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STUDY PLAN 

Sodium Glucoheptonate: 

Daphnia sp., 48-Hour Acute Immobilization Test 

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: 41402586

 
Harlan Study Number: 41402586 Study Plan Page 2 

CONTENTS

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

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

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

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

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

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

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

Quality Assurance .................................................................................................................. 5

Reporting ............................................................................................................................... 5

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 ..................................................... 11

3.9 Statistical Analysis ................................................................................................ 12

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: 
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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 

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. 

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. 
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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 the freshwater 

invertebrate Daphnia magna. The toxicity will be expressed as the median effect concentration 

(EC50) for immobilization. 

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

(LOEC) will also be determined if applicable. 

Daphnia magna is a freshwater invertebrate 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 (2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

 Method C.2 of Commission Regulation (EC) No. 440/2008 

 EPA Ecological Effects Test Guidelines OPPTS 850.1010 “Aquatic Invertebrate Acute 

Toxicity Test, Freshwater Daphnids” 
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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 

For evaluation of the quality of the Daphnia magna clone and the experimental conditions, 

potassium dichromate will be tested as a positive control twice a year to demonstrate satisfactory 

conditions of the test. 

The reference item will be supplied by a commercial supplier and will be stored as specified by 

the supplier. 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Immobilization: Those organisms which are not able to swim within 15 seconds 

after gentle agitation of the test container are considered to be 

immobile. 

EC50: The calculated concentration of test item which results in 50% 

immobilization 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 with young daphnids of the species Daphnia magna Straus, which 

is the preferred test species recommended by the test guideline. A clone of this species was 

originally supplied by the University of Sheffield/UK. The clone (defined by the supplier as 
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clone 5) is successfully bred at Harlan Laboratories Ltd under temperature and light conditions 

identical to those of the test. 

During the culturing period, the parental daphnids will be fed an algal suspension and a Tetramin 

suspension. Details will be documented in the culturing records. Culture conditions ensure that 

reproduction will be by parthenogenesis. 

The animals will be single housed in 150 ml glass beakers containing Elendt M7 medium (see 

Appendix 1). 

To obtain young daphnids for the test which are equal to or less than 24 hours old, any young 

daphnids will be removed from the culture one day prior to initiation of the test such that the 

young daphnids produced overnight are at the required age. 

The daphnids will not be fed during the test. 

3.3 Test Water 

The test water parameters are defined in Appendix 2. The parental daphnids are cultivated in 

water of the same quality (in respect to pH, main ions, and total hardness). 

3.4 Dosage 

The test item will be dissolved or dispersed in the test water once at the start of the test (static 

test). 

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

3.5 Materials 

Glass test vessels containing at least 125 mL of test medium will be used. 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 organisms. 
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The test will be performed in a temperature-controlled room. The water temperature will be 

maintained at 18 - 22°C 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 

A total of 20 daphnids per treatment will be used and divided into four replicates. The following 

conditions will be met: 

Total volume per replicate ≥125 mL 

Volume / daphnid ≥25 mL 

Loading rate / L ≤40 daphnids 

The daphnids will be randomly distributed to the test vessels at initiation of the test. 

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. 

The duration of the test will be 48 hours. 

The test will be conducted with no medium renewal (static test). 

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 preferably cover the range causing 0 to 100% immobility. 

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 only this concentration and the corresponding control. 

The control group will be exposed to the test water alone. Data from the control group may be 

shared with similar concurrent studies. 

The test will be carried out without adjustment of pH. 
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3.8 Evaluations 

3.8.1 Test organism observations 

The number of immobilised Daphnia in each vessel will be recorded after 24 and 48 hours. 

Those organisms not able to swim within 15 seconds after gentle agitation of the test beaker will 

be considered to be immobile. 

Any abnormal behaviour or abnormal appearance of the daphnids (sub lethal effects other than 

immobility) will also be recorded at 24 and 48 hours. 

3.8.2 Water quality criteria 

The temperature is recorded daily and pH and dissolved oxygen measurements are made for each 

test vessel at the start and end of the test. In addition the temperature will be recorded hourly in 

one of the control vessels using a data logger. 

The appearance of the test media will be recorded daily. 

3.8.3 Analysis of Test Item Concentrations 

3.8.3.1 Sampling 

Samples of the test media will be taken at initiation and termination of both the range-finding 

and definitive tests for analysis. Where possible duplicate samples will be taken at each 

occasion, these samples may also be analyzed should the need arise. 

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. 

For the purposes of the definitive test, the replicate test and control vessels will be pooled to give 

one sample. 

Final details of the samples to be taken and the sample size, etc, will be documented in the data. 
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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 48-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. 

3.9 Statistical Analysis 

The 24 and 48-Hour EC50 values and associated 95% confidence limits will be calculated using 

an appropriate statistical method which will be documented in the data. All EC50 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. 
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3.10 Validity Criteria 

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

In the control, not more than 10% of the daphnids should show immobilization or other signs of 

disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). 

The dissolved oxygen concentration at the end of the test should be ≥3 mg/L in the control and 

test vessels. 
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4 REFERENCES 

INTERNATIONAL STANDARDS ORGANISATION (ISO) (1996) Standard 6341: Water 

quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, 

Crustacea) - Acute toxicity test. Geneva: International Standards Institute. 
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APPENDICES 

Appendix 1 Reconstituted Water – Elendt M7 Medium 

Elendt M7 medium will be prepared by dissolving the following analytical grade salts and 

additives in deionised or purified water at the following concentrations: 

Main Compounds 

mg/L 

Trace Elements1

μg/L 

Vitamins 

μg/L 

CaCl2.2H2O 294 B H3BO3 125 Thiamine HCl 75 

MgSO4.7H2O 123 Fe FeSO4.7H2O2 50 Cyanocobalamine (B12) 1.0 

KCl 5.8 Mn MnCl2.4H2O 25 Biotin (B6) 0.75 

NaHCO3 65 Li LiCl 12.5 

Na2SiO3.9H2O 10 Rb RbCl 12.5 

NaNO3 0.27 Sr SrCl2.6H2O 12.5 

KH2PO4 0.14 Br NaBr 3.1 

K2HPO4 0.18 Mo Na2MoO4.2H2O 6.3 

 Cu CuCl2.2H2O 1.6 

 Zn ZnCl2 6.3 

 Co CoCl2.6H2O 2.5 

 I KI 2.5 

 Se Na2SeO3 1.0 

 V NH4VO3 0.3 

 EDTA Na2EDTA.2H2O 625 

Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3) 

Alkalinity: 0.9 mmol/L 

The pH of the prepared media will be in the range of 7.9 ± 0.3 and will be stored at 

approximately 21 oC. If necessary the pH may be adjusted with NaOH or HCl. 

 

1

 Concentrations of the target elements in bold; the source compounds are given in the next column 2

 Stock solutions of FeSO4.7H2O and Na2EDTA.2H2O are combined and autoclaved immediately 
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Appendix 2 Reconstituted Water – Test Water 

Prior to the start of the study, the test water (specifications see below) will be aerated until 

oxygen saturation is reached. Aeration will not be performed during the experiment. The pH of 

the test water after equilibration will be in the range of 7.6 - 8.0. If necessary the pH may be 

adjusted with NaOH or HCl. The pH will normally not vary by more than 1.5 during the test. 

The ratio of Ca:Mg and Na:K will be 4:1 and 10:1, respectively, based on molarity. 

Reconstituted test water according to ISO 6341 (ISO, 1996) will be used in the study. It will 

consist of analytical grade salts dissolved in purified water with a conductivity of < 5μS cm-1 to 

obtain the following nominal concentrations: 

Ingredients / 

Properties 

Concentration 

mmol/L mg/L 

CaCl2.2H2O 2.0 294 

MgSO4.7H2O 0.5 123 

NaHCO3 0.75 65 

KCl 0.075 5.8 

Water Hardness 2.5 250 as CaCO3

Alkalinity 0.8 - 

In order to comply with the water characteristics of the OPPTS guideline, the reconstituted water 

will be diluted 50:50 with deionized reverse osmosis water prior to use to reduce the total water 

hardness.