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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 d
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
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)

Test material


Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Aerobic biodegradation of the test item was determined by measuring oxygen consumption in an inoculum containing aerobic mixed bacterial culture originated from a municipal wastewater treatment plant and maintained at laboratory conditions. The bacterial culture was not adapted to the test item.

Source of inoculum:
Type of inoculum: aerobic mixed bacterial culture
Source: Municipal wastewater treatment plant, Esztergom-Kertváros
Date of arriving: 03 July 2017
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The amount of oxygen consumed by the microorganisms when metabolising the test item is called Biochemical Oxygen Demand (BOD). The BOD of test item is determined by the manometric respiratory - OxiTop® system. The principle of the determination of BOD by OxiTop® - system is based on the measurement of the consumption of oxygen by a pressure sensor in the top of the tightly closed test vessels. Test vessels with the test item solution and mixed bacterial culture, so called inoculum, are under continuous stirring in an incubator at 21 ± 1 °C for 28 days. The carbon dioxide which is evolved during the biodegradation is absorbed by sodium hydroxide granulates. The reduced amount of oxygen causes definite pressure changes in the test vessels which are measured by the apparatus. The amount of oxygen taken up by microorganisms during biodegradation of the test item is expressed as a percentage of theoretical oxygen demand (ThOD).
The percentage of biodegradability of the test item is calculated on the base of quotient of 28-day biochemical oxygen demand (BOD28) and theoretical oxygen demand. The pass level for the biodegradability is 60% of ThOD for Manometric Respirometry method. The pass value has to be reached in 10 days (10-d) window within the 28-day period of the test. The 10-d window begins when the degree of biodegradation has reached 10% ThOD and must end before day 28 of the test. Biodegradability classification of the test item is made according to OECD 301 guideline, which says that chemicals which reach the pass level up to 28-d period are deemed to be readily biodegradable.

Results and discussion

Test performance:
Preparation of inoculum test culture
Before the test 300 ml stock culture was filtered through a filter with 0.2 mm pore size to remove the bigger insoluble particles and then the dry substance content of the filtrate was determined. The measured value was 6.49 g/L. For preparing the inoculum culture 169 ml mineral medium was added to 31 ml filtered stock culture. The calculated dry mass of inoculum culture was 1 g/L.
Preparation of test vessels:
Following test vessels were prepared and analysed for the study:
- Test item (in two replicates)
- Blank control (in two replicates)
- Reference solution (positive control)
- Reference and test item solution (toxicity control)
- Sterilized solution of test item (abiotic control)
% Degradation
% degradation (O2 consumption)
Remarks on result:
other: mean of two replicates
Details on results:
The measured BOD values for test item, positive and toxicity controls were corrected with the results of blank controls. Next table below summarizes the biodegradation data (BOD28/ThOD, %) measured at the end of the test:
Test vessel biodegradability:
Test item solution (mean of two replicates) biodegradability: 14%
Reference solution (positive control) biodegradability: 66%
Reference and test item solution (toxicity control) biodegradability: 53%
Sterilized solution of test item (abiotic control) biodegradability: 4%

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
According to OECD guideline the test item should be classified as readily biodegradable if the percentage biodegradation reaches 60% of theoretical oxygen demand within 28 day period in a 10-day window. Based on ready biodegradability testing results (14%) the Methyl-guanidyl-thiazole dihydrochloride monohydrate is not readily biodegradable.