REPORT 

Sodium Glucoheptonate CAS No. 31138-65-5: 

Acute Toxicity to Rainbow Trout 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

S Harris 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Harcros Chemicals Inc. 

3452 Corporate Drive 

Dalton GA 30720 

UNITED STATES OF AMERICA 

41402585 

15 June 2015 

Page 1 of 41 
Harlan Study Number: 41402585 Report Page2 

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 

Deviations from Study Plan ................................................................................................... 8 

Archiving ............................. .-................................................................................................. 8 

1 INTRODUCTION AND PURPOSE ..................................................................................... 9 

1.1 Guidelines I Regulations .......................................................................................... 9 

2 TEST ITEM ......................................................................................................................... 10 

3 MATERIALS AND METHODS ......................................................................................... 11 

3.1 Definitions and Abbreviations ............................................................................... 11 

3.2 Test System ............................................................................................................ 11 

3.3 Test Water .............................................................................................................. 11 

3.4 Procedure ............................................................................................................... 12 

3.4.1 Range-finding Test .................................................................................. 12 

3.4.2 Definitive Test ......................................................................................... 12 

3.4.3 Evaluations .............................................................................................. 13 

3.4.4 Statistical Analysis .................................................................................. 14 

3.5 Validation Criteria ................................................................................................. 14 

4 RESULTS ............................................................................................................................ 15 

4.1 Range-finding Test ................................................................................................ 15 

4.2 Definitive Test ....................................................................................................... 15 

4.2.1 Verification of Test Concentrations ........................................................ 15 

4.2.2 Mortality Data ......................................................................................... 15 

4.2.3 Sub-Lethal Effects ................................................................................... 16 

4.2.4 Validation Criteria ................................................................................... 16 

4.2.5 Water Quality Criteria ............................................................................. 16 

4.2.6 Observations on Test Item Solubility ...................................................... 17 

5 CONCLUSION .................................................................................................................... 18 

6 REFERENCES .................................................................................................................... 19 

TABLES ........................................................................................................................................ 20 

FIGURES ...................................................................................................................................... 23 

APPENDICES ............................................................................................................................... 24 
Harlan Study Number: 41402585 Report Page3 

LIST OF TABLES 

Table 1 

Table 2 

Table 3 

Cumulative Mortality Data in the Range-finding Test ............................................... 20 

Cumulative Mortality Data in the Definitive Test ...................................................... 21 

Water Quality Measurements ..................................................................................... 22 

LIST OF FIGURES 

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

Using a Testo Temperature Logger .............................................................. _ .............. 23 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Certificate of Analysis ........................................................................................... 24 

Typical Water Quality Characteristics .................................................................. 25 

Analytical Investigations ....................................................................................... 28 

Monitoring Authority Statement of GLP Compliance ......................................... .41 
Harlan Study Number: 41402585 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

Harlan Study Number: 41402585 

Study Title: Sodium Glucoheptonate CAS No. 3113 8-65-5: 

Acute Toxicity to Rainbow Trout 

This study 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); and are in accordance with, and implement, the 

requirements of Directives 2004/9/EC and 2004/1 0/EC. 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHL W, MAFF and METI). 

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

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

Study Director: S Harris 

Date: 

......... ~ 1 5 JUN 2015 
Harlan Study Number: 41402585 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 41402585 

Study Title: Sodium Glucoheptonate CAS No. 31138-65-5: 

Acute Toxicity to Rainbow Trout 

The general facilities and activities are inspected at least once a year and the results are reported 

to the relevant responsible person and management. 

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

these audits and inspections are given below. 

Reported to the 

Dates and Types of QA Inspections relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

22 July 2014 Study Plan NIA 22 July 2014 

Verification 

07 April 2015 Process - based Test Item Preparation 07 April 2015 

16 April2015 Process - based Test System 16 April2015 

Preparation and 

Exposure 

01 April2015 Process - based Assessment of 01 April2015 

Response 

16 April2015 Process - based Chemical Analysis 16 April2015 

08 June 2015 Report Audit N/A 08 June 2015 

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

Quality Assurance: '-\ . f'r\ Af:-0 AN \F\ 

Date: 

..................... Y~ 

1 2 JUN 2015 
Harlan Study Number: 41402585 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D MMullee 

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

······ ..... (~M!t .............. . Date: 

2 2 JUN 2015 
Harlan Study Number: 41402585 Report Page 7 

SUMMARY 

Introduction 

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

(Oncorhynchus my kiss). 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.1 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 a preliminary range-finding test, ten fish were exposed to an aqueous solution of the 

test item, at a single concentration of 100 mg active ingredient (ai)/L for a period of 96 hours at a 

temperature of approximately 14 °C under semi-static test 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 

Analysis of the 100 mg ai/L test preparations at 0 and 72 hours (fresh media) and 96 hours (old 

media) showed measured test concentrations to range from 89% to 98% of nominal. However, 

analysis of the 24 hour old test preparation showed that a measured concentration of 77% of 

nominal was obtained. Given that preliminary work conducted, along with the range finding test 

showed that the test item was stable in the test system, this result was considered to be 

anomalous and was considered not to have had an effect on the outcome of the study. 

Consequently, the results are based on nominal test concentrations only. 

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC5o values based on the 

nominal test concentrations of greater than 100 mg ai/L. The No Observed Effect Concentration 

was 1 00 mg ai/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1 00 mg ai/L. 
Harlan Study Number: 41402585 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Animal Welfare 

16 March 2015 

17 April 2015 

Page 8 

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 Amendment Regulations 2012. The conduct of the study may 

be 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. 

Deviations from Study Plan 

Deviation No. I The study plan states that feeding will be discontinued 48 hours prior to 

the start of the study, however, during the range finding test, feeding was discontinued 24 hours 

prior to the start of the study. Given that the correct time was employed in the definitive test, 

this was considered not to have had an impact on the outcome or integrity of the test. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan, all raw data (paper and electronic) 

and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for 

five years after which instructions will be sought as to further retention or disposal. Further 

retention or return of the data will be chargeable to the Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41402585 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 I Regulations 

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

internationally accepted guidelines and recommendations: 

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

• Method C.1 of Commission Regulation (EC) No. 440/2008. 

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

Toxicity Test, Freshwater and Marine". 
Harlan Study Number: 41402585 Report Page 10 

2 TESTITEM 

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is 

given in Appendix 1. 

Identification: 

Physical state/ Appearance: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

Intended use/ Application: 

Sodium Glucoheptonate CAS No. 31138-65-5 

Amber colored liquid 

T-461 

39.94% 

Not supplied 

Room temperature in the dark 

Not supplied 
Harlan Study Number: 41402585 Report Page 11 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

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

mortality within the exposure period. 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

3.2 Test System 

The highest test concentration at which no significant toxic 

effect on the test organisms is observed within the exposure 

period. 

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

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 23 March 2015. Fish were maintained in a glass fiber tank with a 

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

31 March 2015 to 07 April 2015. 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 9.7 mg 0 2/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 no mortality in the 7 days prior to the start of the test and 

the fish had a mean standard length of 3.9 em (sd = 0.1) and a mean weight of 0.72 g (sd = 0.08) 

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

0.25 g bodyweight/liter. 

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 

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

dechlorination and softening, are given in Appendix 2. 
Harlan Study Number: 41402585 Report Page 12 

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

total water hardness of 134 mg/L as CaC03, alkalinity of 112.4 mg/L as CaC03, Total Organic 

Carbon content of 2.0 mg C/L, chemical oxygen demand of 1.36 mg 0 2/L and conductivity of 

297 flS/cm. The chlorine content ranged from 0.008 to 0.03 mg Ch/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 

3.4.1 Range-finding Test 

The test concentration to be used in the definitive test was determined by a preliminary rangefinding test. 

In the range-finding test fish were exposed to a single nominal test concentration of 100 mg ai/L 

as data obtained from the Daphnia magna study (Harlan Study Number 41402586) indicated that 

toxicity was not expected at this level. The test item was dissolved directly in test water. 

A nominal amount of test item (5007.5 mg) was dissolved in test water and the volume adjusted 

to 20 liters to give the 100 mg ai/L test concentration. The test concentration was corrected for a 

test item purity of 39.94%. 

The prepared concentration was stirred using a flat bladed mixer for approximately 1 minute to 

ensure homogeneity. 

In the range-finding tests three fish were placed in each test and control vessel and maintained in 

a temperature controlled room at approximately 14 °C 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 under 

static test conditions. Each 25-30 liter test and control vessel contained 20 mL of test media and 

was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sublethal effects of exposure were determined by visual inspection of the test fish. 

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

A sample of the 100 mg ai/L 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. The 0-Hour samples 

were stored frozen prior to analysis. 

3.4.2 Definitive Test 

Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 

100 mg ai/L to confirm that at the maximum concentration given in the OECD/EC Test 

Guidelines, no mortalities or sub-lethal effects of exposure were observed. 
Harlan Study Number: 41402585 Report Page 13 

3.4.2.1 Experimental Preparation 

A nominal amount oftest item (5007.5 mg) was dissolved in test water and the volume adjusted 

to 20 litres to give the 100 mg ai/L test concentration. The test concentration was corrected for a 

test item purity of39.94%. 

The prepared concentration was stirred using a flat bladed mixer for approximately 1 minute to 

ensure homogeneity. 

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

analysis at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) (see Appendix 3). 

3.4.2.2 Exposure Conditions 

As in the range-finding test, 25-30 liter glass exposure vessels containing 20 liters oftest media 

were used for each control and test concentration. At the start of the test ten 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 approximately 14 ac 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 prevent the build up of nitrogenous waste products. 

