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Toxicity to birds

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Endpoint:
short-term toxicity to birds: acute oral toxicity test (LD50-only)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.2100 (Avian Acute Oral Toxicity Test)
Deviations:
yes
Remarks:
Deficiencies were noted for species selection, regurgitation, measurement of food consumption and fasting period.
Principles of method if other than guideline:
99.3% purity
GLP compliance:
yes
Dose method:
other: Intubated directly into the crop or proventriculus of each bird
Analytical monitoring:
no
Vehicle:
yes
Remarks:
1% carboxymethyl cellulose aqueous solution
Test organisms (species):
Poephila guttata
Details on test organisms:
Test birds were housed indoors by dosage group in batteries of pens manufactured by Prevue Pet Products, Inc. (Model No. F060). Each pen had floor space that measured approximately 29 X 26 cm with a ceiling height of 31 cm. External walls, ceilings and floors were constructed of coated wire while sidewalls were plastic. Each pen contained one or more perches. Each dosage group was assigned five pens. One pen contained one male and one female. Birds were assigned to pens by indiscriminate draw. Each group of birds was identified by pen number.
Birds were maintained at ambient room temperature. The continuously monitored average temperature for this study was 26.4°C ± 0.7°C (SD) with a continuously monitored average relative humidity of 39% ± 9% (SD). The photoperiod (maintained by a time clock) was approximately 0.25 hour dim light/eight hours light/0.25 hour dim light/15.5 hours dark during acclimation and throughout the test. The light source was fluorescent lights that closely approximated noonday sunlight. The quarter hour of dim light prior to the light and dark phases allowed birds to settle. The birds were exposed to an average of approximately 177 lux of illumination at bird height in caging. Housing and husbandry practices were
conducted so as to adhere to the guidelines established by the National Research Council.
Acclimation period: At least 3 weeks
Diet: Commercially available finch food and millet
Water: Tap water, ad libitum
Post exposure observation period:
14 days
No. of animals per sex per dose and/or stage:
5
Control animals:
yes, concurrent vehicle
Nominal and measured doses / concentrations:
292, 486, 810, 1350, and 2250 mg/kg
Details on examinations and observations:
Birds were observed for clinical signs of toxicity, body weight effects, and mortality for 14 days after dosing. All mortalities were subjected to gross necropsy. After consultation with the Sponsor it was decided that gross necropsies on birds surviving until test termination were not necessary given the lack of persistent toxicity.

Body weights were measured individually at the initiation of the test and on Days 3, 7 and 14 of the test. Given the nature of the feed presented to the birds and their hulling and wasteful behavior, no attempts were made to quantify the amount of feed consumed.

All mortalities were subjected to gross necropsy. Gross necropsies included but were not limited to, a general examination of the exterior of the bird and an examination of the thoracic and abdominal cavities, including cardiovascular and respiratory systems, liver, spleen, gastro-intestinal tract, and urogenital system. After consultation with the Sponsor it was decided that gross necropsies on birds surviving until test termination were not necessary given the lack of persistent toxicity.
Details on reproductive parameters:
Not examined
Reference substance (positive control):
no
Key result
Dose descriptor:
LD50
Effect level:
> 1 350 mg/kg bw
Conc. / dose based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Single dose
Mortality and sub-lethal effects:
There were no mortalities in the control group and all control birds were normal in appearance and behavior throughout the test. In addition, there were no mortalities in the 292, 486 and 1350 mg/kg treatment groups. There was 10% (1 of 10) mortality noted at the 810 and 2250 mg/kg dosage levels,

All birds in the 292 and 486 mg/kg treatment groups were normal in appearance and behavior for the duration of the test.

At the 810 mg/kg dosage level, one of ten birds was noted as regurgitating on the day of dosing. On Day 1 of the test a different bird was found dead without displaying prior signs of toxicity. All other birds were normal in appearance and behavior for the duration of the test.

At the 1350 mg/kg dosage level signs of toxicity were first noted approximately 20 minutes after dosing when two birds were noted with a ruffled appearance, with one of the two also exhibiting lethargy. A total of five birds were noted with signs of toxicity on the day of dosing. Additionally, on the day of dosing four of ten birds were noted as regurgitating. Signs of toxicity seen at the 1350 mg/kg dosage level were ruffled appearance and lethargy. All birds were normal in appearance and behavior from Day 1 of the test until test termination.

At the 2250 mg/kg dosage level signs of toxicity were first noted 4 minutes after dosing when one bird was noted with a ruffled appearance. Thirty-five minutes after dosing the only mortality in the 2250 mg/kg dosage level was noted when a female was found dead. On the day of dosing seven of ten birds were noted as regurgitating and a total of six birds were noted with signs of toxicity. Signs of toxicity seen at the 2250 mg/kg dosage level were prostrate posture, loss of righting reflex, ruffled appearance and lethargy. All surviving birds were normal in appearance and behavior from Day 1 of the test until test termination.
Further details on results:
When compared to the control group, there were no apparent treatment related effects upon body weight at any interval for males or females at the 292, 486, 810, 1350, and 2250 mg/kg dosage levels.

The two mortalities that occurred during the course of the study were subjected to gross necropsy. The mortality in the 810 mg/kg dosage level was noted with an empty upper gastrointestinal tract, grit only in gizzard and pale kidneys. The mortality in the 2250 mg/kg level was noted with a mostly empty gastrointestinal tract, grit only in gizzard and kidneys that were dark in color. The bird was also noted with intracranial bleeding with the largest area on the back right quadrant of the skull. Although the bird was noted with intracranial bleeding, a treatment related death could not be precluded.
Validity criteria fulfilled:
yes
Conclusions:
An LD50 value can be set at greater than 1350 mg/kg, a level with no mortalities and where the majority of birds did not regurgitate. The no-observed effect level was 486 mg/kg, based on the incidence of sublethal effects.
Executive summary:

The test substance was administered to fasted zebra finch (Poephila guttata) in an acute oral toxicity study according to EPA guideline OPPTS 850.2100. Five zebra finch/sex/dose received single oral nominal doses of 0, 292, 486, 810, 1350, and 2250 mg/kg at a dose volume of 5 mL/kg in 1% carboxymethyl cellulose aqueous solution. Birds were observed for clinical signs of toxicity, body weight effects, and mortality for 14 days after dosing. Mortalities were examined for gross pathological changes. An LD50 value can be set at greater than 1350 mg/kg, a level with no mortalities and where the majority of birds did not regurgitate. The no-observed effect level was 486 mg/kg, based on the incidence of sublethal effects.

Endpoint:
short-term toxicity to birds: acute oral toxicity test (LD50-only)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 205 (Avian Dietary Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
98.4% purity
Dose method:
feed
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and analysis of diet:
The test diets were prepared by mixing the test substance directly into the ration, without the use of solvent or carrier. Test diets were prepared by first blending the test substance into a small amount of ration in a Waring® blender, and then blending the mixture into the diet in a Hobart (Model Number AS200T) mixer. An amount of diet sufficient to last the five day exposure period was prepared on the day of test initiation for each treatment and control group. Diets were presented to the birds at initiation of the test, and added to the feeders as needed during the five day exposure period.

The dietary concentrations were not adjusted for purity of the test substance. Therefore all dietary concentrations are reported as parts per million of the test substance as received. Nominal dietary test concentrations used in this study were 0, 325, 650, 1300, 2600 and 5200 ppm. Nominal dietary test concentrations during the second phase of this study were 0 and 5200 ppm.

Samples of the test diets were collected during the first phase of the study to verify the test concentrations administered and to confirm the stability and homogeneity of the test substance in the diets. Homogeneity of the test substance in the diet was evaluated by collecting six samples from the 325 and 5200 ppm test diets at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. The homogeneity samples also served as verification samples. One verification sample was collected from the control diet and two verification samples were collected from each remaining treatment group at preparation on Day 0. At the end of the exposure period (Day 5) one sample was collected from the control group and two samples were collected from each treatment group to determine stability of the test substance in the diet. The samples were collected from feed remaining in the feeders.

On Day 0 of Phase II one verification sample was collected from the control group, and two verification samples were collected from the 5200 ppm treatment group. Samples were transferred immediately to Wildlife International Ltd. analytical chemistry for analysis.
Test organisms (species):
Colinus virginianus
Details on test organisms:
All northern bobwhite (Colinus virginianus) were 10 days of age and appeared to be in good health at initiation of the test. The birds used for the first phase were hatched at Wildlife International Ltd. on July 21, 1997. The birds used for the second phase were hatched at Wildlife International Ltd. on September 15, 1997. All birds for each phase were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the time of hatch until initiation of the test. Pre-test mortality during acclimation of chicks for Phase I was less than 2%. There were no mortalities during the pre-test acclimation period for the Phase II chicks.
Total exposure duration (if not single dose):
5 d
Post exposure observation period:
3 days
No. of animals per sex per dose and/or stage:
10 chicks per test substance dose and each control group contained 20 chicks
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
325, 650, 1300, 2600, and 5200 ppm
Details on test conditions:
During acclimation and testing, all birds were housed indoors by test group in batteries of thermostatically controlled brooding pens manufactured by Beacon Steel Products Co. (Model No. B735Q). Birds were assigned to pens by indiscriminate draw. Each pen had floor space that measured approximately 72 x 90 cm. Ceiling height was approximately 23 cm. External walls, ceilings and floors were constructed of galvanized steel wire and sheeting.

During both phases of the test each treatment group was assigned two pens that contained five chicks each. The control group for each phase was assigned four pens that contained five chicks each. Birds were assigned to pens by indiscriminate draw. Each group of birds was identified by pen number and test concentration. Individual birds were identified by numbered leg bands.

During both phases of the test the average temperature in the brooding compartment of the pens was 38°C ± 1°C (SD). Average ambient room temperature was 27.4°C ± 0.5% (SD), with an average relative humidity of 51.7% ± 11.3% (SD) during Phase I and 25.0°C ± 0.7°C (SD), with an average relative humidity of 51.0% ± 10.0% (SD) during Phase II. The air handling system in each study room was designed to vent up to fifteen room air volumes every hour and replace them with fresh air.

The photoperiod (maintained by a time clock) was sixteen hours of light per day during acclimation and throughout the test. The light source was fluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of approximately 207 lux of illumination (approximately 19 foot-candles) during Phase I and an average of approximately 471 lux of illumination (approximately 44 foot-candles) during Phase II. Housing and husbandry practices were based on guidelines established by the National Institutes of Health.
Details on examinations and observations:
During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, all birds were observed at least twice daily. A record was maintained of all mortality, signs of toxicity and abnormal behavior.

Individual body weights were measured at the initiation, at the end of the exposure period on Day 5, and at termination on Day 8 of each test phase. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined for each treatment and control group. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds.

At test termination of Phase II, birds from the control and 5200 ppm treatment group were examined macroscopically for signs of lesions.

The no-observed-effect concentration was determined by evaluation of the mortality, clinical signs, body weight and feed consumption and necropsy data.
Reference substance (positive control):
no
Key result
Duration (if not single dose):
5 d
Dose descriptor:
NOEC
Effect level:
5 200 other: ppm
Conc. / dose based on:
test mat.
Remarks on result:
other: no effects at highest dose tested
Mortality and sub-lethal effects:
There were no mortalities in the control group during either phase of the test. There were no mortalities or overt signs of toxicity in any of the treatment groups in Phase I or Phase II of the test.

During Phase I of the test a single control bird was noted with a lesion on the right foot on Days 7 and 8 of the test. The lesion appeared to be the result of toepicking. This bird was otherwise normal in behavior and appearance. All other birds in the control group from each phase of the test were normal in appearance and behavior throughout the test period.

On Day 3 of Phase I, a bird in the 1300 ppm level was noted with a severe ventral head curl which caused the bird to roll and tumble, making it difficult to walk. Although the severity of the curl was reduced the following day and the bird no longer rolled or tumbled, the condition persisted for the duration of the test. Ventral head curl is generally thought to result from an injury to the neck and this condition was not considered treatment related. Additionally, this bird was noted as ruffled in appearance and with foot lesions due to toepicking during the final two days of observation. All other birds in the 325 through 5200 ppm treatment groups during Phase I and all birds in the 5200 ppm treatment group during Phase II were normal in behavior and appearance throughout the test period.

There were no apparent treatment related effects on body weight or feed consumption during either test phase at any of the concentrations tested.

All birds from the control and 5200 ppm group were found to be in good general condition, with no lesions observed.
Validity criteria fulfilled:
no
Conclusions:
The dietary LC50 value for northern bobwhite exposed to the test substance was determined to be greater than 5200 ppm, the highest concentration tested. Therefore, the no mortality concentration and the no observed effect concentration (NOEC) were both 5200 ppm.
Executive summary:

In Phase I of this study, groups of 10 bobwhite chicks, Colinus virginianus, were fed diets containing 0, 325, 650, 1300, 2600, or 5200 ppm of the test substance for 5 days and then allowed a 3-day recovery period. The control group contained 20 chicks. In Phase II of this study, another group of 10 chicks was fed a diet containing 5200 ppm of the test substance. As was the case in Phase I, the control group contained 20 chicks. Mortality, clinical signs of toxicity, body weight and feed consumption, and necropsy examinations were conducted at the end of the study. The test was conducted according to OECD guideline 205.

 

There were no apparent treatment related effects on body weight or feed consumption during either test phase at any of the concentrations tested. All birds from the control and 5200 ppm group were found to be in good general condition, with no lesions observed. The dietary LC50 value was determined to be greater than 5200 ppm, the highest concentration tested.

Endpoint:
short-term toxicity to birds: acute oral toxicity test (LD50-only)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 205 (Avian Dietary Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
98.4% purity
Dose method:
feed
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and analysis of diet:
The test diets were prepared by mixing the test substance directly into the ration, without the use of solvent or carrier. Test diets were prepared by first blending the test substance into a small amount of ration in a Waring® blender, and then blending the mixture into the diet in a Hobart (Model Number AS200T) mixer. An amount of diet sufficient to last the five day exposure period was prepared on the day of test initiation for each treatment and control group. Diets were presented to the birds at initiation of the test, and added to the feeders as needed during the five day exposure period.

The dietary concentrations were not adjusted for purity of the test substance. Therefore all dietary concentrations are reported as parts per million of the test substance as received. Nominal dietary test concentrations used during the first phase of this study were 0, 325, 650, 1300, 2600 and 5200 ppm. Nominal dietary test concentrations during the second phase of this study were 0 and 5200 ppm.

Samples of the test diets were collected during the first phase of the study to verify the test concentrations administered and to confirm the stability and homogeneity of the test substance in the diets. Homogeneity of the test substance in the diet was evaluated by collecting six samples from the 325 and 5200 ppm test diets at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. The homogeneity samples also served as verification samples. One verification sample was collected from the control diet and two verification samples were collected from each remaining treatment group at preparation on Day 0. At the end of the exposure period (Day 5), one sample was collected from the control and two samples were collected from each treatment group to determine stability of the test substance in the diet. The samples were collected from feed remaining in the feeders.

On Day 0 of Phase II one verification sample was collected from the control group, and two verification samples were collected from the 5200 ppm treatment group. Samples were transferred immediately to Wildlife International Ltd. analytical chemistry for analysis.
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
All mallards (Anas platyrhynchos) were 10 days of age and appeared to be in good health at initiation of the test. The birds were obtained from Whistling Wings, Hanover, Illinois. The birds used during the initial test were hatched on July 21, 1997 and received at Wildlife International Ltd. on July 22, 1997. The birds used for the second phase of the test were hatched September 15, 1997 and received at Wildlife International Ltd. on September 16, 1997. All birds for each Phase were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day of receipt until initiation of the test.

There were no mortalities during the pre-test acclimation period for the Phase I and Phase II ducklings.

Throughout acclimation and testing all test birds were fed a game bird ration formulated to Wildlife International Ltd.'s specifications. Water, from the town of Easton public water supply, and feed were provided ad libitum during acclimation and during the test. The birds received no form of antibiotic medication during acclimation or the test.

During acclimation and testing, all birds were housed indoors by test group in batteries of thermostatically controlled brooding pens manufactured by Safeguard Products, Inc. Each pen had floor space that measured approximately 62 x 92 cm. Ceiling height was approximately 25.5 cm. External walls, ceilings and floors were constructed of vinyl-coated wire grid.

During both phases of the test each treatment group was assigned two pens that contained five ducklings each. The control group for each phase was assigned four pens that contained five ducklings each. Birds were assigned to pens by indiscriminate draw. Each group of birds was identified by pen number and test concentration. Individual birds were identified by numbered wing bands.

During the first test phase the average temperature in the brooding compartment of the pens was 30°C ± 1°C (SD). The average brooder compartment temperature during the second phase of the test was 29°C ± 1°C (SD). Average ambient room temperature was 25.5°C ± 0.7°C (SD), with an average relative humidity of 71.3% ± 13.6% (SD) during the first phase and 24.2 °C ± 0.8°C (SD), with an average relative humidity of 57% ± 9% (SD) during the second phase of the test. The air handling system in each study room was designed to vent up to fifteen room air volumes every hour and replace them with fresh air.

The photoperiod (maintained by a time clock) was sixteen hours of light per day during acclimation and throughout both test phases. The light source was fluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of approximately 161 lux of illumination (approximately 15 foot-candles) during Phase I and an average of approximately 302 lux of illumination (approximately 28 foot-candles) during Phase II. Housing and husbandry practices were based on guidelines established by the National Institutes of Health.
Total exposure duration (if not single dose):
5 d
Post exposure observation period:
3 days
No. of animals per sex per dose and/or stage:
10 duckingsd in each treatment group and 20 ducklings in each control group
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
325, 650, 1300, 2600, and 5200 ppm
Details on test conditions:
The initial phase of the test consisted of a geometric series of five test concentrations and a
control group. The second phase of the test consisted of one test concentration and a control group.

Twenty mallard ducklings were assigned to each of the control groups and ten mallard ducklings were assigned to each of the treatment groups by indiscriminate draw. The birds were housed in
brooding pens containing five ducklings each. Nominal dietary concentrations used during the first phase of this study were 0, 325, 650, 1300, 2600 and 5200 parts per million (ppm). Nominal dietary concentrations used during the second phase of the study were 0 and 5200 ppm. The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor.

During both phases of the test, each group was fed the appropriate test or control diet for five days. Following the five-day exposure period all groups were given untreated feed for three days.
Details on examinations and observations:
During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, all birds were observed at least twice daily. A record was maintained of all mortality, signs of toxicity and abnormal behavior. At test termination of Phase II, birds from the control and 5200 ppm treatment group were examined macroscopically for signs of lesions.

Individual body weights were measured at the initiation, at the end of the exposure period on Day 5, and at termination on Day 8, of each test phase. Average feed consumption values during the exposure periods (Days 0-5) and the post-exposure observation periods (Days 6-8) were determined for each treatment and control group. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds.
Reference substance (positive control):
no
Key result
Duration (if not single dose):
5 d
Dose descriptor:
NOEC
Effect level:
5 200 other: ppm
Conc. / dose based on:
test mat.
Remarks on result:
other: no effects at highest dose tested
Mortality and sub-lethal effects:
There were no mortalities in the control group during either phase of the test. All control birds were normal in appearance and behavior throughout the test. In addition, there were no mortalities among birds in the 325, 650, 1300, 2600 and 5200 ppm treatment groups during the first phase of the test or among birds in the 5200 ppm treatment group during the second phase of the test. All birds in those groups were normal in appearance and behavior for the duration of the test.

When compared to the control group, there were no apparent treatment related effects on body weight among birds in the 325, 650 and 1300 ppm treatment groups during Phase I of the test. However, there was a slight decrease in body weight gain among birds at the 2600 ppm concentration and a more pronounced reduction in body weight gain at the 5200 ppm concentration during the exposure period (Days 0-5), that appeared to be concentration responsive.

During the second phase of the test, there was a decrease in body weight gain at the 5200 ppm concentration during the exposure period that appeared to be treatment related. This reduction in weight gain at the 5200 ppm level was similar to the reduction in weight gain observed during the first phase of the test at this test concentration.

There were no apparent treatment related effects on feed consumption at the 325, 650, 1300 and 2600 ppm test concentrations during the first phase of the test. A slight decrease in feed consumption was noted in the 5200 ppm treatment group during the exposure period of the first test phase. Feed consumption by the 5200 ppm treatment group appeared to be comparable to the control during the second phase of the test.

At test termination of Phase II, birds from the control and 5200 ppm treatment group were examined macroscopically for signs of lesions. All birds from the control and 5200 ppm group were found to be in good general condition, with no lesions observed.
Conclusions:
The dietary LC50 value for mallards exposed to the test substance was determined to be greater than 5200 ppm, the highest nominal concentration tested. The no mortality concentration was 5200 ppm. Based on a slight decrease in body weight gain among birds in the 2600 ppm treatment group during the first phase of the test, the no-observed-effect concentration was 1300 ppm.
Executive summary:

In Phase I of this study, groups of 10 ducklings, Anas platyrhynchos, were fed diets containing 0, 325, 650, 1300, 2600, or 5200 ppm of the test substance for 5 days and then allowed a 3-day recovery period. The control group contained 20 chicks. In Phase II of this study, another group of 10 ducklings was fed a diet containing 5200 ppm of the test substance. As was the case in Phase I, the control group contained 20 ducklings. Mortality, clinical signs of toxicity, body weight and feed consumption, and necropsy examinations were conducted at the end of the study. The test was conducted according to OECD guideline 205.

