4-[(4-HYDROXYPYRIMIDIN-2-YL)AMINO]BENZONITRILE

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-15 to 2016-04-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).

Deviations:

no

Qualifier:

according to guideline

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).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15GC2731
- Expiration date of the lot/batch: 2016-07-23 (re-test date)
- Purity test date: 2015-03-29 (certificate release date)
- Analytical purity: >97.0% (based on chromatographic purity HPLC: 99.8%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 0.01 g/L

Oxygen conditions:

aerobic

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.
- 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 conc.:

19.5 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- 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: 22.1-22.7°C
- pH: 7.51-7.81, 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 7.64 to 7.56 at start of the test using 1M HCl) (test item A: from 7.66 to 7.60 at start of the test using 1M HCl)
- Aeration of dilution water: The test solutions were continuously stirred during the test.
- Continuous darkness: yes

TEST SYSTEM
- 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
- 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.

CONTROL AND BLANK SYSTEM
- 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, less than 25% degradation occurred within 14 days (4% based on ThCO2). Therefore, the test item appears to have an inhibiting effect on microbial activity at the tested target concentration of 19.5 mg/L, corresponding to 12 mg TOC/L.
- 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 40 mg/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 (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

Parameter:

% degradation (CO2 evolution)

Value:

ca. 1

Sampling time:

28 d

Remarks on result:

other: mean of 2 bottles

Remarks:

absolute difference of 1 between two 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% (72%) within 14 days, confirming suitability of the activated sludge.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of JNJ-40370226-AAA (T002487) (1% and 0%, based on ThCO2, in bottle A and B, respectively). Biodegradation of at least 60% in a 10-day window within the 28-day period of the test was not reached. In the toxicity control, less than 25% biodegradation occurred within 14 days (4%, based on ThCO2). Therefore, the test item appears to have an inhibiting effect on microbial activity at the tested target concentration of 19.5 mg/L, corresponding to 12 mg TOC/L, which was considered to have had no effect on the outcome of this study (the criterion for ready biodegradability). Since all criteria for validity of the test were met, this study was considered to be valid.

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 T002487 was determined according to OECD Guideline 301B and EU Method C.4-D. Under the conditions of the test, T002487 was determined to be not readily biodegradable within 28 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

JNJ-40370226-AAA (T002487) was designated as not readily biodegradable (1% and 0% based on ThCO2 in bottle A and B, respectively), 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, was not reached. In the toxicity control, less than 25% biodegradation occurred within 14 days (4%, based on ThCO2). Therefore, the test item was found to have an inhibiting effect on microbial activity, which was considered to have had no effect on the outcome of this study (the criterion for ready biodegradability). Since all criteria for validity of the test were met, this study was considered to be valid.

In conclusion, JNJ-40370226-AAA (T002487) 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 was not reached.