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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
Specific details on test material used for the study:
- Lot/batch No.: 407940
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge : Maxéville's wastewater treatment plant, mainly treating domestic wastewater (98%) or Toul's wastewater treatment plant (both are located in France). Sampling the 4th of June
- Preparation of inoculum: The day of sampling, the sludge was washed by 3 successive centrifugations (1100 g for 10 min) after resuspension of the pellet in the mineral medium and filtration on a 100 µm porosity stainless steel sieve.
- Storage conditions: activated sludges are used 24 hours after sampling
Duration of test (contact time):
ca. 28 d
Initial conc.:
14.9 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium composed as detailed in OECD guideline 301 B (1992) paragraphs 5 and 6.
- Additional substrate: no
- Test temperature: 22°C +/- 2°C
- pH: no data
- Suspended solids concentration: ≤ 30 mg/L in the final mixture
- Continuous darkness: no

- Number of culture flasks/concentration: 2 replicates for the test item
- Method used to create aerobic conditions: flow of 50 mL/min of air exempted of CO2 throughout the study
- Details of trap for CO2: the produced carbon dioxide is absorbed into the barium hydroxide solution (250 to 300 mL) in the flask placed close to the test mixture, and is measured by titrimetric analysis.

- Sampling frequency: D1, D5, D7, D11, D14, D18, D21, D25 and D28
- Sampling method: the barium hydroxide flask close to the test mixture is replaced by another barium hydroxide solution, and the CO2 is measured by titrimetric analysis.

- Negative control (Inoculum control): mineral medium + inoculum : 2 replicates
- Positive control (reference item = sodium acetate) : mineral medium + inoculum + sodium acetate : 1 replicate.
- Toxicity control: mineral medium + inoculum + sodium acetate + test item
Reference substance:
acetic acid, sodium salt
Test performance:
Study validity:
- The degradation percent of the reference substance = 76% at the 14th day, which is above the 60% threshold.
- The CO2 quantity produced by the inoculum control was 30.1 mg at the end of the study (28th day). This result is below the admitted threshold of 40 mg.
- The toxicity control assay showed a degradation percent of 54% of the reference substance following a 14 days period, so the test item was not considered toxic to the microbial populations in the inoculum.

The study was considered as valid.
Key result
% degradation (CO2 evolution)
ca. 61
Sampling time:
28 d
Results with reference substance:
76% of biodegradation at the 14th day
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable, but failing 10-day window
The test item was considered to be readily biodegradable, with a biodegradation of 61% after 28 days without fulfillling the 10-day window criterion;
Executive summary:

The study was performed of assess the ready biodegradability of the test item Sepisol Fast Yellow MG-F in an aerobic aqueous media, according to the OCDE Guidelines for Testing of Chemicals (1992) No. 301B "CO2 evolution (Modified Sturm Test)", references as method C.4 -C of Council Regulation (EC) No. 440/2008.

Activated sludge from wastewater treatment plant, mainly treating domestic wasterwaters at 98%, was used.

14.9 mg/L of the test material (corresponding to a TOC of 10 mg/L) was added to the mineral medium. The assay was performed in duplicate.

The theoric CO2 ( ThCO2) quantity was calculated to be 36.7 mg. No organic solvents were used to facilitate the dispersion of the test item.

Each test vessel was connected to a series of 3 absorption bottles, 2 of them containing 4g/L of barium hydroxyde solution and one containing a sodium hydroxide solution. The test was started by bubbling CO2 free air through the suspensions.

CO2 evolution from the test suspensions, inoculum blanks and the reference substance (sodium acetate) were followed in parallel.

A toxicity check was included.

For measurement of evolved CO2, the barium hydroxide absorber closely to the test vessel was disconnected and titrated.

Under the test condition, the biodegradation percentage of the test material (10 mg/L of TOC) has been determined as 61% after a 28 days period of incubation, without fulfilling the 10 -day window criterion. The % of biodegradation of the reference substance, sodium acetate, is greater than 60 % (76%) at the 14th day. According the guideline OCDE 301B, the test item is considered to be readily biodegradable, but failing the 10 -day window.

Description of key information

Test method OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test) and Regulation (EC) n°440/2008, test method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test) was followed for the determination of the endpoint.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

According to the CLP, annex I - part 4 - subsection, a substance is considered rapidly degradable in the environment if in 28 -day ready biodegradation test based on oxygen depletion or carbon dioxide generation, a 60% of degradation (theoretical maximum) is at least achieved. These levels of biodegradation must be achieved within 10 days of the start of degradation which point is taken as the time when 10 % of the substance has been degraded, unless the substance is identified as an UVCB or as a complex, multiconstituent substance with structurally similar constituents. In this case, and where there is sufficient justification, the 10-day window condition may be waived and the pass level applied at 28 days.


The ready biodegradability of Sepisol Fast Yellow MG-F has been assessed in an aerobic aqueous media, according to the OECD test guideline N° 301B (CO2 evolution).


Under the test condition, the biodegradation percentage was of 61% at the end of the 28 days period of incubation, without fulfilling the 10 -day window criterion.


As the Sepisol Fast Yellow MG-F is an UVCB substance consisting at 96.5% of structural similar constituents (same groups but with different sites of branching) it is anticipated that a sequential biodegradation of the individual constituents is taking place, then the 10-day window can be omitted to interpret the results of the test.


This is supported by the results of the ready biodegradability test performed on the Sepisol Fast Yellow MG-DPG (performed at the same period, by the same laboratory, according to the same guideline) which was readily biodegradable, fulfilling the 10 -day window.