3.4.3 Evaluations 

3.4.3.1 Test Organism Observations 

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. 

3.4.3.2 Water Quality Criteria 

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 dissolved oxygen concentration were measured 

using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna 

Instruments HI 93510 digital thermometer. 
Harlan Study Number: 41402585 Report Page 14 

The temperature was also monitored in the control hourly using a Testa temperature logger. 

3 .4.3 .3 Verification ofT est Concentrations 

Water samples were taken from the control and the 100 mg ai/L test vessel at 0 and 72 hours 

from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples 

were stored frozen prior to analysis. 

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old 

media) were taken and stored frozen for further analysis if necessary. 

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

3.4.4 Statistical Analysis 

An estimate of the LCso values was given by inspection of the mortality data. 

3.5 Validation Criteria 

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

• In the control, not more than one of the fish should die or show signs of stress during the 

96 hours. 

• The dissolved oxygen concentration at the end of the test should be 2:60% of Air 

Saturation Values (ASV) in the control and test vessels. 
Harlan Study Number: 41402585 Report Page 15 

4 RESULTS 

4.1 Range-finding Test 

Cumulative mortality data from the exposure of rainbow trout to the test item during the rangefinding test are given in Table 1. There were no sub-lethal effects of exposure during the rangefinding test. 

The results showed no mortalities at the test concentration of 100 mg ai/L. 

Based on this information, a single test concentration of 1 00 mg ai/L was selected for the 

definitive test. This experimental design conforms to a "Limit test" to confirm that at the 

maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal 

effects of exposure were observed. 

Chemical analysis of the test preparations at 0 and 24 hours (see Appendix 3) showed that near 

nominal concentrations were achieved, indicating that the test item was stable under test 

conditions. 

4.2 Definitive Test 

4.2.1 Verification of Test Concentrations 

Analysis of the 100 mg ai/L test preparations at 0 and 72 hours (fresh media) and 96 hours (old 

media) showed measured test concentrations to range from 89% to 98% of nominal. However, 

analysis of the 24 hour old test preparation showed that a measured concentration of 77% of 

nominal was obtained. Given that preliminary work conducted, along with the range finding test 

showed that the test item was stable in the test system, this result was considered to be 

anomalous and was considered not to have had an effect on the outcome of the study. 

Consequently, the results are based on nominal test concentrations only (see Appendix 3). 

4.2.2 Mortality Data 

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

definitive test are given in Table 2. 

There were no mortalities in 10 fish exposed to a test concentration of 100 mg ai/L for a period 

of96 hours. Inspection of the mortality data gave the following results: 
Harlan Study Number: 41402585 Report Page 16 

Time (h) LCso (mg ai/L) 

3 >100 

6 >100 

24 >100 

48 >100 

72 >100 

96 >100 

The No Observed Effect Concentration (NOEC) was considered to be 100 mg ai/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1 00 mg ai/L. 

4.2.3 Sub-Lethal Effects 

At the 6-Hour time point, 1 of the 10 fish in the 100 mg ai/L test concentration was observed to 

be sitting on the bottom of the tank. Given that this effect did not continue for the rest of the test, 

it was considered to be due to natural causes and therefore this was considered not to have an 

effect on the outcome of the test. 

4.2.4 Validation Criteria 

The test was considered to be valid given that none of the control fish died or showed signs of 

stress during the test and that the oxygen concentration at the end of the test was ~60% of ASV 

in the control and test vessels. 

4.2.5 Water Quality Criteria 

The results of the water quality measurements are given in Table 3. Temperature was 

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

differences for oxygen concentration or pH. 

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

using a Testo temperature logger (see Figure 1) ranged from 13 °C to 14 °C. A temperature of 

18 °C was recorded at the start of the test, however, this was considered to be due to the logger 

being moved from storage at a higher temperature and not able to acclimatize. Data taken at 

0 hours shows that the temperature of the media was 14 °C, therefore this was considered not to 

have had an impact on the outcome or integrity of the test. 
Harlan Study Number: 41402585 Report Page 17 

4.2.6 Observations on Test Item Solubility 

Throughout the test, the test preparations were observed to be clear straw colored solutions. 
Harlan Study Number: 414025 85 Report Page 18 

5 CONCLUSION 

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

has been investigated and based on the nominal concentrations, gave a 96-Hour LC5o of greater 

than 100 mg ai/L. The No Observed Effect Concentration was 100 mg ai/L. 
Harlan Study Number: 41402585 Report Page 19 

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: 41402585 Report Page 20 

TABLES 

Table 1 Cumulative Mortality Data in the Range-finding Test 

Nominal Cumulative Mortality 

Concentration (Initial Population = 3) 

