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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 July 2012 - 4 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
TEST SUBSTANCE PREPARATION
Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
-Activated sludge from the municipal wastewater treatment plant of Mannheim, Germany was collected on 02 July 2012 from the aeration tank of the plant. The activated sludge suspension was sieved with a fine woven mesh (mesh size about 1 mm).
-This suspension was pre-aerated over night at room temperature. The next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dry matter.
-An aliquot of this suspension was added to the test vessels to obtain a sludge concentration of 1.5 g/L dry substance.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.8 - 21.1 °C
pH:
7.1 - 7.2
Dissolved oxygen:
Oxygen concentration during aeration: >2 mg/L
Oxygen concentration immediately before measurement: >7 mg/L
Nominal and measured concentrations:
62.5, 125, 250, 500 and 1000 mg/L as nominal concentration based on test substance without correction of purity or composition.
Details on test conditions:
TEST GROUPS
-4 replicates for the control.
-2 replicates for each test substance concentration.
-2 replicates for each reference substance concentration.

TEST CONDITIONS
Test vessels: glass beakers (nominal volume 1 L)
Test volume: 500 mL
Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
Duration of the measurement of oxygen consumption: 8 - 10 min
Sludge concentration in the test vessel: 5 g/L

EXPERIMENTAL PROCEDURE
Preparation of the test
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with about 234 mL deionized water. Aliquots of reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. Finally, 16 mL synthetic medium was dosed to each test vessel with test substance and reference substance. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary.
After addition of 250 mL of inoculum suspension, the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test or reference substance.
After 3 hours incubation, the mixtures in the test vessels were placed subsequently for oxygen measuring.
The oxygen uptake was measured for a period of 8 to 10 minutes or until the oxygen concentration fell below 2 mg/L.

EVALUATION
The consumption rate (mg O2/L x 6 minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L x h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.

STATISTICS
The consumption rates were used for the determination of the ECx by the probit method based on Finney with the software TOXRAT Professional 2.10.

VALIDITY CRITERIA
1. The coefficient of variation of the oxygen consumption of the blank controls should be ≤ 30 % O2 consumption
2. The EC50 of 3,5-dichlorophenol should be in the range of 2 - 25 mg/L
3. The mean oxygen uptake of the blank controls should be not less than 20 mg/g (h).
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The following effect concentrations (mg/L) were obtained:

Toxicity Response Based on nominal
Variable effect concentration (mg/L)
EC10 > 1000
EC20 > 1000
EC50 > 1000
EC80 > 1000

No monotone concentration response exists. Therefore the LCx calculation is not meaningful and the EC10 value is assumed to be greater than 1000 mg/L.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 11.9 mg/L (in the required range of 2 - 25 mg/L in 3 hours).
These results indicate that the microorganisms responded normally to toxicant stress.

Table 1 Measurement of the Oxygen Consumption

Assay Identification

BC1

BC2

BC3

BC4

RS1

RS2

RS3

RS4

RS5

RS6

TS1

TS2

TS3

TS4

TS5

TS6

TS7

TS8

TS9

TS10

O2 conc. Start (mg/L)

7.4

7.5

7.5

7.4

7.7

7.7

8.1

8.1

8.7

8.7

8.0

8.0

7.0

7.1

7.6

7.6

8.1

8.1

8.0

8.0

O2 conc. End (mg/L)

5.5

5.5

5.5

5.5

6.0

6.0

7.0

7.0

8.5

8.5

6.0

6.0

5.0

5.0

5.5

5.5

6.0

6.0

6.0

6.0

O2 Consumption Rate (mg/L)

1.9

2.0

2.0

1.9

1.7

1.7

1.1

1.1

0.2

0.2

2.0

2.0

2.0

2.1

2.1

2.1

2.1

2.1

2.0

2.0

O2 Consumption in Minutes

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

6.0

O2 Consumption Rate (mg/L x h), RT

19

20

20

19

17

17

11

11

2

2

20

20

20

21

21

21

21

21

20

20

Specific O2 Consumption Rate (mg/g x h)

13

13

13

13

11

11

7

7

1

1

13

13

13

14

14

14

14

14

13

13

Calc. of Inhibition of Respiration (%), IT

-

-

-

-

13

13

44

44

90

90

-3

-3

-3

-8

-8

-8

-8

-8

-3

-3

BC 1 - 4 = Blank Controls

RS 1 - 2 = Reference Substance concentration 1 mg/L

RS 3 - 4 = Reference Substance concentration 10 mg/L

RS 5 - 6 = Reference Substance concentration 100 mg/L

TS 1 - 2 = Test Substance concentration 62.5 mg/L

TS 3 - 4 = Test Substance concentration 125 mg/L

TS 5 - 6 = Test Substance concentration 250 mg/L

TS 7 - 8 = Test Substance concentration 500 mg/L

TS 9 - 10 = Test Substance concentration 1000 mg/L

The respiration rate was calculated in mg O2/L x h. The oxygen consumption was measured over a period of 8 - 10 minutes. A suitable period from the plotted track within a linear range of the graph was used for the determination of the respiration rate.

 

IT= (1 - (RT/ RTB) x 100

IT= inhibition of the total respiration (%)

RT= oxygen consumption in the test assay (mg O2/L x h)

RTB= mean value oxygen consumption of all blank controls (mg O2/L x h)

Validity criteria fulfilled:
yes
Conclusions:
In an activated sludge respiration inhibition test, the EC50 was determined to be >1000 mg/L and the NOEC was determined to be = 1000 mg/L.
Executive summary:

The potential of the test material to inhibit the respiration rate of microbial inoculum was investigated in accordance with the standardised guidelines OECD 209 and EU Method C.11.

During the study, activated sludge from a municipal wastewater treatment plant was exposed to the test material for 3 hours at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg/L. The respiration rate was calculated from the oxygen consumption and under the conditions of the study the EC50 value was determined to be >1000 mg/L and the NOEC was determined to be = 1000 mg/L.

Description of key information

In an activated sludge respiration inhibition test, the EC50 was determined to be > 1 000 mg/L and the NOEC was determined to be = 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The potential of the test material to inhibit the respiration rate of microbial inoculum was investigated in accordance with the standardised guidelines OECD 209 and EU Method C.11. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, activated sludge from a municipal wastewater treatment plant was exposed to the test material for 3 hours at nominal concentrations of 62.5, 125, 250, 500 and 1 000 mg/L. The respiration rate was calculated from the oxygen consumption and under the conditions of the study the EC50 value was determined to be > 1 000 mg/L and the NOEC was determined to be = 1000 mg/L.

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