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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:
2019-01-16 - 2019-03-19
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C23 H17 F2 N9
Test material form:

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.

The aerobic activated sludge used for this study was deposited for 30 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System
The test flasks prepared according paragraph 6.6 were incubated at 22°C ± 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System. The test flasks were closed gas-tight by a measuring head.
Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.

Surrounding Type: Climatised chamber
Temperature: 22°C ± 1°C
Light Conditions: Darkness
pH-Value of Test Solutions: 7.6 (measured at the start of the test)
7.4 to 8.2 (measured at the end of the test)

Recording: Test conditions (temperature) were recorded continuously with suitable instruments,documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and were not reported.) Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume

In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water.
The pH of the solution was 7.6.

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThODNO3) was considered for the evaluation of the results. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation.
Fluorazopyrimidine never reached 10% biodegradation. The mean biodegradation at test end after 28 days was 2% (ThODNH4) and 1% (ThODNO3).
Therefore, Fluorazopyrimidine is considered to be not readily biodegradable based on ThODNH4 and ThODNO3.

BOD5 / COD results

Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 89% after 14 days and to 99% after 28 days of incubation.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
The degradation rate of Fluorazopyrimidine never reached 10%.
Therefore, Fluorazopyrimidine is considered to be not readily biodegradable.

The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.