Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 APR 2005 to 28 APR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guideline and compliant to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Reference substance copper (II) sulphate pentahydrate instead of the prescribed 3,5-dichlorophenol. Test performed as limit test (one treatment replicate)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German Chemikaliengesetzt and Directive 88/320/EEC
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test concentrations 1000 mg/L
Pretreatment The test item was weighed out directly into an Erlenmeyer flask, filled with 250 mL autoclaved tap water and stirred for 24 h.
Dispersion treatment Agitation
CONTROL 2 control samples without test item were included, one at the beginning and one at the end of the test.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Non adapted activated sludge from the municipal sewage treatment plant of D-31157 Sarstedt
The sludge was washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L ± 10 %.
Date of receipt: 2005-04-27
Dry sludge concentration: 4.12 g/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
21 °C
pH:
6.95 for the activated sludge
Nominal and measured concentrations:
Treatment: 1000 mg/L nominal concentration
Control (1 and 2): 0 mg/L
Details on test conditions:
REFERENCE ITEM Copper (II) sulphate pentahydrate p.a. MERCK
CAS RN 7758-99-8
Lot A265990
Purity 99.9 %
Expiry date 2005-11-30
Test concentrations 58 - 100 - 180 mg/L
Range of validity (EC50) 53 - 155 mg/L

TEST METHOD Static
Test duration 3 h
Replicates One per concentration and two per control
Test vessels 500 mL Erlenmeyer flasks, DIN 12380
Volume of the study medium 500 mL

Nutrient solution Synthetic waste water according to OECD 209

Application:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Ventilation Permanent

The pH-value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The oxygen depletion of the control, reference and test item was measured after 3 h incubation by means of oximeter and recorded for at least 3 minutes on a flat bed recorder.
Room temperature: continuously recorded by a hgrothermograph

A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L.
Based on the results of the preliminary test the study was carried out with a limit test concentration of 1000 mg/L.
2 control replicates without test item were included.
A fresh sample of activated sludge taken from the sewage plant Sarstedt was used for the test procedure. It was washed twice and diluted with autoclaved tap water to a dry sludge concentration of 4.0 g/L +/- 10 %.
The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8. The respiration rates of all samples were determined subsequently to an incubation period of 3 h.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate, CAS RN 7758-99-8
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
Details on results:
After stirring for 24 h the test item was mostly dispersed. Only part of the test item stuck to the side of the Erlenmeyer flask.
For details see "Any other information on results incl. tables"!
Results with reference substance (positive control):
In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 107 mg/L
This is in the recommended confidence range of 53 - 155 mg/L.
Reported statistics and error estimates:
The EC50-value of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows) rel. 8.02, 2002, SPSS CORPORATION. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

Table 1: Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations:

 

Concentration
[mg/L]

Respiration rate
[mgO
2/L x h]

Inhibition
[%]

Test item

1000

30.8

5

Control 1

-

33.2

-

Control 2

-

31.6

-

Reference item

180

8.4

74

 

100

16.8

48

 

58

25.6

21

Difference of the control respiration rates: 5 %

Validity criteria fulfilled:
yes
Conclusions:
In a respiration inhibition test with acitvated municipial sludge (OECD 209) the following effect concentration was determined for the submission substance:
EC50 (3h) > 1000 mg/L (nominal concentration)
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal limit concentration 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. No inhibition was observed at the tested limit concentration.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity was determined. The EC50- value for the reference item was 107 mg/L. Validity criteria of the test guideline were fulfilled.

The test item is not toxic at concentrations <= 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-29 to 2011-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC
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: None, limit concentration of 1000 mg/L
- Controls: 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 sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %
- Dry sludge concentration: 3.14 g/L (corresponding to 1.57 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
21.4 °C
pH:
7.31 (activated sludge)
7.42 (synthetic waste water)
Salinity:
None, freshwater
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): 6
- 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) :
Determination of respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS

- Range finding study

Inhibition of Respiration (Preliminary Tests)
Nominal Test Item Concentration
[mg/L] Inhibition[%]
10 -4
100 -1
1000 -2





Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
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: 110 mg/L (95 % CI = 105 - 117 mg/L)
Reported statistics and error estimates:
The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods

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

19.6

12.5

-

-

2

18.8

12.0

3

18.0

11.5

4

18.8

12.0

5

18.0

11.5

6

18.0

11.5

Reference Item
58

1

15.6

16

19

2

15.6

16

3

14.0

24

100

1

10.4

44

47

2

9.2

50

3

10.0

46

180

1

4.8

74

73

2

5.2

72

3

5.2

72

Test Item
1000

1

17.2

7

6

2

17.6

5

3

17.2

7

EC-Values with Confidence Interval for the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item is > 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The inhibition of respiration for the test item replicates ranged from 5 to 7 % .

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 110 mg/L.

Validity criteria of the test guideline were fulfilled.

Table 1:           EC-Values with Confidence Interval for the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

                                                         n.d. = not determinable

The NOEC of the test item is 1000 mg/L.

The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

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:
2011-08-29 to 2011-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC
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: None, limit concentration of 1000 mg/L
- Controls: 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 sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %
- Dry sludge concentration: 3.14 g/L (corresponding to 1.57 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
21.4 °C
pH:
7.31 (activated sludge)
7.42 (synthetic waste water)
Salinity:
None, freshwater
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): 6
- 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) :
Determination of respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS

- Range finding study

Inhibition of Respiration (Preliminary Tests)
Nominal Test Item Concentration
[mg/L] Inhibition[%]
10 -4
100 -1
1000 -2





Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
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: 110 mg/L (95 % CI = 105 - 117 mg/L)
Reported statistics and error estimates:
The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods

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

19.6

12.5

-

-

2

18.8

12.0

3

18.0

11.5

4

18.8

12.0

5

18.0

11.5

6

18.0

11.5

Reference Item
58

1

15.6

16

19

2

15.6

16

3

14.0

24

100

1

10.4

44

47

2

9.2

50

3

10.0

46

180

1

4.8

74

73

2

5.2

72

3

5.2

72

Test Item
1000

1

17.2

7

6

2

17.6

5

3

17.2

7

EC-Values with Confidence Interval for the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item is > 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The inhibition of respiration for the test item replicates ranged from 5 to 7 % .

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 110 mg/L.

Validity criteria of the test guideline were fulfilled.

Table 1:           EC-Values with Confidence Interval for the test item

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

1000

-

EC20

> 1000

-

EC50

> 1000

n.d.

EC80

> 1000

-

                                                         n.d. = not determinable

The NOEC of the test item is 1000 mg/L.

The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

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:
From 27 APR 2005 to 28 APR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guideline and compliant to GLP
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (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
Remarks:
Reference substance copper (II) sulphate pentahydrate instead of the prescribed 3,5-dichlorophenol. Test performed as limit test (one treatment replicate)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to German Chemikaliengesetzt and Directive 88/320/EEC
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test concentrations 1000 mg/L
Pretreatment The test item was weighed out directly into an Erlenmeyer flask, filled with 250 mL autoclaved tap water and stirred for 24 h.
Dispersion treatment Agitation
CONTROL 2 control samples without test item were included, one at the beginning and one at the end of the test.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Non adapted activated sludge from the municipal sewage treatment plant of D-31157 Sarstedt
The sludge was washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L ± 10 %.
Date of receipt: 2005-04-27
Dry sludge concentration: 4.12 g/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
21 °C
pH:
6.95 for the activated sludge
Nominal and measured concentrations:
Treatment: 1000 mg/L nominal concentration
Control (1 and 2): 0 mg/L
Details on test conditions:
REFERENCE ITEM Copper (II) sulphate pentahydrate p.a. MERCK
CAS RN 7758-99-8
Lot A265990
Purity 99.9 %
Expiry date 2005-11-30
Test concentrations 58 - 100 - 180 mg/L
Range of validity (EC50) 53 - 155 mg/L

TEST METHOD Static
Test duration 3 h
Replicates One per concentration and two per control
Test vessels 500 mL Erlenmeyer flasks, DIN 12380
Volume of the study medium 500 mL

Nutrient solution Synthetic waste water according to OECD 209

Application:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Ventilation Permanent

The pH-value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The oxygen depletion of the control, reference and test item was measured after 3 h incubation by means of oximeter and recorded for at least 3 minutes on a flat bed recorder.
Room temperature: continuously recorded by a hgrothermograph

A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L.
Based on the results of the preliminary test the study was carried out with a limit test concentration of 1000 mg/L.
2 control replicates without test item were included.
A fresh sample of activated sludge taken from the sewage plant Sarstedt was used for the test procedure. It was washed twice and diluted with autoclaved tap water to a dry sludge concentration of 4.0 g/L +/- 10 %.
The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8. The respiration rates of all samples were determined subsequently to an incubation period of 3 h.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate, CAS RN 7758-99-8
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
Details on results:
After stirring for 24 h the test item was mostly dispersed. Only part of the test item stuck to the side of the Erlenmeyer flask.
For details see "Any other information on results incl. tables"!
Results with reference substance (positive control):
In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 107 mg/L
This is in the recommended confidence range of 53 - 155 mg/L.
Reported statistics and error estimates:
The EC50-value of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows) rel. 8.02, 2002, SPSS CORPORATION. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

Table 1: Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations:

 

Concentration
[mg/L]

Respiration rate
[mgO
2/L x h]

Inhibition
[%]

Test item

1000

30.8

5

Control 1

-

33.2

-

Control 2

-

31.6

-

Reference item

180

8.4

74

 

100

16.8

48

 

58

25.6

21

Difference of the control respiration rates: 5 %

Validity criteria fulfilled:
yes
Conclusions:
In a respiration inhibition test with acitvated municipial sludge (OECD 209) the following effect concentration was determined for the submission substance:
EC50 (3h) > 1000 mg/L (nominal concentration)
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal limit concentration 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. No inhibition was observed at the tested limit concentration.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity was determined. The EC50- value for the reference item was 107 mg/L. Validity criteria of the test guideline were fulfilled.

The test item is not toxic at concentrations <= 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Description of key information

In a respiration inhibition test (PY154) with activated municipal sludge (OECD 209) the following effect concentration was determined for the submission substance:

EC50 (3h) > 1000 mg/L (nominal concentration)

The NOEC of the test item (PY120) is>1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Key value for chemical safety assessment

Additional information