2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

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:

Experimental starting date 17-01-2017, Experimental completion date 16-02-2017

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).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
Batch/Lot number 1609501020
CAS no 4203-89-8
Purity 94.5%
The concentrations cited in this report refer to the as-received sample of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (12-01-2017) 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, 0.40 g 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). The inoculum was not pre-adapted to the test substance.

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:

Reference substances and chemicals
Sodium acetate anhydrous was used as a reference substance in the Closed Bottle test. This compound was purchased from Sigma-Aldrich, St Louis, US. The substance data were submitted by the supplier.

Reference compound acetic acid, sodium salt
CAS reg. No. 127-09-3
Purity >99%
Batch/lot number BCBP8197V
Appearance white crystals
The silica gel Davisil grade 636, pore size 60A, 35-60 mesh particle size was purchased from Sigma-Aldrich. All other chemicals used were of reagent grade quality.

Deionized water
Deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.

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

Nutrients, and stocks
The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 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.
Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the 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 a stock solution of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing mineral salts medium and inoculum, 10 bottles containing mineral salts medium, inoculum, and silica gel, 10 bottles containing mineral salts medium, inoculum, and silica gel with test substance, 6 bottles with mineral salts medium, and sodium acetate. 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.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

74

Sampling time:

28 d

Details on results:

RESULTS AND DISCUSSION
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate is 2.2 g oxygen/g test substance. This ThOD was calculated by assuming formation of ammonia, water and carbon dioxide. It was also assumed that the ThOD of the impurities (5.5%) is identical to the ThOD of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate. The ThOD of sodium acetate is 0.8 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 2-(1-oxa- 4-azaspiro[4.5]dec-4-yl)ethyl methacrylate 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 7.3 at the start of the test. The pH of the media at day 28 was 7.1 (test) and 7.3 (control and control with silica gel). Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 0.7 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 81%.
Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 81%

Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (day)		Oxygen concentration (mg/L)
		Ocs	Ot	Oc	Oa
0		8.7	8.7	8.7	8.7
		8.7	8.7	8.7	8.7
Mean (M)		8.7	8.7	8.7	8.7
7		8.4	7.1	8.4	4.3
		8.4	7.1	8.4	4.3
Mean (M)		8.4	7.1	8.4	4.3
14		8.3	6.6	8.2	3.8
		8.3	6.7	8.1	4
Mean (M)		8.3	6.7	8.2	3.9
21		8.1	5.6	8
		8	5.3	8
Mean (M)		8.1	5.4	8
28		8.1	4.8	7.9
		8	5.1	8
Mean (M)		8.1	5	8

Ocs Mineral salts medium, inoculum and silica gel.

Ot Mineral salts medium, inoculum, test material (2.0 mg/L) and silica gel.

Oc Mineral salts medium, and inoculum.

Oa Mineral salts medium, inoculum and sodium acetate (6.7 mg/L).

Oxygen consumption (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 (%)

	Oxygen Concentration (mg/L)		Biodegradation (%)
Time (days)	Test Substance	Acetate	Test Substance	Acetate
0	0	0	0	0
7	1.3	4.1	30	76
14	1.6	4.4	36	81
21	2.7		61
28	3.1		70

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

2-(1-Oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate was biodegraded by 74% at day 28 in the Closed Bottle test.
Over 60% biodegradation was not achieved in a period of 14 days immediately following the attainment of 10% biodegradation. The window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Hydrolysis of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate however results in the formation of cyclohexanone, diethanolamine and methacrylate. The biodegradation kinetics (lag period, growth rate, yield etc.) of the hydrolysis products are not necessarily same. It is thus possible that individual hydrolysis products meet the time window criterion whereas the biodegradability curve of 2-(1-oxa-4- azaspiro[4.5]dec-4-yl)ethyl methacrylate suggests that the substance is not readily biodegradable. The time window is therefore an inappropriate pass/fail criterion as described for mixtures of chemicals by Richterich and Steber (2001).
2-(1-Oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate should therefore be classified as readily biodegradable.

Executive summary:

In order to assess the biotic degradation of 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate, 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.

2-(1-Oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be noninhibitory to the inoculum. 2-(1-Oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate was biodegraded by 74% at day 28 in the OECD 301 D Closed Bottle test. The substance should therefore be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 0.7 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 81% 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

The substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater