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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2017 - 27 June 2017
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))
Version / remarks:
July 22, 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
updated on 1 March 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
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Test mixtures: At the start of the test defined amounts of 5 x 300 mg test item were administered (measured into empty containers) directly. The containers were filled up with water and synthetic sewage, just before the inoculation.
- Controls:
Blank Controls: In the main test eight controls (containing water, synthetic sewage and inoculum, but without addition of the test or reference item), four at the start and four at the end of the test series were investigated.
Reference Control: In the main test the reference item 3,5-dichlorophenol was tested at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L).
Abiotic Control: The investigation of abiotic controls in the main experiment was considered as not necessary based on the preliminary information about the test item and based on the results of the preliminary test, where abiotic controls (with three parallels) were tested at the highest test item concentration (1000 mg/L) and no remarkable abiotic oxygen consumption was noticed.
Nitrification Control: In order to check whether the sludge nitrifies and at what rate, mixtures (same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included (with three parallels) in the preliminary experiment and in the main experiment.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary on 26 April 2017 (one day before the main test)
- Preparation of inoculum for exposure:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis).
(In the test containers the final concentration of suspended solids was 1.5 g per litre.)
The activated sludge was not used on the day of the collection but it was continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge.
- Initial biomass concentration: 3 g/L (on dry weight basis)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
NA
Test temperature:
The noticed temperatures in the environmental room varied between: 20.0 - 20.8°C.
pH:
The pH of the activated sludge inoculum was checked after preparation (pH: 7.43).
Dissolved oxygen:
6.59 - 8.31 mg O2/L
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
nominal concentration: 1000 mg/L
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, size, headspace, fill volume: glass, 300 mL volume
- Aeration: yes, with compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 4 at the start and 4 at the end
- No. of vessels per abiotic control (replicates): 3 (only tested in preliminary experiment)
- Sludge concentration (weight of dry solids per volume): 3 g per litre (on dry weight basis)
- Nutrients provided for bacteria: synthetic sewage feed
- Nitrification inhibitor used: yes; N-allylthiourea

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

OTHER TEST CONDITIONS
- Adjustment of pH: no

TEST CONCENTRATIONS
- Range finding study
In the preliminary range-finding test three test item concentrations were tested (10, 100 and 1000 mg/L). In addition, the nitrification potential of sludge was examined with additional control mixture that contained N-allylthiourea at 11.6 mg/L.
- Results used to determine the conditions for the definitive study: In the preliminary experiment inhibitory effect of the test item was not noticed (~ 4 % inhibition) at the nominal concentration of 1000 mg/L. In the preliminary test the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurred. In the main test the test item concentration of 1000 mg/L was examined.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
Key result
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
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
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 total respiration
Details on results:
- Blank controls oxygen uptake rate:
The specific respiration rate of the blank controls (without the test substance or reference substance) was 46.00 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour with a coefficient of variation of 6.98 %.
- Nitrification controls: With the application of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 69.00 mg/Lh, the heterotrophic respiration (RH) was 67.57 mg/Lh, the nitrification respiration (RN): 1.43 mg/Lh was calculated according to the following equation: RN= RT-RH. The obtained 1.43 mg/Lh was considered as not significant difference within a biological variability range of the applied test system, that lower than the 5 % of RT (3.45 mg/Lh) in blank controls.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 9.12 mg/L, (95 % confidence limits: 7.53 – 11.05 mg/L).

Table 1: The Oxygen Consumption Rate (R), Q1, Q2 and the applied Δt values in the Blank Control 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

7.10

2.23

4

73.05

69.00

CBB

7.21

2.07

5

61.68

CBC

7.54

2.38

4.5

68.80

CBD

7.20

2.27

4

73.95

CBE

7.05

2.21

4

72.60

CBF

7.13

2.32

4

72.15

CBG

7.21

2.17

4.5

67.20

CBH

7.18

2.49

4.5

62.53

6

T1A

1000

7.30

2.09

4.5

69.47

68.46

T1B

7.29

2.64

4

69.75

T1C

7.41

2.43

4.5

66.40

T1D

7.52

2.27

4.5

70.00

T1E

7.24

2.24

4.5

66.67

Table 2.: The Oxygen Consumption Rate (R), Q1, Q2 and the applied Δt valuesin the Control 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

7.10

2.23

4

73.05

69.00

CBB

7.21

2.07

5

61.68

CBC

7.54

2.38

4.5

68.80

CBD

7.20

2.27

4

73.95

CBE

7.05

2.21

4

72.60

CBF

7.13

2.32

4

72.15

CBG

7.21

2.17

4.5

67.20

CBH

7.18

2.49

4.5

62.53

2

CNA

11.6

7.45

2.45

4.5

66.67

67.57

CNB

7.08

2.42

4

69.90

CNC

7.52

2.56

4.5

66.13

3

R1A

2

7.26

2.31

5

59.40

60.60

R1B

7.31

2.17

5

61.68

R1C

7.09

2.03

5

60.72

4

R2A

7

7.14

2.16

7

42.69

44.17

R2B

7.17

2.29

7

41.83

R2C

7.32

2.12

6.5

48.00

5

R3A

24.5

7.11

5.28

10

10.98

11.08

R3B

7.25

5.54

10

10.26

R3C

7.29

5.29

10

12.00

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.).

Table 3.: The Specific Respiration Rate (RS) in the Blank Control 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

48.70

46.00

CV(%)= 6.98

CBB

41.12

CBC

45.87

CBD

49.30

CBE

48.40

CBF

48.10

CBG

44.80

CBH

41.69

6

T1A

1000

46.31

45.64n.s.

 

CV(%)= 2.58

T1B

46.50

T1C

44.27

T1D

46.67

T1E

44.44

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

Table 4 : The Specific Respiration Rate (RS)in the Control Groups

Test group
No.

Identification

Concentration
(mg/L)

Specific Respiration Rate
(mg O2/gh)

Average RS
(mg O2/gh)

1, 7

CBA

0.00

48.70

46.00

CV(%)= 6.98

CBB

41.12

CBC

45.87

CBD

49.30

CBE

48.40

CBF

48.10

CBG

44.80

CBH

41.69

2

CNA

11.6

44.44

45.04

 

CV(%)= 1.54

CNB

46.60

CNC

44.09

3

R1A

2

39.60

40.40

 

CV(%)= 1.89

R1B

41.12

R1C

40.48

4

R2A

7

28.46

29.45

 

CV(%)= 7.57

R2B

27.89

R2C

32.00

5

R3A

24.5

7.32

7.39

 

CV(%)= 7.89

R3B

6.84

R3C

8.00

 

Table 5 : 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

2.07

3

R1(A-C)

Reference Item

2

12.17

4

R2(A-C)

Reference Item

7

35.98

5

R3(A-C)

Reference Item

24.5

83.94

6

T1(A-E)

Test Item

1000

0.78

 

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

In a 3-hour test the influence of the test item on the activity of the activated sludge was determined according OECD TG 209 by measuring the respiration rate under defined conditions.

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 the 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. Defined amounts of the test item were added (measured) directly into the test vessels.

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. The test was performed without pH adjustment. All validity criteria of the study were met.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. 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 specific respiration rates were compared with the blank control values using 2 Sample t-Test (α=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.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to section 13 for "Read-Across justification".
Reason / purpose for cross-reference:
read-across source
Key result
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
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
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 total respiration

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of the calcium salt of the target substance were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test 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

The toxicity to microorganisms was addressed with read-across approach to the calcium salt of the target substance.

In a 3-hour test the influence of the analouge substance on the activity of the activated sludge was determined according OECD TG 209 by measuring the respiration rate under defined conditions.

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 the 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. Defined amounts of the test item were added (measured) directly into the test vessels.

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. The test was performed without pH adjustment. All validity criteria of the study were met.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. 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 specific respiration rates were compared with the blank control values using 2 Sample t-Test (α=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.