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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-26 to 2017-09-26
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 (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
dated May 30, 2008 (updated on 1stMarch 2016)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Ecological Effects Test Guidelines of the United States Environmental Protection Agency (EPA 712-C-014): OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test"
Version / remarks:
January 2012.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

TEST ITEM
- Method: Plastic flasks used for test item (to avoid reaction of the test item with the laboratory equipment), test solution with a final volume of 300 mL (300 mg test item measured directly into the empty test containers, 50 mL synthetic sewage-water mixture filled into test containers just before the inoculation)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

CONTROLS
Because of more parallel running studies, the corresponding controls (blank, reference and nitrification) were commonly used.

Blank control: Containing water, synthetic sewage and inoculum, but without addition of the test or reference item
Abiotic control (only in preliminary test): Tested at the highest test item concentration (1000 mg/L); the abiotic control group in the preliminary experiment did not show oxygen uptake therefore in the main test abiotic control group was not included
Reference control: 3,5-dichlorophenol at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L)
Nitrification control: Containing 11.6 mg/L N-allylthiourea


At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added first to the blank controls (CBA, thereafter CBB CBC and CBD the “start” CB group), then in appropriate time intervals to the nitrification controls, the test solutions of the reference item and the test item and finally to the “end” CB group (CBE, CBF, CBG and CBH). In time intervals of approximately 12 minutes (an arbitrary but convenient interval) a maximum of four test vessels were started
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Activated sludge, microorganisms from a domestic waste water treatment plant
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant for domestic sewage in Balatonfüred, Hungary on 25 September 2017 (one day before the test)
- Preparation of inoculum for exposure: Coarse particles removed by settling for 10 minutes, upper layer of finer solids decanted; washed by centrifugation after adding isotonic saline solution, supernatant liquid phase decanted, solid material re-suspended in isotonic saline solution with shaking and again centrifuged (wash steps repeated twice); final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined
- Method of cultivation: Calculated amount of wet sludge was suspended in isotonic saline solution; activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 24 hours and fed with 50 mL synthetic sewage/L activated sludge; pH: 7.32 (adjustment not necessary); foaming was not significant
- Initial biomass concentration: Concentration equivalent to about 3 g per litre (on dry weight basis)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
20 ± 2 °C
pH:
Test item: 6.50 - 7.89
Blank control: 7.69 - 8.29
Nitrification control: 7.77 - 8.11
Reference control: 7.41 - 8.19
Dissolved oxygen:
Test item: 7.03 - 7.67 mg O2/L
Blank control: 7.20 - 7.77 mg O2/L
Nitrification control: 7.07 - 7.71 mg O2/L
Reference control: 7.16 - 7.85 mg O2/L
Nominal and measured concentrations:
Because of the directly added test item the obtained results were referred to the nominal test item concentration.

Test item: 1000 mg/L
Blank control: 0 mg/L
Nitrification control: 11.6 mg ATU/L
Reference control: 2, 7, 24.5 mg 3,5-DCP/L
Details on test conditions:
TEST SYSTEM
- Test vessel: plastic flasks (test item), glass flasks (controls)
- Material, size, headspace, fill volume: 300 mL
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per blank control (replicates): 8 (number of the blank control containers was set up to the number of the O2 electrodes)
- No. of vessels per reference control (replicates per concentration): 3
- No. of vessels per nitrification control: 3
- Sludge concentration (weight of dry solids per volume): 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added
- Nutrients provided for bacteria: Fed with 50 mL synthetic sewage/L activated sludge before start of experiment, Synthetic Sewage used for test solutions
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- Particulate matter: No
OTHER TEST CONDITIONS
- Adjustment of pH: No
EFFECT PARAMETERS MEASURED :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: None (limit test)
- Range finding study: yes
- Test concentrations: one limit concentration of 1000 mg/L
- Results used to determine the conditions for the definitive study: three test item concentrations (10, 100 and 1000 mg/L) were tested; reference item 3,5-dichlorophenol was tested at three concentrations (at the nominal test concentrations of 2, 7 and 24.5 mg/L, with three parallels each); nitrification potential of sludge was examined with additional control mixture that contained N-allylthiourea at 11.6 mg/L; abiotic control group in the preliminary experiment did not show oxygen uptake therefore in the main test abiotic control group was not included; main test was performed in one set of test vessels, without ATU addition, because of a differentiation between heterotrophic respiration and nitrification was considered as not necessary
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
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 heterotrophic respiration
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 heterotrophic respiration
Key result
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 heterotrophic respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption (e.g. of test material to the walls of the test container): No (plastic flasks used for test item)
- Blank controls oxygen uptake rate: 56.36 mg O2/Lh

VALIDITY OF THE TEST
The study was considered as valid because of the specific respiration rate of the blank controls (without the test substance or reference substance) was 37.57 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 2.71 %. (In the test containers the final concentration of suspended solids was 1.5 g per litre.) The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 16.82 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary). (Additionally, the difference between “start” and “end” blank control respiration rates (CBA-BD and CBE-BH beginning and end of the exposure period, respectively) were minimal (2.7 %), within the biological variability of the applied system.)
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 3-hour EC50 = 16.82 mg/L (95 % confidence limits: 10.21-31.42 mg/L)
Reported statistics and error estimates:
The EC50 value of the reference item was calculated using Probit analysis. The analysis was done using the IBM® SPSS® Statistics, Version 24 (2016) statistical software program. Since one concentration (as a limit concentration) of the test item was tested, this concentration level of the test item was compared to the blank control using the 2 Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics, Version 24 (2016) software.

Table 1: The Oxygen Consumption Rate (R), Q1, Q2 and the applied Δt valuesin the Controls and Test Item Treatment Groups

Test group
No.

Identifi-cation

Concentration
(mg/L)

Oxygen concentration
(mg O2/L)

Δt (min)

Oxygen Consumption Rate (R)
(mg O2/Lh)

Average R
(mg O2/Lh)

Q1

Q2

1, 7

CBA

0.00

7.39

2.00

5.5

58.80

56.36

CBB

7.17

2.02

5.5

56.18

CBC

7.34

2.21

5.5

55.96

CBD

7.22

2.43

5

57.48

CBE

7.14

2.44

5

56.40

CBF

7.40

2.06

6

53.40

CBG

7.20

2.01

5.5

56.62

CBH

7.01

2.34

5

56.04

2

CNA

11.6 mg
ATU/L

7.23

2.07

5.5

56.29

56.36

CNB

7.24

2.11

5.5

55.96

CNC

7.39

2.18

5.5

56.84

3

R1A

2 mg
3,5-DCP/L

7.43

2.14

6

52.90

51.07

R1B

7.38

2.21

6

51.70

R1C

7.04

2.18

6

48.60

4

R2A

7 mg
3,5-DCP/L

7.04

2.05

6.5

46.06

45.37

R2B

7.12

2.12

7

42.86

R2C

7.02

2.30

6

47.20

5

R3A

24.5 mg
3,5-DCP/L

7.58

5.41

10

13.02

13.04

R3B

7.31

5.19

10

12.72

R3C

7.44

5.21

10

13.38

6

T1A

1000 mg
Test Item/L

7.03

2.35

5

56.16

54.50

T1B

7.15

2.29

5.5

53.02

T1C

7.31

2.24

5.5

55.31

T1D

7.37

2.44

5.5

53.78

T1E

7.22

2.25

5.5

54.22

Q1: the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);

Q2: the oxygen concentration at the end of the selected section of the linear phase (mg/L);

Δt: the time interval between these two measurements (min.).

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

Table 2: The Specific Respiration Rate (RS)in the Controls and Test ItemTreatment Groups

Test group
No.

Identification

Concentration
(mg/L)

Specific Respiration Rate
(mg O2/gh)

Average RS
(mg O2/gh)

1, 7

CBA

0.00

39.20

37.57

CV(%)= 2.71

CBB

37.45

CBC

37.31

CBD

38.32

CBE

37.60

CBF

35.60

CBG

37.75

CBH

37.36

2

CNA

11.6 mg
ATU/L

37.53

37.58

 

CV(%)= 0.78

CNB

37.31

CNC

37.89

3

R1A

2 mg
3,5-DCP/L

35.27

34.04

 

CV(%)= 4.35

R1B

34.47

R1C

32.40

4

R2A

7 mg
3,5-DCP/L

30.71

30.25

 

CV(%)= 4.96

R2B

28.57

R2C

31.47

5

R3A

24.5 mg
3,5-DCP/L

8.68

8.69

 

CV(%)= 2.53

R3B

8.48

R3C

8.92

6

T1A

1000 mg
Test Item/L

37.44

36.33n.s.

 

CV(%)= 2.28

T1B

35.35

T1C

36.87

T1D

35.85

T1E

36.15

n.s.: Statistically not significant significantly different compared to the control (2 Sample t-Test; α = 0.05)

3,5-DCP:  3,5-dichlorophenol

ATU: N-allylthiourea

Table 3: The Percentage Inhibition (IT) of Total Oxygen Consumption

Test group
No.

Identification

Test group

Concentration
(mg/L)

Inhibition (IT), of total oxygen consumption
(%)

1, 7

CB (A-H)

Blank Control

0.00

2

CN (A-C)

Nitrification Control

11.6 mg ATU/L

-0.01#

3

R1(A-C)

Reference Item

2 mg 3,5-DCP/L

9.39

4

R2(A-C)

Reference Item

7 mg 3,5-DCP/L

19.50

5

R3(A-C)

Reference Item

24.5 mg 3,5-DCP/L

76.86

6

T1(A-E)

Test Item

1000 mg Test Item/L

3.31

#: the 0.01 % negative value, calculated by Excel was taken into consideration as zero

3,5-DCP:  3,5-dichlorophenol

ATU: N-allylthiourea

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. All validity criteria were fulfilled.
Executive summary:

To evaluate the influence of the test item on the activity of activated sludge, a Sludge Respiration Inhibition Test was conducted in compliance with GLP criteria and according to OECD Guideline 209 (Carbon and Ammonium Oxidation), EU Method C.11 and US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. The test item could adsorb to glassware. Thus, in the main experiment plastic tubes were used at the test item treatments in order to avoid reaction of the test item with the laboratory equipment. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. All validity criteria of the study were met. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L. The EC50 value was determined as: EC50 > 1000 mg/L. The specific respiration rates were compared with the blank control values using 2 Sample t-Test (a=0.05). No statistical significant differences were observed in the comparison with the blank control values, consequently based on the results of this study the NOEC can be statistically and biologically determined as 1000 mg/L.

Description of key information

The EC10 and EC50 values of test item was determined as higher than 1000 mg/L. The NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

To evaluate the influence of the test item on the activity of activated sludge, a Sludge Respiration Inhibition Test was conducted in compliance with GLP criteria and according to OECD Guideline 209 (Carbon and Ammonium Oxidation), EU Method C.11 and US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. All validity criteria of the study were met. The EC50 value was determined as > 1000 mg/L and the NOEC was statistically and biologically determined as 1000 mg/L.