Benzonitrile, 4-[(4-chloro-2-pyrimidinyl)amino]-

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-06 to 2016-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

See study report

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FC2164
- Expiration date of the lot/batch:23 June 2017
- Purity test date:24-06-2015 (date of certificate of analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until expiry date
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: not indicated

OTHER SPECIFICS:
- CAS n°: 244768-32-9
- Solubility in water: <0.01 g/L
- Analytical purity: 102.2% (w/w HPLC).
- Impurities (identity and concentrations): HPLC purity: <0.1% (w/w)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
- Controls: blank controls: test medium without test item and treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: predominantly domestic sewage treatment plant, "Waterschap Aa en Maas", 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ~105°C to determine the amount of suspended solids
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per L of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L of sludge

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

- at start 7.7 and 7.8.
- after the 3 hours between 7.5 and 8.1.

Dissolved oxygen:

Dissolved Oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

Nominal and measured concentrations:

Nominal concentrations:
combined limit/range-finding test: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass
- Type (delete if not applicable): open
- Aeration: adjusted such that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Highest loading rate: tested in triplicate
- Lower loading rates: one replicate.
- Abiotic control: 1 replicate tested at 1000 mg/L.
- Sludge concentration (weight of dry solids per volume): initial loading ca. 3.0 g dw/L
- Nutrients provided for bacteria: no
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
* CaCl2.2H2O 211.5 mg/L
* MgSO4.7H2O 88.8 mg/L
* NaHCO3 46.7 mg/L
* KCl 4.2 mg/L

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: no defintive study required

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate:39.94 (mg O2/L.h)
- Coefficient of variation of oxygen uptake rate in control replicates: 6
- NOELR: 1000 mg/L

Results with reference substance (positive control):

3-h EC50 = 4.2 mg/L (95% Confidence limits 2.6 – 6.9)
This is within the recommended range of 2-25 mg/L confirming suitability of the activated sludge used.

Reported statistics and error estimates:

For the reference item the calculation of ELRx/EC50 value was based on non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the substance.
NOELR determniation was based on Two-sample t-test Procedure α=0.05, one-sided smaller

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test JNJ-4754724-AAA (T002488) was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

The influence of JNJ-4754724-AAA (T002488) on the respiration rate of activated sludge was investigated after a contact time of 3 hours.U nder the conditions of this present test JNJ-4754724-AAA (T002488) was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).  The ELR50 was above 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L