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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:
25 May 2010 to 13 May 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with an internationally recognised guideline.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): TFO
- Substance type: organic
- Physical state: liquid
- Lot/batch No.:
- Expiration date of the lot/batch: TFOS285001
- Storage condition of test material: room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 0, 4.27, 9.39, 20.7, 45.5, 100 mg/L

Samples at 0.0954 and 10 mg/L were analysed in the presence and in the absence of algal cells.

- Sampling method: complete contents of the sample bottle were analysed.

- Sample storage conditions before analysis: samples analysed immediately after being taken therefore no storage required. Duplicate samples were stored in a refrigerator in case further analysis was required.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

The method of preparation used during the definitive test was based on the results of a feasibility trial and formulation trials and a range finding test conducted as part of this study.

On Day 0 of the definitive test, the test substance (68, 31 or 28 μL; corrected for purity and specific gravity) was dispersed in dilution medium (1 or 2 L) in a volumetric flask to prepare test concentrations of 100, 45.5 and 20.7 mg/L. Aliquots (455 and 206 mL) of the test media at 20.7 mg/L were then further diluted (1 L) in dilution medium to prepare the remaining test concentrations of 9.39 and 4.27 mg/L. On Day 1 of the definitive test, only the media at nominal concentrations of 20.7, 9.39 and 4.27 mg/L were required.

In an attempt to minimise losses by volatilisation, the medium at each concentration was individually prepared, and the analytical samples were taken and the test organisms were added before proceeding with the preparation of the next concentration.

- Controls: test medium

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: daphnia
- Strain: Straus
- Source: research institute
- Age at study initiation: less than 24 hours

Study design

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

Test conditions

264 as CaCO3,
Test temperature:
20.8 to 21.8°C
Dissolved oxygen:
99-105 % ASV
Not applicable to a freshwater study.
Nominal and measured concentrations:
(mg/L) 0 hours 24 hours 48 hours Geometric mean % of nominal concentration
Control nd nd nd nd nd
4.27 1.65 1.74 2.62 2.13 50
9.39 4.11 4.16 5.88 5.05 54
20.7 12.1 12.2 ns 12.1 59
45.5 18.4 19.0 ns 18.7 41
100 62.3 62.8 ns 62.5 63

nd = none detected
ns = not sampled as all Daphnia were immobile after 24 hours exposure

The overall geometric mean measured levels of TFO, 2.13, 5.05, 12.1, 18.7 and 62.5 mg/L have been used in the calculation of test results.
Details on test conditions:
- Test vessel: vials
- Type: closed
- Material, size, headspace, fill volume: glass , completely filled and sealed, leaving no headspace.
- Aeration:
- Renewal rate of test solution: media was removed after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: ca. 26 mL medium per organism

- Source/preparation of dilution water: deionised, reverse osmosis water.

- Adjustment of pH: none
- 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: no information provided

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel.
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: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: after 48 hours under sealed, static conditions, all of the Daphnia at 100 mg/L
were immobile and all Daphnia at 0.1, 1.0 and 10 mg/L were mobile. Based on these results, the definitive test employed nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/L.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
24 h
Dose descriptor:
Effect conc.:
7.82 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% confidence limit 5.05 & 12.1 mg/L
48 h
Dose descriptor:
Effect conc.:
3.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% confidence limit 2.13 & 5.05 mg/L
Dose descriptor:
Effect conc.:
2.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable as a reference substance was not used.
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application (version 1.3), SAS 9.1.3 (SAS Institute, 2002). Test results were expressed in terms of the overall mean measured concentrations.

Any other information on results incl. tables

The measured concentrations of TFO ranged between 39 and 63% of their nominal values in freshly prepared media and between 41 and 69% of their nominal values in samples of expired (24-hour-old) media (between 101 and 110% of the starting values). Although precautions were taken to minimise losses via volatilisation during the preparation of the test media, the values achieved were considered acceptable given the nature of the test substance. As the levels achieved were maintained throughout the test, the use of a sealed test system was considered successful.

After 48 hours, the lowest measured concentration at which 100% immobility occurred was 5.05 mg/L and the highest concentration at which no immobilisation was noted was 2.13 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
The 48-hour EC50 for immobilisation was 3.28 mg/L (95% confidence limits of 2.13 and 5.05 mg/L) and the ‘no-observed effect concentration’ was 2.13 mg/L.

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