Cholest-5-en-3-β-yl acetate

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:

16 Jan 2018 - 15 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International 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"

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cholest-5-en-3-β-yl acetate

EC Number:

210-066-4

EC Name:

Cholest-5-en-3-β-yl acetate

Cas Number:

604-35-3

Molecular formula:

C29H48O2

IUPAC Name:

(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-yl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White crystalline powder
- Storage condition: At room temperature

Specific details on test material used for the study:

Solubility in water: Insoluble

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant "Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L.
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 15 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: According to OECD 301 Guideline.
- Test temperature: 22-23°C
- pH: 7.6 (at start, after adjustment); 7.7 (at day 14); 7.5-7.6 (at day 28)
- pH adjusted: yes, at start of test.
- Continuous darkness: yes, the test media were excluded from light and the test vessels were made of brown coloured glass.

TEST SYSTEM
- Culturing apparatus: 2 L brown coloured glass bottles
- Number of culture flasks/concentration: 2 for test suspensions, 2 for inoculum blank, 1 for positive control, 1 for toxicity control.
- Method used to create aerobic conditions: continuous aeration with CO2 free synthetic air. A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- CO2 trap: 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.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: titration of Ba(OH)2 in the CO2-absorber bottles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum
- Positive control: containing reference item and inoculum
- Toxicity control: containing test item, reference item and inoculum

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

45

Sampling time:

28 d

Remarks on result:

other: Mean of 2 test bottles

Details on results:

ThCO2 of the test substance was calculated to be 2.98 mg CO2/mg.

The relative biodegradation values calculated from the measurements performed during the test period revealed 40% and 50% biodegradation of Cholesteryl acetate (based on ThCO2), 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, more than 25% biodegradation occurred within 14 days (61%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Between nominal day 24 and 25 there was a period without aeration because of clogged tubing. This was considered by the Test Facility to have no impact on the outcome and the overall integrity of the study, or the interpretation of the study results and conclusions.

BOD5 / COD results

Results with reference substance:

Sodium acetate was biodegraded by 90% within 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Interpretation of results:

not readily biodegradable

Conclusions:

Cholesteryl acetate was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The 'ready' biodegradability of Cholesteryl acetate was investigated in the Carbon Dioxide (CO2) Evolution Test (Modified Sturm Test), according to OECD Guideline 301 B and GLP principles. A test concentration of ca. 15 mg/L test item, corresponding to 12 mg TOC/L, was used. Based on the results, biodegradation of the test substance was 40% and 50% in two replicates after 28 days. The pass criterion for ready biodegradation (60% biodegradation within a 10 -day window) was not met. Therefore, Cholesteryl acetate is considered not readily biodegradable.

The test met all criteria for acceptability, and thus is considered reliable without restrictions.