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STUDY PLAN 

Sodium Glucoheptonate: 

Acute Toxicity to Rainbow Trout 

Study Director: S Harris 

Test Facility: Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Sponsor: Harcros Chemicals Inc. 

3452 Corporate Drive 

Dalton GA 30720 

UNITED STATES OF AMERICA 

Harlan Study Number: 41402585

 
Harlan Study Number: 41402585 Study Plan Page 2 

CONTENTS

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

SIGNATURES ................................................................................................................................ 3

GOOD LABORATORY PRACTICE ............................................................................................. 4

GENERAL INFORMATION ......................................................................................................... 5

Proposed Schedule ................................................................................................................. 5

Animal Welfare ...................................................................................................................... 5

Distribution List ..................................................................................................................... 5

Study Plan Amendments and Deviations ............................................................................... 5

Quality Assurance .................................................................................................................. 6

Reporting ............................................................................................................................... 6

Archiving ............................................................................................................................... 6

1 INTRODUCTION AND PURPOSE ..................................................................................... 7

1.1 Guidelines / Regulations .......................................................................................... 7

2 TEST AND REFERENCE ITEMS ....................................................................................... 8

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

2.2 Reference Item ......................................................................................................... 8

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

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

3.2 Test System .............................................................................................................. 8

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

3.4 Dosage ..................................................................................................................... 9

3.5 Materials .................................................................................................................. 9

3.6 Experimental Conditions ......................................................................................... 9

3.7 Study Design .......................................................................................................... 10

3.8 Evaluations ............................................................................................................ 11

3.8.1 Test organism observations ..................................................................... 11

3.8.2 Water quality criteria ............................................................................... 11

3.8.3 Analysis of Test Item Concentrations ..................................................... 12

3.9 Statistical Analysis ................................................................................................ 13

3.10 Validity Criteria ..................................................................................................... 13

4 REFERENCES .................................................................................................................... 14

APPENDICES ............................................................................................................................... 15
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SIGNATURES 

Study Director: S Harris 

 .................................................. 

 Date: 

Test Facility 

Management: C Mead 

 .................................................. 

 Date: 

For the Sponsor: Harcros Chemicals Inc. 

 

 ................................................. 

 Date: 
Harlan Study Number: 41402585 Study Plan Page 4 

GOOD LABORATORY PRACTICE 

The associated study will be 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 as revised 1997 (ENV/MC/CHEM(98)17); and are in accordance with, and 

implement, the requirements of Directives 2004/9/EC and 2004/10/EC. 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHLW, MAFF and METI). 
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GENERAL INFORMATION 

Proposed Schedule 

Experimental Starting Date: 31 August 2014 

Experimental Completion Date: 30 September 2014 

Draft Report Date: 31 October 2014 

Animal Welfare 

The study is designed and will be 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 Procedure) Act 1986 Amendment Regulations 2012. This study plan is 

subject to review and the conduct of the study may be included as part of the Harlan Laboratories 

Ltd, Shardlow, UK Ethical Review Process. 

The study will also be 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. 

Distribution List 

The original Study plan will be retained in the study file. Copies of the study plan along with any 

amendments will be distributed as follows: 

Sponsor: Harcros Chemicals Inc. 

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK 

Study Plan Amendments and Deviations 

Any intended change to the study plan will result in an amendment to study plan approved by the 

Study Director and also signed by test facility management and the Sponsor. Amendments will 

be distributed to all recipients of the study plan. 

Deviations (unplanned changes) from the study plan will be documented and acknowledged by 

the Study Director. 
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Quality Assurance 

Quality Assurance will audit and inspect study-related procedures and will report any audit and 

inspection results in writing to the Study Director and Test Facility Management. 

Reporting 

A GLP compliant report will be presented. A draft report will be sent to the Sponsor for review 

and comments before issue of the final report. 

Archiving 

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

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

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

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

No data will be discarded without contacting the Sponsor to obtain their written consent. 
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1 INTRODUCTION AND PURPOSE 

The purpose of this study is to assess the acute toxicity of the test item to freshwater fish. The 

toxicity will be expressed as the median lethal concentration (LC50). 

The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration 

(LOEC) will also be determined if applicable. 

The species used will be Rainbow Trout (Oncorhynchus mykiss) a freshwater species 

representative of a wide variety of natural habitats, and can therefore be considered as an 

important non-target organism in freshwater ecosystems. 

The dispersal of the test item in the surrounding medium is considered to represent the most 

probable route of exposure in the environment. 

1.1 Guidelines / Regulations 

The test methods described are designed to be compatible with the procedures indicated by the 

following internationally accepted guidelines and recommendations: 

 The OECD Guidelines for Testing of Chemicals (1992) No 203 "Fish, Acute Toxicity 

Test" 

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

 EPA Ecological Effects Test Guidelines OPPTS 850.1075 “Fish Acute Toxicity Test, 

Freshwater and Marine” 
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2 TEST AND REFERENCE ITEMS 

2.1 Test Item 

The test item will be supplied by or on behalf of the Sponsor including the following information 

if known; description, batch, purity, expiry / retest date, storage conditions and safety 

precautions to be taken. 

2.2 Reference Item 

In the interest of animal welfare, a positive control using a reference item will not be performed. 

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. 

NOEC (No Observed Effect 

Concentration) : 

The highest test concentration at which no significant toxic 

effect on the test organisms is observed within the exposure 

period. 

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

3.2 Test System 

The study will be performed using Rainbow Trout (Oncorhynchus mykiss) supplied by an 

accredited live fish supplier, details of which will be documented in the data. 

Juvenile fish < 3.0 g will be used and the longest should not be more than twice the length of the 

shortest. The fish will be maintained at a temperature of 12 ± 2.0 o

C. 

Following a 48-Hour settling in period, mortalities are recorded and the following rejection 

criteria applied. 

> 10% in 7 days: rejection of entire batch 

5-10% in 7 days: continue acclimatisation for an additional 7 days 
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< 5% in 7 days: acceptance of batch 

The fish will be acclimatised for a minimum period of 12 days at Harlan Laboratories Ltd, with 

at least 7 days acclimatisation prior to test start in water of the quality and temperature to be used 

in the test. 

The fish will be fed commercial fish food, details of which will be documented in the data, 

regularly on demand. Feeding will be discontinued 48 hours prior to testing. The diet and 

diluent water are considered not to contain any contaminant that would affect the integrity or 

outcome of the study. 

Animals will be maintained in glass or fibreglass tanks at a loading rate not exceeding 0.8 g body 

weight per liter in static systems. This loading rate may be exceeded in flow-through systems. 

Aeration will be supplied to provide  80% Air Saturation Value (ASV). 

3.3 Test Water 

The animals will be maintained in dechlorinated laboratory tap water (see Appendix 1). 

3.4 Dosage 

The test item will be dissolved in the test water on a daily basis (semi-static test). 

Details of the method of preparation will be documented in the data. 

3.5 Materials 

Glass test vessels containing an appropriate amount of test medium. The test vessels will be 

covered to reduce evaporation and to avoid entry of dust into the solutions. The size of the test 

vessels and the volume of test solution will be documented in the data. 

The test vessels will be labelled with the study number and all necessary information to ensure 

unique identification. 

3.6 Experimental Conditions 

The environmental parameters will be as used to maintain the stock animals. Auxiliary aeration 

will not normally be supplied to the test vessels, however for ethical reasons if the dissolved 

oxygen levels are measured to be less than 60% ASV then gentle aeration may be provided. 
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The test will be performed under temperature-controlled conditions at 12 ± 2.0 oC with a 

maximum deviation of ± 1 °C during the test. 

A 16-hour light to 8-hour dark cycle will be applied with a 20 – 30 minute transition period. 

3.7 Study Design 

Seven fish will be used at each test concentration and in the control. 

The loading rate will not be greater than 0.8 g bodyweight per liter of test medium. 

Animals will be selected at random from the total number collected. The animals will not be 

individually identified. The animals will not be fed during the test. 

The test will be conducted with daily medium renewal (semi-static test). The actual method of 

test media renewal will be documented in the data. 

The test will be started by addition of the test organisms to the test vessels within 30 minutes of 

the test media being added to the test vessels. 

At least five concentrations of the test item arranged in a geometric series will be tested. The 

spacing factor between the test concentrations will preferably not exceed 2.2. The actual test 

concentrations are assigned according to preliminary range-finding tests or available toxicity 

data and will be documented in the data prior to the start of the definitive test. If in the rangefinding test a flat concentration-effect relationship is determined and, thus, a large concentration 

range will have to be tested, a spacing factor exceeding 2.2 (e.g., 3.2) will be applied. 

The chosen test concentrations should permit the determination of the 96-Hour LC50 and, as far 

as possible, span the range of 0 to 100% mortality. Concentrations of the test item higher than 

1000 mg/L will usually not be tested. 

If no effect is observed in the preliminary range-finding test at 1000 mg/L, a Limit Test may be 

conducted using a single test vessel at this concentration together with a control vessel. 

The control group will be exposed to the test water alone. In the interests of animal welfare, data 

from the control group may be shared with similar concurrent studies. 

The pH of the test preparations will not be adjusted. 
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3.8 Evaluations 

3.8.1 Test organism observations 

The fish are inspected after 3, 6, 24, 48, 72 and 96 hours and at intermediate times when 

appropriate. Mortalities are recorded and any abnormalities in appearance or behaviour shown 

by surviving fish are summarised. The criteria of death are absence of respiratory movement and 

the lack of response to physical stimulation. Dead fish are removed when observed. 

During the test, the principles mentioned in the OECD Humane Endpoints Guidance Document 

No. 19 (OECD, 2002) will be taken into consideration. Test fish, which are found in moribund 

condition, will be humanely killed before test end due to reasons of animal welfare. For the 

calculation of the LC50 value the sacrificed fish will be taken into account as dead fish. 

The length and weight of a representative batch of fish will be determined e.g. fish from the 

stock tank prior to the start of the test or the control fish at the end of the test 

3.8.2 Water quality criteria 

Measurements of pH, dissolved oxygen and temperature in each surviving test vessel (prior to 

and post test media renewal) will be recorded daily. In addition the temperature will be recorded 

hourly in the control vessel using a data logger. 

The appearance of the test media will be recorded daily. 
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3.8.3 Analysis of Test Item Concentrations 

3.8.3.1 Sampling 

Samples of the test media will be taken at 0 hours (fresh media), 24, 48 and 72 hours (fresh and 

old media) and 96 hours (old media) of the definitive test for analysis. In the range-finding test, 

samples of the test media will be taken at 0 hours and after 24 or 96 hours. Where possible 

duplicate samples will be taken at each occasion, these samples may also be analyzed should the 

need arise. 

Samples will be taken from the control and each surviving test group or at the observation point 

when all fish are dead in a certain test concentration. 

Analysis of samples taken from the range-finding test is performed in order to determine the 

stability of the test item under test conditions over the exposure period. The test concentrations 

provided for analysis will be documented by the Study Director in the data. 

Final details of the samples to be taken and the sample size, etc, will be documented in the data. 

3.8.3.2 Storage and sample preparation 

All samples will be analyzed immediately after sampling or stored at approximately -20°C until 

analysis. 

If the test item has been shown to be unstable under the storage conditions, the samples will be 

analyzed immediately after sampling without prior storage or measures will be taken (e.g. pH 

adjustment or addition of organic solvents) in order to maintain the stability of the test item until 

analysis. 

3.8.3.3 Analysis of samples 

The test item formulations will be analyzed for concentration and stability by Harlan 

Laboratories Ltd, Shardlow, UK Analytical Services. 

The test item concentrations will be measured in at least one of the duplicate test medium 

samples. However, the samples from test concentrations below the 96-Hour NOEC will not 

normally be analyzed. The remaining samples may be analyzed, if necessary. 

The analytical method (eg HPLC, LC-MS, GC, AAS or photospectrometry) will either be 

provided by the Sponsor and adapted, if necessary, to the analytical conditions at Harlan 

Laboratories Ltd or developed by Harlan Laboratories Ltd. Adaptation or development of the 

analytical method will not be performed under GLP. Details about the analytical method used 

will be given in an Appendix of the final report. The accuracy of the analytical method will be 

verified by recoveries spanning the concentration range to be analyzed within this study. 

Analytical pre-experiments may be performed within a different Harlan Laboratories study. 
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3.9 Statistical Analysis 

The LC50 values and associated 95% confidence limits for all appropriate observation times will 

be calculated using an appropriate statistical method which will be documented in the data. All 

LC50 values will be rounded to two significant figures. 

The No Observed Effect Concentration (NOEC) is the highest test concentration at which no 

significant toxic effect on the test organisms is observed within the exposure period. 

The Lowest Observed Effect Concentration (LOEC) is the lowest test concentration at which a 

significant toxic effect on the test organisms is observed within the exposure period. If the test 

has been conducted as a limit test then it is not possible to determine a LOEC. 

3.10 Validity Criteria 

For the test to be valid, the following criteria should be fulfilled: 

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 ≥60% of Air Saturation 

Values (ASV) in the control and test vessels. 
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4 REFERENCES 

OECD (2002) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as 

Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Series on 

Testing and Assessment, No. 19. OECD Publishing.
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APPENDICES 

Appendix 1 Test Water 

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

Series 500) and a proportion of the incoming water softened (Elga Nimbus 1248D Duplex water 

softener) and then remixed with the main supply to give a water hardness of approximately 

140 mg/L as CaCO3 and pH 6.0 - 8.5. After dechlorination and softening the water will then be 

passed through a series of computer controlled heat exchangers to achieve the required 

temperature. When a "single pass" system is not possible, the water will be recirculated via a 

suitable purification filter. 

Chemical analysis of the water used in testing will include the following elements and 

limitations on maximum concentrations: 

Substance Maximum Concentration 

Particulate matter 20.0 mg/L 

Chemical Oxygen Demand (COD) 5.0 mg/L 

Total Organic Carbon (TOC) 2.0 mg/L 

Boron and fluoride <100.0 mg/L 

Residual chlorine 0.003 mg/L 

Un-ionized ammonia 0.020 mg/L 

Aluminum, arsenic, chromium, cobalt, copper, iron, lead, 

nickel, and zinc 

0.001 mg/L 

Cadmium, mercury, and silver <0.100 mg/L 

Total organophosphorus pesticides 0.050 mg/L 

Total organochlorine pesticides + PCBs or organic 

chlorine 

0.050 mg/L 

Specific conductivity <1.0 mohms 

Water hardness will be measured at the beginning of the test, daily chlorine analysis will be 

performed.