Registration Dossier

Administrative data

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th March 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
30 May 1988
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
4 April 1984
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Light pink tinted powder
Specific details on test material used for the study:
Physical state/Appearance: Lightly pink tinted powder
Batch: Batch ref Y12511/002/001, bottle no. 287981)
Purity: 99.8% w/w

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: extracted into sampling tubes

Test solutions

Details on test solutions:
Test solutions were prepared by the direct addition of a known weight of UL125 to a total volume of 500 ml aerated deionised water, synthetic sewage and activated sludge. Nominal 1.0, 3.2, 10, 32 and 100 mg/l concentrations of UL125 were prepared together with twocontrol culture flasks. Four flasks containing the reference substance 3,5-dichlorophenol at nominal concentrations of 3.2, 10, 32 and 100 mg/l, plus one flask at 100 mg/l with no activated sludge, were also prepared.

Each flask contained an excess of synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/l, an appropriate quantity of either UL125 or 3,5-dichlorophenol stock solution and aerated deionised water to give a final flask contents volume of 500ml.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Buckland sewage treatment works, Newton Abbot, Devon, UK
- Pretreatment: the activated sludge was settled and the concentrated sludge was washed with mineral medium comprising 0.035 g of NaCl, 0.02g of CaCl2.2H2O and 0.01g of MgSO4.7H20 per litre of deionised water. the washed settled sludge was fed with 50ml of OECD synthetic sewage feed per litre of sludge per day and aerated at room temp until it was used in the test.
- Initial biomass concentration:total filterable solids concentration was determined on the day of the test and was found to be 4628 mg/l

Study design

Test type:
static
Water media type:
freshwater
Remarks:
Deionised, aerated water
Total exposure duration:
3 h

Test conditions

Test temperature:
20 +/- 2 degrees centrigrade
pH:
6.67 to 8.44
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Aeration: yes
- Sludge concentration (weight of dry solids per volume): total filterable solids concentration was determined on the day of the test and was found to be 4628 mg/l
- Nutrients provided for bacteria: synthetic sewage was prepared containing the follow constituents per litre of deionised wate - 15.2g of peptone, 10.5g of meat extract, 2.9g of urea, 0.7g of NaCl, 0.4g of CaCl2.2H2O, 0.2g of MgSO4.7H2O and 2.8g of K2HPO4.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: non
- Photoperiod: continuous illumination
- Temperature: 20 ± 2 °C
- Test concentrations: Positive Control (3,5-Dichlorophenol) - 100 (no activated sludge) , 100, 32, 10 & 3.2 mg/l. 2 control cuture flasks. Nominal 1.0, 3.2, 10, 32 and 100 mg/l UL125 flasks.
Reference substance (positive control):
yes
Remarks:
3,5-dicholorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Details on results:
In test flaskes dosed with UL125, all test concentrations showed inhibition less than or equal to 10%. Particulate matter was observed in 100, 32, and 10 mg/l test flasks which was considered to be undissolved UL125.

The Median effective concentration (EC50) is defined as the concentration, estimated rom data obtained, resulting in a 50% reduction in the respiration rate of activated sludge within the period of the test, therefore - 3 hour EC50 => 100 mg/l

The 20 and 80% effect Concentrations (EC20 and EC80) are defined as the concentrations, estimated from data obtained, resulting in 20 and 80% reduction respectively in the respiration rate of activated sludge within the period of the test, therefore - 3 hour EC20 > 100 mg/l and 3 hour EC80 > 100 mg/l

the 10% effect concentration (EC10) is defined as the concentation, estimated from the data obtained, resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentrage inhibition less than or equal to 10% was within the expected experimental variability of the test and was considered not to be an effect of the UL125. The No Obeserved Effect concentration (NOEC) is therefore equivalent to the EC10 in this test. Therefore 3 hour NOEC = 100 mg/l
Results with reference substance (positive control):
The reference substance 3,5-Dichlorophenol, caused substantial inhibition of the respiration rate of the activated sludge. From the resulted obtained, the 3 hour EC50 value was estimated to be 18 mg/l. this is within the expected normal range of 5 to 30 mg/l indicating the sludge was responding normally and confirmng the viability of the sludge organisms.

The respiration rates in the two control flasks (culture control) were within 15% of each other. Therefore, the mean control respiration rate was used in calculation of the percentage inhibition.

The Abiotic flask results shows 3,4-dichlorophenol did not cause abiotic depletion of oxygen concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes