REPORT 

+++ 

£NV IGO 

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Acute Toxicity to Rainbow Trout 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Study Number: 

Study Completion Date: 

D Sacker 

Envigo Research Limited 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL33410 

UNITED STATES OF AMERICA 

41401173 

15 January 2016 

On 21 September 2015 the legal entity Harlan Laboratories Limited was renamed as Envigo Research 

Limited. No other changes to the legal entity have occurred. 

Page 1 of38 
Study Number: 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 INTRODUCTION AND PURPOSE ..................................................................................... 8 

1.1 Guidelines I Regulations .......................................................................................... 8 

2 TEST ITEM ........................................................................................................................... 9 

3 MATERIALS AND METHODS ......................................................................................... 10 

3.1 Definitions and Abbreviations ............................................................................... 10 

3.2 Test Species ........................................................................................................... 10 

3.3 Test Water .............................................................................................................. 10 

3.4 Procedure ............................................................................................................... 11 

3 .4.1 Range-finding Test .................................................................................. 11 

3.4.2 Definitive Test ......................................................................................... 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 of Test 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 ............................................................................................................................... 22 
Study Number: 41401173 Report Page 3 

LIST OF TABLES 

Table 1 

Table 2 

Table 3 

Table 4 

Cumulative Mortality Data in the Range-finding Test ............................................... 18 

Sub-lethal Effects ofExposure in the Range-finding Test ......................................... 19 

Cumulative Mortality Data in the Definitive Test ...................................................... 20 

Water Quality Measurements ..................................................................................... 21 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Certificate of Analysis ........................................................................................... 22 

Typical Water Quality Characteristics .................................................................. 23 

Analytical Investigations ....................................................................................... 26 

Monitoring Authority Statement of GLP Compliance .......................................... 38 
Study Number: 41401173 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Study Number: 41401173 

Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7]: 

Acute Toxicity to Rainbow Trout 

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory 

Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are 

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice 

(revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the 

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

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHLW, MAFF and METI). 

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

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

Study Director: D Sacker 

Date: 

...... ~ 1 5 JAN 2016 
Study Number: 41401173 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Study Number: 41401173 

Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7]: 

Acute Toxicity to Rainbow Trout 

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

to the relevant responsible person and management. 

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

these audits and inspections are given below. 

Reported to the 

Dates and Types of QA Inspections relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

15 April2014 Study Plan N/A 15 April2014 

Verification 

16 November 2015 Process - based Test Item Preparation 16 November 2015 

16 November 2015 Process - based Test System 16 November 2015 

Preparation and 

Exposure 

18 November 2015 & Process - based Assessment of 18 November 2015 & 

20 November 2015 Response 20 November 2015 

19 October 2015 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 that this report reflects the raw data and the procedures followed. 
Study Number: 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 of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as 

Method C.1 of Commission Regulation (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 of 96 hours at a temperature of 

14 °C under continuous flow-through test conditions. The number of mortalities and any sublethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the 

start of exposure and then daily throughout the test until termination after 96 hours. 

Results 

Analysis of the test preparations 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 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. 
Study Number: 41401173 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

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 of the United Kingdom's Animals 

(Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the study may 

be reviewed, as part of the Envigo Research Limited, Shardlow, UK Ethical Review Process. 

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

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

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

used in safety evaluation. 

Deviations from Study Plan 

Deviation No.1 

The Study Plan states that particulate matter concentration, COD, TOC, hardness, alkalinity and 

conductivity will be determined at the start of the test, however, due to a technical oversight this 

was not conducted at the start of the test, instead, data from dechlorinated water samples tested 

in July 2015 and January 2016 are presented in Section 3.3 of this report. This deviation was 

considered not to have adversely affected the results of the test. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan 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 data will be discarded without contacting the Sponsor to obtain their written consent. 
Study Number: 41401173 Report Page 8 

1 INTRODUCTION AND PURPOSE 

This report contains a description of the methods used and results obtained during a study to 

investigate the acute toxicity of the test item to rainbow trout (Oncorhynchus my kiss). 

Rainbow trout is a freshwater fish representative of a wide variety of natural habitats, and can 

therefore be considered as an important non-target organism in freshwater ecosystems. 

1.1 Guidelines I Regulations 

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

internationally accepted guidelines and recommendations: 

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

• Method C.l of Commission Regulation (EC) No. 440/2008. 
Study Number: 41401173 Report Page 9 

2 TESTITEM 

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

given in Appendix 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% 

Expiry Date: 26 March 2016 

Storage Conditions: Room temperature in the dark 
Study Number: 41401173 Report Page 10 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

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

mortality within the exposure period. 

LOEC (Lowest Observed 

Effect Concentration): 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

3.2 Test Species 

The lowest test concentration at which a significant toxic effect 

on the test organisms is observed within the exposure period. 

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. 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 dusk transition periods. 

The water temperature 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 of the definitive test. There was no mortality in the 7 days prior to the start of the test and 

the fish had a mean standard length of 4.0 em (sd = 0.3) and a mean weight of 0.88 g (sd = 0.15) 

at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.07 

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 of the study. 

3.3 Test Water 

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

maintain the stock fish. 

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

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

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

water was passed through a series of computer controlled plate heat exchangers to achieve the 
Study Number: 41401173 Report Page 11 

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

dechlorination and softening, are given in Appendix 2. 

Dechlorinated laboratory water tested in July 2015 and January 2016 and had a particulate 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 

Test Methods for the Analysis of Waters (British Standards Institution, 1993). 

3.4 Procedure 

Due to the potentially light sensitive nature of the test item, all test item preparation was 

performed under 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 of test 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 of 0.50 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 of 96 hours under dynamic test 

conditions. Each 25 liter test and control vessel contained 20 L of test media and was covered to 

reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of 

exposure were determined by visual inspection of the test fish. 

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

A sample of each test concentration was taken immediately for chemical analysis at 0, 72 and 96 

hours in order to determine the stability of the test item under test conditions. 
Study Number: 41401173 Report Page 12 

3.4.2 Definitive Test 

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

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

no mortalities or sub-lethal effects of exposure were observed. 

3 .4.2.1 Experimental Preparation 

A nominal amount of test item (12 g) was dissolved in deionized reverse osmosis water and the 

volume adjusted to 1 liter to give the 12 g/L stock solution. The stock solution was sonicated for 

5 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 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 delivered to the test vessel to give the required test concentration of 100 mg/L. 

The concentration and stability of the test item in the 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 of test media were used for each control and test concentration. At the start 

of the test seven 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 14 °C in a temperature 

controlled room in the dark for a period of 96 hours. The test solutions were not aerated. The 

fish were not individually identified and received no food during exposure. 

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

3.4.3 Evaluations 

3.4.3.1 Test Organism Observations 

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

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

movement and response to physical stimulation. 

3.4.3.2 Water Quality Criteria 

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

the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi 
Study Number: 41401173 Report Page 13 

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

digital thermometer. 

3.4. 3.3 Verification of Test 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 if necessary. 

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

3.4.4 Statistical Analysis 

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

3.5 Validation Criteria 

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

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

96 hours. 

• The dissolved oxygen concentration at the end of the test should be greater than or equal 

to 60% of Air Saturation Values (equivalent to 6.1 mg 02/L) in the control and test 

vessels. 
Study Number: 41401173 Report Page 14 

4 RESULTS 

4.1 Range-finding Test 

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

The results showed no mortalities at the test concentrations of 1 0 and 1 00 mg/L. The sub-lethal 

effect observed in the 100 mg/L test concentration was believed to have resulted from aggressive 

behavior of the other fish as the affected fish had physical damage to the tail. The sub-lethal 

effect was therefore considered not to be related to exposure to the test item. 

Chemical analysis of the test preparations at 0, 72 and 96 hours (see Appendix 3) indicated that 

the test item was stable under test conditions. 

Based on this 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 of Test Concentrations 

Analysis of the 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 of the test preparation at 

0 hours showed a measured concentration of 48% of nominal, however, evaluation of the stock 

solution and test flow rates suggests the analytical result to be anomalous. Given that the 0-Hour 

analytical result was suspected to be anomalous and as all other measured concentrations 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. 

4.2.2 Mortality Data 

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

definitive test are given in Table 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 of the definitive test showed the No Observed Effect Concentration (NOEC) was 

100 mg/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. 
Study Number: 41401173 Report Page 15 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L. 

4.2.3 Sub-Lethal Effects 

There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 

100 mg/L for a period of 96 hours. 

4.2.4 Validation Criteria 

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

stress during the test and that the oxygen concentration at the end of the test was greater than or 

equal to 60% of ASV (6.1 mg 0 2/L) in the control and test vessels. 

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 or pH. 

4.2.6 Observations on Test Item Solubility 

At the start and throughout the test the test solutions were observed to be clear colorless 

solutions. 
Study Number: 41401173 Report Page 16 

5 CONCLUSION 

The acute toxicity of the test item to the freshwater fish rainbow trout has been investigated and 

based on the nominal test concentrations gave a 96-Hour LC5o of greater than 1 00 mg/L. The No 

Observed Effect Concentration was 1 00 mg/L. 
Study Number: 41401173 Report Page 17 

6 REFERENCES 

BRITISH STANDARDS INSTITUTION (1993) Determination of Alkalinity. In: Field and Onsite Test Methods for the Analysis of Waters, (BS 1427), pp 33-35. 
Study Number: 41401173 Report Page 18 

TABLES 

Table 1 Cumulative Mortality Data in the Range-finding Test 

Nominal Cumulative Mortality 

Concentration (Initial Population = 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 0 
Study Number: 41401173 Report Page 19 

Table 2 Sub-lethal Effects of Exposure 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 on the side 0 0 0 0 0 1* 

*Fish appeared to have a damaged tail, possibly from aggressive behavior of other fish, not from toxicity. 
Study Number: 4140 1173 Report Page 20 

Table 3 Cumulative Mortality Data in the Definitive Test 

Cumulative Mortality (Initial Population = 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 0 
Study Number: 41401173 Report Page 21 

Table 4 Water Quality Measurements in the Definitive Test 

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 14 
Study Number: 41401173 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

... 

vuntVf\1=1 

Sodium Erythorbate •ceGt•• K-

.. ,~.If"~ ~;~.' .-,';. .. 4 .ft._ 

lp,...ntae• :•~ ..., :·"" "'"' _.,., ~tCWlc•tliDn rT 

... ..,. HU,M .. t$-( .. li'IJ;t 1{- .':o.l-'~' ~;.,, 

(11CI ............. Fee) 

~.Aid .. ::p-.-'1"':• mt. ... 

~-tlf>tl,.... ','""' -~· Glt- Cj>,, 

~ifllC '!t~'!!!.~~~"" 4 &'!· "!~ 

~· ,.. ........ ,..,. ................. 

,..,,.,__,..r,._ "~'''< •<r"'n.~•-- 1an~ .. ·~·-"-"""oM ~. -·- '#o-<" ... ,...\'fl"- ~ ·,~-~· ~- '·~~·'t": ----.... .,,.4 ' ...... ~_ ........... ~·-"'- ffo"'"' ,.~~\~"'....,* -~- .. ,..,."""_...-.,."*'-- .-... ~,_.,..,._,nu,<W~-•.&."*""'.illlr i;!f''.W j·'&\1-..-,.,_ii'f'-~- ,,.,~~<'f,."'t.»""b-~~,..... ....... _ ..j!~""'-"""' ~-

IJ--" 

P*.,_"i J•..- t.J4~ < 

~ 1~~t:<;::r•,. 

Page 22 
Study Number: 41401173 Report Page 23 

Appendix 2 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 

Chlordane A 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 or value for non-mandatory parameters (indicators and 

disinfectants). 
Study Number: 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 

Gross Alpha 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 

Heptachlor epoxide ~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) 

Number - 1 1.89 

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 or value for mandatory parameters and specified limit or value for non-mandatory parameters (indicators and disinfectants). 
Study Number: 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 

Trichloroethene ~gil 8 0.000 0.000 0.000 10 0 0.00 

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) 

CaCo3 - 0 0.00 

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 or value for non-mandatory parameters (indicators and 

disinfectants). 

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

2 Acceptable to customers and no abnormal change. 

3 No numerical limit applies. 

4 No abnormal change. 

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

the TID in 2012. 
Study Number: 41401173 Report Page 26 

Appendix 3 Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The test item concentration in the test samples was determined by high performance liquid 

chromatography with UV detection (HPLC/UV) using an external standard. The test item gave a 

chromatographic profile consisting of a single peak. 

The method was developed and implemented by the Department of Analytical Services, Envigo 

Research Limited, Shardlow, UK. 

1.2 Test Item 

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

1.3 Analytical Standard 

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

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The samples were analyzed on the day of receipt. 

1.4.2 Reagents and Solvents 

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

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

Solvents: 

Reagents: 

acetonitrile, Fisher Scientific, HPLC grade. 

tetrabutylammonium, Sigma-Aldrich,~ 99.0% 

acetic acid, Fisher Scientific, analytical reagent 
Study Number: 41401173 Report Page 27 

Analytical Investigations 

1.4.3 Preparation of Calibration 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 of this solution 

was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range 

of 5 to 100 mg/L. A second series of calibration standards was similarly prepared in the range of 

10 to 125 mg/L. These solutions were used to determine the recovery and test sample 

concentrations. 

1.4.4 Preparation of Linearity 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 of this solution 

was then diluted with 5% (v/v) acetic acid in water to obtain calibration standards in the range 

of 5 to 100 mg/L. A second series of calibration standards was similarly prepared in the range of 

10 to 125 mg/L. These standards were used to evaluate the linearity of the analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked 

with the test item and the recovery was assessed. The test item (nominal 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 of this 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 

without the addition of the test item (synthetic control) was also analysed. 

1.4.6 Preparation of Test Samples 

The test samples were analyzed on the day of receipt. An aliquot of acetic acid (5 mL) was 

added to the test samples (95 mL). See Table 1 for more information on preparation volumes. 
Study Number: 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 mm id.) 

Mobile phase Mobile Phase A: 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 

Injection volume 

Flow rate 1 mL/minute 

Column temperature 

Autosampler temperature 

Detection wavelength 280nm 

Retention time approximately 3.2 minutes 

1.4.8 Calculations 

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

The calibration data is presented in Table 2 and Figure 1. From the calibration curve, the 

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

equation: 

X= __ 

-b+~b 2 -4·a·(c- y) _;_____ _____ _ (1) 

2·a 

where x: 

y: 

a, b, c: 

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

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

constants 

* TBAB solution= 4 g oftetrabutylammonium bromide in 200 mL acetonitrile 
Study Number: 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 of the test item in the test sample [ mg/L] 

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

sample preparation factor 

recovery rate[%] (If not correcting for recovery then R is taken as 100) 

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

equation: 

R =-c-·100 

cfort 

where R: 

c: 

Cfort: 

(3) 

recovery rate [%] 

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

fortified concentration of the test item in the spiked recovery sample [ mg/L] 

The percentage of nominal concentration of the test item in the test samples was calculated using 

the following equation: 

% nominal = _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg/L] 
Study Number: 41401173 Report Page 30 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

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

validation was later conducted 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 of the Analytical Method 

Specificity 

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

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

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

the specificity of the method by retention time was confirmed. 

Linearity 

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

The data was found to have a quadratic correlation within the calibration range of 0 to 124 mg/L. 

The R2 fit of the calibration curve to the data was 1.0000, and was considered to be acceptable. 

Accuracy (Recovery) and Precision 

A set of recovery samples accurately fortified at a relevant concentration of test item was 

prepared five-fold and analyzed. The results obtained for the concentration of the test item in the 

recovery samples are presented in Table 4. 

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

The test sample results were not corrected for recovery. 

Limit of Quantification 

The limit of quantification (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 

determined to be 2.6 mg/L. 

2.2.2 Test Samples 

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

in Table 5. 

Typical chromatograms are shown in Figures 2 to 4. 
Study Number: 41401173 Report Page 31 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable correlation to the fit of a quadratic equation. 

The analytical procedure was found to have acceptable recoveries of test item in test medium. 

The method of analysis was validated and proven to be suitable for use. 
Study Number: 41401173 Report Page 32 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Final Nominal 

Concentration of Test Sample Volume Final Volume Preparation Concentration of Test 

Item Factor Item 

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

Control 95 100 1.1 -

100 95 100 1.1 95 

not applicable 

Table 2 Calibration Standard Data 

Concentration of Test Item 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 106 
Study Number: 41401173 Report Page 33 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Determined 

Nominal Sample Concentration %of Nominal Timepoint Concentration Preparation of Test 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 

reversed to correct the error. 

Determined 

Nominal Sample Concentration Percentage of 

Timepoint Concentration Preparation of Test Item 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 I 0 and 100 mg/L analytical results at 72 and 96 hours have been reversed. 
Study Number: 41401173 Report 

Analytical Investigations 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Determined Mean 

Concentration Concentration Sample Concentration Analytical 

of of Test Item in Preparation of Test Item in Recovery the Spiked Factor the Spiked Test Item 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 

Table 5 Results for Test Samples in the Definitive Test 

LOQ 

* 

Nominal Timepoint Concentration 

Cnom 

[hours] [mgiL] 

0 Control 

100 

24 Control 

100 

48 Control 

100 

72 Control 

100 

96 Control 

100 

Limit of Quantification 

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, stored frozen prior to analysis 

Determined 

Concentration 

of Test Item 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 

-

96 
Study Number: 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 

100 120 140 
Study Number: 41401173 Report 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (100 mg/L) 

~ vwo, Signlol A. 280 nm 

stand.,<<l a 1100 mg-1 

Figure 3 Chromatogram of Biological Control Sample, 24 Hour 

Page 36 
Study Number: 41401173 

Analytical Investigations 

Report 

Figure 4 Chromatogram of Test Sample (100 mg/L), 24 Hour 

Page 37 
Study Number: 41401173 Report 

Appendix 4 Monitoring Authority Statement of GLP 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 

Shard low 

Derbyshire 

DE72 2GD 

Note: This statement has been issued following a 

company name change. 

DATE OF INSPECTION 

TEST TYPE(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 statement constitutes a Good Laboratory Practice Instrument (as defined in the 

UK Good Laboratory Practice Regulations 1999) . 

• Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority 

Page 38 

On 21 September 2015 the legal entity Harlan Laboratories Limited was renamed as Envigo Research Limited. No 

other changes to the legal entity have occurred.