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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:
August 2017 - February 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600.000 inhabitant equivalents. Sampling date of activated sludge was on 7 August 2017. The dry solid content of the activated sludge was 3.9 g/L. It was determined by weight measurements after drying at 105°C for 4 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
measured carbon content in test item 42.3%
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.
11.5 mL activated sludge was filled up to 1500 mL with 1488.5 mL mineral medium corresponding to 30 mg/L dry solids. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, the absorber wash bottles were filled with 0.2 M NaOH and the test substance was added into the three test vessels and into the toxicity control vessel. The reference compound was added into the reference vessels and into the toxicity control vessel. The aeration rate was kept at a rate of 1 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days. The determination by counting the gas bubbles over a defined period using a stop watch was made on day 10. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days.
At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2-absorption flasks was determined as 3.44 mg/L. The IC in the reactors at the beginning of the test was 0.27 mg/L. On the 3rd, 7th, 10th, 14th, 21st and 28th day 6 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.
Reference substance:
benzoic acid, sodium salt
A stock solution of 10.0 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were added into the reference vessels corresponding to a concentration of mg/L organic carbon.
Test performance:
Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.27mg/L) were found.
The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 9 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.
The temperature was 20.3 – 22.9°C throughout the whole study and therefore within the required range.
The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 3.0 – 4.9 bubbles/second).
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: Average for two replicates (third replicate considered outlier)
the overall result of a degradation >60% is shown in all three replicates
Details on results:
The degradation extents of the test item replicates developed very parallel before acidification (day 28). After acidification in two of three replicates the degradation development went on and reached degradation extents between 73.1 and 83.7%, in one of the three replicates the degradation extent stayed almost the same and was 60.1%. To follow the validity criteria of a difference of extremes <20% this test vessel replicate was regarded as an outlier and was not considered in the calculation of the test end mean degradation extent. Nevertheless the overall result of a degradation >60% was shown in all three replicates.

The degradation extent of the test item at the end of the test was 78.4% (28 d after acidification, mean of two replicates).
The test item did not met the 10-d-window (60% of ThCO2 within a 10-d window), however as the substance is identified as an UVCB the 10-day window condition may be waived and the pass level applied at 28 days.
Results with reference substance:
The reference compound sodium benzoate reached the pass level for ready biodegradability within 3 days (66.2%).

The degradation extent in the toxicity control was 63.5% within 14 days, the test substance had no inhibitory effect on the inoculum.

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The degradation extent of the test item at the end of the test was 78.4% (after 28 days).
The test item did not met the 10-d-window (60% of ThCO2 within a 10-d window), however as the substance is identified as an UVCB the 10-day window condition may be waived and the pass level applied at 28 days.
Therefore, the substance is considered to be readily biodegradable.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:

Additional information