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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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:
yes
Remarks:
One slight deviation to the validity criteria without any incidence on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
None.
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
INOCULUM
The inoculum was obtained on 3 August 2015 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage. On arrival at the laboratory, the activated sludge (around 10L) was centrifuged (1000g / 10 minutes) and the solid sewage inoculant portion was re-suspended in 10L French source water (Volvic®). 500 mL synthetic sewage feed was then added. In order to obtain the required sludge solids concentration of 3 g/L: the solid content of the sludge was determined by removing a sub-sample of homogenised sludge and drying in an oven at approximately 105°C. The suspended solids content was adjusted until the recommended guideline value of 3 g/L ± 10% is achieved. Sewage sludge was maintained under aerated conditions for a maximum of 4 days and fed daily with synthetic sewage feed at a rate of 25 mL/L.

SYNTHETIC SEWAGE FEED
The synthetic sewage feed was prepared to contain the following constituents per liter:

- 16 g peptone
- 11 g meat extract
- 3 g urea
- 0.7 g sodium chloride
- 0.4 g calcium chloride dihydrate CaCl2, 2H2O
- 0.2 g magnesium sulfate heptahydrate MgSO4, 7H2O
- 2.8 g di-potassium hydrogen phosphate K2HPO4

The final volume was filled up with ultra-pure water.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Remarks on exposure duration:
None.
Post exposure observation period:
None
Hardness:
Not reported.
Test temperature:
21.1°C - 22.4°C (min - max)
pH:
Not reported.
Dissolved oxygen:
Not applicable (respiration rate measure)
Salinity:
Freshwater
Conductivity:
Not reported.
Nominal and measured concentrations:
Nominal concentrations of test item (definitive test): 104.5, 188.1, 338.8, 610.5 and 1100 mg/L
Nominal concentrations of 3,5-dichlorophenol: 0.1, 0.44, 1.98, 8.90 and 44.00 mg/L
Details on test conditions:
A parent solution of N-allylthiourea in ultra-pure water at a concentration of 2.3 g/L was prepared (232 mg was weighed and poured in a 100 mL volumetric flask). The volume was made with ultra-pure water; aliquots were then added to the appropriate test vessel to give the required addition rates.
The reference item, 3,5-DCP, was tested at the following nominal concentrations: 0.1, 0.44, 1.98, 8.90 and 40.00 mg/L. A 3,5-dichlorophenol stock solution (1 g/L) was prepared by dissolving the reference item (100 mg) directly in previously heated water to aid dissolution, the volume was then made up to 100 mL with ultra-pure water. Aliquots were then added to the appropriate test vessel to give the required reference item addition rates.
The range-finding test was conducted at the following nominal concentrations of test item: 10, 100 and 1000 µL/L together with two controls. In order to convert results from µL/L to mg/L, the test item density was estimated by weighing different volumes pipetted: a density of 1.10 was estimated, nominal concentrations are thus converted as 11, 110 and 1100 mg/L.
The definitive test was conducted using a range of concentrations deduced from the preliminary range-finding test. In order to obtain both a NOEC and EC50, two controls and five treatment concentrations in a geometric series were prepared at the following concentrations: 95, 171, 308, 555 and 1000 µL/L converted as 104.5, 188.1, 338.8, 610.5 and 1100 mg/L.
For both range-finding and definitive tests, the test item was dispersed directly in water to the appropriate test vessel to give the required test item addition rates. Single replicate vessels were prepared per concentration (including the controls).
At the start of the different tests, 16 mL of synthetic sewage feed was made up to 250 mL with ultra-pure water. 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L suspended solid was added to the first control vessel. Thereafter, in time intervals of about fifteen minutes the procedure was repeated with test vessels, except that the 16 mL synthetic sewage was added to the appropriate amounts of reference/test item or ATU in ultra-pure water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum. The second control vessel was prepared at last.
The addition of the inoculum was considered as the start of the 3-hour incubation time period. All solutions were incubated at 20 +/- 2°C in darkness in a temperature controlled laboratory, mixed by stirring (around 500-700 rpm) and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the test period, a well-mixed sample of each solution (following their order of preparation) was poured into a BOD bottle (250 mL capacity) after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration falls below 1 mg/L. The temperature was monitored during test period.

Reference substance (positive control):
no
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: No inhibition far above water solubility limit. No hazard identified.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other: No inhibition far above water solubility limit. No hazard identified.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: No inhibition far above water solubility limit. No hazard identified.
Details on results:
The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time and expressed as mg O2/L/h for the linear part of the graphs. Inhibitions of total and heterotrophic respirations were calculated compared to the control. Results for the definitive test showed that the test item is not likely to inhibit total, heterotrophic or nitrification respirations, neither at its water solubility limit (115 mg/L), nor far above (1100 mg/L).
Results with reference substance (positive control):
The 3h-EC50 for 3,5-dichlorophenol to activated sludge were estimated under the conditions of the test as follows:

- 13.2 mg/L for total respiration
- 14.4 mg/L for heterotrophic respiration
- 8.0 mg/L for nitrification respiration.

This test met the validity criteria of the guideline:

- The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: the maximum was determined to be 15.9%
- The 3h-EC50 for the reference item was in the guideline range 2 to 25 mg/L for total respiration (13.2 mg/L), in the range 5 to 40 mg/L for heterotrophic respiration (14.4 mg/L) and in the range 0.1 to 10 mg/L for nitrification respiration (8.0 mg/L)
Reported statistics and error estimates:
For mathematical reasons, no ECx nor NOEC/LOEC could be statistically determined. It was graphically determinable that the test item did not inhibit total or heterotrophic respiration up to the highest concentration tested. A fortiori, the test item did not inhibit nitrification respiration either. Therefore NOEC was estimated be superior or equal to 1100 mg test item/L and EC50 was estimated to be >1100 mg/L, under the conditions of this test.

Table 1: Respiration Rates and Percentage Inhibition: Reference item (3,5-dichlorophenol)

Oxygen measurements (mg O2/L)

Time (min)

Control 1

Nominal concentration (mg/L)

Control 2

0.1

0.44

1.98

8.9

40

0

8.31

7.84

7.88

7.47

7.30

7.78

7.84

1

7.36

7.35

6.75

6.61

6.88

7.74

6.37

2

6.91

6.90

6.27

6.14

6.22

7.61

5.8

3

6.47

6.44

5.77

5.69

5.58

7.49

5.22

4

6.01

5.96

5.28

5.22

4.92

7.37

4.65

5

5.55

5.49

4.81

4.77

4.27

7.24

4.07

6

5.1

5.01

4.3

4.31

3.60

7.14

3.51

7

4.65

4.53

3.82

3.85

2.95

7.03

2.93

8

4.18

4.06

3.32

3.39

2.30

6.89

2.36

9

3.75

3.57

2.84

2.92

1.66

6.79

1.79

10

3.25

3.10

2.35

2.45

1.00

6.65

1.22

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

Respiration Rate

(mg O2/g/h)

% Inhibition

T0

T+"x" min

Control 1

7.36

3.25

9.0

27.40

18.27

Control 2

6.37

1.22

9.0

34.33

22.89

0.10*

7.35

3.10

9.0

28.33

18.89

8.2

0.44

6.75

2.35

9.0

29.33

19.55

5.0

1.98

6.61

2.45

9.0

27.73

18.49

10.2

8.90*

6.88

1.00

9.0

39.20

26.13

-27.0

40.00

7.74

6.65

9.0

7.27

4.85

76.5

*this time point was considered as outlier value

Table 2: Respiration Rates and Percentage Inhibition: Reference item (3,5 -dichlorophenol) + Nitrification inhibitor (ATU)

Oxygen measurements (mg O2/L)

Time (min)

Control 1

Nominal concentration (mg/L)

Control 2

0.1

0.44

1.98

8.9

40

0

8.36

8.04

8.15

8.14

7.84

8.14

8.06

1

7.21

7.03

7.30

7.26

6.94

8.15

6.84

2

6.79

6.59

6.88

6.83

6.54

8.04

6.35

3

6.4

6.17

6.47

6.47

6.13

7.94

5.88

4

6

5.72

6.04

6.05

5.74

7.82

5.41

5

5.57

5.3

5.62

5.66

5.33

7.72

4.93

6

5.14

4.89

5.21

5.25

4.93

7.60

4.47

7

4.75

4.46

4.79

4.85

4.53

7.49

3.99

8

4.33

4.03

4.36

4.44

4.11

7.37

3.52

9

3.92

3.60

3.94

4.02

3.69

7.29

3.04

10

3.5

3.16

3.53

3.62

3.30

7.15

2.56

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

Respiration Rate

(mg O2/g/h)

% inhibition

T0

T+"x" min

Control 1

7.21

3.50

9.0

24.73

16.49

Control 2

6.84

2.56

9.0

28.53

19.02

0.10*

7.03

3.16

9.0

25.80

17.20

3.1

0.44

7.30

3.53

9.0

25.13

16.75

5.6

1.98

7.26

3.62

9.0

24.27

16.18

8.9

8.90*

6.94

3.30

9.0

24.27

16.18

8.9

40.00

8.15

7.15

9.0

6.67

4.45

75.0

*this time point was considered as outlier value

Table 3: Respiration Rates and Percentage Inhibition: Test item (Range-finding test)

Oxygen measurements (mg O2/L)

 

Time (min)

Control 1

Nominal concentration (mg/L

Control 2

11

110

1100

0

8.79

6.86

8.10

7.01

7.48

1

6.77

6.70

5.50

4.70

5.79

2

6.28

6.15

4.93

4.12

5.11

3

5.74

5.62

4.31

3.57

4.44

4

5.22

5.07

3.80

2.98

3.76

5

4.73

4.52

3.25

2.40

3.06

6

4.21

3.97

2.69

1.82

2.39

7

3.67

3.41

2.12

1.24

1.70

8

3.16

2.88

1.56

0.66

1.02

9

2.62

2.32

0.99

0.10

0.35

10

2.10

1.77

0.43

0.04

0.04

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

% inhibition

T0

T+"x" min

Control 1

6.77

2.10

9.0

31.13

Control 2

5.79

1.02

7.0

40.89

11.00

6.70

1.77

9.0

32.87

8.7

110.00

5.50

1.56

7.0

33.77

6.2

1100.00

4.70

1.24

6.0

34.60

3.9

 

Table 4: Respiration Rates and Percentage Inhibition: Test item + Nitrification inhibitor (Range-finding test)

Oxygen measurements (mg O2/L)

 

Time (min)

Control 1

Nominal concentration (mg/L)

Control 2

11

110

1100

0

7.06

7.90

8.33

7.93

7.65

1

6.50

6.67

5.99

5.57

6.54

2

6.04

6.15

5.51

5.13

6.01

3

5.52

5.65

5.04

4.68

5.47

4

4.98

5.15

4.57

4.23

4.93

5

4.46

4.63

4.08

3.77

4.41

6

3.91

4.13

3.61

3.32

3.86

7

3.37

3.62

3.13

2.87

3.33

8

2.83

3.11

2.64

2.41

2.80

9

2.28

2.59

2.14

1.95

2.25

10

1.73

2.08

1.65

1.48

1.71

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

% inhibition

T0

T+"x" min

Control 1

6.50

1.73

9.0

31.80

Control 2

6.54

1.71

9.0

32.20

11.00

6.67

2.08

9.0

30.60

4.4

110.00

5.99

1.65

9.0

28.93

9.6

1100.00

5.57

1.48

9.0

27.27

14.8

Table 5: Respiration Rates and Percentage Inhibition: Test item (Definitive test)

Oxygen measurements (mg O2/L) - Test item

Time (min)

Control 1

Nominal concentration (mg/L)

Control 2

104.5

188.1

338.8

610.5

1100

0

8.9

7.14

6.6

7.33

7.41

ND

8

1

8.13

5.57

4.67

4.68

4.38

1.45

6.24

2

6.51

5.03

4.06

4.07

3.72

0.86

5.58

3

6.00

4.47

3.47

3.46

3.05

0.18

4.97

4

5.44

3.92

2.87

2.84

2.39

ND

4.31

5

4.89

3.37

2.26

2.23

1.71

ND

3.69

6

4.35

2.82

1.66

1.60

1.04

ND

3.04

7

3.77

2.26

1.04

0.98

0.38

ND

2.39

8

3.22

1.71

0.45

0.36

ND

ND

1.75

9

2.67

1.14

ND

ND

ND

ND

1.11

10

2.09

0.58

ND

ND

ND

ND

0.46

ND: Not Determined

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

Respiration Rate

(mg O2/g/h)

% inhibition

T0

T+"x" min

Control 1

6.51

2.09

8.0

33.15

22.10

Control 2

6.24

1.11

8.0

38.48

25.65

104.5

5.57

1.14

8.0

33.23

22.15

7.2

188.1

4.67

1.04

6.0

36.30

24.20

-1.4

338.8

4.68

1.60

5.0

36.96

24.64

-3.2

610.5

4.38

1.04

5.0

40.08

26.72

-11.9

1100

1.45

0.86

1.0

35.40

23.60

1.2

 

Table 6: Respiration Rates and Percentage Inhibition: Test item + Nitrification inhibitor (Definitive test)

Oxygen measurements (mg O2/L) - Test item + N-allylthiourea

Time (min)

Control 1

Nominal concentration (mg/L)

Control 2

104.5

188.1

338.8

610.5

1100

0

6.85

6.98

7.72

8.01

7.20

7.32

7.66

1

6.37

5.49

5.56

5.92

4.78

4.75

5.85

2

5.83

4.92

5.03

5.42

4.17

4.16

5.27

3

5.29

4.38

4.49

4.95

3.58

3.53

4.68

4

4.75

3.82

3.95

4.46

2.97

2.91

4.10

5

4.2

3.25

3.4

3.97

2.37

2.29

3.51

6

3.65

2.69

2.84

3.49

1.76

1.66

2.92

7

3.09

2.12

2.31

3.0

1.16

1.04

2.32

8

2.54

1.55

1.76

2.5

0.55

0.42

1.73

9

1.99

0.98

1.20

2.01

ND

ND

1.12

10

ND

0.42

ND

1.50

ND

ND

0.53

ND: Not Determined

Percentage Inhibition

Treatment

Oxygen measurements

Minutes retained

Respiration Rate
(mg O2/L/h)

Respiration Rate

(mg O2/g/h)

% inhibition

T0

T+"x" min

Control 1

6.37

1.99

8.0

32.85

21.90

Control 2

5.85

1.12

8.0

35.48

23.65

104.5

5.49

0.98

8.0

33.83

22.55

1.0

188.1

5.56

1.20

8.0

32.70

21.80

4.3

338.8

5.92

1.50

9.0

29.47

19.65

13.7

610.5

4.78

1.16

6.0

36.20

24.13

-6.0

1100

4.75

1.04

6.0

37.10

24.73

-8.6

Validity criteria fulfilled:
yes
Remarks:
except O2 consumption in the reference item assay, which was 19.2 which is slightly less than 20 mg oxygen / h / g of activated sludge (dry weight of suspended solids). This slight deviation was not considered critical on the outcome of the study.
Conclusions:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol does not inhibit bacterial total, heterotrophic or nitrification respiration. No hazard is identified.
Executive summary:

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010).

The test was conducted at the following nominal concentrations: 95, 171, 308, 555 and 1000 µL/L.In order to convert results from µL/L to mg/L, the test item density was estimated by weighing different volumes pipetted: a density of 1.10 was estimated. Nominal concentrations are thus converted as 104.5, 188.1, 338.8, 610.5 and 1100 mg/L. A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 0.10, 0.44, 1.98, 8.9 and 40 mg/L.

The inhibition of oxygen uptake by micro-organisms oxidising organic carbon was separately expressed from that by micro-organisms oxidising ammonium by measurement of the rates of uptake of oxygen in the absence and presence of N-allylthiourea (ATU) (the percentage inhibition of the rate of oxygen uptake was calculated by comparison of the rate of oxygen uptake in the presence of the test item with the mean oxygen uptake rate of the corresponding controls both in the presence and absence of N-allylthiourea).

The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrationsversustime and expressed as mg O2/L/h for the linear part of the graphs (then converted in mg O2/g of activated sludge dry weight of suspended solids/h).

For mathematical reasons, no ECx nor NOEC/LOEC could be statistically determined. It was graphically determinable that the test item did not inhibit total or heterotrophic respiration up to the highest concentration tested.A fortiori, the test item did not inhibit nitrification respiration either. Therefore NOEC was estimated be superior or equal to 1100 mg test item/L and EC50was estimated to be >1100 mg/L, under the conditions of this test.[FCT1] [WN2] 

The 3h-EC50for 3,5-dichlorophenol to activated sludge were estimated under the conditions of the test as follows. They fulfilled the validity criteria of the OECD 209 guideline:

- 13.2 mg/L for total respiration

- 14.4 mg/L for heterotrophic respiration

- 8.0 mg/L for nitrification respiration

2,2'-isopropylidenebis(p-phenyleneoxy)diethanol does not inhibit bacterial total, heterotrophic or nitrification respiration. No hazard is identified.

Description of key information

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010).

The test was conducted at the following nominal concentrations: 95, 171, 308, 555 and 1000 µL/L.In order to convert results from µL/L to mg/L, the test item density was estimated by weighing different volumes pipetted: a density of 1.10 was estimated. Nominal concentrations are thus converted as 104.5, 188.1, 338.8, 610.5 and 1100 mg/L. A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 0.10, 0.44, 1.98, 8.9 and 40 mg/L.

The inhibition of oxygen uptake by micro-organisms oxidising organic carbon was separately expressed from that by micro-organisms oxidising ammonium by measurement of the rates of uptake of oxygen in the absence and presence of N-allylthiourea (ATU) (the percentage inhibition of the rate of oxygen uptake was calculated by comparison of the rate of oxygen uptake in the presence of the test item with the mean oxygen uptake rate of the corresponding controls both in the presence and absence of N-allylthiourea).

The prepared vessels were aerated for 3 h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrationsversustime and expressed as mg O2/L/h for the linear part of the graphs (then converted in mg O2/g of activated sludge dry weight of suspended solids/h).

For mathematical reasons, no ECx nor NOEC/LOEC could be statistically determined. It was graphically determinable that the test item did not inhibit total or heterotrophic respiration up to the highest concentration tested.A fortiori, the test item did not inhibit nitrification respiration either. Therefore NOEC was estimated be superior or equal to 1100 mg test item/L and EC50was estimated to be >1100 mg/L, under the conditions of this test.[FCT1] [WN2] 

The 3h-EC50for 3,5-dichlorophenol to activated sludge were estimated under the conditions of the test as follows. They fulfilled the validity criteria of the OECD 209 guideline:

- 13.2 mg/L for total respiration

- 14.4 mg/L for heterotrophic respiration

- 8.0 mg/L for nitrification respiration

2,2'-isopropylidenebis(p-phenyleneoxy)diethanol does not inhibit bacterial total, heterotrophic or nitrification respiration. No hazard is identified.

Key value for chemical safety assessment

Additional information