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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 28, 2018
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))
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
Note: In the range finding test, only the highest tested concentration of 1000 mg/L caused 50.74 % inhibition of the total respiration rate in activated sludge after 3 hours. Therefore, in accordance with the study plan and OECD 209, further testing (i.e., a main test) was required and conducted using the test item concentrations 62.5, 125, 250, 500 and 1000 mg/L and two controls (3 replicates per treatment and the controls, respectively). An abiotic control was not applied since the abiotic control applied in the range-finder proved that the test item did not show any oxygen depleting characteristics. Starting at a concentration of 500 mg/L, cesium tetrafluoroaluminate inhibited the respiration of the inoculum significantly.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2016-05-31
Analytical monitoring:
yes
Details on sampling:
The respiration rates of the samples were measured 3 hours after the start of the incubation of respective vessels.
Vehicle:
no
Details on test solutions:
The required mass to obtain the accurate test concentrations was applied to the dilution water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Fresh samples of activated sludge from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany were taken. The plant is mainly fed with municipal wastewater.
- Preparation of inoculum for exposure: The sludge was left alone to settle and the supernatant was decanted. The sludge was washed one time with isotonic solution. A small amount of the sludge was weighed and dried. A stock solution of the washed activated sludge was prepared in chlorine-free tap water with a mixed liquor suspended solids level of about 3 - 4 g dry mass sludge/liter (main test 3.9 g/L).
- Pretreatment: The sludge stock solution was fed with synthetic sewage feed (25 ml/L) and was incubated and aerated overnight at 20 ± 2 °C in the dark.
- Initial biomass concentration: Activated sludge was adjusted to a mixed liquor suspended solids level of 3.6 g dry mass sludge/liter. The mixed liquor suspended solids level in the test vessel was nominally 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
test start:
test end:
pH:
pH at test end (3 h):
control:
treatments:
Reference:
Nominal and measured concentrations:
Nominal test concentrations in the main test: 0 (control), 62.5, 125, 250, 500 and 1000 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L Erlenmeyer flasks
- Fill volume: total volume 500 mL
- Aeration: stirred and aerated (air flow 0.5 to 1 L/min)
- No. of replicates: 3 per treatment and the controls
- Nutrients provided for bacteria:
synthetic sewage for sludge stock feeding according to OECD 209: 160 g/L Peptone, 110 g/L Meats extract, 30 g/L Urea, 28 g/L K2HPO4, 7 g/L NaCl, 4 g/L CaCl2 x 2H2O, 2 g/L MgSO4 x 7H2O
synthetic sewage for test assay feeding according to OECD 209: 16 g/L Peptone, 11 g/L Meats extract, 3 g/L Urea, 2.8 g/L K2HPO4, 0.7 g/L NaCl, 0.4 g/L CaCl2 x 2H2O, 0.2 g/L MgSO4 x 7H2O
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+)
- Particulate matter:
OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED: respiration rate (total oxygen uptake including heterotrophic and nitrification uptake) after 3 h
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg test item/L
- Results used to determine the conditions for the definitive study: 50.74 % inhibition of total respiration rate after 3 h at 1000 mg test item/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Cesium tetrafluoroaluminate
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
226 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Cesium tetrafluoroaluminate
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
374 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Cesium tetrafluoroaluminate
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
982 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Cesium tetrafluoroaluminate
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Cesium tetrafluoroaluminate
Basis for effect:
inhibition of total respiration
Details on results:
Validity: with 23 mg O2/g activated sludge (dry weight of suspended solids) in an hour, the oxygen uptake rate in blank controls was ≥ 20 mg O2/g in an hour, with 13 %, the coefficient of variation of oxygen uptake rate in control replicates is ≤ 30 %, with 24.7 mg/L, the EC50 (3 hours) of the reference substance 3,5-dichlorophenol is in the accepted range of 2 to 25 mg/L.
Results with reference substance (positive control):
An EC50 of the reference item 3,5-dichlorophenol for respiration inhibition between 2 and 25 mg/L was observed.
Reported statistics and error estimates:
For each endpoint, the NOEC, LOEC, and, if possible, the EC10, EC20, EC50 and EC80 were determined. A LOEC was calculated by using ANOVA followed by Williams’ test. For trend analysis, a Contrasts test (Monotonicity of Concentration/Response) for metric data was applied. The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal test item concentrations. For statistical calculations, the computer program ToxRat® was applied.
Validity criteria fulfilled:
yes
Remarks:
. All validity criteria concerning the control activity, the reference item and the comparability of the controls were fulfilled.
Conclusions:
Effects of cesium tetrafluoroaluminate on the respiration inhibition of activated sewage sludge were determined in a GLP-conform 3-h activated sludge respiration inhibition test according to OECD 209. The mean total microbial respiration rates of the activated sludge incubated with 62.5, 125, 250, 500 and 1000 mg/L cesium tetrafluoroaluminate (nominal, analytically verified based on Cs), ranged from 11.3 to 22.8 mg O2/(g*h) after 3 h. Starting at a concentration of 500 mg/L, cesium tetrafluoroaluminate inhibited the respiration of the inoculum significantly. The NOEC was found to be 250 mg/L cesium tetrafluoroaluminate (nominal). The EC10, EC20 and EC50 were found to be 226, 374 and 982 mg cesium tetrafluoroaluminate/L (nominal), respectively. The EC80 was found to be higher than the highest concentration tested (i.e. > 1000 mg cesium tetrafluoroaluminate/L). All validity criteria of the test were met.

Description of key information

In a reliable GLP- and guideline-conform activated sewage sludge respiration inhibition test of cesium fluoroaluminate according to OECD 209, the 3h-EC10 and EC50 values were determined with 226 and 982 mg cesium fluoroaluminate/L (nominal), respectively (Simon, 2018). Hence, cesium fluoroaluminate seems to have some potential for toxicity to aquatic microorganisms.

Key value for chemical safety assessment

Additional information