Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Type: mixed population of aquatic microorganisms
- Origin: secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2017-01-30
- Pre-treatment: separation of coarse particles by filtration, aeration of the resulting inoculum for 1 day
- Effluent concentration of reaction mixture: 5 mL/L (5.8 mL/1.16 L)

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Pre-treatment of the test item
- 2.3 mg of test item were weighed out on aluminium foil and added to the test flasks, filled with ca. 300 mL of mineral medium
- inoculum was added and the flasks volume was made up to 1.16 L with mineral medium
- the flasks were closed free from air bubbles with glass stoppers.
Initial concentration of test item: 2.0 mg/L (2.3 mg/1.16 L)

Pre-treatment of the toxicity control
- 2.3 mg of test item were weighed out on aluminium foil and added to the test flasks, filled with ca. 300 mL of mineral medium
- additionally 3.4 mL of the reference compound stock solution were added
- inoculum was added and the flasks volume was made up to 1.16 L with mineral medium
- the flasks were closed free from air bubbles with glass stoppers.
Initial concentration of test item: 2.0 mg/L (2.3 mg/1.16 L)
Initial concentration reference compound: 2.9 mg/L (3.4 mg/1.16 L)

Exposure conditions
- Test volume: 1.16 L
- Incubation time: 28 days
- Incubation temperature: 22 ± 2 °C


The following flasks were used:
− Test suspension (18 flasks) A measured volume of mineral medium containing a known concentration of the test item (as the nominal sole source of organic carbon) and inoculum.
− Reference control (10 flasks) In order to check the procedure, the reference compound sodium benzoate was tested in parallel to the normal test runs.
− Inoculum blank (18 flasks) A measured volume of mineral medium containing only inoculum.
− Toxicity control (10 flasks) Toxic effects of the test item were determined in toxicity control flasks containing test item, reference compound and inoculum.

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, all determinations were carried out in duplicate.
The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start and at the end of the test. The oxygen consumed by nitrification was calculated. This oxygen consumption by nitrification was subtracted from the respective measurements.

Reference substance:

benzoic acid, sodium salt

Remarks:

Acros Organics, purity: 99.9 %, Batch no. A0357641

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

28 d

Details on results:

1 % degradation after 7 days
4 % degradation after 14 days
6 % degradation after 21 days
No nitrification was observed in the flasks with test item.

Results with reference substance:

Sodium benzoate showed 82 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

oxygen depletion (blank control) < 1.5 mg dissolved oxygen/L after 28 days; reference compound ≥ 60 % ready biodegradability within 14 days; toxicity control exhibited degradation rates > 25 % within 14 days

Interpretation of results:

not readily biodegradable

Conclusions:

Macrolex Orange R showed 5 % degradation after 28 days. Therefore, Macrolex Orange R is considered to be “Not Readily Biodegradable“

Executive summary:

The study was conducted in accordance with the Council Regulation (EC) 440/2008 Method C.4-E “Closed Bottle Test” (2008), which is in all essential parts identical with OECD Guideline 301 D (adopted July 1992).

The test item Macrolex Orange R in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions for 28 days at 22 ± 2°C in the dark. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage theoretical oxygen demand (ThOD). Reference control, inoculum blank and toxicity control were run in parallel.

Macrolex Orange R showed: 5 % degradation after 28 days. Therefore, Macrolex Orange R is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 82 % degradation after 14 days.

The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start and at the end of the test. The oxygen consumed by nitrification was calculated. This oxygen consumption by nitrification was subtracted from the respective measurements.

No nitrification was observed in the flasks with test item.

Description of key information

The study was conducted in accordance with the Council Regulation (EC) 440/2008 Method C.4-E “Closed Bottle Test” (2008), which is in all essential parts identical with OECD Guideline 301 D (adopted July 1992).

The test item Macrolex Orange R in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions for 28 days at 22 ± 2°C in the dark. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage theoretical oxygen demand (ThOD). Reference control, inoculum blank and toxicity control were run in parallel.

Macrolex Orange R showed: 5 % degradation after 28 days. Therefore, Macrolex Orange R is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 82 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed