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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:
31 Jan 2013
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:
2010
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): DERIPHAT 160 C
- Physical state: Solid/ white (flakes)
- Batch No.: 4986V1
- Expiration date of the lot/batch: 03 Jun 2014
- Homogeneity: Homogeneous
- Water solubility: Miscible
- Storage conditions: Ambient at room temperature
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
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, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from the municipal wastewater treatment plant of Mannheim, Germany was collected on 30 Jan 2013 from the aeration tank of the plant.
- Preparation of inoculum for exposure: 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. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dry matter.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.9 - 21.2 °C
pH:
- At test start: 7.1 - 7.2
- At test end: 7.9 - 8.1
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L (based on test substance without correction of purity or composition).
- Nominal concentrations based on active ingredient: 0 (control), 18.7575, 37.5, 75, 150 and 300 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessels: Glass-beakers (nominal volume 1L)
- Test volume: 500 mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- No. of vessels per reference substance concentrations: 2
- Sludge concentration in the test: 1.5 g/L

TEST MEDIUM
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
- Oxygen concentration during aeration: > 2 mg/L
- Oxygen concentration immediately before measurement: > 7 mg/L

TEST PROCEDURE
The test substance and controls were directly added to the vessels, after whicih the pH-values in all test vessels were checked. An adjustment was not necessary. After addition of the 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 content of oxygen at the start of the measurements was > 7 mg/L. The total oxygen consumption of were measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15 (BC = blank control; RS = reference substance; TS = test substance). The oxygen consumption of the blank control BC4+6 were measured at last. No abiotic control was tested.

EFFECT PARAMETERS MEASURED
The consumption rate (mg O2/L*x minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The oxygen uptake was measured for a period of 8 to 10 minutes or until the oxygen concentration fell below 2 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
171 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
Inhibition of the respiration rate was observed from 150 mg a.i./L (~39%) which increased up to ~90% inhibition at 300 mg a.i./L. See 'Any other information on results incl. tables' for an overview of the results.
Results with reference substance (positive control):
The 3-h EC50 value of 3,5-dichlorophenol was 7.1 mg/L.
Reported statistics and error estimates:
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.

Table: Measured data of oxygen content of the test assays at the start and end of the evaluation time

 

Blank control

Reference substance

62.5 mg/L;

18.75 mg a.i./L

125 mg/L;

37.5 mg a.i./L

250 mg/L;

75 mg a.i./L

500 mg/L;

150 mg a.i./L

1000 mg/L;

300 mg a.i./L

Assay identification

BC1

BC2

BC3

BC4

BC5

BC6

RS1

RS2

RS3

RS4

RS5

RS6

TS1

TS2

TS3

TS4

TS5

TS6

TS7

TS8

TS9

TS10

TS11

TS12

TS13

TS14

TS15

O2 conc. Start [mg/L]

7.6

7.7

7.1

7.6

7.4

7.3

6.6

7.8

8

8.3

9.1

8.9

7.2

6.5

7.7

7.2

7.4

4.4

5.5

7.6

8

5.4

6.4

7.4

8.2

7.8

8.2

O2 conc. End [mg/L]

5.5

5.5

5.4

5

5.5

5.3

4.7

6

7

7.6

8.9

8.8

4.5

5

5.8

4.5

4.5

2.6

3.4

4.5

4.5

4

5.4

6

8

7.6

8

O2 consumption rate [mg/L]

2.1

2.2

1.7

2.6

1.9

2

1.9

1.8

1

0.7

0.2

0.1

6

4

6

6

6

5

6

6

6

6

6

6

6

6

6

O2 consumption in minutes

6

6

6

6

6

6

6

6

6

6

6

6

2.7

1.5

1.9

2.7

2.9

1.8

2.1

3.1

3.5

1.4

1

1.4

0.2

0.2

0.2

O2 consumption rate (RT) [mg/L*h]

21

22

17

26

19

20

19

18

10

7

2

1

27

23

19

27

29

18

21

31

35

14

10

14

2

2

2

Specific O2 consumption rate [mg/g*h]

14

15

11

17

13

13

13

12

7

5

1

1

18

15

13

18

19

15

14

21

23

9

7

9

1

1

1

Calculation of inhibition respiration (IT) [%]

-

 

 

 

 

 

9

14

52

66

90

95

-30

-10

9

-30

-39

-6

-1

-49

-68

33

52

33

90

90

90

The respiration rate was calculated in mg O2/L*h.

Table: Summarised results of respiration rate decrease by the test substance after 3 h

Treatment (mg/L)

Treatment (mg a.i./L)

Mean

Std. Dev

n

% Decrease

0 (control)

0 (control)

20.833

3.0605

6

0

62.5

18.75

20.834

3.0606

3

-10.4

125

37.5

20.835

3.0607

3

-24.8

250

75

20.836

3.0608

3

-39.2

500

150

20.837

3.0609

3

39.2

1000

300

20.838

3.0610

3

90.4

ADDITIONAL EFFECT VALUES, not corrected for 30% active ingredient

- EC10: 330 mg/L (95% C.L.: 288.5 - 379.1 mg/L)

- EC20: 400 mg/L (95% C.L.: 359.9 - 439.2 mg/L)

- EC50: 570 mg/L (95% C.L.: 529.9 - 603.9 mg/L)

- EC80: 800 mg/L (95% C.L.: 717.6 - 901.9 mg/L)

Validity criteria fulfilled:
no
Remarks:
the study could still be regarded as valid however, see 'Any other information on materials and methods incl. tables'.

Description of key information

The 3-h EC50 and EC10 values are 171 and 100 mg a.i./L, respectively, as observed in a respiration inhibition study with activated sludge of domestic sewage.

Key value for chemical safety assessment

EC50 for microorganisms:
171 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

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 (BASF, 2013). In this study, non-adapted activated sludge from a domestic sewage was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours, corresponding to 0 (control), 18.75, 37.5, 75, 150 and 300 mg/L based on active ingredient. 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 the respiration rate was observed from 150 mg a.i./L (~39%) which increased up to ~90% inhibition at 300 mg a.i./L. Based on these results, the 3-h EC50 and EC10 values were determined at 171 mg a.i./L and 100 mg a.i./L, respectively.