REPORT 

CAS# 111-44-4: 

Algal Inhibition Test 

Data Requirements/Test Guidelines: 

• 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 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

H Vryenhoef 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Champion Technologies, Inc 

3200 Southwest Freeway 

Suite 2700 

Houston 

Texas 

USA 77027 

UNITED STATES OF AMERICA 

41201182 

24 July 2013 

Page 1 of44 
Harlan Study Number: 41201182 Report Page2 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 

No claim of confidentiality, on any basis whatsoever, is made for any information contained in 

this document. 

Submitter: 

Date: 
Harlan Study Number: 41201182 Report 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

Harlan Study Number: 

Study Title: 

41201182 

CAS# 111-44-4: 

Algal Growth Inhibition Test 

Page 3 

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 2004/1 O/EC.17). 

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: HVryenhoef 

...!1£,.~~---······ Date. 2 4 JUL 2013 
Harlan Study Number: 41201182 Report Page4 

CONTENTS 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ................................................... 2 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE .................................................... 3 

CONTENTS .................................................................................................................................... 4 

QUALITY ASSURANCE STATEMENT ...................................................................................... 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS ...................................................................... 7 

SUMMARY .................................................................................................................................... 8 

Introduction ............................................................................................................................ 8 

Methods ................................................................................................................................. 8 

Results ................................................................................................................................. 8 

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

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

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

1 INTRODUCTION AND PURPOSE ................................................................................... 1 0 

1.1 Guidelines I Regulations ........................................................................................ 1 0 

2 TEST ITEM ......................................................................................................................... 10 

3 MATERIALS AND METHODS ......................................................................................... 11 

3.1 Test Species ........................................................................................................... 11 

3.2 Culture Medium ..................................................................................................... 12 

3.3 Procedure ............................................................................................................... 12 

3.3.1 Range-finding test .................................................................................... 12 

3.3.2 Definitive test. .......................................................................................... 13 

3.3.3 Evaluation of data .................................................................................... 14 

3.4 Validation Criteria ................................................................................................. 16 

4 RESULTS ............................................................................................................................ 17 

4.1 Range-finding Test ................................................................................................ 17 

4.2 Definitive Test ....................................................................................................... 17 

4.2.1 Validation criteria .................................................................................... 17 

4.2.2 Growth data ............................................................................................. 18 

4.2.3 Observations on cultures .......................................................................... 19 

4.2.4 Observations on test item solubility ........................................................ 19 

4.2.5 Physico-chemical measurements ............................................................. 19 

4.2.6 Verification of test concentrations ........................................................... 20 

5 CONCLUSION .................................................................................................................... 20 

6 REFERENCES ..................................................................................................................... 20 

TABLES ........................................................................................................................................ 21 

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

Table 2 

Table 3 

Table 4 

Table 5 

Test ......................................................................................................................... 21 

Cell Densities and pH Values in the Definitive Test.. ........................................... 22 

Daily Specific Growth Rates for the Control Cultures in the Definitive Test.. ..... 23 

Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test 

(0- 72 Hour) .......................................................................................................... 24 

Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test 

(0- 96 Hour) .......................................................................................................... 25 

FIGURES ...................................................................................................................................... 26 

Figure 1 

Figure 2 

Mean Cell Densities v Time for the Definitive Test .............................................. 26 

Temperature Measurements Taken Throughout the Test Period ........................... 27 
Harlan Study Number: 41201182 Report Page 5 

APPENDICES ............................................................................................................................... 28 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Appendix 5 

Certificate of Analysis ........................................................................................... 28 

Positive Control ..................................................................................................... 29 

Culture Medium ..................................................................................................... 30 

Analytical Investigations ....................................................................................... 31 

Monitoring Authority Statement of GLP Compliance .......................................... 44 
Harlan Study Number: 41201182 Report 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 

Study Title: 

41201182 

CAS# 111-44-4: 

Algal Growth Inhibition Test 

Page 6 

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 relevant 

Dates and Types of QA Inspections the Study Director 

and Test Facility 

Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

02 August 2011 General Study Plan N/A 02 August 2011 

Verification 

22 April 2013 Process - based Test Item Preparation 22 April 2013 

22 April 2013 Process - based Test System Preparation 22 April 2013 

22 April 2013 Process - based Exposure 22 April 2013 

25 April 2013 Process - based Assessment of Response 25 April 2013 

26 April 20 13 Process - based Chemical Analysis 26 April 2013 

09 May 2013 Report Audit N/A 09 May 2013 

The complete study plan (including study specific supplement) was verified for GLP compliance 

at report audit. 

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

Quality Assurance: 

2 3 JUL 2013 
Harlan Study Number: 41201182 Report Page 7 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D M Mullee 

Harlan Laboratories Ltd., Shardlow Business Park, London Road, Shardlow, Derbyshire, UK 

Date: 

················~············· 2 4 JUL 2013 
Harlan Study Number: 41201182 Report Page 8 

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 and the US EPA 

Draft Ecological Effects Test Guideline OPPTS 850.5400. 

Methods 

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

aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for 96 hours, 

under constant illumination and shaking at a temperature of24 ± 1 °C. 

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

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

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 

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on growth 

rate and yield of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. 

The range-finding test indicated that the test item was non-toxic at concentrations up to 100 mg/L. 

Therefore, the definitive test was a limit test with an exposure concentration of 100 mg/L. 

Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range 

from 99% to 100% of nominal and so the results are based on nominal test concentrations only. 
Harlan Study Number: 41201182 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Archiving 

7 March2013 

19 April 2013 

Page 9 

Unless instructed otherwise by the Sponsor, all original data 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. 
Harlan Study Number: 41201182 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. 

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 760/2009 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 

2 TESTITEM 

The integrity of the supplied data relating to the identity, purity and stability of the test item is the 

responsibility of the Sponsor. A Certificate of Analysis, provided by the Sponsor is provided in 

Appendix 1. 

Identification: 

Description: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

CAS# 111-44-4 

Clear colorless liquid 

45296HKV 

99.7% 

7 June 2013 

Room temperature in the dark 
Harlan Study Number: 41201182 Report Page 11 

3 MATERIALS AND METHODS 

Biomass: 

ECx: 

Growth: 

Growth Rate (Average Specific 

Growth Rate): 

Yield: 

LOEC (Lowest Observed 

Effect Concentration): 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

Section-by-Section Growth 

Rate: 

AUC (Area Under the Growth 

Curve): 

3.1 Test Species 

Measurement variable (fluorescence or 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 (or solvent control, if applicable) within a stated 

exposure period. 

The increase in biomass over the test period. 

The logarithmic increase in biomass during the exposure 

period. 

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 (or 

solvent control, if applicable). 

The highest test concentration at which no statistically 

significant inhibition of growth is determined relative to the 

control (or solvent control, if applicable). 

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

solvent control (if applicable). 

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

(days 0-1, 1-2, 2-3 and, 3-4 where appropriate). 

Biomass integral calculated as area under the growth curve. 

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 pm) and constant illumination at 24 ± 1 °C until the algal 

cell density was approximately 104 - 10 cells/mL. 
Harlan Study Number: 41201182 Report Page 12 

A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item. 

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

between 13 February 2012 and 17 February 2012. 

3.2 Culture Medium 

The culture medium used for the range-finding and definitive test was the same as that used to 

maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to provide a 

sufficient supply of C02 and to counteract the increase in pH due to algal growth in an enclosed 

system (Herman et a/1990). 

The culture medium is defined in Appendix 3. 

3.3 Procedure 

3.3.1 Range-finding test 

The test concentration to be used in the definitive test was 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 mg/L for a period of 

96 hours. 

The test was conducted in 250 mL glass conical flasks each completely filled with test preparation 

and sealed with ground glass stoppers. Two replicate flasks were used for each control and test 

concentration. The test item was dissolved directly in culture medium. 

An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 

1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further 

stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was 

separately inoculated with algal suspension (15.2 mL) to give the required test concentrations of 

0.1 0, 1.0, 10 and 100 mg/L. 

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

adequate mixing and homogeneity. 

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

At the start of the range-finding test a sample of each test and control culture was removed and 

the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then 

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 96 hours. 

After 96 hours the cell density of each flask was determined using a Coulter® Multisizer Particle 

Counter. 
Harlan Study Number: 41201182 Report Page 13 

A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order to 

determine the stability of the test item under test conditions. All samples were stored at 

approximately -20 oc prior to analysis. 

3.3.2 Definitive test 

Based on the result of the range-finding test a "limit test" was conducted at a concentration of 

100 mg/L. 

3.3 .2.1 Experimental Preparation 

For the purpose of the definitive test, the test item was dissolved directly in culture medium. 

An amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 

2 liters to give a 100 mg/L stock solution. This stock solution was inoculated with algal 

suspension (32.4 mL) to give the required test concentration of 100 mg/L. 

The stock solution and the prepared concentration 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 and 96 hours (see Appendix 4). 

3.3 .2.2 Exposure conditions 

As in the range-finding test 250 mL glass conical flasks were used. Six flasks were used for the 

control and the 100 mg/L treatment group. 

