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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 February 2010 to 8 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009
Analytical monitoring:
no
Details on sampling:
- Concentrations:
Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L

- Sampling method:
As 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.


Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
For the purpose of the test, the test material was dispersed in water.

Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.

The control group was maintained under identical conditions but not exposed to the test material.

- Controls:
For the purpose of the test a reference item, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ) was used. Two stock solutions of 50 and 160 mg/L were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. Aliquots (10 and 100 mL) of the 160 mg/L stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/L. Similarly, a 100 mL aliquot of the 50 mg/L stock solution was used to prepare the 10 mg/L concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.








Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Method of cultivation:
A mixed population of activated sewage sludge micro-organisms was obtained on 3 February 2010 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 for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample was 7.4 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 105°C 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 3.7 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference item and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
Hardness:
Water with a total hardness of approximately 140 mg/L as CaCO3.
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21 ± 1°C.
pH:
The pH of the control, reference item and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
pH range for test items: 7.5 - 7.9
Dissolved oxygen:
Measured in the control, reference item and test material preparations using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
Nominal and measured concentrations:
Nominal concentrations of 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 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 approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. Finally a second control was prepared.

As 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 approximately 6.5 mg O2/L and 2.5 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.

The test was conducted under normal laboratory lighting in a temperature controlled room at 21 ± 1°C.

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. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 (see attached background material).

- Total organic carbon: 1.018 mg/L
- Chlorine: 0.307 mg/L
- Conductivity: 386.192 μS/cm at 20°C
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
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 litre of water with the aid of ultrasonication.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.

TEST CONCENTRATIONS
- Range finding study: No range finding study.

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:
growth inhibition
Remarks on result:
other: No 95% CL
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: No 95% CL
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.

The following results were derived:
3 hour EC20: >1000 mg/L
3 hour EC50: >1000 mg/L
3 hour EC80: > 1000 mg/L
3 hour NOEC: 1000 mg/L

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

Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 3%.

The validation criteria for the control respiration rates and reference item EC50 values were therefore satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 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.

Observations made at 0 hours, 30 minutes and 3 hours (see Table 3) showed that the control vessels contained a dark brown dispersion and the reference item vessels contained a dark brown dispersion with no undissolved reference item visible.

Observations of the test material vessels made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentration of 10 mg/L contained a clear colourless water column with very small particles of test material visible throughout and the test concentration of 32 mg/L contained a clear colourless water column with particles of test material visible throughout. The test concentration of 100 mg/L contained a very pale brown dispersion with particles of test material visible throughout and the test concentration of 320 mg/L contained a pale brown dispersion with particles of test material visible throughout. The test concentration of 1000 mg/L contained a brown dispersion with particles of test material visible throughout.

Observations of the test material vessels made after 30 minutes and 3 hours contact time showed that the test concentration of 10 mg/L contained a dark brown dispersion with no undissolved test material visible and the test concentration of 32 mg/L contained a dark brown dispersion with few particles of test material visible throughout. The test concentrations of 100, 320 and 1000 mg/L contained a dark brown dispersion with particles of test material visible throughout.




Results with reference substance (positive control):
Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol only (Figure 1 - attached background material).

The following results were derived for 3,5-dichlorophenol
3 hour EC20: 2.7 mg/L
3 hour EC50: 8.4 mg/L (95% Confidence limits 6.6 - 11 mg/L)
3 hour EC80: 26 mg/L
3 hour NOEC: 2.3 mg/L

Reported statistics and error estimates:
Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 3%.

The validation criteria for the control respiration rates and reference item EC50 values were therefore satisfied.

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal

Concentration

(mg/L)

Initial O2

Reading

(mg O2/L)

Measurement Period

(minutes)

Final O2 Reading

(mg O2/L)

O2Consumption Rates

(mg O2/L/min)

% Inhibition

Control

R1

6.0

6

2.5

0.58

-

 

R2

5.2

6

1.9

0.55

-

Test material

10

4.7

4

2.4

0.58

[3]

 

32

5.0

4

2.7

0.58

[3]

 

100

4.8

5

1.8

0.60

[6]

 

320

6.1

6

2.8

0.55

3

 

1000

5.6

6

2.3

0.55

3

3,5-dichlorophenol

3.2

6.2

8

2.7

0.44

22

 

10

7.4

10

5.1

0.23

59

 

32

8.0

10

7.1

0.09

84

[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

7.4

7.8

 

R2

7.6

7.8

Test material

10

7.5

7.7

 

32

7.5

7.7

 

100

7.6

7.8

 

320

7.6

7.9

 

1000

7.7

7.9

3,5-dichlorophenol

3.2

7.4

8.0

 

10

7.4

8.1

 

32

7.4

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 Time

3 Hours

Contact Time

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 water column with very small particles of test material visible throughout[*]

Dark brown dispersion with no undissolved test material visible

Dark brown dispersion with no undissolved test material visible

 

32

Clear colourless water column with particles of test material visible throughout*

Dark brown dispersion with few particles of test material visible throughout

Dark brown dispersion with few particles of test material visible throughout

 

100

Very pale brown dispersion with particles of test material visible throughout*

Dark brown dispersion with particles of test material visible throughout

Dark brown dispersion with particles of test material visible throughout

 

320

Pale brown dispersion with particles of test material visible throughout*

Dark brown dispersion with particles of test material visible throughout

Dark brown dispersion with particles of test material visible throughout

 

1000

Brown dispersion with particles of test material visible throughout*

Dark brown dispersion with particles of test material visible throughout

Dark brown dispersion with particles of test material visible throughout

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

 

10

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

 

32

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item visible

Dark brown dispersion, no undissolved reference item 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.
Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of 21 ± 1°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3 -hourour EC50of 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.

Description of key information

3 hour EC50 > 1000 mg/L; 3 hour NOEC = 1000 mg/L, OECD 209, EU Method C.11, EPA OPPTS 850.6800,  Youngs (2010) 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 3 hours at a temperature of 21 ± 1°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3 -hourour EC50of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/L.

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