Harcros Chemicals Inc. 

5200 Speaker Road 

Kansas City, KS 66106 cppf CBTC 

Telephone : 913/621-7747 

Fax: 913/621-7746 

E-mail: OrganicsCS@Harcros.com 20I3 ;.rr: 266 AK I0= 21 

Date 04/24/13 

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: Acute Toxicity to Rainbow Trout 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 Acute Toxicity to Rainbow Trout Test on D-gluco-Heptonic 

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

This study was conducted to fulfill the Acute Toxicity to Rainbow Trout requirements of testing 

category C as set forth in Table 2 of 40 CFR 799.50870). 

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 III III 

CONTAINS NO CB! 
REPORT 

harlair 

Sodium Glucoheptonate: 

Acute Toxicity To Rainbow Trout (Oncorhynchus mykiss) 

Data Requirements/Test Guidelines: 

• OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute 

Toxicity Test". 

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

• US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1075 "Fish 

Acute Toxicity Test, Freshwater and Marine". 

Study Director: S Harris 

Test Facility: Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Sponsor: Harcros Chemicals Inc. 

3452 Corporate Drive 

Dalton GA 30720 

UNITED STATES OF AMERICA 

Harlan Study Number: 

Study Completion Date: 

41104694 

08 April 2013 

Page 1 of37 
p. 3 

Harlan Study Number: 41104694 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 

Animal Welfare 8 

Archiving 8 

1 INTRODUCTION AND PURPOSE 9 

1.1 Guidelines / Regulations 9 

2 TEST ITEM 9 

2.1 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 Tests 11 

3.4.2 Definitive Test 12 

4 RESULTS 14 

4.1 Range-finding Tests 14 

4.2 Definitive Test 14 

4.2.1 Mortality Data 14 

4.2.2 Sub-Lethal Effects 15 

4.2.3 Observations on Test Item Solubility 15 

4.2.4 Physico-Chemical Measurements 15 

4.2.5 Verification of Test Concentrations 15 

5 CONCLUSION 16 

6 REFERENCES 16 

TABLES 17 

Table 1 Cumulative Mortality Data in the Range-finding Tests 17 

Table 2 Sub-lethal Effects of Exposure in the Range-finding Tests 18 

Table 3 Cumulative Mortality Data in the Definitive Test 19 

FIGURES : 20 

Figure 1 Temperature Measurements Recorded Over the 96-Hour Test Period Using a 

Testo Temperature Logger 20 

APPENDICES 21 

Appendix 1 Typical Water Quality Characteristics 21 

Appendix 2 Analytical Investigations 23 
p. 4 

Harlan Study Number: 41104694 Report Page 3 

Appendix 3 Physico-Chemical Measurements 36 

Appendix 4 Monitoring Authority Statement of GLP Compliance 37 
p. 5 

Harlan Study Number: 41104694 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: 

41104694 

Sodium Glucoheptonate: 

Acute Toxicity To Rainbow Trout {Oncorhynchus 

mykiss) 

Date: 0 8 APR 2013 
Harlan Study Number: 41104694 Report Page 5 

QUALITY ASSURANCE STATEMENT 

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

Harlan Study Number: 41104694 

Study Title: Sodium Glucoheptonate: 

Acute Toxicity To Rainbow Trout {Oncorhynchus 

mykiss) 

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 

11 October 2011 General Study Plan 

Verification 

NA 11 October 2011 

08 January 2013 Process - based Test Item Preparation 08 January 2013 

10 January 2013 Process - based Test System 

Preparation 

10 January 2013 

07 January 2013 Process - based Exposure 07 January 2013 

10 January 2013 Process - based Assessment of 

Response 

10 January 2013 

11 January 2013 Process - based Chemical Analysis 11 January 2013 

02 April 2013 Report Audit NA 02 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: ^ 

0 8 APR 20K 
p. 7 

Harlan Study Number: 41104694 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D M Mullee 

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

Date: g APR 2013 
p. 8 

Harlan Study Number: 41104694 Report Page 7 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the test item to rainbow trout 

{Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD 

Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as 

Method C.l of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological 

Effects Test Guidelines OPPTS 850.1075 "Fish Acute Toxicity Test, Freshwater and Marine". 

Methods 

Following preliminary range-finding tests, fish were exposed, in groups of ten, to an aqueous 

solution of the test item over a range of concentrations of 100, 180, 320, 560, and 1000 mg 

active ingredient (ai)/L for a period of 96 hours at a temperature of 13 °C to 14 °C under semistatic conditions. The number of mortalities and any sub-lethal effects of exposure in each test 

and control vessel were determined 3 and 6 hours after the start of exposure and then daily 

throughout the test until termination after 96 hours. 

Results 

The 96-Hour LC50 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 1000 mg ai/L test preparation at 0,24,48, 72 and 96 hours showed measured test 

concentrations to range from 91% to 114% of nominal and so the results are based on nominal 

test concentrations only. The fresh test preparation at 48 hours was measured to be 184% of 

nominal, however, as the corresponding old preparation at 72 hours was near nominal it is 

considered that this result was anomalous and has no impact on the outcome of the test. 
p. 9 

Harlan Study Number: 41104694 Report Page 8 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

05 March 2012 

08 March 2013 

Animal Welfare 

The study was designed and conducted to cause the minimum suffering or distress to the animals 

consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, 

Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals 

(Scientific Procedures) Act 1986. The conduct of title study may be retrospectively reviewed, as 

part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process. 

The study was conducted in accordance with the UK Home Office Guidance document on 

Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on 

recognition, assessment and use of clinical signs as humane endpoints for experimental animals 

used in safety evaluation. 

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: 41104694 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 rainbow trout (Oncorhynchus mykiss). 

Rainbow trout is a freshwater fish 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 (1992) No 203 "Fish, Acute Toxicity Test". 

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

• US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1075 "Fish Acute 

Toxicity Test, Freshwater and Marine". 

2 TEST ITEM 

2.1 Test Item 

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

the responsibility of the Sponsor. 

Identification: Sodium Glucoheptonate 

Description: dark brown liquid 

Batch: 921000100 

Purity: 49.5% w/w 

Expiry / retest date: 09 February 2015 

Storage conditions: room temperature in the dark 
p. 11 

Harlan Study Number: 41104694 Report Page 10 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

LC50: The calculated concentration of test item which results in 50% 

mortality within the exposure period. 

NOEC (No Observed Effect The highest test concentration at which no significant toxic 

Concentration): effect on the test organisms is observed within the exposure 

period. 

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

3.2 Test Species 

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were 

obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and 

maintained in-house since 03 December 2012. Fish were maintained in a glass fiber tank with a 

"single pass" water renewal system. Fish were acclimatized to test conditions from 04 January 

2013 to 14 January 2013. The lighting cycle was controlled to give a 16 hours light and 8 hours 

darkness cycle with 20 minute dawn and dusk transition periods. 

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of 

greater than or equal to 10.3 mg O2/L. These parameters were recorded daily. The stock fish 

were fed commercial trout pellets which was discontinued approximately 48 hours prior to the 

start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and 

the fish had a mean standard length of 4.3 cm (sd = 0.2) and a mean weight of 0.91 g (sd = 0.14) 

at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.46 

g bodyweight/Iiter. 

The diet and diluent water are considered not to contain any contaminant that would affect the 

integrity and outcome of the study. 

3.3 Test Water 

The test water used for both the range-finding and definitive tests was the same as that used to 

maintain the stock fish. 

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite 

Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with 

a total hardness of approximately 140 mg/L as CaC03. After dechlorination and softening the 

water was passed through a series of computer controlled plate heat exchangers to achieve the 
p. 12 

Harlan Study Number: 41104694 Report Page 11 

required temperature. Typical water quality characteristics for the tap water as supplied, prior to 

dechlorination and softening, are given in Appendix 2. 

At the start of the definitive test the diluent had a particulate matter concentration of 0.1 mg/L, 

total water hardness of 144 mg/L as CaCOa, alkalinity of 101.2 mg/L as CaCCh, Total Organic 

Carbon content of less than the limit of quantisation, determined to be 1,0 mg C/L, chemical 

oxygen demand of 2.09 mg O2/L and conductivity of 438 u-S/cm. The chlorine content ranged 

from 0.000 to 0.091 mg CI2/L. The water hardness and alkalinity were determined using the 

methods described in Field and On-site Test Methods for the Analysis of Waters (British 

Standards Institution, 1993). 

3.4 Procedure 

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

milligrams of active ingredient (ai) per liter. 

3.4.1 Range-finding Tests 

The test concentrations to be used in the definitive test were determined by preliminary rangefinding tests. 

In the initial range-finding test fish were exposed to a single test concentration of 1000 mg ai/L 

as information from the Acute Toxicity to Daphnia Magna study (Harlan Study Number 

41104695) suggested that toxicity was not expected at this level, The test item was dissolved 

directly in dechlorinated tap water. 

An amount of test item (40.40 g) was dissolved in dechlorinated tap water and the volume 

adjusted to 20 liters to give the 1000 mg ai/L test concentration. Mortalities were observed at 

this concentration so it was considered appropriate to conduct a second range finding test. 

In the second range finding test fish were exposed to a series of test concentrations of 1.0, 10 and 

100 mg ai/L. The test item was dissolved directly in dechlorinated tap water. 

Amount of test item (40.40, 404.40 and 4040.40 mg) were dissolved in dechlorinated tap water 

and the volume adjusted to 20 liters to give the 1.0,10 and 100 mg ai/L test concentrations. 

In the range-finding tests 3 fish were added to each 20 liter test and control vessel and 

maintained at 13 °C to 14 °C in a temperature controlled room with a photoperiod of 16 hours 

light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of up to 

96 hours under static test conditions. 

The control groups were maintained under identical conditions but not exposed to the test item. 
p. 13 

Harlan Study Number: 41104694 Report Page 12 

Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any 

mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish. 

A sample of each test concentration was taken for chemical analysis at 0 and 24 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 tests the following test concentrations were assigned to 

the definitive test: 100, 180, 320,560 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 dechlorinated tap 

water. Prior to weighing, the test item was boiled to remove any crystals that had formed. 

Amounts of test item (4040, 7272, 12930,22626 and 40400 mg) were dissolved in dechlorinated 

tap water and the volume adjusted to 20 liters to give the test concentrations of 100, 180, 320, 

560 and 1000 mg ai/L respectively. The test media was stirred with a flat bladed mixer to ensure 

complete dissolution. Prior to addition of the test animals, the pH of the test media was adjusted 

to pH 7. 

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

analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 (fresh media) hours (see 

Appendix 3), 

3.4.2.2 Exposure Conditions 

As in the range-finding tests, 20 liter glass exposure vessels were used for each test 

concentration. At the start of the test 10 fish were placed in each test vessel at random, in the 

test preparations. The test vessels were then covered to reduce evaporation and maintained at 

13 °C to 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours 

darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test 

vessels received no auxiliary aeration. The fish were not individually identified and received no 

food during exposure. 

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

A semi-static test regime was employed in the test involving a daily renewal of the test 

preparations to ensure that the concentrations of the test item remained near nominal and to 

prevent the build up of nitrogenous waste products. 
p. 14 

Harlan Study Number: 41104694 Report Page 13 

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours 

after the start of exposure. The criteria of death were taken to be the absence of both respiratory 

movement and response to physical stimulation. 

3.4.2.3 Physico-Chemical Measurements 

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout 

the test. The measurements at 0 hours, and after each test media renewal at 24,48 and 72 hours, 

represent those of the freshly prepared test preparations while the measurements taken prior to 

each test media renewal, and on termination of the test after 96 hours, represent those of the used 

or 24-Hour old test preparations. The pH and oxygen were measured using a Hach HQ30d Flexi 

handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital 

thermometer. The temperature was also monitored in the control hourly using a Testo 

temperature logger. 

3.4.2.4 Verification of Test Concentrations 

Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24, 

48, 72 (old and fresh media) and 96 (fresh media) hours for quantitative analysis. The 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. Only samples of the NOEC were analyzed. 

The method of analysis, recovery and test preparation analyses are described in Appendix 3. 

3.4.2.5 Evaluation of Data 

An estimate of the LC50 values was given by inspection of the mortality data. 
p. 15 

Harlan Study Number: 41104694 Report Page 14 

4 RESULTS 

4.1 Range-finding Tests 

Cumulative mortality data from the exposure of rainbow trout to the test item during the rangefinding tests are given in Table 1 and sub-lethal effects of exposure are given in Table 2. 

The results showed no mortalities at the test concentrations of 1.0, 10 and 100 mg ai/L. 

However, mortalities were observed at 1000 mg ai/L 

After approximately 3 hours exposure in the initial range finding test 3 out of 3 fish at 

1000 mg ai/L were observed to exibit a loss of equilibrium. Due to animal welfare implications 

(Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for 

the following observational time point. 

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 1.0, 10 and 100 mg ai/L test preparations at 0 and 24 hours (see 

Appendix 2) snowed measured concentrations ranging from 99% to 126% of nominal indicating 

that the test item was stable under test conditions. 

4.2 Definitive Test 

4.2.1 Mortality Data 

Cumulative mortality data from the exposure of rainbow trout to the test item during the 

definitive test are given in Table 3. 

There were no mortalities in 10 fish exposed to a series of test concentrations of 100, 180, 320, 

560 and 1000 mg ai/L for a period of 96 hours. Inspection of the mortality data gave the 

following results: 

Time (h) LC5o (mg ai/L) 

3 >1000 

6 >1000 

24 >1000 

48 >1000 

72 >1000 

96 >1000 
p. 16 

Harlan Study Number: 41104694 Report Page 15 

The results of the definitive test showed the highest test concentration resulting in 0% mortality 

to be greater than or equal to 1000 mg ai/L, the lowest test concentration resulting in 100% 

mortality to be greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) was 

1000 mg ai/L. 

In the initial range finding test, mortalities were observed at 1000 mg ai/L. Due to a technical 

oversight the pH of the test preparations was not adjusted to within specification. Adjustments 

to pH were made during the definitive test and hence it was considered likely that the mortalities 

observed in the initial range finding test were due to a pH effect rather than a true toxic effect. 

4.2.2 Sub-Lethal Effects 

There were no sub-lethal effects of exposure observed in 10 fish exposed to a test concentration 

of 1000 mg ai/L for a period of 96 hours. 

4.2.3 Observations on Test Item Solubility 

Throughout the test, the 100 and 180 mg ai/L test preparations were observed to be light yellow 

solutions increasing in color with concentration, and the 320, 560 and 1000 mg ai/L test 

preparations were observed to be dark brown solutions increasing in color with concentration. 

4.2.4 Physico-Chemical Measurements 

The results of the physico-chemical measurements are given in Appendix 3. Temperature was 

maintained at 13 °C to 14 °C throughout the test, while there were no treatment related 

differences for oxygen concentration or pH. 

The oxygen concentration in some of the test vessels was observed to have an air saturation 

value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic 

air bubbles in the media super-saturating the diluent and was considered not to have had an 

impact on the outcome or integrity of the test as no adverse effects were observed in the control 

group. 

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

using a Testo temperature logger (see Figure 1) ranged from 12 °C to 14 °C. 

4.2.5 Verification of Test Concentrations 

Analysis of the 1000 mg ai/L test preparation at 0, 24, 48, 72 and 96 hours showed measured test 

concentrations to range from 91% to 114% of nominal and so the results are based on nominal 

test concentrations only. The fresh test preparation at 48 hours was measured to be 184% of 
p. 17 

Harlan Study Number: 41104694 Report Page 16 

nominal, however, as the corresponding old preparation at 72 hours was near nominal it is 

considered that this result is anomalous and has no impact on the outcome of the test. 

5 CONCLUSION 

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) 

has been investigated and gave a 96-Hour LC50 of greater than 1000 mg ai/L. The No Observed 

Effect Concentration was 1000 mg ai/L. 

6 REFERENCES 

BRITISH STANDARDS INSTITUTION (1993) Determination of Alkalinity. In: Field and Onsite Test Methods for the Analysis of Waters, (BS 1427), pp 33-35. 

BRITISH STANDARDS INSTITUTION (1993) Determination of Calcium and Total Hardness. 

In: Field and On-site Test Methods for the Analysis of Waters, (BS 1427), pp 35-36. 
Harlan Study Number: 41104694 Report 

p. 18 

Page 17 

TABLES 

Table 1 Cumulative Mortality Data in the Range-finding Tests 

Nominal 

Concentration 

(mg ai/L) 

Cumulative Mortality 

(Initial Population = 3) 

Nominal 

Concentration 

(mg ai/L) 3 Hours 6 Hours 24 Hours 48 Hours 72 Hours 96 Hours 

Control* 0 0 0 0 0 0 

1.0 0 0 0 0 0 0 

10 0 0 0 0 0 0 

100 0 0 0 0 0 0 

1000 3** 3 3 3 3 3 

* The results observed for the controls during both range-finding tests were observed to be identical and 

therefore were not separately tabulated. 

** After 3 hours all of the fish were observed to have a loss of equilibrium. Due to approach of the substantial 

severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at 

the 3 hour time point. 
Harlan Study Number: 41104694 Report 

p. 19 

Page 18 

Table 2 Sub-lethal Effects of Exposure in the Range-finding Tests 

Nominal 

Concentration 

(mg ai/L) 

Sub-lethal Effects 

Nominal Time (Hours) 

Concentration 

(mg ai/L) 

Sub-lethal Effects 

3 6 24 48 72 96 

Control* No abnormalities detected 3/3 3/3 3/3 3/3 3/3 3/3 

1.0 No abnormalities detected 3/3 3/3 3/3 3/3 3/3 3/3 

10 No abnormalities detected 3/3 3/3 3/3 3/3 3/3/ 3/3 

100 No abnormalities detected 3/3 3/3 3/3 3/3 3/3 3/3 

1000 Loss of equilibrium 3/3** A/D A/D A/D A/D A/D 

* The results observed for the controls during both range-finding tests were observed to be identical and 

therefore were not separately tabulated. 

** After 3 hours all of the fish were observed to have a loss of equilibrium. Due to approach of the substantial 

severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at 

the 3 hour time point. 

A/D = All fish dead 
Harlan Study Number: 41104694 Report Page 19 

Table 3 Cumulative Mortality Data in the Definitive Test 

Nominal 

Concentration 

(mg ai/L) 

Cumulative Mortality (Initial Population = 10) % 

Mortality 

Nominal 

Concentration 

(mg ai/L) 3 

Hours 

6 

Hours 

24 

Hours 

48 

Hours 

72 

Hours 

96 

Hours 

96 

Hours 

Control 0 0 0 0 0 0 0 

100 0 0 0 0 0 0 0 

180 0 0 0 0 0 0 0 

320 0 0 0 0 0 0 0 

560 0 0 0 0 0 0 0 

1000 0 0 0 0 0 0 0 

Harlan Study Number: 41104694 Report 

p. 22 

Page 21 

APPENDICES 

Appendix 1 Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

REPORTING PERIOD: 01/01/2010 - 31/12/2010 

PARAMETER ANALYSIS 

UNITS 

NO. 

SAMPLES 

TAKEN 

CONCENTRATION OR VALUE 

(ALL SAMPLES) PCV+ 

SAMPLES 

CONTRAVENING PCV PARAMETER ANALYSIS 

UNITS 

NO. 

SAMPLES 

TAKEN MINIMUM AVERAGE MAXIMUM 

PCV+ 

NUMBER PERCENT 

1,2 Dichloroethane ug/1 40 <0.080 <0,080 <0.080 3 0 0,00 

Aluminium 33 <5.000 <6.684 12.000 200 0 0.00 

Amelryn ug/l 8 <0.003 <0.003 <0.003 0.1 0 0,00 

Antimony \igl\ 8 0.180 0.531 1.200 5 0 0,00 

Arsenic ug/l 8 <0.370 <0.370 <0.370 10 0 0,00 

Atrazlne ug/l 8 <0.003 <0,003 <0.003 0.1 0 0.00 

Azlnphos methyl ug/l 8 <0.004 <0.004 <0.004 0,1 0 0.00 

Benzene 40 <0.060 <0.060 <0.060 1 0 0,00 

Benzo 3,4-Pyrene M9'l 8 <0.001 <0.001 O.001 0.01 0 0.00 

Boron mg/l 40 <0.017 0.020 0.038 1 0 0.00 

Bfomadl ug/i 8 <0.004 <0.004 <0.004 0.1 0 0 00 

Bromate Mafl 8 <0.600 <0.600 <0.600 10 0 0.00 

Cadmium 8 0.080 0,121 0.160 5 0 0.00 

Carbenazfm ug/l 8 <0.002 <0.002 <0.002 0.1 0 0.00 

Carbetamide ugi 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Carbophenophlon ugfl 8 <0.012 <0.012 <0.012 0,1 0 0.00 

Chlorfenvinphos MS" 8 <0.005 <0.005 <0.005 0.1 0 0,00 

Chlorotoluron Mg/l 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Chlorpyriphos Ethyl pg/l 8 <0.010 <0.010 <0.010 0.1 0 0.00 

Chlorpyrlphos Methyl Mfl" 8 <0.015 <0.015 <0.015 0.1 0 0.00 

Chromium ug/i 6 <0.700 <0.700 0.700 50 0 0.00 

Colour mg/l Pt/Co 38 <0.400 <0.608 2.600 20 0 0.00 

Copper mg/l 8 <0.003 <0.032 0,130 2 0 0.00 

Cyanazlne Mfl/I 8 <0.005 <0.005 O.005 0.1 0 0.00 

Cyanide ug/l 40 <0.300 0.740 1 600 50 0 0.00 

Demeton-S-Methyl Mfl/I e <0.006 <0.006 <0.006 0.1 0 0.00 

Dlazlnon Mfl/I 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Dlchlorvos Mfl/I 8 <0.008 <0.008 <0.008 0.1 0 0.00 

Dlflufenican ug/i 8 «=0.005 <0.005 <0.005 0.1 0 0.00 

Dimelhoate Mfl/I 8 <0,005 <0.005 <0.005 0.1 0 0.00 

Dluron ug/l 8 <0.003 <0.003 <0.003 0.1 0 0.00 

E.coll No./100 ml 204 0.000 0.000 0,000 0 0 0.00 

Enterococcl No. /100 ml 8 0.000 0.000 0.000 0 0 0,00 

Ethofumesate Mfl/I 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Fenpropidln Mfl/I 8 <0.0D4 <0.004 <0.004 0.1 0 0.00 

Fenpropimorph Mfl/I 3 <0.002 <0.002 <0.002 0.1 0 0.00 

Fluoride mg/l 8 0.270 0.331 0.414 1.5 0 0.00 

Flutriafol Mfl/I 8 <0.007 <0.007 <0.007 0.1 0 0,00 

Heptenophos Mfl/I 8 <0.005 <0.005 <0.005 0,1 0 0.00 

Iron Mfl/I 76 <10.000 <26.579 488.000 200 1 1.32 

Isoproturon Mfl/I 8 <0.002 <0.002 <0.002 0.1 0 0.00 

Lead Mfl/I 8 <0.500 <2,288 10.000 25 0 0,00 

Llnuron Mfl/I 8 <0.005 <0.005 <0.005 0,1 0 0.00 

Malathion Mfl/I 6 <0.004 <0.004 <0.004 0.1 0 0.00 

Manganese Mfl/I 38 <1.800 <1.987 5.600 50 0 0,00 

Mercury Mfl/I 40 <0.012 0.018 0.199 1 0 0.00 

Metaldehyde Mfl/I 32 0.004 0.009 0.027 0.1 0 0.00 

Metamltron Mfl/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Metazachlor Mfl/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Methabenzthlazuron Mfl/I 8 <0.003 <0 003 <0.003 0,1 0 0.00 

Mevinphos MS" 8 <0.004 <0.004 <0.004 0.1 0 0,00 

Monollnuron Mfl/I 8 <0.004 <0.004 <0.004 0.1 0 0.00 

+PCV Prescribed concentration or value for mandatory parameters and specified limit or value for non-mandatory parameters (Indicators and 

disinfectants). 
Harlan Study Number: 41104694 

Typical Water Quality Characteristics 

Report 

p. 23 

Page 22 

WATER SUPPLY ZONE ZDB13 ALVASTON 

REPORTING PERIOD: 01/01/2010 - 31/12/2010 

PARAMETER ANALYSIS 

UNITS 

NO. 

SAMPLES 

TAKEN 

CONCENTRATION OR VALUE 

(ALL SAMPLES) PPV+ 

SAMPLES 

CONTRAVENING PCV PARAMETER ANALYSIS 

UNITS 

NO. 

