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Environmental fate & pathways

Biodegradation in water: screening tests

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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)
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 organisms
- 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: 2017-07-24
- Concentration of inoculum: 30 mg/L suspended solids

Pre-treatment of the inoculum
− Before use, inoculum was stored for three days 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
− 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 the 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
- 25 mg of the reference compound were weighed out and were 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 the test item and 25 mg of the 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-value of test solutions: 7.4 - 8.1
Reference substance:
benzoic acid, sodium salt
Remarks:
Acros Organics, 99.9 %, batch no. A0357641
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
1 % degradation after 7 days
0 % degradation after 14 days
0 % degradation after 21 days
Results with reference substance:
The reference compound sodium benzoate showed 91 % degradation after 14 days.
Validity criteria fulfilled:
yes
Remarks:
biodegradability reference compound ≥ 60 % within 14 d; degradation rate toxicity control > 25 % within 14 d; difference biodegradation in parallels with test item <= 20%, oxygen uptake of inoculum blank ≤ 60 mg/L, pH values independent
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
After 28 d Special Brilliant Blue FFR showed 0 % degradation, thus it is considered to be not readily biodegradable.
Executive summary:

This study was designed to assess the ready biodegradability of Special Brilliant Blue FFR. It was conducted 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 Special Brilliant Blue FFR in a mineral medium was inoculated at a constant temperature (22 ± 1 °C) for 28 d under aerobic conditions in the dark. During this period, degradation was followed by continuous automated BOD determinations. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of theoretical oxygen demand (ThOD).

The endogenous activity of the inoculum was checked running parallel blanks in triplicate with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel in duplicate 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. The determination of biodegradation of test item was carried out in triplicate. At the end of incubation, the pH was measured in the flasks.

Special Brilliant Blue FFR showed 0 % degradation after 28 days. Therefore, Special Brilliant Blue FFR is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 91 % 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

A study was conducted to assess the ready biodegradability of Special Brilliant Blue FFR in accordance with EC 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 Special Brilliant Blue FFR in a mineral medium was inoculated at a constant temperature (22 ± 1 °C) for 28 d 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 in triplicate with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel in duplicate 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. The determination of biodegradation of test item was carried out in triplicate.

Special Brilliant Blue FFR showed 0 % degradation after 28 days. Therefore, Special Brilliant Blue FFR is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 91 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The reference compound sodium benzoate showed 91 % degradation after 14 days.