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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 to 17 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(1S)-1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol
EC Number:
700-699-9
Cas Number:
877397-65-4
Molecular formula:
C8H7Cl2FO
IUPAC Name:
(1S)-1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol
Specific details on test material used for the study:
The test substance, PF-00968603, was supplied by Pfizer Limited, Analytical Research & Development, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK.

Material name: PF-00968603

The test substance was received at Brixham Environmental Laboratory on 26 May 2010 and assigned the Brixham test substance number 10-0146. The test substance (Batch Sample Ref E010010678) was supplied as a white crystalline solid.

The sample was stored at ambient temperature, in the container in which it was received until required for testing, when an appropriate subsample was provided for the test operator.

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Vehicle:
yes
Details on test solutions:
Test substance PF-00968603 was added to reverse osmosis (RO) water to make a nominal 5000 mg/L stock solution. This solution was stirred overnight, but the test substance did not dissolve. Therefore, the test solutions were prepared by the direct addition of a known weight of PF-00968603 to a total volume of 500 mL reverse osmosis (RO) water, synthetic sewage and activated sludge.

Reference substance A nominal 500 mg/L stock solution of the reference substance, 3,5-dichlorophenol, was prepared in RO water. This stock solution was observed to be clear and colourless and its pH was measured as 7.5 before use.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge
Activated sludge was obtained from Totnes Sewage Works, Totnes, Devon, UK, 1 day prior to the exposure of test and reference substances. This works treats sewage of predominantly domestic origin.

The activated sludge was settled, the supernatant decanted and the concentrate washed with tap water. The washed settled sludge was fed with 50 mL of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test.

The total filterable solids concentration was determined on the day of the test and was found to be 5300 mg/L. The pH was measured as 5.2, and was subsequently adjusted to 7.5 with 2 M sodium hydroxide prior to use.

Synthetic sewage
A synthetic sewage mixture described by the OECD guideline was prepared containing the following constituents per litre of water: 16.0 g of peptone, 11.0 g of meat extract, 3.0 g of urea, 0.7 g of NaCl, 0.4 g of CaCl2.2H2O, 0.2 g of MgSO4.7H2O and 2.8 g of K2HPO4. 4

Study design

Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
20 +/- 2oC
pH:
7.5
Nominal and measured concentrations:
PF-00968603 was added to reverse osmosis (RO) water to make a nominal 5000 mg/L stock solution.
Reference substance A nominal 500 mg/L stock solution of the reference substance, 3,5-dichlorophenol, was prepared in RO water.
Nominal 10, 32, 100, 320 and 1000 mg/L concentrations of PF-00968603 were prepared together with two control culture flasks. Five flasks containing the reference substance, 3,5-dichlorophenol, at nominal concentrations of 3.2, 10, 32 and 100 mg/L, and an abiotic flask at 100 mg/L were also prepared.
Details on test conditions:
This test measures the respiration rate of an activated sludge 3 hours after feeding an excess, but standard amount, of a synthetic sewage and compares this with the respiration rate of the same activated sludge in the presence of the test chemical. 3,5-dichlorophenol is used as a reference substance as it has known inhibitory effects on respiration and ensures that the batch of sludge used in the test shows a normal level of sensitivity.

Nominal 10, 32, 100, 320 and 1000 mg/L concentrations of PF-00968603 were prepared together with two control culture flasks. Five flasks containing the reference substance, 3,5-dichlorophenol, at nominal concentrations of 3.2, 10, 32 and 100 mg/L, and an abiotic flask at 100 mg/L were also prepared. The experimental design is shown in Table 1.

Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/L, an appropriate quantity of either PF-00968603 or 3,5-dichlorophenol stock solution and RO water to give a final flask contents volume of 500 mL. The pH of each flask was measured at the start and end of the test. After measuring the pH in the flasks containing PF-00968603 at the start of the test, the probe was rinsed into the flasks with a few mL of RO water to avoid removing un-dissolved test substance.

Flasks were set up in batches of six and aerated at 20 ± 2˚C for 3 hours. Each batch included a control flask and five test or reference substance flasks. The temperatures of the flask contents were measured at the end of the 3 hours aeration using a mercury-in-glass thermometer.

The respiration rate of each culture was measured after 3 hours and compared with the mean respiration rate of the two control cultures. The rate of oxygen uptake was measured in glass sample tubes into which microcathode oxygen electrodes were inserted. The electrodes were connected to an interface unit, which converted the current produced by the electrodes into dissolved oxygen readings. These were in turn transferred to a computer, which calculated the respiration rate in each flask over the linear part of the curve and compared it to the mean of the two control cultures using the system software. The rates of oxygen uptake were expressed as mg O2/L/h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 631 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
The respiration rates of the flasks dosed with the test substance or the reference substance were expressed as percentages of the mean of the respiration rate of the control flasks, from which the percentage inhibition was derived.

The EC50 value was estimated by the computer software.

In test flasks dosed with PF-00968603, inhibition was observed in the 1000 and 320 mg/L test concentrations.

Particulate material was observed in all the flasks containing PF-00968603, (except the 10 mg/L test concentration), which was considered to be undissolved PF-00968603.

The median effective concentration (EC50) is defined as the concentration, estimated from the data obtained resulting in a 50% reduction in the respiration rate of activated sludge within the period of the test, therefore,

3 hour EC50 = 631 mg/L
The 20% and 80% effect concentrations (EC20 and EC80) are defined as the concentrations, estimated from the 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 = 250 mg/L
3 hour EC80 >1000 mg/L

The 10% effect concentration (EC10) is defined as the concentration, estimated from the data obtained, resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage 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 PF-00968603. The no observed effect concentration (NOEC) is therefore equivalent to the EC10 in this test, therefore, 3 hour NOEC = 100 mg/L

The temperatures of the flask contents after the 3 hours aeration were all within the range 20 ± 2°C.
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 results obtained the 3 hour EC50 value was estimated to be 11 mg/L. This is within the expected normal range of 2 to 25 mg/L indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other. Therefore, the mean control respiration rate was used in calculation of the percentage inhibition.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
3 hour EC50 631 mg/L
3 hour EC20 EC20 = 250 mg/L
3 hour EC80 EC80 >1000 mg/L
3 hour NOEC 100 mg/L
Executive summary:

Sponsor Pfizer EH&S, Pfizer Inc., 50 Pequot Avenue, New London, Connecticut 06320, USA Sponsor contact F Mastrocco International telephone: (1) 516 567 8732 Test facility, location of raw data and final report Brixham Environmental Laboratory, AstraZeneca UK Limited, Freshwater Quarry, Brixham, Devon, TQ5 8BA, UK Test facility contact R H Swarbrick International telephone: (44) 1803 882882 International fax: (44) 1803 882974 Test substance common name

PF-00968603

Guideline OECD Guidelines for the Testing of Chemicals. Test Guideline 209, Activated Sludge, Respiration Inhibition Test. Adopted 22 July 2010. Subject Effect on the respiration of activated sludge Test type Aerobic Length of test 3 hours Test dates 16 to 17 August 2010 Nominal test temperature 20 ± 2°C 3 hour EC50 631 mg/L  3 hour EC20 3 hour EC80 EC20 = 250 mg/L EC80 >1000 mg/L 3 hour NOEC 100 mg/L Comments Results are based on nominal concentrations

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