 

There were no mortalities in the control group during either phase of the test. All control birds were normal in appearance and behavior throughout the test. In addition, there were no mortalities among birds in the 325, 650, 1300, 2600 and 5200 ppm treatment groups during the first phase of the test or among birds in the 5200 ppm treatment group during the second phase of the test. All birds in those groups were normal in appearance and behavior for the duration of the test.

 

When compared to the control group, there were no apparent treatment related effects on body weight among birds in the 325, 650 and 1300 ppm treatment groups during Phase I of the test. However, there was a slight decrease in body weight gain among birds at the 2600 ppm concentration and a more pronounced reduction in body weight gain at the 5200 ppm concentration during the exposure period (Days 0-5), that appeared to be concentration responsive.

 

During the second phase of the test, there was a decrease in body weight gain at the 5200 ppm concentration during the exposure period that appeared to be treatment related. This reduction in weight gain at the 5200 ppm level was similar to the reduction in weight gain observed during the first phase of the test at this test concentration.

 

There were no apparent treatment related effects on feed consumption at the 325, 650, 1300 and 2600 ppm test concentrations during the first phase of the test. A slight decrease in feed consumption was noted in the 5200 ppm treatment group during the exposure period of the first test phase. Feed consumption by the 5200 ppm treatment group appeared to be comparable to the control during the second phase of the test.

At test termination of Phase II, birds from the control and 5200 ppm treatment group were examined macroscopically for signs of lesions. All birds from the control and 5200 ppm group were found to be in good general condition, with no lesions observed.

 

The dietary LC50 value for mallards exposed to the test substance was determined to be greater than 5200 ppm, the highest nominal concentration tested. The no mortality concentration was 5200 ppm. Based on a slight decrease in body weight gain among birds in the 2600 ppm treatment group during the first phase of the test, the no-observed-effect concentration was 1300 ppm.

Endpoint:
short-term toxicity to birds: acute oral toxicity test (LD50-only)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
EPA OPP 71-1 (Avian Acute Oral Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
98.4% purity
Dose method:
gavage
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
Corn oil
Details on preparation and analysis of diet:
The test substance was dispersed in corn oil. The concentration of the test substance in the diluent was adjusted to provide a constant volume to body weight dose for all treatment birds. The dose and LD50 value reported were not corrected for purity of the test substance. The nominal doses used in this study were 100, 562.5, 1125 and 2250 mg/kg.

Samples of the dosing solutions were collected prior to the dosing procedure during each phase to verify solution concentration and assess homogeneity and stability of the dosing solution. Samples were transferred to the Wildlife International Ltd. analytical laboratory for immediate analysis or stored frozen prior to analysis.
Test organisms (species):
Colinus virginianus
Details on test organisms:
Test birds were housed indoors by dose group in batteries of pens manufactured by GQF Manufacturing Co. (Model No. 0010). Birds were assigned to pens by indiscriminate draw. Each pen had floor space that measured approximately 78 x 51 cm. Floors were sloped so that ceiling
height ranged from approximately 20 to 25 cm. External walls, ceilings and floors were constructed of galvanized wire while side walls were constructed of galvanized sheeting. Each dose group was assigned two pens. One pen contained five males and the other five females. Each group of birds was identified by pen number. Birds were maintained at ambient room temperature. The photoperiod (maintained by a time clock) was eight hours of light per day during acclimation and throughout each test phase. The light source was fluorescent lights which closely approximate the spectrum of noon-day sunlight. Environmental conditions during each test phase are summarized as follows:

Dose Mean Room Temperature Relative Humidity Illumination

100 mg/kg 23.5 ± 0.6°C (SD) 22± 13% 131 lux (12.2 ft. candles)
562.5 mg/kg 23.8 ± 0.6°C (SD) 22± 13% 94 lux (8.7 ft. candles)
1125 mg/kg 25.3 ± 0.9°C (SD) 26±9% 170 lux (15.8 ft. candles)
2250 mg/kg 25.4 ± 1.4 °C (SD) 64± 16% 133 lux (12.4 ft. candles)

Housing and husbandry practices were based on guidelines established by the National Research Council.

Test birds were purchased reproductively immature and maintained separately by sex at Wildlife International Ltd. under conditions that would not facilitate reproduction. All birds within a dose level and its concurrent control were from the same hatch, pen-reared and phenotypically
indistinguishable from wild birds. Birds were assigned to one test group and one control group for each test phase. Each group contained five males and five females. Individual birds within each pen were identified by colored leg bands. All test birds were acclimated to the caging and facilities for approximately 6 to 15 weeks prior to the initiation of a given phase of the test.

During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation until termination of each phase all birds were observed at least once daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior.

Throughout acclimation and testing all test birds were fed a game bird ration formulated to Wildlife International Ltd.'s specifications. Water, from the town of Easton public water supply, and feed were provided ad libitum during acclimation and during the test. The birds were fasted for approximately 18 to 23 hours prior to dosing dependent on test phase. The birds received no form of antibiotic medication during the test.
Post exposure observation period:
14 days
No. of animals per sex per dose and/or stage:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Nominal and measured doses / concentrations:
100, 562.5, 1125, and 2250 mg/kg (Nominal)
Details on test conditions:
Birds were acclimated for at least 6 weeks prior to test initiation. The birds were fasted for at least 18 hours prior to dosing. At initiation of each test phase, a single dose of the test substance in diluent (com oil) was orally intubated directly into the crop or proventriculus of each bird using a stainless steel cannula. Each bird was individually weighed and dosed on the basis of milligrams of test substance per kilogram of body weight. The control birds received a corresponding volume of diluent only. All treatment and control birds received a constant dose volume of eight milliliters per kilogram of body weight.
Details on examinations and observations:
During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation until termination of each phase all birds were observed at least once daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior.

Body weights were measured individually at the initiation of each test phase and on Days 3, 7, and 14. Average feed consumption was determined for the treatment group and the control group for Days 0-3, 4-7, and 8-14 for each test phase. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds.
Reference substance (positive control):
no
Key result
Duration (if not single dose):
1 d
Dose descriptor:
LD50
Effect level:
> 2 250 mg/kg bw
Conc. / dose based on:
test mat.
Remarks on result:
other: no effects at highest dose tested
Key result
Duration (if not single dose):
1 d
Dose descriptor:
NOEL
Effect level:
562.5 mg/kg bw
Basis for effect:
body weight
Mortality and sub-lethal effects:
There were no mortalities in any of the control groups and all control birds were normal in appearance and behavior throughout the test period. In addition, no mortalities occurred and no overt signs of toxicity were noted in the 100, 562.5 or 1125 mg/kg treatment groups. In the 2250 mg/kg treatment group, one death occurred on Day 3 of the test, resulting in a 10% (1/10) mortality rate. On the morning of Day 3, a single male in the treatment group was noted displaying depression, wing droop and prostrate posture. Approximately five hours later the bird was found dead. Gross necropsy of this bird revealed picking around the uropygial gland with some dried blood on the tail feathers; the kidneys were also noted as pale. While the signs of picking indicate some penmate aggression, a treatment related cause of death could not be precluded. All other birds at the 2250 mg/kg treatment group were normal in appearance and behavior for the duration of the test.

When treatments were compared with concurrent controls, there were no statistically significant treatment related effects on body weight among females in the 100 mg/kg dose group (P < 5%), and among males at doses ≤ 2250 mg/kg (P < 5%). Using the pooled control, there were again no
statistically significant treatment related effects on body weight among females in the 100 mg/kg dose group, or among males at doses ≤ 1125 mg/kg. Using body weight as a parameter, females were more sensitive than males. The NOEC for females was 100 mg/kg using both concurrent controls (t-test) and pooled controls (Dunnett's method). The NOEC for males was 2250 and 1125 mg/kg for concurrent and pooled controls, respectively.

There were no apparent effects upon feed consumption in the 100 and 562.5 mg/kg dose groups at any feed consumption interval. A marked reduction in feed consumption was noted in the 1125 and 2250 mg/kg dose groups for the period from Day 0 to Day 3, but feed consumption was comparable to the control groups at all other feed consumption intervals.
Conclusions:
The acute oral LD50 value for northern bobwhite quail exposed to the test substance as a single oral dose was greater than 2250 mg/kg, the highest dose tested. Based upon the single mortality in the 2250 mg/kg dose group, the no mortality level was 1125 mg/kg. Based upon effects on body weight at dose levels of 562.5 mg/kg and above, the no observed effect dose was 100 mg/kg.
Executive summary:

Groups of 5 male and 5 female bobwhite quail,Colinus virginianus, were administered by gavage, 0, 100, 562.5, 1125, or 2250 mg/kg of the test substance in corn oil according to EPA guideline OPP-71 -1.

There were no mortalities in any of the control groups and all control birds were normal in appearance and behavior throughout the test period. In addition, no mortalities occurred and no overt signs of toxicity were noted in the 100, 562.5 or 1125 mg/kg treatment groups. In the 2250 mg/kg treatment group one death occurred on Day 3 of the test, resulting in a 10% (1/10) mortality rate. On the morning of Day 3, a single male in the treatment group was noted displaying depression, wing droop and prostrate posture. Approximately five hours later the bird was found dead. Gross necropsy of this bird revealed picking around the uropygial gland with some dried blood on the tail feathers; the kidneys were also noted as pale. While the signs of picking indicate some penmate aggression, a treatment related cause of death could not be precluded. All other birds at the 2250 mg/kg treatment group were normal in appearance and behavior for the duration of the test.

 

When treatments were compared with concurrent controls, there were no statistically significant treatment related effects on body weight among females in the 100 mg/kg dose group (P < 5%), and among males at doses ≤ 2250 mg/kg (P < 5%). Using the pooled control, there were again no statistically significant treatment related effects on body weight among females in the 100 mg/kg dose group, or among males at doses ≤ 1125 mg/kg. Using body weight as a parameter, females were more sensitive than males. The NOEC for females was 100 mg/kg using both concurrent controls (t-test) and pooled controls (Dunnett's method). The NOEC for males was 2250 and 1125 mg/kg for concurrent and pooled controls, respectively.

 

There were no apparent effects upon feed consumption in the 100 and 562.5 mg/kg dose groups at any feed consumption interval. A marked reduction in feed consumption was noted in the 1125 and 2250 mg/kg dose groups for the period from Day 0 to Day 3, but feed consumption was comparable to the control groups at all other feed consumption intervals.

 

The acute oral LD50 value for northern bobwhite quail exposed to the test substance as a single oral dose was greater than 2250 mg/kg, the highest dose tested. Based upon the single mortality in the 2250 mg/kg dose group, the no mortality level was 1125 mg/kg. Based upon effects on body weight at dose levels of 562.5 mg/kg and above, the no observed effect dose was 100 mg/kg.

Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 206 (Avian Reproduction Test)
GLP compliance:
yes
Specific details on test material used for the study:
98.4% purity
Dose method:
feed
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and analysis of diet:
Test diets were prepared by mixing the test substance into a premix that was used for weekly preparation of the final diet. Control diet and three test concentrations were prepared weekly and presented to the birds on Wednesday of each week. Dietary concentrations were not adjusted for purity of the test substance and are presented as ppm. Homogeneity of the test substance in the diet was evaluated by collecting six samples each from the treatment groups on Day 0 of Week l. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. During Weeks 2, 3, 4, 8, 12, 16 and 20 of the test, a single sample was collected from the control diet and duplicate samples were collected from each treatment group diet to measure/verify test concentrations. Additionally, a single control and duplicate treatment diet samples were collected from the hoppers on Day 7 of Week I to assess stability of the test substance under actual test conditions. The diet samples were stored frozen prior to transfer to the Wildlife International Ltd. analytical chemistry for analysis.

The analyses of diet samples were performed at Wildlife International Ltd. using high performance liquid chromatography (HPLC) with UV detection. Homogeneity, verification, and stability samples were collected and submitted for analysis. A subsample of avian diet (10. 0 g) was weighed into a cellulose thimble. All samples were then placed in a soxhlet apparatus, acetonitrile (~300 mL) was added to the associated round bottom flasks, and extracted for ~3.0 hours at ~60°C. After extraction, each soxhlet apparatus was rinsed with acetonitrile and the sample brought to a volume of 500 ml in a volumetric flask. Samples were then diluted into the range of the calibration curve with acetonitrile. Concentrations of the test substance in extracts of the samples were determined by high performance liquid chromatography using a Hewlett-Packard Model 1090 High Performance Liquid Chromatograph (HPLC) equipped with a diode array detector (DAD). HPLC separations were achieved using a Hypersil Cl8 BDS analytical column (250 mm x 4.6 mm I.D., 5 µm particle size).
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
Pen-reared mallard ducks that were apparently healthy and phenotypically indistinguishable from wild birds, were purchased from Whistling Wings, Inc., Box 1, 113 Washington Street, Hanover, Illinois 61041. All birds were from the same hatch and were 23 weeks of age at test initiation (first day of exposure to test diet). Birds ranged in weight from 846 to 1280 grams at test initiation. The birds were approaching their first breeding season and had not been used in previous testing. At test initiation all birds were examined for physical injuries and general health. Birds that did not appear healthy were replaced with suitable extra birds which had been acclimated with the study birds and came from the same lot. Sex of the birds was determined by a visual examination of the plumage. Adult birds were identified by individual leg bands and randomly assigned to a pen and treatment group. Each pen was identified with a unique number, and groups of pens were identified by project number and concentration. All eggs laid during the study were marked with a permanent ink marking pen for identification. Hatchlings were identified by wing bands so that the ducklings could be traced to their parental pen of origin.
Total exposure duration (if not single dose):
21 wk
Post exposure observation period:
6 weeks (Final incubation, hatching, and 14-day offspring rearing period)
No. of animals per sex per dose and/or stage:
16
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
150, 450, and 1350 ppm
Details on test conditions:
The test substance was administered ad libitum in the diet to groups of 16 pairs of young adult mallard ducks (23-weeks old at test initiation) approaching their first breeding season. Mallard ducks received the test substance at nominal dietary concentrations of 150, 450 and 1350 ppm for approximately 21 weeks. A control group was maintained concurrently with the treatment groups.

For the first nine weeks of the test the birds were held under a photoperiod of seven hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 10 to induce egg laying. The adults continued on a photoperiod of 17 hours of light per day until adult termination. Eggs were collected daily from the onset of egg production and set weekly for incubation. The first eggs were set during Week 12. Weekly throughout the laying period, eggs were collected from every other pen for egg shell thickness measurements. In addition, effects upon egg production and quality, and hatchling health and survivability were examined.

Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult mallard duck study room during the course of the test was 22.4 °C ± 1.5°C (SD) with an average relative humidity of 64% ± 16% (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air. The photoperiod in the adult mallard duck room was maintained by a time clock The photoperiod during acclimation and the first nine weeks of the test was seven hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week l0 and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 157 lux (~14 ft. candles) of illumination provided by fluorescent lights which closely approximated noon-day sunlight. Hatchlings were placed in batteries of brooding pens manufactured by Safeguard Products Inc. Each pen measured approximately 62 x 92 x 25. 5 cm high. The pens were constructed of vinyl coated wire mesh and stainless steel sheeting. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38° C from the time of hatching until the birds were 5 to 7 days of age. At that time, thermostats were reset to maintain an average temperature of approximately 29°C. The average ambient room temperature was 24.5°C ± l.5°C (SD) with an average relative humidity of 74%± 14% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day.

In an evaluation of this study, EPA indicated the floor space was less than minimally recommended for this species. In response, Wildlife International issued the following statement to EPA:

“While it would intuitively seem that ‘bigger’ would be better, that is not the case when housing birds designed to be ‘as close to wild type as possible’. For mallards, a bigger cage can result in more injuries, rather than less. These birds need sufficient room to stretch and make normal comfort movements, but not so much room as to encourage any attempt at flight. Cage size also impacts the ability to catch the birds for necessary handling without undue stress. A cage sized so that the bird can be easily caught gently efficiently limits the amount of chase and handling time.

It also needs to be recognized that maintain birds ‘as close to wild type as possible’ means the birds typically used are a ‘flighting’ type bird that tend to be more ‘high strung’ and reactive and consequently more prone to self (or cage mate) inflicted injury. While environmental enrichment, treatment of injuries (bandaging, suturing, etc.) and separation of incompatible pen mates are part of routine husbandry at Wildlife International, we continue to pursue ways to minimize impact upon birds”.

In light of the information provided, Wildlife International requested EPA re-evaluate the validity of this study. No additional comments were received from EPA.
Details on examinations and observations:
All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. A record was maintained of all mortalities and observations. At the conclusion of the exposure period all surviving adult birds were euthanized by cervical dislocation, necropsied and disposed of by incineration. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have had on egg production.

For each pen, feed consumption was measured weekly throughout the test, except the last interval, where feed consumption was measured over a two-day period. Feed consumption was determined by weighing the freshly filled hopper on Day 0, recording the amount of any additional diet added during the week and weighing the hopper and remaining feed at the end of the feeding period (Day 7). No attempt was made to quantify the amount of feed wasted by the birds, as the wasted feed is normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate.
Details on reproductive parameters:
Eggs were collected daily and stored in a cold room maintained at a mean temperature of 13.7°C ± 0.6 °C (SD) with a mean relative humidity of approximately 76% ± 6% (SD). Eggs to be incubated were washed to reduce the possibility of pathogen contamination before storing them in the cold room. Eggs were washed in a commercial egg washer (Kuhl Egg Washer) with a chlorine based detergent (Kuhl Super CD). Water in the washer was warmed to approximately 45°C. The eggs were placed in the wash water and soaked for approximately 15 seconds. The washer's circulation motor was then turned on for approximately three minutes. The eggs were removed from the washer, allowed to cool to approximately room temperature and rinsed with fresh water. The eggs were then ready for storage in the cold room.

Eggs were set for incubation on a weekly basis. The eggs were placed in the incubator where the temperature was maintained at an average 37.5°C ± 0.0°C (SD) with an average wet bulb temperature of 30.0°C ± 0.0°C (SD) (relative humidity of approximately 56%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement that was designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50° off of vertical in one direction to 50° off of vertical in the opposite direction (total arc of rotation was 100°) every two hours through Day 24 or 25 of incubation. The eggs were transferred to the hatcher on Day 24 or 25. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 °C ± 0.1°C (SD), and the average wet bulb temperature was raised to 33.4°C ±0.3 °C (SD) (relative humidity of approximately 76%).
Reference substance (positive control):
no
Key result
Duration (if not single dose):
21 wk
Dose descriptor:
NOEC
Effect level:
1 350 other: ppm
Conc. / dose based on:
test mat.
Basis for effect:
reproductive parameters
Remarks on result:
other: no effects at the highest dose tested
Mortality and sub-lethal effects:
No mortality or overt signs of toxicity
Effects on reproduction:
There were no effects on reproduction at the highest dose tested
Validity criteria fulfilled:
yes
Conclusions:
There were no statistically significant (p < 0.05) treatment related effects upon reproductive performance in the 150, 450 or 1350 ppm treatment groups. Based upon no statistically significant (p < 0.05) treatment related effects upon reproductive performance, the no observed effect concentration (NOEC) for mallards exposed to the test substance in the diet during this study was 1350 ppm, the highest concentration tested.
Executive summary:

Mallards were exposed to the test substance at dietary concentrations of 0, 150, 450 or 1350 ppm for 21 weeks according to OECD guideline 206. There were no treatment related mortalities or overt signs of toxicity at any of the concentrations tested. No effects upon adult body weight or feed consumption were noted in the 150 or 450 ppm treatment groups. However, in the 1350 ppm treatment group, feed consumption was reduced for the first week of the study, but subsequently increased during Weeks 3 to 7. This was the probable cause of the transient loss of body weight observed at the 1350 ppm test concentration during the first two weeks of the study. This appears to have been an initial palatability effect of no significance for survival or reproduction. There were no statistically significant (p < 0.05) treatment related effects upon reproductive performance in the 150, 450 or 1350 ppm treatment groups. Based upon no statistically significant (p < 0.05) treatment related effects upon reproductive performance, the no observed effect concentration (NOEC) for mallards exposed to the test substance in the diet during this study was 1350 ppm, the highest concentration tested.

Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 206 (Avian Reproduction Test)
Deviations:
no
Remarks:
See "Any other information on materials and methods" section for a list of the protocol deviations
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Dose method:
feed
Analytical monitoring:
yes
Remarks:
See "Any other information on materials and methods" for information on the analytical monitoring methods.
Vehicle:
yes
Remarks:
basal diet
Details on preparation and analysis of diet:
Test diets were prepared by mixing the technical test substance into a premix that was used for weekly preparation of the final diet. Control diet and each of the three treated diets were prepared weekly and presented to the birds on Tuesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active substance (ppm a.s.).

Analysis: Samples were extracted with acetonitrile. Concentrations of the test substance in extracts of the samples were determined by high performance liquid chromatography using a Waters Alliance High Performance Liquid Chromatograph (HPLC) equipped with a Waters 486 variable wavelength detector (VWD). High performance liquid chromatographic separations were achieved using a YMC ODS-AM analytical column (150 mm x 4.6 mm I.D., 3 μm particle size).
Test organisms (species):
Colinus virginianus
Details on test organisms:
Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council. The adult birds were housed indoors in batteries of pens manufactured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330), measuring approximately 25 x 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. Sisal ropes were added to each pen for animal enrichment from the time of photostimulation to termination. Each pen was equipped with feed and water troughs. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water troughs were changed and water added as necessary to provide potable water (generally every 2-3 days).
Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult northern bobwhite study room during the course of the test was 21.7 ± 0.7°C (SD) with an average relative humidity of 68 ± 9% (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air.

The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeks of the test was eight hours or less of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 250 lux (~ 23 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight.

The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Cargill Animal Nutrition, Shippensburg, PA. The basal ration contained at least 27% protein and 2.5% crude fat, and no more than 3.8% crude fiber.

The basal diet contained approximately 1.0% calcium, derived from feedstuffs and the 0.62% limestone used in the formulation of the basal diet by Cargill. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.4%) (4). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone.

Water was supplied by the town of Easton public water supply. All offspring received a water-soluble vitamin and electrolyte mix in their water.

Age of test animals: 42 weeks of age at the initiation of the test
Total exposure duration (if not single dose):
47 wk
Remarks:
Acclimation - 21 weeks. 2. Pre-photostimulation - 7 weeks. 3. Pre-egg laying (with photostimulation) - 2 weeks. 4. Egg laying - Approximately 11 weeks 5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 6 weeks.
Post exposure observation period:
Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 6 weeks.
No. of animals per sex per dose and/or stage:
16
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
300 ppm a.s./313 ± 11.1 ppm a.s.
600 ppm a.s./604 ± 22.4 ppm a.s.
1200 ppm a.s./1260 ± 26.4 ppm a.s.
Details on examinations and observations:
During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations.

At the conclusion of the exposure period, all surviving adult birds were euthanized by asphyxiation with carbon dioxide gas, necropsied, and disposed of by incineration.

Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8 and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production.

Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week and weighing the feeder and remaining feed at the end of the feeding period (Day 7). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a “feed-saver” lip. Feed wastage was further reduced by placement of a piece of wire grid on the top of the feed. The wire grid allowed to birds to feed unencumbered, but prevented the birds from scooping or pushing feed out of the feeder. The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption.
Details on reproductive parameters:
Eggs were collected daily from all pens, when available. The eggs were stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.1 ± 0.1°C (SD) with a mean relative humidity of approximately 90 ± 0% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot.

At the end of the weekly interval, all eggs were removed from the cold room, counted and eggs selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility of pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 20 g of potassium permanganate and 19 ml of 37% commercial grade formalin in a porcelain bowl at the base of the airtight cabinet.

All eggs not discarded or used for egg shell thickness measurements were placed in a NatureForm Incubator/Hatcher (Model No. NMC 4000). In the incubator the temperature was maintained at an average 37.4 ± 0.0°C (SD) with an average relative humidity of 55 ± 0% (SD). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 45° off of vertical in one direction to 45° off of vertical in the opposite direction (total arc of rotation was 90°) every two hours through Day 21 of incubation. Eggs were candled on Day 11 of incubation to determine embryo viability and on Day 21 to determine embryo survival.

On Day 21 of incubation, the eggs were placed in a Petersime Hatcher (Model No. S6H) and allowed to hatch. Pedigree baskets constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 ± 0.0°C (SD), and the average wet bulb temperature was 33.3 ± 0.0°C (SD) (relative humidity of approximately 77%).

All hatchlings, unhatched eggs and egg shells were removed from the hatcher on Day 25 or 26 of incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were leg banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. At 14 days of age, the average body weight by parental pen of all surviving chicks was determined. The chicks were euthanized by asphyxiation with carbon dioxide and disposed of by incineration.

Hatchlings were housed in batteries of brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 x 90 X 23 cm high. The external walls and ceilings of each pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of galvanized wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38° C from the time of hatching until the birds were 14 days of age. The average ambient room temperature was 25.8 ± 0.7°C (SD) with an average relative humidity of 50 ± 14% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day.

Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered pens during odd numbered weeks (1,3,5, etc.) and from each of the even numbered pens during the even numbered weeks (2,4,6, etc.). The eggs were opened at the waist, the contents removed, and the shells thoroughly rinsed with water. The shells were then allowed to air dry for at least two weeks at room temperature. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometer. Measurements were made to the nearest 0.002 mm.
Reference substance (positive control):
no
Key result
Duration (if not single dose):
20 wk
Dose descriptor:
NOEC
Effect level:
1 200 other: ppm a.s.
Conc. / dose based on:
test mat.
Remarks on result:
other: no effects observed at highest dose tested
Mortality and sub-lethal effects:
While no mortalities occurred in the control group or in either the 600 or 1200 ppm a.s. treatment groups, two incidental mortalities occurred during the study in the 300 ppm a.s. treatment group. The first mortality was the female in Pen 118 of the 300 ppm a.s. treatment group that was found dead on Day 6 of Week 12. Prior to being found dead, the female was noted to have a head lesion with swelling. At necropsy, was emaciated, with feather loss and a loss of muscle mass, prominent keel and no fat in the coronary groove. The bird had an extensive head lesion with necrotic margins and the cranium was exposed. Internally, the kidneys were pale, liver margins pale, spleen small and pale and the gastrointestinal tract was primarily empty. The female’s ovary was developing. Necropsy of the female’s penmate indicated slight feather loss, but the male was otherwise unremarkable.

The second incidental mortality was the male in Pen 123 of the 300 ppm a.s. treatment group that was euthanized on Day 2 of Week 13. Prior to being euthanized, the male was noted as thin (weighing 144 g), with a head lesion, depression, ruffled appearance and moribund. At necropsy the male had feather loss and extensive head lesions with necrotic margins, multiple abscesses with caseous necrosis and the cranium was exposed. The male was emaciated, with a loss of muscle mass, prominent keel and no fat in the coronary groove. The male’s right testis was small, ~ 1.25 cm. Necropsy of the male’s penmate indicated feather loss and a small abscess with caseous necrosis on the head, but was otherwise unremarkable.

No other mortalities occurred during the course of the study. Due to the timing of the mortalities, the lack of concentration responsiveness and the nature of the lesions observed at necropsy, the mortalities that occurred were considered to be unrelated to treatment.

No overt signs of toxicity were observed at any of the concentrations tested. Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear. Such signs included feather loss, wing, foot, back and head lesions, bruising and swelling. Additional clinical observations included lameness, a ruffled appearance and two females noted as thin, one in each of the 300 and 600 ppm a.s. treatment groups. The male in Pen 148 of the 600 ppm a.s. treatment group suffered a leg fracture during body weight measurements. The break was splinted and healed. No other injuries occurred. With the exception of incidental findings, all birds appeared normal throughout the study.

All surviving adults were subjected to gross necropsy following adult termination. All findings observed were considered unrelated to treatment.

There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences between the control group and the 300, 600 or 1200 ppm a.s. treatment groups were observed at any of the body weight intervals.

