Tagetes minuta, 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 2016-12-27 to 2017-04-20

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

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch N° 2563305, provided by Sponsor
- Expiration date of the lot/batch: March 07, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At 20 ± 5 °C, in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The amounts of test item and reference item were directly weighed into the test flasks. Silicone oil AR20 was used as an emulsifying agent.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Bensheim, Germany.

The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap 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.

Duration of test (contact time):

60 d

Initial conc.:

102 mg/L

Based on:

other: Test Item loading rate

Initial conc.:

301.8 mg/L

Based on:

ThOD

Remarks:

Oxygen demand (ThOD NH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- 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 temperature: 22°C ± 1°C
- pH: 7.5 to 8.0
- pH adjusted: no
- Continuous darkness: yes


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
- Test performed in closed vessels due to significant volatility of test substance:

SAMPLING
O2 consumption measured daily.

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

Value:

52

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

53

Sampling time:

60 d

Details on results:

The mean biodegradation of 10% of TAGETE OIL was reached at day 3 (ThODNH4). At the end of the 10-day window at day 13, the degradation of TAGETE OIL was 39% (ThODNH4) and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 52% and the plateau of biodegradation was not reached, therefore the test was prolonged to 60 days incubation time. The plateau of biodegradation was reached at day 34 and remained at this level till the end of the test. At test end after 60 days the biodegradation of the test item was 53% (mean, ThODNH4).
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.
Results for all flasks are illustrated in the attached figure 1.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 82% after 14 days and to 79% after 28 days of incubation.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

pH-Values at the End of the Test

Flask	No. Treatment	pH-value
1	TAGETE OIL	7.5
2	TAGETE OIL	7.5
3	Inoculum control	7.5
4	Inoculum control	7.5
5	Reference item (procedure control)	8

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 60 Days

Time (days)	Flask
	1	2	3	4	5	6	7
1	5	5	0	5	50	0	65
2	10	5	5	10	80	0	90
3	40	40	10	10	105	0	125
4	55	55	10	10	120	0	140
5	60	65	10	10	130	0	150
6	70	75	10	10	135	0	170
7	80	85	15	15	145	0	180
8	90	90	15	15	150	0	195
9	100	100	15	15	155	0	205
10	110	105	15	15	155	0	205
11	125	115	15	15	155	0	220
12	130	120	15	20	160	0	225
13	140	130	20	20	160	0	240
14	140	130	20	20	160	0	240
15	150	135	20	20	160	0	250
16	155	140	20	20	160	0	255
17	160	145	20	25	160	0	265
18	170	150	20	25	160	0	275
19	170	155	20	30	160	0	280
20	175	160	20	30	160	0	285
21	175	160	20	30	160	0	285
22	180	170	20	35	165	0	290
23	185	170	20	35	165	0	290
24	185	170	20	35	165	0	290
25	190	180	20	35	165	0	295
26	190	180	20	35	165	0	295
27	190	180	25	35	165	0	295
28	190	180	25	35	165	0	295
29	190	180	25	35	165	0	295
30	195	180	25	35	165	0	300
31	195	185	25	35	165	0	300
32	200	185	25	40	165	0	300
33	205	190	25	40	165	0	300
34	205	195	25	40	165	0	305
35	210	195	25	45	165	0	305
36	210	195	25	45	165	0	305
37	210	195	25	45	165	0	305
38	210	195	25	45	165	0	305
39	210	195	25	45	165	0	305
40	210	195	25	45	165	0	305
41	210	195	25	45	165	0	305
42	210	195	25	45	165	0	305
43	210	195	25	45	165	0	310
44	210	195	25	45	165	0	310
45	210	195	25	45	165	0	310
46	210	195	25	50	165	0	315
47	210	195	25	50	165	0	315
48	210	195	25	50	165	0	315
49	210	195	25	50	165	0	315
50	210	195	25	55	165	0	315
51	210	195	25	55	165	0	315
52	210	195	25	55	165	0	315
53	210	195	25	55	165	0	315
54	210	195	25	55	165	0	315
55	210	195	30	55	165	0	315
56	210	195	30	55	165	0	315
57	210	195	30	55	165	0	315
58	210	195	30	55	165	0	315
59	210	195	30	55	165	0	315
60	210	195	30	55	165	0	315

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4:

¹ ThOD_NH4 of TAGETE OIL: 2.957 mg O2/mg test item

² ThOD_NH4 of sodium benzoate: 1.666 mg O2/mg reference item

	Percentage Biodegradation
Time (Days)	TAGETE OIL^1		Sodium Benzoate ^2	Toxicity control ^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	1	1	28	13
2	1	0	42	17
3	10	10	55	24
4	15	15	64	28
5	17	18	70	30
6	20	22	73	34
7	21	23	76	35
8	25	25	79	38
9	28	28	82	40
10	31	30	82	40
11	36	33	82	43
12	37	34	83	44
13	40	37	82	47
14	40	37	82	47
15	43	38	82	49
16	45	40	82	50
17	45	41	80	51
18	49	42	80	54
19	48	43	79	54
20	50	45	79	55
21	50	45	79	55
22	50	47	80	56
23	52	47	80	56
24	52	47	80	56
25	54	51	80	57
26	54	51	80	57
27	53	50	79	56
28	53	50	79	56
29	53	50	79	56
30	54	50	79	57
31	54	52	79	57
32	55	51	77	57
33	57	52	77	57
34	57	54	77	58
35	58	53	76	57
36	58	53	76	57
37	58	53	76	57
38	58	53	76	57
39	58	53	76	57
40	58	53	76	57
41	58	53	76	57
42	58	53	76	57
43	58	53	76	58
44	58	53	76	58
45	58	53	76	58
46	57	52	74	59
47	57	52	74	59
48	57	52	74	59
49	57	52	74	59
50	56	52	73	58
51	56	52	73	58
52	56	52	73	58
53	56	52	73	58
54	56	52	73	58
55	55	51	71	58
56	55	51	71	58
57	55	51	71	58
58	55	51	71	58
59	55	51	71	58
60	55	51	71	58

Biodegradation in the Toxicity Control

In the toxicity control containing both, the test item and the reference item sodium benzoate, 47% biodegradation was noted within 14 days and 56% biodegradation after 28 days of incubation.

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.

Validity Criteria of the Study

- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 30 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 7.5 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 the plateau and at the end of the test was less than 20%. The difference of duplicate values at day 13 (end of 10-day window) and day 34 (plateau) was 3% and 4% at test end at day 60. 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 47% at day 14; the test item was not inhibitory.

All validity criteria are fulfilled.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of the test item TAGETE OIL reached 52%, following the ThODNH4, after 28 days of incubation and 53%, on Day 60. The 10-Day window failed. According to the test guideline, the test item is not considered readily biodegradable.

Executive summary:

The aim of this study was to determine the effects of the test item TAGETE 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).

The mean biodegradation of 10% of TAGETE OIL was reached at day 3 (ThODNH4). At the end of the 10-day window at day 13, the degradation of TAGETE OIL was 39% (ThODNH4) and therefore the 10 day window criterion was not passed.

The mean biodegradation at day 28 was 52%. According to the OECD guideline 301F, the test item is not considered as readily biodegradable.

Nevertheless, the plateau of biodegradation was not reached, therefore the test was prolonged to 60 days incubation time. The plateau of biodegradation was reached at day 34 and remained at this level until the end of the test. At test end after 60 days the biodegradation of the test item was 53% (mean, ThODNH4).

The oxygen demand of the inoculum control was not greater than 60 mg O2/L within 28 days. The pH-value of the test item flasks at the end of the test was within the range of pH 6.0 to 8.5.

The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation, thus confirming the suitability of the aerobic activated sludge used.

The difference of duplicate values for the degradation of the test item at the end of the 10-day window, at the plateau and at the end of the test was less than 20%.

The biodegradation was 47% at day 14 in the toxicity control; the test item was not inhibitory.

All the validity criteria were fulfilled.

Description of key information

The degradation rate of the test item TAGETE OIL reached 52%, following the ThODNH4, after 28 days of incubation. Therefore, TAGETE OIL is considered to be not readily biodegradable.

Moreover, the biodegradation rate of the substance was 53% at the end of the test, at day 60.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A reliable experimental study was available for that endpoint. The registered substance 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 mean biodegradation of 10% of TAGETE OIL was reached at day 3 (ThODNH4). At the end of the 10-day window at day 13, the degradation of TAGETE OIL was 39% (ThODNH4) and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 52%. According to the OECD guideline 301F, the test item is not considered as readily biodegradable.

Nevertheless, the plateau of biodegradation was not reached, therefore the test was prolonged to 60 days incubation time. The plateau of biodegradation was reached at day 34 and remained at this level until the end of the test. At test end after 60 days the biodegradation of the test item was 53% (mean, ThODNH4).

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 comply with the requirements of the guideline. This study is considered valid for that endpoint.