United States Prevention, Pesticides EPA712-C-96-328
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4000
Background for
Nontarget Organism
Testing of Microbial Pest
Control Agents
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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 885.4000 Background for nontarget organism testing of mi-
crobial pest control agents.
(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 guideline is OPP guideline 154A-1.
(b) Overview. The purpose of nontarget organism testing is to de-
velop data necessary to assess potential hazard of microbial pest control
agents (MPCAs) to terrestrial wildlife, aquatic animals, plants, and bene-
ficial insects. The test standards and definitions in this guideline apply
to OPPTS Series 885, Group D.
(1) Approach. The Agency has concluded that at least some test data
on terrestrial and aquatic organisms should usually be evaluated, regardless
of the pesticide's site of outdoor application and apparent potential for
exposure. These data would be necessary for the following reasons:
(i) When a microorganism is applied as a pesticide, great numbers
are placed in the environment apart from its host, at a discrete point in
time (day of application), and spread over living and nonliving components
of the target site. Often, there will be spread to adjacent areas, due to
drift. Hence, in terms of numbers of nontarget organisms exposed, number
of different species exposed, and the degree of exposure (number of micro-
organisms per nontarget organism), exposure may be greater than under
natural conditions. In addition, data on toxic or pathogenic effects are es-
sential for hazard assessment purposes when terrestrial or aquatic orga-
nisms are likely to be exposed to a MPCA, especially when no fate data
will be required by the Agency in the first tier of testing.
(ii) Pathogenicity and toxicity appear to be the major effects of con-
cern regarding exposure of terrestrial and aquatic organisms to microbial
pesticides. Therefore, the Agency has developed guidelines that will allow
hazard assessment of pathogenicity and toxicity problems to be made. The
Agency desires a high level of confidence that no unreasonable adverse
environmental effects will result from actual use of MPCAs. Toward this
end, the guidelines in Tier I reflect a maximum hazard approach to testing.
Negative results from tests using this approach would provide a high de-
gree of confidence that no unreasonable adverse effects are likely to occur
from the actual use of MPCAs.
(iii) If unacceptable adverse effects are identified in Tier I tests, Tier
II tests are performed to attempt to quantify levels of the MPCA to which
the susceptible nontarget species may be exposed. Prior to registration of
MPCAs, applicants would submit Tier I data on nontarget organisms. En-
vironmental expression data (Tier II) may also be required on a case-by-
case basis for certain MPCAs which are determined to present unique con-
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cerns. In addition, on a case-by-case basis, definitive Tier II data showing
that the MPCA will not survive or persist in the environment to which
it is applied, can be submitted as support for a request for waiver (40
CFR 158.45) of some or all of Tier I testing requirements. 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.
(iv) If the results from Tier II tests show that the MPCA persists
or survives in the environment at significant levels, Tier III studies are
designed to show effects of chronic exposure to these levels on fish and
wildlife. If it is indicated that there may still be a problem, Tier IV studies
(simulated or actual field studies) may be able to determine if there is
a problem under actual use conditions.
(2) Major issues—(i) Maximum hazard dosage levels. Unlike envi-
ronmental levels of chemical pesticides, which generally decrease follow-
ing application, the environmental levels of MPCAs and any associated
toxins may, at least temporarily, increase when the product is effective.
Therefore, the maximum hazard dose for Tier I testing will be based on
some safety factor times the maximum amount of active ingredient (MPCA
or its toxin) expected to be available to terrestrial and aquatic plants and
animals in the environment. The target hosts (e.g. insects) are likely to
contain the highest concentration of the MPCA that will be available to
nontarget terrestrial wildlife and aquatic animals following a pesticide ap-
plication.
(A) Avian wildlife will be exposed, most commonly, through the diet
(via infected insects) or through the respiratory tract (via spray drift or
aerosolization). The maximum amount of MPCA a bird in the wild may
consume is difficult to determine, but as much a 1 x 109 units/mL is pos-
sible. Due to anatomical constraints, the Agency recognizes that dosing
at this level cannot always be achieved. Thus, the recommended daily oral
or injected dose should be calculated as follows:
MDD (units) = [MPCA] in TGAI x 5 mL/kg BW x weight of bird (kg)
where
MDD = maximum daily dose expressed as units per volume
[MPCA] = concentration of MPCA
TGAI = technical grade of the active ingredient
BW = body weight
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Therefore, for a product whose TGAI contains 1 x 109 units/mL and
using a 25 g bobwhite quail, the maximum daily dose would be:
(1 x 109 units/mL) (5 mL/kg) (0.025 kg) = 1.25 x 108 units of MPCA
(B) This dose should be administered over a 5-day period so that
the total dose the bird would receive orally, over a 5-day period, would
be 6.25 x 108 units.
(C) Maximum doses for the respiratory administration should be cal-
culated in a similar manner except that the dosing volume should be re-
duced from 5 mL/kg to 0.2 mL/kg.
(D) Maximum hazard aquatic exposures must, in some way, account
for the fact that fish and aquatic invertebrates, are less mobile than terres-
trial species and less able to avoid the pesticide. In addition, under condi-
tions of nutrient influx or the presence of alternate hosts or target pests
in aquatic ecosystems, aquatic organisms may be exposed to elevated num-
bers of microbial pesticides. It is recommended that the maximum hazard
exposure be in the range of 1 x 106 units of MPCA per milliliter of water
or in a concentration l,000x the calculated concentration after direct appli-
cation to a 6-in layer of water at label rates if the microorganism produces
a toxin. Aquatic exposure will simultaneous expose fish by the dietary
route.
(E) The Agency realizes that it would be very difficult to establish
specific LC50, ED50, or LD50 values (e.g. LD50 = 1,000 mg/kg) and
95 percent confidence limits for most MPCAs whose mechanism of action
is pathogenicity, because test data are not likely to exhibit a log-probit
dose-response relationship that is typical of chemical pesticides. Therefore,
data that establishes an LC50, ED50, or LD50 that is greater than the
maximum hazard dosage level (e.g. LD50 > 1,000 mg/kg) would often be
adequate for the purposes of hazard assessment. In most cases, testing at
one maximum hazard dosage level is expected to be sufficient to evaluate
effects for these MPCAs. MPCAs that are toxin-producing are more likely
to produce a log-probit response. In most cases multiple groups would
be necessary in order to quantify the hazard of these organisms. If there
are no effects at the maximum hazard dose, low doses will not be nec-
essary.
(ii) Maximum hazard routes of administration. (A) Various
routesof administration (dosing) are provided for in these guidelines and
are chosen to reflect "natural" exposure routes. The Agency believes that
these routes—oral and respiratory for birds, aquatic and food exposure for
aquatic organisms, and the oral route for insects—can best define the haz-
ard to nontarget organisms in the wild.
(B) Parenteral dosing, such as intravenous and intraperitoneal injec-
tion, would provide a high degree of confidence that a particular microbial
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pesticide would not cause adverse effects, if negative. Positive results, on
the other hand, given the complex and undefined components (exogenous
protein, metabolic byproducts, etc.) of microbial pesticide preparations and
the environmentally unrealistic nature of the route, would be difficult to
translate to effects on species in the environment. Due to the high degree
of confidence an injection test gives, it is being suggested as an alternate
exposure route in OPPTS 885.3050 whenever the microbial dosing prepa-
ration is sufficiently free from exogenous protein and other contaminating
substances so that the test will not be confounded.
(iii) Age of the test animals. The Agency considers that sufficient
immunological and physiological differences exist between immature ani-
mals and mature animals to suggest that immature animals are potentially
more susceptible to infection and possibly to the effects of any toxin pro-
duced by the MPCA. The Agency has developed age guidelines for the
test animals in Tier I tests, and recommends the use of immature animals
in keeping with the hazard approach to testing.
(iv) Methods for detecting MPCAs. Unlike toxicity tests where mor-
tality can usually be determined by observation, infectivity tests often re-
quire sophisticated assessment methods for detecting sublethal pathogenic
effects. These methods may include serological or nucleic acid technology.
(v) Detailed test protocols. No standard, widely accepted, laboratory
validated, test protocols are available at this time to evaluate the safety
of MPCAs to terrestrial and aquatic animals. In the meantime, the draft
and final protocols, as they become available, may be obtained on request
from the Biopesticides and Pollution Division of OPP.
(vi) Length of tests. (A) The guidelines provide that the duration
of all Tier I tests be about 30 days long. This should permit time for
incubation, infection, and manifestation of effects in the test organisms
for most MPCAs. Some test species, notable nontarget insects, may be
difficult to culture and the test duration has been adjusted accordingly.
Recommended test durations are included for each testing guideline.
(B) Various authors have proposed test duration times for toxicity
and pathogenicity tests ranging from 14 to 35 days (see paragraphs (g)(9),
(g)(ll), and (g)(24) of this guideline). The Agency realizes that the test
duration period may be unnecessarily long, or may not be sufficiently long
enough to detect effects such as viral diseases that recur after prolonged
intervals of latency, e.g. Herpes zoster (under paragraph (g)(5) of this
guideline). At the present time, however, the Agency is not aware of an
accurate method to predict whether a virus detected in a test organism
will manifest latent effects. The Agency invites comments on the proposed
test duration period and the probability of encountering MPCAs with latent
effects.
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(vii) Control groups. Appropriate control groups are addressed in
the recommended guidelines for each test.
(c) Terrestrial wildlife—(1) Approach. These guidelines call for two
tests on birds for all MPCAs: an avian acute oral toxicity and pathogenicity
test (OPPTS 885.4050) and an avian respiratory pathogenicity test (OPPTS
885.4100). The avian acute oral toxicity and pathogenicity test would pro-
vide data on any toxic effects to avian wildlife from exposure to the micro-
organism or any toxin it may produce. This test would also provide data
on pathogenic effects following an acute exposure either by the oral (or
injection) route. The duration of the study would be about 30 days to allow
for an incubation period prior to onset of symptoms.
(i) The avian respiratory pathogenicity test would provide data on
the pathogenic effects of the MPCA on birds following exposure due to
drifts or aerosolation. The guidelines for the duration of the test and gross
necropsies are similar to the avian acute oral toxicity and pathogenicity
test.
(ii) In both the acute dose and inhalation tests, gross necropsy,
histopathological examination and culture and isolation should be per-
formed on exposure site tissues and other organs showing anatomical or
physiological abnormalities. In some cases, such as viruses, there is a pref-
erence for certain cell or tissue types. In cases where tissue preferences
are known or suspected, those tissues should be examined whether or not
gross anatomical or physiological changes are seen.
(2) Tier progression—(i) Tier I. (A) If no toxic or pathogenic effects
are observed after exposing birds to the MPCA via two different routes
of administration (oral and respiratory) at the maximum hazard dosage
levels, no further testing of birds would be indicated. If toxic or pathogenic
effects are observed at the maximum hazard dosage levels, Tier II, envi-
ronmental expression tests (OPPTS 885.5200, 885.5300, and 885.5400),
would be indicated. In some cases, a subchronic test may serve to better
understand the effects served at the Tier I level and might alleviate the
need for Tier II testing.
(B) Data on wild mammal toxicity and pathogenicity (OPPTS
885.4150) are required on a case-by-case basis when data indicate that
there is considerable variation in the sensitivity of different mammalian
species to the effects of a MPCA or where wild mammals would be heav-
ily exposed to the MPCA under normal use. The toxicity and pathogenicity
data in OPPTS 885.3050 through 885.3650 for evaluating hazard to hu-
mans and domestic animals are normally adequate to indicate hazard to
wild mammals. If no toxic or pathogenic effects are observed in these
tests, no further testing of wild mammals would follow. If any effects
are observed in tests on wild mammals, Tier II, environmental expression
testing (OPPTS 885.5200, 885.5300, and 885.5400) would be indicated.
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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.
(ii) Tier II. The data outlined in Tier II are described in the environ-
mental expression testing guidelines (OPPTS 885.5200, 885.5300, and
885.5400). If the expression characteristics preclude exposure of the
MPCA to nontarget birds and mammals, no further testing of these animals
would be indicated. If Tier II tests indicate that birds and mammals will
be exposed to the MPCA, testing at Tier III would follow.
(iii) Tier III. (A) The types of effects reported in the Tier I tests
would determine which Tier III tests would apply. If adverse effects are
reported in Tier I tests, and Tier II tests indicate exposure, Tier III testing
would be required. If reproductive or fertility effects, or oncogenicity are
reported in tests in OPPTS 885.3650 for evaluating hazards to humans
and domestic animals, a long-term avian pathogenicity and reproduction
test would apply. This test would provide data on pathogenic effects of
the MPCA on birds during a critical period in their life—breeding and
reproduction. It would also provide data on the effects of the MPCA on
avian reproduction. If no pathogenic or reproductive effects are observed,
the Agency would, at this time, review all the data and determine if deci-
sions regarding registration can be made.
(B) Pathogenic effects occurring at Tier III and beyond raise serious
questions concerning the registration of any MPCA. Also, testing at Tier
IV, simulated and actual field testing for mammals and birds (OPPTS
885.4900) may not be feasible, since it may not be possible to confine
the MPCA to a test area and prevent it from contaminating adjacent areas.
In such a case, simulated field testing may be possible but would neces-
sitate a very complex design.
(iv) Tier IV. Simulated and actual field testing (OPPTS 885.4900)
would provide data on the pathogenic effects of the MPCA on birds and
mammals following field applications at actual label use rates. This test
would be indicated when pathogenic effects are reported in Tier III testing
(OPPTS 885.4600) at levels equal to actual or expected field residue expo-
sure levels, and when the Agency is reasonably confident that quarantine
methods will prevent MPCA dispersal to pen, large-pen, or full-scale field
tests) should be discussed with the Agency before beginning the study.
Protocols for such studies must be submitted to the Agency and must re-
ceive Agency approval prior to test initiation.
(3) Major issues. In the process of developing the guidelines for ter-
restrial animals, the Agency recognized many important areas that require
outside input and comment. The Agency invites scientific input and com-
ments on the following issues of concern:
(i) In vivo testing. (A) The guidelines outline in vivo testing of birds
and mammals, in vitro testing may be considered in the future. Wolf, under
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paragraphs (g)(30) and (g)(31) of this guideline a two-pronged testing ap-
proach for safety testing of baculoviruses has been suggested, using both
in vivo and tissue culture testing. There are established or permanent cell
lines for duck embryo fibroblasts, chicken embryo fibroblasts, and rep-
resentative mammalian cell lines from a bat, rabbit, mouse, and deer.
Ignoffo (under paragraph (g)(10) of this guideline) reported that at least
12 viruses—including all major viral types—have been tested in vitro in
either avian egg embryo fibroblasts (chicken or turkey), fish, or mamma-
lian cell lines. Virus multiplication or cytopathic effects were reported for
one nuclear polyhedrosis virus in chicken embryo cells and human amnion
tissue, and for one noninclusion virus in chicken embryo cells and mouse
sarcoma tissue. In contrast, no effects were observed in vivo when rabbits
and mice were injected or fed the latter virus. More recently it has been
shown (under paragraph (c)(4)(i) of this guideline) that Autographica
californica NPV can penetrate the nucleus of three poikilothermic verte-
brate cell lines, although no productive infection was demonstrated.
(B) The Agency is not convinced at this time that the results of in
vitro tests can be used exclusively to determine potential adverse effects
to individual terrestrial animals (e.g. endangered species) or populations
of terrestrial animals in the environment.
(ii) Test substance. (A) Microorganisms used as pesticides could be
applied in any one of a combination of naturally existing forms. It is pref-
erable that the test organism be exposed to the most infectious form when-
ever infectivity is the primary hazard of concern. Similarly, when toxicity
(e.g. a microbial toxin) is the hazard of concern, the test organism should
be exposed to a form of the MPCA in which the toxin would be produced
in the greatest amount and most readily available. Unfortunately, there is
no easy way to determine which is the most infectious or toxic form of
the microorganism to the test organisms. The route of administration may
also play an important role in determining which form should be tested.
For example, if the route of administration is intravenous, the active vege-
tative cells of a bacterium, or the infectious hemolymph may be more
appropriate than vegetative cells or polyhedryda, respectively.
(B) For these guidelines, testing the technical grade of the active in-
gredient applies in all tests except the simulated and actual field testing
(OPPTS 885.4900), where the use of the formulated product applies in
order to simulate or reproduce actual field use. The Agency realizes that
in some cases the technical grade of the active ingredient and the formu-
lated product may be identical.
(iii) Route of administration. (A) These guidelines outline testing
by oral gavage or by injection and via the respiratory tract. It is important
to note that the administration of test material to 14- to 28-day old birds
by oral gavage will likely require the use of small needles or cannulae
with ball-tipped ends in order to prevent injury to the birds. It has been
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reported under paragraph (g)(9) of this guideline that the following groups
of terrestrial animals have been tested in vivo for effects caused by
entomopathogens:
Routes of Administration in Terrestrial Animals
Group Route
Mammals—primarily laboratory popu- Diet, oral, inhalation, subcutaneous,
lations dermal application, intradermal,
intraperitoneal, intravenous, intra-
cerebral, intranasal, intramuscular,
eye application.
Birds—chickens and laboratory popu- Oral, diet, intraperitoneal (chickens)
lations of quail and ducks that are
phenotypically similar to wild spe-
cies
Since the gut normally provides such a radically different environment
from that in the rest of the bird or mammal body, and since insectivorous
birds and mammals can be expected to ingest large quantities of actively
growing microorganisms when they feed on diseased insects, the Agency
believes that the oral route would be appropriate.
(B) Inhalation, or rather intranasal or intratracheal instillation has
been chosen as the second exposure route because birds may be exposed
by this route during spraying operations or by the MPCA made airborne
through the effects of wind or animal movement during feeding or other
activities. In addition, the respiratory tract is a major portal of disease
acquisition in avian species.
(C) The Agency recognizes that a combination of administrations in
one test (e.g. oral and intravenous or intraperitoneal injection) may be pos-
sible. It would certainly be in keeping with the maximum hazard testing
philosophy and would reduce testing time and expense. However, com-
bined exposures could unduly traumatize the test animals so as to cause
mortality, or in some other way cause spurious results.
(iv) Avian test species. (A) These guidelines provide that young bob-
white quail or mallard ducks be tested in Tier I tests. Birds between 14
and 28 days of age at the beginning of the test period should be used
in the avian oral toxicity and pathogenicity test and in the avian inhalation
pathogenicity test. Within a given test, all birds should be the same age.
(B) The Agency prefers bobwhite quail, but will accept ringneck
pheasants, and mallard ducks as acceptable test species for avian acute
toxicity tests of chemical pesticides.
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(C) In support of testing immature birds in Tier I, the Agency notes
that insects are vital to immature birds during the first 2 or 3 weeks of
life and make up a much larger proportion of their diet during this time
than at other times in their life. Thus, they are functionally insectivorous
birds at this age. Also, for the purposes of pathogenicity testing, the Agen-
cy feels that sufficient immunological and physiological differences exist
between immature birds and adult birds to warrant considering the imma-
ture bird as potentially more susceptible to infective challenge and so pro-
poses their use in the maximum hazard testing approach.
(v) Selection of dose levels. For Tier I tests, the Agency suggests
that a maximum hazard dosage be administered. For all testing, the maxi-
mum dose should be no less than the maximum hazard dose as defined
in the testing guidelines (OPPTS 885.4000(h) and OPPTS 885.4050,
885.4100, 885.4150, 885.4200, 885.4240, 885.4280, 885.4300, 885.4340,
and 885.4380). If the MPCA produces significant toxic or pathogenic ef-
fects at the maximum hazard dose level, testing at lower doses would be
indicated. Sufficient doses and test organisms would be required to deter-
mine an LD50 value, if possible.
(vi) Protocols. Interim protocols for some ecological effects testing
have been developed by EPA's Office of Research and Development. Al-
though these protocols have not been validated, they are available on re-
quest from EPA in order to provide guidance for applicants and testing
laboratories in developing protocols for testing microbial pesticides on
nontarget organisms.
(d) Aquatic animals (1) Approach. (A) The Agency has considered
several criteria that could be used to determine the extent of testing for
effects an aquatic animals in Tier I—the site of application and resulting
potential for aquatic exposure, the natural geographic distribution of the
microorganism, the natural population level of the microorganism com-
pared with population levels likely after application, and the ability of the
MPCA to survive and replicate after application.
(B) While all of these criteria are important, the Agency has chosen
site of application and its resulting potential for aquatic exposure as the
key criteria for establishing the extent of initial effects testing far MPCAs.
The rationale for selecting these criteria is that they directly address the
most critical issue regarding potential hazard—likelihood of exposure. Fur-
thermore, other criteria would be implicitly considered in connection with
the criterion for site of application.
(C) The Agency recognizes that considerable judgment will be re-
quired to properly employ site of application as a criterion. While many
uses obviously entail direct application to water (e.g. mosquito control and
aquatic weed control), the Agency also intends that less obvious or border-
line uses will be considered aquatic uses. Some examples that fall into
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the latter category are applications to forests, drainage ditches, riverbanks,
and partially aquatic crops such as rice. Widespread applications to major
crops such as cotton, soybeans, and corn could also warrant expanded test-
ing if these crops are grown near bodies of water. To the extent possible,
the Agency will rely on its experience with the classical chemical pes-
ticides in distinguishing between terrestrial and aquatic use patterns in bor-
derline situations.
(2) Tier progression (i) Tier I. (A) For MPCAs applied in terrestrial
use patterns (where direct aquatic exposure is not anticipated), one fresh-
water fish (OPPTS 885.4200) and one freshwater aquatic invertebrate
(OPPTS 885.4240) should be tested to assess toxicity and pathogenicity.
For MPCAs applied directly to fresh, estuarine, or marine waters, one ad-
ditional fish species and one additional invertebrate species should be test-
ed in Tier I. These tests should be conducted as 30-day static renewal
bioassays using one or a combination of methods to administer the pes-
ticide (e.g. aqueous or dietary) These tests should be designed to simulta-
neously assess both toxicity and pathogenicity as to detect and quantify
the microbial agent in the test animal. The concentration of MPCA in the
water or food must be monitored to ensure that the test organisms are
exposed to a sufficient MPCA level throughout the test period.
(B) No further testing would be indicated if:
(7) Results of the Tier I tests indicate no toxic or pathogenic effects.
(2) Host range testing indicates that the MPCA has a narrow host
range such that crossover into nontarget aquatic invertebrates is unlikely.
(3) If toxic or pathogenic effects are observed, environmental expres-
sion testing (Tier II) would generally be required. In some cases, a sub-
chronic test may serve to better understand the effects observed at the
Tier I level and might alleviate the need for Tier II testing.
(C) If host range testing implies crossover into nontarget aquatic in-
vertebrates, additional aquatic invertebrate species (those expected to be
susceptible or likely to be exposed) would have to be tested in Tier I,
or as an alternative, Tier II testing would have to be conducted. If tests
on these additional species indicate toxic or pathogenic effects, testing at
Tier II would be indicated; if otherwise, no further testing would be nec-
essary.
(ii) Tier II. The data for Tier II are described in environmental ex-
pression testing (OPPTS 885.5200, 885.5300, and 885.5400). If the envi-
ronmental expression characteristics do not indicate exposure of the MPCA
to nontarget fish or aquatic invertebrates, no further testing of these ani-
mals would be indicated. If Tier II tests indicate that fish and aquatic inver-
tebrates will be exposed to the MPCA, testing at Tier 111 is indicated.
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(iii) Tier III. (A) Whereas Tier I tests are designed to screen MPCAs
using a maximum hazard testing scheme, Tier III tests are intended to
evaluate and quantify the actual hazard associated with the MPCA more
precisely. The types of effects reported in Tier I tests would help determine
which Tier III tests would be required. If only toxic effects are observed
in Tier I tests, OPPTS Series 850 (Ecological Effects Test Guidelines)
would apply, and further testing would proceed. If pathogenic effects or
both pathogenic and toxic effects are observed in Tier I, tests that could
be indicated in Tier III are the following:
(7) Additional acute or subacute tests of fish or aquatic invertebrates
to evaluate the spectrum of susceptible nontarget species, or determine the
susceptible routes of exposure, or determine the dose-response relationship
between the pesticidal agent and susceptible nontarget organism.
(2) Aquatic invertebrate range testing (OPPTS 885.4650) and fish life
cycle testing (OPPTS 885.4700).
(3) Aquatic ecosystem disruption studies (OPPTS 885.4750).
(B) If results of Tier III tests indicate no pathogenic effects, no further
testing would be indicated. Conversely, if results of Tier III tests, along
with environmental fate data, indicate toxic or pathogenic effects, simu-
lated or actual field testing may be warranted.
(3) Major issues. This section identifies and discusses issues regard-
ing aquatic testing of MPCAs that may require further research and devel-
opment. Most of the issues stem from there being no standard widely ac-
cepted test protocols available to evaluate the effects of MPCAs on nontar-
get aquatic animals. There are some potential hazards associated with the
use of MPCAs that the Agency recognizes and for which practical methods
of evaluation are not available. The role of in vitro testing and Tier IV
testing is also discussed.
(i) Issues associated with Tier I protocol. Useful Tier I test proto-
cols would simultaneously assess toxicity and pathogenicity in aquatic ani-
mals. The maximum hazard test philosophy would be exerted in terms
of treatment level, method of pesticide administration, and age of the test
animal.
(A) Conduct of Tier I tests. (7) A Tier I test should be conducted
as a static renewal bioassay. The microorganisms should be administered
as a suspension in the water (aqueous exposure), in the diet in the form
of diseased host insects or treated feed, or as a combination of both routes
of exposure.
(2) If any test animals die during the test, the cause of death (e.g.
toxicity, pathogenicity) should be determined, if possible, and reisolation
of the microorganism from test organism tissues should be attempted. This
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information would be used to determine what further tests, if any, are war-
ranted. Exposure and observation should extend for at least 30 days for
fish and 21 days for aquatic invertebrates. Individual test animals should
be removed periodically, if necessary, throughout the test period and at
test termination for examination to assess pathogenicity.
(3) If a sublethal infection is observed in test animals prior to test
termination, it may be necessary to continue the observation period in
order to more adequately assess the significance of the infection (e.g. will
it be lethal?). Several published studies address certain aspects of the pro-
tocol, under paragraph (g) of this guideline.
(ii) Discussion of Tier I aquatic organism tests—(A) Test orga-
nisms. (7) The guidelines provide that the species tested be selected from
the list of species recommended with the exception of goldfish (warmwater
species—bluegill sunfish, channel catfish, and fathead minnow; coldwater
species—rainbow trout, brook trout, coho salmon). These species are desir-
able test organisms for several important reasons: They are used to evalu-
ate chemical pesticides, and EPA has considerable background data on
these species; standard methods for the care and handling of these species
are available; and the species are widely distributed, are generally avail-
able, and have a variety of food habits and habitat requirements.
(2) Consideration should be given to testing species representative
of the geographic region or ecosystem where the MPCA is to be applied.
Species likely to prey upon or scavenge the diseased target host animals
should be tested when appropriate.
(3) Unless there are other overriding considerations, the rainbow trout
should be used as the freshwater fish test species. It is a desirable test
animal because: It is partially insectivorous; no one species has been
shown to be preferable in terms of sensitivity to MFC As; there is consider-
able background data on this species pertaining to its microbial diseases;
and standard tissue culture procedures are available for this species (under
paragraph (g)(xix) and (g)(xx) of this guideline).
(4} Use of young fish (3 to 6 mon old) is preferable since they would
be more likely to display a lethal pathogenic effect, whereas older fish
may become carriers.
(5) Due to the broad phylogenetic spectrum from which the investiga-
tor may choose, it is difficult to select the most appropriate aquatic inverte-
brate. Generally, a test organism that is phylogenetically closest to the
target host should be chosen. Such a test organism would be the most
likely to be susceptible to infection by the MPCA. It would be appropriate
to choose an aquatic insect (e.g. caddisfly) as the nontarget aquatic inverte-
brate test species when evaluating a MPCA whose target host is an insect.
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(6} Daphnia, a Cladoceran, has the advantage of having considerable
background data for comparative purposes. Pound (under paragraph
(g)(18) of this guideline) exposed the entomopathogen Mattesia to
Daphnia and observed a bioconcentration effect. This resulted from the
filter feeding habits of Daphnia and is a desirable feature in terms of assur-
ing that the test animal ingests the microorganism. Both Daphnia and cer-
tain other aquatic insects have the advantage of a short like cycle or aquat-
ic phase, and both undergo periods of natural stress and potential suscepti-
bility to the microorganism as a consequence of molting.
(B) Method of MPCA administration. (7) Two methods of pesticide
administration should be considered:
(/) Suspension in the test water (aqeous exposure).
(//) Dietary, in the form of diseased target host animals or incorpora-
tion of the MPCA into a standard feed.
(2) When possible, both routes should be used simultaneously in a
single test to ensure that the most appropriate route of exposure has been
tested and to ensure a maximum challenge. Different pathogens may be
capable of infection by different routes of exposure so that no single route
may adequately screen all microorganisms. Because each of the proposed
routes has certain advantages and disadvantages, multiple routes of expo-
sure would be extremely beneficial and cost effective in screening MPCAs.
Addition of the microorganism directly to the test water is a rou-
tine procedure. It simulates the type of natural exposure that could occur
immediately after application of a MPCA. It also simulates the routes of
exposure by which many known pathogenic agents infect fish and aquatic
invertebrates. However, care must be taken to assure that a high concentra-
tion of microorganisms be maintained in the test system and that this high
concentration does not lower water quality to an unacceptable level. There-
fore, the static revewal method is recommended. Use of this method will
ensure that high MPCA concentrations and acceptable water quality can
be maintained.
(4) Dietary exposure also simulates certain natural conditions. It is
perhaps the most important means of infection for the normal hosts of
entomopathogenic agents (under paragraph (d)(4)(xv) of this guideline),
and its use in evaluating effects an nontarget fish and aquatic invertebrates
is logical. This route offers a further advantage: It increases the possibility
of exposing the test animals to a different life stage of the microorganism
than may be present in the formulated product if diseased target hosts
(e.g. insects) are used as the feed.
(5) Finally, oral intubation of fish is another possible route of expo-
sure, and is one that has been used to evaluate microorganism effects in
fish. This route has the advantage of assuring that a known amount of
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test material is ingested. This advantage does not outweigh the risk of
injury or undue stress that could result from using this method, so that
the oral intubation method, though acceptable, is not recommended.
(C) Test substance. The substance to be tested will depend in part
on the method of pesticide administration used in the study. It is essential
to test the most challenging form of the microorganism (in terms of patho-
genicity or toxicity). It is equally important to test the form of the micro-
organism to which nontarget aquatic animals are most likely to be exposed.
These objectives should be achievable through the use of multipleof ad-
ministration, provided it is known which form is most challenging and
which form is most likely to be encountered by the nontarget animal. The
technical grade of the active ingredient should be used for all exposures.
The formulated product should be tested if it is to be applied directly to
water.
(D) Selection of treatment concentrations. (7) Treatment concentra-
tions must be related to the number of microorganisms to which aquatic
animals nay be exposed under actual use conditions. In keeping with the
maximum hazard philosophy, treatment concentrations must be relatively
high. Consideration must be given to the level of exposure resulting from
direct application as well as exposure resulting from consumption of dis-
eased target host organisms (usually insects). Exposure in terms of fre-
quency and number of microorganisms could be extremely high in the
latter case.
(2) The highest feasible concentrations should be used in all expo-
sures. At a minimum, the concentration for aqueous exposure IxlO6 units/
mL f^O or 100 x the theoretical concentration present in 6 in of water
immediately after a direct application of the MPCA, at label rates, to 6
in of water, whichever is greater and attainable. Because the use of such
a high concentration may be limited by its adverse effect on water quality
such as oxygen depletion and production of metabolic wastes by the micro-
organisms, treated water in the test vessels should be renewed frequently
enough to maintain water quality and microorganism concentration.
(E) Test duration. (7) Exposure and observation must be extended
to at least 30 days (unless test animals die) to allow time for any potential
infection, microorganism replication, or pathogenic or toxic effects to
manifest themselves. If a sublethal infection is observed, the test should
be extended to evaluate the significance of the infection. Similarly, if test
animals begin to die near the end of the 30-day period, the test should
be continued to determine the fate of the remaining test population.
(2) The 30-day test duration was selected on the basis of past research
under paragraph (g)(xvii) of this guideline and the recommendation of
Summers et al. (under paragraph (g)(23) of this guideline). Certain factors
may dictate that this period be modified. For example, if infection and
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death of target hosts is normally not evident for many days (i.e. 20 to
30), it would be logical to lengthen the period of exposure for the test
animals. Conversely, a shorter period of exposure may be warranted in
tests using animals with short life cycles (i.e. Daphnia or mysid shrimp).
(F) Observation and examination of test animals. (7) Daily obser-
vations are required to record mortalities and note any behavioral, patho-
genic, or toxic effects. Test organisms must be examined for infection or
any microorganism-related effects periodically throughout the study and
at test termination. The most difficult aspect of this requirement is the
verification of the presence or absence of an infection. The general meth-
ods of assessment that may be required to make this determination include
histopathology, serology, and nucleic acid hybridization and reisolation
and identification of the microorganism from organ tissue. These methods,
and the situations in which their use may be appropriate, were presented
in paragraph (c)(4) of this guideline under the general discussion of nontar-
get organism hazard testing.
(2) Undeen and Maddox (under paragraph (g)(26) of this guideline)
used the following criteria in their work with Nosema algerae to distin-
guish between a true infection and microorganisms observed in the test
animal. In a true infection:
(/) Both vegetative forms and spores had to be present in the test
animal.
(//) The number of spores recovered had to exceed the number in-
jected by lOOx. This type of approach may be useful for other microorga-
nisms.
(iii) Issues associated with Tier III test protocols. (A) The aquatic
invertebrate host embryo larvae, fish life cycle, and aquatic ecosystem tests
in Tier III (OPPTS 885.4650, 885.4700, and 885.4750) are similar to the
protocols that are referenced for these types of tests in OPPTS Series 850
(Ecological Effects Test Guidelines). Generally accepted standard proto-
cols for conducting these studies with MPCAs have not been developed.
In fact, few, if any, such tests have ever been conducted with MPCAs,
and the Agency recognizes at the outset that new and different test designs
and test parameters may be more appropriate than modified OPPTS Series
850 tests. Research and methods development are in progress and need
to be completed in this area before the Agency can publish specific rec-
ommendations concerning protocols and Tier progression.
(iv) Issues associated with in vitro testing. (A) The Agency recog-
nizes that there are in vitro tests available to assess the infectivity of cer-
tain microorganisms, one of which is tissue culture for viruses. Cell lines
are established for several species offish, and such a test might be a useful
means of assessing infectivity in certain situations. However, the relation-
ship between effects demonstrated by in vitro tests and effects likely to
15
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occur under in vivo situations is uncertain. For example, Ignoffo, under
paragraph (g)(9) of this guideline, states that "Tissue, completely non-
susceptible in the intact organism, may support viral multiplication when
explanted into a culture media.'' Therefore, the results obtained from tissue
culture tests could be useless in accurately predicting environmental haz-
ard. Another potential drawback of tissue culture studies is that, often,
no host cell culture (e.g. insect cell culture) has been developed. Such
a study would have no positive control group and the validity of a negative
result would always be subject to some doubt.
(B) The Agency has concluded that, at the present time, in vitro stud-
ies such as tissue culture cannot be substituted for the in vivo studies pro-
vided in Tier I. At the same time, the Agency recognizes the potential
value of these studies for the following purposes:
(7) As a relatively inexpensive and rapid means to screen for potential
infectivity in a broad spectrum of species.
(2) As a test to support or check the results of in vivo tests. A provi-
sion for cell culture studies is included in Tier III of the testing scheme.
(v) Issues associated with Tier IV testing. (A) The Agency recog-
nizes the possible shortcomings in using simulated or actual field tests
(Tier IV) as the final test of the safety of an MPCA. If an agent has
progressed through the Tier system and requires a field test, it must have
displayed significant adverse effects in some or all of the previously con-
ducted laboratory tests. This fact might argue against the use of a field
test, since such a test could release potentially hazardous microorganisms,
with the potential to proliferate in the environment and pose widespread
environmental risk, unless adequate quarantine measures could be taken.
Before any Tier IV field test is to be undertaken, the applicant should
discuss its plans with the Agency concerning potential hazards. If the
Agency determines that a Tier IV field test would pose an unacceptable
risk, the MPCA would not likely be acceptable for registration.
(B) The Agency recognizes the potential value of Tier IV simulated
or actual field tests as a further check on the safety of MPCAs that dem-
onstrate a hazard in Tier I tests, or that demonstrate a hazard that could
be adequately controlled by quarantine methods in the field. These tests
could be conducted concurrently with full-scale efficacy testing, and the
Agency would strongly encourage such tests. This would provide the op-
portunity to evaluate pesticidal effects (both direct and indirect) on a much
broader spectrum of nontarget species, under more natural exposure condi-
tions than is possible in Tier I testing.
(vi) Assessment of other potential hazards: opportunistic infec-
tions and latent viruses. (A) Opportunistic infections in nontarget aquatic
animals are recognized by the Agency to be a potential hazard. A similar
concern is noted for latent viruses. Research indicates that aquatic animals
16
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may be rendered significantly more susceptible to microbial infection, (e.g.
by viruses and bacteria) when stressed by such factors as Aroclor 1254,
copper, temperature, salinity, pesticides, and other pollutants. This in-
creased susceptibility raises several important questions:
(7) What is the likelihood of an opportunistic infection (from an
MPCA) occurring in a nontarget aquatic animal?
(2) What is the significance of the effect of opportunistic infections
on individuals and populations?
(3) Will the proposed Tier I test adequately screen MPCAs for poten-
tial opportunistic effects? Or could a MPCA be noninfective in a
Tier I test, but infect stressed nontarget animals?
(4) Will a latent virus be detected by a Tier I test and, if so, how
can its significance be assessed?
(B) There is far too little background information and research on
MPCAs to suggest an answer to the first question. However, the Agency
believes that the potential for this type of problem should not be ignored.
The Agency is confident that sublethal infections produced in Tier I tests
can be detected if the proper methods of detection are employed. However,
the potential for an apparently noninfective agent (in Tier I testing) to
infect stressed animals is unknown. At present, the Agency is not aware
of any practical, generally accepted, routine screening test that could be
used in Tier I to determine the potential for such an occurrence. If a sub-
lethal infection is observed in Tier I, further testing may be warranted.
A microorganism/stress interaction test is proposed in Tier III as a means
of assessing sublethal infections, but further research is needed to develop
the protocol for such a test. With regard to latent viral infections, the
Agency is not aware of a standard method to evaluate the potential for
a latent virus to reactivate and cause adverse effects in aquatic animals.
Further research is required.
(vii) Oncogenic effects. The Agency recognizes the potential for on-
cogenic effects that are associated with viruses and mycotoxins. The prob-
ability of oncogenicity in nontarget aquatic animals, as a result of exposure
to a viral pesticide, is unknown. At this time, the Agency is unaware of
any standard method that could be used to screen for such an effect. Fur-
ther research is required to develop an appropriate test and determine when
its use is justified.
(e) Nontarget plant testing—(1) Approach. The plant testing
scheme proposed herein is based on the Tier testing scheme for testing
other nontarget organisms. Tier I screening tests incorporate maximum
hazard single dosing using a route of exposure most likely to show any
potential plant toxicity or pathogenicity. The duration of the test should
be sufficient to allow for manifestation of a delayed pathogenic response.
17
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Tier II testing examines population dynamics to quantity persistence and
survival of the MPCA in the environment. 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. Tier III testing is designed
to record a dose response and determine if there is a minimum infective
dose for any adverse effects identified in Tier I tests. Tier IV testing, if
still needed for risk assessment, will include both exposure and hazard
testing under simulated or actual field conditions.
(2) Major issues, (i) Diseases of commercially important plants have
been intensively studied for decades and many plant pathogens have been
identified and subsequently well characterized. Some plant pathogens have
a very narrow host range and may attack only one species of plant, other
plant pathogens may attack a wide range of plant species, and still other
microorganisms have never been identified in association with disease in
plants.
(ii) A thorough taxonomic description of the MPCA should allow de-
termination of its similarity to known plant pathogens. MFC As that are
similar to plant pathogens with very narrow host ranges may only need
to be tested for adverse effects against plants similar to known hosts.
MPCAs that are similar to wide range plant pathogens may need additional
testing to identity the complete host range. A knowledge of the mode of
action may assist in determining the extent of testing needed for potential
plant pathogens. Finally, MPCAs that do not resemble any known plant
pathogen may require little, if any, plant testing. Microbial herbicides are
designed to be toxic or pathogenic to their target plants. This class of
MPCAs will require close scrutiny to ensure that nontarget plants are not
unreasonably affected, whereas microbial insecticides generally would not
be expected to have phytopathogenic properties, and would not require
as much testing,
(iii) A second factor in determining the extent of plant testing is the
anticipated exposure of plants to the MPCA as determined by the use pat-
tern, dissemination, and the persistence/survival in the environment. For
example, MPCAs that will not be disseminated to, or do not survive in,
aquatic environments will not need testing in aquatic plants. A related fac-
tor is whether the MPCA is to be used within its area of natural occur-
rence. Where an MPCA is proposed for use in an area where it does not
naturally occur, additional plant testing may be warranted.
(iv) Another factor in selecting species of plants to be tested is that
of susceptibility to plant diseases. Genetically diverse groups of plants are
generally less susceptible as a species to any given plant pathogen since
there is a greater chance that a variety of the species will be resistant
to the disease. The most important group of genetically identical
(monoculture) plants are the commercial agricultural crops. These plants
18
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should be given priority in testing for plant pathogenicity because of both
their potential susceptibility and their commercial importance.
(f) Nontarget insects—(1) Terrestrial insects—(i) Approach. As-
sessment of potential nontarget insect hazard from uses of MPCAS is made
difficult by a number of factors:
(A) Most MPCAs will be specifically selected and/or designed for
their ability to control pest insects. Nontarget insects are the organism
group most at risk, being relatively closely related to the target organism
in most cases.
(B) While there are few nontarget insects that have been shown to
be economically important to humans, there are many nontarget insects
which have an important rule in ecological processes and may benefit hu-
mans indirectly.
(C) Unlike chemical pesticides, many microbials will exert their effect
through pathogenicity as well as toxicity. The acute, short duration, Tier
I tests, which should suffice for hazard evaluation for some chemical pes-
ticides, will not be appropriate for microbial agents. Adequate assessment
of pathogenicity will demand time to evaluate the MPCA for infectivity
and for its ability to reproduce or develop in the test insect.
(D) The host range is an important factor in hazard evaluation for
a MPCA. A problem here is that extrapolation, even across species lines,
is often not dependable. For this reason, the Agency will provide for test-
ing with representatives from a number of ' 'beneficial insect'' taxa. Infor-
mation from these tests will be used in conjunction with host range data
(developed during efficacy testing) to develop a clearer idea of the overall
insect host range.
(E) The Agency is aware that Tier I testing may be more extensive
in some cases than the baseline data requirements in OPPTS Series 850.
However, there should be very few microbials which require effects testing
beyond the Tier I level.
(F) The tier-testing scheme for MPCAs is based on a fairly extensive
first tier. The purpose of the Tier I testing is to assess toxicity and patho-
genicity of the MPCA to the honey bee and to three species of predaceous
and parasitic insects. Selection of the predator/parasite species to be tested
should take into account such factors as the likelihood of exposure to the
MPCA, phylogenetic proximity of the test species to target pest species,
and similar relationships. A rationale for selection should be developed
by the registrant.
(ii) Tier progression—(A) Tier I. Under these guidelines, toxicity/
pathogenicity tests on the honey bee and insect predators and/or parasites
are indicated for all MPCAs except Bts. Selection of predator and parasite
19
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species for testing is made by the registration applicant. Rationale for se-
lection is to be provided by the registrant. The main purpose of the Tier
I testing is to determine presence of toxic or pathogenic effects an rep-
resentatives of a few major orders of beneficial insects. As noted above,
the representative test species selected, in addition to the honey bee, should
be of some importance in the ecosystem to be exposed to the microbial
control agent. Data derived from Tier I testing will be used in conjunction
with available information on use pattern, host range (specificity), fate,
and other similar factors, to assess potential for adverse effects. If data
indicate no potential for affects, no further testing would be indicated. If
the results of Tier I testing indicate toxic and/or pathogenic effects, Tier
II testing (environmental expression) would follow. In some cases, a sub-
chronic test may lead to a better understanding of the effects observed
at the Tier I level and might alleviate the need for Tier II testing. The
Agency should be consulted before making these decisions.
(B) Tier II. The data for Tier II are described in environmental ex-
pression testing (OPPTS 885, Group E) of these guidelines. If expression
characteristics preclude exposure, no further testing would be indicated.
If data indicate that nontarget insects will be exposed to the MPCA, the
registration applicant should consult with the Agency regarding possible
Tier III testing.
(C) Tier III. For all MPCAs, Tier III consists of advanced tests spe-
cifically responding to adverse effects identified in earlier Tier testing.
Such tests may be simulated or actual field tests, but further research is
needed to develop the protocols for such testing. In any case, Tier III
testing would be preceded by consultation with the Agency.
(2) Aquatic insects. Tier I testing, as outlined in the "Aquatic Ani-
mal Tier Testing Scheme for Microbial Pest Control Agents" (under para-
graph (d) of this guideline) will include toxicity/pathogenicity testing with
Daphnia, or a species of aquatic insect, or both, depending on use pattern.
Detection of pathogenicity/toxicity in Tier I testing will automatically lead
to expanded testing which, if the impacted site is fresh water, will most
likely involve testing with aquatic insects.
(g) References. The following references can provide useful back-
ground information in developing protocols.
(1) Brusca, J. et al., Autographa californica nuclear polyhedrosis virus
efficiently enters but does not replicate on poikilothermic vertebrate cells.
Intervirology 26: 207-222 (1986).
(2) Committee on Methods for Toxicity Tests with Aquatic Orga-
nisms. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates,
and Amphibians. U.S. Environmental Protection Agency Ecological Re-
search Series, EPA 660/3-75-009 (1975).
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(3) Couch, J.A. et al, Environmental Significance of Baculovirus in-
fections in estuarine and marine shrimp. Annals of the New York Academy
of Science 196:528-536 (1975).
(4) Couch, J.A. and Courtney L., Interaction of chemical pollutants
and virus in a crustacean: A novel bioassay system. Annals of the New
York Academy of Science 198:4977-5048 (1977).
(5) Fenner, F. et al., The Biology of Animal Viruses. Second Edition.
Academic Press, NY (1974).
(6) Friend, M and Trainer, D.O., Experimental DDT-Duck hepatitis
virus interaction studies. Journal of Wildlife Management 38:887-895
(1974).
(7) Friend, M. and Trainer, D.O., Experimental Dieldrin-Duck hepa-
titis virus interaction studies. Journal of Wildlife Management 38:896-902
(1974).
(8) Hetrick, P.M. et al., Increased susceptibility of rainbow trout to
infectious hematopoietic necrosis virus after exposure to copper. Applied
and Environmental Microbiology 37:198-201 (1979).
(9) Ignoffo, C.M., Effects of entomopathogens on vertebrates. Annals
of the New York Academy of Science 217:141-164 (1973).
(10) Ignoffo, C.M., Evaluation of in vivo specificity of insect viruses.
in: Baculoviruses for Insect Pest Control: Safety Considerations, M. Sum-
mers et al. (eds.) American Society of Microbiology, Washington, DC
(1975).
(11) Ignoffo, C.M. et al., An Evaluation of the Risks to Mammals
of the Use of an Entomopathogenic Fungus, Nomuraea rileyi, as a Micro-
bial Insecticide. Pages 354-359, in: Baculoviruses for Insect Pest Control.
Safety Considerations. Selected papers from EPA-USDA Working Sympo-
sium American Society of Microbiology. Washington, DC (1975).
(12) Lightner, D.V. et al., Testing Penaeid shrimp for susceptibility
to an insect nuclear polyhedrosis virus. Environmental Entomology 2:611-
613 (1973).
(13) Macek, K.J. et al., Considerations in assessing the potential for,
and significance of biomagnification of chemical residues in aquatic food
chains. ASTM, STP. Pages 251-268, in: American Society for Testing
and Materials, Philadelphia (1979).
(14) Mann, J.A., Diseases and parasites of fishes: An annotated bibli-
ography of books and symposia, 1904-1977. Fish Disease Leaflet 53.
USDI. Fish and Wildlife Service, Washington, DC (1978).
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(15) McLeese, D.W. et al, Structure-lethality relationships for
phenols, anilines and other aromatic compounds in shrimp and clams.
Chemosphere 2:53-57 (1979).
(16) Narayan, K. et al., Nonsusceptibility of common carp, Cyrinus
carpio L. to nuclear polyhedrosis virus Baculovirus amsacta of groundnut
red hairy caterpillar. Madras Agricultural Journal 642:411-412 (1977).
(17) Nordlund, D.A. and Lewis, W.J., Terminology of chemical re-
leasing stimuli in intraspecific and interspecific interactions. Chemical
Ecology 2:211-220 (1976).
(18) Pound, J.G., Safety and potential hazards of the entomopathogen
Mattesia trogodermae to nontarget species. Ph.D. dissertation, University
of Wisconsin (1977).
(19) Savan, M. et al., A study of two species offish inoculated with
spruce budworm nuclear polyhedrosis virus. Wildlife Diseases 15:331-334
(1979).
(20) Schwartz, J.J., Prevalence of pathogenic pseunomonad bacteria
isolated from fish in a warmwater lake. Transactions of the American Fish
Society 103:114-116(1974).
(21) Slesin, L. and Sandier, R., Categorization of chemicals under
the Toxic Substances Control Act. Ecology Law Quarterly 7:359-396
(1978).
(22) Snieszko, S.F., The effects of environmental stress on outbreaks
of infectious diseases of fishes. Journal of Fish Bioliogy 6:197-208 (1974).
(23) Summers, M. et al., (eds.) Bacullviruses for Insect Pest Control:
Safety Considerations. Selected papers from EPA-USDA Working Sympo-
sium. American Society of Microbiology. Washington, DC (1975).
(24) Summers, M. et al., (eds.) Guidelines for Safety Testing of
Baculoviruses. Pages 179-184 in: Baculoviruses for Insect Pest Control:
Safety Considerations. Selected Papers from EPA-USDA Working Sympo-
sium, American Society of Microbiology. Washington, DC (1975).
(25) Surtees, G., Epidemiology of microbial control on insect pest
populations. International Journal of Environmental Studies 2:195-201
(1971).
(26) Undeen A.H. and Maddox J.V., The infection of nonmosquito
hosts by injection with spores of the microsporidan Nosema algerae. Jour-
nal of Invertebrate Pathology 22:258-265 (1973).
(27) U.S. Environmental Protection Agency. Registration of Pes-
ticides in the United States: Proposed Guidelines, Subdivision E Hazard
Evaluation: Wildlife and Aquatic Organism FEDERAL REGISTER 43:
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29724-29737 (1978).(28) Van Essen, F.W. and Anthony, D.W., Suscepti-
bility of nontarget organisms to Nosema alqerae (Microsporida:
Nosematidae) a parasite of mosquitoes. Journal of Invertebrate Pathology
28:77-85.
(29) Weiss, B. and Laties, V.G., Assays for Behavioral Toxicity: A
Strategy for the Environmental Protection Agency. Pages 213-215 in: Test
Methods for Definition of Effects of Toxic Substances on Behavior and
Neuromotor Function, Neuro-Behavioral Toxicology, Vol. 1. Suppl. 1.
ANKHO International Inc. Report No. EPA 560/1179-010 (1979).
(30) Wolf, K., Evaluation of the Exposure of Fish and Wildlife to
Nuclear Polyhedrosis and Granulosis Viruses. Pages 109-111 in:
Baculoviruses for Insect Pest Control: Safety Considerations. Selected Pa-
pers from EPA-USDA Working Symposium, American Society of Micro-
biology. Washington, DC (1975).
(31) Wolf, K., Evaluation of the exposure of fish and wildlife to nu-
clear polyhedrosis and granulosis viruses, in: Baculoviruses for Insect Pest
Control: Safety Considerations, M. Summers et al. (eds.) American Society
of Microbiology. Washington, DC (1975).
(32) Wolf, K. and Quimby, M.C., Fish Cell and Tissue Culture. Pages
253-305 in: Fish Physiology. Vol. 3. W.S. Moor and D.J. Randall (eds.)
Academic Press, New York (1969).
(33) Wolf, K. and Quimby, M.C., Towards a practical fail-safe system
of managing poikilothermic vertebrate cell line culture. In Vitro 8:316-
321 (1973).
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