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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:
06 December 2016 - 16 December 2016
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: assay 101.1%
- Source and lot/batch No.of test material: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (retest date)
- Purity test date: 2016-09-19 (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: <0.1 g/L
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, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.
- 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:
no
Total exposure duration:
3 h
Remarks on exposure duration:
During which aeration and stirring took place
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, this did not effect the validity of the test.
pH:
-Before addition of sludge: 7.3 - 7.4
-After the 3 hour exposure: 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 applicable
Conductivity:
not applicable
Nominal and measured concentrations:
Combined limit/range-finding test: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all glass open bottles/vessels
- Type (delete if not applicable): open
- Aeration: Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes. 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): highest loading rate was tested in triplicate, lower loading rates consisted of one replicate
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per abiotic control (replicates): 1 replicate
- Biomass loading rate: 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; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) 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
- Range finding study: yes (combined limit/range-finding test)
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: no definitive test was performed
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: 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: 26.99 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 14
Results with reference substance (positive control):
-3-h EC50 = 5.89 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:
-For the reference item calculation of the EC50 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.
- For 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, α=0.05, one-sided, smaller).
-All 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 = 230 mg T003421/L. Under the conditions of this present test JNJ-42808389-AAA (T003421) 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 guideline study (Desmares-Koopmans, 2017) it is concluded that the test item, JNJ-42808389-AAA (T003421) is not toxic to waste water bacteria under the conditions of the activated sludge respiration inhibition test (carbon and ammonium oxidation), since the NOELR is >= 1000 mg/L and the ELR50 is above 1000 mg/L. Results of this test can be considered reliable without restriction.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information