cis-4-tert-butylcyclohexyl acetate

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 12-12-2012 to 11-01-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to slightly modified OECD, EU and ISO Test Guidelines with GLP statement. All validity criteria were fulfilled and no major deviations were observed.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviation. Ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviation. Ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).

Qualifier:

according to guideline

Guideline:

other: ISO 10707 (1994) Water quality - Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (Closed Bottle test).

Deviations:

yes

Remarks:

minor deviation. Ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

18 September 2012

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge (07-12-2012) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Initial conc.:

2.7 other: mg O2/mg

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Test temperature: 22-24°C
- pH: The pH of the media was 7.3 at the start of the test. The pH of the medium at day 28 was 7.2 (control with silica gel, and test) and 7.3 (control).
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles containing only inoculum (not agitated), 6 bottles containing inoculum and sodium acetate (not agitated), 10 bottles containing inoculum and test substance (agitated), and 10 bottles with 0.2 g silica gel containing inoculum (agitated).
- Method used to create aerobic conditions: no data
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: Oxygen electrode
- Test performed in closed vessels: yes
- Test performed in open system: no

SAMPLING
The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 (bottles containing only inoculum and bottles with 0.2 g silica gel containing inoculum)
- Abiotic sterile control: no
- Procedure control: yes (bottles containing inoculum and sodium acetate)
- Toxicity control: no. Inhibition of the degradation of a well-degradable compound was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant.
- Other: none

STATISTICAL METHODS:
None

Reference substance:

acetic acid, sodium salt

Preliminary study:

None

Test performance:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Remarks on result:

other: Over 60% biodegradation was achieved in a period of approximately 10 days immediately following the attainment of 10% biodegradation.

Details on results:

Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 72.

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	9.0	9.0	9.0	9.0
	9.0	9.0	9.0	9.0
	9.0	9.0	9.0	9.0
7	8.5	8.1	8.4	4.5
	8.5	8.0	8.5	4.7
	8.5	8.1	8.5	4.6
14	7.7	4.9	7.7	3.9
	7.7	5.4	7.8	3.8
	7.7	5.2	7.8	3.9
21	7.8	4.2	7.7
	7.7	3.9	7.6
	7.8	4.1	7.7
28	7.7	3.7	7.6
	7.7	3.4	7.6
	7.7	3.6	7.6

O_cs      Mineral nutrient solution with inoculum in bottles with silica gel. Content of bottles was agitated.

O_t       Mineral nutrient solution with test material (2.0 mg/L). Content of bottles was agitated.

O_c       Mineral nutrient solution with only inoculum.

O_a       Mineral nutrient solution with sodium acetate (6.7 mg/L) and inoculum.

Table 5.2.1/2: Oxygen consumption (mgL) and the percentages biodegradation of the test substance (BOD/ThOD) and reference substance (BOD/ThOD) in the closed bottle test

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.4	3.9	7	72
14	2.5	3.9	46	72
21	3.7		69
28	4.1		76

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The substance should be classified as readily biodegradable (76% biodegradation after 28 days, in the 10-day window).

Executive summary:

This closed bottle test was performed according to slightly modified OECD (OECD TG 301), EU and ISO Test Guidelines with GLP statement, to assess the readily biodegradation of the test substance, in an aerobic aqueous medium.

Administration was accomplished by dosing 0.034 g of test substance on 11.3 g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with aluminum foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Use was made of 10 bottles containing only inoculum (not agitated), 6 bottles containing inoculum and sodium acetate (not agitated), 10 bottles containing inoculum and test substance (agitated), and 10 bottles with 0.2 g silica gel containing inoculum (agitated). The contents of bottles were agitated with magnetic stirrers at 600 rpm. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The substance was biodegraded by 76% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 10 days immediately following the attainment of 10% biodegradation.

The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 72% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

In conclusion, the substance should be classified as readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

This study is a screening test, performed according to slightly modified OECD Guideline 301D without GLP statement. Raw data were not available in this report, therefore the validity criteria cannot be verified.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviation. Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: The inoculums consisting of relatively low numbers of microorganisms were derived from an activated sludge plant and the river Rhine.

Duration of test (contact time):

42 d

Initial conc.:

2 mg/L

Based on:

test mat.

Key result

Parameter:

% degradation (O2 consumption)

Value:

62

Sampling time:

28 d

Remarks on result:

other: with sludge

Key result

Parameter:

% degradation (O2 consumption)

Value:

73

Sampling time:

42 d

Remarks on result:

other: with sludge

Parameter:

% degradation (O2 consumption)

Value:

49

Sampling time:

28 d

Remarks on result:

other: with river

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

42 d

Remarks on result:

other: with river

Details on results:

See table 5.2.1/1 in "Any other information on results incl. tables".
The parent substance was biodegraded 62% at day 28 in the Closed Bottle test inoculated with sludge. Using river water biodegradation percentages of 49 and 66 at day 28 and 42, respectively, were found. Biodegradation results were also obtained with hydrolysis products: acetate and 4-tert-Butylcyclohexanol. See table 5.2.1/1 in "Any other information on results incl. tables".

Table 5.2.1/1: Biodegradation percentages of the parent substance, 4 -tert-butylcyclohexanol and acetate in Closed Bottle tests inoculated with activated sludge and river water

Substance	Inoculum	Biodegradation at day
		7	14	21	28	42
Parent substance	Sludge	0	23	45	62	73
Parent substance	River	0	13	40	49	66
4-tert-Butylcyclohexanol	Sludge	-1	15	57	77
4-tert-Butylcyclohexanol	River	-1	7	50	67
Acetate	Sludge	72	72
Acetate	River	74	78

Validity criteria fulfilled:

not specified

Remarks:

raw data were not provided in this report.

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The parent substance was biodegraded 62% at day 28 in the Closed Bottle test inoculated with sludge. Using river water biodegradation percentages of 49 and 66% at day 28 and 42, respectively, were found. Raw data were not available therefore the 10-d window cannot be evaluated. Biodegradation results were also obtained with the hydrolysis products, acetate and 4-tert-Butylcyclohexanol: acetate was biodegraded 72% and 78% at day 14 with sludge and river water, respectively (meeting the 10-d window) and 4-tert-butylcyclohexanol was biodegraded 77% and 67% at day 28 with sludge and river water, respectively (the 10-d window cannot be evaluated). The quaternary carbon does not hamper the biodegradation of the parent substance.

Executive summary:

This study was performed according to slightly modified OECD Guideline 301D without GLP statement.

The incentive of this investigation is to determine the biodegradability of the parent substance and its hydrolysis products, acetate and 4-tert-Butylcyclohexanol. The substances have been exposed to inocula under aerobic conditions for a period of at least 28 days. The inoculums consisting of relatively low numbers of microorganisms were derived from an activated sludge plant and the river Rhine.

The parent substance and the hydrolysis product 4 -tert-Butylcyclohexanol were administrated with the use of silica gel. Only the upper layer of the silica gel was brought into contact with the test substance. Subsequently, silica gel was administrated to each test bottle. The final concentration of all test substances in the BOD bottles was always 2.0 mg/L. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L. The concentration of sodium acetate in the bottles was 6.7 mg/L. The tests were performed in 0.3 L biological oxygen demand (BOD) bottles with glass stoppers. Use was made of 3 bottles containing only inoculum and series of 3 bottles for each test substance with the respective inoculum. The activated sludge was diluted to 2.0 mg DW/L in the nutrient medium and the river water was used undiluted. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark with the temperature ranging between 22 and 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode.

The parent substance was biodegraded 62% at day 28 in the Closed Bottle test inoculated with sludge. Using river water biodegradation percentages of 49 and 66 % at day 28 and 42, respectively, were found. Raw data were not available therefore the 10-d window cannot be evaluated. Biodegradation results were also obtained with the hydrolysis products acetate and 4-tert-Butylcyclohexanol: acetate was biodegraded 72% and 78% at day 14 with sludge and river water, respectively (meeting the 10-d window) and 4-tert-butylcyclohexanol was biodegraded 77% and 67% at day 28 with sludge and river water, respectively (the 10-d window cannot be evaluated). The quaternary carbon does not hamper the biodegradation of the parent substance.

Raw data were not available in this report, therefore the validity criteria cannot be verified.

In conclusion, based on data lacking, the parent substance and its hydrolysis products cannot be considered as readily biodegradable meeting the 10-d window.

Description of key information

OECD Guideline 301 D, EU Method C.4-E, ISO 10707, GLP, key study, validity 1:

76% biodegradation after 28 days within the 10-day window.

The registered substance is readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Two Closed Bottle tests are available to assess the biodegradation in water of the registered substance.

The first study, considered as a valid key study, was performed according to slightly modified OECD (OECD TG 301), EU and ISO Test Guidelines with GLP statement, to assess the biodegradability of the registered substance in an aerobic aqueous medium.

The substance did not cause a reduction in the endogenous respiration at day 7, therefore, the substance is considered to be non-inhibitory to the inoculum. The substance was biodegraded by 76% at day 28 within the 10 days window.

The second study, considered as a not assignable supporting study, was performed according to slightly modified OECD Guideline 301D without GLP statement, to assess the biodegradability of the registered substance and its hydrolysis products, acetate and 4 -tert-Butylcyclohexanol, in an aerobic aqueous medium, using two types of inoculum (activated sludge and river water). The registered (parent) substance was biodegraded 62% at day 28 with sludge. Using river water, biodegradation percentages of 49 and 66 % at day 28 and 42, respectively, were found. Raw data were not available therefore the 10-d window cannot be evaluated. For hydrolysis products, acetate was biodegraded 72% and 78% at day 14 with sludge and river water, respectively (meeting the 10-d window) and 4 -tert-butylcyclohexanol was biodegraded 77% and 67% at day 28 with sludge and river water, respectively (the 10-d window cannot be evaluated). The quaternary carbon does not hamper the biodegradation of the parent substance. Based on the lack of datapoints in this study, it's not possible to verify that the parent substance and its hydrolysis products passed the 10 days window and validity criteria cannot be verified.

According to the key study, the registered substance is considered readily biodegradable. The supporting study demonstrates that hydrolysis products (acetate and 4-tert-butylcyclohexanol) are also biodegraded over 60% within 28 days. Acetate can be considered as readily biodegradable but insufficient data are available to assess whether the 10 days window criteria for 4-tert-butylcyclohexanol was met.