Paraquat-dichloride

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to birds: acute oral toxicity test (LD50-slope)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Dec 2013 to 29 Jan 2014

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 2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2100 (Avian Acute Oral Toxicity Test)

Version / remarks:

2012

GLP compliance:

yes

Dose method:

gavage

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

reverse osmosis deionized water

Details on preparation and analysis of diet:

DOSE PREPARATION AND DOSING
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% ion content. Therefore, all dosages and the LD50 value are reported as milligrams of ion of test substance per kilogram of body weight. 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 20 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 ion 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. Due to the nature of the test substance capsule dosing was not an option and dosing by gavage was chosen as the method of administration. The dosages initially chosen were established based on an estimated LD50 value of 54 mg ion/kg bw. Stage 1 of 7.64, 28.1, 104 and 382 mg ion/kg bw were thus established. Stage 2 dosage levels were 5.02, 6.37, 8.08, 10.3, 13.0, 16.5, 20.9, 26.6, 33.7 and 42.8 mg ion/kg bw. Stage 3 dosage levels were 14.0, 17.7, 22.5, 28.6 and 36.3 mg ion/kg bw.

DOSING SOLUTIONS SAMPLING AND ANALYSIS
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. The samples taken for Stage 3 of the test were analysed.

ANALYTICAL METHOD
- Method: The analysis of the test substance in dosing solutions was based upon methodology developed by the test facility. Samples were diluted with acidified reverse osmosis (RO) water. Concentrations of the test substance in extracts of the samples were determined by high performance liquid chromatography using an Agilent Series 1200 high performance liquid chromatograph (HPLC) equipped with an Agilent Series 1200 variable wavelength detector (VWD). Chromatographic separations were achieved using an Ultra Quat analytical column (150 mm x 4.6 mm I.D., 5 μm particle size).
- Calibration standards: Calibration standards of the test substance, ranging in concentration from 1.00 to 10.0 mg a.i./L, were analyzed with the samples. A calibration curve was constructed for each set of analyses. The peak areas and the theoretical concentrations of the calibration standards were fit with least-squares regression analysis to a linear function. The concentration of test substance in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation.
- Limit of Quantification (LOQ): The LOQ for these analyses was set at 1.00 mg/L based upon the product of the lowest analytical standard (1.00 mg a.i./L) and the dilution factor of the matrix blank extract (1.00), divided by test substance purity (99.7%). Measured values greater than or equal to the LOQ were reported.
- Matrix blank: Along with the sample analyses, one matrix blank sample was analyzed to determine possible interferences. No interferences were observed at or above the LOQ in the matrix blank during the sample analyses.
- Fortified samples: Samples of acidified RO water were fortified at 1010 and 10100 mg/L and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded a mean procedural recovery of 101%. This value corresponds to the sample set analyzed on Day 0. Sample measured concentrations were not corrected for the mean procedural recovery from the sample set.

HPLC OPERATING CONDITIONS
- Instrument: Agilent Series 1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1200 Variable Wavelength Detector (VWD).
- Analytical column: Ultra Quat (150 mm x 4.6 mm I.D., 5 μm particle size)
- Flow rate: 1.000 mL/minute
- Column temperature: 30 °C
- Mobile phase: Channel A: 95% Ultra Quat Reagent; Channel B: 5.0% Acetonitrile
- Injection volume: 50.00 μL
- Test substance peak retention time: Approximately 9.2 minutes
- Analytical wavelength: 257 nm

Test organisms (species):

other: Taeniopygia guttata

Details on test organisms:

TEST ORGANISM
- Common name: Zebra finch
- Age: The birds were approximately three to four months of age at time of receipt.
- Weight at test initiation: 1.3 to 15.8 g
- Sexes used: Males and females; males and females were identified by the phenotypic plumage.
- Disease free: Yes; All birds were in adult plumage and appeared to be in good health at initiation of the test.
- Kept according to standard practices: Yes

Limit test:

no

Remarks:

Single oral dose exposure

Post exposure observation period:

14-day post-dosing period

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

- Control: 5
- Test substance treatments: 1

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

- Nominal test dosage Stage 1: 7.64, 28.1, 104 and 382 mg ion/kg bw
- Nominal test dosage Stage 2: 5.02, 6.37, 8.08, 10.3, 13.0, 16.5, 20.9, 26.6, 33.7 and 42.8 mg ion/kg bw
- Nominal test dosage Stage 3: 14.0, 17.7, 22.5, 28.6 and 36.3 mg ion/kg bw

Details on test conditions:

ACCLIMATION
- Acclimation period: All test birds were acclimated to the study room and to the test caging for at least seven weeks prior to test initiation. Birds were kept in a sexually quiescent state by maintaining them under a shortened photoperiod and keeping the sexes separated.

FEEDING (ACCLIMATION AND TEST)
At the start of acclimation all test birds were fed a commercially available finch food Kaytee Forti-diet Pro Health Finch. During the acclimation period the birds were transitioned to ZuPreem FruitBlend Flavor diet (a pelleted diet). During the test the birds were fed ZuPreem FruitBlend Flavor diet, size xs. During acclimation and test, grit (Kaytee Hi-Cal Grit) was provided to aid with the birds’ digestion. 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: Yes; the birds were fasted for approximately 2-3 hours prior to dosing.

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: 2-3

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: 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 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.
- Compliant to good husbandry practices: Yes; housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council
- Caging: group

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 10
- For treated: 1

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: 1
- For vehicle control: 3

TEST CONDITIONS
- Temperature: Birds were maintained at ambient room temperature. The continuously monitored average temperature for this study was 18.9°C ± 2.1°C (SD) with a maximum of 22.4°C, a minimum of 15.7°C and a coefficient of variability of 11.1%.
- Relative humidity (%): The continuously monitored average relative humidity was 26.3% ± 9.5% (SD) with a maximum of 55.8%, a minimum of 14.5% and a coefficient of variability of 36.3%.
- Photoperiod: 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 throughout the test.
- Lighting type: 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.
- Light intensity: The birds were exposed to an average of approximately 284 lux of illumination.

Details on examinations and observations:

GENERAL 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 three 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):

14 d

Dose descriptor:

LD50

Effect level:

33.8 mg/kg bw

Conc. / dose based on:

other: pure test substance

Basis for effect:

body weight

Remarks on result:

other: 95% C.I.: 23.8 - 51.8 mg pure substance/kg bw

Remarks:

recalculated value, expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation.

Duration (if not single dose):

14 d

Dose descriptor:

LD50

Effect level:

24.5 mg/kg bw

Conc. / dose based on:

other: test substance cation species

Basis for effect:

body weight

Remarks on result:

other: 95% C.I.: 17.2 - 37.5 mg ion/kg bw

Remarks:

original value presented in study

Mortality and sub-lethal effects:

An overview of the results on mortality and sub-lethal effects is presented in 'Any other information on results incl. tables'.

MORTALITIES
There was a single mortality (1 of 10) in the control group. There were no mortalities in the 5.02, 6.37, 7.64, 8.08, 10.3, 13.0, 14.0, 16.5, 17.7 and 26.62 mg ion/kg bw dosage groups. There was 50% (1 of 2) mortality among the birds at the 22.5 and 36.3 mg ion/kg bw dosage level and 100% mortality at the 20.9, 28.1, 28.6, 33.7, 42.8, 104 and 382 mg ion/kg bw dosage levels.

CLINICAL OBSERVATIONS
No incidences of regurgitation were noted for the control birds or for any treatment birds. In the control group one bird was found dead on Day 7 of the test. All other control birds were normal in appearance and behavior for the remainder of the test. All birds in the 5.02, 6.37, 7.64, 8.08, 10.3, 13.0, 14.0, 16.5 and 17.7 mg ion/kg bw dosage groups were normal in appearance and behavior throughout the test. Signs of toxicity or mortality occurred at the 20.9, 22.5, 26.6, 28.1, 28.6, 33.7, 36.3, 42.8, 104 and 382 mg ion/kg bw dosage levels. Signs of toxicity were observed on the day of dosing, Day 1, Day 2 and Day 3 of the test. Signs of toxicity included ruffled appearance, lethargy, loss of coordination, lower limb weakness, prostrate posture, loss of righting reflex and depression. All surviving birds were normal in appearance and behavior from the afternoon of Day 2 until termination. Mortalities were noted on the day of dosing, the morning of Day 1 and on the morning of Day 3. All surviving birds were normal in appearance and behavior from Day 4 to test termination.

BODY WEIGHT
From Day 0 to Day 3, all control birds exhibited a change in body weight from -0.2 to 1.7 grams. The surviving treatment birds exhibited a change in body weight from Day 0 to Day 3 of the test which ranged from -0.2 to 2.0 grams. Body weight changes for all intervals were comparable among the control and most surviving treatment birds.

FOOD CONSUMPTION
For all surviving birds, there were no apparent treatment related effects upon the amount of feed consumed for any of the feed consumption intervals.

NECROPSY
Gross necropsies were performed on all of the mortalities. Gross necropsies were also performed on all treatment birds surviving to test termination and three of the control birds surviving to test termination. For the birds surviving to test termination, there were no remarkable findings.

Further details on results:

The slope of the dose responsive curve was 6.89 and the Chi-square value was 8.83. No mortalities occurred at dosages up to 17.7 mg ion/kg bw.

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: Summary of Signs of Toxicity from a Zebra Finch Acute Oral Toxicity Study with the test substance 

Stage	Nominal dosage (mg ion/kg bw)	First noted signs of toxicity after dosing	Signs of toxicity(a)	Last day with signs of toxicity	Day of mortality
Stage 1	7.64	NA	NA	NA	NA
	28.1	3 hr 6 min	11,14,M	0	1
	104	1 hr 7 min	4,11,14,M	0	0
	382	1hr 5 min	4,11,14,M	0	0
Stage 2	5.02	NA	NA	NA	NA
	6.37	NA	NA	NA	NA
	8.08	NA	NA	NA	NA
	10.3	NA	NA	NA	NA
	13.0	NA	NA	NA	NA
	16.5	NA	NA	NA	NA
	20.9	5 hr 54 min	11,14,M	0	1
	26.6	5 hr 58 min	11	0	NA
	33.7	2 hr 52 min	1,6,11,M	0	0
	42.8	2 hr 51 min	1,5,11,M	0	0
Stage 3	14.0	NA	NA	NA	NA
	17.7	NA	NA	NA	NA
	22.5	Day 1	13,M	3	1
	28.6	5 hrs 49 min	11,13,14,M	2	1&3
	36.3	4 hrs 4 min	11,M	0	1

NA = Not applicable

a) sign of toxicity: 1 = depression, 4 = loss of coordination, 5 = prostrate posture, 6 = loss of righting reflex, 11 = ruffled appearance, 13 = lower limb weakness, 14 = lethargy, M = mortality (not counted towards last day with signs of toxicity)

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 24.5 mg test substance cation/kg bw = 33.8 mg pure test substance/kg bw, accordingly, the 95% C.I. is calculated to be 23.8 - 51.8 mg pure test substance/kg bw.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

The acute oral LD50 value for zebra finch exposed to the test substance as a single oral dose was calculated to be 24.5 mg ion/kg bw with a 95% confidence interval of 17.2 to 37.5 mg ion/kg bw. This value corresponds to a recalculated value of 33.8 mg pure substance/kg bw (95% C.I.: 23.8 - 51.8 mg pure substance/kg bw). The slope of the dose responsive curve was 6.89 and the Chi-square value was 8.83. No mortalities occurred at dosages up to 17.7 mg ion/kg bw, corresponding to 24.4 mg pure substance/kg bw.

Executive summary:

The acute toxicity to birds of the test substance was determined in a LD50-slope test design study according to OECD TG 223 and in compliance with GLP. In this study, zebra finches were exposed (1 pair per dose) to a single oral dose containing the test substance over subsequent stages of 14 days each. The first stage was Stage 1, with one zebra finch per dosage level at four dosage levels. The second stage (Stage 2) was one zebra finch per dosage level at ten dosage levels 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 that bracketed the estimation of the LD50 determined from Stages 1 and 2. Mortality data from Stages 1, 2 and 3 were combined to estimate an LD50 value. The nominal test dosages ranged from 5.02 mg ion/kg bw to 382 mg ion/kg bw. 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 acute oral LD50 value for zebra finch exposed to the test substance as a single oral dose was calculated to be 24.5 mg ion/kg bw with a 95% confidence interval of 17.2 to 37.5 mg ion/kg bw. This value corresponds to a recalculated value of 33.8 mg pure substance/kg bw (95% C.I.: 23.8 - 51.8 mg pure substance/kg bw).

