p. 1 

Harcros Chemicals Inc. 

5200 Speaker Road 

Kansas City, KS 66106 

RECEIVED 

OPPT CBIC 2 

Telephone: 913/621-7747 

Fax: 913/621-7746 

E-mail: OrganicsCS@Harcros.com 

2M3 JUN IU AMIO-t* 

CBIC Control Number 

Date 06/12/13 354719 

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: Daphnia, 48 hour Acute Immobilization 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 Daphnia, 48 hour Acute Immobilization Test on D-glucoHeptonic acid, monosodium salt, (2.xi.)- [CAS# 31138-65-5). 

This study was conducted to fulfill the Acute Toxicity to Daphnia 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: Peter Radford 

James Grantham 

Randy Buckner 

John Miles 

File 

Regards, 
REPORT 

harlan 

Sodium Glucoheptonate: 

Daphnia sp., 48-Hour Acute Immobilization Test 

Data Requirements/Test Guidelines: 

• OECD Guidelines for Testing of Chemicals (April 2004) No 202, 

"Daphnia sp., Acute Immobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.1010 "Aquatic 

Invertebrate Acute Toxicity Test, Freshwater Daphnids" 

Study Director: S Harris 

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: 41104695 

Study Completion Date: 1 May 2013 

Page 1 of 41 
P. 3 

Harlan Study Number: 41104695 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 Test Water. 10 

3.4 Procedure 11 

3.4.1 Range-finding Test 11 

3.4.2 Definitive Test 11 

3.4.3 Post-Study Experiment 13 

4 RESULTS 15 

4.1 Range-finding Test 15 

4.2 Definitive Test 15 

4.2.1 Immobilization Data 15 

4.2.2 Observations on Test Item Solubility 15 

4.2.3 Physico-Chemical Measurements 16 

4.2.4 Verification of Test Concentrations 16 

4.3 Post-Study Experiment 16 

5 CONCLUSION 16 

TABLES 17 

Table 1 Cumulative Immobilization Data in the Range-finding Test 17 

Table 2 Cumulative Immobilization Data in the Definitive Test 18 

Table 3 Cumulative Immobilization Data in the Post-Study Experiment 19 

FIGURES 20 

Figure 1 Testo Temperature Data 20 

APPENDICES 21 

Appendix 1 Reconstituted Water - Elendt M7 Medium 21 

Appendix 2 Positive Control 23 

Appendix 3 Reconstituted Water 24 

Appendix 4 Analytical Investigations 25 
P-4 

Harlan Study Number: 41104695 Report Page 3 

Appendix 5 Physico-Chemical Measurements 40 

Appendix 6 Monitoring Authority Statement of GLP Compliance 41 
p. 5 

Harlan Study Number: 41104695 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). 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHLW, MAFF and METI). 

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

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

Study Director: S Harris 

Harlan Study Number: 

Study Title: Sodium Glucoheptonate: 

Daphnia sp., 48-Hour Acute Immobilization Test 

41104695 

Date :

 0 1 MAY 2013 
Harlan Study Number: 41104695 Report Page 5 

QUALITY ASSURANCE STATEMENT 

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

Harlan Study Number: 

Study Title: 

41104695 

Sodium Glucoheptonate: 

Daphnia sp., 48-Hour Acute Immobilization 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 General Study Plan 

Verification 

N/A 02 August 2011 

10 January 2013 Process - based Test Item Preparation 10 January 2013 

10 January 2013 Process - based Test System 

Preparation 

10 January 2013 

15 January 2013 Process - based Exposure 15 January 2013 

10 January 2013 Process - based Assessment of 

Response 

10 January 2013 

11 January 2013 Process - based Chemical Analysis 11 January 2013 

08 April 2013 Report Audit N/A 08 April 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: 30 APR 2013 
Harlan Study Number: 41104695 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D M Mullee 

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

Date: 
P. 8 

Harlan Study Number: 41104695 Report Page 7 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The 

method followed was designed to be compatible with the US EPA Draft Ecological Effects Test 

Guidelines OPPTS 850.1010 (April 1996) and the OECD Guidelines for Testing of Chemicals 

(April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of 

Commission Regulation (EC) No. 440/2008. 

Methods 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were 

exposed to an aqueous solution of the test item at concentrations of 100, 180, 320, 560 and 

1000 mg active ingredient (ai)/L for 48 hours at a temperature of 20 °C to 21 °C under static test 

conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours. 

Results 

The 48-Hour ECso for the test item to Daphnia magna based on nominal test concentrations was 

greater than 1000 mg ai/L. The No Observed Effect Concentration was 1000 mg ai/L. 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range 

from 80% to 83% of nominal value and so the results are based on nominal test concentrations 

only. 

This study was submitted to the U.S. Environmental Protection Agency (EPA) by 

Harcros Chemicals as part of a TSCA Section 4 test rule. Submittal of the study to 

EPA does not authorize any entity to cite to or refer to the study without express 

authorization from Harcros Chemicals. Harcros Chemicals retains data compensation 

rights under all statutory and regulatory programs, including, but not limited to, EU 

REACH and Korea-REACH. Possession of a copy of the study, in whole or in part, 

does not constitute "legitimate possession" of the study and does not create any rights 

to use the study or the data presented herein for any chemical registration (such as, 

but not limited to, the REACH registration program) or any other commercial 

purpose. In addition, Harcros Chemicals expressly retains all rights for first 

publication of the data and the information presented herein. In the event anyone 

seeks the right to cite or refer to the data, please contact Harcros Chemicals. 
p. 9 

Harlan Study Number: 41104695 Report Page 8 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

21 February 2012 

14 March 2013 

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: 4II04695 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 acute toxicity of the test item to Daphnia magna. 

Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, 

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 (April 2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.1010 "Aquatic 

Invertebrate Acute Toxicity Test, Freshwater Daphnids" 

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

the responsibility of the Sponsor. 

2 TEST ITEM 

Identification: 

Description: 

Sodium Glucoheptonate 

dark brown liquid 

Batch: 

Purity: 

Expiry / retest date: 

Storage conditions: 

921000100 

49.5% w/w 

9 February 2015 

room temperature in the dark 
p. 11 

Harlan Study Number: 41104695 Report Page 10 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Immobilization: Those organisms which are not able to swim within 15 seconds 

after gentle agitation of the test container are considered to be 

immobile. 

EC50: The calculated concentration of test item which results in 50% 

immobilization within the exposure period. 

NOEC (No Observed Effect The highest test concentration or loading rate at which no 

Concentration): significant toxic effect on the test organisms is observed within 

the exposure period. 

Treatment: Comprises test item treatments, control (test water only) and 

solvent control (if applicable). 

3.2 Test Species 

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory 

cultures. 

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see 

Appendix 1) in a temperature controlled room at approximately 20 °C. The lighting cycle was 

controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk 

transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus 

subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction 

was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such 

that the young daphnids produced overnight were less than 24 hours old. These young were 

removed from the cultures and used for testing. The diet and diluent water are considered not to 

contain any contaminant that would affect the integrity or outcome of the study. 

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the 

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

conducted between 6 June 2012 and 8 June 2012. 

3.3 Test Water 

For both the range finding and definitive tests, reconstituted water was used 

(Appendix 3). The theoretical hardness of reconstituted water is 250 mg/L as CaCC>3. 

Therefore, in order to comply with the water characteristics of the OPPTS guideline, the 

prepared water was diluted in a ratio of 50:50 with deionized reverse osmosis water prior to use 

to reduce the total water hardness. 
p. 12 

Harlan Study Number: 41104695 Report Page 11 

At the start of the definitive test the diluent had a total water hardness of 148 mg/L as CaC03 and 

an alkalinity of 26 mg/L as CaCC>3. The alkalinity was determined using the methods described 

in Field and On-site Test Methods for the Analysis of Waters (British Standards Institution, 

1993). 

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. 

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations 

of 0.10, 1.0, 10, 100 and 1000 mg ai/L. The test item was dissolved directly in reconstituted 

water. 

An amount of test item (1980 mg) was dissolved in reconstituted water and the volume adjusted 

to 1 liter to give the 1000 mg ai/L test concentration. Serial dilutions were made in reconstituted 

water to give the remainder of the test concentrations. In error, the test concentration was 

corrected for a water content of 49.5% instead of 50.5%, however, as this difference was so 

small, this error was considered not to have had an effect on the outcome of the test. 

