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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.3, 10, 16, 25, 40 and 63 mg/L
- Sampling method: Samples were taken at ca. 20 and 23 hours following the start of dosing samples at 63 and 16 mg/L, respectively, to check the actual exposure concentrations. At the start of the exposure period samples from all concentrations. After 48 hours of exposure and at the end of the exposure period, samples at 6.3, 10, 16 and 25 mg/L.
- Sample storage conditions before analysis: Not applicable, all samples were analyzed on the day of sampling.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Source: Bio International, Horn, The Netherlands
- Length at study initiation: 2.16 +- 0.15 cm
- Weight at study initiation: 0.32 +- 0.11 g

ACCLIMATION
- Acclimation period: at least 12 days after delivery
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
75 mg CaCO3/L
Test temperature:
21.0 - 21.2 °C
pH:
7.8 - 8.1
Dissolved oxygen:
8.9 - 9.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: Control, 6.3, 10, 16, 25, 40 and 63 mg/L
Measured concentrations: 4.2, 8.0, 10.6, 20, 33, 53 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 30 L (50x30x25 cm) tank
- Type: closed. Sealed with a thin film of silicone and covered by a removable glass plate.
- Aeration: No
- No. of organisms per vessel: 7
- Biomass loading rate: 0.012 g fish/L/day
- Type of flow-through: The test substance was dosed via a computer controlled system consisting of separate dispensers (Gilson). Via this system the dosed volumes of pure test substance entered a mixing flask separately from the tap water supply. The tap water was supplied via a flow meter. The flow rate of the tap water was between 7.3 and 7.5 liters per hour. In the mixing flask the dosed volume and the tap water was mixed under continuous stirring. The air space above the test solutions was connected with the air space above the mixing vessels to minimize loss of test substance by volatization (closed system). The whole system was checked daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water

OTHER TEST CONDITIONS
- Photoperiod: 16 h light daily

EFFECT PARAMETERS MEASURED:
Mortality (after 3, 24, 48, 72 and 96 h), pH, temperature and O2 (daily).

RANGE-FINDING STUDY:
- Test concentrations: 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: All fish exposed to the concentration of nominal 100 mg/L died within 216 hours of exposure. The fish exposed to nominally prepared test concentrations of 0.1 to 10 mg/L became not affected during the 96-hour test period. The analytical results of the samples taken at 10 mg/L showed that the actual test item concentration decreased from 6.18 to 4.39 mg/L. Hence, the average concentration measured corresponded with 5 mg/L during the 24-hour period between renewal of the test solution. Hence, the 96h-LC50 was expected to be between test item concentrations of 5 and 100 mg/L (see table 1).
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence interval: 17-26 mg/L)
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence interval: 13-20 mg/)
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence interval: 13-20 mg/L)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
8.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Mortality of control: None
- Any observations that might cause a difference between measured and nominal values: Observations during dosing showed that there was no complete mixing of test substance in the mixing vessels in spite of vigorously stirring. Further, small air bubbles were observed in the tube used for dosing of nominal 6.3 mg/L. This probably accounted for the lower recoveries at this concentration.
Reported statistics and error estimates:
The LC50 was determined using:
1. The maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).
2. If there was no concentration between the highest concentration (A) at which 100% mortality occurred and the lowest concentration (B) at which 0% mortality occurred, the LC50 was calculated as (AB)1/2, with A and B being limits of the 95% confidence interval.
Sublethal observations / clinical signs:

Range Finding Study

Table 1: Incidence and total mortality

Concentration of test item

Nominal (mg/L)

Initial number of fish

Cumulative mortality

Total Mortality (%)

2.5 h

24 h

48 h

72 h

96 h

0.1

3

0

0

0

0

0

0

1.0

3

0

0

0

0

0

0

10

3

0

0

0

0

0

0

100

3

3

3

3

3

3

100

 

Main Study

Table 2: Incidence and total mortality

Concent.(mg/L)
Test item
nominal

Initial
number
of fish

Cumulative mortality

Total
Mortality
(%)

3 h

24 h

48 h

72 h

96 h

Blank -control

7

0

0

0

0

0

0

6.3

7

0

0

0

0

0

0

10

7

0

0

0

0

0

0

16

7

0

0

0

0

0

0

25

7

0

3

6

6

7

100

40

7

0

7

7

7

7

100

63

7

7

7

7

7

7

100

Table 3: Average exposure concentration of the test item on concentrations measured during the study

Test item concentration

Nominal (mg/L)

Measured concentration t=0h (mg/L)

Measured concentration t=48 h (mg/L)

Measured concentration t=96h (mg/L)

Average recovery

(mg/L)            

(%)

6.3

4.2

2.6

3.0

3.3 ± 0.8

52

10

8.0

4.8

6.3

6.4 ± 1.6      

64

16

10.6

7.1

8.5

8.7 ± 1.8

54

25

20

20.9

26.3

22.4 ± 3.4

90

40

33

n.m.

n.m.

33

83

63

53

n.m.

n.m.

53

84

n.m. Not measured, fish had already died.

Validity criteria fulfilled:
yes
Conclusions:
An 96-h acute toxicity study on zebrafish was conducted under flow-through conditions. The test substance concentrations were monitored during the test, and the results were based on measured concentrations. The 96h-LC50 was determined to be 14 mg/L.
Executive summary:

The acute toxicity of the test item was assessed in a 96-h acute toxicity study in Zebrafish according to OECD guideline 203. Zebrafish (7 fish/ concentration) were exposed to nominal test item concentrations of 6.3, 10, 16, 25, 40 and 63 mg/L for 96 hours under flow-through conditions (7.3-7.5 L/h). The test concentrations were analytically verified. Samples for determination of actual test concentrations were taken at the start, after 48 hours of exposure and at the end of the test period. The standard deviations decreased with increasing exposure concentrations from 25% to 15% of the average measured concentrations. The toxicity parameters were based on the average measured concentrations. The 96h-LC50 and NOEC were determined to be 14 mg/L and 8.7 mg/L, respectively.

Description of key information

An 96-h acute toxicity study on zebrafish was conducted under flow-through conditions according to OECD guideline 203. The test substance concentrations were monitored during the test, and the results were based on measured concentrations. The 96h-LC50 was determined to be 14 mg/L (reference 6.1.1-1).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
14 mg/L

Additional information

The acute toxicity of the test item was assessed in a 96-h acute toxicity study in Zebrafish according to OECD guideline 203. Zebrafish (7 fish/ concentration) were exposed to nominal test item concentrations of 6.3, 10, 16, 25, 40 and 63 mg/L for 96 hours under flow-through conditions (7.3-7.5 L/h). The test concentrations were analytically verified. Samples for determination of actual test concentrations were taken at the start, after 48 hours of exposure and at the end of the test period. The standard deviations decreased with increasing exposure concentrations from 25% to 15% of the average measured concentrations. The toxicity parameters were based on the average measured concentrations. The 96h-LC50 and NOEC were determined to be 14 mg/L and 8.7 mg/L, respectively (reference 6.1.1-1).