United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-018

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3030:

Honey Bee Toxicity of 

Residues on Foliage
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 11

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 (lb/A) and for a pesticide pound(s) of active ingredient applied per 

acre (lb 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 (lb a.i./A).

(d) General considerations—

(1) Summary of test. The honey bee (Apis mellifera) 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 RT25) in test populations at an expressed rate of application (lb 

a.i./A).

(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 (CO2) or nitrogen 

gas (N2), and placed in holding cages. Exposure to CO2 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.
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(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, 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 (m2) (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 (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 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.
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(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 (RT25). A test for comparing two paired populations (e.g., paired ttest) 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)(1) of this guideline) should be used in the event of control mortality. Additional 

discussion about measurement 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. 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

Test type Toxicity of residues on foliage

Test duration 24 h for each aged residue interval (3, 8, and 24 h 

aged residue intervals are tested; additional residue 

intervals may be appropriate).

Temperature 25 - 35 oC

Relative humidity 50 – 80%

Lighting Darkness, except during transfer of bees to treatment 

cages and observations

Test chamber Metal, plastic, wire mesh, or cardboard

Test substance application 15-g or 500-cc portions of treated foliage (hand sprayer 

used to apply test solution to foliage) placed in a test 

cage 

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

status

Number of bees per chamber 25 (minimum)

Number of bees per treatment and control 150 (minimum)

Number of treatments 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 postapplication treatment.

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

Measure of Effect or Measurement Endpoint 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.
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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)(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) 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 (lb 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 

treatments).

(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 

lighting.

(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 

RT25 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 al., 1977. Bee Research Investigations. Dept. of Entomology, 

Washington State University, unpublished, 22 pp.

(3) Lagier, R.F. et al., 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, 

WA.

(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.
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(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.