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Toxicity to birds

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Reference
Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Mar 1998 to 08 Sep 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 71-4 (Avian Reproduction Test)
Version / remarks:
1982
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard Practice for Conducting Avian Reproduction Test, Draft No. 9
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Dose method:
feed
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and analysis of diet:
DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: An amount of test material was measured based upon the desired ppm concentration and a given volume of feed. The test substance was sprinkled directly onto the feed and mixed in the large bulk mixer for 30 minutes. Residual amounts of test material in the measuring beaker were rinsed into the diet mix with approximately 10 mL of acetone. Control diet preparations were mixed in the large bulk mixer for 30 minutes. Like amounts of acetone as used in the test material diet mixes were added to the control preparations. Acetone added to the diet mix preparations was allowed to air dry before administration of the test feed.

DIET VERIFICATION
Replicates of four test concentrations (0 ppm, 100 ppm, 300 ppm and 900 ppm) were analyzed for diet verification for the definitive portion of the study. Replicates of four test concentrations (0 ppm, 100 ppm, 300 ppm and 900 ppm) were analyzed for diet verification for the definitive portion of the study. No test substance was found in the 0-ppm samples. Mean concentrations found in the 100 ppm feed mixture samples was 101.82 ppm (SD=19.47). Mean concentrations in the 300 ppm feed mixture samples was 307.14 ppm (SD=34.33) and mean concentrations in the 900 ppm feed mixture samples was 908.75 ppm (SD=73.72). These mean concentrations were very close, although slightly above, the nominal level. The variability between samples reflects the physical nature of the test substance, which determines its mixing characteristics. The test material was a crystalline powder-like substance with very small, hard and spherical particles. Talcum-like powders usually distribute evenly in feed and adhere well to feed particles; however, the hard spherical physical characteristics and, therefore, the behavioral characteristics of the test substance are quite different, which resulted in a higher degree of heterogeneity among samples. Given the well documented mixing procedures and the mean concentrations in the samples, the data indicate that the mallards in the study received very close to, or just slightly more than, the nominal exposure.

HOMOGENEITY
Replicates of two test concentrations (100 ppm and 900 ppm) were analyzed for homogeneity verification for the definitive portion of the study. The mean concentrations for the 100-ppm mixture was 123 ppm (SD=73.31, Range= 61.79-229.62) and the mean concentration for the 900-ppm mixture was 1053 ppm (SD=495.50, Range = 638.45-1765.15). Again, following the discussion above, the mean concentrations were very close, although slightly above, the nominal level and inter-sample variability was relatively high.

STORAGE STABILITY
A storage stability sample was collected from the 100 and 900 ppm treatment levels of one batch of diet prepared during the definitive study for ambient and freezer storage stability analysis. Ambient temperature samples were analyzed at intervals of 0, 14, 28 and 90 days and frozen samples were analyzed at intervals of 0, 1, and 2 months and again at the end of the treated feed phase of the study which was an approximate 11 month interval. Ambient samples most accurately reflect feeding conditions in this study as feed mixes were prepared frequently, stored at ambient temperature and kept no longer than 21 days
before being replaced with a new mixture. Because of the heterogeneity in the measured concentration among samples, interpretation of the decline in test substance concentration was difficult. To facilitate the interpretation of the data, the means were interpolated for the ppms for the two concentration levels between Day 0 and Day 28. We determined a straight line "best fit" for the data. These data indicate that the 100 ppm feed mix sample mean at Day 0 was at a concentration of approximately 76 ppm and by 21 days had declined to approximately 68 ppm, a reduction in concentration of about 10.5%. The 900 ppm feed mix sample mean concentration at Day 0 was approximately 600 ppm and declined to approximately 560 ppm by Day 21, a reduction of approximately 6.7%. The frozen storage stability samples demonstrated less inter-sample variability, making data interpretation more straightforward. The 100 ppm feed mix mean concentration declined approximately 25% over the 11-month period. The 900 ppm feed mix mean sample concentration of the test substance demonstrated no reduction over the 11-month test period.
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: Mallard duck
- Justification for test species: The Mallard Duck is generally recognized as an acceptable model of waterfowl for use in toxicity tests of pesticides and the U.S. Environmental Protection Agency recommends it as a test species. A substantial data base exists on the response of this species to pesticides. Dietary exposure to pesticides is considered appropriate because birds in nature may ingest pesticides along with their food items.
- Age at test initiation: The birds were from a single hatch, 25 weeks and 1 day of age at experimental start. Experimental start occurred prior to their first breeding season.
- Weight at test initiation: The weight range for males and females was 800 - 1327 g at experimental start.
- Sexes used / mixed or single sex: Mixed; male and female in equal proportions
- Disease free: yes, pen-reared, apparently healthy and phenotypically indistinguishable from wild birds. The birds were inspected for health by a veterinarian prior to the start of treated feed.
- Bird Identification: Birds were individually identified with numbered leg bands.


Limit test:
no
Total exposure duration (if not bolus):
149 d
Remarks:
Exposure period was 21 weeks and 2 days.
Post exposure observation period:
Post-exposure observations were made of the offspring up to 2 weeks post-hatching. The total experimental study duration was 27 weeks.
No. of animals per sex per dose and/or stage:
16
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
- Nominal doses: 0 ppm (control), 100 ppm, 300 ppm and 900 ppm of test substance in the test diet.
- Measured doses: 0 (control), 101.82, 307.14 and 908.75 ppm of test substance in the test diet. See 'Details on preparation and analysis of diet' for a detailed description on the verification of the test doses.
Details on test conditions:
ACCLIMATION
- Acclimation period: 18 days
- Acclimation conditions: The study room was maintained between 12.8° C and 23.3 °C. The average temperature was 19.0 °C with a SD of 1.7 °C. Relative humidity (RH) was recorded between 62 and 99% with an average of 87.9% and a SD of 7.5%. Light was provided by fluorescent lights which closely approximate the natural sunlight spectrum. Light exposure was at an average of 6.7 Foot Candles as measured at the lowest level cages.
- Housing: Poly carbonate-coated, galvanized, welded-wire cages.
- Feeding: Purina Game Bird Ration (Layena) was used for feed. Water and feed were provided ad libitum during acclimation and during the test period. Ducklings were fed Purina Startena game ration ad libitum. Water was supplied through automatic waters ad libitum. No medication or treated feed was offered to the ducklings or was included in any of the game ration.
- Health: Birds were observed daily for any signs of illness or disease upon the arrival.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description husbandry adults: Birds were kept in pairs, one male and one female per cage for the acclimation and experimental phases of the study. The cage dimensions were 76 cm deep, 83 cm wide and 44 cm in height, with a floor slope so eggs roll forward and out of the cage onto the collection tray.
- Description husbandry offpsring: The ducklings were housed in brooder units measuring approximately 46 cm long x 141 cm wide x 46 cm high. The pens were constructed of epoxy-coated wire mesh. Heat lamps suspended over the pens were set to maintain a temperature of approximately 38 °C from the time of hatching until the birds were 14 days of age. The photoperiod for the ducklings was maintained at 17 hours of light and 7 hours of dark. The brooder heat lamps, however, did provide intermittent infra-red light throughout the 24 hour period. Ducklings were housed by hatch and by treatment level in one cage. The brooder cages were maintained in the brooder facility. The room was maintained at an average temperature of 37.2 °C.
- Assignment: Each bird was indiscriminately assigned a unique number by leg band. Cages were numbered, and birds were randomly assigned to cages. First, male birds were selected in numerical order by tag number and assigned to a cage based on a set of randomly generated cage numbers. Females were assigned to cages by matching them to males of close weight. Once the birds were assigned to a cage (in pairs), they were randomly assigned to a treatment group or the control group.

TEST CONDITIONS
- Test conditions egg storage: The temperature range in the egg storage refrigerator was 4.4 °C - 9.4 °C with an average temperature of 6.1 ° C and a SD of 0.7 °C. The humidity was maintained at an average of 55% and a SD of 3.2%.
- Test conditions incubator: The temperature range for incubation for this study was 37.2 - 37.6 °C with an average temperature of 37.4 °C and a SD of 0.1 °C. The relative humidity range for incubation for this study was 41 - 72% with an average of 56.3% and a SD of 7.5%.
- Test conditions hatcher: The temperature range for the hatcher for this study was 35.9 °C to 37.3 °C with an average of 37.0 °C and a SD of 0.3 °C. The humidity range for the hatcher for this study was 70 to 79% RH with an average of 75.4% and a SD of 2.5%.
- Photoperiod: 7 hrs light, 17 hrs dark per 24 hour interval during acclimation and for the first 8 weeks of treated feed. During Week 9, the lights were then increased gradually over a 6-day period to 17 hours of light per 24 hour interval. The light interval remained at 17 hours of light per 24 hour interval until the end of the treated feed portion of the study.

RANGE FINDING STUDY
A range-finding portion of the study was defined in the protocol to include data collection through four weekly sets of eggs incubated through 14 days and then checked for fertility. The treatment levels were set at 0 ppm, 90 ppm, 270 ppm, 810 ppm and 2430 ppm. For whatever reason, the test system never laid the requisite number of eggs in order to start treated feed. Treated feed was finally started in anticipation of eggs but after two weeks, few pairs had laid eggs. The decision was made to terminate the range finding portion of the study and initiate the definitive study replicating the treatment levels in an earlier Northern Bobwhite study,
Details on examinations and observations:
MORTALITY
- Time schedule for examinations: Observations were made at least once daily for any mortalities that may have occurred.

BODY WEIGHT
- Time schedule for examinations: Body weights were measured at the start of acclimation, start of treated feed, start of photostimulation and adult termination.
- Remarks: Weights for each interval were statistically compared against the control group.

FEED CONSUMPTION
- Time schedule for examinations: Feed consumption was measured weekly for each pair of birds during the treated feed portion of the study for a total of 21 weeks and t\vo days for a total of 22 feeding intervals.
- Remarks: Total feed consumption for each treatment group was statistically compared to the control group.

BEHAVIORAL OBSERVATIONS
- Time schedule for examinations: Birds were observed once daily during acclimation and during the treatment period of the study.
- Remarks: Any behavior other than normal maintenance or feeding behavior was noted.

PATHOLOGY
- Time schedule for examinations: Gross pathological examinations were performed on all birds succumbing prior to adult termination and on all birds surviving the test.
Details on reproductive parameters:
- Number of eggs laid: Eggs were collected on a daily basis and marked using a graphite pencil with information that included cage of origin, date collected, and treatment group. These eggs were then stored in in a refrigerator until placed into the incubator.
- Number of eggs cracked: Eggs were candled prior to being placed into the incubator. Those eggs that were cracked were recorded as cracked and then discarded. Those eggs not cracked were placed into the incubator in egg trays segregated by treatment group and grouped by cage of origin.
- Number of eggs taken for eggshell thickness: Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered cages during odd numbered weeks (1, 3, 5, etc.) and from each of the even numbered cages during the even numbered weeks (2, 4, 6, etc.). The eggs were cleaned of their contents, and allowed to air dry for a minimum of one week. The egg shells were then measured five points around the equator using a micrometer which measured to the nearest 0.001 mm.
- Number of fertile eggs: On day 14 of incubation, eggs were candled for fertility. Those eggs not fertile were documented and discarded. The fertile eggs were placed back into the incubator.
- Number of viable embryos: On day 21 of incubation, eggs were candled for viability. Those eggs not viable were documented and discarded. The viable eggs were returned to the incubator. On Day 24 the eggs were transferred to the hatcher. Eggs were placed into the hatcher in hatching trays that segregate eggs by treatment group and were grouped by cage of origin. Eggs remained in the hatcher for approximately 2 days and were allowed to hatch over an approximate 28 hour interval.
- Number of ducklings hacthed: Upon hatch, ducklings were weighed, banded with a wing tag having a unique number, and weighed to the neirest tenth of a gram.
- Behavioral observations: Ducklings were observed once daily for behavior and mortality.
- 14-Day survivors: Those ducklings surviving to the fourteenth day were euthanized on Day 14 and weighed.
Reference substance (positive control):
no
Key result
Duration (if not bolus):
27 wk
Dose descriptor:
NOEC
Effect level:
300 mg/kg diet
Conc. / dose based on:
act. ingr.
Basis for effect:
body weight
Mortality and sub-lethal effects:
MORTALITY
There was one mortality in the adult duck population during Week 15 of the treated feed portion of the study. In the 100 ppm group the female mallard in Cage 1 died. Post mortem notations indicated blocked and infected intestines. The bird was emaciated, weighing 782 grams at time of post mortem. The bird was not sexually active at the time of death and had not laid any eggs during the study.

CLINICAL OBSERVATIONS
All birds were noted to be normal in appearance and behavior. No overt signs of treatment-related toxicity were observed in the study.

POST-MORTEM EXAMINATIONS
All surviving adults were subjected to a post-mortem examination following adult termination. By the time of euthanasia, the majority of the birds had discontinued egg laying. At the time of euthanasia the 0 ppm group had two cages where both birds were noted to be sexually active and had produced eggs in set 10; the 100 ppm group had three cages; the 300 ppm group had five cages and the 900 ppm group had four cages. The 900 ppm group had three additional cages where both birds were noted to be sexually active but did not produce eggs during set 10. There were notations in the 300 ppm group noting hardened ovaries and/or eggs in three of the female birds. One additional female in the 300 ppm group was noted to have a clear fluid in its intestinal cavity. All other notations referenced whether or not the individual bird appeared sexually active.
Effects on reproduction:
- Number of Eggs Laid and Number of Eggs Set: There were no significant differences detected between any of the treatment groups and the corresponding control group.
- Number of Eggs Cracked/Number of Eggs Laid: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- Number of Fertile Eggs/Number of Eggs Set: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- Number of Viable Embryos/Number of Fertile Eggs: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- Number of Eggs Hatched/Number of Viable Embryos: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- Number of 14 Day Survivors/Number of Eggs Hatched: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- Hatch Weights: There were no significant differences detected between any of the treatment groups and the corresponding controls.
- 14 Day Survivor Weights: A significant difference was detected between each treatment group mean when compared to the corresponding control group mean. The mean for the 900 ppm group was higher than the mean for any other group. The control group mean was lower than the mean for any other group. The difference is not considered to be a dose response effect.
- Eggshell Thickness: There were no significant differences detected between any of the treatment groups and the corresponding controls.
Results on the reproductive parameters are tabulated in 'Any other information on results incl. tables'.
Reported statistics and error estimates:
Upon completion of the test, statistical analyses were completed to determine whether significant (p < 0.05) differences existed between the control group and any of the treatment groups. The unit of analysis was defined as the individual cage (pair) within each group, except for adult body weight where the unit of analysis was the individual bird. Data sets were tested for normality using a Chi-square test and for homogeneity of variance using a Bartlett's test or Levene's test. Proportional data, if not normal, was Arcsine transformed. If the data were normal and variances were homogenous the parameters listed below were analyzed with ANOVA and a Dunnett's post hoc test (equal sized groups) or an ANOVA and then a Tukey's post-hoc test (unequal sized groups) for pair-wise comparisons. If data were not normal or were heterogeneous, they were analyzed with a Kruskal Wallis' ANOVA by Ranks followed by a Dunn's Multiple Comparison. All statistical measures were made based upon standard sources (Finney, 1971; TOXSTAT, 1994; Williams, 1971; and
Williams, 1972).

Table: Mean Adult Body Weight (g) from a Mallard Reproduction Study with the test substance 

Experimental Group

Sex

Weight 1: Start of Acclimation

Change

Weight 2: Start of Test Feed

Change

Weight 3: Start of Photostimulation

Change

Weight 4: at Adult Termination

Total Change Weight 2-4

Control

Male

1022.6

111.8

1134.3

26.2

1160.5

65.2

1225.7

91.4

Female

964.3

41.6

1005.9

67.9

1073.8

55.9

1129.6

123.8

100 ppm

Male

1011.1

80.1

1091.1

62.0

1153.1

38.7

1197.4

103.5

Female

956.2

42.5

998.7

38.6

1037.3

75.8

1112.3

112.3

300 ppm

Male

1006.3

107.5

1113.8

4.3

1118.1

59.4

1177.5

63.8

Female

937.7

52.6

990.3

89.8

1080.1

33.6

1113.6

123.3

900 ppm

Male

1020.6

75.9

1096.5

-30.7

1065.8*

43.5

1109.3*

12.8

Female

947.1

80.2

1027.3

60.6

1087.9

44.0

1131.9

104.6

* Significantly different from the control group. (p<0.05)

 

Table: Mean Feed Consumption ( g / cage / week )from a Mallard Duck Reproduction Study with the test substance

Experimental group

Feed week

1

2

3

4

5

6

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Control

1315

1257

1303

1471

1184

1365

1553

1752

2156

2135

2162

1928

2106

2112

2055

2181

1871

2055

1935

1860

459

100 ppm

1144

1161

1132

1306

1239

1268

1364

1664

1992

2066

2084

1875

2009

2046

2081

2137

1913

1923

2024

1858

426

300 ppm

1252

1227

1254

1384

1270

1283

1473

1647

2101

2153

2078

1785

2086

2155

2171

2200

2039

1984

2037

1999

417

900 ppm

1234

1204

1237

1474

1205

1565

1424

1740

2090

2197

2090

1818

2137

1947

2043

2161

2002

2037

2101

1990

433

No significant difference was detected between groups (P > 0.05)

Week 22 is a two-day Interval

 

Table: Summary of Reproductive Performance From a Mallard Duck Reproduction with the test substance 

Reproductive parameter

Experimental Group

Control

100 ppm

300 ppm

900 ppm

Number of replicates*

16

14

12

16

Total Eggs Laid

718

580

558

551

Eggs Cracked

44

39

25

22

Eggs Set into Incubator

616

492

480

479

Fertile Eggs

567

453

457

427

Viable Embryos

536

412

421

378

Hatchlings

458

334

361

324

14 Day Old Survivors

420

320

345

306

Total egg laying days: 70

* Number of laying pair

 

Table: Summary of Reproductive Performance From a Mallard Duck Reproduction with the test substance Normalized as Proportions

Reproductive parameter

Experimental Group

Control

100 ppm

300 ppm

900 ppm

Number Eggs Laid *

0.64

0.59

0.66

0.49

Number Eggs Set Into Incubator *

0.55

0.50

0.57

0.43

Eggs Cracked / Eggs Laid

0.06

0.07

0.04

0.04

Fertile Eggs / Eggs Incubated

0.92

0.92

0.95

0.89

Viable Embryos / Fertile Eggs

0.95

0.91

0.92

0.89

Hatchlings / Viable Embryos

0.85

0.81

0.86

0.86

14 Day Old Survivors / Hatchlings

0.92

0.96

0.96

0.94

Hatchlings / Eggs Incubated

0.74

0.68

0.75

0.68

14 Day Old Survivors / Eggs Incubated

0.68

0.65

0.72

0.64

• These paramenters computed as number per day per hen. All other computed as

total / total (for example: total eggs cracked I total eggs laid).

 

Table: Mean Body Weight (g) of Hatchlings and 14 Day Old Survivors From a Mallard Duck Reproduction with the test substance

Experimental Group

Hatchlings

14 Days Old Survivors Group

No.

Mean

Std Dev

No.

