Reaction mass of α-3,3-trimethyl-(αS,1R)-cyclohexanemethanol and α-3,3-trimethyl-(αR,1S)-cyclohexanemethanol

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:

12 December 2012 to 25 January 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 301 D with GLP statement. Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound); and river water instead of an effluent/extract/mixture was used as inoculum

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301): - Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification; - River water instead of an effluent/extract/mixture was used as inoculum.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

18 September 2012

Specific details on test material used for the study:

PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 1 mm Hg at 50 °C
- Estimated solubility in water: 278 mg/L at 25 °C

Oxygen conditions:

aerobic

Inoculum or test system:

other: River water without particles was used as inoculum

Details on inoculum:

- Source of inoculum/activated sludge: River water was sampled from the Rhine near Heveadorp, The Netherlands (7-12-2012).
- The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained 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, and 0.25 mg FeCl3•6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Test temperature: 22-24 °C
- Continuous darkness: Yes
- Deionized water containing no more than 0.01 mg/L copper was prepared in a water purification system.

TEST SYSTEM
- Culturing apparatus: 0.3 L Biological oxygen demand (BOD) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel dosed with test substance, and 6 bottles containing sodium acetate and inoculum.
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Eutech Cyberscan pH11 pH meter (Eutech Instruments, Nijkerk, The Netherlands). The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: No

OTHERS:
- 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 biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Remarks:

6.7 mg/L

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Value:

37

Sampling time:

28 d

Details on results:

- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of test substance is 3.0 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of test substance to microorganisms degrading acetate is not relevant. At day 7, a slight inhibition of the endogenous respiration was detected. Retardation of the onset of biodegradation due to the "high" initial concentration of the test substance can therefore not be excluded.
- Test conditions: The pH of the media was 8.1 at the start of the test. The pH of the river water at day 28 was 7.8 (test and control) and 8.0 (control with silica gel). Temperatures were within the prescribed temperature range of 22 to 24°C.
- Biodegradability: Test substance is biodegraded by 37 % at day 28 in the Closed Bottle test. Since, in the prolonged Closed Bottle test the test substance is biodegraded 66% at day 42 it should be classified as inherently biodegradable. The biodegradation of the test substance in excess of 60% at day 42 also allows classification as not persistent (enhanced biodegradability testing).

Results with reference substance:

The ThOD of sodium acetate is 0.8 mg/mg. Sodium acetate was degraded by 78% of its theoretical oxygen demand after 14 days.

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
Mean (M)	9.0	9.0	9.0	9.0
7	8.2	8.3	8.4	4.5
	8.4	8.4	8.3	4.5
Mean (M)	8.3	8.4	8.4	4.5
14	7.6	7.3	7.7	3.4
	7.7	7.3	7.7	3.5
Mean (M)	7.7	7.3	7.7	3.5
21	7.7	7.5	7.6	-
	7.7	6.5	7.8	-
Mean (M)	7.7	7.0	7.7	-
28	7.4	5.2	7.6	-
	7.5	5.4	7.4	-
Mean (M)	7.5	5.3	7.5	-
42	7.0	3.1	-	-
	6.9	2.8	-	-
Mean (M)	7.0	3.0	-	-

 

O_csRiver water with nutrients and silica gel but without test material.

O_tRiver water with nutrients, test material (2.0 mg/L), and silica gel.

O_cRiver water with nutrients.

O_aRiver water with nutrients and sodium acetate (6.7 mg/L).

 

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, the test substance (BOD/ThOD) and sodium acetate (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	-0.1	3.9	0	72
14	0.4	4.2	7	78
21	0.7	-	12	-
28	2.2	-	37	-
42	4.0	-	66	-

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.5 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20 %. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 78. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test substance was biodegraded by 37% at day 28 in the Closed Bottle test. Since, in the prolonged Closed Bottle test the test substance is biodegraded by 66% at day 42 it should be classified as inherently biodegradable. The biodegradation of the test substance in excess of 60% at day 42 also allows classification as not persistent (enhanced biodegradability testing).

Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed and the biodegradability was measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD Guideline 301 D and in compliance with the OECD principles of GLP.

 

The test substance was exposed to river water at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21 and 28 days. The degradation of the test material was assessed by the measurement of oxygen consumption. 

The calculated theoretical oxygen demand (ThOD) of the test substance is 3.0 mg/mg. The test substance caused a slight reduction in the endogenous respiration at day 7. Inhibition of the onset of the biodegradation of the test substance can therefore not be excluded. The test is valid as shown by an endogenous respiration of 1.5 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 78% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met: oxygen concentrations > 0.5 mg/L in all bottles during the test period.

 

The test substance was biodegraded by 37% at day 28 in the Closed Bottle test. In the enhanced (prolonged) Closed Bottle test this test substance was biodegraded by 66% at day 42. The biodegradation reached at the last day of the test demonstrates that the test substance is inherently biodegradable and should be classified as not persistent.

 

Description of key information

The test substance was biodegraded by 37% at day 28 in the Closed Bottle test. In the enhanced (prolonged) Closed Bottle test this substance was biodegraded by 66% at day 42. The biodegradation reached at the last day of the test demonstrates that the test substance is inherently biodegradable and should be classified as not persistent.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

In order to assess the biotic degradation, a ready biodegradability test was performed and the biodegradability was measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD Guideline 301 D and in compliance with the OECD principles of GLP.

The calculated theoretical oxygen demand (ThOD) of test substance is 3.0 mg/mg. The test substance did cause a slight reduction in the endogenous respiration at day 7. Inhibition of the onset of the biodegradation of the test substance can therefore not be excluded. The test is valid as shown by an endogenous respiration of 1.5 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 78% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met: oxygen concentrations > 0.5 mg/L in all bottles during the test period.

 

The test substance was biodegraded by 37% at day 28 in the Closed Bottle test. In the enhanced (prolonged) Closed Bottle test this substance was biodegraded by 66% at day 42. The biodegradation reached at the last day of the test demonstrates that the test substance is inherently biodegradable and should be classified as not persistent.