mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

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:

9 October 1986 to 17 November 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-1 (Avian Acute Oral Toxicity Test)

Version / remarks:

October 1982

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For each dose group 50 g of a dispersion in 0.5 % aqueous carboxymethylcellulose (= CMC; bidistilled water was used) were prepared separately. The concentrations of the test compound were 10, 5, 2.5, 1.25 % or 0.625 % (w/w). The compound was mixed into the CMC preparation with an ultrasonic stirrer. The mixing procedure was then continued on a magnetic stirrer during the drawing of samples for the administration.

Dose method:

gavage

Analytical monitoring:

yes

Remarks:

The analytical determinations were performed by HPLC.

Vehicle:

yes

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: Yes
"Ssniff" experimental diet, special mixture for quails in pellets with the following analytical data (diet partly for housing and for adaptation period and test):
24.7 % crude protein
3.6 % crude fat
2.7 % crude fiber
6.8 %crude ash
1.04 % calcium
0.82 % phosphorus
0.20 % sodium
0.40 % methionine
1.00 % lysine
Content of additives
13 500 I.U. vitamin A
2 000 I.U. vitamin D3
12 mg vitamin E
- Type, identity and function of solvent/vehicle: 0.5 % aqueous carboymethylcellulose.
- Amount of vehicle: 0.5 %
- Residues in the diet: The feed used in the study is regularly assayed for contaminants.
- Analyses of drinking water: The drinking water is regularly assayed for contaminants by the municipal authorities of Frankanthal and the Technical Services of BASF Aktiengesellschaft. In view of the aim and duration of the study there are no special requirements exceeding the specifications of the drinking water.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite quail
- Age at test initiation: Age at the beginning of the test about 7 months (hatched April 4, 1986).
- Weight at test initiation: The birds were weighed individually shortly before the beginning of the test and were allocated to the test groups by a randomisation plan on the basis of their body weights prepared by following a standard laboratory method.
- Sexes used / mixed or single sex: Male and female birds; these could be determined properly due to the coloration of the animals at that age.
- Disease free: Yes
- Medical treatment: No
- Kept according to standard practices: Yes

Limit test:

no

Remarks:

Single bolus dose by gavage directly into the crop.

Post exposure observation period:

14 days

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

Each group consisted of 5 males and 5 females

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

Nominal concentrations: 62.5, 125, 250, 500 and 1 000 mg/kg.

Details on test conditions:

ACCLIMATION
- Acclimation period: Adaptation to the housing conditions of a group consisting of a sufficient number of birds in an iron wire pen of sufficient size with a stainless steel wire mesh bottom from Sept. 9, 1986 onward. Adaptation to the test cages from Oct. 24 to Nov. 3, 1986 (beginning of the test).
- Acclimation conditions: During adaptation and test period about 20 °C; Relative humidity: Adaptation and test period generally about 50 to 60 %; during adaptation to the housing conditions 10 hours light, 14 hours dark. During adaptation to the test cages and during the test period 12 hours light, 12 hours dark; warm-light fluorescent lamps.
- Feeding: The birds were offered a commercial poultry diet ad libitum throughout maintenance before the study and during the test with the exception of a fasting period of about 24 hours prior to dosing. Municipal water was available ad libitum throughout bird maintenance and the study.
- Health (any disease or mortality observed): None

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Stainless steel wire mesh cages with wire mesh floors (0.59 x 0.45 x 0.26 m); floor area about 0.26 m^2 for 5 birds each; males and females are caged separately.
- Caging: Group

NO. OF BIRDS PER STAGE OR REPLICATE
- For vehicle control: 5 males and 5 females.
- For treated: 5 males and 5 females per dose

NO. OF STAGES OR REPLICATES PER GROUP
- For vehicle control: One replicate per dose.
- For treated: One replicate per dose.

TEST CONDITIONS
- Temperature: 20 ºC
- Relative humidity (%): 50 - 60 %
- Photoperiod: 12 hours light, 12 hours dark; warm-light fluorescent lamps.

RANGE FINDING STUDY
- Results used to determine the conditions for the definitive study: The initial range finding study with 2 male and 2 female birds/dose indicated that the LD50 could be expected at about 500 mg/kg body weight.
The EPA protocol requires a minimum of five doses spaced by a constant factor for calculating the LD50. If the LD50 is expected to be greater than 2 000 mg/kg a smaller number of doses can be used, but at least one dose level without mortality should be included. In addition, it is desirable that the No Observable Effect Level should be determined. In view of these requirements and the results of the range finding study the doses were determined.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Mortality: Twice on day zero, daily thereafter for the 14 day observation period.
Clinical signs: Twice on day zero, daily at least on work days thereafter for the 14 day observation period.

FOOD CONSUMPTION
- Time schedule for examinations: Mean feed consumption (g/bird/day) calculated from the daily feed consumption/cage separately for male and female birds.

BODY WEIGHT
- Time schedule for examinations: Individual body weights were determined and the group means were calculated separately for male and female birds on day zero and 14 of the study.

PATHOLOGY
- Dose groups that were examined: All sporadic deaths; all remaining birds at termination of the study.

Reference substance (positive control):

no

Key result

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single bolus dose by gavage directly into the crop.

Key result

Dose descriptor:

LD50

Remarks:

Males and females

Effect level:

ca. 500 mg/kg bw/day (nominal)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single bolus dose by gavage directly into the crop.

Dose descriptor:

LD50

Remarks:

Males

Effect level:

ca. 600 mg/kg bw/day (nominal)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single bolus dose by gavage directly into the crop.

Dose descriptor:

LD50

Remarks:

Females

Effect level:

ca. 500 mg/kg bw/day (nominal)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single bolus dose by gavage directly into the crop.

Mortality and sub-lethal effects:

MORTALITY
No deaths occurred in the control group or in the three lowest dose groups (62.5; 125 and 250 mg/kg).
Highest dose tested causing no mortality was 250 mg/kg body weight.
Minimum dose tested causing 100 % mortality was 1 000 mg/kg weight.
The LD50 for males plus females is about 500 mg/kg body weight:
- For males: About 600 mg/kg body weight;
- For females: About 500 mg/kg body weight.

CLINICAL SIGNS
In the control group and in the 62.5 or 125 mg/kg groups all birds were in good health during the whole study.
In the 250 mg/kg group one male bird showed apathy on the day of administration and on day one males and females had soft faeces.
In the higher dose groups (500 or 1 000 mg/kg) severe toxic signs such as apathy, prone and side position, ruffled feathers, difficulties in walking (ataxia) and diarrhoea were seen.
The survivors had completely recovered from day 3 onward (on days 5 and 6 no checks for toxic signs were made).

BODY WEIGHT
There was no statistically significant reduction in body weight in the surviving birds of all test groups (male and female) on day 14 at the end of the study.
The body weights of the birds that had died during the study are not reported as they are considered not to be relevant for the results of the study.

FOOD CONSUMPTION
The initial feed consumption/bird/day was reduced - dose dependent - in the males and females in the three highest dose groups (250, 500 and 1 000 mg/kg body weight).
From day 4 onward the feed uptake was in the normal range in all dose groups.
The mean total feed consumption/bird/day during the 14-day observation period was in the same order of magnitude in all test groups with surviving birds and in the control group with the exception of a marginal reduction in the feed uptake of the two female survivors in the 500 mg/kg group.

PATHOLOGY
- Results: All birds were examined macroscopically.
- Surviving birds: No adverse effects
- Birds that had died (500 and 1 000 mg/kg): General congestive hyperemia or no adverse effects detectable

Reported statistics and error estimates:

The statistical evaluation of the body weights was performed by one-way analyses (ANOVA) followed by Dunnets test. Dunnets test: A multiple comparison
procedure for comparing several treatments with a control , C.W. Dunnet 1955, JASO 50 and "New tables for multiple comparisons with a control", C.W. Dunnet 1964, Biometrics 20. The statistical evaluation was performed with the computer systems of the Department of Toxicology using the INSTEM-Toxicology-data system (program TOXREP).
The LD50 values were calculated by a computer program following the probit model according to: Finney, D.J.,Probit Analysis, Cambr. Univ. Press, 1971.

Cumulative Mortality

Test Group	Dose Level (mg/kg)	Day of the Study
		0*		0**		1		2		3		4		5		6
		M	F	M	F	M	F	M	F	M	F	M	F	M	F	M	F
0	0 (Carrier control)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	62.5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	125	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	250	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	500	0	0	0	0	1	0	1	1	1	3	1	3	1	3	1	3
5	1000	0	0	0	0	2	4	5	5	5	5	5	5	5	5	5	5
Test Group	Dose Level (mg/kg)	Day of the Study
		7		8		9		10		11		12		13		14
		M	F	M	F	M	F	M	F	M	F	M	F	M	F	M	F
0	0 (Carrier control)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	62.5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	125	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	250	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	500	1	3	1	3	1	3	1	3	1	3	1	3	1	3	1	3
5	1000	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

* About 2 to 4 hours after administration.

** About 3 to 5 hours after administration.

Five male (m) and 5 female (f) birds/test group.

Stability

The stability of aqueous solutions was verified for a period of 4 days by analytical determination in a previous acute study on fish.

 

Homogeneity

The homogeneity of the preparations is ensured because they were mixed thoroughly.

 

Concentration Control

An analytical concentration control was performed for all dose levels as they had to be prepared separately (one determination per concentration).

 

Nominal Concentration (mg/kg)	Analytically Detected Concentration (mg/kg)	% of Nominal Concentration (Mean)
6250	5164	82.6
12500	11116	88.9
25000	31526	126.1
50000	54022	108.0
100000	101304	101.3

 

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study, the LD50 for the bobwhite quail is:
- For males plus females: About 500 mg/kg body weight.
- For males only: About 600 mg/kg body weight
- For females only: About 500 mg/kg body weight
The NOEL was 125 mg/kg body weight.

Executive summary:

The toxicity of the test material to birds was assessed according to EPA Test Guideline OPP 71-1 and in compliance with GLP using bobwhite quail.

The test material (dispersed in 0.5 % aqueous CMC preparation) was tested for its acute toxicity to the bobwhite quail after administration by gavage of single doses of 0, 62.5, 125, 250, 500 and 1 000 mg/kg body weight followed by a 14-day observation period.

Mortality: No deaths occurred in the control group or in the three lowest dose groups (62.5, 125 and 250 mg/kg). Highest dose tested causing no mortality was 250 mg/kg bodyweight. Minimum dose tested causing 100 % mortality was 1 000 mg/kg body weight.

Clinical signs: In the control group and in the 62.5 or 125 mg/kg groups all birds were in good health. In the 250 mg/kg group one male bird showed apathy on the day of administration and on day one males and females had soft faeces. In the higher dose groups (500 and 1 000 mg/kg) severe toxic signs such as apathy, prone and side position, ruffled feathers, ataxia and diarrhoea were diagnosed. The survivors had completely recovered from day 3 onward.

Feed consumption: The initial feed consumption/bird/day was reduced – dose dependent - in the males and the females in the three highest dose groups (250, 500 and 1 000 mg/kg body weight). From day 4 onward the feed uptake was in the normal range in all dose groups. The mean total feed consumption/bird/day during the 14-day observation period was in the same order of magnitude in all test groups with surviving birds and in the control group with the exception of a marginal reduction in the feed uptake of the two female survivors in the 500 mg/kg group.

Body weight: There was no statistically significant reduction in body weight in the surviving birds on day 14 at the end of the study.

Gross post-mortem examination: Surviving birds had no adverse effects. Birds that had died (500 and 1000 mg/kg) showed general congestive hyperemia or no adverse effects.

Under the conditions of the study, the LD50 for the bobwhite quail is:

- For males plus females: About 500 mg/kg body weight.

- For males only: About 600 mg/kg body weight

- For females only: About 500 mg/kg body weight

The NOEL was 125 mg/kg body weight.

Reference 2

Endpoint:

short-term toxicity to birds: acute oral toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 August 1994 to 22 September 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-1 (Avian Acute Oral Toxicity Test)

Version / remarks:

October 1982, and draft revised guideline dated March 1988.

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Corn oil

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: The diet had the following composition:
38.00 % w/w wheat
30.00 % w/w maize
10.00 % w/w Provimi 66
10.00 % w/w soya 48 %
5.00 % w/w wheatfeed
5.50 % w/w limestone
0.30 % w/w Serla-Bondex FP
1.25 % w/w layer supplement

- Preparation of doses: A single preparation in the vehicle was made at five dose concentrations so that all birds received the same dose volume per unit of bodyweight. The test material was ground in a mortar with the vehicle until a smooth paste was formed. The formulation was then gradually made up to volume and mixed using a high shear homogeniser.
Dose concentrations were 2.60, 3.64, 5.10, 7.14 and 10.0 % w/v. Immediately after preparation, a 20 mL sample was taken from each dose suspension and stored frozen.
- Type, identity and function of solvent/vehicle: Corn oil
- Amount of vehicle: All birds were dosed at a rate of 10 mL/kg.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite quail
- Age at test initiation: Adults, approximately eleven months of age on arrival.
- Weight at test initiation: At the start of the pre-treatment period (Day -15) all birds were within the bodyweight range 181 - 227 g.
- Sexes used / mixed or single sex: Mixed
- Disease free: On arrival, the birds were given aureomycin, an antibiotic, in water (120 mg/L) for ten days in order to eliminate any subclinical bacterial infection.
- Kept according to standard practices: Yes
- Other : Birds 7904 and 7905 were replacement birds used due to excessive weight loss in the original birds.

Limit test:

no

Remarks:

Single bolus dose.

Post exposure observation period:

14 days

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

Five male and five female birds per dose

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

260, 364, 510, 714 or 1 000 mg test material/ kg bodyweight.

Details on test conditions:

ACCLIMATION
- Acclimation period: The birds were acclimatised for 15 days prior to dosing
- Acclimation conditions: Yes, same as test.
- Feeding: Food was offered ad libitum, with the exception of an overnight starvation period of approximately 22 hours prior to dosing. Food hoppers and their contents were weighed at the beginning and end of each time period. Food consumption was calculated as the difference between these two values.
- Health (any disease or mortality observed): No
- Fasting period before study: Yes. Overnight starvation period of approximately 22 hours prior to dosing.
- The birds were given a single dose of the test material or vehicle by oral intubation using a disposable syringe and a Ch 10 Nelaton plastic catheter. Care was taken to ensure that the bird had ingested all the dose material before being returned to its cage.

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: 22 h

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The birds were housed by sex in groups of two or three according to treatment in tiered cages measuring 0.31 x 0.39 x 0.24 min a building which provided appropriate environmental conditions for the species. Each cage was made of plastic coated steel wire mesh and contained an automatic drinker and food hopper.
- Caging: Group . The birds were allocated to treatment groups on the basis of bodyweight, with the aim of all treatment groups having similar mean bodyweights and bodyweight distributions.

NO. OF BIRDS PER STAGE OR REPLICATE
- For vehicle control: One group of five male and five female adult birds
- For treated: Groups of five male and five female adult birds

NO. OF STAGES OR REPLICATES PER GROUP
- For vehicle control: One
- For treated: One per dose

TEST CONDITIONS
- Temperature: 19 - 21 ºC
- Relative humidity (%): 72 %
- Photoperiod: artificial lighting provided 10 hours continuous light and 14 hours dark.
- Ventilation: Ventilation fans were adjusted as necessary.

RANGE FINDING STUDY
- Test concentrations: 250, 500 or 1 000 mg/kg
- Test conditions: Pairs of birds were dosed.
- Results used to determine the conditions for the definitive study: Yes. At 1 000 mg/kg both birds died. dose levels were selected in compliance with the guideline.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Birds were observed daily during the study and at frequent intervals during the post treatment period. Mortalities, bird health and clinical signs were recorded at each observation.

BODY WEIGHT
- Time schedule for examinations: Individual bodyweights on Days -15, -7, 0 (immediately prior to dosing), 7 and 14.

FOOD CONSUMPTION
- Time schedule for examinations: Group mean food consumption over Days -15 to -8, -7 to -1, 1 to 7 and 8 to 14.

PATHOLOGY
- Dose groups that were examined: Any bird which died during the study was examined post mortem. At termination of the study, post mortem examination was carried out on all control birds and all ten birds from the highest surviving dose group.

Details on reproductive parameters:

None

Reference substance (positive control):

no

Key result

Dose descriptor:

LD50

Effect level:

648 mg/kg bw

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Single bolus dose.

Remarks:

95 % confidence limits 574 - 728 mg/kg bw

Key result

Dose descriptor:

NOEL

Effect level:

364 mg/kg bw

Conc. / dose based on:

test mat.

Basis for effect:

mean feed consumption

Remarks on result:

other: Single bolus dose.

Mortality and sub-lethal effects:

MORTALITY
There was a single mortality (Bird 982) in Group 4 (test material at 510 mg/kg) during Day 1.
In Group 5 (test material at 714 mg/kg), three male birds (101, 103 and 107) and four female birds (102, 106, 108 and 110) died during Day 1.
All birds died in Group 6 (1 000 mg/kg). Four male birds (1110, 113, 117 and 119) and four female birds (7 904, 114, 118 and 1202 died on Day 1, the remaining two birds; 1150 and 1162 died on Day 2.

CLINICAL SIGNS
- Results:
All control birds and Group 2 (test material at 260 mg/kg) remained in good health throughout the study.
Unsteadiness and ruffled feathers were observed in all birds in Group 4 following dosing. By Day 4 all surviving birds had recovered and remained in good health until termination.
In Group 5 (test material at 714 mg/kg), all birds showed unsteadiness and ruffled feathers following dosing.
All birds in Group 6 (1 000 mg/kg) showed unsteadiness and ruffled feathers from shortly after dosing until death.

ABNORMAL BEHAVIOUR
- Results:
In Group 3 (test material at 364 mg/kg) all birds showed subdued behaviour following dosing but had recovered before the end of Day 1.
Subdued behaviour was observed in all birds in Group 4 following dosing.
In Group 5 (test material at 714 mg/kg), all birds showed subdued behaviour following dosing; survivors remained subdued on Days 4 and 5 with full recovery by the end of Day 5.
All birds in Group 6 (1 000 mg/kg) showed subdued behaviour from shortly after dosing until death.

BODY WEIGHT
- During the acclimatisation period, bodyweight changes were variable. Analyses of variance (Williams 1971, 1972) were carried out for bodyweights on Days 7 and 14, excluding Group 6 (test material at 1 000 mg/kg) where there was 100 % mortality and Group 5 where insufficient data were available.
- For the males at Day 7, the Williams' test indicated that the 510 mg/kg test material was significantly different (p < 0.05), when compared with the control group. For the females, at Day 7, Williams' test showed that the 364 g/kg and 510 mg/kg test material dose groups were significantly different when compared with the control group (respectively p <0.05 and p <0.01). There were no statistically significant treatment-related effects for either sex at Day 14.

FOOD CONSUMPTION
- A small reduction was observed in food consumption over Days 1 - 7 in Groups 3 - 5 (test material at 364 - 714 mg/kg). Values obtained for consumption for Group 6 (test material at 1 000 mg/kg) are based on limited data and therefore no conclusive statement can be made regarding food consumption.

PATHOLOGY
- There were no abnormalities in any birds examined.

Reported statistics and error estimates:

The LD50 value was determined using a logistic model (Berkson 1944) for which 95 % confidence limits were estimated by the likelihood ratio method (Williams 1986).

The mean of bodyweights recorded at Day 7 and at Day 14 were analysed for each sex separately. The mean pre-dose bodyweight (Day -15, -7 and 0) was included as a covariate in these analyses as this improved precision (covariate efficiency > 100 %). Comparisons between the treated groups and the control were carried out using Williams' test (Williams 1971, Williams 1972) for a dose-related trend.
For dose group 714 mg/kg group there was insufficient data for the analyses of the bodyweights at Day 7 and at Day 14. For the 1 000 mg/kg dose group no bodyweight data was available for Day 7 and for Day 14. Therefore, the dose groups, 714 mg/kg and 1 000 mg/kg doses, were excluded from the further statistical analyses and this for both sexes.
For the males at Day 7, the Williams' test indicated that the 510 mg/kg dose was significantly different (p < 0.05), when compared with the control group. For the females, at Day 7, a statistically significant difference (p <0.001) was found between the groups. The Williams' test showed that the 364 mg/kg and 510 mg/kg dose groups were significantly different when compared with the control group (respectively p < 0.05 and p < 0.01). There were no statistically significant treatment-related effects for either sex at Day 14.

Distribution of Mortalities

Group	Treatment (mg/kg)	No. of Birds	Day of Study		Total
			1	2
1	0	10
2	260	10
3	364	10
4	510	10	1		1
5	714	10	7		7
6	1000	10	8	2	10

Group Mean Bodyweights and Bodyweight Changes (g)* 

Group	Treatment (mg/kg)	Sex	Days of Study
			Bodyweight					Bodyweight Change
			-15	-7	0	7	14	-15 to 0	-7 to 0	0 to 7	7 to 14
1	Control	M	198	194	181	194	194	-4	-13	+13	0
	0	F	212	208	200	212	210	-4	-8	+12	-2
2	Test material	M	198	189	180	184	189	-9	-9	+4	+5
	260	F	214	202	198	214	212	-12	-4	+16	-2
3	Test material	M	198	188	180	184	191	-10	-8	+4	+7
	364	F	210	214	198	201	205	4	-16	+3	+4
4	Test material	M	199	184	181	181	190	-15	-3	0	+9
	510	F	212	209	201	194^4	209^4	-3	-8	-10	+15
5	Test material	M	196	187	182	163^2	181^2	-9	-5	-23	+18
	714	F	213	212	199	172^1	200^1	-1	-13	-25	+28
6	Test material	M	197	198	189	-	-	+1	-9	-	-
	1000	F	213	215	200	-	-	+2	-15	-	-

*Bodyweight changes are based on the means of the birds surviving between the relevant time points.

Superscript indicates the number of birds weighed.

 

Group Mean Food Consumption (g/bird/day)

Group	Treatment (mg/kg)	Sex	Days of Study
			-15 to 0	-7 to 0	0 to 7	7 to 14
1	Control	M	16	17	20	21
	0	F	21	23	26	28
2	Test material	M	15	16	18	21
	260	F	19	22	22	26
3	Test material	M	15	17	16	21
	364	F	20	19	18	25
4	Test material	M	13	17	15	19
	510	F	18	20	15	22
5	Test material	M	14	15	12	22
	714	F	21	23	13	24
6	Test material	M	15	16	9	-
	1000	F	20	21	24	-

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of the study the acute oral toxicity (LD50) of test material to the Bobwhite quail was calculated to be 648 mg/kg. The no-effect level for mortality was 364 mg/kg.

Executive summary:

The acute oral toxicity of the test material was assessed according to the United States Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Series 71 -Avian and Mammalian Testing § 71-1 Avian single-dose oral LD50 test and in compliance with GLP using the Bobwhite quail.

Groups of five male and five female adult birds were given a single oral dose, by intubation, of either 260, 364, 510, 714 or 1 000 mg test material/ kg bodyweight. A similar sized control group was dosed in the same way receiving the vehicle, corn oil, alone. Birds were observed for 14 days following dosing. Observations included mortality, clinical signs, bodyweight and food consumption.

Ten mortalities occurred at 1 000 mg/kg, seven at 714 mg/kg and one at 510 mg/kg. Clinical signs of toxicity, including subdued behaviour, unsteadiness and ruffled feathers were observed in birds treated with the test material at 364 mg/kg and above.

Significantly lower bodyweights on Day 7 (females) at 364 mg/kg and 510 mg/kg were observed.

Food consumption was reduced in Groups 3 - 5 over Days 1 - 7.

No abnormalities were observed in any birds examined at post mortem examination.

Under the conditions of the study the acute oral toxicity (LD50) of test material to the Bobwhite quail was calculated to be 648 mg/kg.  The no-effect level for mortality was 364 mg/kg.

Reference 3

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2016 to 26 January 2017

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2300 (Avian Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Deviations:

no

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Corn oil and acetone.

Details on preparation and analysis of diet:

DIET PREPARATION
Test diets were prepared by mixing the test material 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 beginning on the 18 July 2016 and presented to the birds on Wednesday of each week. Dietary concentrations were adjusted for the purity of the test material and are presented as parts per million active ingredient (ppm a.i.).

Premixes were prepared on 18 July 2016 and 08 August 2016. Nominal preparation was as follows:
Control: 8918.0 g ration + 202 mL corn oil
- 80 ppm a.i.: 23.7908 g test material + 8894.2 g ration + 202 mL corn oil
- 200 ppm a.i.: 59.477 g test material + 8858.5 g ration + 202 mL corn oil
- 400 ppm a.i.: 118.954 g test material + 8799.0 g ration + 202 mL corn oil
For each of the premixes, an appropriate amount of test material was ground with a mortar and pestle. Once the test material was ground the needed amount was weighed in a tared weigh boat on an analytical or top loading balance. Basal ration, 4500.0 g, was weighed into a tared mixing bowl on a top loading balance. An aliquot of the basal ration was held for later use (retained ration).
A portion of the retained basal ration was placed in a blender and the test material was added to the blender. The weigh boat was rinsed three times with some of the retained ration and the rinsate was added to the blender and the contents were blended for at least two minutes. After blending, the contents were transferred to the mixing bowl with the basal ration. The blender was rinsed two times with portions of the retained ration and the rinsate was added to the mixing bowl. Corn oil was measured in a graduated cylinder and added to the basal ration in the mixing bowl. The remaining retained ration was also added to the mixing bowl. The bowl contents were mixed on a stand mixer for approximately 15 minutes. The remaining amount of basal ration needed was weighed on a top-loading balance and added to the mixing bowl. Mixing then continued for an additional 25 minutes (the first premix was mixed for five and 15 minutes; homogeneity results indicated that additional mixing time would be required and subsequent premixes were prepared by mixing for 15 and 25 minutes).
After mixing, 1800.0 gram aliquots of the premix were weighed on a top-loading balance, placed in appropriately labelled plastic bags, reweighed and stored frozen, unless used immediately for preparation of final diet.
As needed, the appropriate premix was incorporated into the final diet as follows:
- 0 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 80 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 200 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 400 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
The diet was mixed for approximately 20 minutes in a Patterson-Kelley® Twin Shell Blender.

Premixes for test material were prepared on 16 August 2016, 18 September 2016, 10 October 2016 and 11 November 2016. Nominal preparation was as follows:
- Control: 8918.0 g ration + 202 mL corn oil + 400 mL acetone
- 80 ppm a.i.: 23.7908 g test material + 8894.2 g ration + 202 mL corn oil + 400 mL acetone
- 200 ppm a.i.: 59.4771 g test material + 8858.5 g ration + 202 mL corn oil + 400 mL acetone
- 400 ppm a.i.: 118.9542 g test material + 8799.0 g ration + 202 mL corn oil + 400 mL acetone
Acetone was added to premixes beginning on 16 August 2016 through the end of the study, to promote a better mix with the test material. An appropriate amount of test material was ground with a mortar and pestle and the amount needed was weighed in a tared weigh boat on an analytical or top loading balance. The test material was transferred to a beaker.
Acetone was measured in a graduated cylinder and was added to the test material with a portion of the acetone used to rinse the weigh boat. The rinse was added to the beaker. A magnetic stir-bar was placed in the beaker and the mixture stirred for at least approximately two minutes on a magnetic stir plate, until the test material dissolved. Corn oil was measured in a graduated cylinder then added to the beaker and the mixture was stirred for at least one additional minute.
Basal ration, 4500.0 g, was weighed into a tared mixing bowl on a top loading balance. The test material mixture was then added to the basal ration in the mixing bowl. The beaker was rinsed with additional acetone measured in a graduated syringe. The rinse was added to the mixing bowl and the contents of the bowl mixed on a stand mixer for approximately 15 minutes. The remainder of the basal ration needed for the premix was then weighed on a top-loading balance and added to the mixing bowl and mixed for an additional 25 minutes.
After mixing, 1800.0 gram aliquots of the premix were weighed on a top-loading balance, placed in appropriately labelled plastic bags, reweighed and stored frozen, unless used immediately for preparation of final diet.
As needed, the appropriate premix was incorporated into the final diet as follows:
- 0 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 80 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 200 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
- 400 ppm a.i.: 1800 g Premix + 50.45 kg ration + 2.750 kg limestone
The diet was mixed for approximately 20 minutes in a Patterson-Kelley® Twin Shell Blender.

DIET SAMPLING
Homogeneity of the test material in the diet was evaluated by collecting six samples from each of the 80 and 400 ppm a.i. treated diets and one sample from the control diet on Day 0 of Week 1.
Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Weeks 1 and 20 to assess stability of the test material under actual test conditions. Additionally, samples were collected from the 200 ppm a.i. treatment group on Day 0 of Week 1 and samples were collected from the control and treatment group diets during Weeks 8, 16 and 20 of the test to measure/verify test concentrations. The diet samples were transferred to the testing facility’s analytical chemistry facility and stored frozen prior to analysis.

ANALYTICAL METHOD
The analysis of test material in avian diet was based upon methodology developed by the testing facility. Samples were extracted with 0.05 M sodium hydroxide in methanol.
- Instrument: Agilent Series 1200 high performance liquid chromatograph (HPLC) equipped with an Agilent Series 1200 variable wavelength detector (VWD)
- Analytical column: Inertsil ODS-2 AM (250 mm x 4.6 mm I.D., 5 μm particle size)
- Flow rate: 1.000 mL/minute
- Column temperature: 40 °C
- Mobile phase: Channel A: 50:50:0.1 (v/v/v) Acetonitrile: HPLC grade water: Phosphoric Acid; Channel B: Acetonitrile
- Gradient profile: At 1 minute: 90 % A and 10 % B; at 7 and 8 minutes: 10 % A and 90 % B; at 8.10 and 12 minutes: 90 % A and 10 % B
- Injection volume: 25.00 µL
- Test material peak retention time: Approximately 6.4 minutes
- Analytical wavelength: 230 nm

Calibration standards of test material, ranging in concentration from 0.500 to 10.0 ppm a.i. were prepared in 40:60 (v/v) methanol: HPLC grade water using a stock solution of test material in methanol.
The stock solution of tets material was prepared by accurately weighing 0.1069 g (corrected for purity) on an analytical balance. The test material was transferred to a 100-mL volumetric flask, and brought to volume using methanol. The primary stock solution (1.00 mg a.i./mL) was diluted in methanol to prepare a 0.100 mg a.i./mL stock solution. The 0.100 mg a.i./mL stock solution was used to prepare the calibration standards in 40:60 (v/v) methanol: HPLC grade water. The set of calibration standards had the following concentrations: 0.500, 1.00, 2.50, 5.00, 7.50 and 10.0 ppm a.i.

Calibration standards were analysed with each sample set. 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 material in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation. Examples of equations used in calculations are as follows:
The detected concentration of test material in each sample was determined from the slope and intercept of the calibration curve and the peak area response of each sample injected using the following equation:

Test material detected concentration = (ppm a.i.) (Peak area response - y intercept) / Slope

Determination of Sample Concentration: The concentration expressed as ppm a.i. for each sample was determined using the following equation:

Test material analysed sample concentration (ppm a.i.) = (Test material detected concentration (ppm a.i.) x extraction volume (mL) x Final dilution) / Initial sample weight (g)

Fortification Recoveries: The percent recovery of the method at each level of fortification is calculated as follows:

% Recovery = (Analysed sample concentration (ppm a.i.) / Fortified concentration (ppm a.i.)) x 100

The limit of detection (LOD) was defined as the lowest analyte concentration divided by the signal to noise ratio times 3 times the dilution factor of the matrix blank sample. The mean LOD for the study was calculated to be 0.123 ppm a.i. The limit of quantitation (LOQ) was defined as the lowest analyte concentration divided by the signal to noise ratio times 10, times the dilution factor of the matrix blank sample. The mean LOQ for the study was calculated to be 0.41 ppm a.i. Measured values greater than or equal to the LOQ were reported.
Along with the sample analyses, eight matrix blanks were analysed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses.
Avian diet samples were fortified at 60.0 and 1000 ppm a.i. using a dry mix technique and analysed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 120, 112, 102, 110, 110, 109, 115 and 108 %. These values correspond to each sample set analysed during the definitive study. Sample measured concentrations were not corrected for the mean procedural recoveries from each sample set.

ANALYTICAL RESULTS
Analysis of the control samples did not show any indication of the presence of the test material or of the presence of a co-eluting material at the characteristic retention time of the test material. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 1 Day 0. Mean concentrations and standard deviations for the two test concentrations were 71.1 ± 16.0 ppm a.i. and 387 ± 148 ppm a.i., respectively. The coefficients of variation were 22.5 and 38.2 %, respectively. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 4 Day 0. Mean concentrations and standard deviations for the two test concentrations were 86.4 ± 25.1 ppm a.i. and 403 ± 92.1 ppm a.i., respectively. The coefficients of variation were 29.0 and 22.9 %, respectively. Diet samples were collected from the 80 and 400 ppm a.i. test concentrations and were analysed to evaluate the homogeneity of the test material in the diet for Week 5 Day 0. Mean concentrations and standard deviations for the two test concentrations were 81.0 ± 3.86 ppm a.i. and 380 ± 11.2 ppm a.i., respectively. The coefficients of variation were 4.76 and 2.96 %, respectively. Samples collected during the test to verify test material concentrations for the 80, 200 and 400 ppm a.i. diets had mean concentrations and standard deviations of 82.4 ± 4.60 ppm a.i., 198 ± 26.9 ppm a.i. and 412 ± 25.1 ppm a.i., respectively. The coefficients of variation were 5.59, 13.6 and 6.08 %, respectively. These values represented 103, 99.0 and 103 % of nominal concentrations, respectively. Analysis of Week 1 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 97.7 86.4 and 76.7 % of the Week 1 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively. Analysis of Week 5 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 89.6, 85.6 and 93.7 % of the Week 5 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively. Analysis of Week 20 Day 7 diet samples collected from feeders after being held at ambient temperature for 7 days averaged 88.9, 91.1 and 80.3 % of the Week 20 Day 0 values for the 80, 200 and 400 ppm a.i. test concentrations, respectively.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Age at test initiation: All birds were 18 weeks of age at test initiation (first day of exposure to test diet).
- Weight at test initiation: Birds ranged in weight from 840 to 1290 grams.
- Sexes used / mixed or single sex: Male and female.
- Cultural background: Pen-reared. At the start of acclimation, the mallard were apparently healthy and phenotypically indistinguishable from wild type.
- Disease free: Yes
- Kept according to standard practices: Yes. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomise pen assignment for each bird. 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. Sex of the birds was determined by a visual examination of the plumage.
- Breeding population: The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing.
- Feed and water: All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to the testing facility’s specifications by Cargill Animal Nutrition, Shippensburg, PA). The basal ration contained at least 27 % protein and 2 % crude fat, and no more than 5 % crude fibre. The basal diet contained approximately 1.12 % calcium, derived from feedstuffs and 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 % 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 %). Offspring received basal diet without test material and without the addition of 5 % supplemental limestone. Water was supplied by the town public water supply. Neither the adults nor offspring received any form of medication in their feed during the test. Feed and water were analysed periodically.

Limit test:

no

Total exposure duration (if not single dose):

21 wk

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

18 animals per sex per dose

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

- Nominal concentrations: 0, 80, 200 and 400 ppm a.i.

Details on test conditions:

ACCLIMATION
- Acclimation period: The test birds were acclimated to the facilities and study pens for four weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behaviour or debilitating physical injuries were not used for the test.

HOUSING AND ENVIRONMENTAL CONDITIONS
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. Sisal rope was added to each pen for animal enrichment at the start of photostimulation.
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.
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 study room during the course of the test was 21.7 (20.3 – 22.6) ± 0.6 °C (SD) with an average relative humidity of 67 (30 – 86) ± 15 % (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 room was maintained by a time clock. The photoperiod during acclimation and the first nine weeks of the test was eight hours or less of light per day with an average light intensity of approximately 303 lux (~ 28 ft. candles). The photoperiod was increased to 17 hours of light per day at the beginning of Week 11 to induce egg laying and was maintained at that length until the adult birds were euthanised. Measurement of light intensity in the study room following photostimulation was approximately 462 lux (~ 43 ft. candles), and during the egg-laying phase of the test the light intensity was approximately 461 lux (~ 43 ft. candles). Throughout the test illumination was provided by fluorescent lights that closely approximated the colour spectrum of noonday sunlight.

STUDY PHASES
The primary phases of the study and their approximate durations were:
1. Acclimation - 3 weeks.
2. Pre-photostimulation - 10 weeks.
3. Pre-egg laying (with photostimulation) – 2 weeks.
4. Egg laying - 10 weeks.
5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) – 6 weeks.

RANGE FINDING STUDY
The test concentrations were selected based upon the results of a pilot reproduction study.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: During the study, all adult birds were observed daily for signs of toxicity or abnormal behaviour.
- Remarks: Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations.

BODY WEIGHT
- Time schedule for examinations: Adult body weights were measured at test initiation, at the end of Weeks 2, 4, 6, 8, and at adult termination.
- Remarks: Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex.

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. Statistical comparisons were made between the control and each treatment group.

Estimated test material intakes, daily dietary dose, for mallards were calculated by treatment group for the pre-egg production period, the egg production period and the overall adult period using the following formula:

Daily Dietary Dose (mg a.i./kg body weight/day) = (Test Concentration (mg a.i./kg) x Daily Feed Consumption (g/bird/day)) / Body Weight (g/bird)

The mean body weight value is the mean of both male and female body weights. For the pre-egg production interval, the body weights were averaged over Weeks 0, 2, 4, 6 and 8. For the egg-production interval body weights were averaged over Weeks 8 and 21 (adult termination). 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.

WATER CONSUMPTION: Not measured

PATHOLOGY
- Dose groups that were examined: All animals
- Remarks: At the conclusion of the exposure period, all adult birds were euthanised by cervical dislocation, necropsied, and disposed of by incineration.

Details on reproductive parameters:

EGG COLLECTION AND STORAGE
Eggs were collected daily from all pens, when available. 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, rinsed with fresh water and allowed to cool to approximately room temperature. The eggs were then stored in a cold room until incubation. The cold room was maintained at a mean temperature of 14.3 ± 0.3 °C (SD) with a mean relative humidity of approximately 82 ± 8 % (SD). All eggs laid in a weekly interval were considered as one lot.

CANDLING AND INCUBATION
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 an egg-candling lamp (Speed King Model No. 32) to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded.
All eggs not discarded or used for egg shell thickness measurements were placed in an incubator (NatureForm Model No. NMC 4000). In the incubator the temperature was maintained at an average of 37.4 ± 0.0 °C (SD) with an average relative humidity of approximately 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 24 of incubation. Eggs were candled on Day 13 - 14 of incubation to determine embryo viability and on Day 20 - 21 to determine embryo survival. Eggs that appeared “clear” at the Day 13 - 14 candling were broken out and examined in an effort to determine fertility.

HATCHING AND BROODING
On Day 24 of incubation, the eggs were placed in an incubator configured for hatching (NatureForm Model No. NMC 4000 or Model # 2340) and allowed to hatch. Pedigree baskets constructed of galvanised 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 37.3 ± 0.0 °C (SD) with an average relative humidity of 60 ± 0 % (SD).
All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 27 or 28 of incubation. The body weights of surviving hatchlings were recorded and the average body weight by pen was determined. Hatchlings were wing 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 individual body weight of each surviving hatchling was recorded. The ducklings were euthanised with carbon dioxide and disposed of by incineration.
Hatchlings were housed in batteries of brooding pens. Each pen measured approximately 62 x 92 x 25.5 cm high. The walls, floors and ceilings of each pen were constructed of vinyl-coated 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 five to seven days of age, when the temperature was adjusted to maintain a temperature of approximately 29 °C.
The average ambient room temperature was 22.4 ± 0.9 °C (SD) with an average relative humidity of 40 ± 11 % (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day.

EGG SHELL THICKNESS MEASUREMENTS
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, 7 & 9) and from each of the even numbered pens during the even numbered weeks (2, 4, 6, 8 & 10). 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 one week 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 micrometre. Measurements were made to the nearest 0.002 mm.

STATISTICAL ANALYSES
Each of the following parameters was analysed statistically:
- Eggs/Hen/Day - The number of eggs laid per female divided by the total number of days of egg production.
- Eggs Cracked of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen.
- Fertile Eggs of Eggs Set – The number of fertile eggs at the Day 14 candling was divided by the number of eggs set, per pen.
- Viable Embryos of Eggs Set - The number of viable embryos at the Day 14 candling was divided by the number of eggs set, per pen.
- Live 3-Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen.
- Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen.
- 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings, per pen.
- Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set, per pen.
- Hatchlings of Fertile Eggs – The number of hatchlings was divided by the number of fertile eggs, per pen.
- 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per pen.
- Hatchlings/Pen/Day - The number of hatchlings per female divided by the total number of days of egg production.
- 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors per pen divided by the total number of days of egg production.
- Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured.
- Offspring's Body Weight - The average body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

21 wk

Dose descriptor:

NOEC

Effect level:

400 other: ppm a.i.

Conc. / dose based on:

act. ingr.

Basis for effect:

other: No effects were seen.

Mortality and sub-lethal effects:

MORTALITY
No adult mortalities occurred during the course of the study.

CLINICAL SIGNS
- Results: No overt signs of toxicity were observed at any of the concentrations tested.
- Remarks: Incidental clinical observations noted during the test, included incidental injuries associated with pen wear such as foot lesions, twisted primary feathers, moulting, and an unkempt appearance and were limited to a few birds. With the exception of incidental-injuries, all birds are normal in appearance and behaviour.

BODY WEIGHT
- Results: There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested.
- Remarks: No statistically significant differences between the control group and the 80, 200 or 400 ppm a.i. treatment groups were observed at any of the body weight intervals.

FOOD CONSUMPTION
- Results: There were no apparent treatment-related effects upon feed consumption at the 80, 200 and 400 ppm a.i. test concentrations.
- Remarks: No statistically significant differences between the control group and any of the treatment groups were observed at any of the feed consumption intervals.

PATHOLOGY
- Results: All findings observed were considered unrelated to treatment.

Effects on reproduction:

EGG SHELL THICKNESS
There were no apparent treatment related effects upon egg shell thickness at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in egg shell thickness in the 80, 200, 400 ppm a.i. treatment groups.

REPRODUCTIVE RESULTS
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 80, 200, 400 ppm a.i. treatment groups.

OFFSPRING BODY WEIGHTS
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 80, 200, 400 ppm a.i. treatment groups.

Reported statistics and error estimates:

STATISTICAL ANALYSES
Upon completion of the test, an analysis of variance (ANOVA) was performed to determine statistically significant differences among groups. Dunnett's multiple comparison procedure was used to compare the four treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation for reproductive parameters.
Additional statistical analyses were performed according to the decision tree currently employed by the EPA and using the Comprehensive Environmental Toxicity Information System (CETIS) software.

Estimated Maximum Mean Daily Dietary Dose of Test Material

Test Interval (Test Weeks)	Test Concentration (ppm a.i.)	Mean Body Weight (g)	Mean Feed Consumption (g/bird/day)	Estimated Daily Dietary Dose* (mg a.i./kg/day)
Pre-egg production (weeks 1 – 10)	0	1053	116	0
	80	1052	111	8.42
	200	1062	118	22.2
	400	1050	115	43.9
Egg production (weeks 11 – 21)	0	1117	164	0
	80	1110	161	11.6
	200	1131	165	29.1
	400	1110	165	59.5
Overall (weeks 1 – 21)	0	1070	141	0
	80	1067	137	10.3
	200	1082	142	26.3
	400	1066	141	53.0

*Results were generated using Excel 2010® in full precision mode. Manual calculations may differ slightly.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study 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 any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Executive summary:

A reproduction study in the mallard was carried out in accordance with the standardised guidelines OECD 206, EPA OPPTS 850.2300 and EPA OPP 71-4 under GLP conditions.

