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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March - 4 April 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to
EU Method C.2 (Acute Toxicity for Daphnia)
according to
ISO 6341 15 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): Aquapel® 203
- Substance type: yellow fatty liquid
- Physical state: liquid
- Analytical purity: 89.9%
- Lot/batch No.: G12FY067
- Expiration date of the lot/batch: 12 June 2008
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: Water Accommodated Fractions (WAFs) prepared at loading rates of 1.0, 10 and 100 mg/l.
- Sampling method: During both tests singular samples for possible analysis were taken from all test concentrations and the control according to the following schedule: Frequency: at t=0 h and t=48 h; Volume: 2 ml from the approximate centre of the test vessels .

- Sample storage conditions before analysis: Samples were pre-treated with acetonitrile on the day of sampling. There after samples were stored at room temperature until analysis.

Test solutions

Details on test solutions:
All test solutions were prepared separately. Loading rates of 1.0, 10 and 100 mg Aquapel® 203 were added per litre of ISO-medium, stirred for 24 hours and subsequently stabilised for 24 hours.
For the first test, the Water Accommodated Fractions (WAFs) were siphoned after stabilisation and used for testing. The WAF prepared at 1.0 mg/l was observed to be clear and colourless, while the WAFs prepared at loading rates of 10 and 100 mg/l were increasingly hazy.
For the second test, the Water Accommodated Fractions were filtered through glass wool after stabilisation. These WAFs were all observed to be clear and colourless. Part of the WAFs was used for pre-incubation of the test vessels (1½ hour). The remaining part of the WAFs was used for testing.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Source: In-house laboratory culture with a known history.
- Age at study initiation (mean and range, SD): < 24 hours
- Peripheral shell growth removed prior to test initiation:
- Method of breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
- Feeding during test: no feeding

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:

Test conditions

180 mg/l expressed as CaCO3
Test temperature:
18.9 - 19.7°C
7.8 - 7.9
Dissolved oxygen:
9.0 - 9.5 mg O2/L
Not applicable
Nominal and measured concentrations:
See any other information on materials and methods below.
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 ml glass vessels, containing 80 ml solution.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest test concentration; 2 for the lower test concentrations
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: Medium: Adjusted ISO medium

- Adjustment of pH: yes
- Photoperiod: 16 hours photoperiod daily
- Light intensity: not mentioned

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for the highest test concentration and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

- Spacing factor for test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/l.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
24 and 48 h
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
First test:
After 48 hours of exposure, no immobility was observed at the clear and colourless 1.0 mg/l WAF. At the 10 and 100 mg/l WAFs a floating layer was observed from 24 hours of exposure onwards. 70 and 100% immobility was observed at these concentrations, respectively. However, it was expected that this immobility was related to mechanical hampering due to the presence of undissolved material rather than by toxicity. Analytical results of the analysis performed on the samples taken from the 100 mg/l WAF showed an initial concentration of 14 mg/l, which was indeed above the water solubility limit for Aquapel® 203. This concentration decreased to 7.9-9.1 mg/l during the test period. Analysis of the samples taken from the clear and colourless 1.0 mg/l WAF showed that the actual concentration was below the Limit Of Detection (LOD; i.e. below 0.04 mg/l) from the start.
Because of the indistinct cause of immobility, the test was repeated using a different method for preparation of the test solutions.

Second test
Measured concentrations:
Based on the biological results, only samples taken from the control and the clear and colourless 100 mg/l WAF were analysed. The initial concentration of the WAF was determined to be 0.22 mg/l. This concentration decreased to 0.085 mg/l during the test period. Based on these results, the average exposure concentration was calculated to be 0.14 mg/l.
No immobility was observed at any of the test concentrations after 48 hours of exposure. No floating layers were observed, indicating that the immobility seen in the first test was indeed caused by mechanical hampering of the daphnids.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality: immobility of 15% (24h) and 65% (48h) at a concentration of 0.56 mg/L; immobility of 75% (24h) and 100% (48h) at a concentration of 1.0 mg/L
- EC50/LC50: The 24h-EC50 was 0.76 mg/l with a 95% confidence interval between 0.67 and 0.90 mg/l.
The 48h-EC50 was 0.51 mg/l with a 95% confidence interval between 0.47 and 0.58 mg/l.
Reported statistics and error estimates:
Determination of the average exposure concentrations
The average exposure concentrations were calculated as , being the geometric means of the concentrations of Aquapel® 203 measured in the samples taken at the start (Ct=0) and the end of the test (Ct=48).

Calculation of EC50
No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration tested).

Applicant's summary and conclusion

Validity criteria fulfilled:
Under the conditions of the present study Aquapel® 203 did not induce acute immobilisation of Daphnia magna at an average exposure concentration of 0.14 mg/l (NOEC).

The 48h-EC50 was beyond 0.14 mg/l and was considered to be beyond the water solubility limit of Aquapel® 203.