United States Prevention, Pesticides EPA712-C-96-332
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4200
Freshwater Fish 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 885.4200 Freshwater fish 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-19.
(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 material to be considered
as the test substance is described in OPPTS 885.0001. In addition, any
substance used to enhance virulence or toxicity should be tested along
with the test substance.
(2) Test organisms, (i) Testing shall be performed on one fish spe-
cies, preferable the rainbow trout if the microbial pest control agent
(MPCA) has only a terrestrial use and direct aquatic exposure is not ex-
pected, or two fish species, preferably the bluegill sunfish and rainbow
trout, when direct aquatic exposure is anticipated. Other species of fish
may be used, but a justification must be supplied based on an increased
susceptibility to the MPCA or ecological considerations that preclude the
use of recommended species.
(ii) The following characteristics should guide species selection:
(A) Fish species likely to prey upon or scavenge the target host orga-
nisms should be tested, when applicable.
(B) Testing of young fish is preferable. Very young (not yet actively
feeding), spawning, or recently spent fish should not be used.
(C) Fish should weigh between 0.5 and 5.0 g and be from the same
year class. The length of the longest fish should be no more than twice
that of the shortest fish.
(iii) Ten fish per group should be used in multiple group testing, 30
fish in single group testing.
(3) Route of exposure, (i) The test substance shall be administered
as a suspension directly into the water (i.e. aqueous exposure).
(ii) Additionally, the MPCA should be administered through the oral
route of exposure, preferably through incorporation in standard fish food
or through the use of infected insects.
(4) Maximum hazard dose, (i) At a minimum, the concentration in
the test water (for aqueous exposure) should, whenever possible, be at
least 106 units/mL or at least l,000x the maximum calculated pesticide
concentration in a 6-in layer of water immediately following a direct appli-
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cation to a 6-in layer of water, whichever is greater and attainable. Meas-
ures should be taken to ensure that the initial concentration of the MPCA
is maintained throughout the test should be described.
(ii) Feed used in the dietary exposure should be supplemented with
the test substance to achieve a microbial concentration per gram of food
of at least lOOx the calculated cell density per milliliter in a 6-in layer
of water immediately following a direct application to a 6-in layer of
water.
(5) Controls, (i) A negative, nondosed control group should be run
concurrently the test groups.
(ii) A control group in which the fish are exposed to sterile filtrate
from production cultures should be performed concurrently with the test
groups.
(6) Test duration. The fish should be observed for a minimum of
30 days after dosing. If symptomatology is present at the 30th day, obser-
vation should be continued until recovery, mortality, or unequivocal
moribundity is established.
(7) Treatment concentrations. A single, group of fish may be tested
at the maximum hazard dose. If deleterious effects, due either to toxicity
or pathogenicity are observed, sequentially lower doses should be tested
as described in paragraph (b)(8) of this guideline.
(8) Determination of LC50 or ID50. (i) The study endpoint must
be chosen to reflect the activity of the specific microorganism under test,
i.e. if an MPCA is expected to produce a toxin and has little or no infectiv-
ity, the appropriate endpoint would be mortality. If, however, the major
mechanism is pathogenicity, a more appropriate endpoint would be overt
symptomatology.
(ii) The data should establish that the freshwater fish LC50, defined
as the dose required to kill 50 percent of the test organisms, or IC50,
defined as the dose necessary to produce overt symptomatology in 50 per-
cent of the test organisms, is greater than the maximum hazard dosage
level. If the LC50 or IC50 is lower than the hazard dose, an LC50 or
IC50 with confidence intervals should be established.
(c) Reporting and evaluation of data. In addition to information
meeting the general reporting requirements of OPPTS 885.0001, a report
of the results of a fish toxicity and infectivity test must include the follow-
ing:
(1) LC50 or IC50 determination, including all associated parameters,
e.g. slope, goodness of fit, etc., along with the raw mortality data.
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(2) Detailed description of the steps taken to determine microorga-
nism dissemination, replication, or survival in any test animals tissues, or-
gans, or fluids.
(3) Detailed description of dilution water, including:
(i) Sterilization method.
(ii) Source.
(iii) Chemical characteristics (e.g. dissolved oxygen content, pH, chlo-
rine content, dissolved salts).
(iv) Pretreatments (if any).
(4) Detailed description of the test, including:
(i) Design.
(ii) Container size.
(iii) Medium (e.g. depth and volume).
(iv) Prophylactic treatments.
(v) Number of organisms per treatment level.
(vi) Loading (weight of organisms per unit volume of medium).
(vii) Lighting, acclimation, and test temperatures (averages and
range).
(viii) Amount of test substance administered by each route of expo-
sure.
(ix) Any unusual feature of the test method.
(5) Detailed descriptions of methods (or references to established
methods) used for all chemical analyses of water for chemical content and
MPCA concentrations.
(6) Detailed description of methods used for all microbial analyses
of water, transport hosts and test organisms, and results of such analyses.
(7) Detailed description of the effects of exposure to the test substance
including:
(i) The criteria used to determine the effects.
(ii) Percentages of test animals that died or showed symptomology.
(iii) A summary of these observations.
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(8) Any additional relevant information about the test or its results
that would assist in the determination of hazard potential.
(d) Tier progression. (1) If toxic or pathogenic effects are observed,
testing at Tier II (environmental expression testing (OPPTS 885.5000,
885.5200, 885.5300, 885.5400,) is required as specified in 40 CFR
158.740(e). In some cases, a subchronic test may serve to better under-
standing of the effects observed at the Tier I level and alleviate the need
for Tier II testing.
(2) Further testing generally is not required if results of this study
do not indicate toxic or pathogenic effects. The Agency may require addi-
tional testing if it determines that there is a potential risk to fish despite
negative Tier I results.
(e) References. The following may contain useful background infor-
mation for developing test protocols:
(1) Standard Methods for Examination of Water and Wastewater. 14th
Ed. American Public Health Association, Washington, DC (1975).
(2) ASTM-Standard E 729-80, Practice for Conducting Acute Tox-
icity Tests with Fishes, Macroinvertebrates, and Amphibians. American
Society for Testing and Materials, 1916 Race Street, Philadelphia, PA
19103.
(3) Committee an Methods for Toxicity Tests with Aquatic Orga-
nisms. Methods for acute toxicity tests with fish, macroinvertebrates, and
amphibians. USEPA Ecological Research Series, EPA 660/3-75-009. 61
pp. (1975).
(4) Hetrick, P.M. et al.Increased susceptibility of rainbow trout to in-
fectious hematopoietic necrosis virus after exposure to copper. Applied and
Environmental Microbiology 37:198-201 (1979).
(5) Huang, E. and J.S. Pagano. Nucleic acid hybridization technology
and detection of proviral genomes. Chapter 13 in: The Atlas of Insect and
Plant Viruses, K. Maramorosch, Ed. Academic Press, NY (1977).
(6) Ignoffo, C.M. et al. Susceptibility of aquatic vertebrates and inver-
tebrates to the infective stage of the mosquito nematode Reesimermis
nielseni. Mosquito News 33:599-602 (1973).
(7) Tamer, M.F. et al. The tissue localization of Aeromonas
salmonicida in rainbow trout, Salmo gairdneri Richardson, following three
methods of administration. Journal of Fish Biology 25: 95-108 (1984).
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