The objective of this study was to evaluate the effects of dietary exposure to the test material upon adult mallard (Anas platyrhynchos) over a five-month period. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to the test material on the number of eggs laid, fertility of the eggs, normal development of eggs including viability and survival of the embryos, hatchability, offspring survival and egg shell thickness were evaluated.

Mallard (72 males and 72 females) were randomly distributed into one control group and three treatment groups. Each treatment and control group contained 18 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 80, 200, and 400 ppm a.i. of the test material for 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test material.

All adult birds were observed daily throughout the test for signs of toxicity or abnormal behaviour. Adult body weights were measured at test initiation, at the end of Weeks 2, 4, 6, 8, and at adult termination. Feed consumption was measured weekly throughout the test. At the beginning of Week 10, 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 24 of incubation, the eggs were placed in an incubator configured for hatching and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the individual body weights of the hatchlings were determined. At 14 days of age, the individual body weight of each surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences among groups.

Under the conditions of this study 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 any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Description of key information

Short-Term Toxicity to Birds: Munk (1994)

Under the conditions of the study, the LD50 for the bobwhite quail is:

- For males plus females: About 500 mg/kg body weight.

- For males only: About 600 mg/kg body weight

- For females only: About 500 mg/kg body weight

The NOEL was 125 mg/kg body weight.

Short-Term Toxicity to Birds: Rodgers (1995)

Under the conditions of the study the acute oral toxicity (LD50) of test material to the Bobwhite quail was calculated to be 648 mg/kg.  The no-effect level for mortality was 364 mg/kg.

Long-Term Toxicity to Birds: Pagano et al. (2017)

Under the conditions of this study 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 any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.

Additional information

Short-Term Toxicity to Birds: Munk (1994)

The toxicity of the test material to birds was assessed according toEPA Test Guideline OPP 71-1 and in compliance with GLP using bobwhite quail.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material (dispersed in 0.5 % aqueous CMC preparation) was tested for its acute toxicity to the bobwhite quail after administration by gavage of single doses of 0, 62.5, 125, 250, 500 and 1 000 mg/kg body weight followed by a 14-day observation period.

Mortality: No deaths occurred in the control group or in the three lowestdosegroups (62.5, 125 and 250 mg/kg). Highestdosetested causing no mortality was 250 mg/kg bodyweight.Minimum dose tested causing 100 % mortality was 1 000 mg/kgbody weight.

Clinicalsigns:In the control group and in the 62.5 or 125 mg/kg groups all birdswerein good health. In the 250 mg/kg group one male bird showed apathy on the day of administration and on day one males and females had soft faeces. In the higherdosegroups (500 and 1 000 mg/kg) severe toxic signs such as apathy, prone andsideposition, ruffled feathers, ataxia and diarrhoea were diagnosed. The survivors had completely recovered from day 3 onward.

Feedconsumption: The initial feed consumption/bird/day was reduced – dose dependent - in the males and the females in the three highest dose groups (250, 500 and 1 000 mg/kg body weight). From day 4 onward the feed uptake was in the normal range in all dose groups. The mean total feed consumption/bird/day during the 14-day observation period was in thesameorder of magnitude in all test groups with surviving birds and in the control group with the exception of a marginal reduction in the feed uptake of the two female survivors in the 500 mg/kg group.

Body weight:There was no statistically significant reduction in body weight in the surviving birds on day 14 at the end of the study.

Gross post-mortem examination:Surviving birds had no adverse effects. Birds that had died (500 and 1000 mg/kg) showed general congestive hyperemia or no adverse effects.

Under the conditions of the study, the LD50 for the bobwhite quail is:

- For males plus females: About 500 mg/kg body weight.

- For males only: About 600 mg/kg body weight

- For females only: About 500 mg/kg body weight

The NOEL was 125 mg/kg body weight.

Short-Term Toxicity to Birds: Rodgers (1995)

The acute oral toxicity of the test material was assessed according to the United States Environmental Protection Agency Pesticide Assessment Guidelines, Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Series 71 -Avian and Mammalian Testing § 71-1 Avian single-dose oral LD50 test and in compliance with GLP using the Bobwhite quail. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of five male and five female adult birds were given a single oral dose, by intubation, of either 260, 364, 510, 714 or 1 000 mg test material/ kg bodyweight. A similar sized control group was dosed in the same way receiving the vehicle, corn oil, alone. Birds were observed for 14 days following dosing. Observations included mortality, clinical signs, bodyweight and food consumption.

Ten mortalities occurred at 1 000 mg/kg, seven at 714 mg/kg and one at 510 mg/kg. Clinical signs of toxicity, including subdued behaviour, unsteadiness and ruffled feathers were observed in birds treated with the test material at 364 mg/kg and above.

Significantly lower bodyweights on Day 7 (females) at 364 mg/kg and 510 mg/kg were observed.

Food consumption was reduced in Groups 3 - 5 over Days 1 - 7.

No abnormalities were observed in any birds examined at post mortem examination.

Under the conditions of the study the acute oral toxicity (LD50) of test material to the Bobwhite quail was calculated to be 648 mg/kg.  The no-effect level for mortality was 364 mg/kg.

Long-Term Toxicity to Birds: Pagano et al. (2017)

A reproduction study in the mallard was carried out in accordance with the standardised guidelines OECD 206, EPA OPPTS 850.2300 and EPA OPP 71-4 under GLP conditions.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the effects of dietary exposure to the test material upon adult mallard (Anas platyrhynchos) over a five-month period. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to the test material on the number of eggs laid, fertility of the eggs, normal development of eggs including viability and survival of the embryos, hatchability, offspring survival and egg shell thickness were evaluated.

Mallard (72 males and 72 females) were randomly distributed into one control group and three treatment groups. Each treatment and control group contained 18 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing 80, 200, and 400 ppm a.i. of the test material for 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test material.

All adult birds were observed daily throughout the test for signs of toxicity or abnormal behaviour. Adult body weights were measured at test initiation, at the end of Weeks 2, 4, 6, 8, and at adult termination. Feed consumption was measured weekly throughout the test. At the beginning of Week 10, 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 24 of incubation, the eggs were placed in an incubator configured for hatching and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the individual body weights of the hatchlings were determined. At 14 days of age, the individual body weight of each surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences among groups.

Under the conditions of this study 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 any of the reproductive parameters measured at the 80, 200 or 400 ppm a.i. test concentrations. The no-observed-effect concentration for mallard exposed to the test material in the diet during the study was 400 ppm a.i. (53.0 mg a.i./kg/day), the highest concentration tested.