4-bromo-2,2-diphenylbutanenitrile

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:

2016-07-18 to 2016-10-28

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 2018-01-11 (retest date)
- Purity test date: 2016-02-18 (certificate of analysis release date)
- Purity: 99.3% (chromatographic purity by GC)

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

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

Test temperature:

Range-finding test:
The temperature continuously measured in the temperature control vessels ranged between 19 and 23°C during the test, and was outside the range prescribed by the study plan.
Evaluation: The validity criteria for the controls and reference item were met and therefore this deviation is considered to have no effect on the results of this test

Final test:
The temperature continuously measured in the temperature control vessels ranged between 20 and 24°C during the test, and was outside the range prescribed by the study plan.
Evaluation: The validity criteria for the controls and reference item were met and therefore this deviation is considered to have no effect on the results of this test.

pH:

Final test:
The pH in all test vessels, before addition of sludge was between 7.3 and 7.5. After the 3 hour exposure period the pH was between 7.8 and 8.3.

Dissolved oxygen:

The aeration was adjusted in such a way 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.

Salinity:

not applicable

Nominal and measured concentrations:

Loading rates for the test substance:
* combined limit/range-finding test : 10, 100 and 1000 mg/L
* range-finding test: 1.0, 10, 100 and 1000 mg/L
* Final test: 3.2, 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

- Test vessel: All glass open bottles/vessels
- Aeration: The aeration was adjusted in such a way 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. 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.
- 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) 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: x 3.2
- Range finding study
- Test concentrations: 1.0, 10, 100 and 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.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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: 24.88 mg O2/g.h
- Coefficient of variation of oxygen uptake rate in control replicates: 17

Results with reference substance (positive control):

- The EC50 of 3,5-dichlorophenol was within the accepted range of 2 to 25 mg/L for total respiration
(3.0 mg/L)

Reported statistics and error estimates:

EC50 and ELR50
For the reference item calculation of the EC50 value was based on non-linear regression analyses (3-parameter normal CDF [cumulative distribution function]) with the percentages of respiration inhibition versus the corresponding concentrations of the item.
For JNJ-1597622-AAA (T000625) no ELR50-value could be calculated because the test item proved to be non-toxic (ELR50 > 1000 mg/L).

NOELR determination:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Dunnett`s Multiple t-test Procedure, alpha=0.05, one-sided smaller).

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge repiration inhibitiontest (according OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 = 1000 mg T000625/L. Under the conditions of this present test, JNJ-1597622-AAA (T000625) was not toxic to activated sludge at a loading rate of 1000 mg/L (NOELR). The results of the study can be considered reliable without restriction.

Description of key information

The study of Desmares-Koopmans (2017), investigating the toxicity of T000625 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was above 1000 mg/L. The 3-h NOELR was >= 1000 mg/L.

Additional information

One key study investigating the toxicity to aquatic microorganisms was available (Desmares-Koopmans, 2017). This study exposed activated sludge of a sewage treatment plant treating predominantly domestic sewage to a series of test substance concentrations (3.2, 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.

JNJ-1597622-AAA (T000625) was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The 3-h ELR50 was above 1000 mg/L.