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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-24 to 2015-07-24
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
according to
other: ISO 10634 (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
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): T001624
- Physical state: solid
- Appearance: white powder
- CAS: 132961-05-8
- Molecular formula: C23H28F2N4O2
- Molecular weight: 430.5 g/mol
Specific details on test material used for the study:
- Analytical purity: 100.0% (base titration)
- Lot/batch No.: I14LC5175
- Expiration date of the lot/batch: 2020-12-11 (retest date)
- Storage condition of test material: at room temperature
- solubility in water: 0.121 g/L
- Stability in water: not indicated
- Molecular weight: 430.5
- Molecular formula: C23H28F2N4O2

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: municipal sewage treatment plant receiving predominantly domestic sewage, 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: The concentration of suspended solids was determined to be 4.5 g/L in the concentrated sludge.

Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
19 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: test water prepared according to test guidelines, analytical grade salts dissolved in tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon. Since T001624 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 L test bottles containing medium with microbial organisms and mineral components (test item bottle A: 37.74 mg; test item bottle B: 37.74 mg and toxicity control bottle: 37.03 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing, the resulting suspension was added quantitatively to the test medium. The test solution were continuously stirred during the test.
- Additional substrate: no
- Test temperature: 22 - 24°C
- pH: 7.6, measured at the start of the test (after addition of concentrated HCl), and on day 28 (7.6-7.9)
- pH adjusted: yes, using 37% HCl
- Aeration of dilution water: A mixture of oxygen (± 20%) and nitrogen (±80%) was passed through a bottle, containing 0.1 - 1 L 0.0125 M Ba(OH)2 solution to trap CO2. The synthetic air was sparged through the scrubbing solutions at a rate of ~1-2 bubbled per second ( ± 30-100 mL/min).
- Continuous darkness: yes

- Test flasks: 2 L glass brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: A mixture of oxygen (~20%) and nitrogen (~80%) was passed through a bottle, containing 0.5 - 1 L 0.0125 M Ba(OH)2 solution to trap CO2. The synthetic air was sparged through the scrubbing solutions at a rate of ~1-2 bubbles per second ( ~30-100 mL/min).
- Details of trap for CO2 and volatile organics if used: 3 CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each bottle. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0,05 M standardized HCl.

- Inoculum blank: yes, two replicates with only inoculum
- Toxicity control: yes, one replicate with test item, reference substance, and inoculum
- Procedure control: yes, 1 replicate with reference item and inoculum
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
% degradation (CO2 evolution)
ca. 4
Sampling time:
28 d
Details on results:
No significant biodegradation of the test item occurred within 28 days of incubation (1% and 7%, based on ThCO2 in bottle A and B respectively). The toxicity controls indicated that the test item did not have an inhibitory effect on the microbial activity.

BOD5 / COD results

Results with reference substance:
The positive control item was biodegraded by at least 60% (62%) within 14 days, confirming suitability of the activated sludge.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
A 28-d ready biodegradability test (OECD 301B, modified sturm test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that JNJ-7941726-AAA (T001624) was not readily biodegradable under the conditions of the test (initial test concentration 19 mg/L, corresponding to 12 mg TOC/L). The results of the test can be considered reliable without restriction.