## REPORT Algal Growth Inhibition Test
DataRequirementsffestGuidelines: • OECD Guidelines for Testing ofChemicals (2006) No 201, "FreshwaterAlga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of CommissionRegulation (EC) No. 761/2009 

### Study Director: TestFacility:

Sponsor:

Harlan StudyNumber: Study Completion Date: HVryenhoef 

### HarlanLaboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK

41206436 23 April 2013

Page 1of44Harlan StudyNumber: 41206436 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 ......................................................................................................... 8 Schedule.................................................................................................................................8 Archiving ...............................................................................................................................8 1 INTRODUCTION AND PURPOSE.....................................................................................9 1.1 Guidelines I

### Regulations..........................................................................................9 2 TEST ITEM ...........................................................................................................................9 3 MATERIALS AND METHODS......................................................................................... 10 3.1 Definitions and Abbreviations............................................................................... 10 3.2 Test Species ........................................................................................................... 10 3.3 Culture Medium..................................................................................................... 11 3.4 Procedure ............................................................................................................... 11 3.4.1 Range-Finding Tests................................................................................ 11 3.4.2 Definitive Test......................................................................................... 12 3.4.3 Evaluation ofData................................................................................... 14 3.5 Validation Criteria................................................................................................. 16 4 RESULTS ............................................................................................................................ 17 4.1 Range-finding Tests............................................................................................... 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 ofTest Concentrations ........................................................ 19 5 CONCLUSION....................................................................................................................20 6 REFERENCES ....................................................................................................................20 TABLES........................................................................................................................................21 Table 1 Cell Densities and Percentage Inhibition ofGrowth from the Initial Rangefinding Test............................................................................................................21 Table 2 Cell Densities and Percentage Inhibition ofGrowth from the Second Rangefinding Test............................................................................................................22 Table 3 Cell Densities and pH Values inthe Definitive Test..........................,..................23 Table 4 Daily Specific Growth Rates for the Control Cultures inthe Definitive Test.......24 Table 5 Inhibition ofGrowth Rate and Yield inthe Definitive Test..................................25 FIGURES ......................................................................................................................................26 Figure 1 Mean Cell Densities v Time for the Definitive Test .............................................26Harlan StudyNumber: 41206436 Report Page3 

### Figure 2 Inhibition ofGrowth Rate Based onNominal Test Concentrations......................27 Figure 3 Inhibition ofYield Based onNominal Test Concentrations .................................28 APPENDICES...............................................................................................................................29 Appendix 1 Appendix 2 Appendix 3 Appendix 4 Positive Control .....................................................................................................29 Culture Medium..................................................................................................... 30 Analytical Investigations .......................................................................................31 Monitoring Authority Statement ofGLP Compliance ..........................................44Harlan StudyNumber: 41206436 Report 

## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: Study Title: 41206436 Algal Growth Inhibition Test Page4

The study described 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). 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 (MHLW, MAFF and METI).

This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affected the integrity ofthe study orthe validity ofthe data. Study Director:



# ..//.-.I..,.A--4········· D;t;?_ 2 3 APR 2013 HVryenhoefHarlan StudyNumber: 41206436 Report Page 5 

## QUALITYASSURANCE STATEMENT Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: Study Title: 41206436 Algal Growth Inhibition Test The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsible person and management. Study-related procedures conducted at the test facility were audited and inspected. The details of these audits and inspections are givenbelow. 

### Reportedto the relevant Study Dates andTypes ofQA Inspections Directorand Test Facility Management Date ofInspection Type ofInspection Phase Inspected Report Date 19 November 2012 Study Plan N/A 19 November 2012 Verification 14 January 2013 Process
- based Test Item Preparation 14 January 2013 14 January 2013 Process
- based Test SystemPreparation 14 January 2013 14 January 2013 Process
- based Exposure 14 January 2013 17 January 2013 Process
- based Assessment ofResponse 17 January 2013 22 January 2013 Process
- based Chemical Analysis 22 January 2013 26 March 2013 Report Audit N/A 26 March 2013 This statement confirms thatthis reportreflects the raw data and theprocedures followed. QualityAssurance: 

# S.£evANHarlan StudyNumber: 41206436 Report Page 6 

## SIGNATURE OF CONTRIBUTING SCIENTISTS Analytical Chemistry: DMMullee Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK

# I~ f, '1 r / . 

### ........................................................ Date: 

## 2 3 APR 2013Harlan StudyNumber: 41206436 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 was designed to be compatible with 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. 76112009. 

### Methods Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of24 ± 1 °C. Samples ofthe algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. 

### Results Exposure ofPseudokirchneriella subcapitatato the test item gave the following results: No Observed Lowest Observed Response ECso 95% Confidence Effect Effect Variable (mg/L) Limits (mg/L) Concentration Concentration (NOEC) (mg/L) (LOEC) (mg/L) Growth Rate 0.068 0.053 

## - 0.086 0.010 0.032 Yield 0.025 0.023 

## - 0.027 0.010 0.032 Analysis of the test preparations for both the Acute Toxicity to Rainbow Trout and Acute Toxicityto Daphnia magna tests (Harlan Study Numbers: 41206434 and 41206435 respectively) showed consistent, near nominal results were obtained. However, analysis of the test preparations at 0 and 72 hours for this study showed variable and inconsistent results were obtained. From a chemical perspective, the test item is a water soluble poly-quaternary ammonium salt and there is a potential in this case for the polymer to bind to any receptive surface, the effects of which being more notable/pronounced at the lower test concentrations employed in the algal study. Given that the test item was known to be stable it was considered thatthe algae were exposedto near nominal concentrations throughout the duration ofthe test.Harlan Study Number: 41206436 Report 

## GENERALINFORMATION Schedule Experimental Starting Date: Experimental Completion Date: 

### Archiving 24 December 2012 14 March 2013 Page 8

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 ofthe data will be chargeable to the Sponsor. No data will be discarded without contacting the Sponsorto obtaintheir written consent.Harlan StudyNumber: 41206436 Report Page 9 

## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe 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. 76112009 

## 2 TESTITEM The integrity ofthe supplied data relating to the identity, purity and stability ofthe test item is the responsibility ofthe Sponsor. Identification: Description: Batch: Purity: Expiry date: Storage conditions:Harlan StudyNumber: 41206436 Report Page 10 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Biomass: ECx:
Growth: Measurement variable (fluorescence or algal cell density) as surrogate measure for algal biomass. The calculated concentration oftest 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 inbiomass overthe testperiod. 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 Species 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, ifapplicable). The highest test concentration at which no statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable). Comprises test item treatments, control (test medium only) and solvent control (ifapplicable). Specific growth rate for each day during the course ofthe test (days 0-1, 1-2, 2-3 and, 3-4 where appropriate).

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures ofPseudokirchneriella subcapitata were obtained from the Culture Collection ofAlgae 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 1 00 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 keptHarlan StudyNumber: 41206436 Report Page 11

under constant agitation by orbital shaker (100- 150 7m) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104

- 10 cells/mL. A positive control (Harlan Study Number: 41205049) used potassium dichromate as the reference item. Details ofthepositive control are given inAppendix 1. The positive control was conducted between 17 September2012 and 21 September 2012. 

### 3.3 Culture Medium The culture medium used for both the range-finding and definitive tests was the same as that used to maintainthe stock culture. The culture medium is defined inAppendix 2. 

### 3.4 Procedure 3.4.1 Range-Finding Tests The test concentrations to be used in the definitive test were determined by preliminary rangefinding tests. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series ofnominal test concentrations of0.10, 1.0, 10 and 100 mg/L for a period of72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium. An amount oftest item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL 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 (450 mL) of each ofthe stock solutions was separately inoculated with algal suspension (4.5 mL) to give the required test concentrations ofO.lO, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing andhomogeneity. The control group was maintainedunder identical conditions butnot exposedto the test item. At the start ofthe range-finding test a sample ofeach test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged withpolyurethane foam bungs 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.Harlan StudyNumber: 41206436 Report Page 12
After 72 hours the cell density ofeach flask was determined using a Coulter® Multisizer Particle Counter. The results ofthe initial range-finding test showed significant inhibition ofgrowth occurred at all test concentrations and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of0.0010, 0.010, 0.10 and 1.0 mg/L for aperiod of72hours. An amount oftest item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series ofdilutions was made to give further stock solutions of 10, 1.0, 0.10, 0.010 and 0.0010 mg/L. An aliquot (450 mL) ofeach of the 0.0010, 0.010, 0.10 and 1.0 mg/L stock solutions was separately inoculated with algal suspension (1.4 mL) to give the required test concentrations of0.0010, 0.010, 0.10 and 1.0 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 butnot exposed to the test item. Exposure conditions inthe secondrange-finding test were the same as those in the initial test. A sample ofeach 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. All samples were stored at approximately -20 °C priorto analysis. 3.4.2 Definitive Test Based on the results ofthe range-finding tests the following test concentrations were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. 3.4.2.1 Experimental Preparation Forthe purpose ofthe definitive test, the test item was dissolved directly in culture medium. An amount oftest item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series ofdilutions was made to give further stock solutions of 10, 1.0, 0.32, 0.10, 0.032 and 0.010 mg/L. An aliquot (900 mL) of each ofthe 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L stock solutions was separately inoculated with algal suspension (14.3 mL) to give the required test concentrations of0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.Harlan StudyNumber: 41206436 Report Page 13

The concentration and stability ofthe test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 3). 3.4.2.2 Exposure Conditions As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 1

00 mL oftest preparation were used for the control and three flasks each containing 100 mL were used for eachtreatment group. The control group was maintained under identical conditions butnot exposedto the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of3.14 x 105 cells per mL. Inoculation of900 mL oftest medium with 14.3 mL ofthis algal suspension gave an initial nominal cell density of 5 x 1 03 cells per mL and had no significant dilution effect onthe final test concentration. The flasks were plugged with polyurethane foam bungs 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. Samples were taken at 0, 22, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. 3.4.2.3 Physico-Chemical Measurements The pH ofthe control and each test preparation was determined at initiation ofthe test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature withinthe incubatorwas recorded daily. 3.4.2.4 VerificationofTest Concentrations Samples were taken from the control (replicates R1- RQ pooled) and each test group (replicates R1
- R3 pooled) at 0 and 72 hours for quantitative analysis. All samples were stored at approximately -20 

# oc prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis ifnecessary. The method ofanalysis, recovery andtestpreparation analyses are described inAppendix 3.Harlan StudyNumber: 41206436 Report Page 14


### 3.4.3 Evaluation ofData 3.4.3.1 Comparison ofGrowth Rates The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

Where: ll N1 Nn tl tn =

= average specific growthrate from time t1to tn cell concentration at t1 cell concentration attn time offirst measurement time ofnth 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 thanthe measured starting value in orderto increase the precision ofthe 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 ofgrowth rate for each replicate test item vessel was calculated using the following equation: I 

## = l..lc -l..lt X 100

r Where: 

### Ir l..lc l..lt l..lc
percentage inhibition ofaverage specific growthrate mean average specific growthrate for the control cultures average specific growthrate for the test cultureHarlan StudyNumber: 41206436 Report Page 15


### 3.4.3.2 Comparison ofYield 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 ofthe test cell concentration at the end ofthe 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 ofyield mean value for yield inthe control group mean value for yield for the treatment group 3.4.3.3 Determination ofECx Values 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 ECso values were calculated, using the simplified method ofevaluating dose-effect experiments (Litchfield and Wilcoxon, 1949). 3.4.3.4 Statistical Analysis One way analysis ofvariance incorporating Bartlett's test for homogeneity ofvariance (Sokal and Rohlf, 1981) and Dunnett's multiple comparisonprocedure 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).Harlan StudyNumber: 41206436 Report Page 16


### 3.5 Validation Criteria The results ofthe test are considered valid ifthe following performance criteria are met: • The cell concentration ofthe control cultures must increase by a factor ofat least 16 over thetestperiod. • The mean ofthe coefficients ofvariation ofthe sectionby section specific growthrates in the control cultures during the course ofthe test (days 0-1, 1-2 and 2-3, for 72-Hourtests) mustnot exceed 35%. • The coefficient ofvariation ofthe average specific growthrate inreplicate control cultures mustnot exceed 7%.Harlan StudyNumber: 41206436 Report Page 17 

## 4 RESULTS 4.1 Range-finding Tests The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding tests are given in Table 1 and Table 2. The results showed no effect on growth at the test concentrations of 0.0010 and 0.010 mg/L. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L. Based on this information test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test. Chemical analysis of the 0.010, 0.10 and 1.0 mg/L test preparations from the second rangefinding test (see Appendix 3) showed measured test concentrations ofless than the LOQ, 101% and 93% ofnominal respectively were obtained at 0 hours. A measured concentration ofless than the LOQ was not unexpected at 0.010 mg/L given that the LOQ ofthe analytical method was determined to be 0.084 mg/L. Analysis of the test preparations at 72 hours showed measured test concentrations ofless than the LOQ were obtained for the 0.010 and 0.10 mg/L test preparations whilst a measured concentration of90% ofnominal was observed at 1.0 mg/L. 

### 4.2 Definitive Test Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 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. Percentage inhibition values are plotted against test concentration inFigure 2 and Figure 3. 4.2.1 Validation Criteria The following data show that the cell concentration ofthe control cultures increased by a factor of 126 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement mustbe at leastby a factor of 16 after 72 hours. Mean cell density ofcontrol at 0 hours Mean cell density ofcontrol at 72 hours 6.05 x 103 cells permL 7.60 x 105 cells per mLHarlan StudyNumber: 41206436 Report Page 18
The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% and hence satisfied the validation criterion given in the OECD Guideline which states the meanmustnot exceed 35%. The coefficient ofvariation for average specific growth rate for the control cultures over the test period (0
- 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states thatthis must not exceed 7%. 4.2.2 Growth Data From the data given in Table 3 and Table 5, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence ofthe test item overthe 72-Hour exposure period. Accordingly the following results were determined from the data: 4.2.2.1 Inhibition ofGrowth Rate ErCto (0
- 72 h) ErC2o (0
- 72 h) ErCso (0
- 72 h) 0.013 mg/L 0.024 mg/L 0.068 mg/L; 95% confidence limits 0.053
- 0.086 mg/L where ErCx is the test concentrationthatreduced growthrate by x%. Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis ofvariance incorporating Bartlett's test for homogeneity ofvariance (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 and 0.010 mg/L test concentration (P:2:0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.010 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.032 mg/L. 4.2.2.2 Inhibition ofYield EyCto (0
- 72 h) EyC2o (0- 72 h) EyCso (0
- 72 h) Where: 0.018 mg/L 0.020 mg/L 0.025 mg/L; 95% confidence limits 0.023
- 0.027 mg/L EyCx is the test concentrationthatreduced yield by x%.Harlan StudyNumber: 41206436 Report Page 19
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 and 0.010 mg/L test concentration**(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 0.010 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.032 mg/L. 4.2.3 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 0.010, 0.032 and 0.10 mg/L, however no intact cells and cell debris was observed to be present in the test cultures at 0.32 and 1.0 mg/L. 4.2.4 Observations on Test Item Solubility At the start ofthe test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 0.010 mg/L test cultures were observed to be green dispersions. The 0.032 mg/L test cultures were observed to be pale green dispersions, the 0.10 mg/L test cultures were observed to be very pale green dispersions, the 0.32 mg/L test cultures were observed to be extremely pale green dispersions whilst the 1.0 mg/L test cultures were observed to be clear colorless solutions. 4.2.5 Physico-Chemical Measurements The pH values ofthe control and each test preparation are given in Table 3. Temperature was maintained at 24 ± 1 °C throughout the test. The pH value of the control cultures (see Table 3) was observed to increase from pH 8.0 at 0 hours to pH 8.6 at 72 hours. The pH deviation inthe control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. 4.2.6 Verification ofTest Concentrations Analysis of the test preparations for both the Acute Toxicity to Rainbow Trout and Acute Toxicityto Daphnia magnatests (Harlan Study Numbers: 41206434 and 41206435 respectively) showed consistent, near nominal results were obtained. However, analysis of the test preparations at 0 and 72 hours for this study (see Appendix 3) showed variable and inconsistent results were obtained. From a chemical perspective, the test item is a water soluble polyquaternary ammonium salt and there is a potential in this case for the polymer to bind to any receptive surface, the effects of which being more notable/pronounced at the lower test concentrations employed in the algal study. Given that the test item was known to be stable itHarlan StudyNwnber: 41206436 Report Page20

was considered that the algae were exposed to near nominal concentrations throughout the duration ofthe test. 

## 5 CONCLUSION The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hourperiod and gave the following results: No Observed Lowest Observed Response ECso (mg/L) 95% Confidence Effect Effect Variable Limits (mg/L) Concentration Concentration (NOEC) (mg/L) (LOEC) (mg/L) Growth Rate 0.068 0.053 

# - 0.086 0.010 0.032 Yield 0.025 0.023 - 0.027 0.010 0.032 

## 6 REFERENCES DUNNETT, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. JAm StatAssoc 50, 1096-1121. LITCHFIELD, J T and WILCOXON, F (1949) A Simplified Method ofEvaluating Dose-Effect Experiments. JPharmacal Exp Ther 96,99-113. 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. Xlfit, ID Business Solutions Ltd.Harlan StudyNumber: 41206436 Report Page 21 

## TABLES Table 1 CellDensities and PercentageInhibition ofGrowth from theInitialRangefinding Test Cell Densities* (cells per mL) Inhibition Values (%) NominalConcentration (mg/L) 0 Hours 72 Hours Growth Rate Yield Control R1 5.81E+03 1.17E+06 R2 5.66E+03 1.42E+06 

### - 

## - Mean 5.74E+03 1.30E+06 0.10 RI 6.40E+03 1.80E+05 R2 5.90E+03 1.37E+05 40 88 Mean 6.15E+03 1.59E+05 1.0 RI 5.55E+03 1.49E+03 R2 5.44E+03 1.28E+03 125 100 Mean 5.50E+03 1.38E+03 10 RI 4.80E+03 1.26E+03 R2 5.31E+03 9.12E+02 128 100 Mean 5.06E+03 1.09E+03 100 RI 5.25E+03 2.59E+03 R2 5.57E+03 2.50E+03 113 100 Mean 5.41E+03 2.54E+03



### * Cell densities represent the mean number ofcells per mL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R1 andR2 = Replicates 1 and 2Harlan StudyNumber: 41206436 Report Page 22

Table 2 CellDensities and Percentage Inhibition ofGrowth from the Second Rangefinding Test Cell Densities* (cells per mL) Inhibition Values(%) Nominal Concentration (mg/L) 0 Hours 72 Hours Growth Rate Yield Control RI 5.76E+03 3.58E+05 R2 5.70E+03 4.43E+05 

### - 

## - Mean 5.73E+03 4.01E+05 0.0010 RI 6.61E+03 5.17E+05 R2 5.47E+03 2.93E+05 2 [1] Mean 6.04E+03 4.05E+05 0.010 R1 5.68E+03 2.99E+05 R2 7.17E+03 5.32E+05 2 [4] Mean 6.42E+03 4.15E+05 0.10 Rl 5.58E+03 1.60E+05 R2 5.10E+03 3.67E+04 32 76 Mean 5.34E+03 9.85E+04 1.0 R1 5.33E+03 1.60E+03 R2 6.94E+03 1.31E+03 134 101 Mean 6.14E+03 1.46E+03



### * Cell densities represent the mean number ofcells per mL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R1 andR2 = Replicates 1and 2 [Increase in growth comparedto controls]Harlan StudyNumber: 41206436 Report Page23
Table 3 Cell Densities and pH Values in the Definitive Test Nominal Concentration pH Cell Densities* (cells per mL) pH (mg/L) Oh Oh 22 h 46 h 72h 72h Control RI 5.92E+03 2.19E+04 1.16E+05 6.74E+05 Rz 5.52E+03 2.49E+04 1.33E+05 8.54E+05 R3 4.74E+03 2.43E+04 1.39E+05 8.08E+05 

