Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in acordance with international guideines and in accordance with GLP. The validity criteria of the guideline was met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Assumed stable (silica gel)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): River Rhine (nr. Hevadorp, NL), sampled on 06 Oct 2016, 3 km downstream from domestic STP.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Stored aerobically prior to use.
- Storage length: 7 days.
- Preparation of inoculum for exposure: Particles removed by sedimentation after 1 day storage.
- Pretreatment: 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.
- Concentration of sludge: N/A
- Initial cell/biomass concentration: N/A
- Water filtered: no (particles removed through sedimentation)
- Type and size of filter used, if any: N/A

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 CONDITIONS
- Composition of medium: natural river water (spiked, per L 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.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22 - 24 °C
- pH: 8.0 (test start and end)
- pH adjusted: No
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Yes, for 7 days prior to test initiation.
- Suspended solids concentration: N/A
- Continuous darkness: Yes
- Other: N/A

TEST SYSTEM
- Culturing apparatus: 0.30 L biological oxygen demand bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles (river water), 10 bottles (river water + silica gel), 10 bottles (river water + test substance), 6 bottles (river water + sodium acetate)
- Method used to create aerobic conditions: River water aerated for 7 days prior to test initiation. Test conducted in closed vessels.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530). The pH was measured using a Eutech Cyberscan pH11 pH meter. The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: Day 0, 7, 14, 21 and 28. Test extended using Day 28 vessels to include Day 42 and 60.
- Sampling method: 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 in the bottles of day 28 using 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
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, river water only
- Abiotic sterile control: No
- Toxicity control: No
- Other:

STATISTICAL METHODS:

Calculation of endogenous respiration;

The endogenous respiration (oxygen depletion in the control) was calculated as follows; Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28) Mc is the mean oxygen level in the control bottle with river water.

Calculation of the theoretical oxygen demand (ThOD);

The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights as follows;

ThOD NH3 (mg O2 / mg) = 16 (2C x 0.5 x (H x Cl x 3N) x 3S x 2.5P x 0.5Na x O) / MW

Calculation of the biochemical oxygen demand (BOD)

Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:

Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma

Mc or cs is the mean oxygen level in the control bottles with and without silica gel n-days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.

The 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.

Calculation of the biodegradation percentages;

The biodegradation was calculated as the ratio of the BOD to the theoretical demand (ThOD).

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

12

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

37

Sampling time:

42 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

48

Sampling time:

60 d

Details on results:

Test item was biodegraded (based on ThOD) by 12 % at Day 28. Test tem is not readily biodegradable. The test item had biodegraded by 48 % at Day 60, indicating that the test item is partially biodegradable.

Results with reference substance:

Sodium acetate was biodegraded by 91 % at Day 14.

Table 1: Oxygen Consumption (mg/L) and the Percentage Biodegradation of the Test Item and Sodium Acetate (based on BOD/ ThOD)

Time (days)	Oxgen Consumption (mg/L)		Biodegradation (%)
	Test Substance	Sodium acetate	Test Substance	Sodium acetate
0	0.0	0.0	0	0
7	0.0	4.8	0	89
14	0.1	4.9	2	91
21	0.3	-	5	-
28	0.7	-	12	-
42	2.2	-	37	-
60	2.9	-	48	-

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was biodegraded by 12% at day 28 in the Closed Bottle test (Table I). In the prolonged Closed Bottle test this test substance was biodegraded by 48% at day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrates that this substance is partially biodegradable.

The validity of the test is demonstrated by an endogenous respiration of 0.9 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 91. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Executive summary:

The biodegradability of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed in an OECD 301D Closed Bottle test.

The test substance was exposed to a relatively low numbers of microorganisms present in river water, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

Bottles containing river water and nutrients with silica gel (carrier substance)

Bottles containing river water and nutrients only (control)

Bottles containing river water, nutrients and test item with silica gel (prepared at a concetration of 2 mg/L)

Bottles containing river water, nutrients and sodium acetate with silica gel (reference substance)

Duplicate bottles were removed for theoretical oxygen demand (ThOD) analysis from each test group at Day 0, 7, 14 and 28 (42 and 60 also for test item group).

The calculated theoretical oxygen demands (ThOD) of the test item and reference substance was 3.0 g/g (mean result of the two known constituents) and 0.8 g/g, respectively, at Day 28. This equated to biodegradation values of 12 and 91 %, respectively. In the prolonged Closed Bottle test this test substance was biodegraded by 48% at Day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrates that this substance is partially biodegradable.

Description of key information

OECD 301D, 2017 - The biodegradability of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed in an OECD 301D Closed Bottle test.

The test substance was exposed to a relatively low numbers of microorganisms present in river water, under aerobic conditions for a period of at least 28 days, with the test substance being the sole carbon and energy source.

Four test groups were prepared;

Bottles containing river water and nutrients with silica gel (carrier substance)

Bottles containing river water and nutrients only (control)

Bottles containing river water, nutrients and test item with silica gel (prepared at a concetration of 2 mg/L)

Bottles containing river water, nutrients and sodium acetate with silica gel (reference substance)

Duplicate bottles were removed for theoretical oxygen demand (ThOD) analysis from each test group at Day 0, 7, 14 and 28 (42 and 60 also for test item group).

The calculated theoretical oxygen demands (ThOD) of the test item and reference substance was 3.0 g/g (mean result of the two known constituents) and 0.8 g/g, respectively, at Day 28. This equated to biodegradation values of 12 and 91 %, respectively. In the prolonged Closed Bottle test this test substance was biodegraded by 48% at Day 60. The test item should therefore not be classified as readily biodegradable. However, the biodegradation reached at day 60 demonstrates that this substance is partially biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Based on the screening test data submitted (OECD 301D, extended to 60 days), the substance can not be concluded as not persistent. Biodegradation was <60 % ThOD at day 28 (not readily biodegradable) and day 60 (potentially persistent).