cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

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:

2016-12-06 to 2016-12-20

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:

yes

Remarks:

The temperature was below the range prescribed by the study plan (20 ± 2°C) during the exposure phase -> 17-22°C. Evaluation: The validity criteria for the controls and reference item were met -> no influence.

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:

The characterization of the test item was conducted in a cGMP environment

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: impurities: R036844 3.30%, unknown <0.10%
- Source and lot/batch No.of test material:
* batch: I15DB1648
* test item: 207556/A
- Expiration date of the lot/batch: 2020-04-19 (retest date)
- Purity test date: 2015-05-27 (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: <0.05 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, 1L test bottles were filled with 200 mL of test item mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24h. 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 of 10, 100 and 1000 mg/L. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Eluate: Not specified
- Differential loading: 10, 100 and 1000 mg/L
- Controls: 3,5-dichlorophenol solution with a final concentration of 1 g/L in Milli-RO water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not specified
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not specified
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not specified

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Micro-organisms in activated sludge
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation: not specified
- Preparation of inoculum for exposure: The sludge was 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 ca. 105°C to determine the amount of suspended solids
- Pretreatment: The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre 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:

not specified

Hardness:

not specified

Test temperature:

17-22°C

pH:

- Before the addition of the sludge: 7.3-7.4
- After the 3 hour exposure period: 7.2-8.1

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 specified

Conductivity:

Not specified

Nominal and measured concentrations:

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass open bottles/vessels.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:
- Aeration: The aeration comprised of a set up in which 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.
- No. of vessels per concentration (replicates): 1000 mg/L in triplicates and 100 and 10 mg/L in duplicates
- No. of vessels per control (replicates): Control with 6 replicates
- No. of vessels per vehicle control (replicates): not specified
- No. of vessels per abiotic control (replicates): abiotic control with one replicate
- Biomass loading rate: 3.0 g/L of sludge
- Weight of dry solids per volume of reaction mixture per unit of time: 27 mg oxygen per one gram of activated sludge per hour

TEST MEDIUM / WATER PARAMETERS
- Nutrients provided for bacteria: To prepare a test system, adjusted ISO-medium, formulated using RO-water which comprised, CaCl2.2H2O(211.5 mg/L), MgSO4.7H2O (88.8 mg/L), NaHCO3 (46.7 mg/L) and KCl (4.2 mg/L) were used.
As a feed a synthetic medium was provided which comprised peptone(16g), meat extract(11g), urea(3g), NaCl(0.7g), CaCl2.2H2O (0.4g), MgSO4.7H2O(0.2g), K2HPO4 (2.8 g), dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes
- Test concentrations: 10 mg/L, 100 mg/L 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: NOELR

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: ELR

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: None
- Effect concentrations exceeding solubility of substance in test medium: Not specified
- Adsorption (e.g. of test material to the walls of the test container): Not specified
- Blank controls oxygen uptake rate: 26.99 mg O2/g.h
- Coefficient of variation of oxygen uptake rate in control replicates: 14

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Other: Results with the reference substance are 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 calculation of the ELR 50 value was based on a 3 parameter logistic cumulative distribution function (CDF) using a non-linear regression analyses with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item. However, no ELR 50 was calculated for the test item since it proved to be non-toxic.

- For the 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, one-sided, smaller).

-These 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 OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant was realised. Under the conditions of this present test JNJ-16479671-AAA (T001095) was not toxic to 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

Based on the study by Desmares-Koopmans (2017) performed according to the guideline OECD 209, EC 440/2008 C.11 and ISO standard 8192, the test item, JNJ-16479671-AAA (T001095) was not found to be toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR₅₀ for this test item was above 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L