### Rt 8.0 8.00E+03 2.03E+04 1.24E+05 6.41E+05 8.6 Rs 6.53E+03 2.48E+04 1.40E+05 7.67E+05**~** 5.62E+03 2.58E+04 1.48E+05 8.20E+05 Mean 6.05E+03 2.37E+04 1.33E+05 7.60E+05 0.010 R1 4.98E+03 9.36E+03 7.86E+04 7.66E+05 Rz 5.20E+03 7.26E+03 7.74E+04 7.63E+05 7.8 8.6 R3 5.31E+03 7.01E+03 5.50E+04 7.58E+05 Mean 5.16E+03 7.88E+03 7.03E+04 7.62E+05 0.032 RI 5.15E+03 3.92E+03 1.94E+04 1.85E+05 Rz 5.39E+03 2.83E+03 1.22E+04 9.12E+04 7.7 8.4 R3 4.37E+03 2.50E+03 1.27E+04 9.50E+04 Mean 4.97E+03 3.08E+03 1.47E+04 1.24E+05 0.10 R1 5.07E+03 2.29E+03 1.15E+04 6.69E+04 Rz 4.59E+03 1.90E+03 9.01E+03 5.52E+04 7.7 8.3 R3 4.70E+03 2.78E+03 8.46E+03 5.13E+04 Mean 4.79E+03 2.33E+03 9.66E+03 5.78E+04 0.32 RI 4.53E+03 2.19E+03 4.61E+03 9.60E+03 Rz 4.98E+03 9.44E+02 2.61E+03 5.60E+03 7.6 8.2 R3 4.11E+03 1.38E+03 3.18E+03 5.41E+03 Mean 4.54E+03 1.50E+03 3.47E+03 6.87E+03 1.0 R1 4.82E+03 1.34E+03 2.37E+03 4.03E+03 Rz 4.75E+03 1.55E+03 1.28E+03 2.50E+03 7.6 8.1 R3 4.62E+03 6.88E+02 1.90E+03 2.77E+03 Mean 4.73E+03 1.19E+03 1.85E+03 3.10E+03



### * Cell densities represent the mean number of cells per mL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R1
-**~** = Replicates 1to 6Harlan StudyNumber: 41206436 Report Page24


### Table4 Daily Specific GrowthRates for the Control Cultures inthe Definitive Test Daily Specific Growth Rate (cells/mL/hour) Day 0-1 Day 1-2 Day2 -3 Control R1 0.067 0.070 0.068 R2 0.073 0.070 0.072 R3 0.072 0.073 0.068 R. 0.064 0.075 0.063 Rs 0.073 0.072 0.065**~** 0.075 0.073 0.066 Mean 0.071 0.072 0.067

R1
-**~** = Replicates 1to 6Harlan StudyNumber: 41206436 Report Page 25


### Table 5 Inhibition ofGrowth Rate and Yield in the Definitive Test Growth Rate Yield Nominal Concentration (cells/mL/hour) (cells/mL) (mg/L) 0-72h % Inhibition 0-72h % Inhibition* Control RI 0.068 6.68E+05 R2 0.071 8.48E+05 R3 0.071 8.03E+05**~** 0.067 

## - 6.33E+05 

## - Rs 0.070 7.60E+05**~** 0.071 8.14E+05 Mean 0.070 7.54E+05 SD 0.002 8.60E+04 0.010 RI 0.070 0 7.61E+05 R2 0.070 0 7.58E+05 R3 0.070 0 7.53E+05 Mean 0.070 0 7.57E+05 0 SD 0.000 4.08E+03 0.032 R1 0.050 29 1.80E+05 R2 0.040 43 8.58E+04 R3 0.041 41 9.07E+04 Mean 0.044 38 1.19E+05 84 SD 0.006 5.32E+04 0.10 R1 0.036 49 6.19E+04 R2 0.033 53 5.06E+04 R3 0.032 54 4.66E+04 Mean 0.034 52 5.30E+04 93 SD 0.002 7.91E+03 0.32 R1 0.009 87 5.07E+03 R2 0.002 97 6.24E+02 R3 0.001 99 1.30E+03 Mean 0.004 94 2.33E+03 100 SD 0.004 2.40E+03 1.0 RI -0.003 104 -7.84E+02 R2 -0.010 114 -2.26E+03 R3 -0.008 111 -1.86E+03 Mean -0.007 110 -1.63E+03 100 SD 0.004 7.61E+02



### * Inaccordance with the OECDtest guideline onlythe mean value for yield for eachtest concentration is calculated R1-**~** =Replicates 1to 6 SD = StandardDeviationHarlan StudyNumber: 41206436 Report Page26 

## FIGURES Figure 1 Mean Cell Densities v Time for theDefinitive Test

l.OOE+06**-r-~-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_"J.,'** ..----------------------------------------------------- __.,..J_- __-

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## --------------------------------------!---------------------------- _,..______ I /**-------------------------------------~----------------------** 

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---- 0.032 mg!L

- -EI- · 0. 10 mg!L 1 / I / ___.__ 0. 32mg!L

- -e- · 1.0 mg!L**-------------------------------------~---------------** -----'--------------- .__

________ ;:_

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### y



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', ! _.._...-.- !**~r_..** 1 vI I**l.OOE+03~-----------------------------+ -----------------------------4** **----------------------------~** l.OOE+04

0 24 48 72 Time(Hours)Harlan StudyNumber: 41206436 Report Figure 2 Inhibition ofGrowth Rate Based on Nominal Test Concentrations 120 r---- . .---..,..' -....,.-.,...., ----ri--,-!..,..!--,!--,-!
---.......--....--,..-.,..., .....,....'
- .................,,.



# ____**Gmw1h_r~----!------!-----!---j---((!--(-------------:---------:------:-----:---:---:--:--!­**

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# ---------------"--------1*--+---:---1---l-+H---------------t--------[-----+-+-++-1-+

0,01 

# irrri,'li 

# rrr:,:::,:::tl,

0.1 Concentration (mgL) Page27Harlan StudyNumber: 41206436 Report Figure 3 Inhibition ofYield Based on Nominal Test Concentrations

0.01 0.1 Concentration (mfifl) Page 28Harlan StudyNumber: 41206436 Report Page29 

## APPENDICES Appendix 1 Positive Control A positive control (Harlan Study Number: 41205049) used potassium dichromate as the reference item at concentrations of0.25, 0.50, 1.0, 2.0 and 4.0 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: ErCso (0
- 72 h) EyCso (0
- 72 h) 1.1 mg/L; 95% confidence limits 1.0- 1.3 mg/L 0.70 mg/L* No Observed Effect Concentration (NOEC) based ongrowthrate: No Observed Effect Concentration (NOEC) based onyield: Lowest Observed Effect Concentration (LOEC) based ongrowthrate: Lowest Observed Effect Concentration (LOEC) based onyield: 0.50 mg/L 0.25 mg/L 1.0 mg/L 0.50 mg/L The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

• It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation ofconfidence limits.Harlan StudyNumber: 41206436 Report Page 30 Appendix2 Culture Medium NaN03 25.5 mg/L MgC}z.6H20 12.16 mg/L CaC}z.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 MnC}zAH20 0.415 mg/L ZnC}z 0.00327 mg/L FeCh.6H20 0.16 mg/L CoC}z.6H20 0.00143 mg/L Na2Mo04.2H20 0.00726 mg/L CuC}z.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 adjustedto 7.5 ± 0.1 with O.lNNaOH orHCL



### *Elga Optima 15+ orElgaPurelab Option R-15 BPHarlan StudyNumber: 41206436 Report Page 31 Appendix 3 Analytical Investigations 

## Analytical Investigations CONTENTS CONTENTS ..................................................................................................................................31 1 MATERIALS AND METHODS.........................................................................................32 1.1 Introduction ...........................................................................................................32 1.2 Testitem................................................................................................................32 1.3 Analytical Standard ...............................................................................................32 1.4 Analytical Procedure .............................................................................................32 1.4.1 Storage ..................................................................................................... 32 1.4.2 Reagents and Solvents.............................................................................32 1.4.3 Preparation ofCalibration Standards.......................................................33 1.4.4 Preparation ofLinearity Standards..........................................................33 1.4.5 Preparation ofSpiked Recovery Samples ...............................................33 1.4.6 Preparation ofTest Samples....................................................................33 1.4.7 Instrumental Parameters ..........................................................................34 1.4.8 Calculations .............................................................................................34 2 RESULTS ............................................................................................................................36 2.1 Range-Finding Test ...............................................................................................36 2.2 Definitive Test ....................................................................................................... 36 2.2.1 Validation oftheAnalytical Method.......................................................36 2.2.2 Test Samples............................................................................................36 3 DISCUSSION...................................................................................................................... 37 TABLES ........................................................................................................................................38 Table 1 Preparation ofTest Samples ..................................................................................38 Table 2 Calibration Standard Data .....................................................................................38 Table 3 Results for Range-Finding Samples ......................................................................39 Table 4 Results for Spiked Recovery Samples...................................................................39 Table 5 Results for Test Samples .......................................................................................40 FIGURES ......................................................................................................................................41 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Calibration Plot......................................................................................................41 Chromatogram ofCalibration Solution (0.5 mg/L)...............................................42 Chromatogram ofBiological Control Sample.......................................................42 Chromatogram ofTest Sample (0.010 mg/L) .......................................................43 Chromatogram ofTest Sample (1.0 mg/L) ...........................................................43Harlan StudyNumber: 41206436 Report Page 32 Analytical Investigations 

## 1 MATERIALSAND METHODS 1.1 Introduction The test item concentration in the test samples was determined by direct injection to an electrospray mass spectrometer (MS) using an external standard. The method was developed by the Department ofAnalytical 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 test samples were stored at approximately -20 °C prior to analysis. 1.4.2 Reagents and Solvents Water: Fisher Scientific, HPLC-MS grade. Test medium: as described in the biological part ofthis study. Solvents: methanol, Romil, UG grade. Reagents: formic acid, Fisher Scientific, analytical reagent grade.Harlan StudyNumber: 41206436 Report Page 33 Analytical Investigations 1.4.3 Preparation ofCalibration Standards 

### The test item (nominal 100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain calibration standards in the range of0.10 to 1.0 mg/L. A second series ofcalibration standards was similarly prepared in the range of0.30 to 1.5 mg/L. These solutions were used to determine the recovery and test sample concentrations. 1.4.4 Preparation ofLinearity Standards 

### The test item (nominal 100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain calibration standards in the range of0.10 to 1.0 mg/L. A second series ofcalibration standards was similarly prepared in the range of0.30 to 1.5 mg/L. These standards were used to evaluate the linearity ofthe analytical system. 1.4.5 Preparation ofSpiked Recovery Samples 

