Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

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:

2016 - 2017

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. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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: +4 ºC in the dark and under nitrogen.
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- 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: Accurate administering of the test substance was accomplished by preparing a solid stock of 3.1 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.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: As above.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a solid via silica gel.

OTHER SPECIFICS: 0.2 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.1 mg/L.

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

River water was sampled from the Rhine near Heveadorp, The Netherlands (06-10-2016). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Rhine near Heveadorp, The Netherlands. Collection procedures not reported.
- Laboratory culture: n/a
- Method of cultivation: n/a
- Storage conditions: The river water was aerated for 7 days before use to reduce the endogenous respiration.
- Storage length: 7 days.
- Preparation of inoculum for exposure: River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. 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.
- Pretreatment: n/a
- Concentration of sludge: n/a
- Initial cell/biomass concentration: n/a
- Water filtered: no (particles removed by sedimentation prior to use).
- Type and size of filter used, if any: n/a

Duration of test (contact time):

60 d

Initial conc.:

2.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Natural water spiked with nutrients at the following rate (per L of 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.
- Additional substrate: n/a
- Solubilising agent (type and concentration if used): Test item coated on to silica gel.
- Test temperature: 22 - 24 ºC.
- pH: 8.0
- pH adjusted: no
- CEC (meq/100 g): n/a
- Aeration of dilution water: Yes, for 7 days prior to test initiation. No aeration during incubation phase.
- Suspended solids concentration: n/a
- Continuous darkness: Yes
- Other: n/a

TEST SYSTEM
- Culturing apparatus: 0.3 L BOD (biological oxygen demand) bottles
- 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 test substance, and 6 bottles containing river water and sodium acetate.
- Method used to create aerobic conditions: Innoculum water aerated for 7 days prior to test initiation. BOD bottles filled with zero head-space.
- 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, 28. Extended to Day 60.
- Sampling method: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at the specified sampling intervals. Day 60 measurements were taken from the Day 28 vessels. A Day 28, analysis was conducted 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 (to allow analysis at Day 60).
- Sterility check if applicable: n/a
- Sample storage before analysis: n/a
- Other: n/a

CONTROL AND BLANK SYSTEM
- Inoculum blank: River water only
- Abiotic sterile control: n/a
- Toxicity control: Sodium acetate at a rate of 6.7 mg/L.
- Other: "vehicle" control using river water and silica gel (0.2 g, as used to introduce test item to the treatment flasks)

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

Remarks:

Batch # BCBP8197V (Sigma-Aldrich)

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

0 d

Parameter:

% degradation (O2 consumption)

Value:

18

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

25

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

33

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

40

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

40

Sampling time:

42 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

43

Sampling time:

60 d

Details on results:

Test item was biodegraded (based on ThOD) by 40 % at Day 28. Test tem is not readily biodegradable. The test item had biodegraded by 43 % at Day 60, indicating that the test item is partially biodegradable.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 91 %.

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	1.2	4.8	18	89
14	1.6	4.9	25	91
21	2.1	-	33	-
28	2.6	-	40	-
42	2.6	-	40	-
60	2.8	-	43	-

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was biodegraded by 40 % at Day 28 in the Closed Bottle test (Table 1). In the prolonged Closed Bottle test this test substance was biodegraded by 43 % 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. A plateau of biodegradation was not reached at the end of the test indicating that the test item could be degraded completely in time.

The validity of the test is demonstrated by an endogenous respiration of 0.9 mg/L at Day 28 (Table 1). 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:

OECD 301D (2017) - The biodegradability of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexen 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 2.1 mg/L)

Bottles containing river water, nutrients and sodium acetate with silica gel (reference substance at a concentration of 6.7 mg/L)

Duplicate bottles were removed for oxygen concentration/ consumption analysis from each test group at Day 0, 7, 14 and 28 ( extended to Day 42 and 60 for the test item group).

Oxygen consumption of the test item at Day 28 and Day 60 was 2.6 and 2.8 mg/L, respectively. With a ThOD of 3.1 mg/L, these consumption values equated to degradation values of 40 and 43 % at Day 28 and 60, respectively. 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.

This closed bottle biodegradation study is acceptable and satifies the validity criteria outlined in OECD 301D.

Description of key information

Not readily biodegradable; OECD 301D, Anon. (2017)

Constituent known to be readily biodegradable (ECHA, 2010)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In a study conducted in accordance with OECD 301D, the biodegradability of the test item 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 2.1 mg/L)

Bottles containing river water, nutrients and sodium acetate with silica gel (reference substance at a concentration of 6.7 mg/L)

Duplicate bottles were removed for oxygen concentration/ consumption analysis from each test group at Day 0, 7, 14 and 28 ( extended to Day 42 and 60 for the test item group).

Oxygen consumption of the test item at Day 28 and Day 60 was 2.6 and 2.8 mg/L, respectively. With a ThOD of 3.1 mg/L, these consumption values equated to degradation values of 40 and 43 % at Day 28 and 60, respectively. 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.

This closed bottle biodegradation study is acceptable and satifies the validity criteria outlined in OECD 301D.