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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study with acceptable restrictions: No analytical purity is given, no analytical monitoring)
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substanz was emulsified using Ultra-Turrax.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge from sewage plant Bempflingen, Germany
- Preparation of inoculum for exposure: Activated sludge was washed with tap water twice, afterwards centrifuged and the dry residue was
determined. The amount of the sludge was weighed per liter, which was corresponded to 4 g dry mass.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not stated
Test temperature:
20 ± 2 °C
pH:
not stated
Dissolved oxygen:
> 6.5 mg/L
Nominal and measured concentrations:
Nominal: 10000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Material: glass; Size: 1 L; Fill volume: 500 mL
- Aeration: continously to reach an oxygen content > 6.5 mg/L
- No. of vessels per concentration: 1
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

EFFECT PARAMETERS MEASURED: Respiration inhibition by decrease of oxygen content was measured in 15-minutes-pulses over a test period of 3 h using an OXI-meter.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorphenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 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:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes, result is in the range of 5 to 30 mg/L
- Relevant effect levels: EC50 = 5 mg/L

An increase about 43% of respiration rate could be observed at the nominal tested concentration of 10000 mg/L in comparison to the control.

Table 1: Respiration rate of control, reference and test substance and inhibition of the respiration rate of test and reference substance (*A negative sign means an increase of the respiration)

 

 Replicates

Concentration [mg/L]

Respiration rate [mg O2/L x h]

Inhibition [%]

Control

1

 

24.0
25.0

-

Referenz

1
2
3

2.5

10

30

22.0

11.0

5.0

10.2

55.1

79.6

Test substance

1

10000

35.0

-43.0*

 *: negative value indicate an increase of respiration

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 - 29 Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health, UK
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Information provided by the Sponsor indicated that the test substance is not sufficiently soluble to prepare an aqueous solution. Therefore, at test inition weights were established by the addition of appropriate volumes (based on specific gravity, 0.99) of test substance to the test vessels containing RO water, synthetic sewage and microbial inoculum (see Table 1).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works, which treats predominantely domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. One day one of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried, preweighed Whatman GF/C filter paper, which was then dried again at ~ 105 °C for at least one hour, allowed to cool in a desiccator before being reweighed. The mixed liquor suspended solids content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge. The mixture was aerated overnight. The mixed liquor suspended solids content of the activated sludge was adjusted to 4 g/L by addition of tap water. Aliquots (200 mL) were added to the test vessels to give a final suspended solids concentration of 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
test start: 19.5 - 19.6 °C
test end: 19.3 - 19.4 °C
pH:
test start: 7.3 - 7.4
test end: 7.6 - 7.9
Nominal and measured concentrations:
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type: closed (loosely covered with aluminium foil)
- Aeration: using a glass aerator connected to a laboratory supply of oil-free compressed air (~ 1 L/min.)
- No. of vessels per concentration: 1 (10, 100 mg/L); 3 (1000 mg/L)
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water
- Culture medium different from test medium: no, according to OECD guideline 209
- Intervals of water quality measurement: The pH and temperature were measured at the start and end of the test.

EFFECT PARAMETERS MEASURED: The respiration rate was measured after a test period of 3 h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
EC10
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:
The test substance showed no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Respiration rates of mixtures containing the test substance showed an increase in respiration rate above that of the controls.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3h) = 6.4 mg/L (4.1 - 9.6 mg/L)

Table 1: Preparation of test solutions

Treatment

Test substance [µL] or reference substance [mL]

Synthetic sewage [mL]

RO water [mL]

Microbial Inoculum [mL]

Control 1

0

16

284

200

Control 2

0

16

284

200

Test substance [mg/L]

 

 

 

 

10

5.1

16

284

200

100

50

16

284

200

1000

505

16

284

200

Reference substance [mg/L]

 

 

 

 

3

3

16

281

200

10

10

16

274

200

32

32

16

252

200

Table 2: Dissolved oxygen concentration and measurement times

Treatment

Initial dissolved oxygen conc. in culture [mg O2/L]

Initial measured dissolved oxygen conc. [mg O2/L]

Final measured dissolved oxygen conc. [mg O2/L]

Measurement time [min]

Control 1

6.9

6.5

2.5

8.5

Control 2

6.9

6.5

2.5

9.0

Test substance [mg/L]

 

 

 

 

10

4.5

4.2

1.5

5.3

100

5.0

4.8

1.5

6.3

1000

5.8

5.5

2.5

5.7

1000

4.9

4.6

1.5

5.8

1000

5.5

4.9

2.0

5.8

Reference substance [mg/L]

 

 

 

 

3

7.0

6.5

3.2

9.5

10

8.0

7.8

5.8

9.9

32

7.4

8.1

7.7

8.1

Table 3: Measurement of respiration rate

Treatment

Specific respiration rate [mg O2/g/h]

% inhibition or stimulation ()

Control 1

17.6

-

Control 2

16.7

-

Test substance [mg/L]

 

 

10

19.1

0 (11)

100

19.6

0 (14)

1000

19.7

0 (15)

1000

20.0

0 (17)

1000

18.8

0 (9)

Reference substance [mg/L]

 

 

3

13.0

24

10

7.6

56

32

1.9

89

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:
14 May 1998
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substanz was emulsified using Ultra-Turrax.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge from sewage plant Bempflingen, Germany
- Preparation of inoculum for exposure: Activated sludge was washed with tap water twice, afterwards centrifuged and the dry residue was
determined. The amount of the sludge was weighed per liter, which was corresponded to 4 g dry mass.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not stated
Test temperature:
20 ± 2 °C
pH:
not stated
Dissolved oxygen:
> 6.5 mg/L
Nominal and measured concentrations:
Nominal: 10000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Material: glass; Size: 1 L; Fill volume: 500 mL
- Aeration: continously to reach an oxygen content > 6.5 mg/L
- No. of vessels per concentration: 1
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

EFFECT PARAMETERS MEASURED: Respiration inhibition by decrease of oxygen content was measured in 15-minutes-pulses over a test period of 3 h using an OXI-meter.
Reference substance (positive control):
yes
Remarks:
3,5 dichlorphenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 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:
EC10
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes, result is in the range of 5 to 30 mg/L
- Relevant effect levels: EC50 = 5 mg/L

An increase about 43% of respiration rate could be observed at the nominal tested concentration of 10000 mg/L in comparison to the control.

Table 1: Respiration rate of control, reference and test substance and inhibition of the respiration rate of test and reference substance (*A negative sign means an increase of the respiration)

 

 Replicates

Concentration [mg/L]

Respiration rate [mg O2/L x h]

Inhibition [%]

Control

1

 

24.0
25.0

-

Referenz

1
2
3

2.5

10

30

22.0

11.0

5.0

10.2

55.1

79.6

Test substance

1

10000

35.0

-43.0*

 *: negative value indicate an increase of respiration

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:
28 - 29 Oct 2008
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:
Department of Health, UK
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Information provided by the Sponsor indicated that the test substance is not sufficiently soluble to prepare an aqueous solution. Therefore, at test inition weights were established by the addition of appropriate volumes (based on specific gravity, 0.99) of test substance to the test vessels containing RO water, synthetic sewage and microbial inoculum (see Table 1).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works, which treats predominantely domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. One day one of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried, preweighed Whatman GF/C filter paper, which was then dried again at ~ 105 °C for at least one hour, allowed to cool in a desiccator before being reweighed. The mixed liquor suspended solids content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge. The mixture was aerated overnight. The mixed liquor suspended solids content of the activated sludge was adjusted to 4 g/L by addition of tap water. Aliquots (200 mL) were added to the test vessels to give a final suspended solids concentration of 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
test start: 19.5 - 19.6 °C
test end: 19.3 - 19.4 °C
pH:
test start: 7.3 - 7.4
test end: 7.6 - 7.9
Nominal and measured concentrations:
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type: closed (loosely covered with aluminium foil)
- Aeration: using a glass aerator connected to a laboratory supply of oil-free compressed air (~ 1 L/min.)
- No. of vessels per concentration: 1 (10, 100 mg/L); 3 (1000 mg/L)
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water
- Culture medium different from test medium: no, according to OECD guideline 209
- Intervals of water quality measurement: The pH and temperature were measured at the start and end of the test.

EFFECT PARAMETERS MEASURED: The respiration rate was measured after a test period of 3 h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
EC10
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:
The test substance showed no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. Respiration rates of mixtures containing the test substance showed an increase in respiration rate above that of the controls.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3h) = 6.4 mg/L (4.1 - 9.6 mg/L)

Table 1: Preparation of test solutions

Treatment

Test substance [µL] or reference substance [mL]

Synthetic sewage [mL]

RO water [mL]

Microbial Inoculum [mL]

Control 1

0

16

284

200

Control 2

0

16

284

200

Test substance [mg/L]

 

 

 

 

10

5.1

16

284

200

100

50

16

284

200

1000

505

16

284

200

Reference substance [mg/L]

 

 

 

 

3

3

16

281

200

10

10

16

274

200

32

32

16

252

200

Table 2: Dissolved oxygen concentration and measurement times

Treatment

Initial dissolved oxygen conc. in culture [mg O2/L]

Initial measured dissolved oxygen conc. [mg O2/L]

Final measured dissolved oxygen conc. [mg O2/L]

Measurement time [min]

Control 1

6.9

6.5

2.5

8.5

Control 2

6.9

6.5

2.5

9.0

Test substance [mg/L]

 

 

 

 

10

4.5

4.2

1.5

5.3

100

5.0

4.8

1.5

6.3

1000

5.8

5.5

2.5

5.7

1000

4.9

4.6

1.5

5.8

1000

5.5

4.9

2.0

5.8

Reference substance [mg/L]

 

 

 

 

3

7.0

6.5

3.2

9.5

10

8.0

7.8

5.8

9.9

32

7.4

8.1

7.7

8.1

Table 3: Measurement of respiration rate

Treatment

Specific respiration rate [mg O2/g/h]

% inhibition or stimulation ()

Control 1

17.6

-

Control 2

16.7

-

Test substance [mg/L]

 

 

10

19.1

0 (11)

100

19.6

0 (14)

1000

19.7

0 (15)

1000

20.0

0 (17)

1000

18.8

0 (9)

Reference substance [mg/L]

 

 

3

13.0

24

10

7.6

56

32

1.9

89

Description of key information

Key value for chemical safety assessment

Additional information

Since no studies investigating the short-term toxicity of Pentaerythritol, mixed esters with linear and branched fatty acids to activated sludge microorganisms are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substance Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6) and Decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9) was conducted. The target substance is characterized as a tetraester of pentaerythritol and linear and branched fatty acids. The source substance Fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol (CAS 11138-60-6) is a triester of trimethylolpropane and C8-C10 fatty acids (even numbered). Decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9) is a tetraester of C5/C7/C9/C10 with pentaerythritol. All substances share similar physico-chemical properties, only differ in the distribution of chain lengths of the fatty acid and do not share any functional group which might increase aquatic toxicity the read-across is suitable to investigate the aquatic toxicity using read-across.

This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13.

The first study investigating the toxicity to activated sludge microorganisms is available for fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol (CAS 11138-60-6). This respiration inhibition test (Kuttler, 1998) with domestic activated sludge according to OECD 209 showed an increase in respiration of 43% at the nominal tested concentration of 10000 mg/L compared to the control. No inhibition of respiration rate was observed. Therefore an EC50 and EC10 (3 h) of > 10000 mg/L (nominal) was derived.

The second study is with the source substance decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9) to activated sludge microorganisms was conducted according to OECD 209 (GLP; Dickinson, 2008). Non-adapted activated sludge obtained from a domestic sewage treatment plant was used as inoculum. Test concentrations of 10, 100 and 1000 mg/L were applied and added directly to the test vessels. The test substance did not inhibit the respiration rate of activated sludge microorganisms at any of the concentrations employed in the test. Hence, the EC50 (3 h) was determined to be > 1000 mg/L based on the nominal test concentration. Therefore, it can be concluded that the test substance will not exhibit effects on the respiration rate of microorganisms.

Based on the available results from structurally related source substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that Pentaerythritol, mixed esters with linear and branched fatty acids will not exhibit effects to activated sludge microorganisms and will not inhibit the degradation in sewage treatment plants.