[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

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:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600.000 inhabitant equivalents. Sampling date of activated sludge was 30 November 2021. The dry solids (d.s.) content of the activated sludge was 4.7 g/L. It was determined by weight measurements after drying at 105°C for 4 hours. (mean of triplicate measurements).

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

expressed as ThOD

Details on study design:

The Manometric Respirometry Test determines the ready biodegradability of test items following their oxygen consumption in closed flasks at a constant temperature (±1°C) in a range of 20 – 24°C. The consumption of oxygen is determined by measuring the negative pressure in the flasks after absorption of the evolved carbon dioxide in sodium hydroxide. A solution or suspension of the test item in a mineral medium, corresponding to 100 mg/L test item giving at least 50 – 100 mg/L of theoretical oxygen demand (ThOD), is inoculated with activated sludge (30 mg d.s./L) for 28 days. The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of the ThOD or the chemical oxygen demand (COD). The pass level for ready biodegradability is 60% of ThOD and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%.
The test method is suitable for water soluble and non-soluble test items that are not toxic under the test conditions.
SOLUTIONS
1.Mineral medium:
A: Potassium dihydrogenphosphate KH2PO4 8.50 g
Dipotassium hydrogenphosphate K2HPO4 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
Ammonium chloride NH4Cl 0.50 g
are dissolved in demineralised water and made up to 1 litre. The pH of the solution should be 7.4.
B: Calcium chloride dihydrate CaCl2 * 2 H2O 36.4 g
is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7 H2O 22.5 g
is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6 H2O 0.25 g
is dissolved in demineralised water, made up to 1 litre and stabilised with one drop of concentrated HCl.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre. Before use, the mineral medium is aerated for at least half an hour and the pH is, if necessary, adjusted to 7.4 ± 0.2.
The mineral medium used for this test was prepared by diluting 50 mL of solution (A) and 5 mL each of solutions (B), (C) and (D) with demineralised water to a total volume of 5 L. The medium was aerated for 35 min. at 29.8 – 22.0°C. The pH was 7.8 and was therefore adjusted to 7.5.

2. Blank
164 mL of the mineral medium with a pH of 7.5 was added into the blank vessels.

3. Test item
Due to the insolubility and for a better distribution of the test item, silicone oil was applied at a concentration of 1% of the final flask volume to improve the bioavailability of the mixture constituents.
A stock solution of 3.645 g/L was prepared with silicone oil. For this purpose, 72.9 mg test item were dissolved in about 9 mL silicone oil in a volumetric flask and sonicated for 30 minutes in an ultrasonic bath (ultrasonic power 100%, without tempering). The volume was then filled up to 20 mL with silicone oil.
1.64 mL of this stock solution and 162.36 mL mineral medium with a pH of 7.5 were added to each test vessel and sonicated for 30 minutes at the above settings. This corresponds to a concentration of 100.2 mg/L ThOD and a silicone oil concentration of 1%.

4.Reference substance
A stock solution of 128.4 mg/L sodium acetate in mineral medium was prepared. 164 mL of this stock solution were added into the reference vessels corresponding to a concentration of 100.2 mg ThOD/L.
The pH was 7.5 and was not adjusted.

5. Toxicity control
1.64 mL of the test item stock solution in silicone oil (3.645 g/L) and 162.36 mL mineral medium were added to the toxicity control vessel and sonicated for 30 minutes as described above. Afterwards, 21.0 mg sodium acetate were added (corresponding to a total concentration of 200.1 mg/L ThOD and 1% silicone oil).

6. Silicone oil control
1.64 mL silicone oil and 162.36 mL mineral medium were added to each silicone oil control vessel and sonicated for 30 minutes (corresponding to a silicone oil concentration of 1%.)

EXPERIMENTAL SET-UP
The systems OxiTop® (WTW / Xylem) and Sensomat (AQUALYTIC®) were used as test systems. They consist of 500 mL narrow-necked glass bottles with rubber quiver inserts for NaOH pellets, which absorb the carbon dioxide produced. The liquid volume was fixed as 164 mL each. Magnetic stir bars were introduced into the bottles which were positioned on magnetic stirrers. The bottles were sealed tightly with the measuring heads with a measuring range of 500 – 1350 hPa. Every 112 minutes the current pressure was measured and stored by each measuring head. At the end of the experiment pressure data were read out via an infrared interface to the controller unit and afterwards data were transferred via a RS232 interface using the Achat OC software of WTW to an Excel file where further data processing was carried out.

PROCEDURE
In total three bottles containing the test item, three bottles containing only inoculum (blank), three bottles containing the reference compound (reference) two bottles containing only silicone oil (silicone oil control) and one bottle containing reference compound and test item (toxicity control) were set up.
The blank, test, reference and toxicity control vessels were filled . After tempering the vessels to the incubation temperature for 2 h 20 min at 23.5 – 23.6°C, 1.045 mL of the inoculum were added into each vessel. Afterwards, the rubber quivers were inserted in the vessels, one sodium hydroxide pellet was added to each quiver, the vessels were sealed tightly with the measuring heads and the test was started. On day 28, the data were read out and the pH in the vessels was measured on the next day.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

83.8

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

10 d

Details on results:

The degradation extent of the test item at the end of the test was 83.8% of ThOD (28 d, mean of three replicates). A degradation value of >60% was reached within a 10-d window.
The test item reached the criteria for ready biodegradability (60% of ThOD within 28 days and within a 10-d window).
The pH in the test vessels at the end of the test was 7.3.

The degradation in the toxicity control on day 14 was >25%. Thus, the test item had no toxic effect on the inoculum according to the validity criteria of OECD 301.
The pH in the toxicity control vessel at the end of the test was 8.3.

The oxygen consumption of the blanks after 28 days was 26.7 mg/L (mean of three replicates).
The pH in the blank vessels at the end of the test was 7.1.

The oxygen consumption of the silicone oil control after 28 days was 26.7 mg/L (mean of three replicates). The oxygen consumption was the same as in the blank vessels, indicating that the silicone oil had no effect on the inoculum and was not degradable.
The pH in the silicone oil control vessels at the end of the test was 7.3 – 7.4.

The temperature was 21.6 – 23.6°C throughout the whole study.

Key result

Parameter:

ThOD

Value:

83.8 other: %

Results with reference substance:

The reference compound sodium acetate reached the pass level of 60% ThOD within 4 days.
The pH in the reference vessels at the end of the test was 8.5.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item reached the criteria for ready biodegradability (60% of ThOD within 28 days and within a 10-d window).

Executive summary:

The test item reached the criteria for ready biodegradability (60% ofThODwithin 28 days and within a 10-d window).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater