United States      Prevention, Pesticides     EPA 712-C-98-419
          Environmental Protection    and Toxic Substances     March 1998
          Agency        (7101)
4»EPA    Product Performance
          Test Guidelines
          OPPTS 810.3400
          Mosquito, Black Fly, and
          Biting Midge (Sand Fly)

     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, or  call 202-512-0132 for disks
or paper copies.  This  guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and  Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 810.3400  Mosquito, black  fly,  and  biting  midge (sand fly)
     (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 95-10 Mosquito,  black
fly, nonbiting midge, and biting midge (sand fly) treatments (Pesticide As-
sessment  Guidelines, Subdivision  G:  Product Performance,  EPA report
540/9-82-026, October 1982).

     (b) Overview. (1) This section is concerned with efficacy data re-
quirements for evaluation of invertebrate control pesticides as  outdoor and
breeding ground treatments against several groups of important insect pests
which pose economic and  public health threats to  man in urban,  rural,
agricultural, and wilderness habitats. The insects for which data consider-
ations are outlined here belong to five families: Culicidae (mosquitoes),
Simuliidae (black  flies), Ceratopogonidae  (biting midges), and nonbiting
midges in the families Chironomidae (midges) and Chaoboridae (phantom

     (2) These insects inhabit a variety of ecological niches and  show di-
verse biological, physiological, and behavioral patterns. In the evaluation
of biologically active compounds, the unique features of each pest species
must be taken into consideration. Members of the 5  families for which
these guidelines  are developed are mostly aquatic or semi-aquatic in their
immature stages. The mature  stages are terrestrial,  dispersing beyond the
immediate vicinity of breeding sources.

     (c) General considerations—(1) Species,  stage, age, and  sex. The
test insect should be identified as to genus  and species. Additional infor-
mation as to  subspecies or  strains should be recorded. Methods and proce-
dures utilized for assessment are dictated by the stage and habitat of the
insect. Pesticides are generally  evaluated against the  larval and/or adult
stages. In certain situations, however, registrants may have to gather data
on the egg  and pupal  stages. In larval evaluation, the instar should be
standardized  and specified. The  age or age range  (if  precise age is not
known) of the adult  test insects should be reported. Reporting of the cal-
endar age of larvae is not essential if the larval instars are specified. The
sex of the adult  test  insects should be  specified. Determination of the sex
of the immature stages is  difficult,  and generally this  information is not

     (2) Plot size. Plots which are suitable in size for commercial applica-
tions should  be used. Plots size can vary from a few  square  feet to  hun-
dreds of acres (hectares), depending on the  objectives  of the test, species,

and type of control desired. Test plot dimensions should be large enough
to avoid drift onto sampling areas of untreated control plots.

     (3) Number of trials. A minimum of 5  large-scare geographically
separated trials are generally necessary, but the number of trials can vary
somewhat due to the accessibility of infestations, fluctuations in pest popu-
lation pressures, behavior,  and other important considerations in the biol-
ogy of the target pest.

     (4) Application techniques and equipments. Aerial or ultra-low vol-
ume applications should be evaluated if these methods are  specified on
the label.

     (5) Evaluation and reporting procedures. The evaluation proce-
dures should be specified in the  presentation of the data. Reports should
include larval counts, bite  counts, percent mortality, knock-down time or
other appropriate measures of determining the effectiveness of the test
product. Results should include the infestation or nuisance reduction, or
preferably both, compared with the control plots or areas. Raw data on
pest counts  or other measures of control should be summarized.  Insect
resistance can be a major  problem when testing a product for mosquito
control, and any evidence  of such should be reported when  tests are run
in areas where resistance  is a known problem.  Other factors of special
concern in determining a product's usefulness are wildlife and fish hazards
(see OPPTS Test Guideline Series 850, Ecological Effects), possible water
or air pollution, effects on  house  paints, and spotting of automobiles. Any
adverse effects should be reported. Repellency from mosquito coils, can-
dles, and torches is dependent upon the concentrations and duration of
smoke or fumes which permeate a limited area.  These types of products
should demonstrate repellency of mosquitoes and other small flying insects
on patios, torches,  or  other confined outdoor  areas where there is little
or no breeze.

     (6) Sampling  techniques. Various species  and the  different  stages
of the same  species require specific sampling techniques. Applicable tech-
niques for assessment of populations of test species or groups should be

     (d) Suggested performance standards. Unless otherwise  specified,
these standards are  presented on  the basis of pre- and post-treatment pest
population counts (including landing, bite, and trap counts)  from treated
as well as the untreated pest  plots and  standard comparative treatments.
All percentages  of control refer to  the  performance of the test product
[as determined by pest insect counts (landing and bite) and other measures
correlated to insect population pressures] against the  vulnerable stage(s)
of the target pest,  when evaluated  according  to a recognized aerial or
ground application treatment program under actual field conditions.

     (1) Culicidae (mosquitoes)—(i) Larvae. A minimum of 95% popu-
lation reduction, based on pre- and post-treatment infestation counts from
tests conducted under actual field conditions.

     (ii) Adults. A minimum of 95% population reduction, based on pre-
and post-treatment infestation counts. When appropriate, laboratory colony
or caged wild  mosquitoes can be used. The tests should be conducted
under actual field conditions.

     (2) Simuliidae (black flies)—(i) Larvae. A minimum of 80% popu-
lation reduction, based on laboratory evaluation techniques (jar, cloth trap,
flushing and draining, and trough) and the single stream technique to deter-
mine effectiveness by comparing larval populations above  and below the
stream treatment point, not more than one day before and one day after

     (ii) Adults. Large scale control operations are evaluated by pre- and
post-treatment population estimates. Landing rate counts  (a  one-minute
count beginning after a 5-minute waiting period is satisfactory) and stand-
ardized sweeps about the head of observer inside and outside the treated
areas provide a useful index to the annoyance rate.

     (3) Ceratoponidae (biting midges)—(i) Larvae. A minimum of 95%
population reduction, based on pre- and post-treatment counts. Populations
of larvae can be sampled by laboratory inspection of suspected breeding
media collected in the field or by an emergence trapping method to deter-
mine the level of control.

     (ii) Adults. A minimum of 95% population reduction, based on pre-
and post-treatment population estimates determined by  landing counts or
light trap collections.