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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
there were minor deviations not affecting the validity of the study
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
DETAILS ON PREPARATION
As the solubility of the test item in the test medium was below 100 mg/L and according to the available information, stability was not given over a period of more than 24 hours, the test solutions were prepared as follows. The load of 795 mg/L (corresponding to a volume of 0.9 mL/L based on a density of 0.883 g/mL) was added to the corresponding amount of dilution water and homogenised for 30 minutes at 30°C in an ultrasonic bath. Afterwards, the solution was stirred for 30 minutes on a magnetic stirrer. Then, the solution was homogenised again using ultrasonic for 15 min and afterwards stirred for 15 minutes. The resulting clouded solution was filtrated through 0.45 µm nylon filters. An aliquot was examined for colloidal particles by testing the solution with a laser for the Tyndall effect. This solution was stated as the 100 % saturated solution. Each day of the test, a new test solution was freshly prepared.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Danio rerio HAMILTON-BUCHANAN
Age: sexually immature young fish, length 2 ± 1 cm
Origin : The original animals were obtained from Umweltbundesamt (UBA) and used for breeding and production of eggs.
The offspring of the original animals was used in the test.

Danio rerio is routinely used for toxicity tests. The test fish are kept following SOP 115 005 04 in the current edition.
Vessels: polyethylene aquaria
Medium: chlorine-free tap water, full composition see annex
Feeding: three times a day with warm-water fish food and daphnids, totalling to about 1-2% of body weight per day
Medium renewal: twice a week
Photoperiod: 12/12 hours, using neon tubes
Temperature: 27 – 28 °C
Dilution Water: Good quality drinking water is used. The composition is analysed yearly by the supplier. The result of this analysis is given in the annex
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
1.08 mmol CaCO3/L
Test temperature:
20.2- 23.2 °C
The temperature deviation during the main study was higher than ±1°C. Because no fish died during husbandry and not in the control and in the test solution during the test, this can be stated as uncritical
pH:
7.3 - 8.1 (variation of 0.8 < 1 unit)
Dissolved oxygen:
7.1 - 9.6 mg O2/L (> 88%)
Nominal and measured concentrations:
Nominal : 795 mg/L
Limit of solubility = approx. 5.4 mg/L, based on the geometric mean of the measured DOC concentrations in the fresh prepared solutions
Details on test conditions:
24 hours before the start of the test, food was withheld from the designated test fish.
Date: 22. – 26. Feb. 2013
Treatment: 795 mg/L
Replicates: one vessel, containing 7 L test solution and 7 fish
Control: one vessel, containing 7 L dilution water and 7 fish
Test design semi-static
Medium renewal every 24 ± 1 hours
Duration 96 hours
Loading 1 fish/L
Vessels: glass aquaria, maximal volume 10 L
Aeration: accomplished with glass tubes, frequency of bubbles 1/s
Feeding: none
Photo period: 12/12 hours using neon tubes
Temperature: 20.2- 23.2 °C
pH adjustment: none
On each day of the test, a fresh test solution was prepared. Due to a writing error in the study plan, the test concentration was 795 mg/L instead of 100 mg/L. As both concentrations are far above the limit of solubility of the test item and the saturated solution was tested, this deviation was stated as uncritical.
Observations were made every 24 hours, measuring pH and O2-concentration of the old and new test solution in each vessel and documenting mortalities or abnormal behaviour. The content of DOC in the test vessels was measured at the beginning and every 24 hours in the fresh and the old solutions. The DOC in the control was measured at the beginning and at the end of the test.
A fish was considered dead, if no visible movement could be observed, and if touching of the caudal peduncle produced no reaction.
The temperature variation during the main study was higher than ±1°C. Because no fish died during husbandry and not in the control and in the test solution during the test, this can be stated as uncritical.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
>= 795 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 795 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
No mortality was observed during the test at the limit of solubility of the substance (approx. 5.4 mg/L), considering loading rate of 795 mg/L.
No abnormal behaviour of the surviving fish was observed.

The mortalities were recorded daily and can be found in the following table:

Nom. Conc. in mg/L

Total of fish used

dead fish

24 h

48 h

72 h

96 h

 

 

new

cum.

new

cum.

new

cum.

new

cum.

0

7

0

0

0

0

0

0

0

0

795

7

0

0

0

0

0

0

0

0

Validity criteria fulfilled:
yes
Remarks:
No mortalities occurred in the control. The concentration of dissolved oxygen stayed above 7.1 mg/L or 88 % throughout the test. The highest variation of the pH-value in the test solutions during the test was 0.8 units.
Conclusions:
96h NOELR ≥ limit of solubility (approx. 5.4 mg/L in the test media) with a loading rate of 795 mg/L
96h LL50 > limit of solubility (approx. 5.4 mg/L in the test media) with a loading rate of 795 mg/L
Executive summary:

As the solubility of the test item in the test medium was below 100 mg/L and stability was not given over a period of more than 24 hours, the fish toxicity test was performed in semi-static conditions. A load of 795 mg/L was added to the corresponding amount of dilution water (drinking water) and homogenised. This solution was stated as a 100 % saturated solution.

The main study was performed using one treatment containing the nominal concentration of 795 mg/L (100 % saturated solution).

The measured DOC concentrations in the new solutions were used for determination of the test item concentration during the test. The limit of solubility of the test item based on the geometric mean of the DOC concentration in the fresh solutions can be determined as approx. 5.4 mg/L.

The tested concentration showed no mortality. No observations were made which might cause doubts concerning the validity of the study outcome.

The following results were determined:

96h NOELR≥limit of solubility (approx. 5.4 mg/L in the test media)

96h LL50 > limit of solubility (approx. 5.4 mg/L in the test media)

Description of key information

The toxicity of the test item to the Danio rerio was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 203.

The 96h NOELR ≥ limit of solubility (approx. 5.4 mg/L in the test media) with a loading rate of 795 mg/L (100% saturated solution)

The 96h LL50 > limit of solubility (approx. 5.4 mg/L in the test media) with a loading rate of 795 mg/L (100% saturated solution)

Key value for chemical safety assessment

Additional information

The toxicity of the test item to fish Danio Rerio was investigated in one GLP-compliant study performed in accordance with standard methods, with minors deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.