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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:
from 2005-12-13 to 2006-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
The solubility of the test material could not be determined but was < 0.005 mg/L. Thus, no further analysis was performed during this study.
Vehicle:
no
Details on test solutions:
- Reconstituted water prepared from deionized water containing the following chemicals: CaCl2 x 2 H2O: 294.00 mg/L; MgCl2 x 7 H2O: 123.25 mg/L; NaHCO3: 64.75 mg/L; KCl: 5.75 mg/L.
- Before exposure of the fish, the test medium (reconstituted water and test material) was freshly prepared: The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. Subsequently the preparation was aerated, and stirred with a magnetic stirrer for further 23 hours. After 24 hours the formulation was given through a nutsch filter (pore size >10 - <16 µm). The filtrate was used for the study.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Origin: West-Aquarium
- Size: 2.0 + 1.0 cm
- Body weight per group: 5.6 and 7.5 g
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
about 15° dH
Test temperature:
23 ± 1 °C
pH:
7.43 to 7.95
Dissolved oxygen:
75 % or higher
Nominal and measured concentrations:
Loading rate of nominal 100mg/L
No measured concentrations.
Details on test conditions:
ACCLIMATIZATION
The fish were acclimatized to the conditions of the laboratory for more than 14 days and were held in reconstituted water for at least for 7 days before testing. During the acclimatization, until one day before the experimental part, the fish were fed with flakes (Tetra Min diet; Tetra GmbH, Melle). Furthermore, the fish got daphnia, or anemia, or frozen mosquito larva daily. During the week before testing, the mortality of the test fish batch was lower than 5 %. At the start of the best, 10 fish per group were randomly introduced into aquaria filled with test medium or reconstituted water, respectively.

ASSIGNMENT
Numbers of zebra fish: Pretest: 4; Control group: 10; Test material group: 10

ENVIRONMENTAL CONDITIONS
During the study, the zebra fish were kept in all-glass aquaria. The study was located in an air-conditioned room in the testing facility. Lighting was controlled by a timer providing a 12 hours light - 12 hours dark regime. The Danio rerio were not fed during the study.

ADMINISTRATION
At the start of the experimental phase, 10 zebra fish per group were introduced into an aquarium (all-glass) with about 9 liters of reconstituted water (control group) or test medium (test material group).

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

LABORATORY TESTS
pH values, and dissolved oxygen (O2) concentration in percent were measured in the control and test material groups at the beginning and at the end of the exposure period. During the experimental part, the temperature was registered in the control vessel with an electronic thermometer containing a maximum and minimum memory display.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
A limit test was performed in accordance with the result of the preliminary test to demonstrate that the test substance at the loading rate of 100 mg/L has no acute toxicity effect on the test fish Danio rerio. A dose group with nominal 100 mg/L of the test item (maximal solubility <0.005 mg/L analytically) revealed no aquatic toxicity in this test system. All fish survived the observation period. Due to the poor water solubility, the analytical 24, 48, 72, and 96 h LC50 values could not be determined. No remarkable observations were made concerning the appearance of the solution of the test material preparation. The test medium was a clear preparation and stayed unchanged throughout the study. The pH values in the treatments and the controls ranged from 7.5 to 7.9, and water temperature was maintained at 23 °C. During the period of the test, the dissolved oxygen concentration was always 75 % or higher, which fulfilled the criteria for a valid test.
Sublethal observations / clinical signs:

No signs of toxicity were seen.

Table 1: Summary symptoms in the Control

Time

3h

24h

48h

72h

96h

Observed fish/time point

10

10

10

10

10

Symptom

No abnormalities detected

10

10

10

10

10

 

Table 2: Summary symptoms in the Test material group

Time

3h

24h

48h

72h

96 h

Observed fish/time point

10

10

10

10

10

Symptom

No abnormalities detected

10

10

10

10

10

In the control and the test material group no clinical findings were observed during the experimental part.

Table 3: Mortality

Concentration mg/L

24h

48h

72h

96h

Control

0/10

0/10

0/10

0/10

100

0/10

0/10

0/10

0/10

All fish survived the observation period.

Validity criteria fulfilled:
yes
Conclusions:
The 96-h LC50 was determined to >100 mg/L.
Executive summary:

The objective of this study was to determine the acute fish toxicity of the test substance using zebrafish (Danio rerio) according to OECD 203 (1992) under GLP conditions. For this purpose, 10 zebra fish per test material group and further 10 zebra fish in a control group were exposed over 96 hours in a limit test, under defined conditions. The fish were observed for signs of toxicity or death. Zebrafish (Danio rerio) were exposed to aqueous test material solutions of nominal 0 (control) and 100 mg/L (limit concentration) in an open static system. The analysis revealed, that the solubility of the test substance in water was < 0.005 mg/L. Because of this low water solubility and the fact that the study was performed as a limit test, the test material concentrations in the aqueous medium at the start and the end of this study could not be quantified. No signs of toxicity were seen in the control group and in the zebra fish exposed to nominal concentrations of 100 mg/L. For the test material, the 96-h LC50 value for fish was determined to be > 100 mg/L.

Description of key information

The acute toxicity of the test substance to Zebrafish (Danio rerio) was determined by an open static test according to OECD 203 under GLP conditions. The 96-h LC50 was determined to > 100 mg/L.

Key value for chemical safety assessment

Additional information

The objective of this study was to determine the acute fish toxicity of the test substance using zebrafish (Danio rerio) according to OECD 203 (1992) under GLP conditions. For this purpose, 10 zebra fish per test material group and further 10 zebra fish in a control group were exposed over 96 hours in a limit test, under defined conditions. The fish were observed for signs of toxicity or death. Zebrafish (Danio rerio) were exposed to aqueous test material solutions of nominal 0 (control) and 100 mg/L (limit cancentration) in an open static system. The analysis revealed, that the solubility of the test substance in water was < 0.005 mg/L. Because of this low water solubility and the fact that the study was performed as a limit test, the test material concentrations in the aqueous medium at the start and the end of this study could not be quantified. No signs of toxicity were seen in the control group and in the zebra fish exposed to nominal concentrations of 100 mg/L. For the test material, the 96-h LC50 value for fish was determined to be > 100 mg/L (reference 6.1.1-1).

In a supporting study, the acute toxicity of the test item was studied in a pre-test for a bioaccumulation study (OECD 305) using solvents to increase exposure. The GLP test was performed according to the METI Guideline: Concentration Test on Chemical Substances in Fish. To achive the exposure concentrations, solvent Tween 80 was used. All concentrations tested were above the water solubility limit of the pure test substance. For the test Danio rerio fish were used. The following concentrations have been applied: 122.1, 113.4, 109.4, 91.5, 81.8, and 72.9 mg/L. The test item does not possess acute toxic potential to fish up to the limit of the water solubility (< 0.0.005 mg/L). Using Tween 80 as solubiliser, toxicity was observed at higher concentrations. The 96 hours LC50 amounts to 113.4 mg/L. The LC50 of 113.4 mg/L obtained under these conditions is clearly above the limit concentration of 100 mg/L given in the OECD TG 203 (reference 6.1.1-2).