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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Commission Directive 92/69/EEC, 1992
not specified
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
not specified
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: nominal 100 mg/L and control
- Sampling intervals: start (0 h), after 48 h (not analyzed) and at test end (96 h)
- Sampling method:
At test start: unfiltrated and filtrated (maximum pore size approx. 1.2 µm) samples from the freshly prepared undiluted supersaturated suspension.

After 48 and 96 h: for analysis of dissolved and very finely suspended test material, samples were taken directly from the approximate center of the aquarium without mixing of the test medium and filtered (maximum pore size approx. 1.2 μm). In addition, undiluted supersaturated suspensions, incubated in parallel without fish under the same conditions as in the actual test were collected (collecting of representative suspension-samples from the actual test itself was not possible since the test item was not homogeneously distributed in the test medium).
Control samples were taken directly out of the aquarium.
Samples were taken in duplicate.
- Sample storage conditions before analysis: The filtrated samples and all samples from the sampling at day 2 were deep-frozen (at about -20 °C) immediately after sampling, all the other samples were analyzed immediately after sampling.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item the supersaturated suspension with a nominal concentration of 100 mg/I was prepared as follows: 500 mg of the test item were suspended in a small volume of test water by using a homogenisator. Then the mixture was transferred quantitatively into an adequate volume of test water to obtain a final concentration of nominal 100 mg/L. This suspension was treated ultrasonically for 15 minutes. Then, the supersaturated suspension was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum concentration of the test item in the suspension. No auxiliary solvent or emulsifier was used. The stirring period of 96 hours was chosen according to the results of a pre-test which showed that during this time no significant loss of the test item was to be expected. After stirring and after another 15 minutes ultrasonic treatment prior to the start of the test, a homogeneous suspension was obtained. The undiluted supersaturated suspension with the nominal concentration of 100 mg/L dissolved and suspended test item was used as test medium.
- Controls: Test water without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the test start, the test substance was very finely and homogeneously suspended in the test medium. After about two hours, test substance particles were observed at the water surface and the bottom of the aquarium.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: zebrafish
- Source: professinal pet shop
- Length at study initiation (mean ± SD): 2.6 ± 0.1 cm
- Weight at study initiation (mean ± SD): 0.2 ± 0.05 g

No medication was applied during holding and acclimatization.
- Acclimation period: > 2 weeks
- Acclimation conditions (same as test or not): same
- Feeding during acclimation: until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, TETRA-Werke, 49324 Melle / Germany)
- Health during acclimation (any mortality observed): During the last five weeks prior to the test, no fish died in the test fish batch and all fish were healthy.

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
2.5 mmol/l as CaCO3
Test temperature:
water temperature ranged between 21 and 22 °C.
pH ranged between 7.9 and 8.1.
Dissolved oxygen:
Oxygen saturation was alwasy higher than 60%.
Nominal and measured concentrations:
nominal loading rate: 100 mg/L
measured test concentrations
- unfiltered sample of the suspensions: 90% – 91% of the nominal value (total mean 90%)
- dissolved and very finely suspended test item (measured in the filtrate of the samples): below the limit of detection (LOD) of approx. 1.0 mg/L.

The biological results are based on the loading rate (nominal concentration) of the test item
Details on test conditions:
- Test vessel: full glass aquarium, containing two liters test medium
- Type (delete if not applicable): open
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

- Source/preparation of dilution water: reconstituted water acc. to ISO 6341
- Alkalinity: 0.8 mM
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: not indicated
- Intervals of water quality measurement: water temperature, pH values and oxygen concentrations were measured at the start of the test and once daily during the test. At the same dates the appearance of the test medium was recorded.

- Adjustment of pH: no
- Photoperiod: 16 hour light to 8-hour dark photoperiod

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): symptoms of intoxication and mortality; recorded at approx. 2, 24, 48, 72 and 96 hours

- Spacing factor for test concentrations: not applicable (limit test)
- Results used to determine the conditions for the definitive study: the test concentration was based on the results of a range-finding test and the results of pre-experiments to the solubility of the test material.
Reference substance (positive control):
not required
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality (fish)
Key result
96 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: mortality and sub-lethal effects
Details on results:
- Behavioural abnormalities:
- Other biological observations: none; all fish survived and no visible abnormalities were observed.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Effect concentrations exceeding solubility of substance in test medium: Yes; the solubility of the test item in test water was below the limit of determination of the analytical method (< 1 mg/L).
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
The results indicate that the test material is not toxic to fish up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

Since no classification criteria according to GHS/CLP (Regulation (EC) No. 1272/2008) are fulfilled, the substance is not classified for environmental hazards.
Executive summary:

Due to the low water solubilty of test item, an unfiltrated supersaturated suspension with a nominal concentration of 100 mg/L was used as the test medium, containing a max. concentration of the test substance.

During the test period the total mean measured concentration of dissolved and suspended test substance in the unfiltrated duplicate samples of the suspension was 90% of the nominal value. Under the test conditions, the concentration was sufficiently constant during the 96-hours test period. The concentration of dissolved and very finely suspended test substance was below the detection limit of approx. 1.0 mg/L.

As no moratlity was observed, the 96-hour LL0 (NOELR) thus a PNEC cannot be determined.

Description of key information

effect value for Danio rerio, based on loading rates (OECD 203, EU C.1)
96-h LL50: >100 mg/L (solubility in test water < 1 mg/L)

Key value for chemical safety assessment

Additional information

96-h LL50 > 100 mg/L for Danio rerio.

The results indicate that the test material is not toxic to freshwater fish up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.