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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:

2020-Sep-02 - 2020-Oct-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Due to UCVB substance with constituents of different water solubility, guidance documents OECD 23 and ASTM d6081-19 were used

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992-Jul-17

Deviations:

yes

Remarks:

Flasks stored at varying temperatures instead of 4°C (range 1.5 - 9.7°C) on days 4, 11, 18, 23, 29. This is considered non-critical

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: information provided by sponsor, Batch no. 0320SA017,
- Purity, including information on contaminants, isomers, etc.: not applicable, UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20± 5 °C) in tightly closed vessel under dry, dark conditions
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: homogeneous, stable under storage conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): carbon content of 72.04% (mean from 72.09 and 71.99%) determined by elemental analysis under non-GLP conditions, performed at laboratory Mikroanalytisches Labor Pascher, An der Pulvermühle 1, 53424 Remagen, Germany
- Preliminary steps: As the test item, due to its properties, is not sufficiently soluble in aqueous media, it was added to the flasks directly, based on the nominal amount of organic carbon calculated from elemental analysis.
To achieve a maximum bioavailability on the surface of the test vessels, the nominal load of the test item was added into glass beakers and dissolved in a minimum amount of acetone. This solution was transferred in 2000 mL-SCHOTT-flasks. The glass beakers were rinsed twice with 5 mL acetone to guarantee complete transfer of test item into the test vessel. Then acetone was evaporated completely using compressed air. Thus, a thin layer of test item on the surface of the test vessels was present.
On the day of the start of the test, CO2-free medium and inoculum was filled into the test flask
- Inoculum concentration: 25 mg/L
- Test start concentration: 20 mg organic carbon/L of test item

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, 67435 NW-Lachen-Speyerdorf.
- Pretreatment: The sludge was filtered through a cloth, washed with test medium (2x) and resuspended in test medium. It was then aerated until use. The dry matter was determined to contain 4.08 g of suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

27.7 mg/L

Based on:

test mat.

Initial conc.:

27.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: see Table 1-2
- Test temperature: 19.4 – 20.7 °C without direct lighting
- pH: 6.8-7.5
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: 2000 mL flasks as test vessels, 100mL scrubber flasks as absorbent vessels. 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 against settling of inoculum.
- Number of culture flasks/concentration: 2, containing test item, mineral medium and inoculum
- Method used to create aerobic conditions: aeration with purified (by activated charcoal), CO2-scrubbed, moistened air
- Details of trap for CO2 and volatile organics if used: 0.25 M NaOH trap, two scrubbers containing 100mL each were connected in series to test vessls

SAMPLING
- Sampling frequency: day 0, 2, 4, 7, 9, 11, 14, 18, 23, 29
- Sampling method: from each fron scrubber flask, 10 samples of 1mL were taken to determine CO2. On day 28, 5mL HCl 2M was added to each test flask to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken
- Sample storage before analysis: not indicated

CONTROL AND BLANK SYSTEM
- Apparatus blanks: 2, containing mineral medium only
- Blank controls: 2, containing mineral medium and inoculum
- Acetone Controls: 2, containing evaporated acetone, mineral medium and inoculum
- Positive control: flasks 2, containing positive control, mineral medium and inoculum
- Abiotic control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and in-oculum

Reference substance:

aniline

Remarks:

stock solution containing 2.1412 g/L in deionised water was prepared and its organic carbon content was measured with 1649.01 mg/L, corresponding to an organic carbon content of the positive control of 77.01 %

Key result

Parameter:

% degradation (CO2 evolution)

Value:

39

St. dev.:

2.3

Sampling time:

28 d

Details on results:

The 10-day-window began on day 12, at its end, 31 % degradation were reached, missing the pass level of 60 % given in the OECD guideline.
- Degradation missed 60 % within 28 days, too.
- Because the test item is a mixture, the 10-day window has not to be taken into account. Therefore, regardless of the 10-day-window the test item is considered as not readily biodegradable within 28 days in this test, as well.

Results with reference substance:

On day 12 the validity criterion was met (calculated with graphical evaluation). The mean degradation of the positive control was 65.4 % on day 14.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The valid experiment showed that the test item is not readily biodegradable in the used system. The degree of biodegradation reached 39 % after 28 days. No observations were made which might cause doubts concerning the validity of the study outcome.

Executive summary:

Degradation behaviour of positive control (mean of 2 replicates) and toxicity control was in the valid range normal. Abiotic degradation was not observed. Both replicates of the test item showed very good correspondence.

For the calculation of the degradation of the positive control the blank control was taken into account.
For the calculation of the degradation of the test item the acetone control was taken into account.

Degradation in the toxicity flask was 40.2 % after 14 days, so the test item can be stated as “not toxic towards the inoculum in a concentration of 27.7 mg/L”.

The results of the test can be considered valid.

Description of key information

The study was performed in accordance with OECD guideline 301B and is considered reliable without restriction (Klimisch 1). Degradation behaviour of positive control (mean of 2 replicates) and toxicity control was in the validrange normal. Abiotic degradation was not observed. Both replicates of the test item showed very goodcorrespondence. The degree of biodegradation reached 39 % after 28 days. Therefore, the substance is considered 'not readily biodegradable'.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater