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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 1993 - 02 August 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
After 30 minutes of aeration at 22˚C, the first sample was taken for oxygen measurement over a period of up to 10 minutes. Thereafter, in 15 minutes intervals, the rest of the samples were submitted to measurement of oxygen concentration.
Sampling was started with the control 1. Thereafter, the measurements were continued with the positive controls and the test article samples. Finally, the oxygen consumption of control 12 was determined,
Details on test solutions:
ANOX BF (stock solution 1):
0.5 g ANOX BF was suspended with 0.5 g Tween 80 and filled to a volume of 1000 ml with bidistilled water. The final tween 80 concentration was 0.01%.

Tween 80 solution (stock solution II):
A Tween 80 solution contained 1 g Tween 80/l was prepared. The final Tween 80 concentration was 0.01%.

3,5-dichlorophenol (stock solution III):
3,5-dichlorophenol was used as a positive standard. Therefore 0.5 g of standard were dissolved according to the OECD Guideline No. 209 in 10 ml 1N NaOH, diluted to about 30ml with bidistilled water. Excess of NaOH was neutralized with 1N H2SO4 to the point of incipient precipitation. Therefore, the mixture was made up to 1 litre. The final pH was 7.8
Test organisms (species):
anaerobic bacteria from a domestic water treatment plant
Details on inoculum:
The test was run with aerobic activated sludge from a domestic waste-water treatment plant (ARA, Fullinsdorf, Ergolz II/Switzerland).

The sludge was centrifuged and the supernatant liquid phase decanted. The solid material was resuspended in tap water and again centrifuged. This procedure was repeated a further two times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (ml) to its dry weight (g) determined. Based on this ratio, a volume of washed sludge suspension corresponding to 4.0g dry material were made up with Soerensen buffer (pH =) to 1 litre. To that mixture, 50ml OECD recommended synthetic sewage feed were added. Thereafter, the sludge was aerated until use. The pH measured was 7.9.

Synthetic sewage feed (filled up to a final volume of 1 litre with bidistilled water):
16g peptone
11g meat extract
3g urea
0.7NaCl
0.4g CaCl2 . 2H2O
0.2g MgSO4 . 7H2O
2.8g K2HPO4
Test type:
other: aerobic
Water media type:
freshwater
Limit test:
no
Total exposure duration:
0.5 h
Hardness:
Not specified
Test temperature:
Not specified
pH:
7.9-8.0
Dissolved oxygen:
0.72 mg/O2/l min (mean)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The respiration rate of the sludge was not inhibited. The inhibition rates ranged from -23% to 2.1% for the concentration range 3.2 mg/l to 100 mg/l. The inhibition at 10mg/l could not be calculated due to the rapid oxygen consumption.

Based on these results the EC50 can be defined as >100mg/l
Results with reference substance (positive control):
At the two lowest concentrations of the standard of 1 mg/l and 3.2 mg/l, the respiration rate was inhibited by 2.1 and 4.9%, respectively. At 10 mg/l, the inhibition was 30.1%. At the two highest concentration of 32 and 50 mg/l, the inhibition rate was 86.0%.
y=56.392 x -11.293 (R=0.948)
(x represents the logarithm of dose of 3,5-dichlorophenol and Y the % inhibition).
According to this equation the EC50 was 12.2 mg/l.
Validity criteria fulfilled:
yes
Conclusions:
The inhibitory effect of ANOX BF on aerobic waste-water bacteria was investigated in a respiration test.

The test article showed no inhibition on the respiration rate up to a test concentration of 100mg/l. The EC50 can be defined as > 100 mg/l.

The EC50 of the reference compound 3,5-dichlorophenol tested under the same conditions was: 12.2 mg/l which in the guideline accepted range.
Executive summary:

The test article showed no inhibition on the respiration rate up to a test concentration of 100mg/l. The EC50 can be defined as > 100 mg/l.

Description of key information

The inhibitory effect of ANOX BF on aerobic waste-water bacteria was investigated in a respiration test.

The test article showed no inhibition on the respiration rate up to a test concentration of 100mg/l. The EC50 can be defined as > 100 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information