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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:
06 Oct - 04 Nov 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
17 Jul 1992
according to
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
GLP compliance:
yes (incl. certificate)
Ministerium für Umwelt und Verkehr, Baden-Württemberg, Germany
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: municipal sludge plant, Pforzheim, Germany
- Storage conditions: The effluent was kept under aerobic conditions in the period between sampling and application.
- Preparation of inoculum for exposure: The inoculum was filtered through a coarse filter (Macherey & Nagel) and the first 200 mL of the filtrate was discarded.
- Pretreatment: The inoculum was shaken during one week, for starvation. The initial number of microorgansims was determined by preparing dilution series of the inoculum in steps of 10E-01 NaCl solution and counting the bacterial colonies after incubation for at least 3 d in Petri dishes and yeast extract.
- Initial cell numbers: 7.97E+04 bacterial cells on average in each test vessel
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: Mineral medium, prepared from 4 stock solutions using ultra pure grade water.
- Solubilising agent: acetone
- Test temperature: 20 ± 2 °C
- Aeration of dilution water: A sufficient volume of ultra pure grade water was prepared in 5 L volumetric flasks. The flasks were filled at first to about three quarters of their volume with water. The water was strongly aerated for approx. 10 minutes to achieve oxygen saturation and allowed to stand for 24 h without aeration at test temperature. The O2 content was measured afterwards.
- Continuous darkness: Yes

- Culturing apparatus: BOD flasks with ground-in-glass stoppers.
- Number of culture flasks/concentration: 3 per measurement date.
- Method used to create aerobic conditions: Aeration of dilution water and inoculum prior to test.
- Measuring equipment: Oxygen concentrations were measured with a WTW Microprocessore Oximeter (OXI 340) and a calibrated electrode.
- Test performed in closed vessels due to significant volatility of test substance: The test was performed in closed bottles.
- Other: Each test vessel was inoculated with 0.5 mL inoculum.

- Sampling frequency: Sampling was performed on Days 0, 7, 14, 21, and 28
- Sampling method: Removal of 3 bottles from each treatment group for analysis of O2 content. On Day 0 only 1 test bottle from each treatment group was measured.
- Other: Each 3-fold test assay (i.e. each test vessel) was measured twice on the day of sampling. On Day 0, only 1 test vessel was sampled and measured twice.

- Inoculum blank: 3 bottles per treatment group: 0 mg/L test item + 0 mg/L reference item. The same amount of acetone as in the test solutions, but without the test substance was added to the test vessels and allowed to evaporate overnight, corresponding to the test item vessels.
- Reference item: 3 bottles per treatment group: 0 mg/L test item + 2 mg/L reference item
- Toxicity control: 3 bottles per treatment group: 1 mg/L test item + 1 mg/L reference item
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
Toxicity control: 20.1% degradation after 28 d.
Results with reference substance:
Degradation of reference item (Benzoic Acid, Sodium salt) after 28 d: 88.0%.

Tab 1: Degradation [%] of test item, reference and toxicity control

Time [d]

% degradation

8- Hydroxyquinoline


Toxicity Control

















The degradation of the reference compound reached 60% by Day 14, confirming the activity of the inoculum.

The criterion for ready biodegradability of > 60% removal of the ThOD within the 28 d period was not reached. Therefore, the test substance cannot be considered as readily biodegradable.

The degradation of the toxicity control was < 25% (required pass level) after 14 d. Therefore, toxic effects of the test substance to the inoculum cannot be excluded. This is not unusual, as the test item acts as a fungicide and bactericide agent.

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test item is not readily biodegradable according to the criteria of the OECD guideline 301 D.

Description of key information

Not readily biodegradable (domestic activated sludge, OECD 301 D)

Toxic effects on microorganisms cannot be excluded (domestic activated sludge, OECD 301 D)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

There is one GLP study available investigating the biodegradability of the test substance in an aerobic, aqueous medium according to the OECD guideline 301 D.

In a closed bottle test, a nominal concentration of 2 mg/L test item was inoculated with 7.9 E+04 cells per test vessel from activated sludge of a municipal sewage treatment plant for 28 d under controlled conditions in the dark. Since the test item is practically insoluble in water, a stock solution was prepared in acetone (60.8 mg/20 mL). One day prior to the test, a defined volume of this stock solution was transferred to each test vessel and the acetone was allowed to evaporate overnight. An inoculum control, a toxicity and reference test were run in parallel. The inoculum control received the same amount of acetone and underwent the same treatment.

Degradation was followed by measuring oxygen concentrations in regular intervals (Days 0, 7, 14, 21, and 28). The % biodegradation was expressed in terms of the biochemical oxygen demand (BOD) with respect to the calculated, theoretical oxygen demand (ThOD).

After 28 d, the degradation of the reference item reached 88.0%, confirming the activity of the inoculum. The degradation of the test item was 6.6% after 28 d, which does not fulfill the criterion for ready biodegradability (> 60% within 28 d). Therefore, the test substance cannot be considered as readily biodegradable. The toxicity control reached a degradation of 20.1%, which is below the recommended pass level (< 25% after 14 d). Hence, toxic effects of the test substance on the activated sludge microorganisms cannot be excluded. However, this is not an unusual finding in the light of the application of the test substance as fungicide and bactericide agent.