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Biodegradation in water: screening tests

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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
Composition 0
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
Test material information:
Composition 1
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Easton Wastewater Treatment Facility, Easton, Maryland. The Easton facility treats predominantly residential wastes.
- Method of cultivation: The sludge was sieved using a 2-mm screen, adjusted to approximately 1000 mg total suspended solids (TSS)/L with mineral media and then aerated at test temperature until use.
- Preparation of inoculum for exposure: A total suspended solids measurement and standard agar plate count were performed on the inoculum on the day of test chamber preparation.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: modified biochemical oxygen demand (BOD) test dilution water, prepared using high quality water
- Test temperature: 19.89-22.24ºC
- pH: 7.48-7.63 (test day 28)
- pH adjusted: no
- Aeration of dilution water: All chambers were aerated with CO2-free air overnight at a rate of approximately 50 mL per minute to purge the systems of CO2.
- Suspended solids concentration: 1006 mg total suspended solids (TSS)/L
- Continuous darkness: not reported

- Culturing apparatus: 4-L bottles
- Number of culture flasks/concentration: 2 (blank and positive controls), 2 (test substance), 1 (toxicity control)
- Method used to create aerobic conditions: The biodegradation test was started by bubbling CO2-free air through each of the test chambers at a rate of approximately 50 mL per minute.
- Measuring equipment: Shimadzu Model TOC-VCSH carbon analyzer
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as potassium carbonate (K2CO3) in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study, using a Shimadzu Model TOC-VCSH carbon analyzer.

- Sampling frequency: days 2, 5, 8, 12, 16, 20, 23 and 27
- Sampling method: the CO2 trap nearest the test chamber was removed and analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test chamber and a new trap was placed on the end of the series.

- Inoculum blank: yes
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
Test performance:
The control chambers evolved an average of 66.7 mg CO2 over the test period, corresponding to approximately 22.2 mg CO2/L test medium. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2 evolution tests. The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 95.3% of theoretical CO2 was evolved. An average percent biodegradation of greater than 60% was achieved by Day 5, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 14.
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The final mean percent biodegradation for Inositol was 94.4% based on the cumulative CO2 measured over the 28-day period of the study. Inositol is considered readily biodegradable since more than 60% TCO2 was achieved within 10 days, surpassing the criteria of 60% TCO2 within a 10-day window of reaching 10% TCO2. The toxicity control achieved > 25% degradation by Day 14 and is considered non-inhibitory at the concentration tested in this study.
Results with reference substance:
An average of 95.3% of theoretical CO2 was evolved with sodium benzoate.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test substance is readily biodegradable (94.4% biodegradation).
Executive summary:

The biodegradability of the test substance was determined by the Carbon Dioxide Evolution Test Method (OECD Guideline 301B). In the CO2 test, inoculated mineral medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air. The CO2 produced from the mineralization of organic carbon within the test chambers was displaced by the flow of CO2-free air and trapped as K2CO3 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation of the test substance occurred. The test contained a blank control group, a reference group, a treatment group and a toxicity control. Each group contained a minimum of two replicate test chambers except for the toxicity control, which was not replicated. The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group test chambers were used to evaluate the test substance at a nominal concentration of approximately 10 mg C/L. The toxicity control was used to evaluate the inhibition of the test substance to the inoculum and was dosed with both the reference (10 mg C/L) and test substances (10 mg C/L). The results indicate that the activated sludge inoculum was active, degrading the reference substance 95.3%. The test substance was not inhibitory to the microbial inoculum. The average cumulative percent biodegradation for the test substance was 94.4% after 28 days.

Description of key information

The test substance reached a maximum mean of 94.4% ThCO2 by day 28.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information