United States Prevention, Pesticides EPA712-C-96-336
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4340
Nontarget Insect
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 885.4340 Nontarget insect 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-23.
(b) Test standards. In addition to satisfying the applicable general
test standards outlined in OPPTS 885.0001, the following apply:
(1) Test substance. The actual form of the material to be regarded
as the test substance is discussed in OPPTS 885.0001. In addition, any
substances used to enhance virulence should be tested along with the test
substance. Whenever feasible, dosage shall be in suitable increments up
to lOOx the LD50 or LC50 of the pathogen in its natural host, or 10-
lOOx the recommended field dosage.
(2) Test species, (i) Testing should be performed on three species
of insects, representing at least two of the following groups—parasitic
dipterans, predaceous hemipterans, predaceous coleopterans, predaceous
mites, predaceous neuropterans, parasitic hymenopterans.
(ii) Selection of test species is the responsibility of the researcher.
Rationale for selection must be provided. More specifically, the following
points apply:
(A) Viruses. Testing shall be performed on three species of insects
representative of those parasites and predators known or suspected to at-
tack the target host or to share the same ecological habitat.
(B) Protozoa. In selecting the three species of nontarget arthropods,
at least one should be a major (i.e. important) parasite of the target host.
Many protozoans have a wide host range. Accordingly, if possible, more
than the minimum (three) species of nontarget organisms should be tested.
(C) Fungi. Assuming testing is justified, appropriate organism test
species should be the major predacious and parasitic regulatory agents
common to the ecosystem where the MPCA will be applied. In addition,
testing could include parasites and/or predators specific to the host of the
fungal agent.
(D) Bacteria. Testing shall be performed on three species of insects
representative of those parasites and predators known or suspected to at-
tack the target host or to share the same ecological habitat.
(3) Controls. A concurrent control group is recommended and should
be treated with microbe-free (or nonviable microbe) material from the cul-
ture system used for propagation of the microbial pest control agent.
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(4) Routes of exposure—(i) Viruses. The best routes of exposure
will depend on the developmental location of the nontarget organism. In-
ternal parasites may be tested with virus-infected hosts, or if they can be
cultured in vitro, the virus can be added to the diet. External stages of
parasites and predators, if they are obligatory, may be fed virus-infected
hosts, and others may be fed virus-contaminated media or virus suspended
in sugar or honey solutions.
(ii) Protozoa. (A) In addition to feeding adult predators and parasites
of the target insect with the resistant stage of the protozoan, immature
stages of the predator or parasite should be exposed. Predators can be fed
hosts infected with the protozoan over a period of time. The predator,
at a prescribed time, should be checked for protozoan infection.
(B) The protocol for parasites is more complex. Protozoan-infected
hosts can be parasitized or parasitized hosts can be fed protozoans. Para-
sites from protozoan-infected hosts should be examined for protozoan in-
fection. The immature stages as well as adults should be examined. If
possible, a primary hymenopterous and dipterous parasite should be exam-
ined.
(C) The best route of infection for adult Hymenoptera or Diptera is
oral acquisition of protozoan spores. Predaceous insects could also be fed
in this manner, but feeding them infected (live) hosts of known age is
more appropriate. In either case, the actual amount of spores consumed
cannot be accurately determined. If infection of the parasite or predator
adult occurs, the possibility for transovarial or transovum transmission
should be examined.
(iii) Fungi. Routes of exposure should simulate field conditions as
much as possible. In the case of entomopathogenic fungi, environmental
conditions (> 90 percent relative humidity) are critical at the time of expo-
sure.
(iv) Bacteria. Best routes of exposure will depend on developmental
location of the nontarget organism. Internal parasites may be tested with
bacteria-infected hosts, or if they can be cultured in vitro, the bacteria
can be added to the diet. External stages may be fed bacteria-infected
hosts, bacteria-contaminated media, or bacteria suspended in sugar or
honey solutions.
(5) Duration of test/endpoints—(i) Viruses. (A) Control and treated
insects should be observed for a duration of at least 30 days after dosing,
or in cases where an insect species cannot be cultured for 30 days, until
control mortality rises above 20 percent. In cases where signs, symptoms
and pathologies are detected, the treated insects should be examined in
detail at late stages of infection, at moribund, and at death. Such tests
need not be prolonged to 30 days, if death of treated insects occurs prior
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to 30 days. In all cases of pathologies in the treated nontarget insects,
it is essential that the etiology of the infection be established.
(B) The end-points should be based on the frank development of
pathologies and on the early mortality of the treated as compared to the
untreated (control) nontarget organisms.
(ii) Protozoa. (A) Test duration should be determined on a case-by-
case basis. The most appropriate end-point for protozoan diseases for de-
termining pathogenic effects is the presence of the vegetative stages
(shizonts or meronts) in the tissues of nontarget insects. The schizonts
within suspected tissues can be detected by making a smear, staining with
Giemsa stain, and examining the slide with oil immersion using a
compound microscope. The nontarget insect should be alive when the tis-
sues are removed for the smear because the shizonts are fragile and are
usually destroyed by other microbes or are distorted upon death of the
host.
(B) Protozoan spores can be used as an indicator of infection. How-
ever, if the infection is light, the few spores could have come from the
inoculum. If spores are abundant (a relative term) and occur in the tissues
of the nontarget insect, it is likely that it is infected.
(C) Another way to confirm infection is to conduct histological stud-
ies of the tissues using standard methods and looking for spores and other
pathological effects. The end-point would be just before death of the orga-
nism or a prescribed period of time.
(D) Death of the nontarget insect is a good end-point if the protozoan
is virulent. However, since most protozoans have chronic effects on their
host, changes in behavior, size, or color could be used as an end-point.
In each case, a microscopic examination to find schizonts or spores is
essential to confirm the presence of the protozoan. Koch's postulates to
confirm the virulence of the isolate should be run.
(iii) Fungi. (A) With entomopathogenic fungi, test duration can be
limited to 8-10 days.
(B) Mortality time, expressed as LT50 (time course of population
mortality), is considered the most reliable parameter for bioassaying fungi
of insects in the laboratory. Pathogenicity should be confirmed by identify-
ing the fungal agent as the original inoculum.
(iv) Bacteria. Control and treated insects should be observed for 21
to 30 days after dosing, if this is possible. In cases where the insect species
cannot be cultured for 21 to 30 days, observation should continue until
control mortality rises above 20 percent.
(c) Reporting and evaluation of data. The reporting provisions are
the same as those specified in OPPTS 885.0001.
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(d) Tier progression. (1) Data derived from Tier I testing will be
used in conjunction with available information on use pattern, host range,
and other similar factors, to assess potential for adverse effects. If data
indicate potential for adverse effects, Tier II testing will be required as
specified in 40 CFR 158.740. In some cases, a subchronic test may serve
to better understand the effects observed at the Tier I level and might
alleviate the need for Tier II testing.
(2) If no toxic or pathogenic effects are observed in this study, addi-
tional testing is ordinarily not necessary.
(e) References. The following references are provided for use in the
development of acceptable test protocols for conducting toxicity/patho-
genicity tests with microbial pesticides:
(1) Andreadis, T.G. Nosema pyrausta infection in Macrocentrus
grandii, a braconid parasite of the European corn borer, Ostrinia nubilalis.
Journal of Invertebrate Pathology 35:229-233 (1980).
(2) Brooks, W.M. Protozoa: Host—parasite—pathogen interrelation-
ships. Miscellaneous Publication of the Entomological Society of America
9:105-111(1973).
(3) Gardner, W.A. et al. Susceptibility of the two-spotted spider mite,
Tetranychus urticae Koch, to the fungal pathogen Hirsutella thompsonii
Fisher, Florida Entomology 65:458-465 (1982).
(4) 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).
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