## REPORT Sodium Glucoheptonate CAS No. 31138-65-5: Acute Toxicity to Rainbow Trout

StudyDirector: TestFacility:

Sponsor:

Harlan StudyNumber: Study Completion Date: S Harris Harlan Laboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK Harcros Chemicals Inc. 3452 Corporate Drive Dalton GA 30720 UNITED STATES OF AMERICA

41402585 15 June 2015

Page 1of41Harlan StudyNumber: 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 INTRODUCTIONAND 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 TestWater.............................................................................................................. 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 ofTest 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 onTest Item Solubility...................................................... 17 5 CONCLUSION.................................................................................................................... 18 6 REFERENCES .................................................................................................................... 19 TABLES........................................................................................................................................20 FIGURES ......................................................................................................................................23 APPENDICES...............................................................................................................................24Harlan StudyNumber: 41402585 Report Page3 

## LIST OF TABLES Table 1 Table 2 Table 3 Cumulative Mortality Datainthe Range-finding Test ...............................................20 Cumulative Mortality Datainthe Definitive Test......................................................21 Water QualityMeasurements .....................................................................................22 

## LIST OF FIGURES Figure 1 Temperature Measurements Recorded Overthe 96-HourTestPeriod Using a Testo Temperature Logger .............................................................._ ..............23 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Certificate ofAnalysis ...........................................................................................24 Typical Water Quality Characteristics ..................................................................25 Analytical Investigations .......................................................................................28 Monitoring Authority Statement ofGLP Compliance ..........................................41Harlan StudyNumber: 41402585 Report Page4 

## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: 41402585 Study Title: Sodium Glucoheptonate CAS No. 31138-65-5: 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 ofDirectives 2004/9/EC and 2004/10/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 (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affectedthe integrity ofthe study orthe validity ofthe data. Study Director: S Harris



### .........


# ~ Date: 

## 1 5 JUN 2015Harlan StudyNumber: 41402585 Report Page 5 

## QUALITYASSURANCE STATEMENT Harlan StudyNumber: 41402585 Study Title: Sodium Glucoheptonate CAS No. 31138-65-5: Acute Toxicityto Rainbow Trout The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsible person andmanagement. Study-relatedprocedures conducted at the test facility were audited and inspected. The details of these audits and inspections are givenbelow. Reportedto the Dates and Types ofQAInspections relevant Study Director andTest Facility Management Date ofInspection Type ofInspection Phase Inspected Report Date 22 July 2014 StudyPlan NIA 22 July 2014 Verification 07 April 2015 Process
- based TestItem 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 thatthis reportreflects the rawdata andthe procedures followed. QualityAssurance: '-\ . f'r\

### Af:-0

## AN\F\



# .....................**Y~** Date: 

## 1 2 JUN 

## 2015Harlan StudyNumber: 41402585 Report Page 6 

## SIGNATURE OF CONTRIBUTING SCIENTISTS Analytical Chemistry: D MMullee Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK



# ······.....



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

# 2 2JUN 

## 2015Harlan StudyNumber: 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 ofChemicals (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 ofthe test item, at a single concentration of 100 mg active ingredient (ai)/L for aperiod of96 hours at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects ofexposure in eachtest and control vessel were determined 3 and 6 hours after the start ofexposure and then daily throughout the test until termination after 96 hours. 

### Results Analysis ofthe 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% ofnominal. However, analysis ofthe 24 hour old test preparation showed that a measured concentration of 77% of nominal was obtained. Giventhat 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 onnominal test concentrations only. Exposure ofrainbow trout (Oncorhynchus mykiss) 

### to the test item gave LC5o

### values based on the nominal test concentrations ofgreater 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 StudyNumber: 41402585 Report 

## GENERALINFORMATION 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 ofthe United Kingdom's Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct ofthe study may be reviewed, as part ofthe Harlan Laboratories Ltd, Shardlow, UKEthical ReviewProcess. 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 ofclinical signs as humane endpoints for experimental animals used in safety evaluation. 

### Deviations from Study Plan DeviationNo.I The study plan states that feeding will be discontinued 48 hours prior to the start ofthe study, however, during the range finding test, feeding was discontinued 24 hours prior to the start ofthe study. Given that the correct time was employed in the definitive test, this was considered not to have had an impact onthe outcome orintegrity ofthe 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 orreturn ofthe data will be chargeable to the Sponsor. No datawill be discarded without contacting the Sponsorto obtain their written consent.Harlan StudyNumber: 41402585 Report Page 9 

## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the acute toxicity ofthe test item to rainbowtrout (Oncorhynchus mykiss). 

### Rainbow trout is a freshwater fish representative ofa wide variety ofnatural habitats, and can therefore be considered as an important non-target organism infreshwater 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 ofChemicals (1992) No 203 "Fish, Acute Toxicity Test". • Method C.1 ofCommission Regulation (EC) No. 440/2008. • US EPA DraftEcological Effects Test Guidelines OPPTS 850.1075 "FishAcute Toxicity Test, Freshwater and Marine".Harlan StudyNumber: 41402585 Report Page 10 

## 2 TESTITEM Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is given inAppendix 1. Identification: Physical state/ Appearance: Batch: Purity: Expiry Date: Storage Conditions: Intendeduse/ Application: Sodium Glucoheptonate CAS No. 31138-65-5 Amber colored liquid T-461 39.94% Not supplied Room temperature in the dark Not suppliedHarlan StudyNumber: 41402585 Report Page 11 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations LCso: The calculated concentration oftest item which results in 50% mortality withinthe 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 8hours darkness cycle with 20 minute dawn and dusk transitionperiods. The watertemperature 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 ofthe definitive test. There was no mortality in the 7 days prior to the start ofthe test and the fish had a mean standard length of3.9 em (sd = 0.1) and a mean weight of0.72 g (sd = 0.08) at the end ofthe definitive test. Based on the mean weight value this gave a loading rate of 0.25 gbodyweight/liter. The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome ofthe study. 

### 3.3 Test Water The test water used for both the range-finding and definitive tests was the same as that used to maintainthe 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 ofapproximately 140 mg/L as CaC03. After dechlorination and softening the water was passed through a series ofcomputer 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 StudyNumber: 41402585 Report Page 12


### At the start ofthe definitive test the diluent had a particulate matter concentration of0.1 mg/L, total water hardness of 134 mg/L as CaC03, 

### alkalinity of 112.4 mg/L as CaC03, 

### Total Organic Carbon content of2.0 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 ofWaters (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 StudyNumber 41402586) indicated that toxicity was not expected atthis level. The test item was dissolved directly intest water. A nominal amount oftest 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 itempurity of39.94%. 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 8hours darkness with 20 minute dawn and dusk transition periods for a period of96 hours under static test conditions. Each 25-30 liter test and control vessel contained 20 mL oftest media and was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sublethal effects ofexposure were determined by visual inspection ofthe test fish. The control group was maintained under identical conditions butnot exposed to the test item. A sample ofthe 100 mg ai/L test concentrationwas taken for chemical analysis at 0 and 24 hours in order to determine the stability ofthe test item under test conditions. The 0-Hour samples were stored frozen priorto analysis. 3.4.2 Definitive Test Based on the results ofthe 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 ofexposure were observed.Harlan StudyNumber: 41402585 Report Page 13
3.4.2.1 Experimental Preparation A nominal amount oftestitem (5007.5 mg) was dissolved intest 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 ofthe 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 ofthe testten 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 of96 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 butnot exposed to the test item. A semi-static test regime was employed in the test involving a daily renewal of the test preparations to preventthe build up ofnitrogenous waste products. 

### 3.4.3 Evaluations 3.4.3.1 Test Organism Observations Any mortalities and sub-lethal effects ofexposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start ofexposure. The criteria ofdeath were taken to be the absence ofboth 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 mediarenewal at 24, 48 and 72 hours, represent those ofthe freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination ofthe test after 96 hours, represent those ofthe 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 StudyNumber: 41402585 Report Page 14
The temperature was also monitored inthe 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 priorto 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 ifnecessary. The method ofanalysis, recovery and testpreparation analyses are described in Appendix 3. 

### 3.4.4 StatisticalAnalysis An estimate ofthe LCso values was givenby inspection ofthe mortality data. 

### 3.5 Validation Criteria The results ofthe test are considered valid ifthe following criteria are met: • In the control, not more than one ofthe fish should die or show signs ofstress during the 96 hours. • The dissolved oxygen concentration at the end of the test should be 2:60% of Air SaturationValues (ASV) in the control and test vessels.Harlan StudyNumber: 41402585 Report Page 15 

## 4 RESULTS 4.1 Range-finding Test Cumulative mortality data from the exposure ofrainbow trout to the test item during the rangefinding test are given in Table 1. There were no sub-lethal effects ofexposure during the rangefinding test. The results showed no mortalities atthe 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 ofexposure were observed. Chemical analysis ofthe 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 ofTest Concentrations Analysis ofthe 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% ofnominal. However, analysis ofthe 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 inTable 2. There were no mortalities in 10 fish exposed to a test concentration of 100 mg ai/L for a period of96hours. Inspection ofthe mortality data gave the following results:Harlan StudyNumber: 41402585 Report Page 16

Time (h) LCso (mg ai/L) 3 >100 6 >100 24 >100 48 >100 72 >100 96 >100 TheNo Observed Effect Concentration (NOEC) was considered to be 100 mg ai/L. Itwas considered unnecessary and unrealistic to test atconcentrations inexcess of 1 00 mg ai/L. 

### 4.2.3 Sub-Lethal Effects At the 6-Hour time point, 1 ofthe 10 fish in the 100 mg ai/L test concentration was observed to be sitting onthe bottom ofthe tank. Giventhat this effect did not continue for the rest ofthe test, it was considered to be due to natural causes and therefore this was considered not to have an effect onthe outcome ofthe test. 

### 4.2.4 Validation Criteria The test was considered to be valid given that none ofthe control fish died or showed signs of stress during the test and that the oxygen concentration at the end ofthe test was**~60%** ofASV inthe control and testvessels. 

### 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 orpH. 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 ofthe 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 ofthe media was 14 °C, therefore this was considered not to have had an impact onthe outcome or integrity ofthe test.Harlan StudyNumber: 41402585 Report Page 17


### 4.2.6 Observations on Test Item Solubility Throughoutthe test, the testpreparations were observedto be clear strawcolored solutions.Harlan StudyNumber: 41402585 Report Page 18 

## 5 CONCLUSION The acute toxicity ofthe 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

### ofgreater than 100 mg ai/L. The No Observed Effect Concentrationwas 100 mg ai/L.Harlan StudyNumber: 41402585 Report Page 19 

# 6 REFERENCES BRITISH STANDARDS INSTITUTION (1993) Determination ofAlkalinity. In: Field and Onsite TestMethods for the Analysis ofWaters, (BS 1427), pp 33-35. BRITISH STANDARDS INSTITUTION (1993) Determination ofCalcium and Total Hardness. In: Field and On-site TestMethods for the Analysis ofWaters, (BS 1427), pp 35-36.Harlan StudyNumber: 41402585 Report Page 20 

## TABLES Table 1 CumulativeMortality Datain the Range-finding Test Nominal CumulativeMortality 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 0Harlan StudyNumber: 41402585 Report Page 21


### Table2 CumulativeMortalityDatain the Definitive Test CumulativeMortality (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 0Harlan StudyNumber: 41402585 Report Page22


### Table 3 Water QualityMeasurements 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 14Harlan StudyNumber: 41402585 Report Page 23 

## FIGURES Figure 1 Temperature Measurements Recorded Over the 96-Hour TestPeriod 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 StudyNumber: 41402585 Report 

## APPENDICES Appendix 1 Certificate ofAnalysis

**8et~** Sodium

1% Water
% %Dextrose CAS# 

## Harcros Chemicals, Inc. Dalton Location 3452 Corporate Drive Dal.ton, GA 30720 CertificateofAnalysis 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/noneHarlan 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 orvalue for mandatory parameters and spec1f1ed llm1t orvalue for non-mandatory parameters (1nd1cators and disinfectants).Harlan StudyNumber: 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 CalcuIated 

### - 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 orvalue for non-mandatory parameters (indicators and disinfectants).Harlan StudyNumber: 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 orvalue for mandatory parameters and specified limit orvalue 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 ofTotal Indicative Dose (TID), which is the regulatory parameter. No samples exceeded the TID in 2011.Harlan StudyNumber: 41402585 Report Page 28 Appendix 3 Analytical Investigations 

## 1 MATERIALSAND 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 of39.94%, and are reported as milligrams ofactive ingredient (ai) per litre. The method was developed by the Department ofAnalytical Services, Harlan Laboratories Ltd, Shardlow, UK. 

### 1.2 Test Item The test item is described inthe biological part ofthis 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 inthe biological part ofthis study. Solvents: methanol, Fisher Scientific, HPLC grade. Reagents: ammonia, Fisher Scientific analytical reagent grade.Harlan StudyNumber: 41402585 Report Page29 Analytical Investigations 

### 1.4.3 Preparation ofCalibration 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 ofthis solution were then diluted with diluent* to obtain calibration standards in the range of0.10 to 1.0 mg ai/L. A second series of calibration standards was similarly prepared in the range of0.25 to 1.5 mg ai/L. These solutions were used to determine the recovery and test sample concentrations. 1.4.4 Preparation ofLinearity 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 ofthis solution were then diluted with diluent* to obtain calibration standards in the range of0.10 to 1.0 mg ai!L. A second series of calibration standards was similarlyprepared in the range of0.25 to 1.5 mg ai/L. These standards were used to evaluate the linearity ofthe analytical system. 1.4.5 Preparation ofSpiked Recovery Samples To demonstrate the validity ofthe analytical procedure, volumes oftest 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 ofthis 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 ofthe active ingredient (synthetic control) was also analyzed. 1.4.6 Preparation ofTest Samples The test samples were thawed with the aid ofsonication. 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 inTable 1.

• diluent: methanol: water: ammonia: test medium (50:40:0.10:10 v/v/v/v)Harlan StudyNumber: 41402585 Report Analytical Investigations 

### 1.4.7 Instrumental Parameter Autosampler: Pump: Detector: Column: Mobile phase: Flowrate: Injectionvolume: Ionizationmode: Polarity: Capillaryvoltage: Gas temperature: Nebulizerpressure: Fragmentorvoltage: Drying gas flow: Dataacquisition 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 30Harlan StudyNumber: 41402585 Report Page 31 Analytical Investigations 1.4.8 Calculations A calibration curve was prepared using a quadratic function ofthe 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 ofthe active ingredient inthe injected sample [mg ai/L] average peak area ofthe active ingredient inthe injected sample [counts] Constants The concentration ofthe active ingredient in the test samples was calculated using the following equation:
c = X ·F R/100 where c: x: 

### F: R: (2) concentration ofthe active ingredientinthe test sample [mg ai/L] concentrationofthe active ingredient inthe injected sample solution [mg 

### ai/L] sample preparation factor recoveryrate [%] (Ifnot 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 concentrationofthe active ingredientinthe spikedrecovery sample [mg ai/L] fortified concentration ofthe active ingredient inthe spiked recovery sample [mg ai/L]Harlan StudyNumber: 41402585 Report Page 32 Analytical Investigations The percentage of nominal concentration of the active ingredient in the test samples was calculatedusing the following equation: % nominal

## =_c_·100 cnom (4) where Cnom: nominal concentrationin the test sample [mg ai/L]Harlan StudyNumber: 41402585 Report Page 33 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validated priorto the analysis ofrange-finding samples; full validation was later conducted specifically at the concentration employed in the definitive test. The results for the range-findingtest samples are summarized inTable 3. 

