## REPORT 

# +++ £NVIGO D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Acute Toxicityto Rainbow Trout

StudyDirector: TestFacility:

Sponsor:

StudyMonitor:

StudyNumber: StudyCompletionDate: D Sacker Envigo Research Limited ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK UnivarUSA Inc. 17425 NEUnionHill Road Redmond WA 98052 UNITED STATES OF AMERICA Greenwich ChemicalConsulting, Inc. 3300PGABoulevard, Suite 635 Palm Beach Gardens FL33410 UNITED STATES OF AMERICA 41401173 15 January 2016
On21 September2015 the legal entity Harlan Laboratories Limited was renamed as Envigo Research Limited. No otherchanges to the legal entity have occurred. Page 1of38StudyNumber: 41401173 Report Page2 

## CONTENTS CONTENTS ....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ....................................................4 QUALITY ASSURANCE STATEMENT......................................................................................5 SUMMARY ....................................................................................................................................6 Introduction............................................................................................................................6 Methods...............................................................................................................................6 Results..................................................................................................................................6 GENERAL INFORMATION .........................................................................................................7 Schedule.................................................................................................................................7 Animal Welfare......................................................................................................................7 Deviations from Study Plan...................................................................................................7 Archiving ...............................................................................................................................7 1 INTRODUCTIONAND PURPOSE..................................................................................... 8 1.1 Guidelines IRegulations..........................................................................................8 2 TEST ITEM ...........................................................................................................................9 3 MATERIALS AND METHODS......................................................................................... 10 3.1 Definitions and Abbreviations............................................................................... 10 3.2 Test Species ........................................................................................................... 10 3.3 TestWater.............................................................................................................. 10 3.4 Procedure ............................................................................................................... 11 3.4.1 Range-finding Test.................................................................................. 11 3.4.2 Definitive Test......................................................................................... 12 3.4.3 Evaluations .............................................................................................. 12 3.4.4 Statistical Analysis .................................................................................. 13 3.5 Validation Criteria................................................................................................. 13 4 RESULTS ............................................................................................................................ 14 4.1 Range-finding Test ................................................................................................ 14 4.2 Definitive Test ....................................................................................................... 14 4.2.1 Verification ofTest Concentrations ........................................................ 14 4.2.2 Mortality Data ......................................................................................... 14 4.2.3 Sub-Lethal Effects................................................................................... 15 4.2.4 Validation Criteria................................................................................... 15 4.2.5 Water Quality Criteria............................................................................. 15 4.2.6 Observations on Test Item Solubility...................................................... 15 5 CONCLUSION.................................................................................................................... 16 6 REFERENCES .................................................................................................................... 17 TABLES........................................................................................................................................ 18 APPENDICES...............................................................................................................................22StudyNumber: 41401173 Report Page 3 

## LIST OF TABLES Table 1 Table 2 Table 3 Table 4 Cumulative MortalityDatainthe Range-finding Test ............................................... 18 Sub-lethal Effects ofExposure inthe Range-finding Test ......................................... 19 Cumulative Mortality Datainthe Definitive Test......................................................20 Water QualityMeasurements .....................................................................................21 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Certificate ofAnalysis...........................................................................................22 Typical Water Quality Characteristics ..................................................................23 Analytical Investigations .......................................................................................26 MonitoringAuthority StatementofGLP Compliance ..........................................38StudyNumber: 41401173 Report Page4 

## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Envigo Research Limited, ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK StudyNumber: 41401173 Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Acute Toxicityto RainbowTrout
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 consideredto have affected the integrity ofthe study orthe validity ofthe data. Study Director: D Sacker



### ......

# ~ Date: 

### 1 5 JAN 2016StudyNumber: 41401173 Report Page 5 

## QUALITYASSURANCE STATEMENT StudyNumber: 41401173 Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: 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 and management. Study-relatedprocedures conducted at the test facility were audited and inspected. The details of these audits and inspections are givenbelow. Reported to the Dates and Types ofQAInspections relevant Study Directorand Test Facility Management Date ofInspection Type ofInspection Phase Inspected ReportDate 15 April2014 StudyPlan N/A 15 April2014 Verification 16 November 2015 Process
- based TestItemPreparation 16 November2015 16 November 2015 Process
- based Test System 16 November2015 Preparation and Exposure 18 November 2015 & Process
- based Assessmentof 18 November2015 & 20 November 2015 Response 20 November2015 19 October2015 Process
- based Chemical Analysis 19 October 2015 07 January 2016 Report Audit NIA 07 January 2016 Verification of the Study Plan Amendment for GLP compliance was performed during the course ofthe study. This statement confirms thatthis reportreflects the raw data and the procedures followed.StudyNumber: 41401173 Report Page 6 

## SUMMARY Introduction A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing ofChemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 ofCommissionRegulation (EC) No. 440/2008/. 

### Methods Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of96 hours at a temperature of 14 °C under continuous flow-through test conditions. The number ofmortalities and any sublethal effects ofexposure in each test and control vessel were determined 3 and 6 hours after the startofexposure andthen daily throughoutthe testuntil termination after 96 hours. 

### Results Analysis ofthe testpreparations at 24, 48, 72 and 96 hours showed measured test concentrations to range from 80 to 96% of nominal. Analysis of the test preparation at 0 hours showed a measured concentration of 48% of nominal, however, evaluation of the stock solution preparation and test flow rates indicates that the test system was correctly dosed and the test system was functioning correctly and therefore suggests the analytical result to be anomalous. Given that the 0-Hour analytical result was suspected to be anomalous and as measured concentrations at 24, 48, 72 and 96 hours were near nominal it was considered justifiable to exclude the 0-Hour measured concentration and to base the results on the nominal test concentration only. Exposure of rainbow trout to the test item gave LC50 values based on the nominal test concentration ofgreaterthan 100 mg/L. The No Observed Effect Concentrationwas 100 mg/L. Itwas consideredunnecessary and unrealistic to test at concentrations inexcess of 100 mg/L.Study Number: 41401173 Report 

## GENERALINFORMATION Schedule Experimental Starting Date: Experimental CompletionDate: 

### Animal Welfare 22 September 2015 30 October 2015 Page 7

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 Envigo Research Limited, 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 partofthe Envigo Research Limited, 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.1 The Study Plan states that particulate matter concentration, COD, TOC, hardness, alkalinity and conductivity will be determined at the start ofthe test, however, due to a technical oversight this was not conducted at the start ofthe test, instead, data from dechlorinated water samples tested in July 2015 and January 2016 are presented in Section 3.3 ofthis report. This deviation was considered notto have adversely affected the results ofthe test. 

### Archiving Unless instructed otherwise bythe Sponsor, the studyplan (general studyplan and study specific supplement), all raw data (paper and electronic) and the final report will be retained in the Envigo Research Limited, 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 datawill be discarded without contacting the Sponsorto obtaintheir written consent.StudyNumber: 41401173 Report Page 8 

## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the acute toxicity ofthe test itemto rainbowtrout (Oncorhynchus my kiss). Rainbow trout is a freshwater fish representative ofa wide variety ofnatural habitats, and can therefore be considered as an importantnon-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 ofChemicals (1992) No 203 "Fish, Acute Toxicity Test". • Method C.l ofCommissionRegulation (EC) No. 440/2008.StudyNumber: 41401173 Report Page 9 

## 2 TESTITEM Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is given inAppendix 1. Identification: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] Description: White granular solid Batch: 14031314 Purity: 99.19% ExpiryDate: 26 March2016 Storage Conditions: Roomtemperature inthe darkStudyNumber: 41401173 Report Page 10 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations LC50: The calculated concentration oftest item which results in 50% mortality withinthe exposure period. LOEC (Lowest Observed Effect Concentration): NOEC (No ObservedEffect Concentration): Treatment: 

### 3.2 Test Species The lowesttest concentration at which a significanttoxic effect on the test organisms is observed withinthe exposureperiod. The highest test concentration at which no significant toxic effect on the test organisms is observed within the exposure period. Comprises test itemtreatments and control (test water only).

The test was carried out using juvenile rainbow trout. Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 7 October 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 14 to 26 October 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusktransitionperiods. The watertemperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.2 mg 0 2 /L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 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 of4.0 em (sd = 0.3) and a mean weight of0.88 g (sd = 0.15) at the end ofthe definitive test. Based on the mean weight value this gave a loading rate of0.07 g ofbodyweight/liter (volume in 24 hours= 86.4liters). The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome ofthe study. 

### 3.3 TestWater 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 (ElgaNimbus 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 theStudyNumber: 41401173 Report Page 11 required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given inAppendix 2. Dechlorinated laboratory water tested inJuly 2015 and January 2016 and had aparticulate matter concentration of 0.4 and 0.0 mg/L, total water hardness of 134 and 136 mg/L as CaC03, alkalinity of 115 and 78.8 mg/L as CaC03, Total Organic Carbon content of 0.36 and 3.1 mg C/L, chemical oxygen demand of 1.7 and 2.9 mg 0 2/L, conductivity of 408 and 324 11S/cm and the chlorine content ranged from 0.000 to 0.012 mg Ch/L respectively. The water hardness and alkalinity were determined using the methods described in Field and On-site TestMethods for the Analysis ofWaters (British Standards Institution, 1993). 

### 3.4 Procedure Due to the potentially light sensitive nature of the test item, all test item preparation was performedunder non-actinic lighting I shielded from the light. 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 nominal test concentrations of 10 and 100 mg/L under continuous flow-through conditions. Nominal amounts oftest item (1.2 and 12 g) were dissolved in deionized reverse osmosis water and the volume adjusted to 1 liter to give the 1.2 and 12 g/L stock solution respectively. The stock solutions were sonicated for 10 minutes and inverted several times to ensure adequate mixing and homogeneity. Dilution water was dosed at a flow rate of 60 mL per minute using Watson Marlow 505S peristaltic pump and the stock solutions at a flow rate of0.50 mL per minute using a Watson Marlow 205U peristaltic pump. The dilution water and stock solutions were combined in separate mixing vessels and delivered to the test vessels to give the required test concentrations of 1 0 and 1 00 mg/L. In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at 14 °C in the dark for a period of96 hours under dynamic test conditions. Each 25 litertest and control vessel contained 20 L oftestmedia and was coveredto reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection ofthe test fish. The control group was maintainedunder identical conditions butnot exposedto the test item. A sample ofeachtest concentrationwas taken immediately for chemical analysis at 0, 72 and 96 hours in orderto determinethe stability ofthe test itemundertestconditions.StudyNumber: 41401173 Report Page 12
3.4.2 Definitive Test Based on the results ofthe range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no mortalities or sub-lethal effects ofexposure were observed. 3.4.2.1 Experimental Preparation A nominal amount oftest item (12 g) was dissolved in deionized reverse osmosis water and the volume adjusted to 1literto give the 12 g/L stock solution. The stock solutionwas sonicated for 5minutes and inverted severaltimes to ensure adequate mixing and homogeneity. Dilution water was dosed at a flow rate of 60 mL per minute using Watson Marlow 505S peristaltic pump and the stock solution at a flow rate of 0.50 mL per minute using a Watson Marlow 205U peristaltic pump. The dilution water and stock solution were combined in a mixing vessel and deliveredto the test vessel to give the requiredtestconcentration of 100 mg/L. The concentration and stability ofthe test item inthe test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours (see Appendix 3). 3.4.2.2 Exposure Conditions Continuous flow-through test conditions were employed in the test in an effort to maintain dissolved test item concentrations. As in the range-finding test, 25 liter glass exposure vessels containing 20 liters oftest media were used for each control and test concentration. At the start ofthe test seven fish were placed in eachtest vessel at random, inthe testpreparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C in a temperature controlled room in the dark for a period of96 hours. The test solutions were not aerated. The fish were notindividually identified andreceived no food during exposure. The control group was maintainedunder identical conditions butnot exposedto the testitem. 3.4.3 Evaluations 3.4.3.1 Test Organism Observations Anymortalities 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 andresponse 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 pH and dissolved oxygen concentration were measured using a Hach FlexiStudyNumber: 41401173 Report Page 13
handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. 3.4.3.3 Verification ofTest Concentrations Water samples were taken from the control and 100 mg/L test vessel at 0, 24, 48, 72 and 96 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis ifnecessary. Themethod ofanalysis, recovery andtestpreparationanalyses are described inAppendix 3. 3.4.4 StatisticalAnalysis An estimate ofthe LC50 values was givenby inspection ofthe mortality data. 

### 3.5 Validation Criteria The results ofthe test are consideredvalid 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 ofthe test should be greater than or equal to 60% of Air Saturation Values (equivalent to 6.1 mg 02/L) in the control and test vessels.StudyNumber: 41401173 Report Page 14 

## 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 1and sub-lethal effects ofexposure are givenin Table 2. The results showed no mortalities at the test concentrations of 1 0 and 1 00 mg/L. The sub-lethal effect observed inthe 100 mg/L test concentration was believedto have resulted from aggressive behavior ofthe other fish as the affected fish had physical damage to the tail. The sub-lethal effectwas therefore considerednotto be related to exposureto the testitem. Chemical analysis ofthe test preparations at 0, 72 and 96 hours (see Appendix 3) indicated that the test item was stable under test conditions. Based onthis information, a single test concentration of 100 mg/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. 

### 4.2 Definitive Test 4.2.1 Verification ofTest Concentrations Analysis ofthe test preparations at 24, 48, 72 and 96 hours (see Appendix 3) showed measured test concentrations to range from 80 to 96% of nominal. Analysis ofthe test preparation at 0 hours showed a measured concentration of48% ofnominal, however, evaluation ofthe stock solution and test flow rates suggests the analytical result to be anomalous. Giventhatthe 0-Hour analytical result was suspected to be anomalous and as all other measured concentrations were near nominal it was consideredjustifiable to exclude the 0-Hour measured concentration and to base the results onthe nominal test concentration only. 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 3. There were no mortalities in 7 fish exposed to a test concentration of 1 00 mg/L for a period of 96 hours. The results ofthe definitive test showed the No Observed Effect Concentration (NOEC) was 100 mg/L. Itwas considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.StudyNumber: 41401173 Report Page 15
Itwas consideredunnecessary and unrealistic to test at concentrations in excess of 100 mg/L. 4.2.3 Sub-Lethal Effects There were no sub-lethal effects ofexposure observed in 7 fish exposedto a test concentration of 100 mg/L for aperiod of96 hours. 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 greater than or equal to 60% ofASV (6.1 mg 0 2/L) inthe control and testvessels. 4.2.5 Water Quality Criteria The results of the water quality measurements are given in Table 4. Temperature was maintained at 14 °C throughout the test, while there were no treatment related differences for oxygen concentration orpH. 4.2.6 Observations on TestItem Solubility At the start and throughout the test the test solutions were observed to be clear colorless solutions.StudyNumber: 41401173 Report Page 16 

## 5 CONCLUSION The acute toxicity ofthe test item to the freshwater fish rainbowtrout has been investigated and based onthe nominal test concentrations gave a 96-Hour LC5oofgreaterthan 1 00 mg/L. The No Observed Effect Concentrationwas 1 00 mg/L.StudyNumber: 41401173 Report Page 17 

# 6 REFERENCES BRITISH STANDARDS INSTITUTION (1993) Determination ofAlkalinity. In: Field and Onsite Test Methods for the Analysis ofWaters, (BS 1427), pp 33-35.StudyNumber: 41401173 Report Page 18 

## TABLES Table 1 CumulativeMortalityDataintheRange-findingTest Nominal CumulativeMortality Concentration (InitialPopulation = 3) (mg/L) 3 Hours 6 Hours 24 Hours 48 Hours 72 Hours 96 Hours Control 0 0 0 0 0 0 10 0 0 0 0 0 0 100 0 0 0 0 0 0StudyNumber: 41401173 Report Page 19
Table2 Sub-lethal Effects ofExposure in the Range-finding Test Nominal Time (Hours) Concentration Sub-lethal Effects (mg!L) 3 6 24 48 72 96 Control No abnormalities detected 3 3 3 3 3 3 10 No abnormalities detected 3 3 3 3 3 3 No abnormalities detected 3 3 3 3 3 2 100 Swimming onthe side 0 0 0 0 0 1*

*Fish appeared to have a damagedtail, possibly from aggressive behavior ofother fish, not from toxicity.StudyNumber: 41401173 Report Page 20
Table3 CumulativeMortalityDataintheDefinitiveTest CumulativeMortality (InitialPopulation = 7) 0 /o Nominal Mortality Concentration (mg/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 0Study Number: 41401173 Report Page 21
Table4 Water QualityMeasurements intheDefinitiveTest Nominal Time (Hours) Concentration 0 Hours 24 Hours 48 Hours (mg!L) pH mg02/L T"C pH mg02/L T°C pH mg02/L T"C Control 7.4 9.6 14 7.4 9.3 14 7.4 9.4 14

100 7.5 9.1 14 7.4 9.0 14 7.5 9.1 14 Nominal Time (Hours) Concentration 72 Hours 96 Hours (mg!L) pH mg 0 2/L T°C pH mg02/L T"C Control 7.4 9.5 14 7.5 9.0 14

100 7.5 9.0 14 7.5 8.6 14StudyNumber: 41401173 Report 

## APPENDICES Appendix 1 Certificate ofAnalysis



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**~ 1~~t:<;::r•,.**  Page 22StudyNumber: 41401173 Report Page 23 Appendix2 Typical Water Quality Characteristics WATER SUPPLY ZONE ZDB13 ALVASTON REPORTING PERIOD: 01/01/2012-31/12/2012 NO. CONCENTRATION OR VALUE SAMPLES PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 1,2 Dichloroethane iJQII 44 <0.080 <0.099 0.130 3 0 0.00 2, 3, 6- TBA IJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 2, 4-DB IJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 2, 4, 5- TCPA IJQ/1 44 <0.005 <0.005 <0.005 0.1 0 0.00 2,4-D IJQII 44 <0.004 <0.004 0.004 0.1 0 0.00 Aldrin iJQII 36 <0.002 <0.003 <0.003 0.03 0 0.00 Alpha-HCH IJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 Aluminium IJQII 26 <5.000 <7.500 19.000 200 0 0.00 Ametryn IJQ/1 44 <0.003 <0.003 <0.003 0.1 0 0.00 Antimony IJQII 8 0.110 0.334 0.460 5 0 0.00 Arsenic iJQII 8 <0.090 <0.143 0.370 10 0 0.00 Asulam IJQ/1 36 <0.003 <0.003 <0.005 0.1 0 0.00 Atrazine IJQ/1 44 <0.003 <0.003 <0.003 0.1 0 0.00 Azinphos methyl iJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Benazolin IJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Bentazone iJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Benzene IJQII 44 <0.050 <0.052 <0.060 1 0 0.00 Benzo 3,4-Pyrene IJQII 8 <0.001 <0.001 <0.001 0.01 0 0.00 Beta- HCH iJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 Boron mg/1 44 <0.012 <0.026 0.036 1 0 0.00 Bromacil IJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Bromate IJQII 8 <0.250 <0.768 1.400 10 0 0.00 Bromoxynil iJQII 44 <0.003 <0.004 <0.004 0.1 0 0.00 Cadmium IJQII 8 0.080 0.119 0.170 5 0 0.00 Caplan IJQII 36 <0.002 <0.003 <0.003 0.1 0 0.00 Carbenazim iJQII 44 <0.002 <0.002 <0.002 0.1 0 0.00 Carbetamide iJQII 44 <0.006 <0.006 <0.006 0.1 0 0.00 Carbophenophion IJQII 44 <0.002 <0.007 <0.012 0.1 0 0.00 ChlordaneA iJQII 36 <0.002 <0.002 <0.002 0.1 0 0.00 Chlorfenvinphos iJQII 44 <0.005 <0.005 <0.005 0.1 0 0.00 Chloride mg/1 44 8.650 18.499 36.000 250 0 0.00 Chlormequat IJQII 12 <0.004 <0.004 <0.004 0.1 0 0.00 Chlorothalonil IJQ/1 36 <0.001 <0.002 <0.003 0.1 0 0.00 Chlorotoluron iJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Chlorpyriphos Ethyl iJQII 44 <0.001 <0.006 <0.010 0.1 0 0.00 Chlorpyriphos Methyl IJQII 44 <0.001 <0.008 <0.015 0.1 0 0.00 Chromium IJQ/1 8 <0.700 <0.700 0.700 50 0 0.00 Cis- Permethrin iJQII 36 <0.001 <0.002 <0.003 0.1 0 0.00 Clopyralid iJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Colour mg/1 Pt/Co 26 <0.300 <1.255 1.600 20 0 0.00 Copper mg/1 8 <0.003 <0.014 0.052 2 0 0.00 Cyanazine IJQII 44 <0.005 <0.005 <0.005 0.1 0 0.00 Cyanide iJQII 44 <2.000 <2.000 <2.000 50 0 0.00 Cyfluthrin iJQII 36 <0.001 <0.002 <0.003 0.1 0 0.00 Cypermethrin iJQII 36 <0.001 <0.002 <0.003 0.1 0 0.00 D.D.E.- Ortho Para IJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 D.D.E. Para Para iJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 D.D.T.- Ortho Para IJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 D.D.T.- Para Para iJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 Delta- HCH iJQII 36 <0.001 <0.002 <0.002 0.1 0 0.00 Deltamethrin IJQII 36 <0.002 <0.003 <0.004 0.1 0 0.00 Demeton-S-Methyl iJQII 44 <0.006 <0.006 <0.006 0.1 0 0.00 Diazinon iJQII 44 <0.006 <0.006 <0.006 0.1 0 0.00 Dicamba IJQII 44 <0.004 <0.004 <0.004 0.1 0 0.00 Dichlobenil IJQII 36 <0.001 <0.001 <0.001 0.1 0 0.00 Dichlorprop iJQII 44 <0.003 <0.003 <0.003 0.1 0 0.00 Dichlorvos IJQII 44 <0.008 <0.008 <0.008 0.1 0 0.00 Dieldrin IJQII 36 <0.002 <0.002 <0.002 0.03 0 0.00 Diflufenican iJQII 44 <0.005 <0.005 <0.005 0.1 0 0.00 +PCV Prescribed concentration or value for mandatory parameters and specified limit orvalue for non-mandatory parameters (indicators and disinfectants).StudyNumber: 41401173 Report Page 24 Typical Water Quality Characteristics

WATER SUPPLY ZONE ZDB13 ALVASTON REPORTING PERIOD: 01/01/2012-31/12/2012 NO. CONCENTRATION OR VALUE SAMPLES PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT Dimethoate

**~g/1** 44 <0.005 <0.005 <0.005 0.1 0 0.00 Disulfoton

**~g/1** 36 <0.001 <0.002 <0.003 0.1 0 0.00 Diuron

**~g/1** 44 <0.003 <0.003 <0.003 0.1 0 0.00 E. coli No. I 100 ml 192 0.000 0.000 0.000 0 0 0.00 Endosulfan A

**~g/1** 36 <0.002 <0.003 <0.003 0.1 0 0.00 Endosulfan B

**~g/1** 36 <0.002 <0.003 <0.003 0.1 0 0.00 Endrin

**~g/1** 36 <0.002 <0.003 <0.003 0.1 0 0.00 Enterococci No. I 100 ml 8 0.000 0.000 0.000 0 0 0.00 EPTC

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Ethofumesate

**~g/1** 44 <0.006 <0.006 <0.006 0.1 0 0.00 Fenitrothion

**~g/1** 36 <0.002 <0.003 <0.004 0.1 0 0.00 Fenoprop

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Fenpropidin

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Fenpropimorph

**~g/1** 44 <0.002 <0.002 <0.002 0.1 0 0.00 Fenvalerate

**~g/1** 36 <0.001 <0.002 <0.003 0.1 0 0.00 Fluoride mg/1 8 0.190 0.280 0.350 1.5 0 0.00 Fluroxypyr

**~g/1** 44 <0.006 <0.006 <0.006 0.1 0 0.00 Flutriafol

**~g/1** 44 <0.007 <0.010 0.048 0.1 0 0.00 Gamma- HCH

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Glyphosate

**~g/1** 12 <0.006 <0.011 0.063 0.1 0 0.00 GrossAlpha Activity Bq/1 44 <0.014 <0.080 0.100 0. 1(oote 5)
- - Gross Beta Activity Bq/1 44 <0.012 <0.068 0.089 1 (note 5)

- - Heptachlor

**~g/1** 36 <0.002 <0.002 <0.002 0.03 0 0.00 Heptachlorepoxide

**~g/1** 36 0.000 0.000 0.000 0.03 0 0.00 Heptenophos

**~g/1** 44 <0.005 <0.005 <0.005 0.1 0 0.00 Hexachlorobenzene

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Hexachlorobutadiene

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 loxynil

**~g/1** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Iron

**~g/1** 52 <1 0.000 <25.423 221.000 200 1 1.92 lsodrin

**~g/1** 36 <0.001 <0.002 <0.003 0.1 0 0.00 lsoproturon

**~g/1** 44 <0.002 <0.002 0.002 0.1 0 0.00 Lead

**~g/1** 8 <0.500 <0.600 1.000 25 0 0.00 Linuron

**~g/1** 44 <0.005 <0.005 <0.005 0.1 0 0.00 Malathion

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Manganese

**~g/1** 26 <1.800 <3.077 18.000 50 0 0.00 MCPA

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 MCPB

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Mecoprop

**~g/1** 44 <0.006 <0.006 <0.006 0.1 0 0.00 Mercury

**~g/1** 44 <0.012 <0.012 <0.012 1 0 0.00 Metaldehyde

**~g/1** 36 <0.003 <0.01 0 0.030 0.1 0 0.00 Metamitron

**~g/1** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Metazachlor

**~g/1** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Methabenzthiazuron

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Methoxychlor

**~g/1** 36 <0.002 <0.003 <0.003 0.1 0 0.00 Mevinphos

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Monolinuron

**~g/1** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Nickel

**~g/1** 8 2.100 3.388 5.200 20 0 0.00 Nitrate mg/1 8 5.460 10.664 13.800 50 0 0.00 Nitrite mg/1 8 <0.007 <0.007 <0.007 0.5 0 0.00 Nitrite/Nitrate Calculated - (note 1) 8 0.110 0.214 0.280 <1 (note 1) 0 0.00 Odour Dilution 53 0.000 0.038 2.000 (note2) 1 1.89 Number - PAH

**~g/1** 8 0.000 0.000 0.000 0.1 0 0.00 Parathion

**~g/1** 44 <0.001 <0.004 <0.006 0.1 0 0.00 PCB 101

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 PCB 118

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 PCB 138

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 PCB 153

**~g/1** 36 <0.001 <0.002 <0.002 0.1 0 0.00 PCB 180

**~gil** 36 <0.002 <0.002 <0.002 0.1 0 0.00 PCB28

**~g/1** 36 <0.001 <0.001 <0.001 0.1 0 0.00 +PCV Prescribed concentration orvalue for mandatory parameters and specified limit or value for non-mandatory parameters (indicators and disinfectants).StudyNumber: 41401173 Report Page 25 Typical Water Quality Characteristics

WATER SUPPLY ZONE ZDB13 ALVASTON REPORTING PERIOD: 01/01/2012-31/12/2012 NO. CONCENTRATION OR VALUE SAMPLES PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT PCB 52

**~gil** 36 <0.002 <0.002 <0.002 0.1 0 0.00 Pendimethalin

**~gil** 44 <0.005 <0.005 <0.005 0.1 0 0.00 Phorate

**~gil** 44 <0.001 <0.005 <0.009 0.1 0 0.00 Phosalone

**~gil** 44 <0.007 <0.007 <0.007 0.1 0 0.00 Pirimicarb

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Pirimiphos-Methyl

**~gil** 44 <0.009 <0.009 <0.009 0.1 0 0.00 Prometryn

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Propachlor

**~gil** 44 <0.005 <0.005 <0.005 0.1 0 0.00 Propazine

**~gil** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Propetamphos

**~gil** 44 <0.007 <0.007 <0.007 0.1 0 0.00 Propiconazole

**~gil** 44 <0.010 <0.01 0 <0.010 0.1 0 0.00 Propyzamide

**~gil** 44 <0.004 <0.004 <0.004 0.1 0 0.00 Selenium

**~gil** 8 <0.200 <0.446 0.770 10 0 0.00 Simazine

**~gil** 44 <0.007 <0.007 <0.007 0.1 0 0.00 Sodium mg/1 8 11.000 21.375 26.000 200 0 0.00 Sulphate mg/1 44 22.400 46.838 77.800 250 0 0.00 Taste Dilution 53 0.000 0.000 0.000 (note2) 0 0.00 Number - T.D.E.- Ortho Para

**~gil** 36 <0.001 <0.002 <0.002 0.1 0 0.00 T.D.E.- Para Para

**~gil** 36 <0.001 <0.002 <0.003 0.1 0 0.00 Tecnazene

**~gil** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Terbutryn

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Tetrachloroethene and

**~gil** 8 0.000 0.000 0.000 10 0 0.00 Trichloroethene Tetrachloromethane

**~gil** 8 <0.018 <0.072 <0.080 3 0 0.00 Total Organic Carbon mg/1 44 <0.500 <1.310 1.530 

### -(note 4)

- 

### - Total Pesticides

**~gil** 44 0.000 0.011 0.063 0.5 0 0.00 Total Trihalomethanes

**~gil** 8 26.850 36.034 50.630 100 0 0.00 Trans - Permethrin

**~gil** 36 <0.001 <0.003 <0.004 0.1 0 0.00 Triadimefon

**~gil** 44 <0.009 <0.009 <0.009 0.1 0 0.00 Triazophos

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Trichlorobenzene

**~gil** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Triclopyr

**~gil** 44 <0.003 <0.003 <0.003 0.1 0 0.00 Trietazine

**~gil** 44 <0.006 <0.006 <0.006 0.1 0 0.00 Trifluralin

**~gil** 36 <0.001 <0.002 <0.002 0.1 0 0.00 Tritium Bq/1 8 <5.000 <5.000 <5.000 100 0 0.00 Turbidity NTU 52 0.090 0.248 1.140 4 0 0.00 Free Chlorine mg/1 192 0.010 0.234 0.610 -(note 3)

- 

### - Total Chlorine mg/1 192 0.070 0.301 0.660 -(note 3)

- - Hardness Total as mg/1 CaC03 1 144.250 144.250 144.250 (note 3) 0 0.00 CaCo3 - Ammonium mg/1 26 <0.018 <0.018 0.021 0.5 0 0.00 Clostridium perfringens No./100 ml 26 0.000 0.000 0.000 0 0 0.00 Coliform Bacteria No./100 ml 192 0.000 >0.521 >100.000 0 1 0.52 Colony Count after 48 No./100 ml 52 0.000 0.308 5.000 -(ote 4) 

### - - hours at 37°C Colony Count after 72 No./100 ml 52 0.000 1.038 15.000 -(note4)


### - - hours at 22°C Conductivity

**~S/cm** at 20oc 26 214.000 344.385 486.000 2500 0 0.00 pH pH value 52 6.930 7.731 8.090 Max9.5, 0 0.00 Min 6.5 +PCV Prescribed concentration or value for mandatory parameters and specified limit orvalue for non-mandatory parameters (indicators and disinfectants). 1 The formula forthe 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 2012.StudyNumber: 41401173 Report Page 26 Appendix3 Analytical Investigations 

## 1 MATERIALSAND METHODS 1.1 Introduction The test item concentration in the test samples was determined by high performance liquid chromatography withUV detection (HPLC/UV) using an external standard. The testitem gave a chromatographicprofile consisting ofa singlepeak. The method was developed and implemented by the Department ofAnalytical Services, Envigo Research Limited, Shardlow, UK. 

### 1.2 Test Item The testitem is described inthe biologicalpart 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 analyzed onthe day ofreceipt. 1.4.2 Reagents and Solvents Water: preparedusing an ELGA Pure Lab OptionR-15 waterpurification system. Test medium: as described inthe biological part ofthis study. Solvents: Reagents: acetonitrile, Fisher Scientific, HPLC grade. tetrabutylammonium,

**Sigma-Aldrich,~** 99.0% acetic acid, Fisher Scientific, analytical reagentStudy Number: 41401173 Report Page 27 Analytical Investigations 1.4.3 Preparation ofCalibration Standards The test item (nominal 100 mg) was dissolved in 5% (v/v) acetic acid in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis solution was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range of5to 100 mg/L. A second series ofcalibration standards was similarly prepared inthe range of 10 to 125 mg/L. These solutions were used to determine the recovery and test sample concentrations. 1.4.4 Preparation ofLinearity Standards The test item (nominal 100 mg) was dissolved in 5% (v/v) acetic acid in water (100 mL) to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis solution was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range of5to 100 mg/L. A second series ofcalibration standards was similarlyprepared inthe range of 10 to 125 mg/L. These standards were usedto evaluatethe 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 100 mg) was initially dissolved in 5% (v/v) acetic acid in test medium to prepare a stock solution with a concentration of 1000 mg/L. A defined volume ofthis stock solution was diluted with 5% (v/v) acetic acid in test medium to obtain spiked recovery samples at a concentration of 100 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium withoutthe addition ofthe test item (synthetic control) was also analysed. 1.4.6 Preparation ofTest Samples The test samples were analyzed on the day ofreceipt. An aliquot of acetic acid (5 mL) was added tothetest samples (95 mL). See Table 1for more information onpreparationvolumes.StudyNumber: 41401173 Report Page 28 Analytical Investigations 1.4.7 Instrumental Parameters HPLC System HP 1260 Series Column: Gemini, C18, 3 !Jill (100 x 4.6 mmid.) Mobile phase Mobile PhaseA: water+ TBAB solution* (90:10 v/v) Mobile Phase B: acetonitrile+ TBAB solution* (90:10 v/v) Gradient Time %A %B (mins) 0 100 0 5 0 100 5.1 100 0 10 100 0 Injectionvolume Flowrate 1mL/minute Columntemperature Autosamplertemperature Detectionwavelength 280nm Retentiontime approximately 3.2 minutes 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 test item 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 testitem inthe injected sample [mg/L] averagepeak area ofthetest item inthe injected sample [counts] constants

* TBAB solution=4 g oftetrabutylammoniumbromide in 200 mL acetonitrileStudyNumber: 41401173 Report Page 29 Analytical Investigations The concentration of the test item in the test samples was calculated using the following equation:
C = X .p R/100 where c: 

### x: F: R: (2) concentration ofthe test item inthe test sample [ mg/L] concentrationofthetestiteminthe injected sample solution [mg/L] samplepreparationfactor recoveryrate[%] (Ifnot correcting for recoverythenR is takenas 100) The recovery rate (R) of the spiked recovery samples was calculated usmg the following equation: R =-c-·100 cfort where R: c: Cfort: (3) recoveryrate [%] determined concentrationofthetestitem inthe spikedrecovery sample [ mg/L] fortified concentrationofthetest iteminthe spikedrecovery sample [ mg/L] Thepercentage ofnominal concentration ofthetest item inthe test samples was calculatedusing the following equation: % nominal = _c_ ·100 cnom (4) where Cnom: nominal concentrationinthetest sample [mg/L]StudyNumber: 41401173 Report Page 30 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validatedpriorto the analysis ofrange-finding samples; full validation was later conducted at a suitable concentration. The results for the range-finding test samples are summarized in Table 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 ofthe test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity ofthe method byretentiontime was confirmed. Linearity The calibration data for the calibration standards ofthe test item is given in Table 2 and Figure 1. The data was found to have a quadratic correlationwithinthe calibrationrange of0 to 124 mg/L. The R2 fit ofthe calibrationcurveto the data was 1.0000, and was consideredto be acceptable. Accuracy (Recovery) and Precision A set of recovery samples accurately fortified at a relevant concentration of test item was preparedfive-fold and analyzed. The results obtained for the concentration ofthe test item inthe recovery samples are presented in Table 4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were notcorrected for recovery. Limit ofQuantification The limit ofquantification (LOQ) was determined by calculating the sample concentration that gave a peak equivalent to ten times the baseline noise. Using this method the LOQ was determinedto be 2.6 mg/L. 2.2.2 Test Samples The results obtained for the concentration ofthe test item found in the test samples are presented inTable 5. Typical chromatograms are shownin Figures 2 to 4.StudyNumber: 41401173 Report Page 31 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 oftest item in test medium. The method ofanalysis was validated and provento be suitable for use.StudyNumber: 41401173 Report Page 32 Analytical Investigations 

## TABLES Table 1 Preparation ofTestSamples Nominal Sample FinalNominal Concentration ofTest SampleVolume FinalVolume Preparation Concentration ofTest Item Factor Item [mg/L] [mL] [mL] F [mg/L] Control 95 100 1.1 

### -

100 95 100 1.1 95 not applicable Table2 Calibration StandardData
Concentration ofTestItem Mean Area [mg/L] [counts] 0 0 4.97 1.823 X 105 9.95 4.205 X 105 24.9 1.200 X 106 49.7 2.547 X 106 99.4 5.651 X 106 124 7.439 X 106StudyNumber: 41401173 Report Page 33 Analytical Investigations Table3 Results forRange-Finding Samples Determined Nominal Sample Concentration %ofNominal Timepoint Concentration Preparation ofTest Item in Concentration Factor Range-Finding Sample
Cnom F c [hours] [mg/L] [mg/L] [%] 0 10 1.1 6.49 65 100 1.1 91.4 91 72 10 1.1 94.6 945* 100 1.1 3.17 3* 96 10 1.1 103 1027* 100 1.1 7.50 s* * The analytical results at 72 and 96 hours indicate that the 10 and 100 mg/L samples were mixed. In the table below the results are re-presented with the 72 and 96-Hour analytical results for the 10 and 100 mg/L samples reversedto correctthe error. Determined Nominal Sample Concentration Percentage of Timepoint Concentration Preparation ofTestItem in Nominal Factor Range-Finding Concentration Sample
Cnom F c [hours] [mg/L] [mg/L] [%] 0 10 1.1 6.49 65 100 1.1 91.4 91 72 10 1.1 3.17** 32 100 1.1 94.6** 95 96 10 1.1 7.5o** 75 100 1.1 103** 103 ** The I0 and 100 mg/L analytical results at 72 and 96 hours havebeenreversed.StudyNumber: 41401173 Report Analytical Investigations Table4 Results for SpikedRecovery Samples Nominal Fortified Determined Mean Concentration Concentration Sample Concentration Analytical of ofTestItem in Preparation ofTestItem in Recovery the Spiked Factor the Spiked TestItem Sample Sample Rate
Cnom Crort F c R [mgiL] [mg!L] [mg!L] [%] 1.1 104 1.1 105 100 102 1.1 106 103 1.1 105 1.1 106 Acceptance Target 80-120 Table5 Results forTest Samples in theDefinitiveTest

LOQ 

### * Nominal Timepoint Concentration

Cnom [hours] [mgiL] 0 Control 100 24 Control 100 48 Control 100 72 Control 100 96 Control 100 Limit ofQuantification not applicable Sample Preparation Factor F 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1
duplicate sample, storedfrozen priorto analysis Determined Concentration ofTestItem in

Test Sample
c [mgiL] <LOQ 47.7 I 10.8* <LOQ

82.7 <LOQ

83.9 <LOQ

79.9 <LOQ

96.4 Page 34

Precision (Relative Standard Deviation of

Recovery) [%]
0.69
<10

Percentage of Nominal Concentration
[%]



## - 48 I 11*



### - 83



### - 84



### - 80



## - 96StudyNumber: 41401173 Report Analytical Investigations 

## FIGURES Figure 1 Calibration Plot 

## :j" 8000000

**~** ......, o; 

### a c( 7000000



## ~ 6<000000 .5000000 4000000 3000000 2000000 1000000 y=H0.8734r+46,507.5Ul!x-45,097.7864 R."= 1.0000

20 40 60 80 Page 35

100 120 140StudyNumber: 41401173 Report AnalyticalInvestigations Figure 2 Chromatogram ofCalibration Solution (100 mg/L)

**~** vwo, Signlol A. 280 nm stand.,<<l a 1100 mg-1

Figure 3 Chromatogram ofBiological Control Sample, 24 Hour Page 36StudyNumber: 41401173 AnalyticalInvestigations Report Figure 4 Chromatogram ofTest Sample (100 mg/L), 24 Hour Page 37StudyNumber: 41401173 Report Appendix4 MonitoringAuthority StatementofGLP Compliance 

## f

# nH) Department



### ~ ofHealth THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC TEST FACILITY

Envigo Research Limited Shardlow Business Park London Road Shardlow Derbyshire DE72 2GD Note: This statement has been issued following a company name change.

DATE OF INSPECTION TESTTYPE(S) Analytical/Clinical Chemistry Environmental Fate Environmental Toxicity Phys.Chem. Testing Mutagenicity

Toxicology

17 June 2015 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 statementconstitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999)
. 

# • Dr. AndrewJ. Gray Head, UK GLP Monitoring Authority Page 38

On 21 September2015 the legal entityHarlanLaboratories Limited was renamed as Envigo ResearchLimited. No otherchanges to the legal entityhave occurred.