Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline compliant GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
6.25, 12.5, 25, 50 and 100 mg test item/L

- Sampling method:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates were pooled. All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions. The concentrations of the test item Sulfosuccinic (FA C12-18)E, 2Na were determined in the duplicate test medium samples from nominal test concentrations of 6.25 to 100 mg/L. From the control samples, one of the duplicate samples was analyzed per sampling time.

- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 150.28 mg of the test item completely in 1500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 10 minutes at room temperature. The test medium of the highest test concentration was used in a series of dilution with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Controls: dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. A slight foaming at beginning of the test was observed. All test media were clear solutions throughout the test period. The test item was stable in the test media over the test period of 72 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT
- Strain:Strain No. 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The water temperature during the test was maintained at 22 °C.

pH:

The pH raised in the control from 7.5 at test start to 8.8 at test end fulfilling the requirement of the OECD guideline, the pH of the control medium should not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

nominal: control and 6.25, 12.5, 25, 50 and 100 mg test item/L. The measured concentrations of Sulfosuccinic (FA C12-18)E, 2Na in the test medium of the test concentrations of 6.25 to 100 mg/L were between 92 and 108% of the nominal values at the start of the test and between 98 and 104% at the end of the test. Thus, the correct dosing of the test item Sulfosuccinic (FA C12-18)E, 2Na was confirmed. The test item was stable in the test medium over the test period of 72 hours. The biological results were related to the nominal concentration of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution.
- Aeration: no
- Initial cells density: 5000 cells/mL
- Control end cells density: not provided
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: start and end of test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: approximately 6700 Lux (range: 6280 to 7180 Lux, measured at nine places in the experimental area)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: The selection of the test concentrations was based on the results of a range-finding test and on results of pre-experiment to determine the solubility of the test item.
- Results used to determine the conditions for the definitive study: not provided

Reference substance (positive control):

yes

Remarks:

performed about every half year

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 12.5 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells:no
- Other:No remarkable observations were made concerning the appearance of the test media. A slight foaming at beginning of the test was observed. All test media were clear solutions throughout the test period.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, measured concentrations within 80-120% of nominal
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.69 mg test item/L (Harlan Laboratories Study D64300), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.64-1.1 mg test item/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression.

For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t test.

In the control, the biomass increased by a factor of 55 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates during 72 hours was 33%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.5%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Executive summary:

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity of Sulfosuccinic (FA C12-18)E, 2Na to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3. The nominal concentrations of the test item of 6.25, 12.5, 25, 50 and 100  mg test item/L were tested in parallel with a control. Dose verification analysis was performed . The measured concentrations of the test item were between 92 and 108 of the nominal values at the start of the test and between 98 and 1ß4% at the end of the test. therefore, the biological results were based on nominal concentrations. In order to determine the growth of the cultures, the cultures were measured with the fluorescent method . The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 10 and 60 mg test item/L based on nominal concentrations.

This information is considered to be relevant and reliable for the further risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

60 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity of Sulfosuccinic (FA C12-18)E, 2Na to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3. The nominal concentrations of the test item of6.25, 12.5, 25, 50 and 100  mg test item/L were tested in parallel with a control. Dose verification analysis was performed . The measured concentrations of the test item were between 92 and 108 of the nominal values at the start of the test and between 98 and 1ß4% at the end of the test. therefore, the biological results were based on nominal concentrations. In order to determine the growth of the cultures, the cultures were measured with the fluorescent method . The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 10 and 60 mg test item/L based on nominal concentrations.

This information is considered to be relevant and reliable for the further risk assessment.