### 2.2 Definitive Test 2.2.1 Validation ofthe 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 ofthe method byretentiontime was confirmed. Linearity The calibration data for the calibration standards ofthe active ingredient is given in 

### Table 2 and Figure 1. The data was found to have a quadratic correlation within the calibration range of 0to 1.6 mg ai/L. The R2 

### fit ofthe calibration curve to the data was 0.9971, and was considered to be acceptable. Accuracy (Recovery) and Precision A set ofrecovery samples accurately fortified at a relevant concentration ofactive ingredient was prepared five-fold and analyzed. The results obtained for the concentration of the active ingredient inthe recovery samples are presented inTable 4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were not corrected for recovery. Limit ofQuantification (LOQ) The limit of quantification (LOQ) was determined as ten times the standard deviation offive synthetic controls. Using this methodthe LOQ was determined to be 1.1 mg ai/L. 2.2.2 Test Samples The results obtained for the concentration ofthe active ingredient found in the test samples are presented inTable 5. Typical chromatograms are shown in Figures 2 to 4.Harlan StudyNumber: 41402585 Report Page 34 Analytical Investigations 

## 3 DISCUSSION The detection system was found to have acceptable correlation to the fit ofa quadratic equation. The analytical procedure was found to have acceptable recoveries of active ingredient in test medium. The method ofanalysis was validated and provento be suitable for use.Harlan StudyNumber: 41402585 Report Page 35 Analytical Investigations 

## TABLES Table 1 Preparation ofTest Samples Nominal Sample Sample FinalNominal Concentration of Volume FinalVolume 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 

### Table2 Calibration StandardData Concentration ofActive MeanArea 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 104Harlan StudyNumber: 41402585 Report Page 36 Analytical Investigations Table 3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration Concentration Concentration ofActive Preparation ofActive of Ingredientin Factor Ingredient in %ofNominal Time Point Active Ingredient SampleVial 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
Table4 Results for Spiked Recovery Samples Nominal Fortified Measured Sample Determined Mean Concentration Concentration Concentration Concentration Analytical of ofActive ofActive Preparation ofActive 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 R Cnom Cfort X c [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 Forreporting purposes, x has been calculated from c retrospectively.Harlan StudyNumber: 41402585 Report Analytical Investigations Table 5 Results for Test Samples Nominal Measured Sample Concentration Concentration of ofActive Preparation Time Point

[hours] 0 (Fresh media) 24 (Old media) 72 (Fresh media) 96 (Oldmedia) 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 ofQuantification not applicable Factor
F 10 100 10 100 10 100 10 100
duplicate sample, stored frozen priorto analysis Forreporting purposes, x has been calculated from c retrospectively. Page 37

Determined Concentration ofActive 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 95Harlan StudyNumber: 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 38Harlan StudyNumber: 41402585 Report Page 39 Analytical Investigations 

### Figure2 Chromatogram ofCalibration 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 ofBiological 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 thantheLOQHarlan StudyNumber: 41402585 Analytical Investigations Report 

### Figure 4 Chromatogram ofTest 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 minHarlan StudyNumber: 41402585 Report 

### Appendix 4 MonitoringAuthority Statement ofGLP Compliance 

### Department ofHealth 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 TESTTYPE(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-:.1t¥ Dr. AndrewJ. Gray •• , Head, UKGLP Monitoring Authority Page41This st udy was submitted to the U.S. Environmental P rotection Agency (EPA) by Harcros Chemicals as panofa TSCA Section 4 test mle. Submittal ofthe study to EPA does not authorize anyentityto cite to orrefer to the sllldy withoutexpress authorizat ion from Harcros Ch emicals. Harcros Chemicals retains data compensation rights under all statutory and regulatory prog rams, including, but not limited to, EU REACH and Korea-REACH. Possession ofa copyofthe study, in whole orin part, does not constitute "l egitimate possession"ofthe studyand does not crea te any rights to use the study or the datapr esented her ein for any chemical registra tion (such as, but not limited to, the REACH registration program) or any othercommercial

purpose. I n addition, Harcros Chemicals expresslyretains all rights for first publication ofthe data and the in formation presented herein. I n the event anyone seeks the right to cite orrefer to the data, please contact Harcros Chemicals.