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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:
31 Oct - 29 Nov 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 Jul 1992
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany. 08 Apr 2015.
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, NW-Lachen-Speyerdorf, Germany. The plant treats mostly domestic sewage. Date of collection: 28 Oct 2016.
- Pretreatment: The sludge was filtered, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use.
- Concentration of sludge: The dry matter was determined as 3700 mg suspended solids/L.
- Initial inoculum concentration: 25.0 mg/L
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Initial conc.:
25.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: Mineral medium prepared from stock solutions a (KH2PO4), b (CaCl2), c (MgSO4*7H2O), and d (FeCl3*6H2O)
- Test temperature: 19.5 - 21.6 °C
- pH: 6.7 - 7.3 (pH at the end of the test, before addition of HCl)
- Suspended solids concentration: 25.0 mg dry matter/L
- Continuous darkness: Yes.

- Culturing apparatus: Schott-flasks and 100 mL scrubber flasks as CO2 absorbend vessels.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The test vessels were aerated for 72 h with purified (by activated charcoal), CO2-free (CO2-scrubbed), moistened air to purge the sysem of CO2.
- Measuring equipment: Carbon analyser TOC multi N/C 2100S, Analytik Jena
- Test performed in open system: Yes.
- Details of trap for CO2 and volatile organics if used: The 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.
- Other: The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.

- Sampling frequency: Samples were taken on Day 0, 2, 4, 7, 9, 11, 14, 18, 23, and 29.
- Sampling method: 1 mL was collected. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
- Other: On Day 28, 5 mL HCl 2 M was 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, containing mineral medium and inoculum
- Apparatus blanks: 2, containing mineral medium only
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and inoculum.
- Positive control: 2, containing positive control, mineral medium and inoculum
Reference substance:
% degradation (CO2 evolution)
Sampling time:
28 d
Results with reference substance:
Degradation of the positive control was 70 % after 9 days.


All validity criteria were fulfilled and the result of the test can be considered valid. The degradation behaviour of the positive control and toxicity control was normal. Abiotic degradation reached 0.7%. Both replicates of the test item showed very good correspondence.


Degradation in the toxicity control was 44% after 14 d. Therefore, the test item can be graded as "not toxic towards the inoculum at a concentration of 25.5 mg/L".

Table 1: Validity criteria for OECD 301B.

Criterion from the guideline


Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.



Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).



The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.



The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.



The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.




Validity criteria fulfilled:
Please refer to table 1 in "Any other information on results incl. tables"
Interpretation of results:
not readily biodegradable

Description of key information

Degradation rate: 0% after 28 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

There is one study available investigating the ready biodegradability of the substance according to the OECD guideline 301 B and GLP.

A nominal concentration of 25.3 mg/L test item (corresponding to 20 mg organic carbon/L) was inoculated with 25.0 mg dry matter/L of activated sludge for 28 d under aerobic conditions. The test vessels were aerated with CO2 free air and the CO2 emitted during the test was trapped in scrubber flasks filled with 0.25 M NaOH. Degradation was followed by determining the carbon dioxide produced in regular intervals. The produced CO2 was calculated from the measured dissolved inorganic carbon (DIC) in the CO2 absorber bottles using a TOC analyser. A toxicity control and apparatus blanks were run in parallel.

After 28 d no biodegradation of the test item was observed. Degradation of the positive control was 70% after 9 d, thereby meeting the validity criteria (> 60%, ≤ 14 d). Abiotic degradation reached 0.7% after 28 d and the toxicity control reached a degradation of 44% after 14 d. Therefore, the test item is considered to be not readily biodegradable and not toxic towards the inoculum at a nominal concentration of 25.5 mg/L test item.