier 2) are usually conducted with the TGAI but may also be conducted with the TEP when bees may be
orally exposed to TEP.

There may be situations in which registrants/applicants submit/request the use of surrogate species such as
aquatic invertebrates to serve as a means of estimating risk to bees when bee-specific data are not available.
At the present time, information is lacking on the ability of toxicity data from other taxa to predict acute or
chronic toxicity of pesticides to adult and larval honey bees. Specifically, it is not known how well pesticide
toxicity data for aquatic invertebrates (e.g., Daphnia, mysid shrimp) or other terrestrial arthropods (e.g.,
parasitic wasps) are correlated with toxicity to honey bees. In addition, predictive toxicity tools based on
chemical structure (e.g., quantitative structure activity relationships [QSARs]) or toxicological mechanisms
are  either  not  available  or  in  the  early  stages  of  development.  Further  exploration  of  these  and  other
predictive toxicity tools is expected as additional data become available on the comparative toxicity of
pesticides with different modes of action to bees and related taxa. As with any recommendations made by
risk assessors for additional toxicity testing, the ultimate decision to require testing is based on discussion
with the risk manager and the need for additional data to inform the regulatory When appropriate, risk assessors
may determine it is appropriate to bridge toxicity data for bees based on other pesticides with the same
mode(s) of action and similarity in chemical structure.
3.4.   USEPA Residue Chemistry Requirements for Pollen and Nectar (Subpart O)
In addition to the bee-specific effects data, other data may be helpful in determining whether a pesticide
may have the potential to cause adverse effects. The next section discusses how residue chemistry data may
be helpful in this regard.

Bees may be impacted if exposed to pesticide residues in various use settings. As noted earlier, pesticide
residues may be transferred to pollen and/or nectar of treated plants and subsequently brought back to hive
where all life stages of bees may be exposed. For some pesticides, the quantification of pollinator-relevant
residues in treated flowering plants should be measured, since pollinators will be exposed to residues from
either current or prior season applications (due to the potential for residues to accumulate in plants and
trees).  Residues  in  edible/transportable-to-hive  parts  of  treated  trees  and  plants,  including  (where
appropriate), but not limited to, guttation water, sap/resins, whole plant tissue (e.g., leaves, stems), as well
as  blooming,  pollen-shedding,  and  nectar  producing  parts  (i.e.,  flowers  and,  if  present,  extra-floral
nectaries) of plants may inform the potential for exposure and subsequent risk.

Measured residues in pollen and nectar can serve as a means though which screening-level RQs may be
refined.  Initially,  risk  estimates  are  based  on  exposure  values  generated  using  conservative  models  or
default values. However, risk estimates can be refined using measured residue values in pollen and/or nectar
collected by bees. Such data may be available by modifying existing residue chemistry data requirements,
such as the magnitude of residue crop trial (OCSPP 860.150052) and the field rotational crop trials (OCSPP
860.190053); alternatively, residue data for pollen and nectar for specific crops and methods of application
may be necessary on a case-by-case basis. Table 3 depicts selected pertinent test requirements from the 40

52 USEPA. 1996. Residue Chemistry Test Guidelines OPPTS 860.1500. Crop Field Trials. EPA 712-C-96.183. August 1996.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-860-residue-chemistry-test-guidelines (last accessed 06/27/2016).
53 USEPA. 1996. Residue Chemistry Test Guidelines OPPTS 860.1900 Field Accumulation in Rotational Crops. EPA 712-C-96-189.
http://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-860-residue-chemistry-test-guidelines (last accessed 06/27/2016).
19

CFR Part158 Subpart O (Residue Chemistry §158.141054).  For studies examining residues in pollen and/or
nectar, protocols should be submitted for EPA review prior to initiating the studies.

Table 3. Requirements for Residue Data Similar to Existing Requirements in 40 CFR Part 158
Subpart O.
Use Pattern
Guideline
Test
Test Note
Study Type
Terrestrial
Number
Aquatic  Greenhouse
Indoor
Residential  substance
No.
Food or
Food
Food
Food
Outdoor
Feed
Magnitude of residue
860.1500
Crop field trials
R
R
R
CR
CR
TEP
a
Field rotational
860.1900
CR
CR
NR
NR
NR
TEP
a
crops
Definitions: R = Required; CR = Conditionally Required; NR = Not Required; TGAI = Technical Grade of the Active Ingredient; TEP = Typical
End-Use Product
a see 40CFR158 §158.1410;  http://www.ecfr.gov/cgi-bin/text-
idx?SID=167bce653abc0770ce9748e9d28ff832&mc=true&node=sp40.24.158.o&rgn=div6 (last accessed 06/27/2016).
Table  4  below  shows  data  that  may  be  necessary  on  a  case-by-case  basis  for  characterizing  risk  from
residues in pollen and nectar. Since risk is a function of both exposure and toxicity, measured residues in
various plant matrices can provide exposure data to refine risk estimates. These data can also be useful in
determining uptake and decline curves for residues of concern in pollen/nectar and for determining the
extent to which a compound is distributed systemically. Alternatively, data on residues in pollen and nectar
may be available through the ecological effect studies where the pesticide is applied under semi- and full-
field  conditions  (Tables  1  and  2).  As  with  other  environmental  fate  studies  where  the  registrant  must
demonstrate that the methods of chemical analysis are appropriate (i.e., reliable and sensitive), to support
residue  analysis  in  pollen  and  nectar,  the  registrant  must  provide  evidence  that  suitable  environmental
chemistry  methods  (ECM)  with  independent  laboratory  validation  (ILV)  have  been  used  to  quantify
residues. It is important to keep in mind that sampling colonies for pollen, nectar, wax and beebread can be
destructive to the colony as food reserves and comb are collected as samples. Depending on the sample
sizes and the frequency of sampling, such efforts may be disruptive to studying the adverse effects on the
bees/colony as a whole. Depending on the study design, separate colonies for collecting residue exposure
data may be needed where pollen traps are used to collect incoming pollen from the legs (corbicula) of
forager bees or nectar from the honey stomach of forager bees. Pollen and/or nectar (beebread and/or honey)
collected from  the  comb  may  require  that  the  desired  sample  is  gouged  from  the  comb  until  sufficient
sample  size  is  obtained.    Additional  information  on  exposure  study  design  elements  to  consider  when
recommending/reviewing such studies is provided in Appendix 5.


54 CFR40. 2016. Title 40 (Protection of Environment), Part 158 (Data Requirements for Pesticides), Subpart O (Residue Chemistry) §158.1410
(Residue chemistry data requirements table. http://www.ecfr.gov/cgi-
bin/retrieveECFR?gp=&SID=fc0257afaa2feb94488f8f1c4af6d3e6&n=pt40.24.158&r=PART&ty=HTML#se40.24.158_12150 (last accessed
06/27/2016).
20

Table 4. Requirements for Residue Data Measured in Pollen and Nectar.
Study
Study Type
Test substance  Test Note No.
Non-Guideline Study (Tier 2)  Field trial of residues in pollen and nectar
TEP
1, 2
Definitions:; TEP = Typical End-Use Product
1.Field studies that quantify pesticide residues in pollen/nectar may be required to refine screening level exposure estimates, depending on the results
and evaluation of Tier 1 studies. Pollen and nectar residue studies may be required if the ratio of the EEC and larval or adult bee acute LD50 >0.4
or the ratio of the EEC and chronic NOAEC >1. Incident data and/or compelling open literature studies can also serve as rationale for requiring
pollen  and  nectar  residue  studies.  These  data  can  be  collected  at  any  point  during  the  tiered  process;  prior  consultation  with  the  Agency  is
recommended to determine when to collect the data, and test protocols must be submitted for Agency review prior to initiation of the study. For
greenhouse use patterns, data are required for crops that require pollination (e.g., tomatoes); for aquatic use patterns, data are required if bees are
likely to be exposed as a result of the proposed use (e.g., riparian vegetation).
2Protocol should be submitted for EPA review prior to initiating study.

4. Non-Conventional Pesticides
Some of the studies noted in this paper may be useful for reviewing effects of other (non-conventional)
pesticides, but how and whether they will be appropriate will require further consideration. Other types of
pesticides  may  vary  from  conventional  pesticides  in  their  use  patterns,  modes  of  action,  likelihood  of
exposure,  and  other  unique  characteristics.  The  following  sections  explain  EPA’s  current  approach  to
evaluating effects of non-conventional pesticides on honey bees and other pollinators.
4.1
Antimicrobial Pesticides
For  antimicrobial  pesticides,  exposure  to  pollinators  may  result  from  compounds  used  as  wood
preservatives  or  any  product  which  can  be  used  “for  beehive  applications  when  the  beehive  (empty  or
occupied) may be treated”. In 40 CFR Part 158 Subpart W55, the Tier 1 studies [honey bee acute contact
data (OCSPP 850.3020) and toxicity of residues to honey bees (OCSPP 850.3030)] are required for all
wood preservatives and conditionally required for products used for beehive applications. The acute contact
study is routinely required of all wood preservatives and for hive treatments. The toxicity of residues study
is intended to provide risk information for hives constructed of treated wood and is rarely required since
EPA’s  Biological  and  Economic  Analysis  Division  (BEAD)  provided  information  that  hives  are  not
constructed of treated wood because the potential exposure of wood preservatives to bees is too high.

The additional studies listed in Table 2 are not applicable to antimicrobial compound use patterns because
exposure from antimicrobial uses is expected to be minimal. Honey bees may rest on treated surfaces, but
would not be attracted to feed or to gather treated materials for transport to the hive.
4.2
Biochemical Pesticides
For  biochemical  pesticides,  exposure  to  pollinators  may  result  primarily  from  foliar  ground  and  aerial
applications, as well as from products used within beehives to control pathogens or parasites. In 40 CFR

55 CFR40. 2016. Title 40 (Protection of Environment), Part 158 (Data Requirements for Pesticides), Subpart W (Antimicrobial Pesticide Data
Requirements), §158.2240 (Non-target organisms.
http://www.ecfr.gov/cgi-bin/text-idx?SID=fc0257afaa2feb94488f8f1c4af6d3e6&node=sp40.24.158.w&rgn=div6  (last accessed 06/27/2016).
21

Part 158.2060 Subpart U56) Tier 1 non-target insect testing (OCSPP 880.435057) is required for all use sites
(except indoor use sites) “depending on pesticide mode of action, method and timing of application and
results of any available efficacy data. Typically, honeybee acute toxicity testing (850.3020) satisfies this
requirement, however, additional non-target insect species” (OCSPP 850.304058; a Tier 3 study)  “may
have to be tested if necessary to address issues raised by use patterns and potential exposure of important
non-target  insect  species  (e.g.  endangered  species).”  The  honey  bee  toxicity  of  foliar  residues  study
(OCSPP 850.303059) is required on a case-by-case basis, dependent upon the route of exposure. In addition,
a honey bee acute oral toxicity as shown in Table 2 may be necessary, on a case-by-case basis, if the active
ingredient is systemic within treated plants and is likely to result in residues in pollen or nectar. The honey
bee  larvae  acute  oral  toxicity  testing  as  shown  in  Table  2  may  be  necessary  on  a  case-by-case  basis
depending upon the route of exposure to honey bee larvae.

Additional non-guideline  chronic toxicity  testing  is  not considered  necessary  due  to the  nature  of  most
biochemical pesticide active ingredients which are applied at relatively low rates and are non-persistent in
the environment. Chronic oral toxicity and chronic contact toxicity studies may be considered in the future,
on  a  case-by-case  basis,  should  a  new  biochemical  pesticide  active  ingredient  be  demonstrated  to  be
persistent, or if chronic exposure resulting from repeated applications would indicate the necessity for such
studies.
5.3
Microbial Pesticides
Pollinators  may  be  exposed  to  microbial  pesticides  through  both  contact  and  oral  routes,  although  the
importance of each route to toxicity or pathogenicity varies among active ingredients. Current microbial
pesticide data requirements that are specific to pollinators are described in 40 CFR Part 158 Subpart V60,
and include Tier 1 honey bee testing (OCSPP 885.438061) and Tier 3 simulated or actual field testing with
insect pollinators (OCSPP 850.304062). Tier 1 testing with honey bees is intended to examine the potential
for both toxic and pathogenic effects, and is required for all aquatic and terrestrial food/feed and non-food
uses, forestry uses, and outdoor residential uses. Tier 3 testing is conditionally required depending on effects
observed in testing at lower tiers.
The Microbial Pesticides Branch (MPB) of the Biopesticides and Pollution Prevention Division (BPPD)
recognizes the importance of considering additional pollinator data requirements with the goal of ensuring
consistency within OPP and improvements to pollinator testing and risk assessment. Additional attention
to bee effects testing is necessary for microbial pesticides due to their unique nature and modes of action.

56 CFR40. 2016. Title 40 (Protection of Environment), Part 158 (Data Requirements for Pesticides), Subpart U (Biochemical Pesticides), §158.2060
(Biochemical  pesticides  nontarget  organisms  and  environmental  fate  data  requirements  table).
http://www.ecfr.gov/cgi-
bin/retrieveECFR?gp=&SID=fc0257afaa2feb94488f8f1c4af6d3e6&n=pt40.24.158&r=PART&ty=HTML#sp40.24.158.u      (last  accessed
06/27/2016).
57 USEPA. 1996. Biochemicals Test Guidelines. OPPTS 880.4350. Nontarget Insect Testing. Office of Chemical Safety and Pollution Prevention
formerly  the  Office  of  Prevention,  Pesticides  and  Toxic  Substances  (7101),  EPA  712-C-96-285.  February  1996.    http://www.epa.gov/test-
guidelines-pesticides-and-toxic-substances/series-880-biochemicals-test-guidelines (last accessed 06/27/2016).
58 Ibid USEPA 2012c
59 Ibid USEPA 2012b
60 CFR40. 2016. Title 40 (Protection of Environment), Part 158 (Data Requirements for Pesticides), Subpart V (Microbial Pesticides) §158.2150
(Microbial
pesticides
nontaget
organism
and
environmental
fate
data
requirements
table)
http://www.ecfr.gov/cgi-
bin/retrieveECFR?gp=&SID=fc0257afaa2feb94488f8f1c4af6d3e6&n=pt40.24.158&r=PART&ty=HTML#se40.24.158_12150  (last  accessed
06/27/2016)
61 USEPA. 1996. Microbial Pesticides Test  Guidelines OPPTS 885.4380. Honey Bee  Testing, Tier  I. Office of  Chemical Safety and Pollution
Prevention formerly the Office of Prevention, Pesticides and Toxic Substances  (7101). EPA 712-C-96-337. http://www.epa.gov/test-guidelines-
pesticides-and-toxic-substances/series-885-microbial-pesticide-test-guidelines (last accessed 06/27/2016).
62 Ibid USEPA 2012c
22

The MPB is considering these data needs and will develop future guidance for microbial pesticide pollinator
data requirements.
5.4
Plant-Incorporated Protectants
Pollinator data requirements for Plant-Incorporated Protectants (PIPs) are currently determined on a case-
by-case basis. The MPB does not anticipate changes to the current approach for PIPs at this time.

23

Appendix 1. Tier 3 Field Study Design Considerations

Tier 3 studies conducted under full-field conditions where bees are free foraging are intended to address
specific uncertainties/risks that have been identified in lower-tier studies. The design of these studies will
depend on the specific questions that need to be answered; therefore, it is not possible to define a single
study design or specific design elements that must be incorporated into every full-field study. Below are
elements that the risk assessor should consider; however, these are not intended to be prescriptive. Some of
these study design elements have also been identified in the EFSA guidance63 document. It is incumbent on
the chemical team to ultimately identify the study design elements that should be considered by the pesticide
registrant/applicant in developing a study protocol that is responsive to the Tier 3 study requirement.
Full-Field Pollinator Study Design Considerations
Application Conditions
  Maximum application rate
  Minimum reapplication interval
  Maximum number of applications
  Use of formulated end-product
  Application method
o  Foliar
o  Soil treatment
o  Seed treatment
o  Combination
  Suitable weather
o  Avoid applications when rain and/or high winds are predicted.
  Season

Test crop
  Attractive test bees
  Long bloom period to address concerns identified at lower tiers
  Large area to ensure majority of foraging on test crop
  Follow standard [local] agriculture practices

Colonies
  Package bees/new equipment to limit incidence of disease; if older colonies are used, they should
be as pest/disease free as possible.
o  Colonies should not be used if they have received any chemical treatments within last 4
weeks. Colonies suspected of having American foulbrood should not be utilized. Other
disease treatments should be reported.
o  Beekeeper standard practice for maintain colony health during study
  All treatments must be uniform across study colonies.

63 Ibid EFSA. 2013.
24

  Queen-right (healthy queen present); sister queens for each replicate.
  Acclimation  period:    2  months  minimum  to  establish  representative  age  distribution  in  newly
established hives;
  Homogeneous colony strength, brood pattern as close as possible
  If existing colonies are to be used; broad spectrum residue analysis in hive products (honey/nectar,
pollen, wax); must document low incidence of diseases/parasites.
  Size of the colonies may vary depending on the focus of the study and when the study is initiated.
Typically, each hive should at least 10,000 bees to cover 10 frames and include at least 5 brood
frames. Excessive food storage should be avoided.
  Colonies can be positioned in plots when test crops are blooming enough to minimize test bees
foraging on plants other than the test crop, e.g., 20-25% bloom

Study Design Considerations
Historically there has been difficulty in controlling the extent to which the free-foraging bees utilize the
treated crop or that treatment groups cross-over (i.e., return to colonies other than their own). Sufficiently
large field plots, if feasible, will overcome the cross-over issue between plots and problems of insuring
exposure to treated crops due to competing vegetation.
  Mean honey bee foraging distance 1.5 – 3 km with extreme distances of 10 km; average surface
area range 7 – 100 km2 (Medrzycki et al. 201364).
o  EFSA  201365 recommends  minimum  of  2  ha  to  provide  sufficient  flowers  and  support
exclusive foraging; Medrzycki et al. 2013 recommends minimum of 5 ha.
  Suitable  crop  that is  representative  of  actual  use;  good  source  of  both  pollen  and  nectar, (e.g.,
phacelia  (Phacelia  tanacetifolia),  canola/oilseed  rape/mustard  (Brassica  napus,  Brassica  rapa,
Sinapis alba, Brassica juncea, or Brassica nigra), buckwheat (Fagopyrum esculentum))
o  Pollinator-attractive
  Account for crops/alternative forage within 3 km of colonies.

Distance of treated crop from other nectar producing plants is essential to insure exposure and must be
documented.
  Pollen traps should be used to demonstrate extent to which bees have foraged on treated crop.
  Pollen identification (palynological analysis) may be used to insure origin of pollen
  Pollen/nectar collection for residue analyses
o  Collected by bees and sampled using pollen traps (corbicular pollen)
o  Collected directly from plants
o  Sampling of nectar forager honey stomachs
o  Sampling comb pollen/nectar

Minimum number of replicate colonies:  6 - 10 per treatment (Medrzycki et al. 2013); the number of
replicates per treatment will depend on the targeted magnitude of effects and desired statistical power.

64 Mwsezycki, P. H. Giffard, P. Aupinel, L. P. Belzunces, M-P. Chauzat, C. Classen, M. E. Colin, T. Dupont, V. Girolami, R. Johnson, Y. LeConte,
J. Lückmann, M. Marzaro, J. Pistorius, C. Porrini, A. Schur, F. Sgolastra, N. S. Delso, J van der Steen, K. Wallner, C. Alaux, D. G. Biron, N. Blot,
G. Bogo, J-L Burnet, F. Delbac, M. Diogon, H. El Alaoui, B. Provost, S. Tosi and C. Vidau. 2013. Standard methods for toxicology research in
Apis mellifera. Journal of Apicultural Research 52(4):  http://www.coloss.org/beebook/I/introduction (last accessed 06/27/2106).
65 Ibid EFSA 2013
25

Study duration should assess at least two brood cycles (42 days) to ensure brood is exposed to residues
stored in the colony (EFSA 2013).

Measurement Endpoints: depend on the risk hypothesis tested and the nature of uncertainties identified in
lower-tier tests. Possible measurement endpoints may include.
Adult Forage Bees
  Adult bee survival/longevity
  Adult bee foraging activity (visual counts of returning foragers; mark-and-recapture; calibrate Dead
Bee Dead Zone traps)
  Queen status over the course of the exposure

Colony health (disease/pest incidence)

Colony Strength
  Brood (quantify number of eggs; larvae, capped cells, pollen, honey/nectar cells)
  Monitoring of brood in a minimum of two staggered cohorts, mid-way and late in the exposure
period
  Adult longevity: measured by using 30 newly-emerged adult bees from each colony (minimum n=6
colonies/treatment) in a controlled laboratory cage experiment monitoring daily mortality
  Newly-emerged bee weights

Other potential endpoints include the following:
Overwintering Success
Fitness measure: Pathogen challenge (e.g., Nosema exposure) newly emerged bees
Assess the ability of colonies to re-queen themselves by removing all queens and determining the success
of each colony in rearing a replacement queen.
Documenting Exposure
  Residue analyses in pollen/nectar
  Residue analyses in bee carcasses
  Residue analyses in wax
  Foliar Residue analysis
  Measure total residues of concern (parent + degradate(s))
  Pollen source (palynology) to ensure bees have been foraging on target crop.

Suitable control bees (residue analyses to demonstrate lack of exposure). Utility of mark-and-recapture to
document drift of bees from treated colonies.

26

Appendix 2. Data Justification Tables for Non-Codified Exposure
and Effects Studies with Bees

The following data justification tables should be considered by OPP risk assessors when recommending
additional  bee  exposure  and  effects  studies  in  support  of  pesticide  review  actions  (e.g.,  Problem
Formulation documents under Registration Review).

Study Title: Tier 1 Honey bee Adult Acute Oral Toxicity
Rationale for Requiring the Data
Terrestrial  invertebrates  are  likely  to  be  impacted if exposed to  pesticides in  various use  settings. With
eusocial  bees,  pesticide  residues  may  be  transferred  to  pollen  and/or  nectar  of  treated  plants  and
subsequently brought back to the hive. Therefore, potential acute effects to adult honey bees and other insect
pollinators from oral exposure to some pesticides could exist. Currently available toxicity studies do not
address possible effects of oral exposure on adult terrestrial insect survival. Because of the potential for
pollen and nectar to be contaminated with pesticide residues, and subsequently brought back to the hive, it
is important to determine the acute oral toxicity of this compound to adult  honey bees and other insect
pollinators.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the honey bee as a surrogate test species. The guidance discusses Tier 1 laboratory-based acute oral toxicity
studies of individual adult bees as a critical component of the screening-level risk assessment process for
examining  potential  adverse  effects  from  specific  routes  of  exposure.  The  guidance  can  be  found  at:
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance.  Additional  guidance  on
the  honey  bee  oral  toxicity  test  design  can  be  found  in  OECD  Test  Guideline  213  (http://www.oecd-
ilibrary.org/docserver/download/9721301e.pdf?expires=1423074617&id=id&accname=guest&checksum
=2F0764FCB4DCF01D32382952A2E995C3)
Practical Utility of the Data
How will the data be used?
The  Tier  1  acute  oral  toxicity  data  on  adult  honey  bees  serve  as  a  foundation  for  the  screening-level
assessment of potential risk non-target organisms such as federally listed threatened or endangered and non-
listed terrestrial invertebrate insects, including pollinators, from acute oral exposures to pesticides. The data
will be used to reduce uncertainties associated with the risk assessment for terrestrial invertebrates and will
improve EPA’s understanding of the potential direct and indirect effects on a broad range of taxa. This study
will also provide information with which to compare whether oral toxicity estimates differ from contact
toxicity estimates obtained from other Tier 1 studies. If acute oral effects data for adult honey bees are not
available, risks to terrestrial insects from acute oral exposure will be assumed.

How could the data impact the Agency’s future decision-making?
The  data  will  inform  the  determination  required  under  FIFRA  or  the  ESA  as  to  whether  continued
registration of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely
to adversely affect listed threatened or endangered species and/or modify their designated critical habitat.
Without these data, EPA may need to presume risk, which will limit the flexibility of pesticide products to
comply with FIFRA and the ESA, and could result in use restrictions.

27

Study Title: Tier 1 Honey bee Larvae Acute Oral Toxicity
Rationale for Requiring the Data
Terrestrial  invertebrates  are  likely  to  be  impacted if exposed to  pesticides in  various  use  settings. With
eusocial  bees,  pesticide  residues  may  be  transferred  to  pollen  and/or  nectar  of  treated  plants  and
subsequently  brought  back  to  the  hive  where developing  larvae and  pupae  may  be  exposed. Therefore,
potential adverse effects to developing bees could result from exposure to pesticide residues. Available
toxicity studies do not address possible effects on brood (larvae and pupae) survival/development. Because
of the potential for pollen and nectar to be contaminated with pesticide residues, and subsequently brought
back to the hive, it is important to determine the acute toxicity of this compound to bee brood.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the honey bee as a surrogate test species. The guidance discusses Tier 1 laboratory-based acute toxicity
studies of individual honey bee larvae as a critical component of the screening-level risk assessment process
for  examining  potential  risks  from  specific  routes  of  exposure.  The  guidance  be  found  at:
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance.  Additional  guidance  on
larval  honey  bee  toxicity  test  design  can  be  found  in  OECD  Test  Guideline  237  (http://www.oecd-
ilibrary.org/docserver/download/9713171e.pdf?expires=1422485600&id=id&accname=guest&checksum
=D8E07C2B1DF77BF096C3B29F55BF86A7).
Practical Utility of the Data
How will the data be used?
The Tier 1 acute toxicity data on honey bee larvae serve as a foundation for the screening-level assessment
of potential risk to non-target organisms including federally listed threatened or endangered and non-listed
terrestrial  invertebrates,  including  pollinators,  and/or  modify  their  designated critical  habitat  from  acute
exposures to pesticides. The data will be used to reduce uncertainties associated with the risk assessment
for  terrestrial  invertebrates  and  will  improve  EPA’s  understanding  of  the  potential  effects  on  terrestrial
species and whether there is a differential sensitivity of larval bees relative to adult bees. If acute effects
data for larvae are not available, risks to terrestrial insects from acute exposure will be assumed.

How could the data impact the Agency’s future decision-making?
The  data  will  inform  the  determination  required  under  FIFRA  or  the  ESA  as  to  whether  continued
registration of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely
to adversely affect listed threatened or endangered species and/or modify their designated critical habitat.
Without these data, EPA may need to presume risk which will limit the flexibility of pesticide products to
comply with FIFRA and the ESA, and could result in use restrictions.

28

Study Title:  Tier 1 Honey Bee Adult Chronic Oral Toxicity
Rationale for Requiring the Data
Terrestrial invertebrates are likely to be impacted if exposed to pesticides in various use settings.  With
eusocial  bees,  pesticide  residues  may  be  transferred  to  pollen  and/or  nectar  of  treated  plants  and
subsequently brought back to the hive. Therefore, potential chronic effects to adult honey bees and other
pollinators from oral exposure to some pesticides could exist. Currently available toxicity studies do not
address possible lethal and sublethal effects of chronic oral exposure on adult terrestrial invertebrates and
will assist in determining whether the sensitivity of adult bees differs from that of earlier life stages. Because
of the potential for pollen and nectar to be contaminated with pesticide residues, and subsequently brought
back to the hive, it is important to determine the chronic oral toxicity of this compound to adult honey bees
and other pollinators.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the  honey  bee  as  a  surrogate  test  species.  The  guidance  discusses  Tier  1  laboratory-based  chronic  oral
toxicity studies of individual adult honey bees as a critical component of the screening-level risk assessment
process  for  examining  potential  risks  from  specific  routes  of  exposure.  The  guidance  can  be  found  at:
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance.  Although  study  design
elements for the chronic 10-day oral toxicity test with honey bees are similar to the OECD TG 213 acute
oral
toxicity
test
(
http://www.oecd-
ilibrary.org/docserver/download/9721301e.pdf?expires=1422484908&id=id&accname=guest&checksum
=C38495D2A570AC2216CFB1F223D24AA7), EPA requires that the proposed protocol for this study be
submitted for review and approval by EPA prior to initiating the test.
Practical Utility of the Data
How will the data be used?
The Tier 1 chronic toxicity data on adult bees serve as a foundation for the screening-level assessment of
potential risk to non-target organisms including federally listed threatened or endangered species and non-
listed terrestrial invertebrates, including pollinators, from chronic oral exposures to pesticides. The data will
be  used to  reduce  uncertainties associated  with  the  risk  assessment  for  terrestrial invertebrates  and  will
improve EPA’s understanding of the potential direct and indirect lethal and sublethal effects on a broad
range of terrestrial species, particularly insect pollinators and to determine whether adult toxicity differs
substantially from other life stages evaluated in other Tier 1 tests. If chronic oral effects data for adults are
not available, risks to terrestrial insects from chronic exposure will be assumed.

How could the data impact the Agency’s future decision-making?
The  data  will  inform  the  determination  required  under  FIFRA  or  the  ESA  as  to  whether  continued
registration of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely
to adversely affect listed threatened or endangered species and/or their designated critical habitat. Without
these data, EPA may need to presume risk which will limit the flexibility of pesticide products to comply
with FIFRA and the ESA, and could result in use restrictions.

29

Study Title:  Tier 1 Honey Bee Larvae Chronic Oral Toxicity
Rationale for Requiring the Data
Terrestrial  invertebrates  are  likely  to  be  impacted  if  exposed  to  pesticides  in  various  use  settings.  For
eusocial  bees,  pesticide  residues  may  be  transferred  to  pollen  and/or  nectar  of  treated  plants  and
subsequently brought back to the hive where larvae and pupae may be exposed. Therefore, potential effects
to developing bees could result from chronic exposure to pesticide residues. Available toxicity studies do
not address possible chronic effects on brood (larvae and pupae) survival. Because of the potential for pollen
and  nectar  to  be  contaminated  with  pesticide residues,  and  subsequently  brought  back  to the  hive,  it is
important to determine chronic larval/pupal toxicity and whether adult emergence is adversely affected.
This  study  will  provide  information  on  whether  honey  bee  larvae  differ  in  sensitivity  from  adult  bees
following chronic exposure.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the honey bee as a surrogate test species. The guidance discusses Tier 1 laboratory-based chronic toxicity
studies of individual honey bee larvae as a critical component of the screening-level risk assessment process
for  examining  potential  risks  from  specific  routes  of  exposure.  The  guidance  can  be  found  at:
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance. Additional information on
larval  honey  bee  toxicity  repeat  exposure  test  design  can  be  found  in  the  OECD  draft  guidance
(http://www.oecd.org/env/ehs/testing/Draft_GD_honeybees_rep_exp_for_2nd_CR_25_November_2013.p
df). Although study design elements for the chronic 21-day toxicity test with honey bee larvae have been
drafted, EPA requires that the proposed protocol for this study be submitted for review and approval by
EPA prior to initiating the test.
Practical Utility of the Data
How will the data be used?
The Tier 1 chronic toxicity data on bee larvae serve as a foundation for the screening-level assessment of
potential risk to non-target organisms including federally listed threatened or endangered and non-listed
terrestrial invertebrates, including insect pollinators, from chronic exposures to pesticides. These data will
be  used to  reduce  uncertainties associated  with  the  risk  assessment  for  terrestrial invertebrates  and  will
improve EPA’s understanding of the potential direct and indirect  lethal and sublethal effects on a broad
range of terrestrial species, particularly insect pollinators. These data will also assist in determining whether
early life stages of the bee differ in their sensitivity to pesticides relative to adults. If chronic effects data
for larvae are not available, risks to terrestrial insects from chronic exposure will be assumed.

How could the data impact the Agency’s future decision-making?
The  data  will  inform  the  determination  required  under  FIFRA  or  the  ESA  as  to  whether  continued
registration of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely
to adversely affect listed threatened or endangered species and/or modify their designated critical habitat.
Without these data, EPA may need to presume risk which will limit the flexibility of pesticide products to
comply with FIFRA and the ESA, and could result in use restrictions.

30

Study Title:  Tier 2 Semi-field Testing for Pollinators (tunnel studies)
Rationale for Requiring the Data
Tier 2 studies are conditional on the outcome of the screening-level assessment where acute and/or chronic risk
levels  of  concern  have  been  exceeded  for  terrestrial  invertebrates.  Terrestrial  invertebrates  are  likely  to  be
impacted if exposed to pesticides in various use settings. For eusocial bees, pesticide residues may be transferred
to pollen and/or nectar of treated plants and subsequently brought back to the hive and may adversely affect
developing brood (egg, larvae, and pupae) and adult bees. Screening-level (Tier 1) studies of individual bees do
not address possible effects and/or exposure to pesticide residues at the colony-level. Because of the potential
for pollen and nectar to be contaminated with pesticide residues, and subsequently brought back to the hive, it
is important to determine whether bee colonies may be negatively affected under relatively controlled exposure
conditions of a semi-field study. In addition to providing effects data, these studies can provide data on pesticide
residues in pollen/nectar of treated plants.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using the
honey bee as a surrogate test species. This guidance describes the tiered testing process and can be found at:
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance,  Additional  information  on
honey  bee  colony  studies  under  semi-field  conditions  can  be  found  in  the  OECD  Guidance  75
(http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono%282007%2922&doc
language=en). Due to the complexities of this study, EPA requires that the proposed protocol for this study be
submitted for review EPA prior to initiating the test.
Practical Utility of the Data
How will the data be used?
Tier 2 colony-level data will be used to assess potential risk to non-target organisms including listed and non-
listed terrestrial social invertebrate species and to determine whether effects observed in the screening-level (Tier
1) laboratory-based studies of individual bees are evident in colony-level studies under semi-field conditions.
The Tier 2 semi-field test of whole colonies is a relatively controlled study, i.e., bees are confined to a specific
area that is designed to represent potential field-level exposure and account for hive dynamics, which are not
achievable from other pollinator studies. This study will be used to determine whether adverse effects to insect
pollinators at the whole colony level, may result for the use of pesticides and will help to refine risk estimates
derived  in  the  screening-level  risk  assessment  for  beneficial  terrestrial  invertebrates.  Measured  residues  in
pollen/nectar  can  also  be  used  to  refine  risk  estimates  derived  from  model-based  or  default  values  in  the
screening-level assessment.

How could the data impact the Agency’s future decision-making?
The data will inform the determination required under FIFRA or the ESA as to whether continued registration of
a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely to adversely affect
federally listed threatened or endangered species or their designated critical habitat. Without these data, EPA
may need to presume risk which will limit the flexibility of pesticide products to comply with FIFRA and the
ESA, and could result in significant use restrictions.
31

Study Title:  Tier 2 Semi-field Testing for Pollinators (colony feeding studies)
Rationale for Requiring the Data
For  eusocial  bees,  pesticide  residues  may  be  transferred  to  pollen  and/or  nectar  of  treated  plants  and
subsequently brought back to the hive and may adversely affect developing brood (egg, larvae, and pupae)
and adult bees. Tier 2 feeding studies are conditional on the outcome of the screening-level assessment where
acute and/or chronic risk levels of concern have been exceeded for terrestrial invertebrates based on Tier 1
studies of individual bees. Feeding studies utilize free foraging bee colonies that are “dosed” with specific
quantities of test material and represent a means of ensuring exposure to the test material through spiked
pollen  and/or  sugar  solutions  fed  to  the  colony  while  still  allowing  the  bees  to  forage  freely.  Since  bee
colonies  are  not  confined  to  enclosures,  colonies  can  be  exposed  for  longer  duration  periods  without
subjecting the bees to stress that typically results from Tier 2 tunnel studies. Available toxicity studies of
individual bees (Tier 1) conducted to support screening-level assessments do not address possible effects
and/or exposure to pesticide residues at the colony-level. It is therefore important to determine whether bee
colonies may be negatively affected where bees are free foraging and have the option to collect/consume
alternative  forage  items  beyond  the  spiked  food.  Since  multiple  dose  levels  can  be  more  readily  tested,
feeding studies can help to define dose-response relationships at the whole colony level.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the  honey  bee  as  a  surrogate.  This  guidance  describes  the  tiered  testing  process  and  can  be  found  at::
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance. Additional information on
honey  bee  colony  feeding  studies  can  be  found  in  the  EPPO  Guidance  170
(www.nationalbeeunit.com/downloadDocument.cfm?id=191).  Although  study  design  elements  for  the
feeding study are available through the EPPO Guidance 170, EPA requires that the proposed protocol for
this study be submitted for review and approval by EPA prior to initiating the test.
Practical Utility of the Data
How will the data be used?
Tier 2 colony feeding data will be used to assess potential risk to non-target organisms including listed and
non-listed terrestrial social invertebrate species. The colony feeding study is designed to represent potential
field-level exposure and account for hive dynamics using longer duration exposure periods than are possible
in Tier 2 tunnel studies. This  study will be used to determine whether potential adverse effects to insect
pollinators at the whole colony level when bees are able to forage naturally beyond the spiked food. Results
from  the  feeding  study  will  help  to  refine  the  screening-level  risk  assessment  for  beneficial  terrestrial
invertebrates that were based on Tier 1 studies on individual bees. Since feeding studies can help to define a
dose-response relationship at the  colony  level,  the  studies  can  provide a  means of  determining  exposure
thresholds below which the likelihood of adverse effects on colonies may be low.

How could the data impact the Agency’s future decision-making?
The  Tier  2  colony-level  data  will  be  used  to  refine  screening-level  risk  estimates  derived  using  Tier  1
laboratory-based data on individual bees. The Tier 2 data will help to inform the determination required
under FIFRA or the ESA as to whether continued registration of a pesticide is likely to result in unreasonable
adverse effects to non-target species or is likely to adversely affect federally listed threatened or endangered
species or their designated critical habitat. Without these data, EPA may need to presume risk which will
limit the flexibility of pesticide products to comply with FIFRA and the ESA, and could result in significant
use restrictions.

32

Study Title:  Tier 3 Field Testing for Pollinators
Rationale for Requiring the Data
Tier 3 studies are conditional on the outcome of the screening-level assessment (Tier 1) where acute and/or
chronic risk levels of concern have been exceeded for terrestrial invertebrates and where Tier 2 studies either
under  semi-field  tunnel  conditions  and/or  feeding  studies  have  indicated  potential  adverse  effects  at  the
colony  level.  Available  toxicity  studies  from  lower-tier  studies  do  not  address  possible  effects  and/or
exposure to pesticide residues at the colony-level under actual pesticide use conditions and where specific
uncertainties regarding the likelihood of exposure and/or effects remain. Full-field studies also provide an
opportunity to measure residues in pollen and nectar as well as various matrices (beebread, honey, wax)
within the colony to obtain a more realistic understanding of exposure.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the  honey  bee  as  a  surrogate.  This  guidance  describes  the  tiered  testing  process  and  can  be  found  at::
http://www2.epa.gov/pollinator-protection/pollinator-risk-assessment-guidance. Additional information on
honey bee colony studies under full-field conditions can be found in the OCSPP 850.3040; useful guidance
is also available through OCSPP 850.2500 (Field Testing of Terrestrial Wildlife; http://www.epa.gov/test-
guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines)  Although design
elements for the full-field colony-level study are available through the 850.3040 and 850.2500, EPA requires
that the proposed protocol for this study be submitted for review and approval by EPA prior to initiating the
test; the protocol should attempt to address specific uncertainties identified in lower-tier studies.
Practical Utility of the Data
How will the data be used?
Tier 3 colony-level data will be used to further characterize potential risk to non-target organisms including
listed and non-listed terrestrial social invertebrate species and to refine screening-level risk estimates that
were based on individual bee responses. The semi-field test is a controlled study that is designed to represent
potential field-level exposure under relatively controlled conditions and account for hive dynamics, which
are not achievable from lower-tier pollinator studies. This study will be used to determine whether adverse
effects to insect pollinators at the whole colony level, may result for the use of pesticides and will help to
refine the screening-level risk estimates for beneficial terrestrial invertebrates. This study will also be used
to determine whether more refined (Tier 3) studies are needed to characterize risk.

How could the data impact the Agency’s future decision-making?
The data will inform the determination required under FIFRA or the ESA as to whether continued registration
of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely to adversely
affect federally listed threatened or endangered species or their designated critical habitat. Without these
data, EPA may need to presume risk which will limit the flexibility of pesticide products to comply with
FIFRA and the ESA, and could result in significant use restrictions.

33

Study Title:  Field trial of residues in pollen and nectar
Rationale for Requiring the Data
Terrestrial invertebrates are likely to be impacted if exposed to pesticides residues in various use settings.
Pesticide residues may be transferred to pollen and/or nectar of treated plants and subsequently brought back
to hive all life stages may be exposed. For some pesticides, the quantification of pollinator-relevant residues
in treated flowering plants is needed, since pollinators will be exposed to residues from either current or prior
season  applications  (due  to  the  potential  for  residues  to  accumulate  in  plants  and  trees).  Residues  in
edible/transportable-to-hive parts of treated trees and plants, including (where appropriate), but not limited
to, guttation water, sap/resins, whole plant tissue (e.g., leaves, stems), as well as blooming, pollen-shedding,
and  nectar  producing  parts  (i.e.,  flowers  and,  if  present,  extra-floral  nectaries)  of  plants  may  inform  the
potential for risk. Studies should be designed to provide residue data for crops and application methods of
concern.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the honey bee as a surrogate. This can be found at:  http://www2.epa.gov/pollinator-protection/pollinator-
risk-assessment-guidance. Since residue studies are intended to provide exposure data in multiple matrices
and  under  specific  application  conditions,  EPA  requires  that  the  protocol  is  submitted  for  review  and
approval by EPA prior to initiation of the study.
Practical Utility of the Data
How will the data be used?
Measured  residue  data  will  be  used  to  refine  conservative  estimates  of  pesticide  exposure  and  reduce
uncertainties associated with the Tier 1 exposure assessment by providing direct measurements of pesticide
concentrations  resulting  from  actual  use  settings.  Measured  residues  may  provide  a  more  realistic
understanding of exposure through contact or ingestion with which to calculate risk quotients for individual
bees  as  well  as  to  characterize  exposure  to  the  colony.  If  measured  residue  data  are  not  available,  risk
estimates  for  terrestrial  insects  will  be  based  on  model  generated  or  default  values  used  to  support  the
screening-level assessment.

How could the data impact the Agency’s future decision-making?
The data will inform the determination required under FIFRA or the ESA as to whether continued registration
of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely to adversely
affect federally listed threatened or endangered species or their designated critical habitat. Without these
data, EPA will have to rely on conservative estimates of exposure which may limit the flexibility of pesticide
products to comply with FIFRA and the ESA, and could result in use restrictions.

34

Study Title: Tier 1 Pollinator Acute Vapor Exposure Toxicity (modification of acute contact toxicity
test)
Rationale for Requiring the Data
Pesticide chemicals can come in the form of solids, liquids or gases. Some pesticides are highly volatile or
are gases (e.g., fumigants). Conducting toxicity testing based on contact and ingestion routes such as might
occur  with  liquid  or  solid  pesticides  is  not  appropriate  for  evaluating  the  toxicity  of  highly  volatile
compounds or gases. If environmentally-relevant concentrations are possible, such as may be the case for
most  pesticides  used  as  fumigants,  evaluation  of  the  impact  on  non-target  species,  such  as  terrestrial
invertebrates including pollinators, provides valuable information for mitigating that risk in the use labeling.
Therefore, to assess the toxicity of highly volatile pesticides and gases to terrestrial invertebrates, an acute
vapor exposure toxicity study is appropriate in lieu of the toxicity testing through other delivery methods.

The Office of Pesticide Programs has made available a guidance regarding ecological testing for bees using
the honey bee as a surrogate. These can be found at: http://www2.epa.gov/pollinator-protection/pollinator-
risk-assessment-guidance.  Design elements from Tier 1 laboratory-based studies of individual adult bees
(OCSPP 850.3020 and OECD TG 213, OECD TG 214) and larval bees (OECD TG 237 as well as draft
OECD guidance on chronic larval bee toxicity testing) may also provide useful information. EPA requires
that the proposed protocol for the inhalation study be submitted to EPA for review and approval prior to
initiating the study.

Practical Utility of the Data
How will the data be used?
Tier 1 data on individual bees serve as a foundations for the screening-level risk assessment process used
to  determine  the  potential  for  a  pesticide  (in  the  form  of  a  gas/vapor)  to  affect  non-target  terrestrial
invertebrates, including pollinators, in their environment. These data will be used to reduce uncertainties
associated with the risk assessment for terrestrial invertebrates and will improve EPA’s understanding of
the potential effects on terrestrial species. If inhalation toxicity data are not available, risks to terrestrial
insects from vapor exposure will be assumed.

How could the data impact the Agency’s future decision-making?
The  data  will  inform  the  determination  required  under  FIFRA  or  the  ESA  as  to  whether  continued
registration of a pesticide is likely to result in unreasonable adverse effects to non-target species or is likely
to  adversely  affect  federally  listed  threatened  or  endangered  species  or  their  designated  critical  habitat.
Without these data, EPA may need to presume risk which will limit the flexibility of pesticide products to
comply with FIFRA and the ESA, and could result in use restrictions.

35

Appendix 3. Laboratory Larval Study Design Elements

Laboratory-based studies with larval bees are described in the OECD Test Guideline 23766; however, this
design is for single dose studies where bees are euthanized after Day 9. An OECD draft guidance67 has been
developed for repeat dose exposure where the study is conducted for 21 days and is intended to extend
through adult bee emergence. Bees are fed treated diets from Day 3 through Day 6 and mortalities are
recorded from Day 4 to Day 8, Day 15 and Day 22. The study provides a NOAEC and/or EC50 for adult
emergence on Day 22 as well. Study conditions described in the draft guidance do not differ substantially
from OECD TG 237; however, some contract labs have had difficulty in achieving control mortality rates
of less than 20%. High mortality rates may in some cases result from contamination of the individual test
wells where fungi and/or bacteria overtake the well. In the development of brood, the digestive tract of the
larvae is not complete until roughly Day 9; once the digestive tract is complete, the organism will defecate
and this process is typically considered the initiation of pupation. The excrement may be the source of
contamination that increases mortality levels in these tests.

Minor modifications in the study design have been effective in reducing control mortality and are typically
intended to reduce the likelihood of well contamination. Protocols submitted for review and approval by
EPA should describe measures taken to reduce the likelihood of contamination in culture wells. One option
is to transfer larvae to clean culture wells on Day 9. During the transfer, individual test organisms can be
carefully cleaned with sterile physiological saline and gently blotted dry.

Concerns  regarding  diet  preparation  for  chemicals  with  limited  solubility  and/or  high  sorption
characteristics should be resolved through discussions with the registrant during protocol review.

Additional  study  designs  for  evaluating  the effects  of  chronic  exposure  on  bee  larval  development and
which extend through adult emergence are under development. Similar to evaluating data for other taxa,
risk assessors should consider the strengths and weaknesses of alternative study designs, i.e., non-guideline
studies, in providing data to evaluate potential effects.


66 Ibid OECD 2013.
67 OECD 2013b. OECD Draft Guidance Document Honey Bee (Apis mellifera) Larval Toxicity Test, Repeated Exposure.
http://www.oecd.org/env/ehs/testing/Draft_GD_honeybees_rep_exp_for_2nd_CR_25_November_2013.pdf
36

Appendix 4. Tier 2 Feeding Study Design Considerations

Study Overview:
The honey bee colony feeding study is designed to evaluate the effects of long-term exposure to various
concentrations  of  a  pesticide  in  a  food  source  for  honey  bees.  The  study  is  intended  as  a  Tier  2  study
consistent with the overall tiered approach of pollinator risk assessment as identified in the 2013 White
Paper and the 2014 Guidance on Assessing Pesticide Risks to Bees. Measurement endpoints relate to colony
strength, specifically in terms of brood abundance, adult population size, and the amount of food stores
(both  pollen  and  nectar)  within  the  hives.  These  measurement  endpoints  are  also  intended  to  provide
information on the concentrations of the pesticide that affect whole colonies due to their long-term exposure
to a pesticide in food sources for honey bees. Although not intended to be prescriptive, the study design
considerations described here are intended to help ensure that a no observed adverse effect level (NOAEL)
can  be  established  and  used  in  conjunction  with  field  residue  studies  to  qualitatively  characterize  the
potential toxicity of a pesticide to honey bees based on corresponding application rates to specific crops.
Past  study  designs  have  included  in-hive  feeders  with  untreated  sucrose  solutions  (control  hives)  or
pesticide-spiked sucrose solutions (treatment hives) over a 6-week period. Colony condition assessments
are observed prior to and during exposure period as well as after overwintering.

Pesticide Treatments:
Past feeding study designs have included feeding spiked sucrose solutions and/or spiked pollen. Depending
on the nature of the pesticide exposure and toxicity, one or both of these dietary media may be used for
pesticide exposure. Generally, pesticide treatments should include a negative control and at least five test
concentrations. Selected concentrations should bracket the Tier 1 estimates for residues in pollen and nectar,
take into consideration measured residue data from field studies and incident reports, and include the lowest
concentration  where  sublethal  effects  were  noted  in  acute  or  chronic  toxicity  studies  with  honey  bees.
Treatment  levels  should  be  chosen  to  ensure  that  a  NOAEL  and  lowest  observed  adverse  effect  level
(LOAEL) are obtained.

The study design should make every effort to minimize variability between the exposure levels for each of
the hives (see potential sources of variability below). It is important to confirm the test material exposure
levels in the food prior to replenishment, as well as confirm test material stability during the study. The
volume/mass  of  the  new  and  old  treatment  solutions/pollen  paddies  at  replenishment  should  also  be
reported.

Initial Hive Conditions:
At initiation, each hive should consist of an appropriate number of bees to ensure that the colony would be
sizable enough to have overwintering success (e.g., 10,000 bees (3 lb package)). The size of the initial
colony will vary based on geographic location; therefore it is important to follow local beekeeping practices.
Each hive should consist of one hive box with an initial 8-frames, and an empty box above for the feeder.
After the exposure phase of the study, hive boxes should be expanded (i.e., adding more frames or boxes)
as appropriate to facilitate food storage and colony growth.

It is expected that the use of new colonies (i.e., single box) earlier in the season and new hive equipment
would  minimize  the  infestation  levels  of  diseases  and  parasites.  The  new  hive  equipment  (e.g.,  plastic
foundation) would minimize exposure to other contaminants as well. The study should report levels of pests
37

(e.g., Varroa mite; small hive beetle; wax moths) and disease (e.g., fungal; bacterial; viral) levels in the
colonies, pre-exposure as well as during the exposure/post-exposure period.

Queen genotype may influence the differential performance of the hives. Colonies should use sister queens
to minimize genotypic variability as much as possible. If a queen dies during the pre-exposure period, she
should be replaced; however queen replacement is not acceptable during the exposure or post-exposure
phases, the hives should be allowed to generate a new queen (i.e., supersede) naturally.

The study design should reduce and equalize the amount of stored food at the start of the exposure period
to minimize this source of variability between the hives and ensure that bees are consuming the sucrose
solution provided during the experiment. The amount of stored food prior to the exposure period can affect
the extent and timing of the exposure to the artificial food source, thus effects on the colony may be delayed
due to delayed consumption of treated food.

Hive Maintenance and Apiculture:
All colonies should be maintained as typical for apicultural practice in the relevant region, including the
application  of antibiotics, pesticide  treatments and supplemental food  that  may  be  required to  maintain
colony vitality. Apicultural practices must be clearly described and applied equally across the hives when
one hive requires intervention. Interventions have the potential to mask the effects of the pesticide  and
should be used judiciously. The study report should note when an intervention is applied.

Ideally the hives should be positioned in an area that provide adequate forage outside of the dearth period
should not require supplemental feeding. However, during the pre-exposure period, supplemental feeding
with both pollen and nectar will provide ample resources for building up the frames with comb and stimulate
brood  production.  In  locations  with  marginal  forage  habitat  (i.e.,  inadequate  supplies  of  pollen  and/or
nectar) for honey bees, planting a bee-attractive crop adjacent to the study sites that is known to provide
both  pollen  and  nectar  (e.g.,  buckwheat,  alfalfa,  clover)  can  also  stimulate  brood  production  and  hive
strength/condition during the pre-exposure and post-exposure periods.

In addition, precautions should be taken to prevent swarming (e.g., adding a box to increase hive size);
details of incidents of swarming must be provided in the study report. Robbing screens should be used,
where  necessary,  to  reduce  the  potential  for  robbing,  this  is  especially  important  during  and  after  the
exposure period.

Test Site Locations and Characteristics:
The  study  should  include  a  sufficient  number  of  replicates  across  sites  to  capture  the  environmental
variability  in  the  geographic  region  (suggested  12  sites).  Each  site  should  contain  one  group  of  hives
containing the control, treated colonies, and one colony used to identify residues collected during foraging.
Hive selection for sites should be grouped (i.e., blocked) by hive strength (e.g., food storage (pollen and
honey), adult population size, brood (egg, larval, pupal) abundance), pre-exposure colony assessments will
help to normalize the hives to reduce within site variability. It is recommended that a visual layout of each
test site is provided to better ascertain the design of the feeding study (e.g., Figure 1).

The study should attempt to minimize inter-site differences in habitat and potential foraging sources. This
may be done by examining land-use/land-cover maps and selecting sites as equivalent as possible in land
use within at least a 3 mile range around the individual sites where bees can be expected to forage. Although
38

the honey bee foraging range can exceed 3 miles, this radius is assumed to provide a reasonable distance to
standardize the land-use/land-cover for each of the sites. The study should include justifications for the
selection  of  sites,  specifically  related  to land-cover  types, and  the  methods  used  to  select  various sites.
During  the  conduct  of  the  study,  the  surrounding  area  should  be  adequately  characterized  in  terms  of
available forage, and potential sources of pesticide contamination. To the extent possible or feasible, the
hives  should  be  placed  in  an  environment  with  minimal  pesticide  use.  For  studies  designed  to  mimic
exposure through contaminated nectar, the exposure period should occur during a period when alternative
sources of floral nectar are low (i.e., dearth) at the selected study site to increase honey bees’ reliance on
the in-hive sucrose solution as their source of nectar. At each site, meteorological data should be recorded,
including temperature, humidity, rainfall, etc.

Within Site Hive Placement, Orientation and Treatment Levels:
Prior to study initiation, colonies should be ranked according to colony strength. At each site, hives should
be grouped with similar colony strength to minimize confounding influence of hive strength within a site.
The orientation of the hives may also contribute to inter-hive variability. Hives that face the north would
be exposed to the least amount of solar radiation. Temperature affects the ability of a bee to fly such that
cold  temperatures  can  inhibit  the  flight  of  forager  bees.  Different  hive  orientations  may  affect  the
productivity  of  hives  and  introduce  variability  into  the  study.  Conversely,  different  hive  orientations
minimize the opportunity for bees to mistake their hive for another hive (i.e., drift). The hives may therefore
be arranged in a semi-circle and treatments would be randomly assigned a position on that semi-circle at
the first site. The order of the hives by treatment would then incrementally rotate from site to site, as in
Figure 1. If the study design can ensure that drift between hives would be negligible if all of the hives have
the same orientation, then all hives could face the same direction to minimize the effect of aspect on colony
performance.

125’
Site 1
30’
Entrance to
the hive
faces away
from the
North
Poll
center
Res

Figure  1.  Potential  layout  of  the  hives  within  site  locations  where  the  treatments  are
randomly assigned a position within the shape of semi-circle, with the center of the semi-
circle  oriented  to  the  South.  The  hive  marked  “Poll  Res”  is  the  designated  hive  for
evaluating the pollen diversity and residues that bees are foraging over the course of the
study. At the next site, the treatment location is rotated one space counter-clockwise. Note
39

that the selected site locations may not necessarily move in a North-South direction, but
rather sites locations are selected based on land-use/land-cover.

In-Hive Observations:
Colony Condition Assessments (CCA’s) should assess all of the frames in each colony for food storage
(pollen  and  honey),  presence/absence  of  queens,  adult  population  size,  brood  (egg,  larval,  pupal)
abundance, brood termination rate, compensation index, and brood index. Where possible, the study design
should  rely  on  digital  image  analysis  or  specifically state  why  a  different  method  will  be used.  Digital
imagery can provide a more reliable estimate of frame area beyond acetate sheets (EFSA report68), and can
reduce  the  amount  of  time  the  hive  is  open. The  study  design  should  provide a  clear  indication  of  the
measurement standard operating procedures for the collection of measurement endpoints considering that
more  one  person  may  be  collecting  measurements.  The  study  directors  should  record  and  report  any
behavioral observations using a standardized approach when performing the CCA’s. The protocol and final
study report should clearly describe the measurement unit and mechanism for assessing each assessment
endpoint and colony descriptor (e.g., strength of the adult population) used in the study.  Furthermore, steps
to  monitor  queen  replacement  (supersedure)  by  the  colony,  such  as  utilizing  marked  queens,  should  also be
conducted.  Care should be taken to ensure that any re-marking of queens during the study does not result in
adverse effects on the queen or colony.

CCA’s should be made during the pre-exposure (3 or more assessments), the exposure period (assessments
every 2 weeks), the post-exposure period prior to overwintering (2 assessments, including once just before
overwintering), and post-overwintering (2 assessments). No assessments should take place during the over-
wintering period. The exact timing of the pre- and post-overwintering assessments will depend upon the
weather  and  geographic  location.  To  reduce  the  potential  impact  of  colony  assessments  on  the  hive,
assessments should be conducted at the same time as normal beekeeping practices that would open the
hives.

Residue Analysis:
The study design should include an additional hive at each location to collect pollen samples for residue
analysis of contaminants (including the test material) that may enter the hives through freely foraged food
sources.  Samples  for  residue  analysis  from  hive  matrices  should  be  taken  to  monitor  residues  of  the
pesticide, relevant metabolites, and other residues of concern in capped honey, royal jelly, corbicular pollen,
and comb pollen at a minimum of four time points during the study. The study report should clarify if
contaminated hive matrices were detected and from which sites the residues were found. Details should be
provided in the final protocol as to the analytical sampling scheme for the various exposure media.

