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Diss Factsheets

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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
EPA OPPTS 835.3110 (Ready Biodegradability)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
EC Name:
Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Cas Number:
Molecular formula:
C17H12N4O3 . C17H12N4O3 . Cr. C12H28N to C14H32N
Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Test material form:
Details on test material:
- color: yellow to orange
Specific details on test material used for the study:
- Storage condition of test material: Storage at room temperature

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 27 June 2016 from the aeration tank of the plant. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 24 hours at 22 ± 2° C.
At the day of exposure the suspension was washed one time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water and the concentration oft the sludge was adjusted to 6.0 g/L dry weight.
Aliquots of 7.5 mL were added to the test vessels to obtain an activated sludge concentration of 30 mg/L dry weight.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
20 mg/L
Based on:
Initial conc.:
ca. 32 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The following test assays were prepared: 2 blank control assays (BC)
2 test substance assays (TS)
1 inhibition control test assay (IH)
1 reference substance assay (RS)
The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
Solution A: KH2PO4 : 8.50 g
K2HPO4 : 21.75 g
Na2HPO4 × 2 H2O : 33.40 g
NH4Cl : 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B: CaCl2 × 2 H2O : 36.40 g
The compound was dissolved with deionized water to 1000 mL

Solution C: MgSO4 × 7 H2O : 22.50 g
The compound was dissolved with deionized water to 1000 mL

Solution D: FeCl3 × 6 H2O : 0.25 g
The compound was dissolved with deionized water to 1000 mL

15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

The Carbon Dioxide Evolution Test was performed in 2 L incubation bottles filled up to a volume of 1.5 L. The bottles were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes. Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.
The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount. The
incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.
The test assays were prepared at the day of exposure. First, the required volumes of deionized water and the solutions of mineral salts were dosed to all test vessels. For preparation of the test vessels with test substance, the required amounts of the test substance aliquots for a test concentration of 20 mg/L TOC were weighed onto small plastic cups and completely added with the plastic cups to the vessels of the test substance assays and to the vessel of the inhibition control. Because of poor water solubility of test substance these test assays were treated for few minutes in an ultrasonic bath to ensure an even distribution of test substance in test medium. Finally enough reference substance stock solution was added to reach 20 mg TOC/L in the reference substance assay and 20 mg TOC/L in the inhibition control, related to aniline.
The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2, if necessary. Aliquots of activated sludge suspension were added to all test vessels, to adjust the concentration of activated sludge to 30 mg/L dry weight. Samples for DIC measurement (validity criterion) from the blank control assays were taken. For
determination of the decrease of dissolved organic carbon (DOC) samples were taken from the test vessels of the blank control and from the test vessel of the reference substance control and the DOC content was determined after centrifugation (approx. 15 minutes at 4000 rpm). At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers.
At the end of exposure, the pH values were measured in each test vessel. For stripping of carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid. The concentration of dissolved organic carbon in the blank controls and reference substance assays were determined. Since the test substance was insufficiently soluble in water, no DOC-measurements could be performed from the test assay of the inhibition control and from the test substance test assays.
The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day.

The TIC- and DOC-analyses were performed as repeat determination, using a TOCanalyzer equipped with an auto sampler (Shimadzu TOC-5000A and/or TOC-L, or TOCCSN). The system works with a combustion/non-disperse infrared gas analysis method. For calibration of the TOC-Analyzer, standard samples were measured before start of measurements to prove the conformity with the calibration curve. The samples for TIC-analysis (absorption solution) were measured without further treatment.
The samples for the DOC-analysis were centrifuged for about 15 minutes at 4000 rpm.
The samples were analyzed on the day of sampling.
The DOC was calculated using the following formula:
TC – IC = TOC*
TC = total carbon
IC = inorganic carbon
TOC = total organic carbon
*Since samples were centrifuged TOC = DOC
Reference substance
Reference substance:

Results and discussion

% Degradation
% degradation (CO2 evolution)
< 10
Sampling time:
28 d
Details on results:
The required pass level for ready biodegradability within a ten day window was not reached.
The degree of biodegradation was calculated as mean of the values from two test assays at the end of exposure.

Measured DIC-concentrations in the blank controls at begin of exposure (mean value): 0.8 mg/L
Amount of produced CO2 in the blank controls at the end of exposure (mean value): 43.3 mg/L
Deviation of the degree of biodegradation of the test substance in the plateau phase should be < 20 %: yes
The degree of biodegradation of the reference substance should be > 60 % CO2/ThCO2 after 14 days: yes
The degree of biodegradation in the inhibition control should be > 25 % CO2/ThCO2 after 14 days: yes
The content of DIC in the blank control at start of exposure at the test concentration of 20 mg/L TOC should be < 1 mg/L: yes
The amount of produced CO2 in the inoculum blank (“blank controls”) at the end of exposure (mean value) should be < 70 mg/L: yes

BOD5 / COD results

Results with reference substance:
Degree of biodegradation of the reference substance after 14 days: 77 % CO2/ThCO2
Degree of biodegradation in the inhibition control after 14 days: 45 % CO2/ThCO2

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
The test substance is not readily biodegradable according to OECD criteria.