United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention
January 2012
        Ecological Effects
        Test Guidelines
        OCSPP 850.2100:
        Avian Acute Oral
        Toxicity Test


     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's  number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and  protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from  the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional  information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 850.2100: Avian acute oral toxicity test.

(a) Scope-

       (1) Applicability.  This guideline is intended to be used to help develop data to submit to
       EPA under  the Toxic  Substances  Control Act (TSCA) (15 U.S.C. 2601,  et seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

       (2) Background.  The source materials used in developing this harmonized OCSPP test
       guideline include the OPPT guideline under 40 CFR 797.2175 Avian Acute Oral Toxicity
       Test; the OPP 71-1 Avian Single-Dose Oral LD50 Test (Pesticide Assessment Guidelines
       Subdivision  E); the Avian Single-Dose Oral LD50 Standard Evaluation Procedure; and
       OPP Pesticides Reregi strati on Rejection Rate Analysis: Ecological Effects.

(b) Purpose.  This  guideline is designed to develop data, specifically both a median lethal  dose
(LDso) and slope  of the dose-response relationship, for acute oral  toxicity to upland game birds
(e.g., northern  bobwhite  (Colinus virginianus)},  water  fowl  (e.g.,  mallard  duck  (Anas
platyrhynchos)),  or passerine  species  (e.g., house  sparrow (Passer domesticus), zebra finch
(Taeniopygia guttatd), red-wing blackbird (Agelaius phoeniceus)) of chemical substances and
mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations.
While the study is specifically  designed to  allow calculation of the  LCso, the study can be used to
obtain information  regarding  sublethal effects  which are used in  Agency evaluations.   This
guideline prescribes methodologies to determine both the LDso and slope of the dose-response in
the same study. The Agency can consider  data generated that provides a LDso value but no slope
information but such a study would be insufficient to meet the purpose of this guideline unless it
is a  limit test (see  paragraph  (d)(3) of this guideline).  The use of a  test based primarily on
lethality is justified  because it  presents  or insures a consistent, unbiased  endpoint for assessment
purposes and has  unambiguous ecological relevance to adverse effects.

(c) Definitions.  The definitions in  the OCSPP 850.2000 guideline  apply to this test guideline.
In addition, the following more specific definition also applies to this guideline:

Observation period is the portion of the test that begins after the test birds have been dosed and
extends at least 14 days, but to continue until overt evidence of toxicity has subsided.

Frank sublethal effects for the purpose  of this study refers to overt or frank toxicological  effects
for birds and  include, but are not limited to, decreased body weight, loss of coordination,  or
lethargy. Less  significant sublethal  effects such as ruffled appearance  or muted color are not
considered frank toxicological  effects.

(d) General considerations—

       1) Summary of the test. Birds are administered the test substance as a single oral dose,
       either by capsule or gavage.  Birds are observed regularly for mortality or  any signs of
       intoxication  throughout the observation period. Birds are weighed  and feed consumption
       is estimated at least weekly  thereafter.  The mortality response pattern is examined and
       subjected  to the appropriate statistical analysis to derive the LDso, confidence limits, and
                                      Page 1  of 19

slope of the dose-response line.  Sublethal effects should also be monitored, for example,
gross appearance  and behavior  of the  birds, weight  changes,  and  changes in food
consumption.   Histopathological  and physiological  changes  should  be monitored.
Moreover,  delayed mortality and  differences in  sensitivities of each  sex should be

(2) General test guidance.

       (i) The general guidance in  OCSPP 850.2000 applies to this guideline except as
       specifically noted herein.  Studies should not be conducted with endangered or
       threatened species.

       (ii)  Successful  adherence to elements in paragraph (h) of this guideline when
       testing  passerine  species  may  depend  upon  special  provisions  regarding
       husbandry, diet, acclimation of wild-caught  birds,  handling and age at dosing.
       For  tests  conducted  with passerine  species it  is  recommended,  prior to test
       initiation, to submit protocols to the regulatory Agency for approval that identify
       husbandry, dietary, holding and acclimation methods, age at dosing and any other
       special provisions planned (see in particular  references in paragraphs (j)(2) and
       (j)(4) through (j)(9) of this guideline).  To  be complete, the protocol should also
       contain information on all of the items discussed in this guideline.

(3) Range-finding test. Unless the approximate toxicity of the test substance is known
already, a range-finding test  should be conducted  to determine the dosage levels of the
test  substance to be used  in the definitive test.  Refer to paragraph (e)(4)(ii) of this
guideline for details on dosage levels for definitive tests.  Procedures  for range-finding
tests may vary, but generally, groups of a few birds are administered three to five widely-
spaced doses.  A series of 2,  20, 200, and 2,000 milligrams per kilogram of body weight
(mg/kg-bw) is suggested.

       (i) If there is  no mortality at the  2,000  mg/kg-bw  dose level,  and the test
       procedures and numbers of birds per dosage are the same as would be used in a
       definitive  test,  and  also  meet the elements of an  acceptable  limit test  (see
       paragraph (d)(4)  of this  guideline),  then  the  range-finding test  may provide
       sufficient information to negate the need for a definitive test.   If mortality does
       occur, than the results of  the range-finding tests may  then  be used to help
       establish the definitive test dose levels.

       (ii) If a test substance is expected to be of low toxicity, it may be useful to first
       conduct a limit test at 2,000 mg/kg-bw as described under paragraph (d)(5) of this
       guideline.  If mortality occurs at this dose level, then further range-finding at
       lower levels is suggested.  The results of the range-finding test then may be used
       to establish the  definitive test dosage levels.

