Reaction mass of (2R,5R)-2-isopropyl-5-methylcyclohexanone and (2S,5R)-2-isopropyl-5-methylcyclohexanone

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 June to 11 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid an conclusive guideline study under GLP

Data source

Reference

Reference Type:

secondary source

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, Bern, 11 January 2011

Test material

Test material information

Constituent 1

Reference substance name:

L-menthan-3-one

EC Number:

237-926-1

EC Name:

L-menthan-3-one

Cas Number:

14073-97-3

Molecular formula:

C10H18O

IUPAC Name:

2-isopropyl-5-methylcyclohexanone, (2S,5R)-

Details on test material:

- Name of test material (as cited in study report): Menthone-L/ Isomenthone-D
- Substance type: Organic chemical, Isoprenoid
- Storage condition of test material: At room temperature at 20 °C ± 5 °C, in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted.
- Concentration of sludge: 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 equivalent to 4 g dry material per litre and stored.
- Storage conditions: During the holding period prior to use, the sludge was aerated at room temperature.
- Storage length: 2 days
- Preparation of inoculum for exposure: Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per litre (dry weight basis).
- Initial cell/biomass concentration: Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per litre.
- Water filtered: No

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

20 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

>= 58 - <= 59 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4 x 2H2O, 0.50 g/L NH4Cl; The pH of this solution was 7.4.
2) 22.50 g/L MgSO4 x 7H2O
3) 36.40 g/L CaCl2 x 2H2O
4) 0.25 g/L FeCl3 x 6H2O, stabilized with one drop of concentrated HCl per litre
To obtain the final test water, 10 mL of stock solution No. 1) and 1 mL each of stock solution nos. 2), 3) and 4) were combined and made up to 1000 mL with purified water. The pH was adjusted from 8.1 to 7.4 with a diluted hydrochloric acid solution.
- Additional substrate: None
- Solubilising agent: No emulsifiers or solvents were used.
- Test temperature: 22 °C, maintained with a built-in thermostat and checked on each working day
- pH: The pH measured in all flasks at the start of the test was found to be 7.4. At the end of exposure (day 28), pH values of 7.5–8.1 were measured.
- pH adjusted: No
- Suspended solids concentration: 30 mg dry material/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: The test flasks (500 mL reaction vessels, labelled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith GmbH, Heidenheim, Germany).
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Electrochemical analysis process: The biodegradation process consumes the dissolved oxygen in the test medium and generates CO2. The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulphate solution.
- Measuring equipment: Oxygen consumption was recorded manually by taking a daily reading at least on each working day.
- Test performed in closed vessels due to significant volatility of test substance: Yes, as required by the guideline
- Details of trap for CO2: As required by the guideline (soda lime)

SAMPLING: Not required according to the guideline as the oxygen consumption is followed by reading of the instruments displaying the quantity of electrochemically resupplied oxygen

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Abiotic sterile control: Not employed
- Toxicity control: 1 replicate (20 mg/L test item corresponding 59 mg ThOD/L plus 100 mg/L reference substance corresponding to 167 mg ThOD/L)
- Other: The reference item sodium benzoate was tested simultaneously under the same conditions as the test item and functioned as a procedure control. A stock solution containing 2.5 g sodium benzoate per litre test water was prepared by completely dissolving 250 mg sodium benzoate in 100 mL test water. From this stock solution, 10 mL aliquots were added to the corresponding test flasks containing test water.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

99.7 % purity, Sigma-Aldrich product no. 71295, Lot no. BCBB4145, expiration date Nov-2015, used at 100 mg/L corresponding to 167 mg ThOD/L

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

52

Sampling time:

28 d

Remarks on result:

other: Replicates 1 and 2

Details on results:

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand (ThOD) of 2.9 mg oxygen/mg test item. The biochemical oxygen demand (BOD) of the test item in the test media significantly increased from exposure day 2 to day 13, corresponding to a mean biodegradation from 0 % to 50 %, respectively. Thereafter the biodegradation increased marginally until test termination. At the end of the 28-day exposure period, the mean biodegradation of the test item amounted to 52 % of the ThOD.

BOD5 / COD results

Results with reference substance:

The results are presented in Table 1 and Table 2 and in the attached Figure. The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg oxygen/mg (ThOD). In the procedure controls, the reference item sodium benzoate was degraded by an average of 89 % 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 93 %.

Any other information on results incl. tables

Table 1: Oxygen Consumption in the Test Flasks

Tim [d]	Cumulative oxygen consumption [mg/L]
	Test item		Inoculum control		Procedure control		Toxicity control
	Replicate No.		Replicate No.		Replicate No.		Replicate No.
	1	2	1	2	1	2	1
0	0	0	0	0	0	0	0
1	1	3	2	4	14	16	17
2	3	4	2	5	91	93	96
3	--	--	--	--	--	--	--
4	--	--	--	--	--	--	--
5	15	11	7	10	134	137	143
6	30	15	7	11	141	143	151
7	32	28	9	12	146	149	165
8	35	32	11	14	151	153	174
9	37	35	11	15	153	156	179
10	--	--	--	--	--	--	--
11	--	--	--	--	--	--	--
12	44	42	13	16	161	164	188
13	47	44	13	20	163	167	191
14	47	45	15	20	164	168	191
15	47	46	15	20	165	170	191
16	48	47	15	20	166	172	192
17	--	--	--	--	--	--	--
18	--	--	--	--	--	--	--
19	49	48	15	21	168	175	193
20	50	49	15	22	169	177	195
21	50	49	15	23	169	178	195
22	51	50	15	24	171	179	197
23	51	51	15	25	171	180	197
24	--	--	--	--	--	--	--
25	--	--	--	--	--	--	--
26	51	51	15	26	171	180	199
27	52	52	15	28	172	181	201
28	52	52	15	28	172	181	201

-- No reading taken

Table 2: Biodegradation in the Test Flasks

Time [d]	Percentage Biodegradation¹
	Test item		Procedure control		Toxicity control
	Replicate No.		Replicate No.		Replicate No.
	1	2	1	2	1
0	0	0	0	0	0
1	-3*	0	7	8	6
2	-1*	1	52	54	41
3	--	--	--	--	--
4	--	--	--	--	--
5	11	4	75	77	60
6	36	10	79	80	63
7	37	30	81	83	68
8	38	33	83	84	72
9	41	37	84	86	74
10	--	--	--	--	--
11	--	--	--	--	--
12	50	47	88	90	77
13	52	47	88	90	77
14	50	47	88	90	77
15	50	48	88	91	77
16	52	50	89	93	77
17	--	--	--	--	--
18	--	--	--	--	--
19	53	51	90	94	77
20	54	52	90	95	78
21	53	51	90	95	78
22	54	52	91	96	79
23	53	53	90	96	78
24	--	--	--	--	--
25	--	--	--	--	--
26	52	52	90	96	79
27	52	52	90	96	79
28	52	52	90	96	79
Mean (day 28)	52		93		not applicable

¹ Corrected for the mean oxygen uptake of the inoculum controls

-- Not determined

* Negative value due to higher oxygen consumption in the inoculum controls than in the test flasks with test item.

Validity of the Test

The results are considered valid since the following criteria are met:

The mean oxygen demand of the inoculated mineral medium (inoculum control) was 22 mg oxygen/L within 28 days (criterion: normally 20–30 mg oxygen/L but not greater than 60 mg oxygen/L). No difference between the degradation rates of the replicate test vessels containing the test item, test water and inoculum was observed at the end of the test (criterion: not more than 20 % deviation at the time the plateau is reached, at the end of the test or at the end of the 10-day window, as appropriate). The percentage degradation of the reference item reached 76 % on day 5 (criterion: at least 60 % of ThOD in a 10-day window by day 14). The pH of all inoculated test vessels was in the range 7.5–8.1 at the end of the exposure period of 28 days (criterion: pH inside the range 6.0–8.5 after 28 days).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Based on oxygen consumption 50 % biodegradation in a 10-day and total 52 % within 28 days, substance to be considered as inherently biodegradable.

Executive summary:

The biodegradation of the test item, consisting of the naturally occurring menthone isomers in a composition of 84.5 % L-menthan-3-one (CAS 14073-97-3) and 15.1 % D-Isomenthone (CAS 1196-31-2), was investigated in a valid, GLP-compliant study using the manometric respirometry method according to the OECD TG 301 F (1992).

The test micro-organism inoculum consisted of non-adapted activated sludge from a municipal sewage water treatment plant. The parameter followed in duplicate for biodegradation estimation was the oxygen consumption measured as oxygen consumption in the test vessels. Readings were made on every working day. The percent biodegradation of the test item in the firmly closed test vessels was calculated based on the Theoretical Oxygen Demand (ThOD) of 2.9 mg oxygen/mg test item.

During the 28 days of incubation at 22 °C the Biochemical Oxygen Demand (BOD) of the test item in the test media significantly increased from exposure day 2 to day 13, corresponding to a mean biodegradation from 0 % to 50 %, respectively. Thereafter the biodegradation increased marginally until test termination. At the end of the 28 day exposure period, the mean biodegradation of the test item amounted to 52 % of the ThOD. The 10-day window was started on day 5 or 6 and ended on day 15 or 16 in the replicates 1 and 2, respectively, when 50 % biodegradation was recorded. Consequently, the test item was found to be biodegradable under the test conditions within 28 days. However, the pass level for ready biodegradability, i.e. biodegradation of at least 60 % of the ThOD in a 10-day window within the 28 day period of the test, was not reached, the substance therefore is to be considered as inherently biodegradable.