Benzenemethanol, 2,6-dichloro-3-fluoro-.alpha.-methyl-, (.alpha.S)-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-03-2011

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:

other: ISO1466

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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.

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 appropriate subsamples were provided for the test operators.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels, and were analysed for the concentration of the test substance. The concentrations of PF-00968603 in the test solutions were determined by the high performance liquid chromatography.

At the end of the 48 hour test period, the replicate test solutions were combined, sampled and analysed in the same manner.

Test solutions

Vehicle:

yes

Remarks:

dilution water

Details on test solutions:

This study was run with nominal PF-00968603 concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 mg/L together with a control.

PF-00968603 was soluble in water at a higher concentration than the test concentration selected. Therefore the test, solutions were prepared without the use of an organic solvent.

A 250 mL volume of a primary stock (50 mg/L) was prepared by the direct addition of PF-00968603 to dilution water. After approximately 30 minutes of ultrasonics this stock solution was observed to be clear and colourless. The test concentrations were prepared from this primary stock, using dilution water, to give final volumes of 100 mL. The control consisted of seawater only. All test solutions were observed to be clear and colourless.

Test organisms

Test organisms (species):

other aquatic crustacea: Tisbe battagliai

Details on test organisms:

The test species was the marine copepod, Tisbe battagliai, obtained from continuous laboratory cultures. The culture was characterised by Laboratorium Voor Ecologie En Systematiek, Vrije Universiteit, Brussels, Belgium.

The stock cultures of Tisbe battagliai were maintained in seawater, identical to the test dilution water, at a temperature of 20 ± 1 °C. The cultures were maintained in 2.5 L plastic vessels and were fed a defined diet of algae Rhodomonas reticulata, strain CCAP 995/2. A photoperiod of 16 hours light:8 hours dark, with 20 minute dusk and dawn transition periods was provided.

Tisbe battagliai 6 ± 2 days old (copepodid stage), obtained from a single culture vessel, were used for testing.

Study design

Test type:

not specified

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

24 and 48 hrs

Test conditions

Test temperature:

20.2 - 20.8ºC

pH:

8.1

Dissolved oxygen:

7.4 – 7.8 mg/L

Salinity:

35 ± 1

Nominal and measured concentrations:

Control and nominal concentrations 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 mg/L

Details on test conditions:

Test procedure and apparatus
Disposable plastic tissue culture cells (nominal volume 5 mL) with loose fitting lids were used as test vessels, with four replicates per test concentration. The test vessels were leached for at least 24 hours prior to use. Each vessel contained 5 mL of test solution.

The test was initiated by the addition of five impartially selected Tisbe battagliai to each test vessel in sequence across the treatments within 50 minutes of the test solution preparation. Each treatment contained a total of 20 Tisbe battagliai . The loading of the Tisbe battagliai in each test vessel was 1 Tisbe battagliai mL-1.

The nominal test solution temperature was 20 ± 1 °C, maintained by control of the room temperature. A photoperiod of 16 hours light:8 hours dark, with 20 minute dusk and dawn transition periods, was provided. The test solutions were not aerated and the Tisbe battagliai were not fed during the course of the study.

Preparation of test solutions
This study was run with nominal PF-00968603 concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 mg/L together with a control.

PF-00968603 was soluble in water at a higher concentration than the test concentration selected. Therefore the test, solutions were prepared without the use of an organic solvent.

A 250 mL volume of a primary stock (50 mg/L) was prepared by the direct addition of PF-00968603 to dilution water. After approximately 30 minutes of ultrasonics this stock solution was observed to be clear and colourless. The test concentrations were prepared from this primary stock, using dilution water, to give final volumes of 100 mL. The control consisted of seawater only. All test solutions were observed to be clear and colourless.

Observation of effects
An assessment of the response of the Tisbe battagliai was made 24 and 48 hours after the commencement of the test. Each Tisbe battagliai was viewed by microscope and mortality was defined as the absence of any movement within a period of 15 seconds.

The numbers of mortalities in the replicates of the control and each test concentration were summed for each time period.

Analytical method
At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels, and were analysed for the concentration of the test substance. The concentrations of PF-00968603 in the test solutions were determined by the high performance liquid chromatography method.

At the end of the 48 hour test period, the replicate test solutions were combined, sampled and analysed in the same manner.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Analytical data
The concentrations of PF-0096603 determined in the test solutions are given in Table 1, together with their limits of quantification. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentrations at the start of the test ranged from 94 to 97% of the nominal values. After 48 hours the measured concentrations ranged from 90 to 96%.

The analysis of the triplicate samples, taken from the 1.56 mg/L exposure concentration, showed consistency throughout the study.

Taking all these factors into consideration, nominal concentrations have been used for the reporting of results.

Biological data
The LC50 is defined as the concentration, calculated from the data obtained, resulting in the death of 50 % of the Tisbe battagliai in the time period specified.

The results obtained (based on nominal concentrations) of PF-00968603 were:

Time LC50 95 % Confidence interval Calculation method

24 hour 35 mg/L 30 – 44 mg/L Moving average angle
48 hour 28 mg/L 43 – 61 mg/L Moving average angle

The NOEC (No Observed Effect Concentration) is defined as the highest tested concentration in which there was no observed effect (mortality) on the Tisbe battagliai within the period of the test, therefore,

48 hour NOEC = 12.5 mg/L

100% mortality occurred in the 50 mg/L concentration, no mortalities were observed in the control.

Physical and chemical data
Dissolved oxygen concentrations ranged from 7.4 to 7.8 mg/L and the pH values were all 8.1. At no time during the course of the study was dissolved oxygen concentration in any of the test vessels less than 60 % (4.4 mg/L) of the air-saturation value. The thermometer readings at 0, 24 and 48 hours were 20.2, 20.8 and 20.4°C.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

48 hour No Observed Effect Concentration (NOEC) based on mortality = 12.5 mg/L

Executive summary:

Sponsor: Pfizer EH&S, Pfizer Inc., 50 Pequot Avenue, New London, Connecticut 06320, USA Sponsor contact F Mastrocco International telephone:   [phone]

Test facility, location of raw data and final report: Brixham Environmental Laboratory, AstraZeneca UK Limited, Freshwater Quarry, Brixham, Devon, TQ5 8BA, UK

Test substance: common name PF-00968603

Subject: Acute toxicity to Tisbe battagliai

Guideline: ISO14669

Source of organisms: Continuous laboratory cultures; 6 ± 2 days old, copepodid stage

Exposure concentrations: Control and nominal concentrations 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 mg/L

Length of test: 48 hours

Test conditions

Temperature: 20.2 - 20.8ºC

Dissolved oxygen: 7.4 – 7.8 mg/L pH 8.1

Photoperiod: 16 hours light:8 hours dark, with 20 min dawn and dusk transition periods

Results based on nominal concentrations of PF-00968603

Time              LC50       Calculation method 95% Confidence Interval

24 hour        35 mg/L           Moving average angle 30 -44 mg/L

48 hour        28 mg/L           Moving average angle 25 -33 mg/L

 48 hour No Observed Effect Concentration (NOEC) based on mortality = 12.5 mg/L