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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
other: experimental data on similar substance
Adequacy of study:
key study
Study period:
19 Oct 2015 to 20 Oct 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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:
- Origin of the test system: aeration tank of the wastewater treatment plant of Mannheim, Germany
- Collection of the test system: 19 Oct 2015
- Arrival in the test facility: 19 Oct 2015
- Preparation of inoculum for exposure: After arrival of the activated sludge suspension in the test facility the 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 Dw.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21.4 - 21.5 °C
pH:
Start: 7.4 - 7.5
After incubation time: 8.0 - 8.1
Dissolved oxygen:
5.6 - 8.8 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L
Details on test conditions:
TEST PREPARATION
The test substance was added in the required amounts according to the test concentrations directly to the test vessels. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water . Synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays, deionized water and synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary.

TEST SYSTEM
- Test vessel: glass-beakers (nominal volume 1L)
- Test volume: 500 mL
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
- Replicates: 6 for the control, 3 for each test substance concentration, 2 for each reference substance concentration
- Duration of measurement during oxygen consumption: about 8 - 10 minutes
- Sludge concentration in the test vessel: 1.5 g/L Dw

OTHER TEST CONDITIONS
- Adjustment of pH: adjustment of pH was not necessary

EFFECT PARAMETERS MEASURED:
- The oxygen consumption was measured over a period of 8 - 10 minutes. The 5.5 - 6.7 minutes from the plotted track within a linear range of the graph was used for the determination of the respiration rate (mg O2/L*h) and compared to a blank control.
- Other: The pH-values in all test vessels were measured at test start and after incubation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% C.L.: 252.6 - 407.0 mg/L
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
Details on results:
A dose-related inhibition of respiration by the test substance was observed, see ‘Any other information on results incl. tables’.
Results with reference substance (positive control):
The 3-h EC50 of the reference substance 3,5-dichlorophenol was determined to be 9.0 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 (Finney, D.J., Probit Analysis; Cambr. Univ. Press, 3rd ed., 1971) with the software TOXRAT Professional 2.10 (Ratte, M.: ToxRat Professional 2.10 (2001-2010). ToxRat Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany).

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

Assay identification

BC1

BC2

BC3

BC4

BC5

BC6

RS1

RS2

RS3

RS4

RS5

RS6

Reference substance

concentration;

[mg/L]

-

-

-

-

-

-

 

1

 

10

 

100

O2 conc. Start

[mg/L]

5.6

6.3

7.4

7.4

7.5

8.0

8.0

7.0

8.3

8.5

8.8

8.7

O2 conc. End

[mg/L]

1.7

2.5

3.6

3.4

3.5

4.3

4.5

3.5

6.6

6.6

8.4

8.2

O2 consumption

rate [mg/L]

3.9

3.8

3.8

4.0

4.0

3.7

3.5

3.5

1.7

1.9

0.4

0.5

O2 consumption in minutes

6.1

6.1

6.0

6.1

6.2

5.9

6.2

6.4

6.0

6.0

5.5

6.5

O2 consumption

rate [mg/L×h]

38

37

38

39

39

38

34

33

17

19

4

5

Specific O2 consumption

rate [mg/g×h]

25

25

25

26

26

25

23

22

11

13

3

3

Calculation of

inhibition

respiration [%]

 

-

11

14

55

50

90

87

 

Assay identification

TS1

TS2

TS3

TS4

TS5

TS6

TS7

TS8

TS9

TS10

TS11

TS12

TS13

TS14

TS15

Test substance

concentration;

[mg/L]

 

62.5

 

125

 

250

 

500

 

1000

O2 conc. Start

[mg/L]

6.5

7.4

7.0

6.2

6.6

7.5

7.1

6.3

7.0

7.7

7.5

6.7

6.7

7.7

7.5

O2 conc. after x minutes [mg/L]

2.5

3.5

3.0

2.5

2.8

3.7

3.3

2.5

3.3

4.7

4.3

3.5

4.1

5.1

4.8

O2 consumption in minutes

6.5

6.0

6.1

6.0

6.1

6.0

6.0

6.2

6.3

6.0

6.0

6.7

6.5

6.0

6.0

O2 consumption

rate [mg/L]

4.0

3.9

4.0

3.7

3.8

3.8

3.8

3.8

3.7

3.0

3.2

3.2

2.6

2.6

2.7

O2 consumption

rate [mg/L×h]

37

39

39

37

37

38

38

37

35

30

32

29

24

26

27

Specific O2

consumption

rate [mg/g×h]

25

26

26

25

25

25

25

25

23

20

21

19

16

17

18

Calculation of

inhibition

respiration [%]

3

-2

-2

3

3

0

0

3

8

21

16

24

37

32

29

BC = blank control, RS = reference substance, TS = test substance

 

Table: Obtained effect concentrations

Toxicity Response

Variable

Based on nominal

concentration

[mg/L]

Confidence Limits

(95%)

lower/upper

EC10

320

252.6/407.0

EC20

570

498.0/656.1

EC50

> 1000

-

EC80

> 1000

-

Validity criteria fulfilled:
yes
Conclusions:
Not toxic to microorganism
IC50 > 100 mg/l

Description of key information

The 3-h EC50 and EC10 values are > 1000 mg/L and 320 mg/L, respectively, as observed in a respiration inhibition study with activated sludge of a predominantly domestic sewage.

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 (Stahl, 2016). In this study, non-adapted activated sludge from a predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 100 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 33% at the highest concentration tested. Based on these findings, the 3-h EC50 and EC10 values were determined at > 1000 mg/L and 320 mg/L, respectively.

Based on the read across considerations same results apply to the target substance