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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Mar 1990
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
Version / remarks:
1984
Deviations:
yes
Remarks:
See 'Deviations' in 'Any other information on materials and methods incl. tables'
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
- For making the test solution, 22.36, 6.98, 2.88, 0.93 and 0.28 mg of the test substance were weighed and added to the test medium. The volume was adjusted to 200 mL with water and aerated for 3 hours.
Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge collected from the sewage treatment plant of CH-4153 Reinach on 20/03/90
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
Nominal and measured concentrations:
- Norminal concentration: 0 (Control), 1.4, 4.65, 14.40, 39.40 and 111.80 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL BOD flasks with gas inlet
- Sludge concentration: 1.7 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated drinking water
- Test medium: same as OECD TG 209 suggested

EFFECT PARAMETERS MEASURED
Oxygen consumption per hour in mg/liter was measured.

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenole
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.
After 3 hours incubation, 7%, -5%, 2%, 2% and 8% inhibition on the oxygen consumption were observed in 1.4, 4.65, 14.40, 39.40 and 111.80 mg/L groups, respectively.
Results with reference substance (positive control):
An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.
After 3 hours incubation, 15%, 35% and 74% of inhibition on oxygen consumption were observed in 3.2, 10 and 32 mg/L 3,5-Dichlorophenole treated groups, respectively. The EC50 value was determined to be 14.2 mg/L.
Reported statistics and error estimates:
The inhibitions were calculated on the basis of the measured time depend oxygen consumption of a Blank and Test solution.

Table 1. Effects on oxygen consumption rate of activated sludge

Nominal Concentration (mg test substance/L)

Concentration (mg/L)

Consumption rate (mg/L/h)

 

Inhibition (%)

Blank 1

0

57.32

4

Blank 2

0

62.57

-4

Reference 1

32.0

15.73

74

Reference 2

10.0

38.97

35

Reference 3

3.2

50.94

15

Test conc 1

111.8

55.40

8

Test conc 2

34.9

58.50

2

Test conc 3

14.4

58.70

2

Test conc 4

14.64

62.80

-5

Test conc 5

1.4

55.91

7
Validity criteria fulfilled:
yes
Conclusions:
In an toxicity study performed in accordance with OECD TG 209, the 3-hour EC50 value was determined to be > 100 mg/L for inhibition of oxygen consumption of the microorganisms in activated sludge.
Executive summary:

A test for inhibitory concentration on aerobic bacteria of the test substance was performed according to OECD TG 209 and GLP principles. Activated sludge collected from sewage treatment plants was placed in test bottles to which the test substance was added (no stock could be made due to low solubility). The final concentrations of the test substance were 0 (blank control),1.4, 6.0, 4.65, 14.4, 34.9 and 111.8 mg/L. 3,5-Dichlorophenole was used as positive control and tested at concentrations of 3.2, 10 and 32 mg/L under the same experimental condition. The filled volume in the test bottle was adjusted to 200 mL with water and aerated for 3 hours. Oxygen consumption per hour was recorded over a 3-hour period at 20 ± 2 °C and the inhibitions calculated on the basis of the measured time dependent oxygen consumption of a blank compared with the test solution. Calculations were based on nominal test concentrations.

After 3 hours incubation, 15%, 35% and 74% of inhibition on oxygen consumption were observed in 3.2, 10 and 32 mg/L 3,5-Dichlorophenole treated groups, respectively. For the test substance treated groups, 8% (in 111.8 mg/L group) or less inhibition was observed. Therefore, the test substance EC20, EC50 and EC80 were all determined to be > 100 mg/L.

Description of key information

3-h EC50 > 100 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Bader 1990

Key value for chemical safety assessment

Additional information

One study is available for this endpoint, which was selected as key study. In this study performed according to OECD TG 209 and in compliance with GLP, activated sludge collected from sewage treatment plants was exposed to the test substance (no stock could be made due to low solubility) at concentrations of 0 (control),1.4, 6.0, 4.65, 14.4, 34.9 and 111.8 mg/L for 3 hours at 20 ± 2 °C. 3,5-Dichlorophenole was used as positive control and tested at concentrations of 3.2, 10 and 32 mg/L under the same experimental condition. The filled volume in the test bottle was adjusted to 200 mL with water and aerated for 3 hours. Oxygen consumption per hour was recorded over a 3-hour period. After 3 hours incubation, 15%, 35% and 74% of inhibition on oxygen consumption were observed in 3.2, 10 and 32 mg/L 3,5-Dichlorophenole treated groups, respectively. For the test substance treated groups, 8% (in 111.8 mg/L group) or less inhibition was observed. Therefore, the test substance EC20, EC50 and EC80 were all determined to be > 100 mg/L.

Metabolites - available information

In addition to the study with the test substance, two OECD TG 209 followed studies are available with degradation product M6 and M7. As the dossier has been prepared to address the test substance itself, these studies are not summarized as endpoint study records but are briefly discussed here. Both 3-h inhibition of total respiration studies (one with M6 and one with M7), performed in compliance with GLP conditions, showed EC50 > 100 mg/L (Weinstock 1993 and Bader 1988).