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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Remarks:
Stability of test substance was measured
Details on sampling:
The test substance was stored as follows: room temperature, dark, air-tight in a desiccator.
Sampled before and after exposure
Vehicle:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Konan-Chubu Water Reclamation Plant
- Preparation of inoculum for exposure: MLSS was measured and adjusted to 3000 mg/L by adding ultra pure water on the day of collection. In addition, the synthetic sewage feed was added to the sludge (50mL synthetic sewage feed/1L sludge) and was stored until use for the exposure. The pH was checked and adjusted by adding NaOH to pH 7.5 -/= 0.5 on the day of exposure.
- Storage conditions: 20 +/- 2°C
- Mixed liquor suspended solid concentration (MLSS): 3180 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Test temperature:
20 +/- 2°C
Nominal and measured concentrations:
1, 5.6, 32, 180, 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass beakers
- Aeration: 0.5 to 1L/min
- No. of vessels per concentration (replicates): 3 (main phase)
- No. of vessels per control (replicates): 6 (main phase)
- Nutrients provided for bacteria: Syntheic sewage feed
- Nitrification inhibitor used: N-allylthiourea
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultra pure water
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 3 hour respiration inhibition (based on oxygen consumption).
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5.62
- Range finding study
- Test concentrations: 1,10, 100 and 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (DCP)
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:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC50
Basis for effect:
inhibition of nitrification rate
Remarks on result:
not determinable
Remarks:
% of inhibition fluctuated largely. Total respiration was greatly attributed to inhibition of hetrotrophic respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Temperature was maintained at 20.3°C

See tables in Any other information on results incl. tables.
Results with reference substance (positive control):
EC50 of DPC = 7.79 mg/L (Total respiration), 9.3 mg/L (Heterotrophic respiration) and 0.137 mg/L (nitrification respiration). Validity criteria met for reference substance.
Reported statistics and error estimates:
NOEC value was determined by an analysis of variance (ANOVA), Williams test. The significant level for test was set a p=0.05

Table 1: pH values

Concentration

(mg/L)

Type of respiration

Total respiration

Type of respiration

Heterotrophic respiration

No.

pH

No.

pH

1*

2*

1*

2*

Control

FB-1

7.4

7.7

FBH-1

7.4

7.8

FB-2

-

7.7

FBH-2

-

7.8

FB-3

7.7

FBH-3

7.8

FB-4

7.7

FBH-4

7.8

FB-5

7.7

FBH-5

7.8

FB-6

7.7

FBH-6

7.8

1.0

FT1-1

7.4

7.7

FTH1-1

7.4

7.9

FT1-2

-

7.6

FTH1-2

-

7.8

FT1-3

7.7

FTH1-3

7.8

5.6

FT2-1

7.4

7.7

FTH2-1

7.4

7.7

FT2-2

-

7.6

FTH2-2

-

7.8

FT2-3

7.6

FTH2-3

7.8

32

FT3-1

7.4

7.6

FTH3-1

7.4

7.8

FT3-2

-

7.6

FTH3-2

-

7.7

FT3-3

7.6

FTH3-3

7.7

180

FT4-1

7.4

7.5

FTH4-1

7.4

7.9

FT4-2

-

7.5

FTH4-2

-

7.7

FT4-3

7.6

FTH4-3

7.7

1000

FT5-1

7.4

7.6

FTH5-1

7.4

7.8

FT5-2

-

7.5

FTH5-2

-

7.8

FT5-3

7.5

FTH5-3

7.7

1* Before adding activated sludge

2* At the end of exposure period

Table 2: Oxygen consumption rates of blank controls

Type of respiration

No.

Concentration

(mg/L)

Time (min)

DO

(mg/L)

R

(mgO2/L/hr)

Average

SD

CV

(%)

(mgO2/L/hr)

Total respiration

FB-1

-

0.75

Q1

3.96

RTB-1

46.8

45.2

1.1

2.4

3.25

Q2

2.01

FB-2

0.75

Q1

3.41

RTB-2

44.9

2.58

Q2

2.04

FB-3

0.75

Q1

3.69

RTB-3

45.3

2.92

Q2

2.05

FB-4

0.75

Q1

2.96

RTB-4

43.3

2.08

Q2

2.00

FB-5

0.75

Q1

3.22

RTB-5

45.2

2.33

Q2

2.03

FB-6

0.75

Q1

4.27

RTB-6

45.6

3.67

Q2

2.05

Heterotrophic respiration

FBH-1

1.00

Q1

4.99

RHB-1

36.6

40.1

-

-

5.83

Q2

2.04

FBH-2

1.00

Q1

5.14

RHB-2

37.6

6.00

Q2

2.01

FBH-3

1.00

Q1

4.71

RHB-3

40.9

4.92

Q2

2.04

FBH-4

1.00

Q1

5.53

RHB-4

40.7

6.17

Q2

2.02

FBH-5

1.00

Q1

4.41

RHB-5

41.3

4.50

Q2

2.00

FBH-6

1.00

Q1

4.20

RHB-6

43.4

4.00

Q2

2.03

Nitrification respiration

-

 

 

 

RNB-1

6.7

5.1

-

-

RNB-2

4.8

RNB-3

5.2

RNB-4

3.2

RNB-5

5.1

RNB-6

5.5

 

Equation:

R=(Q1-Q2)/Δtx60

RNB=RTB-RHB (Average)

Table 3: Oxygen consumption rates, percent inhibitions of test mixtures (Total respiration)

Type of respiration

No.

Concentration

(mg/L)

Time (min)

DO

(mg/L)

R

(mgO2/L/hr)

Average

(mgO2/L/hr)

Inhibition

Average

(%)

Total respiration

 

FT1-1

1.0

0.75

Q1

 4.06

RTT1-1

48.5

46.2

ITT1

-7.3

-2.3

3.25

Q2

 2.04

FT1-2

0.75

Q1

 3.63

RTT1-2

45.3

-0.2

2.83

Q2

 2.06

FT1-3

0.75

Q1

 3.28

RTT1-3

44.9

0.7

2.42

Q2

 2.03

FT2-1

5.6

0.75

Q1

 2.02

RTT2-1

45.6

44.5

ITT2

-0.9

1.5

2.58

Q2

0.63 

FT2-2

0.75

Q1

 3.74

RTT2-2

44.3

2.0

3.08

Q2

 2.02

FT2-3

0.75

Q1

 3.17

RTT2-3

43.7

3.3

2.33

Q2

 2.02

FT3-1

32

0.75

Q1

 2.71

RTT3-1

36.4

40.8

ITT3

19.5

9.8

3.67

Q2

 0.94

FT3-2

0.75

Q1

 1.88

RTT3-2

41.3

8.6

2.42

Q2

 0.73

FT3-3

0.75

Q1

 2.91

RTT3-3

44.6

1.3

1.92

Q2

 2.04

FT4-1

180

0.75

Q1

 3.16

RTT4-1

34.8

31.1

ITT4

23.0

31.1

2.75

Q2

 2.00

FT4-2

0.75

Q1

2.91 

RTT4 -2

28.1

37.8

2.67

Q2

 2.01

FT4-3

0.75

Q1

 3.69

RTT4-3

30.5

32.5

4.67

Q2

 2.04

FT5-1

1000

0.75

Q1

 1.97

RTT-5-1

32.2

31.0

ITT5

28.8

31.5

3.08

Q2

 0.72

FT5-2

0.75

Q1

 2.67

RTT5-2

33.8

25.2

1.92

Q2

 2.01

FT5-3

0.75

Q1

 3.77

RTT5-3

26.9

40.5

4.67

Q2

 2.01

 

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RTT/RTB (Average)} x 100

Table 4: Oxygen consumption rates, percent inhibitions of test mixtures (heterotropic respiration)

Type of respiration

No.

Concentration

(mg/L)

Time (min)

DO

(mg/L)

R

(mgO2/L/hr)

Average

(mgO2/L/hr)

Inhibition

Average

(%)

Heterotrophic respiration

 

FTH1-1

1.0

1.00

Q1

5.67

RHT1-1

40.2

39.3

IHT1

-0.2

2.1

6.42

Q2

2.04

FTH1-2

1.00

Q1

5.02

RHT1-2

38.0

5.2

5.75

Q2

2.01

FTH1-3

1.00

Q1

4.76

RHT1-3

39.6

1.2

5.17

Q2

2.01

FTH2-1

5.6

1.00

Q1

4.26

RHT2-1

38.1

38.2

IHT2

5.0

4.8

4.50

Q2

2.04

FTH2-2

1.00

Q1

5.76

RHT2-2

37.7

6.0

6.92

Q2

2.04

FTH2-3

1.00

Q1

4.51

RHT2-3

38.7

3.5

4.83

Q2

2.04

FTH3-1

32

1.00

Q1

2.20

RHT3-1

28.8

30.9

IHT3

28.2

23.0

3.50

Q2

1.00

FTH3-2

1.00

Q1

4.84

RHT3-2

31.1

22.4

6.42

Q2

2.03

FTH3-3

1.00

Q1

4.65

RHT3-3

32.7

18.5

5.83

Q2

2.02

FTH4-1

180

6.13

Q1

6.13

RHT4-1

23.9

26.2

IHT4

40.4

34.7

2.54

Q2

2.54

FTH4-2

5.19

Q1

5.19

RHT4-2

26.4

34.2

2.00

Q2

2.00

FTH4-3

4.75

Q1

4.75

RHT4-3

28.3

29.4

2.00

Q2

2.00

FTH5-1

1000

5.22

Q1

5.22

RHT-5-1

36.0

28.6

IHT5

10.2

28.7

2.07

Q2

2.07

FTH5-2

5.51

Q1

55.1

RHT5-2

25.0

37.7

2.00

Q2

2.00

FTH5-3

5.42

Q1

5.42

RHT5-3

24.8

38.2

2.01

Q2

2.01

 

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RHT/RHB (Average)} x 100

Table 5: Oxygen consumption rates, percent inhibitions of test mixtures (nitrification respiration)

Type of respiration

No.

Concentration

(mg/L)

R

(mgO2/L/hr)

Average

(mgO2/L/hr)

Inhibition

Average

(%)

Nitrification respiration

 

-

1.0

RNT1-1

9.2

6.9

IHT1

-80.4

-35.9

RNT1-2

6.0

-17.6

RNT1-3

5.6

-9.8

-

5.6

RNT2-1

7.4

6.3

IHT2

-45.1

-24.2

RNT2-2

6.1

-19.6

RNT2-3

5.5

-7.8

-

32

RNT3-1

5.5

9.9

IHT3

-7.8

-93.4

RNT3-2

10.4

-103.9

RNT3-3

13.7

-168.6

-

180

RNT4-1

8.6

4.9

IHT4

-68.6

3.3

RNT4-2

1.9

62.7

RNT4-3

4.3

15.7

-

1000

RNT-5-1

3.6

2.4

IHT5

29.4

53.6

RNT5-2

5.2

-2.0

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RNT/RNB (Average)} x 100

Validity criteria fulfilled:
yes
Conclusions:
The 3h EC50 value was calculated to be >1000 mg/L for total respiration inhibition. The NOEC was estimated to be 5.6 mg/L for total respiration inhibition.

Description of key information

OECD guideline, GLP compliant study. Activated sludge was exposed to the test substance for 3 hours during the test. % of respiration inhibition (total, heterotrophic, nitrification) was recorded.

EC50 value of >1000 mg/L and NOEC value of 5.6 mg/L were calculated for total respiration inhibition.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
5.6 mg/L

Additional information