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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2011 - 2 December 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
according to guideline
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
The batch of FAT 41043/A tested was a red powder (solid) with a purity of 99.8 %.
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 25 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and 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 pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.
Test temperature:
Between 19.6 and 21.3 °C.
pH before addition of sludge: 7.6 - 7.8
pH after the 3 hour exposure period: 7.5 - 8.2 (8.4, with N-allylthiourea)

Dissolved oxygen:
The oxygen concentration at the start of exposure was at least 7.9 mg O2/l.

(Mean) Respiration rates:
Control: 36 mg O2/l/h
10 mg/l: 40 mg O2/l/h
100 mg/l: 41 mg O2/l/h
1000 mg/l: 37 mg O2/l/h
Nominal and measured concentrations:
Nominal concentrations, three loading rates: 10, 100 and 1000 mg/l.
Details on test conditions:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass
- Aeration: During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 10 (1), 100 (1) and 1000 mg/l (3 replicates)
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.5 g/l suspended solids in final test mixture

- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
(loading rate)
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg FAT 41043/A per litre (Two Sample t-Test: α=0.05 Toxstat).

Furthermore, there was no significant oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was slightly higher than the total inhibition.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (8.1 mg/l).
Reported statistics and error estimates:
The percentage inhibition for the reference substance was plotted against the logarithm of the concentrations. The EC50 was determined using linear regression analysis. The 95 % confidence limits of the EC50 are given. The EC50 for the test substance was above the loading rate tested (1000 mg/l).

NOEC estimation:
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the blank- control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.
Validity criteria fulfilled:
Under the conditions of this present test FAT 41043/A was not toxic to wastewater (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

Description of key information

The short-term toxicity to fish of FAT 41043/A was investigated according with OECD Guideline Nr. 209, EU Method C.11 and GLP principles. Under the conditions of the present test FAT 41043/A was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for microorganisms could not be reached.