REPORT 

Acute Toxicity To Rainbow Trout (Oncorhynchus mykiss) 

Data Requirements/Test Guidelines: 

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

Toxicity Test". 

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

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

SParr 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

41206435 

26 April2013 

Page 1 of38 
Harlan Study Number: 41206435 Report Page2 

CONTENTS 

CONTENTS .................................................................................................................................... 2 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE .................................................... 3 

QUALITY ASSURANCE STATEMENT ...................................................................................... 4 

SIGNATURE OF CONTRIBUTING SCIENTISTS ...................................................................... 5 

SUMMARY .................................................................................................................................... 6 

Introduction ............................................................................................................................ 6 

Methods ................................................................................................................................. 6 

Results .................................................................................................................................... 6 

GENERAL INFORMATION ......................................................................................................... 7 

Schedule ................................................................................................................................. 7 

Animal Welfare ...................................................................................................................... 7 

Archiving ............................................................................................................................... 7 

1 INTRODUCTION AND PURPOSE ..................................................................................... 8 

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

2 TEST ITEM ........................................................................................................................... 8 

2.1 Test Item .................................................................................................................. 8 

3 MATERIALS AND METHODS ........................................................................................... 8 

3.1 Definitions and Abbreviations ................................................................................. 8 

3.2 Test Species ............................................................................................................. 9 

3.3 · Test Water ................................................................................................................ 9 

3.4 Procedure ............................................................................................................... 10 

3.4.1 Range-finding Test .................................................................................. 10 

3.4.2 Definitive Test ......................................................................................... 10 

4 RESULTS ............................................................................................................................ 13 

4.1 Range-finding Test ................................................................................................ 13 

4.2 Definitive Test ....................................................................................................... 13 

4.2.1 Verification of Test Concentrations ........................................................ 13 

4.2.2 Mortality Data ......................................................................................... 13 

4.2.3 Sub-Lethal Effects ................................................................................... 14 

4.2.4 Observations on Test Item Solubility ...................................................... 14 

4.2.5 Physico-Chemical Measurements ........................................................... 14 

5 CONCLUSION .................................................................................................................... 15 

6 REFERENCES .................................................................................................................... 16 

TABLES ........................................................................................................................................ 17 

Table 1 Cumulative Mortality Data in the Range-finding Test .......................................... 17 

Table 2 Cumulative Mortality Data in the Definitive Test.. ............................................... 18 

FIGURES ...................................................................................................................................... 19 

Figure 1 Concentration-Mortality Curve ............................................................................. 19 

Figure 2 Toxicity Curve ....................................................................................................... 20 

APPENDICES ............................................................................................................................... 21 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix4 

Typical Water Quality Characteristics .................................................................. 21 

Analytical Investigations ....................................................................................... 23 

Physico-Chemical Measurements .......................................................................... 3 7 

Monitoring Authority Statement of GLP Compliance .......................................... 38 
Harlan Study Number: 41206435 Report 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

Harlan Study Number: 

Study Title: 

41206435 

Acute Toxicity To Rainbow Trout (Oncorhynchus 

mykiss) 

Page3 

The study described was performed in compliance with UK GLP standards (Schedule 1, Good 

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

Regulations are in accordance with GLP standards published as OECD Principles on Good 

Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17). 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHL W, MAFF and METI). 

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

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

Study Director: SParr 
Harlan Study Number: 41206435 Report Page4 

QUALITY ASSURANCE STATEMENT 

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

Harlan Study Number: 

Study Title: 

41206435 

Acute Toxicity To Rainbow Trout (Oncorhynchus 

mykiss) 

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

to the relevant responsible person and management. 

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

these audits and inspections are given below. 

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 

19 November 2012 Study Plan N/A 19 November 2012 

Verification 

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

10 January 2013 Process - based Test System 1 0 January 2013 

Preparation 

07 January 20 13 Process - based Exposure 07 January 2013 

10 January 2013 Process - based Assessment of 10 January 2013 

Response 

11 January 2013 Process - based Chemical Analysis 11 January 2013 

05 April2013 Report Audit N/A 05 April2013 

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

Quality Assurance: 

....... 1:-::.f!..~l. 

2 4 APR 2013 
Harlan Study Number: 41206435 Report Page 5 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: DMMullee 

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

/J 'I If 1 2 0 l013 ...... (/...~ ............................. . 

Date: 
Harlan Study Number: 41206435 Report Page6 

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, fish were exposed, in groups of seven, to an aqueous 

solution of the test item over a range of concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for a 

period of 96 hours at a temperature of 13 °C to 14 °C under semi-static test conditions. The 

number of mortalities and any sub-lethal effects of exposure in each test and control vessel were 

determined 3 and 6 hours after the start of exposure and then daily throughout the test until 

termination after 96 hours. 

Results 

Analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old 

media) showed the following mean measured test concentrations: 

Nominal Concentration Measured Concentrations Mean Measured Concentration 

(mg/L) (mg/L) (mg/L) 

1.8 1.7-2.4 2.1 

3.2 2.9-4.1 3.5 

5.6 5.3-6.8 6.1 

10 9.3 - 12 11 

The 96-Hour LCso based on mean measured test concentrations was 6.5 mg/L with 95% 

confidence limits of 4.6 - 10 mg!L. The Lowest Observed Effect Concentration was considered 

to be 3.5 mg!L. The No Observed Effect Concentration was 2.1 mg/L. 
Harlan Study Number: 41206435 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Animal Welfare 

11 February 2013 

08 March 2013 

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 Harlan Laboratories Ltd, 

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

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

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

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

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

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

used in safety evaluation. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), all raw data (paper and electronic) and the final report will be retained iJ1 the Harlan 

Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as 

to further retention or disposal. Further retention or return of the data will be chargeable to the 

Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41206435 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 mykiss). 

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

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

1.1 Guidelines I Regulations 

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

internationally accepted guidelines and recommendations: 

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

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

2 TESTITEM 

2.1 Test Item 

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

the responsibility of the Sponsor. 

Identification: 

Description: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

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): 

The lowest test concentration at which a significant toxic effect 

on the test organisms is observed within the exposure period. 
Harlan Study Number: 41206435 

NOEC (No Observed .Effect 

Concentration): 

Treatment: 

3.2 Test Species 

Report Page 9 

The highest test concentration at which no significant toxic 

effect on the test organisms is observed within the exposure 

period. 

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

solvent control (if applicable). 

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

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

maintained in-house since 4 February 2013. Fish were maintained in a glass fiber tank with a 

"single pass" water renewal system. Fish were acclimatized to test conditions from 20 February 

2013 to 4 March 2013. The lighting cycle was controlled to give a 16 hours light and 8 hours 

darkness cycle with 20 minute dawn and dusk transition periods. 

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

greater than or equal to 10.5 mg 02/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 1 mortality in the 7 days prior to the start of the test and the 

fish had a mean standard length of 4.6 em (sd = 0.2) and a mean weight of 1.17 g (sd = 0.21) at 

the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.41 g 

bodyweight/liter (static volume). 

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

integrity and outcome ofthe study. 

3.3 Test Water 

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

maintain the stock fish. 

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

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

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

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

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

dechlorination and softening, are given in Appendix 1. 
Harlan Study Number: 41206435 Report Page 10 

3.4 Procedure 

3.4.1 Range-finding Test 

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

In the range-finding test fish were exposed to a series of nominal test concentrations of 

1.0, 10 and 100 mg/L. 

An amount of test item ( 4000 mg) was dissolved in dechlorinated tap water and the volume 

adjusted to 1 liter to give a 4000 mg/L stock solution. Aliquots (5.0, 50 and 500 mL) of the 

4000 mg/L stock solution were then each separately added to a final volume of 20 liters of 

dechlorinated tap water to give the 1.0, 10 and 100 mg/L test concentrations respectively. 

The stock solution was inverted several times, and the prepared concentrations were stirred using 

a flat-bladed mixer for approximately 1 minute to ensure adequate mixing and homogeneity. 

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

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

8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under 

static test conditions. 

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

Data from the control group was shared with similar concurrent studies. 

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

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

A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to 

determine the stability of the test item under test conditions. All samples were stored at 

approximately -20 °C prior to analysis. Only concentrations within the range to be used for the 

definitive test were analyzed. 

3.4.2 Definitive Test 

Based on the results of the range-finding test the following test concentrations were assigned to 

the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L. 

3.4.2.1 Experimental Preparation 

An amount of test item (1 000 mg) was dissolved in dechlorinated tap water and the volume 

adjusted to 1 liter to give a 1000 mg/L stock solution. Aliquots (20, 36, 64, 112 and 200 mL) of 
Harlan Study Number: 41206435 Report Page 11 

the 1000 mg/L stock solution were then each separately added to final volumes of 20 liters of 

dechlorinated tap water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L test concentrations 

respectively. 

The stock solution was inverted several times, and the prepared concentrations were stirred using 

a flat-bladed mixer for approximately 1 minute to ensure adequate mixing and homogeneity. 

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

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

(see Appendix 2). 

3.4.2.2 Exposure Conditions 

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

At the start of the test 7 fish were placed in each test vessel at random, in the test preparations. 

The test vessels were then covered to reduce evaporation and maintained at 13 oc to 14 °C in a 

temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 

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

aerated via narrow bore glass tubes. 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. 

Data from the control group was shared with similar concurrent studies. 

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

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

prevent the build-up of nitrogenous waste products. 

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

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

movement and response to physical stimulation. 

3.4.2.3 Physico-Chemical Measurements 

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

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

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

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

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

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

thermometer. 
Harlan Study Number: 41206435 Report Page 12 

3.4.2.4 Verification of Test Concentrations 

Water samples were taken from the control and all surviving test groups at 0 and 72 hours (fresh 

media) and at 24 and 96 hours (old media) for quantitative analysis. The 0, 24 and 72 hour 

samples were stored at approximately -20 °C prior to analysis with the 96 hour samples. 

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

media) were taken and stored at approximately -20 °C for further analysis if necessary. 

The method of analysis, recovery and test preparation analyses are described in Appendix 2. 

3.4.2.5 Evaluation of Data 

The LC50 values and associated confidence limits at 24 and 48 hours were calculated by the 

maximum-likelihood probit method (Finney, 1971) using the ToxCalc computer software 

package (ToxCalc, 1999). 

Probit analysis is used where two or more partial responses to exposure are shown. 
Harlan Study Number: 41206435 Report Page 13 

4 RESULTS 

4.1 Range-finding Test 

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

range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the 

range-finding test. 

The results showed no mortalities at the test concentration of 1.0 mg/L. However, mortalities 

were observed at 10 and 100 mg/L. 

Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for 

the definitive test. 

Chemical analysis of the test preparations at 0 and 24 hours (see Appendix 2) showed measured 

concentrations of 99% to 106% of nominal indicating that the test item was stable under test 

conditions. 

4.2 Definitive Test 

4.2.1 Verification of Test Concentrations 

Analysis of the test preparations at 0, 24, 72 and 96 hours (see Appendix 2) showed the test item 

to be stable between media renewal time points (i.e., 0 to 24 hours and 72 to 96 hours). However, 

variation was observed when the 0 hour fresh media to the 72 hour fresh media were compared, 

and when the 24 hour old media to the 96 hour old media were compared. It was therefore 

considered justifiable to calculate the LC5o values in terms of the mean-measured test 

concentrations: 

Nominal Concentration Measured Concentrations Mean Measured Concentration 

(mg/L) (mg/L) (mg/L) 

1.8 1.7-2.4 2.1 

3.2 2.9-4.1 3.5 

5.6 5.3-6.8 6.1 

10 9.3- 12 11 

4.2.2 Mortality Data 

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

definitive test are given in Table 2 and the relationship between percentage mortality and 

concentration at 96 hours is given in Figure 1. 
Harlan Study Number: 41206435 Report Page 14 

Time (h) LCso (mg/L) 95% Confidence limits 

(mg/L) 

3 >11 -

6 >11 -

24 >11 -

48 7.0 5.0- 12 

72 6.5 4.6- 10 

96 6.5 4.6- 10 

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

to be 2.1 mg/L and the lowest test concentration resulting in 100% to be greater than 11 mg/L. 

The Lowest Observed Effect Concentration (LOEC) was considered to be 3.5 mg/L. The No 

Observed Effect Concentration (NOEC) was 2.1 mg/L. 

The relationship between the median lethal concentration (LC50) and time 1s presented 

graphically in Figure 2. 

4.2.3 Sub-Lethal Effects 

There were no sub-lethal effects of exposure observed in the test. 

4.2.4 Observations on Test Item Solubility 

The test preparations were observed to be clear colorless solutions for the duration of the test. 

4.2.5 Physico-Chemical Measurements 

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

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

differences for oxygen concentration or pH. 

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

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

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

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

group. 
Harlan Study Number: 41206435 Report Page 15 

5 CONCLUSION 

Based on the mean measured test concentrations of the test media the acute toxicity of the test 

item to rainbow trout gave a 96-Hour LC5o value of 6.5 mg/L with 95% confidence limits of 

4.6- 10 mg/L. The Lowest Observed Effect Concentration was considered to be 3.5 mg/L. The 

No Observed Effect Concentration was 2.1 mg/L. 
Harlan Study Number: 41206435 Report Page 16 

6 REFERENCES 

FINNEY, D.J. (1978) Statistical Methods in Biological Assay. 3rd ed. London: Charles Griffin. 

TOXCALC VERSION 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, 

USA. 
Harlan Study Number: 41206435 Report Page 17 

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 

1.0 0 0 0 0 0 0 

10 0 0 1 3 3 3 

100 0 0 3 3 3 3 
Harlan Study Number: 41206435 Report Page 18 

Table 2 Cumulative Mortality Data in the Definitive Test 

Nominal Mean Cumulative Mortality (Initial Population = 7) 0/o 

Concentration Measured Mortality 

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

(mg/L) Hours Hours Hours Hours Hours Hours Hours 

Control - 0 0 0 0 0 0 0 

1.0 • 

- 0 0 0 0 0 0 0 

1.8 2.1 0 0 0 0 0 0 0 

3.2 3.5 0 0 0 1 1 1 14 

5.6 6.1 0 0 0 2 3 3 43 

10 11 0 0 1 6 6 6 86 

• No Mean Measured concentration determined as only the No Observed Effect Concentration (1.8 mg/L) and above 

were analyzed. 
Harlan Study Number: 41206435 Report Page 19 

FIGURES 

Figure 1 Concentration-Mortality Curve 

99.9 

99 

90 

70 

50 

30 

10 

0.1 

0.01 ~----~~--------------~------------------------~ 

1 10 100 

Concentration (mg!L) 
Harlan Study Number: 41206435 Report Page 20 

Figure 2 Toxicity Curve 

10 

10 100 

Lc50 Value (mg!L) 
Harlan Study Number: 41206435 Report Page 21 

APPENDICES 

Appendix 1 Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

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

NO. CONCENTRATION OR VALUE SAMPLES 

PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV 

UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

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

Aluminium ~g/1 38 <5.000 <6.684 12.000 200 0 0.00 

Ametryn ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Antimony ~g/1 8 0.180 0.531 1.200 5 0 0.00 

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

Atrazine ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Azinphos methyl ~g/1 8 <0.004 <0.004 <0.004 0.1 0 0.00 

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

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

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

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

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

Cadmium ~g/1 8 0.080 0.121 0.160 5 0 0.00 

Carbenazim ~g/1 8 <0.002 <0.002 <0.002 0.1 0 0.00 

Carbetamide ~g/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Carbophenophion ~g/1 8 <0.012 <0.012 <0.012 0.1 0 0.00 

Chlorfenvinphos ~g/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

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

Chlorpyriphos Ethyl ~g/1 8 <0.010 <0.010 <0.010 0.1 0 0.00 

Chlorpyriphos Methyl ~g/1 8 <0.015 <0.015 <0.015 0.1 0 0.00 

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

Colour mg/1 Pt/Co 38 <0.400 <0.608 2.600 20 0 0.00 

Copper mg/1 8 <0.003 <0.032 0.130 2 0 0.00 

Cyanazine ~g/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Cyanide ~g/1 40 <0.300 0.740 1.600 50 0 0.00 

Demeton-S-Methyl ~g/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Diazinon ~g/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

DichloNos ~g/1 8 <0.008 <0.008 <0.008 0.1 0 0.00 

Diflufenican ~g/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Dimethoate ~g/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Diuron ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

