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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 26 September 2017 Experimental completion date 03 January 2018
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
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
EC Number: 500-215-4
CAS Number: 68475-94-5
Batch: 52611021
Purity: not supplied
Physical state/Appearance: clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: room temperature in the dark
Vehicle:
no
Details on test solutions:
Range-Finding Test
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 a Hach HQ40d Flexi handheld meter.
The control group was maintained under identical conditions but not exposed to the test item.

Test Item Preparation
The test item was dispersed directly in water.
Nominal amounts of test item (5.0, 16 and 50 mg (5 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 10, 32 and 100 mg/L (5 replicates of each).
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter.
The control group was maintained under identical conditions but not exposed to the test item.

Additional Test
The test item was dispersed directly in water.
Nominal amounts of test item (160 mg (5 replicates)) 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 concentration of 320 mg/L (5 replicates).
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter.
The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 26 September 2017 for the range-finding test, on 11 December 2017 for the definitive test and on 2 January 2018 for the additional test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Test Water
The test water used for the range-finding, definitive and additional tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory
substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
In the range finding test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.3 and the pH of the synthetic sewage stock used for the range finding test was pH 7.3. In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.3 and the pH of the synthetic sewage stock used for the definitive test was pH 7.2. The pH values were measured using a Hach HQ40d Flexi handheld meter.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 20 °C
pH:
7.1 - 7.4
The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
Dissolved oxygen:
5.89 - 8.00 mg O2/L
The oxygen concentrations in all vessels were measured after 30 minutes contact time.
Nominal and measured concentrations:
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.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 32 and 100 mg/L.
Based on the results of the definitive test the following test concentration was assigned to the additional test: 320 mg/L.
Details on test conditions:
Range-Finding Test
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 15 minutes. The pH of this stock solution was measured to be pH 6.2 and was adjusted to pH 7.1 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.

Definitive Test
Reference Item Preparation
For the purpose of the definitive 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 15 minutes. The pH of this stock solution was measured to be pH 6.3 and adjusted to pH 7.1 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 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 (9 liters) was fed synthetic sewage (450 mL). The pH of the sample on the day of the test was 7.3 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.

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 in a 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 in Section 3.4.2.1. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 °C and 22 °C.

Additional Test
Based on the results from the definitive test it was considered necessary to perform an additional test in order to confirm the NOEC obtained for the test item. Based on the results of the definitive test the following test concentration was assigned to the additional test: 320 mg/L.
Reference Item Preparation
For the purpose of the additional 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 5.7 and 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 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.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorephenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 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 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 concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentration of 100 mg/L. It was not possible to perform statistical analysis on the 1000 mg/L test vessel due to due to the oxygen consumption rate not being determined due to the initial oxygen reading falling below 1.5 mg O2/L which was considered to be due to the insoluble nature of the test item coating the BOD probe.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC for the test item. Test concentrations of 10, 32 and 100 mg/L were selected for the definitive test.

Validation Criteria for the Definitive Test
• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 6.8 mg/L.
• The specific respiration rate of the controls was 27.85 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 5.9%.
All validation criteria for the study were therefore satisfied.

Inhibition of Respiration Rate for the Definitive Test
No statistically significant toxic effects were shown at all of the test concentrations employed. Based on these results the NOEC after 3 hours exposure was 100 mg/L. It was therefore considered necessary to perform an additional test in order to determine whether the NOEC was higher than100 mg/L.
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.

Validation Criteria for the Additional Test
• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.8 mg/L.
• The specific respiration rate of the controls was 29.89 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 2.5%.
All validation criteria for the study were therefore satisfied.

Inhibition of Respiration Rate for the Additional Test
Statistically significantly toxic effects (P < 0.05) were shown at the test concentration of 320 mg/L. Based on these results the NOEC after 3 hours exposure was confirmed as being 100 mg/L.
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.2 to 8.9 mg/L.

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

 

Nominal Concentration(mg/L)

 

Initial O2 Reading (mgO2/L)

 

Measurement Period (minutes)

 

Final O2 Reading (mgO2/L)

O2

Consumption Rates

(mg O2/L/hour)

 

Inhibition (%)

 

 

 

 

 

Control

R1

4.2

3

1.9

46.00

-

R2

4.2

3

2.1

42.00

-

R3

3.3

2

2.0

39.00

-

R4

3.7

2

2.3

42.00

-

R5

5.5

5

2.2

39.60

-

R6

5.4

5

1.9

42.00

-

 

 

 

 

 

 

 

 

 

Test Item

10 R1

2.2

1

1.5

42.00

[1]

10 R2

4.3

4

1.9

36.00

14

10 R3

5.1

5

1.7

40.80

2

10 R4

4.9

4

2.2

40.50

3

10 R5

4.7

4

2.1

39.00

7

32 R1

3.6

2

2.3

39.00

7

32 R2

7.2

10

4.0

19.20

54*

32 R3

4.3

3

2.2

42.00

[1]

32 R4

4.3

4

1.7

39.00

7

32 R5

3.8

3

1.8

40.00

4

 

Nominal Concentration (mg/L)

 

Initial O2 Reading (mg O2/L

 

Measurement Period (minutes)

 

Final O2 Reading (mgO2/L)

O2

Consumption Rates

(mg O2/L/hour)

 

Inhibition(%)

 

 

 

 

Test Item

100R1

3.9

3

2.0

38.00

9

100 R2

3.2

2

2.1

33.00

21

100 R3

3.1

2

1.8

39.00

7

100 R4

3.4

2

2.1

39.00

7

100 R5

3.7

2

2.3

42.00

[1]

 

 

3,5-Dichlorophenol

3.2

5.6

7

2.3

28.29

32

10

6.6

10

3.7

17.40

58

32

7.6

10

6.8

4.80

89

R= Replicate

*Result considered to be erroneous compared to other replicate vessels therefore omitted from determination of the NOEC

[ ] = Increase in respiration rate as compared to controls

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Additional Test

 

NominalConcentration(mg/L)

 

Initial O2Reading (mgO2/L)

 

Measurement Period (minutes)

 

Final O2Reading (mgO2/L)

O2

ConsumptionRates

(mg O2/L/hour)

 

Inhibition (%)

 

 

 

 

 

Control

R1

4.3

3

2.0

46.00

-

R2

4.4

3

2.1

46.00

-

R3

5.1

4

2.1

45.00

-

R4

5.2

4

2.2

45.00

-

R5

4.9

4

2.0

43.50

-

R6

5.2

4

2.3

43.50

-

 

 

 

 

Test Item

320 R1

6.0

8

2.0

30.00

33

320 R2

5.7

7

2.1

30.86

31

320 R3

5.8

7

2.3

30.00

33

320 R4

5.2

6

2.0

32.00

29

320 R5

5.5

7

1.8

31.71

29

 

 

3,5-

Dichlorophenol

3.2

6.0

7

2.0

34.29

24

10

6.9

10

3.2

22.20

50

32

7.8

10

6.8

6.00

87

R = Replicate

Validity criteria fulfilled:
yes
Conclusions:
The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.
The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.2 to 8.9 mg/L.
Executive summary:

Introduction

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 10, 32 and 100 mg/L (5 replicates) for a period of 3 hours at a measured temperature of between 19 °C and 22 °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 No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.2 to 8.9 mg/L.

Description of key information

The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.2 to 8.9 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information