Reference 2

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Nov 1981 to 29 Apr 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Version / remarks:

Proposed Guidelines for Registering Pesticides in the United States 40 CFR 163.71-4 Avian Reproduction, July 1978

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

no

Vehicle:

no

Details on preparation and analysis of diet:

Three pre-mix concentrates to be utilized in the preparation of final diets were prepared as follows:

- Concentrate A: 17.2197 grams technical test substance + 130 mL corn oil + 5862.72 grams Breeder Ration.
- Concentrate B: 51.8390 grams technical test substance + 130 mL corn oil + 5828.16 grams Breeder Ration.
- Concentrate C: 172.7971 grams technical test substance + 130 mL corn oil + 5707.20 grams Breeder Ration.

For each concentrate, the required amount of corn oil was first incorporated into the appropriate amount of game bird breeder ration in the Hobart mixer. The required amount of technical test substance was then weighed on a Mettler analytical balance. Approximately 300 g of the pre-mixed breeder ration was then placed in a
quart, stainless steel Waring blender container, and blended to a fine powder. After the breeder ration was allowed to cool for at least 10 minutes, the required amount of technical test substance was slowly added with the blender running and blended at high speed for five minutes. No more than 30 mL of technical test substance was added to a single 300 g. aliquot of breeder ration. The mixture was then added to the remaining breeder ration in the Hobart mixer and the final concentrate was mixed for twenty minutes. All concentrates to be utilized during the course of the study were prepared at the beginning of the study. The concentrates were then divided into weekly aliquots and frozen. Weekly, the appropriate concentrates were removed from the freezer, and incorporated into the final diet as follows:

- 10 ppm: 500 grams Concentrate A [0.4536 grams test substance cation] Q.S. 45.359 kilograms Breeder Ration
- 30 ppm: 500 grams Concentrate B [1.3608 grams test substance cation] Q.S. 45.359 kilograms Breeder Ration
- 100 ppm: 500 grams Concentrate C [4.5359 grams test substance cation] Q.S. 45.359 kilograms Breeder Ration

Each final diet was mixed for twenty minutes in a Patterson-Kelley twin shell dry blender. Diets were prepared on Tuesday of each week and presented to the birds on that day. The basal diet was Wildlife International, Ltd. 's game bird breeder ration containing 19.4% protein, 6.7% fat, and 3.8% fiber. Samples of the control diet and each of the test diets were taken on Day 0 and Day 8 of Weeks 1, 4, 8, 12, and 16 of the study. Samples were frozen immediately after collection and shipped, on dry ice, to the sponsor for analysis.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Age at test initiation 6 months
- Sexes used / mixed or single sex: Mixed
- Cultural background: Pen-reared bobwhite quail, previously untreated, and phenotypically indistinguishable from wild birds
- Disease free: Yes
- Breeding population: Birds approaching their first breeding season

Limit test:

no

Total exposure duration (if not single dose):

18 wk

Remarks:

10 weeks of adult exposure prior to egg laying, 8 weeks of adult exposure during egg laying

Post exposure observation period:

8 weeks offspring obervation period

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

12

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

- Nominal concentrations: 0 (control), 10, 30 and 100 ppm test substance cation

Details on test conditions:

FEEDING
Groups received the appropriate diets ad libitum.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The birds were housed indoors in Beacon (Model B754) battery finishers measuring 72 x 90 x 33 cm high, with one drake and one hen per pen. External walls of the finishers were constructed of wire grid, while the ceilings and common walls were constructed of galvanized sheeting. Floors of the finishers were constructed of 1 x 1/2 inch wire mesh grid coated with plastic to reduce egg breakage.
- Suitable to avoid crowding stress: yes
- Caging: group , one male and one female

NO. OF BIRDS PER STAGE OR REPLICATE
- For vehicle control: 24
- For treated: 24

TEST CONDITIONS
- Temperature: The average temperature was 18.3 ± 2.2 °C
- Humidity: 74 % relative humidity
- Photoperiod: 8 hours light, 16 hours dark in the first 7 weeks. The photoperiod was then increased to 17 hours light per day until study termination.
- Light intensity: The birds received twelve footcandles of illumination throughout the study, provided by florescent lights which closely approximate noon-day sunlight.
- Ventilation: Temperatures below 0 °C and above 32.2 °C were prevented through the use of cooling ventilators, exhaust fans, and insulation.