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

Control 0 0 0 0 0 0 

100 0 0 0 0 0 0 
Harlan Study Number: 41402585 Report Page 21 

Table 2 Cumulative Mortality Data in the Definitive Test 

Cumulative Mortality (Initial Population= 10) % Nominal Mortality Concentration 

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

Hours Hours Hours Hours Hours Hours Hours 

Control 0 0 0 0 0 0 0 

100 0 0 0 0 0 0 0 
Harlan Study Number: 41402585 Report Page22 

Table 3 Water Quality Measurements 

Nominal Time (Hours) 

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

(mg ai/L) pH mgOiL T"C pH mgOiL T°C 

Control 7.7 10.1 13 7.8 8.6 14 

100 7.7 10.2 13 7.8 8.4 14 

Nominal Time (Hours) 

Concentration 24 Hours (Fresh Media) 48 Hours (Old Media) 

(mg ai/L) pH mg02/L T"C pH mg02/L T°C 

Control 7.6 9.9 14 7.7 8.5 14 

100 7.6 10.5 14 7.7 8.6 14 

Nominal Time (Hours) 

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

(mg ai/L) pH mg02/L T"C pH mg02/L T°C 

Control 7.5 10.1 14 7.8 9.9 14 

100 7.6 10.2 14 7.7 8.6 14 

Nominal Time (Hours) 

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

(mg ai/L) pH mg02/L T"C pH mg02/L T°C 

Control 7.6 9.9 13 7.8 8.6 14 

100 7.6 9.9 14 7.8 8.4 14 
Harlan Study Number: 41402585 Report Page 23 

FIGURES 

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

Testo Temperature Logger 

20 

18 

16 

14 

6 

4 

2 

0~~--~~~~~~--~~--~~--~~--~~~--~~--~~--~~--~~~ 

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 

Time (Hours) 
Harlan Study Number: 41402585 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

8et~ Sodium 

1% 

Water 

% 

%Dextrose 

CAS# 

Harcros Chemicals, Inc. 

Dalton Location 

3452 Corporate Drive 

Dal.ton, GA 30720 

Certificate of Analysis 

Mother Liquor CAS# 31138 .. 65-5 

Only byHPLC 

Record HPLC 

0~ 10 ppm 

1321 0 ppm 

1062 9 n 

TM 1019 35 50 

TM 1.300 

TM 1079 so 

1335 0 traee 

Page 24 

10.96% 

ppm 

l.l13ppm 

9.1 

49.1 

50.9% 

0/none 
Harlan Study Number: 41402585 Report Page 25 

Appendix 2 Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

REPORTING PERIOD: 01/01/2011 - 31/12/2011 

NO. CONCENTRATION OR VALUE SAMPLES 

PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV 

UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

1,2 Dichloroethane ~g/1 40 <0.080 <0.080 <0.080 3 0 0.00 

2, 3, 6- TBA ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

2, 4-DB ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

2, 4, 5- TCPA ~g/1 32 <0.002 <0.004 <0.005 0.1 0 0.00 

2,4-D ~g/1 32 <0.002 <0.003 0.004 0.1 0 0.00 

Aldrin ~g/1 40 <0.003 <0.003 <0.003 0.03 0 0.00 

Alpha-HCH ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Aluminium ~g/1 38 <5.000 <9.737 91.000 200 0 0.00 

Ametryn ~g/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Antimony ~g/1 8 0.200 0.465 0.700 5 0 0.00 

Arsenic ~g/1 8 <0.370 <0.371 0.380 10 0 0.00 

Atrazine ~g/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Azinphos methyl ~g/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Benazolin ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Bentazone ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Benzene ~g/1 40 <0.060 <0.060 <0.060 1 0 0.00 

Benzo 3,4-Pyrene ~g/1 8 <0.001 <0.001 <0.001 0.01 0 0.00 

Beta- HCH ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Boron mg/1 40 <0.017 <0.027 0.032 1 0 0.00 

Bromacil ~g/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Bromate ~g/1 8 <0.600 <0.713 1.100 10 0 0.00 

Bromoxynil ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Cadmium ~g/1 8 0.130 0.161 0.210 5 0 0.00 

Caplan ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Carbenazim ~g/1 32 <0.002 <0.002 <0.002 0.1 0 0.00 

Carbetamide ~g/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

Carbophenophion ~g/1 32 <0.012 <0.012 <0.012 0.1 0 0.00 

Chlordane A ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Chlorfenvinphos ~g/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Chloride mg/1 40 9.960 20.732 59.400 250 0 0.00 

Chlormequat ~g/1 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Chlorothalonil ~g/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Chlorotoluron ~g/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Chlorpyriphos Ethyl ~g/1 32 <0.010 <0.010 <0.010 0.1 0 0.00 

Chlorpyriphos Methyl ~g/1 32 <0.015 <0.015 <0.015 0.1 0 0.00 

Chromium ~g/1 8 <0.700 <0.700 0.700 50 0 0.00 

Cis - Permethrin ~g/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Clopyralid ~g/1 32 <0.001 <0.003 <0.004 0.1 0 0.00 

Colour mg/1 Pt/Co 38 <0.500 <1.587 2.200 20 0 0.00 

Copper mg/1 8 0.003 0.074 0.410 2 0 0.00 

Cyanazine ~g/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Cyanide ~g/1 40 <0.580 <1.833 5.900 50 0 0.00 

Cyfluthrin ~g/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Cypermethrin ~g/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

D. D. E.- Ortho Para ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

D.D.E. Para Para ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

D.D.T. - Ortho Para ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

D.D.T.- Para Para ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Delta- HCH ~g/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Deltamethrin ~g/1 40 <0.004 <0.004 <0.004 0.1 0 0.00 

Demeton-S-Methyl ~g/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

Diazinon ~g/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

Dicamba ~g/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Dichlobenil ~g/1 40 <0.001 <0.001 <0.001 0.1 0 0.00 

Dichlorprop ~g/1 32 <0.002 <0.003 <0.003 0.1 0 0.00 

Dichlorvos ~g/1 32 <0.008 <0.008 <0.008 0.1 0 0.00 

Dieldrin ~g/1 40 <0.002 <0.002 <0.002 0.03 0 0.00 .. +PCV Prescnbed concentration or value for mandatory parameters and spec1f1ed llm1t or value for non-mandatory parameters (1nd1cators and 

disinfectants). 
Harlan Study Number: 41402585 Report Page 26 

Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

REPORTING PERIOD: 01/01/2011 - 31/12/2011 

NO. CONCENTRATION OR VALUE SAMPLES 

PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV 

UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

Diftufenican IJQ/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Dimethoate j.JQ/1 32 <0.005 <0.006 <0.020 0.1 0 0.00 

Disulfoton IJQ/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Diuron j.JQ/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

E. coli No./100 ml 204 0.000 0.000 0.000 0 0 0.00 

Endosulfan A j.JQ/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Endosulfan B j.JQ/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Endrin IJQ/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Enterococci No./100 ml 8 0.000 0.000 0.000 0 0 0.00 

EPTC j.JQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Ethofumesate j.Jg/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

Fenitrothion j.Jg/1 40 <0.004 <0.004 <0.004 0.1 0 0.00 

Fenoprop j.JQ/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Fenpropidin j.JQ/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Fenpropimorph IJQ/1 32 <0.002 <0.002 <0.002 0.1 0 0.00 

Fenvalerate j.Jg/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Fluoride mg/1 8 0.220 0.348 0.500 1.5 0 0.00 

Fluroxypyr j.Jg/1 32 <0.002 <0.004 <0.006 0.1 0 0.00 

Flutriafol j.JQ/1 32 <0.007 <0.007 <0.007 0.1 0 0.00 

Gamma-HCH IJQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Gross Alpha Activity Bq/1 16 <0.014 <0.069 0.105 0.1 5 - -

Gross Beta Activity Bq/1 16 0.022 0.057 0.089 1 5 - - Heptachlor j.Jg/1 40 <0.002 <0.002 <0.002 0.03 0 0.00 

Heptachlor epoxide j.JQ/1 40 0.000 0.000 0.000 0.03 0 0.00 

Heptenophos j.JQ/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Hexachlorobenzene j.JQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Hexachlorobutadiene IJQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Iron j.JQ/1 76 <10.000 <25.276 83.000 200 0 0.00 

loxynil j.JQ/1 32 <0.001 <0.002 <0.003 0.1 0 0.00 

lsodrin j.Jg/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

lsoproturon j.Jg/1 32 <0.002 <0.002 <0.002 0.1 0 0.00 

Lead IJQ/1 8 <0.500 <2.163 5.700 25 0 0.00 

Linuron j.JQ/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Malathion j.Jg/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Manganese j.JQ/1 38 <1.800 <2.561 12.000 50 0 0.00 

MCPA j.JQ/1 32 <0.002 <0.003 0.004 0.1 0 0.00 

MCPB j.JQ/1 32 <0.002 <0.003 <0.004 0.1 0 0.00 

Mecoprop j.JQ/1 32 <0.002 <0.006 0.012 0.1 0 0.00 

Mercury j.Jg/1 40 <0.012 <0.012 <0.012 1 0 0.00 

Metaldehyde j.JQ/1 32 0.004 0.008 0.018 0.1 0 0.00 

Metamitron j.JQ/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Metazachlor IJQ/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Methabenzthiazuron j.Jg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Methoxychlor j.JQ/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Mevinphos j.JQ/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Monolinuron IJQ/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Nickel j.JQ/1 8 3.000 4.338 7.600 20 0 0.00 

