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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 19 September 2017 and 09November 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:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Triazolone
Batch: 0727/16
Purity: 99%
Physical state/Appearance: off white powder
Expiry Date: 28 April 2019
Storage Conditions: room temperature in the dark
Analytical monitoring:
no
Remarks:
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This exception with regard to GLP has been reflected in the GLP compliance statement.
Vehicle:
yes
Remarks:
Deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Details on test solutions:
Range-Finding Test
Test Item Preparation
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0 if necessary.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 180, 320, 560, 1000 and 1800 mg/L.

Test Item Preparation The test item was dispersed directly in water.
Nominal amounts of test item (90, 160, 280, 500 and 900 mg (3 replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 180, 320, 560, 1000 and 1800 mg/L (3 replicates of each).
The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0.
Test organisms (species):
activated sludge of a predominantly industrial sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 19 September 2017 for the range-finding test and on 08 November 2017 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation of Inoculum The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL).
pH of the sample on the day of the test was measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.1 - 20.6”C
pH:
7.3
Dissolved oxygen:
<1 mg/L
Nominal and measured concentrations:
Nominal Cocentrations
Details on test conditions:
Experimental Design and Study Conduct 3.4.1 Range-Finding Test 3.4.1.1 Test Item Preparation The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 and 8.0 if necessary.
The exposure conditions for each flask were as described below.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group was maintained under identical conditions but not exposed to the test item.

Reference Item Preparation
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 6.2 and was adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
The exposure conditions for each flask were as described below.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 180, 320, 560, 1000 and 1800 mg/L.

Test Item Preparation
The test item was dispersed directly in water.
Nominal amounts of test item (90, 160, 280, 500 and 900 mg (3 replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 180, 320, 560, 1000 and 1800 mg/L (3 replicates of each).
The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group was maintained under identical conditions but not exposed to the test item.

Reference Item Preparation
For the purpose of the test a reference item, 3,5-Dichlorophenol was used. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 6.1 and adjusted to pH 7.3 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added to a final volume of 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Two additional control vessels were then prepared prior to the test item vessels as described above. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of between 20 °C and 21 °C.
Reference substance (positive control):
yes
Remarks:
A reference item, 3,5-Dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum.
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
930 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.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Range-Finding Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2. Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 3.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain an EC50 value for the test item. Based on this information test concentrations of 180, 320, 560, 1000 and 1800 mg/L were selected for the definitive test.

Definitive Test

The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 4. Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in Table 5.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 4.1% and the specific respiration rate of the controls was 27.87 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied. The validation criterion for the reference item EC50 value was also satisfied.

Inhibition of Respiration Rate The following results were derived:

Triazolone 3,5-Dichlorophenol
ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC10 160 - 1.1 -
EC20 250 - 1.7 -
EC50 930 - 6.3 4.7 – 8.4
EC80 - - 24 -

It was not possible to determine an EC80 value or the 95% confidence limits for the test item as no concentration tested resulted in greater than 61% inhibition.
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Results with reference substance (positive control):
See Above

Table 2 Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

 Nominal Concentration (mg/L)    Dissolved Oxygen Concentration (mg O2/L)  Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
 Control  R1  7.78  87
   R2  7.80  88
   R3  5.82  65
   R4  5.78  66
 Test Item  10  7.17  81
   100  6.59  74
   1000 R1  7.40  83
   1000 R2  6.99  79
   1000 R3  7.23  81
 3,5 -Dichlorophenol  3.2  827  93
   10  4.85  88
   31  8.66  97

Table 3 Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)

Initial O2Reading

(mg O2/L)

Measurement

Period

(minutes)

Final O2

Reading

(mg O2/L)

O2

Consumption

Rates

(mg O2/L/hour)

% Inhibition

Control

R1

5.1

4

1.9

48.0

-

R2

5.3

4

2.2

46.5

-

R3

5.2

4

1.9

49.5

-

R4

4.5

3

2.0

50.0

-

Test Item

10

5.2

4

2.0

48.0

1

100

5.5

4

2.5

45.0

7

1000 R1

6.9

10

3.1

22.8

53

1000 R2

7.1

10

3.3

22.8

53

1000 R3

7.3

10

3.3

24.0

51

3,5-

Dichlorophenol

3.2

6.3

8

2.0

32.3

34

10

7.3

10

4.0

19.8

59

32

8.1

10

6.8

7.8

84

Table 4          Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Nominal Concentration (mg/L)

Dissolved Oxygen

Concentration

(mg O2/L)

Expressed as a Percentage of a

Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Control

R1

7.22

81

R2

5.62

63

R3

7.81

88

R4

6.94

78

R5

7.13

80

R6

7.33

82

Test Item

180 R1

6.21

70

180 R2

6.81

77

180 R3

7.77

87

320 R1

5.94

67

320 R2

6.71

75

320 R3

8.11

91

560 R1

5.79

65

560 R2

8.02

90

560 R3

7.76

87

1000 R1

7.00

79

1000 R2

6.83

77

1000 R3

6.21

70

1800 R1

6.06

68

1800 R2

7.34

82

1800 R3

6.97

78

3,5-Dichlorophenol

3.2

6.24

70

10

8.73

98

32

8.77

99

Table 5           Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal

Concentration

(mg/L)

Initial O2Reading

(mg O2/L)

Measurement

Period

(minutes)

Final O2

Reading

(mg O2/L)

O2

Consumption

Rates

(mg O2/L/hour)

%

Inhibition

Control

R1

4.1

4

1.5

39.00

-

R2

4.3

4

1.5

42.00

-

R3

5.2

5

1.8

40.80

-

R4

3.5

2

2.1

42.00

-

R5

4.8

4

1.9

43.50

-

R6

4.9

4

2.0

43.50

-

Test Item

180 R1

6.0

6

2.3

37.00

11

180 R2

5.9

6

2.2

37.00

11

180 R3

6.0

7

1.9

35.14

16

320 R1

6.4

9

2.0

29.33

30

320 R2

5.7

7

2.0

31.71

24

320R3

6.2

8

1.9

32.25

23

560 R1

6.2

10

1.9

25.80

38

560 R2

6.4

10

2.3

24.60

41

560 R3

6.7

10

2.6

24.60

41

1000 R1

6.9

10

3.4

21.00

50

1000 R2

6.9

10

3.5

20.40

51

1000 R3

6.7

10

3.3

20.40

51

1800 R1

7.2

10

4.4

16.80

60

1800 R2

6.9

10

4.0

17.40

58

1800 R3

7.2

10

4.5

16.20

61

3,5-

Dichlorophenol

3.2

6.3

10

1.8

27.00

35

10

7.2

10

4.4

16.80

60

32

8.3

10

7.4

5.40

87

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 930 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 180, 320, 560, 1000 and 1800 mg/L (3 replicates) for a period of 3 hours at a measured temperature of between 20.1 °C and 20.6 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-Dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of 930 mg/L.

The reference item gave a 3-Hour EC50value of 6.7 mg/L, 95% confidence limits 4.7 to 8.4 mg/L

Description of key information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 180, 320, 560, 1000 and 1800 mg/L (3 replicates) for a period of 3 hours at a measured temperature of between 20.1 °C and 20.6 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-Dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of 930 mg/L.

The reference item gave a 3-Hour EC50value of 6.7 mg/L, 95% confidence limits 4.7 to 8.4 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
930 mg/L
EC10 or NOEC for microorganisms:
160 mg/L

Additional information