United States Prevention, Pesticides EPA712-C-96-344
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4700
Fish Life Cycle Studies,
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 885.4700 Fish life cycle studies, Tier III.
(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 har-
monized OPPTS test guideline is OPP guideline 154A-28.
(b) Test standards. Data must be derived from tests that satisfy the
general test standards in OPPTS 885.0001, and the following:
(1) Test substance. The actual form of the test material to be used
is described in OPPTS 885.0001. In addition, any substances used to en-
hance virulence should be tested along with the test substance.
(2) Duration of test. The fish life-cycle tests require that the test
animals be cultured in the presence of the microbial pest control agent
(MPCA) during one complete life cycle, i.e. one stage of the life cycle
to the same stage of the next generation.
(3) Test organisms, (i) Choose the species most likely to be affected
by the MPCA being tested. If a microorganism that is closely related
(within the same family) to the MPCA causes disease in a certain fish
species, that species should be chosen as the test organism.
(ii) Microbial pest control agents that are expected to enter freshwater
ecosystems should be tested on freshwater fish. If an estuary is likely to
be impacted, marine fish should be used.
(iii) In the event that a species cannot be identified using the fore-
going criteria, suggested species are as follows:
(A) Freshwater species—Brook trout (Salvelinus fontinalis); fathead
minnow (Pimephales promelas}; bluegill sunfish (Lepomis marochirus};
channel catfish (Ictalurus punctatus).
(B) Marine/esturine species—sheepshead minnow (Cyprinodon
variegatus); Atlantic silverside (Menidia menidid).
(4) Dose levels. The dose levels shall be equal to that expected to
be found in the aquatic environment calculated from application rates with
appropriate adjustments to take into account the environmental survival
and multiplication characteristics of the MPCA as determined by Tier II
testing.
(5) Test procedure. Refer to the references under paragraph (e) of
this guideline for general guidance in design of fish life cycle tests. The
applicant should consult with the Agency for testing details.
(6) Test duration. For test species with life cycles of 1 year or less,
the test should begin with newly-hatched larvae and continue until 30 days
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after progeny hatch. When fish with life cycles that are greater than
1 year, begin the test with fish that are about to commence their first
spawn and continue until 90 days after the progeny hatch.
(c) Reporting and evaluation of data. In addition to the information
specified in OPPTS 885.0001, the test report shall contain the following
information on the nontarget test organism:
(1) Reproductive effects.
(2) Effects an the growth of larvae and juveniles.
(3) Detailed records of spawning, egg numbers, fertility, and fecun-
dity.
(4) Estimated no observed effect level.
(5) Mortality data.
(6) Statistical evaluation of effects.
(7) Locomotion, behavioral, physiological, and pathological effects.
(8) Definition of the criteria used to determine effects.
(9) Summary of observed signs of pathogenicity or other effects.
(10) Isolation, identification, and enumeration of microorganisms re-
sponsible for any observed pathogenic effects.
(11) Stage of life cycle in which effects occurred.
(d) Tier progression. (1) Further testing at OPPTS 885.4900 may
be required if pathogenic effects are observed.
(2) Additional testing at higher tiers ordinarily is required if patho-
genic effects are observed.
(e) References. The following may contain useful information for de-
veloping acceptable protocols:
(1) American Public Health Association. Standard Methods for Exam-
ination of Water and Wastewater. 16th edition. American Public Health
Assoc., Washington, D.C. (1985), pp. 1268.
(2) Biesinger, K.E. Procedure for Daphnia magna tests in standing
system. Environmental Protection Agency, Environmental Research Lab-
oratory, Duluth, MN (1974).
(3) Biesinger, K.E. Procedure for Daphnia magna chronic tests in
flowing system. Environmental Protection Agency, Environmental Re-
search Laboratory, Duluth, MN (1974).
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(4) Biesinger, K.E. Culturing methods for Daphnia and certain other
cladocerans. Environmental Protection Agency, Environmental Research
Laboratory, Duluth, MN (1974).
(5) Hansen, D.J. et al. Life-cycle toxicity test using sheepshead min-
nows (Cyprinodon variegatus}. Pp. 109-116 in Bioassay Procedures for
the Ocean Disposal Permit Program. Environmental Protection Agency,
Office of Research and Development EPA-600/9-78-010 (1978).
(6) Nimmo, D.E. et al. Entire life-cycle toxicity test using mysids
(Mysidopsis bahia) in flowing water. Pp. 64-68 in Bioassay Procedures
for the Ocean Disposal Permit Program. Environmental Protection Agency,
Office of Research and Development EPA-600/9-78-010 (1978).
(7) Schimmel, S.C. and D.J. Hansen. Sheepshead minnow Cyprinodon
variegatus: an estuarine fish suitable for chronic (entire life-cycle) bio-
assays. Proceedings of the 28th Annual Congress of the Southeastern As-
sociation of Game-Fish Comm. Pp. 392-398 (1974).
(8) National Water Quality Laboratory Committee on Aquatic Bio-
assays. Recommended bioassay procedure for fathead minnow Pimephales
promelas (Rafinesqui) chronic tests. National Water Quality Laboratory,
Duluth, MN, 13 pp. (1971) (Revised January 1972.)
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