Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 13 March 2018; Experimental completion date 13 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: ESA (1,3,2-Dioxathiolane, 2,2-dioxide)
CAS Number: 1072-53-3
Batch: ESA-170926-160
Purity: 100.0%
Physical state/Appearance: white solid
Expiry Date: 25 June 2018
Storage Conditions: frozen at approximately -20 ºC in the dark
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was dissolved directly in water.
A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 20 minutes 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.
The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0.

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 12 March 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

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 7.3 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 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.

* Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
approximately 20 °C
pH:
7.3 - 7.9
Dissolved oxygen:
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.
Nominal and measured concentrations:
nominal test concentrations of 10, 100 and 1000 mg/L.
Details on test conditions:
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. Two additional control vessels were then prepared 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 approximately 20 °C.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol,
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
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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 concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Based on these results and after discussion with the Sponsor, it was considered not necessary to perform a definitive test.

Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.9 mg/L.
• The specific respiration rate of the controls was 27.67 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 2.4%.
All validation criteria for the study were therefore satisfied.

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 EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.9 mg/L.

 Inhibition of RespirationRate

 The following results were derived:

 

ESA (1,3,2-Dioxathiolane, 2,2-dioxide)

3,5-Dichlorophenol

ECx(3 Hours) (mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours) (mg/L)

95% Confidence Limits (mg/L)

EC10

>100

-

2.1

-

EC20

>1000

-

3.0

-

EC50

>1000

-

8.9

7.0 - 11

EC80

>1000

-

27

-

NOEC

100

-

-

-

 

pH Values of the Test Item Stock Solutions prior to theAdditionof Inoculum in the Range-FindingTest

 

Nominal Concentration (mg/L)

pH

Prior to Adjustment

After Adjustment

 

 

Test Item

25

3.7

7.3

250

2.7

7.9

2500

*

7.0

 

 *pH meter did not give a reading as the pH of the stock solution was out of range for the pH meter. The pH reading on the meter dropped before showing that the pH was out of range indicating that the stock solution was acidic. The manufacturer’s manual for the pH meter indicates that the pH meter will not take readings below pH 2. Therefore it is reasonable to assume that the pH of the 2500 mg/L stock solution was below pH 2

Dissolved Oxygen Concentrations of the TestPreparationsafter 30 Minutes Contact Time in the Range-FindingTest

 

 

Nominal Concentration (mg/L)

Dissolved Oxygen Concentration (mg O2/L)

Dissolved Oxygen Saturation Level of 8.9 mg O2/L

(%)

 

 

 

Control

R1

5.75

65

R2

5.68

64

R3

6.38

72

R4

6.01

68

 

 

 

 

Test Item

10

6.73

76

100

6.81

77

1000 R1

5.99

67

1000 R2

6.03

68

1000 R3

6.14

69

 

 

3,5-Dichlorophenol

3.2

6.72

76

10

7.28

82

32

7.68

86

 R  = Replicate

Oxygen Consumption Rates and Percentage InhibitionValuesafter 3 Hours Contact Time in the Range-FindingTest

 

 

Nominal Concentration (mg/L)

 

Initial O2Reading (mgO2/L)

 

Measurement Period (minutes)

 

Final O2Reading (mgO2/L)

O2

ConsumptionRates

(mg O2/L/hour)

 

Inhibition(%)

 

 

 

Control

R1

3.5

2

2.1

42.00

-

R2

4.2

3

2.2

40.00

-

R3

5.5

5

2.0

42.00

-

R4

4.2

3

2.1

42.00

-

 

 

 

 

Test Item

10

3.9

3

1.6

46.00

[11]

100

4.1

3

2.1

40.00

4

1000 R1

5.0

5

2.1

34.80

16

1000 R2

5.2

5

2.3

34.80

16

1000 R3

5.5

6

2.1

34.00

18

 

 

3,5-

Dichlorophenol

3.2

5.1

6

1.9

32.00

23

10

6.6

10

3.2

20.40

51

32

7.6

10

6.6

6.00

86

 

 R  = Replicate

-    = Notapplicable

[ ] = Increase in respiration rate as compared to controls

 

 

 

 

 

 

 

 

 

 

 

 

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.
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 solution 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 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.

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.

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

The reference item gave a 3-Hour EC50 value of 8.9 mg/L, 95% confidence limits 7.0 to 11 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)".

Activated sewage sludge was exposed to an aqueous solution 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 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.

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.

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

The reference item gave a 3-Hour EC50 value of 8.9 mg/L, 95% confidence limits 7.0 to 11 mg/L.

Key value for chemical safety assessment

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

Additional information