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

     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

     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,    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 885.4000   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-

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-

    (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)


    MDD  = maximum daily dose expressed as units per volume

    [MPCA] = concentration of MPCA

    TGAI  = technical grade of the active ingredient

    BW = body weight

     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

     (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-

     (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

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

     (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

     (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.

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

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

   reported under paragraph (g)(9) of this guideline that the following groups
   of terrestrial  animals  have been tested  in  vivo for effects  caused by

                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-
   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.


     (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

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-

     (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.


     (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

     (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


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.


     (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


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

    (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


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

     (//) The number of spores  recovered had to exceed the number in-
jected by  lOOx. This type of approach may be useful for other microorga-

     (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


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


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.


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

     (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


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


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).


     (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

     (7) Friend, M.  and Trainer,  D.O., Experimental Dieldrin-Duck hepa-
titis virus interaction studies. Journal of Wildlife Management 38:896-902

     (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

     (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).


     (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
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