Reaction Mass of 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-octamethyldecahydro-1,6-methanonaphthalen-1-ol

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:

From 2018-01-09 to 2018-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD 301F guideline and under GLP conditions. The validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Specific details on test material used for the study:

None

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Species/Origin: Aerobic activated sludge (microorganisms from a domestic waste¬water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
- Conditioning: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
The washed activated sludge was pre-conditioned in test water for a maximum of 2 days.

Duration of test (contact time):

60 d

Initial conc.:

3.021 other: mg oxygen per mg test item

Based on:

ThOD

Remarks:

NH4

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Surrounding Type: Climatic chamber
- Temperature: 22 °C ± 1°C
- Light Conditions: Darkness
- pH-Value: 6.5 to 6.7 (measured at the start of the test and adjusted to 7.4 with Sodium Hydroxide) 7.2 to 7.8 (measured at day 63)
- Reconstituted Water: The following stock solutions with analytical grade salts was prepared:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; the pH should be 7.4 ± 0.2.
b) 22.5 g MgSO4 x 7 H2O filled up with pure water to 1000 mL volume
c) 36.4 g CaCl2 x 2 H2O filled up with pure water to 1000 mL volume
d) 0.25 g FeCl3 x 6 H2O filled up with pure water to 1000 mL volume
In order to avoid precipitation of iron hydroxide during storage, one drop of concentrated HCl per litre was added to stock solution d) before storage. 10 mL of stock solution a) and 1 mL of the stock solutions b) - d) were combined and filled up to a final volume of 1000 mL with pure water.
- Solubilising agent: Silicone oil AR20 was used as an emulsifying agent. (1%)

TEST SYSTEM
- The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Number of culture flasks/concentration: 2
The test flasks were closed gas-tight by a measuring head. An appropriate CO2-absorber (KOH as an aqueous solution) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

SAMPLING
O2 consumed daily by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 (activated sludge + silicone oil + test water)
- Abiotic sterile control: 1
- Toxicity control: 1
- Other: Procedure control : (Ref. Item + activated sludge + silicone oil + test water)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No data.

Test performance:

No data.

Parameter:

% degradation (O2 consumption)

Value:

54

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

68

Sampling time:

60 d

Details on results:

TEST ITEM: The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThODNH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThODNH4).The mean biodegradation after 28 days was 54% (ThODNH4) and 68% (ThODNH4) after 60 days.
TOXICITY CONTROL: In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% (ThODNH4) biodegradation was noted within 14 days, 53% (ThODNH4) biodegradation after 28 days and 57% (ThODNH4) after 60 days of incubation.
According to the test guideline, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 85% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	PATCHOULI EO FRACTION PATCHOULOL RICH	7.3
2	PATCHOULI EO FRACTION PATCHOULOL RICH	7.2
3	Inoculum control	7.5
4	Inoculum control	7.5
5	Reference item (procedure control)	7.8

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 60 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	0	0	0	0	60	0	60
2	5	5	5	0	85	0	85
3	5	10	5	5	105	0	100
4	20	15	5	5	120	0	120
5	25	35	5	5	130	0	130
6	30	45	10	5	135	0	135
7	40	65	10	10	140	0	145
8	55	75	10	10	145	0	150
9	70	85	10	10	150	0	160
10	85	95	15	10	150	0	160
11	90	105	15	10	150	0	170
12	95	115	15	10	155	0	175
13	100	120	15	10	155	0	180
14	110	130	20	10	160	0	190
15	115	140	20	10	160	0	195
16	120	145	20	15	160	0	200
17	120	155	25	15	165	0	205
18	125	160	25	15	165	0	210
19	130	170	25	20	165	0	215
20	135	175	25	20	170	0	225
21	140	185	25	20	170	0	230
22	140	190	25	20	170	0	240
23	150	195	25	20	170	0	245
24	150	200	25	20	170	0	250
25	155	205	25	20	170	0	250
26	160	205	25	20	175	0	260
27	160	210	25	20	175	0	265
28	165	215	25	20	175	0	275
29	170	220	25	20	180	0	285
30	170	220	25	20	180	0	285
31	170	225	25	20	180	0	285

32	175	225	25	20	180	0	285
33	175	230	25	20	180	0	285
34	175	230	25	20	180	0	285
35	175	235	25	20	185	0	290
36	175	240	25	20	185	0	290
37	180	245	25	20	190	0	290
38	180	245	25	20	190	0	290
39	180	245	25	20	190	0	290
40	185	245	25	20	190	0	290
41	185	245	25	20	190	0	290
42	190	245	25	20	190	0	290
43	190	250	25	20	190	0	290
44	190	250	25	20	195	0	290
45	190	250	30	20	195	0	290
46	195	255	30	20	195	0	290
47	195	255	30	20	195	0	290
48	195	255	30	20	195	0	290
49	195	255	30	20	195	0	290
50	195	255	30	20	195	0	290
51	200	260	30	20	195	0	290
52	200	260	30	20	195	0	290
53	200	260	30	20	195	0	290
54	200	260	30	20	195	0	290
55	200	260	30	20	195	0	290
56	200	260	30	20	195	0	290
57	205	260	30	20	195	0	290
58	205	260	30	20	195	0	290
59	210	260	30	20	200	0	295
60	210	260	30	20	200	0	295

Flasks 1 and 2: PATCHOULI EO FRACTION PATCHOULOL RICH

Flasks 3 and 4: inoculum control

Flask5: reference(procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4:

Time	Percentage Biodegradation^1
(Days)	PATCHOULI EO FRACTION PATCHOULOL RICH^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	35	13
2	1	1	48	17
3	0	2	59	20
4	5	3	67	24
5	7	10	73	26
6	7	12	75	27
7	10	18	76	28
8	15	21	79	29
9	20	24	82	32
10	24	26	81	31
11	25	30	81	33
12	27	33	83	34
13	29	34	83	35
14	31	37	85	37
15	33	40	85	38
16	34	41	83	38
17	33	43	85	39
18	34	45	85	40
19	35	47	83	41
20	37	49	86	43
21	38	52	86	44
22	38	53	86	46
23	42	55	86	47
24	42	57	86	48
25	43	58	86	48
26	45	58	89	50
27	45	60	89	51
28	47	61	89	53
29	48	63	92	55
30	48	63	92	55
31	48	65	92	55

32	50	65	92	55
33	50	66	92	55
34	50	66	92	55
35	50	68	95	56
36	50	69	95	56
37	52	71	98	56
38	52	71	98	56
39	52	71	98	56
40	53	71	98	56
41	53	71	98	56
42	55	71	98	56
43	55	73	98	56
44	55	73	101	56
45	54	72	100	56
46	56	73	100	56
47	56	73	100	56
48	56	73	100	56
49	56	73	100	56
50	56	73	100	56
51	57	75	100	56
52	57	75	100	56
53	57	75	100	56
54	57	75	100	56
55	57	75	100	56
56	57	75	100	56
57	59	75	100	56
58	59	75	100	56
59	60	75	103	57
60	60	75	103	57

           ¹ThOD_NH4of PATCHOULI EO FRACTION PATCHOULOL RICH: 3.021 mg O₂/mg test item
           ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Biodegradation in the Toxicity Control

Percentage Biodegradation:	In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% (ThODNH4) biodegradation was noted within 14 days, 53% (ThODNH4) biodegradation after 28 days and 57% (ThODNH4) after 60 days of incubation.
Conclusion:	According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Abiotic Control

Oxygen Demand:

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test item and toxicity control.

Validity criteria

Inoculum Control:	The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
pH-Value:	The pH-value of the test item flasks at day 63 was 7.2 to 7.3 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
Reference Item:	The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.
Test Item:	The difference of duplicate values for the degradation of the test item at the end of the 10-day window, at day 28 and at day 60 of the test was less than 20%. The difference of duplicate values at days 17, 28 and 60 differed by 10%, 14% and 5%, respectively. The validity criterion was fulfilled.
Toxicity Control:	If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on ThODNH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 37% at day 14; the test item was not inhibitory.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

With a mean biodegradation rate (ThOD-NH4) of 54% after 28 days of incubation, the test item is considered not readily biodegradable.

Executive summary:

The test item Patchouli EO Fraction Patcoulol Rich was investigated for its ready biodegradability in a manometric respirometry test, according to the OECD 301F guideline and under GLP compliance. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThOD_NH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThOD_NH4).The mean biodegradation after 28 days was 54% (ThOD_NH4) and 68% (ThOD_NH4) after 60 days.

Therefore, the test item is considered not readily biodegradable according to test guideline.

The reference item sodium benzoate was sufficiently degraded to 67% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% biodegradation was noted within 14 days and 53% biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Description of key information

With a mean biodegradation rate (ThOD-NH4) < 60% after 28 days of incubation, the test item is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The test item Patchouli EO Fraction Patcoulol Rich was investigated for its ready biodegradability in a manometric respirometry test, according to the OECD 301F guideline and under GLP compliance. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The mean biodegradation of 10% of PATCHOULI EO FRACTION PATCHOULOL RICH was reached at day 7 (ThOD_NH4). At the end of the 10-day window at day 17, the mean degradation of PATCHOULI EO FRACTION PATCHOULOL RICH was 38% (ThOD_NH4).The mean biodegradation after 28 days was 54% (ThOD_NH4) and 68% (ThOD_NH4) after 60 days.

Therefore, the test item is considered not readily biodegradable according to test guideline.

The reference item sodium benzoate was sufficiently degraded to 67% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% biodegradation was noted within 14 days and 53% biodegradation after 28 days of incubation. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradationwas >25% within 14 days.