diethyl 1,4-cyclohexanedicarboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06.09.2005 to 09.11.2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO Guideline n°14593 (CO2 in Sealed Vessels – Headspace Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

the test material is not volatile (VP 0.2 [Pa] at 25°C, Water solubility: 1280 [mg/l], Henry Cte < 0.1 [Pa m3/mol]

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge microorganisms was obtained on 14 September 2005 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.

- Preparation of inoculum for exposure: the sample of activated sewage sludge was filtered through filter paper 1 (100 micrometer). The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 4 mg/L dry weight activated sludge.

Duration of test (contact time):

28 d

Initial conc.:

31.69 mg/L

Based on:

test mat.

Initial conc.:

20.04 other: mg Carbon/liter

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: as recommended by OECD TG 301 & 310 (The culture medium was prepared with an original solution of 0.2 g/L FeCl3,6H2O instead of 0.25 g/L. The final concentration was 0.2 mg/L instead of 0.25 mg/L. The consequence was an iron deficiency for the microorganisms. In spite of this deficiency, the activity of the microorganisms is correct and the test material was degraded. The deviation was considered to have had not influence on the study).
- Solubilising agent (type and concentration if used): none
- Test temperature: 23 +/- 2°C
- Suspended solids concentration: 4 mg/l
- Continuous darkness: no (diffused light for 28 days)
- Other: The test material was dispersed in 3 L mineral medium at the concentration of 31.74 mg/L (corresponding to 20.04 mg C/L). The solution was stirred at room temperature during 24 hours. Control test was prepared in the same way without addition of test material. Fructalate has a measured water solubility of 1280 mg/L and a measured vapour pressure of 0.2 Pa at 25°C. Hence it is water soluble and not significantly volatile.

TEST SYSTEM
- Culturing apparatus: 250ml glass culture vessels filled to 75ml (total volume when full: 320 ml. All vessels containing 75 ml of solution were sealed using Teflon lined silicon septa and incubated at 23 +/- 2°C with constant shaking at approximately 150 rpm (Kuhner ISF-1-W) in diffused light for 28 days.
- Number of culture flasks/concentration: triplicates
- Measuring equipment: Shimadzu TOC Vcph analyser.


SAMPLING
The samples taken on Days 0, 4, 7, 11, 13, 19, 21, 25, 28 were analysed for CO2 immediately. For each sample, 0.5 ml H3PO4 85% was added and the culture vessels replaced at test temperature for at least 1 hour. The IC was directly measured in the headspace by Shimadzu TOC Vcph analyser. The IC concentration in the sample was measured using a non-dispersive infrared detector utilising ultra-pure air (CO2 free) as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3) and sodium hydrogenocarbonate (NaHCO3).


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Reference substance: sodium benzoate
- Toxicity control: yes (test substance + sodium benzoate)

Reference substance:

benzoic acid, sodium salt

Remarks:

(Riedel de Haen batch n°02720)

Preliminary study:

None

Test performance:

Due to a technical problem, biodegradation results of Day 13 could not be measured/taken in account.

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

69.4

Sampling time:

28 d

Details on results:

See "Any other information on results incl. results" and "Illustration" for detailed results.

Results with reference substance:

Results with reference substance are valid as more than 60% of biodegradation were reached on day 3 (before Day 14).

RESULTS:Total inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values for the test and standard materials and toxicity control are given in Table 2 and the biodegradation curves are presented in “Illustration”.

The mean TIC in control vessels on day 28 was 1.40 mg/L equivalent to 7.0% of the organic carbon added initially as test material to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test material attained 69.39% degradation after 28 days and therefore can be considered to be readily biodegradable.

The toxicity test attained 84.79% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Sodium Benzoate attained 93.34% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

 

TABLE 1 = Inorganic Carbon Values on Each Analysis Occasion

Days	Control		Test material			Reference		Toxicity control
	Control	(mg TIC)	Fructalate		(mg TIC)	Sodium benzoate	(mg TIC)	Fructalate+ Sodium Benzoate TOX	(mg TIC)
	Sample 1	Sample 2	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 1	Sample 2
0	0.038	0.047	0.049	0.050	0.054	0.048	0.049	0.044	0.042
4	0.075	0.073	0.181	0.181	0.179	1.327	1.251	1.362	1.388
7	0.092	0.128	0.230	0.238	0.275	1.382	1.403	1.507	1.522
11	0.090	0.090	0.543	0.674	0.503	1.463	1.462	1.852	1.759
13(*)	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
19	0.106	0.096	1.166	0.922	1.234	1.507	1.546	0.991	2.406
21	0.104	0.096	1.214	1.212	0.853	1.507	1.500	2.141	2.399
25	0.103	0.100	0.645	1.156	1.250	1.512	1.265	2.776	2.548
28	0.108	0.103	1.180	0.993	1.271	1.503	1.503	2.634	2.663

(*) technical problems

 

TABLE 2: PERCENTAGE BIODEGRADATION VALUES

Days	% BIODEGRADATION
	Test material	Reference	Toxicity control
	Fructalate	Sodium benzoate	Test material + Sodium benzoate
0	0.60	0.44	0.04
4	7.10	81.13	43.37
7	9.14	85.62	46.81
11	32.16	91.63	57.19
13(*)	0(*)	0(*)	0(*)
19	66.97	95.17	53.27
21	66.09	93.67	72.34
25	60.90	85.91	85.36
28	69.39	93.34	84.79

(*) technical problems

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 69% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 310.

Executive summary:

Introduction

This study investigates the ready biodegradability of the test material (Fructalate) when exposed to sewage sludge micro-organisms under aerobic conditions. The method followed the recommendations of ISO Guideline N° 14593: "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO₂ headspace test) (adopted 1999)" and of the OECD Guideline for Testing of Chemicals N° 310 (adopted March 23^rd, 2006).

 

Methods

The test material, at a concentration of 20.04 mg C/l, was exposed to activated sewage sludge micro-organisms (treating predominantly domestic sewage) with culture medium in sealed culture vessels in diffused light at 23°C ± 2°C for 28 days.

 

The degradation of the test material was assessed by the determination of carbon dioxide produced on Days 0, 3, 7, 10, 14, 17, 21, 24 and 28. Control solutions withinoculumand the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

Results

The test material attained 69% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 310.

The test material is not considered as toxic to sludge microorganisms as toxicity control attained > 25% degradation after 14 days (more than 40% of biodegradability was obtained on day 4).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

other information

Study period:

From 12 December 1994 to 9 January 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD guideline No. 301D referenced as Method C.4-E of Commission Directive 92/69/EEC with GLP statement. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

1994-01-31

Specific details on test material used for the study:

Container: metal canister
Storage conditions: Room temperature until 1st December
1994, thereafter room temperature in the dark.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Key result

Parameter:

% degradation (O2 consumption)

Value:

34

Sampling time:

28 d

Details on results:

The test material attained 34% degradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and
conditions of DECO Guideline No. 3010. The toxicity control, containing both test material and sodium benzoate, attained 47% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the study. The standard material (sodium benzoate) attained 81% degradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Table 1: Dissolved oxygen measurements

Test series		Dissolved oxygen (mg O2/L)
		day
		0	3	6	9	11	16	18	28
Culture medium with inoculum	R1	8.90	8.50	8.40	8.35	8.20	8.00	8.00	7.90
	R2	8.90	8.55	8.50	8.35	8.25	8.05	8.00	7.85
Standard material: sodium benzoate (3 mg/L) with inoculum	R1	8.90	7.10	6.80	6.30	5.00	4.50	4.25	3.90
	R2	8.90	7.10	6.70	6.10	5.00	4.35	4.40	3.70
Test material (2 mg/L) with inoculum	R1	8.90	8.00	7.90	7.70	7.55	7.20	7.05	6.40
	R2	8.90	8.20	7.80	7.60	7.60	7.10	7.00	6.55
Test material (2 mg/L) plus sodium benzoate (1.5 mg/L) with inoculum	R1	8.90	7.10	6.95	7.00	6.00	5.80	5.25	4.80
	R2	8.90	7.10	7.30	7.10	6.20	5.85	5.20	4.75

Table 2: Oxygen depletion and mean percentage biodegradation values

Test series			day
			3	6	9	11	16	18	28
Culture medium with inoculum	Mean O2 depletion (mg O2/L)		0.375	0.450	0.550	0.675	0.875	0.900	1.025
Standard material: sodium benzoate (3 mg/L) with inoculum	O2 depletion (mg O2/L)	R1	1.425	1.650	2.050	3.225	3.525	3.750	3.975
		R2	1.425	1.750	2.250	3.225	3.675	3.600	4.175
	% degradation (mean)		28	34	43	64	72	74	81
Test material (2 mg/L) with inoculum	O2 depletion (mg O2/L)	R1	0.525	0.550	0.650	0.675	0.825	0.950	1.475
		R2	0.325	0.650	0.750	0.625	0.925	1.000	1.325
	% degradation (mean)		11	15	17	16	21	24	34
Test material (2 mg/L) plus sodium benzoate (1.5 mg/L) with inoculum	O2 depletion (mg O2/L)	R1	1.425	1.500	1.350	2.225	2.225	2.750	3.075
		R2	1.425	1.150	1.250	2.025	2.175	2.800	3.125
	% degradation (mean)		21	20	20	32	33	42	47

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

The test material attained 34% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.

Executive summary:

Methods

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (992) 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).

Procedures

The test material was exposed to sewage sludge micro-organisms at a concentration of 2 mg/l with culture medium in sealed culture vessels in the dark at 21°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 34% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.

Description of key information

OECD Guideline 310, ISO Guideline 14593, GLP, key study, validity 1:

69% biodegradation after 28 days of incubation and 10 day window validation criterion fulfilled. 

Readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Two studies are available, according to international guidelines, to determine the readily biodegradation of the test substance.

The two results obtained are not going in the same direction.

For the first biodegradability study (Firmenich, 2005), according to ISO guideline n°14593 and OECD Guideline n°310 with GLP statement, the substance appears to be readily biodegradable with 69% degradation after 28 days of incubation and satisfied the 10 day window validation criterion.

For the second biodegradability study (SPL, 1995), according to OECD Guideline n°301D with GLP statement, the substance appears to be not readily biodegradable with 34% degradation after 28 days of incubation.

A biodegradability test is very stringent. A positive biodegradability test is sometimes difficult to obtain because it's dependent on the quality of the inoculum used. Therefore, even if results are different, a positive result is sufficient to conclude on the readily biodegradation potential of the substance.For this reason, the study performed by Firmenich in 2005 is assessed as the key study and the negative result has no value under REACH, no flag is assigned.

According to the key study, the test substance can be considered to be readily biodegradable.