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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-20 to 2012-02-21
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
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: No vehicle, directly weighing, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1000 mg/L (actual limit concentration in the final test medium)
- Controls: Test medium without test or reference substance. 6 replicates: Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the municipal sewage treatment plant at Hildesheim D-31137 is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: washed twice and adjusted with chlorine free tap water in order to achieve a dry sludge concentration of 3.0 g/L ± 10 %.
The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.68 g/L (corresponding to 1.34 g/L suspended solids in the test vessel)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
nominal 18 - 22 °C, actual measured 20 - 22 °C
pH:
7.40 (activated sludge)
7.50 (synthetic waste water)
Salinity:
None, freshwater
Nominal and measured concentrations:
Test concentrations nominal: control and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control. reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Range finding study

Inhibition of Respiration (Preliminary Tests)

Nominal Test Item Concentration Pretreatment Inhibition [%]
[mg/L]
10 Stirring overnight 4
100 -16
1000 9
1000 -1
1000 -4










Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 107 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculations. Before running the One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-value of the reference item was calculated by by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.

The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel, MICROSOFT CORPORATION.

EC-Values with Confidence Interval of the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
                   of Control, Reference and Test Item Concentrations

Reference and Test Item Concentration
[mg/L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Specific Respiration Rate
Rs
[mg O2/g
×h]


Inhibition

[%]

Mean Inhibition

[%]

Control

1

26.8

10.0

-

-

2

29.2

10.9

3

29.6

11.0

4

28.0

10.4

5

28.0

10.4

6

28.8

10.7

58

1

22.4

21

22

2

22.0

23

3

22.0

23

100

1

15.2

46

46

2

14.8

48

3

15.6

45

180

1

7.6

73

74

2

7.6

73

3

7.2

75

1000

1

32.0

-13

-15

2

33.2

-17

3

32.8

-15

Raw Data and Oxygen Uptake Rates of Test Item. Control and Reference Item

Test Solution / Concentration

[mg/L]

Repl.

Oxygen Concentration*
[mg O2/L]

Measured distance**
[mm/3 min]

Oxygen uptake
rate
[mg O2/L
×h]

Control

1

8.52

67

26.8

2

7.85

73

29.2

3

7.04

74

29.6

4

8.30

70

28.0

5

8.22

70

28.0

6

7.95

72

28.8

Reference Item

58

1

7.39

56

22.4

2

7.62

55

22.0

3

7.16

55

22.0

100

1

7.65

38

15.2

2

7.40

37

14.8

3

8.24

39

15.6

180

1

8.90

19

7.6

2

9.16

19

7.6

3

8.93

18

7.2

Test Item

1000

1

7.44

80

32.0

2

6.94

83

33.2

3

7.04

82

32.8

Validity criteria fulfilled:
yes
Conclusions:
The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-02-20 to 2012-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 242 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: No vehicle, directly weighing, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1000 mg/L (actual limit concentration in the final test medium)
- Controls: Test medium without test or reference substance. 6 replicates: Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the municipal sewage treatment plant at Hildesheim D-31137 is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: washed twice and adjusted with chlorine free tap water in order to achieve a dry sludge concentration of 3.0 g/L ± 10 %.
The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.68 g/L (corresponding to 1.34 g/L suspended solids in the test vessel)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
nominal 18 - 22 °C, actual measured 20 - 22 °C
pH:
7.40 (activated sludge)
7.50 (synthetic waste water)
Salinity:
None, freshwater
Nominal and measured concentrations:
Test concentrations nominal: control and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control. reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Range finding study

Inhibition of Respiration (Preliminary Tests)

Nominal Test Item Concentration Pretreatment Inhibition [%]
[mg/L]
10 Stirring overnight 4
100 -16
1000 9
1000 -1
1000 -4










Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 107 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculations. Before running the One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-value of the reference item was calculated by by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.

The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel, MICROSOFT CORPORATION.

EC-Values with Confidence Interval of the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
                   of Control, Reference and Test Item Concentrations

Reference and Test Item Concentration
[mg/L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Specific Respiration Rate
Rs
[mg O2/g
×h]


Inhibition

[%]

Mean Inhibition

[%]

Control

1

26.8

10.0

-

-

2

29.2

10.9

3

29.6

11.0

4

28.0

10.4

5

28.0

10.4

6

28.8

10.7

58

1

22.4

21

22

2

22.0

23

3

22.0

23

100

1

15.2

46

46

2

14.8

48

3

15.6

45

180

1

7.6

73

74

2

7.6

73

3

7.2

75

1000

1

32.0

-13

-15

2

33.2

-17

3

32.8

-15

Raw Data and Oxygen Uptake Rates of Test Item. Control and Reference Item

Test Solution / Concentration

[mg/L]

Repl.

Oxygen Concentration*
[mg O2/L]

Measured distance**
[mm/3 min]

Oxygen uptake
rate
[mg O2/L
×h]

Control

1

8.52

67

26.8

2

7.85

73

29.2

3

7.04

74

29.6

4

8.30

70

28.0

5

8.22

70

28.0

6

7.95

72

28.8

Reference Item

58

1

7.39

56

22.4

2

7.62

55

22.0

3

7.16

55

22.0

100

1

7.65

38

15.2

2

7.40

37

14.8

3

8.24

39

15.6

180

1

8.90

19

7.6

2

9.16

19

7.6

3

8.93

18

7.2

Test Item

1000

1

7.44

80

32.0

2

6.94

83

33.2

3

7.04

82

32.8

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

Description of key information

The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations.
NOEC (3h) >= 1000 mg/L (nominal) for activated sludge (OECD 209)

Key value for chemical safety assessment

Additional information

For two ‘red disazo condensation pigments’(CAS 68516-75-6 and CAS 71566-54-6) the influence on the respiration of microorganisms were investigated in GLP guideline studies according to OECD 209 [DR.U.NOACK-LABORATORIEN, 2012; Experimental Toxicology and Ecology, BASF SE, 2012]. Both studies conducted with activated sludge from municipal sewage treatment plants, which predominantly receives domestic sewage. Up to the highest test concentration of 1000 mg/L (nominal) no inhibition on respiration observed after 3 hours of exposure.