United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
EPA 712-C-96-148
April 1996
&EPA Ecological Effects Test
OPPTS 850.3030
Honey Bee Toxicity of
Residues on Foliage

Public Draft"

This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP, or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and

OPPTS 850.3030 Honey bee toxicity of residues on foliage.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this test
guideline is OPP 141-2 Honey Bee—Toxicity of Residues on Foliage
(Pesticide Assessment Guidelines, Subdivision L—Hazard Evaluation;
Nontarget Insects) EPA report 540/09-82-019, 1982.
(b)	Purpose. This guideline is designed to develop data on the resid-
ual toxicity to honey bees of chemical substances subject to environmental
effects test regulations. The Agency will use these and other data to assess
acute hazards to bees.
(c)	Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. In addition, the following definitions
apply to this guideline:
Test substance is the specific form of a chemical or mixture of chemi-
cals that is used to develop the data. For the purposes of this test, test
substance is a representative end-use product.
(d)	Test procedures—(1) Summary of test, (i) Test bees may be
obtained directly from hives or from frames kept in an incubator. The
test substance is applied to the test crop (alfalfa is preferred) at typical
label rate(s). At predetermined posttreatment time intervals, treated foliage
is harvested and test bees are caged on the foliage and on control (un-
treated) foliage. Bees are monitored and observed for mortality and signs
of intoxication during the exposure period. Mortality is determined after
24 h of exposure to the treated foliage.
(ii) A test is unacceptable if more than 20 percent of the control bees
die during the test.
(2) Definitive test—(i) Pesticide application and harvest of foliage.
A single application of the test substance will be made to each of nine
alfalfa plots. Plots should be at least 1 m2, in alfalfa grown according
to standard agricultural practices. Applications should be made with a hand
sprayer. Three control plots should be maintained and treated identically
to the treatment plots, with the exception of pesticide application. After
residues have aged for the appropriate time period, alfalfa foliage will be
harvested from the test plots and returned to the laboratory.
(ii) Exposure to test substance. On the day of test initiation, young
bees should be collected from the incubator or directly from the hive, im-
mobilized with CO2 or N2, and placed in holding cages. To initiate the
test, harvested foliage is chopped, mixed, and divided into 15-g portions.

Each portion is placed into a single test chamber. Bees in the holding
cages are again immobilized, and introduced into the test chambers until
each chamber contains at least 25 bees.
(iii)	Controls. (A) A negative (untreated) control is required during
the test. Control plots should be treated identically to treatment plots, ex-
cept for pesticide application. Control and test bees should be kept under
the same environmental conditions.
(B) A concurrent positive control with a substance of known toxicity
is not required. However, a quarterly or semiannual test with a laboratory
standard (reference toxicant) is recommended as a means of detecting pos-
sible interlaboratory or temporal variation. A laboratory standard is also
recommended when there is any significant change in source of bees.
(iv)	Number of animals tested. Six replicates should be assigned
to each treatment and control group, with a minimum of 25 bees for each
(v)	Dosages and posttreatment intervals. At a minimum, the test
substance should be evaluated at the maximum label rate. Multiples of
the maximum rate may be evaluated if desired. Residues should be allowed
to weather in the field for a specific time period prior to collection of
foliage for testing. For purposes of comparison, test samples could be col-
lected 3, 8, and 24 h after application. If mortality rates of bees exposed
to 24-h-old residues is greater than 25 percent, sampling at 24-h intervals
should continue until mortality of bees exposed to the treated foliage is
not significantly greater than control mortality.
(vi)	Duration of test. The definitive test consists of the exposure of
bees to the treated foliage followed by an observation period of 24 h.
(vii)	Observations. (A) Environmental conditions following pesticide
application will be monitored at the field site. Environmental information
to be collected will include temperature, precipitation, relative humidity,
wind speed, and estimated cloud cover.
(B)	Bees should be observed for mortality and toxicological responses
at least once within the first 4 h after exposure and at test termination.
Prior to the evaluation at test termination, observations should be made
without disturbing or removing bees from the test chambers; for these ob-
servations, estimates of mortality and effects are sufficient. Dead bees
should not be removed from the test chambers until the test is terminated.
(C)	Throughout the test period, all signs of intoxication, other abnor-
mal behavior, and mortality should be recorded and reported by dosage
level and by time of occurrence. Signs of intoxication are those behaviors
apparently due to the test chemical and may include a wide variety of
behaviors, such as ataxia, lethargy, and hypersensitivity. All signs of in-

toxication and any other abnormal behavior, that may or may not be attrib-
uted to the test substance, should be reported.
(e)	Test conditions—(1) Test species—(i) Selection. (A) Honey bee,
Apis mellifera, is the test species. Bees may be obtained from on-site colo-
nies or from a commercial apiary. All control and treatment bees used
in a test should be from the same source.
(B)	Bees used in the test should be in apparent good health. Only
bees from disease-free colonies should be used, and they should be kept
in conditions conforming to proper cultural practices.
(C)	Test should be conducted on worker bees 1 to 7 days old at test
initiation. No acclimation period is necessary. Bees used in the test should
be assigned randomly to treatments and controls.
(D)	During holding and testing, bees should be shielded from exces-
sive activity or other disturbance. Bees should be handled only as much
as is necessary to conform to test procedures.
(ii) Diet. (A) A 50 percent sugar/water solution should be provided
ad libitum throughout the holding and test periods. Purified or distilled
water should be used for the sugar solution.
(2) Facilities, (i) Tests should be conducted indoors with bees being
maintained in small test chambers. Test chambers may be constructed of
metal, plastic, wire mesh, or cardboard, or a combination of these mate-
rials. Chambers must be constructed so that a vial containing sugar water
may be attached.
(ii) Testing is done indoors to control lighting and other environ-
mental variables. Temperature should be maintained between 25 and 35
°C, with relative humidity between 50 and 80 percent. It is recommended
that test bees be maintained in the dark except during dosing and observa-
(f)	Reporting. The report should include, but not necessarily be lim-
ited to, the following information:
(1)	Name and address of the facility performing the study and the
dates of the study.
(2)	Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.
(3)	The test and, if used, control substances identified by name,
Chemical Abstracts Service (CAS) number or code number, source, lot
or batch number, strength, purity, and composition or other appropriate
(4)	A description of the methods used, including:

(A)	Description of housing conditions, including type, size, and mate-
rial of pens, and the approximate test room temperature, humidity, and
(B)	Methods of application, foliage collection, and placement of
foliage in test chambers.
(C)	Methods of assigning bees to test chambers.
(D)	Frequency, duration, and methods of observations.
(5)	A description of the test system used, including the scientific
name of the test species, number used, condition, age at test initiation,
and source of test bees.
(6)	A description of the dosages, numbers of bees and replicates per
dose, and method and time of administration. The reported results should
include, 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 dose level and control.
(7)	A description of all circumstances that may have affected the qual-
ity or integrity of the data.
(8)	The name of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the names of all supervisory
personnel involved in the study.
(9)	The signed and dated reports of each of the individual scientists
or other professionals involved in the study, including each person who,
at the request or direction of the testing facility or sponsor, conducted
an analysis or evaluation of data or specimens from the study after data
generation was completed.
(10)	The locations where all raw data and the final report are stored.
(11)	The statement prepared and signed by the quality assurance unit.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	Johansen, C. et al. Bee Research Investigations. Dept. of Ento-
mology, Washington State University, unpublished, 22 pp. (1977).
(2)	Lagier, R.F. et al. Adjuvants Decrease Insecticide Hazard to
Honey Bees. College of Agriculture Research Center, Washington State
University Bulletin 801, 7 pp. (1974).
(3)	U.S. Environmental Protection Agency, Standard Evaluation Pro-
cedure, Honey Bee—Toxicity of Residues on Foliage, EPA Report No.
540/09-85-003 (1985).