United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.2200:
        Avian  Dietary
        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 850.2200: Avian dietary 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  797.2050  Avian Dietary Toxicity
       Test; OPP 71-2 Avian Dietary LC50 Test (Pesticide Assessment Guidelines Subdivision
       E); Avian Dietary LC50 Test Standard Evaluation Procedure; OECD 205, Avian Dietary
       Toxicity  Test;  and ASTM  E 857-05el,  Standard Practice  for Conducting Subacute
       Dietary Toxicity Tests with Avian Species.

(b) Purpose. This guideline is intended for use in developing data, specifically both a median
lethal concentration (LCso) and slope of the concentration-response, on the dietary toxicity to
young northern bob white  (Colinus virginianus) and mallard (Anas platyrhynchos) 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 specific guidance for the testing of northern bobwhite and
mallard, which are the Agency's preferred test species. The Agency will use these data to assess
acute hazard to  birds.   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 definitions also apply to this guideline:

       Exposure period is the 5-day period during which test birds are offered a diet containing
       the test substance.

       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.
       Post-exposure period is the portion  of the test that  begins with the test birds  being
       returned from a treated diet to the basal diet. This period  is typically 3 days in duration,
       but may be extended if birds  continue to die or demonstrate other toxic effects.

       Test periodic the combination of the exposure  period and the post-exposure period,  or
       the entire duration of the test.

(d) General considerations—

       (1) Summary of the test.  Birds are administered the test substance in their daily diet for
       five  consecutive days.   Birds are observed  regularly for  mortality and  any signs  of
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intoxication (e.g., abnormal behavior) during the exposure period and for a period of at
least 3  additional days until  the  test is  completed.  Food consumption is  estimated
throughout the test.  Body weights are determined prior to initiation of exposure and at
the end  of the  test period (at test termination).   The  mortality response pattern is
examined  and subjected  to the  appropriate statistical   analysis  to derive the LCso,
confidence limits, and slope of the  concentration-response  line. Sublethal effects are also
monitored, for example, gross appearance and behavior of the birds, body weights and
feed consumption. Histopathological and physiological changes should be monitored.

(2) General test guidance.  The  general guidance in the OCSPP  850.2000 guideline
applies to this guideline except as specifically noted herein.

(3) Range-finding test. Unless the approximate toxicity  of the test  substance is known
already, a range-finding test should  be conducted to help determine the test  substance
concentrations to be used in  the  definitive test.  Refer to paragraph (e)(4)(ii) of this
guideline for details on concentrations for definitive tests. Procedures for range-finding
tests may vary, but generally,  groups of a few birds are fed three to five widely-spaced
concentrations for 5 days.  A series of 5,  50, 500, and 5,000 parts per million (ppm) of
diet (or milligrams per kilogram of diet mg/kg-diet) is suggested.

       (i) If there is no mortality  at the  5,000 ppm level, and the test procedures and
       numbers  of birds  per concentration  level are the same as would be used  in a
       definitive test,  and also meet the  elements of  an  acceptable  limit test  (see
       paragraph (d)(5)  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 concentrations.

       (ii)  If a test substance is expected  to be of low toxicity, it may be useful to first
       conduct a limit test at  5,000  ppm as described under paragraph (d)(5) of this
       guideline.  If mortality occurs at this concentration 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 concentration levels.

(4) Definitive test.  The  goal  of  the definitive test is to determine the concentration-
response curve for avian mortality after dietary  exposure, and to establish the  subacute
median lethal concentration (LCso)  (and its 95 percent (95%) confidence limits, as well as
the slope of the concentration-response curve and its standard  error). The definitive test
consists of a minimum of five dietary  levels of the test substance,  plus appropriate
controls. The dietary 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.

(5) Limit test. For test substances expected to have relatively low toxicity, in place of a
definitive test a limit test may be conducted  with a single dietary level of 5,000 ppm test
substance or the maximum expected environmental residue  concentration, whichever is
higher,  plus a control group.   For  pesticides,  if the expected environmental residue
concentration exceeds 5,000  ppm the  test  should be  conducted at   a  higher level
equivalent  to the maximum expected environmental concentration (EEC) on food items
                                Page 2 of  19

       (see paragraph (e)(4)(ii)(B) of this guideline).  Based on the results of the limit test, the
       subacute dietary LCso may be reported as greater than the limit concentration (i.e., 5,000
       ppm) 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 treatment group; third except for the number of treatment levels
       the test  procedures  and duration are the same as in the definitive  test;  fourth the
       concentration level in the limit treatment diet is confirmed by  chemical analysis under
       test conditions; and  fifth for pesticides, the limit dose is 5,000 ppm or the maximum
       expected  environmental  residue concentration,  whichever  is  higher.    Signs  of
       intoxication, if any, should be  reported.  Conduct the full definitive  test  when any
       mortality is observed at the limit concentration. If sublethal effects are  suspected in the
       study then 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, then 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
       the test substance in the diet for use in testing.   The Agency should be contacted prior to
       testing with nanomaterials.
       (2) Test duration. The definitive and limit tests consist of 5 days of exposure to the test
       substance in the  diet (exposure period) followed  by at least  3 days of additional
       observation (post-exposure period) while the test birds are receiving an untreated diet.  If
       any test birds die during the second or third day of the post-exposure period, if toxic signs
       are evident  on the third day  of the  post-exposure period, or  if  the test substance  is
       expected to have delayed effects, the test period should be extended until there are two
       successive post-exposure  days  without  mortality  and  one  day without  signs  of

       (3) Test organism—

              (i) Species.  Data on both an upland game bird and 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
              game bird, and the mallard (Anasplatyrhynchos  (L.)) for  a waterfowl. In addition
              to these  species, which  are tested  at  a minimum, additional species, such as
              pigeon (Columba livid); Japanese quail  (Coturnix coturnix japonica); ring-necked
              pheasant (Phasianus colchicus);  and red-legged partridge  (Alectoris rufa\ may
              also be used as upland game birds.  The Agency will use these and other data to
              assess acute hazards and risks to birds.  Other sources  should be consulted for
              appropriate husbandry  standards  for use of these other species in this test.  For
              tests with species other than the northern bobwhite or mallard, it is important that
              they are responsive to the conditions of the test  and cannot  avoid exposure to the
              test substance through fasting.  See paragraph (e)(3)(iii)  of this guideline for
              additional guidance.
                                       Page 3 of 19

(ii) Source.
       (A) 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 hatch.  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 also be certified as disease-free or
       as bred from disease-free stocks.

       (B) If northern bobwhites are purchased, it  is preferable that they be
       obtained  as eggs which then are hatched and reared in the testing facility.
       During incubation, a temperature of 39 degrees Celsius (°C)  and relative
       humidity of 70% are recommended for northern bobwhite. It is feasible to
       purchase live  young northern bobwhite  chicks if they can  be  obtained
       locally, however, young northern bobwhite may suffer adverse effects if
       shipped by air or other  commercial  means.  Young mallard  ducklings
       normally can be shipped without undue adverse effects.

(iii) Age.

       (A) Young birds that are not too old to be able to avoid eating during the
       exposure phase of the test (i.e. 5 days). This is typically achieved if at the
       beginning of the exposure period mallards are 5  days old and northern
       bobwhites are 10 to 14 days old.  If additional test species are used, it is
       important that the test animals are not able to survive for five days without
       eating. This will insure that the animals cannot fast during the test, and
       that the test actually measures the toxicity of the test substance, rather than
       avoidance.  Prior to conducting any testing on alternate species, confirm
       through studies or a literature search that the test animals  are  not  old
       enough to survive without feeding for five days.

       (B) For a satisfactory test  all birds used in a test should be  the same age,
       plus or minus (±1) a day.  Due to the difficulty of sexing young birds, the
       sex of the test animals is not determined.

(iv) Acclimation.  Test birds should be acclimated to test facilities  and basal diet
for a  minimum  of 3  days  for  mallard  and 7  days  for  northern bobwhite.
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.  During the 3-day
period preceding testing, the health of the  populations should be monitored and
mortalities recorded.  Birds should not be used for a test if more than 5% of the
individuals in  the total test population die during this time.  Birds should not have
                         Page 4 of 19

       been selected in any way for genetic resistance to toxic substances or have been
       used in a previous test, either in a treatment or 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 as little as possible to conform to test procedures.

       (vii) Diet and feeding.

              (A) A standard commercial game bird (for northern bobwhite) or duck (for
              mallard) starter mash,  or the nutritional equivalent, should be used for diet
              preparation. Guidance for recommended nutritional values is provided in
              Table 1.

            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. For bobwhite only, an antibiotic demonstrated to fully depurate in
              72 hours may be added to the drinking water up to 72 hours before test
              exposure, if necessary, for birds up through 10 days  of age.  It may not be
              possible to obtain food that is completely free of pesticides,  heavy metals,
              and other contaminants; however, 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 hydrocarbons.

