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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:
27 Feb 2018 - 29 Mar 2018
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)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10634
Version / remarks:
"Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Storage conditions: freshly obtained sludge was used immediately
- Pretreatment: Sludge was washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 3.2 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9.6 mg/L.
- Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Duration of test (contact time):
28 d
Initial conc.:
ca. 15 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PREPARATION OF TEST SOLUTIONS
Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2- litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 30.09 mg; test item bottle B: 30.01 mg and toxicity control bottle: 30.00 mg). To this end, small watch glasses were used to transfer the weighed amounts of test item to the respective test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.

PREPARATION OF TEST BOTTLES
At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.


TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301, made with tap-water purified by reverse osmosis and subsequently passed over activated carbon.
- Test temperature: 22-23°C
- pH: at start 7.6; on day 14 (Procedure control and Toxicity control only): 7.8; on day 28: 7.4-7.5.
- pH adjusted: yes, in Blank Control bottle B (before: 7.7; after: 7.6)
- Continuous darkness: yes, test media were excluded from light and the test vessels were made of brown coloured glass.

TEST SYSTEM
- Culturing apparatus: 2 L brown coloured glass bottles
- Number of culture flasks/concentration: 2 for test suspensions, 2 for inoculum blank, 1 for procedure control, 1 for toxicity control.
- Method used to create aerobic conditions: continuous aeration with CO2 free synthetic air. A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum
- Procedure control: containing reference item and inoculum
- Toxicity control: containing test item, reference item and inoculum.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
2
Sampling time:
28 d
Remarks on result:
other: Mean of two test bottles
Details on results:
ThCO2 of test item was calculated to be 2.98 mg CO2/mg.

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of test item (3% and 0%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (33%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.
Results with reference substance:
Sodium acetate was biodegraded by 84% within 14 days.

Table 1: Comparison of Biodegradation of the Test Item in Bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

2

0

0

0

0

5

0

0

0

0

8

0

0

0

0

12

0

0

0

0

15

0

0

0

0

19

0

0

0

0

23

0

0

0

0

292)

1

0

1

1

292)

1

0

1

1

292)

3

0

2

3

1): Absolute difference in biodegradation between bottles A and B

2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The 'ready' biodegradability of the test item was investigated in the Carbon Dioxide (CO2) Evolution Test (Modified Sturm Test), according to OECD Guideline 301 B and GLP principles. A test concentration of ca. 15 mg/L test item, corresponding to 12 mg TOC/L, was used. Based on the results, biodegradation of the test item was 0% and 3% in two replicates after 28 days (Mean: 2%). The pass criterion for ready biodegradation (60% biodegradation within a 10 -day window) was not met. Therefore, the test item is considered not readily biodegradable.

The test met all validity criteria, therefore the study is considered to be reliable without restrictions.

Description of key information

4,4'-methylenebis-N-sec-butylaniline was found to be not readily biodegradable in a OECD 301B test.

Key value for chemical safety assessment

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

Additional information

The 'ready' biodegradability of the test item was investigated in the Carbon Dioxide (CO2) Evolution Test (Modified Sturm Test), according to OECD Guideline 301 B and GLP principles. A test concentration of ca. 15 mg/L test item, corresponding to 12 mg TOC/L, was used. Based on the results, biodegradation of the test item was 0% and 3% in two replicates after 28 days (Mean: 2%). The pass criterion for ready biodegradation (60% biodegradation within a 10 -day window) was not met. Therefore, the test item is considered not readily biodegradable.

The test met all validity criteria, therefore the study is considered to be reliable without restrictions.

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