Evernia furfuracea, 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 2017-07-11 to 2017-09-22

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Remarks:

Signed on 2016-01-14

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 during 5 days

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:
According to the study monitor, 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 "OAKMOSS ABSOLUTE” 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 21.2 to 22.0 °C (constant temperature +/- 1°C).
- Mineral medium - pH: 7.5
- Inoculum pH: 6.8
- Dissolved oxygen: 9.0 mg/L
- Suspended solids concentration: 3.27 g/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: WTW Oxitop
- Number of flasks/concentration: 3 flasks (test item + inoculum + AR20 1%)
- 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+siliconee oil AR 20) were run in parallel.
Moreover, a control with silicone oil AR 20 1% was performed to check that silicone was non-toxic to bacteria and non-biodegradable.

The pH of the contents was measured at the start and the end of the experiment.

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 = 66.10 and 66.14 for the measurement 1 and the measurement 2 respectively
- % hydrogen = 7.58 and 7.69 for the measurement 1 and the measurement 2 respectively
- % nitrogen = 0.94 and 0.90 for the measurement 1 and the measurement 2, respectively
- % oxygen = 25.05 and 24.82 for the measurement 1 and the measurement 2 respectively

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD NH4

Value:

54.97

St. dev.:

9.42

Sampling time:

28 d

Remarks on result:

other: Mean value (n=2)

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD NH4

Value:

63.7

St. dev.:

14.12

Sampling time:

60 d

Remarks on result:

other: Mean value (n=2)

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD NO3

Value:

53.87

St. dev.:

9.23

Sampling time:

28 d

Remarks on result:

other: Mean value (n=2)

Parameter:

% degradation (O2 consumption)

Value:

62.43

St. dev.:

13.83

Sampling time:

60 d

Remarks on result:

other: Mean value (n=2)

Details on results:

The test item “OAKMOSS ABSOLUTE” contains nitrogen according to the elementary analysis, the evaluation of biodegradation has to be based in the following expressed as ThODNH4 and ThODNO3.
Following the ThODNH4, the degradation for the 10-day window beginning was reached on day 2, but at the end of the 10-day window (reaching at least 60%), on day 12, the mean degradation was 43.56%, therefore the 10-day window failed.
The degradation rate of the test item “OAKMOSS ABSOLUTE” reached 54.97%, following the ThODNH4, after 28 days of incubation.
According toIn agreement with 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 “OAKMOSS ABSOLUTE” reached 63.70%, following the ThODNH4.

Following the ThODNO3, the degradation for the 10-day window beginning was reached on day 2, but at the end of the 10-day window (reaching at least 60%), on day 12, the mean degradation was 42.69%, therefore the 10-day window failed.
The degradation rate of the test item “OAKMOSS ABSOLUTE” reached 53.87%, following the ThODNO3, after 28 days of incubation.
With the study monitor’s agreement, 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 “OAKMOSS ABSOLUTE” reached 62.43%, following the ThODNO3

According to the OECD guideline 301F, the test item is not considered to be readily biodegradable, nevertheless, on day 60, the degradation rate of the test item “OAKMOSS ABSOLUTE” reached 62.43%, following the ThODNO3.
From the Day 42, the degradation rate of the test item reached 60.00% and 62.43% at the end of the test: 60 days.

7.2.5 Biodegradation of the Toxicity Control
A biodegradation of 47.48% and 45.55% 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 62.78% and 62.46%.

A biodegradation of 46.83% and 44.92% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThODNO3.
At the end on the test, 60 days of incubation, the results were respectively 61.93% and 61.61%.

Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge micro-organisms because degradation was superior to 25% within 14 days (validity criterion)

Results with reference substance:

The reference item “Sodium benzoate” was degraded up to 84.34% and 87.13%( mean value: 85.74%) after 14 days and 88.93% in both replicates after 60 days of incubation at the end of the test.
Therefore, the suitability of the used aerobic sludge inoculum is confirmed.

