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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2004 to 06 May 2004
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:
1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
1986
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Activated Sludge Respiration Inhibition Test, Annex to EEC Directive 88/302/EEC
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test material was added in the required amount to the test vessel with about 180 mL deionised water. The mixture was stirred for 22 ± 2 hours to obtain an optimal solution.
Test organisms (species):
activated sludge
Details on inoculum:
- Activated sludge was from the laboratory of a wastewater plant treating municipal sewage.
- The inoculum was washed, brought to a concentration of 5 g/L dry substance and aerated during the night.
- 50 mL were added to a total volume of 250 mL to obtain a concentration of 1 g/L dry substance in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Test temperature:
20 ± 2 °C
pH:
7.2 - 8.1 (after correction)
Nominal and measured concentrations:
Nominal: 100, 500 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer-vessel (nominal volume 250 mL)
- Fill volume: 250 mL
- Oxygen concentration during aeration: > 2.5 mg/L
- Oxygen concentration immediately before measurement: > 6.5 mg/L
- Duration of the measurement of oxygen consumption: 8 - 10 min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 3
- No. of vessels per reference material concentration (replicates): 1
- Sludge concentration: 1.0 g/L
- Synthetic medium: 8 mL/vessel, 100-fold concentrated OECD medium

OTHER TEST CONDITIONS
- Adjustment of pH: yes

EFFECT PARAMETERS MEASURED: Oxygen consumption

TEST CONCENTRATIONS
- 100, 500 and 1000 mg/L

VALIDITY CRITERIA
- Deviation of blank controls should be < 15%
- EC50 of the reference material (3,5-dichlorophenol) should be in the range of 5 - 30 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
180 min
Dose descriptor:
other: EC20
Effect conc.:
ca. 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
180 min
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
VALIDITY CRITERIA
- Deviation of blank controls was < 15%
- EC50 of the reference material (3,5-dichlorophenol) was in the range of 5 - 30 mg/L
- The test is therefore valid
Results with reference substance (positive control):
- After 180 minutes incubation the following results were recorded:
- EC20: < 1 mg/L
- EC50: About 5.2 mg/L
- EC 80: About 43 mg/L

Table 1: Summary of Results

 

Blank control

Test material

Reference material

1

2

3

Mean

1

2

3

1

2

3

Concentration (mg/L)

-

-

-

-

1000

500

100

1

10

100

Added volume of stock solution (mL/vessel)

-

-

-

-

-

-

-

0.5

5.0

50.0

Direct addition (mg/vessel)

-

-

-

-

250.0

125.0

25.0

-

-

-

Synthetic medium (mL/vessel)

8

8

8

-

8

8

8

8

8

8

Inoculum (mL/vessel)

50

50

50

-

50

20

50

50

50

50

Filled up to the total volume with deionised water (mL/vessel)

250

250

250

-

250

250

250

250

250

250

Oxygen concentration start (mg O2/L)

6.9

7.0

6.8

-

7.4

7.5

7.3

7.0

8.5

8.8

Oxygen concentration after 6 min (mg O2/L)

4.3

4.6

4.3

-

5.4

5.4

4.9

5.1

7.5

8.6

Oxygen consumption rate (mg O2/L*6 min)

2.6

2.4

2.5

2.5

2.0

2.1

2.4

1.9

1.0

0.2

Oxygen consumption rate (mg O2/L*h)

26

24

25

25

20

21

24

19

10

2

Specific oxygen consumption rate (mg O2/g*h)

26

24

25

25

20

21

24

19

10

2

Change in oxygen consumption rate compared with the blank control (%)

-

-

-

-

20

16

4

24

60

92

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study the EC50 of the test material was > 1000 mg/L.
Executive summary:

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guidelines OECD 209 and ISO 8192-1986, under GLP conditions.

The determination of the Inhibition of Oxygen Consumption by Activated Sludge was assessed in the Activated Sludge Respiration Inhibition Test. Activated sludge from laboratory wastewater plant treating municipal sewage was used at a concentration of 1 g/L.

The inoculum and the test material (at 100, 500 and 1000 mg/L), or reference material (3,5-dichlorophenol at 1, 10 or 100 mg/L)) were incubated for 180 minutes at 20 ± 2°C and oxygen consumption was measured.

Under the conditions of this study the EC50 of the test material was > 1000 mg/L. All of the validity criteria of the study were met.

Description of key information

Under the conditions of the study the EC50 of the test material was determined to be > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The toxicity of the test material to microorganisms was investigated in accordance with the standardised guidelines OECD 209 and ISO 8192-1986, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The determination of the Inhibition of Oxygen Consumption by Activated Sludge was assessed in the Activated Sludge Respiration Inhibition Test. Activated sludge from laboratory wastewater plant treating municipal sewage was used at a concentration of 1 g/L.

The inoculum and the test material (at 100, 500 and 1000 mg/L), or reference material (3,5-dichlorophenol at 1, 10 or 100 mg/L)) were incubated for 180 minutes at 20 ± 2°C and oxygen consumption was measured.

Under the conditions of this study the EC50 of the test material was > 1000 mg/L. All of the validity criteria of the study were met.