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 6.18 x 105 cells per mL. Inoculation of 2 litres of test medium with 32.4 mL of this 

algal suspension gave an initial nominal cell density of 1 x 104 cells per mL and had no significant 

dilution effect on the final test concentration. 

The flasks were 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 96 hours. 

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a 

Coulter® Multisizer Particle Counter. 
Harlan Study Number: 41201182 Report Page 14 

3.3.2.3 Physico-chemical measurements 

The pH of the control and 100 mg/L test preparation was determined at initiation of the test and 

after 96 hours exposure. The pH was measured using a WTW pH 320 pH meter. The 

temperature within the incubator was recorded hourly using the incubators' internal pt-100 

temperature probe and Multicom Software Package (see Figure 2). 

3.3.3 Evaluation of data 

3.3.3.1 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 

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 = f.lc- f.lt X 100 r 

f.lc 

where: 

percentage inhibition of average specific growth rate 

mean average specific growth rate for the control cultures 

average specific growth rate for the test culture 
Harlan Study Number: 41201182 Report Page 15 

3 .3 .3 .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: 

where: 

percentage inhibition of yield 

mean value for yield in the control group 

mean value for yield for the treatment group 

3.3.3.3 Comparison of biomass integral 

The biomass integral (area under the growth curve) was calculated using the following equation: 

where: 

area 

nominal cell concentration at start of test 

measured cell concentration at t1 

measured cell concentration at tn 

time of first measurement after beginning of test 

time of nth measurement after beginning of test 

Percentage inhibition of the biomass integral for each replicate test item vessel was calculated 

using the following equation: 
Harlan Study Number: 41201182 Report 

where: 

percentage inhibition of the biomass integral 

mean biomass integral for the control cultures 

biomass integral for the test culture 

3.3.3.4 Determination ofECx values 

Page 16 

ECx values were determined by inspection of the growth rate, yield and biomass integral data after 

96 hours. 

3.3.3.5 Statistical analysis 

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) 

was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the 

control and the 100 mg/L test concentration 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). 

3.4 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 after 

72 hours (OECD/EC Guidelines) and by a factor of at least 100 after 96 hours 

(EPA/OPPTS Guidelines). 

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

the control cultures during the course of the test (0-24, 24-48 and 48-72 hours) must not 

exceed 35% (OECD Guidelines). 

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

after 72 hours must not exceed 7% (OECD Guidelines). 

• The coefficient of variation of the cell density values in replicate control cultures must not 

exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines). 
Harlan Study Number: 41201182 Report Page 17 

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 given in Table 1. 

The results showed no effect on growth at the test concentrations of 0.1 0, 1.0, 10 and 100 mg/L. 

Based on this information a single test concentration, six replicates of 1 00 mg/L, was selected for 

the definitive test. 

Chemical analysis of the 100 mg/L test preparation at 0 and 96 hours (see Appendix 4) showed a 

measured test concentration of 109% of nominal was obtained indicating that the test item was 

stable over the test duration. 

4.2 Definitive Test 

Cell density values determined at each sampling time and pH values at 0 and 96 hours are given 

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

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

inhibition values are given in Table 4. Growth rate, yield and biomass integral values for the 

control and test cultures after 96 hours and percentage inhibition values are given in Table 5. 

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

4.2.1 Validation criteria 

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

83 after 72 hours and by a factor of 130 after 96 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 and the 

EPA/OPPTS Guidelines that states the enhancement must be at least by a factor of 100 after 

96 hours. 

Mean cell density of control at 0 hours 

Mean cell density of control at 72 hours 

Mean cell density of control at 96 hours 

1.04 x 104 cells per mL 

8.69 x 105 cells per mL 

1.35 x 106 cells per mL 

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

cultures over the test period (0-72 hours) was 28% 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%. 
Harlan Study Number: 41201182 Report Page 18 

The coefficient of variation of the cell density values in replicate control cultures was 9% after 

72 hours and 1% after 96 hours and hence satisfied the validation criterion which states that this 

must not exceed 20%. 

4.2.2 Growth data 

From the data given in Tables 2, 4 and 5, it is clear that the growth rate (r), yield (y) and biomass 

integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence 

of the test item at a nominal test concentration of 100 mg/L over the 96-Hour exposure period. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1 00 mg/L. 

Accordingly the following results were determined from the data: 

4.2.2.1 Inhibition of growth rate 

ErCIO (0 - 72 h) 

ErC2o (0 - 72 h) 

ErCso (0 - 72 h) 

ErClO (0- 96 h) 

ErC2o (0- 96 h) 

ErCso (0 - 96 h) 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

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

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and 

100 mg/L test group using a Student's t-test 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 decreases 

in growth rate (P~0.05), between the control and 100 mg/L test group and therefore the "No 

Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L. 

4.2.2.2 Inhibition of yield 

EyC10 (0 - 72 h) 

EyC2o (0 - 72 h) 

EyCso (0 - 72 h) 

EyCIO (0 - 96 h) 

EyC2o (0- 96 h) 

EyCso (0 - 96 h) 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

where EyCx is the test concentration that reduced yield by x%. 
Harlan Study Number: 41201182 Report Page 19 

Statistical analysis of the yield data at 72 and 96 hours was carried out as in Section 4.2.2.1. 

There were no statistically significant decrease in yield (P~0.05), between the control and 

100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on 

yield was 1 00 mg/L. 

4.2.2.3 Inhibition of biomass integral 

EbC10 (0 - 72 h) 

EbC2o (0 - 72 h) 

EbCso (0 - 72 h) 

EbC10 (0 - 96 h) 

EbC2o (0- 96 h) 

EbCso (0 - 96 h) 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

: >100 mg/L 

where EbCx is the test concentration that reduced biomass integral (area under the growth curve) 

byx%. 

Statistical analysis of the biomass integral data at 72 and 96 hours was carried out as in 

Section 4.2.2.1. There were no statistically significant decrease in biomass integral (P~0.05), 

between the control and 100 mg/L test group and therefore the "No Observed Effect 

Concentration" (NOEC) based on biomass integral was 100 mg/L. 

4.2.3 Observations on cultures 

All test and control cultures were inspected microscopically at 96 hours. There were no 

abnormalities detected in any of the control or test cultures. 

4.2.4 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 96-Hour test period all control and test cultures were observed to be bright green 

dispersions. 

4.2.5 Physico-chemical measurements 

The pH values of the control and 100 mg/L 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 7.5 at 

0 hours to pH 10.5 at 96 hours. This increase was considered to be due to the amount of carbon 

dioxide required by the large number of algal cells in the log phase of growth (see Figure 1) 

exceeding the transfer rate of C02 from the gaseous phase to the aqueous phase. In this situation 

C02 required for photosynthesis and growth would be derived from bicarbonate in solution which 

results in an increase in the pH of the culture. The increase in pH after 96 hours was in excess of 
Harlan Study Number: 41201182 Report Page 20 

that recommended in the Test Guidelines (1.5 pH units after 96 hours). This was considered to 

have had no adverse effect on the results of the study given that the increase in cell concentration 

in the control cultures exceeded the validation criterion given in the Test Guidelines. 

4.2.6 Verification of test concentrations 

Analysis of the test preparations at 0 and 96 hours (see Appendix 4) showed measured test 

concentrations to range from 99% to 100% of nominal and so the results are based on nominal test 

concentrations only. 

5 CONCLUSION 

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

and gave EC5o values based on growth rate and yield of greater than 100 mg/L. The No Observed 

Effect Concentration was 100 mg/L. 

6 REFERENCES 

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

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

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

SOKAL, R Rand ROHLF, F J (1981) Biometry. New York: W H FREEMAN and Company. 
Harlan Study Number: 41201182 Report Page 21 

TABLES 

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

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

Nominal Concentration 

(mg!L) 0 Hours 96 Hours Growth Rate Yield/Biomass 

Integral 

Control R, 1.02E+04 8.68E+05 

R2 1.01E+04 9.65E+05 - -

Mean 1.01E+04 9.16E+05 

0.10 R, 8.58E+03 5.37E+05 

R2 9.60E+03 1.25E+06 [2] 2 

Mean 9.09E+03 8.93E+05 

1.0 R, 9.22E+03 1.04E+06 

R2 1.45E+04 1.31E+06 [2] [29] 

Mean 1.18E+04 1.18E+06 

10 R, 8.40E+03 1.41E+06 

R2 7.82E+03 1.73E+06 [17] [72] 

Mean 8.11E+03 1.57E+06 

100 R, 5.98E+03 1.33E+06 

R2 7.33E+03 1.21E+06 [17] [39] 

