D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

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:

2016-06-15 to 2016-06-17

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: I15AB0305
- manufacture date: 2015-01-24
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: 2015-09-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS:
- purity: 99.7%
- appearance: white powder
- Water solubility: not available

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- 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 (see also paragraph 6.5). 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

Post exposure observation period:

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

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 18 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.4 and 8.0.

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:

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

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.
- replicates:
* Highest loading rate: triplicate
* Lower loading rates: 1 replicate
* 1000 mg/L and abiotic control: 1 replicate
* blank control: 6 replicates
- 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
- Range finding study: yes
- Test concentrations: loading rates of 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: combined limit/range-finding test

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: 48.90 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 13

Results with reference substance (positive control):

- 3h-EC50 (mg/L) value for 3,5-dichlorophenol: 6.3 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 value was based on 3 parameter normal CDF non-linear regression analysis with the percentages of respiration inhibition versus the corresponding concentrations of the item.
For JNJ-42808415-AAA (T003422) no ELR50-value could be calculated because the test item proved to be non-toxic (ELR50 > 1000 mg/L)
- NOELR: An effect was considered to be significant if statistical analysis of the data obtained for the test
concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided smaller). No inhibition of the respiration rate was observed at any of the tested concentrations. Therefore, the NOELR was considered to be the highest test concentration. 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 OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 > 1000 mg/L. In conclusion, JNJ-42808415-AAA (T003422) 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 T003422 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was >1000 mg/L.