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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 08 July 2014 from the aeration tank of the plant. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 24 hours at 22 ± 2 °C.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Initial conc.:
ca. 38 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The following test assays were prepared:
2 blank control assays (BC)
2 test substance assays (TS)
1 inhibition control test assay (IH)
1 reference substance assay (RS)

The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
Solution A:
KH2PO4 : 8.50 g
K2HPO4 : 21.75 g
Na2HPO4 × 2 H2O : 33.40 g
NH4Cl : 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B:
CaCl2 × 2 H2O : 36.40 g
The compound was dissolved with deionized water to 1000 mL

Solution C:
MgSO4 × 7 H2O : 22.50 g
The compound was dissolved with deionized water to 1000 mL

Solution D: FeCl3 × 6 H2O : 0.25 g
The compound was dissolved with deionized water to 1000 mL

15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

The Carbon Dioxide Evolution Test was performed in 2 L incubation bottles filled up to a volume of 1.5 L. The bottles were connected to two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes. Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.

The TIC-value of the freshly prepared sodium hydroxide solution was determined and considered by the calculation of biogenic produced carbon dioxide amount. The incubation bottles were stirred on magnetic stirrers; the aeration was performed with carbon dioxide free air at a flow of approximately 800 mL per hour.

The test assays were prepared at the day of exposure. First, the required volumes of deionized water and the solutions of mineral salts were dosed to all test vessels. For preparation of the test vessels with test substance, the required amounts of the test substance aliquots for a test concentration of 20 mg/L TOC were weighed onto small glass plates (microscope cover slips) and completely added with glass plates to the vessels of the test substance assays and to the vessel of the inhibition control. Because of poor water solubility of test substance these test assays were treated for few minutes in an ultrasonic bath to ensure an even distribution of test substance in test medium. Finally enough reference substance stock solution was added to reach 20 mg TOC/L in the reference substance assay and 20 mg TOC/L in the inhibition control.

The pH-values in the test vessels were measured and adjusted to 7.4 ± 0.2, if necessary. Aliquots of activated sludge suspension were added to all test vessels, to adjust the concentration of activated sludge to 30 mg/L dry weight. Samples for DICmeasurement (validity criterion) from the blank control assays were taken. For determination of the decrease of dissolved organic carbon (DOC) samples were taken from the test vessels of the blank control and from the test vessel of the reference substance control and the DOC content was determined after centrifugation (approx. 15 minutes at 4000 rpm). At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers.

At the end of exposure, the pH values were measured in each test vessel. For stripping of carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid. The concentration of dissolved organic carbon in the blank controls and reference substance assays were determined. Since the test substance was insufficiently soluble in water, no DOC-measurements could be performed from the test assay of the inhibition control and from the test substance test assays.
The aeration was continued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day.
Reference substance:
% degradation (CO2 evolution)
> 10 - < 20
Sampling time:
28 d
Details on results:
The degree of biodegradation after an exposure period of 28 days was 10-20 % CO2/ThCO2 in this test.
The degree of biodegradation was calculated as mean of the values from two test assays at the end of exposure.
Based on the rate of biodegradation of 10-20 % CO2/ThCO2 at the end of exposure the test substance can be evaluated as poorly biodegradable in this test.
Results with reference substance:
Degree of biodegradation of aniline after 14 days: 81% CO2/ThCO2

Degree of biodegradation in the inhibition control after 14 days: 32% CO2/ThCO2

Validity criteria fulfilled:

Description of key information

Poorly biodegradable.

Key value for chemical safety assessment

Additional information

The biodegradation potential of the substance was evaluated in an experimental study according to OECD guideline 301 B under consideration of GLP. The initial substance concentration was 20 mg/L TOC which is equivalent to approximately 38 mg/L test substance. After 28 days the degree of biodegradation was 10 to 20% CO2/ThCO2. The required pass level for ready biodegradability within a ten days window was not reached. The substance was not readily biodegradable under the conditions of this test. The substance is poorly biodegradable.