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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 1997 - 31 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Frequency: At the preparation and before renewal of water (2-3 days later), total 5 times during exposure
- Sampling Volume: 0.75 mL (1 vessel / group)
Vehicle:
no
Details on test solutions:
Preparation of the test solution (100 mg/L): Eight hundred mL of dilution water was put in a 1.0 L glass beaker and require amount of the stock solution was added.
Preparation of the stock solution (10000 mg/L): Twenty-five hundred mg of the test substance was weighed and mixed with pure water, then adjusted to 250 mL.
Dilution water was used for control group.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: Subcultured in the testing laboratory, Originally from the National Institute for Environmental Studies (Japan) on 18 July 1995
- Age at study initiation: = 24 hours old
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: 0.1 - 0.2 mg C (Organic Carbon)/ day / animal

PRECULTURE TO OBTAIN TEST ANIMALS
Parental daphnids which deliver test animals were breeded following condition:
- Breeding water: Dilution water
- Population density: 35 or 70 daphnids / 3 L breeding water (Juveniles), 35 daphnids / 3 L breeding water (Adults)
- Temperature: 20 ± 1 °C
- Photoperiod: 16-hr Light / 8-hr Dark, Room light
- Type and amount of food: Chlorella vulgaris, 0.1 - 0.2 mg C (Organic Carbon)/ day / animal
- Juveniles were removed twice a week or more
- Parental daphnids were selected from the batch without presence of males or ephippia, and showed low mortality (= 10 % for 2 weeks)

OTHER
- Confirming the reproducibility of the test conditions:
- Reference substance: Potassium dichromate (Reagent grade)
- Results: 48-hr EiC50 = 0.23 mg/L



Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
No data
Test temperature:
20 ± 1 °C (19.5 - 20.7 °C)
pH:
7.5 - 8.0
Dissolved oxygen:
6.1 - 8.2 mg/L (= 60 % of the O2 saturation conc. (8.8 mg/L at 20.0 °C))
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Measured concentrations: 84.3 - 97.4 mg/L (84 - 97 % of nominal) at the preparation, 80.0 - 96.9 mg/L (80 - 97 % of nominal) before renewal of water
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material: Glass
- Size: 1 L
- Fill volume: 800 mL
- Thermostatic bath: PVC Water bath (Thermostat: COOLNIT CL-80F, TAITEC Co., Ltd.)
- Thermomater: 2455 02, Yokogawa Electric Corporation
- D.O. meter: DOL-10, DKK Corporation
- pH meter: HM-40V, TOA Electronics Ltd.
- Renewal rate of test solution: 3 times/week, the whole quantity
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated water (Yokohama-city tap water, removed the residual chlorine with the activated carbon and aerated sufficiently)
- Hardness: 65 mg/L (as CaCO3)
- pH: 8.2

OTHER TEST CONDITIONS
- Photoperiod: 16-hr Light / 8-hr Dark
- Light intensity: Room light

OBSERVATIONS AND MEASUREMENTS:
- Parental daphnids (recorded daily):
- No. of living daphnids
- No. of immobilized
- No. of daphnids with the presence of eggs in the brood pouch
- Size and state
Dead animals were removed after recording

- Juveniles:
- No. of living and dead juveniles
- No. of aborted eggs and ephippia (at the renewal of water)
- Time (day) to the first brood production
Juveniles were removed after recording

- Test solutions: Temperature, pH and Dissolved oxygen concentration were measured at the preparation and before renewal of water (2-3days later), 4 times in exposure duration.


ACUTE IMMOBILIZATION TEST
- Results: 48 h-EiC50: > 1000 mg/L

Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
MORTALITY OF THE PARENTAL DAPHNIDS
- Control group: 3 % at the end of the exposure (meet criteria for the study validity (20 % or less))
- 100 mg/L group: 3 % at the end of the exposure

TIME (DAY) TO FIRST BROOD RELEASE
- Control group: 8 days after exposure in all vessels (meet criteria for the study validity (within 9 days))
- 100 mg/L group: 8 days after exposure

THE MEAN CUMULATIVE NUMBER OF JUVENILES PRODUCED PER PARENTAL DAPHNID
- Control group: 86 animals for 21 days (meet criteria for the study validity (40 animals))
- 100 mg/L group: 80 animals for 21 days

SIZES OF PARENTAL DAPHNIDS
No difference was observed between 100 mg/L group and the control group during the exposure period.

EPHIPPIA
No ephippia was observed in 100 mg/L group and the control group during exposure period.

There were no environmental factors which might have affected the reliability of the test.
Reported statistics and error estimates:
The toxicological values were calculated by nominal concentration because the measured concentrations of the test substance at the preparation and before renewal were kept within ± 20 % of the nominal concentration.

