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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:
2000-03-28 till 2000-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
December 29, 1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
For the analytical measurement samples from the freshly prepared test medium from the nominal concentrations of 0.010, 0.032, and 0.100 g/L were taken at the start of the study. For the determination of the maintenance of the test material concentration during the exposure period a samples were taken from the medium at the end of the exposure period.
Vehicle:
no
Details on test solutions:
The test media (reconstituted water and test material) were freshly prepared. Therefore, the flask with the test material and the vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain: CRL/ZF 1
- Source: Bio International B.V. NL-5845 ZH St. Anthonis
- Length at study initiation: 2.0 ± 1.0 cm

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: Tetra Min® diet (Tetra Werke, D-49304 Melle).
- Health during acclimation: mortality below 5%

FEEDING DURING TEST
No feeding during test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
212 mg CaCO3/L
Test temperature:
23 - 24 °C
pH:
7.44 to 7.91
Dissolved oxygen:
90.2 to 99.6 %
Nominal and measured concentrations:
Nominal concentrations: 10, 18, 32, 56, 100 mg/L
Measured concentrations: 3.5, not measured, 20, not measured, 65 mg/L at the start; 3.5, not measured, 15, not measured, 50 mg/L after 96 h
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquariae
- Type: closed with parafilm® and with a glass plate
- Material, fill volume: glass, 8L
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconsituted water according to OECD 203 was used.
For the reconstituted water the following chemicals (analytical grade) were dissolved in fully demineralized water:
Macro nutrients (mg/L):
CaCl2 x 2H2O 294
MgSO4 x 7H2O 123
NaHCOs 65
KCl 5.8
After preparation the reconstituted water was aerated for 24 hours. The proportion Ca:Mg ions is 4:1 and Na:K ions 10:1.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours light - 12 hours dark regime

EFFECT PARAMETERS MEASURED
The behavior and general condition of all fish were checked immediately after the introduction into the test medium or reconstituted water, after three and six hours, and daily thereafter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: not available
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: see Table 1 below
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no

Validation criteria:
- No mortality was observed in the control.
- Further, all test conditions remained within the ranges prescribed by the protocol.

The control analysis showed a recovery of about 64 % for the two nominal test concentrations of 0.032 and 0.100 g/L at the start of the study. At the nominal concentrations of 0.010 g/L, the analytically determined test material concentration was 35 %. At the end of the study, the analytical concentration in the two highest nominal test concentrations was about 48 %. This amounts to about 75 % of the initial analytical concentration of 0.065 g/L. Thus the test material concentration could not be maintained as at > 80 % of the initial concentration throughout the experimental part.

Table 1: Symptoms observed at the different time points and nominal concentrations

Concentration                           00 mg/L

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

7

7

7

7

Symptom

No abnormalities detected

7

7

7

7

7

7

 

Concentration                           10 mg/L

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

7

7

7

7

Symptom

staying at the water surface

7

7

7

7

7

7

 

Concentration                          18 mg/L

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

7

6

6

6

Symptoms

staying at the water surface

6

6

6

6

6

6

at the bottom of the aquarium

1

1

1

 

 

 

abdominal position

 

 

1

 

 

 

no movement

3

 

 

 

 

 

 

Concentration                           32 mg/L

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

7

6

6

6

Symptoms

staying at the water surface

7

7

7

6

6

5

at the bottom of the aquarium

 

 

 

 

 

1

fish lying on the side

1

1

1

2

2

2

tumbling at swimming

 

 

 

1

1

1

no movement

1

 

 

 

 

 

Concentration                           56 mg/L 

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

3

1

0

0

Symptoms

staving ai the water surface

1

3

1

 

 

 

at the bottom of the aquarium

4

4

2

1

 

 

fish lying on the side

2

6

3

1

 

 

 

Concentration                            100 mg/L

Time (hours)

3

6

24

48

72

96

Observed fish/time point

7

7

0

0

0

0

Symptoms

staying at the water surface

5

6

 

 

 

 

at the bottom of the aquarium

2

1

 

 

 

 

fish lying on the side

5

7

 

 

 

 

 

 

Table 2: Mortality observed at the different time points and nominal concentrations

Concentration [mg/L]

24 hrs

48 hrs

72 hrs

96 hrs

0

0/7

0/7

0/7

0/7

10

0/7

0/7

0/7

0/7

18

0/7

1/7

1/7

1/7

32

0/7

1/7

1/7

1/7

56

4/7

6/7

7/7

7/7

100

7/7

7/7

7/7

7/7

 

Validity criteria fulfilled:
yes
Conclusions:
The test substance has acute toxicity effect on zebrafish under the present conditions of the test, LC50(96h): 17 mg/L.
Executive summary:

The acute toxicity of the test item in Zebrafish was performed according to EU Method, Part C.1. and the OECD guideline No. 203. Groups of Brachydanio rerio were exposed to different concentrations of the test material in reconstituted water. The animals were observed for signs of intoxication or death up to 96 hours after introduction in the test medium or the reconstituted water. After a pretest, the final study was performed with concentrations of 10, 18, 32, 56 and 100 mg test material/L reconstituted water and one control group. Analytical controls were carried out with 10, 32 and 100 mg/L. Initially, the recovery of the low concentration was 35% of the nominal test material concentration and the recovery of the both higher concentrations was about 64 %. At the end of the study, the recovery of the low concentration was 35% and the analytical determined concentrations of the two higher concentrations were about 46,9 % and 50% of the nominal concentrations. A nominal concentration of 0.100 g/L revealed an initial analytical concentration of 0.065 g/L. This amounts to about 75 % only. Thus the test material concentration could not be maintained at > 80 % of the initial concentration throughout the experimental part as recommended.Signs of intoxication were seen immediately after introduction up to the end of the study. These were tumbling at swimming, staying near the water surface, swimming at the bottom of the aquarium, abdominal position, lying on the side, and no movement. Deaths occurred from 23 hours up to 72 hours after start of the exposure in all concentrations exempt the control and 10 mg/L test item. The calculated 96 h LC50 based on mean measured concentrations was 17 mg/L and the NOEC 3.5 mg/L.

Description of key information

The test substance has acute toxicity effect on zebrafish under the present conditions of the test, LC50(96h): 17 mg/L (reference 6.1.1.-1).

Key value for chemical safety assessment

LC50 for freshwater fish:
17 mg/L

Additional information

The acute toxicity of the test item in Zebrafish was performed according to EU Method, Part C.1. and the OECD guideline No. 203.Groups of Brachydanio rerio were exposed to different concentrations of the test material in reconstituted water. The animals were observed for signs of intoxication or death up to 96 hours after introduction in the test medium or the reconstituted water. After a pretest, the final study was performed with concentrations of 10, 18, 32, 56 and 100 mg test material/L reconstituted water and one control group. Analytical controls were carried out with 10, 32 and 100 mg/L. Initially, the recovery of the low concentration was 35% of the nominal test material concentration and the recovery of the both higher concentrations was about 64 %. At the end of the study, the recovery of the low concentration was 35% and the analytical determined concentrations of the two higher concentrations were about 46,9 % and 50% of the nominal concentrations. A nominal concentration of 0.100 g/L revealed an initial analytical concentration of 0.065 g/L. This amounts to about 75 % only. Thus the test material concentration could not be maintained at > 80 % of the initial concentration throughout the experimental part as recommended.Signs of intoxication were seen immediately after introduction up to the end of the study. These were tumbling at swimming, staying near the water surface, swimming at the bottom of the aquarium, abdominal position, lying on the side, and no movement. Deaths occurred from 23 hours up to 72 hours after start of the exposure in all concentrations exempt the control and 10 mg/L test item. The calculated 96 h LC50 based on mean measured concentrations was 17 mg/L and the NOEC 3.5 mg/L (reference 6.1.1 -1).