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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

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:
other: see 'Remark'
The reliability is rated 2 because the study followed a standard guideline of reference (OECD 301 B), which describes a procedure designed to evaluate this endpoint. The results were reviewed f or reliability and assessed as valid. However the study was not conducted under GLP conditions.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
anilino(phenylimino)methanaminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
EC Number:
Molecular formula:
anilino(phenylimino)methanaminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): JAUNE MG DPG
- Substance type: Organic salt

Study design

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 in France).
- 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):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
15.2 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation
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
Reference substance:
acetic acid, sodium salt

Results and discussion

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 61% 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.
% Degradation
% degradation (CO2 evolution)
ca. 91
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
76% of biodegradation at the 14th day

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test item was considered to be readily biodegradable, with a biodegradation of 91% after 28 days.
Executive summary:

The study was performed of assess the ready biodegradability of the test item Sepisol Fast Yellow MG-DPG 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.

15,2 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 91% after a 28 days period of incubation. The % of biodegradation of the reference substance, sodium acetate, is greater than 60 % (76%) at the 14thdays. According the guideline OCDE 301, the test item is considered to be readily biodegradable.