United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.3030:
        Honey Bee Toxicity of
        Residues on Foliage


     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's  number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from  the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline,  but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 850.3030: Honey bee toxicity of residues on foliage.

(a) Scope—

       (1) Applicability. This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances Control Act  (TSCA)  (15  U.S.C. 2601, et seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)  (7 U.S.C. 136, et seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

       (2) Background.   The source materials used in developing this test guideline  include
       OPP 141-2 Honey Bee Toxicity of Residues on Foliage (Pesticide Assessment Guidelines
       Subdivision  L); and  the Honey  Bee—Toxicity of Residues  on  Foliage Standard
       Evaluation Procedure.

(b) Purpose. This  guideline  is intended for use in developing  data on the residual toxicity to
honey bees of chemical substances and mixtures ("test  chemicals" or "test substances") subject
to environmental effects test regulations. This guideline describes an acute toxicity test in which
honey bees are  exposed to the test substance applied to foliage.   The Environmental Protection
Agency will use data from this test  in assessing the acute residual  hazards to bees that a test
substance may present in the environment.

(c) Definitions.  The definitions in OCSPP 850.3000  apply  to this  guideline.  In addition, the
more specific definitions in this paragraph also apply:

       Acute Residual  Toxicity is the adverse effects occurring over a period of time (hours or
       days) from a single dose of the test substance to foliage.

       Dose is the amount of test substance applied. Dose is expressed as a mass, pounds of test
       substance  per acre (Ib/A) and for a pesticide pound(s) of active ingredient applied per
       acre  (Ib  a.i./A). The dose used in this test should be the maximum, single application
       dose allowable according to the end-use product labeling.

       Mortality:  an animal is recorded as dead when it is completely immobile.

       RT25 is the residual time needed to reduce the activity of the test substance and bring bee
       mortality down to 25 percent (25%)  in cage  test exposures to field-weathered spray
       deposits (see paragraph (e)(2) of this  guideline). The time period determined  by this
       toxicity value is considered to be time that the test substance  is expected to remain toxic
       to bees in  the field from the residual exposure  of the test substance on vegetation at an
       expressed rate of application (Ib a.i./A).

(d) General considerations—

     (1) Summary of  test. The honey bee (Apis melliferd) foliar residue  study is a laboratory
     test designed to determine  the  length of time over which field-weathered foliar residues
     remain toxic to honey bees.  The test substance (e.g., a representative end-use product) is
     applied to crop foliage, the foliage is harvested at predetermined intervals post-application,
     and test bees are caged on the treated foliage for 24 hours.   Results are expressed in terms
     of the length  of time following application,  during  which residues continue to cause

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     significant mortality (24-h RJ^s) in test populations at an expressed rate of application (Ib

     (2) General  test guidance.  The general guidance in OCSPP 850.3000  applies to  this
     guideline except as specifically noted herein.

     (3) Definitive test.  The goal of the definitive test is to determine the 24-h RT25, length of
     time post-application that residues of the test substance on foliage are toxic to honey bees.
     For  this determination, three treatment  levels (three  different time intervals between
     application and harvest) are typically used.  At a minimum, the test substance should be
     evaluated  at  the maximum label rate.  Multiples of the maximum label rate may be
     evaluated  if desired.  A summary of test conditions is provided in Table 1 in paragraph (g)
     of this guideline, and validity elements for an acceptable definitive test are listed in Table 2
     in paragraph (h) of this guideline.

(e) Test standards—

       (1) Test substance.  The substance to be tested is the specific form of a chemical or
       mixture  of chemicals that is used to develop the data.  For pesticides, test substance is a
       representative end-use  product.   OCSPP 850.3000  lists the  type  of information  that
       should be known  about  the test substance  before testing, and discusses methods for
       preparation of test substances.

       (2) Test duration.  The test consists of the placement of treated foliage into cages with
       bees followed by an observation period  of 24 hours.   The treated foliage is harvested at
       various time intervals (3, 8 and 24 hours) post-application.  If mortality of bees exposed
       to the foliage harvested 24 hours after the application is greater than 25%, weathered,
       treated foliage samples should continue to be taken every 24 hours (i.e.., 48, 72, 96,  120
       hours, etc.  after the application) and bees exposed to these  additional  samples for 24
       hours until mortality of bees exposed to the treated foliage is 25% or less.

       (3) Test organism—

             (i) Species. Honey bee, Apis mellifera, is the test species.

             (ii) Source. Bees may be obtained from on-site colonies or from a commercial
             apiary.  All control and treatment bees used  in a test should be from the same
             source and  race.  Collection  in early spring or  late autumn should be avoided, as
             the  bees have a  changed physiology during this  time.  If  tests have  to be
             conducted during these times, bees can be emerged in an incubator and reared for
             one  week with "bee bread" (pollen collected from the comb) and sucrose solution.

             (iii) Age. The test should be conducted using young adult worker bees that are of
             a similar age and feeding status.

             (iv)  Acclimation. No acclimation period is usually necessary.

             (v) Health  status.  Bees used in the test should be in apparent good health. Only
             bees from apparently disease-free  colonies should be used,  and they  should be

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       kept in conditions conforming to proper culture practices.  Bees  treated with
       chemical substances, such as antibiotics, anti-varroa, etc., should not be used for
       toxicity tests for four weeks from the time of the end of the last treatment.

       (vi) Care and  handling.  During holding and testing, bees should be shielded
       from excessive activity or other disturbance.  Bees should be handled only as
       much as is necessary to conform to test procedures.

       (vii) Diet and feeding. A 50% weight/volume (w/v) solution of sugar/water (500
       grams/liter)  should be  provided ad  libitum  throughout the  holding and  test
       periods. Purified or distilled water should be used for the sugar solution.

(4) Administration of test substance.  On the day of test initiation or the evening before
exposure, young bees should be collected from frames kept in the incubator or directly
from the hive,  immobilized with cold temperatures, carbon dioxide gas (€62) or nitrogen
gas (N2), and placed in holding cages.  Exposure to CC>2 and N2  gases should be kept to
the minimum  amount necessary to  immobilize the bees. Dead bees should be rejected
and replaced by  healthy bees  before  starting the test.  A portion of treated foliage,
prepared as described in paragraph  (e)(4)(i) of this guideline, is placed into  a  test
chamber.  Bees in the holding cages are again immobilized, and introduced into the test
chambers until each  replicate test chamber contains at least 25 bees. Test bees are also
caged at the same time on control (untreated) foliage.  Bees are monitored and observed
for mortality and signs  of intoxication during the exposure period.

       (i) Preparation of treated foliage.  The test substance is applied to the test crop
       (alfalfa is preferred)  at maximum label rate(s).  At predetermined post-treatment
       time intervals, treated foliage is harvested (90 gram (g) or 3,000 cubic centimeter
       (cc) allotment), and returned immediately to the laboratory to be chopped, mixed,
       and divided into 15-g or 500-cc portions and placed in test cages. Foliage should
       be collected,  using a random sampling scheme, from the  top of the  canopy (first
       15-centimeter (cm) portion)  of the plants and chopped into approximately 2.5 cm
       lengths. Each portion is placed into a single test chamber.

       (ii) Treatment levels and post-treatment intervals.  At  a minimum, the  test
       substance should be  evaluated at the maximum label  rate.  Multiples of the
       maximum label rate may be evaluated if desired.  Residues are allowed to weather
       in the field for a specific time period prior to collection of foliage for testing. Test
       samples are collected at 3, 8, and 24 hours after application. If mortality rate of
       bees exposed to the 24-hour-old residues are expected or observed to be greater
       than 25%, sampling of test plots at 24-h intervals, followed by exposure of bees,
       should  continue until mortality of bees exposed to the weathered foliage  is 25% or
       less. If so indicated by prior knowledge of the properties of the test substance, the
       post-treatment intervals may be spaced at some alternative time series.

(5) Controls.

       (i) Paired negative (untreated) controls are included in the test. Control plots are
       treated identically to treatment plots, except for applications of the test substance.
       Control and test bees are kept under the same environmental conditions.
                                Page 3 of 11

       (ii) A test is not acceptable if more than 20% of the control bees die during the
       test period.

       (iii) A concurrent positive control or reference test with a substance of known
       toxicity is not a  condition for an  acceptable  test.   However, a quarterly or
       semiannual test with a laboratory standard (reference toxicant) is recommended as
       a means of detecting possible interlaboratory or temporal variation.  A laboratory
       standard is also recommended when there is any significant change in source of

(6) Number of test  organisms  and  replicates.   In  the  definitive test, six replicates
should be assigned to each treatment and control group, with a minimum of 25 bees for
each replicate.  Test  organisms are impartially  or randomly assigned to dose levels in
such a manner that the test results show no significant bias from the distributions.

(7) Facilities, apparatus and supplies—

       (i) Facilities.  Tests should be conducted indoors  to control lighting and other
       environmental variables, with bees being maintained in small test chambers.

       (ii) Test  chambers.  Test chambers may be constructed of metal, plastic, wire
       mesh, or cardboard, or a  combination of these  materials.   Chambers should be
       constructed so that a vial containing sugar water may be attached.

       (iii) Field plots and harvest of foliage.

       (A) A single application  of the test substance should be made to each of nine
       alfalfa plots.   Plots should be at least  1  square  meter (m )  (10.8 square feet) in
       alfalfa grown according to standard agricultural practices. Applications should be
       made with a hand  sprayer. Nine test substance treatment plots are used to obtain
       three  plots for harvesting at each  of  three time intervals,  at a minimum (see
       paragraph (e)(4)(ii) of this guideline). After test substance residues have aged for
       the appropriate time period, alfalfa foliage sufficient to place in  six treatment
       cages (90 g or 3,000 cc total),  should be harvested from three test plots using a
       random sampling scheme, and  returned   immediately to the laboratory for
       processing and placement in test cages.  The foliage from each of the three plots
       per time  interval  is chopped, mixed and then impartially allocated into 15-g or
       500-cc portions for placement in each of six replicate  test chambers.   An
       alternative procedure is to apply the test  substance  at three different initial times
       and harvest the alfalfa at the same time.  Additionally, three control plots are
       maintained and treated identically to the treatment plots,  with the exception of test
       substance application.  At each of the three  time intervals  (additional  time
       intervals may  be appropriate; see paragraph (e)(4)(ii) of this guideline) the three
       control plots are harvested, using a random sampling scheme, to obtain sufficient
       foliage to place in six control cages.

       (B) If the validity or integrity  of the study  is affected  by weather (specifically,
       precipitation and/or temperature) then the tests should be replicated over time to

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       reduce the variability due to weather conditions.  Precipitation and temperature
       are two extremely important factors in the breakdown of pesticide residue.

(8) Environmental conditions.  Environmental parameters in the laboratory during the
test should be maintained as follows:

       (i) Temperature and humidity. Temperature should be maintained between 25
       and 35 degrees Celsius (°C), with relative humidity between 50% and 80%.

       (ii) Lighting and photoperiod.  It is recommended that test bees be maintained in
       the dark except during transfer to test cages and observations.

(9) Observations—

       (i) Analysis for  test  substance concentrations.   Test  substance residues on
       treated foliage are measured in ppm fresh weight. Foliage residue sampling and
       analysis should be made immediately after the  application when test substance
       has  dried and on harvested foliage (3, 8, 24 h  post-application, or longer as
       needed) used for bee mortality testing.

       (ii) Field site conditions.  Environmental conditions should be monitored at the
       field site during and after test substance application. Environmental  information
       to be collected should include temperature, precipitation, relative humidity, wind
       speed, and estimated cloud cover.

       (iii) Measures of Effect—

              (A) Mortality.  For a given weathered residue treatment or control, bees
              should be  observed for mortality at least once during the first 4 h after
              exposure and at test termination (24 hours).  Dead bees should not be
              removed from the test chambers until the test is terminated.

              (B) Appearance and behavior. For a given weathered residue treatment
              or control, bees should be observed for all signs of intoxication and any
              other abnormal behavior once during the first 4 h after exposure and at test
              termination.  Observations should be recorded by treatment level and by
              time of occurrence.  Signs of intoxication are those behaviors apparently
              due to the test  substance and may include a wide variety of behaviors,
              such as ataxia, lethargy,  and hypersensitivity. Prior to the evaluation at
              test termination,  observations  should be made without  disturbing or
              removing bees from the test chambers; for these observations, estimates of
              mortality and effects are sufficient.
                               Page 5 of 11

(f) Treatment of results—

       (1) Descriptive summary statistics—

              (i) Environmental  conditions.  Data  should  be summarized in tabular form,
              showing the range and mean temperature, precipitation, relative humidity, and
              wind speed.

              (ii) Mortality.  Data should be summarized in tabular form, showing for each
              weathered  age  of foliage treatment  and control the  number of bees initially
              exposed, mortality at each observation time, and the percent mortality.

              (iii) Appearance  and behavior.  Data should be summarized in tabular form,
              showing for each weathered  age  of foliage,  appearance and behavior at each
              observation time, and the percent mortality.

       (2) Residual Time (RTis).  A test for comparing two  paired populations (e.g., paired t-
       test)  should  be performed to detect significant difference of treatments from controls.
       The 24-h RT25 is estimated using linear interpolation; Abbott's correction (see paragraph
       (j)(l) of this guideline) should be used in the event  of control mortality.  Additional
       discussion about measurement endpoints  and statistical  procedures is found in OCSPP

(g) Tabular summary of test conditions.  Table 1  lists the important conditions that should
prevail during the definitive test. Meeting these conditions will greatly increase the likelihood
that the completed test will be acceptable or valid.
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Table 1.—Summary of Test Conditions for Honey Bee Toxicity of Residues on Foliage
Test type
Test duration
Relative humidity
Test chamber
Test substance application
Age of test bees
Number of bees per chamber
Number of bees per treatment and control
Number of treatments
Measure of Effect or Measurement Endpoint
Toxicity of residues on foliage
24 h for each aged residue interval (3, 8, and 24 h
aged residue intervals are tested; additional residue
intervals may be appropriate).
25 - 35 °C
50 - 80%
Darkness, except during transfer of bees to treatment
cages and observations
Metal, plastic, wire mesh, or cardboard
15-g or500-cc portions of treated foliage (hand sprayer
used to apply test solution to foliage) placed in a test
Young adult worker bees of similar age and feeding
25 (minimum)
150 (minimum)
Minimum of 3 treatment groups (3, 8 and 24 h post
application of maximum label rate) plus paired negative
control(s) if mortality is <25% for the 24 h post-
application treatment.
50% sugar/water (w/v) solution ad libitum
RT25 based upon mortality at 24 hours after bees are
caged on foliage
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 2 occurred. This list should not be misconstrued as limiting the
reason(s) that a test could be found unacceptable or invalid.  However, except for the conditions
listed in Table 2 and in OCSPP 850.3000, it is unlikely a study will be rejected when there are
slight variations from guideline environmental conditions and study design unless  the  control
organisms are significantly affected, the precision of the test is reduced, the power  of a test to
detect differences is reduced, and/or significant biases are introduced in defining the magnitude
of effect on measurement endpoints as compared to  guideline conditions.  Before departing
significantly from this guideline, the investigator should contact the Agency to discuss the reason
for the departure and the effect the change(s) will have on test acceptability.  In the test report, all
departures from the  guideline  should be identified, reasons  for these  changes given, and  any
resulting effects on test endpoints noted and discussed.
                                      Page 7 of 11

     Table 2.—Test Validity Elements for Honey Bee Toxicity of Residue on Foliage Test
1. Test bees were not of similar age and feeding status.

2. More than 20% of the test bees in any control treatment were dead at the end of the test.

3. All bees in a test were not from the same source.

4. Concurrent negative (untreated) controls were not included in the test.

5. Environmental conditions (temperature, precipitation, relative humidity, wind speed and cloud cover) at
the field site were not monitored.

6. Test organisms were not impartially or randomly assigned to test chambers.	

(i) Reporting—

       (1) Background information.  Background information to be supplied  in the report
       consists at a minimum of those background information items listed in paragraph (j)0) of
       OCSPP 850.3000.

       (2) Guideline deviations.  Include a description of any deviations from the test guideline
       or any occurrences which may have influenced the results of the test.

       (3) Test substance.

              (i) End-use product (name, state or form, source), its purity (for pesticides, the
              identity   (common  name,  IUPAC  and  CAS  names, CAS  number)   and
              concentration  of active   ingredient(s)),  and  known physical and  chemical
              properties that are pertinent to the test.

              (ii) Storage conditions of the test substance.

              (iii) Methods of preparation of test  substance  for application onto foliage, the
              maximum label rate, and the actual application rate (Ib a.i./A) with the finished
              spray volume per acre.

              (iv) If residue analysis  is performed  on foliage, describe the stability of the test
              substance under storage conditions.

       (4) Test organisms.

              (i) Scientific name, race, and source.

              (ii) Culture method and conditions.

              (iii) Health status of colonies used  for collection of test bees (e.g., any adult
              diseases,  use  and  application  date(s)  of any  prophylactic  or  preventative

              (iv) Collection method and date of collection.