Percentage (%) Biodegradation during 60 days for Toxicity Control and
Test item (“OAKMOSS ABSOLUTE”) with ThOD_NH4= 2.103 mgO₂/mg Test item

Time (Days)	Tox Control		Test item
	Flask 7	Flask 36	Flask 32	Flask 19	Average
0	0,00	0,00	0,00	0,00	0,00
1	10,88	11,34	10,70	0,67	5,68
2	18,01	18,01	15,85	13,19	14,52
3	20,27	21,17	18,56	17,85	18,20
4	23,75	25,11	20,34	21,00	20,67
5	28,30	29,65	27,71	25,00	26,35
6	31,92	31,92	31,26	27,93	29,59
7	35,14	34,18	35,96	30,59	33,28
8	36,93	37,25	39,78	32,41	36,10
9	39,03	39,35	42,89	34,19	38,54
10	41,13	41,13	45,33	35,30	40,32
11	43,75	43,11	47,96	36,41	42,19
12	45,52	43,91	49,63	37,50	43,56
13	46,66	44,73	49,88	38,66	44,27
14	47,48	45,55	51,09	39,77	45,43
15	47,97	46,36	52,29	40,21	46,25
16	49,09	47,48	53,94	41,10	47,52
17	50,05	48,12	54,41	41,77	48,09
18	50,23	48,62	55,62	42,21	48,91
19	51,34	49,73	56,32	43,10	49,71
20	51,99	50,38	57,74	43,76	50,75
21	51,86	50,57	58,03	45,19	51,61
22	52,18	51,54	58,50	46,62	52,56
23	52,99	51,71	58,76	47,35	53,05
24	53,64	52,03	59,71	47,35	53,53
25	53,80	52,51	59,95	47,11	53,53
26	54,29	52,68	60,68	48,31	54,49
27	54,30	53,02	60,69	47,85	54,27
28	55,58	53,65	61,63	48,31	54,97
29	55,90	54,29	63,05	49,74	56,40
30	57,35	54,78	63,29	50,93	57,11
31	57,69	54,79	64,26	50,47	57,36
32	58,34	55,12	64,75	50,48	57,62
33	58,81	55,59	64,97	50,71	57,84
34	58,81	56,24	65,45	51,18	58,31
35	59,33	57,07	65,26	51,47	58,36
36	59,48	57,55	66,43	51,69	59,06
37	59,18	57,89	65,99	51,24	58,61
38	59,35	58,06	66,24	51,50	58,87
39	59,35	58,06	67,19	51,50	59,34
40	60,62	58,36	68,58	51,94	60,26
41	60,14	58,53	69,79	51,72	60,75
42	60,47	58,53	70,74	51,72	61,23
43	60,32	59,03	70,99	52,45	61,72
44	60,94	59,33	71,44	51,94	61,69
45	61,13	59,84	71,25	52,70	61,97
46	61,43	60,14	71,69	53,15	62,42
47	61,75	60,14	71,69	52,67	62,18
48	60,83	60,83	72,23	52,26	62,24
49	61,95	60,98	72,45	52,48	62,47
50	62,25	61,28	72,42	52,92	62,67
51	61,80	61,15	73,18	52,73	62,96
52	61,80	61,80	73,18	52,73	62,96
53	61,80	61,80	73,18	52,73	62,96
54	62,42	61,45	73,62	53,18	63,40
55	62,61	61,97	73,43	53,46	63,45
56	62,61	61,97	73,43	53,46	63,45
57	62,31	62,31	73,47	53,02	63,24
58	62,48	62,16	73,24	52,80	63,02
59	63,26	61,97	73,91	53,46	63,69
60	62,78	62,46	73,69	53,72	63,70

Percentage (%) Biodegradation during 60 days for Toxicity Control and
Test item (“OAKMOSS ABSOLUTE”) with ThOD_NO3= 2.146 mgO₂/mg Test item

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

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 (7.0 to 12.7 mg/L)	Silicon Control (12.7 to 18.3 mg/L)
		Control (8.5 to 16.9 mg/L)	Control (22.5 to 26.8 mg/L)
Biodegradability of the reference item within 14 days	> 60%	Yes(84.34% to 87.13%)
Biodegradability of the toxicity control within 14 days	> 25%	ThODNH4	ThODNO3
		Yes(45.55% to 47.48%)	Yes(44.92% to 46.83%)
Variation between the test item replicates at 28 days	< 20%	Yes (17.13%)
pH of the inoculum control at 60 days	6 to 8.5	Yes(7.5)

 

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

According to the OECD guideline 301F, the test item is not considered to be readily biodegradable. Nevertheless, on day 60, the degradation rate of the test item reached 63.70% following the ThODNH4 and 62.43%, following the ThODNO3.

Executive summary:

The aim of this study was to determine the biodegradability of the test item “OAKMOSS ABSOLUTE”, according to the OECD 301F guideline, within an experimental period of 28 days by respirometric method. Since a plateau was not reached after 28 days of incubation, 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 theoretical Oxygen Demand (ThOD).

The results of the study were as follow:

The degradation rate of the test item reached 54.97%, following the ThOD_NH4, after 28 days of incubation and 63.70% on Day 60.

The degradation rate of the test item reached 53.87%, following the ThOD_NO3, after 28 days of incubation and 62.43% on Day 60.

Treatment	Test item (“OAKMOSS ABSOLUTE”)
% Biodegradation at Day 28	ThODNH4	Mean (Standard deviation)	ThODNO3	Mean (Standard deviation)
	61.63	54.97% (9.42)	60.39	53.87 (9.23)
	48.31		47.34
% Biodegradation at Day 60	73.69	63.70% (14.12)	72.21	62.43 (13.83)
	53.72		52.64
pH at Day 60	7.6

According to the OECD guideline 301F, the test item is not considered to be readily biodegradable. Nevertheless, on day 60, the degradation rate of the test item “OAKMOSS ABSOLUTE” was above 60%, following the ThOD-NH4 and the ThOD-NO3.

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 (7.0 to 12.7 mg/L)	Silicon Control (12.7 to 18.3 mg/L)
		Control (8.5 to 16.9 mg/L)	Control (22.5 to 26.8 mg/L)
Biodegradability of the reference item within 14 days	> 60%	Yes(84.34% to 87.13%)
Biodegradability of the toxicity control within 14 days	> 25%	ThODNH4	ThODNO3
		Yes(45.55% to 47.48%)	Yes(44.92% to 46.83%)
Variation between the test item replicates at 28 days	< 20%	Yes (17.13%)
pH of the inoculum control at 60 days	6 to 8.5	Yes(7.5)

 

All the validity criteria were successful.

Description of key information

Based on a read-across from an experimental GLP study performed on an analogue substance according to the OECD 301F guideline, the registered substance is regarded as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A reliable study on the registered substance was not available. Therefore, in order to assess the ready biodegradability of the registered substance, the results from an experimental GLP study performed on the analogue substance Oakmoss absolute (Absolute obtained from lichen of Evernia prunastri (Parmeliaceae) by extraction with a mixture of polar and apolar solvents followed by an extraction with ethanol solvent) have been used.

In this study, the test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days,according to the OECD 301F guideline, further extended 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 was expressed as percentage of theoretical Oxygen Demand (ThOD).

The results of the study were as follow:

The degradation rate of the test item reached 54.97%, following the ThOD_NH4, after 28 days of incubation and 63.70% on Day 60.

The degradation rate of the test item reached 53.87%, following the ThOD_NO3, after 28 days of incubation and 62.43% on Day 60.

According to the OECD guideline 301F, the test item is not considered to be readily biodegradable. Nevertheless, the test item can be regarded as non-persistant because its degradation rate was above 60% after 60 days of incubation.

Moreover, the test item was found to be not inhibitory at the tested concentration on the aerobic activated sludge micro-organism because degradation was above 25% after 14 days of incubation.

All validity criteria were successful and the study respected the requirements of the guideline. This study was therefore considered acceptable for that endpoint and the read-across justification is provided in the iuclid study record.