Matricaria recutita, 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 2017-07-24 to 2017-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not applicable.

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)

Remarks:

2017-03-13

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:

- Species/Origin: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Bensheim, Germany.
- Conditioning: The aerobic activated sludge used for this study was deposited for 45 minutes, afterwards it was 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 until use. This suspension was used for the experiment.

Duration of test (contact time):

60 d

Initial conc.:

102 mg/L

Based on:

test mat.

Remarks:

Test Item Loading Rate (initial concentration in medium C0)

Initial conc.:

301.7 mg/L

Based on:

ThOD

Remarks:

ThOD(NH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Surrounding Type:Climatised chamber
- Temperature: 22°C ± 1°C
- Light Conditions: Darkness
- pH-Value of Test Solutions: 7.5 to 7.6 (measured at test start), 7.2 to 8.7 (measured at the end of the test)
- Composition of medium: Aliquots of washed sludge suspension were mixed with reconstitued 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-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 AR20 was used as an emulsifying agent. (1%)


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 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

Preliminary study:

No data.

Test performance:

No data.

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD(NH4)

Value:

25

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD(NH4)

Value:

49

Sampling time:

60 d

Details on results:

TEST ITEM: At day 28, the mean biodegradation was 25% and the plateau of degradation was not reached. Therefore the study was prolonged till day 60. At day 60 at test end the mean degradation was 49%.
TOXICITY CONTROL: In the toxicity control containing both, the test item and the reference item sodium benzoate, 38% biodegradation was noted within 14 days and 55% biodegradation after 28 days of incubation. At test end after 60 days incubation, the degradation was 64%. 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.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 84% after 14 days and to 92% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

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	5	0	0	0	20	0	15
2	5	5	5	0	30	0	25
3	10	10	5	5	105	0	110
4	15	15	5	5	120	0	125
5	20	20	5	5	125	0	135
6	25	25	5	5	130	0	140
7	30	30	10	10	130	0	145
8	35	35	10	10	135	0	150
9	40	40	10	10	140	0	155
10	45	45	10	10	145	0	160
11	45	50	10	10	150	0	170
12	50	55	15	10	150	0	175
13	55	55	15	10	155	0	185
14	55	55	15	10	155	0	195
15	60	60	15	15	160	0	200
16	65	60	15	15	160	0	205
17	65	65	15	15	160	0	210
18	70	65	15	15	160	0	215
19	75	70	15	15	160	0	220
20	75	70	15	15	165	0	225
21	80	75	15	15	165	0	235
22	80	75	15	15	165	0	240
23	85	75	15	15	165	0	245
24	85	80	15	15	170	0	250
25	85	80	15	15	170	0	260
26	85	85	15	15	170	0	265
27	90	85	15	15	170	0	270
28	90	90	15	15	170	0	275
29	90	90	15	15	170	0	280
30	95	95	15	20	175	0	285

31	110	110	20	20	175	0	290
32	115	120	20	20	175	0	305
33	120	120	20	20	175	0	305
34	120	125	20	20	175	0	305
35	125	125	20	20	175	0	305
36	125	130	20	20	175	0	310
37	130	130	20	20	175	0	310
38	130	135	20	20	175	0	315
39	135	135	20	20	175	0	315
40	135	135	20	20	175	0	315
41	140	135	20	20	175	0	315
42	140	135	20	20	175	0	315
43	145	140	20	20	175	0	315
44	145	140	20	20	175	0	315
45	150	140	20	20	175	0	315
46	155	145	20	20	175	0	315
47	155	145	20	20	175	0	315
48	155	145	20	20	175	0	315
49	155	145	20	20	175	0	320
50	155	150	20	20	175	0	320
51	160	150	20	20	180	0	320
52	160	150	20	20	180	0	320
53	165	155	20	20	180	0	320
54	165	155	20	20	180	0	320
55	165	155	20	20	180	0	325
56	165	160	20	20	180	0	325
57	165	160	20	20	185	0	325
58	170	160	20	20	185	0	325
59	170	160	20	20	185	0	325
60	170	165	20	20	185	0	325

Flasks 1 and 2: Blue Chamomilla;      

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
(Days)	Blue Chamomilla^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	2	0	12	3
2	1	1	16	5
3	2	2	59	22
4	3	3	68	25
5	5	5	71	27
6	7	7	74	28
7	7	7	71	28
8	8	8	74	29
9	10	10	77	30
10	12	12	80	32
11	12	13	83	34
12	13	14	81	34
13	14	14	84	36
14	14	14	84	38
15	15	15	86	39
16	17	15	86	40
17	17	16	86	41
18	18	16	86	42
19	20	18	86	43
20	20	18	89	44
21	22	20	89	46
22	22	20	89	47
23	23	20	89	48
24	23	21	92	49
25	23	21	92	51
26	23	23	92	53
27	25	23	92	54
28	25	25	92	55
29	25	25	92	56
30	26	25	93	56

31	30	30	92	57
32	32	33	92	60
33	33	33	92	60
34	33	35	92	60
35	35	35	92	60
36	35	36	92	61
37	37	36	92	61
38	37	38	92	62
39	38	38	92	62
40	38	38	92	62
41	40	38	92	62
42	40	38	92	62
43	42	39	92	62
44	42	39	92	62
45	43	39	92	62
46	45	41	92	62
47	45	41	92	62
48	45	41	92	62
49	45	41	92	63
50	45	43	92	63
51	47	43	94	63
52	47	43	94	63
53	48	44	94	63
54	48	44	94	63
55	48	44	94	64
56	48	46	94	64
57	48	46	97	64
58	50	46	97	64
59	50	46	97	64
60	50	48	97	64

 ¹ThOD_NH4of Blue Chamomilla: 2.956 mg O₂/mg test item
 ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Validity Criteria of the Study

Inoculum Control:	The oxygen demand of the inoculum control (medium and inoculum) was 15 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 the end of the test was 7.0 and 7.2 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 10-day window, at day 28 and at the end of the test was less than 20%. The difference of duplicate values at day 19, day 28 and day 60 differed by 2%, 0% and 2%, respectively.
Toxicity Control:	If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 38% at day 14; the test item was not inhibitory.

The validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is considered not readily biodegradable based on ThODNH4 after 28 days. Moreover, 60% degradation was not reached after 60 days of incubation.

Executive summary:

The test item Blue Chamomilla 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.

At day 28, the mean biodegradation was 25% and the plateau of degradation was not reached. Therefore the study was prolonged till day 60. At test end at day 60, the mean degradation was 49%.

The test item is considered not readily biodegradable based on ThODNH4 after 28 days. Moreover, 60% degradation was not reached after 60 days of incubation.

The reference item sodium benzoate was sufficiently degraded to 84% after 14 days and to 92% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 38% biodegradation was noted within 14 days and 55% biodegradation after 28 days of incubation. At test end after 60 days incubation, the degradation was 64%. 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.

The oxygen demand of the inoculum control (medium and inoculum) was not greater than 60 mg O₂/L within 28 days as required by the test guideline.

pH values were in the range required in the guideline. Therefore, all validity criteria of the study were fulfilled.

Description of key information

The registered substance is considered to be not readily biodegradable after 28 days, and reached a degradation of 49% after 60 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

For that endpoint, an experimental study on the registered substance was available.

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.

At day 28, the mean biodegradation was 25% and the plateau of degradation was not reached. Therefore the study was prolonged till day 60. At test end at day 60, the mean degradation was 49%. Therefore, the test item is considered not readily biodegradable based on ThODNH4 after 28 days. Moreover, 60% degradation was not reached after 60 days of incubation.

All validity criteria were fulfilled. Moreover, the reference item sodium benzoate was sufficiently degraded, thus confirming the suitability of the aerobic activated sludge inoculum used.

In addition, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days in the toxicity control.

This study respected the requirements of the test guideline and was considered acceptable to fulfill that ebdpoint.