Mean*

Std Dev

Control

458

36.4

3.4

420

189.7

56.0

100 ppm

334

35.7

3.6

320

225.3

44.8

300 ppm

361

35.3

3.3

345

222.6

41.5

900 ppm

324

35.2

3.6

306

229.5

49.0

No significant differences between control group and treatment groups. ( p > 0.05)* All treatment group means were higher than that of the control and statistically different from the control

Table: Mean Egg shell Thickness Measurements (mm) From a Mallard Duck Reproduction with the test substance

Experimental Group

No. of Eggs Measured

Shell Thickness Mean

Standard Deviation

Control

56

0.353

0.023

100 ppm

49

0.357

0.021

300 ppm

53

0.361

0.021

900 ppm

50

0.353

0.035

No significant differences between control group and treatment groups. (p > 0.05)

Two eggs taken for eggshell thickness measurement In the Control Group were not measured.

These data are representative of individual eggshell measurements and may differ slightly from the statistical data which is representative of cage means.

Validity criteria fulfilled:
not specified
Conclusions:
No mortality or toxicity was observed in mallard ducks exposed to the test substance at a dietary concentration of 100 ppm, 300 ppm or 900 ppm for 21 weeks and two days. A significant difference was detected in male body weights when comparing the 900 ppm treatment group mean to the corresponding control group mean at two weight intervals; the start of photostimulation and at euthanasia. In both cases the 900 ppm treatment group mean was lower than that of the control. There were no reproductive effects at any treatment level. The reproductive no observable effect concentration (NOEC) during the study was, therefore, 300 ppm.
Executive summary:

The repeated dose and reproductive effects of dietary exposure of the test substance in mallard duck over a 7-months was investigated in a GLP-compliant avian reproduction study in accordance with EPA OPP 71-4. In this study, mature mallard ducks (Anas platyrhynchos; 16/sex/dose) received the test substance at nominal dietary concentrations of 100, 300 and 900 ppm for 21 weeks and 2 days, based on a range-finding study. A control group receiving basal diet was maintained concurrently with the treatment groups. Birds were kept in pairs one male and one female per cage. Body weight was measured 4 times during acclimation and the definitive test. Feed consumption was measured weekly for each pair of birds. Birds where observed daily for signs of behavioural abnormality and mortality. Gross pathological examination were performed on all birds succumbing prior to adult termination and on all birds surviving the test. Eggs were collected on a daily basis and marked. Eggs were candled prior to being placed in an incubator. Cracked eggs were recorded as cracked and discarded. Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered cages during odd numbered weeks and from each of the even numbered changes on even numbered weeks. The eggshells were then measured at 5 points around the equator. On day 14 of incubation, eggs were candled for fertility. Those that were not fertile were discarded. On day 21 of incubation eggs were candled for viability, those deemed not viable were discarded. Upon hatch ducklings were weighed and banded with a unique wing tag. Ducklings were housed in brooder units measuring 46 cm long x 141 cm wide x 46 cm high. Ducklings were fed purina startena game ration ad libitum. Ducklings were observed daily for behaviour and mortality. Surviving ducklings were euthanized on the 14th day and weighed.

No mortality or toxicity was observed in Mallard ducks exposed to the test substance at a dietary concentration of 100, 300 or 900 ppm for 21 weeks and two days. A significant difference was detected in male body weights when comparing the 900 ppm treatment group mean to the corresponding control group mean at two weight intervals; the start of photo stimulation and at euthanasia. In both cases the 900 ppm treatment group mean was lower than that of the control. There were no reproductive effects at any treatment level. The reproductive NOEC during the study was therefore 300 ppm.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

The reproductive 149-d NOEC to mallard duck (Anas platyrhynchos) during the study was determined to be 300 ppm, EPA OPP 71-4, Brewer, 1998

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:
300 mg/kg food

Additional information

Table: Overview of available data on the toxicity to birds

Study

Species

Guideline / GLP

Endpoint

Effect Value

Comment

Reference

Avian reproduction study

Anas platyrhynchos

EPA OPP 71-4 / GLP

149-d NOEC

300 mg/kg food

 

Brewer, 1998