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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:
From 2014-08-05 to 2014-08-14
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The reliability is rated 2 because the study followed a standard guideline of reference (OECD 202), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid. The study was not conducted under GLP conditions.
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: 0, 3.7, 6.3, 11, 18, 31 and 53 (% v/v saturated solution) for the main test
- Sample storage conditions before analysis: -25 +/- 5°C

- Concentrations: 0, 1, 3.5, 10, 35 and 100 % v/v (nominales)
- Sampling method: at 0 and 48 hours for quantitative analysis and pooled.
- Sample storage conditions before analysis: approximately -25 +/- 5°C prior to analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method of preparation: Saturation of the experimental solution:
100 mg of test item was dispersed in 1 liters test medium
Stirring for 24 hours.
Filtration through a PTFE of 0.22 μm to remove undissolved test item
Obtention of a 100% v/v saturated solution.
Series of dilution to give further test concentration for the range finding study and the main test.

- Application: daphnies were placed in test tube containing 10 mL of the saturated solution test

- Controls: control group maintained under identical conditions but not exposed to the test item
- Vehicle: none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain: Daphnia magna Straus clone 5
- Source: in-house subcontracting laboratory cultures
- Age at study initiation (mean and range, SD): less or equal to 24h at beginning of test and at least from the first litter
- Method of breeding: synthetic medium as defined by the standard NF EN ISO 6341

- Acclimation period: no

QUARANTINE (wild caught): not applicable
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
225 mg/L +/- 50 mg/L (CaCO3)
Test temperature:
see table 1
Dissolved oxygen:
see table 2
Nominal and measured concentrations:
see table 3
Details on test conditions:
- Test vessel: glass test tube of 10 mL
- Type : closed
- Material, size, headspace, fill volume: gauged test tube 10mL
- Aeration: no
- Renewal rate of test solution: no renewal
- No. of organisms per vessel: 5 organisms
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates

- Source/preparation of dilution water: ultrapure water
- Test medium: according to NF EN ISO 6341
For 10 L
NaHCO3 2,00 g +/- 0,1 g
CaCl2, 2H2O 2,60 g +/- 0,1 g
MgCl2, 6H2O 1,48 g +/- 0,1 g
K2SO4 0,26 g +/- 0,01 g
ultrapure water to 10 L
Aeration for 12h min.
Kept 30 days max ambient air
- pH 7,8 +/- 0,5
- hardness: 225 mg/L+/- 50 mgL (CaCO3)
- Ca/mg ratio: 4/1 +/- 0.5
- dissolved oxygen > 7 mg/L

- Spacing factor for test concentrations: 1.7
- Range finding study: yes
percentage of organisms immobilisation after 48h in tested concentrations (% of stock solution at 100 mg/L)
100%: 100%
35%: 90%
10%: 30%
3.5%: 10%
1%: 0%
- Test concentrations: 53 mg/L, 31 mg/L, 18 mg/L, 11 mg/L, 6.3 mg/L, 3.7 mg/L
- Results used to determine the conditions for the definitive study: cumulative immobilization data from the range-finding study
Reference substance (positive control):
Potassium dichromate (K2Cr207)
48 h
Dose descriptor:
Effect conc.:
ca. 2.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
ca. 6.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: (95% CL 5.6 - 7.9)
48 h
Dose descriptor:
Effect conc.:
ca. 15.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Behavioural abnormalities: none oberved
- Abnormal responses: none observed
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 24h: 1.1 mg/L (2014-08-11)
Validity criteria fulfilled:
Under experimental conditions, EC50-48h = 6.6 mg/L (determined with measured concentrations in test solutions)
Executive summary:


A study was performed to assess the acute toxicity of the test item to Daphnia magna.The method followed was designed to be compatible with OECD Guidelines. for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test".


Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 3.7, 6.3, 11, 18, 31, and 53 mg/L for 48 hours at a temperature of 18 to 22°C under static test conditions.

The number of immobilized Daphnia were recorded after 24 and 48 hours.


Exposure of Daphnia magna to the test item gave the following results based on the measured test concentrations:

- EC50 – 48 hr = 6.6 mg/L (IC95% 5.6 - 7.9mg/L)

- NOEC = 2.2 mg/L

Description of key information

LC50(48h) for freshwater invertebrates (Daphnia magna): 6.6 mg/L (OECD TG 202; semi static).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
6.6 mg/L

Additional information

The EC50 (48 hours) was 6.6 mg/L (measured concentrations) with 95% confidence limits of 5.6 -7.9 mg/L.

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