STATISTICS
- LC50: Probit analysis
- EC50: Probit analysis
- NOEC and LOEC: F test, Student's t-test

In this study, LC50 could not be calculated because mortality in 50% or more was not observed in the highest concentration.
In this study, ErC50 could not be calculated because the inhibition in 50% or more was not observed in the highest concentration.

Table 5-1. Cumulative Numbers of Dead Parental Daphnia

Nominal Conc.

Days

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Cont.

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

1

1

1

1

100 mg/L

0

0

0

0

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Table 5-2. Mortality (%) of Parental Daphnia

Nominal Conc. Days
1 2 4 7 14 21
Cont. 0 0 0 0 3 3
100 mg/L 0 0 3 3 3 3

Table 6. Time (day) to First Brood Production

Vessel No. Nominal Concentration (mg/L)
Control 100
1 8 8
2 8 8
3 8 8
4 8 8
Mean 8.00 8.00

Table 7. Mean Cumulative Numbers of Juveniles Produced per Adult (¿F1/P)

Nominal Conc. Days
0 6 7 9 10 12 14 16 19 21
Cont. 0.0 0.0 0.0 0.7 10.0 10.1 29.4 61.1 83.7 85.7
100 mg/L 0.0 0.0 0.0 0.3 10.9 10.9 31.8 67.3 80.4 80.4

Table 8. Calculated LC50 Values for Parental Daphnia

Exposure Period (day) LC50 (mg/L) 95 % Confidence Limits (mg/L) Statistical Method
14 > 100 -- ~ -- --
21 > 100 -- ~ -- --

Table 9. Calculated ErC50 Values for Inhibition of Reproduction

Exposure Period (day) ErC50 (mg/L) 95 % Confidence Limits (mg/L) Statistical Method
14 > 100 -- ~ -- --
21 > 100 -- ~ -- --

Table 10. Mean cumulative numbers of juveniles produced per adult in control and test vessels after 21 days

(by Dunnett multiple comparison procedure, Two-sided test)

Vessel No. Nominal Concentration, mg/L
Control 100
1 87.2 78.5
2 89.1 75.1
3 89.7 84.3
4 76.8 83.8
Mean 85.7 80.4
S.D. 6.0 4.4
Inhibition rate (%) 6.2
Significant difference

*: Indicates a significant difference (a = 0.05) from the control.

(There was no sign in this test)

**: Indicates a significant difference (a = 0.01) from the control.

(There was no sign in this test)

Calculation of the Cumulative Number of Juveniles Produced per Parental daphnid

The cumulative number of juveniles produced per parental daphnid (TF) and the mean cumulative number of juveniles produced per parent daphnid (TF) were calculated according to following formula:

ne

TF = ¿{(2×Fn) / (Pn-1 +Pn)}

n=1

n: Frequency of observation for juveniles

Fn: Number of surviving juveniles at n- times

Pn: Number of parental daphnids at n- times

Pn-1: Number of parental daphnids at (n-1)- times

P0: Number of parenatl daphnids at the day before exposure

ne: Final observation time

m

TF = ¿TFn / m

n=1

TFn: Cumulative number of juveniles produced per parental daphnid (per vessel)

m: Replicates per concentaration (Number of vessels: 4)

Validity criteria fulfilled:
yes
Conclusions:
No toxic effect of diacetone alcohol was observed at 100 mg/L (limit test). The 21-day NOEC was 100 mg/L for Daphnia magna under the conditions of the study.
Executive summary:

The effect of 21 -day exposure on reproduction of Daphnia magna was investigated as a chronic study, which was conducted in a semi-static system according to OECD TG 211. This study was well controled under GLP regulation. A limit test was performed at 100 mg/L. pH varied between 7.5 and 8.0, dissolved oxgen concentration was between 6.1 and 8.2 mg/L and the temperature was 20 ±1 °C. The mean cumulative number of juveniles produced per parental daphnid at control and 100 mg/L goups were 86 and 80 animals for 21 days, respectively. Only one dead parent were found per treatment group at the end of the test. Therefore The 21-day NOEC was 100 mg/L under the conditions of the study.

Description of key information

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
100 mg/L

Additional information

One long-term (21-day) toxicity study was performed to assess the toxicity of DAA to Daphnia magna (Mitsubishi Chemical Safety Institute, Ltd., 1997). The effect of 21 -day exposure on reproduction of Daphnia magna was conducted in a semi-static system according to OECD TG 211, and conducted under GLP. A limit test was performed at 100 mg/L. pH varied between 7.5 and 8.0, dissolved oxygen concentration was between 6.1 and 8.2 mg/L and the temperature was 20 ±1 °C. The mean cumulative number of juveniles produced per parental daphnid at control and 100 mg/L groups were 86 and 80 animals for 21 days, respectively. The No effect concentration is expected to be higher than 100 mg/L. Therefore the 21 -day NOEC of 100 mg/L obtained under the conditions of the study is taken as a worst case scenario in the framework of the environmental risk assessment.