3-ethoxyandrosta-3,5-dien-17-one

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:

21 March 2016 - 25 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1999

Deviations:

no

Test guideline 4

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:

3-ethoxyandrosta-3,5-dien-17-one

EC Number:

619-264-9

Cas Number:

972-46-3

Molecular formula:

C21H30O2

IUPAC Name:

3-ethoxyandrosta-3,5-dien-17-one

Test material form:

solid: particulate/powder

Details on test material:

Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)

Specific details on test material used for the study:

Solubility in water: not available
Stability in water: not available

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Storage length: not indicated
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (46 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: 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.
- Concentration of sludge: 3.7 g suspended solids per litre in the concentrated sludge
- Water used: tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Duration of test (contact time):

28 d

Initial test substance concentration

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: mineral medium according to OECD 301 guideline
- Test temperature: 22 - 23°C.
- pH: start: 7.6; end: 7.6-8.0
- pH adjusted: no
- Aeration of dilution water: continuously
- Suspended solids concentration: 10 mL supernatant liquid/L (see 'Details on inoculum' section for details)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with synthetic air (CO2 < 1 ppm; at a rate of approx. 30-100 mL)
- Measuring equipment: 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). Phenolphthalein (1% solution in ethanol) was used as pH-indicator.
- Details of trap for CO2 and volatile organics if used: 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.The CO2 produced in each bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. 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.

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 suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Positive control: yes, 1 replicate
- Toxicity control: yes, 1 replicate

STATISTICAL METHODS: none

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.6%

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

7

Sampling time:

28 d

Remarks on result:

other: Result is based on the mean value of % degradation seen in duplicate bottles tested (10% and 4%).

Details on results:

ThCO2 of the test item was calculated to be 2.92 mg CO2/mg. ThCO2 of the reference item (sodium acetate) was calculated to be 1.07 mg CO2/mg.
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of Diona-R. (10% and 4%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (28%, 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 (see attached illustration).
For tables containing full results, see attached background material (Tables.jpg).

BOD5 / COD results

Results with reference substance:

74% biodegradation after 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria, see 'Overall remarks' section.

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of the modified Sturm test, acoording to OECD 301B guidelines, the test item was found to biodegrade for 10% and 4% after 28 days of incubation, respectively in the two replicates tested. Therefore, the test substance is not considered to be readily biodegradable.

Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and GLP principles, the test item did not reach the pass level of 60% for ready biodegradability, neither within the 10 -d window nor after 28 days of incubation. Therefore, it is designated as not readily biodegradable. All the criteria for acceptability of the test were met, so the study is considered valid without restrictions.