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Environmental fate & pathways

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:
2016-07-04 to 2016-08-04
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Composition 0
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
other: ISO International Standard 9439 “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:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Analytical purity: 98.0 to 102.0% (99.7% based on chromatographic purity HPLC and chromatographic Purity GC)
- Batch n°: I15AB0305
- Expiration date: 2017-01-23 (re-test date)
- Purity test date: 2015-02-19 (certificate of analysis release date)

- Storage condition: at room temperature
- Stability under storage conditions: Analysis of stability, homogeneity and concentration of the test item under test conditions were not
performed as part of this study.
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', Heeswijk-Dhinter, 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 (44 minutes) and the supernatant liquid was used as inoculum.
- Pretreatment: no
- Concentration of sludge: the concentration of suspended solids was determined to be 3.4 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.:
18 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
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.
* 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: 21.4-22.9°C
- pH: 7.5-8.0, 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: no
- Aeration of dilution water: The test solutions were continuously stirred during the test.
- Continuous darkness: yes

- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration:
* 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/positive control: yes, 1 replicate with reference item and inoculum
Reference substance:
acetic acid, sodium salt
Test performance:
- In the toxicity control more than 25 % degradation occurred within 14 days (36% based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.
- The positive control item was biodegraded by at least 60% (73%) within 14 days.
- The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 1%).
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (53.7 mg CO2 per 2 litres of medium, corresponding to 26.9 mg CO2/L).
- The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Since all criteria for validity of the test were met, this study was considered to be valid.
Key result
% degradation (CO2 evolution)
ca. 1
Sampling time:
28 d
Remarks on result:
other: mean of 2 bottles
absolute difference of 1 between two bottles
Details on results:
The criterion for ready biodegradability (biodegradation of at least 60% in a 10-day window within the 28-dayperiod of the test) was not met.
Results with reference substance:
The positive control item was biodegraded by at least 60% (73%) within 14 days, confirming suitability of the activated sludge.
Validity criteria fulfilled:
On day 15 a temporary breakdown in aeration was noted. Evaluation: This relative short breakdown (<1 day) was considered to have no effect on the outcome of this study; all validity criteria were met.
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-42808415-AAA (T003422) was not readily biodegradable under the conditions of the test (initial concentrations 18 mg/L). The test substance showed only 0% and 1% biodegradation (test bottle A and B, respectively, based on ThCO2). The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.

Description of key information

One study (Desmares-Koopmans, 2016) is included in this dossier and regarded as a key study (Klimisch score of 1).  The biodegradability of T003422 was determined according to OECD Guideline 301B and EU Method C.4 -C (Desmares-Koopmans, 2016). Under the conditions of the test, T003422, was determined to be not biodegradable within 28 days.

Key value for chemical safety assessment

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

Additional information

The ready biodegradability of T003422 over 28 days was determined in a carbon dioxide (CO2) evolution test (modified Sturm test) (Desmares-Koopmans, 2016). The test item was tested in duplicate at 18 mg/L, corresponding to 12 mg TOC/L. The inoculum was unadapted activated sludge from a predominantly domestic waste water treatment plant. T003422 was found to be not readily biodegradable under the test conditions within the 28-d exposure period.