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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 24 July 2018 and 29 August 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: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Chemical Name Reaction mass of Magnesium dioxide(oxo)silane and Silicon dioxide (MgSiO3, Si and SiO2)
Batch: Y180510A
Purity: 95.0%
Physical state/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test Item Preparation:
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 (three replicates for the test concentration of 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 sealed with nescofilm prior to being 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 (three replicates at 1000 mg/L).
The pH of the test item dispersions were measured after stirring using a Hach HQ40d Flexi handheld meter.

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.

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 7.3 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 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 stock solution was inverted several times to ensure homogeneity of the solution.
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 28 August 2018 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 (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.6 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 three 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
pH:
7.5-7.9
Dissolved oxygen:
4.7-7.6 mg O2/L
Nominal and measured concentrations:
Test item:
Nominal concentration: 10, 100 and 1000 mg/L

Details on test conditions:
Test Water:
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

Synthetic Sewage:
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
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range-finding test was pH 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter.

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 prior to the test item vessels being prepared. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of 21 °C.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 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 any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol

Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 5.2 mg/L.
• The specific respiration rate of the controls was 24.00 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 0%.
All validation criteria for the study were therefore satisfied.
Results with reference substance (positive control):
The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 5.2 mg/L.

Inhibition of Respiration Rate

The following results were derived:

 

Mg-SiO

3,5-Dichlorophenol

ECx(3 Hours) (mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours) (mg/L)

95% Confidence Limits (mg/L)

EC10

>1000

-

0.67

-

EC20

>1000

-

1.1

-

EC50

>1000

-

5.2

3.7-7.3

EC80

>1000

-

24

-

NOEC

1000

-

-

-

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.

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)

Dissolved Oxygen Saturation Level of 8.9 mg O2/L
(%)

Control

R1

6.88

77

R2

7.02

79

R3

7.87

88

R4

7.30

82

Test Item

10

6.06

68

100

6.22

70

1000 R1

7.12

80

1000 R2

7.54

85

1000 R3

8.01

90

3,5-Dichlorophenol

3.2

7.69

86

10

8.17

92

32

8.17

92

R   = Replicate

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

5.2

6

1.6

36.00

-

R2

5.1

5

2.1

36.00

-

R3

4.9

4

2.5

36.00

-

R4

4.7

4

2.3

36.00

-

Test Item

10

5.0

5

2.0

36.00

0

100

5.2

5

2.1

37.20

[3]

1000 R1

5.3

5

2.4

34.80

3

1000 R2

5.3

5

2.3

36.00

0

1000 R3

5.5

6

2.0

35.00

3

3,5-Dichlorophenol

3.2

6.4

10

3.0

20.40

43

10

6.8

10

4.3

15.00

58

32

7.6

10

6.9

4.20

88

R  = Replicate

-   = Not applicable

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration
(mg/L)

pH

0 Hours

3 Hours

Control

R1

7.4

7.5

R2

7.4

7.5

R3

7.3

7.5

R4

7.5

7.5

Test Item

10

7.5

7.5

100

7.4

7.5

1000 R1

7.3

7.5

1000 R2

7.6

7.6

1000 R3

7.4

7.5

3,5-Dichlorophenol

3.2

7.4

7.9

10

7.5

7.7

32

7.4

7.6

R = Replicate

Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

Nominal Concentration
(mg/L)

Observations on Test Preparations

0 Hours*

30 Minutes
Contact Time

3 Hours
Contact Time

Control

R1

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

R2

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

R3

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

R4

Pale yellow/brown dispersion

Dark brown dispersion

Dark brown dispersion

Test Item

10

Pale yellow/brown dispersion, particles of test item visible dispersed throughout and floating at surface

Dark brown dispersion, few particles of test item visible floating at the surface

Dark brown dispersion, few particles of test item visible floating at the surface

100

Brown/black dispersion, particles of test item visible dispersed throughout, floating at surface and settled on bottom of vessel

Very dark brown dispersion, few particles of test item visible dispersed throughout and floating at the surface

Very dark brown dispersion, few particles of test item visible dispersed throughout and floating at the surface

1000 R1

Black dispersion, film of test item visible floating at surface

Black dispersion, particles of test item visible floating at the surface

Black dispersion, particles of test item visible floating at the surface

1000 R2

Black dispersion, film of test item visible floating at surface

Black dispersion, particles of test item visible floating at the surface

Black dispersion, particles of test item visible floating at the surface

1000 R3

Black dispersion, film of test item visible floating at surface

Black dispersion, particles of test item visible floating at the surface

Black dispersion, particles of test item visible floating at the surface

3,5-Dichlorophenol

3.2

Pale yellow/brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

10

Pale yellow/brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

32

Pale yellow/brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

*Observations made prior to the addition of activated sewage sludge

R   = Replicate

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 greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 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

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of 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 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 5.2 mg/L, 95% confidence limits 3.7 to 7.3 mg/L confirming the acceptability of the inoculum used in the test system.

Description of key information

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

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of 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 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 5.2 mg/L, 95% confidence limits 3.7 to 7.3 mg/L confirming the acceptability of the inoculum used in the test system.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

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