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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Details on test solutions:
An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 60 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (80 and 250 ml) of the 200 mg/l stock solution and aliquots (80 and 250 ml) of the 2000 mg/l stock solution were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l. 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:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.1 measured using a WTW pH/Oxi 340I pH and dissolved oxygen 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC 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 3.9 g/l prior to use.
Limit test:
no
Total exposure duration:
3 h
Test temperature:
21±1°C.
pH:
The pH values measured during the test period are given in table 2
Nominal and measured concentrations:
32, 100, 320 and 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL darkened glass Biological Oxygen Demand (BOD) bottle
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature


OTHER TEST CONDITIONS
- Light intensity: Normal laboratory lighting


EFFECT PARAMETERS MEASURED : The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L).

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: respiration
Results with reference substance (positive control):
The reference material gave a 3-Hour EC50 value of 7.6 mg/L, 95% confidence limits 6.0 – 9.6 mg/L.

Table1     Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/l)

Measurent Period

(minutes)

Final O2Reading

(mg O2/l)

O2Consumption Rates

(mg O2/l/min)

% Inhibition

Control

R1

7.3

10

3.3

0.40

-

 

R2

7.0

10

2.9

0.41

-

Test Material

10

6.7

10

2.6

0.41

[1]

 

32

6.1

8

2.7

0.43

[6]

 

100

6.9

10

2.9

0.40

1

 

320

6.7

10

2.7

0.40

1

 

1000

6.7

10

2.5

0.42

[4]

3,5-dichlorophenol

3.2

7.3

8

4.8

0.31

23

 

10

7.8

10

6.3

0.15

63

 

32

8.4

10

7.9

0.05

88

Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

Table 2: pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration

(mg/l)

pH

0 Hours

3 Hours

Control

R1

7.3

7.9

 

R2

7.6

7.9

Test Material

10

7.6

7.8

 

32

7.6

7.9

 

100

7.7

7.9

 

320

7.5

7.8

 

1000

7.5

7.7

3,5-dichlorophenol

3.2

7.4

8.1

 

10

7.5

8.0

 

32

7.5

8.2

R1– R2= Replicates 1 to 2

Table 3: Observations on the test preparations throughout the test period

Nominal

Concentration

(mg/l)

Observations on Test Preparations

0 Hours

30 Minutes

Contact Ti

3 Hours

Contact Ti

Control

R1

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Dark brown dispersion

Test Material

10

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

32

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

100

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

320

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

 

1000

Clear colourless solution*

Dark brown dispersion, no undissolved test material visible

Dark brown dispersion, no undissolved test material visible

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible


R1– R2= Replicates 1 to 2

*Observations made prior to the addition of synthetic sewage and activated sewage sludge.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Description of key information

Calcium sulfate has no inhibitory effect on sewage sludge microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

An reliable OECD guideline Activated Sludge Respiration Inhibition (ASRI) study has been performed on calcium sulfate dihydrate. Sewage sludge microorganisms were exposed to nominal concentrations of 32, 100, 320 and 1000 mg/L for 3 h. The 3 h EC50 value was >1000 mg/L and the NOEC was 1000 mg/ (the highest dose tested)