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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: 41402586Harlan 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 .................................................................................................................... ........... 15Harlan Study Number: 41402586 Study Plan Page 3



## SIGNATURES

Study Director: S Harris

.................................................. Date:

Test Facility Management: C Mead

.................................................. Date:

For the Sponsor: Harcros Chemicals Inc.

................................................. Date:Harlan Study Number: 41402586 Study Plan Page 4



## GOOD LABORATORY PRACTICE

The associated study will be performed in co mpliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (S I 1999/3106 as amended by SI 2004/0994)). These regulations are in accordance with GLP st andards published as OECD Principles on Good Laboratory Practice as revised 1997 (ENV/MC/C HEM(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 regul ations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI).Harlan Study Number: 41402586 Study Plan Page 5



## 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 st udy 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 pl an 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 reci pients of the study plan.

Deviations (unplanned changes) from the study plan will be documente d and acknowledged by the Study Director.



### Quality Assurance

Quality Assurance will audit and inspect study-rela ted 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 re port will be sent to the Sponsor for review and comments before issue of the final report.Harlan Study Number: 41402586 Study Plan Page 6



### Archiving

Unless instructed otherwise by the Sponsor, the st udy plan, all raw data (p aper 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 wi ll be chargeable to the Sponsor.

No data will be discarded without contacting the Sponsor to obtain their written consent.Harlan Study Number: 41402586 Study Plan Page 7



## 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 (EC 50 ) 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 invertebra te 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 surroundi ng medium is considered to represent the most probable route of exposure in the environment.



### 1.1 Guidelines / Regulations

The test methods described are de signed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 The OECD Guidelines for Testing of Ch emicals (2004) No 202 "Daphnia sp., Acute

Immobilisation Test"

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

 EPA Ecological Effects Te st Guidelines OPPTS 850.1010 “Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids”Harlan Study Number: 41402586 Study Plan Page 8



## 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 th e 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 ar e not able to swim within 15 seconds after gentle agitation of the test container are considered to be

immobile.

EC 50 : 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 concentrati on at which no significant toxic effect on the test organisms is observed within the exposure period.

Treatment: Comprises test item treatm ents 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 Sheffi eld/UK. The clone (defined by the supplier asHarlan Study Number: 41402586 Study Plan Page 9

clone 5) is successfully bred at Harlan Laborat ories Ltd under temperat ure and light conditions

identical to those of the test.

During the culturing period, the parental daphnids w ill be fed an algal suspension and a Tetramin suspension. Details will be documented in the cultu ring records. Culture conditions ensure that reproduction will be by parthenogenesis.

The animals will be single housed in 150 ml gla ss 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 preparati on will be documented in the data.



### 3.5 Materials

Glass test vessels containing at least 125 mL of te st medium will be used. The test vessels will be covered to reduce evaporation and to avoid entry of dust into the solu tions. The size of the test

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

The test vessels will be labell ed with the study number and all necessary information to ensure unique identification.



### 3.6 Experimental Conditions

The environmental parameters will be as us ed to maintain the stock organisms.Harlan Study Number: 41402586 Study Plan Page 10

The test will be performed in a temperature-c ontrolled room. The water temperature will be

maintained at 18 - 22°C with a maximu m deviation of ±1°C during the test.

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



### 3.7 Study Design

A total of 20 daphnids per treatmen t 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 th e test vessels at initi ation 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 adde d 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 ar ranged in a geometric seri es will be tested. The spacing factor between the test concentrations will preferably not exceed 2.2. The actual test concentrations are assigned according to prelim inary 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 teste d, a spacing factor exceeding 2.2 (e.g., 3.2) will be applied.

The chosen test concentrations should preferably c over the range causing 0 to 100% immobility. Concentrations of the test item higher th an 1000 mg/L will usually not be tested.

If no effect is observed in the preliminary ra nge-finding test at 1000 mg/L, a Limit Test may be conducted using only this concentrati on 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 c oncurrent studies.

The test will be carried out without adjustment of pH.Harlan Study Number: 41402586 Study Plan Page 11



### 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 leth al effects other than immobility) will also be r ecorded at 24 and 48 hours.

3.8.2 Water quality criteria

The temperature is recorded dail y and pH and dissolved oxygen m easurements are made for each

test vessel at the start and end of the test. In addition the temperat ure 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 in itiation and termination of both the range-finding and definitive tests for analysis. Where po ssible 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 ov er the exposure period. The test concentrations provided for analysis will be documen ted by the Study Director in the data.

For the purposes of the definitive test, the replicat e test and control vessels will be pooled to give one sample.

Final details of the samples to be taken and the sa mple size, etc, will be documented in the data.Harlan Study Number: 41402586 Study Plan Page 12

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 unstabl e under the storage conditions, the samples will be analyzed immediately after sampling without prio r storage or measures will be taken (e.g. pH adjustment or addition of organic solvents) in order to maintain th e stability of the test item until analysis.

3.8.3.3 Analysis of samples

The test item formulations will be analyzed for concentr ation 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 co ncentrations 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 necessa ry, 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 GL P. 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 EC 50 values and associated 95% confidence limits will be calculated using an appropriate statistical method which will be documented in the data. All EC 50 values will be rounded to two signi ficant figures.

The No Observed Effect Concentration (NOEC) is the highest test concentration at which no significant toxic effect on the test organism s 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.Harlan Study Number: 41402586 Study Plan Page 13



### 3.10 Validity Criteria

For the test to be valid, the followi ng criteria should be fulfilled:

In the control, not more than 10% of the daphni ds should show immobilization or other signs of disease or stress (e.g., discolorati on 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.Harlan Study Number: 41402586 Study Plan Page 14



## 4 REFERENCES

INTERNATIONAL STANDARDS OR GANISATION (ISO) (1996) Standard 6341: Water quality - Determination of the inhibition of th e mobility of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test . Geneva: International Standards Institute.Harlan Study Number: 41402586 Study Plan Page 15



## APPENDICES

Appendix 1 Reconstituted Water – Elendt M7 Medium

Elendt M7 medium will be prepared by disso lving the following analyt ical grade salts and additives in deionised or purified wa ter at the following concentrations:

Main Compounds mg/L Trace Elements 1

μg/L Vitamins μg/L

CaCl 2 .2H 2 O 294 B H 3 BO 3 125 Thiamine HCl 75 MgSO 4 .7H 2 O 123 Fe FeSO 4 .7H 2 O 2 50 Cyanocobalamine (B 12 ) 1.0

KCl 5.8 Mn MnCl 2 .4H 2 O 25 Biotin (B 6 ) 0.75

NaHCO 3 65 Li LiCl 12.5

Na 2 SiO 3 .9H 2 O 10 Rb RbCl 12.5

NaNO 3 0.27 Sr SrCl 2 .6H 2 O 12.5

KH 2 PO 4 0.14 Br NaBr 3.1

K 2 HPO 4 0.18 Mo Na 2 MoO 4 .2H 2 O 6.3

Cu CuCl 2 .2H 2 O 1.6

Zn ZnCl 2 6.3

Co CoCl 2 .6H 2 O 2.5

I KI 2.5

Se Na 2 SeO 3 1.0

V NH 4 VO 3 0.3

EDTA Na 2 EDTA.2H 2 O 625

Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO 3 ) Alkalinity: 0.9 mmol/L

The pH of the prepared media will be in th e range of 7.9 ± 0.3 and will be stored at approximately 21 oC. If n ecessary the pH may be adju sted 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 FeSO 4 .7H 2 O and Na 2 EDTA.2H 2 O are combined and autoclaved immediatelyHarlan Study Number: 41402586 Study Plan Page 16

Appendix 2 Reconstituted Water – Test Water

Prior to the start of the study, th e 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 th e range of 7.6 - 8.0. If necessary the pH may be adjusted with NaOH or HCl. The pH will norma lly 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 pur ified water with a conductivity of < 5μS cm-1 to obtain the following nominal concentrations:

Ingredients / Properties Concentration mmol/L mg/L

CaCl 2 .2H 2 O 2.0 294 MgSO 4 .7H 2 O 0.5 123

NaHCO 3 0.75 65

KCl 0.075 5.8

Water Hardness 2.5 250 as CaCO 3 Alkalinity 0.8 -

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

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

hardness.