United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-025

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.2100:

Avian Acute Oral

Toxicity Test
i

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.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 19

OCSPP 850.2100: Avian acute oral toxicity test.

(a) Scope-

(1) Applicability. This guideline is intended to be used to help develop data to submit to 

EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and 

the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

(2) Background. The source materials used in developing this harmonized OCSPP test 

guideline include the OPPT guideline under 40 CFR 797.2175 Avian Acute Oral Toxicity 

Test; the OPP 71-1 Avian Single-Dose Oral LD50 Test (Pesticide Assessment Guidelines 

Subdivision E); the Avian Single-Dose Oral LD50 Standard Evaluation Procedure; and 

OPP Pesticides Reregistration Rejection Rate Analysis: Ecological Effects.

(b) Purpose. This guideline is designed to develop data, specifically both a median lethal dose 

(LD50) and slope of the dose-response relationship, for acute oral toxicity to upland game birds 

(e.g., northern bobwhite (Colinus virginianus)), water fowl (e.g., mallard duck (Anas 

platyrhynchos)), or passerine species (e.g., house sparrow (Passer domesticus), zebra finch 

(Taeniopygia guttata), red-wing blackbird (Agelaius phoeniceus)) of chemical substances and 

mixtures (“test chemicals” or “test substances”) subject to environmental effects test regulations. 

While the study is specifically designed to allow calculation of the LC50, the study can be used to 

obtain information regarding sublethal effects which are used in Agency evaluations. This 

guideline prescribes methodologies to determine both the LD50 and slope of the dose-response in 

the same study. The Agency can consider data generated that provides a LD50 value but no slope 

information but such a study would be insufficient to meet the purpose of this guideline unless it 

is a limit test (see paragraph (d)(3) of this guideline). The use of a test based primarily on 

lethality is justified because it presents or insures a consistent, unbiased endpoint for assessment 

purposes and has unambiguous ecological relevance to adverse effects.

(c) Definitions. The definitions in the OCSPP 850.2000 guideline apply to this test guideline. 

In addition, the following more specific definition also applies to this guideline:

Observation period is the portion of the test that begins after the test birds have been dosed and 

extends at least 14 days, but to continue until overt evidence of toxicity has subsided.

Frank sublethal effects for the purpose of this study refers to overt or frank toxicological effects 

for birds and include, but are not limited to, decreased body weight, loss of coordination, or 

lethargy. Less significant sublethal effects such as ruffled appearance or muted color are not 

considered frank toxicological effects.

(d) General considerations―

1) Summary of the test. Birds are administered the test substance as a single oral dose, 

either by capsule or gavage. Birds are observed regularly for mortality or any signs of 

intoxication throughout the observation period. Birds are weighed and feed consumption 

is estimated at least weekly thereafter. The mortality response pattern is examined and 

subjected to the appropriate statistical analysis to derive the LD50, confidence limits, and 
Page 2 of 19

slope of the dose-response line. Sublethal effects should also be monitored, for example, 

gross appearance and behavior of the birds, weight changes, and changes in food 

consumption. Histopathological and physiological changes should be monitored. 

Moreover, delayed mortality and differences in sensitivities of each sex should be 

assessed.

(2) General test guidance. 

(i) The general guidance in OCSPP 850.2000 applies to this guideline except as 

specifically noted herein. Studies should not be conducted with endangered or 

threatened species.

(ii) Successful adherence to elements in paragraph (h) of this guideline when 

testing passerine species may depend upon special provisions regarding 

husbandry, diet, acclimation of wild-caught birds, handling and age at dosing. 

For tests conducted with passerine species it is recommended, prior to test 

initiation, to submit protocols to the regulatory Agency for approval that identify

husbandry, dietary, holding and acclimation methods, age at dosing and any other 

special provisions planned (see in particular references in paragraphs (j)(2) and 

(j)(4) through (j)(9) of this guideline). To be complete, the protocol should also 

contain information on all of the items discussed in this guideline.

(3) Range-finding test. Unless the approximate toxicity of the test substance is known 

already, a range-finding test should be conducted to determine the dosage levels of the 

test substance to be used in the definitive test. Refer to paragraph (e)(4)(ii) of this 

guideline for details on dosage levels for definitive tests. Procedures for range-finding 

tests may vary, but generally, groups of a few birds are administered three to five widelyspaced doses. A series of 2, 20, 200, and 2,000 milligrams per kilogram of body weight 

(mg/kg-bw) is suggested.

(i) If there is no mortality at the 2,000 mg/kg-bw dose level, and the test 

procedures and numbers of birds per dosage are the same as would be used in a 

definitive test, and also meet the elements of an acceptable limit test (see 

paragraph (d)(4) of this guideline), then the range-finding test may provide 

sufficient information to negate the need for a definitive test. If mortality does 

occur, than the results of the range-finding tests may then be used to help 

establish the definitive test dose levels.

(ii) If a test substance is expected to be of low toxicity, it may be useful to first 

conduct a limit test at 2,000 mg/kg-bw as described under paragraph (d)(5) of this 

guideline. If mortality occurs at this dose level, then further range-finding at 

lower levels is suggested. The results of the range-finding test then may be used 

to establish the definitive test dosage levels.

