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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:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
July 2010
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Weighed amounts of the test item were added to amber test bottles containing 200 mL Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period
- Controls: test medium without test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- 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 / Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. 250 ml sludge was dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The initial pH was 5.8 on the day of testing. The sludge was buffered to a pH of 6.6 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge.
- Initial biomass concentration: 3.0 g/L (dry weight)
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
1.8 mM (180 mg CaCO3/L)
Test temperature:
20 - 22 °C
test start: 7.4- 7.7 (before addition of sludge); test end: 6.4 - 7.5
Dissolved oxygen:
>60% saturation (> 5 mg/L at 20°C) at test start
Nominal and measured concentrations:
nominal: 10, 100 and 1000 mg/L (combined limit/range-finding test)
Details on test conditions:
- Test vessel: all glass bottles/vessels
- Type (delete if not applicable): open
- Aeration: clean, oil-free air
- No. of vessels per concentration (replicates): 10 and 100 mg/L: 1; 1000 mg/L: 3
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L (dry weight)
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L.
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): dissolved oxygen concentration after 3 hours contact time, continuously recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- Spacing factor for test concentrations: 10
- Justification for using fewer concentrations than requested by guideline: in conformity with the guideline a combined limit/range-finding test was performed
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L plus an abiotic control at 1000 mg/L
- Results used to determine the conditions for the definitive study: in conformity with the guidelines, a definitive test was not necessary since in the combined limit/range-finding test the test item showed no relevant inhibition of the respiration rate at the highest tested concentration
Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat. (total fraction)
Basis for effect:
inhibition of total respiration
Key result
3 h
Dose descriptor:
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat. (total fraction)
Basis for effect:
inhibition of total respiration
Details on results:
- Blank controls oxygen uptake rate: 24 mg O2/g.h (range 19-28; SD 3.42, n=6)
- 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: EC50 (3 h): 3.8 mg/L (95% confidence limit 1.8-5.7 mg/L); the result is within the guidelines-recommended range of 2–25 mg/L
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 non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
No EC50 value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L).
NOEC 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).

Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

The combined limit/range-finding test showed no inhibition on the respiration rate at a concentration of 10 and 100 mg/L and an average of 4% inhibition of the respiration rate at 1000 mg/L (see attached document "S-600 - Respiration Rates and Inhibition.pdf"). The inhibition of the respiration rate observed at 1000 mg/L was not statistically significant.  

There was no oxygen uptake from abiotic processes.

Validity criteria fulfilled:
The specific respiration rates in the controls were ≥20 mg O2/g/h and the coefficient of variation was ≤ 30 %

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC50 > 1000 mg/L, NOEC ≥ 1000 mg/L (OECD 209)

Key value for chemical safety assessment

Additional information

EC50 for microorganisms > 1000 mg/L

NOEC for microorgansims ≥ 1000 mg/L

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