4-methylcyclohexanone

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:

2015-11-02 until 2016-03-24

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

storage below 30°C

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): sewage treatment plant
Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany
- Date of sampling: 24th November 2015
- Storage conditions: No storage, left for settlement for ca. 1 h.
- Preparation of inoculum for exposure: Supernatant discarded. Concentration of suspended solids was measured in the remaining sludge.The concentration was adjusted to 3.8 g/L and verified by dry mass measurement.
- Pretreatment: Kept aerobic until use
- Concentration of sludge in the test: 29.6 mg dry mass/litre

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Initial conc.:

53.3 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

-Sodium benzoate was used as a reference item. The concentration in the vessels with reference item (procedural control, toxicity control) was 100 mg per litre mineral test medium (25 mg/250 mL). The solution of the reference substance in the mineral medium equivalent to a concentration of 100 mg/L was prepared using an aqueous stock solution (10 g/L ). The aqueous stock solution was prepared by dissolving 505 mg of the reference substance in 50 mL deionised water. 2.5 mL of the aqueous stock solution were then filled up to 250 mL with inoculated mineral medium.
The test item concentration in the test assays were 20 mg per litre mineral test medium (5 mg/250 mL). 20 mg of test item has a theoretical oxygen demand (ThOD) of 53.3 mg. No emulsifiers or solvents were used.
Due to the unknown water solubility of the test item, the required amount to ensure a final concentration of 20 mg/L (5 mg per 250 mL) was added directly on a weight basis via Teflon discs. Subsequently, the mineral medium was added to the vessels.
A further solution containing both test and reference item at the same concentrations as in the individual solutions (test item 20 mg/L, reference item 100 mg/L) was prepared to determine the possible toxicity of the test item against the inoculum. For this, the required amount of test item was added directly on a weight basis via Teflon discs. Subsequently, the required volume of aqueous reference stock solution and mineral medium were added to the vessels.
Further flasks with mineral medium only were prepared for inoculum controls. Abiotic controls were also prepared to measure any possible abiotic degradation; a solution of the test item at about 20 mg/L was sterilized by the addition of 1 mL/L HgCl2 (10 g/L) .
In all test assays except of the abiotic controls, 1.93 mL of the inoculum stock solution were transferred for inoculation and the pH was measured. Since the start values were within the acceptable range of 7.4 ± 0.2, no adjustment was necessary.

Reference substance:

benzoic acid, sodium salt

Test performance:

The manometric respirometry test was carried out according to the OECD guideline 301 F (1992) and the Council Regulation (EC) No 440/2008. The study was carried out in closed bottles and is acceptable for volatile substances where the oxygen consumption by mineralization is measured. The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls.

Key result

Parameter:

% degradation (O2 consumption)

Value:

72

St. dev.:

4

Sampling time:

28 d

Details on results:

The percent degradation of the test item in the test media in the 10-day-window within the 28 day period was 73 % (SD = 3.3 %) based on ThOD. The 10-day-window started for BOD at day 9 – 10 where biodegradation had reached 10%. The percent degradation of the test item in the test media was found to be 72 % (SD = 4.0 %) based on ThOD at the end of 28 days of incubation. At day 15, a plateau was reached and no further degradation occurred within the 28 days of the test.

Results with reference substance:

The reference item was degraded by an average of 74 % by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 85 %.

See under "Other information on materials and methods incl. tables"

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Since the threshold value of 60 % degradation based on THOD was surpassed in the 10-day-window within the 28-d period, the test item is deemed readily biodegradable under the test conditions. Both toxicity and procedural controls were valid.

Executive summary:

The ready biodegradability of the test substance was investigated at a concentration of 20 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22°C in the dark over 28 days under continuous stirring. The manometric respirometry test was carried out according to the OECD 301 F (1992) and Regulation (EC) No 440/2008 of 30 May 2008. The OECD 301F is carried out in closed bottles. A loss of substance due to volatilization does not consume oxygen and the fact that test substance is readily biodegradable, shows the test design is suitable. The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls.

The average percent biodegradation of the test item in the test media was found to be 73 % (SD = 3.3 %) at day 19 – 20, within a 10-day-window which started at day 9 – 10. At day 9 – 10 biodegradation had reached 10% degradation. Since the threshold value of 60 % was surpassed in the 10-day-window within the 28-d period, the test item is considered readily biodegradable under the test conditions.

In the toxicity control, containing both the test item and the reference item sodium benzoate, biodegradation was greater than 25 %. According to guidelines this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. Oxygen consumption in the toxicity control was similar to the procedural controls containing only sodium benzoate. In the procedural controls, the reference substance sodium benzoate was degraded by an average of 74 % by exposure day 14 and reached an average biodegradation of 85 % by the end of the test (day 28), thus confirming suitability of the activated sludge. The difference of extremes of replicate values of the removal of the test item at the end of the test is less than 20 %. Therefore, the test can be considered as valid.

Description of key information

Ready biodegradability of the test substance was investigated according to the OECD 301 F (1992) at a concentration of 20 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22°C in the dark over 28 days under continuous stirring in closed bottles (Simon, 2016). The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls. The average percent biodegradation of the test item in the test media was found to be 73 % (SD = 3.3 %) at day 19 – 20, within a 10-day-window which started at day 9 – 10. At day 9 – 10 biodegradation had reached 10% degradation. Since the threshold value of 60 % was surpassed in the 10-day-window within the 28-d period, the test item is considered readily biodegradable under the test conditions. The study was valid in the toxicity and procedural controls.

A second study investigating the biodegradation of 94 aromatic, 15 hydroaromatic and 14 aliphatic compounds was reported in the literature (Pitter, 1975). The study was not conducted according to a standard guideline method, and was not performed under GLP, however it was carried out according to generally accepted scientific principles and thus assigned a Klimisch rating of 2. The study found under the test conditions that 4 -methylcyclohexanone was biodegradable, and is consistent with the results of Simon (2016), and as a result is used as supporting information.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable