Sodium 6,14-diethyl-1,1,1,2,2,18,18,19,19,19-decafluoro-8,12-dioxo-10-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)-4,7,13,16-tetraoxanonadecane-9-sulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 JAN 2013 - 17 JUL 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 201: Alga, Growth Inhibition Test, adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the
Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).C.3. Freshwater algae and cyanobacteria, growth inhibition test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10.0, 17.8, 31.6, 56.2, 100.0 mg/L (nominal)
- Sampling method: Samples of the test item concentrations and the control were taken at the start of the study and after 72 hours.
- Sample storage conditions before analysis: The samples obtained were mixed with Acetonitrile proportionally 1:1 and immediately deep frozen at -20 ± 2 °C. One replicate per concentration was stored at 6 ± 2 °C from 2013-05-14. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium (reconstituted water and test item) was prepared freshly. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 μm. The filtrate was used for the study.
- Controls: reconstituted water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the test media in the control flasks without algae. All test media were clear solutions and remained clear throughout the entire test period.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae (Desmodesmus subspicatus)
- Strain: strain 86.81 SAG
- Source (laboratory, culture collection): The strain was supplied by: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, 37073 Göttingen, Germany. This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Age of inoculum (at test initiation): At the start of the experimental part, an algae stock suspension was prepared from the preculture containing about 10000 algae cells/mL.
- Method of cultivation: Prior to start the study, the algae were grown in reconstituted water as preculture and the growth rate was determined (growth rate 38.6).

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): The pre-culture for the experimental part was set up under the same conditions as in the final study.
- Any deformed or abnormal cells observed: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

250 mg/L

Test temperature:

22.3 - 22.8 °C

pH:

7.82 - 8.95

Nominal and measured concentrations:

nominal: 0, 10.0, 17.8, 31.6, 56.2, 100.0 mg/L
initially mean measured: < LOQ, 8.49, 16.2, 28.6, 50.3, 94.7 mg/L
measured (72 h): < LOQ, 8.43, 14.5, 28.2, 43.3, 94.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type (delete if not applicable): The flasks were covered with air permeable steristoppers.
- Material, size, headspace, fill volume: 100 mL of test solutions
- Initial cells density: 10000 cells/mL
- Control end cells density: 59283 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Reconstituted water was used as the vehicle.
- Detailed composition if non-standard medium was used:
Concentrations given in mg/L:
NH4Cl: 15.00000
MgCl2 * 6 H2O: 12.00000
CaCl2 * 2 H2O: 18.00000
MgSO4 * 7 H2O: 15.00000
KH2PO4: 1.60000

FeCl3 * 6 H2O: 0.06400
Na2EDTA * 2 H2O: 0.10000

H3BO3: 0.18500
MnCl2 * 4 H2O: 0.41500
ZnCl2: 0.00300
CoCl2 * 6 H2O: 0.00150
CuCl2 * 2 H2O: 0.00001
Na2MoO4 * 2 H2O: 0.00700

NaHCO3: 50.00000

The pH of the reconstituted water after aeration is approximately 8.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not specified
- Photoperiod: The flasks were continuously illuminated for the exposure period.
- Light intensity and quality: The light intensity was between 4440 and 8880 Lux and within ± 15% from the average. The illumination was achieved by fluorescent tubes (Philips Master TL5 - 80 W/840 HO) installed above a rotating panel with the flasks.
- Other: The study was performed in an air-conditioned room (09/2108) at Non-Clinical Safety. For reproduction, the algae were agitated during the entire study. Therefore, the test vessels were continuously shaken by a rotating panel (supplier shaking device SM25: Edmund Bühler).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : During the exposure period of 72 h the cell density was measured daily. Volumes of the algal suspension not impairing the study were taken from the test item preparations and the controls with algae after exposure times of 24, 48, and 72 hours. The required volume depended on the cell density.
To determine the morphological effect of the test item on the algal cells, after 72 hours additional samples were taken from the control and at least one concentration with a clearly reduced cell density. The morphological appearance of the treated algal cells in comparison to the controls was assessed by microscope.
- Determination of cell concentrations: The cell densities in the samples were determined with an electronic particle counter (Coulter Z2) and documented.

TEST CONCENTRATIONS
- Range finding study: The test item concentrations for the study were selected on the basis of the results of a preliminary study.
- Results used to determine the conditions for the definitive study: Stability testing of the test item at a nominal concentration of 100 mg/L in reconstituted water for daphnids in open vessels revealed a recovery rate of 83.2% after 72 hours. Therefore, the study was performed in open vessels.

Reference substance (positive control):

yes

Remarks:

The sensitivity of the test system has to be demonstrated periodically. Potassium dichromate (Art. 104864) was tested as positive control (Gado, 2013).

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: not determinable

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

86.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: not determinable

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: not determinable

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: not determinable

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: No morphological changes of the treated algal cells in comparison to the control algal cells were observed.
- Any stimulation of growth found in any treatment: A slight stimulation of growth was determined for each treatment group except the highest treatment group of nominal 100 mg/L. As this stimulation of growth was only slight and not dose related, it can be considered to be an effect of narural biological variation.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Under the experimental conditions given in the reference study performed in November 2012, the test item potassium dichromate showed 72h EC values which are within the recommended range (Yield EC50 0.20 – 0.75 mg/L and Growth rate EC50 of 0.60 –1.03 mg/L) and sensitivity of the test system was demonstrated.

Reported statistics and error estimates:

The statistical values and evaluations were carried out with the PC-program ToxRat Professional. The measured cell counts and the growth rate of the preculture were calculated with the PCprogram
Algendat.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD TG 201 and EU Method C.3. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines and the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
The test material, solved in reconstituted water, was tested in an open static test system. Under the conditions of the present study, the 72h ErC50 and the 72h EyC50 value were determined to be > 100 mg/L nominal. The 72-hour ErC10 was determined to be > 100 mg/L and the 72-hour EyC10 was calculated to be 86.8 mg/L, respectively.

Executive summary:

The objective of this study was to determine the influence of the test item on the growth and growth rate of the unicellular green algal species Desmodesmus subspicatus. The study design included one control group with six replicates and five test item groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10000 cells/mL at the start of the experimental phase. Stability testing of the test material at a nominal concentration of 100 mg/L  in open vessels revealed a recovery rate of 83.2% after 72 hours. Therefore, the study was performed in open vessels. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control groups. Analytical controls of the test item preparation were performed at the start of the study and after 72 hours.
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201 and EU Method C.3.

The analytically determined test item concentration immediately after preparation of the medium was 95-85% and 72 hours thereafter 95-77% of the nominal concentration, respectively.

During the experimental phase of the study, the test item concentration could be maintained within ± 20% of the initial analytical concentration. Therefore, the EC values were calculated with the initial analytical concentration.

Accordingly, the following EC values for Desmodesmus subspicatus were determined for the test material:

E_yC₁₀ (72h) = 86.8 mg/L (95% confidence interval: not determinable)

E_yC₅₀ (72h) > 100 mg/L (95% confidence interval: not determinable)

E_rC₁₀ (72h) > 100 mg/L (95% confidence interval: not determinable)

E_rC₅₀ (72h) > 100 mg/L (95% confidence interval: not determinable)

Description of key information

Toxicity to aquatic algae and cyanobacteria: EC₁₀ (72h) growth rate > 100 mg/L and EC₅₀ (72h) growth rate > 100 mg/L (initial analytical concentration) for the freshwater green algae Desmodesmus subspicatus (static, OECD 201, GLP)

Additional information

The objective of this study was to determine the influence of the test item on the growth and growth rate of the unicellular green algal species Desmodesmus subspicatus. The study design included one control group with six replicates and five test item groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10000 cells/mL at the start of the experimental phase. Stability testing of the test material at a nominal concentration of 100 mg/L  in open vessels revealed a recovery rate of 83.2% after 72 hours. Therefore, the study was performed in open vessels. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control groups. Analytical controls of the test item preparation were performed at the start of the study and after 72 hours.
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201 and EU Method C.3.

The analytically determined test item concentration immediately after preparation of the medium was 95-85% and 72 hours thereafter 95-77% of the nominal concentration, respectively.

During the experimental phase of the study, the test item concentration could be maintained within ± 20% of the initial analytical concentration. Therefore, the EC values were calculated with the initial analytical concentration.

Accordingly, the following EC values for Desmodesmus subspicatus were determined for the test material:

E_yC₁₀(72h) = 86.8 mg/L (95% confidence interval: not determinable)

E_yC₅₀(72h) > 100 mg/L (95% confidence interval: not determinable)

E_rC₁₀(72h) > 100 mg/L (95% confidence interval: not determinable)

E_rC₅₀(72h) > 100 mg/L (95% confidence interval: not determinable)