Sodium 1,4-dicyclohexyl sulphonatosuccinate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protect against frost, protect against direct sunlight (at Biochem agrar: at about 20 °C, dark and dry)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no pre-treatment reported

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 56.4, 112.9, 225.8, 451.5, 903.0 mg active matter/L
- Sampling method: Samples of the test solution were taken from the bulk solution at the test start and spent test solutions were sampled as composite specimens per treatment group. Specimens were stabilised with methanol (v:v, 1:1) and were stored deep frozen (≤ -18°C) until they were analysed. Concentrations of the test item in test solutions at test start (0 h) and at test end (72 hours) were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by adding the test item to test medium.
The stock solution was stirred for 5 minutes on a magnetic stirrer. The highest test concentration was prepared by solution of the test item directly in test medium.
Stock solution and test vessels were prepared in the following way:
- a stock solution (stock A) was prepared by weighing 1500.0 mg test item into a graduated flask and bringing to a volume of 500 mL (= 3000.0 mg/L test item)
- 0.24 mL algal inoculum (measured 208.5 x 104 cells/mL in the stock culture) were added to a 100 mL test volume to result in an initial biomass of 5 x 103 cells/mL

- Controls: blank control

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none

- Test concentration separation factor: 2

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus HILSE
- Strain: 86.81 SAG
- Source (laboratory, culture collection): purchased from MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
- Age of inoculum (at test initiation): The algae were taken from the log phase of the culture
- Method of cultivation: Stock cultures used in the test grew in culturing vessels prior to being used in the test for 4 days. After determination of the initial biomass, the stock culture was kept in the dark for a maximum of 0.5 hours. They contained an initial biomass of 5000 Desmodesmus cells per millilitre of test solution.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.7 – 23.2°C.

pH:

7.44 to 8.22

Nominal and measured concentrations:

Nominal: 0 (control), 56.4, 112.9, 225.8, 451.5, 903.0 mg active matter/L
Measured concentrations of the test item in test solutions were within a range of 96 to 99% of nominal values at test start and after 72 hours the concentrations ranged from 99 to 102% in spent test solutions.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask with air-permeable stoppers
- Type (delete if not applicable): air-permeable stoppers
- Material, size, headspace, fill volume: 100 mL test volume
- Renewal rate of test solution (frequency/flow rate): static
- Initial cells density: 5000 cells/mL test solution in each replicate
- Control end cells density:324000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): none

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: AAP test medium (according to OECD 201)


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: continuous cold white fluorescent light; light intensity was measured once before test start: light intensity: on average 75 µE·m-2·s-1 measured at 400-700 nm differences from the selected light intensity over the test area did not exceed the range ± 15%


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 24, 48 and 72 hours after test start, the biomass (number of cells per millilitre) in all test vessels including control was determined by direct counting (actual microscopic cell count using a Neubauer counting chamber).


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable


CULTURING APPARATUS
-Details on culturing apparatus used: Stock cultures used in the test grew in culturing vessels prior to being used in the test for 4 days. After determination of the initial biomass, the stock culture was kept in the dark for a maximum of 0.5 hours. They contained an initial biomass of 5 x 103 Desmodesmus cells per millilitre of test solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 903 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 903 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Other:
- Any stimulation of growth found in any treatment: yes, 112.9 and 225.8 mg active matter/L
- 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):

- Results with reference substance valid? yes
- EC50: 1.11 mg/L

Reported statistics and error estimates:

A non-regression analysis was performed using individual replicate responses, not treatment group means.
From average specific growth rates and yield, recorded in a series of test solutions, effect concentrations of ErC10, ErC20 and ErC50, (average specific growth rate) and EyC10, EyC20 and EyC50 (yield) was determined using concentrations-response modelling (estimated parameters of the 3-param. normal CDF = cumulative distribution function). To determine a LOEC and to derive a NOEC for effects on growth rate, it was necessary to compare treatment means using analysis of variance (ANOVA) techniques. Shapiro-Wilk´s Test on Normal Distribution was performed. The mean for each concentration was compared with control means using an appropriate multiple comparison test method. Dunnett`s Multiple t-test was used, if analysis of contrasts did not reveal a linear trend. Williams’s t-test was used if variance-homogeneity requirements were fulfilled. As a test for homogeneity of variances, Levene’s test was used.

Statistical analysis was performed using the software ToxRat Professional (Version 3.3, 20.10.2018).

Measured concentrations of the test item in test solutions were within a range of 96 to 99% of nominal values at test start and after 72 hours the concentrations ranged from 99 to 102% in spent test solutions.

Validity criteria fulfilled:

yes

Conclusions:

72h-ErC10 > 903 mg active matter/L
72h-ErC50 > 903 mg active matter/L

Executive summary:

In the study from Warning (2021) the toxicity of an aqueous solution of dicyclohexyl sodium sulfosuccinate (CAS 23386- 52- 9) on Desmodesmus subspicatus was determined in an 72 - hour algal growth inhibition test according to OECD 201. The tested concentrations ranged from 56.4 to 903 mg active matter/L. Measured concentrations of the test item in test solutions were within a range of 96 to 99% of nominal values at test start and after 72 hours the concentrations ranged from 99 to 102% in spent test solutions. The ErC10 and ErC50 were > 903 mg active matter, i.e., higher than the highest tested concentration.

The results are regarded as relevant and reliable for the risk assessment.

Description of key information

ErC50 = > 903 mg a.i./L

Key value for chemical safety assessment

EC50 for freshwater algae:

903 mg/L

EC10 or NOEC for freshwater algae:

903 mg/L

Additional information

For this endpoint there are two studies available.

The Klimisch 1 study from Warning (2021) resulted in an ErC10 and ErC50 of > 903 mg active matter/L

The supporting Klimisch 2 study from Targia (1993) did not show a dose response relationship. Due to the lack of inhibitory effects, no NOEC was defined. Hence, based on the obtained data, the 72 -hour and 96-hour ErC50 is > 960 mg a.i./L. The study from Targia (1993) had a slidely lower control growth rate than requested by OECD 201 (2011). Therefore, this study was considered to be supportive.

Both studies demonstrate that the registered substance is not toxic to freshwater algae.