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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:
from 2nd December 2009 to 13th january 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
not specified
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Batch n. LT 092300125
CAS n. 139-40-2
Chemical formula: c9H16ClN5
Molecular weight: 229.7
Content of active ingredinet (Certificate of analysis): 98.8%
Certificate of analysis date: 3rd November 2009
Production date: 30th October 2009
Expiry date: 29th October 2014
Appaerance/color: white powder
Solubility/miscibility in water (at 20°C): 5.0mg/L
Vapor pressure: 3.9 x 10^-3 hPa
Aggregate state at room temperature: solid
Storage conditions: room temperature
stability in application conditions: sufficient for the purpose.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
The source of the inoculum was the sewage treatment plant of the Taverne di Corciano (Perugia) treating predominately domestic sewage from the town. A fresh sample of activated sludge (3 liters approximately) was collected from the aeration tank of the plant on December the 2th and immediately transported to the laboratory. The activated sludge was transferred in a beaker and let to settle. About 20 minutes after the supernatant was discarded (about 500 mL) and the same volume of tap water was added. The beaker was placed in a thermostatic bath at 22±2 °C and kept stirring by a blades stirrer for about 1 hour. The activated sludge was let to settle and after 20 minutes about the supernatant was discarded (about 700 mL), the same volume of tap water was added and the beaker was placed in the thermostat bath again. The procedure above reported was repeated once again. A 30 mL aliquot of the decanted activated sludge was taken to perform the determination of the suspended solids (SS) and the count of the colonies forming unit (CFU)
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
0.032 g/L
Based on:
other: TOC(Total Organic Carbon)/Liter
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of the test:
3 litres of the test inoculum were transferred in each amber flask. Each flask, containing a magnetic stirrer bar, was placed on a magnetic stirrer plate and connected to the air-flow system by a silicone tube. The air was carbon dioxide free air (type lperpure). The system was left to flux through the flasks for one day without connecting the drechsels, containing the adsorbent agent, and without the test and the reference items adding. This process was performed to remove the greatest amount of the endogen carbon dioxide from the inoculum.

Test and Reference items adding to the inoculum
The amounts of the test substances and the reference substance corresponding to 15 mg TOC(Total Organic Carbon)/L ( Test substance=95.7 mg and reference substance= 153.9 mg) were dispersed in a small quantity of the inoculum and then added directly in to the flasks. A solution of Ba (OH)2 0.0125 M (adsorbent agent) was prepared, 100 mL of this solution were transferred in each drechsel. Three drechsels were connected each other and the 3 in-line drechsels were Connected to the air flow system, downstream of the flasks, by a silicone tube.
Sampling and measurements:
6 flasks were preparred as follows:
2 flasks containing the microbial inocolum with the test substance
2 flasks containing the microbial inocolum with reference substance
2 flasks containing only the microbial inocolum
At the beginning of the test the suspended solids (SS), the count of the colonies forming unit CFU) and the pH were performed on the test inoculum.
Each sampling point, within 5 days interval in the first 10 days and every 4-5 days until the end of e test, the CO2 produced from each flask was determined.
At the end of the test the suspended solids (SS), the count of the colonies forming unit (CFU) and the pH were performed on each test flask.
the suspended solids was performed according to the Sop BT 219, while the count of the colonies forming unit (CFU) was performed according to the Sop BT 228 as follows:
2 nitrocellulose filters (0 45 µm) were placed in a heater, set at 105°C, for 1 hour then they were set to cool in a dryer for 30 minutes The filters were weighed to sign their initial weights and placed in the vacuum filtration apparatus: 10 mL of tap water were made to pass through the filter, then 10 mL of decanted activated sludge and 10 mL of tap water 3 times again until the drip ,as completed The filters were placed at 105°C in a heater and left to dry for 3 hours, then they were set to cool in the dryer for 30 minutes and weighed for their final weights The suspended solids content in the decanted activated sludge was 9.130 g/L. 1 mL aliquot of the decanted activated sludge was taken to perform the count of the colonies forming unit according to the
SOP BT 228

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
not performed
Test performance:
During the test substance application, propazine technical made a film when mixed in a small aliquot of inocolum (used to add the substance in the flask). After the test item adding in the flasks its adsorbtion to the glass of the reactors was observed, this effect disappeared soon and the substance became available to the micro-organisms.
The result of the CFU counts, showed that the test item had no letal effect on the bactirial population of the inocolum since the values obtained were close to the blank inocolum values, however it was not possible evaluate if the test item has inhibitory effect on the specific biodegradation process.
% Degradation
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
Test item biodegradation values:
Days Actual biodegradation (%) Total biodegradation (%)
4 1.7 1.7
5 0.4 2.1
7 0.0 2.1
9 0.2 2.3
13 0.9 3.2
18 1.0 4.2
21 0.8 5.0
25 0.6 5.6
28 0.4 6.0

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: the test item propazine technical was evaluated not to be ready biodegradable by modified sturm test according to the OECD guideline 301-B, adopted 17th July 1992
According to the results of the present study, the test item propazine technical is not ready biodegradable since the biodegradation value reached 6.0% after 28days (pass level:60%).