SAMPLES 

TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

Nickel 8 2.300 3.288 5.800 20 0 0.00 

Nitrate mg/l 8 9.940 13.105 16.700 50 0 0.00 

Nitrite mg/l 8 <0.003 O.005 <0,009 0.5 0 0.00 

Nitrite/Nitrate Calculated -1 8 0.200 0.276 0.370 <V 0 0.00 

Odour Dilution 42 0.000 0.024 1.000 -2 1 2.38 

Number 

PAH yg/I 8 0.000 0.001 0.002 0.1 0 0.00 

Parathion wi 6 <0.006 <0.006 <0.006 0.1 0 0.00 

Pendimethalin 8 <0.005 O.005 <0.005 0.1 0 0.00 

PH pH value 76 7.020 7.497 7.870 Max 9.5, 0 0 00 

Min 6.5 

Phorate pgi 8 <0.009 <0.009 <0.009 0.1 0 0.00 

Phosalone ug/l 8 <0.007 <0.007 <0,007 0.1 0 0.00 

PWmlcarft rafl 8 <0.003 <O.O03 <0.003 0.1 0 0.00 

Plrimlphos-Methyl Mg/i 8 <0.009 <0.009 <0.009 0.1 0 0.00 

Prometryn M9/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Propachlor M9/l 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Propazin^ ug/l 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Propetamphos von 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Propiconazole MS/l 8 <0.010 <0.010 <0.010 0.1 0 0.00 

Propyzamjde U0/J 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Selenium Mfll 8 <0.220 <0.546 0.740 10 0 0.00 

Simazine Mfl/I 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Sodium mg/l 8 14.000 25.250 30.000 200 0 0.00 

Sulphate mg/l 40 23.800 50.345 99.800 25 0 0.00 

Taste Dilution 

Number 

42 0.000 0.000 0.000 2 0 0.00 

Terbutryn Mfl/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Tetrachloroethene and 

Trichloroatriene Mfl/I 8 0.000 0.000 0.000 10 0 0.00 

TelracWorometharte (jgd 8 <0.018 <0.021 <0.030 3 0 0.00 

Total Pesticides Mfl/I 32 0.000 0.009 0.027 0.5 0 0.00 

Total Trihalomethanes Mfl/I 8 19.440 25.449 31.580 100 0 0.00 

Triadlmefon Mfl/I 8 <0.009 <0.009 <0.009 0.1 0 0.00 

Trlallate Mfl/I 7 <0.008 <0.014 <0.050 0.1 0 0.00 

Triazophos Mfl/I 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Trletazine Mfl/I 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Turbidity NTU 76 <0.030 <0.135 3.400 4 0 0.00 

Free Chlorine mg/l 204 0.010 0.207 0.630 _» . -

Total Chlorine mg/l 204 0.040 0.270 0.710 3 

- -

Ammonium mg/l 38 <0.009 <0.012 0,049 0.5 0 0.00 

Chloride mg/l 40 9.660 21.052 50,800 250 0 0.00 

Clostridium perfringens No. /100 ml 38 0.000 0.000 0.000 0 0 0.00 

Coliform bacteria No. /100 ml 204 0.000 0.000 0,000 0 0 0.00 

Colony Count after 48 

hours at 37'C No./100 ml 76 0.000 0.789 31.000 Jt 

- -

Colony Count after 72 

hours at 22'C No./100 ml 76 0.000 2.039 85.000 _A 

- -

Conductivity uS/cm at 20"C 33 315.000 419.000 486.000 2500 0 0.00 

Gross Alpha Activity Bq/I 8 0.032 0.069 0.090 0.1 s 

Gross Beta Activity Bq/I 8 O.001 <0.036 0.053 1

s 

Total Organic Carbon mg/l 40 <0.130 0.920 2.020 _4 

- -

+PCV Prescribed concentration or value tor mandatory parameters and specified limit or value for non-mandatory parameters (indicators and 

disinfectants). 

1 The formula for the calculated Nitrate/Nitrite level Is [Nltrate]/50 + [Nitrite]/3. This number has no units. 

2 Acceptable to customers and no abnormal change. 

3 No numerical limit applies. 

4 No abnormal change. 

5 Guidance levels only. Monitored for the calculation of Total Indicative Dose (TID), which is the regulatory parameter. No samples exceeded 

the TID In 2010. 
p. 24 

Harlan Study Number: 41104694 Report Page 23 

Appendix 2 Analytical Investigations 

Analytical Investigations 

CONTENTS 

CONTENTS 23 

1 MATERIALS AND METHODS 24 

1.1 Introduction 24 

1.2 Test Item 24 

1.3 Analytical Standard 24 

1.4 Analytical Procedure 24 

1.4.1 Storage 24 

1.4.2 Reagents and Solvents 24 

1.4.3 Preparation of Calibration Standards 25 

1.4.4 Preparation of Linearity Standards 25 

1.4.5 Preparation of Spiked Recovery Samples 25 

1.4.6 Preparation of Test Samples 25 

1.4.7 Instrumental Parameters 26 

1.4.8 Calculations 26 

2 RESULTS 28 

2.1 Range-Finding Test 28 

2.2 Definitive Test 28 

2.2.1 Validation of the Analytical Method 28 

2.2.2 Test Samples 28 

3 DISCUSSION 29 

TABLES 30 

Table 1 Preparation of Test Samples 30 

Table 2 Calibration Standard Data 30 

Table 3 Results for Range-Finding Samples 31 

Table 4 Results for Spiked Recovery Samples 31 

Table 5 Results for Test Samples 32 

FIGURES 33 

Figure 1 Calibration Plot 33 

Figure 2 Chromatogram of Calibration Solution (1.0 mg ai/L) 34 

Figure 3 Chromatogram of Biological Control Sample 34 

Figure 4 Chromatogram of Test Sample (1000 mg ai/L) 35 
p. 25 

Harlan Study Number: 41104694 Report Page 24 

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-l 5 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: 41104694 

