(4R,6R)-4-Hydroxy-2,2,6-trimethylcyclohexanone / 60046-50-6

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:

29 November 1999 to 28 January 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study followed the OECD Guideline for Testing of Chemicals No. 301 D, 1992, and the "Closed Bottle Test" according to Commission Directive 92/69/EEC, Part C.4-E.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Department of the Interior

Test material

Test material information

Constituent 1

Reference substance name:

(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexan-1-one

EC Number:

700-052-0

Cas Number:

60046-50-6

Molecular formula:

C9H16O2

IUPAC Name:

(4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexan-1-one

Details on test material:

- Name of test material (as cited in study report): Actinol
- Physical state: White solid
- Analytical purity: 99.5 %
- Lot/batch No.: 410016
- Expiration date of the lot/batch: 31 July 2000
- Stability under test conditions: max 5 days
- Storage condition of test material: At room temperature, protected from sunlight
- Other:

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 49.3 °C
- Boiling point:
- Vapour pressure:
- Henry's law constant (for volatie substances):
- Water solubility (under test conditions): 66.6 g/L +/- 0.9 g/L
- Solubility in organic solvents:
- log Pow: 1.1
- pKa:
- Base or acid catalysis of test material:
- UV absorption:
- Stability of test material at room temperature:
- pH dependance on stability:


OTHER PROPERTIES (if relevant for this endpoint)
- Adsorption characteristics:
- Toxicity to microorganisms:
- Other:

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

The study was performed with aerobic activated sludge from a waste water treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic waste water. The sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material resuspended in tap water. This procedure was repeated twice. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration of 4 g dry material per liter (+/- 10 %). During holding, the sludge was aerated at room temperature in the dark until use. An exact volume of 0.7 mL sludge (filtered through cotton wool) was added per 1000 mL final test medium.
The test medium was prepared from the following stock solutions:

a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x H2O, 0.5 g NH4Cl dissolved in purified water and made up to 1000 mL with purified water. The pH was 7.4.
b) 22.5 g MgSO4 x 7 H2O dissolved in purified water and made up to 1000 mL with purified water.
c) 36.4 g CaCl2 x 2 H2O dissolved in purified water and made up to 1000 mL with purified water.
d) 0.25 g FeCl3 x 6 H2O dissolved in purified water and made up to 1000 mL with purified water. In order to avoid preparation of this solution immediately before use, one drop of concentrated HCl per liter was added.

1 mL each of stock solutions a-d) was combined and filled to a final volume of 1000 mL with purified water. The test medium was aerated for twenty minutes and allowed to stand for twenty hours at the test temperature (22 +/- 2 °C). The dissolve oxygen concentration was 8.8 mg/L.

Duration of test (contact time):

35 d

Initial test substance concentration

Initial conc.:

2 mg/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The test concentrations were as shown in Table 1. Wide-necked reagent bottles (with conical shoulder) were cleaned with 5-10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per liter of 1 % w/v sulfuric acid) by shaking well to coat the bottle walls. After following to stand for 15 minutes, the wash liquid was removed and the bottles were thoroughly rinsed with tap water and purified water.

The bottles were completely and bubble-free filled with the test solutions and tightly stoppered. The number of flasks was 16 (test substance), 16 (procedure control), 16 (inoculum control) and 8 (toxicity control).

The test duration was 35 days since the biodegradation curve of the test substance showed that biodegradation had started but the plateau had not been reached.

The test temperature was 20-21 °C, the temperature was measured on each day of sampling in an additional vessel filled with water and incubated under the same conditions as the test flasks.

The pH measured at the start of the test was 7.3.

The oxygen concentrations ranged from 8.4 to 8.7 mg/L at the beginning of the test.

The test flasks were incubated in the dark.

The oxygen concentration was measured in an airtight system with O2-electrode (under constant stirring at about 500 rpm) using a WTW OXI 539 meter (Wissenschaftlich Technische Werkstätten, Weilheim/Germany). Sampling dates were exposure days 0,2,5,7,9,14,21,28,35 for test substance and inoculum control, days 0,2,5,7,9,14,21,28 for procedure control, and days 0,7,14,21,28 for toxicity control. Measurements were performed in duplicate for test substance, procedure and inoculum controls. Toxicity controls were measured in duplicate on days 0,14 and once on days 7,21.

Reference substance

Reference substance:

other: sodium benzoate supplied by Fluka (BioChemika), Analysis no. 343188/1 20297, 99.5 % purity, expiration date September 2000

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

46

Sampling time:

35 d

Details on results:

The BOD levels of the test flasks containing Actinol increased during the exposure time from 0.017 to 1.121 mg O2/mg test substance. Biodegradation of Actinol started between days 21 and 28. After 28 days of incubation, biodegradation reached 25 % and further increased to 46 % at the end of the test at day 35. Therefore, the test substance can be considered as biodegradable under the present test conditions. However, the pass level for ready biodegradability, i.e., a BOD of at least 60 % of the ThOD in a 10-day window within the 28-day period of the test, was not reached.

BOD5 / COD results

Results with reference substance:

In the procedure controls, biodegradation of the reference substance sodium benzoate reached a plateau of 78-80 % on Day 9, thus confirming the suitability of the used activated sludge inoculum. Biodegradation in the toxicity control containing both, the test and the reference substances, reached on average 40 % within 14 days and 63 % after 28 days of incubation. The pronounced increase of the biodegradation rate between day 21 and 28 of the test was obviously due to the start of Actinol degradation. Thus, according to the test guidelines, Actinol can be assumed to be not inhibitory on activated sludge microorganisms under the present test conditions because degradation was clearly above 25 % within 14 days.

Any other information on results incl. tables

no overall remarks

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test substance can be considered as biodegradable under the present test conditions. However, the pass level for ready biodegradability, i.e., a BOD of at least 60 % of the ThOD in a 10-day window within the 28-day period of the test, was not reached.

Executive summary:

The test substance Actinol was investigated for its ready biodegradability in a "Closed Bottle Test" according to the Commission Directive 92/69/EEC, Part C.4 -E, 1992, and the OECD Guideline for Testing of Chemicals No. 301 D, 1992. In acordance with the testing guildelines the test was prolonged beyond 28 days since the biodegradation curve of the test substance showed that biodegradation had started but the plateau had not been reached at Day 28. The closed bottle test design was chosen since it could not be ruled out that the test substance is volatile.

Under the present test conditions, biodegradation of Actinol started between Days 21 and 28. After 28 days of incubation, biodegradation reached 25 % and further increased to 46 % at the end of the test (Day 35). The test substance can be considered as biodegradable under the present conditions. However, the pass level for ready biodegradability, i.e., a BOD of at least 60 % of the ThOD in a 10 -day window within the 28 -day period of the test, was not reached.

In the procedure controls, biodegradation of the reference material sodium benzoate reached a plateau of 78 -80 % on Day 9, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control, containing both, Actinol and sodium benzoate at about the same concentrations used in the test substance flask and the procedure controls, the biodegradation rate reached on average 40 % within 14 days and 63 % after 28 days of exposure. Thus, according to the guidelines Actinol can be assumed to be not inhibitory to activated sludge microorganisms under the present test conditions.