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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Apr 2004 to 22 Apr 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1993
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1987
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
1986 (Method B)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Appearance: White, powdered solid
- Water-solubility: 29 mg/L
- Homogeneity: homogeneous
- Storage conditions: at room temperature
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was added in the required amount to the test vessel with about 180 mL deionized water. The mixture was stirred for 22 ± 2 hours to obtain an optimal solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from laboratory wastewater plant treating municipal sewage.
- Preparation of inoculum for exposure: 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 the desired dry substance concentration.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.0 - 7.7
Dissolved oxygen:
6.7 - 8.8
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 100, 500 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer-vessel (nominal volume 250 mL)
- Test volume: 250 mL
- Replicates: 3 for the control, 1 for each test concentration, 1 for each reference substance concentration
- Synthetic medium: 8 ml/vessel 100-fold concentrated OECD medium
- Duration of measurement during oxygen consumption: 8 - 10 minutes
- Sludge concentration in the test vessel: 1 g/L Dw

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted after addition of synthetic medium, before the inoculum was added and the test started

EFFECT PARAMETERS MEASURED:
- The oxygen concentration (mg O2/L) was measured at the test start and after 3 hours. The duration of the measurement of oxygen consumption was 8 - 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 750 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
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:
Inhibition of the total respiration was observed at all test concentrations, starting from 12% inhibition at 100 mg/L and increasing to 12% at 500 mg/L and 23% inhibition at 1000 mg/L, respectively. Effect concentrations exceeding solubility of substance in test medium: EC20, EC50 and EC80 were all above the water solubility of the test substance (29 mg/L).
Results with reference substance (positive control):
The 3-h EC50 of the reference substance was determined to be about 7 mg/L.
Reported statistics and error estimates:
The nominal concentrations which indicate a respiration inhibition of 20, 50 and 80 % were taken from the graph of the inhibition curve.

Table: measured data on oxygen consumption in the test vessels

 

Vessel no.:

 

BC1

 

BC2

 

BC3

mv

BC

 

TS1

 

TS2

 

TS3

 

RS1

 

RS2

 

RS3

Concentration of test substance (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

50

50

50

50

50

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

 

250

 

250

 

250

 

-

 

250

 

250

 

250

 

250

 

250

 

250

Oxygen concentration start (mg O2/L)

7.3

7.3

6.7

-

6.7

7.0

7.1

6.9

8.6

8.8

Oxygen concentration after 6 min (mg O2/L)

4.6

4.6

4.2

-

4.7

4.8

4.8

4.5

7.5

8.5

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

2.7

2.7

2.5

2.6

2.0

2.2

2.3

2.4

1.1

0.3

Oxygen consumption rate (mg O2/L*h)

27

27

25

26

20

22

23

24

11

3

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

27

27

25

26

20

22

23

24

11

3

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

-

-

-

-

23

15

12

8

58

88

 BC = blank control, mv = mean value, TS = test substance, RS = reference substance

ADDITIONAL EFFECT VALUES

- 3-h EC20: ca. 750 mg/L (nominal)

- 3-h EC80: > 1000 mg/L (nominal)

Validity criteria fulfilled:
yes
Conclusions:
The inhibition of the degradation activity of activated sludge is not anticipated when introduced to biological treatment plants in appropriate low concentrations.

Description of key information

The inhibition of the degradation activity of activated sludge is not anticipated when introduced to biological treatment plants in appropriate low concentrations.

Key value for chemical safety assessment

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria. In this study non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 100, 500 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. Inhibition of respiration rates increased concentration-dependently reaching 23% at the highest concentration tested. Based on these findings, the 3-h EC50 and EC20 values were determined at >1000 mg/L and ca. 750 mg/L, respectively.