Diquat dibromide

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to birds: acute oral toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 2013 to 28 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 223 (Avian Acute Oral Toxicity Test)

Version / remarks:

July 2012

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2100 (Avian Acute Oral Toxicity Test)

Version / remarks:

2012

Deviations:

not applicable

GLP compliance:

yes

Dose method:

gavage

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
The test substance was dispersed in reverse osmosis deionized water. The reverse osmosis deionized water had been dyed blue with a food grade colorant to aid in the determination of regurgitation. Individual doses were prepared based upon bird weights to provide a constant weight to body weight dosage for all treatment birds. The dosages were adjusted to 100% cation content. Therefore, all dosages and the LD50 value are reported as milligrams of cation of test substance per kilogram of body weight.

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
Results of the homogeneity and stability of the test material in Diet was provided in Table 1 in 'Any other information on materials and methods incl. tables'.

Samples of each dosing solution were collected for chemical analysis on the day of dosing. Three samples from the high and low dosing solutions were taken at approximately 10-second intervals during each stage while the solutions were being continuously mixed for verification and homogeneity. One sample was taken from all other dosage levels for verification.
Samples were diluted with 0.1% phosphoric acid. The concentration of cation in dilutions of the samples were determined by HPLC. The methodology was developed by the test facility. Calibration standards of the cation, ranging in concentration from 1.00 to 10.0 μg a.i./mL, were analyzed with the samples. A linear regression equation was generated using the peak area responses versus the respective concentrations of the calibration standards. The concentration of test substance in the samples was determined by substituting the peak area responses of the samples into the linear regression equation. The method limit of quantitation (LOQ) for these analyses was set at 50.0 ppm a.i. based upon the product of the lowest analytical standard 1.00 μg a.i./mL and the dilution factor (50) of the matrix blank extract. Measured values greater than or equal to the LOQ were reported. The method limit of quantitation (LOQ) for these analyses was set at 50.0 ppm a.i. based upon the product of the lowest analytical standard 1.00 μg a.i./mL and the dilution factor (50) of the matrix blank extract. Measured values greater than or equal to the LOQ were reported.
 
HPLC parameters
- Column: Zorbax SB-Phenyl (250 mm x 4.6 mm I.D., 5 μm particle size)
- Flow rate: 1.200 mL/minute
- Oven temperature: 40˚C
- Wavelength: 290 nm

Test organisms (species):

other: Taeniopygia guttata

Details on test organisms:

TEST ORGANISM
- Common name: Zebra finches
- Health condition: Adult plumage and appeared to be in good health at initiation of the test
- Age at time of receipt: Four to eight months old
- Weight at test initiation: 11.2 - 16.0 g
- Sexes used / mixed or single sex: Males and females were identified by the phenotypic plumage. Birds were randomly assigned to test pens. Birds were kept in a sexually quiescent state by maintaining them under a shortened photoperiod and keeping the sexes separated.

Limit test:

no

Total exposure duration (if not single dose):

30 d

Post exposure observation period:

14 days

No. of animals per sex per dose and/or stage:

- Stage 1: One zebra finch per dosage level
- Stage 2: One zebra finch per dosage level
- Stage 3: Wwo zebra finches per dosage level

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

- Nominal concentrations (stage 1): 0 (negative control), 1.41, 5.21, 19.2 and 70.7 mg cation/kg bw
- Nominal concentrations (stage 2): 0 (negative control), 3.43, 4.35, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0, and 29.2 mg cation/kg bw
- Nominal concentrations (stage 3): 0 (negative control), 20.3, 25.8, 32.8, 41.6 and 52.8 mg cation/kg bw

Details on test conditions:

ACCLIMATION
- Acclimation period: At least nine weeks prior to test initiation
- Acclimation conditions: In the same room as test
- Feeding: At the start of acclimation all test birds were fed a commercially available finch food and golden sunburst millet sprays. During the acclimation period the birds were transitioned to a pelleted diet. During acclimation and test, grit was provided to aid with the birds’ digestion.
- Water and medication: Water from the town of Easton public water supply and feed were provided ad libitum during acclimation and during the test, except during periods of fasting prior to testing. The birds received no form of antibiotic medication during acclimation or during the test.
- Fasting period before study: Approximately 2 - 3 hours

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Approximately 29 x 26 cm with a ceiling height of 31 cm. External walls, ceilings and floors were constructed of coated wire with 1 cm openings between the wires while sidewalls were fiberglass. Each pen contained a cuttlebone and perches constructed of wood doweling, plastic or cement composite.
- Suitable to avoid crowding stress: yes
- Husbandry practices: Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council.
- Caging: Individual

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 10
- Stage 1: One zebra finch per dosage level
- Stage 2: One zebra finch per dosage level
- Stage 3: Wwo zebra finches per dosage level

TEST CONDITIONS
- Temperature: 20.0 ± 0.9°C (18.2 °C - 21.4 °C)
- Relative humidity (%): 25.1 ± 11.0% (12.6% - 62.2%)
- Photoperiod: Approximately 0.25 hour dim light/eight hours light/0.25 hour dim light/15.5 hours dark throughout the test. The quarter hour of dim light prior to the light and dark phases allowed birds to settle.
- Light intensity: Approximately 236 lux of illumination. (The light source was fluorescent lights that closely approximated noonday sunlight.)
- Feed: During the test, the birds were fed with the same pelleted diet (size xs) as in the acclimation period

METHOD OF APPLYING TEST SUBSTANCE
At the experimental start, a single dose of the test substance in diluent was orally intubated directly into the crop or proventriculus of each bird using a stainless steel 14 gauge cannula. The blue dye was used to improve detection of any regurgitation of the dose. Each bird was individually weighed and dosed on the basis of milligrams cation of test substance per kilogram of body weight Each control bird received the diluent in the same volume to body weight ration as the treatment birds.