       (viii) Water.  Only clean  unmedicated water  should be  offered during the 96
       hours preceding the exposure period and during the  test  period.   Clean  water
       should be available ad libitum.  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.  The test substance is administered in the diet with
ad libitum feeding.

       (i) Preparation of diet treatments.
                                Page 5 of 19

       (A) The test substance is mixed into the diet in a manner that will result in
       even distribution of the test substance throughout the diet.  Diets  can be
       mixed by commercial, mechanical food mixers.  For many test substances,
       it is recommended that treated diets be mixed under a hood.  Mashes and
       test substances should be mixed freshly just prior to the beginning of the
       test.   Certain volatile or other test substances may require preparation of
       fresh diets at frequent intervals.  The Agency should be contacted prior to
       testing with  nanomaterials.   Analysis  of the  diet  for test  substance
       concentrations is conducted as described under  paragraph (e)(9)(i)  of this
       (B) If possible, test substance should be added to the diet without the use
       of a diluent. If a diluent is needed, the preferred diluent is distilled water,
       but water should  not be used as a diluent for  test substances  known to
       hydrolyze readily.  When a test substance is  not water soluble,  it may be
       dissolved in a reagent grade evaporative diluent  (e.g. acetone or methylene
       chloride) and  then mixed with the  test diet.    The diluent should be
       completely evaporated  prior to feeding.  Other acceptable diluents may be
       used;  these include table grade corn oil, propylene glycol, and gum arable
       (acacia).  If a diluent is used, it should not comprise more  than  2% by
       weight of the treated diet, and an equivalent  amount of diluent  should be
       added to control diets for untreated birds.

(ii) Treatment concentrations.

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

       (B) For a  limit  test, there is single  dietary level, plus  the appropriate
       control (see paragraph (d)(5) of this guideline). A limit dose of 5,000 ppm
       is  used unless environmental residues are expected to result in a  higher
       dietary concentration.  To calculate the dietary  limit concentration (mg
       a.i./kg-diet) for  spray  applications  of pesticides Equation  1  of  this
       guideline can be used  where a use is  limited to a single application per
       year and Equation 2 of this guideline can be used where there are multiple
       applications per year.   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
                         Page 6 of 19

              Biotransformation), a compilation of actual measured pesticide residue
              values  on plants  (see references  in  paragraphs  (j)(3) and (j)(4) of this
              guideline).   If there are  multiple  uses  this  study is  supporting for
              registration, the limit concentration for the study should be based on the
              one resulting in the highest dietary exposure.
       for a pesticide use with a single application per year:

  Dietary Limit Concentration (mga.i. I kg - diet} = (ApRate}(240)Equation 1

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

                                                        ' 0.693 T
 Dietary  Limit Concentration =
(ApRate\2W\ e\^We
Equation 2
ApRate = maximum single application rate (in Ib a.i./acre);
Halflife = the foliar halflife (default is 35 days);
Interval = the minimum application interval (in days);
/' = application event from 1 to n;
n = total  number of applications

(5) Controls.

       (i) Every test includes a negative control group.  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
       added to the diets of control birds.  If a vehicle is used in preparation of the test
       diets, the same vehicle should be added to the diets of control birds in the highest
       concentration used for test diets.

       (ii)  Controls serve as  a monitor of bird husbandry practices and 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 unacceptable if more than 10% of the negative control birds die
       during the test period.

(6) Number of test organisms and replicates.

       (i) The minimum number of birds for each dietary test substance level and the
       control is 10 young birds.   Birds  may be divided  into two replicates with a
       minimum of five birds per replicate.  Equal numbers of birds should be used for

                                Page 7 of 19

       each treatment level.  When a test substance is known or expected to result in high
       experimental variation, it may be appropriate or necessary to use additional birds.

       (ii) For a  satisfactory test, birds should be randomly assigned to treatment and
       control pens without respect to sex.  Randomization may be done either at the
       initiation of the acclimation  period or at the time when the birds are weighed at
       the beginning  of the  exposure period.  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 for housing young birds  should have a floor area of at least
              300 square  centimeters per bird  (cm2/bird) (approximately  50 square
              inches  per bird (in2/bird)) for northern bobwhite  quail and at least 600
              cm2/bird (approximately 100 in2/bird) for mallards.

              (B) Construction materials.  Tests should be conducted with birds being
              maintained in commercial brooder 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 young birds.

              (C) Pen placement  to prevent cross-contamination  of feed.  Where
              feasible, it is recommended that pens not be  stacked upon each other.  If
              pens are stacked, only  one test substance is  allowed in any single stack.
              Pens should be randomly arranged,  whether or not  in a stack, with respect
              to  dose  levels  and controls.   Pens, such  as   stacked, unmodified,
              commercial pens with external feeders,  that allow  food to be spilled  from
              one pen to a  lower pen,  should be avoided.  Any modifications that
              prevent cross contamination  of concentration levels are acceptable.  For
              example, commercially available, 30 cm (1 ft) long chick feeders may be
              placed  inside  the pens and be  covered with  1.27  cm (0.5  in)  mesh
              hardware cloth over the food, for northern bobwhite.  The  same feeders
              covered with approximately 2.5 cm (1  in)  mesh wire are appropriate for
              mallards.  For either species, external  feeders can be  covered with the
              appropriate size wire mesh and a solid  piece of metal extended from the
              bottom of the cage to a point exterior to the  feeder.  Spillage may occur,
              but the  added metal will prevent food from spilling  into another feeder.
                                Page 8 of 19

              (D)   Pen  placement  to  prevent  cross-contamination  of  volatile
              substances.  In order to avoid cross-contamination for test substances that
              volatilize or  otherwise forms aerosols or vapors in the air; no more than
              one test substance is tested in a room.

              (E) 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, take care to minimize disturbance of the birds.

       (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—

       (i)  Temperature.   Testing is done indoors  to  control temperature  and other
       environmental variables.   Pens  should  be heated,  preferably by thermostatic
       control. A temperature gradient in the pen of approximately 38 degrees Celcius
       (°C) to approximately 22 °C will allow young birds to seek a proper temperature.
       Temperature requirements for young birds typically decline over this range from
       birth through the first several weeks of life.

       (ii) Humidity.  Relative humidity is not as critical as some other variables, but
       ideal test conditions  include a relative humidity between 45 and 70%.

       (iii) Lighting and photoperiod. A photoperiod of 14 hours light and 10 hours
       dark provides sufficient light to  promote the feeding of the young birds  and  is
       recommended.  Continuous lighting is also acceptable.  Lighting may be either
       incandescent or fluorescent. Pens and lights should be positioned so that all pens
       will receive similar illumination.
                               Page 9 of 19

       (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 diet—

              (A)  Sampling.  Samples of treated diets are analyzed to confirm proper
              dietary concentration of the test  substance under test conditions using
              analytical methods that are validated before beginning the test as described
              in the OCSPP 850.2000 guideline.  Representative samples of test feed
              should be taken at a minimum from  feeders of the high, middle, and low
              test concentrations, at the beginning and end of the test.

              (B) Stability. If it is observed that the stability or homogeneity of the test
              substance in the diet cannot be maintained, care should be taken  in  the
              interpretation of the results of the study and note made that the results may
              not be reproducible.  For a satisfactory test, test substance concentration in
              the diet should be maintained at least at 80% of the nominal concentration
              throughout the exposure period (i.e., first 5 days of the test).

       (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 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.  Most commercial feed companies provide both  the
       analysis and the list of supplements on the basal feed label.  The analysis should
       include percent by weight of protein, fat, fiber, ash, calcium, and phosphorus.

       (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 constant basis in at
              least one representative location.

       (v) Measures of effect—

              (A) Monitoring of birds. Observation of test birds should be made, at a
              minimum,  3 times on the first day of the exposure period.   Observations
              also should be made at least daily throughout the remainder of the test
              period; twice daily observations are recommended, where feasible.
                               Page 10 of 19

                    (B) Mortality, intoxication, and other abnormal behavior. Throughout
                    the test period, all  signs of intoxication,  other abnormal behavior, and
                    mortality are identified, enumerated and recorded by treatment level 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,
                    etc.  Record all signs of intoxication and any other abnormal behavior,
                    such as excessive aggression,  toe-picking, etc.  that may or may not be
                    attributed to the test substance.  Among survivors, remission of signs of
                    intoxication and cessation of abnormal behavior is identified and recorded
                    by treatment level  and by day.  An  estimate  of the number of birds
                    exhibiting such  signs should be recorded for each treatment level.

                    (C) Body  weight.   Average  body weight  of  birds is determined and
                    recorded for each pen within  each  test substance treatment and control
                    group at the beginning and end of the exposure period and at the end of
                    the normal  3-day post-exposure period.  If the study is continued beyond 8
                    days,  body weight  data  should be  recorded at least weekly and at the
                    termination of the test.  Body weight data at 72 hours before the  exposure
                    period are not essential, but would provide valuable base-line data.

                    (D) Food consumption.  Measure and record food consumption daily in
                    control  pens and pens  with  the low,  middle,  and high test  substance
                    concentration levels for both the exposure period and  the normal 3-day
                    post-exposure period. For other pens, estimate food consumption for the
                    exposure period  and for the  post-exposure period.   If  the  study  is
                    continued beyond 8  days measure and  record food consumption weekly.
                    Estimate and record any significant amount of food spilled onto litter pans.

                    (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 test substance treatment
              level and control group by observation day should be summarized in tabular form.

              (iii)  Body weight.  For a given observation  time, calculate the average  body
              weight for a given pen using Equation 3.  Calculate the change in average body
              weight for each pen between observation periods (see Equation 4)  and  calculate
              the total  change  in average  body  weight between  test initiation  and test
                                     Page 11 of 19

       termination (Equation 4  where time j is test  termination and  time  /'  is test
       initiation).  Calculate and plot treatment mean body weight change and standard
       error by observation interval to assess effects on the pattern of weight change.
       Calculate and plot treatment mean total body weight change and standard error.

                                 wt = wt/n                             Equation 3

       wt = average weight of a bird in a given pen at observation time t;

       w t = total weight of birds in the pen at time t;

       n = total number of birds in the pen at time t.

                               di-j  = Wj -Wi                           Equation 4


       di-j = difference or change in average bird weight for a pen between observation
       time /' andy;

       Wi = average weight per bird in the pen at time /';

       Wj = average weight per bird in the pen 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 pen.  Tabulate among  survivors, remission of signs of intoxication
       and cessation of abnormal behavior by test substance  treatment level, pen,  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 test substance treatment level and pen.

(2)  Percent mortality.  Calculate the cumulative percent  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) LCso value.  At test termination, if no birds die at the limit concentration, the
       subacute dietary LCso is considered to be greater than the limit concentration (i.e.,

                               Page 12  of 19

            > dietary limit concentration).   This is because  the  Binomial theorem
       predicts that when 10 organisms are tested, the probability of seeing no mortality
       if the true LCso is at or below the limit concentration is <0.001.  Conversely the
       probability of seeing one or more dead birds if the true LCso is at or below the
       limit concentration is >0.999.

       (ii) Proportion of mortality (p ).  The Binomial Theorem can also provide both
       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)(2) of this guideline).   For small sample sizes the
       interval may be large.  For example, for no mortalities in 10  birds (p =0), the
       upper 99% confidence bound on the estimate of p  is (0.00, 0.41) and  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  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 5,000 ppm or the
              maximum  EEC,  whichever is greater), a definitive LCso  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; 3) there  are also
              no frank sublethal effect(s) at the limit dose; 4) the concentration level in
              the  limit treatment diet is confirmed  by  chemical analysis under  test
              conditions; 5)  except for  the  number of  treatment levels the  test
              procedures  and duration are the  same  as  in  the definitive test; 6) for
              pesticides, the limit dose was 5,000 ppm or equivalent to the maximum
              expected environmental concentration on food items, whichever is  higher.
(4) Multiple-dose definitive test—

       (i) Concentration-response curve, slope and LCso.   Statistical procedures are
                               Page 13 of 19

              employed to calculate the LCso (standard error and 95% confidence limits).  If a
              concentration-response curve model (e.g., probit) was fit to the data to determine
              the LCso, 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) NOEC.  While calculation of a NOEC and LOEC  is usually not part of this
              test design for acute tests, reporting these values is useful when testing pesticides
              and industrial chemicals.
              (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 treatment levels, Table 2 also lists the
important conditions that will 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 Subacute Dietary Toxicity Test
Test duration
Exposure period
Light quality
Pen size
Number of pens
Test species
Age of test organisms at test initiation
Sex of test organisms
Number of birds per concentration level
Concentration levels
Administration of test substance
Measures of Effect (Measurement Endpoint)
Minimum of 8 days
Five days
Gradient in a pen of 22 °C to 38 °C
Ambient: incandescent or fluorescent
14 hours light: 10 hours dark
>300 cm2 (approximately 50 in2) per bird for northern
bobwhite and >600 cm2 (approximately 100 in2) per
bird for mallards
One or two per dose level
Northern bobwhite and mallard are preferred
Northern bobwhite: 10-14 days
Mallard: 5 days
Typically both sexes should be tested; however it is not
practical to determine the sex of young birds
Minimum of 10, randomly assigned
Minimum of five, plus control for definitive test
(for limit test, the limit concentration (5,000 ppm or
higher) plus a control)
Through diet
Death (LC50), body weight, food consumption and
other signs of clinical toxicity
(h) Test validity elements. This test would be considered to be 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
                                      Page 14 of 19

when there are slight variations from guideline environmental conditions and study design unless
the 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, communication between the investigator and the
Agency is important in order 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 are identified,
reasons for these changes given, and any resulting effects on test endpoints noted and discussed.

       Table 3.—Test Validity Elements for Avian Subacute Dietary Toxicity Test
1.  Birds were not randomly assigned  to treatment and control pens.

2.  More than 10% of the control birds died or became moribund during the test.

3.  Concentrations of the test substance were not satisfactorily maintained in the diet (levels should be at
least 80% of the nominal concentration) throughout the exposure period (i.e., the first 5 days).

4.  Birds were not administered the test substance in their daily diet for 5 consecutive days.

5.  A minimum of 10 young birds were not used for each dietary concentration of the test substance.

6.  The test substance was not administered in the diet.

7.  In the definitive test a minimum of five dietary levels of the test substance, plus appropriate controls,
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,  stock solutions,  and  the
              treatment concentrations used in the range-finding and definitive test, or limit test.
                                       Page 15 of 19

       (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

       (v) Name of toxicant used for positive control (if applicable), dietary treatment

(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) Age of birds (in days) at the beginning of the test.

       (iv) Average body weight for each pen at the beginning of the test, at the end of
       the exposure period, and at the end of the test.

       (v) Acclimation procedures and conditions, including housing and environmental
       conditions (if different from test  conditions),  feeding  procedures,  and health
       observations (including mortality).

(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 (see 850.2000 (h) Reference toxicants) tests.

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

       (ii) Description of feed and water  dispensing apparatus.

       (iii) Description of placement of pens to eliminate cross-contamination, due either
       to food cross-contamination or volatility of test substance. Include a description
       of any modifications of food dispensers that prevent cross-contamination.

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

       (v) Detailed description of basal diet, including source/type, percent 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.

       (vi) 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.

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

                                Page 16 of 19

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

       (ix) Methods of assigning birds to test pens and test pens to treatment levels.

       (x)  Date of initial exposure of birds to the test substance, dates of exposure,
       duration of test, including length of exposure period (in days) and length of the
       post-exposure period on "clean" feed (in days).

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

       (xii) Methods and  frequency  of measuring test substance to confirm dietary
       exposure levels of the test substance and in control feed.

       (xiii)  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).

       (xiv) 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

       (xv) Estimated food consumption per pen daily in control pens and pens with the
       low, middle, and high test concentration levels. For other pens,  food consumption
       should be estimated for the exposure  period and for the post-exposure period.
       Indicate whether food was regurgitated or spilled and in which treatments.
(6) Results.
       (i) Tabulation of dietary test substance analytical  results by pen and treatment
       (provide raw data).  Include a statement about the stability and homogeneity of
       the test substance in the diet throughout the exposure period.

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

       (iv) For  a limit test, tabulation for the limit concentration treatment  and the
       control the number of dead birds in each pen at each observation time during the
       test (provide the raw data).
                               Page 17 of 19

              (v) For the definitive test, tabulation by test pen and treatment of the number of
              dead birds at each observation time during the test (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 test  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 by test pen, treatment gross
              morphology of dead birds and samples  of surviving birds at test termination
              (provide raw data).

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

              (x)  For a limit test,  provide conclusion  about the  LCso 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 method(s)  used for point estimates, including
              software  package, for determining the LCso value,  fitting the dose-response
              model, and the basis for the choice of method. 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)  Conover, W.  1980.  Practical Nonparametric Statistics, 2nd Edition. John Wiley &
       Sons, Inc., New York, NY. 493 pp.

       (3) 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.
                                      Page 18 of 19

(4) 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.

(5)  Organization  for  Economic  Cooperation  and   Development  (OECD),  1984.
Guidelines for testing of chemicals, Guideline 205, Avian Dietary Toxicity Test, Adopted
April 1984.

(6)  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.

(7) U.S. Environmental Protection Agency, 1985.  Hazard Evaluation Division Standard
Evaluation Procedure: Avian Dietary LC50 Test.  Office of Pesticides Programs, U.S.
Environmental Protection Agency, Washington, D.C. EPA-540/9-85-008, June 1985.
                              Page 19 of 19