Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

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:

2018-06-26 to 2018-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene
Synonym: KALAMA® Ocimene PQ
CAS No.: 3338-55-4
Lot No.: A170524D
Purity: Limonene: 20.1%
Cis-beta-Ocimene: 71.1%
Aggregate State at Room Temperature: Liquid
Color: Clear, almost colorless (according to CoA)
Chemical Structure / Molecular Weight : C10H16 / 136.23 g/mol
Theoretical Oxygen Demand: ThODNH4 : 3.289 mg oxygen per mg test item
Expiry / Retest Date: June 06, 2019
Storage Conditions at Test Facility: At 20°C ± 5°C, in the dark under inert gas (Nitrogen)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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 Rossdorf, Germany.

Conditioning: The aerobic activated sludge used for this study was deposited for 60 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.

Details on study design:

Test Conditions

Surrounding Type: Climatised chamber

Temperature: 22°C ± 1°C

Light Conditions: Darkness

pH-Value of Test Solutions: 7.4 (measured at the start of the test), 7.4 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 range do normally not result in major disturbances of the test performance and were not reported.)


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 7.4.
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.
The pH was adjusted to pH 7.4 with NaOH solution.

Course of the Test

Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks. For a better distribution, 2.4 mL silicon oil AR 20 per flask was added.

Incubation: The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

Test Duration: 28 days

Reference substance:

benzoic acid, sodium salt

Test performance:

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 7.5 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 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 4%. 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 38% at day 14; the test item was not inhibitory.

Key result

Parameter:

% degradation (O2 consumption)

Value:

45

Sampling time:

28 d

Details on results:

Biodegradation of Test Item

Percentage Biodegradation: 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 10-day windows began on day 4 after application, the mean value was calculated to be 10% biodegradation (ThODNH4). Therefore, the end of the 10-day window was at day 14. The mean biodegradation percentage based on ThODNH4 at the end of the 10-day window was 34%; the criterion of the 10 day window was not passed. The mean biodegradation percentage at the end of the 28-day exposure period was 45% (ThODNH4). The silicone oil added to the test vessels achieved its purpose. No clotting of the silicone oil or any other inhibiting effects on the dispersion of the test substances were recorded.

Conclusion: The degradation rate of Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene never reached 60%. Therefore, Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene is considered to be not readily biodegradable.

Biodegradation in the Toxicity Control

Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item sodium benzoate, 38% (ThODNH4) biodegradation was noted within 14 days and 51% (ThODNH4) biodegradation after 28 days of incubation.

Conclusion: 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.

Abiotic Control
Oxygen Demand: The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test item and toxicity control.

Results with reference substance:

Reference Item
The information concerning the reference item was provided by the Manufacturer.
Name: Sodium Benzoate
Batch No.: SLBW2610
Recommended Retest Date: November 2021
Purity: 100.0%
Aggregate State at Room Temperature: Solid
Colour: White
Quality Release Date: November 08, 2017
Empirical Formula / Molecular Weight: C7H5O2Na / 144.10 g/mol
Theoretical Oxygen Demand: ThODNH4: 1.666 mg oxygen per mg sodium benzoate
Storage Conditions at Test Facility: At 20°C ± 5°C, in the desiccator in the dark

Biodegradation of Reference Item Sodium Benzoate

Percentage Biodegradation: The reference item sodium benzoate was sufficiently degraded to 72% after 14 days and to 75% after 28 days of incubation.

Conclusion: 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 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	5	5	0	0	55	0	55
2	5	5	5	5	80	0	80
3	10	10	5	5	90	0	105
4	40	40	10	5	115	0	130
5	65	60	10	5	120	0	145
6	80	70	10	10	125	0	155
7	85	80	10	10	130	0	160
8	95	85	10	10	130	0	170
9	100	90	10	10	130	0	175
10	120	100	10	10	135	0	185
11	125	105	15	10	135	0	190
12	130	110	15	15	135	0	195
13	135	115	15	15	135	0	200
14	135	120	15	15	140	0	205
15	140	125	15	15	140	0	215
16	145	130	15	15	140	0	220
17	150	135	15	15	140	0	225
18	150	135	15	15	140	0	230
19	155	140	15	15	140	0	235
20	155	145	15	15	140	0	245
21	160	145	15	15	140	0	255
22	160	150	15	15	140	0	260
23	160	150	15	15	140	0	260
24	165	155	15	15	145	0	265
25	165	155	15	15	145	0	270
26	170	155	15	15	145	0	270
27	170	160	15	15	145	0	275
28	170	160	15	15	145	0	275

Flasks 1 and 2: Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene

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)	Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	2	1	32	11
2	0	0	43	15
3	2	1	49	20
4	10	10	62	24
5	17	16	65	27
6	21	18	67	29
7	23	21	69	30
8	26	22	69	32
9	27	24	69	33
10	33	27	72	35
11	34	27	71	35
12	35	28	69	36
13	36	30	69	37
14	36	31	72	38
15	38	33	72	40
16	39	34	72	41
17	41	36	72	42
18	41	36	72	43
19	42	37	72	44
20	42	39	72	45
21	44	39	72	47
22	44	40	72	48
23	44	40	72	48
24	45	42	75	49
25	45	42	75	50
26	47	42	75	50
27	47	43	75	51
28	47	43	75	51

¹ThOD_NH4of Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene: 3.289 mg O₂/mg test item
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene	7.5
2	Ocimene PQ: Reaction mass of (Z)-3,7-dimethylocta-1,3,6,-triene and dipentene	7.5
3	Inoculum control	7.5
4	Inoculum control	7.4
5	Reference item (procedure control)	7.6

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Ocimene PQ is considered to be not readily biodegradable.

Executive summary:

The ready biodegradation of Ocimene PQ was determined in a Manometric test system according to OECD guideline 301F.The degradation rate of Ocimene PQ never reached 60%.Therefore,Ocimene PQ is considered to be not readily biodegradable.

Description of key information

The ready biodegradation of Ocimene PQ was determined in a Manometric test system according to OECD guideline 301F.The degradation rate of Ocimene PQ never reached 60%.Therefore,Ocimene PQ is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater