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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Source and lot/batch No.of test material: KN53200656
- Expiration date of the lot/batch: 18 Nov 2017
- Purity: 100% (sum of isomers, multiconstituent substance)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 13 February 2017 from the aeration tank of the plant.
- Preparation of inoculum for exposure: 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. At the day of exposure the suspension was washed one time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drinking water.
- Concentration of sludge: adjusted to 6.0 g/L dry weight.
- Water filtered: yes
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Initial conc.:
ca. 27 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium according to guideline
- Test temperature: 20 - 22 °C
- pH: 7.4 ± 0.2,
- pH adjusted: yes
- Continuous darkness: yes

- Culturing apparatus: 2L incubation bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: 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.
- Measuring equipment: TOCanalyzer equipped with an auto sampler (Shimadzu TOC-5000A and/or TOC-L, or TOCCSN
- Details of trap for CO2 and volatile organics if used: 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.

- Sampling frequency: at day 0, 2, 6, 8, 10, 14, 17, 21, 24, 27 and 28d
- Sampling method: For stripping of carbon dioxide, dissolved in the test medium, each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid.

- Abiotic sterile control: yes
Reference substance:
Key result
% degradation (CO2 evolution)
60 - 70
Sampling time:
28 d
Remarks on result:
mean value: 67%
Details on results:
5%: 10d
14%: 14d
46%: 21d
67%: 28d
Results with reference substance:
The percentage degradation of the functional control reached the pass level of 60% after 10 days and came to 92% after 28 days.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable, but failing 10-day window
The conclusion that the test substance was readily biodegradable in this carbon dioxide evolution test based on the quantitative determination of the formed carbon dioxide in the test substance assays by comparison with the calculated maximal theoretical carbon dioxide production. The test substance is a mixture of structure similar compounds. Such test substances are multi-component substrates and are typically characterized by different kinetic degradation steps. Therefore the 10 days window is not applied and the test substance can be classified as „readily biodegradable“ if the required pass level of the test guideline OECD 301B (60 % CO2/ThCO2) was reached.

Description of key information

Readily biodegradable: 67% CO2 evolution in 28 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One study investigating the ready biodegradability of Bis(methylcyclohexyl) phthalate (CAS 27987-25-3) is vailable. The study was performed according to OECD guideline 301B under GLP conditions (BASF 2017). Non-adapted municipal activated sludge was used as inoculum. The test substance was applied at a concentration of 20 mg TOC/L in duplicates. A biodegradation of 60 -70% was observed after 28 days. The 10-day-window was narrowly missed; however, according to “OECD guidelines for the testing of chemicals – Revised introduction to the OECD guidelines for testing of chemicals, Section 3 – Part 1: Principles and strategies related to the testing of degradation of organic chemicals degradation” published by OECD (2006) the ready biodegradability data for mixtures should be evaluated carefully. Such test substances are typically characterized by different kinetic degradation steps. Therefore, the 10-day window should not be used for the interpretation of the results. Hence, Thus, due to a biodegradation of > 60% after 28 days, the test substance can be considered as readily biodegradable.