Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized

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:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17-Jul-92

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

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:

no

Principles of method if other than guideline:

- Principle of test: To measure the biodegradabilty of the test item over 28 days, with an extension to 60 days to test persistence of the test item.
- Short description of test conditions: Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301) were introduced; a) 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), and b) river water instead of an effluent/extract/mixture was used as inoculum.
- Parameters analysed / observed: N/A

GLP compliance:

yes

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): Rhine near Heavedrop, The Netherlands (06-10-2016). The nearest sewage treatment plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Not reported, assuemd ambient
- Storage length: 8 days
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating. 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.
- Pretreatment: N/A
- Concentration of sludge: N/A
- Initial cell/biomass concentration: N/A
- Water filtered: no
- 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: River water from the Rhine spiked with 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 per litre.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used):N/A
- Test temperature: 22 - 24 °C
- pH: 8.0 (starting pH and on day 28)
- pH adjusted: no
- CEC (meq/100 g): not measured
- Aeration of dilution water: 8 days before use
- Suspended solids concentration: N/A
- Continuous darkness: yes
- Other: Ammonium chloride was not added to the river water to prevent nitrification.

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and test substance (2.0 mg/L), and 6 bottles containing river water and sodium acetate (6.7 mg/L).
- Method used to create aerobic conditions: 8 days of aeration before use
- 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:
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: 0, 7, 14, 21, 28, 42 and 60 days.
- Sampling method: 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: ambient temperature, in the dark
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: river water only and river water and silica gel
- Abiotic sterile control: N/A
- Toxicity control: Sodium acetate
- Other: N/A

STATISTICAL METHODS:
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.

ThODNH3(mgO2 /mg)= 16(2C+12 (H-Cl-3N)+3S+2(0.5*P)+(0.5*Na-O))/ MW

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 oxygen demand (ThOD).

Reference substance:

other: sodium acetate

Key result

Parameter:

% degradation (DOC removal)

Value:

68

Sampling time:

60 d

Key result

Parameter:

% degradation (DOC removal)

Value:

41

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

18

Sampling time:

14 d

Details on results:

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 %. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Key result

Parameter:

ThOD

Value:

2.8 g O2/g test mat.

Remarks on result:

other: test item

Key result

Parameter:

ThOD

Value:

0.8 g O2/g test mat.

Remarks on result:

other: reference material

Results with reference substance:

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

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

Time (days)	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	8.1	7.9	8.1	3.2
	8.1	7.8	8.2	3.6
Mean (M)	8.1	7.9	8.2	3.4
14	7.8	7.0	7.8	3.1
	7.8	6.6	7.9	2.9
Mean (M)	7.8	6.8	7.9	3.0
21	7.6	5.5	7.6
	7.8	5.8	7.7
Mean (M)	7.7	5.7	7.7
28	7.7	5.5	7.8
	7.7	5.2	7.8
Mean (M)	7.7	5.4	7.8
42	7.4	4.2
	7.3	4.4
Mean (M)	7.4	4.3
60	7.1	3.3
	6.9	3.0
Mean (M)	7.0	3.2

Oc           River water with nutrients

Ocs        River water with nutrients with silica gel but without test material.

Ot           River water with nutrients, test substance (2.0 mg/L).

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

Table II. Oxygen consumption (mg/L) and the percentages biodegradation of the test item, 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	0.2	4.8	4	89
14	1.0	4.9	18	91
21	2.0		36
28	2.3		41
42	3.1		55
60	3.8		68

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

but not classified as persistent based on the enhanced biodegradability testing scheme.

Conclusions:

The test item was biodegraded by 41% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test the test substance is biodegraded by 68% at day 60. A biodegradation in excess of 60% within the 60-day test period allows classification of the item as not persistent.

Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed (slightly modified OECD 301D) using spiked river water (spiked 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 per litre).  Dissolved oxygen  content was used as the measurand.

 

The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The test item biodegraded by 41% at day 28 in the Closed Bottle test. In the pro­longed Closed Bottle test, the test item was biodegraded by 68% at day 60 (enhanced biodegradability testing). A biodegradation percentage of >60 within a 60-day time period allows classification of the test item as not persistent.

 

The results were considered valid.

Description of key information

Biodegradation: not readily biodegradable (41% biodegradation at day 28), not persistent (68% biodegradation at day 60), OECD 301D, (Anon, 2017).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

In order to assess the biotic degradation, a ready biodegradability test was performed (slightly modified OECD 301D) using spiked river water (spiked 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 per litre). 

The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The test item biodegraded by 41% at day 28 in the Closed Bottle test. In the pro­longed Closed Bottle test, the test item was biodegraded by 68% at day 60 (enhanced biodegradability testing). A biodegradation percentage of >60 within a 60-day time-period allows classification of the test item as not persistent.

The results were considered valid.