RANGE FINDING STUDY (non-GLP)
- Test concentrations: 11.7, 43.2, 159 and 587 mg cation/kg (One bird each dosage.)
- Results used to determine the conditions for the definitive study: The bird in the 11.7 mg cation/kg dosage level did not regurgitate the dose while the other three birds did. The bird in the 11.7 mg cation/kg dosage level died. Based on the mortality at the 11.7 mg cation/kg dosage level the initial test dosages for Stage 1 of 1.41, 5.21, 19.2 and 70.7 mg cation/kg were thus established. Stage 2 dosage levels were 3.43, 4.35, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0 and 29.2 mg cation/kg. Stage 3 dosage levels were 20.3, 25.8, 32.8, 41.6 and 52.8 mg cation/kg.

Details on examinations and observations:

OBSERVATIONS
During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. On the day of dosing (Day 0), all birds were observed at least 60 minutes following dosing and multiple times after the completion of dosing to determine the onset of clinical signs of toxicity and any indication of regurgitation. All birds were observed twice daily for the remainder of the test. A record was maintained of all signs of toxicity and abnormal behavior.

BODY WEIGHT
Body weights were measured individually at the initiation of the test and on Days 3, 7 and 14 of the test. For body weight determination, birds are caught by knowledgeable, trained personnel. The key to minimizing stress on a bird is to capture a bird efficiently but gently, and to hold the birds “caged” in the hand so as to not allow escape, but to minimize pressure.

FOOD CONSUMPTION
Average estimated feed consumption was determined by pen for each dosage group and the control group for approximately 24-hour intervals from Days 0 to 1, Days 1 to 2, and Days 2 to 3. Average estimated feed consumption was then measured from Days 3 to 7, Days 7 to 10 and Days 10 to 14. Given the nature of feed presented to the birds and their seed hulling and wasteful behavior, feed consumption measurements were done to qualitatively evaluate feeding and feeding behavior. The accuracy of feed consumption values may have been affected by unavoidable wastage of feed by birds. No attempts were made to quantify the amount of wasted feed, as it is normally scattered and mixed with water and excreta.

NECROPSY
Gross necropsies were performed on all of the mortalities and on four control birds surviving to test termination and all treatment birds surviving to test termination. The 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.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

30 d

Dose descriptor:

LD50

Effect level:

57.7 mg/kg bw

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Recalculated value, expressed as pure substance

Duration (if not single dose):

30 d

Dose descriptor:

LD50

Effect level:

30.9 mg/kg bw

Conc. / dose based on:

act. ingr.

Remarks:

cation

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Mortality and sub-lethal effects:

An overview of the results is provided in Table 3 - Table 5 in 'Any other information on results incl. tables'
- Mortality: There were no mortalities in the control group (0 of 10). There were also no mortalities in the 1.41, 3.43, 4.35, 5.21, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0, 25.8 and 29.2 mg cation/kg dosage groups. There was 50% (1 of 2) mortality among the birds at the 20.3 and 41.6 mg cation/kg dosage level, 67% mortality (2 of 3) at the 32.8 mg cation/kg dosage level and 100% mortality at the 19.2, 52.8 and 70.7 mg cation/kg dosage levels.
- Clinical Observations: No incidences of regurgitation were noted for the control birds. Regurgitation was noted among some of the birds at the 32.8 and 52.8 mg cation/kg dosage levels of stage 3 of the test. One of the two birds originally dosed at the 32.8 and 52.8 mg cation/kg dosage levels regurgitated the dose. Two more birds were dosed at the two levels and both birds retained the dose at the 32.8 mg cation/kg dosage level and one of two regurgitated at the 52.8 mg cation/kg dosage level. No other treatment birds were noted as regurgitating.
All birds in the control group and in the 1.41, 3.43, 4.35, 5.21, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0 and 29.2 mg cation/kg dosage groups were normal in appearance and behavior throughout the test. Signs of toxicity or mortality occurred at the 19.2, 20.3, 25.8, 32.8, 41.6, 52.8 and 70.7 mg cation/kg dosage levels. Signs of toxicity were first observed on the day of dosing or on the morning of Day 1. Signs of toxicity included ruffled appearance, lethargy, prostrate posture, loss of righting reflex, minor muscle fasciculation and depression. All surviving birds were normal in appearance and behavior from the afternoon of Day 2 until termination. All mortalities occurred on the day of dosing or were noted on the morning of Day 1 of the test.

- Body Weights: From Day 0 to Day 3, all control birds exhibited a gain in body weight (0.8 to 1.6 grams). The surviving treatment birds also exhibited a gain in body weight from Day 0 to Day 3 of the test which ranged from 0.2 to 2.7 grams. Body weight changes for all intervals were comparable among the control and most surviving treatment birds.

- Feed Consumption: The control group had feed consumption values on Day 0 to Day 1 of 2.6 to 5.2 g, on Day 1 to Day 2 of 3.5 to 4.7 g and on Day 2 to Day 3 of 3.3 to 4.3 g. Among the surviving treatment birds (total of 18 birds) 4 birds for Day 0 to Day 1, 5 birds from Day 1 to Day 2 and 3 birds from Day 2 to Day 3 had a feed consumption values that were less than the minimum control value for that period. For all other feed consumption intervals, there were no apparent treatment related effects upon feed consumed among the surviving birds.

- Necropsy: Gross necropsies were performed on all of the mortalities. Gross necropsies were also performed on all treatment birds surviving to test termination and four of the ten control birds surviving to test termination. Of the birds surviving to test termination the only findings noted were for the birds in the 1.41 and 5.21 mg cation/kg dosage levels and for 3 of the 4 control birds necropsied. These birds were noted with intracranial bleeding and the bird in the 1.41 mg cation/kg dosage level was also noted with a subcutaneous bruise.

Reported statistics and error estimates:

The mortality data was analyzed using the computer program of SEDEC. The program was designed to calculate the LD50 value and the 95% confidence interval by probit analysis. No statistical analyses were applied to separate mean responses among treatment groups for the endpoints of food consumption and body weight.

Table 3. Cumulative mortality

Experimental group (mg cation/kg bw)

Number Dead / Number exposed

Day of test

Sex

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Total

Control

0

M

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

F

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

Stage 1

1.41

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

5.21

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

19.2

M

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

70.7

F

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

Stage 2

3.43

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

4.35

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

5.52

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

7

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

8.88

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

11.3

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

14.3

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

18.1

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

23

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

29.2

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

Stage 3

20.3

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

20.3

F

0/1

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/2

25.8

M

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

25.8

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/2

32.8

M

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

32.8

F*

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

32.8

M

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

32.8

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

2/3*

41.6

M

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

41.6

F

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

0/1

1/2

52.8

M

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

52.8

F*

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

52.8

M*

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

52.8

F

0/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

1/1

2/2*

 * Bird noted as regurgitating, regurgitating birds were not counted in the total mortality count and were not used in the calculation of the LD50 value.

Table 4. Summary of sign of toxicity

Stage	Nominal dosage (mg cation/kg)	First noted signs of toxicity after dosing	Signs of toxicity*	Last day with signs of toxicity	Day of mortality
Stage 1	1.41	NA	NA	NA	NA
	5.21	NA	NA	NA	NA
	19.2	Day 1	M	1	1
	70.7	1hr 52 min	11,14,M	1	1
Stage 2	3.43	NA	NA	NA	NA
	4.35	NA	NA	NA	NA
	5.52	NA	NA	NA	NA
	7.00	NA	NA	NA	NA
	8.88	NA	NA	NA	NA
	11.3	NA	NA	NA	NA
	14.3	NA	NA	NA	NA
	18.1	NA	NA	NA	NA
	23.0	NA	NA	NA	N A
	29.2	NA	NA	NA	NA
Stage 3	20.3	Day 1	M	1	1
	25.8	Day 1	11	1	NA
	32.8	4 hrs 8 min	6,11	2	1
	41.6	2 hrs 24 min	11,M	1	1
	52.8	2 hrs 19 min	1,5,11,14,16,M	1	0 & 1

NA = Not applicable

* Sign of toxicity: 1 = depression, 5 = prostrate posture, 6 = loss of righting reflex, 11 = ruffled appearance, 14 = lethargy, 16 = minor muscle fasciculation, M = mortality

Table 5. Summary of gross pathological findings

Stage	1	1	3	3	3	3	3	3	3	3
Treatment groups (mg cation/kg)	19.2	70.7	20.3	32.8	32.8	41.6	52.8	52.8	52.8	52.8
Pen	Z11	Z12	Z54	Z57	Z63	Z59	Z61	Z62	Z65	Z66
White plaques in pericardium	-	-	X	-	-	-	-	-	X	X
Left lung: some consolidation	-	-	-	-	-	-	-	-	-	X
Spleen pale	-	-	X	-	-	X	-	-	-	X
Liver pale	-	X	-	-	X	-	-	-	-	X
Liver mottled	-	X	-	-	-	-	-	-	-	-
White plaques covering the liver	-	-	X	-	-	-	-	-	X	X
White plaques in abdominal cavity	-	-	-	-	-	-	-	-	-	X
White plaques covering gizzard	-	-	-	-	-	-	-	-	-	X
GI tract primarily empty	-	-	X	-	X	-	X	X	-	X
GI tract contains yellowish fluid, otherwise primarily empty	-	-	-	-	-	X	-	-	-	-
Kidneys pale	-	-	X	X	-	X	-	-	X	X
Kidneys mottled	-	-	X	-	-	X	-	-	-	X

 

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 30.9 mg cation/kg bw = 57.7 mg registered substance/kg bw

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the acute oral LD50 value for zebra finch was calculated to be 30.9 mg cation/kg with a 95% confidence interval of 18.7 to 77.7 mg cation/kg.

Executive summary:

The effect of the test substance on the zebra finch (Taeniopygia guttata) were determined in an acute toxicity test. The test was conducted according OECD TG 223 and OCSPP 850.2100 guideline. This study was in compliance with GLP criteria. The test organisms (three different stages) exposed to the test substance via gavage.

 Stage 1 with one zebra finch per dosage level at four dosage levels (1.41, 5.21, 19.2 and 70.7 mg cation/kg bw). The second stage (Stage 2) was one zebra finch per dosage level at ten dosage levels (3.43, 4.35, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0, and 29.2 mg cation/kg bw) and was conducted using dosages that were equally spaced along a log scale around the working estimate of the LD50 value obtained from Stage 1. Stage 3 was conducted with two zebra finches at each of five dosages (20.3, 25.8, 32.8, 41.6 and 52.8 mg cation/kg bw) that bracketed the estimation of the LD50 determined from Stages 1 and 2. In Stage 3 two of the five levels had 50% regurgitating among the two birds originally dosed so at those level a total of 4 birds were dosed to define the extent of regurgitation at these levels. Mortality data from Stages 1, 2 and 3 were combined to estimate an LD50 value. Zebra finches were randomly assigned to pens. The birds were fasted for more than 2 hours but less than 3 hours prior to dosing for each stage. At the start of each stage, a single dose of the test substance in reverse osmosis and deionized water that was dyed blue with a food grade colorant was orally intubated directly into the crop or proventriculus of each bird. Each bird was individually weighed and dosed on the basis of milligrams of cation of test substance per kilogram of body weight (mg cation/kg). From test initiation until termination, all birds were observed at least twice daily including an observation period of at least 60 minutes following dosing to observe any occurrence of regurgitation. A record was maintained of all mortality, signs of toxicity, and abnormal behavior. Body weights were measured individually at the initiation of each stage and on Days 3, 7 and 14 of the stage. Feed consumption was determined by pen for approximately 24-hour intervals from Day 0 to Day 1, Day 1 to Day 2 and Day 2 to Day 3. Average feed consumption was then determined from Day 3 to Day 7, Day 7 to Day 10 and Day 10 to Day 14.

 

The results indicated that there were no mortalities in the control group (0 of 10). There were also no mortalities in the 1.41, 3.43, 4.35, 5.21, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0, 25.8 and 29.2 mg cation/kg dosage groups.There was 50% (1 of 2) mortality among the birds at the 20.3 and 41.6 mg cation/kg dosage level, 67% mortality (2 of 3) at the 32.8 mg cation/kg dosage level and 100% mortality at the 19.2, 52.8 and 70.7 mg cation/kg dosage levels. No incidences of regurgitation were noted for the control birds.Regurgitation was noted among some of the birds at the 32.8 and 52.8 mg cation/kg dosage levels of stage 3 of the test.No other treatment birds were noted as regurgitating.All birds in the control group and in the 1.41, 3.43, 4.35, 5.21, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0 and 29.2 mg cation/kg dosage groups were normal in appearance and behavior throughout the test. Signs of toxicity or mortality occurred at the 19.2, 20.3, 25.8, 32.8, 41.6, 52.8 and 70.7 mg cation/kg dosage levels.From Day 0 to Day 3, all control birds exhibited a gain in body weight (0.8 to 1.6 g). The surviving treatment birds also exhibited a gain in body weight from Day 0 to Day 3 of the test which ranged from 0.2 to 2.7 grams. Body weight changes for all intervals were comparable among the control and most surviving treatment birds. The control group had feed consumption values on Day 0 to Day 1 of 2.6 to 5.2 grams, on Day 1 to Day 2 of 3.5 to 4.7 grams and on Day 2 to Day 3 of 3.3 to 4.3 grams. Among the surviving treatment birds (total of 18 birds) 4 birds for Day 0 to Day 1, 5 birds from Day 1 to Day 2 and 3 birds from Day 2 to Day 3 had a feed consumption values that were less than the minimum control value for that period. For all other feed consumption intervals, there were no apparent treatment related effects upon feed consumed among the surviving birds.Gross necropsies were also performed on all treatment birds surviving to test termination and four of the ten control birds surviving to test termination. Of the birds surviving to test termination the only findings noted were for the birds in the 1.41 and 5.21 mg cation/kg dosage levels and for 3 of the 4 control birds necropsied. These birds were noted with intracranial bleeding and the bird in the 1.41 mg cation/kg dosage level was also noted with a subcutaneous bruise.

 Based on the findings, the acute oral LD50 value for zebra finch was calculated to be 30.9 mg cation/kg with a 95% confidence interval of 18.7 to 77.7 mg cation/kg.

Reference 2

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar 2003 to 20 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 206 (Avian Reproduction Test)

Version / remarks:

1984

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Version / remarks:

1982

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Version / remarks:

1996

Deviations:

not specified

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

no

Vehicle:

yes

Remarks:

feed

Details on preparation and analysis of diet:

DIET PREPARATION
- Nutrient analysis of basal diet: The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet. 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 mallard (2.75%).
- Preparation of doses: 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 each of the four treated diets were prepared weekly beginning on March 27, 2003 and presented to the birds on Thursday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.).

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the 10 and 80 ppm a. i. treated diets, two samples from each of the 20 and 40 ppm a.i. treated diets and one sample from the control diet on Day 0 of Week 1. Control and treatment group diet samples were also collected from the bin feeders on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, samples were collected from the control and treatment group diets during Weeks 4 and 8 of the test to measure/verify test concentrations. The diet samples were stored frozen prior to being transferred to analysis.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Source: Approximately 111 pairs of pen-reared mallard were purchased from a commercial supplier. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing.
- Age at test initiation: Approximately 35 weeks of age
- Weight at test initiation: 790 - 1317 g
- Sexes used / mixed or single sex: Sex of the birds was determined by a visual examination of the plumage. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird.
- Acclimation: At the start of acclimation, the mallards were apparently healthy and phenotypically indistinguishable from wild type.
- Health condition check: Immediately prior to test initiation, all potential study birds were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study.

Limit test:

no

Total exposure duration (if not single dose):

9 wk

Remarks:

3 weeks pre-egg laying exposure followed by 6 weeks exposure during egg production

No. of animals per sex per dose and/or stage:

1

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal concentration: 0 (negative control), 10, 20, 40, and 80 ppm a.i.

