Betaines, coco alkyldimethyl(3-sulfopropyl)

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-20 - 2011-09-22 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 3, Degradation and Accumulation, guideline No. 301 B: "Ready Biodegradability: CO2 Evolution Test" adopted July 17, 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.4. "Biodegradation: determination of the 'ready' biodegradability, C.4-C: Carbon dioxide (CO2) evolution test (Modified Sturm Test).

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ISO Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995)

Deviations:

no

GLP compliance:

yes

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

Test material form:

liquid

Remarks:

clear colourless

Details on test material:

Density 1.0256 g/cm³ at 20°C
Solubility in vehicle: Water No, fully miscible
Molecular formula: C17H37NO3S
Molecular weight: 335.54 (average)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: stable

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Laboratory culture, treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.2 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (58 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

39.5 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

The test concentration was 39.5 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the composition and the molecular formula.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
Stock solutions of mineral components
A)8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 20.1 and 21.9°C
- pH: 7.7 (start of test), 7.5 - 7.6 (end of test)
- pH adjusted: no

TEST SYSTEM
Preparation of bottles
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Type and number of bottles:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Preparation At the start of the test (day 0) test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
- Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul), Merck, Darmstadt, Germany).
- Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
- Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.
Measurements and recording
pH: At the start of the test (day 0)and on the 28th day before of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 18 - <= 35

Sampling time:

28 d

Remarks on result:

other: Values are derived from bottles A and B.

Remarks:

The test item was biodegraded significantly (35 and 18%) during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test the test item was not readily biodegradable.

Details on results:

See tables below

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Interpretation of results:

not readily biodegradable

Remarks:

The relative biodegradation values calculated from the measurements performed during the test period revealed 35 and 18% biodegradation of the test item, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

Conclusions:

The study was conducted under GLP according to OECD 301B on the registered substance itself. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, all criteria for acceptability of the test were met, this study was considered to be valid. The test item was biodegraded significantly (35 and 18%) during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test the test item was not readily biodegradable.

Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with the test item under GLP.

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard

9439, 1999 and ISO Standard 10634, 1995.

The test substance consisted of the test item (50.0%) in water (information obtained by the sponsor). The test item was fully miscible in water. Therefore, the test item was added directly to the test media using a pipette. The test substance was tested in duplicate at 39.5 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the composition and the molecular formula. The Theoretical CO2 production (ThCO2) of The test item was calculated to be 1.12 mg CO2/mg. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29^thday).

The study consisted of six bottles:

2 inoculum blanks (no test substance),

2 test bottles (the test item),

1 positive control (sodium acetate) and

1 toxicity control (the test item plus sodium acetate).

The relative biodegradation values calculated from the measurements performed during the test period revealed 35 and 18% biodegradation of the test item, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, the test item was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, the test item was designated as not readily biodegradable.