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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:
May 07 to june 05 1998
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: ISO, GLP study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
Constituent 2
Reference substance name:
Test material form:
solid - liquid: suspension
migrated information: dispersion
Details on test material:
- Name of test material (as cited in study report): Azacyclonol-Base
- Physical state: white-beige powder
- Purity: 98.3 % (w/w)
- Batch No.: 960050 v. 10.09.1996
- ThTOC: 79.5 % (0.795 mg/C/mg substance)
- ThCO2: 2.96 mg CO2/mg substance
- Toxicity to bacteria (OECD 209): No toxicity in the test concentration
- Water solubility: not soluble at 20 °C
- Storage conditions: refrigerator 7°C +/- 2, protected from light and moisture

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Origin: municipal sewage treatment plant, D-31137 Hildesheim
- Type of inoculum: non adapted activated sludge
- Pre-treatment: The activated sludge is maintained in an aerobic condition by aeration for 4 hours and then homogenized with a mixer. The sludge is decanted and the supernatant (30 ml) is subsequently used to initiate inoculation.
- Colony forming units of the inoculum: 8.6. 10^8 [CFU/L] (CFU-Determination NON-GLP-state)
- Colony forming units in the test vessels: 8.6. 10^6 [CFU/L] (CFU-Determination NON-GLP-state)
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
30 mg/L
Based on:
test mat.
Initial conc.:
23.9 mg/L
Based on:
other: ThTOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Reference substance
Reference substance:
benzoic acid, sodium salt
- Purity: 99.9 % - Test concentration: 35 mg/L - ThC02: 2.14 mg C02/mg - ThTOC: 0.583 mgC/mg - Carbon content in the vessel: 20.4 mg C/L

Results and discussion

Test performance:
The identity of the test substance was checked on March 09, 1998.
ThC02 was calculated based on the sum formula.
The following test solutions were prepared in 5 L brown glass bottles as incubation vessels:
- two incubation vessels for the test compound concentration (FT1, FT2)
- one incubation vessel for the reference compound (Fc)
- two incubation vessels for monitoring the activity of the inoculum (FB1, FB2)
- one incubation vessel for the toxicity control (FI)

Necessary amounts of nutrient media and inoculum placed in each of the incubation vessels. Vessels were then connected to the system for the production ofC02 free air and aerated for 24h. After 24h, C02 trapping vessels were connected to the air exits of the incubation vessels via a series of 3 gas wash botties.
Test and reference substance concentrations were placed into the incubation vessels, the vessels made up to 3 L with C02 free aqua bidest. and connected to the system for the production of C02 free air.
Incubation: temperature range between 22 ± 2 °C in a water bath. All vessels were stirred.

On the 28th day 1 ml concentrated HCI was added to each of the vessels. Aeration was continued for a further 24h.
On the 29th day the quantity of C02 released was determined.
% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
10 d
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
- Control group a maximum of 32.8 mgC02/L was formed after 28 days. The concentration of C02 from the blank did not exceed 70 mg/L. The quality criterion of the guidline is fullfilled.
-Functional control: The percentage degradation reached the pass level of> 60 % after 14 days with a degradation rate of 75 % at this point of time. The quality criterion of the guideline is fulfilled.
- Toxicity control containing both test substance and reference compound a biodegradation of 33 % occured within 15 days. The biodegradation came to a maximum of 35 % in the test period. The biodegradation of the reference substance seemed to be not inhibited by the test substance.
- Test substance did not reach the 10 % level (begin of biodegradation). The pass level of a biodegradation> 60 % was not reached neither in a 10d-window nor after 28 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
Azacyclonol must be regarded to be not ultimate aerobic biodegradable in the 10 d-window and after 28 days.
The biodegradability potential of the free base is taken as reference for the biodegradability of Azacyclonol HCl as HCl is an inorganic substance.