Nitrate mg/1 8 8.740 11.689 13.700 50 0 0.00 

Nitrite mg/1 8 <0.005 <0.006 <0.009 0.5 0 0.00 

Nitrite/Nitrate Calcu Ia ted -

1 8 <0.170 <0.250 0.370 <1' 0 0.00 

Odour Dilution 76 0.000 0.000 0.000 2 0 0.00 Number -

PAH IJQ/1 8 0.000 0.000 0.000 0.1 0 0.00 

Parathion IJQ/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

PCB 101 j.JQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

PCB 118 j.JQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

PCB 138 IJQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

PCB 153 j.JQ/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

PCB 180 j.JQ/1 40 <0.002 <0.002 <0.002 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: 41402585 Report Page 27 

Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

REPORTING PERIOD: 01/01/2011-31/12/2011 

NO. CONCENTRATION OR VALUE SAMPLES 

PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV 

UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

PCB 28 J..lg/1 40 <0.001 <0.001 <0.001 0.1 0 0.00 

PCB 52 J..lg/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Pendimethalin J..fg/1 32 <0.005 <0.005 0.006 0.1 0 0.00 

Phorate J..lg/1 32 <0.009 <0.010 <0.020 0.1 0 0.00 

Phosalone J..lg/1 32 <0.007 <0.007 <0.007 0.1 0 0.00 

Pirimicarb J..fg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Pirimiphos-Methyl J..lg/1 32 <0.009 <0.009 <0.009 0.1 0 0.00 

Prometryn J..lg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Propachlor J..lg/1 32 <0.005 <0.005 <0.005 0.1 0 0.00 

Propazine J..fg/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Propetamphos J..lg/1 32 <0.007 <0.007 <0.007 0.1 0 0.00 

Propiconazole J..lg/1 32 <0.010 <0.010 <0.010 0.1 0 0.00 

Propyzamide J..fg/1 32 <0.004 <0.004 <0.004 0.1 0 0.00 

Selenium J..lg/1 8 <0.220 <0.745 2.100 10 0 0.00 

Simazine J..lg/1 32 <0.007 <0.007 <0.007 0.1 0 0.00 

Sodium mg/1 8 13.000 22.000 30.000 200 0 0.00 

Sulphate mg/1 40 25.000 51.744 112.000 250 0 0.00 

Taste Dilution 76 0.000 0.000 0.000 2 0 0.00 

Number -

T.D.E.- Ortho Para J..lg/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

T.D.E.- Para Para J..lg/1 40 <0.003 <0.003 <0.003 0.1 0 0.00 

Tecnazene J..lg/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Terbutryn J..lg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Tetrachloroethene and J..lg/1 8 0.000 0.000 0.000 10 0 0.00 

Trichloroethene 

Tetrachloromethane J..lg/1 8 <0.018 <0.018 <0.018 3 0 0.00 

Total Organic Carbon mg/1 40 <0.260 0.996 1.900 -

4 - -

Total Pesticides J..lg/1 40 0.000 0.007 0.020 0.5 0 0.00 

Total Trihalomethanes J..fg/1 8 16.560 33.423 53.160 100 0 0.00 

Trans - Permethrin J..lg/1 40 <0.004 <0.004 <0.004 0.1 0 0.00 

Triadimefon J..lg/1 32 <0.009 <0.009 <0.009 0.1 0 0.00 

Triazophos J..lg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Trichlorobenzene J..lg/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Triclopyr J..lg/1 32 <0.003 <0.003 <0.003 0.1 0 0.00 

Trietazine J..fg/1 32 <0.006 <0.006 <0.006 0.1 0 0.00 

Trifluralin J..lg/1 40 <0.002 <0.002 <0.002 0.1 0 0.00 

Turbidity NTU 76 <0.070 <0.125 0.330 4 0 0.00 

Free Chlorine mg/1 204 0.000 0.204 0.540 -

3 - -

Total Chlorine mg/1 204 0.040 0.248 0.590 -

3 - - Ammonium mg/1 38 <0.015 <0.019 <0.021 0.5 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 No. /100 ml 76 0.000 3.039 149.000 -

4 - - hours at 37•c 

Colony Count after 72 No./100 ml 76 0.000 1.553 76.000 -

4 - - hours at 22•c 

Conductivity J..fS/cm at 2o•c 38 282.000 411.711 504.000 2500 0 0.00 

pH pH value 76 7.020 7.414 8.110 Max 9.5, 0 0.00 Min 6.5 

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

disinfectants). 

1 The formula for the calculated Nitrate/Nitrite level is [Nitrate]/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 2011. 
Harlan Study Number: 41402585 Report Page 28 

Appendix 3 Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The active ingredient concentration in the test samples was determined by direct injection into 

the mass spectrometer (HPLC-MS) using an external standard. 

All test sample concentrations were corrected for a purity content of 39.94%, and are reported as 

milligrams of active ingredient (ai) per litre. 