Reducing Sources of Variability:
To the extent possible, it is important to control the sources of variability in the feeding study. Some sources
of variability include, but are not limited to, the following:


68 EFSA Panel on Plant Protection Products and their Residues (PPR); Scientific Opinion on the science behind the development of a risk
assessment of Plant Protection Products on bees (Apis mellifera, Bombus spp. and solitary bees). EFSA Journal 2012; 10(5) 2668. [275 pp.]
doi:10.2903/j.efsa.2012.2668. Available online:  http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/2668.pdf
(last accessed 06/27/16).
40

  Inter-site differences in microclimate, habitat (percent crop, forest, residential within the foraging
range of the colony), and available forage
  Pre-exposure variability or inadequate equalization of hive strength in hive parameters of adult
bees, stored honey, stored pollen, capped brood, and open brood
  Infection/infestation by diseases or parasites
  Typical apicultural practices
  Orientation of the hives and consequent differences in aspect as it relates to microclimate
  Differences in queen genotype as it relates to colony phenotype across the colonies
  Variability in data collection techniques, especially when the data are collected based on visual
estimation of area of frame that is covered and by multiple people
  Variation in pesticide residue levels in hives within treatment groups
  Amount of stored food in each hive immediately prior to exposure
  Differences in consumption rates of the supplied sucrose solution
  Exposure to external sources of contaminants via collection of pollen and nectar outside of the
hives
  Variability in hive maintenance by beekeepers

Given the many potential sources of variability in the measurement endpoints related to a study at the colony
level with honey bees in a field setting, each of these sources of variability can individually or collectively
affect  the  interpretation  of  the  study  results.  It  is  recommended  that  the  study  address  these  and  other
potential sources of variability and attempt to minimize them in the feeding study design.

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Appendix 5. Residue Field Study Design Considerations

Objectives
These studies can be designed to meet one or more of the following objectives:
1)  To quantify pesticide parent (and degradates of concern) residues in pollen and nectar of crops to
which the assessed pesticide has been applied;
2)  To determine the extent of year-to-year pesticide “carryover” in pollen and nectar;
3)  To estimate dissipation rates of pesticides in pollen and nectar following application; and
4)  To quantify pesticide (and degradates of concern) residues in leaves/flowers of the treated crop
over time and related these to concentrations in pollen and in nectar.

Due to the differing objectives of such residue  studies, protocols should be submitted and reviewed by
EFED prior to the conduct of the residue study.

Expected utility of data
While it may be difficult to refine estimates of contact exposure, dietary exposure estimates can be refined.
Studies of pesticide concentrations in nectar and/or pollen may be used to further characterize pesticide
(and degradates of concern) exposures to bees and in doing so, provide a means of refining screening-level
dietary-based RQs.
Residue  data  may  be  collected  in  multiple  plant  matrices  (e.g.,  foliage,  intact  flowers  or  associated
structures, pollen and/or nectar).  For systemic pesticides, data describing pesticide concentrations over
time (i.e., uptake and decline curves) in leaves of treated crops are useful because they may allow EPA to
expand its understanding of the relationship between pesticide concentrations in leaves and in pollen and
in nectar. For example, if a reliable relationship between pesticide concentrations in pollen and nectar and
leaves can be established, data available from magnitude of residue studies (e.g., residues in foliage and
edible fruits in studies already submitted to EPA) may be useful in characterizing exposures to bees and
may serve as protective estimates of exposure in pollen/nectar. Another example of a potential use of the
uptake and decline in leaves may allow EPA to evaluate impacts of changes to application timing (e.g., pre-
application intervals) on residue levels in pollen and nectar.  These studies may also be useful in determining
the potential for year to year accumulation in pesticide concentrations in pollen and nectar or accumulation
in bee-relevant matrices of rotational crops.

Site selection and replication
To the extent possible, for selected crop groupings, sites should be selected such that soils and regions are
representative of where the crop is grown and where the pesticide is used. A minimum of three separate
study sites are desired; soil type could be important if the pesticide is systemic and used as a soil application
or seed treatment. Site-specific factors that may lead to variability in pollen, nectar and leaf concentrations
of  a  pesticide  should  be  considered  when  selecting  sites.  Potential  sources  of  variability  among  sites
containing the same crop include soil characteristics and weather. The selected sites should not have prior
uses of the assessed pesticide for prior to the study.  However, in situations where prior treatments may
have occurred, the duration between a prior treatment and the study initiation should consider the half-life
of the parent compound and the potential presence of degradates in the soil. Each site should include at
42

least 3 replicates, represented by separate plots within the same field (block). The number of plants sampled
within  a  replicate  should  be  clearly  described  in  the  protocol.  Control  (reference)  treatments  may  be
included, but are not necessary in these residue study designs if assessing background contamination is not
a concern.

Pesticide application
The protocol should specify the treatment frequency and application method. Applications should be at
maximum label rate, maximum number of applications, and minimal reapplication interval consistent with
the product label. If the label allows for pre-bloom application, the study design should include pre-bloom
application.

Sample collection
A single sample may be collected from multiple plants within the same plot, thus a sample would represent
a composite. Minimally, the following plant matrices are recommended for collection: pollen, leaves, floral
and extra-floral nectar. When nectar or pollen for a species is not feasible given the biology of the species,
whole flowers or flower parts may be collected to represent pollen (e.g., anthers) and nectar concentrations.
Prior to conducting the study, justification for using these surrogate structures should be provided in the
protocol submission.
Depending on the species of plant, pollen and/or nectar samples may be readily collected directly from the
plant as discussed above; however, it may also be appropriate to use bees to sample these matrices where
pollen is in turn collected using pollen traps while nectar is collected from the honey stomach of returning
forager  bees.  The  use  of  hive  matrices  (e.g.,  bee  bread,  pollen  stores,  and  stored  nectar)  are  not
recommended  as  they  may  not  be  representative  of  the  current  exposure  period,  and  may  incorporate
dilution and/or degradation.
When  information  on  dissipation  rates  are  desired  there  are  additional  sampling  considerations.  The
pesticide residue levels in plant tissues are expected to rise to a certain level and then decline over time.
The frequency of leaf, pollen, or nectar residue sampling should minimally include 4 different samples, and
across a time frame sufficient to define the pattern of residue uptake and decline over time (e.g., enough to
establish the DT50). For dissipation in leaves, it is recommended that sampling of leaves begin on the day
of application, followed by regular sampling after application. If the crop is a perennial or a biennial species
and a clear declining trend in concentrations is not observed during the first year, sampling should continue
into the second year (no additional application of the active ingredient is needed the second year).
With respect to leaf sampling, the selection of individual leaves should consider the hypothesis being tested.
If a compound accumulates in leaves (i.e., xylem only transported compounds), sampling the youngest
leaves will provide a measure of current transport; whereas, sampling oldest leaves will account for a longer
period of transport of the chemicals as well as any degradation occurring. Where applicable, based on the
plant form (e.g., trees, palms, or vines) a composite of leaf samples should be taken from across the lower,
middle, and upper portion of the individual plant, and for large leaves isolating a region of leaf tissue (e.g.,
terminal leaflet of a compound leaf, cross section at middle of a large leaf) to reduce bias in the sampling
regime.
43

For soil-applied pesticides, it is recommended that residues also be quantified in soils prior to and after
pesticide application. If information on year-to-year carryover is of concern, then multi-year sampling of
soil and/or bee-relevant matrices should be conducted.

Detection limits
Analysis for the parent compound as well as major degradates is recommended. Appropriate environmental
chemistry methods (ECM) with independent laboratory validation (ILV) should be provided to support the
analysis method that demonstrate adequate limits of detection (LOD) and limits of quantification (LOQ).
Ecological effects studies should be considered in the process of selecting analytical methods to obtain
appropriate levels of detection and quantification for the studies. In addition, when reporting residue data,
it should be clearly stated that the residues for pollen and nectar are based on fresh weight and those for
leaves are in terms of dry weight.

Data Reporting
Residue data are not typically normally distributed and outliers are common.  Box and whisker plots may
be  a  means  of  visualizing  data.    The  protocol  should  specify  how  residue  levels  below  the  level  of
quantification (LOQ) are captured in the distribution (e.g., ½LOQ or utilize the LOQ). All raw data should
be submitted electronically (e.g., in spreadsheet format) to facilitate data analysis and interpretation.

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