Lupine, L. albus, 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:

March 21, 2016 to April 21, 2016

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 (incl. QA statement)

Remarks:

14 January 2016

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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) - Tank 2.
- 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 recantation in a mineral medium until sludge was considered free from exces 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):

28 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 soluble in water; It was directly diluted with mineral medium and sludge was added directly in each test vessels (100 mg/L). The test medium was the mineral medium, a weakly saline aqueous medium. The test item was tested at 100 mg/L in mineral medium.

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301F guideline
- Test temperature: The test was performed in darkness between 20 to 24 °C (constant temperature +/- 1°C).
- Mineral medium - pH: 7.5
- Inoculum pH: 6.2
- Dissolved oxygen: 8.9 mg/L
- Suspended solids concentration: 3.07 g/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: WTW Oxitop (FCBA reference: ACQU 1011)
- Number of culture flasks/concentration: 3 flasks (test item 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.
- 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 60.2 mg/L + inoculum) and toxicity control (sodium benzoate + inoculum + test item) were run in parallel.

SAMPLING
- Sampling frequency: 0 to 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; 3 flasks (inoculum only)
- Reference item: 2 flasks (Sodium benzoate and inoculum)
- Toxicity control: Yes; 2 flasks (test item, sodium benzoate and inoculum)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

None

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

following the ThOD-NH4

Value:

105.16

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

following the ThOD-NO3

Value:

75.09

Sampling time:

28 d

Details on results:

Biodegradation of the test item:
- The test Item contains nitrogen, therefore the evaluation of biodegradation is based in the following expressed as ThOD-NH4 and ThOD-NO3.
- The 10-day window begins on day 1 (17.28 % of biodegradation) and ends on day 4 (70.79 % of biodegradation) based on ThOD-NH4. Therefore, the 10-day window was reached.
- The degradation rate of the test item reached 105.16 % (between 100.25% and 109.25%), following the ThOD-NH4, after 28 days of incubation.
- The 10-day window begins on day 1 (12.34 % of biodegradation) and ends on day 6 (63.66 % of biodegradation) based on ThOD-NO3. Therefore, the 10-day window was reached.
- The degradation rate of the test item reached 75.09 % (between 71.58% and 78.01%), following the ThOD-NO3, after 28 days of incubation.
According to the OECD guideline 301F, the test item is considered as readily biodegradable whatever the nitrification (ThOD-NH4 or ThOD-NO3)

Biodegradation of the Toxicity Control:
- A biodegradation of 99.5 and 81.5 % after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThOD-NH4 and ThOD-NO3 respectively.
- At the end of the test, 28 days of incubation, the results were respectively 100.9% and 82.7 %. Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge microorganism because degradation was >25 % within 14 days (validity criterion).

Results with reference substance:

The reference item 'Sodium benzoate" was degraded up to 82.21% after 28 days.
Therefore, it confirms the suitability of the used aerobic sludge inoculum.

Table 5.2.1/2: pH values

Treatment	Measuring system	pH at the start	pH at the end of the test (28 days)
Inoculum Control	12	7.5	7.4
	4	7.5	7.4
	8	7.5	7.4
Reference Item	5	7.5	7.7
	1	7.5	7.7
Test Item	10	7.5	7.1
	11	7.5	7.2
	9	7.5	7.1
Toxicity Control	6	7.6	7.3
	7	7.6	7.3

 

Table 5.2.1/3: Percentage (%) Biodegradation during 28 days for Toxicity Control and Test item with ThOD_NH4=1.221 mg O₂/mg test Item

Time (Days)	Toxicity Control		Test item
	Measuring system 6	Measuring system 7	Measuring system 10	Measuring system 11	Measuring system 9	Average
0	0,00	0,00	0,00	0,00	0,00	0,00
1	34,86	34,86	18,43	16,13	17,28	17,28
2	48,58	49,48	38,08	34,64	36,94	36,55
3	61,62	61,62	55,36	51,92	54,22	53,84
4	74,21	74,66	72,73	69,21	70,43	70,79
5	81,89	82,79	86,87	81,14	84,41	84,14
6	86,00	87,35	91,07	86,16	90,25	89,16
7	88,73	90,97	96,04	89,49	95,22	93,58
8	92,11	93,01	98,94	92,38	98,12	96,48
9	93,49	94,84	100,63	94,89	99,81	98,44
10	95,10	96,45	101,91	96,18	101,91	100,00
11	96,03	97,83	103,60	97,87	103,60	101,69
12	96,70	98,95	104,01	99,92	104,01	102,65
13	96,97	100,12	105,32	100,41	105,32	103,69
14	97,23	101,72	104,97	100,87	106,61	104,15
15	98,58	103,07	106,61	101,69	106,61	104,97
16	98,19	102,68	105,90	101,80	107,53	105,08
17	98,43	102,92	105,51	102,24	107,97	105,24
18	98,49	102,98	105,62	100,71	108,90	105,08
19	98,28	103,22	105,24	100,33	109,34	104,97
20	98,10	102,59	104,91	99,18	108,19	104,10
21	97,68	102,17	104,15	99,24	109,06	104,15
22	98,58	102,17	105,79	99,24	109,06	104,70
23	98,37	102,86	105,41	100,49	108,68	104,86
24	98,40	102,89	105,46	99,73	108,74	104,64
25	98,19	102,68	105,90	100,16	109,99	105,35
26	98,86	102,91	105,49	98,94	109,58	104,67
27	98,68	103,18	104,34	100,25	109,25	104,61
28	98,68	103,18	105,98	100,25	109,25	105,16

Table 5.2.1/3: Percentage (%) Biodegradation during 28 days for Toxicity Control and Test item with ThOD_N03=1.710 mg O₂/mg test Item

Time (Days)	Toxicity Control		Test item
	Measuring system 6	Measuring system 7	Measuring system 10	Measuring system 11	Measuring system 9	Average
0	0,00	0,00	0,00	0,00	0,00	0,00
1	28,57	28,57	13,16	11,52	12,34	12,34
2	39,82	40,56	27,19	24,74	26,37	26,10
3	50,51	50,51	39,53	37,08	38,71	38,44
4	60,83	61,20	51,93	49,42	50,29	50,55
5	67,13	67,86	62,03	57,93	60,27	60,08
6	70,49	71,60	65,03	61,52	64,44	63,66
7	72,73	74,57	68,58	63,90	67,99	66,82
8	75,51	76,24	70,64	65,96	70,06	68,89
9	76,64	77,74	71,85	67,76	71,27	70,29
10	77,95	79,06	72,77	68,67	72,77	71,40
11	78,71	80,19	73,98	69,88	73,98	72,61
12	79,27	81,11	74,27	71,35	74,27	73,29
13	79,49	82,07	75,20	71,70	75,20	74,04
14	79,70	83,38	74,95	72,03	76,12	74,37
15	80,80	84,49	76,12	72,61	76,12	74,95
16	80,48	84,17	75,61	72,69	76,78	75,03
17	80,68	84,37	75,34	73,00	77,10	75,15
18	80,73	84,42	75,42	71,91	77,76	75,03
19	80,56	84,61	75,15	71,64	78,07	74,95
20	80,41	84,10	74,91	70,82	77,25	74,33
21	80,07	83,75	74,37	70,86	77,88	74,37
22	80,80	83,75	75,54	70,86	77,88	74,76
23	80,63	84,32	75,26	71,75	77,60	74,87
24	80,66	84,34	75,30	71,21	77,64	74,72
25	80,48	84,17	75,61	71,52	78,54	75,22
26	81,04	84,36	75,32	70,64	78,25	74,74
27	80,89	84,58	74,50	71,58	78,01	74,70
28	80,89	84,58	75,67	71,58	78,01	75,09

Table 5.2.1/4: Validity criteria conformity

Criteria	Validity for OECD 301F	Validity for test
Variation between the test item replicates at the end of the test	< 20 %	Yes (4.3%) with ThOD-NH4 Yes (4.3%) with ThOD-NO3
Biodegradability of the toxicity control within 14 days	> 25 %	Yes (97.23% and 101.72%) with ThOD-NH4 Yes (79.70% and 83.38%) with ThOD-NO3
Oxygen uptake of the inoculum control in 28 days	< 60 mg/L	Yes (16.9 to 25.4 mg/L)
Biodegradability of the reference item within 14 days	> 60 %	Yes (81.98 %)
pH of the inoculum control at 28 days	6 to 8.5	Yes (7.1 - 7.7)

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under conditions of OECD Guideline 301F, the test item is considered as readily biodegradable. Furthermore, the 10-day window was reached under the test conditions.

Executive summary:

The ready biodegradability of the test item has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301F with GLP compliance.

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. 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 60.2 mg/L+ inoculum) and toxicity control (sodium benzoate + inoculum + test item) were run in parallel. 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 degradation rate of the test item reached 105.16 % and 75.09%, following the ThOD-NH4 and the ThOD-NO3 respectively, after 28 days of incubation. The 10-day window begins on day 1 (17.28 % of biodegradation) and ends on day 4 (70.79 % of biodegradation) based on ThOD-NH4 and begins on day 1 (12.34 % of biodegradation) and ends on day 6 (63.66 % of biodegradation) based on ThOD-NO3. Therefore, the 10-day window was reached.

 

In addition, a biodegradation of 100.9% and 82.7% was noted at the end of the test in the Toxicity control (containing both of the test item and the reference item) based on ThOD-NH4 and ThOD-NO3 respectively. Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge microorganism because degradation was > 25 % within 14 days (validity criterion). The reference item 'Sodium benzoate" was degraded up to 82.21% after 28 days. Therefore, it confirms the suitability of the used aerobic sludge inoculum.

 

Under conditions of OECD Guideline 301F, the test item is considered as readily biodegradable.

Description of key information

The substance is readily biodegradable according to the criteria of OECD 301 F guideline.

The degradation rate of the test item reached 105.16 % and 75.09%, following the ThOD-NH4 and the ThOD-NO3 respectively, after 28 days of incubation.

The pass level of 60% was reached in approximately less than 4 and 6 days based on ThOD-NH4 and on ThOD-NO3 respectively. Therefore, the 10-day window was reached.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The ready biodegradability of the test item has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301F with GLP compliance.

 

The degradation rate of the test item reached 105.16 % and 75.09%, following the ThOD-NH4 and the ThOD-NO3 respectively, after 28 days of incubation. The 10-day window begins on day 1 (17.28 % of biodegradation) and ends on day 4 (70.79 % of biodegradation) based on ThOD-NH4 and begins on day 1 (12.34 % of biodegradation) and ends on day 6 (63.66 % of biodegradation) based on ThOD-NO3. Therefore, the 10-day window was reached.

 

A biodegradation of 99.5 and 81.5% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item) based on ThOD-NH4 and ThOD-NO3 respectively. At the end of the test, the results were respectively 100.9% and 82.7 %. Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge microorganism because degradation was >25 % within 14 days (validity criterion). The reference item 'Sodium benzoate" was degraded up to 82.21% after 28 days. Therefore, it confirms the suitability of the used aerobic sludge inoculum.

 

Under conditions of OECD Guideline 301F, the test item is considered as readily biodegradable.