Pepper (Piper), P. nigrum, 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 2017-03-23 to 2017-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 No.: 1003131757, provided by the sponsor
- Expiration date of the lot/batch: May 07, 2018
- Certificate of Analysis Date: March 03, 2017

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: microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, 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 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.

Duration of test (contact time):

60 d

Initial conc.:

102.7 mg/L

Based on:

test mat.

Remarks:

corresponding to an oxygen demand of about 326.3 mg/L (ThODNH4)

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: 7.4 to 7.9
- 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


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:

acetic acid, sodium salt

Preliminary study:

No data.

Test performance:

No data.

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

34

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

53

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNH4

Value:

72

Sampling time:

60 d

Details on results:

The mean biodegradation of 10% of Pepper black oil was reached at day 4. At the end of the 10-day window at day 14, the mean degradation of Pepper black oil was 34% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 53%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 38, when the mean degradation was 60%.
Afterwards an increase of degradation could be determined till day 59, when the plateau of biodegradation was reached; the mean degradation was 72% at day 59. Afterwards the degradation of Pepper black oil remained constant and was 72% at test end after 60 days.
Results for all flasks are illustrated in the attached figure 1.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 77% after 14 days, to 77% after 28 days and to 78 % after 60 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	Pepper black oil	7.4
2	Pepper black oil	7.4
3	Inoculum control	7.6
4	Inoculum control	7.6
5	Reference item (procedure control)	7.9

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	0	0	0	0	45	0	60
2	5	10	5	5	85	0	90
3	30	40	5	5	105	0	110
4	50	55	5	10	115	0	120
5	60	60	10	10	125	0	130
6	70	70	10	10	135	0	135
7	80	80	10	15	140	0	145
8	90	90	10	15	145	0	155
9	100	100	15	20	150	0	165
10	105	105	15	20	150	0	175
11	110	110	15	20	150	0	180
12	115	120	15	20	155	0	190
13	120	130	15	20	155	0	200
14	130	135	20	25	155	0	205
15	135	145	20	25	155	0	210
16	140	155	20	25	160	0	215
17	145	160	20	25	160	0	220
18	150	165	20	25	160	0	225
19	155	170	25	25	160	0	230
20	160	175	25	25	160	0	235
21	165	180	25	25	160	0	240
22	170	185	25	25	160	0	245
23	175	185	25	25	160	0	245
24	180	190	25	30	160	0	250
25	185	190	25	30	160	0	255
26	190	195	25	30	160	0	255
27	195	195	25	30	160	0	255
28	200	200	25	30	160	0	255
29	200	200	25	30	160	0	255
30	200	205	25	30	160	0	255
31	205	210	25	30	160	0	255
32	210	210	25	30	160	0	255
33	210	215	25	30	160	0	255
34	210	215	25	30	160	0	255
35	210	220	25	30	160	0	255
36	210	220	25	30	160	0	255
37	215	225	25	30	160	0	255
38	215	230	25	30	160	0	255
39	220	230	25	30	160	0	255
40	220	235	25	35	160	0	255
41	220	235	25	35	160	0	255
42	225	235	25	35	160	0	255
43	225	240	25	35	160	0	255
44	230	240	25	35	160	0	255
45	230	245	25	35	160	0	255
46	235	245	25	35	160	0	255
47	235	245	25	35	160	0	255
48	240	250	25	35	160	0	255
49	240	250	25	35	160	0	255
50	245	250	25	35	160	0	255
51	245	255	25	35	165	0	255
52	245	255	25	35	165	0	255
53	250	255	25	35	165	0	255
54	250	260	25	35	165	0	260
55	250	260	25	35	165	0	260
56	250	265	25	35	165	0	260
57	255	265	25	35	165	0	260
58	255	265	25	35	165	0	260
59	255	270	25	35	165	0	260
60	255	270	25	35	165	0	260
Flasks 1 and 2: Pepper black oil
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^1
(Days)	Pepper black oil^1		Sodium Benzoate^2	Toxicity
				control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	26	12
2	0	2	46	17
3	8	11	58	21
4	13	15	62	23
5	15	15	67	24
6	18	19	73	25
7	21	21	74	27
8	24	24	77	29
9	25	25	77	30
10	27	27	77	32
11	28	29	77	33
12	30	32	80	35
13	31	35	80	37
14	33	35	77	37
15	34	38	77	38
16	36	41	80	39
17	37	42	80	40
18	39	44	80	41
19	40	45	78	41
20	41	46	78	42
21	43	48	78	43
22	44	49	78	44
23	46	49	78	44
24	46	50	77	45
25	48	50	77	46
26	50	52	77	46
27	51	52	77	46
28	53	53	77	46
29	53	53	77	46
30	53	55	77	46
31	54	56	77	46
32	56	56	77	46
33	56	58	77	46
34	56	58	77	46
35	56	59	77	46
36	56	59	77	46
37	57	61	77	46
38	57	62	77	46
39	59	62	77	46
40	58	63	76	45
41	58	63	76	45
42	59	63	76	45
43	59	65	76	45
44	61	65	76	45
45	61	66	76	45
46	62	66	76	45
47	62	66	76	45
48	64	68	76	45
49	64	68	76	45
50	66	68	76	45
51	66	69	78	45
52	66	69	78	45
53	67	69	78	45
54	67	71	78	46
55	67	71	78	46
56	67	72	78	46
57	69	72	78	46
58	69	72	78	46
59	69	74	78	46
60	69	74	78	46
^1ThODNH4of Pepper black oil: 3.178 mg O2/mg test item
^2ThODNH4of 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, 37% biodegradation was noted within 14 days and 46% 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 27.5 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 the end of the test was 7.4 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 (day 14), at day 28, at the plateau (day 59) and at the end of the test and was less than 20%. The difference of duplicate values was not greater than 5% for any time point.

- 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 37% 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:

Pepper black oil is considered to be not readily biodegradable after 28 days, but reached a degradation of 72% after 60 days.
All the validity criteria were fulfilled.

Executive summary:

The test item Pepper black oil was investigated for its ready biodegradability in a manometric respirometry test over a period of 60 days according to the OECD 301F guideline.

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.

The mean biodegradation of 10% of Pepper black oil was reached at day 4. At the end of the 10-day window at day 14, the mean degradation of Pepper black oil was 34% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 53%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 38, when the mean degradation was 60%.

Afterwards an increase of degradation could be determined till day 59, when the plateau of biodegradation was reached; the mean degradation was 72% at day 59. The degradation of Pepper black oil remained constant and was 72% at test end after 60 days.

Pepper black oil is considered to be not readily biodegradable after 28 days, but reached a degradation of 72% after 60 days.

Validity criteria:

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.

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 in the toxicity control was 37% at day 14; the test item was not inhibitory.

All the validity criteria were fulfilled.

Description of key information

Based on an experimental study on Pepper black oil according to guidance OECD 301F, the substance is considered as not readily biodagradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The test item Pepper black oil was investigated for its ready biodegradability in a manometric respirometry test over a period of 60 days according to the OECD 301F guideline. Pepper black oil is considered to be not readily biodegradable after 28 days, but reached a degradation of 72% after 60 days.