United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-019

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3020:

Honey Bee Acute

Contact Toxicity Test
i

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.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.
Page 1 of 12

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 LD50 (Pesticide Assessment Guidelines 

Subdivision L); the Honey Bee—Acute Contact LD50 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 

(μg) of test substance per bee (μg/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.

LD50 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 LD50 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 2 of 12

(e)(2) of this guideline. The mortality pattern is examined and subjected to the 

appropriate statistical analysis to derive the LD50, slope, and confidence limits. Results 

are expressed as LD50 in micrograms (μg) 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 μg 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 

(LD50) (and its 95 percent (95%) confidence limits), as well as the slope of the doseresponse 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 μg of active 

ingredient per bee (25 μg a.i./bee). If the expected environmental contact residue level 

exceeds 25 μg 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 doseresponse 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 3 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 4 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 (CO2) or nitrogen gas (N2), 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)(1) 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 (μL) 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 LD05 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 μg/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 lb 

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 5 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  0.128ApRate135 Equation 1

  

  



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



















































 

 



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

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

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

n 

i

n erval

halflife Contact Limit Dose ApRate e

1

1 int 0.6931

0.128 135 Equation 2

ApRate = maximum single application rate (in lb a.i./acre);

Halflife = the foliar halflife (default is 35 days); 

Interval = the minimum application interval (in days);

i = 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 6 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 (oC), 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 7 of 12

(3) Limit test—

(i) LD50 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., LD50 > 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 

LD50 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 LD50 is at or below the limit dose is >0.999. 

Therefore, if <1 mortality occurs among the 25 bees tested, the 48-h LD50 is 

reported as greater than the limit dose (i.e., 48-h LD50 >25 μg/bee for pesticides).

(ii) Proportion of mortality ( pˆ ). Assuming mortality follows the binomial 

distribution, an estimate of the true proportion of mortality ( pˆ ) 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 ( pˆ = 0), the 99% confidence interval on the estimate of pˆ

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 ( pˆ = 0.04), the 99% confidence interval on 

the estimate of pˆ 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 pˆ 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 8 of 12

(4) Multiple-dose definitive test―

(i) Dose-response curve, slope and LD50. Statistical procedures are employed to 

calculate the 48-h LD50 (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 LD50, 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.

Table 1.—Summary of Test Conditions for the Honey Bee Acute Contact Toxicity

Test

Test type Acute contact

Test duration 48 h (96 h, if mortality increases >10% between 

24 and 48 h)

Temperature 25 - 35 oC 

Relative humidity 50 – 80%

Lighting Darkness, except during dosing and observations

Test chamber Metal, plastic, wire mesh, or cardboard

Test solution application Microapplicator or dusting apparatus

Age of test bees Young adult worker bees of similar age and 

feeding status

Number of bees per chamber Variable

Number of bees per treatment and control 25 (minimum)

Number of dose levels Minimum of 5 in a geometric series, a negative 

control, and a solvent control (if solvent used)

Feeding 50% sugar/water (w/v) solution ad libitum

Measure of Effect or Measurement Endpoint 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 9 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)(1) 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 10 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 11 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 LD50 values, with 95% 

confidence limits, at each recommended observation time.

(viii) Description of statistical method used, including software package, for determining 

LD50 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 al., 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 al., 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, 2

nd 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. 
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(8) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard 

Evaluation Procedure, Honey Bee—Acute Contact LD50 Test. Office of Pesticides 

Programs, Washington, D.C., EPA-540/9-85-002.