Fenugreek, 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 2018-01-17 to 2018-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 301F guideline.

Justification for type of information:

Details on study plan is provided in this study record.

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 applicable.

GLP compliance:

yes (incl. QA statement)

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:

- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant will be supplied by a municipal sewage treatment plant (e.g. 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.
The washed activated sludge will be pre-conditioned in test water for a maximum of 2 days.

Duration of test (contact time):

>= 28 - <= 60 d

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 to 7.6 with Sodium Hydroxide) 7.4 to 7.8 (measured at day 63)
- Recording: Test conditions (temperature) will be recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range will normally not result in major disturbances of the test performance and will not be recorded).

TEST WATER
- 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 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 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.

COURSE OF THE TEST
- Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks.
For a better distribution of the test item, silicone oil AR 20 at a concentration of about 1% of the final flask volume was be added to all flasks in order to improve the bioavailability of the mixture constituents.
- 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.
- Test Duration: 60 days (pH was measured at day 63)

TEST PARAMETERS
- Measurements of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
- Temperature: The temperature was recorded by means of the automated software AMR Wincontrol©.
- pH-Value: pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at day 63, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

TEST SYSTEM
- 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

Remarks:

Sodium Benzoate

Preliminary study:

No data.

Test performance:

No data.

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4/ThODNO3

Value:

10

Sampling time:

1 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

60

Sampling time:

11 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

61

Sampling time:

11 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

70

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

72

Sampling time:

28 d

Details on results:

The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThODNH4, ThODNO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThODNH4) and 60% (ThODNO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThODNH4) and 70% (ThODNO3), and at test end after 60 days 82% (ThODNH4) and 80% (ThODNO3).
Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4 and ThODNO3.

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.

pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	Fenugreek Absolute	7.4
2	Fenugreek Absolute	7.6
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	30	30	0	0	60	0	55
2	55	55	5	0	85	0	95
3	80	75	5	5	105	0	125
4	95	100	5	5	120	10	155
5	110	115	5	5	130	20	200
6	120	130	10	5	135	25	210
7	130	140	10	10	140	30	225
8	135	150	10	10	145	30	230
9	145	160	10	10	150	35	240
10	150	170	15	10	150	35	250
11	155	175	15	10	150	35	265
12	160	175	15	10	155	35	275
13	165	175	15	10	155	40	280
14	170	175	20	10	160	45	280
15	175	175	20	10	160	45	280
16	175	180	20	15	160	45	280
17	180	180	25	15	165	45	280
18	180	180	25	15	165	45	280
19	185	180	25	20	165	45	280
20	185	185	25	20	170	50	285
21	190	185	25	20	170	50	285
22	190	190	25	20	170	50	285
23	195	190	25	20	170	50	285
24	195	195	25	20	170	50	285
25	195	195	25	20	170	50	285
26	195	195	25	20	175	55	285
27	200	200	25	20	175	55	285
28	205	200	25	20	175	55	285
29	210	205	25	20	180	55	285
30	210	205	25	20	180	55	285
31	210	205	25	20	180	55	285
32	210	205	25	20	180	55	285
33	210	205	25	20	180	55	285
34	215	205	25	20	180	55	285

35	215	210	25	20	185	55	285
36	215	210	25	20	185	55	285
37	220	210	25	20	190	60	285
38	220	215	25	20	190	60	285
39	220	215	25	20	190	60	285
40	220	215	25	20	190	60	285
41	220	215	25	20	190	60	285
42	220	215	25	20	190	60	285
43	220	215	25	20	190	60	285
44	220	215	25	20	195	65	285
45	220	220	30	20	195	65	285
46	220	220	30	20	195	65	285
47	220	220	30	20	195	65	285
48	225	225	30	20	195	65	285
49	230	230	30	20	195	65	285
50	230	230	30	20	195	65	285
51	230	230	30	20	195	65	285
52	230	230	30	20	195	65	285
53	230	230	30	20	195	65	285
54	230	230	30	20	195	65	285
55	230	230	30	20	195	65	285
56	230	230	30	20	195	65	285
57	230	230	30	20	195	65	285
58	230	230	30	20	195	65	285
59	230	230	30	20	200	65	285
60	230	230	30	20	200	65	285

Flasks 1 and 2: Fenugreek Absolute

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 ThOD_NH4:

Time	Percentage Biodegradation^1
(Days)	Fenugreek Absolute^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	12	12	35	13
2	21	21	48	22
3	30	28	59	29
4	36	39	67	36
5	41	45	73	47
6	44	50	75	48
7	47	53	76	51
8	49	57	79	52
9	53	61	82	55
10	54	64	81	57
11	56	66	81	60
12	58	66	83	63
13	60	66	83	64
14	61	65	85	63
15	63	65	85	63
16	62	66	83	63
17	63	65	85	62
18	63	65	85	62
19	64	64	83	61
20	64	66	86	63
21	66	66	86	63
22	66	68	86	63
23	68	68	86	63
24	68	70	86	63
25	68	70	86	63
26	68	70	89	63
27	70	72	89	63
28	72	72	89	63
29	74	74	92	63
30	74	74	92	63
31	74	74	92	63