### To demonstrate the validity ofthe analytical procedure, volumes oftest medium were spiked with the test item and its recovery was assessed. The test item (nominal 100 and 160 mg) was initially dissolved in (50:50 v/v) methanol:test medium to prepare stock solutions with concentrations of 1 000 and 1600 mg/L. These stock solutions were further diluted with (50:50 v/v) methanol:test medium to produce solutions of32 and 100 mg/L. A defined volume of these stock solutions were diluted with test medium to obtain spiked recovery samples at concentrations of0.32 and 1.0 mg/L. Five replicates at each concentration level were prepared and subjected to the same treatment as the test samples. In 

### addition, test medium without the addition ofthe test item (synthetic control) was also analyzed. 1.4.6 Preparation ofTest Samples 

### The test samples were thawed with the aid ofsonication and centrifuged (4500 rpm, 10 minutes) to remove algal cells. The test samples were diluted with (50:50 v/v) methanol:test medium as shown in Table 1.Harlan StudyNumber: 41206436 Report Analytical Investigations 1.4.7 Instrumental Parameters Autosampler: Pump: Detector: Column: Mobile phase: Flowrate: Injection volume: Ionization mode: Polarity: Capillary voltage: Gas temperature: Nebulizerpressure: Fragmentor voltage: Drying gas flow: Data acquisition mode: SIM ion:
1.4.8 Calculations Agilent 1200 Series Agilent 1200 Series Agilent 1200 Series MSD no column water:methanol:formic acid (500:500:0.5, v/v/v) 0.5 mL/minute 10 )lL ESI positive 1000

## v 350

## oc 40 psi 100V 10 L/min Single Ion Monitoring (SIM) 102.2 m/z Page 34

A calibration curve was prepared using a quadratic function ofthe 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+~b**2** -4·a·(c- y) X = ----'-------'-------'--'- (1) 2·a where x: y: a, b, c: concentration ofthe test item ininjected sample [ mg/L] average peak area ofthe test item ininjected sample [counts] constantsHarlan StudyNumber: 41206436 Report Page 35 Analytical Investigations 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) concentrationofthe test item inthe test sample [ mg/L] concentrationofthetest item inthe injected sample solution [mg/L] sample preparation factor recoveryrate [%] (Ifnot correcting for recovery then R is taken as 100) 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 ofthe test item inthe spikedrecovery sample [mg/L] fortified concentration ofthe test item inthe spikedrecovery sample [mg/L] The percentage ofnominal concentration ofthe test item inthe test samples was calculated using the following equation: %nominal=_c_ ·100 cnom (4) where Cnom: nominal concentration inthe test sample [mg/L]Harlan StudyNumber: 41206436 Report Page 36 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validatedpriorto the analysis ofrange-finding samples; full validation was later conducted specifically over the test concentration range to be employed in the definitive test. The results for the range-finding test samples are summarized inTable 3. 

### 2.2 Definitive Test 2.2.1 Validation ofthe Analytical Method Linearity The calibration data for the calibration standards ofthe 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 1.51 mg/L. The R2 fit ofthe calibration curve to the data was 0.9998, 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 ofthe test item inthe recovery samples are presented inTable 4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were not corrected for recovery. Limit ofQuantitation (LOQ) The limit ofquantitation (LOQ) was determined by calculating the concentration equivalent to ten times the standard deviation of five synthetic controls. Using this method the limit of quantitation was determined to be 0.084 mg/L. 2.2.2 Test Samples The results obtained for the concentration ofthe test item found inthe test samples are presented in Table 5. Typical chromatograms are shown inFigures 2 to 5.Harlan StudyNumber: 41206436 Report Page 37 Analytical Investigations 

## 3 DISCUSSION The detection system was found to have acceptable correlation to the fit ofa quadratic equation. The analytical procedure was found to have acceptable recoveries oftest item in test medium. The method ofanalysis was validated and provento be suitable for use. Analysis of the test preparations for both the Acute Toxicity to Rainbow Trout and Acute Toxicity to Daphnia magna

### tests (Harlan Study Numbers: 41206434 and 41206435 respectively) showed consistent, near nominal results were obtained. However, analysis of the test preparations at 0 and 72 hours for this study (see Appendix 3) showed variable and inconsistent results were obtained. From a chemical perspective, the test item is a water soluble polyquaternary ammonium salt and there is a potential in this case for the polymer to bind to any receptive surface, the effects of which being more notable/pronounced at the lower test concentrations employed in

### the algal study.Harlan StudyNumber: 41206436 Report Page 38 Analytical Investigations 

## TABLES Table 1 Preparation ofTest Samples Nominal Sample Sample Final Nominal FinalVolume Preparation Concentration ofTest Concentration of Volume Factor Item Test Item

[mg/L] [mL] [mL] F [mg/L] Control 5 10 2 

### -

0.010 5 10 2 0.0050 0.032 5 10 2 0.016 0.10 5 10 2 0.05 0.32 5 10 2 0.16

1.0 5 10 2 0.50 not applicable Table 2 Calibration Standard Data Concentration ofTest Item Area [mg/L] [counts] 0 1.904 X 104 0.103 3.536 X 104 0.302 5.989x 104 0.514 8.894 X 104 0.706 1.122 X 105 1.027 1.528 X 105 1.512 2.098 X 105Harlan StudyNumber: 41206436 Report Analytical Investigations 

### Table3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration of Concentration Preparation Concentration Time point

[hours] 0
72

LOQ TestItem in ofTestItem in Range-Finding SampleVial Sample X

Cnom [mg/L] [mg/L] 0.010 <LOQ 0.10 0.0505 1.0 0.463 0.010 <LOQ 0.10 <LOQ 1.0 0.450
Limit ofQuantitation not applicable 

### Table4 Results for Spiked Recovery Samples Nominal Fortified Measured Sample Factor ofTest Item in Range-Finding Sample F c [mg!L] 2 <LOQ 2 0.101 2 0.925 2 <LOQ 2 <LOQ 2 0.900

Determined Analytical Concentration Concentration Concentration Preparation Concentration Recovery of ofTestItem in ofTestItem in Factor ofTestItem in Rate TestItem the Spiked the Sample the Spiked Sample Vial Sample Cnom Crort X F c R [mg!L] [mg/L] [mg!L] [mg!L] [%]

0.32 0.324 0.168 2 0.336 0.166 2 0.332 0.171 2 0.341 105 0.173 2 0.345 0.174 2 0.348 1.0 1.01 0.473 2 0.946 0.510 2 1.02 0.505 2 1.01 99 0.494 2 0.987 0.515 2 1.03 Acceptance Target 80-120 Forreportingpurposes, x hasbeen calculated from c retrospectively. Page 39

%ofNominal Concentration
[%]



## - 101 93



## -

- 90

Precision (Relative Standard Deviation of

Recovery) [%]
1.9

3.4
<10Harlan StudyNumber: 41206436 Report Analytical Investigations Table4 Results for Test Samples Nominal Measured Sample Concentration Concentration Preparation Time point

[hours] 0

72

LOQ of ofTestItem in TestItem in Sample Vial Test Sample Cnom X [mg/L] [mg/L] Control <LOQ/<LOQ

### •

0.010 <LOQ/<LOQ

### • 0.032 <LOQ/<LOQ

### • 0.10 0.137/<LOQ* 0.32 0.215/0.0960° 1.0 0.44110.340° Control <LOQ/<LOQ• 0.010 0.107/<LOQ• 0.032 <LOQ/<LOQ

### • 0.10 0.148/<LOQ• 0.32 0.0985/0.0795. 1.0 0.510/0.342" Limit ofQuantitation not applicable Forreportingpurposes, x hasbeen calculated from c retrospectively. Factor
F 2 2 2 2 2 2 2 2 2 2 2 2

"Duplicate samples stored at approximately -20 °C priorto analysis. Page40

Determined Concentration ofTestItem in %ofNominal

Test Sample Concentration
c [mg/L] [%] <LOQ/<LOQ* -1-

### • <LOQ/<LOQ

### • -1-. <LOQ/<LOQ

### • -1-. 0.274/<LOQ

### • 274/<LOQ* 0.429/0.192

### • 134/60° 0.882/0.680

### • 88/68° <LOQ/<LOQ• -1-. 0.213/<LOQ• 2130/<LOQ

### • <LOQ/<LOQ

### • -1-

### • 0.295/<LOQ• 295/<LOQ

### • 0.197/0.159• 62/50° 1.02/0.683. 102/68

### •Harlan StudyNumber: 41206436 Analytical Investigations 

## FIGURES Figure 1 Calibration Plot**~** 250000 .l!l ·c: 2 (IS !!? 200000 <(**~** (IS (I) a. 150000
100000
50000 Report

y=-8.680E+03x2 + 1.392E+05x + 1.904E+04 R2 =9.998E-01

0**+-------------r------------,------------~--**

0.0 0.5 1.0 1.5
Concentration (mg/L) Page 41Harlan StudyNumber: 41206436 Report Page 42 Analytical Investigations Figure 2 Chromatogram ofCalibration Solution (0.5 mg/L)

MSD1 TIC, MSFile (S:\ANASER\/S\41206<f:i6\EC!ilJi!rs\ECAOJ55\017-1701.D) ES-APl, SIM, Frag: 100

28000 26000 24000 22000 20000 18000 18000 14000**~~----------~~~----------------------~** 12000**·~~~~~·-,-,-,· ~-,-,~~·** **~,-~~~~,_~-,~-,~~~--,-,-~~** 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 mi Figure 3 Chromatogram ofBiological Control Sample*

MSD1 TIC,**MifFiie(S~ANASER\/S\41206436\ECAL955\ECi\T955\004-0401D)E&Af'~SIM,** Frag: 100

28000 26000 24000 22000 20000

# .~~~~**18000~~~--------------------~==========~--------------------------------------------~** 18000

14000 12000 0.3 0.4 0.5 0.6 0.7 0.8 0.9 mi

*Background interference level ofthe 102.2 m/z response. Not test item.Harlan StudyNumber: 41206436 Report Page 43 Analytical Investigations Figure 4 Chromatogram ofTest Sample (0.010 mg/L)*

,---------,MS ,

28000
28000
24000
22000
20000
18000
18000
14000
12000**~-,-··..,..-~,** ,- ' 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Figure 5 Chromatogram ofTest Sample (1.0 mg/L)

28000
28000
24000
22000
20000
18000
18000
14000
12000 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 miHarlan StudyNumber: 41206436 Report 

### Appendix 4 MonitoringAuthority Statement ofGLP Compliance 

# 'nH). 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 Shardlow 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).

Dr. AndrewJ. Gray Head, UK GLP Monitoring Authority 

# - Page44