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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-16 to 2016-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: I15FB2768
- Expiration date of the lot/batch: 01 July 2017 (retest date)
- Purity: 100% (based on base titration assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
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 25 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.
- 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
Test temperature:
Ranged between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:
pH between 7.2 and 7.3. After the 3 hour exposure period the pH was between 7.1 and 8.4.
Dissolved oxygen:
DO 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.
Nominal and measured concentrations:
Nominal concentrations:
combined limit/range-finding test: 10, 100 and 1000 mg/L
Details on test conditions:
- Test vessel: All glass
- Type (delete if not applicable): open
- Aeration: adjusted such 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.
- Highest loading rate: triplicate,
- Lower loading rates: one replicate. In addition, a blank (6 replicates) and an abiotic control were included.
- Sludge concentration (weight of dry solids per volume): initial loading ca. 3.0 g dw/L
- Nutrients provided for bacteria: no
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
* 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: 10
- Range finding study: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: no definitive study



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
Details on results:
- Blank controls oxygen uptake rate: 63 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 6
Results with reference substance (positive control):
EC50 value: 6.779 mg/L
Reported statistics and error estimates:
EC50: non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
NOELR: Two-sample t-test Procedure α=0.05, one-sided smaller
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test JNJ-1806792-AAA (T001325) was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).
The 3-h ELR50 was above 1000 mg/L.
These results can be considered reliable without restrictions

Description of key information

The study of Desmares-Koopmans (2016), investigating the toxicity of T001325 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage.

Under the conditions of this present test JNJ-1806792-AAA (T001325) was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).  The 3-h ELR50 was above 1000 mg/L. These results can be considered reliable without restrictions

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information