REPORT 

Algal Growth Inhibition Test 

Data Requirementsffest 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 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

HVryenhoef 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

41206436 

23 April 2013 

Page 1 of44 
Harlan Study Number: 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 of Test Concentrations ........................................................ 19 

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

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

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

Table 1 Cell Densities and Percentage Inhibition of Growth from the Initial Rangefinding Test ............................................................................................................ 21 

Table 2 Cell Densities and Percentage Inhibition of Growth from the Second Rangefinding Test ............................................................................................................ 22 

Table 3 Cell Densities and pH Values in the Definitive Test .......................... , .................. 23 

Table 4 Daily Specific Growth Rates for the Control Cultures in the Definitive Test ....... 24 

Table 5 Inhibition of Growth Rate and Yield in the Definitive Test.. ................................ 25 

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

Figure 1 Mean Cell Densities v Time for the Definitive Test ............................................. 26 
Harlan Study Number: 41206436 Report Page3 

Figure 2 Inhibition of Growth Rate Based on Nominal Test Concentrations ...................... 27 

Figure 3 Inhibition of Yield Based on Nominal 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 of GLP Compliance .......................................... 44 
Harlan Study Number: 41206436 Report 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

Harlan Study Number: 

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 (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: 

.. //.-.I..,.A--4········· D;t;?_ 

2 3 APR 2013 

HVryenhoef 
Harlan Study Number: 41206436 Report Page 5 

QUALITY ASSURANCE STATEMENT 

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

Harlan Study Number: 

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 relevant responsible person and management. 

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

these audits and inspections are given below. 

Reported to the 

relevant Study 

Dates and Types of QA Inspections Director and Test 

Facility 

Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

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 System Preparation 14 January 2013 

14 January 2013 Process - based Exposure 14 January 2013 

17 January 2013 Process - based Assessment of Response 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 that this report reflects the raw data and the procedures followed. 

Quality Assurance: S. £evAN 
Harlan Study Number: 41206436 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: DMMullee 

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

f, 

I~ '1 

r / . ........................................................ 

Date: 

2 3 APR 2013 
Harlan Study Number: 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 of 24 ± 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. 

Results 

Exposure of Pseudokirchneriella subcapitata to 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 

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 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 

that the algae were exposed to near nominal concentrations throughout the duration of the test. 
Harlan Study Number: 41206436 Report 

GENERAL INFORMATION 

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 of the data will be 

chargeable to the Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41206436 Report Page 9 

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. 76112009 

2 TESTITEM 

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

the responsibility ofthe Sponsor. 

Identification: 

Description: 

Batch: 

Purity: 

Expiry date: 

Storage conditions: 
Harlan Study Number: 41206436 Report Page 10 

3 MATERIALS AND 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 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. 

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, 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). 

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 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 kept 
Harlan Study Number: 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 ofthe positive control are given in Appendix 1. The positive control was 

conducted between 17 September 2012 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 maintain the stock culture. 

The culture medium is defined in Appendix 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 of nominal test concentrations of 0.1 0, 1.0, 10 and 100 mg/L for a 

period of 72 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 of test 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 of the 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 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 

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. 
Harlan Study Number: 41206436 Report Page 12 

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

Counter. 

The results of the initial range-finding test showed significant inhibition of growth occurred at all 

test concentrations and so a second range-finding test was conducted by exposing 

Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010, 0.10 and 

1.0 mg/L for a period of72 hours. 

An amount of test 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, 0.10, 0.010 and 0.0010 mg/L. An aliquot (450 mL) of each 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 but not exposed to the test item. 

Exposure conditions in the second range-finding test were the same as those in the initial test. 

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

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

approximately -20 °C prior to analysis. 

3.4.2 Definitive Test 

Based on the results of the 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 

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

An amount of test 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, 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 of 0.010, 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 Study Number: 41206436 Report Page 13 

The concentration and stability of the 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 of test preparation were used for the control and three flasks each containing 100 mL 

were used for each 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 3.14 x 105 cells per mL. Inoculation of 900 mL of test medium with 14.3 mL of this 

algal suspension gave an initial nominal cell density of 5 x 1 03 cells per mL and had no 

significant dilution effect on the 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 of the control and each test preparation was determined at initiation of the test and after 

72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature 

within the incubator was recorded daily. 