INCUBATION AND OFFSPRING PROCEDURE
- Egg collection and incubation: Eggs were collected daily and marked according to the pen from which they were taken. The eggs were placed in a Kuhl egg washer (Model No. F4220) and soaked for fifteen seconds, then washed for three minutes at approximately 46.1 °C in a bactericide (Kuhl "Super C D" chlorine base detergent) to reduce E. coli contamination. The clean eggs were then stored 13.9 °C and 82 percent relative humidity. Weekly, the eggs were placed in a Petersime Incubator (Model No. S2) for incubation. All eggs were candled prior to incubation to detect egg shell cracks, on Day 14 of incubation to measure embryo viability and to remove any E. coli contaminated eggs, and on Day 21 to measure embryo survival. On day 25 of incubation, the eggs were placed in a Petersime Hatcher (Model S20) and allowed to hatch.
- Chick incubation: Throughout incubation the temperature was maintained at 37.5° with a wet bulb humidity index of 29.4 °C. The incubator was equipped with a pulsator fan and blades that produce a mild breathing air movement that eliminated intracabinet temperature and humidity variation during incubation.The incubator was equipped with automatic egg rotation, assuring that each egg was rotated from 50° off of vertical in one direction to 50° off of vertical in the opposite direction (total arc of rotation is 100°) each hour through Day 25 of incubation. When the eggs were transferred to the hatcher, rotation was discontinued, the temperature was lowered 37.2 °C and the wet bulb humidity index was raised to 33.3 °C. All hatchlings, unhatched eggs and egg shells were removed from the hatcher on Day 28 of incubation, and the average body weight of the hatchlings was determined. Hatchlings were then housed according to the appropriate parental dose grouping in battery brooders until 14 days of age. Battery brooder temperature was maintained at approximately 37.8 °C from hatching to Day 7 of brooding and then lowered to approximately 23.9 °C for the remainder of the brooding period. During brooding the basal diet was Wildlife International, Ltd.'s game bird starter ration. Starter ration and water were available ad libitum. At 14 days of age the ducklings were removed from the brooding units and the average body weight determined. One day each week throughout egg laying, all eggs layed that day in each experimental group and the controls were weighed to the nearest one-tenth of a gram. Weekly throughout egg laying, when available, one egg from each pen in each experimental group and the controls was randomly selected for eggshell thickness measurement. The eggs were first opened at the waist. The contents were thoroughly washed out, and the shells were dried for one week at room temperature. The average thickness of the dried shell plus the membrane at the waist was determined by measuring at five points around the circumference of the egg using an eggshell thickness micrometer calibrated in 0.01 mm units.

Details on examinations and observations:

All adult birds were observed daily throughout the study, and a record maintained of all mortalities, signs of toxicity or abnormal behavior. Body weights were recorded at initiation, on Weeks 2, 4, 6, 8, and at termination of the study. Body weights were not recorded during egg laying because of the adverse effect handling has on egg production. Feed consumption was measured every two weeks throughout the study.

Details on reproductive parameters:

The following reproduction parameters were evaluated: eggs laid, eggs cracked, eggs set, viable embryos, live three-week embryos, hatchlings, body weight of the hatchlings, 14-day old survivors, body weight of the 14-day old survivors, egg weight and eggshell thickness.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

18 wk

Dose descriptor:

NOEL

Effect level:

41.4 mg/kg diet

Conc. / dose based on:

other: pure test 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):

18 wk

Dose descriptor:

NOEL

Effect level:

30 mg/kg diet

Conc. / dose based on:

other: test substance cation species

Basis for effect:

reproductive parameters

Remarks on result:

other: Original value as presented in the study report

Mortality and sub-lethal effects:

For details on cumulative mortality and sub-lethal effects see 'Any other information on results incl. tables'
MORTALITY
Mature mallard duck received the test substance at dietary concentrations of 10 ppm, 30 ppm, and 100 ppm cation for the duration of the study. One mortality occurred in the 100 ppm cation treatment group during Week 18. This hen was observed exhibiting symptoms of lethargy, reduced reaction to external stimuli (sound and movement), and lower limb weakness one day prior to death. Gross necropsy revealed a ruptured uterus with massive peritonitis. No other mortalities occurred in the control group or in any test group during the course of the study.

