United States Prevention, Pesticides EPA712-C-96-333
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4240
Freshwater Aquatic
Invertebrate 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 885.4240 Freshwater aquatic invertebrate 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-20.
(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 regarded
as the test substance is described in OPPTS 885.0001. In addition, any
substances used to enhance the virulence or toxicity of the MPCA should
be tested along with the test substance.
(2) Test organisms, (i) For microbial pest control agents (MPCAs)
having terrestrial use patterns, where direct aquatic exposure is not ex-
pected, one species of benthic invertebrate should be tested. For MPCAs
where direct aquatic exposure is anticipated, testing shall be performed
on two aquatic invertebrate species, one of which is planktonic and the
other benthic.
(ii) The species of aquatic invertebrate selected should bear as close
a taxonomic relationship to the target host as possible.
(iii) Aquatic invertebrate species likely to prey upon or scavenge the
diseased target host organisms should be tested, when applicable.
(iv) Larval stages of invertebrates should be used whenever possible.
(v) Twenty invertebrates/group should be used if there are multiple
test groups. Fifty invertebrates should be used for single group testing.
(3) Controls, (i) A negative, nondosed control group should be per-
formed concurrently with the test groups.
(ii) A control group in which the invertebrates are exposed to sterile
filtrate from production cultures should be performed concurrently with
the test groups.
(4) Method of pesticide administration, (i) The test substance shall
be administered as a suspension directly into the water (i.e. aqueous expo-
sure).
(5) Maximum hazard dose. 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 con-
centration in a 6-in layer of water, immediately following a direct applica-
tion to a 6-in layer of water, whichever is greater and attainable. The
initial concentration of MPCA should be maintained throughout the test
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and the method used for maintenance of the dose level should be de-
scribed.
(6) Test duration. The test duration should be at least 21 days. If
pathogenicity and/or toxicity are apparent at the 21st day, observation
should continue until recovery, mortality, or unequivocal moribundity is
established.
(7) Treatment concentrations. A single group may be tested at the
maximum hazard concentration. If deleterious effects, due either to toxicity
or pathogenicity, are observed, sequentially lower doses should be tested
as described in paragraph (b)(8)(i) through (ix) of this guideline.
(8) Determination of LC50 or ID50. (i) Satisfactory data must estab-
lish whether or not the test substance is pathogenic to the test organisms
during a sufficiently long period of exposure and observation.
(ii) If the test substance produces a toxin, the data must establish
either:
(A) A definitive LC50 value with 95 percent confidence intervals.
(B) That the LC50 is greater than the highest dose.
(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 an aquatic invertebrate toxicity and infectivity test must
include:
(1) Raw data and EC50 calculations including 95 percent confidence
intervals.
(2) A detailed description of the steps taken to determine microorga-
nism dissemination, replication, or survival in the test animal tissues, or-
gans, or fluids.
(3) A detailed description of dilution water, including source, chemi-
cal characteristics (e.g. dissolved oxygen content, pH, dissolved salts),
method of sterilization, and pretreatment, (if any).
(4) A detailed description of the test, including:
(i) Design.
(ii) Container size.
(iii) Medium (e.g. depth and volume).
(iv) Pretreatments, if any.
(v) Method of exposing organisms to the test substance.
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(vi) Number of organisms per treatment.
(vii) Lighting, acclimation, and test temperatures (averages and
range).
(viii) Amount of test substance administered.
(ix) Any unusual feature of the test method.
(5) Detailed descriptions of methods (or references to established
methods) used for chemical analyses of water for chemical content and
MFC A concentrations.
(6) Detailed descriptions of methods used for all microbial analyses
of water and test organisms, and the results of such analyses.
(7) Detailed description of the effects of exposure to the test sub-
stance, including:
(i) The criteria used to determine the effects.
(ii) Statement of percentages of organisms that died or showed effects
of treatment.
(iii) A summary of these observations, including changes in life cycle
(duration, fecundity, and morphology).
(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, and 885.5400)) shall be required. In some cases a
subchronic test may serve to better understanding of the effects observed
at the Tier I level and alleviate the need for Tier II testing.
(2) If no toxic or pathogenic effects are observed, no further testing
at higher tiers ordinarily is required, except as noted in paragraph (d)(3)
of this guideline.
(3) If host spectrum or beneficial insect tests indicate a broad host
spectrum such that hazard toward aquatic invertebrates is indicated, either:
(i) Additional aquatic invertebrate species must be tested as described
in paragraphs (b) through (d) of this guideline.
(ii) Testing at Tier II, environmental expression (OPPTS 885.5000,
885.5200, 885.5300, and 885.5400)) is required.
(4) If toxic or pathogenic effects are observed in tests conducted in
accordance with paragraph (d)(3)(i) of this guideline, testing at Tier II en-
vironmental expression (OPPTS 885.5000, 885.5200, 885.5300, and
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885.5400)) is required. If not, no further tier testing is ordinarily required.
The Agency may require additional testing, however, if it determines that
there is a potential risk to aquatic invertebrates despite negative Tier I
results.
(e) References. The following references may contain useful back-
ground information for developing test protocols:
(1) Standard Methods for Examination of Water and Wastewater. 14th
Ed. American Public Health Association, Washington, DC (1975). pp.
1193.
(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) Committees on Methods for Toxicity Tests with Aquatic Orga-
nisms. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates,
and Amphibians. USEPA Ecological Research Series, EPA 660/375-009.
pp. 61.
(4) 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).
(5) Ignoffo, C. M. et al. Susceptibility of aquatic vertebrates and in-
vertebrates to the infective stage of the mosquito nematode, Reesimermis
nielseni. Mosquito News 33:599-602 (1973).
(6) Lightner, D.V. et al. Testing Penaeid shrimp for susceptibility to
an insect Nuclear Polyhedrosis virus. Environmental Entomology 2:611-
613 (1973).
(7) Pagano, J.S. and E. Huang. The application of RNA-DNA
cytohybridization to viral diagnostics. In: Viral Immunodiagnosis. E.
Kurstak and R. Morisset, eds. Academic Press, NY (1974).
(8) Reynolds, G.J. Enzyme labelled antibody in histopathology.
Qualityline Winter 1978/1979:2-10 (1978).
(9) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds.
Baculoviruses for Insect Pest Control: Safety Considerations. Selected pa-
pers from EPA-USDA Working Symposium, American Society for Micro-
biology, Washington, DC (1975).
(10) Undeen A.H. and J.V. Maddox. The infection of nonmosquito
hosts by injection with spores of the microsporidan Nosema algerae. Jour-
nal of Invertebrate Pathology 22:258-265 (1973).
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(11) Van Essen, F.W. and D.W. Anthony. Susceptibility of nontarget
organisms to Nosema algerae (Microsporida: Nosematidae), a parasite of
mosquitoes. Journal Invertebrate Pathology 28:77-85 (1976).
(12) Weber, C.E. (ed.) Biological field laboratory methods for meas-
uring the quality of surface waters and effluents. USEPA Environmental
Monitoring Series, EPA-670/473-001 (1973).
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