3.4.2.4 Verification of Test 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 if necessary. 

The method of analysis, recovery and test preparation analyses are described in Appendix 3. 
Harlan Study Number: 41206436 Report Page 14 

3.4.3 Evaluation of Data 

3.4.3.1 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: 

ll 

N1 

Nn 

tl 

tn 

= 

= 

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

Where: 

Ir 

l..lc 

l..lt 

l..lc 

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: 41206436 Report Page 15 

3.4.3.2 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 

3.4.3.3 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 of evaluating dose-effect experiments (Litchfield and Wilcoxon, 1949). 

3.4.3.4 Statistical Analysis 

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

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

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

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

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

computer software package (SAS, 1999- 2001). 
Harlan Study Number: 41206436 Report Page 16 

3.5 Validation Criteria 

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

• The cell concentration of the control cultures must increase by a factor of at least 16 over 

the test period. 

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

the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) 

must not exceed 35%. 

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

cultures must not exceed 7%. 
Harlan Study Number: 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 of less than the LOQ, 101% 

and 93% of nominal respectively were obtained at 0 hours. A measured concentration of less 

than the LOQ was not unexpected at 0.010 mg/L given that the LOQ of the analytical method 

was determined to be 0.084 mg/L. Analysis of the test preparations at 72 hours showed 

measured test concentrations of less than the LOQ were obtained for the 0.010 and 0.10 mg/L 

test preparations whilst a measured concentration of90% of nominal 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 in Figure 2 and Figure 3. 

4.2.1 Validation Criteria 

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

of 126 after 72 hours. This increase was in line with the OECD Guideline that states the 

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

Mean cell density of control at 0 hours 

Mean cell density of control at 72 hours 

6.05 x 103 cells per mL 

7.60 x 105 cells per mL 
Harlan Study Number: 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 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 3% and hence satisfied the validation criterion given in the OECD 

Guideline which states that this 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 of the test item 

over the 72-Hour exposure period. 

Accordingly the following results were determined from the data: 

4.2.2.1 Inhibition of Growth 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 concentration that reduced growth rate by x%. 

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

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

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

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

differences between the control 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 of Yield 

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 concentration that reduced yield by x%. 
Harlan Study Number: 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 of the 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 of the 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 in the 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 of Test Concentrations 

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. Given that the test item was known to be stable it 
Harlan StudyNwnber: 41206436 Report Page20 

was considered that the algae were exposed to near nominal concentrations throughout the 

duration of the test. 

5 CONCLUSION 

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

investigated over a 72-Hour period 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 Stat Assoc 50, 1096-1121. 

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

Experiments. J Pharmacal 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 Study Number: 41206436 Report Page 21 

TABLES 

Table 1 Cell Densities and Percentage Inhibition of Growth from the Initial Rangefinding Test 

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

Nominal Concentration 

(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 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 
Harlan Study Number: 41206436 Report Page 22 

Table 2 Cell Densities and Percentage Inhibition of Growth 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 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: 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 of the cell counts from 

3 counts for each of the replicate flasks. 

R1 - ~ = Replicates 1 to 6 
Harlan Study Number: 41206436 Report Page24 

Table 4 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 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 1 to 6 
Harlan Study Number: 41206436 Report Page 25 

Table 5 Inhibition of Growth 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 

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

R1- ~ =Replicates 1 to 6 

SD = Standard Deviation 
Harlan Study Number: 41206436 Report Page26 

FIGURES 

Figure 1 Mean Cell Densities v Time for the Definitive Test 

l.OOE+06 -r-~-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_-:_"J.,' .. -----------------------------------------------------__ .,..J_- __ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ -__ ..,_ I ---+--- Control ............................................................................... , .............................................................................. .. _____________________________________ J ___________________________________ _ 

I -------------------------------------1-------------------------------- 7"""""" 

I / --------------------------------------!---------------------------- _,.. _____ _ I / 

-------------------------------------~---------------------- ---.1----------

- -6-· 0.010 mg!L 

---- 0.032 mg!L 

- -EI- · 0. 10 mg!L 

1 / 

I / ___.__ 0. 32 mg!L 

.__ ________ - -e-· 1. 0 mg!L ;:_ ___ ~ -------------------------------------~--------------- I -----'--------------- / 

I / 

I / I : I -------------------------------------t·------------------·--................................ 1........... -------;---------------------- : I I : I I 

I / 

I / i I 

I / _____________________________________ .l........................................................ .. ............... ..:... .. .L. ............................................................. ---

-------------------------------------.S.------------------------- -----------~-'------------------------------ ----- ' 7 .................................................................................. t............................................ ------------,~---------------------------- -------

-------------------------------------t------------------- ------------?-t-------------------------- ---------- -------------------------------------+---------------- --------------}'-----1------------------------ ------------ 1 / I --------------------------------------l------------- --------------,..--------+------------------- -------------;'-- 1 / I / ' ' / -------------------------------------+------- .................................. .,1.. ........................ 1--------------- ......................... , ............ .. ; / I / 

I / I / .............................................................................. .1. .................................. L ................................ J ............................................ L ........................ .. I I I / 

l.OOE+05 

/ I / 

/ I / I I I / ............ .:.. ...................... , .................................................... ~-- ..................... .L ............................................. .. 

I I I / 

I / / I / 

I / / 

I I I / ' / ' / i/ y 

-----------__ ::::::::::::::::::::::::t::::::::::::::::::::--:_:::::::;:..,~{j:::::::::::::::::::::::::::::::::::::: /1 / I 

::--::_:::::::::::::~::::~:::~:i:::::::::::::::--: __ ::::;:~:::::::~:::::::::::::::::::::::::::::::::::::-

------::;.-~-:::::. ..................................... t----------- ·----·-/---------------~----------------------------- ------ _.._.. I / I -------------------------------------t------- -------;-----------·--------1------------------ ...... I / I ...... I / I 

.... "'a...,...... --------------------+- ------7!"'----------·----------------1---- , ' / ............ I / I 

---------~"= ............. ::::.~-,------- -t-7t:............................................. -------·+---------------.................................. ;:: 

' ' V I _.._.. ' ' /• I --- ' '~ I I _.._.._.. , I I _.._.._.. ---------------------""-- -----------~--------- ...................................................... e:::r-..... -------·----------------------·---- ', ! _.._...- I 

' I _.._.._.._.. ~ 

', ! _.._...-.- ! ~r_.. 1 

vI I 

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

l.OOE+04 

0 24 48 72 

Time (Hours) 
Harlan Study Number: 41206436 Report 

Figure 2 Inhibition of Growth Rate Based on Nominal Test Concentrations 

120 r----.---..,..' . -....,.-.,...., ----ri --,-! ..,..! --,!--,-! ---....... --.... --,..-.,..., .....,....' - ............. ....,,. 

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

---------------:---------r---r--i---r(trr--------------:---------!f---:-----t---:---;--~·-;--

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

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

0,01 

i rrri,'li r rr:,:::,:::tl, 

0.1 

Concentration (mgL) 

Page27 
Harlan Study Number: 41206436 Report 

Figure 3 Inhibition of Yield Based on Nominal Test Concentrations 

0.01 0.1 

Concentration (mfifl) 

Page 28 
Harlan Study Number: 41206436 Report Page29 

APPENDICES 

Appendix 1 Positive Control 

A positive control (Harlan Study Number: 41205049) used potassium dichromate as the 

reference item at concentrations of 0.25, 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 on growth rate: 

No Observed Effect Concentration (NOEC) based on yield: 

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

Lowest Observed Effect Concentration (LOEC) based on yield: 

0.50 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 of confidence limits. 
Harlan Study Number: 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 

Na2EDT A.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.lN NaOH or HCL 

* Elga Optima 15+ or Elga Purelab Option R-15 BP 
Harlan Study Number: 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 of Calibration Standards ....................................................... 33 

1.4.4 Preparation of Linearity Standards .......................................................... 33 

1.4.5 Preparation of Spiked 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 ofthe Analytical Method ....................................................... 36 

2.2.2 Test Samples ............................................................................................ 36 

3 DISCUSSION ...................................................................................................................... 37 

TABLES ........................................................................................................................................ 38 

Table 1 Preparation of Test 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 of Calibration Solution (0.5 mg/L) ............................................... 42 

Chromatogram of Biological Control Sample ....................................................... 42 

Chromatogram ofTest Sample (0.010 mg/L) ...................................................... .43 

Chromatogram of Test Sample (1.0 mg/L) .......................................................... .43 
Harlan Study Number: 41206436 Report Page 32 

Analytical Investigations 

1 MATERIALS AND 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 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 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 Study Number: 41206436 Report Page 33 

Analytical Investigations 

1.4.3 Preparation of Calibration 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 of 0.10 to 1.0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 mg/L. These solutions were used to determine 

the recovery and test sample concentrations. 

1.4.4 Preparation of Linearity Standards 

The test item (nominal 100 mg) was dissolved in (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 of 0.10 to 1.0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 mg/L. These standards were used to evaluate 

the linearity ofthe 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 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 of 0.32 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 of the test item (synthetic control) was also analyzed. 

1.4.6 Preparation of Test Samples 

The test samples were thawed with the aid of sonication 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 Study Number: 41206436 Report 

Analytical Investigations 

1.4. 7 Instrumental Parameters 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Flow rate: 

Injection volume: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

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 of the form shown in equation (1). 

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

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

equation: 

-b+~b 2 -4·a·(c- y) X = ----'-------'-------'--'- (1) 

2·a 

where x: 

y: 

a, b, c: 

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

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

constants 
Harlan Study Number: 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) 

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

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

sample preparation factor 

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

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

equation: 

R =-c-·100 

cfort 

where R: 

c: 

(3) 

recovery rate [%] 

determined concentration of the test item in the spiked recovery sample 

[mg/L] 

fortified concentration of the test item in the spiked recovery sample 

[mg/L] 

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

the following equation: 

%nominal= _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg/L] 
Harlan Study Number: 41206436 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 over the test concentration range 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 

Linearity 

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

The data was found to have a quadratic correlation within the calibration range of 

0 to 1.51 mg/L. The R2 fit of the 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 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 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 of the test item found in the test samples are presented 

in Table 5. Typical chromatograms are shown in Figures 2 to 5. 
Harlan Study Number: 41206436 Report Page 37 

Analytical Investigations 

3 DISCUSSION 

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

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

The method of analysis was validated and proven to be suitable for use. 

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 Study Number: 41206436 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 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 of Test 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 105 
Harlan Study Number: 41206436 Report 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined Concentration 

of Concentration Preparation Concentration 

Time point 

[hours] 

0 

72 

LOQ 

Test Item in of Test Item in 

Range-Finding Sample Vial 

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 of Quantitation 

not applicable 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample 

Factor of Test 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 of Test Item in of Test Item in Factor of Test Item in Rate 

Test Item 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 

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

Page 39 

%of Nominal 

Concentration 

[%] 

-

101 

93 

-

-

90 

Precision 

(Relative 

Standard 

Deviation of 

Recovery) 

[%] 

1.9 

3.4 

<10 
Harlan Study Number: 41206436 Report 

Analytical Investigations 

Table 4 Results for Test Samples 

Nominal Measured Sample 

Concentration Concentration Preparation 

Time point 

[hours] 

0 

72 

LOQ 

of of Test Item in 

Test Item 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 of Quantitation 

not applicable 

For reporting purposes, x has been 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 prior to analysis. 

Page40 

Determined 

Concentration 

of Test Item in %of Nominal 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 Study Number: 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 41 
Harlan Study Number: 41206436 Report Page 42 

Analytical Investigations 

Figure 2 Chromatogram of Calibration 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 of Biological 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 Study Number: 41206436 Report Page 43 

Analytical Investigations 

Figure 4 Chromatogram of Test 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 of Test 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 mi 
Harlan Study Number: 41206436 Report 

Appendix 4 Monitoring Authority Statement of GLP 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 

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). 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority -

Page44