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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 2016-04-15
Reliability:
1 (reliable without restriction)
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: I15FB3131
- Expiration date of the lot/batch: 2016-09-30 (retest date)
- Purity test date: 2017-06-11

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
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 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.
-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
Test temperature:
The temperature continuously measured in the temperature control vessels remained 21°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 7.7 and 7.9. After the 3 hour exposure period the pH was between 7.4 and 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 applicable
Nominal and measured concentrations:
Nominal concentrations:
- Range-finding test: 10, 100, 1000 mg/L
- Final test: 100, 180, 320, 560 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: 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)

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: 1.8
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: ELR10
Effect conc.:
56 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: ELR50
Effect conc.:
190 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: 62.88 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates did not exceed 30% at the end of the definitive test (7%).
- A NOELR could not be determined
Results with reference substance (positive control):
EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (4.1 mg/L for final test)
Reported statistics and error estimates:
ELRx /EC50 value was based on the 3-parameter normal cumulative distribution function (CDF) using a nonlinear regression analysis with the respiration rate versus the logarithms of the corresponding loading rates/concentrations of the test/reference item.

NOELR: Welsh-t-test after Bonferroni-Holm procedure α=0.05, one-sided smaller
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 EC50 = 190 mg T001141/L. The results of the study can be considered reliable without restriction.

Description of key information

Based on the guideline study (Desmares-Koopmans, 2017), the EC50 of the test item, JNJ-119379-AAA (T001141) was 190 mg/L, in the activated sludge respiration inhibition test. The results of the study can be considered reliable without restriction.

Key value for chemical safety assessment

EC50 for microorganisms:
190 mg/L
EC10 or NOEC for microorganisms:
56 mg/L

Additional information