&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-024
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.2200:
Avian Dietary
Toxicity Test
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NOTICE
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.
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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
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(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
intoxication.
(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.
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(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
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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
Calcium
Phosphorus
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.
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(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
guideline.
(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
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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.
where:
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 =
v'=1
(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
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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.
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(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.
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(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.
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(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
humidity.
(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
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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
where:
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
where:
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.,
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> 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
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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
Temperature
Light quality
Photoperiod
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
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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.
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(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.
(v) Name of toxicant used for positive control (if applicable), dietary treatment
levels.
(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.
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(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
control.
(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).
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(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
concentration.
(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
method.
(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.
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(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.
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