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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:
29 Mai - 27 Jun 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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)
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant, Edenkoben
- Pretreatment: the sludge was filtrated, washed twice with tap water, then re-suspended in test medium, aerated for ≥ 12 h. The dry matter was determined with 4920 mg suspended solids/L.
- Concentration of sludge: 25 mg dw/L
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: organic carbon
Initial conc.:
34.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: according to guideline
- Test temperature: 22 ± 2 °C
- pH: 6.76 - 7.47
- Aeration: flasks (with medium and inoculum) were aerated for 24 h with purified, CO2-free, moistened air to purge the system of CO2

- Culturing apparatus: 2000 mL Schott-flasks as test vessels, 100 mL scrubber flasks as absorbent vessels (glassware cleaned with the cleaning agent Mucasol® and then rinsed with tap water, diluted HCL, tap water and deionised water)
- Number of culture flasks/concentration: 2
- Measuring equipment: IC measurement was conducted using the carbon analyser TOC multi N/O 2100S, Analytik Jena
- Test performed in closed vessels: no
- Details of trap for CO2: emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask

- Sampling method and frequency: from each front scrubber flask, 9 samples (sample volume 1 mL) were taken in order to determine the emitted CO2 (on days 0, 4, 6, 10, 12, 14, 18, 24 and 29). The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. On day 28, 5 mL 2 M HCl were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1
- Positive control (with reference substance): 2

Reference substance:
82% organic carbon content
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The lag phase lasted until day 8 and 22 % degradation was determined at the end of the study (pass level of 60 % biodegradation given in the OECD guideline was not reached).
Results with reference substance:
69% degradation of the reference substance after 10 d.

Table 1: % biodegradation identified in positive control, test item, abiotic controla and toxicity control vessels.

Day  Positive Control 1 Positive Control 2 Positive Control Mean Test 1  Test 2  Test Mean Abiotic Control Toxicity Control
4 45.6 39.5 42.6 7.2 -1.4 2.9 -0.6 15.8
6 54.4 56.7 55.5 6.9 3.4 5.1 -1.0 33.1
10 64.4 73.6 69.0 24.3 7.7 16.0 -0.3 37.1
12 66.8 76.8 71.8 21.4 10.6 16.0 0.6 41.1
14 66.6 81 73.8 21.1 14.7 17.9 0.2 48.5
18 69.3 81.4 75.3 26.7 16.1 21.4 0.0 51.8
24 71.5 81.4 76.5 28.2 17.1 22.6 -2.0 51.8
29 75.2 79.9 77.5 28.1 14.9 21.5 -6.2 51.9

Degradation in the toxicity control was over 25% after 14 d (49%). Therefore, the test item can be considered as not toxic towards the inoculum under tested conditions.

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable under tested conditions

Description of key information

Not readily biodegradable (22%, based on CO2 evolution, OECD 301 B).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One study investigates the biodegradability of the substance (CAS 72869-86-4) according to the OECD guideline 301 B and GLP. A non-adapted, activated sludge from a domestic sewage treatment plant was used as inoculum and was exposed to an initial nominal test substance concentration of 34.2 mg/L (equivalent to 20 mg/L organic carbon). After 28 d, 22% degradation of the tested substance (based on CO2 evolution) was observed. Therefore the substance is not readily biodegradable under tested conditions. A toxicity control containing both, reference substance (aniline) and test substance did not indicate inhibitory effects to the inoculum, as 49% biodegradation took place within 14 d.