&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-019
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.3020:
        Honey Bee Acute
        Contact Toxicity Test

-------
                                     NOTICE

     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.requlations.qov 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 850.3020: Honey bee acute toxicity.

(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 harmonized test guideline
       include the OPP 141-1 Honey Bee Acute Contact LDso (Pesticide Assessment Guidelines
       Subdivision L); the Honey Bee — Acute Contact LDso Standard Evaluation Procedure;
       and OECD 214, Honeybees, Acute Contact Toxicity Test.

(b) Purpose. This guideline is intended for use in developing data on the acute contact toxicity
to honey bees  of chemical substances  and mixtures ("test chemicals" or "test  substances")
subject to environmental effects test regulations.   The Agency  will use data  from this test in
assessing the acute hazard a test substance may present to bees.  While the study is specifically
designed to allow calculation of the LC50, the study can be used  to obtain information regarding
sublethal effects which are used in Agency evaluations.

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

       Acute contact  toxicity is the adverse effects occurring within a maximum period of 96
       hours of a topical application of a single dose of test substance.

       Dose is the amount of test substance applied.  Dose is expressed as  a mass, microgram
       (jig) of test substance per bee (jig/bee).

       Frank sublethal effects refers to overt or frank toxicological effects and include, but are
       not  limited to  lethargy, abnormal behavior, or ataxia. Less significant sublethal effects
       such as excessive grooming are not considered frank toxicological effects.
            is the empirically derived dose of the test substance that is expected to result in
       mortality of 50 percent (50%) of a population of bees which is treated  with a  single
       contact dose level under the conditions of the test.

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

(d) General considerations —

       (1) Summary of test. The honey bee acute contact LDso test, using the honey bee (Apis
       mellifera), is an acute, one-time dose laboratory study designed to determine the quantity
       of test substance that  causes 50% mortality in a test population of bees. Test bees may be
       obtained  directly from  hives  or from  frames  kept in  an incubator.   Test  bees are
       immobilized and randomly assigned to the  various  dosage levels and controls.   Test
       substance  is administered as a single topical dose, either via microapplicator (topical
       drop) or via whole body  exposure to  impregnated  dust.  Bees  are closely monitored
       within the first 4 hours (h) after treatment, and then  observed for mortality and signs of
       intoxication at 24 and 48 h, with  extended observations to 96 h as indicated in paragraph
                                      Page  1 of 12

-------
(e)(2)  of this  guideline.   The mortality pattern  is  examined and  subjected  to  the
appropriate statistical analysis to derive the LDso, slope, and confidence limits.  Results
are expressed as LD50  in micrograms  (ug)  of test substance per bee.   The complete
mortality pattern, along with signs of intoxication, should be reported.

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

(3) Range finding test.  If the approximate toxicity of the test substance is unknown, a
range-finding test may be conducted to determine the dosage levels of the test substance
to be used in the definitive test.  Refer to paragraph (d)(4) of this guideline for details on
dosage levels for definitive tests. If a test substance is expected to be of low toxicity, it
may be useful to first conduct a limit test at 25 ug per bee under paragraph (d)(5) of this
guideline.  If mortality occurs at this level,  then further range-finding  at lower levels
should be performed. The results of the range-finding test may then be used to establish
the definitive test dosage levels. Results of range-finding tests should be reported along
with the results of the definitive test, if range-finding tests are conducted.

(4) Definitive test. The goal of the definitive test is to determine the dose-response curve
for honey bee mortality after 48 h acute  contact, and to establish the median lethal dose
(LDso) (and its 95 percent (95%) confidence limits), as well as the slope of the dose-
response  curve, its  associated  standard error and 95% confidence limits.   For this
determination, a minimum of five dosage levels of the test substance should be used.  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.

