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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:
2018-01-23 to 2018-02-22
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 guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to guideline
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R,2S,3S,4R)-3-((5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid
EC Number:
Cas Number:
Molecular formula:
(1R,2S,3S,4R)-3-((5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid
Test material form:
solid: particulate/powder
Details on test material:
- Name of the test material (as cited in study reports): JNJ-63757109-AAA (T003690)
- Physical state: solid (powder)
- Appearance: white to slightly yellow power
Specific details on test material used for the study:
- Analytical purity: 99.9% (Titration)
- Source and lot/batch No.of test material: I17BD0751
- Expiration date of the lot/batch: 2019-02-24 (retest date)
- Purity test date: 2017-11-14 (certificate of analysis release date)

- Storage condition of test material: At room temperature
- Solubility in water: <0.01 g/L (21.6°C)

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.
- Storage conditions: Sludge was kept under continuous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
- Pretreatment: no
- Concentration of sludge: The concentration of suspended solids was determined to be 3.0 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 test substance concentrationopen allclose all
Initial conc.:
20.5 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
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.
* 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: 22-23°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 aerated and stirred during the test. On nominal day 23 there was a short breakdown in aeration for test item bottle B. Aeration was immediately restored. Evaluation: Such a short breakdown in aeration has no impact on the validity or outcome of the study.
- Continuous darkness: yes

- Culturing apparatus: 2-L 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).
- 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
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
- In the toxicity control more than 25 % degradation occurred within 14 days (26% based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.
- 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 slightly exceeds 40 mg/L (81.1 mg CO2 per 2 litres of medium, corresponding to 40.6 mg CO2/L). Since the CO2 release in the blank was still well below 70 mg/L this does not constitute a breach of the validity criterion.
- 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).

% Degradation
Key result
% degradation (CO2 evolution)
ca. 2.5
Sampling time:
28 d
Remarks on result:
other: mean of test 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.

BOD5 / COD results

Results with reference substance:
The positive control item was biodegraded by at least 60% (83%) 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 T003690 was not readily biodegradable under the conditions of the test (initial concentration 20.5 mg/L). The test substance showed only 2% and 3% 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.