
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




                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
  
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  NOTICE
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  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.
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  	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.  
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  	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.
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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 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 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.
            (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 (m[2]) (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 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 ([o]C), with relative humidity between 50% and 80%. 
            (ii) Lighting and photoperiod.  It is recommended that test bees be maintained in the dark except during transfer to test cages and observations.
	(9) Observations--
            (i) Analysis for test substance concentrations.  Test substance residues on treated foliage are measured in ppm fresh weight.  Foliage residue sampling and analysis should be made immediately after the application when test substance has dried and on harvested foliage (3, 8, 24 h post-application, or longer as needed) used for bee mortality testing.
            (ii) Field site conditions.  Environmental conditions should be monitored at the field site during and after test substance application.  Environmental information to be collected should include temperature, precipitation, relative humidity, wind speed, and estimated cloud cover. 
		(iii) Measures of Effect--
                  (A) Mortality.  For a given weathered residue treatment or control, bees should be observed for mortality at least once during the first 4 h after exposure and at test termination (24 hours).  Dead bees should not be removed from the test chambers until the test is terminated.  
                  (B) Appearance and behavior.  For a given weathered residue treatment or control, bees should be observed for all signs of intoxication and any other abnormal behavior once during the first 4 h after exposure and at test termination.  Observations should be recorded by treatment level and by time of occurrence.  Signs of intoxication are those behaviors apparently due to the test substance and may include a wide variety of behaviors, such as ataxia, lethargy, and hypersensitivity.  Prior to the evaluation at test termination, observations should be made without disturbing or removing bees from the test chambers; for these observations, estimates of mortality and effects are sufficient.



(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 t-test) should be performed to detect significant difference of treatments from controls.  The 24-h RT25 is estimated using linear interpolation; Abbott's correction (see paragraph (j)(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.
	

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 [o]C
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 post-application 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.

	Table 2. -- Test Validity Elements for Honey Bee Toxicity of Residue on Foliage Test
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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. 

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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.
		(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 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.
	(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.
