Sage, Salvia officinalis, ext.

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 2017-12-08 to 2018-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on March 2017

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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 set to deposit for 15 minutes, then 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.:

102.9 mg/L

Based on:

test mat.

Remarks:

Corresponding to 306.8 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight.
- Reconstitued pure water: Analytical grade salts were added to pure water to prepare the following stock solutions:
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-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
- Solubilising agent: silicone oil AR 20 at a concentration of about 1% of the final flask volume were added to all flasks in order to improve the bioavailability of the mixture constituents.
- Test temperature: 22°C ± 1°C
- pH: 8.0 (measured at the start of the test and adjusted to pH 7.6 with Hydrochloric Acid), 7.2 to 7.6 (measured at the end of the test)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The closed test flasks were incubated in a climatised chamber under continuous stirring.
- Number of culture flasks/concentration: 2
The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) 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.
- Measuring equipment: BSB/BOD-Sensor- System, Aqualytic Dortmund, Germany

SAMPLING
O2 consumption measured daily.

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:

34

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

53

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

60

Sampling time:

35 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

72

Sampling time:

60 d

Details on results:

The mean biodegradation of 10% of Sage dalmatian oil was reached at day 4 (ThODNH4). At the end of the 10-day window at day 14, the mean degradation of Sage dalmatian oil was 34% (ThODNH4) and therefore the 10 day window criterion was not passed. After 28 days the mean biodegradation was 53% (ThODNH4) and the test was prolonged until day 60. Therefore, Sage dalmatian oil is considered to be not readily biodegradable based on ThODNH4. Still 72% were reached after 60 days
Results for all flasks are illustrated in the attached figure 1.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 86% after 14 days, to 94% after 28 and 108% 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	Sage dalmatian oil	7.5
2	Sage dalmatian oil	7.4
3	Inoculum control	7.2
4	Inoculum control	7.2
5	Reference item (procedure control)	7.6

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	75	0	80
3	30	30	5	0	95	0	100
4	50	50	10	5	120	0	125
5	55	65	10	5	130	0	135
6	75	75	15	10	140	0	145
7	80	80	15	10	145	0	155
8	90	85	15	10	150	0	165
9	95	90	15	15	155	0	180
10	105	105	15	15	155	0	195
11	105	110	15	15	160	0	205
12	110	110	20	15	160	0	215
13	115	115	20	15	160	0	225
14	120	125	20	15	165	0	235
15	125	130	20	15	165	0	245
16	130	135	20	15	165	0	245
17	135	140	20	15	170	0	250
18	140	145	20	15	170	0	255
19	140	150	25	15	175	0	260
20	145	155	25	20	180	0	265
21	150	160	25	20	180	0	270
22	155	165	25	20	185	0	275
23	160	170	25	20	185	0	280
24	170	175	25	20	185	0	280
25	170	180	25	20	185	0	280
26	170	185	25	20	185	0	285
27	175	190	25	20	185	0	285
28	175	195	25	20	185	0	290
29	180	200	30	20	190	0	290
30	185	205	30	20	190	0	295
31	190	205	30	20	190	0	295
32	195	210	30	20	190	0	295
33	195	215	30	20	190	0	295
34	200	215	30	20	195	0	295
35	200	225	30	20	195	0	295
36	205	225	30	20	195	0	295
37	210	225	30	20	195	0	295
38	215	225	30	20	195	0	295
39	220	225	30	20	195	0	295
40	220	230	30	20	195	0	295
41	225	230	30	20	200	0	295
42	225	230	30	20	200	0	295
43	230	230	30	20	200	0	295
44	230	235	30	20	200	0	295
45	230	240	30	20	200	0	295
46	230	240	30	20	200	0	295
47	230	240	30	20	200	0	295
48	230	240	30	20	200	0	295
49	230	240	30	20	200	0	295
50	235	240	30	20	200	0	295
51	235	240	30	20	200	0	295
52	235	245	30	20	205	0	295
53	240	245	30	20	205	0	295
54	240	245	30	20	210	0	295
55	240	245	30	20	210	0	295
56	240	245	30	20	210	0	295
57	240	245	30	20	210	0	295
58	240	245	30	20	210	0	295
59	245	245	30	20	210	0	295
60	245	245	30	20	210	0	295

Flasks 1 and 2: Sage dalmatian oil

Flasks 3 and 4: inoculum control

Flask 5: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)	Sage dalmatian oil^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	35	13
2	1	1	42	16
3	9	9	54	21
4	14	14	65	25
5	15	19	71	27
6	20	21	74	28
7	22	22	77	30
8	25	24	80	32
9	26	25	81	35
10	29	30	81	38
11	29	31	84	40
12	30	30	83	42
13	32	32	83	44
14	33	35	86	46
15	35	37	86	48
16	36	39	86	48
17	38	40	89	49
18	40	42	89	50
19	39	43	90	51
20	40	44	92	51
21	41	45	92	52
22	43	47	94	53
23	44	48	94	54
24	48	50	94	54
25	48	52	94	54
26	48	53	94	55
27	49	55	94	55
28	49	57	94	56
29	50	58	96	56
30	52	59	96	57
31	53	59	96	57
32	55	61	96	57
33	55	62	96	57
34	57	62	99	57
35	57	66	99	57
36	58	66	99	57
37	60	66	99	57
38	61	66	99	57
39	63	66	99	57
40	63	67	99	57
41	65	67	102	57
42	65	67	102	57
43	66	67	102	57
44	66	69	102	57
45	66	71	102	57
46	66	71	102	57
47	66	71	102	57
48	66	71	102	57
49	66	71	102	57
50	68	71	102	57
51	68	71	102	57
52	68	72	105	57
53	70	72	105	57
54	70	72	108	57
55	70	72	108	57
56	70	72	108	57
57	70	72	108	57
58	70	72	108	57
59	71	72	108	57
60	71	72	108	57

¹ThOD_NH4of Sage dalmatian oil: 2.982 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, 46% biodegradation was noted within 14 days, 56% biodegradation after 28 days and 57% biodegradation after 60 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.

Abiotic Control

The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use 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 25 mg O2/L and thus not greater than 60 mg O2/L within 60 days as required by the test guideline.
pH-Value:	The pH-value of the test item flasks at the end of the test were 7.5 and 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 test was less than 20%. The difference of duplicate values at day 60 differed by 1%. The validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Sage Dalmaian oil is considered to be not readily biodegradable after 28 days, bu reached a degradation of 72% after 60 days.

Executive summary:

The test item Sage dalmatian oil was investigated for its ready biodegradability in a manometric respirometry test over a period of 60 days. 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 Sage dalmatian oil was reached at day 4 (ThOD_NH4). At the end of the 10-day window at day 14, the mean degradation of Sage dalmatian oil was 34% (ThOD_NH4) and therefore the 10 day window criterion was not passed.Due to the fact that the substance is identified as an UVCB or as a complex, multi-constituent substance with structurally similar constituents, the 10-day window condition may be waived and the pass level applied at 28 days according to section 4.1.2.9.5 of CLP COMMISSION REGULATION (EU) No 286/2011 of 10 March 2011. After 28 days the mean biodegradation was 53% (ThOD_NH4) and the test was prolonged until day 60. The mean biodegradation at test end after 60 days was 72% (ThOD_NH4).

Therefore, Sage dalmatian oil is considered to be not readily biodegradable based on ThOD_NH4. Still 72% were reached after 60 days.

Validity criteria:

The oxygen demand of the inoculum control (medium and inoculum) was 25 mg O₂/L and thus not greater than 60 mg O₂/L within 60 days as required by the test guideline. The pH-value of the test item flasks at the end of the test were 7.5 and 7.4, and therefore within the range of pH 6.0 to 8.5 as required by the atest guideline. 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. The difference of duplicate values for the degradation of the test item at the end of the test was less than 20%. The difference of duplicate values at day 60 differed by 1%.

The validity criterion was fulfilled.

Description of key information

According to the test guideline, the registered substance is considered not readily biodegradable because the biodegradation was 53% after 28 days. Nevertheless, the biodegradation reached 72% after 60 days of test.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater