Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 April 2005, 27 April 2005,and 9 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in 2005 according to OECD Method 209 and in accordance with GLP. Study material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material is a extremely light tan colored powder which was received at testing laboratory on 15 February 2005 and stored at room temperature in the dark.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
A reference material, 3,5-dichlorophenol was used. Stock solutions were prepared and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2, 10 and 32 mg/L concentration. The volumetric flasks containing the reference material were inverted several times to ensure homogeneity of the solutions.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Test material was dissolved in water with the aid of high shear mixing and dilutions to give a 8000 mg/L ,1000 and 100 mg/L stock solutions. An aliquot of the 100 mg/L stock solution was dispersed with synthetic sewage, activated sewage sludge and water, to give the required concentration of 32 mg/L. Similarly, aliquots were prepared for the additional test concentrations of 100, 320, 1000 and 3200 mg/L. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions. Preparation of the test solutions were performed as quickly as possible at the start of the study.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Method of cultivation: activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire,UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was 7.9
- Initial biomass concentration: suspended solids determined to be equal to 4.0 g/L prior to use
The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated, partly softened and then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
total hardness of approximately 100 mg/L as CaC03.
Test temperature:
21 C
pH:
pH of test preparations at end of exposure period:
controls 8.1
test article: 7.9 to 8.1
positve control: 8.2-8.4
Dissolved oxygen:
The test vessels will be aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 -1 litre/min.
Salinity:
Not determined
Nominal and measured concentrations:
Nominal as no sample analysis completed.
Details on test conditions:

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 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 material was dissolved directly in water. A reference material, 3,5-dichlorophenol, 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.
- Results used to determine the conditions for the definitive study:
- no significant effects on respiration at the test concentrations of 1.0, 10 and 100 mg/L
-14% inhibition of respiration was observed at 1000 mg/L and could be the No Observed Effect Level
- In order to possibly obtain a 3-Hour EC50 value for the test material, it was considered justifiable to use a range of test concentrations with a test concentration in excess of 1000 mg/L The test concentration range of 32, 100, 320, 1000 and 3200 mg/L was therefore selected for the definitive test.

- Definitive Study Study Details:
- Starting at time 0, it is important that the control, reference and test solutions were prepared as quickly as possible and added the conical flasks which were then aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5-1 litre/minute.
Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the test ot reference material being added.
- After 30 minutes contact time, a sample was taken and poured into a darkened glass Biological Oxygen Demand (BOD) bottle. The rate of respiration was measured using a dissolved oxygen meter fitted with a BOD probe. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace for approximately 10 minute period between approximately8.2 mg 02/L and 1.7 mg 02/L when the respiration rate is low. After measurement the contents of the BOD bottle were returned to the test vessel. This procedure was repeated after 3 hours contact time. The oxygen consumption for each culture after 30 minutes and 3 hours contact time is measured and recorded by a chart recorder. 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21 C. Observations will be made on the test preparations during the exposure period for reasons such as precipitation/settlement of the test material.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 800 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:
An initial experiment was performed but the results from this experiment were considered to be inconsistent after 30 minutes contact time as significant inhibition was observed at the test concentrations of 100 and 1000 mg/L and no significant inhibition at the test concentration of 320 mg/L. It was therefore considered appropriate to repeat this experiment and not report the results obtained from this experiment.
Results with reference substance (positive control):
The reference material gave a 3-Hour EC50 value of 10 mg/L.
Reported statistics and error estimates:
As replication of test vessels is not a requirement of the testing guideline statistical analysis of the data can not be performed.
EC50 values are rounded to 2 significant figures.

Any other information on results incl. tables

BMS 296796 -02: EC50 (30 minutes): not obtained for the test material* (*36% inhibition was noted at the highest test concentration of 3200 mg/l)

3,5-dichlorophenol EC50 (30 minutes):16 mg/l

Variation in respiration rates of controls 1 and 2 after 30 minutes:+/- 2%.

The No Observed Effect Concentration (NOEC) after 30 minutes was 1000 mg/L.

BMS 296796-02: EC50 (3 hours): 2800 mg/L

3,5-dichlorophenol EC50 (3 hours): 10 mg/L

Variation in respiration rates of controls 1 and 2 after 3 hours:+/-1%.

The No Observed Effect Concentration (NOEC) after 3 hours was 1000 mg/L.

The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.

Observations made throughout the study period showed that at all test concentrations employed no undissolved test material was visible.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Valid as the two control respiration rate was 1% (within 15% of each other)and the EC50 for 3,5-dichlorophenol was 10 mg/L (within the range of 5 to 30 mg/L.)
Conclusions:
The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 2800 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 2800 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The aquatic test was considered valid and assigned a reliability rating of 1 (reliable without restriction).