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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of Experiment: October 23, 1996 - Completion of Experiments: November 01, 1996
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:
adopted April 04, 1984
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SAMPLING AND ANALYSIS:
After 30 minutes of aeration at 21.5 °C, the first sample was taken for oxygen measurement over a period of about 10 minutes. Thereafter, in about 10-minute intervals the rest of the samples was submitted to measurement of oxygen concentration. The starting oxygen concentration was > 6.5 mg O2/l for all flasks.
Sampling was started with the control flask 1. Thereafter, the measurements were continued with the reference compound (flasks 2 - 6), control flask 7 (additional control), and the test article samples (flasks 8 - 12). Finally, the oxygen consumption of control flask 13 was determined.
Vehicle:
no
Details on test solutions:
Please refer to "Details on test conditions" and tables in "Any other information in MM"
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The test was performed with aerobic activated sludge from a domestic waste water treatment plant (ARA Ergolz II, Fullinsdorf /Switzerland).
- Preparation of sludge:
The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre, were made up with Soerensen buffer (pH 7). To this mixture, 50 ml OECD recommended synthetic sewage feed per litre was added daily, starting three days prior to use, and the sludge was aerated until use. Before use, the pH of the activated sludge was adjusted to 7.8 with 0.5 mol/l H2S04.
Synthetic sewage feed: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 filled up to a final volume of 1 l with bidistilled water.
Test type:
static
Water media type:
other: bidistilled water
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Not specified.
Test temperature:
The test was performed at 21.5 °c.
pH:
Before use, the pH of the activated sludge was adjusted to 7.8 with 0.5 mol/l H2S04.
Dissolved oxygen:
Not specified.
Salinity:
Not specified.
Conductivity:
Not specified.
Nominal and measured concentrations:
The study examined nominal test article concentrations in the range from 3.2 mg/I to 100 mg/I.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL glass flasks
- Aeration: The test was carried out in glass flasks (500 ml) aerated with an air-flow of about 0.2 I/minute instead of 0.5 I/minute to prevent possible foam development
- Sludge concentration (weight of dry solids per volume): please refer to "Details on inoculum".

TEST CONCENTRATIONS
- Test concentrations: from 3.2 to 100 mg/L

For details on filling the flasks, please refer to section "Any other information on materails and methods incl. tables"
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol used as a reference compound.
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Key result
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
The respiration rate of aerobic waste water microorganisms was practically not inhibited (-1.8 % to 2.1 %), when exposed to the test article concentrations in the range from 3.2 to 100 mg/I. Based on this the EC50 and EC20 (30 minutes) can be defined as
>100 mg/I.
Results with reference substance (positive control):
The respiration rate of the activated sludge was only slightly inhibited (3.3 % and 5.3 %) at the two lowest concentrations of 1.0 and 3.2 mg/I. At 10, 32 and 50 mg/I, the respiration rate was inhibited by 11.5, 66.7 and 73.4 %, respectively.
The EC50 (30 minutes) determined by the Logit model (Ref. 1) was calculated to be 23.7 mg/I with the 95 % confidence limits of 23.1 - 24.4 mg/I.
The dose response line was: Logit = -4.37 + 1.380 x log (dose). The 95 % confidence limits for the slope were 1.379 - 1.380.

Flasks were filled up to 500 ml final volume according to the scheme given below:

Flask

Identification

Synth.

sewage

feed

(ml)

Activ.

sludge1

 (ml)

Stock solution

Water

 

 

(ml)

Final

conc.

 

(mg/l)

I*

 (ml)

II**

 (ml)

1

Control

16

200

-

-

284

-

2

3,5-dichlorophenol

16

200

-

50

234

50

3

3,5-dichlorophenol

16

200

-

32

252

32

4

3,5-dichlorophenol

16

200

-

10

274

10

5

3,5-dichlorophenol

16

200

-

-3.2

280.8

3.2

6

3,5-dichlorophenol

16

200

-

1.0

283

1.0

7

Control

16

200

-

-

284

-

8

SCARLET RN 1165

16

200

100

 

184

100

9

SCARLET RN 1165

16

200

50

 

234

50

10

SCARLET RN 1165

16

200

32

 

252

32

11

SCARLET RN 1165

16

200

10

 

274

10

12

SCARLET RN 1165

16

200

3.2

 

280.8

3.2

13

Control

16

200

-

-

284

-

Influence of SCARLET RN 1165 on oxygen consumption of activated sludge.

Flask No.

Chemical

Concentration of test article (mg/l)

Oxygen consumption (mg O2/l min.)

Inhibition (%)

Starting pH

1

Control

 

0.391

 

7.9

13

Control

 

0.420

 

8.1

Mean

Deviation (%)

0.406

 

 

7.4

 

 

8

SCARLET RN 1165

100

0.407

-0.4

8.0

9

SCARLET RN 1165

50

0.397

2.1

8.1

10

SCARLET RN 1165

32

0.407

-0.4

8.1

11

SCARLET RN 1165

10

0.410

-1.1

8.1

12

SCARLET RN 1165

3.2

0.413

-1.8

8.1

Influence of 3,5-dichlorophenol on oxygen consumption of activated sludge.

Flask No.

Chemical

Concentration of test article (mg/l)

Oxygen consumption (mg O2/l min.)

Inhibition (%)

Starting pH

1

Control

 

0.391

 

7.9

13

Control

 

0.420

 

8.1

Mean

Deviation (%)

0.406

 

 

7.4

 

 

8

3,5-dichlorophenol

50

0.108

73.41

7.9

9

3,5-dichlorophenol

32

0.135

66.71

7.9

10

3,5-dichlorophenol

10

0.359

11.51

7.9

11

3,5-dichlorophenol

3.2

0.384

5.31

8.0

12

3,5-dichlorophenol

1

0.392

3.31

8.0

1 Used for Logit estimation

Validity criteria fulfilled:
yes
Conclusions:
When SCARLET RN 1165 was assessed for its toxicity to waste water bacteria, the following result was obtained:
EC50 (30 minutes): >100 mg/I
EC20 (30 minutes): >100 mg/I
Executive summary:

The inhibitory effect of SCARLET RN 1165 on aerobic waste water microorganisms of activated sludge was investigated in a respiration test.

This study examined nominal test article concentrations in the range from 3.2 mg/l to 100 mg/l.

The respiration rate of aerobic waste water microorganisms was practically not inhibited (-1.8 to 2.1 %) when exposed to the test article concentrations in the range from 3.2 to 100 mg/I. The EC50 and EC20 (30 minutes) were therefore determined to be >100 mg/l.

The EC50 (30 minutes) for the reference compound 3,5-dichlorophenol, which was tested under the same conditions as the test article, was found to be at 23.7 mg/l and therefore lies within the range of 5 - 30 mg/l recommended by the test guidelines.

Description of key information

When SCARLET RN 1165 was assessed for its toxicity to waste water bacteria, the following result was obtained:

EC50 (30 minutes) >100 mg/I

EC20 (30 minutes) >100 mg/I

Key value for chemical safety assessment

Additional information