REPORT 

Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 

(para-Chlorobenzotrifluoride (PCBTF)): 

Algal Growth Inhibition Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

Study Completion Date: 

H Vryenhoef 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

PCBTF Testing Consortium 

c/o Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

Mr. John V. Massingale 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

41304731 

21 April2015 

Page 1 of47 
Harlan Study Number: 41304731 Report Page2 

CONTENTS 

CONTENTS .................................................................................................................................... 2 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE .................................................... 4 

QUALITY ASSURANCE STATEMENT ...................................................................................... 5 

SIGNATURE OF CONTRIBUTING SCIENTISTS ...................................................................... 6 

SUMMARY .................................................................................................................................... ? 

Introduction ............................................................................................................................ 7 

Methods ............................................................................................................................... 7 

Results .................................................................................................................................. 7 

GENERAL INFORMATION ......................................................................................................... 9 

Schedule ................................................................................................................................. 9 

Deviations from Study Plan ................................................................................................... 9 

Archiving ............................................................................................................................... 9 

1 INTRODUCTION AND PURPOSE ................................................................................... 10 

1.1 Guidelines I Regulations ........................................................................................ 10 

2 TEST ITEM ......................................................................................................................... 11 

3 MATERIALS AND METHODS ......................................................................................... 12 

3.1 Definitions and Abbreviations ............................................................................... 12 

3.2 Test System ............................................................................................................ 12 

3.3 Test Water .............................................................................................................. 13 

3.4 Procedure ............................................................................................................... 13 

3 .4.1 Preliminary Media Preparation Trial.. ..................................................... 13 

3.4.2 Range-Finding Test ................................................................................. 13 

3.4.3 Definitive Test ......................................................................................... 14 

3.4.4 Evaluations .............................................................................................. 15 

3.4.5 Data Analysis ........................................................................................... 16 

3.5 Validation Criteria ................................................................................................. 18 

4 RESULTS ............................................................................................................................ 19 

4.1 Range-finding Test ................................................................................................ 19 

4.2 Definitive Test ....................................................................................................... 19 

4.2.1 Verification ofTest Concentrations ........................................................ 19 

4.2.2 Growth Data ............................................................................................ 19 

4.2.3 Validation Criteria ................................................................................... 20 

4.2.4 Observations on Cultures ........................................................................ 21 

4.2.5 Water Quality Criteria ............................................................................. 21 

4.2.6 Observations on Test Item Solubility ...................................................... 21 

5 CONCLUSION .................................................................................................................... 22 

6 REFERENCES .................................................................................................................... 23 

TABLES ........................................................................................................................................ 24 

FIGURES ...................................................................................................................................... 28 

APPENDICES ............................................................................................................................... 31 
Harlan Study Number: 41304731 Report Page 3 

LIST OF TABLES 

Table 1 

Table 2 

Table 3 

Table 4 

Cell Densities and Percentage Inhibition of Growth from the Rangefinding Test ................................................................................................................. 24 

Cell Densities and pH Values in the Definitive Test.. ................................................ 25 

Daily Specific Growth Rates for the Control Cultures in the Definitive 

Test .............................................................................................................................. 26 

Inhibition of Growth Rate and Yield in the Definitive Test ....................................... 27 

LIST OF FIGURES 

Figure 1 

Figure 2 

Mean Cell Densities v Time for the Definitive Test.. ................................................. 28 

Inhibition of Growth Rate Based on 0-Hour Measured Test 

Concentrations ............................................................................................................ 29 

Figure 3 Inhibition ofYield Based on 0-Hour Measured Test Concentrations ........................ 30 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Appendix 5 

Appendix 6 

Certificate of Analysis ........................................................................................... 31 

Positive Control ..................................................................................................... 32 

Culture Medium ..................................................................................................... 33 

Preliminary Media Preparation Trial ..................................................................... 34 

Analytical Investigations ....................................................................................... 35 

Monitoring Authority Statement of GLP Compliance .......................................... 4 7 
Harlan Study Number: 41304731 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Harlan Study Number: 41304731 

Study Title: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifluoride (PCBTF)): 

Algal Growth Inhibition Test 

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory 

Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994) ). These Regulations are 

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice 

(revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the 

requirements of Directives 2004/9/EC and 200411 0/EC. 

These principles are compatible with Good Laboratory Practice regulations specified by 

regulatory authorities throughout the European Community, the United States (EPA and FDA), 

and Japan (MHL W, MAFF and METI). 

This report fully and accurately reflects the procedures used and data generated. There were no 

circumstances considered to have affected the integrity of the study or the validity of the data. 

Study Director: H Vryenhoef 

.. 4~.L/ ...... . Date-: . 2~ 1 APR 2015 
Harlan Study Number: 41304731 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 41304731 

Study Title: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifluoride (PCBTF)): 

Algal Growth Inhibition Test 

The general facilities and activities are inspected at least once a year and the results are reported 

to the relevant responsible person and management. 

Study-related procedures conducted at the test facility were audited and inspected. The details of 

these audits and inspections are given below. 

Reported to the 

relevant Study 

Dates and Types of QA Inspections Director and Test 

Facility 

Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

07 November 2014 Study Plan N/A 07 November 2014 

Verification 

26 January 2015 Process - based Test Item Preparation 26 January 2015 

06 January 2015 Process - based Test System Preparation 06 January 2015 

and Exposure 

06 January 2015 Process - based Assessment of Response 06 January 2015 

20 January 2015 Process - based Chemical Analysis 20 January 2015 

12 March 2015 Report Audit N/A 12 March 2015 

Verification of Study Plan Amendment for GLP compliance was performed during the course of 

the study. 

This statement confirms that this report reflects the raw data and the procedures followed. 

Quality Assurance:~ \~~'C. 

....... ~ ................... . Date: 2 0 APR 2015 
Harlan Study Number: 41304731 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: DMMullee 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Date: 

2 1 APR 2015 
Harlan Study Number: 41304731 Report Page 7 

SUMMARY 

Introduction 

A study was performed to assess the effect of the test item on the growth of the green alga 

Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines 

for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth 

Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009. 

Methods 

Information provided by the Sponsor indicated the water solubility of the test item to be in the 

range of 29 - 34 mg/L. A preliminary media preparation trial indicated that a dissolved test item 

concentration of approximately 57 mg/L was obtained from a saturated solution method of 

preparation indicating this to be the limit of water solubility of this item under test conditions. 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to 

solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated 

solution (equivalent to 0-Hour measured concentrations of 0.37, 0.84, 3.7, 16 and 51 mg/L) (five 

replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a 

temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess 

(100 mg/L) oftest item in culture medium using a magnetic stirrer at a rate such that a 5% vortex 

was formed for 24 hours. After the stirring period any undissolved test item was removed by 

filtration (0.2 j.lm Sartorius Sartopore filter, first approximate 2 liters discarded in order to precondition the filter) to produce a 100% v/v saturated solution of the test item (equivalent to a 

0-Hour measured concentration of 51 mg/L). This saturated solution was then further diluted as 

necessary, to provide the remaining test groups. 

Samples of the algal populations were removed daily and cell concentrations determined for each 

control and treatment group, using a Coulter® Multisizer Particle Counter. 

Due to the volatile nature of the test item, testing was conducted in completely filled (i.e., no 

headspace ), stoppered test vessels in order to minimize possible losses due to volatilization. 

Following the recommendations of published data (Herman et al 1990) in order to prevent 

inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate 

was added to the culture medium to provide a source of carbon dioxide for algal growth. 

Results 

Analysis of the test preparations at 0 hours showed measured test concentrations of 0.37, 0.84, 

3.7, 16 to 51 mg/L were obtained from the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test 

preparations respectively. There was no significant change in the measured concentrations at 24, 

48 and 72 hours (85% to 98% of the 0-Hour measured concentrations) and so the results are 

based on 0-Hour measured test concentrations only. 
Harlan Study Number: 41304731 Report Page 8 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on 

the 0-Hour measured test concentrations: 

Response 95% Confidence No Observed Effect Lowest Observed 

ECso (mg/L) Concentration (NOEC) Effect Concentration Variable Limits (mg/L) (mg!L) (LOEC) (mg/L) 

Growth Rate 6.2 4.8 - 8.1 0.84 3.7 

Yield 2.2 1.8 - 2.6 0.84 3.7 
Harlan Study Number: 41304731 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Deviations from Study Plan 

29 December 2014 

23 January 2015 

There were no deviations (unplanned changes) from the study plan. 

Archiving 

Page 9 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), all raw data (paper and electronic) and the final report will be retained in the Harlan 

Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as 

to further retention or disposal. Further retention or return of the data will be chargeable to the 

Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41304731 Report Page 10 

1 INTRODUCTION AND PURPOSE 

This report contains a description of the methods used and results obtained during a study to 

investigate the effect of the test item on the growth of the green alga Pseudokirchneriella 

subcapitata. 

Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary 

producers found in natural waters and can therefore be considered as an important non-target 

organism in freshwater ecosystems. 

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water 

soluble test items, a modification of the standard method for the preparation of aqueous media 

was performed. An approach endorsed by several important regulatory authorities in the EU and 

elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of 

the test item in cases where the test item is of high purity and is poorly soluble in water and in 

the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was 

prepared by stirring an excess (100 mg/L) of test item in culture medium for a period of24 hours 

prior to removing any undissolved test item present by filtration (0.2 ~-tm Sartorius Sartopore, 

first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated 

solution of the test item. 

1.1 Guidelines I Regulations 

This study was designed to be compatible with the procedures indicated by the following 

internationally accepted guidelines and recommendations: 

• OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and 

Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission 

Regulation (EC) No 761/2009 

• OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances 

and Mixtures. OECD Series on Testing and Assessment, No. 23. OECD Publishing. 
Harlan Study Number: 41304731 Report Page ll 

2 TESTITEM 

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is 

given in Appendix 1. 

Identification: 

Physical state/ Appearance: 

Batch: 

Purity: 

Expiry date: 

Storage conditions: 

Intended use/ Application: 

Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 

(para-Chloro benzotrifluoride (PCB TF)) 

Clear colorless liquid 

2014-02-08 

99.77% 

17 February 2016 

Room temperature in the dark 

Chemical intermediate 
Harlan Study Number: 41304731 Report Page 12 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Biomass: 

Growth: 

Measurement variable (algal cell density) as surrogate measure 

for algal biomass. 

The calculated concentration of test item that results in an x% 

reduction of the respective growth parameter relative to the 

control within a stated exposure period. 

The increase in biomass over the test period. 

Growth Rate (Average Specific The logarithmic increase in biomass during the exposure 

Growth Rate): period. 

Yield: 

LOEC (Lowest Observed 

Effect Concentration): 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

Section-by-Section Growth 

Rate: 

3.2 Test System 

The value of the measurement variable at the end of the 

exposure period minus the measurement variable's value at the 

start of the exposure period, calculated to express biomass 

increase during the test. 

The lowest test concentration at which a statistically significant 

inhibition of growth is determined relative to the control. 

The highest test concentration at which no statistically 

significant inhibition of growth is determined relative to the 

control. 

Comprises test item treatments and control (test medium only). 

Specific growth rate for each day during the course of the test 

(days 0-1, 1-2 and 2-3). 

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid 

cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae 

and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, 

Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of 

culture medium (Section 3.3). The master cultures were maintained in the laboratory under 

constant aeration and constant illumination at 21 ± 1 °C. 

Prior to the start of the test sufficient master culture was added to approximately 100 mL 

volumes of culture media contained in conical flasks to give an initial cell density of 

approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept 

under constant agitation by orbital shaker (100- 150 rrm) and constant illumination at 24 ± 1 oc 

until the algal cell density was approximately 104 - 10 cells/mL. 
Harlan Study Number: 41304731 Report Page 13 

A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference 

item. Details of the positive control are given in Appendix 2. The positive control was 

conducted between 14 October 2014 and 31 October 2014. 

3.3 Test Water 

The culture medium used for both the range-finding and definitive tests was the same as that 

used to maintain the stock culture. For the purposes of testing an additional 500 mg/L of sodium 

bicarbonate was added to counteract the increase in pH due to algal growth in an enclosed 

system (Herman et a/ 1990). At the request of the Sponsor the pH of the culture medium was 

adjusted to pH 7.0 after the addition ofthe sodium bicarbonate. 

The culture medium is defined in Appendix 3. 

3.4 Procedure 

3.4.1 Preliminary Media Preparation Trial 

Information provided by the Sponsor indicated the water solubility of the test item to be in the 

range of29- 34 mg/L based on the U.S. EPA's citation of a Merck Index of unknown reliability. 

Based on this information the test item was categorized as being a 'difficult substance' as 

defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances 

and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to 

determine the solubility of the test item under test conditions (see Appendix 4). 

3.4.2 Range-Finding Test 

The results obtained from the preliminary media preparation trial conducted indicated that a 

dissolved test item concentration of approximately 57 mg/L could be obtained using a saturated 

solution method of preparation. 

The test concentrations to be used in the definitive test were determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata 

cells to a series of nominal test concentrations of 0.1 0, 1.0, 10 and 100% v/v saturated solution 

(equivalent to 0-Hour measured concentrations of 0.067, 0.60, 5.0 and 50 mg/L) for a period of 

72 hours. 

A nominal amount of test item (1090 mg) was dispersed in 10.9liters of culture medium with the 

aid of magnetic stirring at a rate such that a 5% vortex was formed for 24 hours. After 24 hours 

the stirring was stopped and any undissolved test item was removed by filtration through a 

0.2 J.lm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition 

the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this 
Harlan Study Number: 41304731 Report Page 14 

saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An 

aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension 

(2.7 mL) to give the required test concentrations ofO.lO, 1.0, 10 and 100% v/v saturated solution 

(equivalent to 0-Hour measured concentrations of0.068, 0.60, 5.0 and 50 mg/L). 

The test was conducted in 250 mL glass conical flasks each completely filled (i.e., no headspace) 

with test preparation and sealed with a ground glass stopper to reduce evaporation and minimize 

losses due to the test items volatile nature. Two replicate flasks were used for each control and 

test concentration. 

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

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 

incubator) at 24 ± 1 oc under continuous illumination (intensity approximately 7000 lux) 

provided by warm white lighting (380 - 730 run) and constantly shaken at approximately 

150 rpm for 72 hours. Previous experience gained has shown that the use of ground glass 

stoppers to seal test vessels is effective in attaining an airtight seal, thus preveting losses of test 

item through volatility. This was demonstrated in this test by the analytical results obtained at 0 

and 72 hours. 

