United States Prevention, Pesticides EPA712-C-96-334
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4280
Estuarine and Marine
Animal Testing, Tier
-------
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."
-------
OPPTS 885.4280 Estuarine and marine animal testing.
(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-21.
(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 material is described in OPPTS 885.0001. In addition, any sub-
stances used to enhance virulence or toxicity should be included.
(2) Test organisms, (i) Toxicity and pathogenicity shall be deter-
mined for one species of shrimp, preferably Paleomonetes vulgaris and
one estuarine or marine fish species.
(ii) Testing of additional estuarine or marine animal species may be
required in Tier I as specified in paragraph (d)(3)(i) of this guideline.
(iii) Estuarine or marine animals likely to prey upon or scavenge the
diseased target host organisms should be tested, when applicable.
(iv) Tests should be conducted using young fish, from the same year
class, weighing between 0.5 and 5.0 gm. Very young (not yet actively
feeding), spawning, or recently spawned fish should not be tested. The
length of the largest fish should be no more than twice that of the shortest
fish.
(3) Controls, (i) A negative, nondosed control should be performed
concurrently with the test groups.
(ii) A control group in which the animals are exposed to the sterile
filtrate from the manufacturing-use product should be performed concur-
rently with the test groups.
(4) Method of pesticide administration, (i) For fish, the microbial
pest control agent (MPCA) will be administered as a suspension directly
into the water and, separately, incorporated into the food.
(ii) For shrimp, the MPCA will be incorporated into food pellets
which are fed to the test organisms.
(5) 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 cell density
in a 6-in layer of water immediately following a direct application to a 6-
in layer of water, whichever is greater and attainable.
-------
(ii) Feed used in the dietary exposure should be supplemented with
the test substance to achieve a microbial concentration at least lOOx the
calculated cell density in a 6-in layer of water immediately following a
direct application to a 6-in layer of water.
(6) Test duration. The test should last at least 30 days. If pathogenic-
ity and/or toxicity is apparent at the 30th day, observations should continue
until recovery, mortality, or unequivocal moribundity is established.
(7) Number of organisms per concentration. The number of test
organisms per group should be 10 for fish and 30 for shrimp.
(8) Treatment concentrations. A single group may be tested at the
maximum hazard dose. If deleterious effects, due either to toxicity or path-
ogenicity, are observed, sequentially lower doses should be tested as de-
scribed in paragraph (b)(9) of this guideline.
(9) Determination of LC50 or ID50. (i) Satisfactory data must estab-
lish an IC50 with 95 percent confidence limits or, that the IC50 is greater
than the highest dose.
(ii) If the test substance produces a toxin, the data must establish
either:
(A) A precise LC50 or value with 95 percent confidence intervals,
or
(B) That the LC50 is greater than the highest dose.
(c) Reporting and evaluation of data. In addition to information
meeting the general requirements of OPPTS 885.0001, a report of the re-
sults of estuarine or marine animal toxicity and pathogenicity tests must
include the following:
(1) LC50 data (if the test substance produces a toxin).
(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) Detailed description of dilution water, including source, chemical
characteristics (e.g., dissolved oxygen content, pH, chlorine content, dis-
solved salts), method of sterilization, and pretreatment (if any).
(4) Other pertinent details, 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 (e.g. placing
test substance in water which contains organisms or placing organisms
in water which contains the test substance).
(vi) Number of organisms per treatment.
(vii) Loading (weight of organisms per unit volume of medium or
unit of surface).
(viii) Lighting.
(ix) Acclimation and test temperatures (average and range).
(x) Salinities.
(xi) Amount of test substance administered.
(xii) Any unusual feature of the test.
(5) Detailed description of methods (or references to established
methods) used for all chemical analyses of water for chemical content and
MFC A concentrations.
(6) Detailed description of methods used in 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) A statement of the percentage of organisms that died or showed
effects from the treatment.
(iii) A summary of these observations.
(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 (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 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 is ordinarily required, except as noted in paragraph (d)(3)
of this guideline.
-------
(3) If efficacy or beneficial insect tests indicate a broad host spectrum
such that susceptibility of estuarine or marine invertebrates is indicated,
either:
(i) Additional estuarine or marine invertebrate species must be tested
following the guidelines in paragraphs (b) through (d) of this guideline,
or
(ii) Testing at Tier II environmental expression (OPPTS 885.5000,
885.5200, 885.5300, 885.5400) is required as specified in 40 CFR
158.740(e).
(4) If toxic or pathogenic effects are observed in tests conducted in
accordance with the requirements of this section, testing at Tier II environ-
mental expression (OPPTS 885.5000, 885.5200, 885.5300, 885.5400) is
required. Otherwise, no further tier testing is required.
(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). pp.
1193.
(2) ASTM Standard E 729-80, Practice for Conducting Static Acute
Toxicity Tests with Larvae of Four Species of Bivalve Molluscs. American
Society for Testing and Materials, 1916 Race Street, Philadelphia, PA
19103.
(3) Bioassay Procedures for the Ocean Disposal Permit Program.
USEPA, Office of Research and Development. EPA-600/9-78-010;pp.
121 (1978).
(4) Banner, L.H. et al. A salt-water flow-through bioassay method
with controlled temperature and salinity, Progress in Fish-Culture 37:126-
129 (1975).
(5) Clark, J.R. and R.L. Clark, eds. Seawater systems for experimental
aquariums. US Interior Department, Fish and Wildlife Service, Bureau of
Sport. Fish. Wild. Research Report No. 63, 192 pp. (1964).
(6) Committee on Methods for Toxicity Tests with Aquatic Orga-
nisms. Methods for acute toxicity tests with fish, macroinvertebrates, and
amphibians. USEPA Ecological Research Series, EPA 660/3-75-009. 61
pp. (Marine and estuarine species listed in this publication are acceptable.)
(1975).
(7) Couch, J.A. et al. Environmental significance of baculovirus infec-
tions in estuarine and marine shrimp. Annals N.Y. Academy of Science
219:528-536 (1975).
-------
(8) Couch, J.A. Design and Test of a Simple System for the Prelimi-
nary Evaluation of Infectivity and Pathogenesis of Insect Virus in a Non-
target Estuarine Shrimp. Journal of Invertebrate Pathology 43:351-357
(1984).
(9) DeBen, E.A. Design and construction of saltwater environment
simulator. Federal Water Quality Administration, Pacific N.W. Water Lab-
oratory, Working Paper 71:1-30 (1970).
(10) Hetrick, P.M. et al. Increased susceptibility of rainbow trout to
infectious hematopoetic necrosis virus after exposure to copper. Applied
Environmental Microbiology 37:198-201 (1979).
(11) Huang, E. and J.S. Pagano. Nucleic acid hybridization tech-
nology and detection of proviral genomes. Chapter 13 in The Atlas of In-
sect and Plant Viruses. K. Maramorosch, ed. Academic Press, NY (1977).
(12) 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).
(13) Lightner, D.V. et al. Testing Penaeid shrimp for susceptibility
to an insect Nuclear Polyhedrosis virus. Environmental Entomology 2:
611-613(1973).
(14) Pagano, J.S., and E. Huang, 1974. The application of RNA-DNA
cytohybridization to viral diagnostics. In: Viral Immuno diagnosis. E.
Kurstak and R. Morisset, eds., Academic Press, Inc., N.Y.
(15) Reynolds, G.J. 1978. Enzyme labelled antibody in histo pathol-
ogy. Qualityline (Winter 1978/1979):2-10.
(16) Shelbourne, HE. 1962. Experimental seawater systems for
rearing fish larvae. Pp.81-93 in Seawater Systems for Experimental
Aquariums. J.R. Clark and R.L. Clark, eds. U.S. Dept. Int., Fish. Wild.
Serv., Bur. Sport Fish. Wild. Res. Rep. No.63. 192 pp.
(17) Strickland, J.D.H., and T.R. Parsons. 1968. A practical handbook
of seawater analysis. Fish Res. Board Can. Bull. No. 167., 311 pp.
(18) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds. 1975.
Baculoviruses for Insect Pest Control: Safety Considerations. Selected pa-
pers from EPA-USDA Working Symposium, American Society for Micro-
biology Washington, D.C.
(19) 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).
-------
(20) 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).
(21) Van Essen, F.W. and D.W. Anthony. Susceptibility of nontarget
organisms to Nosema algerae (Microsporida: Nosematidae), a parasite of
Mosquitoes. Journal of Invertebrate Pathology 28:77-85 (1976).
(22) Weber, C.E. (ed.) Biological field laboratory methods for meas-
uring the quality of surface waters and effluents. USEPA, Environmental
Monitoring Series, EPA-670/4-73/001 (1973).
------- |