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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:
2015-03-15 to 2016-04-14
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
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

Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: White, slight beige amorphous, crystalline, micropowder
Specific details on test material used for the study:
- Source and lot/batch No.of test material: I15BB0491
- Expiration date of the lot/batch: 2021-02-04 (retest date)
- Purity test date: 2015-02-06

- Storage condition of test material: at room temperature

- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: white to slightly grey powder

specific details on test material used for the study:
- Analytical purity: 99.8% (base titration: >=98.0%)

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 allowed to settle (76 minutes) and the supernatant liquid was used as inoculum at the amount of 10mL/L of mineral medium.
- Pretreatment: no
- Concentration of sludge: the concentration of suspended solids was determined to be 4.0 g/L in the concentrated sludge.
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
19.5 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: 36.40 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: 21.1-22.7°C
- pH: 7.5-7.8, 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 (toxicity control: from 7.7 to 7.6 at start of the test using 1M HCl)
- Aeration of dilution water: The test solutions were aerated and stirred continuously 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 the bottles of the inoculum blank and test supsension. 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
- Positive control: yes, 1 replicate with reference item and inoculum

TOC content of test item:
TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma, Steinheim, Germany) as total carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as inorganic carbon (IC) standard. The Total Organic Carbon (TOC) content of the test item was determined to be 62.89%.
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 (32 % 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% (72%) 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 40mg/L (57.7 mg CO2 per 2 litres of medium, corresponding to 28.9 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 (mili-RO water), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
% Degradation
Key result
% degradation (CO2 evolution)
ca. 1 - ca. 2
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
The positive control item was biodegraded by at least 60% (72%) 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-117676-AAA (T000263) was not readily biodegradable under the conditions of the test (initial concentration 19.5 mg/L) as the pass level for ready biodegradability was not reached (i.e. biodegradation of at least 60% in a 10-day window period within the 28-day period). The test substance showed only 1,5 % biodegradation (mean of 2 bottles, based on % ThCO2). The test substance did not inhibit microbial activity at the concentration used in the test and the system was demonstrated to be healthy. The results of the test can be considered reliable without restriction.