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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
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

Test material form:
Details on test material:
Purity: 98.9%

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Community Sewerage Plant, Bharath Electronics Limted, Vidyaranyapuram, Bangalore, INDIA.
- Preparation of inoculum for exposure: This effluent was allowed to settle for one hour and decanted. The decanted effluent was then used in the test
- Bacterial population: The bacterial population in the inoculum was determined as colony forming units (CFU/mL) by diluting the inoculum up to 10e-4 dilution and then plating on nutrient agar plates.
- Preconditioning: The decanted effluent was preconditioned by aerating for 6 days at 21.3 to 23.3°C.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
33.33 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: A - Potassium dihydrogen orthophosphate, Dipotassium hydrogen orthophosphate, Disodium hydrogen orthophosphate dihydrate, Ammonium chloride (These four constituents were dissolved in and made up to 1000 mL using Milli-Q water. The pH of the solution was 7.53); B - Calcium chloride dihydrate (This was dissolved in and made up to 1000 mL using Milli-Q water); C - Magnesium sulphate heptahydrate (This was dissolved in and made up to 1000 mL using Milli-Q water); D - Iron (III) chloride hexhydrate (This was dissolved in and made up to 1000 mL using Milli-Q water).
The test medium for test flasks, was prepared by mixing 24 mL of solution A with 1920 mL of Milli-Q water, then 2.4 mL each of solution B, C and D was added and finally the volume was made up to 2400 mL with Milli-Q water in each flask. Similarly, 3000 mL of test medium was prepared by mixing 30 mL of solution A with 2400 mL of Milli-Q water, then 3 mL each of solution B, C and D was added and finally the volume was made up to 3000 mL with Milli-Q water.
- Test temperature: 20.9-23.0°C
- pH: 7.53 to 7.58

- Culturing apparatus: 5-L flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The flasks were aerated with CO2-free air at 43 to 50 mL/minute, overnight to purge the system of carbon dioxide
- Measuring equipment: Bubble flow meter

- Inoculum blank: 2400 mL mineral salts medium, 300 mL additional mineral salts medium and 300 mL inoculum
- Procedure control: 2400 mL mineral salts medium, 300 mL reference standard solution (75.2 mg reference standard in 300 mL mineral salts medium) and 300 mL inoculum
- Toxicity control: 2400 mL mineral salts medium, 300 mL inhibition suspension (50 mg test item and 37.6 mg reference standard in 300 mL mineral salts medium) and 300 mL inoculum
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Cumulative CO2 production in the controls was 106.61 mg CO2/3L that was typical for this type of test and inoculum source and was within the acceptable range for this assay system.
% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
6 d
% degradation (CO2 evolution)
Sampling time:
14 d
Key result
% degradation (CO2 evolution)
Sampling time:
29 d
Details on results:
The degradation of sodium benzoate was also rapid in the presence of test substance and had achieved 63.23% of its CO2 after 14 days. These results show that the test substance did not cause any inhibitory effect on the test system at this concentration.

BOD5 / COD results

Results with reference substance:
The degradation of sodium benzoate was rapid and had achieved 18.87% of its CO2 after 2 days, 38.52% after 4 days, 79.43% after 14 days and 99.49% after 29 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Cumulative CO2 production by the mixtures containing test substance had achieved 12.67% of ThCO2 after 6 days, 18.62% after 14 days and 30.74% after 29 days.
Executive summary:

The present study was conducted to determine the ready biodegradability of the test substance. The ready biodegradability was tested using the CO2 Evolution Test (Modified Sturm Test) as per the OECD Guideline No. 301B.

The test substance was added to two test vessels at the concentration of 33.33 mg/L of mineral medium [equivalent to 14.62 mg Carbon (C)/L]. Two control treatments containing only the inoculum, one positive control treatment containing inoculum plus reference standard (sodium benzoate), and one toxicity control treatment containing the inoculum plus test substance and reference standard were also tested. All the treatments were prepared with inoculum from a secondary effluent treatment plant receiving predominantly domestic sewage.

Treatment mixtures were aerated for 29 days with carbon dioxide (CO2) free air. The CO2 released by each treatment mixture was trapped in a series of bottles containing barium hydroxide, which were connected to the outlet of each test vessel. The residual barium hydroxide was measured on the 2, 4, 7, 10, 14, 18, 22, 25 and 29 days after the initiation of the test by titration. The pH of the treatment mixtures was measured on Day 28.

Sodium benzoate had biodegraded by 38.52% at Day 4 and 79.43% by Day 14 in the absence of the test substance meeting the validity criteria of the test. Test mixture containing sodium benzoate and the test substance had biodegraded by 63.23% at Day 14 showing that the test substance was not inhibitory at this concentration.

The cumulative CO2 production by mixtures containing only the test substance had achieved 30.74% degradation of test substance during the treatment period. the test substance had biodegraded by 1.33% by Day 2 and by 18.62% at Day 14. Based on the pass levels (60% bio-degradation in the 10-day window period), the test substance cannot be considered as readily biodegradable since only 30.74% degradation was achieved during the test period of 28 days. However, it may be considered biodegradable as there was 30.74% degradation during the treatment period.