Santalum austro-caledonicum, 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:

guideline study

Remarks:

All validity criteria was 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)

Remarks:

2017-03-13

Specific details on test material used for the study:

None

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Test Species: Aerobic activated sludge (microorganisms from a domestic waste¬water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
- Conditioning: The aerobic activated sludge used for this study 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 overnight. This suspension was used for the experiment.

Duration of test (contact time):

60 d

Initial conc.:

103.1 mg/L

Based on:

other: Test Item Loading Rate (initial concentration in medium C0)

Initial conc.:

304 mg/L

Based on:

ThOD

Remarks:

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: 7.4 to 7.9
- 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 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

Value:

59

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

78

Sampling time:

60 d

Details on results:

The mean biodegradation of 10% of the test item was reached at day 6. At the end of the 10-day window at day 16, the mean degradation of the test item was 38% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 59%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 30, when the mean degradation was 61%. Afterwards an increase of degradation could be determined till day 59, when the plateau of biodegradation was reached; the mean degradation was 78% at day 59. Afterwards the degradation of the test item remained constant and was 78% at test end after 60 days.

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.

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	45	0	60
2	10	5	5	5	85	0	95
3	10	10	5	5	105	0	115
4	15	20	5	10	115	0	130
5	25	25	10	10	125	0	135
6	40	40	10	10	135	0	145
7	55	55	10	15	140	0	150
8	65	75	10	15	145	0	160
9	75	90	15	20	150	0	170
10	85	105	15	20	150	0	175
11	90	115	15	20	150	0	180
12	100	125	15	20	155	0	185
13	105	135	15	20	155	0	185
14	110	140	20	25	155	0	190
15	115	150	20	25	155	0	195
16	120	155	20	25	160	0	200
17	125	165	20	25	160	0	205
18	130	175	20	25	160	0	210
19	135	180	25	25	160	0	215
20	145	185	25	25	160	0	215
21	150	190	25	25	160	0	220
22	155	195	25	25	160	0	220
23	165	200	25	25	160	0	225
24	170	205	25	30	160	0	230
25	175	210	25	30	160	0	235
26	185	215	25	30	160	0	240
27	185	220	25	30	160	0	240
28	195	220	25	30	160	0	240
29	195	220	25	30	160	0	240
30	200	225	25	30	160	0	245

31	210	225	25	30	160	0	245
32	215	230	25	30	160	0	250
33	220	235	25	30	160	0	255
34	220	240	25	30	160	0	255
35	225	240	25	30	160	0	260
36	230	245	25	30	160	0	260
37	230	245	25	30	160	0	260
38	235	250	25	30	160	0	260
39	235	250	25	30	160	0	260
40	240	250	25	35	160	0	260
41	240	250	25	35	160	0	260
42	245	250	25	35	160	0	260
43	250	255	25	35	160	0	260
44	255	255	25	35	160	0	260
45	255	255	25	35	160	0	260
46	255	255	25	35	160	0	260
47	255	255	25	35	160	0	260
48	260	255	25	35	160	0	260
49	260	255	25	35	160	0	260
50	260	255	25	35	160	0	260
51	260	255	25	35	165	0	260
52	265	255	25	35	165	0	260
53	265	255	25	35	165	0	260
54	270	255	25	35	165	0	260
55	270	255	25	35	165	0	260
56	270	255	25	35	165	0	260
57	270	255	25	35	165	0	260
58	275	255	25	35	165	0	260
59	275	260	25	35	165	0	260
60	275	260	25	35	165	0	260

Flasks 1 and 2: test substance

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)	Test item		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	2	0	26	13
2	2	0	46	19
3	2	2	58	23
4	2	4	62	26
5	5	5	67	26
6	10	10	73	28
7	14	14	74	29
8	17	20	77	31
9	19	24	77	32
10	22	29	77	33
11	24	32	77	34
12	27	35	80	35
13	29	38	80	35
14	29	38	77	35
15	31	42	77	36
16	32	43	80	37
17	34	47	80	38
18	36	50	80	39
19	36	51	78	40
20	40	52	78	40
21	41	54	78	41
22	43	56	78	41
23	46	57	78	42
24	47	58	77	43
25	49	60	77	44
26	52	61	77	45
27	52	63	77	45
28	55	63	77	45
29	55	63	77	45
30	57	65	77	46
31	60	65	77	46

32	62	66	77	47
33	64	68	77	48
34	64	69	77	48
35	65	69	77	49
36	67	71	77	49
37	67	71	77	49
38	69	73	77	49
39	69	73	77	49
40	70	72	76	48
41	70	72	76	48
42	71	72	76	48
43	73	74	76	48
44	74	74	76	48
45	74	74	76	48
46	74	74	76	48
47	74	74	76	48
48	76	74	76	48
49	76	74	76	48
50	76	74	76	48
51	76	74	78	48
52	78	74	78	48
53	78	74	78	48
54	79	74	78	48
55	79	74	78	48
56	79	74	78	48
57	79	74	78	48
58	81	74	78	48
59	81	75	78	48
60	81	75	78	48

¹ThOD_NH4of test item: 2.949 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, 35% biodegradation was noted within 14 days and 45% 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.

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 16), 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 11% for any time point.

The validity criterion was fulfilled.

- 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 35% 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 registered substance is considered to be not readily biodegradable after 28 days, but reached a degradation of 78% after 60 days.

Executive summary:

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, and further prolongated to 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.

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 Santalum austro-caledonicum ext was reached at day 6. At the end of the 10-day window at day 16, the mean degradation of Santalum austro-caledonicum ext was 38% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 59%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 30, when the mean degradation was 61%. Afterwards an increase of degradation could be determined till day 59, when the plateau of biodegradation was reached; the mean degradation was 78% at day 59. Afterwards the degradation of Santalum austro-caledonicum ext remained constant and was 78% at test end after 60 days.

The degradation rate of the test item did reach 60% after 30 days, but not within 28 days.

Therefore, the registered substance is considered to be not readily biodegradable after 28 days, but reached a degradation of 78% after 60 days.

The reference item sodium benzoate was sufficiently degraded, thus confirming the suitability of the aerobic activated sludge inoculum used.

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 in the toxicity control.

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 reached a mean degradation rate of 59% at 28 days and is therefore not considered as readily biodegradable according to the test guideline. However, the substance reached a mean degradation rate of 60% after 30 days of incubation and at the end of the test at day 60, the mean biodegradation was 78%.

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 the test item was reached at day 6. At the end of the 10-day window at day 16, the mean degradation of the test item was 38% and therefore the 10 day window criterion was not passed. The mean biodegradation at day 28 was 59%; therefore the test was prolonged till day 60. 60% biodegradation or more was reached at day 30, when the mean degradation was 61%. Afterwards an increase of degradation could be determined till day 59, when the plateau of biodegradation was reached; the mean degradation was 78% at day 59. Afterwards the degradation of the test item remained constant and was 78% at test end after 60 days.

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.

Therefore, this study is considered valid for that endpoint.