Essential oil of Cistus ladaniferus L (Cistaceae) obtained from stems and 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:

2016-11-08 to 2017-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD 301F guideline. All the validity criteria were successful.

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 (incl. QA statement)

Remarks:

14 October 2013

Specific details on test material used for the study:

None

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Remarks:

Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature

Details on inoculum:

- Source of inoculum/activated sludge: The inoculum was a fresh sample of activated sludge and was collected from the aeration tank of a sewage treatment plant receiving predominantly domestic sewage.
- Sample site: Water treatment plant "STEP de Lille", 33290 Blanquefort (France).
- Laboratory culture: A filtration through a fine sieve (about 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in a mineral medium until sludge was considered free from excess substrate or inhibitor.
- Inoculum preconditioning: Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST ITEM PREPARATION:
- the test item was poorly soluble in water and in order to improve the dissolution the test item was dissolved in silicone oil AR 20 up to final concentration of 1 % v/v. Around 25 mg of test item was stirred with 2.5 mL silicone oil AR 20 by magnetically stirring until completely dissolved. Then, this solution was directly supplemented or completed into 250 mL with mineral medium and sludge directly in each test vessels.
The test medium was the mineral medium, a weakly saline aqueous medium
The test item "CISTUS ORGANIC OIL” was tested at 100 mg/L in mineral medium containing 1 % v/v of silicone oil AR 20.

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301F guideline
- Test temperature: The test was performed in darkness between 22.0 to 22.5 °C (constant temperature +/- 1°C).
- Mineral medium - pH: 7.6
- Inoculum pH: 6.9
- Dissolved oxygen: 9.2 mg/L
- Suspended solids concentration: 3.65 g/L
- Continuous darkness: Yes

TEST DETAILS
- Culturing apparatus: WTW Oxitop
- Number of culture flasks/concentration: 3 flasks (test item, silicone oil 1% and inoculum)
- Test performed in closed vessels: Yes
- A measured volume of inoculated medium containing a known concentration of the test item (100 mg/L) as the nominal sole source of Carbon, was stirred in a closed vessel at a constant temperature up to 28 days. The experiment was prolonged up to 60 days.
- Details of trap for CO2: Microbial respiration consumes O2 and releases CO2 which were absorbed by soda lime pellets present in the headspace of flasks. This produced a decrease in pressure in the flask proportional to the amount of oxygen taken up by the microbial population during biodegradation, which was expressed as a percentage of ThOD (Theoretical Oxygen Demand).
- Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without the test item. Furthermore, in order to check the procedure, a reference item control (sodium benzoate + inoculum) and a toxicity control (sodium benzoate + inoculum + test item+ silicone oil AR 20) were run in parallel. Moreover, a control with silicone oil AR 20 1% was performed to check that silicon was non toxic to bacteria and non biodegradable.
The pH of the contents was measured at the start and the end of the experiment.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; 3 flasks (inoculum only)
- Inoculum silicone control: 3 flasks (inoculum silicone oil AR 20 1% v/v)
- Reference item: 2 flasks (Sodium benzoate and inoculum)
- Toxicity control: Yes; 2 flasks (test item, silicone oil AR 20 1% v/v, sodium benzoate and inoculum)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

An elementary analysis of the test item (non GLP data) has been performed and gave the following results:
- % carbon = 81.16 and 81.32 for the measurement 1 and the measurement 2 respectively
- % hydrogen = 10.74 and 10.76 for the measurement 1 and the measurement 2 respectively
- % nitrogen = Absence for the measurement 1 and 2
- % oxygen = 7.61 and 7.57 for the measurement 1 and the measurement 2 respectively

Key result

Parameter:

% degradation (O2 consumption)

Value:

55.07

St. dev.:

6.48

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

63.31

St. dev.:

3.8

Sampling time:

60 d

Details on results:

Biodegradation of the test item:
The test item “CISTUS ORGANIC OIL” contains no nitrogen according to the elementary analysis, the evaluation of biodegradation has to be based in the following expressed as ThODNH4.
The degradation for the 10-day window beginning was reached on day 3, but at the end of the 10-day window (reaching at least 60%), on day 13, the mean degradation was 38.00% following the ThODNH4, therefore the 10-day window failed.
The degradation rate of the test item “CISTUS ORGANIC OIL” reached 55.07%, following the ThODNH4, after 28 days of incubation.
According to the study monitor, and as it had been anticipated in the study plan, the testing period was extended to 60 days. After 60 days, the degradation rate of the test item “CISTUS ORGANIC OIL” reached 63.31%, following the ThODNH4.
According to the OECD guideline 301F, the test item is not considered to be biodegradable, nethertheless, on day 60, the degradation rate of the test item “CISTUS ORGANIC OIL” reached 63.31%, following the ThODNH4. From the 42nd day, the degradation rate of the test item reached 60.37% to 63.31% at the end of the test: 60 days.

Biodegradation of the Toxicity Control:
A biodegradation of 41.25% and 40.49% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThODNH4.
At the end on the test, 60 days of incubation, the results were respectively 64.29% and 66.05%.
Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge micro-organism because degradation was superior to 25% within 14 days (validity criterion)

Results with reference substance:

The reference item “Sodium benzoate” was degraded up to 82.91% and 78.72%( mean value: 81.82%) after 14 days and 77.89% in the both replicates after 60 days of incubation at the end of the test. Therefore, it confirms the suitability of the used aerobic sludge inoculum.

Table 5.2.1/2: pH values

Treatment	Flask number	pH at the start	pH at the end of the test (60 days)
Inoculum Control	3	7.6	7.4
	9	7.6	7.4
	1	7.6	7.4
Reference Item	5	7.6	7.9
	8	7.6	7.9
Test Item	17	7.6	7.5
	18	7.6	7.5
	20	7.6	7.5
Toxicity Control	22	7.6	8.0
	16	7.6	8.0
Inoculum silicon Control	2	7.5	7.4
	24	7.5	7.4
	13	7.5	7.4

 

Table 5.2.1/3: Percentage (%) Biodegradation during 60 days for Toxicity Control and Test item (“PYCNOGENOL”) with ThOD_NH4=1.524 mgO₂/mg Test item

Time (Days)	Tox Control		Test item
	Flask 22	Flask 16	Flask 17	Flask 18	Flask 20	Average
0	0,00	0,00	0,00	0,00	0,00	0,00
1	6,03	5,66	0,00	0,00	0,00	0,00
2	13,45	15,94	0,47	0,00	0,00	0,16
3	15,58	17,34	7,09	4,74	2,86	4,89
4	18,78	19,83	11,83	10,42	7,09	9,78
5	21,97	22,67	17,04	15,63	11,83	14,83
6	25,39	25,39	21,30	19,42	15,63	18,78
7	28,40	28,40	24,63	23,19	19,42	22,41
8	30,92	30,41	29,84	27,45	22,72	26,67
9	33,51	32,50	33,37	31,01	25,34	29,91
10	35,67	34,41	36,26	32,90	27,69	32,28
11	36,93	36,67	38,61	35,25	30,07	34,64
12	38,33	38,08	40,49	37,13	31,49	36,37
13	39,49	39,24	42,37	38,68	32,96	38,00
14	41,25	40,49	44,39	40,02	34,64	39,68
15	42,40	41,15	45,26	41,57	35,85	40,89
16	43,47	42,47	47,03	42,66	36,95	42,21
17	44,55	43,30	48,14	44,10	37,72	43,32
18	46,56	44,55	49,82	45,78	39,06	44,89
19	47,71	45,19	51,68	46,98	39,58	46,08
20	48,79	45,77	53,46	48,42	40,69	47,52
21	50,05	46,28	54,81	49,76	41,70	48,76
22	51,63	46,85	56,25	50,87	42,47	49,87
23	52,46	47,43	56,69	52,65	43,58	50,97
24	53,72	48,19	57,69	54,33	44,59	52,21
25	55,05	48,76	58,13	55,78	45,36	53,09
26	55,88	49,59	58,57	56,22	46,47	53,75
27	57,21	50,42	59,34	56,99	47,24	54,53
28	58,54	51,00	59,78	57,76	47,68	55,07
29	59,55	52,01	60,45	58,10	49,36	55,97
30	59,95	52,41	59,98	58,30	49,56	55,95
31	60,55	53,01	60,45	58,77	50,71	56,64
32	61,38	53,59	60,89	58,87	50,81	56,85
33	62,14	54,60	61,22	59,88	51,81	57,64
34	62,54	55,50	61,42	59,74	52,35	57,84
35	63,29	56,25	61,42	60,42	52,69	58,18
36	63,54	56,76	62,10	60,42	53,36	58,62
37	63,95	57,66	61,63	60,28	53,90	58,60
38	64,20	58,17	62,30	60,95	54,57	59,27
39	64,52	58,74	62,40	61,39	55,01	59,60
40	64,78	59,25	62,40	61,39	55,34	59,71
41	65,10	59,57	62,84	61,83	55,11	59,92
42	65,35	60,33	62,84	62,16	56,12	60,37
43	65,18	60,65	62,60	61,93	56,22	60,25
44	65,61	61,33	62,84	62,84	57,12	60,93
45	65,43	61,91	62,94	62,94	57,22	61,03
46	65,76	62,49	63,37	63,37	57,66	61,47
47	65,76	62,99	63,37	63,37	58,00	61,58
48	65,76	63,49	63,71	63,71	58,00	61,81
49	65,58	63,82	63,14	63,47	58,10	61,57
50	65,58	64,07	63,47	63,47	58,10	61,68
51	65,58	64,57	63,47	63,81	58,77	62,02
52	65,58	64,83	63,81	64,48	58,77	62,35
53	64,90	64,90	63,58	64,25	58,87	62,23
54	64,90	65,40	63,58	64,58	59,54	62,57
55	64,90	65,40	63,58	65,26	59,88	62,90
56	64,90	66,16	64,25	65,26	59,88	63,13
57	64,73	65,98	64,01	65,02	59,98	63,00
58	64,46	65,97	64,00	65,00	59,96	62,99
59	64,73	66,23	64,35	65,36	60,65	63,45
60	64,29	66,05	63,76	65,44	60,74	63,31

Table 5.2.1/4: Validity criteria conformity

Criteria	Validity for OECD 301F	Validity for the test
Oxygen uptake of the inoculum control in 28 days and up to 60 days	< 60 mg/L	Day 28	Day 60
		Yes	Yes
		Silicon Control 12.7 & 15.5 mg/L	Silicon Control 22.5 to 24.0 mg/L
		Control 19.7 to 24.0 mg/L	Control 26.8 to 36.6 mg/L
Variation between the test item replicates at the end of the test	< 20%	Yes (11.8%)	Yes (3.8%)
Biodegradability of the reference item within 14 days	> 60%	Yes(78.72% to 82.91%)
Biodegradability of the toxicity control within 14 days	> 25%	Yes(40.49% to 41.25%)
pH of the inoculum control at 60 days	6 to 8.5	Yes(7.4 to 8.0)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of the test item “cistus organic oil” reached 55.07%, following the ThODNH4, after 28 days of incubation and 63.31%, at Day 60.
Under conditions of OECD Guideline 301F, the test item “CISTUS ORGANIC OIL” is not considered as readily biodegradable.

Executive summary:

The aim of this study was to determine the effects of the test item “CISTUS ORGANIC OIL”, according to the OECD 301F guideline, ready biodegradability within an experimental period of 28 days by respirometric method. According to the study monitor’s decision, the experiment was prolonged up to 60 days.

The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected by blank inoculum, run in parallel) was determined from the change in pressure in apparatus and will be expressed as percentage of theorical Oxygen Demand (ThOD).

 

Ready biodegradability of the test item "CISTUS ORGANIC OIL" was performed according to the OECD 301 F guideline.

 

 

Results are presented as follows:

Treatment	Reference item (Sodium benzoate)		Test item (“CISTUS ORGANIC OIL”)
% Biodegradation at Day 28		Mean (Standard deviation)		Mean (Standard deviation)
	80.25	79,25% (1.41)	59.78	55.07% (6.48)
	78.25		57.76
			47.68
% Biodegradation at Day 60	77.89	77.89% (0.00)	63.76	63.31% (3.80)
	77.89		65.44
			60.74
pH at Day 60	7.9		7.5

 

The degradation rate of the test item “CISTUS ORGANIC OIL” reached 55.07%, following the ThOD_NH4, after 28 days of incubation. The 10-Day window failed.

According to the OECD guideline 301F, the test item is therefore not considered as readily biodegradable. Nevertheless, at day 60, the degradation rate of the test item “CISTUS ORGANIC OIL” was above 60% (63.31%), following the ThOD_NH4.

Validity criteria conformity:

Criteria	Validity for OECD 301F	Validity for the test
Oxygen uptake of the inoculum control in 28 days and up to 60 days	< 60 mg/L	Day 28	Day 60
		Yes	Yes
		Silicon Control 12.7 & 15.5 mg/L	Silicon Control 22.5 to 24.0 mg/L
		Control 19.7 to 24.0 mg/L	Control 26.8 to 36.6 mg/L
Variation between the test item replicates at the end of the test	< 20%	Yes (11.8%)	Yes (3.8%)
Biodegradability of the reference item within 14 days	> 60%	Yes(78.72% to 82.91%)
Biodegradability of the toxicity control within 14 days	> 25%	Yes(40.49% to 41.25%)
pH of the inoculum control at 60 days	6 to 8.5	Yes (7.4 to 8.0)

 

All the validity criteria were successful.

Description of key information

According to the OECD guideline 301F, the registered substance is not considered as readily biodegradable.

Indeed, the degradation rate reached 55.07%, following the ThOD_NH4, after 28 days of incubation. Nevertheless, the degradation rate of the test item reached 63.31%, following the ThOD_NH4 at day 60.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

For that endpoint, a reliable experimental study performed on the registered substance was available. This study intended to determine the ready biodegradability of the test item “CISTUS ORGANIC OIL”, according to the OECD 301F guideline and in compliance with GLP.

The amount of oxygen taken up by the microbial population during the biodegradation of the test item (corrected by blank inoculum, run in parallel) was determined from the change in pressure in apparatus and was expressed as percentage of theorical Oxygen Demand (ThOD).

 

The degradation rate of the test item “CISTUS ORGANIC OIL” reached 55.07%, following the ThOD_NH4, after 28 days of incubation and 63.31%, at Day 60.

 

All the validity criteria of the guideline were successful in the study. This study was therefore considered acceptable for that endpoint.