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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2016 - 16 December 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
according to
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
according to
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Source and lot/batch No.of test material: I16CB1581
- Expiration date of the lot/batch:04 April 2019
- Purity test date: 07 April 2016
- Radiochemical purity: Not specified
- Specific activity: Not specified
- Locations of the label: Not specified
- Expiration date of radiochemical substance: Not specified

- Storage condition of test material: At room temperature
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: Explained in next sections
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Explained in next sections

- Treatment of test material prior to testing: Not specified
- Preliminary purification step (if any): Not specified
- Final dilution of a dissolved solid, stock liquid or gel: Not specified
- Final preparation of a solid: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: Not specified
Analytical monitoring:
Details on sampling:
Not specified
not specified
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 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 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: control with 6 replicates and additional abiotic control with highest loading rate with one replicate
- 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: 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:
Water media type:
Limit test:
Total exposure duration:
3 h
Remarks on exposure duration:
during the exposure aeration and stirring took place
Post exposure observation period:
not specified
not specified
Test temperature:
The temperature continuously measured in the temperature control vessels ranged between 17 and 22°C during the test, and was slightly outside the range prescribed by the study plan. However, there was no significant impact on the test results.
-Before addition of 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.
not specified
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 vessel: All glass open bottles/vessels
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: not specified
- Aeration: Yes. 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.
- No. of vessels per concentration (replicates): 1000 mg/L in triplicate, 10 and 100 mg/L with one replicate
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): not specified
- No. of vessels per abiotic control (replicates): abiotic control at the highest loading rate with 1 replicate
- Sludge concentration (weight of dry solids per volume): 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
- Nutrients provided for bacteria: synthetic medium comprising of 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.
- Biomass loading rate: initial loading ca. 3.0 g dw/L
- Source/preparation of dilution water: Milli-RO water
- Particulate matter: not specified
- Adjustment of pH: not specified
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: not specified
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Spacing factor for test concentrations: not specified
- Justification for using fewer concentrations than requested by guideline: not specified
- Range finding study: Yes
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: No
Reference substance (positive control):
3,5 dichlorophenol
Key result
3 h
Dose descriptor:
Effect conc.:
1 000 mg/L
Key result
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- 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
- Relevant effect levels: 5.84 - 23.43 mg O2/g.h
- Other: The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration
Reported statistics and error estimates:
-For the reference item, EC50 value was based on a 3-parameter 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, since the test item proved to be non-toxic this was not calculated for the test item

- 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:
Under the conditions of this present test JNJ-39453349-AAA (T003066) was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.

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-39453349-AAA (T003066), was not found to be toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).The ELR50for this test item was above 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information