Each prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity, and the pH of each was adjusted to pH 7. 

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained 

in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 8 hours 

darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 

250 mL test and control vessel contained 200 mL of test media and was covered to reduce 

evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded. 

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

A sample of each test concentration was taken for chemical analysis at 0 and 48 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. Only concentrations within the range to be used for the 

definitive test were analyzed. 

3.4.2 Definitive Test 

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

the definitive test: 100, 180, 320, 560 and 1000 mg ai/L. All test sample concentrations were 

corrected for a water content of 50.5%. 
Harlan Study Number: 41104695 Report 

p. 13 

Page 12 

3.4.2.1 Experimental Preparation 

For the purpose of the definitive test the test item was dissolved directly in reconstituted water. 

An amount of test item (10100 mg) was dissolved in reconstituted water and the volume adjusted 

to 5 liters to give the 1000 mg ai/L test concentration. Aliquots (100, 180, 320 and 560 mL) of 

the 1000 mg ai/L test concentration were added to a final volume of 1 liter to give the remaining 

test concentrations of 100, 180,320 and 560 mg ai/L. 

Each stock prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity and the pH of each was adjusted to pH 7. 

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

analysis at 0 and 48 hours (see Appendix 4). 

3.4.2.2 Exposure Conditions 

In the definitive test 250 mL glass jars containing 200 mL of test preparation were used. At the 

start of the test 5 daphnids were placed in each test and control vessel at random, in the test 

preparations. Four replicate test and control vessels were prepared. The test vessels were then 

covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 

21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk 

transition periods with a light intensity ranging from 645 to 682 lux. The daphnids were not 

individually identified, received no food during exposure and the test vessels were not aerated. 

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

The test preparations were not renewed during the exposure period. Any immobilization or 

adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The 

criterion of effect used was that Daphnia were considered to be immobilized if they were unable 

to swim for approximately 15 seconds after gentle agitation. 

3.4.2.3 Physico-Chemical Measurements 

Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen 

concentrations and pH were recorded at the start and termination of the test. The pH and 

dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter 

whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. 
Harlan Study Number: 41104695 Report 

p. 14 

Page 13 

3-4.2.4 Verification of Test Concentrations 

Water samples were taken from the control and each test group (replicates Ri - R* pooled) at 0 

and 48 hours for quantitative analysis. Samples were stored at approximately -20 °C prior to 

analysis. 

Duplicate samples were taken and stored at approximately -20 °C for further analysis if 

necessary. 

Samples at the No Observed Effect Concentration and above only were analyzed. 

The method of analysis, recovery and test preparation analyses are described in Appendix 4. 

3.4.2.5 Evaluation of Data 

An estimate of the EC50 values was given by inspection of the immobilization data. 

3.4.3 Post-Study Experiment 

Whilst significant immobilization was observed to have occurred at 1000 mg ai/L in the range 

finding test, no immobilization occurred at any test concentration employed in the definitive test. 

It was therefore considered appropriate to conduct a post-study experiment in order to confirm 

the results obtained. 

In the post-study experiment Daphnia magna were exposed to a series of nominal test 

concentrations of 0.10, 1.0, 10, 100 and 1000 mg ai/L. The test item was dissolved directly in 

reconstituted water. 

An amount of test item (1980 mg) was dissolved in reconstituted water and the volume adjusted 

to 1 liter to give the 1000 mg ai/L test concentration. Serial dilutions were made in reconstituted 

water to give the remainder of the test concentrations. 

Each prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity, and the pH of each was adjusted to pH 7. 

In the post-study experiment 10 daphnids were placed in each test and control vessel and 

maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 

8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 

250 mL test and control vessel contained 250 mL of test media and was covered to reduce 

evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded. 

The control group was maintained under identical conditions but not exposed to the test item. 
p. 15 

Harlan Study Number; 41104695 Report Page 14 

A sample of each test concentration was taken for chemical analysis at 0 and 48 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. Only concentrations of the NOEC and above were 

analyzed 
p. 16 

Harlan Study Number: 41104695 Report Page 15 

4 RESULT S 

4.1 Range-finding Test 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

range-finding test is given in Table 1. 

No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg ai/L. 

However, immobilization was observed at 1000 mg ai/L. 

Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg ai/L were 

selected for the definitive test. 

Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 4) showed that 

measured concentrations between 83% and 94% of nominal were obtained indicating that the test 

item was stable under test conditions. 

4.2 Definitive Test 

4.2.1 Immobilization Data 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

definitive test are given in Table 2. 

There was no immobilization in 20 daphnids exposed to a range of test concentrations of 100, 

180, 320, 560 and 1000 mg ai/L for a period of 48 hours. Inspection of the immobilization data 

gave the following results: 

Time (h) EC50 (mg ai/L) 

24 >1000 

48 >1000 

The No Observed Effect Concentration after 24 and 48 hours exposure was 1000 mg ai/L. 

4.2.2 Observations on Test Item Solubility 

Throughout the duration of the test, the test preparations were observed to range from an 

extremely pale amber colored solution in the 100 mg ai/L test group to a dark amber solution in 

the 1000 mg ai/L test group. 
p. 17 

Harlan Study Number: 41104695 Report Page 16 

4.2.3 Physico-Chemical Measurements 

The results of the physico-chemical measurements are given in Appendix 5, Temperature was 

maintained at approximately 20 °C to 21 °C throughout the test. 

The water temperature which was also recorded in the control vessel approximately every hour 

using a Testo temperature logger (see Figure 1) ranged from 20 °C to 21 °C. 

4.2.4 Verification of Test Concentrations 

Analysis of the test preparations at 0 and 48 hours (see Appendix 4) showed measured test 

concentrations to range from 80% to 83% of nominal value and so it was considered justifiable 

to estimate the EC$o values in terms of the nominal test concentrations only. 

4.3 Post-Study Experiment 

Cumulative immobilization data from the exposure of Daphnia Magna to the test item during the 

post-study experiment are given in Table 3. 

No immobilization was observed at the test concentrations of 0.10, 1.0, 10, 100 and 

1000 mg ai/L. 

Chemical analysis of the 1000 mg ai/L test preparation at 0 and 48 hours (see Appendix 4) 

showed near nominal concentrations were obtained indicating that the test system was correctly 

dosed. It was therefore considered that the immobilization observed in the range finding test was 

due to contamination rather than a true toxic effect. 

5 CONCLUSION 

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been 

investigated and gave a 48-Hour EC50 of greater than 1000 mg ai/L. The No Observed Effect 

Concentration was 1000 mg ai/L. 
Harlan Study Number: 41104695 Report Page 17 

TABLES 

Table 1 Cumulative Immobilization Data in the Range-finding Test 

Nominal 

Concentration 

(mg ai/L) 

Cumulative Immobilized Daphnia 

(Initial Population: 10 Per Replicate) Nominal 

Concentration 

(mg ai/L) 24 Hours 48 Hours 

Control 0 0 

0.10 0 0 

1.0 0 1 

10 0 0 

100 0 0 

1000 5 10 
Harlan Study Number: 41104695 Report Page 18 

Table 2 Cumulative Immobilization Data in the Definitive Test 

Nominal 

Concentration 

(mg/L) 

Cumulative Immobilized Daphnia 

Nomina (Initial Population: 5 Per Replicate) l 

Concentration 

(mg/L) 24 Hours 48 Hours 

Nominal 

Concentration 

(mg/L) 

Ri R2 Rs R< Total % Ri R2 R3 R4 Total % 

Control 0 0 0 0 0 0 0 0 0 0 0 0 

100 0 0 0 0 0 0 0 0 0 0 0 0 

180 0 0 0 0 0 0 0 0 0 0 0 0 

320 0 0 0 0 0 0 0 0 0 0 0 0 

560 0 0 0 0 0 0 0 0 0 0 0 0 

1000 0 0 0 0 0 0 0 0 0 0 0 0 

Ri -Rt = Replicates 1 to 4 
Harlan Study Number: 41104695 Report 

p. 20 

Page 19 

Table 3 Cumulative Immobilization Data in the Post-Study Experiment 

Nomina] 

Concentration 

(mg ai/L) 

Cumulative Immobilized Daphnia 

(Initial Population: 10 Per Replicate) Nomina] 

Concentration 

(mg ai/L) 24 Hours 48 Hours 

Control 0 0 

0.10 0 0 

1.0 0 0 

10 0 0 

100 0 0 

1000 0 0 

p. 22 

Harlan Study Number: 41104695 Report Page 21 

APPENDICES 

Appendix 1 Reconstituted Water - Elendt M7 Medium 

Solution Concentration of 

stock solution (mg/L) 

(I) H3B03 57190 

(II) MnCl 2

.4H2 0 7210 

(HI) LiCI 6120 

(IV) RbCl 1420 

(V) SrCI2

.6H20 3040 

(VI) NaBr 320 

(VII) Na 2Mo04.2H2 0 1260 

(VIII) CuCI2

.2H20 335 

(IX) ZnCl2 260 

(X) CoCl2

.6H20 200 

(XI) KI 65 

(XII) Na2Se03 43.8 

(XIII) NH4VO3 11.5 

(XIV) Na2EDTA.2H20 5000 

FeS04.7H20 1991 

An aliquot (dependant on the volume of medium required) of each stock solution was added to a 

final volume of deionized reverse osmosis water to give stock solution A and stored at 

approximately 21 °C. 
Harlan Study Number: 41104695 

Reconstituted Water - Elendt M7 

Report 

p. 23 

Page 22 

Macro Nutrient Stock Solutions 

Solution Concentration of 

stock solution (g/L) 

(XV) CaCl2

.2H20 293.80 

(XVI) NaHC03 64.80 

(XVII) MgS04.7H20 246.60 

(XVIII) Na 2Si03.9H2 0 50.00 

(XIX) KC1 58.00 

(XX) NaN03 2.74 

(XXI) K2HP04 1.84 

(XXII) KH2P04 1.43 

Vitamin Nutrients 

Solution Concentration of 

stock solution (mg/L) 

(XXIII) Thiamine hydrochloride 750 

Cyanocobalamine (vitamin Bi 2 ) 10 

D(+) biotin (vitamin H) 7.5 

The final medium was prepared by adding an aliquot of stock solution A along with aliquots of 

each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the 

required amount (final volume) of deionized reverse osmosis water. 

The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21 °C. 
Harlan Study Number: 41104695 Report 

p. 24 

Page 23 

Appendix 2 Positive Control 

A positive control (Harlan Study Number; 41203341) used potassium dichromate as the 

reference item at concentrations of 0.32,0.56, 1.0, 1.8 and 3.2 mg/L. 

Exposure conditions for the positive control were similar to those in the definitive test. 

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed 

Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test 

concentrations gave the following results: 

Time (h) ECso (mg/L) 95% Confidence limits 

(mg/L) 

24 0.75 0.56 - 1.0 

48 0.45 0.42 - 0.48 

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L 

respectively. The No Observed Effect Concentration is based upon zero immobilization at this 

concentration. 

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

for this reference item. 

* HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for 

Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol U, 714-719. 
p. 25 

Harlan Study Number: 41104695 Report Page 24 

Appendix 3 Reconstituted Water 

Stock Solutions 

a) 

b) 

c) 

d) 

CaCl2.2H20 

MgS04.7H2 0 

NaHC03 

KC1 

11.76 g/L 

4.93 g/L 

2.59 g/L 

0.23 g/L 

ii) Preparation 

An aliquot (25 mL) of each of solutions a-d was added to each liter (final volume) of 

deionized water with a conductivity of <5 jiS cm"'. The reconstituted water had a pH of 

7.8 ± 0.2 adjusted (if necessary) with NaOH or HC1 and was aerated until the dissolved 

oxygen concentration was approximately air-saturation value. 

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as 

CaC03. 
p. 26 

Harlan Study Number: 41104695 Report Page 25 

Appendix 4 Analytical Investigations 

Analytical Investigations 

CONTENTS 

CONTENTS 25 

1 MATERIALS AND METHODS 26 

1.1 Introduction 26 

1.2 Test Item 26 

1.3 Analytical Standard 26 

1.4 Analytical Procedure 26 

1.4.1 Storage 26 

1.4.2 Reagents and Solvents 26 

1.4.3 Preparation of Calibration Standards 27 

1.4.4 Preparation of Linearity Standards 27 

1.4.5 Preparation of Spiked Recovery Samples 27 

1.4.6 Preparation of Test Samples 27 

1.4.7 Instrumental Parameters 28 

1.4.8 Calculations 29 

2 RESULTS 31 

2.1 Range-Finding Test 31 

2.2 Definitive Test 31 

2.2.1 Validation of the Analytical Method 31 

2.2.2 Test Samples 31 

3 DISCUSSION 33 

TABLES 34 

Table 1 Preparation of Test Samples 34 

Table 2 Calibration Standard Data 34 

Table 3 Results for Range-Finding Samples 35 

Table 4 Results for Post-Study Range-Finding Samples 35 

Table 5 Results for Spiked Recovery Samples 35 

Table 6 Results for Test Samples 36 

FIGURES 37 

Figure 1 Calibration Plot 37 

Figure 2 Chromatogram of Calibration Solution (1.0 mg ai/L) 38 

Figure 3 Chromatogram of Biological Control Sample 38 

Figure 4 Chromatogram of Test Sample (1000 mg ai/L) 39 
p. 27 

Harlan Study Number: 41104695 Report Page 26 

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 liter. 

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-15 water purification system. 

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

Solvents: methanol, Fisher Scientific, HPLC grade. 

Reagents: ammonia, Fisher Scientific, Analytical reagent. 
Harlan Study Number: 41104695 

Analytical Investigations 

Report 

p. 28 

Page 27 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 200 mg) 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 10 mg/L. Defined volumes of this solution were then diluted with diluent* 

to obtain calibration standards in the range of 0.10 to 1.0 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 200 mg) 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 10 mg/L. Defined volumes of this solution were then diluted with diluent* 

to obtain calibration standards in the range of 0.10 to 1.0 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 200 mg) was initially 

dissolved in test medium to prepare a stock solution with a concentration of 1000 mg ai/L. 

Defined volumes of this stock solution were diluted with test medium to obtain spiked recovery 

samples at concentrations of 100 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 diluted into the calibration 

range initially in test medium and then further diluted with methanol:water:ammonia (50:50:0.10 

v/v/v) as shown in Table 1, and then analyzed directly using the instrumental procedure. 

* Diluent: methanol:water:ammonia:test medium (50:40:0.10:10 v/v/v/v) 
p. 29 

Harlan Study Number: 41104695 Report Page 28 

Analytical Investigations 

1.4.7 Instrumental Parameters 

Autosampler: Agilent 1200 Series 

Pump: Agilent 1200 Series 

Detector: Agilent 1200 Series MSD 

Column: none, direct injection 

Mobile phase: waterrmethanol.-ammonia (500:500:1, v/v/v) 

Flow rate: 0.5 mL/minute 

Injection volume: 10 uJL 

Column temperature: room temperature 

Ionization mode: ESI 

Polarity: negative 

Capillary voltage: 4000 V 

Gas temperature: 350 °C 

Nebulizer pressure: 40 psi 

Fragmentor voltage: 100 V 

Drying gas flow: 12 L/min 

Data acquisition mode: Single Ion Monitoring (SIM) 

SIM ions: 225 m/z 
p. 30 

Harlan Study Number: 41104695 Report Page 29 

Analytical Investigations 

1.4.S Calculations 

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

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

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

equation: 

x = 

_-b + ^b2-4-a-(c-y) 

2-a 

0) 

where x: 

ya, b, c: 

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

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

constants 

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

equation: 

c = R/100 

where c 

x 

F 

R 

• F (2) 

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) 

'fort 

where R: recovery rate [%] 

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

ai/L] 

Cf0rt: 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: 
p. 31 

Harlan Study Number: 41104695 Report Page 30 

Analytical Investigations 

% nominal = —— • 100 (4) 

where 

Cnom: nominal concentration in the test sample [mg ai/L] 
Harlan Study Number: 41104695 

Analytical Investigations 

Report 

p. 32 

Page 31 

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 employed in the 

definitive test. The results for the range-finding test samples are summarized in Table 3. 

2.2 Definitive Test 

2.2.1 Validation of the Analytical Method 

Specificity 

The biological control samples and an analyzed analytical blank showed no significant 

interfering response at the retention time of the test item. The standard solutions contained a 

peak specific for the test item whose area changed accordingly with known concentration, hence 

the specificity of the method by retention time was confirmed. 

Linearity 

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

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

1.6 mg ai /L. The R2 fit of the calibration curve to the data was 0.9985, 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 5. 

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

Limit of Quantitation (LOQ) 

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

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

quantitation was determined to be 0.047 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 6. 
p. 33 

Harlan Study Number: 41104695 Report Page 32 

Analytical Investigations 

Typical chromatograms are shown in Figures 2 to 4. 

2.3 Post-Study Experiment 

2.3.1 Test Samples 

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

in Table 6. 
p. 34 

Harlan Study Number: 41104695 Report Page 33 

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. 
p. 35 

Harlan Study Number: 41104695 Report Page 34 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal 

Concentration of 

Test Item 

Sample 

Volume 

Final Volume 

Sample 

Preparation 

Factor 

Final Nominal 

Concentration of Test 

Item 

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

Control 1 100, then 5 -> 

50 1000 -

1000 1 100, then 5 -> 

50 1000 1.0 

Table 2 Calibration Standard Data 

Concentration of Test Item Area 

[mg ai/L] [counts] 

0 4.161 x 102 

0.0104 1.074xl04 

0.271 2.235 xlO4 

0.522 3.988 x 104 

0.812 5.975 x 104 

1.04 8.017 xlO4 

1.62 1.167 x10s 
p. 36 

Harlan Study Number: 41104695 Report Page 35 

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 

X F c 

[hours] (mg ai/L] [mg ai/L] [mg ai/L] t%l 

0 100 0.937 100 93.7 94 

1000 0.907 1000 907 91 

48 100 0.937 100 93.7 94 

1000 0.832 1000 832 83 

Table 4 Results for Post-Study 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 

X F c 

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

0 1000 0.916 1000 916 92 

48 1000 0.884 1000 884 88 

Table 5 Results for Spiked Recovery Samples 

Nominal 

Concentration 

of 

Test Item 

Fortified 

Concentration 

of Test Item in 

the Spiked 

Sample 

Measured 

Concentration 

of Test Item in 

the Sample 

Vial 

X 

Sample 

Preparation 

Factor 

F 

Determined 

Concentration 

of Test Item in 

the Spiked 

Sample 

c 

Analytical 

Recovery 

Rate 

R 

Precision 

(Relative 

Standard 

Deviation of 

Recovery) 

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

100 107 1.12 100 112 

107 0.999 100 99.9 

107 0.974 100 97.4 94 8.2 

107 1.03 100 103 

107 0.894 100 89.4 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
p. 37 

Harlan Study Number: 41104695 Report Page 36 

Analytical Investigations 

Table 6 Results for Test Samples 

Time Point 

Nominal 

Concentration 

of 

Test Item in 

Test Sample 

Measured 

Concentration 

of Test Item in 

Sample Vial 

Sample 

Preparation 

Factor 

Determined 

Concentration 

of Test Item in 

Test Sample 

% of Nominal 

Concentration 

c

nom X F c 

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

0 Control 

1000 

<LOQ 

0.830 

1000 

1000 

<LOQ 

830 83 

48 Control 

1000 

<LOQ 

0.802 

1000 

1000 

<LOQ 

802 80 

LOQ = Limit of Quantitation 

not applicable 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41104695 Report Page 37 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

Concentration (mg ai/L) 
Harlan Study Number: 41104695 

Analytical Investigations 

Report 

Figure 2 Chromatogram of Calibration Solution (1.0 mg ai/L) 

Page 38 

M301 TIC,MSRle(SVWSERSW1 «6S^C/t-«3W4-1401 CO ESAH.SIM,Frag 100 

12000' 

1CD00-

6300-

TOO4000 

2000-

— ] 1 I 1 1 | 1 1 ! 1 r-— I ( 1 I 1 | 1 I > | 1 I 1 | i < < | 1 • I | • I I"* 

02 04 06 OB 1 12 U If i IB frifl 

Figure 3 Chromatogram of Biological Control Sample 

MSD1 HQNBRle(SVWfER^411M695eX£63K)07-CI7O2CO BAB.afAFraff 100 

1CO0O' 

