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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 23, 2017 to September 26, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
see ''Principle of method if other than guideline''
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
see ''Principle of method if other than guideline''
Principles of method if other than guideline:
The following deviation from the guideline was documented:
Temperature range was 19.4 – 21.6 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range, this is considered as uncritical concerning the outcome of the study.
The deviation was assessed and signed by the study director on 27. Oct. 2017.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 % (UVCB); Appearance: amber, homogeneous liquid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Specification:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage.
Source:
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Pre-Treatment:
The sludge was filtrated, washed with tap water (2x), then washed with and resuspended in test medium. It was then aerated until use. The dry matter was determined with 4120 mg suspended solids/L.

Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC/L
Remarks:
30.3 mg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparations:
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its DOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.
On the day of the start of the test, CO2-free medium and inoculum was filled into the test flask.

Apparatus:
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. 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.
Magnetic stirrers were used to prevent deposition of inoculum.
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.

CO2 Determination:
From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
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.
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
20
Sampling time:
28 d
Details on results:
If degradation in the toxicity flask is below 25 % after 14 days, the test substance can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 39.6 % after 14 days, the test isubstance can be stated as “not toxic towards the inoculum in a concentration of 30.5 mg/L”.
For pure substances ready biodegradability is defined in the guidelines as degradation surpassing 60 % within 10 days after reaching a level of 10 %.
Because the test substance is a mixture, the 10-day window has not to be taken into account. Degradation did not reached 60% within 28 days, though.
Results with reference substance:
Degradation behaviour of positive control and toxicity control was normal.
- Aniline was used as readily bio-degradable positive control. A stock solution containing 2101 mg/L in deionised water was prepared and its organic carbon content was measured with 1570.5 mg/L, corresponding to an organic carbon content of the positive control of 74.8 %.
- Abiotic degradation reached 1.6 %. Both replicates of the test item showed very good correspondence.

Validity criteria:

Parameter

Criterion

Found

Assessment

IC content of test item solution in medium

5% of TC

0 %

valid

CO2emitted by the controls

<70 mg/L

11.5 mg/L

valid

Difference within replicates

20%

2.8 %

valid

Degradation of positive control > 60%

14 days

9 days

valid

Degradation in the toxicity flask on day 14

> 25%

39.6 %

valid

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered not readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance according to OECD Guideline 301B (CO2 evolution test) and EU Method C.4 C, in compliance with GLP. The substance was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 30.3 mg/L). Aniline was chosen as positive control. Activated sludge was used as inoculum (25.0 mg dry matter/L). The test was run for 28 d. All validity criteria were met. Degradation of the positive control was 70% after 9 d. As degradation in the toxicity flask was 39.6% after 14 d, the test substance can be stated as not toxic towards the inoculum at a concentration of 30.5 mg/L. The degree of biodegradation reached 16% at the end of 10 d window and 20% at the end of the test. Under the study conditions, the test substance was considered not readily biodegradable (Muckle, 2017).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance according to OECD Guideline 301B (CO2 evolution test) and EU Method C.4 C, in compliance with GLP. The substance was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 30.3 mg/L). Aniline was chosen as positive control. Activated sludge was used as inoculum (25.0 mg dry matter/L). The test was run for 28 d. All validity criteria were met. Degradation of the positive control was 70% after 9 d. As degradation in the toxicity flask was 39.6% after 14 d, the test substance can be stated as not toxic towards the inoculum at a concentration of 30.5 mg/L. The degree of biodegradation reached 16% at the end of 10 d window and 20% at the end of the test. Under the study conditions, the test substance was considered not readily biodegradable (Muckle, 2017).