Analytical Investigations 

Report 

p. 26 

Page 25 

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 further diluted into the 

calibration range initially in test medium and then further diluted with diluent* 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) 
Harlan Study Number: 41104694 

Analytical Investigations 

Report 

p. 27 

Page 26 

1.4.7 Instrumental Parameters 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Agilent 1200 Series 

Agilent 1200 Series 

Agilent 1200 Series MSD 

none, direct injection 

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

Flow rate: 

Injection volume: 

Column temperature: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

0.5 mL/minute 

10 uL 

room temperature 

ESI 

negative 

4000 V 

350 °C 

40 psi 

100 V 

12 L/min 

Data acquisition mode: Single Ion Monitoring (SIM) 

SIM ions: 225 m/z 

1.4.8 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 1. From the calibration curve, the 

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

equation: 

-b + ^jb2-4-a-(c-y) (1) 

X

~ 2-a 

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

y: average peak area of the test item in injected sample [counts] 
Harlan Study Number; 41104694 

Analytical Investigations 

Report 

p. 28 

Page 27 

a, b, c: 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 

(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 = 

•'fort 

TOO (3) 

where R: 

c: 

recovery rate [%] 

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

ai/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 

nominal concentration in the test sample [mg ai/L] 
Harlan Study Number: 41104694 

Analytical Investigations 

Report 

p. 29 

Page 28 

2 RESULTS 

2.1 Range-Finding Test 

The analytical methodology was not validated prior to the analysis of range-finding samples; full 

validation was later conducted specifically at the test concentrations 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 0 to 1.7 mg/L. 

The R2 fit of the calibration curve to the data was 0.9973, 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. 

Limit of Quantitation (LOQ) 

The limit of quantitation (LOQ) was determined by calculating ten times the standard deviation 

of the response seen in five controls. Using this method the limit of quantitation was determined 

to be 0.056 mg/L. 

2.2.2 Test Samples 

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

in Table 5. Typical chromatograms are shown in Figures 2 to 4. 
p. 30 

Harlan Study Number: 41104694 Report Page 29 

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. 
Harlan Study Number: 41104694 

Analytical Investigations 

Report 

p. 31 

Page 30 

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 

Not Applicable 

Table 2 Calibration Standard Data 

Concentration of Test Item Area 

[mg ai/L] [counts] 

0 8.453 x 103 

0.119 2.001 x 104 

0.288 3.080 x 104 

0.593 5.212 xlO4 

0.864 6.423 x 104 

1.19 8.572 x 104 

1.73 1.024 x10s 
p. 32 

Harlan Study Number: 41104694 Report Page 31 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal 

Concentration 

of 

Test Item in 

Range-Finding 

Sample 

Measured 

Concentration 

Sample 

Preparation 

Determined 

Concentration 

Time Point 

Nominal 

Concentration 

of 

Test Item in 

Range-Finding 

Sample 

of Test Item in 

Sample Vial 

Factor of Test Item in 

Range-Finding 

Sample 

% of Nominal 

Concentration 

X F c 

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

0 1.0 0.123 10 1.23 123 

10 1.13 10 11.3 113 

100 1.10 100 110 110 

24 1.0 0.126 10 1.26 126 

10 0.993 10 9.93 99 

100 1.05 100 105 105 

Table 4 Results for Spiked Recovery Samples 

Nominal 

Concentration 

of 

Test Item 

Fortified 

Concentration 

of Test Item in 

the Spiked 

Sample 

Cfort 

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] [%] [%1 

100 105 1.08 100 108 

105 1.03 100 103 

105 1.07 100 107 100 4.5 

105 1.09 100 109 

105 0.975 100 97.5 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41104694 

Analytical Investigations 

Report Page 32 

Table 5 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 

*-nom X F c 

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

0 

(fresh) 

Control 

1000 

<LOQ 

0.557/0.992* 

1000 

1000 

<LOQ 

557/992* 57/99* 

24 

(old) 

Control 

1000 

<LOQ 

0.714/1.14* 

1000 

1000 

<LOQ 

714/1140* 71/114* 

24 

(fresh) 

Control 

1000 

<LOQ 

0.956 / 0.907* 

1000 

1000 

<LOQ 

946 / 907 95/91* 

48 

(old) 

Control 

1000 

<LOQ 

0.659/0.932* 

1000 

1000 

<LOQ 

659 / 932* 66/93* 

48 

(fresh) 

Control 

1000 

<LOQ 

0.681/ 1.84* 

1000 

1000 

<LOQ 

681 / 1840* 68/ 184* 

72 

(old) 

Control 

1000 

<LOQ 

0.629 / 0.925* 

1000 

1000 

<LOQ 

629/925* 63 / 93* 

72 

(fresh) 

Control 

1000 

<LOQ 

0.809/1.11* 

1000 

1000 

<LOQ 

809/ 1110* 81/111* 

96 

(old) 

Control 

1000 

<LOQ 

0.791/0.929* 

1000 

1000 

<LOQ 

791/929* 79/93* 

LOQ = Limit of Quantitation 

not applicable 

* = duplicate sample stored at approximately -20 °C prior to analysis 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41104694 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

Report Page 33 
Harlan Study Number: 41104694 

Analytical Investigations 

Report 

p. 35 

Page 34 

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

MSD1TK^MSH[e{SVmS7^10me^L57SO2^2901I9 B*fl,StMR8S 100 

£000 

4000 

2000 

\2 

—T— 

1.6 

Figure 3 Chromatogram of Biological Control Sample 

MSOI TIC, fv6 Rle (SW*EB*^11OMWB2^5O0M)6O1 D) E&W.SlMFraff 100 

soooaxo7X0-

6000-

5000-

4X0-

3D00-

2D00 

1000: 

06 08 u 15 

Response seen in test media control subtracted from controls and test samples 
p. 36 

Harlan Study Number: 41104694 Report Page 35 

Analytical Investigations 

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

~1— ' ' • 1—'

 1 —'—1— i '— ' 1—1—1 1 —I— 1 — ' ' 1—' ' '—I '— • '—I '—•—'—1— ' ' " 1

~ 

02 OA 06 Q8 1 12 U 15 nfn 
p. 37 

Harlan Study Number; 41104694 Report Page 36 

Appendix 3 Physico-Chemical Measurements 

Nominal 

Concentration 

(mg ai/L) 

Time (Hours) Nominal 

Concentration 

(mg ai/L) 

0 Hours (Fresh Media) 24 Hours (Old Media) 24 Hours (Fresh Media) Nominal 

Concentration 

(mg ai/L) pH mg 

02/L 

% 

ASV* T°C pH mg 

02/L 

% 

ASV* T°C pH mg Oj/L 

% 

ASV T°C 

Control 7.0 11.0 105 13 7.0 9.1 88 14 7.0 11.2 109 14 

100 7.1 11.0 105 13 7.1 9.2 89 14 7.1 11.4 111 14 

180 7.1 11.0 105 13 7.1 9.3 90 14 7.0 11.4 111 14 

320 7.0 10.8 103 13 6.9 8.9 86 14 7,1 11,1 108 14 

560 7.0 10.5 100 13 7.1 8.7 84 14 7.1 11.1 108 14 

1000 7.1 10.3 98 13 7.1 8.4 82 14 7.1 10.7 104 14 

Nominal 

Concentration 

(mg ai/L) 

Time (Hours) Nominal 

Concentration 

(mg ai/L) 

48 Hours (Old Media) 48 Hours (Fresh Media) 72 Hours (Old Media) 

Nominal 

Concentration 

(mg ai/L) pH mg 

Oj/L 

% 

ASV* T°C PH mg 

Oi/L 

% 

ASV* T°C pH mg 02/L 

% 

ASV* T°C 

Control 7.4 9.2 89 14 7.0 11.4 111 14 7.2 9.3 90 14 

100 7.3 9.1 88 14 7.1 11.4 111 14 7.2 9.3 90 14 

180 7.3 9.1 88 14 7.1 11.3 110 14 7.2 9.3 90 14 

320 7.3 8.7 84 14 7.0 11.2 109 14 7.1 9.0 87 14 

560 7.3 8.6 83 14 7.1 U . l no 14 7.2 8,8 85 14 

1000 7.3 8.3 81 14 7.1 10.8 105 14 7.2 8.6 83 14 

Nominal 

Concentration 

(mg ai/L) 

Time (Hours) Nominal 

Concentration 

(mg ai/L) 

72 Hours (Fresh Media) 96 Hours (Old Media) 

Nominal 

Concentration 

(mg ai/L) pH mg 

Oj/L 

% 

ASV* T°C pH mg 

Oj/L 

% 

ASV* T°C 

Control 7.0 11.0 105 13 7.4 9.2 89 14 

100 7.0 11.3 110 14 7.4 9.2 89 14 

180 7.1 11.2 107 13 7.4 9.2 89 14 

320 7.0 11.1 108 14 7.4 8.8 85 14 

560 7.2 11.2 107 13 7.4 8.7 84 14 

1000 7.0 10.8 103 13 7.4 8.6 83 14 

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value 
Harlan Study Number: 41104694 Report Page 

Appendix 4 Monitoring Authority Statement of GLP Compliance 

(DHj Department 

V_ / 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) 

Harlan Laboratories Ltd Analvticai/Cfinirai 

Shardlow Business Park rhSuJ TM 

London Road . , -

c

1

h

f

m 

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 (913) 621-7733 

JAKE BOOREM 

Hf)RCROS CHEMICALS INC 

5200 SPEAKER RD 

KANSAS CITY. KS 66106 

UNITED STATES US 

SHIP DATE: 25APR13 

ACTWGT: O.S LB MAN 

CAD: O970415^CAFE26OB 

BILL THIRD PARTY 

TO(DCO)(7407M)(OPPT) 

US EPA TSCA SECTION 4 

1200 PENNSYLVANIA AVE NW 

WASHINGTON DC 20460 

(202) 564-9930 

__REF: POSTAGE 

Will FedEx 

Express 

FRI - 26 APR 3:00P 

m\ 5408 6000 0873 STANDARD OVERNIGHT 

XC RDVA 20460 DC-US DCA