CLINICAL SIGNS
No overt symptoms of toxicity were observed during the course of the study. Except for the mortality mentioned previously, all birds in both the control and treatment groups were normal in appearance and behavior throughout the test period.

ADULT BODY WEIGHT AND FEED CONSUMPTION
When subjected to a one way analysis of variance there was no statistically significant (p < 0.05) difference in the body weights of the adult birds at any treatment level. There was a slight, though statistically significant (p < 0.01) reduction in feed consumption by the adult birds at the 100 ppm cation treatment level during the first and seventh feed consumption interval, and a very slight, but statistically significant (p < 0.05) reduction in feed consumption during the sixth and eighth feed consumption interval at the 100 ppm cation concentration level.

Effects on reproduction:

For details on reproductive effects see 'Any other information on results incl. tables'
- At the 10 ppm cation treatment level no statistically significant (p < 0.05) effect was observed on any reproductive parameter.
- At the 30 ppm cation treatment level a statistically significant (p < 0.05) effect was observed on the body weights of 14-day old survivors. However, the observed effect was minor, and does not appear to be biologically meaningful. No other statistically significant differences were observed.
- At the 100 ppm cation treatment level a marked and statistically significant (p < 0.01) effect was observed on the number of eggs laid, the percent of viable eggs of eggs set, the number of normal hatchlings and the number of theoretical 14-day old survivors per pen. In conclusion, under the conditions of a One-Generation Reproduction Study, the test substance had no effect on the reproductive success of mallard ducks at dietary concentrations of up to 30 ppm cation.

Reported statistics and error estimates:

The statistical analysis of the data generated during the course of a One-Generation Reproduction Study conducted in mallard duck with the test substance of two types. The analysis on the data consisting of body weight and other "measurement variables" is of the traditional analysis of variance type, partitioning variability into different sources. The analysis on the egg data and other "count variables" is based on Cochran 1s concept of extraneous variability for the binomial distribution. To facilitate the utilization of Cochran's analysis, which is based on percentage, raw numbers, such as the number of eggs laid per pen, must be converted to a percentage. These raw numbers are divided by a theoretical maximum, which, in the case of the eggs laid, is based on the hypothetical production of one egg per hen per day for the duration of the egg laying period. To analyze the number of 14-day old survivors on a per pen basis, the number of hatchlings per pen was multiplied by the percent of 14-day old survivors of hatchlings per week to yield theoretical 14-day old survivors per pen.

Table: Body weight and feed consumption data of the adults

Week	Controls		Test substance (cation in ppm)
			10		30		100
	B.W.	F.C.	B.W.	F.C.	B.W.	F.C.	B.W.	F.C.
0	1176	-	1158	-	1128	-	1154	-
2	1228	119	1185	101	1146	103	1178	97**
4	1229	120	1210	113	1186	113	1206	114
6	1231	114	1208	108	1184	112	1207	123
8	1184	76	1159	68	1126	66	1167	79
10	-	134	-	129	-	128	-	139
12	-	154	-	153	-	136	-	132*
14	-	148	-	141	-	138	-	121**
16	-	161	-	139	-	139	-	129*
18	1193	138	1196	150	1193	132	1232	127

B.W. - Body weight in grams. The body weight data are presented as a group mean.

F.C. - Feed consumption in grams. Feed consumption data are presented as the group mean feed consumption per bird per day.

* Statistically Significant Difference (p < 0.05)

** Statistically Significant Difference (p < 0.01)

 

Table: Reproductive data

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
Eggs laid	454	496	439	186**
Hens in Production	11	12	12	12
Eggs Cracked	6	8	3	3
Eggs Set	366	395	348	111
Viable Embryos	333	356	303	72
Live Three-Week Embryos	312	334	284	64
Normal Hatchlings	211	221	204	49**
14-Day Old Survivors	205	213	198	49**
Eggs Laid/ Hen in 8 Weeks (a)	38	41	37	16
Eggs Laid/ Hen in Production	41	41	37	16
14-Day Old Survivors/Hen	17	18	17	4
14-Day Old Survivors/Hen in Production	19	18	17	4

a) Based on 12 hens

** Statistically Significant Difference (p < 0.01)

 

Table: Reproductive Success Data

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
Eggs Laid	454	496	439	186**
Eggs Laid of Theoretical Maximum (%) (a)	68	74	65	28
Eggs Cracked Of Eggs Laid (%)	1	2	1	2
Viable Embryos Of Eggs Set (%)	91	90	87	65**
Live Three-Week Embryos Of Viable Embryos (%)	94	94	94	89
Normal Hatchlings Of Live Three-Week Embryos (%)	68	66	72	73
14-Day Old Survivors Of Normal Hatchlings (%)	97	96	97	100
14-Day Old Survivors Of Eggs Set (%)	56	54	57	44
Normal Hatchlings Of Theoretical Maximum (%)(b)	44	46	43	10**
14-Day Old Survivors Of Theoretical Maximum (%) (b)	43	44	41	10**

a) Theoretical maximum= 672 eggs or 1 egg per hen per day for laying period.

b) Theoretical maximum= 420 chicks.

** Statistically Significant Difference (p < 0.01)

 

Table: Body weight data - Hatchlings

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
No. Of Chicks Weighed	208	219	203	49
Mean Body Weight (g)	36	36	36	34

 

Table: Body weight data – 14-Day old survivors

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
No. Of Chicks Weighed	205	213	198	49
Mean Body Weight (g)	216	218	205	248

 

Table: Egg weight data

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
No. Of Eggs Weighed	58	84	68	26
Mean Egg Weight (g)	60.9	61.5	58.4	56.1

 

Table: Eggshell Thickness data

Parameter	Test substance (cation in ppm)
	Control	10 ppm	30 ppm	100 ppm
No. Of Eggs Measured	83	93	88	72
Mean Shell Thickness (mm)	.367	.372	.373	.373

 

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 30 mg test substance/kg bw = 41.4 mg pure test substance/kg bw.

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of a One-Generation Reproduction Study, the test substance had no effect on the reproductive success of mallard ducks at dietary concentrations of up to 30 ppm cation. This corresponds to a recalculated NOEL value of 41.4 mg pure test substance/kg.

Executive summary:

The reproductive effects of the test substance to birds was determined in a long-term toxicity study with mallard duck in accordance with 40 CFR 163.71-4 and in compliance with GLP criteria. The test substance was fed at dietary concentrations of 10 ppm, 30 ppm, and 100 ppm test substance cation

to mature bobwhite quail during 10 weeks, followed by an 8 week egg laying period. The observed reproductive parameters included eggs laid, eggs cracked, viable embryos, live three-week embryos, normal hatchlings, and 14-day old survivors. No biologically significant impairment of reproductive success was observed up to 30 ppm cation. In the highest treatment group, a marked and statistically significant effect was observed on the number of eggs laid, the percent of viable eggs of eggs set, the number of normal hatchlings and the number of theoretical 14-day old survivors per pen. Also, a slight change in feed consumption in the adults was observed. Based on these findings, the no effect level of the test substance in mallard duck was 30 ppm cation. This corresponds to a recalculated NOEL value of 41.4 mg pure test substance/kg.

Description of key information

14-d LD50 was determined at 33.8 mg pure test substance/kg bw (recalculated) in zebra finches (Taeniopygia guttata), LD50-slope test, OECD 223, Hubbard 2014

10-wk NOEC was determined at 41.4 mg pure test substance/kg diet (recalculated) in mallard duck (Anas platyrhynchos), CFR 163.71-4, Beavers, 1982

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

41.4 mg/kg food

Additional information

Table: Overview of available data on the toxicity to birds

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Taeniopygia guttata	OECD 223 / GLP	14-d LD50	33.8 mg/kg bw (recalculated)	LD50-slope test design, 3 stages	Hubbard, 2014
Anas platyrhynchos	CFR 163.71-4 / GLP	10-wk NOEL	41.4 mg/kg (recalculated)		Beavers, 1982