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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2017 to 05 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Concentrations: 100. 320 and 1000 mg/L

Samples were not stored prior to analysis
Vehicle:
no
Details on test solutions:
The test item was dispersed directly in water.
Nominal amounts of test item (50, 160 and 500 mg (five 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 temperatures between 20 C and 21 C, 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 100, 320 and 1000 mg/L (five replicates of each).
The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter (see Table 6) and adjusted to between pH 7.0 to pH 8.0 if necessary.
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.
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
03 March 2017 for the range-finding test and on 04 April 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Test temperature:
between 20 and 21°C
pH:
6.4 -8.4 : after sturring and prior to addition of inoculum

7.-3 -7.4 at end of exposure period
Dissolved oxygen:
5.5 - 7.0 mg O2/L
Salinity:
Not applicable as freshwater study
Nominal and measured concentrations:
Range-finding study: 10, 100 and 1000 mg/L

Main study: 100, 320 and 1000 mg/L
Details on test conditions:
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 were prepared as described in Section 3.3.2.1. 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 20 and 21°C.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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:
No statistically significant toxic effects were shown at the test concentration of 100 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 320 and 1000 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 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.
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):
EC50: 8.9 mg/L (3 hours)

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 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 a NOEC for the test item. Based on this information test concentrations of 100, 320 and 1000 mg/L were selected for the definitive test.

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 6.90% and the specific respiration rate of the controls was 25.83 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

The validation criterion for the reference item EC50value was also satisfied

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

Nominal
Concentration
(mg/L)

Initial O2
Reading
(mg O2/L)

Measurement Period
(minutes)

Final O2Reading
(mg O2/L)

O2Consumption Rates
(mg O2/L/hour)

% Inhibition

Control

R1

5.0

5

1.9

37.20

-

R2

5.4

6

1.8

36.00

-

R3

5.1

5

1.8

39.60

-

R4

4.9

5

1.8

37.20

-

R5

5.2

4

2.3

43.50

-

R6

6.0

6

2.1

39.00

-

Test Item

100 R1

2.6

1

1.9

42.00

[8]

100 R2

4.7

4

2.2

37.50

3

100 R3

4.3

4

1.9

36.00

7

100 R4

3.9

3

2.0

38.00

2

100 R5

4.0

3

2.1

38.00

2

320 R1

4.1

4

2.0

31.50

19

320 R2

4.5

4

2.4

31.50

19

320 R3

4.7

5

2.0

32.40

16

320 R4

4.7

6

1.7

30.00

23

320 R5

5.4

7

1.9

30.00

23

 


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

 


Nominal
Concentration
(mg/L)

Initial O2
Reading
(mg O2/L

Measurement Period
(minutes)

Final O2Reading (mg O2/L)

O2Consumption Rates
(mg O2/L/hour)

% Inhibition

Test Item

1000R1

7.5

10

6.1

8.40

78

1000 R2

7.8

10

6.5

7.80

80

1000 R3

7.9

10

6.4

9.00

77

1000 R4

8.0

10

6.5

9.00

77

1000 R5

8.0

10

6.4

9.60

75

3,5-dichlorophenol

3.2

5.8

7

2.1

31.70

18

10

7.0

10

4.1

17.40

55

32

7.8

10

7.0

4.80

88


 


R1– R6= Replicates 1 to 6

[Increase in respiration rate as compared to controls]

 

R1– R5= Replicates 1 to 5

 

 

Validity criteria fulfilled:
yes
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 100, 320 and 1000 mg/L (5 replicates) for a period of 3 hours at measured temperatures of between 20 and 21°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 No Observed Effect Concentration (NOEC) after 3 hours exposure of 100 mg/L.

The reference item gave a 3-Hour EC50value of 8.9 mg/L, 95% confidence limits 7.2 to 11 mg/L.

Description of key information

A single high quality study, conducted under GLP conditions to OECD Guideline 209 was vailable to assess the substance's inhibitory effects on the respiration of activated sewage sludge.  No statistically significant toxic effects were shown at the test concentration of 100 mg/L, however statistically significantly toxic effects were shown at the test concentrations of 320 and 1000 mg/L.  Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information