(5) Limit test. For test substances expected to have relatively low toxicity, in  place of a
definitive test a limit test may be conducted with a single contact dose level, the limit
dose, plus a control group.  In these situations, it is only necessary to ascertain that the
48-hr LD50 is  above  the limit  dose (i.e..,  48-h LD50 > limit dose).  In a honey bee acute
contact limit test at least 25 bees are exposed to the "limit dose" with the same number of
bees in appropriate  controls.   For most pesticides the limit  dose  is 25  ug of active
ingredient per bee (25 ug a.i./bee).   If the  expected environmental contact residue level
exceeds 25 ug a.i./bee the test should be conducted at a higher dose which for  pesticides
is equivalent  to  twenty times the  maximum expected dermal contact environmental
concentration  (see paragraph (e)(4)(ii)(B) of this guideline). Except for the number of
treatment groups, an acceptable limit test follows the same  test procedures, is the same
duration  and has  the same number of controls as the definitive test (see Table 1 of this
guideline). Signs of intoxication, if any, should be reported.  At test termination if two or
more mortalities occur among the 25 bees tested at the limit dose then a definitive dose-
response test should be conducted. If frank sublethal effects are observed at the  limit dose
beyond the allowable mortality or observation of a sublethal effect(s) (e.g., one  bee out of
25 either died or exhibited sublethal effects),  then a full definitive test may be necessary.
For pesticides, if frank sublethal effects are observed in more than one bee and the limit
dose tested  was: 1)  less  than twenty times  the  maximum expected EEC, then a full
definitive study is necessary; or 2) was at least twenty times the maximum EEC, but there
                                Page 2 of 12

-------
       is other evidence or data that indicate a risk to terrestrial invertebrates, e.g., pesticide use
       incident data, then a full definitive test is necessary.

(e) Test standards—

       (1) Test substance. The substance to be tested should be technical grade unless the test
       is designed to test a specific formulation, mixture or end-use product. For pesticides, if
       more than one active ingredient constitutes a technical product, then the technical grade
       of each active ingredient should be tested separately, in addition to the combination, if
       applicable.  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 administration of the test substance followed
       by an observation period of 48 h.  If mortality increases by more than 10% between 24
       and 48 h, the test duration should be extended up to a maximum of 96 h provided that
       control mortality does not exceed 20%.

       (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 a sucrose
              solution.

              (iii) Age.  The test is 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
              kept in conditions conforming  to  proper  cultural 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.
                                       Page 3 of 12

-------
(4) Administration of test substance.  On the day of test initiation or the evening before,
young bees should be collected from the incubator or directly from the hive, immobilized
with cold temperatures, or carbon dioxide gas (€62) or nitrogen gas (TS^), and placed in
holding cages.  Exposure to CO2 and N2 gases should be kept to the minimum amount
which immobilizes the bees.  Dead bees should be rejected and replaced by healthy bees
before starting the test.   To initiate the test,  bees  in the  holding cages are  again
immobilized, and distributed into treatment groups of at least 25  bees.  A single dose is
applied to the dorsal  side of the thorax of each bee via microapplicator.  Alternatively,
test bees may be treated via a dusting apparatus (see paragraph (j)(l) of this guideline).

       (i) Preparation of dosing mixture.

              (A) A  solvent is generally  used to administer the test  substance.  The
              solvent of choice is  acetone, although other volatile organic solvents of
              low toxicity (e.g., dimethylformamide, dimethylsulfoxide) have been used
              successfully in cases where acetone was not suitable.  Maximum dosage
              volume should not exceed 5 microliters (uL) per bee, to allow for adequate
              volatilization of the solvent.

              (B) If a dusting apparatus is used, for a satisfactory test a nontoxic dust
              diluent is necessary as a carrier.

       (ii) Treatment levels.

              (A) A minimum of five dosage levels of the test substance should be used
              in the definitive test.  These dosage levels should be spaced geometrically
              in  such a manner so that the entire dose-response curve is characterized
              (curve  between LD0s and LD90).  The dosage levels should be spaced so
              that at least three of the doses result in partial mortalities, and that at least
              one of these three doses kills greater than 50%, and at least one  kills less
              than 50% of the bees.   For some test substances (e.g., shallow  slope), it
              may be necessary to use more than five dose levels to  achieve these
              results.

              (B) For a limit test,  there  is  single  contact dose,  plus the appropriate
              control (see paragraph (d)(5) of this guideline). A limit dose of 25  ug/bee
              is used unless environmental residues are expected  to result in a  higher
              acute contact dose. To calculate the acute contact limit dose (mg a.i./bee)
              for spray  applications of pesticides Equation 1  of this guideline can be
              used for pesticide uses with a single application and Equation 2  for a use
              with multiple  applications  per year.  The dietary residue estimates  are
              based  on a  nomogram that  relates food  item  residues to  pesticide
              application rate; the highest residue level expected is with the broadleaf
              plants/small insects category (nomogram value of 135, assuming  a 1 Ib
              a.i./acre application rate).   The nomogram is based  on an EPA database
              called    UTAB    (Uptake,    Translocation,     Accumulation,    and
              Biotransformation),  a compilation of actual measured pesticide residue
              values  on plants  (see references in paragraphs (j)(4) and (j)(5) of this
              guideline).   If there  are  multiple  uses this  study  is  supporting  for
                                Page 4 of 12

-------
              registration,  the  limit dose for the study should  be based on  the  one
              resulting in  the highest dose.   The average weight  of one honeybee is
              assumed to be 0.128 grams.
                   Contact Limit Dose = Q.l2&(ApRate)(l35)               Equation 1
      Contact Limit Dose = 0.128
(ApRate\\35i e
Equation 2
                    ApRate = maximum single application rate (in Ib a.i./acre);
                    Halflife = the foliar halflife (default is 35 days);
                    Interval = the minimum application interval (in days);
                    / = application event from 1 to n; and
                    n = total number of applications.
(5) Controls.

       (i) Two concurrent controls are included in the test: a negative control  and a
       solvent (or vehicle) control.  Control bees are from the same source as the test
       groups and are kept under the same environmental conditions  as treated bees.  The
       test procedures should  be  the same for control  and treated  bees, except  that
       negative controls  receive no treatment, and solvent (vehicle) controls are treated
       only with the solvent (vehicle). Solvent control bees should  receive a volume of
       solvent equal to the largest volume administered to the test bees.  The use of
       shared controls is acceptable for concurrent  tests as long as  the same solvent or
       vehicle is used for all the tests.

       (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
       bees.

(6) Number of test organisms and replicates. In the  definitive test, a minimum of 25
bees should be used for each dosage level and for each control. Bees at a treatment level
may be divided into replicates if desired.  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.  Tests should  be conducted indoors to control
lighting  and  other environmental  variables, with bees being maintained in small  test
chambers. Test chambers may be constructed of metal, plastic, wire mesh, or cardboard,


                                Page 5 of 12

-------
       or a combination  of these materials.   Chambers should  be constructed so that a vial
       containing sugar water may be attached.

       (8) Environmental  conditions.  Environmental conditions 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 dosing and observations.

       (9) Observations—

             (i)  Environmental conditions.  Temperature and relative humidity  should be
             recorded throughout the test.

             (ii) Measures of effect.  Bees  should be observed for mortality and any other
             adverse effects at approximately 4, 24,  48, and  if applicable, 96 hours after
             dosing.  All signs of intoxication, other abnormal behavior, and mortality should
             be recorded throughout the test  period and reported by dosage 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.  All signs  of intoxication and any other abnormal behavior
             that may or may not be attributed to the test substance  should be reported.  Dead
             bees should not be removed from the test chambers  until the test is terminated.

(f) Treatment of results—

       (1) Descriptive summary statistics—

             (i)  Environmental conditions.  Data should be  summarized in tabular form,
             showing the range of temperature and relative humidity during the test, the mean
             temperature and relative humidity and standard deviation.

             (ii) Mortality.  The number  of initial bees at each treatment and control and the
             number of  dead bees should  be  summarized in tabular form by observation time
             and treatment, and replicate if applicable.

             (iii) Appearance and behavior. Number of bees with abnormal appearance or
             behavior symptoms should be summarized in tabular form by  symptom, time of
             observation, and treatment, and replicate if applicable.

       (2) Percent—

             (i)  Mortality.  Calculate  the percent of mortality at  each treatment level and
             control by observation time.

             (ii) Appearance and behavior.  Calculate the percent  effected at each treatment
             level and control by symptom, and observation time.
                                      Page  6 of 12

-------
(3) Limit test-
       (i) LDso value.  At test termination (48 hours), if one or fewer bees are dead at the
       limit dose, the acute contact LD50 is considered to be greater than the limit dose
       (i.e., LDso > limit dose).  This is because the Binomial Theorem predicts that
       when 25 bees are tested, the probability of seeing <1 dead bee if the true 48-h
       LDso is at or below the limit dose is <0.001. Conversely the probability of seeing
       2 or more dead  bees if the true 48-h LDso is at or below the limit dose is >0.999.
       Therefore, if <1 mortality occurs  among the  25 bees tested,  the  48-h LDso  is
       reported as greater than the limit dose (i.e., 48-h LDso >25 jig/bee for pesticides).

