Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14/11/2008 to 26/11/2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No further information required.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Sample storage conditions before analysis: Room temperature in the dark

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test material was dissolved directly in water.
An amount of test material (2000 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. An aliquot (250 ml) of this stock solution was dispersed (in triplicate) 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 1000 mg/l. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the stock solution.

In the range-finding test an amount of test material (2000 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200, 20 and 2.0 mg/l stock solutions. An aliquot (250 ml) of the 2.0 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 1.0 mg/l. Similarly, aliquots (250 ml) of the 20, 200 and 2000 mg/l stock solutions were used to prepare the test concentrations of 10, 100 and 1000 mg/l.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.
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 material being added. Finally a second control was prepared.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations made throughout the test period showed that at the test concentration of 1000 mg/I no undissolved test material was visible.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source:
A mixed population of activated sewage sludge micro-organisms was obtained on 14 November 2008 for the range-finding test and on 26 November 2008 for the definitive test 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 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter WTW pH 320 pH 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 4.0 g/l prior to use.

Study design

Test type:
other: Aerobic
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Not performed

Test conditions

Hardness:
approximately 140 mg/l as CaCO3
Test temperature:
The test was conducted in a temperature controlled room at 21±1°C.
pH:
The pH of the samples was 8.0-8.5 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter WTW pH 320 pH meter.
Dissolved oxygen:
where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l.
Salinity:
n/a
Nominal and measured concentrations:
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. Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume:
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

- Aeration:
The mixtures were aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests 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.

- Pesticides: maximum 0.0001
- Metals: Various metals are present in the water at differing concentrations
- Alkalinity: Average pH of water is 7.719
- Conductivity: 421.385 µS/cm at 20°C

OTHER TEST CONDITIONS
- Adjustment of pH:
The pH of the samples was approximately 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter WTW pH 320 pH meter.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED:
Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
The test concentrations to be used in the definative test were determined by a preliminary range-finding test.

- Justification for using less concentrations than requested by guideline:
The definative study was performed as a limit test.

- Range finding study
- Test concentrations:
An amount of test material (2000 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200, 20 and 2.0 mg/l stock solutions. An aliquot (250 ml) of the 2.0 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 1.0 mg/l. Similarly, aliquots (250 ml) of the 20, 200 and 2000 mg/l stock solutions were used to prepare the test concentrations of 10, 100 and 1000 mg/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

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

A reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367), was included in the range-finding test at concentrations of 3.2 and 32 mg/I in order to confirm the suitability of the inoculum.

- Results used to determine the conditions for the definitive study:
No significant effect on respiration was observed at any of the test concentrations employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/I was selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
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
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
n/a
Results with reference substance (positive control):
- Results with reference substance valid:
The reference material gave a 3-Hour EC50 value of 7.3 mg/l, 95% confidence limits 5.6 - 9.5 mg/l.
A reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367), was included in the range-finding test at concentrations of 3.2 and 32 mg/l in order to confirm the suitability of the inoculum.

- Relevant effect levels:
95% confidence limits were calculated for the EC50 values for the reference material only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
Reported statistics and error estimates:
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.
The validation criteria for the control respiration rates and reference material EC50 values were satisfied.

Any other information on results incl. tables

Oxygen Consumption Rates and Percentage Inhibition Values inthe Definitive Test after 3 Hours Contact Time:

Nominal Concentration (mg/l) Initial O2 reading (mg O2 /l) Measurement period (Mins) Final O2 reading (mg O2 /l) O2 Consumption Rates (mg O2 /l) % Inhibition
Control R1 5.8 6 2.6 0.53
R2 5.5 6 2.0 0.58
Test Material 1000 R1 4.9 6 1.7 0.53 5
1000 R2 5.8 7 2.0 0.54 3
1000 R3 5.3 6 1.9 0.57 [3]
3,5-dichlorophenol 3.2 6.4 10 2.4 0.40 28
10 7.5 10 5.3 0.22 60
32 8.3 10 7.6 0.07 87

R1 - R3 = Replicates 1 to 3

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The validation criteria for the control respiration rates and reference material EC50 values were satisfied.
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:

In an Assessment of the inhibitory effect on the respiration of activated sewage sludge study (Harlan project number: 0959/0233) the test material had an EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

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

The validation criteria for the control respiration rates and reference material EC50values were 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 throughout the test period showed that at the test concentration of 1000 mg/l no undissolved test material was visible.