United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
EPA 712-C-96-140
April 1996
&EPA Ecological Effects Test
OPPTS 850.2200
Avian Dietary Toxicity

Public Draft"

This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP, or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and

OPPTS 850.2200 Avian dietary toxicity test.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline 40 CFR 797.2050 Avian Dietary Toxicity
Test; OPP 71-2 Avian Dietary LC50 Test (Pesticide Assessment Guide-
lines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Orga-
nisms) EPA report 540/09-82-024, 1982; and OECD 205, Avian Dietary
Toxicity Test.
(b)	Purpose. This guideline designed to develop data on the dietary
toxicity to bobwhite and mallard of chemical substances and mixtures sub-
ject to acute environmental effects test regulations. This guideline gives
specific guidance for the testing of bobwhite and mallard, which are EPA's
preferred test species. However, other species, such as pigeon, Columba
livia, Japanese quail, Coturnix coturnix japonica, ring-necked pheasant,
Phasianus colchicus, and red-legged partridge, Alectoris rufa, are also ac-
ceptable. The Agency will use these and other data to assess the acute
hazard to birds and to provide an indication of potential chronic hazard
that these chemicals may present to the environment.
(c)	Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. In addition, the following definitions
apply to this guideline:
Acclimation is the physiological or behavioral adaptation of test ani-
mals to environmental conditions and basal diet associated with the test
Basal diet is the food or diet as it is prepared or received from the
supplier, without the addition of any carrier, diluent, or test substance.
Exposure period is the 5-day period during which test birds are of-
fered a diet containing the test substance.
Hatch is eggs or young birds that are the same age and that are de-
rived from the same adult breeding population, where the adults are of
the same strain and stock.
LC50 is the empirically derived concentration of the test substance
in the diet that is expected to result in mortality of 50 percent of a popu-
lation of birds which is exposed exclusively to the treated diet under the
conditions of the test.
Postexposure 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 period is the combination of the exposure period and the
postexposure period, or the entire duration of the test.
Test substance is the specific form of a chemical or mixture of chemi-
cals that is used to develop the data.
(d) Test procedures—(1) Summary of test, (i) Birds should be accli-
mated for at least 7 days after they have been obtained.
(ii)	Test birds should be randomly assigned to the various treatment
levels and controls.
(iii)	Definitive test concentrations should be established, possibly re-
quiring a range-finding test to be conducted first.
(iv)	The test substance should be mixed thoroughly and evenly into
the diet. Three treatment levels should be analyzed for test substance con-
(v)	Birds should be weighed at the beginning and the end of the expo-
sure period.
(vi)	Birds should be observed regularly for mortality or abnormal be-
havior and any findings should be reported.
(vii)	Food treated with the test substance should be replaced by un-
treated food (basal diet) after 5 days of exposure. Food consumption dur-
ing the exposure period should be carefully estimated on a pen by pen
(viii)	Food consumption should be estimated for the postexposure pe-
riod and birds should be weighed at the end of 8 days. Additional weights
and food consumption estimates should be determined if the test period
is longer than the typical 8 days.
(ix)	The mortality pattern should be examined, and a statistical analy-
sis should be conducted. The LC50, 95 percent confidence limits, and the
dose-response slope should be reported. A test for heterogeneity of data
should be conducted.
(x)	Treated or positive control birds should be sacrificed and disposed
of properly. Negative control birds may be kept as breeding stock, but
should not be used in any other tests.
(xi)	The material to be tested should the highest purity available (tech-
nical grade or analytically pure) and the degree of purity should be re-
ported along with the percentage of each impurity. If specifically required,
a particular substance or mixture should be tested.

(xii) A test is unacceptable if more than 10 percent of the control
birds die during the test.
(2)	Prerequisite data. These include water solubility, vapor pressure,
and the chemical stability of the test substance in water and light to ensure
uniform mixtures and the stability of test concentrations.
(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 test substance concentrations to be used in the definitive
test. (Refer to paragraph (d)(4)(iii) of this guideline for details on con-
centrations 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 concentration series of 5, 50, 500,
and 5,000 ppm is suggested. The results of the range-finding test then
may be used to establish the definitive test concentrations.
(4)	Definitive test—(i) Controls. (A) A concurrent control is required
during every test. The control birds should be from the same hatch as
the test groups. Control and test birds should be kept under the same exper-
imental 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 carrier is used in preparation of the test diets,
the same carrier should be added to the diets of control birds in the highest
concentration used for test diets. The use of shared controls is acceptable
for concurrent tests as long as the same carrier is used for all the tests.
(B)	Test acceptability criteria are as follows:
(7) A test is not acceptable if more than 10 percent of the control
birds die during the test period.
(2)	There must be evidence that the concentration of the substance
being tested has been satisfactorily maintained in the diet (it should be
at least 80 percent of the nominal concentration) throughout the first 5
days of the test period.
(3)	The lowest treatment level should not result in compound-related
mortality or other observable effects.
(C)	A positive control (e.g. dieldrin standard) may be run, but is not
required for each test. However, a quarterly or semiannual laboratory
standard (positive control) is recommended as a means of detecting pos-
sible interlaboratory or temporal variation. A laboratory standard is also
recommended when there is any significant change in food, housing, or
source of birds.
(ii) Number of animals tested. In the definitive test, a minimum
of 10 birds should be used for each dietary concentration of the test sub-
stance. A minimum of 20 birds should be used for the negative or carrier

control; 30 or more control birds are preferable. If a positive control or
laboratory standard is used, 10 or more birds should also be used for each
concentration of the positive control. When a test substance is known or
expected to result in high experimental variation, it may be appropriate
or required to use additional birds.
(iii)	Concentrations and dosage-mortality data. A minimum of five
concentrations of the test substance should be used in the definitive test.
These concentrations should be spaced geometrically. The recommended
spacing is for each concentration to be at least 60 percent of the next
higher dose (less than 1.67 times the next lower dose). If concentrations
are spaced more widely than is recommended, then at least three con-
centrations should result in mortality between, but not including, 0 percent
and 100 percent. For any concentration spacing, at least one concentration
should kill more than 50 percent (including 100 percent) and at least one
concentration should kill less than 50 percent (including 0 percent) of the
birds in a pen. For some test substances, it may be necessary to use more
than five concentrations to achieve these results. The lowest test concentra-
tion should not result in test substance-related mortality or other observable
(iv)	Duration of test. The definitive test should include 5 days of
exposure to the test substance in the diet (exposure period) followed by
at least 3 days of additional observation (postexposure period) while the
test birds are receiving an untreated diet. If any test birds die during the
second or third day of the postexposure period or if toxic signs are evident
on the third day of the postexposure period, the test period should be ex-
tended until 2 successive mortality-free days and 1 day free of toxic signs
occur, or until 21 days after beginning the test, whichever comes first.
(v)	Observations of record. (A) Throughout the test period, all signs
of intoxication, other abnormal behavior, and mortality should be recorded
and reported by dose level and by day. Signs of intoxication are those
behaviors apparently due to the test chemical and may include a wide
array of behaviors, such as labored respiration, leg weakness, hemorrhage,
convulsions, ruffled feathers, etc. 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 should be reported.
Among survivors, remission of signs of intoxication and cessation of ab-
normal behavior should be recorded by dose level and by day. When dif-
ferential signs of intoxication are observed within a dose level, an estimate
of the number of birds exhibiting such signs should be recorded. Observa-
tion 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.

(B)	Average body weights of birds should be recorded and reported
for each pen within each treatment and control group at the beginning
of the exposure period and the end of the normal 3-day postexposure pe-
riod of each test. Body weights 72 h before the exposure period are not
required, but would provide valuable base-line data. Average food con-
sumption should be measured in control pens and pens with the second
lowest and second highest concentration levels either daily or every other
day. Any significant amount of food spilled onto litter pans should be
estimated and reported. For all other pens, average food consumption
should be measured for both the exposure period and the normal 3-day
postexposure period. If the study is continued beyond 8 days, body weight
and food consumption data should be recorded weekly.
(C)	Gross pathology examinations are not required, but they may pro-
vide valuable information on target site, mode of action, etc.
(5) Analytical measurements—(i) Statistical analysis. (A) A statis-
tical analysis should be conducted by transforming the dietary concentra-
tions to logarithmic values and the mortality pattern to probits. Other ac-
ceptable methods that will result in a theoretically straight line through
±2 standard deviations from the LC50 value may be used. The LC50 value
and slope of the transformed concentration-response curve should be deter-
mined for mortality at the end of test period. Probit analysis by calcula-
tions or graphical probit methods are preferred. Any standard method that
is used should provide the slope of the transformed concentration-response
curve as well as the LC50 value. A statistical test for goodness-of-fit (e.g.
X2 test) also should be performed. When mortality at the level of
5,000 ppm, the highest recommended treatment level, is less than 50 per-
cent and the LC50 cannot be calculated, the LC50 should be reported as
greater than 5,000 ppm, and the no-effect level reported as well.
(B) All methods used for statistical analysis should be described com-
(ii) Analysis for test substance concentrations. (A) Samples of
treated diets should be analyzed to confirm proper dietary concentration
of the test substance. Analyses should be conducted at the beginning of
the exposure period with samples from high, middle, and low concentra-
tions. If not already available, data should be generated to indicate whether
or not the test substance degrades or volatilizes. If the test substance is
known or found to be volatile or labile to the extent that 25 percent or
more loss occurs over a 5-day period, then a second series of analyses
of the same concentrations previously analyzed should be conducted at
the end of the exposure period.
(B) The assay method used to determine actual concentrations should
be reported.

(C) If it is observed that the stability or homogeneity of the test sub-
stance in the diet cannot be maintained, care should be taken in the inter-
pretation of the results and note made that the results may not be reproduc-
(iii) Analysis of basal diet. A nutrient analysis of the basal diet
should be included in the test report. For commercially prepared basal
diets, the list of ingredients supplied by the company is normally sufficient
if it is detailed. The composition of any vitamin or other supplements
should also be reported.
(e) Test conditions—(1) Test species—(i) Selection. (A) An upland
bird, bobwhite quail, Colinus virginianus (L.), and a waterfowl, mallard
duck, Anas platyrhynchos L., are the preferred test species. Birds may be
reared in the laboratory or purchased from a breeder. If bobwhite are pur-
chased, 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 °C and relative humidity of 70 percent are recommended for bob-
white. It is feasible to purchase live young bobwhite chicks if they can
be obtained locally, however, young bobwhite may suffer adverse effects
if shipped by air or other commercial means. Young mallard ducklings
normally can be shipped without undue adverse effects.
(B)	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. Birds should be
phenotypically indistinguishable (except for size) from wild stock. It is
recommended that birds be obtained from flocks that have been outbred
periodically with genetically wild stock in order to maintain a genetic com-
position that approximates the natural heterogeneity of the species.
(C)	Birds used in the test should be in apparent good health. De-
formed, abnormal, sick, or injured birds should not be used. During the
72-h 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 percent of the total test population die during the 72 h
immediately preceding the exposure period. Purchased birds should be cer-
tified as disease-free or as bred from disease-free stocks. 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 control group.
(D)	Young birds should be tested to ensure that test birds must feed
during the exposure phase of the test. Mallards should be 5 to 10 days
old and bobwhite should be 10 to 14 days old at the beginning of the
exposure period. All treatment and control birds in a test should be the
same age ± 1 day. The exact age should be recorded and reported.

(E)	Test birds should be acclimated to test facilities and basal diet
for a minimum of 7 days. Acclimation to test pens may be either in the
actual pens used in the test or in identical pens. Birds used in the test
should be assigned randomly to treatment and control pens without respect
to sex. Randomization may be done either at the initiation of the acclima-
tion period or at the time when the birds are weighed at the beginning
of the exposure period.
(F)	Birds should be shielded from excessive noise, activity, or other
disturbance during holding, acclimation, and testing. Birds should be han-
dled only as much as is necessary to conform to test procedures.
(ii) Diet. (A) A standard commercial game bird (for bobwhite) or
duck (for mallard) starter mash, or the nutritional equivalent, should be
used for diet preparation. Antibiotics or other medication should not be
used in the diet before or during the test. For bobwhite only, an antibiotic
demonstrated to fully depurate in 72 h may be added to the drinking water,
if necessary, for birds up through 10 days of age. Only clean unmedicated
water should be offered during the 96 h preceding the exposure period
and during the test period. It may not be possible to obtain food that is
completely free of pesticides, heavy metals, and other contaminants. Diets
should be analyzed periodically, and should be selected to be as free from
contaminants as possible. A nutrient analysis and list of the ingredients
in the diet should be included with the test report.
(B)	The test substance should be mixed into the diet in a manner
that will result in even distribution of the test substance throughout the
diet. If possible, the 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 methyl-
ene chloride) and then mixed with the test diet. The diluent should be
completely evaporated prior to feeding. Other acceptable diluents may be
used, if necessary, and include table grade corn oil, propylene glycol, and
gum arabic (acacia). If a diluent is used, it should not comprise more than
2 percent by weight of the treated diet, and an equivalent amount of diluent
should be added to control diets for untreated birds.
(C)	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. Analysis of the diet
for test substance concentrations is required under paragraph (d)(5)(ii) of
this guideline.

(D) Clean water should be available ad libitum. Water bottles or auto-
matic watering devices are recommended. If water pans or bowls are used,
water should be changed at least once a day.
(2) Facilities, (i) Tests should be conducted with birds being main-
tained 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 adsorb 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. Pens for housing
young birds should have a floor area of at least 300 cm2/bird (approxi-
mately 50 in2/bird) for bobwhite quail and at least 600 cm2/bird (approxi-
mately 100 in2/bird) for mallards. Pens should be disassembled (if feasible)
and should be cleaned thoroughly between tests. Steam cleaning of cages
is recommended. Cages may be brushed thoroughly, 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 necessary to control disease vectors, 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. Pens should not be cleaned during a test.
(ii)	Pens should be kept indoors to control lighting, temperature, and
other environmental variables. Pens should be heated, preferably by
thermostatic control. A temperature gradient in the pen of approximately
38 °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. Relative
humidity is not as critical, but the test room should be maintained at a
relative humidity of 45-70 percent. A photoperiod of 14 h light and
10 h dark is recommended. Other light/dark cycles should not be used,
but continuous lighting is acceptable. Lighting may be either incandescent
or fluorescent. Pens and lights should be positioned so that all pens will
receive similar illumination. The facilities should be well ventilated.
(iii)	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. If a test substance volatilizes or otherwise forms aerosols or
vapors in the air, no more than one test substance should be tested in
a room in order to avoid cross-contamination. Pens should be randomly
arranged, whether or not in a stack, with respect to dose levels and con-
trols. 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 con-
centration 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
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.
(f) Reporting. (1) The test report should include the following infor-
(i)	Name of test, sponsor, test laboratory and location, principal
investigator(s), and actual dates of beginning and end of test.
(ii)	Name of species tested (including scientific name), age of birds
(in days) at the beginning of the test, average body weights for birds in
each pen at the beginning of the test, the end of the exposure period and
end of the test, and individual weights of all birds that die during the
(iii)	Description of housing conditions, including type, size, and mate-
rial of pen, pen temperatures, approximate test room humidity,
photoperiod, and lighting intensity.
(iv)	Detailed description of the basal diet, including source, diluents
(if used), and supplements (if used). A nutrient analysis of the diet should
be included in the test report.
(v)	Detailed description of the test substance including its chemical
name(s), source, lot number, composition (identity of major ingredients
and impurities), and known physical and chemical properties that are perti-
nent to the test (e.g. physical state, solubility, etc.).
(vi)	The number of concentrations used, nominal and (where required)
measured dietary concentration of test substance in each level, assay meth-
od used to determine actual concentrations, number of birds per concentra-
tion and for controls, and names of toxicants used for positive controls
(if applicable).
(vii)	Acclimation procedures and methods of assigning birds to test
(viii)	Frequency, duration, and methods of observation.
(ix)	Description of signs of intoxication and other abnormal behavior,
including time of onset, duration, severity (including death), and numbers
affected in the different dietary concentrations and controls each day of
the test period.

(x)	Estimated food consumption per pen daily or every other day in
the second highest and second lowest concentration and control pens. For
other pens, food consumption should be estimated for the exposure period
and for the postexposure period.
(xi)	Location of raw data storage.
(xii)	Results of range finding tests (if conducted).
(xiii)	The calculated LC50 value, 95 percent confidence limits, slope
of the concentration-response curve, the results of the goodness-of-fit test
(e.g. X2 test), and a description of statistical methods used. The same sta-
tistics are to be provided for positive controls (when used). The methods
used for statistical analysis should be described completely.
(xiv)	Anything unusual about the test, any deviation from these proce-
dures, and any other relevant information.
(2) In addition to the above information required in every report, the
following information should be available upon request:
(i)	A general description of the support facilities.
(ii)	A description of the quality control/quality assurance program,
including the average quality level for the program element performing
the test, procedures used, and documentations that these levels have been
(iii)	The names, qualifications, and experience of personnel working
in the program element performing the test, including the study director,
principal investigator, quality assurance officer, as well as other personnel
involved in the study.
(iv)	Standard operating procedures for all phases of the test and equip-
ment involved in the test.
(v)	Sources of all supplies and equipment involved in the test.
(vi)	Originals or exact copies of all raw data generated in performing
the test.