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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2013 - 28 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates). The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 3401 pH and dissolved oxygen meter and adjusted to between pH 7.0 and 8.0 if necessary.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): oily film visible on surface
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A mixed population of activated sewage sludge micro-organisms was obtained on 30 May 2013 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 deg C overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was allowed to settle and 2 liters of the supernatant was removed to increase the suspended solids concentration. The remaining 8 liters was fed synthetic sewage sludge (400 mL). The pH of the sample on the day of the test was 7.2 measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
The initial suspended solids concentration before removal of the supernatent was equal to 2.5 g/L. After removal of the supernatant and aeration overnight, the suspended solids concentration was repeated and was equal to 4.2 g/L. The activated sewage sludge sample was diluted with dechlorinated tap water (3 liters) and the suspended solids concentration was repeated and was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 +/- 2 deg C
pH:
7.0-7.3
Dissolved oxygen:
2.8-5.8 mg O2/L
Nominal and measured concentrations:
Nominal: 10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: yes, 0.5-1.0 L/min
- No. of vessels per concentration (replicates): 1 for 10 & 100 mg/L and 3 for 1000 mg/L
- No. of vessels per control (replicates):
- Biomass loading rate:

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

OTHER TEST CONDITIONS
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
- Range finding study
- Test concentrations: 0, 10, 100 or 1000 mg/L
- Results used to determine the conditions for the definitive study: Definitve study was not conducted based on the results of the range-finding study
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
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: oily film seen on surface during test
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3-h EC50 = 6.0 mg/L

The dissolved oxygen concentrations after 30 minutes contact time in several of the vessels were below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation to the study plan was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 deg. C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3-Hour EC50 value of 6.0 mg/L, 95% confidence limits 4.6-7.9 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999/02/25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(1984)
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Qualifier:
according to
Guideline:
other: EEC Commission Directive 87/302/EEC
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, date of inspection 23 March 1998
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test substance (500 mg) was dispersed in approx. 250 mL of water and subjected to ultrasonication for approx. 30 min. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and dechlorinated tap water were added to a final volume of 500 mL to give the test concentration of 1000 mg/L.
- Controls: same conditions but without addition of test substance
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: A mixed population of activated sewage sludge micro-organisms was obtained on 25. February 1999 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK.
- Method of cultivation: The sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and was used on the day of collection. The pH of the sample was 7.5.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
21 °C
Nominal and measured concentrations:
Nominal concentrations: control - 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: aerated with compressed air at a rate of approx. 0.5-1 L/min
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter

EFFECT PARAMETERS MEASURED: respiration rate after 30 min and 3 h

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control - 100 - 1000 mg/L
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at all the test concentrations employed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
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:
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:
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:
- Effect concentrations exceeding solubility of substance in test medium: The effect concentrations exceeded the water solubility of the test substance.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (30 min): 15 mg/L; EC50 (3 h): 8.0 mg/L

The validation criteria for the control respiration rates and reference material EC50 values have been satisfied. The test resulted in an EC50 (3 h) of greater than 1000 mg/L.

Table 1: Oxygen consumption rates and percentage inhibition values in the definitive study after 3 h contact time

(RS = Reference substance; - = increase in respiration rate as compared to controls)

Nominal concentration [mg/L]

Initial O2 reading [mg O2/L]

Measurement period [min]

Final O2 reading [mg O2/L]

O2 Consumption rates [mg O2/L/min]

% Inhibition

Control R1

6.5

10

1.4

0.51

-

Control R2

6.4

10

1.4

0.50

-

1000 R1

6.1

10

1.2

0.49

3

1000 R2

7.2

10

2.8

0.44

13

1000 R3

6.5

10

1.4

0.51

-1

RS R1

6.4

10

1.8

0.46

9

RS R2

7.7

10

6.1

0.16

68

RS R3

8.0

10

7.2

0.08

84

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 April 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Remarks:
Steinbeis-Transferzentrum, Angewandte und Umwelt-Chemie an der Fachhochschule Reutlingen, Reutlingen, Germany
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
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
Test temperature:
20 ± 2°C
Dissolved oxygen:
> 6.5 mg/l
Nominal and measured concentrations:
Nominal: 10000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 500 ml fill volume
- Aeration: Continously to reach an oxygen content > 6.5 mg/l
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration inhibition by decrease of oxygen content, measured with a
xt-Schreiber in 15-minutes-pulses.
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):
- Relevant effect levels: EC50 = 5 mg/l is in the range 5 to 30 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. The EC50 (3h) was determined as > 10000 mg/l.

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)

 

 

Concentration [mg/l]

Respiration rate [mg O2/L x h]

Inhibition [%]

Control

K1

 

24.0
25.0

-

Referenz

R1
R2
R3

2.5

10

30

22.0

11.0

5.0

10.2

55.1

79.6

Test substance

T1

10000

35.0

-43.0*

 

Endpoint:
toxicity to microorganisms
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
07 February 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions
Principles of method if other than guideline:
Cell multiplication inhibition test according to Bringman and Kuehn (1980), Water Research 14
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 10 mg/l (nominal concentration) of the test substance in acetone was prepared and used for the test.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1% v/v (final concentration in the system)
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: Pseudomonas putida, strain NCIMB9494, obtained as a freeze-dried culture from National Collections of Industrial and Marine
Bacteria Ltd, Aberdeen, UK
- Method of cultivation: The freeze-dried culture was rehydrated in 0.5 ml of nutrient broth (Oxoid Ltd). A loop of this suspension was streaked onto a nutrient agar (Oxoid Ltd) slope in a universal bottle. This was incubated at 25°C for 24 hours, and then stored at laboratory temperature, until use
as stock culture.
- Preparation of inoculum for exposure: 18-20 hours before the start of the test 4 ml of test medium concentrate were added to 46 ml of deionised
water in a sterile conical flask. A loop of Pseudomonas putida stock culture was added to this growth medium solution, and then incubated overnight at 25°C on an orbital shaker (150 rpm).
- Pretreatment: After incubation the cells were diluted by addition of fresh growth medium solution at 25°C to an optical density which gave an
absorbance of 0.8 (± 0.05) at 600 nm (4 cm cells). This was used as the test inoculum.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
6 h
Nominal and measured concentrations:
Nominal: 10 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Concial flasks contained 50 ml volume
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Basalt salt solution was prepared dissolved in 1 Litre of deionised water. This solution was autoclaved at 121°C for 15 minutes. A glucose solution was prepared containing 6.25 g of glucose dissolved in 1 Litre of deionised water. This solution was autoclaved at 121°C for 15 minutes. Equal volumes of the salt solution and the glucose solution were mixed together with a sterile flask to form a growth mediumconcentrate.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Growth inhibition after 6 hours by measurements of the optical density of
each flask content at 600 nm
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol
Duration:
6 h
Dose descriptor:
EC10
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
6 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test material did not completely
dissolve in the test system.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 96% inhibition of growth at 18 mg/L solution of 3,5 dichlorophenol

The nominal 10 mg/l solution of the test substance gave no inhibition of growth. The EC10 and EC50 (6h) for the test substance to Pseudomonas putida was > 10 mg/l (nominal) test material.

Table 1: Optical density results

Flasknumber

Contents

Mean of optical density at 600 nm (4 cm cells)

Minus value for uninoculated solution

% inhibition

1-3

Control

0.602

0.569

 

4-6

18 mg/l 3,5 dichlorophenol

0.025

 

96

7-9

0.1% Acetone

0.589

0.579

 

13-15

10 mg/l PentaerythritolC7-C10 tetra ester

0.630

0.614

-

16

Blank uninoculated

0.033

 

 

17

0.1% Acetone uninoculated

0.010

 

 

19

10 mg/l PentaerythritolC7-C10

tetra ester uninoculated

0.016

 

 

 

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: analogue substance
Justification for type of information:
Toxicity to microorganisms does not need to be investigated because available data indicate that structural variation does not influence test results or adverse effect profile (see read-across and category justifications attached in Section 13).
Reason / purpose:
read-across source
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

Description of key information

One reliable study is available. In this study activated sewage sludge was exposed to aqueous dispersions of an analogue substance (EC 613 -848 -7) at up to 1000 mg/L with the addition of synthetic sewage as a respiratory substrate for 3 hours. Based on the rate of respiration, the EC50 (3 h) was > 1000 mg/L and the NOEC (3 h) was 1000 mg/L. The substance is not harmful to microorganisms.

Key value for chemical safety assessment

Additional information

One reliable study is available. In this study (Roulstone, 2013) performed under GLP according to OECD TG 209, activated sewage sludge was exposed to an aqueous dispersion of the analogue test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 deg. C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol. The 3-Hour EC50 value was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

The results of this study are supported by data available for the read across substances fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The substances with the CAS No. 131459-39-7 and EC 270 -290 -3 are structural analogues of one of the read-across substances (CAS No. 68424-30-6) and can therefore can also be used for read-across to EC 613 -848 -7.

The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

One study, investigating the toxicity to microorganisms, was available for Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234 -392 -1, CAS-No. 11138-60-6). This respiration inhibition test (Kuttler, 1998) with activated sludge (domestic) according to OECD 209 showed an increase about 43% of respiration rate at the nominal tested concentration of 10000 mg/L in comparison to the control. No inhibition of respiration rate was observed. Therefore the EC50 and EC10 (3h) can be stated as > 10000 mg/L (nominal).

In a static GLP-study conducted to OECD 209 (Mead, 1999) 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS-No. 131459-39-7), was mixed with activated sludge of predominantly domestic sewage. No effect on respiration rate was observed at the tested concentration (EC50 (3h) > 1000 mg/L, NOEC (3h) ≥ 1000 mg/L). Due to the low water solubility of the test substance, no toxicity was observed within the water solubility under the conditions tested.

In addition, in a third study (Comber and Coleman), a cell multiplication inhibition test with Pseudomonas putida according to Bringman and Kuehn (1980) for Fatty acids, C5-10, esters with pentaerythritol (EC 270 -290 -3, CAS-No. 68424-31-7), no inhibition on the growth was observed at the tested concentration (EC10 and EC50 (6h) > 10 mg/L (nominal)). As the test material in this study not completely dissolved in the test system, the tested concentration demonstrated the worst case.