Cyclohexane-1,3-dione

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:

The study was conducted between 1 Feb 2008 and 29 Feb 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21-08-2007 Date of Signature: 15-10-2007

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane-1,3-dione

EC Number:

207-980-0

EC Name:

Cyclohexane-1,3-dione

Cas Number:

504-02-9

Molecular formula:

C6H8O2

IUPAC Name:

cyclohexane-1,3-dione

Details on test material:

- Name of test material (as cited in study report): 1,3 cyclohexane dione
- Substance type: Powder
- Physical state: Solid
- Analytical purity: 1,3 Cyclohexane Dione >97% W/W
- Impurities (identity and concentrations): Sodium Chloride 1%W/W Maximum
Water 0.5%W/W Maximum
- Composition of test material, percentage of components: 1,3 Cyclohexane Dione 99.2% W/W
Sodium Chloride 0.25%W/W
Water 0.4%W/W

- Purity test date: 18 November 2009
- Lot/batch No.: 184731091224
- Expiration date of the lot/batch: 18 May 2010
- Radiochemical purity (if radiolabelling): N/A
- Stability under test conditions: Stable
- Storage condition of test material: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage sludge microorganisms was obtained on 1st February 2008 from the final effluent stage of the Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The sample of effluent was filtered through coarse filter paper (first approximate 200ml discarded) prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

1.5 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

N/A

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

28

Sampling time:

28 d

Remarks on result:

other: Samples taken at 0, 3, 5, 7, 11, 14, 18, 21, 24 and 28 days

Details on results:

The test material attained 28% degradation after 28 days

Any other information on results incl. tables

Dissolved oxygen measurements for the test material, standard material and toxicity control preparations together with the controls on Days 0, 3, 5, 7, 11, 14, 18, 21, 24 and 28 are given in Table 1.

Table 1 Dissolved Oxygen Measurements

Test Series		Dissolved Oxygen (mg O2/l)
		Day
		0	3	5	7	11	14	18	21	24	28
Culture Medium with Inoculum	^R1	8.55	8.40	8.35	8.30	8.10	8.05	8.00	7.95	7.80	7.90
	R2	8.55	8.40	8.40	8.20	8.10	7.95	7.95	8.00	7.90	7.80
Sodium Benzoate (3.0 mg/l) with Inoculum	R1	8.50	5.70	5.45	5.30	5.30	4.90	4.60	4.60	4.70	4.70
	R2	8.40	5.70	5.20	5.10	5.00	4.50	4.90	4.70	4.50	4.40
Test Material (1.5 mg/l) with Inoculum	R1	8.45	8.25	8.05	7.80	7.70	7.00	7.05	7.05	7.00	6.90
	R2	8.40	8.30	7.95	8.05	7.80	7.20	7.10	7.05	6.90	6.90
Test Material (1.5 mg/l) plus Sodium Benzoate (1.5mg/l) with Inoculum	R1	8.40	6.95	6.75	6.70	6.30	5.60	5.60	5.60	5.25	5.30
	R2	8.40	7.00	6.80	6.70	6.40	5.95	5.80	5.60	5.50	5.40

R₁and R₂= Replicates 1 and 2

Oxygen depletion values and the mean percentage degradation values (% of ThOD NO₃for the test material and the standard material) are given in Table 2.

Table 2Oxygen Depletion and Mean Percentage Biodegradation Values

Test Series			Day
			3	5	7	11	14	18	21	24	28
Culture Medium with Inoculum	Mean O2Depletion (mg O2/l)		0.150	0.175	0.300	0.450	0.550	0.575	0.575	0.700	0.700
Sodium Benzoate (3.0 mg/l) with Inoculum	O2Depletion (mg O2/l)	^R1	2.650	2.875	2.900	2.750	3.050	3.325	3.325	3.100	3.100
		R2	2.550	3.025	3.000	2.950	3.350	2.925	3.125	3.200	3.300
	% Degradation (mean)		52	59	59	57	64	62	64	63	64
Test Material (1.5 mg/l) with Inoculum	O2Depletion (mg O2/l)	R1	0.050	0.225	0.350	0.300	0.900	0.825	0.825	0.750	0.850
		R2	0.050	0.275	0.050	0.150	0.650	0.725	0.775	0.800	0.800
	% Degradation (mean)		0	9	7	8	26	26	27	26	28
Test Material (1.5 mg/l) plus Sodium Benzoate (1.5 mg/l) with Inoculum	O2Depletion (mg O2/l)	R1	1.300	1.475	1.400	1.650	2.250	2.225	2.225	2.450	2.400
		R2	1.250	1.425	1.400	1.550	1.900	2.025	2.225	2.200	2.300
	% Degradation (mean)		24	27	25	29	38	39	40	42	43

R₁and R₂= Replicates 1 and 2

The mean percentage degradation values are presented graphically in Figure 1. (see illustration)

The calculation of Theoretical Oxygen Demand (ThOD) values is given in Appendix 2.

The oxygen depletion of the inoculated control did not exceed 1.5 mg O₂/l after 28 days, the residual oxygen concentration in the test bottles remained at 4.4 mg O₂/l or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.

The test material attained 28% degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.

Variation in degradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between control and test vessels.

The toxicity control attained 38% degradation after 14 days and 43% degradation after 28 days therefore confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

The standard material, sodium benzoate, attained 64% degradation after 14 and 28 days thereby confirming the suitability of the test method and culture conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

1,3 Cyclohexanedione cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D

Executive summary:

Introduction.A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301D, "Ready Biodegradability; Closed Bottle Test” referenced as Method C.4-E of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (o).

Methods.The test material, at a concentration of 1.5 mg/l, was exposed to sewage treatment micro-organisms with culture medium in sealed culture vessels in the dark at approximately 20C for 28 days. The degradation of the test material was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.The test material attained 28% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.