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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 2008-11-24 to 2008-12-26
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
O.J. No. L383A, 29.12.92
analysis to confirm test substance concentration not carried out
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
analysis to confirm test substance concentration not carried out
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: 2008-9-16
Purity/assay: >95%
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At each concentration the test substance (4.3, 9.4, 20.7, 45.5 and 100 mg) was mixed with Dimethylsulphoxide (DMSO 0.1mL) and rinsed into volumetric flasks using dilution medium. These aqueous prearations were stirred overnight before being left to stand for four hours. An aliquot (500mL) was removed mid-vessel to provide the water accommodated fraction (WAF) used in the test.
- Controls: Elendt M4 and solvent (dimethylsulphoxide) control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethyl sulphoxide
- Concentration of vehicle in test medium (stock solution and final test solution): Nominal concentrations of the aqueous mixtures from which the WAFs were prepared were 4.27, 9.39, 20.7, 45.5 and 100 mg/L .
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not discussed in report.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Strain: Not stated
- Source: Daphnids used in this study were cultured in-house and were obtained from a strain originating from the National Institute for Applied Chemical Research (IRCHA), France.
- Age at study initiation (mean and range, SD): Less than 24 hours
- Method of breeding: Parthenogenisis
- Feeding during test: no feed was given during the exposure period

- Acclimation period: Not stated
- Acclimation conditions (same as test or not): Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2 ℃.The culture medium was renewed three times each week.
- Type and amount of food: Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given
- Feeding frequency: Daily
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
Equivalent to 244 mg CaCO3/L
Test temperature:
19.9 - 20.4 °C
7.30 - 7.65
Dissolved oxygen:
95 - 103 % ASV
Nominal and measured concentrations:
Nominal concentrations 4.27, 9.39, 20.7, 45.5 and 100 mg/L
No chemical analysis was performed
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): Glass dishes loosely covered
- Material, size, headspace, fill volume: containing 100 mL of medium
- Aeration: No supplementary aeration was employed
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: 20 mL medium per organism

- Source/preparation of dilution water: The medium was prepared in deionised, reverse osmosis water
- Total organic carbon: Not stated
- Particulate matter: At the lowest concentration tested (4.3 mg/L) the test media was a colourless solution. All other concentrations appeared as a colourless solutions with particulate matter at base of the media. The quantity of particulate matter appeared to be concentration related after 48 hours.
- Alkalinity: 47 mg/L as CaCO3
- Culture medium different from test medium: Both culture and test media stated to be Elendt M4
- Intervals of water quality measurement: Not stated

- Adjustment of pH: None recorded
- Photoperiod: A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase
- Light intensity: Not stated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study

- Spacing factor for test concentrations: 2.2
- Range finding study
A range finding test was followed by a main test (originally planned as the definitive test) and the definitive test. The main test was considered to be invalid due to abnormally high levels of floating daphnia in the control group. The range finding test employed nominal test concentrations of 10 and 100 mg/L. After 48 hours, all surviving Daphnia at 100 mg/L were pale and 30% were immobile, at 10 mg/L, 10% of the Daphnia were immobile
Reference substance (positive control):
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Behavioural abnormalities: After 48 hours, 10% immobility had occurred in the test medium prepared at the nominal concentration of 100 mg/L. At the interim concentration of 20.7 mg/L, 15% immobility occurred. Since no immobility occurred at 45.5 mg/L, the result at 20.7mg/L was not considered to be treatment related or of biological significance.
- Effect concentrations exceeding solubility of substance in test medium: WAFs used
Results with reference substance (positive control):
- Results with reference substance valid? Reference substannce not used
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal loading rate based on the nominal concentrations of the aqueous mixtures from which the test media (WAFs) were prepared. 95% confidence limits were unavailable.

The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is usually considered to be significant.
Validity criteria fulfilled:
not applicable
The test item was not found to be acutely toxic to Daphnia magna when tested as a WAF of a maximum loading rate of 100mg/L. Therefore the 48-hour EC50 of test item for the immobilisation of Daphnia magna could not be determined but must be >100 mg/L.

The ‘no-observed effect concentration’ of test item with Daphnia Magna was 100 mg/L
Executive summary:

The acute toxicity of test item to Daphnia magna was assessed under static exposure conditions. The study was conducted in accordance with OECD 202. Groups of twenty Daphnia, less than 24 hours old, were exposed for 48 hours to water accommodated fractions (WAF) of test item, prepared from aqueous mixtures with initial nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were prepared in Elendt M4 medium and to aid dissolution/dispersion, a solvent (Dimethylsulphoxide) and overnight stirring were employed. Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. After 48 hours, at most only 15% immobility occurred at an interim concentration of 20.7 mg/L and this was not considered to be treatment related or of biological significance. Based on these findings the following values have been estimated: 48 -hour EC50 value : >100 mg/L; “No observed effect concentration” : 100 mg/L.

Description of key information

The acute toxicity of test item to Daphnia magna was assessed under static exposure conditions in accordance with OECD 202. 48-hour EC50 value was eatimated to be >100 mg/L; “No observed effect concentration” was 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L
EC50/LC50 for marine water invertebrates:
100 mg/L

Additional information