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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 04, 2017 to November 08, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

see 'Principles 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 – 21.5°C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range this is considered as not critical to the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS No.: 162492-01-5 (or 264888-31-5); EINECS-No.: 500-734-6; Batch No.: DR0072912; Appearance: transparent viscous liquid, Purity: 100 % (UVCB); Homogeneity: homogeneous

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Specification:
Activated sludge from a biologic sewage treatment plant was used as inoculum. 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 re-suspended in test medium. It was then aerated until use. The dry matter was determined as 4520 mg suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

35.5 mg/L

Based on:

TOC

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.

Experimental Parameters:

Flask volume 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test substance, mineral medium and inoculum
Abiotic control: 1, containing test substance, mineral medium and HgCl2
Toxicity control: 1, containing test substance, positive control, mineral medium and inoculum
Inoculum concentration: 25.0 mg/L
Temperature: 19.4 – 21.5 °C
Duration: 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L.

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.

Sampling:

From each front scrubber flask, 9 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 10, 14, 18, 24 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:

5

Sampling time:

28 d

Details on results:

All validity criteria were met.
- Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation reached 1.5 %. Both replicates of the test substance showed very good correspondence.
- 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 31.3 % after 14 days, the test substance can be stated as “not toxic towards the inoculum in a concentration of 35.5 mg/L”.

Results with reference substance:

69 % after 10 days

Validity:

Parameter	Criterion	Found	Assessment
IC content of test substance solution in medium	≤5% of TC	0 %	valid
CO2emitted by the controls	< 70 mg/L	9.5 mg/L	valid
Difference within replicates	≤20%	2.6 %	valid
Degradation of positive control > 60%	≤14 days	10 days	valid
Degradation in the toxicity flask on day 14	> 25%	31.3 %	valid

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the degradation of the test substance did not reach the pass level of 60% in the course of the test, therefore the test substance is considered as not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance according to OECD Guideline 301B and EU Method C.4 – C (CO2 evolution test), in compliance with GLP. The test substance was tested at a nominal concntration of 20 mg organic carbon/L (corresponding to 35.5 mg test substance/L) in test medium. Activated sludge was used as inoculum and the test substance in a mineral medium was incubated under aerobic conditions in the dark through 28 d. The amount of DOC in the test solution due to the inoculum was kept as low as possible compared to the amount of organic carbon due to the test substance. Degradation was followed by determination of the carbon dioxide produced with a TOC analyser. All validity criteria were met. Degradation of the positive control aniline was 69% after 10 d. The degree of biodegradation of the test substance reached 5% after 28 d and therefore, the degradation did not reach the pass level of 60% in the course of the study. Under the study conditions, the test substance is considered as not readily biodegradable (Muckle, 2018).

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 and EU Method C.4 – C (CO2 evolution test), in compliance with GLP. The test substance was tested at a nominal concntration of 20 mg organic carbon/L (corresponding to 35.5 mg test substance/L) in test medium. Activated sludge was used as inoculum and the test substance in a mineral medium was incubated under aerobic conditions in the dark through 28 d. The amount of DOC in the test solution due to the inoculum was kept as low as possible compared to the amount of organic carbon due to the test substance. Degradation was followed by determination of the carbon dioxide produced with a TOC analyser. All validity criteria were met. Degradation of the positive control aniline was 69% after 10 d. The degree of biodegradation of the test substance reached 5% after 28 d and therefore, the degradation did not reach the pass level of 60% in the course of the study. Under the study conditions, the test substance is considered as not readily biodegradable (Muckle, 2018).