(3-methoxy-2-methylpropyl)benzene

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:

2014-09-24 to 2014-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

No additional information

Oxygen conditions:

aerobic

Inoculum or test system:

other: River water without particles was used as inoculum

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): River water was sampled from the Rhine near Heveadorp, The Netherlands (18-09-2014).
- 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 not added to the river water 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 river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate
- 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

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

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

77

Sampling time:

28 d

Remarks on result:

other: Over 60% biodegradation was achieved within a period of 12 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances

Details on results:

- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test substance is 2.8 mg/mg.
- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. 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.
- Test conditions: The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 7.8 (test) and 7.9 (controls). Temperatures were within the prescribed temperature range of 22-24 °C.
- Biodegradability: The test substance was biodegraded by 77% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved within a period of 12 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances (14-day time window can be applied to the Closed Bottle test where measurements are limited to prevent the test becoming too unwieldy). Hence, the test substance is classified as readily biodegradable.

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	8.8	8.8	8.8	8.8
	8.8	8.8	8.8	8.8
Mean (M)	8.8	8.8	8.8	8.8
7	8.2	6.4	8.3	5.1
	8.2	6.6	8.2	4.8
Mean (M)	8.2	6.5	8.3	5.0
14	8.0	4.8	8.1	4.0
	8.1	4.5	8.1	3.7
Mean (M)	8.1	4.7	8.1	3.9
21	7.9	3.8	7.9	-
	7.7	4.0	7.9	-
Mean (M)	7.8	3.9	7.9	-
28	7.6	3.4	7.6	-
	7.7	3.3	7.7	-
Mean (M)	7.7	3.4	7.7	-

 

O_cs River water with nutrients and silica gel.

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

O_c River water with nutrients.

O_a River 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	1.7	3.3	30	61
14	3.4	4.2	61	78
21	3.9	-	70	-
28	4.3	-	77	-

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.1 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance was biodegraded by 77% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved within a period of 12 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances (14-day time window can be applied to the Closed Bottle test where measurements are limited to prevent the test becoming too unwieldy). Hence, the test substance is classified as readily biodegradable.

Executive summary:

This study was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test substance by the Closed Bottle Test.

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 test is valid as shown by an endogenous respiration of 1.1 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

 

The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Test substance was biodegraded by 77% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved within a period of 12 days immediately following the attainment of 10% biodegradation. Hence, this 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:

2014-09-24 to 2014-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

No additional information

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge (18-09-2014) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating 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 bottles to 2.0 mg/L (van Ginkel and Stroo, 1992).

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 medium with inoculum and silica gel, 10 bottles containing medium with inoculum and silica gel with test substance, 6 bottles containing sodium acetate and medium with inoculum, and 6 bottles containing test substance, sodium acetate and medium with 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

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

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

69

Sampling time:

28 d

Remarks on result:

other: Over 60% biodegradation was achieved after 13 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances

Details on results:

- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) of the test substance is 2.8 mg/mg.
- Toxicity: Inhibition of the degradation of an easily-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. 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.
- Test conditions: 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 (test) and 7.3 (controls). Temperatures were within the prescribed temperature range of 22-24 °C.
- Biodegradability: The test substance was biodegraded by 69% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved after 13 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances (14-day time window can be applied to the Closed Bottle test where measurements are limited to prevent the test becoming too unwieldy). Hence, the test substance is classified as readily biodegradable.

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	8.8	8.8	8.8	8.8
	8.8	8.8	8.8	8.8
Mean (M)	8.8	8.8	8.8	8.8
7	8.4	8.4	8.3	4.0
	8.4	8.4	8.4	4.4
Mean (M)	8.4	8.4	8.4	4.2
14	8.3	6.4	8.3	4.2
	8.3	7.3	8.2	3.9
Mean (M)	8.3	6.9	8.3	4.1
21	8.2	4.9	8.1	-
	8.3	5.3	8.2	-
Mean (M)	8.3	5.1	8.2	-
28	8.1	4.4	8.0	-
	8.1	4.2	8.0	-
Mean (M)	8.1	4.3	8.0	-

 

O_cs Mineral nutrient solution without test material but with inoculum and silica gel.

O_t Mineral nutrient solution with test material (2.0 mg/L), silica gel, and inoculum.

O_c Mineral nutrient solution with only inoculum.

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

 

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	0.0	4.2	0	78
14	1.4	4.2	25	78
21	3.2	-	57	-
28	3.8	-	69	-

 

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 0.8 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance was biodegraded by 69% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved after 13 days immediately following the attainment of 10% biodegradation. The test substance therefore fulfilled the 14-day time window criterion for ready biodegradable substances (14-day time window can be applied to the Closed Bottle test where measurements are limited to prevent the test becoming too unwieldy). Hence, the test substance is classified as readily biodegradable.

Executive summary:

This study was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test substance was exposed to activated sewage sludge micro-organisms 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 substance was assessed by the measurement of oxygen consumption.

 

The test is valid as shown by an endogenous respiration of 0.8 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

 

The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Test substance was biodegraded by 69% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved after 13 days immediately following the attainment of 10% biodegradation. Hence, this substance should be classified as readily biodegradable.

Description of key information

OECD Guideline 301D, GLP, key study, validity 1:

77% biodegradation after 28d, satisfied the 14d window criterion.

Readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

To assess the biodegradation potential of the registered substance, two valid experimental studies were performed according to OECD Guideline 301D with GLP compliance, in the same laboratory. The registered substance (at 2 mg/L) was exposed to river water in the first study (considered as the key study based on the best result of biodegradation, see below) and to activated sewage sludge micro-organisms in the second study (considered as the supporting study). The degradation of the test substance was assessed by the measurement of oxygen consumption. In both studies, the test substance did not cause a reduction in the endogenous respiration and therefore is considered to be non-inhibitory of the inoculum. The test substance was biodegraded to a level of 77% at day 28 in river water and 69% at day 28 in secondary activated sludge, fulfilling the 14 -day time window criterion for ready biodegradable substances in both studies. Hence, the registered substance is readily biodegradable.