(4) Definitive test. The goal of the definitive test  is to determine a dose-response curve
for avian mortality after oral dosing and an observation period  of at least 14 days to
establish the acute LD50 (standard error and 95 percent (95%) confidence limits), as well
as the slope of the dose-response  curve (and its standard error and 95% confidence
                                Page 2 of 19

       limits).   The  definitive test consists of a  minimum  of five  dose levels  of the  test
       substance, plus appropriate controls.  The  dosage levels  are  confirmed by  chemical
       analysis under test conditions.  A summary of test conditions is  provided in Table 2  and
       validity elements  for an acceptable definitive test are  listed in Table  3.  The Agency
       should be contacted prior to testing with nanomaterials.

       (5) Limit test.  For test substances expected to have relatively  low toxicity, a limit  test
       may be conducted with a single dose level at 2,000 mg/kg-bw or the maximum expected
       environmental residue  concentration, whichever is higher, plus a control  group.   For
       pesticides, if the expected environmental residue concentration exceeds 2,000 mg/kg-bw
       the  test should be conducted  at a higher level equivalent to  the  maximum  expected
       environmental concentration (EEC) on food items (see  paragraph  (e)(4)(ii)(B) of  this
       guideline).  Based on the results of the limit  test, the acute oral LDso may be reported as
       greater than the limit dose provided that the  following conditions are met: first the limit
       treatment group and control group each  contain  a minimum  of  10 birds; second no
       mortality or frank sublethal effects occurs in the limit  dose group;  third except for the
       number of dosage levels the test procedures and duration are the  same as in the definitive
       test; fourth the dosage level is confirmed by  chemical analysis under test conditions;  and
       fifth,  for pesticides, the limit  dose  is  2,000 mg/kg-bw  or  the  maximum  expected
       environmental residue concentration, whichever is  higher.  Clinical signs of toxicity, if
       any, should be reported. Conduct the full definitive test when any mortality is observed
       at the limit dose.  If sublethal effects are suspected in the study than the Agency should
       be consulted for discussion on the appropriate dose and conduct of the limit test.

(e) Test standards—

       (1) Test substance. The substance to be tested should be technical grade unless the  test
       is designed to test a specific formulation,  mixture, or end-use product. For pesticides, if
       more  than one active ingredient constitutes  a technical product the technical grade of
       each active  ingredient should be tested  separately, in addition to  the  combination, if
       applicable.  The OCSPP 850.2000 guideline lists the type of information that should be
       known about the test substance before testing and  discusses methods for preparation of
       test substances.

       (2) Test duration. The definitive and limit tests consist of the administration of the  test
       substance followed by an observation period  of at least 14 days.   If mortality occurs
       during the last 3 days of the 14-day period,  or if signs  of intoxication are not clearly in
       remission, or if the test substance is expected to have  delayed  effects, then extend the
       observation period until mortality or signs of  intoxication are not observed for 72 hours.

       (3) Test organism—

              (i) Species. Data on both a passerine species and either an upland game bird  or a
              waterfowl  are generally  required for 40 CFR Part 158.   These test protocols  and
              standards  describe  tests  specific to  using the northern  bobwhite  (Colinus
              virginianus (L.)), for an upland bird, the mallard (Anas platyrhynchos L.) for a
              waterfowl.   At this time, there  is  no identified  preferred passerine  species;
              examples of potential passerine species that have  been used  in acute oral testing
                                       Page 3 of 19

include the house sparrow (Passer domesticus), zebra finch (Taeniopygia guttata)
and  red-wing blackbird (Agelaius phoeniceus).  Regardless  of the passerine
species chosen, a protocol should  be submitted to the  Agency  prior to test
initiation  for review.   In addition  to these  species, pigeon  (Columba livid).,
Japanese  quail  (Coturnix coturnix japonica), ring-necked pheasant (Phasianus
colchicus), and red-legged partridge (Alectoris ntfd), have also be used as upland
game birds. The Agency will use these and other data to assess acute hazards and
risks  to birds.   Appropriate husbandry  standards for species  used in this test
should be consulted, examples include references in paragraphs Q)(l), G)(2) and
(j)(4) through (j)(9) of this guideline.

(ii) Source. Birds may be reared in the laboratory or purchased from a breeder.
For a satisfactory test, all control and treatment birds used in a test should be from
the same  source and breeding population and be in  the  same  plumage.  Birds
should be obtained  only from  sources  whose colonies  have  known breeding
histories.  Test  birds should be phenotypically indistinguishable (except for size)
from wild stock.  It is recommended that birds be obtained from flocks that have
been  out  bred  periodically  with genetically wild stock in order to  maintain  a
genetic composition that approximates the natural heterogeneity of the species.
Birds purchased from a breeder should be certified as disease-free or as bred from
disease-free stocks.   The Agency recommends against the use of wild-caught
birds.  However, where suitable cultured supplies of passerine species may not be
available,  protocols submitted in advance should detail why wild stock must be
used and have the necessary elements identified in this guideline addressed (see
paragraph (d)(2)(ii) of this guideline).

(iii) Age, sex, and size.

       (A) Typically, test birds  should be young adults  of both sexes, not yet
       mated, and are  at least 16 weeks old at the time of  dosing.  A less
       preferred alternative  is for the use of first-year birds that may have been
       mated, as long as the birds are brought completely out of production
       through reduced light cycles.  In addition, there may be situations where in
       order to address concerns associated with a chemical-specific  pattern  of
       use younger or older birds (e.g., altricial nestlings), or birds of only one
       sex (e.g., breeding females),  are more appropriate for testing.  The need
       for this  testing should be determined on a case-by-case basis after the
       basic testing is completed.

       (B) For a satisfactory test, all upland and waterfowl birds used in a test
       should be the same  age,  plus  or minus (±)  1  week.  Currently, due  to
       culturing and/or husbandry practices  of Passeriformes, passerine  birds
       used in a test may not be ± 1 week; however this may change if husbandry
       practices evolve.  More consistent responses may be attained if the range
       of body weights is no greater than ±10% of the mean body weight for the
       test population.
                         Page 4 of 19

(iv) Acclimation.  Test birds should be acclimated to test facilities and basal diet
for a minimum of 14 days.  Acclimation to test pens may be either in the actual
pens used in the test or in identical pens.

(v) Health status.  Birds  used in  the test should be in  apparent good health.
Deformed, abnormal, sick,  or injured birds should not be used.  Birds should not
be used for a test if total mortality during the  14-day acclimation period is more
than 5% for cage-reared birds  or more than 10% for  wild-caught birds.   Birds
should  not have  been selected in any  way for genetic resistance  to  toxic
substances.  Birds should  not have been used in a previous test, either in a
treatment or a control group.

(vi) Care and handling.  During holding, acclimation, and testing, birds should
be shielded from excessive  noise, activity,  or other disturbance.  Test birds should
be handled only as much as is necessary to conform to test procedures.

(vii) Diet and feeding.

       (A) A standard commercial game bird (for northern bobwhite) or duck (for
       mallard) feed, or the nutritional equivalent, should be  used as the diet.
       Guidance for recommended nutritional values for these species is provided
       in Table 1.  Passerine  species may require more complex diets.   Feed
       should be withheld from all test groups for a minimum of 15 hours prior to
       administration of the test substance but feed should  be available ad libitum
       during the study.  This fasting period should be decreased, as appropriate,
       for the selected passerine species.

       Table 1.—Recommended nutritional values for feed
Nutritional Component
Crude protein
Crude fiber
Crude fat
Recommended Range (%)
27 to 29
3.5 to 5.0
2.5 to 7.0
2.6 to 3.6
0.9 to 1.1
       (B) Feed should not be used past its normal shelf life. Antibiotics or other
       medication should not be used in the diet during the acclimation period or
       the test.  It may not be possible to obtain feed that is completely free of
       pesticides, heavy metals,  and other contaminants.  Therefore, diets should
       be analyzed periodically, as  described under paragraph (e)(9)(ii) of this
       guideline, and selected to be as free from contaminants as possible.  Extra
       precautions should be taken when fish meal or oil is a major ingredient,
       since  fish are  often  contaminated with  high  levels  of  chlorinated
                         Page 5 of 19

       (viii)  Water.    Clean water  should be  available ad libitum.   Only clean,
       unmedicated water should be offered during the acclimation and testing  periods.
       Water bottles or automatic watering devices are recommended.  If water pans or
       bowls are used, water should be changed at least once a day.

(4) Administration of test substance.  After acclimation under paragraph (e)(3)(iv) of
this guideline, feed should be withheld from all test groups for a minimum of 15 hours
prior, except potentially for Passeriformes, which may  require less time to clear the
digestive system  of confounding substances,  to  administration of the test substance.
Dosing by  gelatin capsule is preferred,  but when  dosing with capsules is not feasible,
doses may  be administered by gavage.  Doses are based  on the individual body weight
(bw) of each bird.  Body weights are typically determined at the time of dosing, but may
be taken, especially when capsules  are  used, within 24 hours prior to  dosing.  Dosing
should be done in the early morning  hours.  The Agency should be contacted  prior to
testing with nanomaterials.

       (i) Preparation of gavage mixtures. If dosing is by gavage and a carrier is  used
       to administer the test substance, the preferred carrier is distilled or deionized
       water unless the test substance is known to hydrolyze readily.  Other acceptable
       carriers include corn oil, propylene glycol,  1% carboxymethyl-cellulose, and  gum
       acacia. Materials with known toxic or emetic properties should not be used.  The
       absence of the crop in many passerine  species  requires  using smaller, softer
       tubing than with waterfowl (see paragraph (j)(12)  of this guideline). The dosing
       volume of test substance plus vehicle in a test should be constant for all birds  with
       respect to  individual  body  weights and  should not  exceed 5  milliliters per
       kilogram of body weight  (mL/kg-bw).  For those unusual test substances that
       might require  a  larger dosing volume (e.g. liquids with low purity), a dosing
       volume up to 8 mL/kg-bw may be used; steps should be taken to ensure that birds
       do not regurgitate the dose. In choosing between the bobwhite quail and mallard,
       it is recommended to use the bobwhite when a dosing volume up to 8 mL/kg-bw
       is needed.

       (ii) Treatment concentrations.

              (A) At a minimum, five dosage levels of the test substance are tested for
              definitive testing, plus the appropriate control. These dosage levels should
              be spaced geometrically in such a manner so that the entire dose-response
              curve (LDio  to LDgo) is  adequately characterized.   Taking into  account
              results of the range-finding test(s), dosage levels should be spaced so that
              at least three doses cause mortality between, but not including,  0% and
              100%.  For a scientifically sound estimate of a point on the curve (e.g.,
              LDso),  responses should immediately  bracket the point estimate  of
              concern.  For some test substances, it may be necessary to use more  than
              five dose levels to achieve these results.

              (B) For a limit test, there  is single dose level, plus the appropriate control
              (see paragraph (d)(5) of this guideline). A limit dose of 2,000 mg/kg-bw
              is used unless  environmental residues are  expected  to result in a higher
                                Page 6 of 19

                     dosage.  Equation 1 of this guideline can be used to calculate the acute
                     avian oral limit dose (mg a.i./kg-bw) for spray  applications of pesticides.
                     The dietary residue estimates are based on a nomogram that relates food
                     item residues to pesticide application rate; for an application rate of 1  Ib
                     a.i./A, the highest residue level expected is with short grass (nomogram
                     value of 240).  The nomogram is based on an EPA database called UTAB
                     (Uptake,  Translocation,   Accumulation,   and  Biotransformation),  a
                     compilation  of actual  measured pesticide residue values on plants (see
                     references in paragraphs (j)(5) and (j)(H) of this guideline).  If there are
                     multiple uses this study is supporting for registration, the limit dose for the
                     study should be based on the  one resulting in the highest  dose.  If the
                     resulting limit  dose  exceeds the digestive capacity of the test  organism,
                     consult with the Agency prior to  conducting the study to determine the
                     appropriate dose to use.
Limit dose(mga,./kg-bw)=  ,    —                 Equation 1
                    for a pesticide use with a single application per year:

                             Cm3X_diet = (ApRate \\. 14)(240)                   Equation 2

                    for a pesticide use with more than one application per year:

                    ,„-,„ = l((^«Xl.l4X240/e-(^)M^ll       Equation 3
ApRate = maximum single application rate (in Ib a.i./acre);
Halflife = the foliar half-life (default is 35 days);
Interval = the minimum application interval (in days);
/' = application event from 1 to n;
n = total  number of applications;
AW = the body weight (in g) of the assessed bird - for pesticides, use 20 grams as this is most
conservative value in screening level assessments;
TW= the body weight (in g) of the test bird;
SF = body weight allometric scaling factor (Mineau et al. scaling factor of 1.15 is the default (see
paragraph (j)(10) of this guideline)); and
1.14 = a  dose conversion factor  assuming that a 20 gram bird consumes 114% of its body weight

       (5) Controls.

              (i) Every test  includes a negative control group where control birds receive a
              sham  dose consisting of the same vehicle  or capsule as received by  the test

                                       Page 7  of 19

       substance dosed birds.  For a satisfactory test, negative control birds should be
       from the same hatch as the test substance dosed groups and be kept under the
       same experimental conditions. The test procedures should be the same for control
       and treated birds, except that no test substance should be administered  to the
       control birds.

       (ii)  Controls  serve as  a  monitor of bird husbandry practices, an indicator of
       possible problems due  to handling, and test substance administration and aid in
       separating treatment related effects from non-treatment  related effects.  Controls
       are  important in  assessing  background  mortality  and disease.   Background
       mortality is never presumed to be negligible.

       (iii) A test  is not  acceptable if more than 10% of the negative control birds die
       during the test period.

       (iv) A concurrent positive control with a reference substance of known toxicity
       may be included in the test, as discussed in the OCSPP 850.2000 guideline.

(6) Number of test organisms.

       (i) The minimum  number of birds per dosage level of the test substance and the
       control is 10 birds. Typically, equal numbers of young  adult birds of both sexes
       are  used.   However,  when necessary to address  chemical- and site-specific
       concerns, the  sex and age of the birds  used for testing may be modified if this is
       justified (e.g., breeding females  may be used when the expected exposure pattern
       indicates they may be at risk).   Equal  numbers of birds should be used for each
       dosage level.

       (ii) Birds used in  the test should be assigned randomly to treatment and control
       pens.  Birds at a dosage level may be divided into two pens of five birds each.  If
       this is done, dividing the groups by sex is recommended. Randomization should
       be done either at the initiation of the acclimation period or at the time of weighing
       just prior to dosing.   The latter is recommended, because it  avoids additional
       handling stress.

(7) Facilities, apparatus  and supplies.  Normal  laboratory equipment and supplies, and
items especially listed in (e)(7)(i) through (e)(7)(iv).

       (i) Facilities. Pens should be kept indoors to control lighting, temperature, and
       other environmental variables.

       (ii) Pens—

              (A)  Size. Pens should have a floor area of at least 500 square centimeters
              (cm ) per bird (approximately 75 square inches (in ) per bird) for northern
              bobwhite and  at least 1,000 cm  per bird  (approximately 150 in per bird)
              for mallards and should be at least  24 centimeters (cm) (approximately 9.5
              inches) high for northern bobwhite and 32 cm (approximately 12.5 inches)
                               Page  8 of 19

              high for mallard.   Appropriate sized pens  for passeriformes potentially
              may be different to accommodate bird size and social behaviors.

              (B)  Construction materials.  Tests should be conducted with birds being
              maintained in  commercial brooder  or  holding  pens  or pens of similar
              construction.  Pens should be  constructed of galvanized metal, stainless
              steel, or perfluorocarbon plastics.  Materials that  are toxic, may affect
              toxicity, or may sorb test substances should not be used.  Wire mesh
              should  be  used for floors and external walls.  Solid  sheeting should be
              used for common walls and ceilings. Wire mesh for floors  should be fine
              enough so  as to not interfere with the normal movement of birds  yet
              coarse enough to allow fecal material to fall through.

              (C)  Cleaning.

                     (1) Between tests, pens should be disassembled (if feasible) and
                     thoroughly  cleaned to  prevent disease  transmission  and  cross-
                     contamination.  Steam cleaning of cages is recommended.  Cages
                     may  be hosed,  brushed thoroughly, and  hosed  again,  as  an
                     alternative method. The use of detergents or bleach is acceptable,
                     but  other chemical disinfectants such as quaternary ammonium
                     compounds should not be used.  When disease vectors have to be
                     controlled, hot or cold  sterilization techniques are recommended,
                     as long as such techniques will not leave chemical residues on the
                     cages. For cold  sterilization, ethylene oxide is recommended.

                     (2) Depending upon the type of pens used,  pens may be cleaned
                     during  a test as  needed to  maintain good  animal husbandry;
                     however,  care should be taken to  minimize  disturbance  of  the

       (iii) Disposal.  After the test is terminated,  treated and  positive control birds
       should be sacrificed and disposed of properly. Negative control birds may be kept
       as breeding stock, but they should not be used in any other tests.

       (iv) Cleaning. All materials  that will come in contact with the test  organisms and
       test substance should be  cleaned before use.   Cleaning procedures should be
       appropriate to remove known or suspected contaminants.

(8) Environmental conditions.  Environmental conditions should be appropriate to  the
study species.  For mallards and northern bobwhite quail environmental conditions during
the test should be  maintained as follows (conditions should  be modified  as appropriate
for passerine species):

       (i)  Temperature.   Testing is done indoors to  control  lighting and other
       environmental variables.   Temperatures for adult birds should  be maintained at
       normal indoor temperatures,  preferably between  15 degrees Celsius  (°C) and 27
       °C (70 to 80 degrees Fahrenheit (°F)).
                                Page 9 of 19

       (ii) Humidity. Relative humidity is not as critical as some other variables, but the
       test room should be maintained at a relative humidity between 45 and 70%.

       (iii) Lighting and photoperiod. A photoperiod of 10 hours light and 14 hours
       dark is recommended in  order to prevent birds from coming into reproductive
       condition for upland and waterfowl species.  However, this may  potentially be
       different for passerine species and should be adjusted as appropriate to ensure
       birds  are not  entering  reproductive condition.    Lighting  may  be  either
       incandescent or fluorescent. Pens and lights should be positioned so that all pens
       will receive approximately equal illumination.

       (iv) Ventilation. It is recommended that ventilation be sufficient to supply  10 to
       15 air exchanges per hour.
(9) Observations—
       (i) Measurement of test substance in dosing media.  Analytical confirmation of
       the dosing media concentration at test initiation is performed as described in the
       OCSPP  850.2000 guideline using  analytical  methods  that are verified before
       beginning the test, to measure the amount of test substance in a sample.

       (ii) Contaminants in  feed.  Contaminated feed may compromise study results,
       therefore,  feed  should  be  analyzed  periodically  to identify  background
       contaminants such as  heavy metals (e.g., arsenic, cadmium, lead, mercury, and
       selenium) and persistent pesticides, especially chlorinated insecticides.  A broader
       pesticide screen to include other chemicals that are known to be acutely toxic to
       birds may be useful.

       (iii) Basal diet composition.  A nutrient analysis of the  basal  diet should be
       included in the test report.  The analysis should include percentages by weight of
       protein,  fat, fiber, ash, calcium, and phosphorus.  In  addition to  these analyzed
       components,  a  list  of expected  amounts  of vitamins,  minerals  or  other
       supplements should also be recorded.  Most commercial feed companies provide
       both the analysis and the list of supplements on the label.

       (iv) Environmental conditions—

             (A) Temperature.  Temperature should be monitored on a constant basis
             in at least one representative location.

             (B) Humidity.  Humidity should be monitored on a continual  basis in at
             least one representative location.

       (v) Measures of effect—

             (A) Monitoring of birds. Birds are monitored closely for the first  60 to
             120 minutes after dosing.  Any regurgitation should  be noted.  Additional
                              Page 10 of 19

                    observations of test birds are made a minimum of three times on the day of
                    dosing and at least  daily (where feasible, twice daily observations are
                    recommended) throughout the remainder of the test period.

                    (B) Mortality, intoxication, and other abnormal behavior. Throughout
                    the test period,  all signs  of intoxication, other abnormal behavior, and
                    mortality are identified, counted,  and recorded  by dosage level, by sex,
                    and by day.  Signs of intoxication are those behaviors apparently due to
                    the test substance and may include a wide array  of behaviors, such  as
                    labored respiration, leg  weakness, hemorrhage, convulsions, and ruffled
                    feathers.    Record  all  signs of  intoxication  and  any  other  abnormal
                    behavior,  such  as  excessive  aggression,  toe-picking,  etc.    Among
                    survivors, remission of signs of intoxication and cessation of  abnormal
                    behavior is identified and recorded by  dosage  level and  by day.   An
                    estimate of the number  of birds exhibiting such signs should be recorded
                    for each dosage level.

                    (C) Body weight.   Individual  body weights of birds are recorded for
                    control  and treated  birds at the  time of calculating the dosage to  be
                    administered  and weekly thereafter  until  the test is concluded.  An extra
                    weighing on  the third day after dosing may provide useful information,
                    especially on anorexia.  Body weights of birds a week prior to dosing are
                    recommended to provide valuable base-line data.

                    (D) Food consumption.  Measure and record food consumption at  least
                    weekly in each pen throughout  the test.   Valuable additional information
                    can be obtained by monitoring food consumption daily, especially for the
                    first few days following  dosing.

                    (E) Gross pathology. Gross pathology examinations are conducted on  all
                    birds  that  die,  as  well as a  sufficient number  of survivors  selected
                    randomly in all  test substance treatment  groups as well as at least three
                    control survivors in order to provide characterization of lesions.

(f) Treatment of results—

       (1) Descriptive summary statistics—

              (i) Environmental conditions. Calculate  descriptive statistics (mean, standard
              deviation,  coefficient   of variation,  minimum, maximum)  temperature  and

              (ii) Mortality.  Cumulative  number of dead for each  dosage  level and control
              group by observation day should be summarized in tabular form.  If birds are
              separated by sex, provide cumulative number of dead birds by sex for each dosage
              level and control group by observation day.

                                      Page 11 of 19

              (iii) Body weight.  Calculate the change in body weight for an individual bird
              between observation periods (see Equation 4) and calculate the total change in
              body weight between test initiation and test termination (Equation 4 where time j
              is test termination and time / is test initiation).  For the control and each test
              substance  dosage level, calculate and plot the mean body weight change and
              standard error by observation interval to assess effects on the pattern of weight
              change.  Calculate and plot for the control and each test  substance dosage level,
              the mean total body weight change and standard error. Determine the mean body
              weight change for males and females separately.