(4) Definitive test. The goal of the definitive test is to determine a dose-response curve 

for avian mortality after oral dosing and an observation period of at least 14 days to 

establish the acute LD50 (standard error and 95 percent (95%) confidence limits), as well 

as the slope of the dose-response curve (and its standard error and 95% confidence 
Page 3 of 19

limits). The definitive test consists of a minimum of five dose levels of the test 

substance, plus appropriate controls. The dosage levels are confirmed by chemical 

analysis under test conditions. A summary of test conditions is provided in Table 2 and 

validity elements for an acceptable definitive test are listed in Table 3. The Agency

should be contacted prior to testing with nanomaterials.

(5) Limit test. For test substances expected to have relatively low toxicity, a limit test 

may be conducted with a single dose level at 2,000 mg/kg-bw or the maximum expected 

environmental residue concentration, whichever is higher, plus a control group. For 

pesticides, if the expected environmental residue concentration exceeds 2,000 mg/kg-bw 

the test should be conducted at a higher level equivalent to the maximum expected 

environmental concentration (EEC) on food items (see paragraph (e)(4)(ii)(B) of this 

guideline). Based on the results of the limit test, the acute oral LD50 may be reported as 

greater than the limit dose provided that the following conditions are met: first the limit 

treatment group and control group each contain a minimum of 10 birds; second no 

mortality or frank sublethal effects occurs in the limit dose group; third except for the 

number of dosage levels the test procedures and duration are the same as in the definitive 

test; fourth the dosage level is confirmed by chemical analysis under test conditions; and 

fifth, for pesticides, the limit dose is 2,000 mg/kg-bw or the maximum expected 

environmental residue concentration, whichever is higher. Clinical signs of toxicity, if 

any, should be reported. Conduct the full definitive test when any mortality is observed 

at the limit dose. If sublethal effects are suspected in the study than the Agency should 

be consulted for discussion on the appropriate dose and conduct of the limit test. 

(e) Test standards―

(1) Test substance. The substance to be tested should be technical grade unless the test 

is designed to test a specific formulation, mixture, or end-use product. For pesticides, if 

more than one active ingredient constitutes a technical product the technical grade of 

each active ingredient should be tested separately, in addition to the combination, if 

applicable. The OCSPP 850.2000 guideline lists the type of information that should be 

known about the test substance before testing and discusses methods for preparation of 

test substances.

(2) Test duration. The definitive and limit tests consist of the administration of the test 

substance followed by an observation period of at least 14 days. If mortality occurs 

during the last 3 days of the 14-day period, or if signs of intoxication are not clearly in 

remission, or if the test substance is expected to have delayed effects, then extend the 

observation period until mortality or signs of intoxication are not observed for 72 hours.

(3) Test organism―

(i) Species. Data on both a passerine species and either an upland game bird or a 

waterfowl are generally required for 40 CFR Part 158. These test protocols and 

standards describe tests specific to using the northern bobwhite (Colinus 

virginianus (L.)), for an upland bird, the mallard (Anas platyrhynchos L.) for a 

waterfowl. At this time, there is no identified preferred passerine species; 

examples of potential passerine species that have been used in acute oral testing
Page 4 of 19

include the house sparrow (Passer domesticus), zebra finch (Taeniopygia guttata)

and red-wing blackbird (Agelaius phoeniceus). Regardless of the passerine 

species chosen, a protocol should be submitted to the Agency prior to test 

initiation for review. In addition to these species, pigeon (Columba livia), 

Japanese quail (Coturnix coturnix japonica), ring-necked pheasant (Phasianus 

colchicus), and red-legged partridge (Alectoris rufa), have also be used as upland 

game birds. The Agency will use these and other data to assess acute hazards and 

risks to birds. Appropriate husbandry standards for species used in this test 

should be consulted, examples include references in paragraphs (j)(1), (j)(2) and 

(j)(4) through (j)(9) of this guideline.

(ii) Source. Birds may be reared in the laboratory or purchased from a breeder. 

For a satisfactory test, all control and treatment birds used in a test should be from 

the same source and breeding population and be in the same plumage. Birds 

should be obtained only from sources whose colonies have known breeding 

histories. Test birds should be phenotypically indistinguishable (except for size) 

from wild stock. It is recommended that birds be obtained from flocks that have 

been out bred periodically with genetically wild stock in order to maintain a 

genetic composition that approximates the natural heterogeneity of the species. 

Birds purchased from a breeder should be certified as disease-free or as bred from 

disease-free stocks. The Agency recommends against the use of wild-caught 

birds. However, where suitable cultured supplies of passerine species may not be 

available, protocols submitted in advance should detail why wild stock must be 

used and have the necessary elements identified in this guideline addressed (see 

paragraph (d)(2)(ii) of this guideline).

(iii) Age, sex, and size. 

(A) Typically, test birds should be young adults of both sexes, not yet 

mated, and are at least 16 weeks old at the time of dosing. A less 

preferred alternative is for the use of first-year birds that may have been 

mated, as long as the birds are brought completely out of production 

through reduced light cycles. In addition, there may be situations where in 

order to address concerns associated with a chemical-specific pattern of 

use younger or older birds (e.g., altricial nestlings), or birds of only one 

sex (e.g., breeding females), are more appropriate for testing. The need 

for this testing should be determined on a case-by-case basis after the 

basic testing is completed.

(B) For a satisfactory test, all upland and waterfowl birds used in a test 

should be the same age, plus or minus (±) 1 week. Currently, due to 

culturing and/or husbandry practices of Passeriformes, passerine birds 

used in a test may not be ± 1 week; however this may change if husbandry 

practices evolve. More consistent responses may be attained if the range 

of body weights is no greater than ±10% of the mean body weight for the 

test population.
Page 5 of 19

(iv) Acclimation. Test birds should be acclimated to test facilities and basal diet 

for a minimum of 14 days. Acclimation to test pens may be either in the actual 

pens used in the test or in identical pens.

(v) Health status. Birds used in the test should be in apparent good health. 

Deformed, abnormal, sick, or injured birds should not be used. Birds should not 

be used for a test if total mortality during the 14-day acclimation period is more 

than 5% for cage-reared birds or more than 10% for wild-caught birds. Birds 

should not have been selected in any way for genetic resistance to toxic 

substances. Birds should not have been used in a previous test, either in a 

treatment or a control group.

(vi) Care and handling. During holding, acclimation, and testing, birds should 

be shielded from excessive noise, activity, or other disturbance. Test birds should 

be handled only as much as is necessary to conform to test procedures.

(vii) Diet and feeding. 

(A) A standard commercial game bird (for northern bobwhite) or duck (for 

mallard) feed, or the nutritional equivalent, should be used as the diet. 

Guidance for recommended nutritional values for these species is provided 

in Table 1. Passerine species may require more complex diets. Feed 

should be withheld from all test groups for a minimum of 15 hours prior to 

administration of the test substance but feed should be available ad libitum

during the study. This fasting period should be decreased, as appropriate, 

for the selected passerine species.

Table 1.—Recommended nutritional values for feed

Nutritional Component Recommended Range (%)

Crude protein 27 to 29

Crude fiber 3.5 to 5.0

Crude fat 2.5 to 7.0

Calcium 2.6 to 3.6

Phosphorus 0.9 to 1.1

(B) Feed should not be used past its normal shelf life. Antibiotics or other 

medication should not be used in the diet during the acclimation period or 

the test. It may not be possible to obtain feed that is completely free of 

pesticides, heavy metals, and other contaminants. Therefore, diets should 

be analyzed periodically, as described under paragraph (e)(9)(ii) of this 

guideline, and selected to be as free from contaminants as possible. Extra 

precautions should be taken when fish meal or oil is a major ingredient, 

since fish are often contaminated with high levels of chlorinated 

hydrocarbons.
Page 6 of 19

(viii) Water. Clean water should be available ad libitum. Only clean, 

unmedicated water should be offered during the acclimation and testing periods. 

Water bottles or automatic watering devices are recommended. If water pans or 

bowls are used, water should be changed at least once a day.

(4) Administration of test substance. After acclimation under paragraph (e)(3)(iv) of 

this guideline, feed should be withheld from all test groups for a minimum of 15 hours 

prior, except potentially for Passeriformes, which may require less time to clear the 

digestive system of confounding substances, to administration of the test substance. 

Dosing by gelatin capsule is preferred, but when dosing with capsules is not feasible, 

doses may be administered by gavage. Doses are based on the individual body weight 

(bw) of each bird. Body weights are typically determined at the time of dosing, but may 

be taken, especially when capsules are used, within 24 hours prior to dosing. Dosing 

should be done in the early morning hours. The Agency should be contacted prior to 

testing with nanomaterials.

(i) Preparation of gavage mixtures. If dosing is by gavage and a carrier is used 

to administer the test substance, the preferred carrier is distilled or deionized 

water unless the test substance is known to hydrolyze readily. Other acceptable 

carriers include corn oil, propylene glycol, 1% carboxymethyl-cellulose, and gum 

acacia. Materials with known toxic or emetic properties should not be used. The 

absence of the crop in many passerine species requires using smaller, softer 

tubing than with waterfowl (see paragraph (j)(12) of this guideline). The dosing 

volume of test substance plus vehicle in a test should be constant for all birds with 

respect to individual body weights and should not exceed 5 milliliters per 

kilogram of body weight (mL/kg-bw). For those unusual test substances that 

might require a larger dosing volume (e.g. liquids with low purity), a dosing 

volume up to 8 mL/kg-bw may be used; steps should be taken to ensure that birds 

do not regurgitate the dose. In choosing between the bobwhite quail and mallard,

it is recommended to use the bobwhite when a dosing volume up to 8 mL/kg-bw 

is needed.

(ii) Treatment concentrations. 

(A) At a minimum, five dosage levels of the test substance are tested for 

definitive testing, plus the appropriate control. These dosage levels should 

be spaced geometrically in such a manner so that the entire dose-response 

curve (LD10 to LD90) is adequately characterized. Taking into account 

results of the range-finding test(s), dosage levels should be spaced so that 

at least three doses cause mortality between, but not including, 0% and 

100%. For a scientifically sound estimate of a point on the curve (e.g., 

LD50), responses should immediately bracket the point estimate of 

concern. For some test substances, it may be necessary to use more than 

five dose levels to achieve these results. 

(B) For a limit test, there is single dose level, plus the appropriate control 

(see paragraph (d)(5) of this guideline). A limit dose of 2,000 mg/kg-bw 

is used unless environmental residues are expected to result in a higher 
Page 7 of 19

dosage. Equation 1 of this guideline can be used to calculate the acute 

avian oral limit dose (mg a.i./kg-bw) for spray applications of pesticides. 

The dietary residue estimates are based on a nomogram that relates food 

item residues to pesticide application rate; for an application rate of 1 lb 

a.i./A, the highest residue level expected is with short grass (nomogram 

value of 240). The nomogram is based on an EPA database called UTAB 

(Uptake, Translocation, Accumulation, and Biotransformation), a 

compilation of actual measured pesticide residue values on plants (see 

references in paragraphs (j)(5) and (j)(11) of this guideline). If there are 

multiple uses this study is supporting for registration, the limit dose for the 

study should be based on the one resulting in the highest dose. If the 

resulting limit dose exceeds the digestive capacity of the test organism, 

consult with the Agency prior to conducting the study to determine the 

appropriate dose to use.

 

 

  















  



1

max ( . ./ )

SF

diet

AW TW

C

Limit dose mga i kg bw Equation 1

where:

for a pesticide use with a single application per year:

 (1.14)240 Cmax ApRate diet  Equation 2

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

   

    

















 

































n

i

n erval

halflife

diet C ApRate e

1

1 int 0.6931

max (1.14) 240 Equation 3

ApRate = maximum single application rate (in lb a.i./acre);

Halflife = the foliar half-life (default is 35 days);

Interval = the minimum application interval (in days);

i = application event from 1 to n;

n = total number of applications;

AW = the body weight (in g) of the assessed bird – for pesticides, use 20 grams as this is most 

conservative value in screening level assessments;

TW = the body weight (in g) of the test bird;

SF = body weight allometric scaling factor (Mineau et al. scaling factor of 1.15 is the default (see 

paragraph (j)(10) of this guideline)); and

1.14 = a dose conversion factor assuming that a 20 gram bird consumes 114% of its body weight 

daily.

(5) Controls.

(i) Every test includes a negative control group where control birds receive a 

sham dose consisting of the same vehicle or capsule as received by the test 
Page 8 of 19

substance dosed birds. For a satisfactory test, negative control birds should be 

from the same hatch as the test substance dosed groups and be kept under the 

same experimental conditions. The test procedures should be the same for control 

and treated birds, except that no test substance should be administered to the 

control birds.

(ii) Controls serve as a monitor of bird husbandry practices, an indicator of 

possible problems due to handling, and test substance administration and aid in 

separating treatment related effects from non-treatment related effects. Controls 

are important in assessing background mortality and disease. Background 

mortality is never presumed to be negligible.

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

during the test period.

(iv) A concurrent positive control with a reference substance of known toxicity

may be included in the test, as discussed in the OCSPP 850.2000 guideline.

(6) Number of test organisms. 

(i) The minimum number of birds per dosage level of the test substance and the 

control is 10 birds. Typically, equal numbers of young adult birds of both sexes 

are used. However, when necessary to address chemical- and site-specific 

concerns, the sex and age of the birds used for testing may be modified if this is 

justified (e.g., breeding females may be used when the expected exposure pattern 

indicates they may be at risk). Equal numbers of birds should be used for each 

dosage level.

(ii) Birds used in the test should be assigned randomly to treatment and control 

pens. Birds at a dosage level may be divided into two pens of five birds each. If 

this is done, dividing the groups by sex is recommended. Randomization should 

be done either at the initiation of the acclimation period or at the time of weighing 

just prior to dosing. The latter is recommended, because it avoids additional 

handling stress.

(7) Facilities, apparatus and supplies. Normal laboratory equipment and supplies, and 

items especially listed in (e)(7)(i) through (e)(7)(iv).

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

other environmental variables.

(ii) Pens―

(A) Size. Pens should have a floor area of at least 500 square centimeters 

(cm2) per bird (approximately 75 square inches (in2) per bird) for northern 

bobwhite and at least 1,000 cm2per bird (approximately 150 in2per bird) 

for mallards and should be at least 24 centimeters (cm) (approximately 9.5 

inches) high for northern bobwhite and 32 cm (approximately 12.5 inches) 
Page 9 of 19

high for mallard. Appropriate sized pens for passeriformes potentially 

may be different to accommodate bird size and social behaviors.

(B) Construction materials. Tests should be conducted with birds being 

maintained in commercial brooder or holding pens or pens of similar 

construction. Pens should be constructed of galvanized metal, stainless 

steel, or perfluorocarbon plastics. Materials that are toxic, may affect 

toxicity, or may sorb test substances should not be used. Wire mesh 

should be used for floors and external walls. Solid sheeting should be 

used for common walls and ceilings. Wire mesh for floors should be fine 

enough so as to not interfere with the normal movement of birds yet

coarse enough to allow fecal material to fall through. 

(C) Cleaning. 

(1) Between tests, pens should be disassembled (if feasible) and 

thoroughly cleaned to prevent disease transmission and crosscontamination. Steam cleaning of cages is recommended. Cages 

may be hosed, brushed thoroughly, and hosed again, as an 

alternative method. The use of detergents or bleach is acceptable, 

but other chemical disinfectants such as quaternary ammonium 

compounds should not be used. When disease vectors have to be 

controlled, hot or cold sterilization techniques are recommended, 

as long as such techniques will not leave chemical residues on the 

cages. For cold sterilization, ethylene oxide is recommended.

(2) Depending upon the type of pens used, pens may be cleaned 

during a test as needed to maintain good animal husbandry; 

however, care should be taken to minimize disturbance of the 

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. Environmental conditions should be appropriate to the 

study species. For mallards and northern bobwhite quail environmental conditions during 

the test should be maintained as follows (conditions should be modified as appropriate 

for passerine species):

(i) Temperature. Testing is done indoors to control lighting and other 

environmental variables. Temperatures for adult birds should be maintained at 

normal indoor temperatures, preferably between 15 degrees Celsius (

oC) and 27 

oC (70 to 80 degrees Fahrenheit (o

F)).
Page 10 of 19

(ii) Humidity. Relative humidity is not as critical as some other variables, but the 

test room should be maintained at a relative humidity between 45 and 70%.

(iii) Lighting and photoperiod. A photoperiod of 10 hours light and 14 hours 

dark is recommended in order to prevent birds from coming into reproductive 

condition for upland and waterfowl species. However, this may potentially be 

different for passerine species and should be adjusted as appropriate to ensure 

birds are not entering reproductive condition. Lighting may be either 

incandescent or fluorescent. Pens and lights should be positioned so that all pens 

will receive approximately equal illumination.

(iv) Ventilation. It is recommended that ventilation be sufficient to supply 10 to 

15 air exchanges per hour.

(9) Observations―

(i) Measurement of test substance in dosing media. Analytical confirmation of 

the dosing media concentration at test initiation is performed as described in the 

OCSPP 850.2000 guideline using analytical methods that are verified before 

beginning the test, to measure the amount of test substance in a sample.

(ii) Contaminants in feed. Contaminated feed may compromise study results, 

therefore, feed should be analyzed periodically to identify background 

contaminants such as heavy metals (e.g., arsenic, cadmium, lead, mercury, and 

selenium) and persistent pesticides, especially chlorinated insecticides. A broader 

pesticide screen to include other chemicals that are known to be acutely toxic to 

birds may be useful.

(iii) Basal diet composition. A nutrient analysis of the basal diet should be 

included in the test report. The analysis should include percentages by weight of 

protein, fat, fiber, ash, calcium, and phosphorus. In addition to these analyzed 

components, a list of expected amounts of vitamins, minerals or other 

supplements should also be recorded. Most commercial feed companies provide 

both the analysis and the list of supplements on the label.

(iv) Environmental conditions―

(A) Temperature. Temperature should be monitored on a constant basis 

in at least one representative location.

(B) Humidity. Humidity should be monitored on a continual basis in at 

least one representative location.

(v) Measures of effect―

(A) Monitoring of birds. Birds are monitored closely for the first 60 to

120 minutes after dosing. Any regurgitation should be noted. Additional 
Page 11 of 19

observations of test birds are made a minimum of three times on the day of 

dosing and at least daily (where feasible, twice daily observations are 

recommended) throughout the remainder of the test period.

(B) Mortality, intoxication, and other abnormal behavior. Throughout 

the test period, all signs of intoxication, other abnormal behavior, and 

mortality are identified, counted, and recorded by dosage level, by sex, 

and by day. Signs of intoxication are those behaviors apparently due to 

the test substance and may include a wide array of behaviors, such as 

labored respiration, leg weakness, hemorrhage, convulsions, and ruffled 

feathers. Record all signs of intoxication and any other abnormal 

behavior, such as excessive aggression, toe-picking, etc. Among 

survivors, remission of signs of intoxication and cessation of abnormal 

behavior is identified and recorded by dosage level and by day. An 

estimate of the number of birds exhibiting such signs should be recorded 

for each dosage level.

(C) Body weight. Individual body weights of birds are recorded for 

control and treated birds at the time of calculating the dosage to be 

administered and weekly thereafter until the test is concluded. An extra 

weighing on the third day after dosing may provide useful information, 

especially on anorexia. Body weights of birds a week prior to dosing are 

recommended to provide valuable base-line data.

(D) Food consumption. Measure and record food consumption at least 

weekly in each pen throughout the test. Valuable additional information 

can be obtained by monitoring food consumption daily, especially for the 

first few days following dosing.

(E) Gross pathology. Gross pathology examinations are conducted on all 

birds that die, as well as a sufficient number of survivors selected 

randomly in all test substance treatment groups as well as at least three 

control survivors in order to provide characterization of lesions.

(f) Treatment of results―

(1) Descriptive summary statistics―

(i) Environmental conditions. Calculate descriptive statistics (mean, standard 

deviation, coefficient of variation, minimum, maximum) temperature and 

humidity.

(ii) Mortality. Cumulative number of dead for each dosage level and control 

group by observation day should be summarized in tabular form. If birds are 

separated by sex, provide cumulative number of dead birds by sex for each dosage 

level and control group by observation day.
Page 12 of 19

(iii) Body weight. Calculate the change in body weight for an individual bird 

between observation periods (see Equation 4) and calculate the total change in 

body weight between test initiation and test termination (Equation 4 where time j

is test termination and time i is test initiation). For the control and each test 

substance dosage level, calculate and plot the mean body weight change and 

standard error by observation interval to assess effects on the pattern of weight 

change. Calculate and plot for the control and each test substance dosage level, 

the mean total body weight change and standard error. Determine the mean body 

weight change for males and females separately.

di j  wj  wi Equation 4

where:

di-j = difference or change in weight for an individual between observation time i and j;

wi = weight of an individual at time i;

wj = weight of an individual at time j.

(iv) Food consumption. Calculate and plot the mean food consumption by

treatment level and observation period.

(v) Appearance and behavior. Number of birds with appearance and behavioral 

symptoms should be summarized in tabular form by time of observation, 

treatment, and sex (if applicable). Tabulate among survivors, remission of signs 

of intoxication and cessation of abnormal behavior by dosage level and by 

observation day.

(vi) Gross pathology. Types of observed pathologies, and the number of dead or 

examined surviving birds with these lesions should be summarized in tabular 

form by treatment and sex.

(2) Percent mortality. Calculate the cumulative percentage of dead birds at each test 

substance treatment level and in the controls at test termination. Test substance treatment 

data should be adjusted to account for any control mortality.

(3) Limit test—

(i) LD50 value. At test termination, if no birds die at the limit dose, the acute oral 

LD50 is considered to be greater than the limit dose (i.e., LD50 > limit dose). This 

is because the Binomial Theorem predicts that when 10 organisms are tested, the 

probability of seeing no mortality if the true LD50 is at or below the limit dose is 

<0.001. Conversely the probability of seeing one or more dead birds if the true 

LD50 is at or below the limit dose is >0.999.

(ii) Proportion of mortality ( pˆ ). Assuming mortality follows the binomial 

distribution, an estimate of the true proportion of mortality ( pˆ ) in the laboratory 

test population as well as confidence bounds on that estimate (see Table A4 of the 

reference in paragraph (j)(3) of this guideline) can be obtained. For small sample 

sizes, the interval may be large. For example, for a limit test resulting in no 
Page 13 of 19

mortality in 10 birds ( pˆ = 0), the 99% confidence interval on the estimate of pˆ is 

(0.00, 0.41) and the 95% confidence interval is (0.00, 0.31). Using the 95% 

confidence interval as an example, the true (unknown) proportion of mortality

will be covered by the calculated confidence interval in 95% of repeated trials. 

For assessing risks, the confidence in the estimated proportion impacted is 

considered in determining acute effects at environmental exposure doses. If the 

uncertainty in pˆ is high at the limit concentration, and the expected environmental 

exposure concentration is close to the limit concentration, risks to threatened and 

endangered species may not be able to be discounted.

(iii) Multiple-dose definitive testing. 

(A) At test termination, if one or more mortalities occur among the 10 

birds at the limit concentration (which was conducted at 2,000 mg/kg-bw 

or the maximum limit dose, whichever is greater), a definitive LD50 test 

should be conducted. If frank sublethal effect(s) are observed in one or 

more birds at the limit dose, despite an absence of mortality, then a full 

definitive test may be necessary. For pesticides, if frank sublethal effect(s)

are observed in one or more birds and the limit dose tested was: 1) less 

than ten times the maximum expected EEC, then a full definitive study is 

necessary; or 2) was at least ten times the maximum EEC, but there is 

other evidence or data that indicate a risk to avian species, e.g., pesticide 

use incident data, then a full definitive test is necessary. 

(B) A multiple-dose definitive LD50 test may be waived if, at test 

termination: 1) the limit treatment group and control group each contain a 

minimum of 10 birds 2) no birds died at the limit dose; 2) and there are 

also no frank sublethal effects observed at the limit dose; 3) except for the 

number of dosage levels the test procedures and duration are the same as 

in the definitive test; 4) the dosage level is confirmed by chemical analysis 

under test conditions and 5) for pesticides, the limit dose was 2,000 

mg/kg-bw or equivalent to the maximum expected environmental 

concentration on food items, whichever is higher.

(4) Multiple-dose definitive test―

(i) Dose-response curve, slope, and LD50. Statistical procedures are employed 

to calculate the LD50 (standard error and 95% confidence limits). If a doseresponse curve model (e.g., probit) was fit to the data to determine the LD50, the 

model parameters (e.g., slope) and their uncertainty estimates (e.g., standard 

error) should be recorded. A statistical test for goodness-of-fit (e.g., chi-square 

test) should also be performed to determine how well the data fit the 

computational model used.

(ii) NOEL. While calculation of a NOEL and LOEL is usually not part of this 

test design, reporting these values is useful when testing both pesticide and 

industrial chemicals. 
Page 14 of 19

(iii) Statistical methods. Statistical procedures for modeling quantal data are 

available and should be used. Additional discussion about endpoints and 

statistical procedures is found in the OCSPP 850.2000 guideline.

(g) Tabular summary of test conditions. Table 2 lists the important conditions that should 

prevail during the definitive test. Except for the number of dose levels, Table 2 also lists the 

important conditions that should prevail during a limit test. Meeting these test conditions will 

greatly increase the likelihood that the completed test will be acceptable or valid for the purposes 

of this test.

Table 2.—Summary of Test Conditions for Avian Acute Oral Toxicity Test

Test duration (observation period) Minimum of 14 days

Temperature 15 oC to 27 oC (59 to 80 oF)

Light quality Ambient: incandescent or fluorescent

Photoperiod 10 hours light: 14 hours dark

Pen size ≥500 cm2(approximately 75 in2) per bird for northern 

bobwhite and ≥1,000 cm2(approximately 150 in2) per 

bird for mallards. Pens for passerines should be 

appropriate for the study species.

Number of pens One or two per dose level. Dividing by sex is 

recommended if using two pens

Test species Northern bobwhite quail and mallard ducks are the 

preferred upland game bird and waterfowl species, 

respectively. Currently, no preferred passerine 

species has been identified. For passerines, 

protocols, which include the selected test species, 

should be submitted prior to test initiation.

Age of test organisms Young adults, not yet mated. Other age groups may

be tested to address a specific concern associated 

with a chemical-specific pattern of use on a case-bycase basis

Sex of test organisms Typically, both sexes should be tested. Just one sex 

may be tested to address a specific concerns 

associated with a chemical-specific pattern of use on 

a case-by-case basis.

Number of birds per dose level Minimum of 10 birds per dose, split evenly by sex if 

both sexes are tested

Dose levels Minimum of five for definitive LD50 test, plus a control 

(for the limit test—the limit dose (2,000 mg/kg-bw or 

higher) plus a control)

Administration of dose By gavage or capsule

Measures of Effect (Measurement Endpoint) Death (LD50), body weight, food consumption, and 

other signs of clinical toxicity

(h) Test validity. This test would be considered unacceptable or invalid if one or more of the 

conditions in Table 3 occurred. This list should not be misconstrued as limiting the reason(s) 

that a test could be found unacceptable or invalid. However, except for the conditions listed in 

Table 3 and in the OCSPP 850.2000 guideline, it is unlikely that a study will be rejected when 

there are slight variations from guideline environmental conditions and study design unless the 
Page 15 of 19

control organisms are significantly affected, the precision of the test is reduced, the power of a 

test to detect differences is reduced, and/or significant biases are introduced in defining the 

magnitude of effect on measurement endpoints as compared to guideline conditions. Before 

departing significantly from this guideline, the investigator should contact the Agency to discuss 

the reason for the departure and the effect the change(s) will have on test acceptability. In the test 

report, all departures from the guideline should be identified, the reasons for the changes given, 

and any resulting effects on test endpoints noted and discussed.

Table 3.—Test Validity Elements for the Avian Acute Oral Toxicity Test

1. Birds were not randomly assigned to treatment and control pens. 

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

3. A minimum of ten birds were not used for each dose level of the test substance and control.

4. The test substance was not orally administered, via either capsule or gavage.

5. In the definitive test a minimum of five dose levels of the test substance, plus an appropriate control, 

were not tested.

(i) Reporting―

(1) Background information. Background information to be supplied in the report 

consists at a minimum of those background information items listed in paragraph (j)(1) of 

the OCSPP 850.2000 guideline.

(2) Guideline deviations. Provide a statement of the guideline or protocol followed. 

Include a description of any deviations from the test guideline or any occurrences which 

may have influenced the results of the test, the reason for these changes, and any 

resulting effects on test endpoints noted and discussed.

(3) Test substance. 

(i) Identification of the test substance: common name, IUPAC and CAS names, 

CAS number, structural formula, source, lot or batch number, chemical state or 

form of the test substance, and its purity (i.e. for pesticides, the identity and 

concentration of active ingredient(s)), radiolabeling if any, location of label(s), 

and radiopurity.

(ii) Storage conditions of the test chemical or test substance and stability of the 

test chemical or test substance under storage conditions if stored prior to use.

(iii) Methods of preparation of the test substance and the treatment doses used in 

the range-finding and definitive test, or limit test.

(iv) If a diluent is used to prepare stock or test substance provide: the name and 

source of the diluent, the nominal concentration(s) of the test substance in the 

stock solution, and the diluent concentration(s) used in the treatments and diluent 

control. Provide a description of the dosing volume of test substance plus diluent 

for all birds with respect to individual body weights.

(v) Name of toxicant used for positive control (if applicable) and dosage levels.
Page 16 of 19

(4) Test organisms. 

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

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

disease status.

(iii) Sex and reproductive history and condition at test initiation. If sexually

mature birds that have been mated are used, describe the process used to bring the 

birds completely out of production.

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

(v) Individual body weights at the beginning of the test (typically determined at 

time of dosing, but may be taken, especially for capsules, within 24 hours prior to 

dosing) and weekly thereafter.

(vi) Acclimation procedures and duration.

(5) Test methods and conditions. Provide a description of the test system and 

conditions used in the definitive or limit test, any preliminary range-finding tests, and any

positive control tests.

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

(ii) Description of housing environmental conditions: temperature, humidity, 

ventilation rate, photoperiod, and lighting source and intensity.

(iii) Detailed description of basal diet, including source/type, percentages by

weight of protein, fat, fiber, ash, calcium, and phosphorus, a list of expected 

amounts of vitamins, minerals or other supplements. Most commercial feed 

companies provide both the analysis and the list of supplements on the label.

(iv) Describe the frequency and sample date(s) for documenting the contaminant 

status (heavy metals, persistent or chlorinated pesticides) of the feed and 

tabulation of the results of the analysis.

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

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

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

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

(ix) Methods and frequency of environmental monitoring performed during the 

definitive or limit study for temperature and humidity.
Page 17 of 19

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

doses.

(xi) For the definitive and limit test, all analytical procedures should be described. 

The accuracy of the method, method detection limit, and limit of quantification 

should be given (described in 850.2000 (b) Definitions).

(xii) Methods and frequency of measuring number of dead birds, observing signs 

of intoxication, including regurgitation, and other abnormal behavior, including 

time of onset, duration, severity, and number affected at each dose level and 

control.

(xiii) Feed consumption per pen at least weekly or as often as measured, if more 

frequently than weekly, along with an estimate of wastage.

(6) Results. 

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

(ii) Environmental monitoring data results (temperature and humidity) in tabular 

form (provide raw data for measurements not made on a continuous basis), and 

descriptive statistics (mean, standard deviation, minimum, and maximum).

(iii) For preliminary range-finding tests, if conducted, tabulate the number and 

percentage of birds that died in each test pen, treatment level and in the control at 

each observation period. Provide a description and count of any other appearance 

or behavioral effects at each treatment level and in the control. Tabulate the 

results of gross pathological examinations in dead birds and samples of surviving 

birds.

(iv) For a limit test, tabulation of the number of dead birds in each pen at each 

observation time during the test for the limit dose and the control (provide the raw 

data).

(v) For the definitive test, the number of dead birds at each observation time 

during the test tabulated by pen and treatment (provide the raw data).

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

control mortality.

(vii) For the limit and definitive test, tabulation by pen, treatment, and observation 

time of abnormal appearance and behavioral signs of toxicity and recovery, if any 

(provide raw data).

(viii) For the limit and definitive test, tabulation of gross morphology of dead 

birds and samples of surviving birds at test termination by pen and treatment 

(provide raw data).
Page 18 of 19

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

(x) For a limit test, provide conclusion about the LD50 being above the limit 

concentration and the lack or presence of other signs of toxic effects at the limit 

dose.

(xi) For the definitive test, where sufficient data exist to fit a model (e.g. probit) 

the slope of the dose-response curve and its standard error and 95% confidence 

limits and any goodness of fit results

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

(xiii) Description of statistical methods used to fit the dose-response model or 

determine point estimates and the LD50 (including software package), and the 

basis for the choice of methods. Provide results of any goodness-of-fit tests.

(xiv) Description of statistical method(s) used for NOEL and LOEL 

determination, including software package, and the basis for the choice of 

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 857-05e1, Standard Practice 

for Conducting Subacute Dietary Toxicity Tests with Avian Species, In Annual Book of 

ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Reapproved 2005. 

(2) Committee on Birds of the Institute of Laboratory Animal Resources, National 

Research Council, 1977. Laboratory Animal Management: Wild Birds. National 

Academy of Sciences, Washington, DC.

(3) Conover, W. 1980. Practical Nonparametric Statistics, 2

nd Edition. John Wiley &

Sons, Inc., New York, NY. 493 pp.

(4) Dorrestein, G, 2003. Diagnostic approaches and management of diseases in captive 

passerines. Seminars in Avian and Exotic Pet Medicine 12(1): 11-20.

(5) Fletcher, J., J. Nellessen, and T. Pfleeger. 1994. Literature review and evaluation of 

the EPA food-chain (Kenaga) Nomogram, an instrument for estimating pesticide residues 

on plants. Environmental Toxicology and Chemistry 13(9):1383-1391.

(6) Grue, C. and L. Franson, 1986. Use of captive starlings to determine effects of 

environmental contaminants on passerine reproduction: pen characteristics and nestling 

food requirements. Bulletin of environmental contamination and toxicology 37(5):655-

663.

(7) Harding, C., 1999. Husbandry and care of passerine birds. Poultry and avian biology 

reviews 10(2): 79-83.
Page 19 of 19

(8) Hayre, M., 1995. Guidelines for housing excruciatingly happy zebra finches. Lab 

Animal 24(6):43-44.

(9) Matheson, S., L. Asher, and M. Bateson, 2008. Larger, enriched cages are associated 

with ‘optimistic’ response biases in captive European starlings (Sturnus vulgaris). 

Applied Animal Behaviour Science 109(2-4):374-383.

(10) Mineau, P., B. Collins, and A. Baril. 1996. On the use of scaling factors to improve 

interspecies extrapolation to acute toxicity in birds. Regulatory Toxicology and 

Pharmacology 24:24-29.

(11) Nellessen, J. and J. Fletcher. 1992. UTAB: A computer database on residues of 

xenobiotic organic chemicals and heavy metals in plants. Journal of Chemical 

Information and Computer Sciences 32:144-148.

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

Science.

(13) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines 

Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Office of Pesticides 

and Toxic Substances, Washington, D.C. EPA-540/9-82-024, October 1982.

(14) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard 

Evaluation Procedure, Avian Single-Dose Oral LD50. Office of Pesticides Programs, 

Washington, DC, EPA-540/9-85-001, June 1985.

(15) U.S. Environmental Protection Agency, 1994. Pesticides Reregistration Rejection 

Rate Analysis: Ecological Effects, Office of Prevention, Pesticides and Toxic Substances, 

Washington, D.C. EPA 738-R-94-035.