Mean 6.66E+03 1.27E+06 

* 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: 41201182 Report Page 22 

Table 2 Cell Densities and pH Values in the Definitive Test 

Nominal Concentration pH Cell Densities* (cells per mL) pH 

(mg/L) Oh Oh 24h 48 h 72h 96h 96h 

Control R1 1.14£+04 6.00£+04 3.04£+05 8.93£+05 1.35£+06 

Rz 1.08£+04 5.03£+04 2.67£+05 7.59£+05 1.35£+06 

R3 1.03£+04 5.72£+04 3.19£+05 9.01£+05 1.38£+06 

~ 7.5 1.05£+04 6.27£+04 3.20£+05 7.96£+05 1.34£+06 10.5 

Rs 1.02£+04 5.98£+04 3.29£+05 8.81£+05 1.35£+06 

~ 9.30£+03 6.73£+04 3.26£+05 9.83£+05 1.35£+06 

Mean 1.04£+04 5.96£+04 3.11£+05 8.69£+05 1.35£+06 

100 R1 1.16£+04 6.22£+04 3.95£+05 1.27£+06 1.23£+06 

Rz 1.13£+04 5.80£+04 3.47£+05 1.09£+06 1.43£+06 

R3 1.13£+04 6.29£+04 3.46£+05 1.20£+06 1.48£+06 

~ 7.7 1.07£+04 6.02£+04 3.52£+05 1.05£+06 1.12£+06 10.6 

Rs 1.08£+04 6.00£+04 3.05£+05 1.01£+06 1.43£+06 

~ 1.05£+04 5.88£+04 3.33£+05 1.02£+06 1.14£+06 

Mean 1.10£+04 6.04£+04 3.46£+05 1.11£+06 1.31£+06 

* 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 - ~ = Replicates 1 to 6 
Harlan Study Number: 41201182 Report Page 23 

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 Day 3-4 

Rt 0.075 0.068 0.045 0.017 

R2 0.067 0.070 0.043 0.024 

R3 0.073 0.072 0.043 0.018 

Control ~ 0.077 0.068 0.038 0.022 

Rs 0.075 0.071 0.041 0.018 

~ 0.079 0.066 0.046 0.013 

Mean 0.074 0.069 0.043 0.019 

R1 - ~ = Replicates 1 to 6 
Harlan Study Number: 41201182 Report Page 24 

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

(0-72 Hour) 

Growth Rate Yield 

Nominal Concentration ( cells/mL/hour) (cells/mL) 

(mg/L) 

0-72h % % Inhibition 0-72h Inhibition* 

Control RI 0.062 8.81£+05 

Rz 0.060 7.48E+05 

R3 0.063 8.91E+05 

Rt 0.061 - 7.85E+05 -

Rs 0.062 8.71E+05 

~ 0.064 9.74E+05 

Mean 0.062 8.58E+05 

SD 0.001 8.09E+04 

100 RI 0.067 [8] 1.26E+06 

Rz 0.065 [5] 1.08E+06 

R3 0.066 [6] 1.18E+06 

Rt 0.065 [5] 1.04E+06 

Rs 0.064 [3] l.OOE+06 

~ 0.064 [3] 1.01£+06 

Mean 0.065 [5] 1.10E+06 [28] 

SD 0.001 1.03E+05 

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

R1- ~ = Replicates 1 to 6 

SD = Standard Deviation 

[Increase in growth as compared to controls] 

Biomass Integral 

( cells/mL/hour) 

% 0-72h Inhibition 

1.89E+07 

1.61£+07 

1.92E+07 

1.81E+07 -

1.93E+07 

2.06E+07 

1.87E+07 

1.51£+06 

2.56E+07 [37] 

2.22E+07 [19] 

2.36E+07 [26] 

2.19E+07 [17] 

2.03E+07 [9] 

2.10E+07 [12] 

2.24E+07 [20] 

1.89E+06 
Harlan Study Number: 41201182 Report Page 25 

Table 5 Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test 

(0- 96 Hour) 

Growth Rate Yield 

Nominal Concentration ( cells/mL/hour) (cells/mL) 

(mg/L) % 0-96h % Inhibition 0-96 h Inhibition* 

Control RI 0.051 1.34£+06 

Rz 0.051 1.34£+06 

R3 0.051 1.37£+06 

Rt 0.051 - 1.33£+06 -

Rs 0.051 1.34£+06 

R.<; 0.051 1.35£+06 

Mean 0.051 1.34£+06 

SD 0.000 1.30£+04 

100 RI 0.050 2 1.22£+06 

Rz 0.052 [2] 1.42£+06 

R3 0.052 [2] 1.47£+06 

Rt 0.049 4 1.11£+06 

Rs 0.052 [2] 1.42£+06 

R.<; 0.049 4 1.13£+06 

Mean 0.051 1 1.29£+06 4 

SD 0.002 1.59£+05 

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

R1 - R.<; = Replicates 1 to 6 

SD = Standard Deviation 

[Increase in growth as compared to controls] 

Biomass Integral 

( cells/mL/hour) 

0-96h % Inhibition 

4.55£+07 

4.12£+07 

4.64£+07 

4.35£+07 -

4.59£+07 

4.85£+07 

4.52£+07 

2.49£+06 

5.53£+07 [23] 

5.22£+07 [16] 

5.54£+07 [23] 

4.78£+07 [6] 

4.94£+07 [9] 

4.68£+07 [4] 

5.11£+07 [14] 

3.76£+06 

Harlan Study Number: 41201182 Report 

Figure 2 Temperature Measurements Taken Throughout the Test Period 

£ 

... 

.. 

= 

25.5 

25.0 

"E 24.5 ... 

!:>. 

e 

... ,... 

24.0 

23.5 

Page 27 

23.0+----------------.----------------.----------------.--------------~ 

0 24 48 72 96 

Time (Hours) 
Harlan Study Number: 41201182 

APPENDICES 

Appendix 1 Certificate of Analysis 

SIGMA-ALDRICH'' 

Report 

aHO Sfmao• Sll'lllll.Sdlt laliiUIJQ 113tii3,UBA 

Bnilil 'MIIIIiiJE UIM: ····~-- lllllll'llllllff....__ 

Qublidll; UIM: .....,.....,.....,_ 

PniGid. Nlnlroot 

;--r·l~'llf'rll'jlll!'ll'lf'r- "'"""' 

Certificate of Analysis 

l"nnlbi liiii'IIIIIIW: 

loil: lltinlbh: 

llrat'd: 

CAS likmllilli': 

MO.~ 

F-a ~· Wii(jllt 

,...._._ {CIIIat) 

~-~) 

ll'llwe ....,..m 

PtiiiDII NIR ~ 

Alii)' (9::) 

J ... a.-., ...... 

Qullill)' CI!M'tbill 

............. Willalnlin us 

0/IDdailll 

liquid 

C!!lnr- ID Sbui!IUe 

C!!lnf- ID SIN!I,.. 

! 118.6¥. 

0/IDdailll 

UJ&Ild 

C!!lnfC!!Inr-

!19.."1 "" 

.. 

_,..,IIIII 

..MIIiilll•llftlh, 

in lllii ~ 

lhll ill.. 

1'116 

ltiiO 

cauwt 

lil'nia al 

Spimil"llllliilim 

ltiiO qtdlll)' ,..._ 

lllloilllllliiiJ 

IXIillllilllllqilall 

II611WIIilllllil6 

llillJid 

ill.. ~11.-. 

llliD lllii pllllllallt ciiii-.IID 

For,......,..-.~ 

ltiiO ~ 

......... Dlll'tlld 

Tldiriclilll Slwiila Aftil--ill ~ ltiiO llllbllilil)' al ltiiO pnllllld filM' ib ........... ..._ S. --llld• al.....,_ or pdilf 

.... '"" ~Iiiii ._ant~* al ..... 

Page 28 
Harlan Study Number: 41201182 Report Page 29 

Appendix 2 Positive Control 

A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item at 

concentrations of 0.01 0, 0.032, 0.1 0, 0.32 and 1.0 mg/L. 

Exposure conditions and data evaluation for the positive control were similar to those in the 

definitive test. 

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

following results: 

No Observed Lowest 

Time Point Response EC50 (mg/L) 95% Confidence Effect Observed Effect 

(Hours) Variable Limits (mg/L) Concentration Concentration 

(NOEC) (mg/L) (LOEC) (mg/L) 

Growth Rate 0.31 0.28 - 0.35 0.10 0.32 

72 Yield 0.26 * 0.10 0.32 

Biomass 0.27 * 0.10 0.32 

Growth Rate 0.35 0.30 - 0.40 0.10 0.32 

96 Yield 0.27 * 0.10 0.32 

Biomass 0.26 * 0.10 0.32 

The results from the positive control with zinc chloride were within the normal ranges for this 

reference item. 

* It was not possible to calculate 95% confidence limits for these EC50 values as the data generated did not fit the 

models available for the calculation of confidence limits. 
Harlan Study Number: 41201182 Report Page 30 

Appendix3 Culture Medium 

NaN03 25.5 mg/L 

MgCb.6H20 12.16 mg/L 

CaCb.2H20 4.41 mg/L 

MgS04.7H20 14.6 mg/L 

K2HP04 1.044 mg/L 

NaHC03 15.0 mg/L 

H3B03 0.186 mg/L 

MnCbAH20 0.415 mg/L 

ZnCb 0.00327 mg/L 

FeCh.6H20 0.160 mg!L 

CoCb.6H20 0.00143 mg/L 

Na2Mo04.2H20 0.00726 mg/L 

CuCb.2H20 0.000012 mg/L 

Na2EDTA.2H20 0.30 mg/L 

The culture medium was prepared using reverse osmosis purified deionized water* and the pH 

adjusted to 7.5 ± 0.1 with O.lNNaOH or HCl. 

For the purposes of the range-finding and definitive test, additional sodium bicarbonate 

(500 mg/L) was added to the prepared culture medium prior to use. 

* Elga Optima 15+ or Elga Purelab Option R-15 BP 
Harlan Study Number: 41201182 Report Page 31 

Appendix 4 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 of this 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 0 hour samples were stored at approximately -20 oc whilst the 96 hour 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: methyl tert-butyl ether, Fisher Scientific, HPLC grade. 

Methanol 
Harlan Study Number: 41201182 Report Page 32 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 100 mg) was dissolved in methyl tert-butyl ether (100 mL) to prepare a 

stock solution with a nominal concentration of 1000 mg/L. This stock solution was further 

diluted with methyl tert-butyl ether to obtain a nominal 100 mg/L calibration standard. A 

duplicate calibration standard was similarly prepared at 100 mg/L. These duplicate calibration 

standards 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 methyl tert-butyl ether (100 mL) to prepare a 

stock solution with a nominal concentration of 1000 mg/L. Defined volumes of this stock 

solution were diluted with methyl tert-butyl ether to obtain standards in the range of 1.0 to 150 

mg/L. A second standard was similarly prepared at a nominal concentration of 100 mg/L. These 

standards were used 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 methanol to prepare a stock solution with a concentration of 10000 mg/L. A defined 

volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a 

concentration of 100 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 0 hour test samples were thawed with the aid of sonication whilst the 96 hour test samples 

were analyzed on the day of receipt. The test samples were extracted directly with 100 mL of 

methyl tert-butyl ether in the same bottle as shown in Table 1. An aliquot of the methyl tert-butyl 

ether layer was taken for analysis. 
Harlan Study Number: 41201182 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: 

Detector temperature: 

Retention time: 

Agilent 6850, with autosampler 

FID 

Rxi-1 ms (Restek) 

(15m x 0.25 mm i.d.; 1 J..Lm film thickness) 

4 mm empty liner 

nitrogen 

15 psi 

injector: 250 oc 

oven:40 oc for 1 minute 

with 20 °C/minute to 150 oc for 1 minute 

splitless 

injection volume: 1 J..LL 

on at 1 minute 

250°C 

approximately 4.6 minutes 

Page 33 
Harlan Study Number: 41201182 Report Page 34 

Analytical Investigations 

1.4.8 Calculations 

A linear response was obtained during the validation of the analytical method, so for the definitive 

test samples a single-point calibration was performed using duplicate calibration standards. The 

mean peak areas of each calibration standard were corrected to a nominal concentration and the 

mean value taken. 

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

equation: 

X = 

Rspi·N 

Real std 

where x: 

Rspl: 

Rca! std: 

N: 

(1) 

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

mean peak area response of the test item in injected sample [counts] 

mean peak area response for the calibration standard, corrected to nominal 

concentration [counts] 

nominal calibration standard concentration [ mg/L] 

The concentration of the test item in the test samples was calculated using 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 

(3) 

where R: recovery rate [%] 

c: determined concentration of the test item in the spiked recovery sample 

[mg/L] 

Cfort: fortified concentration of the test item in the spiked recovery sample 

[mg/L] 

The percentage of nominal concentration of the test item in the test samples was calculated using 

the following equation: 
Harlan Study Number: 41201182 Report Page 35 

Analytical Investigations 

% nominal = _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg/L] 
Harlan Study Number: 41201182 Report Page 36 

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 specifically at the test concentration to be employed in the 

definitive test. 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 and an analyzed analytical blank 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 linear correlation within the calibration range of 0 to 169 mg/L. The R2 

fit of the calibration curve to the data was 0.9999, 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 Quantitation (LOQ) 

The limit of quantitation (LOQ) was determined by calculating the sample concentration that gave 

a peak equivalent to ten times the baseline noise. Using this method the limit of quantitation was 

determined to be 0. 099 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 4. 
Harlan Study Number: 41201182 Report Page 37 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable linearity. 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: 41201182 Report Page 38 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Sample Final Nominal 

Final Volume Preparation Concentration of Test Concentration of Volume Factor Item Test Item 

[mg/L] [mL] [mL] F [mg/L] 

Control 100 100 1 100 

100 100 100 1 100 

Table 2 Linearity Data 

Concentration of Test Item Area 

[mg/L] [counts] 

0 0 

1.124 5.660 X 104 

2.810 1.385 X 105 

5.620 2.749 X 105 

11.240 5.460 X 105 

28.100 1.364 X 106 

56.200 2.696 X 106 

112.400 5.426x 106 

115.200 5.616 X 106 

134.880 6.521 X 106 

168.600 8.035 X 106 
Harlan Study Number: 41201182 Report Page 39 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined Concentration 

of Concentration Preparation Concentration 

Tesi Iiem in of Test Item in Factor of Test Item in % ofNominal Time point Range-Finding Sample Vial Range-Finding Concentration 

Sample Sample 

X F c Cnom 

[hours] [mg/L] [mg!L] [mg/L] [%] 

0 100 109 1 109 109 

96 100 109 1 109 109 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Analytical 

Concentration Concentration Concentration Preparation Concentration Recovery Precision 

of of Test Item in of Test Item in Factor of Test Item in Rate (Relative 

Test Item the Spiked the Sample the Spiked Standard 

Sample Vial Sample Deviation of 

Recovery) 

Cnom Cfort X F c R 

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

100 115 126 1 126 

126 1 126 

127 1 127 110 0.78 

126 1 126 

128 1 128 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41201182 Report 

Analytical Investigations 

Table 5 Results for Test Samples 

Time point 

[hours] 

0 

96 

LOQ 

Nominal Measured 

Concentration Concentration 

of of Test Item in 

Test Item in Sample Vial 

Test Sample 

Cnom X 

[mg/L] [mg!L] 

Control <LOQ 

100 100 

Control <LOQ 

100 98.6 

Limit of Quantitation 

not applicable 

Sample 

Preparation 

Factor 

F 

1 

I 

1 

I 

For reporting purposes, x has been calculated from c retrospectively. 

Page 40 

Determined 

Concentration 

of Test Item in %of Nominal Test Sample Concentration 

c 

[mg!L] [%] 

<LOQ - 100 100 

<LOQ -

98.6 99 
Harlan Study Number: 41201182 

Analytical Investigations 

FIGURES 

Figure 1 Linearity 

2 9000000 

·;:: 

2- 8000000 co 

!!! 

<( 7000000 ..><: 

co 

Ql 

a.. 6000000 

5000000 

4000000 

3000000 

2000000 

1000000 

0 

0 

Report 

y = 48062x + 8914.5 

R2 = 0.9999 

50 100 

Page 41 

150 200 

Concentration (mg/L) 
Harlan Study Number: 41201182 Report Page 42 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (100 mg/L) 

600 j - .$tandard de<ec<o< t ~oo 

' Reten~ion Tim : 

'a. 300 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 

l I I I I I 

I I l I I 

I I I I 

I I , I I 

- ~- __ _!_ _____ ----1 ••. - _ _ ___ , _________ L ________ L _ _ _ _ _ _ _ l _ _ ______ , _________ I_ .• 

I I l I 

I I ' I 

I I I I 

l I I I 

' I I I 

I I I I 

I I I I 

I I I I 

I I I I 

I I I I _______ -'- ________ L _______ J. _- •• _____ ~ 

________ .J ________ I 

······~" ' . ' 

Figure 3 Chromatogram of Biological Control Sample 

~ 300 
Harlan Study Number: 41201182 Report Page 43 

Analytical Investigations 

Figure 4 Chromatogram of Test Sample (100 mg/L) 

600j ~ de<ec<o< ~ , !iT149161 dp Ohr 

Reten~ion Tim : 

' ' ' ' ' ' ' ' ' 

' ' ' --- -,---------,..-------- ~-

' ' ' 

' ~ 

: ~ 

~ 300 1--------- ---------- ' -------------, ' ' --------;- -------, .. ·- ·----------- ----------·-, 

l. ____ .J ________ , _________ L I --- --

_______ ,v_, 

200 
Harlan Study Number: 41201182 Report 

Appendix 5 Monitoring Authority Statement of GLP Compliance 

i oii) Department ~ · ofHealth 

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 Ltd 

Shardlow Business Park 

London Road 

Shard low 

Derby 

DE72 2GD 

DATE OF INSPECTION 

10 July 2012 

TEST TYPE(S) 

Analytical/Clinical 

Chemistry 

Environmental Toxicity 

Environmental Fate 

Mutagenicity 

Phys/Chem. Tests 

Toxicology 

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). 

~~~ -:30 /u /tz.... 

Page 44 

Dr. Andrew J. Gray -

Head, UK GLP Monitoring Authority • •