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle 

Counter. 

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to 

determine the stability of the test item under test conditions. 

3.4.3 Definitive Test 

Based on the results of the range-finding test the following test concentrations were assigned to 

the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution. 

3 .4 .3 .1 Experimental Preparation 

A nominal amount of test item (11 00 mg) was dispersed in 11 liters of culture medium with the 

aid of magnetic stirring at a rate such that a 5% vortex was formed for 24 hours. After 24 hours 

the stirring was stopped and any undissolved test item was removed by filtration through a 

0.2 J.lm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition 

the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this 

saturated solution to give stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An 

aliquot (2 liters) of each of the stock solutions was separately inoculated with 5.2 mL of algal 

suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated 

solution (equivalent to 0-Hour measured concentrations of0.37, 0.84, 3.7, 16 and 51 mg/L). 
Harlan Study Number: 41304731 Report Page 15 

The stock solutions and each of the prepared concentrations were inverted several times to 

ensure adequate mixing and homogeneity. 

The concentration and stability of the test item in the test preparations were verified by chemical 

analysis at 0, 24, 48 and 72 hours (see Appendix 5). 

3.4.3.2 Exposure Conditions 

As in the range-finding test 250 mL glass conical flasks each completely filled (i.e., no 

headspace) with test solution were used. Of the five test replicates prepared at the start of the 

test: (a) one replicate was kept sealed (with no prior sampling) until 24 hours, at which time 

samples were taken for both cell density determination and chemical analysis; this replicate was 

then discarded; (b) one test replicate was kept sealed (with no prior sampling) until 48 hours, at 

which time samples were taken for both cell density determination and chemical analysis; this 

replicate was then discarded; (c) three test replicates were kept sealed (with no prior sampling) 

until 72 hours, at which time samples were taken for both cell density determination and 

chemical analysis. Therefore, each of the test replicates containing treated test solutions was 

used only once for sampling before being discarded. 

Of the nine control replicates prepared at the start of the test: (a) three replicates were kept sealed 

(with no prior sampling) until 24 hours, at which time samples were taken for cell density 

determination then discarded; (b) three replicates were kept sealed (with no prior sampling) until 

48 hours, at which time samples were taken for cell density determination then discarded; and (c) 

three replicates were kept sealed (with no prior sampling) until 72 hours, at which time samples 

were taken for both cell density determination and chemical analysis. 

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

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell 

density of 1.16 x 106 cells per mL. Inoculation of 2 liters of test medium with 5.2 mL of this 

algal suspension gave an initial nominal cell density of 3 x 1 03 cells per mL and had no 

significant dilution effect on the final test concentration. 

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 

incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) 

provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 

150 rpm for 72 hours. 

3.4.4 Evaluations 

3.4.4.1 Test Organism Observations 

The 0-Hour cell density was determined by electronic particle counting (Coulter® Multisizer 

Particle Counter) of the pre-culture and division by the dilution factor. Samples were taken at 
Harlan Study Number: 41304731 Report Page 16 

24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle 

Counter. 

To determine the potential effect of the test item on the appearance of algal cells, a sample was 

removed from each test and control culture (replicates pooled) at the end of the test. The shape 

and size of the algal cells was inspected microscopically and any abnormalities recorded. 

3.4.4.2 Water Quality Criteria 

The pH of the control and each test preparation was determined at initiation of the test and after 

72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The 

temperature within the incubator was recorded daily. 

3 .4 .4 .3 Verification of Test Concentrations 

Samples were taken from the control and each test group from the bulk test preparation at 

0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples 

were taken at 0 and 72 hours and stored frozen for further analysis if necessary. 

A sample of the vessel sacrificed for cell density determination at 24 and 48 hours was taken for 

chemical analysis. 

The method of analysis, recovery and test solution analyses are described in Appendix 5. 

3.4.5 Data Analysis 

3.4.5.1 Comparison of Growth Rates 

The average specific growth rate for a specified period is calculated as the logarithmic increase 

in biomass from the equation: 

Where: 

average specific growth rate from time t1 to tn 

cell concentration at t1 

cell concentration at tn 

time of first measurement 

time of nth measurement 
Harlan Study Number: 41304731 Report Page 17 

The average specific growth rate over the test duration was calculated for each replicate control 

and test item vessel using the nominally inoculated cell concentration as the starting value rather 

than the measured starting value in order to increase the precision of the calculation. 

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for 

the control cultures and the results examined in order to determine whether the growth rate 

remained constant. 

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the 

following equation: 

I = Jlc -J..tt X 100 r 

Where: 

Ir 

llc 

llt 

llc 

percentage inhibition of average specific growth rate 

mean average specific growth rate for the control cultures 

average specific growth rate for the test culture 

3.4.5.2 Comparison of Yield 

Yield is calculated as the increase in biomass over the exposure period using the following 

equation: 

Where: 

y 

No 

Nn 

yield 

cell concentration at the start of the test 

cell concentration at the end of the test 

For each test concentration and control the mean value for yield along with the standard 

deviation was calculated. Percentage inhibition of yield was calculated using the following 

equation: 
Harlan Study Number: 41304731 Report 

Where: 

percentage inhibition of yield 

mean value for yield in the control group 

mean value for yield for the treatment group 

3.4.5.3 Determination ofECx Values 

Page 18 

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) 

was plotted against test concentration (logarithmic axis) and a line fitted by computerized 

interpolation using the Xlfit software package (IDBS). ECx values were then determined from 

the equation for the fitted line. 

Where appropriate 95% confidence limits for the EC50 values were calculated, using the 

simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 

3.4.5.4 Statistical Analysis 

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal 

and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments 

with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours 

for the control and all test concentrations to determine any statistically significant differences 

between the test and control groups. All statistical analyses were performed using the SAS 

computer software package (SAS, 1999 - 2001 ). 

3.5 Validation Criteria 

The results of the test are considered valid if the following performance criteria are met: 

• The cell concentration of the control cultures must increase by a factor of at least 

16 over the test period. 

• The mean of the coefficients of variation of the section by section daily growth rates in 

the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not 

exceed 35%. 

• The coefficient of variation of the average specific growth rate in replicate control 

cultures must not exceed 7%. 
Harlan Study Number: 41304731 Report Page 19 

4 RESULTS 

4.1 Range-finding Test 

The cell densities and percentage inhibition of growth values from the exposure of 

Pseudokirchneriella subcapitata to the test item during the range-finding test are gtven m 

Table 1. 

The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated 

solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution. 

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution 

were selected for the definitive test. 

Chemical analysis of the test preparations at 0 hours (see Appendix 5) showed measured test 

concentrations to range from 0.067 to 50 mg/L whilst concentrations in the range of 0.059 to 

48 mg/L were observed at 72 hours (74% to 97% of the 0-Hour measured concentrations). 

4.2 Definitive Test 

4.2.1 Verification of Test Concentrations 

Analysis of the test preparations at 0 hours (see Appendix 5) showed measured test 

concentrations of 0.37, 0.84, 3.7, 16 to 51 mg/L were obtained from the 1.0, 3.2, 10, 32 and 

100% v/v saturated solution test preparations respectively .. There was no significant change in 

the measured concentrations at 24, 48 and 72 hours (85% to 98% of the 0-Hour measured 

concentrations) and so the results are based on 0-Hour measured test concentrations only as 

recommended in Section 39 of the OECD Test Guideline No. 201. 

4.2.2 Growth Data 

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given 

in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate 

and yield values for the control and test cultures after 72 hours and percentage inhibition values 

are given in Table 4. 

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage 

inhibition values are plotted against test concentration in Figure 2 and Figure 3. 

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of 

Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item 

over the 72-Hour exposure period. 

Accordingly the following results were determined from the data: 
Harlan Study Number: 41304731 

4.2.2.1 Inhibition of Growth Rate 

: 1.8 mg/L 

:2.9 mg!L 

Report 

ErCIO (0 - 72 h) 

ErC2o (0 - 72 h) 

ErCso (0 - 72 h) : 6.2 mg/L; 95% confidence limits 4.8 - 8.1 mg/L 

where ErCx is the test concentration that reduced growth rate by x%. 

Page 20 

Statistical analysis of the growth rate data was carried out for the control and all test 

concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity 

of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing 

several treatments with a control (Dunnett, 1955). There were no statistically significant 

differences between the control, 0.37 and 0.84 mg/L test concentrations (P~0.05), however all 

other test concentrations were significantly different (P<0.05) and, therefore the "No Observed 

Effect Concentration" (NOEC) based on growth rate was 0.84 mg/L. Correspondingly the 

"Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.7 mg/L. 

4.2.2.2 Inhibition of Yield 

:0.87 mg/L 

: 1.2 mg/L 

EyCIO (0 - 72 h) 

EyC2o (0 - 72 h) 

EyCso (0 - 72 h) : 2.2 mg/L; 95% confidence limits 1.8 - 2.6 mg/L 

Where: 

EyCx is the test concentration that reduced yield by x%. 

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no 

statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations 

(P~0.05), however all other test concentrations were significantly different (P<0.05) and, 

therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.84 mg/L. 

Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 

3.7 mg/L. 

4.2.3 Validation Criteria 

The following data show that the cell concentration of the control cultures increased by a factor 

of 190 after 72 hours. This increase was in line with the OECD Guideline that states the 

enhancement must be at least by a factor of 16 after 72 hours. 

Mean cell density of control at 0 hours 

Mean cell density of control at 72 hours 

3.00 x 103 cells per mL 

5.96 x 105 cells per mL 
Harlan Study Number: 41304731 Report Page 21 

The mean coefficient of variation for section by section specific growth rate for the control 

cultures was 22% and hence satisfied the validation criterion given in the OECD Guideline 

which states the mean must not exceed 35%. 

The coefficient of variation for average specific growth rate for the control cultures over the test 

period (0 - 72 h) was 2% and hence satisfied the validation criterion given in the OECD 

Guideline which states that this must not exceed 7%. 

4.2.4 Observations on Cultures 

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there 

were no abnormalities detected in the control or test cultures at 0.37, 0.84, 16 and 51 mg/L, 

however some enlarged/swollen cells were observed to be present in the test cultures at 

3.7 mg/L. 

4.2.5 Water Quality Criteria 

The pH values of the control and each test preparation are given in Table 2. Temperature was 

maintained at 24 ± 1 °C throughout the test. 

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.9 at 

0 hours to pH 9.4 at 72 hours. The pH deviation in the control cultures was equal to 1.5 pH units 

after 72 hours and therefore was within the limits given in the Test Guidelines. 

4.2.6 Observations on Test Item Solubility 

At the start of the test all control and test cultures were observed to be clear colorless solutions. 

After the 72-Hour test period all control, 0.37 and 0.84 mg/L test cultures were observed to be 

green dispersions. The 3.7 mg/L test cultures were observed to be pale green dispersions whilst 

the 16 and 51 mg/L test cultures were observed to be clear colorless solutions. 
Harlan Study Number: 41304731 Report Page 22 

5 CONCLUSION 

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been 

investigated over a 72-Hour period and based on the 0-Hour measured test concentrations gave 

the following results: 

Response 95% Confidence No Observed Effect Lowest Observed 

ECso (mg!L) Concentration (NOEC) Effect Concentration Variable Limits (mg/L) (mg/L) (LOEC) (mg/L) 

Growth Rate 6.2 4.8 - 8.1 0.84 3.7 

Yield 2.2 1.8 - 2.6 0.84 3.7 
Harlan Study Number: 41304731 Report Page 23 

6 REFERENCES 

DUNNETT, C.W. (1955) A Multiple Comparison Procedure for Comparing Several Treatments 

With a Control. JAm Stat As soc 50, 1096-1121. 

ENVIRONMENT DIRECTORATE, ORGANISATION FOR ECONOMIC CO-OPERATION 

AND DEVELOPMENT (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of 

Difficult Substances and Mixtures. 

EUROPEAN CENTRE FOR ECOTOXICOLOGY AND TOXICOLOGY OF CHEMICALS 

(ECETOC) Monograph No. 26 (1996) Aquatic Toxicity Testing of Sparingly Soluble, Volatile 

and Unstable Substances. 

HERMAN et al. (1990) Impact of volatile aromatic hydrocarbons, alone and in combination, on 

growth ofthe freshwater alga Selenastrum capricornutum. Aquatic Toxicology 18, 87- 100 

LITCHFIELD, J.T. and WILCOXON, F. (1949) A Simplified Method of Evaluating Dose-Effect 

Experiments. J Pharmacal Exp Ther 96,99-113. 

MAYER, P., NYHOLM, N., VERBRUGGEN, E. M. J., HERMENS, J. L. M. and TOLLS, J. 

(2000) Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. 

Environmental Toxicology and Chemistry, 19, pp. 2551-2556. 

SAS/STAT Proprietary Software Release 8.02 (1999- 2001), SAS Institute Inc, Cary, NC, USA. 

SOKAL, R.R. and ROHLF, F.J. (1981) Biometry. New York: W H FREEMAN and Company. 

Xlfit, ID Business Solutions Ltd. 
Harlan Study Number: 41304731 Report Page 24 

TABLES 

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test 

Cell Densities* (cells per mL) Inhibition Values(%) 

Nominal Concentration 

(% v/v Saturated Solution) 0 Hours 72 Hours Growth Rate Yield 

R1 3.00£+03 5.31£+05 

Control Rz 3.00£+03 5.94£+05 - -

Mean 3.00£+03 5.62£+05 

R1 3.00£+03 6.40£+05 

0.10 Rz 3.00£+03 5.12£+05 0 [2] 

Mean 3.00£+03 5.76£+05 

RI 3.00£+03 5.65£+05 

1.0 Rz 3.00£+03 4.63£+05 3 9 

Mean 3.00£+03 5.14£+05 

R1 3.00£+03 7.87£+04 

10 Rz 3.00£+03 6.20£+04 40 88 

Mean 3.00£+03 7.03£+04 

R1 3.00£+03 3.63£+03 

100 Rz 3.00£+03 2.99£+03 99 100 

Mean 3.00£+03 3.31£+03 

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 

3 counts for each of the replicate flasks. 

R1 and R2 = Replicates 1 and 2 

[Increase in growth compared to controls] 
Harlan Study Number: 41304731 Report Page 25 

Table 2 Cell Densities and pH Values in the Definitive Test 

Nominal Concentration pH Cell Densities* (cells per mL) 

(% v/v Saturated Solution) Oh Oh 24 h 48 h 72h 

Rt 3.00E+03 1.31E+04 1.18E+05 5.72E+05 

Rz 3.00E+03 1.16E+04 1.09E+05 5.50E+05 Control 7.9 

R3 3.00E+03 1.31E+04 1.11E+05 6.67E+05 

Mean 3.00E+03 1.26E+04 1.13E+05 5.96E+05 

Rt 3.00E+03 1.22E+04 1.23E+05 6.52E+05 

1.0 Rz 3.00E+03 6.10E+05 

7.7 

R3 3.00E+03 6.29E+05 

Mean 3.00E+03 1.22E+04 1.23E+05 6.30E+05 

Rt 3.00E+03 1.20E+04 8.41E+04 4.69E+05 

3.2 Rz 3.00E+03 6.29E+05 

7.6 

R3 3.00E+03 4.96E+05 

Mean 3.00E+03 1.20E+04 8.41E+04 5.32E+05 

Rt 3.00E+03 6.13E+03 2.97E+04 1.30E+05 

Rz 3.00E+03 1.37E+05 10 7.5 

R3 3.00E+03 1.39E+05 

Mean 3.00E+03 6.13E+03 2.97E+04 1.35E+05 

Rt 3.00E+03 2.75E+03 5.42E+03 6.19E+03 

32 Rz 3.00E+03 5.49E+03 7.5 

R3 3.00E+03 6.31E+03 

Mean 3.00E+03 2.75E+03 5.42E+03 5.99E+03 

Rt 3.00E+03 1.55E+03 3.74E+03 5.63E+03 

100 Rz 3.00E+03 4.63E+03 

7.4 

R3 3.00E+03 6.69E+03 

Mean 3.00E+03 1.55E+03 3.74E+03 5.65E+03 

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 

3 counts for each of the replicate flasks. 

R1 - R3 = Replicates 1 to 3 

pH 

72h 

9.4 

9.1 

9.2 

8.1 

7.7 

7.5 
Harlan Study Number: 41304731 Report Page 26 

Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive Test 

Daily Specific Growth Rate (cells/mL/hour) 

Day 0-1 Day 1-2 Day 2-3 

Rt 0.062 0.092 0.066 

R2 0.056 0.093 0.067 Control 

R3 0.062 0.089 0.075 

Mean 0.060 0.091 0.069 

R1 - R3 =Replicates 1 to 3 
Harlan Study Number: 41304731 Report Page 27 

Table 4 Inhibition of Growth Rate and Yield in the Definitive Test 

Nominal Concentration Growth Rate (cells/mL/hour) Yield (cells/mL) 

(% v/v Saturated Solution) 0-72h % Inhibition 0-72h % Inhibition* 

RI 0.073 - 5.69E+05 

R2 0.072 5.47E+05 

Control R3 0.075 - 6.64E+05 -

Mean 0.073 5.93E+05 

SD 0.002 6.22E+04 

RI 0.075 -3 6.49E+05 

R2 0.074 -1 6.07E+05 

1.0 R3 0.074 -1 6.26E+05 [6] 

Mean 0.074 -1 6.27E+05 

SD 0.001 2.12E+04 

RI 0.070 4 4.66E+05 

R2 0.074 -1 6.26E+05 

3.2 R3 0.071 3 4.93E+05 11 

Mean 0.072 2 5.29E+05 

SD 0.002 8.59E+04 

RI 0.052 29 1.27E+05 

R2 0.053 27 1.34E+05 

10 R3 0.053 27 1.36E+05 78 

Mean 0.053 27 1.32E+05 

SD 0.001 4.67E+03 

R1 0.010 86 3.19E+03 

R2 0.008 89 2.49E+03 

32 R3 0.010 86 3.31E+03 99 

Mean 0.009 88 2.99E+03 

SD 0.001 4.44E+02 

R1 0.009 88 2.63E+03 

R2 0.006 92 1.63E+03 

100 R3 0.011 85 3.69E+03 100 

Mean 0.009 88 2.65E+03 

SD 0.003 1.03E+03 

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is 

calculated 

R1 - R3 = Replicates 1 to 3 

SD = Standard Deviation 

[Increase in growth compare to controls] 
Harlan Study Number: 41304731 Report 

FIGURES 

Figure 1 Mean Cell Densities v Time for the Definitive Test 

l.OOE+06 -r--r---------------------------~---------_-__ -_-__ -_..,+------------------------------------------_-__ -_-__ -_-__ -_-__ -_..,1,.. __ -_-__ -_-__ -_-_-__ -_-__ -_-__ -_-__ -_-_-__ -_-__ -_-_-__ -_-__ -_-__ ., 

--A- Control ------------.!.-------------------------------------..:-------------------------------------- ' ' - -A- 0. 3 7 mg!' ·------------t-------------------------------------1-------------------------------------- 'L 0 0 

·-------------~--------------------------------------.,-------------------------------------

--- 0.84mg/l ! ! /', ------------+-------------------------------------1--------------------------------7.; -- --8- 3.7mg!L ! ! /. -4- 16 mg/L ·------------t·------------------------------------l------------------------~7,(. --

1 I / - -e- 51 mg/L ·------------4--------------------------------------+-------------------:>': --- ----------- 1 : / I I / : : / 

: : / -------------------------------------+-------------------------------------1----------y : : / I I / I I / I I / ! 1/ 

' / I I / 

:::::::::::::::::::::::::::::::::::::±::::::::::::::::::::::::::::::::L~_:i--:_:::::::::::::::::::::::~~::::::: 

--------------------------------------f.------------------------------.1____ ------------------------L----------- 1 I. : / -------------------------------------+----------------------------~ --- ---f---------------------"""'---------------

1 h : / -------------------------------------+------------------------7,: --- -----1-----------------..4.-------------------

l h : / -------------------------------------+----------------------z --- ---------1-----------/L----------------------- ~ h I / -------------------------------------.l------------------ -- ...................................... J ..................... L ................ ______________________ _ I I / 

l.OOE+OS 

I I / ' '/ -------------------------------------+------------- - -----------------':;$------------------------------------- : / I : / I I / I 

-------------------------------------..:..................... ---------------·-~---------1-------------------------------------- l / I : / I 

: / : : / : I / : 

/ : I / : ' / ' 

......... :::t::::::::;:~:::::::::::::::::::::::::j::::::================================ : / : ------------------------- -----------r----7-------------------------------,--------------------------------------

l.OOE+04 

_____________ .l ...... Jt' ....................................................................................... J ........................................................................................................ ... '/ ' ----------------- ---------------~------------------------------------~------------------------------- / : / I ~~ ---------:;:.-=---------T-------------------------------· --1-----------------------::;:-..--~--------

// : : ~~~ 

------ -:::.:-;;;-~---------------------t------------------- / I I -~ ----------------~-;;:-c::: ____________________________ _ 

/ I /1 / I / I / I / I 

........ ---------------------------------+--- --------------------;;,.,·-------~--------------------------------------

................ // I ........ , / : ' / ' .............. : / : .................................................................... , ............................... -t---------7L .................................................................. 1 ........................................................................................................ ... ....._ I / I 

...................... !// ! 

Cf' ! 

' ' ' ' ' ' ' ' ' ' ' ' ' ' l.OOE+03+-------------------~·--------------------~·~-------------------4 

0 24 48 72 

Time (Hours) 

Page 28 
Harlan Study Number: 41304731 Report Page29 

Figure 2 Inhibition of Growth Rate Based on 0-Hour Measured Test Concentrations 

100~--~--~~~~~~---,---r~-r~~~--~---r~~~~ 

80 

70 

~~~,~ ]i'' · , ::: r~: . ili!11 

60 I I I I I I I I I I I I I I I 

---------.. ------I--- __ ,_-- .. -- ----------.. - ---1--- ... - _,_ -1- _,_--------- ----- _,_--- .. ---I--_,_ _ _, __ ,_ -l1 I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I t I F 

:::: I I I t I I I I I I I I I I 

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I I I I I I I I I I I 

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:9 

..s:::: 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I 1 I I I I 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I ' ' .s 40 ~ 

F I I I I I I I I I I I I I I I I I I I -'- _ L_L _I ___________ i ______ , ____ J_ _ _ _ _l_ -'- _L_o_ J - _____ , ____ J. __ -L __ L _ _J- -'- -'-

1 I I I I I F I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I I I 

I I I I ---- _____ J ______ L ____ I ___ J __ 

I I I I 

I I I I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I I 

30 I I I I I I I I I I I I I I I I I I I -~- -~-~- -~---------- -;------ -~--- 1---T-- T- -~- -~--~- -~---------- ----- -~---- T-- -~-- -~-- "'i- -~- -~-

1 I I I I I I I I I I I I I 

I I I I I I I I I I I I I 

I I I I I I I I I I I I I 

I I I I I I I I I I I I I 

I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I 

20 I I I I I I I I I I I I I I I I 

----------r------ r--- -~---.,-- - -r-r- r -~----------- r----- ~---- -.--- r- -r- -~- -r -~- -.---------- ------.-----r -- r- -r-.,- -~- -.- 1 I I I I I I I I I I I I I I I I 

I I I I I I I I I I 

I I I I I I I I I I 

I I I I I I I I I I 

I I I I I I I I I I I 

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10 I I I I I I I I I 0 I 

----------t-- ----r- -- -• r------r---- -.--- ~-- ~- -1- -r- -o-..., I I I I I I I I I I I I 

------.-----t 

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I I I I I I I I I 

I I I I I I I I I 

I I I I I I I I I 

0 I I I I I I I I I 

----l-------1-----1---+- -l---1--1--1--1 

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------1----+ 

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I I I I I I I 

I I I I I I I 

I I I I I I 

-10 

0.1 10 100 

Concentration (mg!L) 
Harlan Study Number: 41304731 Report Page 30 

Figure 3 Inhibition of Yield Based on 0-Hour Measured Test Concentrations 

100r---~--~~'~' I I 

~,~,~----~--~~,~,~~~==~~~--~~~~ I I I I 

I I I I I I I 

I I I I I I I 

Yield , : : : , : ' : , : : , : , , , , , , : ----------r------ -r---- -.--- -·-- -r-- ;--r- -.- -·------------·-------·----_,_--"'I -.-- .. - -<- -·- -.------------.------ ----.,--- -- --,.- - .. - I I I I I 0 I I I I I I I I I It I I I I 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I I 

0 I 0 I 0 I I I I I I I I I I I I 

I I I I I I I I I I I I I I I I 

I I I I I I I I I I I I I I I --------r---- --·----.----, --.---.--.--,--·------------·-------·---- ·---.. ---.---.--;-...,-.,- ----- -----,--- --.- 1 I I I I I I I I I I I I 0 0 

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I I I I I I 

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I I I I I I I I I I I I I 

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-------~---- -·- ---'----· --r-!-T i : -----·- _..! --:--r-rrl ___ J_ _________ J_ __ - - - _J_ 

0 I I I I I I 

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10 _______ I ______ 1 _____ 1_ __, __ J_...J _ _I_J __________ J __________ J ___ -- __ J._ __ J _ 

-----'-------'- _______ I ______ , _____ I __ j __ _, __ J_...J_...!_J __________ J______ -- __ J._ __ J_ 

I I I I I I 

' I I I 

: : +: : 

10 100 

Concentration (mg'L) 
Harlan Study Number: 41304731 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

70 W&st 41)th Sire&! 

New York, NY 10018 

Phone: (646) 366.Q400 

Fa>c (646!36f>.0595 

CERTIFICATE OF ANALYSIS 

Product Name: 

Quality: 

Batch Number: 

Test/Property 

Acidity, ppm (spec. ion probe) 

Alkalinity. ppm as NaOH 

Appearance 

Assay,% 

Color (APHA) 

Non Volatile Residue, Wt % 

Specific Gravity (20C/25C) 

Water content, ppm 

Parachlorobenzotrifluoride-OOON 

SMC Spec. 900 

2014·02-08 

Specification 

3.0 PPM MAXIMUM 

10.0 PPM MAXIMUM 

Clear liquid, free of suspended 

material 

99.00 %MINIMUM 

20 APHA MAXIMUM 

0.0020% MAXIMUM 

1.3300 TO 1.3500 GICC 

150 PPM MAXIMUM 

CERTIFIED TRUE AND CORRECT W!Jl 

Approval Date: 2117/2014 

$pee Revision Date: 

Page 31 

Result 

1.7 

0.0 

Clear liquid, frflft of suspended 

material 

99.77 

10 

0.0013 

1.3340 

78 

Print Date: 11/5114 
Harlan Study Number: 41304731 Report Page 32 

Appendix 2 Positive Control 

A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference 

item at concentrations of0.050, 0.13, 0.32, 0.80 and 2.0 mg/L. 

Six flasks each completely filled with test preparation were used for the control, and three flasks 

each completely filled with test preparation were used for each test concentration. The flasks 

were sealed with ground glass stoppers and incubated at 24 ± 1 °C under continuous illumination 

(intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and 

constantly shaken at approximately 150 rpm for 72 hours. The culture medium used was 

prepared with the addition of500 mg/L NaHC03. 

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the 

following results: 

ErCso (0 - 72 h) 

EyCso (0 - 72 h) 

1.2 mg/L; 95% confidence limits 0.97 - 1.4 mg/L 

0.52 mg/L; 95% confidence limits 0.45 - 0.60 mg/L 

No Observed Effect Concentration (NOEC) based on growth rate: 

No Observed Effect Concentration (NOEC) based on yield: 

Lowest Observed Effect Concentration (LOEC) based on growth rate: 

Lowest Observed Effect Concentration (LOEC) based on yield: 

0.32 mg/L 

0.32 mg/L 

0.80 mg/L 

0.80 mg/L 

The results from the positive control with potassium dichromate were within the normal ranges 

for this reference item. 
Harlan Study Number: 41304731 Report Page 33 

Appendix3 Culture Medium 

NaN03 25.5 mg/L 

MgCh.6H20 12.16 mg/L 

CaCh.2H20 4.41 mg/L 

MgS04.1H20 14.6 mg/L 

K2HP04 1.044 mg/L 

NaHC03 15.0 mg/L 

H3B03 0.186 mg/L 

MnChAH20 0.415 mg/L 

ZnCh 0.00327 mg/L 

FeCh.6H20 0.160 mg/L 

CoCh.6H20 0.00143 mg/L 

Na2Mo04.2H20 0.00726 mg/L 

CuCh.2H20 0.000012 mg/L 

Na2EDTA.2H20 0.30 mg/L 

The culture medium was prepared using reverse osmosis purified deionized water*. For the 

purposes of the definitive test, additional sodium bicarbonate (500 mg/L) was added to the 

prepared culture medium prior to adjusting the pH to 7.0 with 0.1N NaOH or HCI. 

* Elga Optima 15+ or Elga Purelab Option R-15 BP 
Harlan Study Number: 41304731 Report Page 34 

Appendix 4 Preliminary Media Preparation Trial 

1. Saturated Solution Preparation 

A nominal amount of test item (1 090 mg) was dispersed, in duplicate, in 10.9 liters of culture 

medium with the aid of magnetic stirring at a rate such that a vortex was formed which 

represented approximately 5% of the overall media column height for periods of either 24 or 

48 hours. After stirring samples were taken for chemical analysis after the following pretreatments: 

• Centrifugation at 10000 g for 30 minutes 

• Centrifugation at 40000 g for 30 minutes 

• Filtration through a 0.2 J..Lm Sartorius Sartopore filter (approximately 1 liter discarded in 

order to pre-condition the filter) 

• Filtration through a 0.2 J..Lm Sartorius Sartopore filter (approximately 2 liters discarded in 

order to pre-condition the filter) 

2. Results 

Stirring Period and Treatment Concentration Found (mg/L) 

24 Hours Centrifuged 10000 g 19.0 

24 Hours Centrifuged 40000 g 11.4 

24 Hours Filtered ~ 1 liter discarded 57.2 

24 Hours Filtered ~ 2 liters discarded 56.7 

48 Hours Centrifuged 10000 g 18.5 

48 Hours Centrifuged 40000 g 9.90 

48 Hours Filtered ~ 1 liter discarded 54.2 

48 Hours Filtered ~ 2 liters discarded 59.0 

3. Discussion 

The results obtained indicated that significantly higher dissolved test item concentrations were 

obtained following removal of any undissolved test item by filtration rather than centrifugation. 

This was considered to be due to losses of test item through volatility during the time taken to 

remove the supernatant after centrifugation. 

Based on this information the test item was prepared using a saturated solution method of 

preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the 

removal of any undissolved test item by filtration through a 0.2 J..Lm Sartorius Sartopore filter 

(first approximate 2 liters discarded) to give a nominal test concentration of approximately 

57 mg/L, the limit of water solubility of this Test item under test conditions. 
Harlan Study Number: 41304731 Report Page 35 

Appendix 5 Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The test item concentration in the test samples was determined by gas chromatography (GC) 

using an external standard. The test item gave a chromatographic profile consisting of a single 

peak. 

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, 

Shardlow, UK. 

1.2 Test Item 

The test item is described in the biological part ofthis study. 

1.3 Analytical Standard 

The test item described in the biological part of this study was also used as the analytical 

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The samples were analyzed on the day of receipt. 

1.4.2 Reagents and Solvents 

Test medium: as described in the biological part of this study. 

Solvents: tetrahydrofuran, Sigma-Aldrich, HPLC grade. 

hexane, Fisher Scientific, pesticide residue grade. 
Harlan Study Number: 41304731 Report Page 36 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 100 mg) was dissolved in hexane (100 mL) to prepare a stock solution 

with a concentration of 1000 mg/L. This stock solution was further diluted with hexane to 

produce a solution of 50 mg/L. Defined volumes of this solution were then diluted with hexane 

to obtain calibration standards in the range of 0.1 to 1.0 mg/L. A second series of calibration 

standards was similarly prepared in the range of 0.25 to 2.5 mg/L. These solutions were used to 

determine the recovery and test sample concentrations. 

1.4.4 Preparation of Linearity Standards 

The test item (nominal 100 mg) was dissolved in hexane (100 mL) to prepare a stock solution 

with a concentration of 1 000 mg/L. Defined volumes of this solution were then diluted with 

hexane to obtain calibration standards in the range of 0.1 to 50 mg/L. A second series of 

calibration standards was similarly prepared in the range of 0.25 to 25 mg/L. These solutions 

were used to determine the test sample concentrations and to evaluate the linearity of the 

analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked 

with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially 

dissolved in tetrahydrofuran to prepare a stock solution with a concentration of 1000 mg/L. This 

stock solution was further diluted with tetrahydrofuran to produce a stock solution of 50 mg/L. 

A defined volume of this stock solution was diluted with test medium to obtain spiked recovery 

samples at a concentration of 0.050 mg!L. Five replicates were prepared and subjected to the 

same treatment as the test samples. In addition, test medium without the addition of the test item 

(synthetic control) was also analyzed. 

1.4.6 Preparation of Test Samples 

The test samples were analyzed on the day of receipt. The test samples were extracted directly 

with hexane in the same bottle. An aliquot of the hexane layer was taken for analysis. See 

Table 1 for more information on preparation volumes. 
Harlan Study Number: 41304731 Report 

Analytical Investigations 

1.4. 7 Instrumental Parameters 

Gas chromatograph: 

Detector: 

Column: 

Inlet liner: 

Carrier gas: 

Pressure: 

Oven temperature 

Program: 

Injection mode: 

Inlet purge time: 

Injection volume 

Injection temperature: 

Detector temperature: 

Retention time: 

HP 5890 Series II, with autosampler 

ECD 

ZB-624 

(60 m x 0.32 mm i.d.;1.8 !Jm film thickness) 

double gooseneck 

nitrogen 

10 psi 

oven: 40 °C, for 0 minutes 

with 15 °C/minute to 220 °C, for 5 minutes 

splitless 

on at 1 minute 

250 oc 

approximately 14.8 minutes 

Page 37 
Harlan Study Number: 41304731 Report Page 38 

Analytical Investigations 

1.4.8 Calculations 

A calibration curve was prepared using a quadratic function of the form shown in equation (1 ). 

The calibration data is presented in Table 2 and Figure 1. From the calibration curve, the 

concentration "x" of the test item in an injected sample was calculated using the following 

equation: 

-b+~b2 -4·a·(c- y) X = ---'----------'--- (1) 

2·a 

where x: 

y: 

a, b, c: 

concentration of the test item in the injected sample [ mg/L] 

average peak area ofthe test item in the injected sample [counts] 

constants 

The concentration of the test item in the test samples was calculated usmg the following 

equation: 

c = X ·F 

R/100 

where c: 

x: 

F: 

R: 

(2) 

concentration of the test item in the test sample [ mg/L] 

concentration of the test item in the injected sample solution [mg!L] 

sample preparation factor 

recovery rate [%] (If not correcting for recovery then R is taken as 1 00) 

The recovery rate (R) of the spiked recovery samples was calculated using the following 

equation: 

R =-c-·100 

cfort 

where R: 

c: 

(3) 

recovery rate [%] 

determined concentration of the test item in the spiked recovery sample 

[mg/L] 

fortified concentration of the test item in the spiked recovery sample 

[mg/L] 
Harlan Study Number: 41304731 Report Page 39 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

The analytical methodology was not validated prior to the analysis of range-finding samples; full 

validation was later conducted at a suitable concentration. The results for the range-finding test 

samples are summarized in Table 3. 

2.2 Definitive Test 

2.2.1 Validation of the Analytical Method 

Specificity 

The biological control samples showed no significant interfering response at the retention time of 

the test item. The standard solutions contained a peak specific for the test item whose area 

changed accordingly with known concentration, hence the specificity of the method by retention 

time was confirmed. 

Linearity 

The calibration data for the calibration standards of the test item is given in Table 2 and Figure 1. 

The data was found to have a quadratic correlation within the calibration range of 0 to 52 mg/L. 

The R2 fit of the calibration curve to the data was 0.9997, and was considered to be acceptable. 

Accuracy (Recovery) and Precision 

A set of recovery samples accurately fortified at a relevant concentration of test item was 

prepared five-fold and analyzed. The results obtained for the concentration of the test item in the 

recovery samples are presented in Table 4. 

The method was considered to be sufficiently accurate and precise for the purposes of this test. 

The test sample results were not corrected for recovery. 

Limit of Quantification 

The limit of quantification (LOQ) was determined as ten times the standard deviation of five 

synthetic controls. Using this method the LOQ was determined to be 0.0099 mg/L. 

2.2.2 Test Samples 

The results obtained for the concentration of the test item found in the test samples are presented 

in Table 5. Typical chromatograms are shown in Figures 2 to 5. 
Harlan Study Number: 41304731 Report Page 40 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable correlation to the fit of a quadratic equation. 

The analytical procedure was found to have acceptable recoveries of test item in test medium. 

The method of analysis was validated and proven to be suitable for use. 
Harlan Study Number: 41304731 Report Page 41 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Sample 

Concentration of Volume Final Volume Preparation 

Test Item Factor 

[% v/v Saturated [mL] [mL] F 

Solution] 

Control 50 10 0.2 

1.0 50 10 0.2 

3.2 50 10 0.2 

10 50 10 0.2 

32 50 100 2 

100 50 100 2 

Table 2 Linearity Data 

Concentration of Test Item Mean Area 

[mg/L] [counts] 

0 1.584 X 104 

0.104 1.149 X 105 

0.264 2.352 X 105 

0.520 3.887 X 105 

1.04 6.535 X 105 

2.64 1.396x 106 

5.20 2.448 X 106 

10.6 4.625 X 106 

26.4 1.068 X 107 

52.0 2.086 X 107 
Harlan Study Number: 41304731 Report Page 42 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined Concentration 

of Concentration Preparation Concentration Expressed as a 

Test Item in of Test Item in Factor of Test Item in percentage of 

Time point Range-Finding Sample Vial Range-Finding the 0-Hour 

Sample Sample Measured 

Concentration 

X F c 

Cnom 

[% v/v 

[hours] Saturated [mg!L] [mg!L] (%) 

Solution] 

0 0.10 0.335 0.2 0.0669 

1.0 3.01 0.2 0.602 NA 10 25.0 0.2 5.00 

100 24.9 2.0 49.8 

72 0.10 0.293 0.2 0.0585 87 

1.0 2.22 0.2 0.444 74 

10 20.3 0.2 4.06 81 

100 24.1 2.0 48.1 97 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Mean 

Concentration Concentration Concentration Preparation Concentration Analytical Precision 

of of Test Item in of Test Item in Factor of Test Item in Recovery (Relative 

Test Item the Spiked the Sample the Spiked Rate Standard 

Sample Vial Sample Deviation of 

Recovery) 

Cnom Crort X F c R 

[mg/L] [mg/L] [mg/L] [mg/L] [%] [%] 

0.286 0.2 0.0572 

0.285 0.2 0.0569 

0.050 0.0529 0.279 0.2 0.0557 107 1.4 

0.283 0.2 0.0566 

0.277 0.2 0.0554 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41304731 Report Page 43 

Analytical Investigations 

Table 5 Results for Test Samples 

Nominal Measured Sample Determined 

Concentration Concentration Preparation Concentration 

of of Test Item in Factor of Test Item in 

Time point Test Item in Sample Vial Test Sample 

Test Sample 

Cnom X F c 

[% v/v 

[hours] Saturated [mg/L] [mg/L] 

Solution] 

0 Control <LOQ 0.2 <LOQ 

1.0 1.86 0.2 0.372 

3.2 4.22 0.2 0.843 

10 18.5 0.2 3.69 

32 7.80 2 15.6 

100 25.4 2 50.7 

24 1.0 1.59 0.2 0.318 

3.2 4.15 0.2 0.830 

10 17.5 0.2 3.50 

32 7.55 2 15.1 

100 24.8 2 49.6 

48 1.0 1.72 0.2 0.344 

3.2 4.05 0.2 0.809 

10 17.0 0.2 3.39 

32 7.50 2 15.0 

100 24.6 2 49.2 

72 Control <LOQ 0.2 <LOQ 

1.0 1.66 0.2 0.332 

3.2 4.07 0.2 0.814 

10 17.3 0.2 3.46 

32 7.55 2 15.1 

100 24.6 2 49.2 

LOQ Limit of Quantification 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41304731 

Analytical Investigations 

FIGURES 

Figure 1 Linearity 

,...__ 

-~ 25000000 

2. 

~ 

" 0 

r;;. 

~ 20000000 

.!<: 

.. 

~ 

15000000 

10000000 

5000000 

Report 

y = -259.3502x2 + 41 0,505.1239x + 179,290.0843 

R2 =0.9997 

0 ~------,--------r-------.-------.--------r-------, 

0 10 20 30 40 50 60 

Concentration (mgl1) 

Page 44 
Harlan Study Number: 41304731 Report 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (5 mg/L) 

0 2 4 6 8 10 

Minutes 

Figure 3 Chromatogram of Biological Control Sample* 

0 2 4 6 

*Response seen determined to be less than the LOQ. 

8 

Minutes 

10 

Page 45 

12 14 16 

12 14 16 
Harlan Study Number: 41304731 Report 

Analytical Investigations 

Figure 4 Chromatogram of Test Sample (1.0% v/v Saturated Solution) 

2 4 6 8 

Minutes 

10 12 14 

Figure 5 Chromatogram of Test Sample (100% v/v Saturated Solution) 

t 2000 

1000 

2 4 6 8 

Minutes 

10 12 14 

Page 46 

16 

1000 

16 
Harlan Study Number: 41304731 Report 

Appendix 6 Monitoring Authority Statement of GLP Compliance 

Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Harlan Laboratories Limited 

Shardlow Business Park 

London Road 

Shardlow 

Derbyshire 

DE722GD 

DATE OF INSPECTION 

TEST TYPE(S) 

Analytical/Clinical Chemistry 

Environmental Fate 

Environmental Toxicity 

Phys.Chem. Testing 

Mutagenicity 

Toxicology 

12 to 14 March 2014 

An inspection for compliance with the Principles of Good Laboratory Practice was 

carried out at the above test facility as part of the UK Good Laboratory Practice 

Compliance Monitoring Programme. 

This statement confirms that, on the date of issue, the UK Good Laboratory 

Practice Monitoring Authority were satisfied that the above test facility was 

operating in compliance with the OECD Principles of Good Laboratory Practice. 

This statement constitutes a Good Laboratory Practice Instrument (as defined in the 

UK Good Laboratory Practice Regulations 1999) . 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority • 

Page47