Details on test conditions:

ACCLIMATION
- Acclimation period: 22 weeks

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The adult birds were housed indoors in batteries of pens measuring approximately 75 X 90 X 45 cm high. The pens were constructed of vinyl-coated wire mesh. Each pen was equipped with a bin feeder. Weekly, sufficient feed for the feeding period was placed in the bin feeder for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the bin feeders as needed. Water was supplied by nipple-type waterers.
- Compliant to good husbandry practices: Yes (Only birds associated with this study were maintained in the study room in order to avoid
excessive disturbances.)
- Suitable to avoid crowding stress: Yes

NO. OF BIRDS PER REPLICATE
- For negative control: 1 Male and 1 Female
- For treated: 1 Male and 1 Female

NO. OF REPLICATES PER GROUP
- For negative control: 20
- For treated: 20

TEST CONDITIONS
- Room temperature: 21.2 ± 1.0 °C
- Relative humidity (%): 58.5 ± 10.5%
- Photoperiod: The photoperiod during acclimation was eight hours or less of light per day. The photoperiod was increased to 17 hours of
light per day at test initiation to induce egg laying and was maintained at that length until the adult birds were euthanized.
- Light intensity: Throughout the test, the birds received a mean of approximately 166 lux ( ~ 15 ft. candles) of illumination provided by fluorescent lights which closely approximated noon-day sunlight.
- Ventilation: 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.
- Others: All adult birds were given feed and water ad libitum during acclimation and testing. Water was supplied by the town of Easton public water supply. The birds did not receive any form of medication in their feed during the test. Feed and water were analyzed periodically in accordance with the test facility's Standard Operating Procedures.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
The test birds were acclimated to the facilities and study pens for approximately 22 weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. A record was maintained of all mortalities and clinical observations.

BODY WEIGHT
- Time schedule for examinations: Individual body weights were measured at test initiation, at the end of Week 2, and at adult termination.

FOOD CONSUMPTION
- Time schedule for examinations: Feed consumption for each pen was measured weekly throughout the test.
- Remarks: 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). 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.

NECROPSY
Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and disposed of by incineration.

Details on reproductive parameters:

EGG COLLECTION AND STORAGE
Eggs were collected and recorded daily from all pens, when available. Eggs were washed to reduce the possibility of pathogen contamination before storing them in the cold room. Eggs were washed by hand or in a commercial egg washer with a chlorine based detergent . 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 stored in a cold room until weighing. The cold room was maintained at a mean temperature of 13.4 ± 0.5°C (SD) with a mean relative humidity of approximately 89 ± 5% (SD).

EGG WEIGHTS
At the end of the weekly interval, all eggs were removed from the cold room and two eggs from each pen (when available) were selected by indiscriminate draw for weighing. The remaining eggs were discarded. Cracked or abnormal eggs were not selected for weighing, unless no other eggs were available from that pen. Eggs selected for weighing were weighed on a balance accurate to 0.1 g, and the weight recorded.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

9 wk

Dose descriptor:

NOEL

Effect level:

74.72 mg/kg diet

Conc. / dose based on:

act. ingr.

Remarks:

the registered substance

Basis for effect:

reproductive parameters

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration (if not single dose):

9 wk

Dose descriptor:

NOEL

Effect level:

40 mg/kg diet

Conc. / dose based on:

act. ingr.

Remarks:

cation

Basis for effect:

reproductive parameters

Remarks on result:

other: Original value presented in study

Mortality and sub-lethal effects:

An overview of the results is provided in Table1, 2 and 7 in ‘Any other information on results incl. tables’.

- Mortalities: While no mortalities occurred in the control group or in the 10, 20 or 80 ppm a.i. treatment groups, a single incidental mortality occurred in the 40 ppm a.i. treatment group. The single mortality in the 40 ppm a.i. treatment group was the female in Pen 1065 that was found dead on Day 4 of Week 7. No clinical signs were noted prior to death. At necropsy, the bird was emaciated, with a loss of muscle mass and prominent keel. Internally, fluid was noted in the pericardium, the spleen was small and pale and the liver was also small. The bird’s intestinal contents were firm, the kidneys pale and the ovary was regressed. The female's penmate was euthanized and the male was unremarkable upon necropsy. No other mortalities occurred during the course of the study. Due to the condition of this bird at necropsy, the mortality was not considered to be related to treatment.

- Clinical Observations: 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, slight wing droop and foot lesions with occasional associated lameness. Other incidental observations noted were dyspnea (labored breathing) observed in one bird over a one week period. Except for incidental findings, all birds were normal in appearance and behavior throughout the study.

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

- Adult Body Weight: There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences in the mean body weight of the male birds were observed between the control group and the 10, 20, 40 or 80 ppm a.i. treatment groups at any of the body weight interval. For the females, no statistically significant differences were observed in mean body weight between the control group and the 10, 20 or 80 ppm a.i. treatment groups at any of the body weight intervals. There was a slight, but statistically significant (p < 0.05), reduction in mean body weight among hens in the 40 ppm a.i. test concentration at adult termination. However, given the variability in body weight change seen in the control group and all treatment groups, the reductions in body weight gain at the 40 ppm a.i. test concentration was not considered to be treatment related.

- Adult Feed Consumption: There were no apparent treatment-related effects upon feed consumption at any of the concentrations tested. When compared to the control group, there were no statistically significant differences observed at any feed consumption interval at the 10, 20 or 80 ppm a.i. test concentrations. However, there was a slight, but statistically significant (p < 0.05) increase in feed consumption at the 40 ppm a.i. test concentration during Week 8. The difference observed was slight, not concentration responsive and not consistent during the test period. Therefore, the difference observed was not considered to be treatment related. The estimated test substance intakes by the test organism from week 1 to week 9 in control, 10, 20, 40 and 80 ppm a.i. were 0, 1.7, 3.2, 6.8 and 13.6 mg/kg body weight/day, respectively. The accuracy of the estimated mean daily dietary dose may be impacted by differences in individual feed consumption, both within and between pens, and feed wastage.

Effects on reproduction:

An overview of the results is provided in Table 3 – Table 6 in ‘Any other information on results incl. tables’.

- Reproductive ResuIts: There were no statistically significant effects upon total egg production in the 10, 20 or 40 ppm a.i. treatment groups. Egg production in the 10 ppm a.i. treatment group was slightly impacted by data from three pens (Pens 1023, 1028 & 1034). Comparable hens were also noted in the control group (Pen 1105), 20 ppm a.i. treatment group (Pens 1052 & 1059), 40 ppm a.i. treatment group (Pens 1063, 1077 & 1078) and in the 80 ppm a.i. treatment group (Pen 1081). At terminal necropsy, these hens all exhibited lesions of extensive egg yolk peritonitis or egg yolk peritonitis and egg remnants in the abdominal cavity, an incidental infection of the abdominal cavity and reproductive tract known to impact egg production. When data from these pens were excluded, egg production was very similar for the control group and the 10, 20 and 40 ppm a.i. treatment groups (0.69, 0.62, 0.67 and 0.61 eggs/hen/day, respectively). However, there was a statistically significant (p < 0.05) reduction in mean number of eggs laid per hen per day (0.47 eggs/hen/day) in the 80 ppm a.i. treatment group that was considered to be related to treatment.

- Egg Weights: There were no apparent treatment related effects upon egg weights at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in egg weights in the 10, 20, 40 or 80 ppm a.i. treatment groups. Eight eggs (two from 10 ppm a.i., two from 20 ppm a.i, three from 40 ppm a.i. and one from 80 ppm a.i.) that were noted as abnormally small or soft shelled were excluded from statistical analyses.

Reported statistics and error estimates:

Upon completion of the test, each of the treatment groups were compared to the control group using Dunnett's Multiple Comparison Procedure to assess the statistical significance of any observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Any pen in which an adult mortality occurred was not used in statistical comparisons of the reproductive data. Analyses were performed on each of the following parameters:
1. Adult Body Weight - Individual body weight was measured at test initiation, at the end of Week 2, and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex.
2. Adult Feed Consumption - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group.
3. Eggs Laid/Hen/Day - The number of eggs laid per female divided by the length of the egg laying period. Statistical comparisons were made between the control and each treatment group.
4. Egg Weights - Two eggs from each pen (when available) were weighed each week. Statistical comparisons were made between the control and each treatment group.

Table 1. Results of Mean Adult Body Weight (g)

Experimental Group	Sex	Week0	Change Week 0 - 3	Week 3	Change Week 3 - 9	Week 9	Total Change
Control	Male	1081	-13	1069	-15	1054	-28
	Female	1007	112	1119	81	1200	193
10 ppm a.i.	Male	1106	8	1114	-39	1075	-31
	Female	984	75	1059	101	1160	176
20 ppm a.i.	Male	1078	-6	1072	-11	1061	-18
	Female	964	75	1038	73	1112	148
40 ppm a.i.	Male	1093	15	ll08	-23	1081	-6
	Female	972	76	1048	33	1091 *	119
80 ppm a.i.	Male	1089	-10	1079	-36	1043	-46
	Female	1002	91	1093	18	1111	109

The means for body weights and body weight changes were calculated and rounded separately.

•statistically different from the control group at p < 0.05 (Dunnett's t-test).

Table 2. Mean Feed Consumption (g/bird/day)

Experimental Group	Week
	1	2	3	4	5	6	7	8	9
Control	121	165	172	197	182	182	178	174	175
10 ppm a.i.	129	179	179	200	186	184	186	186	182
20 ppm a.i.	120	155	168	190	181	174	185	185	181
40 ppm a.i.	120	164	171	209	187	170	204	206 *	189
80 ppm a.i.	139	183	187	214	192	176	187	182	175

 *Statistically different from the control group at p < 0.05 (Dunnett's t-test).

Table 3. Weekly egg production

Experimental group	Week
	1	2	3	4	5	6	7	8	9	Total
Control	8	38	113	127	128	120	122	114	103	873
10 ppm a.i.	10	22	67	87	101	98	101	101	78	665
20 ppm a.i.	6	28	81	103	111	114	118	105	98	764
40 ppm a.i.	4	17	65	101	114	96	77	103	79	656
80 ppm a.i.	11	36	65	79	88	78	89	78	64	588

Table 4. Mean eggs/hen/day by week

Experimental					Week
Group	1	2	3	4	5	6	7	8	9	Mean	SD
Control	0.06	0.27	0.81	0.91	0.91	0.86	0.87	0.81	0.74	0.69	0.17
10 ppm a.i.	0,07	0.16	0.48	0.62	0.72	0.70	0.72	0.72	0.56	0.53	0.29
20 ppm a.i.	0.04	0.20	0.58	0.74	0.79	0.81	0.84	0.75	0.70	0.61	0.23
40 ppm a.i.	0.03	0.13	0.49	0.76	0.86	0.72	0.58	0.77	0.59	0.55	0.22
80 ppm a.i.	0.08	0.26	0.46	0.56	0.63	0.56	0.64	0.56	0.46	0.47	0.22 *

*Statistically different from the control group at p < 0.05 (Dunnett's t-test).

Table 5. Mean Eggs/Hen/Day by Week Minus Hens with Lesions of Extensive Egg Yolk Peritonitis or Egg Yolk Peritonitis and Egg Remnants at Terminal Necropsy

Experimental Group	Number of hens with EYP^a	Weeks
		1	2	3	4	5	6	7	8	9	Mean	SD
Control	1	0.06	0.28	0.81	0.93	0.95	0.89	0.91	0.83	0.76	0.71	0.14
10 ppm a.i.	3	0.08	0.18	0.56	0.72	0.85	0.82	0.85	0.84	0.65	0.62	0.20
20 ppm a.i.	2	0.05	0.22	0.64	0.82	0.87	0.90	0.93	0.82	0.78	0.67	0.13
40 ppm a.i.	3	0.04	0.13	0.53	0.85	0.96	0.81	0.63	0.88	0.66	0.61	0.15
80 ppm a.i.	1	0.08	0.27	0.49	0.59	0.62	0.57	0.65	0.56	0.47	0.48	0.22**

a - lesions of severe egg yolk peritonitis or eye yolk peritonitis and egg remnants in abdominal cavity noted at terminal necropsy.

**Statistically different from the control group at p < 0.05 (Dunnett's t-test).

Table 6. Mean egg’s weights¹(g)

Experimental Group	Weeks									Mean	SD
	1	2	3	4	5	6	7	8	9
Control	53.4	57.4	56.2	58.6	58.4	59.7	58.8	59.9	59.7	58. 5	3.3
10 ppm a.i.	55.2	54.7	57.2	58.3	56.9	57.1	57.0	58.4	57.9	56.8	3.7
20 ppm a.i.	55.3	52.0	56.0	574	57.4	57.9	57.4	57.2	56.9	57.0	2.5
40 ppm a.i.	57.7	56.0	56.0	56.9	57.7	57.6	57.6	58.4	58.2	57.5	3.9
80 ppm a.i.	55.7	56.8	56.4	56.9	56.4	57.6	57.5	57 .3	57.8	56.9	4.0

1 Does not include eggs that were noted as abnormal.

The above differences from the control group were not statistically significant (Dunnett's t-test).

Table 7. Summary of Gross pathological observation

	Males- Treatment group (ppm a.i.)					Females- Treatment group (ppm a.i.)
	Control	10	20	40	80	Control	10	20	40	80
Number of birds	20	20	20	19	20	20	20	20	19	20
External - feather loss	0	1	0	0	0	2	2	1	0	2
External - foot lesions	8	8	11	13	4	15	15	14	14	14
Respiratory - right lung lobe undeveloped	0	0	1	0	0	0	0	0	0	0
Spleen - pale	0	0	0	0	0	1	0	0	0	0
Spleen - margins rounded	0	0	0	0	0	1	0	0	0	0
Spleen - enlarged	0	0	0	0	0	3	1	0	0	1
Liver - pale, mottled and firm	0	0	0	0	0	0	I	0	0	0
Abdominal cavity - retained yolk sac	0	0	2	1	0	0	0	0	0	0
Abdominal cavity - egg remnants	-	-	-	-	-	1	2	0	3	1
Abdominal cavity - old egg yolk peritonitis	-	-	-	-	-	6	1	1	3	2
Abdominal cavity - egg yolk peritonitis	-	-	-	-	-	5	2	2	4	13
Abdominal - extensive egg yolk peritonitis	-	-	-	-	-	0	2	1	0	1
Abdominal cavity - - 30 ml yellow fluid	-	-	-	-	-	0	1	1	0	0
Reproductive - egg remnants	-	-	-	-	-	0	2	1	0	0
Reproductive - fluid-filled cysts on infundibulum	-	-	-	-	-	1	0	1	0	1
Reproductive - hemorrhagic follicle	-	-	-	-	-	2	1	0	0	1
Reproductive - cystic follicles	-	-	-	-	-	0	0	0	0	3
Reproductive - persistent right oviduct	-	-	-	-	-	1	0	0	1	0
Reproductive - right oviduct developed and containing egg remnant	-	-	-	-	-	0	0	1	0	0
Reproductive - ovary regressing	-	-	-	-	-	2	0	1	3	3
Reproductive - ovary regressed Reproductive - testes small - 4.5 cm	1	0	1	1	3	1	1	1	0	1
Reproductive - right testis small, - 4.0 cm	1	0	1	0	0	-	-	-	-	-
Not remarkable	11	12	6	5	15	0	2	3	3	0

 

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 40 mg cation/kg diet = 74.72 mg registered substance/kg diet

Validity criteria fulfilled:

yes

Conclusions:

There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight, feed consumption or egg weight at any of the concentrations tested. Additionally, there were no treatment related effects upon egg production at the 10, 20 or 40 mg a.i. (as cation)/kg diet treatments. However, at the 80 mg a.i. (as cation)/kg diet treatment, there was a statistically significant reduction in egg production. Based upon the effect observed in the 80 mg a.i. (as cation)/kg diet treatment group, the NOEL was determined to be 40 mg a.i. (as cation)/kg diet.

