United States       Prevention, Pesticides     EPA712-C-96-312
          Environmental Protection    and Toxic Substances     February 1996
          Agency         (7101)
&EPA    Microbial Pesticide
          Test Guidelines
          OPPTS 885.5400
          Expression in a Marine
          or Estuarine

     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
(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,    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."

OPPTS 885.5400   Expression in a marine or estuarine environment.
     (a) Scopeó(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  155A-12.

     (b) Test  standards.  This guideline  is  to  be  used with  OPPTS

     (1) Tests shall be conducted in a simulated marine or estuarine envi-
ronment (e.g.,  aquarium with bottom sediment) to  determine  whether the
microbial  pest control agent (MPCA) is able to  survive, persist, and/or
replicate in  a marine or  estuarine  environment consisting of seawater or
brackish water  and bottom  sediment representative  of the proposed use
site.  The following parameters  should be varied to  determine their effect
on the survival and growth of the MPCA population:

     (i)  Temperature.

     (ii) pH.

     (iii) Nutrients.

     (iv) Salinity.

     (v) Sunlight.

     (vi) Oxygen content.

     (vii) Turbulence.

     (2) The values selected for each parameter listed should be  selected
to approximate the conditions expected at the intended use site.

     (3) Specialized laboratory studies designed to determine the MPCA's
growth  requirements  (e.g., temperature, pH, sunlight, and oxygen) may
supplement the study described  in paragraph (d)(l)(i) of OPPTS 885.5000.
Specialized  laboratory studies may demonstrate that the MPCA  will be
unable to survive and persist in  a marine or estuarine environment. In such
instances,  the Agency will consider studies on an individual basis in lieu
of the study  described in this guideline.

     (c) Test substance.  A typical end-use product  or the technical grade
of the active ingredient shall be tested.

     (d) Test duration. Data to  establish a population decline curve should
be collected at intervals until two half-life determinations have been made
or until  data establish that the microbial agent population is able to main-

tain itself in a  marine or estuarine  environment at or above  the  level
present immediately after test initiation.

     (e) Reporting and evaluation of data. The reporting and evaluation
provisions are the same as those set forth in OPPTS 885.5000.  In addition,
any changes in morphology of the microorganism  in  response in to
changes in salinity should be reported.

     (f) Tier progression. If results of this study and use pattern informa-
tion indicate that the MPCA is likely to  enter and is able to persist in
a marine or estuarine environment such that the susceptible nontarget orga-
nisms  tested in  Tier I is likely to be exposed, the appropriate testing in
Tier III (OPPTS 885.4450 through 885.4850) is required as specified by
40 CFR 158.740.

     (g) References. The following references contain  useful for develop-
ing acceptable protocols:

     (1) Anthony, D.W. et al. Field tests with Nosema algerae Vavra and
Undeen (Microsporida, Nosematidae) against Anopheles albinamus  Wiede-
mann in Panama. Miscellaneous Publications  of the Entomological  Society
of America 11:17-28 (1978).

     (2) Brand, R.J. et al. Methods for assessing field persistence of Bacil-
lus thuringiensis spores. Journal of Invertebrate  Pathology 25:199-208

     (3) EPA. Impact of the use of microorganisms on the aquatic environ-
ment. EPA publication 660-3-75-001. Technical Publications  Office, En-
vironmental Protection Agency, National Environmental Research  Center,
Corvallis, OR 97330 (1975).

     (4)  Hostetter,  D.L. et al.  Persistence of formulations  of Bacillus
thuringiensis spores and crystals on eastern red cedar  foliage  in Missouri
Journal of the Kansas Entomological Society 48:189-193 (1975).

     (5)  Ignoffo,  C.N.  et  al.  Stability  of  Bacillus  thuringiensis  and
Baculovirus  heliothis  on  soybean foliage.  Environmental Entomology

     (6)  Kaya,  H.K.  Persistence  of spores  of Pleistophora schuber
(Onidospora: Microsporida) in the field and their application in microbial
control. Journal of Invertebrate Pathology 26:329-332 (1975).

     (7) Pinnock, D.E. et al. The field persistence of Bacillus thuringiensis
spores. Journal of Invertebrate Pathology 18:405-411 (1971).

     (8) Sinclair, J.L. and M. Alexander.  Role of resistance to starvation
in bacterial survival  in  sewage and  lake water. Applied Environmental
Microbiology 48:410-415 (1984).

    (9) Young, S.Y. Pre- and posttreatment assessment of virus levels.
Selected papers from EPA-USDA Working Symposium. M. Summers, R.
Engler, L. Falcon, and P. Vail (eds.) American Society of Microbiology