5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

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:

2015-06-17 to 2015-09-18

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

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: A14JB3414
- Expiration date of the lot/batch: 2016-10-01 (retest date)
- Purity test date: 2014-10-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark, desiccated
- Stability under test conditions: stable

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. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 to 26 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.
- 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:

no

Total exposure duration:

3 h

Post exposure observation period:

After 3 h of exposure, oxygen consumption was recorded for approximately 10 minutes.

Test temperature:

Combined limit/range-finding test:
The temperature was outside the range prescribed by the protocol (20 ± 2°C) during the exposure phase, the temperature was 16.9-22.8°C.
Evaluation: The validity criteria for the controls and reference item were met and therefore this deviation is considered not to have influenced the sludge.

Final test: The temperature was slightly above the range prescribed by the protocol (20 ± 2°C) during the exposure phase, the temperature was 19.6-23.1°C.
Evaluation: The validity criteria for the controls and reference item were met and therefore this deviation is considered not to have influenced the sludge.

pH:

First combined limit/range-finding test:
* pH before addition of sludge in controls and reference item: 7.6-7.7
* pH after the 3-h exposure period in controls and reference item: 7.4-8.4

* pH before addition of sludge in the test item replicates: 7.6-9.2
* pH after the 3-h exposure period in the test item replicates: 7.7-9.1

Second combined limit/range-finding test:
* pH before addition of sludge in controls and reference item: 7.6
* pH after the 3-h exposure period in controls and reference item: 7.8-8.3

* pH before addition of sludge in the test item replicates: 7.6-8.9/9.2
* pH after the 3-h exposure period in the test item replicates: 7.8-8.3

Two combined limit/range-finding tests were performed (with loading rates of 10, 100 and 1000 mg/L). In the first combined limit/range finding test a strong pH increase (up to 9.2 at 1000 mg/L) as well as a strong increase of the inhibition of the respiration rate (96% at 1000 mg/L) was observed. Therefore, an additional limit/range-finding test was performed to investigate the intrinsic toxic effect of the test item. The results of both tests were comparable (96% inhibition in the 1st test and 100% inhibition in the 2nd test at 1000 mg/L, respectively). Thus, the inhibition of the respiration rate was caused by the intrinsic toxicity of the test item instead of the effect of pH. Therefore, the final test was performed without pH adjustment.

Final test:
* pH before addition of sludge in controls and reference item: 7.7
* pH after the 3-h exposure period in controls and reference item: 7.4-8.2

* pH before addition of sludge in the test item replicates: 7.6-9.3
* pH after the 3-h exposure period in the test item replicates: 7.5-8.9

Dissolved oxygen:

dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C)

Salinity:

not applicable

Nominal and measured concentrations:

Loading rates for the test substance:
- Combined limit/range-finding test: 10, 100, 1000 mg/L
- Final test: 10, 32, 100, 320, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Aeration: After 3-hour contact time, oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- Number of replicates: 5 (final test)
- Number of control replicates: 6
- Biomass loading rate: initial loading ca. 3.0 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
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. Oxygen consumption was monitored for approximately 10 min after the 3-h exposure period. Respiration rate was calculated from the measurements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations range finding study: loading rates of 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

230 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% C.L.: 120-440 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: ELR10

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% C.L.: 3.7-130 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
- Blank controls oxygen uptake rate: 45 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 7

Results with reference substance (positive control):

3-h EC50 = 2.05 and 3.1 mg/L (for the range-finding and limit test respectively).
This is within the recommended range of 2-25 mg/L confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The 3-h EC50 values were based on Probit Analysis.
The NOEC determination was based on the Welch-t test for inhomogeneous variances with Bonferroni-Holm Adjustment, alpha=0.05, one sided, smaller.

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test (according OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 = 230 mg T000990/L. In conclusion, JNJ-119743-AAA (T000990) was not toxic to activated sludge at or below a loading rate of 100 mg/L. Statistically significant effects were observed at loading rates of 320 mg/L and 1000 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

The study of Desmares-Koopmans (2016), investigating the toxicity of T000990 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was 230 mg/L. The 3-h ELR10 and the 3-h NOELR were 72 and 100 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

230 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

One key study investigating the toxicity to aquatic microorganisms was available (Desmares-Koopmans, 2016). This study exposed activated sludge of a sewage treatment plant treating predominantly domestic sewage to a series of test substance concentrations (10, 32, 100, 320 and 1000 mg/L) during 3 hours in a static test design, at a temperature of 20°C. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 h. The criteria for a valid test were fulfilled. The inhibitory effect of the test item T000990 on the respiration rate of aerobic waste water microorganisms of activated sludge resulted in a 3-h ELR50 of 230 mg/L. The 3-h NOELR and 3-h ELR10 were determined to be 100 and 72 mg/L respectively.