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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2016 - 02 February 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to
other: ISO International Standard 9439
Version / remarks:
Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).
according to
other: ISO International Standard 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:
Specific details on test material used for the study:
- Analytical purity: assay 101.1%
- Source and lot/batch No.of test material: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (retest date)
- Purity test date: 2016-09-19 (certificate of analysis release date)

- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: <0.1 g/L
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant receiving predominantly domestic sewage, 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands.
- Storage conditions: sludge was kept under continuous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (32 minutes) and the supernatant liquid was used as inoculum.
- Pretreatment: No
- Concentration of sludge: the concentration of suspended solids was determined to be 5 g/L in the concentrated sludge.
- Water filtered: tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon
Duration of test (contact time):
28 d
Initial conc.:
22.5 mg/L
Based on:
act. ingr.
Initial conc.:
12 mg/L
Based on:
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.
* mineral stock solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.5 gNH4Cl dissolved in 1 L Milli-Q water, pH 7.4 ± 0.2
* mineral stock solution B: 22.50 g MgSO4.7H2O dissolved in 1 L Milli-Q water
* mineral stock solution C: 36.4 g CaCl2.2H2O dissolved in 1 L Milli-Q water
* mineral stock solution D: 0.25 g FeCl3.6H2O dissolved in 1 L Milli-Q water
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium
- Additional substrate: no
- Test temperature: 21.9-23.5°C
- pH: 7.6-8.0, measured prior to testing in each test flask before addition of inoculum, and again in each test flask at the end of the incubation period
- pH adjusted: yes
- Aeration of dilution water: The test solutions were continuously aerated and stirred during the test.
- Continuous darkness: yes

- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration: 2
* test substance and inoculum: 2 replicates
* inoculum blank: 2 replicates
* positive control: 1 replicate
* toxicity control: 1 replicate

- 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). The initial suspension of unspiked test medium and inoculum was aerated with this CO2-free air overnight to purge the system of CO2 prior to testing. This CO2-free air was also used for aeration during the test.
- Measuring equipment: CO2-evolution was determined through titration of the remaining Ba(OH)2 with 0.05 M standardized HCl.
- Details of trap for CO2 and volatile organics if used: 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.

- Sampling frequency: every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day
- Sampling method: the absorber bottle closest to the incubation system was sampled each time, the second and third bottle were moved one position closer to the system and a new bottle was added at the end
- On the 28th day, pH of test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. Bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

- 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:
acetic acid, sodium salt
Test performance:
-In the toxicity control, more than 25% biodegradation occurred within 14 days (41%, based on ThCO2).
-The positive control item was biodegraded by at least 60% (76%) within 14 days.
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: mean of bottle A and B
Details on results:
- The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met.
Results with reference substance:
- The positive control item was biodegraded by at least 60% (76%) within 14 days.
Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Based on this guideline study the test substance, JNJ-42808389-AAA (T003421) was designated as not readily biodegradable, as the pass level for ready biodegradability, i.e. biodegradation of at least 60% in a 10-day window within the 28-day period of the test (biodegradation observed in the test were 3% and 1%) was not reached.

Description of key information

Based on the guideline study (Desmares-Koopmans, 2017) it is concluded that the test item, JNJ-42808389-AAA (T003421) is not readily biodegradable since the criteria for biodegradability were not met under the conditions of the carbon dioxide (CO2) evolution test (modified sturm test). Results of this study can be considered reliable without restriction.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information