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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:
2016-04-04 to 2016-08-04
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
* Batch n°: I15FB3131
* Charles River test item: 207223/A
- Expiration date of the lot/batch: 2017-06-11 (retest date)
- Purity test date: 2015-09-04
- Analytical purity: 100% (based on base titration assay)

- 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-Dinther, 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 subsenquently 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: 7.5-7.8, 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 using 1M HCl
- 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 (33% 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% (61%) within 14 days.
- The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 3%).
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (39.3 mg CO2 per 2 litres of medium, corresponding to 19.7 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, IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: mean of 2 bottles
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% (61%) within 14 days, confirming suitability of the activated sludge.
Validity criteria fulfilled:
A different batch of the reference substance was used in the test. Evaluation: The purity of the new batch was comparable to the batch stated in the study plan (i.e. 99.1% vs. 99.6%). The study integrity was not adversely affected by the deviation.
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-119379-AAA (T001141) was not readily biodegradable under the conditions of the test (initial concentration 18 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 16 % 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.

Description of key information

Based on a guideline study (Desmares-Koopmans, 2016), it can be concluded that the substance is not biodegradable as per the conditions of the 28-d ready biodegradability test (modified sturm test). It must be noted that the test substance did not inhibit microbial activity at the concentration used. The results of the study can be considered reliable without restriction.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information