32	74	74	92	63
33	74	74	92	63
34	76	74	92	63
35	76	76	95	63
36	76	76	95	63
37	78	76	98	63
38	78	78	98	63
39	78	78	98	63
40	78	78	98	63
41	78	78	98	63
42	78	78	98	63
43	78	78	98	63
44	78	78	101	63
45	77	79	100	62
46	77	79	100	62
47	77	79	100	62
48	79	81	100	62
49	81	83	100	62
50	81	83	100	62
51	81	83	100	62
52	81	83	100	62
53	81	83	100	62
54	81	83	100	62
55	81	83	100	62
56	81	83	100	62
57	81	83	100	62
58	81	83	100	62
59	81	83	103	62
60	81	83	103	62

¹ThOD_NH4of Fenugreek Absolute: 2.444 mg O₂/mg test item
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NO3:

Time	Percentage Biodegradation^1
(Days)	Fenugreek Absolute^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	12	12	35	13
2	20	21	48	22
3	29	28	59	28
4	35	38	67	35
5	40	44	73	46
6	43	49	75	48
7	46	52	76	51
8	48	56	79	52
9	52	59	82	54
10	53	62	81	56
11	55	64	81	59
12	57	64	83	62
13	59	64	83	63
14	60	63	85	62
15	62	63	85	62
16	61	64	83	62
17	62	63	85	61
18	62	63	85	61
19	63	62	83	61
20	63	64	86	62
21	65	64	86	62
22	65	66	86	62
23	66	66	86	62
24	66	68	86	62
25	66	68	86	62
26	66	68	89	62
27	68	70	89	62
28	70	70	89	62
29	72	72	92	62
30	72	72	92	62
31	72	72	92	62

32	72	72	92	62
33	72	72	92	62
34	74	72	92	62
35	74	74	95	62
36	74	74	95	62
37	76	74	98	62
38	76	76	98	62
39	76	76	98	62
40	76	76	98	62
41	76	76	98	62
42	76	76	98	62
43	76	76	98	62
44	76	76	101	62
45	75	77	100	61
46	75	77	100	61
47	75	77	100	61
48	77	79	100	61
49	79	81	100	61
50	79	81	100	61
51	79	81	100	61
52	79	81	100	61
53	79	81	100	61
54	79	81	100	61
55	79	81	100	61
56	79	81	100	61
57	79	81	100	61
58	79	81	100	61
59	79	81	103	61
60	79	81	103	61

¹ThOD_NO3of Fenugreek Absolute: 2.502 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, 63% (ThOD_NH4) biodegradation was noted within 14 and 28 days (62% based on ThOD_NO3) and 62% after 60 days (ThOD_NH4) as well as 61% (ThOD_NO3).

Abiotic Control

The oxygen demand in the abiotic control started at day 44 and reached a maximum of 65 mg/L during the test duration of 60 days.

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 7.4 to 7.6 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, at day 28 and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 11 was 10%, at day 28 0% and at day 60 2%. 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 total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 63% (ThODNH4) and 62% (ThODNO3) at day 14; the test item was not inhibitory.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The degradation rate of Fenugreek Absolute reached 60% within the 10-day window and after 28 and 60 days.
Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThODNH4 and ThODNO3.

Executive summary:

The test item Fenugreek Absolute 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.

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThOD_NO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

 

The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThOD_NH4, ThOD_NO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThOD_NH4) and 60% (ThOD_NO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThOD_NH4) and 70% (ThOD_NO3), and at test end after 60 days 82% (ThOD_NH4) and 80% (ThOD_NO3).

Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThOD_NH4and ThOD_NO3.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 63% (ThOD_NH4) biodegradation was noted within 14 and 28 days (62% based on ThOD_NO3) and 62% after 60 days (ThOD_NH4) as well as 61% (ThOD_NO3).

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.

Description of key information

The registered substance is regarded as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The test item Fenugreek Absolute 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.

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThOD_NO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.

 

The mean biodegradation of 10% of Fenugreek Absolute was reached at day 1 (ThOD_NH4, ThOD_NO3). At the end of the 10-day window at day 11, the mean degradation of Fenugreek Absolute was 61% (ThOD_NH4) and 60% (ThOD_NO3) and therefore the 10 day window criterion was passed. The mean biodegradation after 28 days was 72% (ThOD_NH4) and 70% (ThOD_NO3), and at test end after 60 days 82% (ThOD_NH4) and 80% (ThOD_NO3).

Therefore, Fenugreek Absolute is considered to be readily biodegradable based on ThOD_NH4and ThOD_NO3.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 63% (ThOD_NH4) biodegradation was noted within 14 and 28 days (62% based on ThOD_NO3) and 62% after 60 days (ThOD_NH4) as well as 61% (ThOD_NO3).

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.