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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 9, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted April 4, 1994
GLP compliance:
not specified
Details on test solutions:
The test article was dissolved in distilled water at a concentration of 500 mg/l.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: aerobic activated sludge from a domestlc waste-water treatment plant (ARA, Sissach/Switzerland).
- Pre-treatment: the sludge was coarsely sieved, centrifugated, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifugated. The procedure was repeated a further two times.
- Preparation of inoculum for exposure: 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 4.0 g dry material were made up with Soerenses buffer (pH = 7) to 1 litre. To that mlxture, 50 ml 0ECD reccomended synthetic sewage feed were added. Thereafter, the sludge was incubated overnight at 20 - 25 °C under aeration.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
ca 22 °C
pH:
7.8 - 7.9
Nominal and measured concentrations:
10 and 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: glass-flasks (> 500 ml).
- Aeration: aeration with 0.2 l/minute.
- No. of vessels per concentration: 1 replicate per concentration.
- No. of vessels per control: 2 replicates.
- No. of vessels per reference substance: 1 replicate per concentration.
- Sludge concentration: 200 ml of activated sludge.

DETERMINATION OF OXYGEN CONCENTRATION
- Measurements: oxygen consumption was measured over a period of 10 minutes.
- Conditions: during measurements, the sample was not aerated but continuously stirred on a magnetic stirrer.

VALIDATION OF THE TESTS
The tests are validated by testing a known standard, i.e. 3,5-dichlorophenol. The IC50-value should be in the range of 5 - 30 mg/l.
Furthermore, the variation between the two control samples (start, end) should not be greater than 15 %.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The aeration of test medium containing test article caused the solutions to foam strongly. The foam was coloured. Under these conditions, the respiration rate of the sludge was moderately inhibited (21.0 %) at the highest concentration of test item (i.e. 100 mg/l).
Results with reference substance (positive control):
IC5 (3h): 12.9 mg/l

Influence of test item on oxygen consumption of activated sludge.

Substance Oxygen consumption (mg O2/l min) Inhibition (%) Starting pH
Control 0.56 -- 7.8
Control 0.63 -- 7.9
Control mean 0.595 ± 0.035 (12.5 %)
Test item at 100 mg/l 0.47 21 7.8
Test item at 10 mg/l 0.48 19.3 7.8
3,5-dichlorophenol 50 mg/l 0.04 93.3 7.8
4,5-dichlorophenol 10 mg/l 0.35 41.2 7.8
5,5-dichlorophenol 3.2 mg/l 0.56 5.9 7.8
Validity criteria fulfilled:
yes
Remarks:
variance between the control samples was 12.5 % and the inhibitory effect of reference substance was withing an acceptable range
Conclusions:
IC50 (3h) > 100 mg/l (nominal)
Executive summary:

The inhibltory effect of test item on aerobic waste-water bacteria was investigated in a respiration test, according to the OECD guideline 209. The test article was dissolved in distilled water; the tested concentrations were 10 and 100 mg/l. Two replicates of negative control were prepared and 3,5-dichlorophenol was used as reference substance.

The test article showed moderate inhibition (21.0 %) of the respiration rate up to a concentration of 100 mg/l. The IC50 was determined to be higher than 100 mg/l.

Conclusion

IC50 (3h) > 100 mg/l (nominal)

Description of key information

IC50 (3h) > 100 mg/l (nominal)

Key value for chemical safety assessment

Additional information

Two tests are available on the inhibltory effect of Acid Red 057 on aerobic waste-water bacteria. The results obtained in the two different experiments are consistent: it can be concluded that the EC50 (3h) is higher than 100 mg/l; the NOEC/EC10 values are expected to be lower than 10 mg/l.

In the key study , the test article was dissolved in distilled water; the tested concentrations were 10 and 100 mg/l. Two replicates of negative control were prepared and 3,5-dichlorophenol was used as reference substance. The test article showed moderate inhibition (21.0 %) of the respiration rate up to a concentration of 100 mg/l, after the 3 hours exposure period; an inhibition of 19.3 % was recorded at the concentration of 10 mg/l.

In the second experiment available, the substance was firstly assayed in a range-finding test: an inhibition of 13 %, 35 % and 45 % at the tested concentration of 10, 100 and 1000 mg/l, respectively, was recorded. The main test was performed only at the concentration of 10 mg/l and, after the exposure period of 3 hours, the O2 consumption rate in the test vessels containing the test item was not impacted respect to those containing negative control.