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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:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations:
32, 100, 320, 1000 and 3200 mg/l

- Sampling method:
As each vessel reached 30 minutes 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.

This procedure was repeated after 3 hours contact time.
Vehicle:
no
Details on test solutions:
Range finding test:
In the range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg/L. The test item was dispersed directly in water.

An amount of test item (4000 mg) was dissolved in water and the volume adjusted to 1000 ml to give a 4000 mg/l stock solution from which a dilution was made to give a 40 mg/l stock solution. Aliquots of either the 40 mg/l or 4000 mg/l stock solution were separately dispersed in synthetic sewage, activated sewage and water to a final volume of 500ml to give the required test concentrations

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

Definitive study:
Based on the results of the range finding study the following test concentrations were used 32, 100, 320, 1000 and 3200 mg/l.
The test material was prepared by direct solution in water.
An amount of test material (6000mg) was dissolved in water and the volume adjusted to give a 6000 mg/l stock solution from which a dilution was made to give a 1000 mg/l stock solution. Aliquots of either the 1000 mg/l or 6000 mg/l stock solution were separately dispersed in synthetic sewage, activated sewage and water to a final volume of 500ml to give the required test concentrations

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

Observations were made on the test material preparations throughout the study period and the pH of the control, reference material and test material preparations was measured at the end of the exposure period prior to measurement of the oxygen consumption rate.

A reference item, 3,5-dichlorophenol, was included in the definitive test at concentrations of 3.2, 10, and 32 mg/L in order to confirm the suitability of the inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test Species:
A mixed population of activated sewage sludge micro-organisms was obtained on 27 October 1999 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, 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 21ºC and was used on the day of collection. The pH of the sample was 7.8 and the suspended solids equal to 4.0g/l prior to use.




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 litre of water.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The test was conducted in a temperature controlled room at 21 ºC.
pH:
The pH of the control, reference item and test item preparations was measured at the end of the exposure period.
Dissolved oxygen:
The oxygen concentrations in all vessels were measured after 30 minutes and 3 hours contact time.
Reference substance (positive control):
yes
Remarks:
3-5, dichlorophenol: 3.2, 10 and 32 mg/L
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 3 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 3 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
It was not possible to obtain an EC50 value for the test material due to the relatively flat response of the activated sewage sludge to the test material. An EC50 (30 minutes) value of 8800mg/l and an EC50 (3 hours) value of 5000mg/l can be obtained by extrapolation of the concentration-response curve, however care should be taken in the interpretation of these results gven that no data points for concentrations in excess of 3200 mg/l were determined from this study.

Based on the results of the range finding test it was considered inappropriate to test at concentrations greater than 3200 mg/l.

Oxygen consumption rates and percentage inhibition vlaues for the control, test and reference materials are given in the attached background information.
The pH at the end of the exposure period and the observations made on the tets material preparations are given in the attached background information.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was
EC50 (30 minutes) = 17mg/l
EC50 (3 hours) = 10mg/l

See attached background information

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (30 minutes and 3 hours) of the test material to municipal sewage microorganisms was > 3200 mg/L. The No Observed Effect Concnetration after 3 hours exposure was 320mg/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 (1984) No. 209 "Activated Sludge, Respiration Inhibition Test .

Methods

Following a preliminary range-finding study, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 32, 100, 320, 1000 and 3200 mg/L for a period of 3 hours at a temperature of 21 °C with the addition of a synthetic sewage as a respiratory substrate.

The rates of respiration was determined after 30 minutes and 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 total respiration of activated sewage sludge gave a 3-Hour EC50value greater than 3200 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.

Description of key information

The effect of the test item on the total respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 3200 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
3 200 mg/L
EC10 or NOEC for microorganisms:
320 mg/L

Additional information

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 (1984) No. 209 "Activated Sludge, Respiration Inhibition Test .

Methods

Following a preliminary range-finding study, activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 32, 100, 320, 1000 and 3200 mg/L for a period of 3 hours at a temperature of 21 °C with the addition of a synthetic sewage as a respiratory substrate.

The rates of respiration was determined after 30 minutes and 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 total respiration of activated sewage sludge gave a 3-Hour EC50value greater than 3200 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/l.