Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

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:

2022-06-16 to 2022-11-14

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:

1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Inoculum of the aqueous phase of non-adapted activated sludge
- Source: Municipal sewage treatment plant, Hildesheim, Germany
- After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic conditions by aeration with CO2 free air until test start (3 days) at test temperature.
- The amount of inoculum used to initiate inoculation was 5.22 ml/L (25.0 mg/L dw)

Duration of test (contact time):

60 d

Initial conc.:

22 mg/L

Based on:

test mat.

Initial conc.:

52.6 mg/L

Based on:

ThOD

Initial conc.:

57.6 mg/L

Based on:

ThOD

Remarks:

with nitrification

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PRE-TREATMENT
- The test item was applied via a stock solution in silicone oil.
- The stock solution was prepared by weighing out of 11.1 g/L test item in a measuring flask and adding approx. 10 mL of silicone oil.
- The stock solution was shaken until the test item was fully dissolved.
- Upon visual checking, the final test solution was clear. 496.5 µL was pipetted into the brown glass bottles.


TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 F
- Test temperature: Nominal: 20 - 24 ± 1 °C; Actual: 21.8 – 22.5 °C
- Photoperiod: Dark

TEST SYSTEM
- Culturing apparatus: Closed bottles
- Number of culture flasks/concentration: 2 for the inoculum control, 2 for the inoculum control with silicone oil, 1 for the functional control, 1 for the functional control with silicone oil, 3 for the test item replicates, 3 for the test item replicates with silicone oil, 1 for the toxicity control, 1 for the toxicity control with silicone oil.
- Method used to create aerobic conditions: Headspace of air
- Measuring equipment: OxiTop OC110 controller with software Achat OC, WTW OxiTop measuring heads, WTW

SAMPLING
- Sampling frequency: daily
- Sampling method: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

43.66

Sampling time:

28 d

Remarks on result:

other: Test item solved in silicone oil for application due to low solubility in water

Key result

Parameter:

% degradation (O2 consumption)

Value:

55

Sampling time:

60 d

Remarks on result:

other: Test item solved in silicone oil for application due to low solubility in water

Details on results:

Degradation kinetics (%) of the test item with silicone oil:
-Day 9: Rep1 9%, Rep2 13%, Rep3 12%
-Day 19: Rep1 16%, Rep2 27%, Rep3 32%
-Day 28: Rep1 26%, Rep2 41%, Rep3 38%
-Day 42: Rep1 38%, Rep2 54%, Rep3 47%
-Day 52: Rep1 42%, Rep2 62%, Rep3 51%
-Day 60: Rep1 44%, Rep2 67%, Rep3 55%


Toxicity control with silicone oil:
- Day 2: 24%
- Day 14: 45%
- Day 28: 49%
- Day 60: 55%

In all test item replicates a formation of oxidized nitrogen lower the inoculum control level was observed. Based on these results the ThOD was used for evaluation.

Results with reference substance:

Biodegradation of the functional control with silicone oil:
- Day 2: 50%
- Day 12: 76%
- Day 28: 78%
- Day 60: 80%

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The study showed that the test item is not readily biodegradable. The test item did not reach its pass level of biodegradation (60%) during the 60-d period.

Executive summary:

The present study was conducted in order to evaluate the biodegradability of the test item by measuring its percentage of biodegradation with inoculum in a period of 60 days. The test method was performed following the OECD Test Guideline 301F and GLP.

The test item concentration was 22 mg/L. It was solved in silicone oil for its application due to its low water solubility. Benzoic acid was used as reference substance in the functional control as well as in the toxicity control. After the 60-d period the biodegradation of the test item was calculated to be 55% and it did not reach a plateau. Therefore, the test item is regarded as not readily biodegradable. 

Description of key information

Not readily biodegradable: 55% after 60 days (OECD 301F)

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The present study was conducted in order to evaluate the biodegradability of the test item by measuring its percentage of biodegradation with inoculum in a period of 60 days. The test method was performed following the OECD Test Guideline 301F and GLP.

The test item concentration was 22 mg/L. It was solved in silicone oil for its application due to its low water solubility. Benzoic acid was used as reference substance in the functional control as well as in the toxicity control. After the 60-d period the biodegradation of the test item was calculated to be 55% and it did not reach a plateau. Therefore, the tes item is regarded as not readily biodegradable.