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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jul – 01 Aug 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
GLP compliance:
yes (incl. QA statement)
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany
Analytical monitoring:
not required
Details on test solutions:
- Method: A test item stock solution of 20.0 g/L was prepared and defined amount of stock solution was added into the test vessels with a total volume of 500 mL to obtain a final concentration of 1000 mg/L.
- Controls: 6 replicates containing activated sludge and test medium.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht, Germany (sampling: 31 Jun 2017)
- Preparation of inoculum for exposure: Before use the activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration in the test.
- Initial biomass concentration: 1.5 g/L dry matter
- Other: The inoculum was not adapted and not pre-conditioned.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20.9 - 21.9 °C
7.4 (test vessels, control vessels, reference vessels)
Dissolved oxygen:
>60 - 70% oxygen saturation
Nominal and measured concentrations:
control and 1000 mg/L (nominal)
Details on test conditions:
- Test vessel: Serum bottle and BOD-bottle
- Type: open
- Material, size, headspace, fill volume: 600 mL Serum bottles with 500 mL volume; aeration was measured after 3 h in BOD bottles
- Aeration: aerated with room air with a membrane pump
- No. of vessels per concentration (replicates): 5 (for 1000 mg/L)
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nutrients provided for bacteria: Nutrients provided for bacteria: Synthetic sewage feed (OECD) prepared with tap water: 16 mL of synthetic sewage obtaining a total volume of 500 mL.
- Nitrification inhibitor used: none

- Source/preparation of dilution water: chlorine-free tap water

- Details on termination of incubation: After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

- Respiration: The oxygen concentration [mg/L] was measured 3 h after addition of the activated sludge suspension (following the starting sequence consisting of the addition of the test/reference items and synthetic sewage and made up to 250 mL with chlorine-free tap water) and recorded for up to 15 min with an oxygen electrode (Oxygen electrode and meter Multi 9430, WTW, Weilheim, Germany).
- Dry matter content: The dry matter content of the sludge suspension was determined at the end of the test, by drying the blank controls at 105 °C overnight.

- Spacing factor for test concentrations: 10
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
3 h
Dose descriptor:
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: No
- Blank controls oxygen uptake rate: 21.3 mg O2 per gram of activated sludge per hour (required: > 20 mg oxygen per 1 g of activated sludge dw).
- Coefficient of variation of oxygen uptake rate in control replicates: 12% (required: ≤ 30%)
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was calculated as 24.28 mg/L and was therefore within the acceptable range of 2 to 25 mg/L.
Reported statistics and error estimates:
The EC50 calculation was performed with the ToxRat software by probit analysis using linear max. likelihood regression (ToxRat Professional Version 3.2.1, ©ToxRat Solutions GmbH). The EC50 is calculated from the slope of the curve.

Table 1: Validity criteria

Criterion from the guideline


Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

21.3 mg oxygen per one gramm sludge per hour


The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.



The EC50 of the reference item (inhibition of total respiration) must be within a range of 2 to

25 mg/L.

24.28 mg/L


Validity criteria fulfilled:
No effects were observed up to and including 1000 mg/L.

Description of key information

NOEC (3 h) = ≥ 1000 mg/L (nominal, OECD 209)

Key value for chemical safety assessment

Additional information

There is one GLP study available investigation the respiration inhibition of activated sludge microorganisms according to OECD guideline 209 (Hydrotox, 2017).

To investigate the inhibitory effects on total respiration, non-adapted activated sludge from a municipal sewage treatment plant was fed with OECD synthetic sewage and inoculated with the nominal concentration of 1000 mg/L substance for 3 h under aerobic conditions. Blank controls (without test item) and sensitivity controls (with a reference item at three concentration levels) were carried out in parallel. After 3 h, the oxygen consumption rate was measured by inserting an oxygen electrode into a BOD bottle and recording oxygen concentration for up to 15 min. No respiration inhibition of the activated sludge compared to the blank controls was determined up to 1000 mg/L test item. Therefore EC50 and LOEC were reported as >1000 mg/L and the NOEC as ≥ 1000 mg/L.