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       (v) Holding period.

       (vi) Age at initiation of exposure to an aged residue treatment.

(5) Test system and conditions. Description of the test system and conditions used in
the definitive test.

       (i) Description of housing conditions: type, size, and material of test cages.

       (ii) Description of any feeding during the test  (if applicable), including: method,
       type of food, source, amount given and frequency.

       (iii) Common and scientific name of treated crop.

       (iv) Plot size, and method and time of administration of test substance on plots.

       (v) Number of aging intervals tested.

       (vi) Time after application to plot of foliage collection (age intervals tested) and
       placement of foliage in test chambers.

       (vii) Plots per aging interval and negative control.

       (viii) Number of bees per test cage.

       (ix) Number of cages (replicates) per aging interval plot and negative control plot.

       (x) Methods used for test cage and treatment  randomization as well as methods
       for impartial assignment of bees to test cages.

       (xi) Exposure duration to a given aged residue and duration of the study.

       (xii) Methods  and frequency of environmental monitoring  performed on treated
       plots  during  administration  of  test  substance  and  weathering  period  for
       temperature  and precipitation,  and  any other known weather conditions that
       would impact initial concentration or stability of residue levels on treated plots.

       (xiii) Methods and frequency of environmental monitoring  performed during the
       definitive study or positive control study for test room temperature, humidity and

       (xiv) For the definitive test, all analytical procedures and  preservation methods
       should be described.  The accuracy of the method, method detection limit, and
       limit of quantification should be given.

(6) Results.

       (i) Laboratory  environmental monitoring data results (test  room  temperature,
       humidity and lighting) in tabular form (provide raw data for measurements  not

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              made on a continuous basis), and descriptive statistics (mean, standard deviation,
              minimum, maximum).

              (ii) Field site environmental monitoring data results (temperature, precipitation,
              wind speed, relative humidity, cloud cover) in tabular form (provide raw data for
              measurements not made on a continuous basis), and descriptive statistics (mean,
              standard deviation, minimum, maximum).

              (iii) For a positive control or reference study the number of dead bees observed at
              24 hours (provide the raw data).

              (iv) For the definitive test, the number of dead bees which were observed at least
              once during the first 4 hours of exposure and at 24 hours (provide the raw data).

              (v) For the definitive  test, a description  of  signs of intoxication  and other
              abnormal  behavior, including time of  onset,  duration,  severity,  and number
              affected at each aged residue treatment and control(s) (provide the raw data).

              (vi) For the definitive and any positive control  or reference studies provide 24-h
              RT2s values.

              (vii) Description of method used, including software package, for determining the
              24-h RT25 value.

              (viii) Results of analysis of variance (ANOVA) to detect significant differences of
              treatment groups from the controls.

(j) References. The references in this paragraph should be consulted  for additional background
material on this test guideline.

       (1)  Abbott, W.S., 1925.  A method of computing  the effectiveness of an insecticide.
       Journal of Economic Entomology 18:265-267.

       (2)  Johansen,  C. et a/.,  1977.  Bee Research Investigations.  Dept. of Entomology,
       Washington State University, unpublished, 22 pp.

       (3)  Lagier, R.F.  et  a/., 1974.  Adjuvants Decrease  Insecticide Hazard to Honey Bees.
       College of Agriculture Research Center, Washington State University Bulletin 801, 7 pp.

       (4)  Mayer, D. and C.   Johansen,  1990.  Pollinator  Protection:  A Bee  &  Pesticide
       Handbook. Wicwas Press. Cheshire, CT.

       (5)  Mayer, D. (approved by),  1996. Standard Operating Procedure (SOPs) - Residue
       Bioassay. The  Bee Group-Irrigated Agriculture Research and Extension Center. Prosser,

       (6)  U.S. Environmental  Protection Agency,   1982.  Pesticide Assessment Guidelines
       Subdivision L Hazard Evaluation: Nontarget Insects.   Office of Pesticides and Toxic
       Substances, Washington, D.C.,  EPA-540/9-82-019.

                                      Page 10 of 11

(7) U.S. Environmental Protection Agency, 1985.  Hazard Evaluation Division Standard
Evaluation Procedure,  Honey Bee—Toxicity  of Residues  on Foliage.    Office  of
Pesticides Programs, Washington, D.C., EPA-540/9-85-003.
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