       (ii) Proportion of mortality (p ).  Assuming mortality  follows the binomial
       distribution,  an  estimate of the true proportion  of mortality  (p ) in the laboratory
       test population as well as confidence bounds on that estimate (see Table A4 of the
       reference in paragraph (j)(3) of this guideline) can be obtained.  For small sample
       sizes the interval may be large.  For example, for  a limit test resulting in no
       mortalities in 25 bees (p = 0), the 99% confidence interval  on the estimate of p
       is  (0.00,  0.19)  and the 95% confidence interval is (0.00, 0.14). For a limit test
       resulting in one mortality in 25 bees (p = 0.04), the  99% confidence interval on
       the estimate  of  p is (0.01, 0.26) and the 95% confidence interval is (0.00, 0.20).
       Using the 95%  confidence interval as an example, the true (unknown) proportion
       of mortality will be covered by  the calculated confidence interval  in 95% of
       repeated  trials.   For assessing risks, the confidence  in the estimated proportion
       impacted is  considered  in determining acute effects at environmental exposure
       doses.   If the  uncertainty in p  is high at  the limit  dose, and  the expected
       environmental exposure dose is close to the limit dose, risks to threatened and
       endangered species may not be able to be discounted.

       (iii) Multiple-dose testing.

             (A) At test termination if two or more mortalities occur among the 25 bees
             tested at the limit dose or there are clinical signs of toxicity,  a definitive
             dose-response  test  should be conducted.  If frank sublethal effects are
             observed at the limit dose beyond the allowable mortality or  observation
             of a sublethal effect(s) (e.g., more than one bee out of 25 either  died or
             exhibited sublethal effects),  then a full definitive test may be necessary.
             For pesticides, if frank sublethal effects are observed in more than one bee
             and  the  limit dose tested was:  1) less than twenty times the maximum
             expected EEC, then a full definitive study is necessary; or 2) was at least
             twenty times the maximum EEC, but there is other evidence  or data that
             indicate  a risk to terrestrial  invertebrates, e.g., pesticide use incident data,
             then a full definitive test is necessary.

             (B)  A  multiple-dose definitive  LD50 test may be waived if  at  test
             termination no more than 1 bee dies at the limit dose, and there are also no
             clinical signs of toxicity at the limit dose.
                                Page 7 of 12

-------
       (4) Multiple-dose definitive test—
              (i) Dose-response curve, slope and LD50.  Statistical procedures are employed to
              calculate the 48-h LDso (standard error and 95% confidence limits) based upon
              mortality. If a dose-response model (e.g., probit) was fit to the data to determine
              the LDso, the model parameters (e.g., slope) and their uncertainty estimates (e.g.,
              standard error) should be recorded.

              (ii) NOEL. While calculation of the NOEL and LOEL is usually not required for
              acute tests, reporting these values is useful when testing pesticide and industrial
              chemicals.   For pesticides, the reporting of a  NOEL  value is  useful  when
              evaluating risk to Federally listed Endangered and Threatened species.

              (iii) Statistical methods.   Statistical procedures for modeling quantal data are
              available and  should  be used.   Additional  discussion  about  endpoints and
              statistical procedures is found in OCSPP 850.3000.

(g) Tabular summary of test conditions.  Table 1 lists the important conditions that should
prevail during the  definitive test.  Except for the dose levels, Table 1 also lists the important
conditions that should prevail during a limit test.  Meeting these conditions will greatly increase
the likelihood that the completed test will be acceptable or valid.
Test
       Table 1.—Summary of Test Conditions for the Honey Bee Acute Contact Toxicity
Test type
Test duration
Temperature
Relative humidity
Lighting
Test chamber
Test solution application
Age of test bees
Number of bees per chamber
Number of bees per treatment and control
Number of dose levels
Feeding
Measure of Effect or Measurement Endpoint
Acute contact
48 h (96 h, if mortality increases >10% between
24 and 48 h)
25 - 35 °C
50 - 80%
Darkness, except during dosing and observations
Metal, plastic, wire mesh, or cardboard
Microapplicator or dusting apparatus
Young adult worker bees of similar age and
feeding status
Variable
25 (minimum)
Minimum of 5 in a geometric series, a negative
control, and a solvent control (if solvent used)
50% sugar/water (w/v) solution ad libitum
LD50 based upon mortality at 48 h (96 h, if
applicable), sublethal effects
(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
                                       Page 8 of 12

-------
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.

       Table 2.—Test Validity Elements for Honey Bee Acute Contact Toxicity 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. The  negative  (untreated)  control [and solvent (or vehicle) control, when a solvent was used] was not
included in the test.

5. 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) Identification of the test substance: common name, IUPAC and CAS names,
              CAS number, structural formula, source, lot or batch number, chemical  state or
              form of the test  substance,  and its purity (i.e. for  pesticides, the identity and
              concentration of active ingredient(s)).

              (ii) Storage conditions of the test chemical or test substance  and stability of the
              test chemical or test substance under storage conditions if stored prior to use.

              (iii) Methods of preparation of the test substance and the treatment doses used in
              the range-finding and definitive test, or limit test.

              (iv) If a vehicle (e.g.., solvent) is used to prepare stock or test solutions provide:
              the name  and source of the  vehicle, the nominal concentration(s) of the test
              substance in the vehicle stock solutions, and the vehicle dose(s) used in the test.

       (4) Test organisms.

              (i) Scientific name, race, and source.

                                       Page 9 of 12

-------
              (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
              treatments).

              (iv) Collection method and date of collection.

              (v) Holding period.

              (vi) Age at test initiation.

       (5) Test system and conditions. Description of the test system and conditions used in
       the definitive or limit test, any  preliminary range-finding tests,  and any positive control
       tests.

              (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) Number of bees per test cage.

              (iv) Number of cages (replicates) per treatment, negative control, and solvent
              control.

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

              (vi) Method of test substance application including the body part and volume of
              test solution applied.

              (vii) Test duration.

              (viii) Methods and frequency of environmental monitoring performed during the
              definitive or limit  study and  any positive controls  for test room  temperature,
              humidity and lighting.

              (ix) For the definitive,  limit,  or positive  control test, all analytical procedures
              should be described.  The accuracy  of the method, method detection limit, and
              limit of quantification should be given.
(6) Results.
       (i) Environmental monitoring data results (test room temperature, humidity and lighting)
       in tabular form (provide raw data for measurements not made on a continuous basis), and
       descriptive statistics (mean, standard deviation, minimum, maximum).

       (ii) For preliminary range-finding tests, if conducted, the number of dead and intoxicated
       bees at each dose level and in the control(s).
                                      Page 10 of 12

-------
       (iii) For the definitive or limit test, the number of dead bees at each observation period at
       each dose level and control(s) (provide the raw data).

       (iv) For  the definitive or limit test, a description of signs of intoxication and other
       abnormal behavior, including time of onset, duration,  severity, and number affected at
       each dose level and control(s) (provide the raw data).

       (v) Graph the dose-response curves at the end of the test.

       (vi) For the definitive study, the slope of the dose-response curve at the end of the test
       and its standard error and 95% confidence limits.

       (vii) For the definitive, limit, and positive control studies provide LDso values, with 95%
       confidence limits, at each recommended observation time.

       (viii) Description of statistical method used, including software package, for determining
       LDso values and the dose-response model  parameters and the basis  for the choice of
       method. Provide results of any goodness-of-fit tests.

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

       (1)  Atkins, E.L., Jr. et a/., 1954. Equipment and technique used in laboratory evaluation
       of pesticide dusts in toxicological  studies with honey  bees.   Journal of Economic
       Entomology 47: 965-969.

       (2)  Atkins, E.L. et a/., 1975.  Toxicity of pesticides and other agricultural chemicals to
       honey bees: Laboratory  studies.   University  of California, Division  of Agricultural
       Sciences, Leaflet 2287, 38 pp.

       (3)  Conover, W. 1980.  Practical Nonparametric Statistics, 2nd Edition.  John Wiley &
       Sons, Inc., New York, NY. 493 pp.

       (4)  European and Mediterranean Plant Protection Organization (EPPO), 1992. Guideline
       on Test Methods for Evaluation  of the  Side-effects of Plant Protection Products on
       Honeybees (No. 170). Bulletin OEPP/EPPO Bulletin 22: 203-215.

       (5)  Mayer,  D.   &  C.  Johansen.   1990.  Pollinator  Protection:  A Bee & Pesticide
       Handbook.Wicwas Press. Cheshire, Conn. p. 161

       (6)  Organization for Economic Co-operation and Development (OECD), 1998.  OECD
       Guidelines for the Testing of Chemicals, TG 214, Honeybees, Acute Contact  Toxicity
       Test. Adopted 21 September, 1998.

       (7)   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 11 of 12

-------
(8)  U.S. Environmental Protection Agency, 1985.  Hazard Evaluation Division Standard
Evaluation  Procedure, Honey Bee—Acute Contact LDso Test.   Office of Pesticides
Programs, Washington, D.C., EPA-540/9-85-002.
                              Page 12 of 12

-------