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

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

Ethofumesate ~g/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

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

Fenpropimorph ~g/1 8 <0.002 <0.002 <0.002 0.1 0 0.00 

Fluoride mg/1 8 0.270 0.331 0.414 1.5 0 0.00 

Flutriafol ~g/1 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Heptenophos ~g/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Iron ~g/1 76 <10.000 <26.579 488.000 200 1 1.32 

lsoproturon ~g/1 8 <0.002 <0.002 <0.002 0.1 0 0.00 

Lead ~g/1 8 <0.500 <2.288 10.000 25 0 0.00 

Linuron i-iQ/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

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

Manganese ~g/1 38 <1.800 <1.987 5.600 50 0 0.00 

Mercury ~g/1 40 <0.012 0.018 0.199 1 0 0.00 

Metaldehyde ~g/1 32 0.004 0.009 0.027 0.1 0 0.00 

Metamitron ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Metazachlor ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Methabenzthiazuron ~g/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

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

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

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

disinfectants). 
Harlan Study Number: 41206435 Report Page 22 

Typical Water Quality Characteristics 

WATER SUPPLY ZONE ZDB13 ALVASTON 

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

NO. CONCENTRATION OR VALUE SAMPLES 

PARAMETER ANALYSIS SAMPLES (ALL SAMPLES) PCV+ CONTRAVENING PCV 

UNITS TAKEN MINIMUM AVERAGE MAXIMUM NUMBER PERCENT 

Nickel IJg/1 8 2.300 3.288 5.800 20 0 0.00 

Nitrate mg/1 8 9.940 13.105 16.700 50 0 0.00 

Nitrite mg/1 8 <0.003 <0.005 <0.009 0.5 0 0.00 

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

Odour Dilution 42 0.000 0.024 1.000 -2 1 2.38 

Number 

PAH IJg/1 8 0.000 0.001 0.002 0.1 0 0.00 

Parathion IJg/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Pendimethalin IJg/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

pH pH value 76 7.020 7.497 7.870 Max 9.5, 0 0.00 

Min 6.5 

Phorate IJg/1 8 <0.009 <0.009 <0.009 0.1 0 0.00 

Phosalone IJg/1 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Pirimicarb IJg/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Pirimiphos-Methyl IJg/1 8 <0.009 <0.009 <0.009 0.1 0 0.00 

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

Propachlor IJg/1 8 <0.005 <0.005 <0.005 0.1 0 0.00 

Propazine IJg/1 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Propetamphos IJg/1 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Propiconazole IJg/1 8 <0.010 <0.010 <0.010 0.1 0 0.00 

Propyzamide IJg/1 8 <0.004 <0.004 <0.004 0.1 0 0.00 

Selenium IJg/1 8 <0.220 <0.546 0.740 10 0 0.00 

Simazine IJg/1 8 <0.007 <0.007 <0.007 0.1 0 0.00 

Sodium mg/1 8 14.000 25.250 30.000 200 0 0.00 

Sulphate mg/1 40 23.800 50.345 99.800 25 0 0.00 

Taste Dilution 42 0.000 0.000 0.000 2 0 0.00 

Number -

Terbutryn IJg/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Tetrachloroethene and IJg/1 8 0.000 0.000 0.000 10 0 0.00 

Trichloroethene 

Tetrachloromethane IJg/1 8 <0.018 <0.021 <0.030 3 0 0.00 

Total Pesticides IJg/1 32 0.000 0.009 0.027 0.5 0 0.00 

Total Trihalomethanes IJg/1 8 19.440 25.449 31.580 100 0 0.00 

Triadimefon IJg/1 8 <0.009 <0.009 <0.009 0.1 0 0.00 

Triallate IJg/1 7 <0.008 <0.014 <0.050 0.1 0 0.00 

Triazophos IJg/1 8 <0.003 <0.003 <0.003 0.1 0 0.00 

Trietazine IJg/1 8 <0.006 <0.006 <0.006 0.1 0 0.00 

Turbidity NTU 76 <0.030 <0.135 3.400 4 0 0.00 

Free Chlorine mg/1 204 0.010 0.207 0.630 -

3 - - Total Chlorine mg/1 204 0.040 0.270 0.710 -

3 - - Ammonium mg/1 38 <0.009 <0.012 0.049 0.5 0 0.00 

Chloride mg/1 40 9.660 21.052 50.800 250 0 0.00 

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

Coliform Bacteria No./100 ml 204 0.000 0.000 0.000 0 0 0.00 

Colony Count after 48 No./100 ml 76 0.000 0.789 31.000 -

4 - - hours at 37°C 

Colony Count after 72 No. /100 ml 76 0.000 2.039 85.000 -

4 - - hours at 22°C 

Conductivity iJS/cm at 20°C 38 315.000 419.000 486.000 2500 0 0.00 

Gross Alpha Activity Bq/1 8 0.032 0.069 0.090 0.1 5 

Gross Beta Activity Bq/1 8 <0.001 <0.036 0.053 15 

Total Organic Carbon mg/1 40 <0.130 0.920 2.020 -

4 - -

+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 2010. 
Harlan Study Number: 41206435 Report Page 23 

Appendix 2 Analytical Investigations 

Analytical Investigations 

CONTENTS 

CONTENTS .................................................................................................................................. 23 

1 MATERIALS AND METHODS ......................................................................................... 24 

1.1 Introduction ........................................................................................................... 24 

1.2 Test Item ................................................................................................................ 24 

1.3 Analytical Standard ............................................................................................... 24 

1.4 Analytical Procedure ............................................................................................. 24 

1.4.1 Storage ..................................................................................................... 24 

1.4.2 Reagents and Solvents ............................................................................. 24 

1.4.3 Preparation of Calibration Standards ....................................................... 25 

1.4.4 Preparation of Linearity Standards .......................................................... 25 

1.4.5 Preparation of Spiked Recovery Samples ............................................... 25 

1.4.6 Preparation of Test Samples .................................................................... 25 

1.4. 7 Instrumental Parameters .......................................................................... 26 

1.4.8 Calculations ............................................................................................. 27 

2 RESULTS ............................................................................................................................ 29 

2.1 Range-Finding Test ............................................................................................... 29 

2.2 Definitive Test ....................................................................................................... 29 

2.2.1 Validation of the Analytical Method ....................................................... 29 

2.2.2 Test Samples ............................................................................................ 29 

3 DISCUSSION ...................................................................................................................... 30 

TABLES ........................................................................................................................................ 31 

Table 1 Preparation of Test Samples ................................................................................. 31 

Table 2 Calibration Standard Data .................................................................................... 31 

Table 3 Results for Range-Finding Samples ..................................................................... 32 

Table 4 Results for Spiked Recovery Samples .................................................................. 32 

Table 5 Results for Test Samples ...................................................................................... 33 

FIGURES ...................................................................................................................................... 34 

Figure 1 

Figure 2 

Figure 3 

Figure 4 

Figure 5 

Calibration Plot ...................................................................................................... 34 

Chromatogram of Calibration Solution (1.5 mg/L) ............................................... 35 

Chromatogram of Biological Control Sample ....................................................... 35 

Chromatogram ofTest Sample (1.8 mg/L) ........................................................... 36 

Chromatogram of Test Sample (10 mg/L) ............................................................ 36 
Harlan Study Number: 41206435 Report Page24 

Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The test item concentration in the test samples was determined by direct injection into an 

electrospray mass spectrometer (MS) using an external standard. 

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

Shardlow, UK. 

1.2 Test Item 

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

1.3 Analytical Standard 

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

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The test samples were stored at approximately -20 °C prior to analysis. 

1.4.2 Reagents and Solvents 

Water: Fisher Scientific, HPLC-MS grade. 

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

Solvents: methanol, Romil, UG grade. 

Reagents: formic acid, Fisher Scientific, analytical reagent grade. 
Harlan Study Number: 41206435 Report Page 25 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to 

prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further 

diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined 

volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain 

calibration standards in the range of 0.10 to 1.0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 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 (50:50 v/v) methanol:test medium (100 mL) to 

prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further 

diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined 

volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain 

calibration standards in the range of 0.1 0 to 1. 0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 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 its recovery was assessed. The test item (nominal 100 mg) was initially 

dissolved in (50:50 v/v) methanol:test medium to prepare a stock solution with a concentration 

of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to 

produce a solution of 100 mg/L. A defined volume of this stock solution was diluted with test 

medium to obtain spiked recovery samples at a concentration of 1.0 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 analyzed. 

1.4.6 Preparation of Test Samples 

The test samples were thawed with the aid of sonication. They were initially diluted with 

methanol and then further diluted into the calibration range with (50:50 v/v) methanol:test 

medium as shown in Table 1. 
Harlan Study Number: 41206435 Report 

Analytical Investigations 

1.4. 7 Instrumental Parameters 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Flow rate: 

Injection volume: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

Data acquisition mode: 

SIM ion: 

Agilent 1200 Series 

Agilent 1200 Series 

Agilent 1200 Series MSD 

no column 

water:methanol:formic acid (500:500:0.5, v/v/v) 

0.5 mL!minute 

10 J!L 

ESI 

positive 

1000V 

350 oc 

40 psi 

100V 

10 Llmin 

Single Ion Monitoring (SIM) 

Page 26 
Harlan Study Number: 41206435 Report Page 27 

Analytical Investigations 

1.4.8 Calculations 

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

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

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

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

constants 

The concentration of the test item in the test samples was calculated using the following 

equation: 

c = X ·F 

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 1 00) 

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

equation: 

R =-c-·100 

cfort 

where R: 

c: 

(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] 
Harlan Study Number: 41206435 Report Page 28 

Analytical Investigations 

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] 
Harlan Study Number: 41206435 Report Page 29 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

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

validation was later conducted specifically over the concentration range to be used in the 

definitive test. The results for the range-finding test samples are summarized in Table 3. 

2.2 Definitive Test 

2.2.1 Validation of the Analytical Method 

Linearity 

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

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

0 to 1.49 mg/L. The R2 fit of the calibration curve to the data was 0.9993, 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 Quantitation (LOQ) 

The limit of quantitation (LOQ) was determined by calculating the concentration equivalent to 

ten times the standard deviation of five synthetic controls. Using this method the limit of 

quantitation was determined to be 0.28 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 5. 
Harlan Study Number: 41206435 Report Page 30 

Analytical Investigations 

3 DISCUSSION 

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

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

The method of analysis was validated and proven to be suitable for use. 
Harlan Study Number: 41206435 Report Page 31 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Sample Final Nominal 

Concentration of Volume Final Volume Preparation Concentration of Test 

Test Item Factor Item 

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

Control 5 10 2 -

1.8 5 10 2 0.90 

3.2 5 10, then 4 0.80 5~10 

5.6 5 10, then 6 0.93 5~15 

10 5 10, then 10 1.0 4~20 

Table 2 Calibration Standard Data 

Concentration of Test Item Area 

[mg/L] [counts] 

0 4.773 X 104 

0.0970 5.609 X 104 

0.298 7.883 X 104 

0.485 9.313 X 104 

0.695 1.125 X 105 

1.490 1.657 X 105 
Harlan Study Number: 41206435 Report Page 32 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined Concentration 

of Concentration Preparation · Concentration 

Test Item in of Test Item in Factor of Test Item in %of Nominal Time Point Range-Finding Sample Vial Range-Finding Concentration 

Sample Sample 

X F c 

Cnom 

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

0 1.0 0.497 2 0.994 99 

10 1.06 10 10.6 106 

24 1.0 0.501 2 1.02 102 

10 1.05 10 10.5 105 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Analytical 

Concentration Concentration Concentration Preparation Concentration Recovery Precision 

of of Test Item in of Test Item in Factor of Test Item in Rate (Relative 

Test Item the Spiked the Sample the Spiked Standard 

Sample Vial Sample Deviation of 

Recovery) 

Cnom Cfort X F c R 

• [mg/L] [mg/L] [mg/L] [mg/L] [%] [%] 

1.0 0.978 0.421 2 0.842 

0.439 2 0.878 

0.436 2 0.872 89 3.2 

0.432 2 0.864 

0.460 2 0.919 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41206435 Report 

Analytical Investigations 

Table 5 Results for Test Samples 

Nominal Measured Sample 

Concentration Concentration Preparation 

Time Point 

[hours] 

0 

(Fresh Media) 

24 

(Old Media) 

72 

(Fresh Media) 

96 

(Old Media) 

LOQ 

of of Test Item in 

Test Item in Sample Vial 

Test Sample 

Cnom X 

[mg/L] [mg/L] 

Control <LOQ 

1.8 0.935 

3.2 0.763 

5.6 0.882 

10 0.933 

Control <LOQ 

1.8 0.855 

3.2 0.723 

5.6 0.890 

10 0.943 

Control <LOQ 

1.8 1.21 

3.2 1.01 

5.6 1.14 

10 1.19 

Control <LOQ 

1.8 1.15 

3.2 1.00 

5.6 1.14 

10 1.19 

Limit of Quantitation 

not applicable 

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

Factor 

F 

2 

2 

4 

6 

10 

2 

2 

4 

6 

10 

2 

2 

4 

6 

10 

2 

2 

4 

6 

10 

Page 33 

Determined 

Concentration 

of Test Item in %of Nominal Test Sample Concentration 

c 

[mg/L] [%] 

<LOQ - 1.87 104 

3.05 95 

5.29 94 

9.33 93 

<LOQ - 1.71 95 

2.89 90 

5.34 95 

9.43 94 

<LOQ - 2.42 134 

4.05 127 

6.82 122 

11.9 119 

<LOQ -

2.29 127 

4.00 125 

6.84 122 

11.9 119 
Harlan Study Number: 41206435 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

:§' 180000 

·c: 

2- 160000 co 

~ 

<( 140000 

-"' co 

Q) 

c.. 120000 

100000 

80000 

60000 

40000 

20000 

0 

0.0 

Report 

y = -1.757E+04x2 + 1.057E+05x + 4.724E+04 

R2 = 9.993E-01 

0.5 1.0 1.5 

Concentration (mg/L) 

Page 34 
Harlan Study Number: 41206435 Report Page 35 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (1.5 mg/L) 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 

Figure 3 Chromatogram of Biological Control Sample* 

55000 

50000 

45000 

«1000 

35000 

30000 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 

• Background interference level oftbe response. Not test item. 
Harlan Study Number: 41206435 Report Page 36 

Analytical Investigations 

Figure 4 Chromatogram of Test Sample (1.8 mg/L) 

Figure 5 Chromatogram of Test Sample (10 mg/L) 

0.1 02 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 
Harlan Study Number: 41206435 Report Page 37 

Appendix 3 Physico-Chemical Measurements 

Nominal Time (Hours) 

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

(mg/L) pH mg % T°C pH mg % T°C pH mg % T°C 02/L ASV* 0 2/L ASV* 02/L ASV* 

Control 8.0 11.3 108 13 8.1 9.6 93 14 8.0 10.8 105 14 

1.0 7.8 10.9 106 14 8.2 9.4 91 14 8.0 10.8 105 14 

1.8 7.8 11.0 107 14 8.2 9.4 91 14 7.9 10.7 104 14 

3.2 7.8 11.1 108 14 8.2 9.6 93 14 7.9 10.5 100 13 

5.6 7.8 11.1 108 14 8.2- 9.3 90 14 7.9 10.5 102 14 

10 7.8 11.1 108 14 8.2 9.3 90 14 7.9 10.5 102 14 

Nominal Time (Hours) 

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

(mg/L) pH mg % T°C pH mg % T°C pH mg % T°C 02/L ASV* 02/L ASV* 02/L ASV* 

Control 8.1 9.7 94 14 8.0 10.7 104 14 8.2 9.9 96 14 

1.0 8.2 9.4 91 14 8.0 10.6 103 14 8.2 9.4 91 14 

1.8 8.2 9.3 90 14 8.0 10.5 102 14 8.2 9.5 92 14 

3.2 8.3 9.4 91 14 8.0 10.5 102 14 8.2 9.4 91 14 

5.6 8.2 9.4 91 14 7.9 10.4 101 14 8.3 9.2 89 14 

10 8.2 9.2 89 14 7.9 10.4 101 14 8.2 9.0 87 14 

Nominal Time (Hours) 

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

(mg/L) pH mg % T°C pH mg % T°C 0 2/L ASV* 0 2/L ASV* 

Control 8.0 10.7 104 14 8.2 9.9 96 14 

1.0 8.0 10.6 103 14 8.2 9.5 92 14 

1.8 8.0 10.5 102 14 8.2 9.5 92 14 

3.2 8.0 10.4 101 14 8.3 9.4 91 14 

5.6 7.9 10.4 101 14 8.3 9.2 89 14 

10 8.0 10.3 98 13 8.3 9.1 88 14 

* ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value 
Harlan Study Number: 41206435 Report 

Appendix 4 Monitoring Authority Statement of GLP Compliance 

Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Harlan Laboratories Ltd 

Shardlow Business Park 

London Road 

Shardlow 

Derby 

DE72 2GD 

DATE OF INSPECTION 

10 July 2012 

TEST TYPE(S) 

Analytical/Clinical 

Chemistry 

Environmental Toxicity 

Environmental Fate 

Mutagenicity 

Phys/Chem. Tests 

Toxicology 

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