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, 

Shardlow, UK. 

1.2 Test Item 

The test item is described in the biological part of this study. 

1.3 Analytical Standard 

The test item described in the biological part of this study was also used as the analytical 

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The samples were stored frozen prior to analysis. 

1.4.2 Reagents and Solvents 

Water: prepared using an ELGA Pure Lab Option R-15 water purification system. 

Fisher Scientific, LC-MS grade. 

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

Solvents: methanol, Fisher Scientific, HPLC grade. 

Reagents: ammonia, Fisher Scientific analytical reagent grade. 
Harlan Study Number: 41402585 Report Page29 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 250 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 ai/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 250 mg) was dissolved in diluent* (100 mL) to prepare a stock solution 

with a concentration of 1 000 mg ai/L. This stock solution was further diluted with diluent* to 

produce a solution of 10 mg ai/L. Defined volumes of this solution were then diluted with 

diluent* to obtain calibration standards in the range of 0.10 to 1.0 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 the recovery was assessed. The test item (nominal 250 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 a concentration of 100 mg ai/L. Five replicates were prepared and subjected to the 

same treatment as the test samples. In addition, test medium without the addition of the active 

ingredient (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 with test medium to 

a nominal concentration of 10 mg ai/L and then further diluted into the calibration range with 

diluent as shown in Table 1. 

• diluent: methanol: water: ammonia: test medium (50:40:0.10:10 v/v/v/v) 
Harlan Study Number: 41402585 Report 

Analytical Investigations 

1.4. 7 Instrumental Parameter 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Flow rate: 

Injection volume: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

Data acquisition mode: 

SIM ions: 

Agilent 1200 Series 

Agilent 1200 Series 

Agilent 1200 Series MSD 

none, direct injection 

methanol: LC-MS water: ammonia (50:50:0.1, v/v/v) 

0.5 mL/minute 

10 J!L 

ESI 

negative 

4000V 

350 oc 

40 psi 

100V 

12 L/min 

Single Ion Monitoring (SIM) 

225.0 m/z 

Page 30 
Harlan Study Number: 414025 85 Report Page 31 

Analytical Investigations 

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 active ingredient in an injected sample was calculated using the 

following equation: 

-b+~b 2 -4·a·(c- y) X=----'--------

(1) 

2·a 

where x: 

y: 

a, b, c: 

concentration of the active ingredient in the injected sample [mg ai/L] 

average peak area of the active ingredient in the injected sample [counts] 

Constants 

The concentration of the active ingredient in the test samples was calculated using the following 

equation: 

c = X ·F 

R/100 

where c: 

x: 

F: 

R: 

(2) 

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

concentration of the active ingredient in the injected sample solution [mg 

ai/L] 

sample preparation factor 

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

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

equation: 

c 

R =-·100 

cfort 

where R: 

c: 

(3) 

recovery rate [%] 

determined concentration of the active ingredient in the spiked recovery 

sample [mg ai/L] 

fortified concentration of the active ingredient in the spiked recovery 

sample [mg ai/L] 
Harlan Study Number: 41402585 Report Page 32 

Analytical Investigations 

The percentage of nominal concentration of the active ingredient in the test samples was 

calculated using the following equation: 

% nominal = _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg ai/L] 
Harlan Study Number: 41402585 Report Page 33 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

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

validation was later conducted specifically at the concentration 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 active ingredient. The standard solutions 

contained a peak specific for the active ingredient 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 active ingredient is given in Table 2 and 

Figure 1. The data was found to have a quadratic correlation within the calibration range of 

0 to 1.6 mg ai/L. The R2 fit of the calibration curve to the data was 0.9971, and was considered 

to be acceptable. 

Accuracy (Recovery) and Precision 

A set of recovery samples accurately fortified at a relevant concentration of active ingredient was 

prepared five-fold and analyzed. The results obtained for the concentration of the active 

ingredient in the recovery samples are presented in Table 4. 

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

The test sample results were not corrected for recovery. 

Limit of Quantification (LOQ) 

The limit of quantification (LOQ) was determined as ten times the standard deviation of five 

synthetic controls. Using this method the LOQ was determined to be 1.1 mg ai/L. 

2.2.2 Test Samples 

The results obtained for the concentration of the active ingredient found in the test samples are 

presented in Table 5. Typical chromatograms are shown in Figures 2 to 4. 
Harlan Study Number: 41402585 Report Page 34 

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 active ingredient in test 

medium. The method of analysis was validated and proven to be suitable for use. 
Harlan Study Number: 41402585 Report Page 35 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Sample Final Nominal 

Concentration of Volume Final Volume Preparation Concentration of 

Active Ingredient Factor Active Ingredient 

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

Control 2 20 10 -

100 2 

20 

100 1.0 

then 1 ~ 10 

not applicable 

Table 2 Calibration Standard Data 

Concentration of Active Mean Area 

Ingredient 

[mg ai/L] [counts] 

0 3.860 X 103 

0.0993 5.308 X 103 

0.263 8.323 X 103 

0.497 1.361 X 104 

0.789 1.728 X 104 

0.993 2.030 X 104 

1.58 2.999 X 104 
Harlan Study Number: 41402585 Report Page 36 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined 

Concentration Concentration Concentration of Active Preparation of Active of Ingredient in Factor Ingredient in %of Nominal Time Point Active 

Ingredient Sample Vial Range-Finding Concentration 

Sample 

Cnom F 

X c 

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

0 100 102 100 102 102 

24 100 99.5 100 99.5 100 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Mean 

Concentration Concentration Concentration Concentration Analytical 

of of Active of Active Preparation of Active Recovery Precision 

Active Ingredient in Ingredient in Factor Ingredient in Rate (Relative 

Standard Ingredient the Spiked the Sample the Spiked Deviation of Sample Vial Sample Recovery) 

F 

Cnom Cfort X c R 

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

83.2 100 83.2 

85.2 100 85.2 

100 100 84.5 100 84.5 85 4.4 

79.5 100 79.5 

89.9 100 89.9 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41402585 Report 

Analytical Investigations 

Table 5 Results for Test Samples 

Nominal Measured Sample Concentration Concentration 

of of Active Preparation 

Time Point 

[hours] 

0 

(Fresh media) 

24 

(Old media) 

72 

(Fresh media) 

96 

(Old media) 

LOQ 

* 

Active Ingredient in 

Ingredient in Sample Vial 

Test Sample 

Cnom X 

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

Control <LOQ 

100 0.776 I 0.894* 

Control <LOQ 

100 0.772 

Control <LOQ 

100 0.984 

Control <LOQ 

100 0.945 

Limit of Quantification 

not applicable 

Factor 

F 

10 

100 

10 

100 

10 

100 

10 

100 

duplicate sample, stored frozen prior to analysis 

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

Page 37 

Determined 

Concentration 

of Active 

Ingredient in % ofNominal 

Test Sample Concentration 

c 

[mg ai/L] [%] 

<LOQ -

77.6 I 89.4* 78 I 89* 

<LOQ - 77.2 77 

<LOQ -

98.4 98 

<LOQ - 94.5 95 
Harlan Study Number: 41402585 Report 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

........ 

·a 

::::> 

'-" 

= a 

< 

-= If 

.35000 

30000 

25000 

15000 

:moo 

y =-1,029 .45l&t2c+ li,OfU512lx +:3,159.6320 

RS=0.$1971 

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

0.00 0.20 0 .. 40 UO OJ!O 1.00 1.20 L40 UO UIO 

Coru::entraiticu.(mdL) 

Page 38 
Harlan Study Number: 41402585 Report Page 39 

Analytical Investigations 

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

MSD1 TIC, MS Ale (S:IANASERVSI41402585\ECAQ1111ECAQ111- 11027.:!701 .~ ES-API, SIM, Fmg: 100 

0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 min 

Figure 3 Chromatogram of Biological Control Sample* 

MSD1 TIC, MS Ale (S:IANASERVSI41402585\ECA.Q1111ECAQ111- 1'01 0-1001.~ ES-AFI, SIM, Fmg: 100 

3500 

3000 

2500 

2000 

1500 

1000 

0.2 0.4 0.8 0.8 1.2 1.4 1.6 1.8 min 

• Less than the LOQ 
Harlan Study Number: 41402585 

Analytical Investigations 

Report 

Figure 4 Chromatogram of Test Sample (100 mg ai/L) 

MSD1 TIC, MS Rle (S:IANASERVS\41402SBSIECAQ111\ECAQ111· 1'1!2();!001.0) ES-API, SIM, F111g: 100 

02 0.4 0.6 0.8 

Page 40 

12 1.4 1.6 1.8 min 
Harlan Study Number: 41402585 Report 

Appendix 4 Monitoring Authority Statement of GLP Compliance 

Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD lABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 200419/EC 

TEST FACILITY 

Harlan Laboratories Limited 

,Shardlow Business Park 

London Road 

Shardlow 

Derbyshire 

DE722GD 

DATE OF INSPECTION 

TEST TYPE(S) 

Analytical/Clinical Chemistry 

Environmental Fate 

Environmental Toxicity 

Phys.Chem. Testing 

Mutagenicity 

Toxicology 

12 to 14 March 2014 

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). 

~-~ 1-:.1 t¥ 

Dr. AndrewJ. Gray •• , 

Head, UK GLP Monitoring Authority 

Page 41 
This study was submitted to the U.S. Environmental Protection Agency (EPA) by 

Harcros Chemicals as pan of a TSCA Section 4 test mle. Submittal of the study to 

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

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