axo 

exo4XX3-

2000-

—i—•—i—'—i—•—'—•—i—•—•—'—i—•—<— 1 i— , —~ " • nTM 1 •— 1 i 1 1 1 i 1 r~ ' i 1 — 

0.2 04 06 08 1 12 14 18 Ij i rti 
Harlan Study Number: 41104695 Report Page 39 

Analytical Investigations 

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

M3D5 TIC, M3 File (SVNi\SS?^ 1016SeS2l£63W2-12Q1 fy 6Sisfl,9M,Ffag 100 

- | 1 1 , 1 1 1 1 1 I 1 , 1 1 1 . | , . r—T . , , , , , , 1 , , , 1 , T—T02 04 06 08 1 VI 14 W 1.6 nin 
Harlan Study Number: 41104695 Report 

p. 41 

Page 40 

Appendix 5 Physico-Chemical Measurements 

Nominal 

Concentration 

(mg ai/L) 

Nominal 0 Hours 24 Hours 48 Hours 

Concentration 

(mg ai/L) PH mg 

Oi/L %ASV* T°C T°C pH mg Oj/L %ASV* T°C 

Control R 7.0 8.8 97 20 21 6.8 8.6 97 21 

R2 

7.0 9.0 99 20 21 6.9 8.6 97 21 

R3 7.0 9.0 99 20 21 6.9 8.6 97 21 

R< 7.0 9.0 99 20 21 6.9 8.6 97 21 

100 Ri 7.0 8.9 98 20 21 7.0 8.5 96 21 

R2 

7.0 8.9 98 20 21 6.9 8.4 94 21 

Rj 7.0 8.9 98 20 21 7.0 8.5 96 21 

R< 7.0 8.9 98 20 21 7.0 8.5 96 21 

180 Ri 7.0 8.9 98 20 21 7.1 8.4 94 21 

R2 

7.0 8.9 98 20 21 7.1 8.4 94 21 

R3 

7.0 8.9 98 20 21 7.1 8.3 93 21 

R4 7.0 8.8 97 20 21 7.1 8.3 93 21 

320 Ri 7.0 8.8 97 20 21 7.2 8.2 92 21 

R2 

7.0 8.3 97 20 21 7.2 8.2 92 21 

R3 7.0 8.8 97 20 21 7.2 8.2 92 21 

Ri 7.0 8.8 97 20 21 7.2 8.2 92 21 

560 Ri 7.0 8.9 100 21 21 7.4 8.1 91 21 

R2 

7.0 8.8 97 20 21 7.3 8.1 91 21 

R3 7.0 8.8 99 21 21 7.3 8.1 91 21 

R, 7.0 8.S 99 21 21 7.3 8.1 91 21 

1000 R> 7.0 8.8 97 20 21 7.4 7.6 85 21 

R2 

7.0 8.8 97 20 21 7.4 7.5 84 21 

Rj 7.0 8.8 97 20 21 7.4 7.6 85 21 

R4 7.0 8.8 97 20 21 7.5 7.6 85 21 

*ASV =* Dissolved oxygen concentration expressed as a percentage of Air Saturation Value 

Ri - R, = Replicates 1 to 4 
Harlan Study Number: 41104695 Report 

p. 42 

Page 41 

Appendix 6 Monitoring Authority Statement of GLP Compliance 

OH | Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY TEST TYPE(S) 

HarianUbpratorlMLtd Analytical/Clinical 

Shardlow Business Park rh a m i«,t« , 

London Road c , ,

C TM . ?? 

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 Monftoring 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). 

-3© /M / t2-. 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority 
p. 43 

ORIGIN ID= KCKA <913> 621-7733 

JAKE BOOREM 

HARCROS CHEMICALS INC 

5200 SPEAKER RD 

KANSAS CITY. KS GS106 

UNITED STATES US 

SHIP DATE: 13JUN13 

ACTWGT: 1.0 LB MAN 

CAD: 0179B01^CAFE2G08 

BILL THIRD PARTY 

TO(DCO)(7407M)(OPPT) 

US EPA TSCA SECTION 4 

1200 PENNSYLVANIA AVE NW 

WASHINGTON DC 20460 

(202) 564-8930 

REF: POSTAGE 

FedEx 

Express 

TRK# 5654 3239 9089 

XC RDVA 

FRI - 14 JUN 10:30A 

PRIORITY OVERNIGHT! 

20460 

DC-US DCA