Reaction mass of 4-methyl-1-oxaspiro[5.5]undec-3-ene and 4-methyl-1-oxaspiro[5.5]undec-4-ene and 4-methylidene-1-oxaspiro[5.5]undecane

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:

9 January 2018 to 7 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

although a minor deviation from guideline is reported (see 'Any other information on materials and methods incl. tables'), this is considered to have not affected the reliability of the test.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992 / use of silica gel method

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

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)

Version / remarks:

1994

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, The Ministry of Health, Welfare and Sport, Den Haag, The Netherlands (d.d. 2014-10-06).

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum: River water was sampled from the Rhine near Heveadorp, The Netherlands (03-01-2018). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Pretreatment: The river water was aerated for 7 days before use to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
- Adaptation: The inoculum was not pre-exposed to the test substance.

Initial conc.:

2 mg/L

Based on:

test mat.

Initial conc.:

5.8 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of the medium: The river water used in the Closed Bottle test was spiked per liter of water with 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 not added to the river water to prevent nitrification.
- Test concentrations: 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 (Davisil grade 636, pore size 60A, 35-60 mesh particle size) 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 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.
- Reference substance: Sodium acetate was added to the bottles at a concentration of 6.7 mg/L (5.36 mg/L based on ThOD). For preparations of reference solutions a stock solution of 1.0 g/L was used.
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers. Use was made of 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. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.
- Toxicity control: 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. 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.
- 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.
- Temperature: The temperature was measured and recorded with a sensor connected to a data logger. Temperatures were within the prescribed temperature range of 22 to 24°C (22.7 to 23.0 °C).
- pH: The pH was measured using a Eutech pH meter. The pH of the media was 8.0 at the start of the test. The pH of the media at day 28 was 8.0 (controls and test).

Reference substance:

acetic acid, sodium salt

Remarks:

(6.7 mg/L based on test mat., 5.36 mg/L based on ThOD)

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Details on results:

ThOD:
ThOD of the test substance, based on molecular formula of the isomers (99.1%), is 2.9 g O2/g. ThOD of sodium acetate is 0.9 mg O2/mg.

Biodegradation:
The test material was not biodegraded (2% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. Lack of biodegradation does not mean that Oxaspirane-819 (multi-constituent) is recalcitrant in nature. The stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Results with reference substance:

The reference compound sodium acetate was biodegraded for 85% at day 14.

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

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.6	8.6	8.6	8.6
	8.6	8.6	8.6	8.6
Mean	8.6	8.6	8.6	8.6
7	8.1	8.1	8.0	4.0
	8.1	8.1	8.1	4.2
Mean	8.1	8.1	8.1	4.1
14	7.8	7.9	7.9	3.3
	7.9	7.9	7.9	3.3
Mean	7.9	7.9	7.9	3.3
21	7.5	7.5	7.4	-
	7.5	7.4	7.5	-
Mean	7.5	7.5	7.5	-
28	7.3	7.3	7.3	-
	7.4	7.3	7.3	-
Mean	7.4	7.3	7.3	-

Ocs: River water with nutrients and silica gel.

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

Oc: River water with nutrients.

Oa: River water with nutrients and sodium acetate (6.7 mg/L).

Table: 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 (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.0	4.0	0	74
14	0.0	4.6	0	85
21	0.0	-	0	-
28	0.1	-	2	-

 

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'.

Interpretation of results:

not readily biodegradable

Conclusions:

The substance is not readily biodegradable under the conditions of the test.

Executive summary:

The biodegradation potential of the substance was examined in a study according to OECD TG 301D (Closed Bottle test) and in compliance with GLP criteria. About 2 mg/L test substance (ca. 6 mg/L based on ThOD) was inoculated for 28 days under aerobic conditions and in the dark with water sampled from the river Rhine. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 0 and after 7, 14, 21, and 28 exposure and the cumulative percentages biodegradation calculated from the oxygen depletion ratio BOD/ThOD. All validity criteria were met. The test substance was biodegraded by 2% at day 28 in the Closed Bottle test. The substance therefore should be classified as not readily biodegradable.

Description of key information

The biodegradation potential of the substance was examined in a study according to OECD TG 301D (Closed Bottle test) and in compliance with GLP criteria. About 2 mg/L test substance (ca. 6 mg/L based on ThOD) was inoculated for 28 days under aerobic conditions and in the dark with water sampled from the river Rhine. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 0 and after 7, 14, 21, and 28 exposure and the cumulative percentages biodegradation calculated from the oxygen depletion ratio BOD/ThOD. All validity criteria were met. The test substance was biodegraded by 2% at day 28 in the Closed Bottle test. The substance therefore should be classified as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater