Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

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:

2018-06-14 to 2018-09-05

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)

Version / remarks:

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

Deviations:

no

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.4D of May 30, Manometric Respirometry Test (EEC
Publication No. L 142/496, May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: Azuril: Reaction mass of 3-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile
- Synonym: KALAMA® Azuril
- CAS No.: 68084-04-8/21690-43-7
- Lot No.: A170421E
- Purity: Azuril 99.35%
- Aggregate State at Room Temperature: Liquid
- Color: Clear, pale yellow (according to data sheet), Clear, almost colorless (according to CoA)
- Chemical Structure / Molecular Weight : C13H19N / 189.30 g/mol
- Theoretical Oxygen Demand: ThODNH4: 2.874 mg oxygen per mg test item ThODNO3: 3.212 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.

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

total amount of oxygen required to oxidise a chemical completely assuming nitrification occurs

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), 7.3 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 from the recommended temperature 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 1N NaOH solution.

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

Test Parameters:

Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.

Temperature: The temperature was recorded by means of the automated software AMR Wincontrol©.

pH-Value: pH-values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ThODNO3

Value:

29

Sampling time:

28 d

Details on results:

Biodegradation of Test Item:

Percentage Biodegradation: Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. 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 14 after application, the mean value was calculated to be 12% biodegradation (ThODNO3). Therefore, the end of the 10-day window was at day 24. The mean biodegradation percentage based on ThODNO3 at the end of the 10-day window was 28%; the criterion of the 10 day window was not passed. The mean biodegradation percentage at the end of the 28-day exposure period was 29% (ThODNO3). If the mean biodegradation was based on ThODNH4, the biodegradation was 33% after 28 days of incubation. 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 Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3- pentenyl)cyclohex-3-ene-1-carbonitrile never reached 60%. Therefore, Azuril: Reaction mass of 3-(4-methyl-3- pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3- pentenyl)cyclohex-3-ene-1-carbonitrile 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, 39% (ThODNH4) biodegradation was noted within 14 days and 52% (ThODNH4) biodegradation after 28 days of incubation (36% and 49% based on ThODNO3).

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:

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	50
2	5	5	5	5	80	0	80
3	10	10	5	5	90	0	95
4	10	10	10	5	115	0	120
5	10	10	10	5	120	0	130
6	15	15	10	10	125	0	135
7	15	15	10	10	130	0	140
8	15	15	10	10	130	0	145
9	15	15	10	10	130	0	150
10	15	20	10	10	135	0	155
11	20	25	15	10	135	0	165
12	25	30	15	15	135	0	175
13	35	35	15	15	135	0	185
14	60	45	15	15	140	0	195
15	70	60	15	15	140	0	205
16	85	75	15	15	140	0	215
17	90	80	15	15	140	0	220
18	90	85	15	15	140	0	225
19	95	90	15	15	140	0	230
20	95	95	15	15	140	0	235
21	100	100	15	15	140	0	240
22	100	105	15	15	140	0	245
23	100	105	15	15	140	0	245
24	100	110	15	15	145	0	250
25	105	110	15	15	145	0	255
26	105	110	15	15	145	0	255
27	105	115	15	15	145	0	260
28	105	115	15	15	145	0	260

Flasks 1 and 2: Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

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
(Days)	Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	2	2	32	11
2	0	0	43	16
3	2	2	49	19
4	1	1	62	24
5	1	1	65	26
6	2	2	67	27
7	2	2	69	28
8	2	2	69	29
9	2	2	69	30
10	2	3	72	31
11	3	4	71	33
12	3	5	69	34
13	7	7	69	36
14	15	10	72	39
15	19	15	72	41
16	24	20	72	43
17	26	22	72	44
18	26	24	72	45
19	27	25	72	46
20	27	27	72	47
21	29	29	72	48
22	29	30	72	49
23	29	30	72	49
24	29	32	75	50
25	31	32	75	51
26	31	32	75	51
27	31	34	75	52
28	31	34	75	52

¹ThOD_NH4of Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile: 2.874 mg O₂/mg test item
²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Percentage Biodegradation of Test Item and of the Toxicity Control based on ThOD_NO3and of Sodium Benzoate based on ThOD_NH4

Time	Percentage Biodegradation
(Days)	Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile^1		Sodium Benzoate^2	Toxicity control^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	2	2	32	10
2	0	0	43	15
3	2	2	49	18
4	1	1	62	22
5	1	1	65	24
6	2	2	67	25
7	2	2	69	26
8	2	2	69	27
9	2	2	69	28
10	2	3	72	29
11	2	4	71	30
12	3	5	69	32
13	6	6	69	34
14	14	9	72	36
15	17	14	72	38
16	21	18	72	40
17	23	20	72	41
18	23	21	72	42
19	24	23	72	43
20	24	24	72	44
21	26	26	72	45
22	26	27	72	46
23	26	27	72	46
24	26	29	75	47
25	27	29	75	48
26	27	29	75	48
27	27	30	75	49
28	27	30	75	49

¹ThOD_NO3of Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile: 3.212 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	Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile	7.5
2	Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile	7.5
3	Inoculum control	7.4
4	Inoculum control	7.3
5	Reference item (procedure control)	7.6

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of Azuril never reached 60%. Therefore, Azuril is considered to be not readily biodegradable.

Executive summary:

The ready biodegradation potential of Azuril was measured using a manometric respiration test according to OECD guidelines 301F. Azuril did not meet the criteria for ready biodegradation under the conditions of this test.

Description of key information

The ready biodegradation potential of Azuril was measured using a manometric respiration test according to OECD guidelines 301F. Azuril did not meet the criteria for ready biodegradation under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThOD_NO3) was considered for the evaluation of the results. 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 14 after application, the mean value was calculated to be 12% biodegradation (ThOD_NO3). Therefore, the end of the 10-day window was at day 24. The mean biodegradation percentage based on ThOD_NO3at the end of the 10-day window was 28%; the criterion of the 10 day window was not passed. The mean biodegradation percentage at the end of the 28-day exposure period was 29% (ThOD_NO3).

If the mean biodegradation was based on ThOD_NH4, the biodegradation was 33% after 28 days of incubation.

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.

The degradation rate of Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile never reached 60%. Therefore, Azuril: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile is considered to be not readily biodegradable.