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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study conducted in compliance with GLP regulations
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
- The activated sludge suspension was sieved with a fine woven mesh (mesh size about 1 mm).
- This suspension was pre-aerated over night at room temperature.
- At the next day the sludge suspension was washed once with tap water and the suspension was adjusted to 3 g/L dry matter.
- An aliquot of this suspension was added to the test vessels to obtain a sludge concentration of 1.5 g/L dry substance.

- The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL demineralized water.
- 16 mL synthetic medium were dosed to each test vessel with test substance.
- After addition of 250 mL of inoculum suspension the incubation was started by aeration of the test vessels with pressured air.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Activated sludge from a municipal wastewater treatment plant of Mannheim, Germany was collected from the aeration tank of the plant.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20.2 – 20.7 °C
Dissolved oxygen:
during aeration: > 2 mg/L
immediately before measurement: > 7 mg/L
Nominal and measured concentrations:
Test concentrations nominal: control, 62.5, 125, 250, 500 and 1000 mg/L
Details on test conditions:
- Test vessel: Glas-beakers
- Type (delete if not applicable): open / closed
- Size, fill volume: 1 L, 500 mL
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4

- Biomass loading rate:

- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

- Adjustment of pH: no
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Results with reference substance (positive control):
The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.

- The coefficient of variation of the four replicates of blank control was 8.3 % O2 consumption.

- The EC50 of 3,5-dichlorophenol was 3.3 mg/L.

In conclusion, the test conformed to all validity criteria and is valid.

Description of key information

The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations.

EC20 (3h) >= 1000 mg/L (nominal) for activated sludge (OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of ‘zinc phthalocyanine sulfonate’ on microorganisms was tested according to OECD 209 (Activated Sludge, Respiration Inhibition Test) with domestic activated sludge [BASF SE, 2012]. Up to the highest test concentration of 1000 mg/L (nominal) no inhibition on respiration was observed after 3 hours of exposure.