Betaines, coco alkyldimethyl(3-sulfopropyl)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-05 - 2018-03-08 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

not affecting the outcome or validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBI-TION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

not affecting the outcome or validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Betaines, coco alkyldimethyl(3-sulfopropyl)

EC Number:

269-225-1

EC Name:

Betaines, coco alkyldimethyl(3-sulfopropyl)

Cas Number:

68201-55-8

Molecular formula:

C7H17NO3S·[CH2]6-16

IUPAC Name:

[3-(dodecyldimethylazaniumyl)propyl]trioxo-λ⁶-sulfanuide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

liquid

Remarks:

Aqueous solution: clear, colourless to yellowish liquid

Details on test material:

- Storage: Room Temperature (20 ± 5°C)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a closed vessel dark and dry at room temperature (15.8 - 23.0°C).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.0032, 0.01, 0.032, 0.1, 0.32, 1 mg/l

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution containing 10.0 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with algal medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

20.9 – 24.0 °C

pH:

7.6 - 7.7

Nominal and measured concentrations:

0 / 0.0032 / 0.01 / 0.032 / 0.1 / 0.32 / 1 mg/L (nominal)
< LOQ / < LOQ / 0.00899 / 0.02912 / 0.09492 / 0.30532 / 0.90952 mg/l (measured, t=0h)
< LOQ / < LOQ / < LOQ / < LOQ / < LOQ / 0.21972 / 0.96893 mg/l (measured, t=72h)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 45 ±1 ml fill volume
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 2580 cells/ml
- Control end cells density: 95457 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, every 24h

TEST CONCENTRATIONS
- Test concentrations: The concentrations to be tested are based on non-GLP pre-tests.
In these pre-tests an inhibition of about 60 % at 0.01 mg/L could be observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Remarks:

test item

Effect conc.:

66.68 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence interval: 49.45 – 89.45 µg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

solid content

Effect conc.:

36.67 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Remarks:

test item

Effect conc.:

26.05 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence interval: 20.16 – 33.66 µg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Remarks:

solid content

Effect conc.:

14.33 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

test item

Effect conc.:

9.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

solid content

Effect conc.:

5.17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

test item

Effect conc.:

30 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

solid content

Effect conc.:

16.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC50

Remarks:

test item

Effect conc.:

36.73 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95%-confidence interval: 19.74 – 66.81 µg/L

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Remarks:

solid content

Effect conc.:

20.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC10

Remarks:

test item

Effect conc.:

16.16 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 95%-confidence interval: 9.87 – 26.48 µg/L

Effect concentrations 12

Duration:

72 h

Dose descriptor:

EC10

Remarks:

solid content

Effect conc.:

8.89 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 13

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

test item

Effect conc.:

30 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 14

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

solid content

Effect conc.:

16.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 15

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

test item

Effect conc.:

93.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

aqueous solution

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 16

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

solid content

Effect conc.:

51.65 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any stimulation of growth found in any treatment: lowest test item concentration
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no precipitation noted
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
72h ErC50 = 0.90 mg/L (0.77 – 1.06 mg/L)
72h EyC50 = 0.42 mg/L (0.35 - 0.51 mg/L)

Any other information on results incl. tables

Results Pre-Test: In this pre-test a clear inhibition of algal growth could be observed at 0.01 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or relevant deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test item resp. Sulfobetaines, coco alkyldimethyl(3-sulfopropyl) towards algae.
One valid experiment was performed.
The study was performed using 6 concentrations ranging from 0.0032 to 1 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.
Significant inhibition of algal growth was observed at the following concentrations:
0.1 / 0.32 / 1 mg/L (0.032 mg/L only for growth rate)
At the start and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS.
At the lowest concentrated treatment, the measured concentration was lower than the LOQ. In the other treatments the measured concentrations lay between 90 % and 95 % of the nominal concentrations at the beginning of the test. At the end of the test, test item was detectable only in the two highest concentrated treatments (69 % and 97 % of the nominal concentration).
That means at the end of the test, the measurable test item concentration was biased by the presence of the test organism (most likely adsorption or ingestion onto the algae cells). Test item was present but partly not measurable in the test solutions.
Photolysis of the test item is less likely, as all test item solutions were equally exposed to 5000 lux. Due to the magnitude of the test item concentration, which could not be detected any more, adsorption onto the glass vessel can also be excluded. So adsorption or ingestion onto the algae cells is the most likely explanation, especially considering the fact that in the highest concentrated treatments no algal cells were visible which could have absorbed the test material.
Because of the growing biomass such effects are inevitable and also described in the OECD guideline in chapter 40 on page 6.
The only possibility to verify that the test material is still present in the test system (not in solution but in the cells), would be a lysis of the cells and an analytical determination of the test item in complete test system. However, the concentrations to be determined would be very low, and the organic material would create a background overlying the test item signal in the LC-MS-MS system. So, the analytical verification of the test material in those concentration ranges is not possible in the presence of algal cells, and the following approach has to be followed:
Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor.
The 72h-EC50s of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50s of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.
The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.1 units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.
The 72h ECr50 of the test item was determined to be 66.68 µg/l, which corresponds to a 72h ECr50 = 36.67 µg/L of the registered substance, Sulfobetaines, coco alkyldimethyl(3-sulfopropyl). Based on the obtained results, Sulfobetaines, coco alkyldimethyl(3-sulfopropyl) must be classified as acutely toxic to the environment (limit value for classification as acutely toxic: 1 mg/l) according to Regulation 1272/2008 and amendments. Further, as it is not biodegradable it should be classified as hazardous to the aquatic environment Cat. 1 (chronic), too.

Executive summary:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on an aqueous solution of the registered substance Sulfobetaines, coco alkyldimethyl(3-sulfopropyl).

One valid experiment was performed.

The study was performed using 6 concentrations ranging from 0.0032 to 1 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations:

0.1 / 0.32 / 1 mg/L (0.032 mg/L only for growth rate)

At the start and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS.

At the lowest concentrated treatment, the measured concentration was lower than the LOQ. In the other treatments the measured concentrations lay between 90 % and 95 % of the nominal concentrations at the beginning of the test. At the end of the test, test item was detectable only in the two highest concentrated treatments (69 % and 97 % of the nominal concentration).

That means at the end of the test, the measurable test item concentration was biased by the presence of the test organism (most likely adsorption or ingestion onto the algae cells). Test item was present but partly not measurable in the test solutions.

Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor.

The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Sulfobetaines, coco alkyldimethyl(3-sulfopropyl), aqueous solution were determined:

 

Table Results of the test item and Sulfobetaines, coco alkyldimethyl(3-sulfopropyl)

Endpoint	NOEC	LOEC	EC10	EC50
Test item
Growth Rate	9.40 µg/L	30.00 µg/L	26.05 µg/L	66.68 µg/L
Yield	30.00 µg/L	93.90 µg/L	16.16 µg/L	36.73 µg/L
Sulfobetaines, coco alkyldimethyl(3-sulfopropyl)
Growth Rate	5.17 µg/L	16.50 µg/L	14.33 µg/L	36.67 µg/L
Yield	16.5 µg/L	51.65 µg/L	8.89 µg/L	20.20 µg/L

 

Sulfobetaines, coco alkyldimethyl(3-sulfopropyl) is classified as hazardous to the aquatic environment Cat. 1 (acute and chronic) according to Regulation 1272/2008 and amendments.