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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 conudcted on 29 October 2014.
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
Principles of method if other than guideline:
None
GLP compliance:
yes
Specific details on test material used for the study:
None
Analytical monitoring:
no
Details on sampling:
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.
Vehicle:
no
Details on test solutions:
Range-Finding Test
A nominal amount of test item (2500 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
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 29 October 2014 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 and was used on the day of collection. The pH of the sample was measured using a Hach 160d 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 one 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
Approximately 20 °C
pH:
The pH of the test item stock solutions were adjusted to between pH 7.0 to pH 8.0.
The pH of the synthetic sewage stock was 7.2;
Dissolved oxygen:
less than 1 mg O2/L.
Salinity:
No data
Nominal and measured concentrations:
Nominal test concentrations of 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 Dissolved Organic Carbon (DOC).

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
dissolved in 1 liter of water with the aid of ultrasonication.

The pH of the synthetic sewage stock was 7.2. The pH value was measured using a Hach 160d handheld meter.

Procedure
Range-Finding Test
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. The test item was dissolved directly in water.

A nominal amount of test item (2500 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

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.

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. Finally two further control vessels were prepared.

The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 ºC.
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
Remarks:
respiration rate
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
Remarks:
respiration rate
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 all 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.

The coefficient of variation of oxygen uptake in the control vessels was 4% and the specific respiration rate of the controls was 20.98 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
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.
Results with reference substance (positive control):
The coefficient of variation of oxygen uptake in the control vessels was 4% and the specific respiration rate of the controls was 20.98 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
Reported statistics and error estimates:
ECX and NOEC
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.

95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Inhibition of Respiration Rate

The following results were derived for the control substance

 

 

3,5-dichlorophenol

ECx (3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

EC10

2.0

-

EC20

2.8

-

EC50

8.1

6.4 - 10

EC80

24

-

 

  pH Values of the Test Item Stock Solutions prior to the Addition of Inoculum in the Range-Finding Test

Nominal
Concentration
(mg/L)

pH

Prior to Adjustment

After Adjustment

Test Item

25

6.9

7.2

 

250

6.1

7.5

 

2500

5.8

7.2

 

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

6.8

76

 

R2

6.6

74

 

R3

5.8

65

 

R4

6.8

76

Test Item

10

5.8

65

 

100

5.9

66

 

1000 R1

5.8

65

 

1000 R2

8.6

97

 

1000 R3

5.7

64

3,5-dichlorophenol

3.2

6.4

72

 

10

7.9

89

 

32

8.6

97

R1– R4= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Timein 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

5.4

6

2.1

33.00

-

 

R2

5.6

6

2.4

32.00

-

 

R3

5.9

7

2.3

30.86

-

 

R4

6.3

8

2.3

30.00

-

Test Item

10

5.3

6

2.1

32.00

[2]

 

100

5.7

7

2.1

30.86

2

 

1000 R1

5.1

6

2.0

31.00

1

 

1000 R2

6.3

8

2.6

27.75

12

 

1000 R3

6.0

8

2.2

28.50

9

3,5-dichlorophenol

3.2

6.2

10

2.1

24.60

22

 

10

6.1

6

4.8

13.00

59

 

32

7.6

10

6.9

4.20

87

[Increase in respiration rate as compared to the controls]

R1– R4= Replicates 1 to 4

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

7.8

 

R2

7.5

7.8

 

R3

7.6

7.8

 

R4

7.6

7.8

Test Item

10

7.6

7.8

 

100

7.6

7.8

 

1000 R1

7.5

7.7

 

1000 R2

7.5

7.8

 

1000 R3

7.5

7.8

3,5-dichlorophenol

3.2

7.5

8.0

 

10

7.5

8.1

 

32

7.6

8.2

R1– R4= Replicates 1 to 4

Observations on the Test Preparations throughout the Test Periodin 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

Dark blue dispersion, no undissolved test item visible

Dark grey dispersion, no undissolved test item visible

Dark grey dispersion, no undissolved test item visible

 

100

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

 

1000 R1

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

 

1000 R2

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

 

1000 R3

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

Dark blue dispersion, no undissolved test item visible

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

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The study on 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.
Executive summary:

A study according to OECD Guideline 209, was performed to assess the effect of the test item on the respiration of activated sewage sludge. Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 20°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. Based on the findings of the study, the 3‑Hour EC50 value was determined to be greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000 mg/L.

Description of key information

The 3-hour EC50 value of the test item in the activated sludge respiration inhibition test was greater than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

A study according to OECD Guideline 209, was performed to assess the effect of the test item on the respiration of activated sewage sludge. Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 20°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. Based on the findings of the study, the 3‑Hour EC50 value was determined to be greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.