(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

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:

From 30 October 2019 to 30 December 2019

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:

yes

Remarks:

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)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

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)

Qualifier:

according to guideline

Guideline:

ISO 10707 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:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Molecular formula: C12H20O1
- Molecular weight: 180.29 g/mol
- Water solubility: 50.3 mg/L at 20°C
- Vapour pressure: 5.0 Pa at 20°C and 8.7 Pa at 25°C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 102.7 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.9 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated by stirring on a magnetic stirrer plate at ambient temperature (± 21 °C) for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

Activated sludge was selected as inoculum based on the non-GLP screening test performed with both activated sludge and river water.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Test bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

- Nutrients, and stocks:
Deionized water used in the Closed Bottle test contained per liter of water 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4.2H2O, 22.50 mg MgSO4·7H2O, 27,51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of test substance per 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 and the content was mixed vigorously. Subsequently, 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.

- Test procedures:
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel (0.2 g silica gel / bottle), 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L 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. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease at day 42 and 60 using the bottles of day 28 and a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

- Analyses:
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.9%; Batch n° BCBP8197V

Preliminary study:

A non-GLP screening test was performed prior the GLP final test in order to determine the most suitable inoculum for the degradation of the test substance, in the Closed Bottle test.
Biodegradation of ≥60% was not found within four weeks with both activated sludge and river water. The test substance should therefore not be classified as readily biodegradable. After four weeks biodegradation increased, reaching >60% after 42 days with both inocula. Percentages of 70 and 76 were achieved at day 56. These results demonstrate that the test substance is not persistent. For the final GLP test it is recommended to use activated sludge as inoculum.

Key result

Parameter:

% degradation (O2 consumption)

Value:

28

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

74

Sampling time:

60 d

Remarks on result:

other: The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60-day test period allows classification of the test item as not persistent.

Details on results:

See tables 5.2.1/1 and 2 in "Any other information on results incl. tables".

- Theoretical oxygen demand (ThOD): The theoretical oxygen demand (ThOD) of the test item used to calculate the biodegradation percentages is 2.93 g oxygen/g test substance. This ThOD was calculated with the molecular formula and by assuming that the ThOD of all constituents is equal to the ThOD of the known constituents. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

- 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 the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

- Test conditions: The pH of the media was 7.4 (control, control with silica gel, test and reference) at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 7.2. The pH of the medium at day 60 was 7.2 for the control and control with silica gel bottles and 7.1 for the test bottles. The temperature ranged from 22.6 to 22.9 °C which is within the prescribed temperature range of 22 to 24°C.

- Biodegradability: The test substance was biodegraded by 28% at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance is biodegraded by 74% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60-day test period allows classification of the test item as not persistent.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80 (see table 5.2.1/2 in "Any other information on results incl. tables").

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

Time (days)	Oxygen concentration (mg/L)
	Oc	Oa	Ocs	Ot
0	8.9	8.9	9.0	9.0
	8.9	8.9	9.0	9.0
Mean (M)	8.90	8.90	9.00	9.00
7	8.1	4.4	8.2	8.1
	8.1	4.4	8.2	8.0
Mean (M)	8.10	4.40	8.20	8.05
14	8.0	3.8	8.0	7.6
	8.0	3.8	8.0	7.6
Mean (M)	8.00	3.80	8.00	7.60
21	7.8		7.8	7.1
	7.8		7.8	7.0
Mean (M)	7.80		7.80	7.05
28	7.7		7.6	5.7
	7.7		7.6	6.2
Mean (M)	7.70		7.60	5.95
42	7.5		7.5	4.0
	7.5		7.5	4.1
Mean (M)	7.50		7.50	4.05
60	7.3		7.3	3.0
	7.3		7.4	3.0
Mean (M)	7.30		7.35	3.00

Oc = Mineral nutrient solution with only inoculum.

Ocs = Mineral nutrient solution with inoculum and silica gel

Ot = Mineral nutrient solution with inoculum, test substance (2.0 mg/L) and silica gel

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

Table 5.2.1/2: Oxygen consumptio (mg/L) and the percentages biodegradation of 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	0.00	0.00	0	0
7	0.15	3.70	3	71
14	0.40	4.20	7	80
21	0.75		13
28	1.65		28
42	3.45		59
60	4.35		74

Validity criteria fulfilled:

yes

Remarks:

Endogenous respiration of 1.2 mg/L at day 28. The differences of the replicate values at day 60 were less than 20%. The biodegradation of sodium acetate at day 14 was 80. Oxygen concentrations >0.5 mg/L in all bottles during the test period.

Interpretation of results:

other: Not readily biodegradable but ultimately (completely) biodegradable.

Conclusions:

The test substance was biodegraded by 28% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable.
In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 74% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60 -day test period allows classification of the test item as not persistent.

Executive summary:

To assess the biotic degradation of the test substance, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item (2 mg/L) was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.6 to 22.9 °C for 60 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, 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 test substance was biodegraded by 28% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 74% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60 -day test period allows classification of the test item as not persistent.

The test is valid as shown by an endogenous respiration of 1.20 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 80% 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.

Description of key information

OECD Guideline 301D, EU Method C4 -E, ISO 10707, GLP, Key study, validity 1:

28% biodegradation after 28 days and 74% biodegradation after 60 days

Not readily biodegradable but ultimately (completely) biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

To assess the biodegradation potential of the registered substance, one experimental key study is available.

This valid study (Nouryon, 2020) was performed on the registered substance according to slighty modified OECD 301D, EU C.4 -E and ISO 10707 Test Guidelines and in compliance with the OECD principles of Good Laboratory Practice. The test substance was exposed to activated sludge which was spiked to a mineral nutrient solution, dosed in closed bottles and incubated in the dark at 22.6 to 22.9 °C for 60 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance 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 test substance was biodegraded by 28% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 74% at day 60. The biodegradation percentage of >60% demonstrates that the test substance is ultimately (completely) biodegradable. Moreover, the biodegradation in excess of 60% within the 60 -day test period allows classification of the test substance as not persistent.