United States       Prevention, Pesticides     EPA712-C-96-336
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA   Microbial Pesticide
          Test Guidelines
          OPPTS 885.4340
          Nontarget Insect
          Testing, Tier

     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 885.4340   Nontarget insect testing.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal  Insecticide, Fungicide,  and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 154A-23.

     (b) Test standards. In addition to satisfying the applicable general
test standards outlined in OPPTS 885.0001, the following  apply:

     (1) Test substance. The actual form  of the material to be regarded
as the test substance is discussed in OPPTS 885.0001.  In addition,  any
substances used to enhance virulence should be tested along with the test
substance. Whenever feasible, dosage shall be in suitable increments up
to lOOx the  LD50  or LC50  of the  pathogen in its natural  host,  or  10-
lOOx the recommended field dosage.

     (2) Test species, (i) Testing should be performed  on three  species
of insects, representing at least  two of the following groups—parasitic
dipterans,  predaceous  hemipterans, predaceous coleopterans, predaceous
mites, predaceous neuropterans, parasitic hymenopterans.

     (ii) Selection of test species is the responsibility of the researcher.
Rationale  for selection must be provided. More specifically, the following
points apply:

     (A) Viruses. Testing shall be performed on three species of insects
representative of those parasites and predators  known or suspected to at-
tack the target host or to share the same ecological habitat.

     (B) Protozoa.  In selecting the three species of nontarget arthropods,
at least one  should  be a major  (i.e. important)  parasite of the target host.
Many protozoans have a wide host range.  Accordingly,  if possible, more
than the minimum (three) species of nontarget organisms  should be tested.

     (C) Fungi. Assuming testing is justified, appropriate  organism  test
species  should  be the major predacious  and parasitic regulatory agents
common to  the ecosystem  where the MPCA will be applied. In addition,
testing could include parasites and/or predators specific to the host of the
fungal agent.

     (D) Bacteria. Testing shall be performed  on three species of insects
representative of those parasites and predators  known or suspected to at-
tack the target host or to share the same ecological habitat.

     (3) Controls. A concurrent control group is recommended and should
be treated with microbe-free (or nonviable microbe) material from the  cul-
ture  system used for propagation of the microbial pest control agent.

     (4) Routes of exposure—(i) Viruses.  The best routes  of exposure
will  depend on the developmental location of the nontarget organism. In-
ternal parasites may be tested with virus-infected hosts, or if they can be
cultured in vitro, the virus can be added to the  diet.  External  stages of
parasites and predators, if they are obligatory, may be fed virus-infected
hosts, and others may be fed virus-contaminated media or virus suspended
in sugar or honey solutions.

     (ii) Protozoa. (A) In addition to feeding adult predators and parasites
of the target insect with the resistant  stage of the protozoan, immature
stages of the predator or parasite should be exposed.  Predators can be fed
hosts infected with  the protozoan over a period of time. The  predator,
at a prescribed time, should be checked for protozoan infection.

     (B) The protocol for parasites  is more complex.  Protozoan-infected
hosts can  be parasitized or parasitized hosts can be fed protozoans. Para-
sites from protozoan-infected hosts should be examined for protozoan in-
fection. The  immature  stages  as well  as  adults should be  examined. If
possible, a primary hymenopterous and dipterous parasite should be exam-

     (C) The best route of infection for adult Hymenoptera or Diptera is
oral  acquisition  of protozoan spores. Predaceous  insects could also be fed
in this manner,  but feeding them infected (live) hosts of known age is
more appropriate. In either case, the actual amount  of spores consumed
cannot be accurately determined.  If infection of the parasite or predator
adult occurs, the  possibility for transovarial or transovum transmission
should be  examined.

     (iii) Fungi. Routes of exposure should simulate field conditions as
much as possible. In the  case  of entomopathogenic fungi, environmental
conditions (> 90 percent relative humidity) are critical at the time of expo-

     (iv) Bacteria. Best routes of exposure will depend on developmental
location of the nontarget  organism. Internal parasites may be tested with
bacteria-infected hosts,  or if they can  be cultured in vitro,  the bacteria
can  be  added to the diet. External stages may be  fed bacteria-infected
hosts, bacteria-contaminated media,  or bacteria  suspended  in  sugar  or
honey solutions.

     (5) Duration of test/endpoints—(i) Viruses. (A) Control and treated
insects should be observed for a duration of at least 30 days after dosing,
or in cases where an insect species  cannot be  cultured for 30 days, until
control mortality rises above 20 percent. In cases where signs, symptoms
and  pathologies are detected, the treated insects should be examined in
detail at late stages of infection,  at  moribund, and  at death. Such tests
need not be prolonged to 30 days, if death of treated insects  occurs prior

to 30 days. In all  cases of pathologies in the treated nontarget insects,
it is essential that the etiology of the infection be established.

     (B)  The  end-points should  be based on the  frank  development of
pathologies and on the early mortality of the treated as compared to the
untreated (control) nontarget organisms.

     (ii)  Protozoa.  (A) Test duration should be determined on a case-by-
case basis. The most  appropriate end-point for protozoan diseases for de-
termining  pathogenic  effects  is the  presence  of the vegetative  stages
(shizonts or meronts) in the tissues of nontarget  insects. The schizonts
within suspected tissues can be detected by making a smear,  staining with
Giemsa  stain,  and  examining  the  slide with  oil  immersion using  a
compound microscope. The nontarget insect should be alive  when the tis-
sues are  removed for  the smear  because the  shizonts are fragile and are
usually destroyed by other microbes  or are distorted upon  death of the

     (B)  Protozoan spores can be used as an indicator of infection.  How-
ever,  if the infection is light, the few spores could have  come from the
inoculum. If spores are abundant (a relative term) and occur  in the tissues
of the nontarget insect, it is likely that it is infected.

     (C)  Another way to confirm infection is to conduct histological stud-
ies of the tissues using standard methods and looking for spores and other
pathological effects. The end-point would be just before death of the orga-
nism or a prescribed period of time.

     (D)  Death of the nontarget insect is a good end-point if the protozoan
is virulent. However,  since most protozoans have chronic  effects on their
host,  changes  in  behavior, size,  or color could be used as  an  end-point.
In each  case,  a microscopic examination to  find  schizonts or spores is
essential  to confirm the presence of the protozoan. Koch's  postulates to
confirm the virulence  of the isolate should be run.

     (iii)  Fungi. (A)  With entomopathogenic fungi, test  duration can be
limited to 8-10 days.

     (B)  Mortality  time, expressed  as LT50  (time course of population
mortality), is considered the most reliable parameter for bioassaying fungi
of insects in the laboratory. Pathogenicity should be confirmed by identify-
ing the fungal  agent as the original inoculum.

     (iv)  Bacteria.  Control and treated insects should be  observed for 21
to 30 days  after dosing, if this is possible. In cases where the insect species
cannot be  cultured for 21  to 30 days, observation  should continue  until
control mortality rises above 20 percent.

     (c) Reporting and evaluation  of data. The reporting provisions are
the same as those specified in OPPTS 885.0001.

     (d) Tier progression. (1) Data derived from Tier I testing will be
used in conjunction with available information on use pattern, host range,
and  other similar factors,  to assess potential for adverse effects. If data
indicate potential for adverse effects, Tier II testing  will be required as
specified in 40 CFR 158.740. In some cases, a subchronic test may serve
to better understand the effects  observed at the Tier I  level and might
alleviate the need for Tier II testing.

     (2) If no toxic or pathogenic effects are observed in this study, addi-
tional testing is ordinarily not necessary.

     (e) References. The following references are provided for use in the
development of  acceptable test  protocols for conducting toxicity/patho-
genicity tests with microbial pesticides:

     (1)  Andreadis,  T.G.  Nosema pyrausta infection  in  Macrocentrus
grandii, a braconid parasite of the European corn borer, Ostrinia nubilalis.
Journal of Invertebrate Pathology 35:229-233 (1980).

     (2) Brooks,  W.M. Protozoa: Host—parasite—pathogen interrelation-
ships. Miscellaneous Publication of the Entomological Society of America

     (3) Gardner, W.A. et al. Susceptibility of the two-spotted spider mite,
Tetranychus  urticae  Koch, to the fungal pathogen Hirsutella thompsonii
Fisher, Florida Entomology 65:458-465 (1982).

     (4) Van Essen,  F.W.  and D.W.  Anthony. Susceptibility  of nontarget
organisms to Nosema  algerae (  Microsporida: Nosematidae  ), a parasite
of mosquitoes. Journal of Invertebrate Pathology 28:77-85 (1976).