Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

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-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

yes

Remarks:

pH was measured two days after last BOD measurement due lab-specific reasons; this has no effect on the results as all validity criteria were met

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test organism
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2016-02-29
- Concentration of inoculum: 30 mg/L suspended solids

Pre-treatment of the inoculum
- inoculum was stored for one day at room temperature under continuous stirring with aeration
- sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant
- an aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
- the calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Pre-treatment of the test item
- 25 mg of test item were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item/L

Pre-treatment of the reference compound
- Reference compound name: sodium benzoate (Acros Organics)
- 25 mg of reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg reference compound/L

Pre-treatment of the toxicity control
- 25 mg of test item and 25 mg of reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound/L

Exposure conditions
- Test volume: 250 mL
- Test apparatus: OxiTopControl System (WTW)
- Mixing: 1 magnetic stirrer per test vessel
- Incubation time: 28 days
- Incubation temperature: 22 +- 1 °C
- pH: 7.2 - 8.0 (measured after 30 d due to lab-specific reasons)
- continuous darkness
- BOD was measured daily
- blanks with inoculum but without test item were run in parallel (3 replicates)

Reference substance:

benzoic acid, sodium salt

Remarks:

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

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

1 % degradation after 7 d
0 % degradation after 14 d
0 % degradation after 21 d

Results with reference substance:

The reference compound sodium benzoate showed 81 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

biodegradation of reference compound >= 60 % within 14 d; degradation of toxicity control > 25 % within 14 d; replicates difference < 20 %; oxygen uptake of inoculum blank <= 60 mg/L; pH values were in between 6.0 and 8.5 at the end of the test

Interpretation of results:

not readily biodegradable

Conclusions:

Macrolex Rot B showed 0 % degradation within 28 d. The reference compound sodium benzoate was degraded to 81 % after 14 d. Thus, Macrolex Rot B is not readily biodegradable.

Executive summary:

A study was performed to assess the ready biodegradability of Macrolex Rot B in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008). This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992).

A suspension of 100 mg/L Macrolex Rot B in a mineral medium, equalling to 50-100 mg ThOD or COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 +- 1 °C) for up to 28 days under aerobic conditions in the dark. During this period, degradation was followed by continuous automated BOD determinations.

The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. A toxicity control (test item and reference compound mixed, one replicate) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms. Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate. At the end of incubation, the pH was measured in the flasks.

Macrolex Rot B showed 0 % degradation after 28 days. Therefore, Macrolex Rot B is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 81 % degradation after 14 days.

The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start of the test. The oxygen consumed by nitrification was not determined after 28 days, because no degradation of the test item was observed.

Description of key information

Macrolex Rot B showed 0 % degradation within 28 d. The reference compound sodium benzoate was degraded to 81 % after 14 d. Thus, Macrolex Rot B is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed