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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 on 29 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Range-Finding TestTest Item PreparationIn 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 dispersed directly in water.A nominal amount of test item (2500 mg) was dispersed 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 dispersion from which dilutions were made to give 250 and 25 mg/L stock dispersions. An aliquot (200 mL) of the 25 mg/L stock dispersion 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 dispersions 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 dispersions were inverted several times to ensure homogeneity.
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 June 2015 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.Preparation of InoculumThe 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 7.7 measured using a Hach HQ160 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 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.* rinsed three 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
Post exposure observation period:
None
Hardness:
No data
Test temperature:
The temperature controlled room was maintained at approximately 20 ºC.
pH:
The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter. The pH ranged from 7.5 to 8.1 at the start and end of the exposure period in the range-finding test.
Dissolved oxygen:
The oxygen concentrations in all vessels were measured after 30 minutes contact time. The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/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.
Salinity:
No data
Nominal and measured concentrations:
Range-finding test: Nominal test concentrations of 10, 100 and 1000 mg/L.
Details on test conditions:
Test WaterThe test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).ProcedureRange-Finding TestTest Item PreparationIn 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 dispersed directly in water.A nominal amount of test item (2500 mg) was dispersed 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 dispersion from which dilutions were made to give 250 and 25 mg/L stock dispersions. An aliquot (200 mL) of the 25 mg/L stock dispersion 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 dispersions 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 dispersions were inverted several times to ensure homogeneity. The pH of the test item stock dispersions were measured using a Hach HQ160 Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0.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 SystemAt 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.EvaluationsObservationsObservations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.pH MeasurementsThe pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.Oxygen ConcentrationThe oxygen concentrations in all vessels were measured after 30 minutes contact time.Measurement of the Respiration RatesAs each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. Data EvaluationCalculation of the Oxygen Uptake RatesThe respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:R = [(Q1 - Q2) / Δt] x 60Where: Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L); Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L); Δt = the time interval between the beginning and end of the selected section of the linear phase (min).The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:RS = R / SSWhere:SS = the concentration of suspended solids in the test mixture (g dry weight/L).Calculation of Percentage of InhibitionThe percentage inhibition was calculated according to the following equation:% inhibition = [1 – (R/Rbc)] x 100Where:Rbc = the mean respiration rate of the blank controls.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Range-Finding TestNo 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.The 3 hours EC10 was > 100 mg/L.The 3 hours EC20 was > 100 mg/L.The 3 hours EC50 was > 1000 mg/L.The 3 hours EC80 was > 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 unnecessary to perform a definitive test in order to obtain a NOEC for the test item.
Results with reference substance (positive control):
The 3 hours EC10 was 1.4 mg/L.The 3 hours EC20 was 2.2 mg/L.The 3 hours EC50 was 8.2 mg/L with 95 % confidence limits of 6.1 - 11 mg/L.The 3 hours EC80 was 31 mg/L.
Reported statistics and error estimates:
ECX and NOECThe 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.The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.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 and all test concentrations 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).

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 4.1% and the specific respiration rate of the controls was 20.93 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

Inhibition of Respiration Rate

 

ZKKETIMIN

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

-

1.4

-

EC20

>100

-

2.2

-

EC50

>1000

 

8.2

6.1 - 11

EC80

>1000

-

31

-

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

Nominal
Concentration
(mg/L)

pH

Prior to Adjustment

After Adjustment

Test Item

25

7.9

-

 

250

8.6

7.7

 

2500

9.7

7.9

  - No adjustment required

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

6.5

73

 

R2

6.4

72

 

R3

7.1

80

 

R4

5.9

66

Test Item

10

6.4

72

 

100

6.2

70

 

1000 R1

6.4

72

 

1000 R2

6.6

74

 

1000 R3

5.6

63

3,5-dichlorophenol

3.2

6.6

74

 

10

7.9

89

 

32

8.5

96

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

5.2

6

1.9

33.00

-

 

R2

5.8

7

2.1

31.71

-

 

R3

6.2

8

2.2

30.00

-

 

R4

5.8

7

2.2

30.86

-

Test Item

10

5.6

7

1.9

31.71

1

 

100

6.0

8

1.9

30.75

2

 

1000 R1

7.0

10

3.5

21.00

33

 

1000 R2

6.5

10

2.9

21.60

31

 

1000 R3

6.5

10

2.9

21.60

31

3,5-dichlorophenol

3.2

6.5

10

2.6

23.40

25

 

10

7.4

10

5.4

12.00

62

 

32

8.3

10

7.1

7.20

77

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

7.5

7.8

 

R2

7.5

7.8

 

R3

7.6

7.9

 

R4

7.6

7.8

Test Item

10

7.7

7.9

 

100

7.7

7.9

 

1000 R1

7.8

8.1

 

1000 R2

7.8

8.0

 

1000 R3

7.8

8.0

3,5-dichlorophenol

3.2

7.6

8.0

 

10

7.6

8.1

 

32

7.7

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

Pale yellow/brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

100

Pale yellow/brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

1000 R1

Pale yellow/brown cloudy dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

1000 R2

Pale yellow/brown cloudy dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

 

1000 R3

Pale yellow/brown cloudy dispersion, no undissolved test item visible

Dark brown dispersion, no undissolved test item visible

Dark brown 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 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 and an EC10 value of greater than 100 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 (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.

 

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 and an EC10value of greater than 100 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 EC50value of 8.2 mg/L, 95% confidence limits 6.1 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)". 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 and an EC10 value of greater than 100 mg/L.  It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 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