                                     dt_j = Wj - wt                          Equation 4
dj-j = difference or change in weight for an individual between observation time /' and 7;
Wj = weight of an individual at time /';
w, = weight of an individual at time/

              (iv) Food consumption.  Calculate and plot the mean food  consumption  by
              treatment level and observation period.

              (v) Appearance and behavior.  Number of birds with appearance and behavioral
              symptoms should be   summarized  in  tabular form by time of observation,
              treatment,  and sex (if applicable).  Tabulate among survivors, remission of signs
              of intoxication  and cessation  of abnormal behavior by dosage level and  by
              observation day.

              (vi) Gross pathology.  Types of observed pathologies, and the number of dead or
              examined  surviving birds with  these lesions  should be  summarized in tabular
              form by treatment and  sex.

       (2) Percent mortality. Calculate the cumulative percentage of dead birds at each test
       substance treatment level and in the controls at test termination. Test substance treatment
       data should be adjusted to account for any control mortality.

       (3) Limit test—

              (i) LDso value. At test termination, if no birds die at the limit dose, the acute oral
              LD50 is considered to be greater than the limit dose (i.e., LD50 > limit dose).  This
              is because the Binomial Theorem predicts that when 10 organisms are tested, the
              probability of seeing no mortality if the true LDso is at or below the limit dose is
              <0.001.  Conversely the probability of seeing one or more dead birds if the true
                   is at or below the limit dose is >0.999.
              (ii) Proportion of mortality ( p ).   Assuming mortality follows the binomial
              distribution,  an estimate of the true proportion of mortality (p ) in the laboratory
              test population as well as confidence bounds on that estimate (see Table A4 of the
              reference in paragraph (j)(3) of this guideline) can be obtained. For small sample
              sizes, the interval may  be large.  For example, for a limit test resulting in no
                                      Page 12 of 19

       mortality in 10 birds (p = 0), the 99% confidence interval on the estimate of p is
       (0.00, 0.41) and the 95% confidence  interval  is  (0.00, 0.31). Using the 95%
       confidence interval as  an example, the true (unknown) proportion of mortality
       will be covered by the calculated confidence interval in 95%  of repeated trials.
       For assessing risks,  the  confidence in  the  estimated  proportion impacted is
       considered in determining acute effects at environmental exposure doses.  If the
       uncertainty in p is high at the limit concentration, and the expected environmental
       exposure concentration is close to the limit concentration, risks to threatened and
       endangered species may not be able to be discounted.

       (iii) Multiple-dose definitive testing.

              (A) At test termination, if one  or more mortalities occur  among the  10
              birds at the limit concentration (which was  conducted at 2,000 mg/kg-bw
              or the maximum limit dose, whichever is greater), a definitive LD50 test
              should be conducted.   If frank  sublethal effect(s) are observed in one or
              more birds at the limit dose, despite an  absence  of mortality, then a full
              definitive test may be necessary. For pesticides, if frank sublethal effect(s)
              are observed in one or more birds and the  limit  dose tested was: 1) less
              than ten times the maximum expected EEC, then a  full  definitive study is
              necessary; or  2) was  at least ten  times the maximum EEC, but there is
              other evidence or data that indicate a risk to avian  species,  e.g., pesticide
              use incident data, then a full definitive test is necessary.

              (B) A multiple-dose  definitive LDso  test  may be waived  if,  at test
              termination: 1) the limit treatment group  and control group each contain a
              minimum of 10 birds  2) no birds died at the limit  dose; 2) and there are
              also no frank sublethal effects observed at the limit  dose; 3)  except for the
              number of dosage levels the test procedures and  duration are the same as
              in the definitive test; 4) the dosage level is confirmed by chemical analysis
              under test conditions  and  5) for  pesticides, the limit dose was 2,000
              mg/kg-bw  or  equivalent  to   the maximum  expected  environmental
              concentration  on food  items, whichever is higher.

(4) Multiple-dose definitive test—

       (i) Dose-response curve, slope, and LDso.  Statistical procedures  are employed
       to calculate the LDso (standard error  and 95% confidence limits).   If a dose-
       response curve model (e.g., probit) was fit to the data to determine the LD50, the
       model  parameters  (e.g.,  slope) and their uncertainty estimates (e.g., standard
       error) should be recorded.  A statistical test for goodness-of-fit (e.g.,  chi-square
       test) should  also  be  performed to  determine  how  well   the  data  fit the
       computational model  used.

       (ii) NOEL.  While calculation of a NOEL and  LOEL is usually not part of this
       test design,  reporting these  values is  useful when testing both  pesticide and
       industrial chemicals.

                               Page 13 of 19

              (iii) Statistical methods.  Statistical procedures for modeling quantal data are
              available  and should  be  used.   Additional  discussion  about  endpoints  and
              statistical procedures is found in the OCSPP 850.2000 guideline.

(g) Tabular summary of test conditions.   Table  2 lists the important conditions that should
prevail during the definitive test.  Except for the number of dose levels, Table 2 also lists the
important conditions that should prevail during a limit test.  Meeting these test conditions will
greatly increase the likelihood that the completed test will be acceptable or valid for the purposes
of this test.
Table 2. — Summary of Test Conditions for Avian Acute Oral Toxicity Test
Test duration (observation period)
Light quality
Pen size
Number of pens
Test species
Age of test organisms
Sex of test organisms
Number of birds per dose level
Dose levels
Administration of dose
Measures of Effect (Measurement Endpoint)
Minimum of 14 days
Ambient: incandescent or fluorescent
10 hours light: 14 hours dark
>500 cm2 (approximately 75 in2) per bird for northern
bobwhite and >1,000 cm2 (approximately 150 in2) per
bird for mallards. Pens for passerines should be
appropriate for the study species.
One or two per dose level. Dividing by sex is
recommended if using two pens
Northern bobwhite quail and mallard ducks are the
preferred upland game bird and waterfowl species,
respectively. Currently, no preferred passerine
species has been identified. For passerines,
protocols, which include the selected test species,
should be submitted prior to test initiation.
Young adults, not yet mated. Other age groups may
be tested to address a specific concern associated
with a chemical-specific pattern of use on a case-by-
case basis
Typically, both sexes should be tested. Just one sex
may be tested to address a specific concerns
associated with a chemical-specific pattern of use on
a case-by-case basis.
Minimum of 1 0 birds per dose, split evenly by sex if
both sexes are tested
Minimum of five for definitive LD50 test, plus a control
(for the limit test — the limit dose (2,000 mg/kg-bw or
higher) plus a control)
By gavage or capsule
Death (LD50), body weight, food consumption, and
other signs of clinical toxicity
(h) Test validity.  This test would be considered unacceptable or invalid if one or more of the
conditions in Table 3 occurred.  This list should not be misconstrued as limiting the reason(s)
that a test could be found unacceptable or invalid.  However, except for the conditions listed in
Table 3 and in the OCSPP 850.2000 guideline, it is unlikely that a study will be rejected when
there are slight variations from guideline environmental conditions and study design unless the
                                      Page 14 of 19

control organisms are significantly affected, the precision of the test is reduced, the power of a
test to detect differences is reduced,  and/or  significant biases are introduced in  defining the
magnitude of effect on measurement  endpoints  as compared to guideline conditions.  Before
departing significantly from this guideline, the investigator should contact the Agency to discuss
the reason for the departure and the effect the change(s) will have on test acceptability. In the test
report, all departures from the guideline should be identified, the reasons for the changes given,
and any resulting effects on test endpoints noted and discussed.

	Table 3.—Test Validity Elements for the Avian Acute Oral Toxicity Test	
1.  Birds were not randomly assigned to treatment and control pens.
2.  More than 10% of the control birds died during the test.
3.  A minimum often birds were not used for each dose level of the test substance and control.
4.  The test substance was not orally administered, via either capsule or gavage.
5.  In the definitive test  a minimum of five dose levels of the test substance,  plus an appropriate control,
were not tested.
(i) Reporting—

       (1) Background information.   Background information to be supplied in the report
       consists at a minimum of those background information items listed in paragraph (j)0) of
       the OCSPP 850.2000 guideline.

       (2) Guideline deviations.  Provide a statement of the guideline or protocol followed.
       Include a description of any deviations from the test guideline or any occurrences which
       may have influenced the results of the test, the reason for these changes, and any
       resulting effects on test endpoints noted and discussed.

       (3) Test substance.

              (i) Identification of the test substance: common name, IUPAC and CAS names,
              CAS number, structural formula, source, lot or batch number, chemical  state or
              form of the  test  substance, and  its purity (i.e. for pesticides,  the identity and
              concentration of  active ingredient(s)), radiolabeling if any,  location of label(s),
              and radiopurity.

              (ii) Storage conditions of the test chemical or test substance and  stability of the
              test chemical or test substance under storage conditions if stored prior to use.

              (iii) Methods of preparation of the test substance and the treatment doses used in
              the range-finding and definitive test, or limit test.

              (iv) If a  diluent is used to prepare stock or test substance provide: the name and
              source  of the diluent,  the nominal concentration(s) of the  test  substance in  the
              stock solution, and the diluent concentration(s) used in the treatments and diluent
              control.  Provide a description of the dosing volume of test substance plus diluent
              for all birds with respect to individual body weights.

              (v) Name of toxicant used for positive control (if applicable) and dosage levels.
                                       Page 15 of 19

(4) Test organisms.

       (i) Name of species tested (including scientific name).

       (ii) Information about the source:  type, name, breeding history, certification of
       disease status.

       (iii) Sex and reproductive history and condition at test initiation.  If sexually
       mature birds that have been mated are used, describe the process used to bring the
       birds completely out of production.

       (iv) Age (in weeks) of all birds at test initiation.

       (v) Individual body weights at the beginning of the test (typically determined at
       time of dosing, but may be taken, especially for capsules, within 24 hours prior to
       dosing) and weekly thereafter.

       (vi) Acclimation procedures and duration.

(5) Test  methods and  conditions.   Provide a description  of the test  system and
conditions used in the definitive or limit test, any preliminary range-finding tests,  and any
positive control tests.

       (i) Description of housing containers: including type, size, and material of pens.

       (ii) Description  of housing  environmental  conditions: temperature,  humidity,
       ventilation rate, photoperiod, and lighting source and intensity.

       (iii) Detailed description of basal  diet, including  source/type, percentages by
       weight of protein, fat,  fiber, ash,  calcium,  and phosphorus,  a  list  of expected
       amounts of vitamins, minerals or  other  supplements.   Most commercial  feed
       companies provide both the analysis and the list of supplements on the label.

       (iv) Describe the frequency and sample date(s)  for documenting the contaminant
       status  (heavy metals,   persistent or  chlorinated  pesticides)  of the  feed and
       tabulation of the results of the analysis.

       (v) Number of birds added to each pen at test initiation.

       (vi) The number of pens per test substance dose level and control.

       (vii) Methods of assigning birds to pens, and pens to dose levels and the control.

       (viii) Date of dosing of test animals and test observation duration.

       (ix) Methods and  frequency of environmental monitoring performed during the
       definitive or limit study for temperature and humidity.

                               Page 16 of 19

       (x)  Methods  and frequency of measuring test  substance to confirm  exposure

       (xi) For the definitive and limit test, all analytical procedures should be described.
       The accuracy of the method, method detection limit, and limit of quantification
       should be given (described in 850.2000 (b) Definitions).

       (xii) Methods and frequency of measuring number of dead birds, observing signs
       of intoxication, including regurgitation,  and other abnormal behavior, including
       time of onset, duration, severity,  and number affected at each dose level  and

       (xiii) Feed consumption per pen at least weekly or as often as measured, if more
       frequently than weekly, along with an estimate of wastage.

(6) Results.

       (i) Tabulation of test substance analytical results by treatment (provide raw data).

       (ii) Environmental monitoring  data results (temperature and humidity) in tabular
       form (provide raw data for measurements not  made on a continuous basis),  and
       descriptive statistics (mean, standard deviation,  minimum, and maximum).

       (iii) For preliminary range-finding tests, if conducted, tabulate the number  and
       percentage of birds that died in each test pen, treatment level and in the control at
       each observation period. Provide a description  and count of any other appearance
       or behavioral  effects at each treatment level  and in the control.  Tabulate the
       results of gross pathological examinations in dead birds and samples of surviving

       (iv) For a limit test, tabulation of the number  of dead birds in  each pen  at each
       observation time during the test for the limit dose and the control (provide the raw

       (v) For the definitive test, the number  of dead birds at each  observation time
       during the test tabulated by pen and treatment (provide the raw data).

       (vi) For the definitive test, tabulation of the treatment percent dead, adjusted for
       control mortality.

       (vii) For the limit and definitive test, tabulation  by pen, treatment, and observation
       time of abnormal appearance and behavioral signs of toxicity and recovery, if any
       (provide raw data).

       (viii) For the limit  and  definitive  test, tabulation of gross morphology of dead
       birds and samples of surviving birds at test termination by  pen and treatment
       (provide raw data).

                               Page 17  of 19

              (ix) Graphs of the dose-response data for percent mortality.

              (x) For a limit  test, provide conclusion about the LD50 being above  the limit
              concentration and the lack or presence of other signs of toxic effects at the limit

              (xi) For the definitive test, where sufficient data  exist to fit a model (e.g. probit)
              the slope  of the dose-response  curve and its standard error and 95% confidence
              limits and any goodness of fit results

              (xii) If determined for the definitive test, the NOEL for mortality.

              (xiii) Description of statistical  methods used to fit the dose-response model or
              determine point estimates and  the LDso (including software package),  and the
              basis for the choice of methods.  Provide results of any goodness-of-fit tests.

              (xiv)  Description  of  statistical  method(s)  used  for  NOEL  and  LOEL
              determination,  including  software package,  and  the  basis  for  the choice of

(j) References.   The following  references  should  be consulted for additional background
material on this test guideline.

       (1) American Society for Testing and  Materials. ASTM E 857-05el, Standard  Practice
       for Conducting Subacute Dietary Toxicity Tests with Avian Species, In Annual Book of
       ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Reapproved 2005.

       (2)  Committee on Birds of the Institute of Laboratory Animal  Resources,  National
       Research  Council,  1977.   Laboratory  Animal Management:  Wild Birds.  National
       Academy of Sciences, Washington, DC.

       (3) Conover, W.   1980.  Practical Nonparametric Statistics, 2nd Edition.  John  Wiley &
       Sons, Inc., New York, NY.  493 pp.

       (4) Dorrestein, G, 2003. Diagnostic approaches and management of diseases in captive
       passerines.  Seminars in Avian and Exotic Pet Medicine 12(1): 11-20.

       (5) Fletcher, J., J. Nellessen, and T.  Pfleeger.  1994.  Literature review and evaluation of
       the EPA food-chain (Kenaga) Nomogram, an instrument for estimating pesticide residues
       on plants.  Environmental Toxicology and Chemistry 13(9): 1383-1391.

       (6)  Grue,  C. and L. Franson,  1986.   Use of captive starlings to  determine effects of
       environmental contaminants on passerine reproduction:  pen characteristics and nestling
       food requirements. Bulletin of environmental contamination and toxicology 37(5):655-

       (7) Harding, C., 1999.  Husbandry and  care of passerine birds.  Poultry and avian biology
       reviews 10(2): 79-83.

                                      Page  18 of 19

(8) Hayre, M., 1995.  Guidelines for housing excruciatingly happy zebra finches.  Lab
Animal 24(6):43-44.

(9) Matheson, S., L. Asher, and M. Bateson, 2008.  Larger, enriched cages are associated
with  'optimistic'  response biases  in captive European starlings (Sturnus vulgaris).
Applied Animal Behaviour Science 109(2-4):374-3 83.

(10) Mineau, P., B. Collins, and A. Baril. 1996. On the use of scaling factors to improve
interspecies  extrapolation to acute  toxicity in  birds.   Regulatory  Toxicology and
Pharmacology 24:24-29.

(11) Nellessen, J. and J. Fletcher.  1992.  UTAB: A computer database on residues  of
xenobiotic  organic chemicals  and  heavy  metals  in  plants.  Journal  of Chemical
Information and Computer Sciences 32:144-148.

(12)  Tully, T.,  M.  Lawton, G  Dorrestein, 2000.  Avian Medicine.   Elsevier Health

(13)  U.S. Environmental Protection  Agency, 1982.  Pesticide Assessment Guidelines
Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Office of Pesticides
and Toxic Substances, Washington, D.C.  EPA-540/9-82-024, October 1982.

(14) U.S. Environmental Protection Agency,  1985. Hazard Evaluation Division Standard
Evaluation Procedure, Avian  Single-Dose  Oral LD50.  Office of Pesticides Programs,
Washington, DC, EPA-540/9-85-001,  June 1985.

(15) U.S. Environmental Protection Agency, 1994.  Pesticides Reregi strati on Rejection
Rate Analysis: Ecological Effects, Office of Prevention, Pesticides and Toxic Substances,
Washington, D.C. EPA 738-R-94-035.
                               Page 19 of 19