There were no apparent treatment-related effects upon feed consumption at the 300, 600 and 1200 ppm a.s. test concentrations. No statistically significant differences between the control group and the 300 or 600 ppm a.s. treatment groups were observed at any of the feed consumption intervals. In the 1200 ppm a.s. treatment group, there were slight, but statistically significant (p < 0.05), increases in feed consumption during Weeks 12, 17 and 18. However, the differences observed were slight and not consistent over time and therefore, were not considered to be related to treatment.
Effects on reproduction:
There were no treatment-related effects upon reproductive performance at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 600 or 1200 ppm a.s. treatment groups. At the 300 ppm a.s. test concentration there was a slight reduction in the number of 14-day old survivors as a percentage of hatchlings that was statistically significant (p < 0.01). However, the difference observed was not concentration dependent and was attributed to several instances of pen mate aggression that occurred during brooding, particularly during Lots E, H and I. In several brooder pens the offspring were observed with lesions of toe or nostril picking, resulting in a higher incidence of mortality. The aggression was limited to individual brooder pens within the week lots impacted and was not considered to be treatment related. There were no other statistically significant differences between the control group and the 300 ppm a.s. treatment group for any other reproductive parameter measured.

There were no apparent treatment related effects upon shell thickness at any of the
concentrations tested. When compared to the control group, there were no statistically significant differences in shell thickness in the 300, 600 or 1200 ppm a.s. treatment groups.

There were no apparent treatment related effects upon offspring body weight at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from the 300, 600 or 1200 ppm a.s. treatment groups.
Validity criteria fulfilled:
yes
Conclusions:
There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon reproductive performance parameters measured at the 300, 600 or 1200 ppm a.s. test concentrations. The no-observed-effect concentration for northern bobwhite exposed to the technical test substance in the diet during this study was 1200 ppm a.s. (equivalent to 110.3 mg a.s./kg/day), the highest concentration tested.
Executive summary:

The objective of this study was to evaluate the effects upon the adult northern bobwhite (Colinus virginianus) of dietary exposure to the technical test substance over a five-month period according to OECD guideline 206. Effects on adult health, weight gain and feed consumption were evaluated. In addition, the effects of adult exposure to the test substance on the number of eggs laid, normal development of eggs, viability of the embryos, percent hatchability, offspring survival and egg shell thickness were evaluated.

 

Each treatment and control group contained 16 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 300, 600 or 1200 ppm a.s. of the technical test substance for 20 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance.

 

All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8 and at adult termination and feed consumption was measured weekly throughout the test. At the beginning of Week 8, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 21 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups.

 

While no mortalities occurred in the control group or in either the 600 or 1200 ppm a.s. treatment groups, two incidental mortalities occurred during the study in the 300 ppm a.s. treatment group. The first mortality was the female in Pen 118 of the 300 ppm a.s. treatment group that was found dead on Day 6 of Week 12. Prior to being found dead, the female was noted to have a head lesion with swelling. At necropsy, was emaciated, with feather loss and a loss of muscle mass, prominent keel and no fat in the coronary groove. The bird had an extensive head lesion with necrotic margins and the cranium was exposed. Internally, the kidneys were pale, liver margins pale, spleen small and pale and the gastrointestinal tract was primarily empty. The female’s ovary was developing. Necropsy of the female’s penmate indicated slight feather loss, but the male was otherwise unremarkable.

 

The second incidental mortality was the male in Pen 123 of the 300 ppm a.s. treatment group that was euthanized on Day 2 of Week 13. Prior to being euthanized, the male was noted as thin (weighing 144 g), with a head lesion, depression, ruffled appearance and moribund. At necropsy the male had feather loss and extensive head lesions with necrotic margins, multiple abscesses with caseous necrosis and the cranium was exposed. The male was emaciated, with a loss of muscle mass, prominent keel and no fat in the coronary groove. The male’s right testis was small, ~ 1.25 cm. Necropsy of the male’s penmate indicated feather loss and a small abscess with caseous necrosis on the head, but was otherwise unremarkable.

 

No other mortalities occurred during the course of the study. Due to the timing of the mortalities, the lack of concentration responsiveness and the nature of the lesions observed at necropsy, the mortalities that occurred were considered to be unrelated to treatment.

 

No overt signs of toxicity were observed at any of the concentrations tested. Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear. Such signs included feather loss, wing, foot, back and head lesions, bruising and swelling. Additional clinical observations included lameness, a ruffled appearance and two females noted as thin, one in each of the 300 and 600 ppm a.s. treatment groups. The male in Pen 148 of the 600 ppm a.s. treatment group suffered a leg fracture during body weight measurements. The break was splinted and healed. No other injuries occurred. With the exception of incidental findings, all birds appeared normal throughout the study.

 

All surviving adults were subjected to gross necropsy following adult termination. All findings observed were considered unrelated to treatment.

 

There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences between the control group and the 300, 600 or 1200 ppm a.s. treatment groups were observed at any of the body weight intervals.

 

There were no apparent treatment-related effects upon feed consumption at the 300, 600 and 1200 ppm a.s. test concentrations. No statistically significant differences between the control group and the 300 or 600 ppm a.s. treatment groups were observed at any of the feed consumption intervals. In the 1200 ppm a.s. treatment group, there were slight, but statistically significant (p < 0.05), increases in feed consumption during Weeks 12, 17 and 18. However, the differences observed were slight and not consistent over time and therefore, were not considered to be related to treatment.

 

There were no treatment-related effects upon reproductive performance at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 600 or 1200 ppm a.s. treatment groups. At the 300 ppm a.s. test concentration there was a slight reduction in the number of 14-day old survivors as a percentage of hatchlings that was statistically significant (p < 0.01). However, the difference observed was not concentration dependent and was attributed to several instances of pen mate aggression that occurred during brooding, particularly during Lots E, H and I. In several brooder pens the offspring were observed with lesions of toe or nostril picking, resulting in a higher incidence of mortality. The aggression was limited to individual brooder pens within the week lots impacted and was not considered to be treatment related. There were no other statistically significant differences between the control group and the 300 ppm a.s. treatment group for any other reproductive parameter measured.

 

There were no apparent treatment related effects upon shell thickness at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in shell thickness in the 300, 600 or 1200 ppm a.s. treatment groups.

 

There were no apparent treatment related effects upon offspring body weight at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from the 300, 600 or 1200 ppm a.s. treatment groups.

 

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon reproductive performance parameters measured at the 300, 600 or 1200 ppm a.s. test concentrations. The no-observed-effect concentration for northern bobwhite exposed to the technical test substance in the diet during this study was 1200 ppm a.s. (equivalent to 110.3 mg a.s./kg/day), the highest concentration tested.

Description of key information

LD50 (zebra finch): >1350 mg/kg; EPA OPPTS 850.2100; Reliability = 1

8-day NOEC (bobwhite): 5200 ppm; OECD 205; Reliability = 1

5-day NOEC (mallard): 5200 ppm; OECD 205; Reliability = 1

LD50 (bobwhite): >2250 mg/kg; EPA OPP 71 -1; Reliability = 1

21-week NOEC (mallard): 1350 ppm; OECD 206; Reliability = 1

21 -week NOEC (bobwhite): 1200 ppm; OECD 206; Reliability = 1

Key value for chemical safety assessment

Short-term EC50 or LC50 for birds:
1 350 mg/kg food

Additional information