Essential oil of Platycladus orientalis (L.) Franco obtained from branches and leaves by steam distillation followed by vacuum distillation

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 2018-08-10 to 2018-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

- Species/Origin:Aerobic activated sludge (microorganisms from a domestic waste¬water treatment plant) was supplied by the sewage treatment plant of Bensheim, 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.

Duration of test (contact time):

28 d

Initial conc.:

102.7 mg/L

Based on:

test mat.

Remarks:

Test Item Loading Rate (initial concentration in medium C0)

Initial conc.:

304.4 mg/L

Based on:

ThOD

Remarks:

ThOD (NH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Solubilising agent (type and concentration if used): Silicon oil AR20 (1%V/V)
- Temperature: 22°C ± 1°C
- Light Conditions: Darkness
- pH-Value of Test Solutions: 7.5 (measured at the start of the test)
- 7.3 to 7.6 (measured at the end of the test)
- Recording: Test conditions (temperature) were 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 do normally not result in major disturbances of the test performance and were not reported.)
- Comopsition of medium :Aliquots of washed sludge suspension were mixed with reconstitued test water.
- Reconstituted Test 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 7.3.
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. The pH of the solution was 7.4.


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.
- 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:

None.

Test performance:

No data.

Parameter:

% degradation (O2 consumption)

Remarks:

ThOD (NH4)

Value:

62

Sampling time:

28 d

Details on results:

Test item :The 10-day windows began on day 3 after application, the mean value was calculated to be 11% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 13. The mean biodegradation percentage based on ThODNH4 at the end of the 10-day window was 40%; the criterion of the 10 day window was no passed. The mean biodegradation percentage at the end of the 28-day exposure period was 62% (ThODNH4).

Toxicity control: In the toxicity control containing both, the test item and the reference item sodium benzoate, 43% (ThODNH4) biodegradation was noted within 14 days and 58% (ThODNH4) biodegradation after 28 days of incubation.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 88% after 28 days of incubation.

Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	5	5	5	5	45	0	55
2	15	5	5	5	75	0	80
3	45	40	10	10	110	0	120
4	60	50	10	10	120	0	130
5	70	65	15	15	125	0	140
6	80	75	15	15	130	0	150
7	90	85	15	15	135	0	155
8	100	95	15	15	135	0	165
9	110	105	15	15	140	0	175
10	120	115	15	15	145	0	180
11	130	125	15	20	150	0	190
12	135	130	20	20	150	0	200
13	140	140	20	20	155	0	215
14	150	145	20	20	160	0	225
15	155	150	20	25	160	0	230
16	165	155	20	25	165	0	240
17	170	160	20	25	165	0	250
18	175	165	20	25	165	0	260
19	180	175	20	25	165	0	265
20	180	180	20	25	165	0	275
21	185	185	20	25	170	0	280
22	185	185	20	25	170	0	285
23	190	190	20	25	170	0	290
24	195	195	20	25	170	0	295
25	200	200	20	25	175	0	300
26	205	205	20	25	175	0	300
27	210	210	20	25	175	0	300
28	210	210	20	25	175	0	300

Flasks 1 and 2: Thuja Oil; 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
(Days)	Thuja Oil^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	23	10
2	3	0	40	16
3	11	10	58	23
4	16	13	63	25
5	18	16	63	26
6	21	20	66	28
7	25	23	69	29
8	28	26	69	31
9	31	30	72	33
10	34	33	75	34
11	37	35	76	36
12	38	36	75	37
13	39	40	78	41
14	43	41	81	43
15	43	42	79	43
16	47	44	82	45
17	48	45	82	47
18	50	47	82	49
19	52	50	82	50
20	52	52	82	53
21	53	53	85	54
22	53	53	85	55
23	55	55	85	56
24	57	57	85	57
25	58	58	88	58
26	60	60	88	58
27	61	62	88	58
28	61	62	88	58

¹ThOD_NH4of Thuja Oil: 2.965 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 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 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 plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 1%. 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 43% at day 14; the test item was not inhibitory.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.

Executive summary:

The test item Thuja Oil was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 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 62%.The test item is considered as readily biodegradable based on ThODNH4 after 28 days.

The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 88% 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, 43% biodegradation was noted within 14 days and 58% 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.

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 degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

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.

The degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.