Executive summary:

The effect of the test substance onegg production in adult mallard ducks (Anas platyrhynchos) was determined in a 9 week reproduction test. The test was conducted according OECD TG 206, FIFRA guideline 71-4 and OCSPP 850.2300 guideline. This study was in compliance with GLP criteria. Mallard ducks (113 males and 111 females) were randomly distributed into one control group and four treatment groups (nominal concentrations: 10, 20, 40 and 80 mg a.i. (as cation)/kg diet). Each treatment and control group contained 20 pairs of birds with one male and one female per pen. 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, at the end of Week 2, and at adult termination. Feed consumption was measured weekly throughout the test. At test initiation, the photoperiod was increased to induce egg production. All eggs were collected daily, and marked with a soft lead pencil or permanent ink corresponding to the pen from which they were collected. Eggs were washed and placed in a cold room for storage. Weekly, two eggs were indiscriminately selected from each pen (when available) for egg weight measurement. Those eggs selected were individually weighed (to the nearest 0.1 g) and the weights recorded. These eggs, and all others, were then discarded. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups.

After 9 weeks exposure, there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight, feed consumption or egg weight at any of the concentrations tested. Additionally, there were no treatment related effects upon egg production at the 10, 20 or 40 mg a.i. (as cation)/kg diet treatment. However, at the 80 mg a.i. (as cation)/kg diet treatment, there was a statistically significant reduction in egg production. Based upon the effect observed in the 80 mg a.i. (as cation)/kg diet treatment group, the NOEL for mallard exposed to the test substance in the diet during the study was 40 mg a.i. (as cation)/kg diet. 

Description of key information

30-d LD50 = 57.7 mg pure test substance/kg bw, Taeniopygia guttata, OECD TG 223, Hubbard 2013

9-wk NOEL = 72.74 mg pure test substance/kg diet, Anas platyrhynchos, reproduction, OECD TG 206, Temple 2004

Key value for chemical safety assessment

Short-term EC50 or LC50 for birds:

57.7 mg/kg bw

Long-term EC10, LC10 or NOEC for birds:

74.72 mg/kg food

Additional information

Acute oral test (Hubbard, 2013)

The effects of the test substance on the zebra finch (Taeniopygia guttata) were determined in an acute toxicity test performed under GLP to OECD TG 223 and OCSPP 850.2100 guideline. Birds were exposed to the test substance via gavage in a staggered approach. Initially, one bird per dose was exposed to 1.41, 5.21, 19.2 and 70.7 mg cation/kg bw. Secondly, one bird per dose was exposed by gavage to 3.43, 4.35, 5.52, 7.00, 8.88, 11.3, 14.3, 18.1, 23.0, and 29.2 mg cation/kg bw. Finally, two birds per dose were exposed by gavage to 20.3, 25.8, 32.8, 41.6 and 52.8 mg cation/kg bw. At two of these doses, 50% regurgitating occurred among the two birds originally dosed and a total of 4 birds were dosed to define the extent of regurgitation at these levels. Mortality data from the test stages were combined to estimate an LD50 value.
Finches were randomly assigned to pens and fasted for 2-3 hours prior to dosing. Test substance was dissolved in deionized water purified by reverse osmosis that was dyed blue with a food grade colorant. Each bird received a single oral dose by gavage directly into the crop or proventriculus of each bird. No mortality occurred in the control group, in the groups receiving up to 18.1 mg cation/kg bw and in the groups dosed at 23, 25.8 and 29.2 mg cation/kg bw. A mortality of 50% occurred among the birds dosed at 20.3 and 41.6 mg cation/kg bw, 67% mortality was observed at 32.8 mg cation/kg and 100% mortality was seen at 19.2, 52.8 and 70.7 mg cation/kg bw. No incidences of regurgitation were noted for the control birds. Regurgitation was noted among some of the birds at the 32.8 and 52.8 mg cation/kg dosage levels, but no other treated birds regurgitated. Signs of toxicity or mortality occurred in the groups dosed at 19.2, 20.3, 25.8, 32.8, 41.6, 52.8 and 70.7 mg cation/kg bw. Gross necropsies were performed on all exposed birds surviving to test termination and four of the ten control birds surviving to test termination. Findings were limited to a few birds in the 1.41 and 5.21 mg cation/kg dose groups and three of the four control birds necropsied. These birds exhibited intracranial bleeding and the bird in the 1.41 mg cation/kg dosage level had a subcutaneous bruise. Based on the observed mortalities, the acute oral LD50 value for zebra finch was calculated to be 30.9 mg cation/kg bw with a 95% confidence interval of 18.7 to 77.7 mg cation/kg. This LD50 was equivalent to 57.7 mg pure test substance/kg bw.

Reproduction test (Temple, 2004)

The effect of the test substance on egg production in adult mallard ducks (Anas platyrhynchos) was determined in a 9 week reproduction test performed under GLP to OECD TG 206 and FIFRA guideline 71-4. Mallard ducks (113 males and 111 females) were randomly distributed into one control group and four treatment groups (nominal concentrations: 10, 20, 40 and 80 mg cation/kg diet). Each treatment and control group contained 20 pairs of birds with one male and one female per pen. The control group was fed plain diet without test substance. All eggs were collected daily, and marked with a soft lead pencil or permanent ink corresponding to the pen from which they were collected. Eggs were washed and placed in a cold room for storage. Weekly, two eggs were indiscriminately selected from each pen (when available) and individually weighed (to the nearest 0.1 g). After 9 weeks of exposure, there were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight, feed consumption or egg weight at any of the concentrations tested. At exposure of 80 mg cation/kg diet, there was a statistically significant reduction in egg production. Based upon the observed effects, the NOEL for mallard exposed to the test substance in the diet was 40 mg cation/kg diet, which is equivalent to 74.72 mg pure test substance/kg diet.