6H-Furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one, 2,3,3a,9a-tetrahydro-3-hydroxy-2-(hydroxymethyl)-7-methyl-, (2S,3S,3aR,9aS)-

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 May- 26 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal sewage treatment plant: 'Waterschap de Maaskant', '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. The concentration of suspended solids was 6.5 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (59 minutes) and the liquid was decanted for use as inoculum at the amount of 7.5 ml/l of mineral medium
- Pretreatment: no special pretreatment
- Concentration of sludge: 7.5 ml supernatant/l of mineral medium.
- Initial cell/biomass concentration: not determined
- Water filtered: yes. Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges.

Duration of test (contact time):

28 d

Initial conc.:

48 other: mg / 2 L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

other: Total Organic Carbon (TOC)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
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.


- Test temperature: between 21.2 and 23.5°C.
- pH: Just before the start of the test 7.6. On day 28: 7.4 - 7.7
- pH adjusted: no
- Aeration of dilution water: During the test period aerated and stirred continuously.
- Suspended solids concentration: 6.5 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant)
- Continuous darkness: yes



TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration:
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).
- Method used to create aerobic conditions: 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 sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- 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 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. 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) was used as pH-indicator. On the 28th day, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%) 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.



SAMPLING
- Sampling frequency: titrations were made on days: 2, 5, 7, 9, 14, 19, 23, 27 and 29.
- Sampling method: titration of whole volume of CO2-absorber


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance:

acetic acid, sodium salt

Remarks:

40 mg/L

Test performance:

Pre-incubation medium
The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80% of total volume).
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.

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. Three CO,-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH), were connected in series to the exit air line of each test bottle.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

9 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

14 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

27 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

29 d

Details on results:

Theoretical CO2 production
The ThCO2 of Anhydrothymidine was calculated to be 1.83 mg CO2/mg.
The ThCO2 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.
The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of Anhydrothymidine.

Results with reference substance:

In the toxicity control more than 25% degradation occurred within 14 days (32%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Description of key information

The test substance was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater