United States       Prevention, Pesticides     EPA712-C-96-337
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA   Microbial Pesticide
          Test Guidelines
          OPPTS 885.4380
          Honey Bee Testing,
          Tier

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                           INTRODUCTION
     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
(OECD).

     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
(7U.S.C. I36,etseq.).

     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     162.140.64.19),    internet:     http://
fedbbs.access.gpo.gov, or call 202-512-0132 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 Guidelines."

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OPPTS 885.4380  Honey bee testing, Tier I.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the  Federal Insecticide,  Fungicide, and  Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 154A-24.

     (b) When required. Data on the toxicity/pathogenicity of a microbial
pest control agent (MPCA) are  required when the proposed use pattern
indicates that honey bees may be  exposed to the pesticide, and for each
manufacturing-use product that may  be used  legally to formulate such an
end-use product. See 40 CFR 158.50 and 158.740 to determine whether
these data must be submitted.

     (c) Test standards. In addition to satisfying  the  applicable general
test standards outlined in OPPTS 885.0001, the following apply:

     (1) Test substance. The actual  form of the material to  be regarded
as the  test substance is discussed in OPPTS 885.0001. In addition, any
substances used  to enhance virulence should be tested along with the test
substance.

     (2) Test species. Testing shall be  performed on the honey bee, Apis
mellifera.

     (3) Age. When the MPCA  may be expected  to affect insect larvae,
test insects should include honey bee larvae.

     (4) Route of exposure. When  the MPCA  may be expected to  act
by a dietary route of exposure or are  particles  of such a size that they
might be  carried back to the hive like pollen,  the honey bees must be
dosed orally. Testing in the hive may  be necessary.

     (5) Controls. A concurrent control group is recommended and should
be treated with microbe-free (or nonviable microbe) material from the cul-
ture system used for propagation of the MPCA.

     (6) Duration of test. Control and treated bees should be observed
for at least 30 days after dosing.

     (d) Reporting and  evaluation  of data. The  reporting requirements
are the same as those specified in OPPTS 885.0001.

     (e) Tier progression  (1) Data  derived  from  Tier I  testing  will be
used in conjunction with available information on use pattern, host range,
and other factors,  to assess potential for adverse effects. If data indicate
that the potential for adverse effects exists, Tier II testing will be required
as specified in 40 CFR  158.740. In some cases,  a subchronic test may

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serve to better understanding of the  effects  observed at the Tier I level
and alleviate the need for Tier II testing.

     (2) If toxic or pathogenic effects are not observed in this study, addi-
tional testing is ordinarily not necessary.

     (f) References. The following references are provided for use in the
development of acceptable test protocols for  conducting a honey bee tox-
icity/pathogenicity test with a microbial pest control agent:

     (1)  Davidson,  E.W.  et al.  Effect  of  Bacillus  sphaericus  strain
SSII-1 on honey bees, Apis mellifera. Journal of Invertebrate Pathology
29:344-346 (1977).

     (2) Menapace, D.M. et al. Adult honey bees are  not  susceptible to
infection by Nosema  locustae. Journal of Economic Entomology 71:304-
306 (1978).

     (3) Morton, H.L. et al. Effect of alfalfa looper nuclear polyhedrosis
virus in honey bees. Journal of Invertebrate Pathology 25:139-140 (1975).

     (4) Hitchcock, J.D. et al. Pathogenicity of Bacillus pulvifaciens to
honey bee larvae of various ages (Hymenoptera: Apidae). Journal of Kan-
sas Entomological Society 52:238-246 (1979).

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