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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb - 13 Jul 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Internal standard solution:
30.0 mg Cyclododecane were dissolved in methanol and made up with methanol to the mark in a 20 ml volumetric flask to prepare a solution of 1500 µg/L. This solution is used as internal standard solution.

Standard solutions:
50.0 mg of the analytical standard were dissolved in acetonitrile and made up with acetonitrile to the mark in a 25 ml volumetric flask (2000 mg/L). 1 ml of this solution was diluted to 20 ml with acetonitrile to prepare a stock solution of 100 mg/L. Defined volumes of this stock solution and an aliquot of the internal standard solution were added to 15 ml water, 2 pellets potassium hydroxide and about 4.5 g sodium sulfate resulting in standard solutions in a range of 0.05 to 12.0 mg test item /I water.
These mixtures were extracted with 4 ml diethylether. The organic extract was used to calibrate the GC-system.
Although the overall method sensitivity may be better, the concentration of the lowest used calibration solution is employed as the limit of quantification.

Sample storage conditions before analysis: Routinely, the samples are analysed immediately. Only in exceptional cases, they are stored overnight deeply frozen and protected from light
Details on test solutions:
To prepare the only test concentration 105.1 mg of the test item were added to 1 litre of dilution water and treated with an ultra turrax for 60 seconds at 8000 rpm.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source: Strain of Bundesgesundheitsamt Berlin
Maintenance and Acclimitisation: A population of parthenogenetic females of synchronized age structure is maintained since more than 15 years in the test facility under constant temperature conditions (20 +/- 1 °C) at a 16:8 hour light-dark photoperiod (illumination: < 1000 lux). The culture water (so-called 'M4 medium') is partly renewed once a week. The Daphnia are exclusively fed with unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period are recorded daily in a semi-quantitative way. The neonates are separated from their parent Daphnia by filtration prior to the acute test.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
The total hardness of the dilution water, measured at test start, was 15.3°dH (= 273.1 mg/L CaCO3).
Test temperature:
21.4 °C
Dissolved oxygen:
8.7 mg/L
Nominal and measured concentrations:
nominal: 100 mg/L
measured: 103.3 mg/L (0 h), 90.4 mg/L (48 h)
Details on test conditions:
Test vessels: 50 ml glass beakers holding 10 neonates in 20 mL of test medium
Experimental design: 1 test concentration plus 1 control; 10 neonates per vessel, 2 replicates per concentration/control; no feeding during the exposure period; static system
Method of initiation: neonates were placed in prepared media
Photoperiod: 16 h light: 8 h dark
Temperature: mean +/- 1 °C (in the temperature range 18-22 °C)
Aeration: none
Method of administration: direct weighing
Medium renewal: none
Duration of exposure: 48h
Criteria of effect: The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.
Reference substance (positive control):
24 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
The results are expressed in terms of nominal concentrations. Recovery rates correspond to 103.3 % of nominal values at 0 hours, and to 90.4 % of nominal values at 48 hours, respectively.
Results with reference substance (positive control):

Validity criteria for the measurement of the daphnia toxicity:

Target condition according to guideline:Actual condition according to the study:Validity criteria met:
Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations for a period of 48 h.

Age of daphnids used: neonates

Exposure time: 48 h

In the control, including the control containing the solubilising agent, not more than 10 % of the daphnids should have been immobilized. (Not more than 10 % of the control daphnids should show immobilisation or other signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water)No daphnids were immobilized under the control conditions after 24 and 48 h.Yes
Test concentrations: At least five test concentrations should be used. They should be arranged in a geometric series with a separation factor preferably not exceeding 2.2. The highest concentration tested should  preferably result in 100 % immobilization, and the lowest concentration tested should preferably give no observable effect.

Test concentrations used: limit test (100 mg/L)

No daphnids were immobilized under the test condition after 24 and 48 h.

Not applicable


Validity criteria fulfilled:
See 'Any other information on results incl. tables'.
The 48h EC50 was determined to be > 100 mg/L, as no immobilisation of daphnia occurred.
Executive summary:

No toxic effects against daphnia at a concentration of 100 mg/l.

Description of key information

The 48h EC50 was determined to be > 100 mg/L, as no immobilisation of daphnia occurred.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
> 100 mg/L

Additional information