Formamide, N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]-

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:

2017-03-22 to 2017-03-24

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: I15EB1917
- Expiration date of the lot/batch: 2017-09-07 (retest date)
- Purity test date: 2017-06-07 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 0.01 g/L

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water it was directly added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). These mixtures were stirred for a short period of time until the adding of 250 mL of activated sludge, resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by 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:

no

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes.

Test temperature:

16 - 23°C which was outside the prescribed range of 20 ± 2°C. However, since the results of the controls and reference items were valid, this did not change the integrity of the study. Evaluation: The validity criteria for the controls and reference item were met and therefore this deviation is considered not to have influenced the conclusion of this study.

pH:

The pH in all test vessels, before addition of sludge was between 7.3 and 7.4. After the 3 hour exposure period the pH was between 7.2 and 8.2.

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

Details on test conditions:

TEST SYSTEM
- 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.
- Number of replicates:
* Highest loading rate: triplicate
* Lower loading rates: 1 replicate
* Abiotic control: 1 replicate
_ 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. The oxygen consumption was recorded
for a period of approximately 10 minutes. after the 3-h exposure period. Respiration rate was calculated from the measurements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
- Range finding study
- Test concentrations: 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

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

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: 23.05 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 11

Results with reference substance (positive control):

-EC50 value for the reference item (3,5 dicholorophenol) was 7.7 mg/L
-This is within the recommended range of 2-25 mg/L confirming suitability of the activated sludge used.

Reported statistics and error estimates:

-EC50 and ELR50: For the reference item calculation of the EC50 value was based on non-linear regression analysis (3- parameter logistic Cumulative Distribution Function) with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
-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 (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
-The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test (according to OECD guidelin 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 > 1000 mg T002078/L. In conclusion, under the conditions of this present test JNJ-28645409-AAA (T002078) was not toxic to activated sludge at or below 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 T002078 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was >1000 mg/L. The 3-h NOELR50 was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L