(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according guideline under GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Remarks:

Statement of compliance

Test material

Test material information

Constituent 1

Reference substance name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

EC Number:

201-210-7

EC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Cas Number:

79-50-5

Molecular formula:

C6H10O3

IUPAC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Details on test material:

- Name of test material (as cited in study report): DL-Lactone
- Physical state: white cristalline mass
- Stability under test conditions: stable
- Storage condition of test material: in refrigerator in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Study design

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, receiving predominantly domestic sewage.
- Laboratory culture: The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.9 g/l in
the concentrated sludge (information obtained from the municipal sewage treatment plant)
The sludge was coarsely sieved and washed twice with tap-water. After washing the sludge was made up to the original volume. A small amount of the
sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids. An amount of the sludge corresponding to approximately
30 mg/l suspended solids was added to the mineral medium. Except for one bottle, an additional adsorption control, in which the amount of the
sludge corresponded to approximately 1000 mg/l suspended solids.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test duration: 28 days (last C02-measurement on the 2gth day). During the test period the test media were aerated and stirred continuously.

Test vessels: 2 litre all-glass brown coloured bottles.

Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ionexchange
cartridges (Milli-Q)

Stock solutions of mineral components: A) 8.50 g KH2P04, 21.75 g K2HP04, 67.20 g Na2HP04.12 H20, 0.50 g NH4Cl dissolved in Milli-Q water and
made up to 1 I, pH 7.4 + 0.2
B) 22.50 g MgS04.7H20 dissolved in Milli-Q water and made up to 1 1.
C) 36.40 g CaCI2.2H20 dissolved in Milli-Q water and made up to 1 1.
D) 0.25 g FeCI3.6H20 dissolved in Milli-Q water and made up to 1 l.

Mineral medium: 1 I mineral medium contains: 10 ml of solution (A), 1 ml of solutions (8) to (D) and Milli-RO water.

Barium hydroxide: 0.01 25 M Ba(OH), stored in a sealed vessel to prevent absorption of C02 from the air.

Synthetic air (C02 < 1 ppm): A mixture of oxygen (21 %) and nitrogen (79%) was passed through a bottle, containing 0.5 - 1 litre
0.0125 M Ba(OH), solution to trap C02 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. 30-1 00 m/lmin).

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

Preliminary study:

Not applicable

Test performance:

On the day of sampling of the sludge a rough indication on the concentration of suspended solids was made. After drying 4 hours at 105°C the
concentration of suspended solids was 4.6 g/l.
Based on this information 14 ml sludge was added per 2 litres of mineral medium to the bottles for the determination of the C02 evolution, resulting
in an amount of the sludge corresponding to approximately 30 mg/l suspended solids.
After drying overnight at 105°C the concentration of suspended solids was 4.3 g/l, which confirmed the concentration of suspended solids in the
bottles for the determination of the CO2 evolution.
A volume of 465 ml sludge was added per 2 litres of mineral medium, in the additional adsorption bottle. This corresponded to approximately
1000 mg/l suspended solids. The heterotrophic microbial colony count of the sludge was determined to be 55 * 10^4 cells/ml.
In the bottles for the determination of the C02 evolution 14 ml sludge was added per 2 litres of mineral medium, thus the test system contained
3.85*10^6 cells/l.
In the additional adsorption bottle 465 ml sludge was added per 2 litres of mineral medium, thus the test system contained 1.28*10^8 cells/l.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

76 - 82

Sampling time:

28 d

Details on results:

The relative degradation values calculated from the measurements performed during the test
period revealed 82 and 76% degradation of dl-Lactone, for the duplicate bottles tested.
Furthermore, more than 60% degradation of dl-Lactone was reached within a 10-day window.
In the toxicity control more than 25% degradation occurred within 14 days (56% based on ThC02).
Thus, dl-Lactone was not inhibitory to microbial activity. The relative degradation values calculated from the measurements performed
during the test period revealed no significant degradation of dl-Lactone in the abiotic control and the adsorption
control.

BOD5 / COD results

Results with reference substance:

The positive control substance was degraded by at least 60% (67%) within 14 days.

Any other information on results incl. tables

Not relevant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The positive control substance was degraded by at least 60% (67%) within 14 days. The difference of duplicate values for %-degradation of dl-Lactone was always less than 20, and the total C02 release in the blank at the end of the test

Interpretation of results:

readily biodegradable

Conclusions:

Dl-Lactone was readily biodegradable under the conditions of the modified Sturm test presently performed.
Furthermore, no significant elimination of dl-Lactone (22 mgll), by abiotic degradation or adsorption by the activated sludge (30 mg/l ss),
was observed. At a higher concentration of dl-Lactone (100 mgll) and activated sludge (1 000 mg/l ss) 18%
adsorption by the activated sludge was observed within 3 hours. Thereafter, no more adsorption of dl-Lactone by the activated sludge was observed.
The DOC measurements in the additional adsorption control, revealed more than 60% degradation (79%) within 14 days, which was in agreement with the biodegradation pattern obtained after C02 determinations.

Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (C02) evolution test (modified Sturm

test) with dl-Lactone.

The theoretical C02 production (ThC02) of dl-Lactone was calculated to be 2.03 mg C02/mg. Dl-Lactone was a white crystalline mass with a purity of 99.6%. Dl-Lactone was tested in duplicate at 43 mg per 2 litres, corresponding to 12 mg TOC/I. The organic carbon content was based on the molecular formula. Furthermore, an additional adsorption control was prepared at 100 mg dl-Lactone/l.

Since dl-Lactone was easily soluble in water the test media were prepared using a stock

solution of 1 g/l in milli-RO water. A weighed amount of 1006.7 mg of dl-Lactone was dissolved

in milli-RO water and made up to 1000 ml. The stock was a clear and colourless solution.

TOC concentration of the stock solution was 542.7 mg/l. This was in the same order of magnitude as the calculated carbon content (55%). Aliquots of 43 ml of the stock solution were added to the test medium, containing the microbial

organisms, of test substance bottles A and B, toxicity control, the abiotic control and the adsorption control.

An aliquot of 200 ml of the stock solution was added to the test medium of the additional adsorption control, resulting in a final dl-Lactone concentration of 100 mg/l. All test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Results of C02 determinations:

The relative degradation values calculated from the measurements performed during the test

period revealed 82 and 76% degradation of dl-Lactone, for the duplicate bottles tested.

Furthermore, more than 60% degradation of dl-Lactone was reached within a 10-day window.

In the toxicity control more than 25% degradation occurred within 14 days (56% based on

ThC02). Thus, dl-Lactone did not inhibit microbial activity.

The relative degradation values calculated from the measurements performed during the test

period revealed no significant degradation of dl-Lactone in the abiotic control and the adsorption control.

Results of DOC analvses:

No significant DOC removal was observed in the abiotic control and the adsorption control (both containing sterilizing agent).

The degradation values calculated from the DOC measurements in the abiotic control and in the

adsorption control, revealed no degradation of dl-Lactone (both containing sterilizing agent).

In the additional adsorption control (dl-Lactone 100 mg/l, inoculum 1000 mg/l ss, no sterilizing

agent) 18% DOC removal was observed after 3 hours. This was the result of adsorption of dl-Lactone by the activated sludge.

Since the DOC concentration of day 1 was approximately the same as after 3 hours, no more adsorption of dl-Lactone by the activated sludge was observed.

The degradation values calculated from the DOC measurements in the additional adsorption

control, revealed more than 60% biodegradation (79%) within 14 days.

This was in agreement with the biodegradation pattern obtained after C02 determinations.

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

In conclusion, dl-Lactone was readily biodegradable under the conditions of the modified Sturm

test presently performed.

Furthermore, no significant elimination of dl-Lactone (22 mg/l), by abiotic degradation or

adsorption by the activated sludge (30 mg/l ss), was observed.

At a higher concentration of dl-Lactone (100 mg/l) and activated sludge (1000 mg/l ss) 18%

adsorption by the activated sludge was observed within 3 hours. Thereafter, no more adsorption

of dl-Lactone by the activated sludge was observed.

The DOC measurements in the additional adsorption control, revealed more than 60%

biodegradation (79%) within 14 days, which was in agreement with the biodegradation pattern obtained after C02 determinations.