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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 February 2017 - 28 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 209 guideline without deviation. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
Adopted: July-2010
Deviations:
yes
Remarks:
According to German Federal Environment Agency on the new OECD TG 209 a minimum preparation of a full test with five concentrations is 3 replicates per test concentration and six replicates for the blank control. 3 replicates is used instead of 5.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: N/D

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A
Analytical monitoring:
no
Details on sampling:
Not required by the guideline
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The required amount of test item was added directly to the test vessel, to acheive the testing concentrations.
- Eluate: N/A
- Differential loading: N/A
- Controls: test medium and organism only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: N/A, sludge was collected from a municipal sewage treatement plant.
- Name and location of sewage treatment plant where inoculum was collected: the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 15 February 2017 (limit test) and 28 February 2017 (full test).
- Method of cultivation: The activated sludge was kept aerated until use at test temperature
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Pretreatment: washing process as indicated above, the microorganism were not adapted.
- Initial biomass concentration: 3g/L dry solids.
Test type:
static
Water media type:
other: Chorine free tap water containing synthetic sewage sludge feed
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Guideline duration.
Post exposure observation period:
None required.
Hardness:
N/D
Test temperature:
20.1 to 21.4°C
pH:
7.3 - 7.8
Dissolved oxygen:
All vessels were stirred and aerated throughout the contact time with room air to assure an oxygen saturation of 60-70% using a membrane pump during 3 hour initial exposure period before the measuring of total respiration for the following 3 hours.
Salinity:
N/A
Conductivity:
N/D
Nominal and measured concentrations:
N/A
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL vessel filled with 250 mL of chlorine-free tap wate and after measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Filled with 500 mL of test medium.
- Aeration:
- No. of vessels per concentration (replicates): 3 replicates at 10, 32, 100, 320 and 1000 mg/L
- No. of vessels per control (replicates): 6
- No. of vesssels per postive control: 1 replicate at 1, 10 and 30 mg/L of 3,5-dichlorophenol
- No. of vessels per vehicle control (replicates): N/A
- No. of vessels per abiotic control (replicates): N/A
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: N/A
- Nutrients provided for bacteria:

16 mL of a litre sewage feed solution made of:

16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2 * 2 H2O
0.2 g MgSO4 * 7 H2O
2.8 g K2HPO4

- Nitrification inhibitor used (delete if not applicable): none
- Biomass loading rate: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Chlorine free tap wate from Hydrotox GmbH, Labor für Ökotoxikologie und Gewässerschutz
- Particulate matter:

OTHER TEST CONDITIONS
- Adjustment of pH: At 320 and 1000 mg/L of test item pH adjustment was require. The starting pH for the 320 mg/L vessels was 8.1 to 8.5, with the pH adjusted to 7.3 to 7.6. For 1000 mg/L the test vessel pH values ranged between 8.7 and 8.9 and were adjusted to 7.3 to 7.5.
- Photoperiod: N/A
- Light intensity: N/A
- Details on termination of incubation: After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer. This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: standard number of concentrations was used.
- Range finding study: A limit test was conducted at 1000 mg/L
- Test concentrations: The definitve test concentrations were: 10, 32,100, 320 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Inhibition at 1000 mg/L was found to be 78 to 90 %. As such the range chosen for the definitive test used a maximum spacing factor of 3.2 and used a maximum concentration of 1000 mg/L.
Reference substance (positive control):
yes
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
454.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
184.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
320 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: N/A
- Effect concentrations exceeding solubility of substance in test medium: Not applicable for the test
- Adsorption (e.g. of test material to the walls of the test container):
- Blank controls oxygen uptake rate: 31.2 to 46.3 mg/(L*h) this is above the validity criteria requirement of 20 mg oxygen per one gram of activated sludge. Therefore this criteria is met.
- Coefficient of variation of oxygen uptake rate in control replicates: 12 % which is below the 30 % CoV required by the guideline validity criteria. Therefore this criteria is met.
Results with reference substance (positive control):
- Results with reference substance valid? The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L
- Relevant effect levels: EC50 of 3,5-dichlorphenol was calculated as 11.27 mg/L
- Other: N/A
Reported statistics and error estimates:
EC50 = 454.2 (95 % CL: 279.681-726.811)
EC10 = 184.7 mg/L (95 % CL: 122.680-277.536 mg/L)
LOEC = 320 mg/L
NOEC = 100 mg/L

Table 1. Respiration rate of activated sludge after application of the test item or positive control

Test item concentration (mg/L)

O2 concentration (mg/L)

Consumption rate

(mg/(l*h))

Inhibition

(%)

Blank 1

7.15

5.59

37.4

-

Blank 2

7.01

5.34

40.1

-

Blank 3

7.29

5.36

46.3

-

Blank 4

6.59

5.29

31.2

-

Blank 5

5.88

4.15

41.5

-

Blank 6

6.57

4.74

43.9

-

1000 mg/L

8.18

7.86

7.7

81

1000 mg/L

8.21

7.99

5.3

87

1000 mg/L

8.48

8.30

4.3

89

320 mg/L

7.70

6.67

24.7

38

320 mg/L

6.36

5.08

30.7

23

320 mg/L

6.40

5.20

28.8

28

100 mg/L

5.89

4.04

44.4

-11

100 mg/L

5.87

4.11

42.2

-5

100 mg/L

6.24

4.52

41.3

-3

32 mg/L

5.80

3.94

44.6

-11

32 mg/L

6.90

5.14

42.2

-5

32 mg/L

6.66

4.95

41.0

-2

10 mg/L

6.95

5.41

37.0

8

10 mg/L

6.90

5.30

38.4

4

10 mg/L

6.32

4.37

46.8

-17

Positive control

1 mg//L 3,5-dichlorophenol

6.91

5.67

29.8

26

10 mg//L 3,5-dichlorophenol

7.93

7.35

13.9

65

30 mg//L 3,5-dichlorophenol

7.94

7.62

7.7

81

Validity criteria fulfilled:
yes
Conclusions:
An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item. This was to assess the inhibition effect of different concentrations of test item to micro-organisms of activated sludge by measuring the respiration rate after adding a standard amount of synthetic sewage feed. The test met all guideline validity criteria.
The results for the inhibition of the total respiration of activated sludge were:
EC50 = 454.2 mg/L (95%-CL: 279.681 – 726.811 mg/L)
EC10 = 184.7 mg/L (95%-CL: 122.860 – 277.536 mg/L)
LOEC = 320.0 mg/L
NOEC ≥ 100.0 mg/L
Executive summary:

An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item. This was to assess the inhibition effect of different concentrations of test item to micro-organisms of activated sludge by measuring the respiration rate after adding a standard amount of synthetic sewage feed.

The respiration rate corresponds to the oxygen respiration of aerobic activated sludge after a contact time of 3 h. The inhibitory effect at a particular concentration is expressed as a percentage of the mean respiration rate of the controls. The EC50 and EC10 values are calculated with the ToxRat software by probit analysis using linear maximum likelihood regression. A known inhibitor (3-5-dichlorophenol) is used as reference item.

To establish if the test item was toxic to sewage sludge micro-organism a limit test was performed where sludge as exposed to 1000 mg/L of test item. If no effects occur the testing need not continue. However, the limit test showed inhibition of >75 % at 1000 mg/L. Therefore, the limit test indicated the requirement for a full definitive test with a series of 5 concentrations with an highest concentration of 1000 mg/L. Five concentrations were selected with a spacing factor of 3.2, these were: 1000, 320, 100, 32 and 10 mg/L. In accordance with the German Question-and-Answer Document by the German Federal Environment Agency on the new OECD Guideline 209 "Activated Sludge, Respiration Inhibition Test (Carbon and ammonium Oxidation)" (Version: 02.03.2012) a minimum preparation of a full test with five concentrations are three replicates per test concentration and six replicates for the blank control. Therefore these number of replicates were used in the defintive test.

The guideline validity criteria were met during the defintive and limit test. The reference item showed the sludge micro-organisms to be of sufficient sensitivity. Thus, the results were valid. The results for the defintive test of inhibition to total respiration were:

EC50 = 454.2 mg/L (95%-CL: 279.681 – 726.811 mg/L)

EC10 = 184.7 mg/L (95%-CL: 122.860 – 277.536 mg/L)

LOEC = 320.0 mg/L

NOEC ≥ 100.0 mg/L

The test item was found to affect total respiration of sewage sludge micro-organisms during an OECD 209 Activated sludge inhibition test with an EC10 of 184.7 mg/L and an EC50 of 454.2 mg/L.

Description of key information

EC50 = 454.2 mg/L (95%-CL: 279.681 – 726.811 mg/L); EC10 = 184.7 mg/L (95%-CL: 122.860 – 277.536 mg/L); NOEC ≥ 100.0 mg/L, Sewage sludge micro-organisms, Brunswick-Titze, A. 2017

Key value for chemical safety assessment

EC50 for microorganisms:
454.2 mg/L
EC10 or NOEC for microorganisms:
184.7 mg/L

Additional information

An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item as a single key study for the endpoint. This was to assess the inhibition effect of different concentrations of test item to micro-organisms of activated sludge by measuring the respiration rate after adding a standard amount of synthetic sewage feed.

The respiration rate corresponds to the oxygen respiration of aerobic activated sludge after a contact time of 3 h. The inhibitory effect at a particular concentration is expressed as a percentage of the mean respiration rate of the controls. The EC50 and EC10 values are calculated with the ToxRat software by probit analysis using linear maximum likelihood regression. A known inhibitor (3-5-dichlorophenol) is used as reference item.

To establish if the test item was toxic to sewage sludge micro-organism a limit test was performed where sludge as exposed to 1000 mg/L of test item. If no effects occur the testing need not continue. However, the limit test showed inhibition of >75 % at 1000 mg/L. Therefore, the limit test indicated the requirement for a full definitive test with a series of 5 concentrations with an highest concentration of 1000 mg/L. Five concentrations were selected with a spacing factor of 3.2, these were: 1000, 320, 100, 32 and 10 mg/L. In accordance with the German Question-and-Answer Document by the German Federal Environment Agency on the new OECD Guideline 209 "Activated Sludge, Respiration Inhibition Test (Carbon and ammonium Oxidation)" (Version: 02.03.2012) a minimum preparation of a full test with five concentrations are three replicates per test concentration and six replicates for the blank control. Therefore these number of replicates were used in the definitive test.

The guideline validity criteria were met during the definitive and limit test. The reference item showed the sludge micro-organisms to be of sufficient sensitivity. Thus, the results were valid. The results for the definitive test of inhibition to total respiration were:

EC50 = 454.2 mg/L (95%-CL: 279.681 – 726.811 mg/L)

EC10 = 184.7 mg/L (95%-CL: 122.860 – 277.536 mg/L)

LOEC = 320.0 mg/L

NOEC ≥ 100.0 mg/L

The test item was found to affect total respiration of sewage sludge micro-organisms during an OECD 209 Activated sludge inhibition test with an EC10 of 184.7 mg/L and an EC50 of 454.2 mg/L.