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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
Study period:
June 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was collected from a sewage treatment works A-2500 Baden, which wastewater catchment is predominantly domestic. On arrival in the laboratory and after sedimentation the clear supernatant was decanted and the sludge was filled up with tap water to its origin volume to reduce the carbon content. The sample was aerated by means of a filtered compressed air before being used for the study.
The suspended solids concentration was determined by filtering a 5 mL sample through a pre-dried and pre-weighed glass filter (Whatman GF/C). The filter with solids were dried at 105 °C and re-weighed and the sludge solids determined by difference.
The concentration of the suspended solids of the sludge was adjusted to give a final concentration in all vessels of nominally 20 mg/L.
The inoculum was not acclimatised or adapted to "SPP100 P7" before exposure to the test substance in this study.
Duration of test (contact time):
> 0 - <= 28 d
Initial conc.:
ca. 15 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The CO2 evolution was determined on Days 1, 2, 4, 6, 8, 10, 15, 20, 25 and 28/29.
Day 0 was the day of the addition of the test substance to the medium. The samples were analysed on the same day, except for Day 28, at which the incubation media were acidified with 1 mL 32 % HCl (Merck KGaA, 64271 Darmstadt, Germany) to release any residual CO2.
CO2 was removed by continuous air flow overnight and absorber solutions were titrated on Day 29.

Pre-incubation: On day –1, to all flasks, containing mineral medium components and deionised water each, an appropriate volume of prepared activated sludge was added. The media were filled up to a total volume of 3000 mL with deionised water, to give a concentration of suspended solids of not more than 30 mg/L in the final inoculated mixture.
The vessels were sealed and aerated overnight with CO2-free air to purge the medium of CO2 prior to the beginning of the study.
Apparatus for carbon dioxide scrubbing and a mixture of CO2-free oxygen and CO2-free nitrogen, from gas cylinders (Linde Gase, A-2492 Eggendorf, Austria), in the correct proportions (20 % O2: 80 % N2) were used. The air flow was regulated for each vessel
individually. Air flow was determined volumetrically in intervals. Adjustments were made as necessary to maintain a flow rate in the range of 30 to 100 mL per minute (1 – 3 bubbles per sec). As an added precaution, a 0.0125 M barium hydroxide solution was used as CO2 absorber.
On Day 0, the relevant vessels were opened to add the test or reference substance. The test substance was added to vessels A and TK, the reference substance was added to vessels PK and TK. The test solutions were continuously stirred during the test. The vessels were incubated in darkness until the end of the study and the medium continually supplied with CO2-free air. Barium hydroxide was used to trap the CO2 formed by the degradation of the test substance. Three absorption bottles each containing 100 mL of 0.0125 M barium hydroxide solution, were connected in series to each 5-litre flask. On the days of CO2 measurement, the barium hydroxide absorbers closest to the test vessels were disconnected. The remaining absorbers were moved up one position and a new absorber containing fresh barium hydroxide solution was placed at the far end of the series. In case a substantial precipitation was seen in the first trap, also the second bottle was analysed.
Test duration: 28 days exposure, with measurement of the last sample on Day 29.
Reference substance:
other: sodium benzoate
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
0 % degradation after 10 d
0 % degradation after 28 d
Results with reference substance:
Points of degradation plot (reference substance):
74 % degradation after 10 d
88 % degradation after 28 d

Positive control

The plateau of biodegradation was reached on about Day 10 and the degradation of the positive reference substance sodium benzoate exceeded the pass level of 60 % on Day 6.

Toxicity control

Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of a relevant inhibition of the microbial activity by the test substance. The final biodegradation was 40.9 %.

Temperature control

Temperatures recorded were between 22.1 ± 0.4 °C.

pH control

The determined pHs are presented in Table 3. No major deviations were seen during the study time.

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
"SPP100 P7" with a nominal concentration of 15 mg carbon/L was not degraded after 28 days of incubation at 22.1 ± 0.4 °C.

Description of key information

"SPP100 P7" with a nominal concentration of 15 mg carbon/L was not degraded after 28 days of incubation at 22.1 ± 0.4 °C.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:

Additional information