Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

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:

The study was conducted between 06 May 2014 and 03 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 310 using the CO2 Headspace Method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test system
A mixed population of sewage sludge micro-organisms was obtained on 06 May 2014 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through course filter paper (first approximate 200 mL discarded).
In order to reduce the inorganic carbon (IC) content of the inoculums, the filtrate was sparged with CO2-free air* for approximately 1 hours whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.2 using 7 M sodium hydroxide and the inoculums allowed to settle for approximately 1 hour prior to removal of an aliquot (2 Litres) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2-free air until use.
*CO2-free air produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb® granules).

Duration of test (contact time):

28 d

Initial conc.:

25.2 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

other: carbon

Details on study design:

Procedure
Preliminary solubility work
Information provided by the Sponsor indicated that the water solubility of the test item was 6.6 mg/L. Therefore preliminary solubility / dispersibility work was performed in order to determine the most suitable method of preparation.

The following preliminary solubility / dispersibility work was performed in order to determine the most suitable method of preparation:
1) Ultrasonication and high shear mixing: An amount of test item (50 mg) was dispersed in 1 L of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 30 minutes. This formed a clear colorless water column with tiny oily globules of test item dispersed throughout and an oily slick on the surface. This was then subjected to high shear mixing (approximately 7500 rpm, 15 minutes) and formed a clear colourless solution.

An amount of test item (50 mg) was dispersed in 500 mL of deionised reverse osmosis purified water with the aid of ultrasonication for 30 minutes. This formed a clear colorless water column with an oily slick on the surface. This was then subjected to high shear mixing (approximately 7500 rpm, 15 minutes) and formed a cloudy homogenous dispersion.

This work confirmed that the test item was insoluble at a suitable concentration in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.

2) Ultrasonication: An amount of test item (2.7 mg) was dispersed in approximately 107 mL of mineral media with the aid of ultrasonication for 15 minutes. This formed a clear colorless media column with an oily layer of test item floating on the surface.

3) Adsoprtion onto an Inert Support: An amount of test item (2.7 mg) was weighed on a filter paper. The filter paper was added to 107 mL of mineral media. This formed a clear colorless media column with an oily slick on the surface and filter paper visible on the bottom of the vessel.

4) Prelimininary Solution in a Volatile Solvent: The addition of a rest solvent stock to glass fibre filter paper was attempted.
An amount of test item (500 mg) was dissolved in acetone (10 mL) and formed a clear colorless solution. An aliquot (43 µL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to approximately 107 mL mineral medium. This formed a clear colorless media column with an oily slick on the surface and filter paper visible on the bottom of the vessel.

From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the most accurate testable dispersion was found to be obtained when using the preliminary solution in a volatile solvent.


Experimental preparation
Test item
An amount of test item (500 mg) was dissolved in 10 mL of acetone to give a 500 mg/10 mL solvent stock solution. An aliquot (54 µL of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to the test vessel containing 107 mL of inoculated mineral medium to give hte required test concentration of 25.2 mg/L, equivalent to 20 mg carbon / L. The test vessels were then sealed using Teflon-lined silicon septa and aluminium crimp caps.
The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 20 mg carbon / L was employed in the study following the recommendations of the test guidelines.

Toxicity control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the study.
Aliquots (54 µL) of the test item stock solution were dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to the test vessel with 107 mL of inoculated mineral medium containing the reference item. The test vessels were then sealed using Teflon lined silicon septa and aluminium crimp caps.
The final concentration in the toxicity control vessels was 25.2 mg test item / L plus 34.3 mg reference item / L, equivalent to 40 mg carbon / L.

Preparation of test system
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, plus a filter paper, 29 replicate vessels.
b) The procedure control containing the reference item (Sodium benzoate) in inoculated mineral medium, plus a filter paper to give a final concentration of 20 mg Carbon / L, 29 replicate vessels.
c) The test item in inoculated mineral medium to give a final concentration of 20 mg carbon / L, 29 replicate vessels.
d) The test item plus the reference item in inoculated mineral medium, to give a final concentration of 40 mg carbon / L to act as a toxicity control, 9 replicate vessels.
A filter paper was added to the inoculums with the prepared inoculums at a final concentration of 100 mL/L.
Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculums control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28).
All vessels were sealed using Teflon lined silicon septa and aluminium crimp caps and incubated in darkness at 20 ± 1 °C with constant shaking at approximately 150 rpm (INFORS Version 2 Multitron ® Incubator).

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

28 d

Details on results:

Definitive test
Validation criteria
The mean TIC in the inoculum control vessels on Day 28 was 0.13 mg/L; equivalent to 0.65 % of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Biodegradation
The test item attained 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
The toxicity control attained 40 % biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Variation in the biodegradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the biodegradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative biodegradation values determined from a single vessel sampled on numerous occasions and as such variation in biodegradation rates on different sampling days was to be expected.

Results with reference substance:

Sodium benzoate attained 87 % biodegradation after 14 days and 93 % biodegradation after 28 days thereby confirming the suitability of the inoculums and test conditions.

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control					Procedure Control					Test Item					Toxicity Control
	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5
0	0.11	0.13	0.11	-	-	0.11	0.11	0.12	-	-	0.13	0.12	0.13	-	-	0.11	0.12	0.10	-	-
2	0.11	0.10	0.10	-	-	1.35	1.41	1.31	-	-	0.12	0.12	0.12	-	-	-	-	-	-	-
6	0.15	0.14	0.13	-	-	1.89	1.82	1.83	-	-	0.15	0.14	0.15	-	-	-	-	-	-	-
8	0.12	0.11	0.12	-	-	1.88	1.93	2.04	-	-	0.14	0.14	0.15	-	-	1.52	1.65	1.59	-	-
10	0.13	0.12	0.12	-	-	1.97	2.04	1.82	-	-	0.15	0.16	0.14	-	-	-	-	-	-	-
14	0.12	0.16	0.13	-	-	2.07	2.00	1.92	-	-	0.15	0.15	0.15	-	-	1.88	1.71	1.93	-	-
16	0.13	0.13	0.12	-	-	1.88	2.04	2.12	-	-	0.17	0.14	0.14	-	-	-	-	-	-	-
21	0.14	0.13	0.17	-	-	1.97	2.05	2.16	-	-	0.15	0.14	0.13	-	-	-	-	-	-	-
28	0.13	0.13	0.14	0.13	0.13	2.22	2.25	1.88	2.01	1.85	0.16	0.15	0.16	0.15	0.14	-	-	-	-	-

R₁– R₅= Replicates 1 to 5

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	59	1	-
6	80	0	-
8	86	1	34
10	85	1	-
14	87	0	40
16	88	1	-
21	89	0	-
28	89	1	-

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Executive summary:

The ready biodegradability of the test item, IFF 215 (Floriane), was assessed according to OECD Test Guideline 310 using the CO2 Headspace Method. The test item attained 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable

Description of key information

The ready biodegradability of the test item, IFF 215 (Floriane) was assessed according to OECD Test Guideline 310 using the Headspace Method. The test item attained 1 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed