Harcros Chemicals Inc. 

5200 Speaker Road 

Kansas City, KS 66106 RECEIVE H 

OPP T CEI O 

Telephone: 913/621-7747 

Fax: 913/621-7746 

E-mail: OrganicsCS@Harcros.com 

2QI3 APR 2U PMII: 10 

Date 04/23/13 

Document Control Office (DCO) (7407M) 

Office of Pollution Control and Toxics (OPPT) 

U.S. Environmental Protection Agency (EPA) 

1200 Pennsylvania Ave. NW 

Washington, D.C. 20460 (202-564-8930) 

Attn: TSCA Section 4 

40 CFR § 799.5087 (Docket # EPA-HQ-OPPT-2007-0531) 

RE: Algal Inhibition Test CASRN 31138-65-5 

To Whom It May Concern: 

In accordance with EPA's final rule, Testing of Certain High Production Volume Chemicals; Second 

Group of Chemicals, published in the Federal Register on January 7, 2011 at 76 fed. Reg. 1067, 

please find enclosed the final report for the Algal Inhibition Test on D-gluco-Heptonic acid, 

monosodium salt, (2.xi.)- [CAS# 31138-65-5]. 

This study was conducted to fulfill the Toxicity to Algae requirements of testing category C as set forth 

in Table 2 of 40 CFR 799.5087(j). 

If any further information is required, please do not hesitate to contact me at (913) 621-7802 or at 

asavini@harcros.com. 

Ann Marie Savini 

Harcros Chemicals Inc. 

Regulatory Specialist 

cc: Howard Stevenson 

James Grantham 

Regards, 

Randy Buckner 

Don Meuschke 

John Miles 

File 

I III III! 

CONTAINS NO CBI 
P. 2 

REPORT 

harlan 

Sodium Glucoheptonate: 

Algal Inhibition Test 

Data Requirements/Test Guidelines: 

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

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

Commission Regulation (EC) No 761/2009 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 

Study Director: H Vryenhoef 

Test Facility: Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Sponsor: Harcros Chemicals Inc. 

3452 Corporate Drive 

DaltonGA 30720 

UNITED STATES OF AMERICA 

Harlan Study Number: 

Study Completion Date: 

41104696 

8 April 2013 

Page 1 of51 

CONTAINS NO CB 
p. 3 

Harlan Study Number: 41104696 Report Page 2 

CONTENTS 

CONTENTS 2 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 4 

QUALITY ASSURANCE STATEMENT 5 

SIGNATURE OF CONTRIBUTING SCIENTISTS 6 

SUMMARY 7 

Introduction 7 

Methods 7 

Results 7 

GENERAL INFORMATION : 8 

Schedule 8 

Archiving 8 

1 INTRODUCTION AND PURPOSE 9 

1.1 Guidelines / 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 test 11 

3.4.2 Definitive test 12 

3.4.3 Evaluation of data 13 

3.5 Validation Criteria 16 

4 RESULTS 17 

4.1 Range-finding Test 17 

4.2 Definitive Test 17 

4.2.1 Validation criteria 17 

4.2.2 Growth data 18 

4.2.3 Observations on cultures 20 

4.2.4 Observations on test item solubility 20 

4.2.5 Physico-chemical measurements 20 

4.2.6 Re-growth test 20 

4.2.7 Verification of test concentrations 20 

5 CONCLUSION 21 

6 REFERENCES 21 

TABLES 22 

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

Test 22 

Table 2 Cell Densities in the Definitive Test 23 

Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive Test 24 

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

(0-72 Hour) 25 

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

(0-96 Hour) 26 

Table 6 pH values in the Definitive Test 27 

FIGURES 28 
P. 4 

Harlan Study Number: 41104696 Report Page 3 

Figure 1 Mean Cell Densities v Time for the Definitive Test 28 

Figure 2 Inhibition of Growth Rate (0 - 72 hours) 29 

Figure 3 Inhibition of Yield (0 - 72 hours) 30 

Figure 4 Inhibition of Biomass Integral (0 - 72 hours) 31 

Figure 5 Inhibition of Growth Rate (0 - 96 hours) 32 

Figure 6 Inhibition of Yield (0 - 96 hours) 33 

Figure 7 Inhibition of Biomass Integral (0 - 96 hours) 34 

Figure 8 Temperature Measurements Taken Throughout the Test Period 35 

APPENDICES 36 

Appendix 1 Positive Control 36 

Appendix 2 Culture Medium 37 

Appendix 3 Analytical Investigations 38 

Appendix 4 Monitoring Authority Statement of GLP Compliance 51 
p. 5 

Harlan Study Number: 41104696 Report Page 4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

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). 

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

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

Study Director: H Vryenhoef 

Harlan Study Number: 

Study Title: Sodium Glucoheptonate 

Algal Inhibition Test 

411044696 

08 APR 2013 
p. 6 

Harlan Study Number: 41104696 Report Page 5 

QUALITY ASSURANCE STATEMENT 

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

Harlan Study Number: 411044696 

Study Title: Sodium Glucoheptonate 

Algal 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. 

Dates and Types of QA Inspections 

Reported to the 

relevant Study 

Director and Test 

Facility 

Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

02 August 2011 

14 January 2013 

14 January 2013 

14 January 2013 

17 January 2013 

22 January 2013 

14 March 2013 

General Study Plan 

Verification 

Process - based 

Process - based 

Process - based 

Process - based 

Process - based 

Report Audit 

N/A 

Test Item Preparation 

Test System Preparation 

Exposure 

Assessment of Response 

Chemical Analysis 

N/A 

02 August 2011 

14 January 2013 

14 January 2013 

14 January 2013 

17 January 2013 

22 January 2013 

14 March 2013 

The complete Study Plan (including Study Specific Supplement) was verified for GLP 

compliance at report audit. 

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

Quality Assurance: 

Date: (j \ APR 20fl' 
p. 7 

Harlan Study Number: 41104696 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D M Mullee 

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

Date :

 0» APR 2013 
p. 8 

Harlan Study Number: 41104696 Report Page 7 

SUMMARY 

Introduction 

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

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

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

Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009 and the US EPA 

Draft Ecological Effects Test Guideline OPPTS 850.5400. 

Methods 

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

aqueous solution of the test item at concentrations of 10, 32, 100, 320 and 1000 mg active 

ingredient (ai)/L (three replicate flasks per concentration) for 96 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: 

Time Point 

(Hours) 

Response 

Variable 

ECi 0 (mg ai/L) 95% Confidence Limits 

(mg ai/L) 

No Observed 

Effect 

Concentration 

(NOEC) (mg ai/L) 

Lowest Observed 

Effect 

Concentration 

(LOEC) (mg ai/L) 

Growth Rate 510 * 32 100 

72 Yield 150 120 - 190 32 100 

Biomass 160 140 - 190 32 100 

Growth Rate 790 * 100 320 

96 Yield 220 180 - 260 10 32 

Biomass 180 140 - 210 32 100 

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

from 93% to 105% of nominal and so the results are based on nominal test concentrations only. 

A re-growth test was performed which showed the test item to be algistatic in effect. 

* It was not possible to calculate 95% confidence limits for the ErCS0 values as the data generated did not fit the 

models available for the calculation of confidence limits. 
p. 9 

Harlan Study Number: 41104696 Report Page 8 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

27 February 2012 

21 November 2012 

Archiving 

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. 
p. 10 

Harlan Study Number: 41104696 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 

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 / Regulations 

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

internationally accepted guidelines and recommendations: 

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

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

Regulation (EC) No 761/2009 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 

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

responsibility of the Sponsor. 

subcapitata. 

2 TEST ITEM 

Identification: 

Description: 

Batch: 

Purity: 

Expiry / Retest Date: 

Storage Conditions: 

Sodium Glucoheptonate 

dark brown liquid 

921000100 

49.5% w/w 

9 February 2015 

room temperature in the dark 
p. 11 

Harlan Study Number: 41104696 Report Page 10 

MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Biomass: 

ECx: 

Growth: 

Growth Rate (Average Specific 

Growth Rate): 

Yield: 

LOEC (Lowest Observed 

Effect Concentration): 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

Section-by-Section Growth 

Rate: 

AUC (Area Under the Growth 

Curve): 

Measurement variable (fluorescence or algal cell density) as 

surrogate measure for algal biomass. 

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

reduction of the respective growth parameter relative to the 

control (or solvent control, if applicable) within a stated 

exposure period. 

The increase in biomass over the test period. 

The logarithmic increase in biomass during the exposure 

period. 

The value of the measurement variable at the end of the 

exposure period minus the measurement variable's value at the 

start of the exposure period, calculated to express biomass 

increase during the test. 

The lowest test concentration at which a statistically significant 

inhibition of growth is determined relative to the control (or 

solvent control, if applicable). 

The highest test concentration at which no statistically 

significant inhibition of growth is determined relative to the 

control (or solvent control, if applicable). 

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

solvent control (if applicable). 

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

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

Biomass integral calculated as area under the growth curve. 

3.2 Test Species 

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 3.2). The master cultures were maintained in the laboratory under 

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

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

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

103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant 
Harlan Study Number: 41104696 Report 

p. 12 

Page 11 

agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal 

cell density was approximately 104 -10 cells/mL. 

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

Details of the positive control are given in Appendix 1. The positive control was conducted 

between 13 February 2012 and 17 February 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 used in the definitive test had a hardness of 

18 mg/L as CaCC>3 and a Total Organic Carbon (TOC) content of less than 1.0 mg C/L. 

The culture medium is defined in Appendix 2, 

3.4 Procedure 

3.4.1 Range-finding test 

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

cells to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg ai/L for a period of 

96 hours. All test concentrations were corrected for atest item water content of 50.5%. 

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 (1010 mg) was dissolved in culture medium and the volume adjusted to 

500 mL to give a 1000 mg ai/L stock solution from which a series of dilutions was made to give 

further stock solutions of 100, 10 and 1.0 mg ai/L. An aliquot (450 mL) of each of the stock 

solutions was separately inoculated with algal suspension (9.7 mL) to give the required test 

concentrations of 1.0, 10, 100 and 1000 mg ai/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 96 hours. 
p. 13 

Harlan Study Number: 41104696 Report Page 12 

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

Counter. 

A sample of each test concentration was taken for chemical analysis at 0 and 96 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 test the following test concentrations were assigned to 

the definitive test: 10, 32, 100, 320 and 1000 mg ai/L. 

3.4.2.1 Experimental Preparation 

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

test concentrations were corrected for a test item water content of 50.5%. 

Amounts of test item (2020 and 646 mg) were each separately dissolved in culture medium and 

the volume adjusted to 1 liter to give 1000 and 320 mg ai/1 stock solutions respectively. A series 

of dilutions was made from these stock solutions to give further stock solutions of 100, 32 and 

10 mg ai/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 

algal suspension (13.5 mL) to give the required test concentrations of 10, 32, 100, 320 and 

1000 mg ai/L. 

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

adequate mixing and homogeneity. 

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

analysis at 0 and 96 hours (see Appendix 3). 

3.4.2.2 Exposure conditions 

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

100 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 6.68 x 105 cells per mL. Inoculation of 900 mL of test medium with 13.5 mL of this 

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

dilution effect on the final test concentration. 

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® 

Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 
Harlan Study Number: 41104696 Report 

p. 14 

Page 13 

7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at 

approximately 150 rpm for 96 hours. 

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

Coulter® Multisizer Particle Counter. 

3.4.2.3 Physico-chemical measurements 

At the request of the Sponsor, at the start of the test the pH of the control and test preparations 

was adjusted to pH 7. The pH of the control and each test preparation was determined at initiation 

of the test and after both 48 and 96 hours exposure. The pH was measured using a WTW pH 320 

pH meter. The temperature within the incubator was recorded hourly using the incubators' 

internal pt-100 temperature probe and Multicom Software Package (see Figure 8). 

3.4.2.4 Re-growth test 

A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the 

test item. Aliquots were removed from each replicate control and test culture (0.25 mL and 

0.50 mL respectively) and the replicates pooled. Fresh sterile culture medium (100 mL) was 

added to each to ensure that the test concentration was reduced to below the inhibiting level. 

The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron® 

Version 2 Incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 

7000 lux) and constantly shaken at approximately 150 rpm for 120 hours. 

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: 

lnN n -ln N 

where: 

Ni 

N„ 

ti 

tn 

average specific growth rate from time ti to t, to 

cell concentration at tj 

cell concentration at tn 

time of first measurement 

time of nt h measurement 
p. 15 

Harlan Study Number: 41104696 Report Page 14 

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, 2-3 and 3-4) 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 r =^xl0 0 

where: 

I r = percentage inhibition of average specific growth rate 

He

 = mean average specific growth rate for the control cultures 

Mt

 = average specific growth rate for the test culture 

3.4.3.2 Comparison of Yield 

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

equation: 

Y = N n - N 0 

where: 

Y = yield 

No = cell concentration at the start of the test 

N n — 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: 

I y = x 100 

where: 

Iy = percentage inhibition of yield 

Yc = mean value for yield in the control group 

Yt = mean value for yield for the treatment group 
p. 16 

Harlan Study Number: 41104696 Report Page 15 

3.4.3.3 Comparison of biomass integral 

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

A =

 N i - N £ x ^ + N, + N 2 -2N 0x(t 2 _ti)+N„,1+N„-2N0x(tn _ ^ 

where: 

A = area 

No = nominal cell concentration at start of test 

Ni = measured cell concentration at ti 

N n = measured cell concentration at t n 

ti = time of first measurement after beginning of test 

t n = time of n* measurement after beginning of test 

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

using the following equation: 

A £ Z A , xl0 0 

Ac 

where: 

IA = percentage inhibition of the biomass integral 

Ac = mean biomass integral for the control cultures 

At = biomass integral for the test culture 

3.4.3.4 Determination of ECX 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 computerised interpolation 

using the Xlfit software package (IDBS). ECX values were then determined from the equation for 

the fitted line. 

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

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

3.4.3.5 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, yield and biomass integral data after 

72 and 96 hours for the control and all test concentrations to determine any statistically significant 
p. 17 

Harlan Study Number: 41104696 Report Page 16 

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

SAS computer software package (SAS 1999 - 2001). 

3.5 Validation Criteria 

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

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

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

(EPA/OPPTS Guidelines). 

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

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

exceed 35% (OECD Guidelines). 

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

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

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

exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines). 
p. 18 

Harlan Study Number: 41104696 Report Page 17 

4 RESULTS 

4.1 Range-finding Test 

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

Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1. 

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

growth was observed to be reduced at 100 and 1000 mg ai/L 

Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg ai/L were selected 

for the definitive test. 

Chemical analysis of the 10, 100 and 1000 mg ai/L test preparations at 0 and 96 hours (see 

Appendix 3) showed measured test concentrations to range from 87% to 129% of nominal 

indicating that the test item was stable over the test duration. 

4.2 Definitive Test 

Cell density values determined at each sampling time are given in Table 2. Daily specific growth 

rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values 

for the control and test cultures after 72 hours and percentage inhibition values are given in Table 

4. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours 

and percentage inhibition values are given in Table 5. pH values determined at 0,48 and 96 hours 

are given in Table 6. 

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, Figure 3, Figure 4, Figure 5, 

Figure 6 and Figure 7. 

4.2.1 Validation criteria 

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

160 after 72 hours and by a factor of 239 after 96 hours. This increase was in line with the OECD 

Guideline that states the enhancement must be at least by a factor of 16 after 72 hours and the 

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

96 hours. 

Mean cell density of control at 0 hours : 1.05 x 104 cells per mL 

Mean cell density of control at 72 hours 1.68 x 106 cells per mL 

Mean cell density of control at 96 hours : 2.51 x 106 cells per mL 

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

cultures over the test period (0-72 hours) was 9% and hence satisfied the validation criterion given 

in the OECD Guideline which states the mean must not exceed 35%. 
p. 19 

Harlan Study Number: 41104696 Report Page 18 

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

period (0 - 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline 

which states that this must not exceed 7%. 

The coefficient of variation of the cell density values in replicate control cultures was 12% after 

72 hours and 3% after 96 hours and hence satisfied the validation criterion which states that this 

must not exceed 20%. 

4.2.2 Growth data 

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

integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of 

the test item over the 96-Hour exposure period. 

Accordingly the following results were determined from the data: 

4.2.2.1 Inhibition of growth rate 

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

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

all test concentrations using one way analysis of variance incorporating Bartletfs test for 

homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure 

for comparing several treatments with a control (Dunnett 1955). After 72 hours there were no 

statistically significant differences between the control, 10 and 32 mg ai/L test concentrations 

(P^0.05), however all other test concentrations were significantly different (PO.05) and, 

therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32 mg ai/L. 

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

100 mg ai/L. After 96 hours there were no statistically significant differences between the control, 

10, 32 and 100 mg ai/L test concentrations (P>0.05), however all other test concentrations were 

significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) 

based on growth rate was 100 mg ai/L. Correspondingly the "Lowest Observed Effect 

Concentration" (LOEC) based on growth rate was 320 mg ai/L, 

ErCI 0 (0 

ErC20 (0 

ErC5 0 (0 

ErC9 0 (0 

ErCio (0 

ErC2 0 (0 

ErC5 0 (0 

ErC9 0 (0 

72 h) 

72 h) 

72 h) 

72 h) 

96 h) 

96 h) 

96 h) 

96 h) 

200 mg ai/L 

330 mg ai/L 

790 mg ai/L* 

>1000mg ai/L 

130 mg ai/L 

210 mg ai/L 

510 mg ai/L* 

>1000 mg ai/L 

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

available for the calculation of confidence limits. 
p. 20 

Harlan Study Number: 41104696 Report Page 19 

4.2.2.2 Inhibition of yield 

EyCjo(0 

EyC2O(0 

EyC30(0 

EyC9() (0 

EyCio(0 

EyC20(0 

EyC50(0 

EyC9Q (0 

72 h) 

72 h) 

72 h) 

72 h) 

96 h) 

96 h) 

96 h) 

96 h) 

470 mg ai/L 

54 mg ai/L 

90 mg ai/L 

220 mg ai/L; 95% confidence limits 180 - 260 mg ai/L 

860 mg ai/L 

50 mg ai/L 

75 mg ai/L 

150 mg ai/L; 95% confidence limits 120 - 190 mg ai/L 

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

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

72 hours there were no statistically significant differences between the control, 10 and 32 mg ai/L 

test concentrations (P>0.05), however all other test concentrations were significantly different 

(P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 

32 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on 

yield was 100 mg ai/L. After 96 hours there were no statistically significant differences between 

the control and 10 mg ai/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 10 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" 

(LOEC) based on yield was 32 mg ai/L. 

4.2.2.3 Inhibition of biomass integral 

EbCio (0-72h) 51 mg ai/L 

EbC2 0 (0 - 72h) 78 mg ai/L 

EbC5 0 (0-72h) 160 mg ai/L; 95% confidence limits 140 - 190 mg ai/L 

EbC9 0 (0 -72h) 500 mg ai/L 

EbCio (0-96h) 49 mg ai/L 

EbC2 0 (0 - 96 h) 78 mg ai/L 

EbC5 0 (0 - 96 h) 180 mg ai/L; 95% confidence limits 140 - 210 mg ai/L 

EbC9 0 (0-96h) 620 mg ai/L 

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

by x%. 

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

Section 4.2.2.1. There were no statistically significant differences between the control, 10 and 

32 mg ai/L test concentrations (P>0.05), however all other test concentrations were significantly 

different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on 

biomass integral was 32 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" 

(LOEC) based on biomass integral was 100 mg ai/L. 
p. 21 

Harlan Study Number: 41104696 Report Page 20 

4.2.3 Observations on cultures 

All test and control cultures were inspected microscopically at 96 hours. After 96 hours there 

were no abnormalities detected in the control or test cultures at 10, 32, 100 and 320 mg ai/L, 

however cell debris was observed to be present in the test cultures at 1000 mg ai/L. 

4.2.4 Observations on test item solubility 

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

cultures were observed to range from clear colorless solutions at 10 mg ai/L through to amber 

colored solutions at 1000 mg ai/L. After the 96-Hour test period all control, 10, 32, 100 and 

320 mg ai/L test cultures were observed to be green dispersions whilst the 1000 mg ai/L test 

cultures were observed to be amber/green dispersions. 

4.2.5 Physico-chemical measurements 

The pH values of the control and each test preparation are given in Table 6. Temperature was 

maintained at 24 ± 1 °C throughout the test. 

The pH value of the control cultures (see Table 6) was observed to increase from pH 7.0 at 

0 hours to pH 8.1 at 48 hours and pH 8.2 at 96 hours. This increase was considered to be due to 

the amount of carbon dioxide required by the large number of algal cells in the log phase of 

growth (see Figure 1) exceeding the transfer rate of CO2 from the gaseous phase to the aqueous 

phase. In this situation CO2 required for photosynthesis and growth would be derived from 

bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH 

after 96 hours was in excess of 1 pH unit after 96 hours. This was considered to have had no 

adverse effect on the results of the study given that the increase in cell concentration in the control 

cultures exceeded the validation criterion given in the Test Guidelines. 

4.2.6 Re-growth test 

Re-growth was observed to have occurred in the control, 10, 32,100 and 320 mg ai/L test cultures 

after 48 hours and in the 1000 mg ai/L test culture after 120 hours. These results indicate that the 

test item was algistatic in effect. 

4.2.7 Verification of test concentrations 

Analysis of the test preparations at 0 and 96 hours (see Appendix 3) showed measured test 

concentrations to range from 93% to 105% of nominal and so the results are based on nominal test 

concentrations only 
p. 22 

Harlan Study Number: 41104696 Report Page 21 

5 CONCLUSION 

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

over a 96-Hour period and gave the following results: 

Time Point 

(Hours) 

Response 

Variable 

EC50 (mg ai/L) 95% Confidence Limits 

(mg ai/L) 

No Observed 

Effect 

Concentration 

(NOEC) (mg ai/L) 

Lowest Observed 

Effect 

Concentration 

(LOEC) (mg ai/L) 

Growth Rate 510 * 32 100 

72 Yield 150 120 - 190 32 100 

Biomass 160 140 - 190 32 100 

Growth Rate 790 * 100 320 

96 Yield 220 180 - 260 10 32 

Biomass 180 140 - 210 32 100 

A re-growth test was performed which showed the test item to be algistatic in effect. 

6 REFERENCES 

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

With a Control. J Am Stat Assoc 50,1096-1121. 

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

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

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

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

Xlfit, ID Business Solutions Ltd. 

* It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the 

models available for the calculation of confidence limits. 
p. 23 

Harlan Study Number: 41104696 Report Page 22 

TABLES 

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

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

Nominal Concentration 

(mg ai/L) 0 Hours 96 Hours Growth Rate 

YielcVBiomass 

Integral 

Control Ri 

R2 

Mean 

1.14E+04 

1.16E+04 

1.I5E+04 

2.22E+06 

2.25E+06 

2.23E+06 

- -

1.0 Ri 1.07E+04 2.27E+06 

R2 

1.04E+04 2.04E+06 0 3 

Mean 1.05E+04 2.16E+06 

10 Ri 1.03E+04 2.09E+06 

R2 

1.00E+04 1.84E+06 0 12 

Mean 1.02E+04 1.97E+06 

100 Ri 1.04E+04 1.67E+06 

R2 

1.07E+04 1.81E+06 4 22 

Mean 1.06E+04 1.74E+06 

1000 Ri 1.06E+04 1.59E+05 

R2 

1.26E+04 1.78E+05 49 93 

Mean 1.16E+04 1.68E+05 

* 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. 

Ki and R2 = Replicates 1 and 2 
p. 24 

Harlan Study Number: 41104696 Report Page 23 

Table 2 Cell Densities in the Definitive Test 

Nominal Concentration 

Cell Densities* (cells per mL) 

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

Control Ri 1.09E+04 5.43E+04 2.80E+05 1.41E+06 2.44E+06 

Rz 1.00E+04 5.09E+04 3.58E+05 1.77E+06 2.45E+06 

R3 

1.00E+04 6.18E+04 3.75E+05 1.85E+06 2.47E+06 

R4 1.09X+04 6.35E+04 3.59E+05 1.62E+06 2.55E+06 

R5 

1.06E-KJ4 6.10E+04 3.60E+05 1.50E-KJ6 2.61E+06 

R* 1.06E-KJ4 6.16E+04 4.14E+05 1.90E-KJ6 2.50E+06 

Mean 1.05E+04 5.88E+04 3.58E+05 1.68E+06 2.51E+06 

10 Ri 1.13E-H)4 4.86E+04 3.13E+05 1.37E+06 2.74E+06 

R2 

1.03E+04 6.35E+04 3.81E+05 1.79E+06 2.80E+06 

R3 

1.31E+04 5.41E+04 2.89E+05 1.57E+06 2.57E+06 

Mean 1.16E+04 5.54E+04 3.28E+05 1.58E+06 2.70E+06 

32 R] 1.27E+04 5.80E+04 2.59E+05 1.43E+06 2.24E+06 

R2 

1.03E-KJ4 5.36E+04 3.14E+05 1.74E+06 2.19E+06 

R3 

1.15E+04 5.28E+04 3.57E+05 1.50E+06 2.14E+06 

Mean 1.15E+04 5.4SE-KM 3.10E+05 1.55E+06 2.19E+06 

100 Ri 1.05E+04 4.87E+04 2.60E+05 1.24E+06 2.06E+06 

R2 

1.1OE+04 4.37E-KJ4 2.93E+05 1.16E+06 2.14E+06 

R3 1.15E+04 4.88E+04 3.00E+05 1.16E+06 1.70E+06 

Mean 1.10E+04 4.71E+04 2.84E+05 1.19E+06 1.97E+06 

320 Ri 1.15E+04 3.20E+04 5.82E+04 3.13E+05 9.14E+05 

R2 

1.07E+04 3.59E+04 7.49E+04 2.90E+05 8.75E+05 

R3 

1.04E+04 3.55E+04 7.26E+04 3.39E+05 1.01E+06 

Mean 1.09E+04 3.45E-KJ4 6.86E+04 3.14E+05 9.31E+05 

1000 Ri 1.07E+04 1.91E-K)4 2.02E+04 4.49E+04 1.29E-KJ5 

R2 

1.09E+04 2.03E+04 2.11E+04 2.60E+04 8.90E+04 

R3 

1.13E+04 2.21E+04 2.06E+04 4.34E+04 7.08E+04 

Mean 1.10E+04 2.05E+04 2.06E+04 3.81E+04 9.64E+04 

* 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. 

R] - Rfi = Replicates 1 to 6 
p. 25 

Harlan Study Number: 41104696 Report Page 24 

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

Daily Specific Growth Rate (cells/mL/hour) 

Day 0 -1 Day 1 - 2 Day2-3 Day 3 - 4 

Control 

Ri 0.070 0.068 0.067 0.023 

Control 

R2 

0.068 0.081 0.067 0.013 

Control 

R3 

0.076 0.075 0.066 0.012 

Control R4 0.077 0.072 0.063 0.019 

R3 

0.075 0.074 0.060 0.023 

Control 

R5 0.076 0.079 0.064 0.011 

Control 

Mean 0.074 0.075 0.065 0.017 

Ri - R« = Replicates 1 to 6 
p. 26 

Harlan Study Number: 41104696 Report Page 25 

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

(0 - 72 Hour) 

Growth Rate Yield Biomass Integral 

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

(mg ai/L) 

0-72h % Inhibition 0-72h % 

Inhibition* 0-72 h % 

Inhibition 

Control Ri 

R2 

RJ 

R* 

R5 

Re 

Mean 

SD 

0.069 

0.072 

0.072 

0.071 

0.070 

0.073 

0.071 

0.001 

-

1.40E+06 

1.76E+06 

1.84E+06 

1.61E+06 

1.49E+06 

1.89E+06 

1.67E+06 

1.97E+05 

-

2.44E+07 

3.05E+07 

3.21E+07 

2.90E+07 

2.75E+07 

3.37E+07 

2.95E+07 

3.33E+06 

-

10 Ri 0.068 4 1.36E+06 2.45E+07 17 

R2 0.072 [1] 1.78E+06 3.16E+07 m 

R3 

0.070 1 1.56E+06 2.65E+07 10 

Mean 0.070 1 1.57E+06 6 2.75E+07 7 

SD 0.002 2.12E+05 3.65E+06 

32 Ri 0.069 3 1.41E+06 2.41E+07 18 

R2 

0.072 [1] 1.73E+06 2.91E+07 2 

R3 

0.070 1 1.49E+06 2.72E+07 8 

Mean 0.070 1 1.54E+06 7 2.68E+07 9 

SD 0.002 1.64E+05 2.49E+06 

100 Ri 0.067 6 1.23E+06 2.17E+07 26 

R2 

0.066 7 1.15E+06 2.14E+07 27 

R3 

0.066 7 1.15E+06 2.17E-HJ7 26 

Mean 0.066 7 1.18E+06 29 2.16E+07 26 

SD 0.001 4.79E+04 1.77E+05 

320 Ri 0.048 32 3.01E+05 5.32E+06 82 

R2 

0.047 34 2.80E+05 5.54E+06 81 

R3 

0.049 31 3.28E+05 6.06E+06 79 

Mean 0.048 32 3.03E+05 82 5.64E+06 81 

SD 0.001 2.43E+04 3.81E+05 

1000 R. 

0.021 70 3.42E+04 8.82E+05 97 

R2 

0.013 82 1.51E+04 7.06E+05 98 

R3 

0.020 72 3.20E+04 9.43E+05 97 

Mean 0.018 75 2.71E+04 98 8.44E+05 97 

SD 0.004 1.04E+04 I.23E+05 

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

Ri - Re - Replicates 1 to 6 

SD = Standard Deviation 

[Increase in growth as compared to controls] 
p. 27 

Harlan Study Number: 41104696 Report Page 26 

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

(0 - 96 Hour) 

Growth Rate Yield Biomass Integral 

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

(mg ai/L) 

0-96 h % Inhibition 0-96 h % 

Inhibition* 0-96 h % Inhibition 

Control Ri 

R2 

R3 

R4 

R5 

R$ 

Mean 

SD 

0.057 

0.057 

0.057 

0.058 

0.058 

0.058 

0.058 

0.001 

-

2.43E+06 

2.44E+06 

2.46E+06 

2.54E+06 

2.60E+06 

2.49E+06 

2.49E+06 

6.65E+04 

-

7.04E+07 

8.09E+07 

8.36E-KJ7 

7.87E+07 

7.67E+07 

8.63E+07 

7.95E+07 

5.61E+06 

-

10 Ri 0.058 0 2.73E+06 7.36E+07 7 

R2 

0.059 [2] 2.79E+06 8.65E+07 [9] 

R3 

0.058 0 2.55E+06 7.59E+07 4 

Mean 0.058 [1] 2.69E+06 [8] 7.87E+07 1 

SD 0.001 1.26E+05 6.89E+06 

32 Ri 0.056 3 2.23E+06 6.79E+07 15 

R2 

0.056 3 2.18E+06 7.59E+07 4 

R3 

0.056 3 2.13E+06 7.06E+07 11 

Mean 0.056 3 2.18E-+06 13 7.15E+07 10 

SD 0.000 5.13E+04 4.09E+06 

100 Ri 0.056 3 2.05E+06 6.12E+07 23 

R2 

0.056 3 2.13E-KJ6 6.08E+07 24 

R3 

0.054 7 1.69E+06 5.58E+07 30 

Mean 0.055 4 1.96E+06 22 5.93E+07 26 

SD 0.001 2.34E+05 2.97E+06 

320 Ri 0.047 19 9.02E+05 1.98E+07 75 

R2 

0.047 19 8.64E+05 1.93E+07 76 

R3 0.048 17 9.95E+05 2.20E+07 72 

Mean 0.047 18 9.20E+05 63 2.03E+07 74 

SD 0.001 6.75E+04 1.41E+06 

1000 Ri 0.027 53 1.19E+05 2.73E+06 97 

R2 

0.023 60 7.81E+04 1.85E+06 98 

R3 

0.020 66 5.95E+04 2.07E+06 97 

Mean 0.023 60 8.54E+04 97 2.22E+06 97 

SD 0.004 3.02E+04 4.60E+05 

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

Ri-Re = Replicates 1 to 6 

SD = Standard Deviation 

[Increase in growth as compared to controls] 
p. 28 

Harlan Study Number: 41104696 Report Page 27 

Table 6 pH values in the Definitive Test 

Nominal Concentration 

(mg ai/L) 

pH 

Nominal Concentration 

(mg ai/L) 

0 Hours 

48 Hours 96 Hours 

Nominal Concentration 

(mg ai/L) 

Pre-adjustment Post-adjustment 

48 Hours 96 Hours 

Control 7.7 7.0 8.1 8.2 

10 7.2 7.0 8.1 8.2 

32 7.4 7.0 8.0 8.1 

100 8.7 7.0 7.9 8.1 

320 9.3 7.0 7.8 8.0 

1000 9.6 7.0 7.7 8.0 
p. 29 

Harlan Study Number: 41104696 Report Page 28 

FIGURES 

Figure 1 Mean Cell Densities v Time for the Definitive Test 

I.O0E-+O71 

1.00E+06-" 

o 

1.00E-H35-

1.00E+04 

1.00E-H33 

48 

Time (Hours) 
p. 30 

Harlan Study Number: 41104696 Report Page 29 

Figure 2 Inhibition of Growth Rate (0 - 72 hours) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

-10 

Growth Rate 

i 

! i : L 

; 

• i i/'. 

i 

' \ i 

i | 

i j I ! 

i i i i 

| 1 | i 

I ! | 

! ; 

• i i i i i . t ... 

i 1 

10 100 

Concentration (mg ai/L) 

1000 
p. 31 

Harlan Study Number: 41104696 Report Page 30 
Harlan Study Number: 41104696 Report Page 31 
p. 33 

Harlan Study Number: 41104696 Report Page 32 

Figure 5 Inhibition of Growth Rate (0 - 96 hours) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

-10 

Growth Rate 

i i 

I 1 

! ! 

i ; 

| I 

[ j 

+ 

i i i i - —1 

10 100 

Concentration (mg ai/L) 

1000 
Harlan Study Number: 41104696 Report Page 33 
p. 35 

Harlan Study Number: 41104696 Report Page 34 

Figure 7 Inhibition of Biomass Integral (0 - 96 hours) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Biomass Integral j j 

] | 

i j 

j i 

j ! / 

i i / 

v.xX/.. 

Xk 

+ \ \ 

i j 

I—i i i i 

10 100 

Concentration (mg ai/L) 

1000 
p. 36 

Harlan Study Number: 41104696 Report Page 35 

Figure 8 Temperature Measurements Taken Throughout the Test Period 

26.0 

Time (Hours) 
p. 37 

Harlan Study Number: 41104696 Report Page 36 

APPENDICES 

Appendix 1 Positive Control 

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

concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. 

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

definitive test. 

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

following results: 

Time Point 

(Hours) 

Response 

Variable 

EC50 (mg/L) 95% Confidence 

Limits (mg/L) 

No Observed 

Effect 

Concentration 

(NOEC) (mg/L) 

Lowest 

Observed Effect 

Concentration 

(LOEC) (mg/L) 

72 

Growth Rate 0.31 0.28 - 0.35 0.10 0.32 

72 Yield 0.26 * 72 0.10 0.32 

Biomass 0.27 * 0.10 0.32 

96 

Growth Rate 0.35 0.30 - 0.40 0.10 0.32 

96 Yield 0.27 * 96 0.10 0.32 

Biomass 0.26 0.10 0.32 

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

reference item. 

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

models available for the calculation of confidence limits. 
p. 38 

Harlan Study Number: 41104696 Report Page 37 

Appendix 2 Culture Medium 

NaN03 25.5 mg/L 

MgCl2.6H20 12.164 mg/L 

CaCl2.2H20 4.41 mg/L 

MgS04.7H20 14.7 mg/L 

K2HP04 

1.044 mg/L 

NaHC03 15.0 mg/L 

H3BO3 0.1855 mg/L 

MnCl2.4H20 0.415 mg/L 

ZnCl2 0.00327 mg/L 

FeCl3.6H20 0.159 mg/L 

CoCl2.6H20 0.00143 mg/L 

Na 2Mo04 -2H2 0 0.00726 mg/L 

CuCl2.2H20 0.000012 mg/L 

Na2EDTA.2H20 0.30 mg/L 

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

adjusted to 7.5 ± 0.1 with O.lNNaOH or HCL 

* Elga Optima 15+ or Elga Purelab Option R-15 BP 
p. 39 

Harlan Study Number: 41104696 Report Page 38 

Appendix 3 Analytical Investigations 

Analytical Investigations 

CONTENTS 

CONTENTS 38 

1 MATERIALS AND METHODS 39 

1.1 Introduction 39 

1.2 Test Item 39 

1.3 Analytical Standard 39 

1.4 Analytical Procedure 39 

1.4.1 Storage 39 

1.4.2 Reagents and Solvents 39 

1.4.3 Preparation of Calibration Standards 40 

1.4.4 Preparation of Linearity Standards 40 

1.4.5 Preparation of Spiked Recovery Samples 40 

1.4.6 Preparation of Test Samples 40 

1.4.7 Instrumental Parameters 41 

1.4.8 Calculations 41 

2 RESULTS 43 

2.1 Range-Finding Test 43 

2.2 Definitive Test 43 

2.2.1 Validation of the Analytical Method 43 

2.2.2 Test Samples 43 

3 DISCUSSION 44 

TABLES 45 

Table 1 Preparation of Test Samples 45 

Table 2 Calibration Standard Data 45 

Table 3 Results for Range-Finding Samples 46 

Table 4 Results for Spiked Recovery Samples 46 

Table 5 Results for Test Samples 47 

FIGURES 48 

Figure 1 Linearity 48 

Figure 2 Chromatogram of Calibration Solution (1.5 mg ai/L) 49 

Figure 3 Chromatogram of Biological Control Sample 49 

Figure 4 Chromatogram of Test Sample (1000 mg ai/L) 50 
p. 40 

Harlan Study Number: 41104696 Report Page 39 

Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The test item concentration in the test samples was determined by direct injection into the mass 

spectrometer (HPLC-MS) using an external standard to calibrate the system. 

All test sample concentrations were corrected for a water content of 50.5%, and are reported as 

milligrams of active ingredient (ai) per litre. 

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 samples were stored at approximately -20 °C prior to analysis. 

1.4.2 Reagents and Solvents 

Water: prepared using an ELGA Pure Lab Option R-l 5 water purification system. 

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

Solvents: methanol, Fisher Scientific, LC-MS grade. 

Reagents: ammonia solution, Fisher Scientific, AR grade. 
p. 41 

Harlan Study Number: 41104696 Report Page 40 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

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

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

produce a solution of 100 mg ai/L. Defined volumes of this solution were then diluted with 

diluent* to obtain calibration standards in the range of 0.10 to 1.25 mg ai/L. A second series of 

calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/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 ai) was dissolved in diluent* (100 mL) to prepare a stock solution 

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

produce a solution of 100 mg ai/L. Defined volumes of this solution were then diluted with 

diluent" to obtain calibration standards in the range of 0.10 to 1.25 mg ai/L. A second series of 

calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/L. These standards 

were used to evaluate the linearity of the analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

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

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

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

solution was further diluted with diluent* to produce a solution of 10 mg ai/L. Defined volumes 

of this stock solution were diluted with test medium to obtain spiked recovery samples at 

concentrations of 1.0 mg ai/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. They were further diluted into the 

calibration range with solvent/mixture as shown in Table 1. 

* Diluent:- methanol:water:testmedia:ammonia (50:40:10:0.1 v/v/v/v). 
p. 42 

Harlan Study Number: 41104696 

Analytical Investigations 

Report Page 41 

1.4.7 Instrumental Parameters 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Agilent 1100 

Agilent 1100 

Agilent 1100 Series MSD 

none, direct injection 

methanol:water:ammonia solution (500:500:1, v/v/v) 

Flow rate: 

Injection volume: 

0.5 mL/minute 

10 uL 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

Data acquisition mode: 

SIM ions: 

ESI 

negative 

4000 V 

350 °C 

40 psi 

100 V 

12 L/min 

Single Ion Monitoring (SIM) 

225 m/z 

1.4.8 Calculations 

A calibration plot was prepared using a linear plot of the form y = mx + a. 

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

equation: 

x = m 

where x: 

y: 

m: 

a: 

(1) 

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

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

slope of the calibration plot [counts per mg ai/L] 

y-axis intercept [counts] 
p. 43 

Harlan Study Number: 41104696 Report Page 42 

Analytical Investigations 

The concentration of the test item in the test samples was calculated using the following equation: 

c

 =dr^*F (2) R/100 

where c 

x 

F 

R 

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

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

sample preparation factor 

recovery rate [%] (If not 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 = — -100 (3) 

C

fort 

where R: recovery rate [%] 

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

[mg/L] 

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

ai/L] 

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

the following equation: 

% nominal = • 100 (4) 

where 

Cnom: nominal concentration in the test sample [mg ai/L] 
p. 44 

Harlan Study Number: 41104696 Report Page 43 

Analytical Investigations 

2 RESULT S 

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 

Specificity 

The biological control samples and analytical blank showed a significant interfering response at 

the mass specific to the test item; however this was taken into consideration when calculating the 

standards, samples and LOQ. The standard response changed accordingly with known 

concentration, hence the specificity was confirmed. 

Linearity 

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

data was found to have a linear correlation within the calibration range of 0 to 1.5 mg ai/L. The 

R

2

 fit of the calibration curve to the data was 0.9993, 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 three times the concentration of 

the response seen in the blank. Using this method the limit of quantitation was determined to be 

2.3 mg ai/L. 

2.2.2 Test Samples 

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

in Table 5. 

Typical chromatograms are shown in Figures 2 to 4. 
p. 45 

Harlan Study Number: 41104696 Report 

Analytical Investigations 

3 DISCUSSION 

Page 44 

The detection system was found to have acceptable linearity. The analytical procedure was found 

to have acceptable recoveries of test item in test medium. The method of analysis was validated 

and proven to be suitable for use. 

/ 
p. 46 

Harlan Study Number: 41104696 Report Page 45 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal 

Concentration of 

Test Item 

[mg ai/L] 

Sample 

Volume 

[mL] 

Final Volume 

[mL] 

Sample 

Preparation 

Factor 

F 

Final Nominal 

Concentration of Test 

Item 

[mg ai/L] 

Control 5 50 10 -

10 5 50 10 1.0 

32 25 

100, 

then 1 -> 10 

40 0.80 

100 5 

50, 

thenl -> 10 

100 1.0 

320 5 

200 

then 1 -> 10 

400 0.80 

1000 1 

100, 

then 1 -» 10 

1000 1.0 

not applicable 

Table 2 Calibration Standard Data 

Concentration of Test Item Area 

[mg ai/L] [counts] 

0 3.335 x 104 

0.104 6.812 xlO4 

0.257 1.140 x10s 

0.522 1.765 x 105 

0.770 2.385 x 105 

1.04 3.177 x 105 

1.03 3.097 x 105 

1,31 3.814 x 105 

1.54 4.481 x 105 
p. 47 

Harlan Study Number: 41104696 Report Page 46 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Time point 

Nominal 

Concentration 

of 

Test Item in 

Range-Finding 

Sample 

Measured 

Concentration 

of Test Item in 

Sample Vial 

Sample 

Preparation 

Factor 

Determined 

Concentration 

of Test Item in 

Range-Finding 

Sample 

% of Nominal 

Concentration 

*-nom 

X F c 

[hours] [mg ai/L] [mg ai/L] [mg ai/L] [%] 

0 10 0.868 10 8.68 87 

100 0.901 100 90.1 90 

1000 0.998 1000 998 100 

96 10 1.29 10 12.9 129 

100 0.981 100 98.1 98 

1000 1.02 1000 1016 102 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Analytical 

Concentration Concentration Concentration Preparation Concentration Recovery Precision 

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

Test Item the Spiked the Sample the Spiked Standard 

Sample Vial Sample Deviation of 

Recovery) 

c

nom Cforl X F c R 

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

1000 1011 0.991 1000 991 

0.890 1000 890 

1.02 1000 1018 98 6.0 

1.02 woo 1021 

1.04 1000 1037 

Acceptance Target 80-120 <io 

For reporting purposes, x has been calculated from c retrospectively. 
p. 48 

Harlan Study Number: 41104696 Report Page 47 

Analytical Investigations 

Table 5 Results for Test Samples 

Time point 

[hours] 

Nominal 

Concentration 

of 

Test Item in 

Test Sample 

[mg ai/L] 

Measured 

Concentration 

of Test Item in 

Sample Vial 

X 

[mg ai/L] 

Sample 

Preparation 

Factor 

F 

Determined 

Concentration 

of Test Item in 

Test Sample 

c 

[mg ai/L] 

% of Nominal 

Concentration 

[%] 

0 Control <LOQ 10 <LOQ -

10 0.939 10 9.39 94 

32 0.780 40 31.2 97 

100 0.975 100 97.5 98 

320 0.818 400 327 102 

1000 1.02 1000 1023 102 

96 Control 

10 

32 

100 

320 

1000 

<LOQ 

0.929 

0.803 

1.01 

0.835 

1.04 

10 

10 

40 

100 

400 

1000 

<LOQ 

9.29 

32.1 

101 

334 

1035 

93 

100 

101 

105 

104 

LOQ = Limit of Quantitation 

not applicable 

For reporting purposes, x has been calculated from c retrospectively. 
p. 49 

Harlan Study Number; 41104696 

Analytical Investigations 

Report Page 48 

FIGURES 

Figure 1 Linearity 

600000 • 

450000 -

400000 -

350000 -

300000 • 

250000 -

200000 • 

150000 -

100000 • 

50000 -

0 

0.0 

y = 264456x+ 38877 

R*= 0.9993 

0.5 1.0 1,5 2.0 

Concentfation{mgai/L) 
p. 50 

Harlan Study Number: 41104696 

Analytical Investigations 

Report Page 49 

Figure 2 Chromatogram of Calibration Solution (1.5 mg ai/L) 

MSD1 TIC, MS Flta (S:\ANASERVSW1104696\ECAL146\ECAL146\024-2401.D} API-ES, SIM. Frafl' 100 

ft 

0 2 0.4 0.6 0.8 1 1 2 14 1.6 1.8 mln 

Figure 3 Chromatogram of Biological Control Sample 

MSDI TIC, IMS Rte(S.lANASERVSWf13488ffieCAL1'ieteCAL!JSl008-3801.Oi API-ES, SIM, Frag- 100 

0 2 0 4 0.6 05 1 1.2 1.4 1.6 18 mln 

Response seen in test media control subtracted from controls and test samples 
p. 51 

Harlan Study Number: 41104696 

Analytical Investigations 

Report Page 50 

Figure 4 Chromatogram of Test Sample (1000 mg ai/L) 

MSD1 TIC, MS Flls (S:\ANASERVSVM1C4696\ECAL146\ECAL146\018-1801.D) API-ES, SIM, Frag: 100 

- | r— . , 1-

0.2 04 06 0n—•— 8 1

1—•—r 1.2 1.4 1 6 1.8 mln 
Harlan Study Number: 41104696 Report 

p. 52 

Page 51 

Appendix 3 Monitoring Authority Statement of GLP Compliance 

(DH1 Department 

V_ '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 TEST TYPE(S) 

Harlan Laboratories Ltd Analytlcaf/Cllnfcal 

Shardlow Business Park * rhaml „.„ , 

London Road Chemistry 

Shardlow Environmental Toxicity 

Derby Environmental Fate 

DE72 2GD Mutagenicity 

Phys/Chem. Tests 

Toxicology 

DATE OF INSPECTION 

10 July 2012 

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 
ORIGIN ID: KCKA C9I3> 621-7733 SHIP DATE: 23APR13 

JAKE BDOREH ACTWGT: 0.7 LB (IAN 

HARCROS CHEMICALS INC CAD: 0978415/CAFE2608 

5200 SPEAKER RD 

KANSAS CITY. KS SS10G BILL THIRD PARTY 

UNITED STATES US 

TO(DCO)(7407M)(OPPT) 

US EPA TSCA SECTION 4 

1200 PENNSYLVANIA AVE NW 

WASHINGTON DC 20460 

(202)664-6930 

REF: POSTAGE 

H 5408 6000 0564 

WED - 24 APR 10:30A 

PRIORITY OVERNIGHT 

XC RDVA 20460 

DC-US DCA