Rectified Essential Oil of Pogostemon cablin (Lamiaceae) with high content in alpha and delta guaiene obtained from leaves by distillation.

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 2018-01-08 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. Validity criteria 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, non-adapted

Details on inoculum:

- Species/Origin: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant 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.

Duration of test (contact time):

60 d

Initial conc.:

3.282 other: mg oxygen per mg test item

Based on:

test mat.

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 6.6 (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
- Culturing apparatus: 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)

Remarks:

ThODNH4

Value:

42.5

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

58

Sampling time:

60 d

Details on results:

TEST ITEM: The mean biodegradation of 10% of PATCHOULI EO FRACTION ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThODNH4). The mean biodegradation after 28 days was 43% (ThODNH4) and at test end after 60 days 58% (ThODNH4).
TOXICITY CONTROL: In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% (ThODNH4) biodegradation was noted within 14 days, 45% (ThODNH4) biodegradation after 28 days and 52% after 60 days of incubation.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 85% after 14 days, to 89% after 28 days and to 103 % after 60 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 ALPHA GUAIENE RICH	7.2
2	PATCHOULI EO FRACTION ALPHA GUAIENE RICH	7.4
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	5	5	5	105	0	105
4	10	10	5	5	120	0	120
5	20	15	5	5	130	0	130
6	35	35	10	5	135	0	140
7	45	60	10	10	140	0	145
8	55	75	10	10	145	0	150
9	60	80	10	10	150	0	155
10	65	85	15	10	150	0	160
11	70	90	15	10	150	0	170
12	75	95	15	10	155	0	175
13	85	105	15	10	155	0	180
14	95	115	20	10	160	0	185
15	100	125	20	10	160	0	190
16	105	135	20	15	160	0	190
17	110	140	25	15	160	0	195
18	115	145	25	15	165	0	200
19	120	150	25	20	165	0	205
20	125	155	25	20	170	0	210
21	130	160	25	20	170	0	215
22	135	160	25	20	170	0	220
23	140	165	25	20	170	0	225
24	145	165	25	20	170	0	230
25	145	165	25	20	170	0	240
26	150	170	25	20	175	0	240
27	155	170	25	20	175	0	245
28	160	170	25	20	175	0	250
29	165	175	25	20	180	0	255
30	165	175	25	20	180	0	255
31	170	180	25	20	180	0	260

32	175	180	25	20	180	0	265
33	175	180	25	20	180	0	265
34	175	180	25	20	180	0	270
35	180	185	25	20	185	0	275
36	180	185	25	20	185	0	275
37	185	190	25	20	190	0	280
38	185	190	25	20	190	0	280
39	190	190	25	20	190	0	285
40	190	190	25	20	190	0	285
41	190	190	25	20	190	0	285
42	195	190	25	20	190	0	285
43	195	195	25	20	190	0	285
44	195	195	25	20	195	0	285
45	195	195	30	20	195	0	285
46	195	200	30	20	195	0	285
47	200	200	30	20	195	0	285
48	200	200	30	20	195	0	285
49	200	200	30	20	195	0	285
50	200	200	30	20	195	0	285
51	200	200	30	20	195	0	285
52	200	205	30	20	195	0	285
53	205	205	30	20	195	0	285
54	205	205	30	20	195	0	285
55	205	210	30	20	195	0	285
56	210	210	30	20	195	0	285
57	210	210	30	20	195	0	285
58	210	215	30	20	195	0	285
59	215	220	30	20	200	0	285
60	215	220	30	20	200	0	285

Flasks 1 and 2: PATCHOULI EO FRACTION ALPHA GUAIENE 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 ThODNH4:

Time	Percentage Biodegradation^1
(Days)	PATCHOULI EO FRACTION ALPHA GUAIENE RICH^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	35	12
2	1	1	48	16
3	0	0	59	20
4	1	1	67	23
5	4	3	73	25
6	8	8	75	26
7	10	15	76	27
8	13	19	79	28
9	15	21	82	29
10	16	21	81	29
11	17	23	81	31
12	19	24	83	32
13	22	27	83	33
14	24	30	85	34
15	25	32	85	35
16	26	35	83	34
17	27	35	82	35
18	28	37	85	36
19	29	38	83	36
20	31	39	86	37
21	32	41	86	38
22	34	41	86	39
23	35	42	86	40
24	37	42	86	41
25	37	42	86	43
26	38	44	89	43
27	40	44	89	44
28	41	44	89	45
29	43	45	92	46
30	43	45	92	46
31	44	46	92	47

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

¹ThOD_NH4of PATCHOULI EO FRACTION ALPHA GUAIENE RICH: 3.282 mg O₂/mg test item
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Biodegradation in the Toxicity Control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% (ThOD_NH4) biodegradation was noted within 14 days, 45% (ThOD_NH4) biodegradation after 28 days and 52% after 60 days of incubation.

Abiotic Control

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 7.4 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 8%, 3% and 1%, 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 34% 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 42.5% after 28 days of incubation, the test item is considered not readily biodegradable.

Executive summary:

The test item Patchouli EO Fraction Alpha Guaiene 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 ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThOD_NH4). The mean biodegradation after 28 days was 43% (ThOD_NH4) and at test end after 60 days 58% (ThOD_NH4). Therefore, the test item is considered not readily biodegradable according to test guideline.

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.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% biodegradation was noted within 14 days and 45% 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.

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 Alpha Guaiene 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 ALPHA GUAIENE 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 ALPHA GUAIENE RICH was 31% (ThOD_NH4). The mean biodegradation after 28 days was 43% (ThOD_NH4) and at test end after 60 days 58% (ThOD_NH4).Therefore, the test item is considered not readily biodegradable according to test guideline.

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.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 34% biodegradation was noted within 14 days and 45% 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.