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## STUDY PLAN

Sodium Glucoheptonate CAS No. 31138-65-5: 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 .......................................

................................................... .................... 9

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

................................................... ..................... 9

3.4 Dosage ............................................

................................................... ...................... 9

3.5 Materials .........................................

................................................... .................... 10

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

................................................... ......... 10

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 complianc e 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 standa rds 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 Laborator y Practice regulations specified by regulatory authorities throughout the European Comm unity, 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: 30 September 2014

Experimental Completion Date: 31 October 2014

Draft Report Date: 30 November 2014



### Distribution List

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

amendments will be distributed as follows:

Sponsor: Harcros Chemicals Inc.

Quality Assurance: Harlan Laboratories Ltd, Shardlo w, UK



### Study Plan Amendments and Deviations

Any intended change to the study plan will result i n an amendment to study plan approved by the Study Director and also signed by test facility man agement 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-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 r eport 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 stu dy 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 Sp onsor 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 to xicity of the test item to the freshwater

invertebrate Daphnia magna . The toxicity will be expressed as the median effe ct 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 invertebrate representative of a w ide 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 m edium 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 compa tible with the procedures indicated by the following internationally accepted guidelines and r ecommendations:

• The OECD Guidelines for Testing of Chemicals (2004) No 202 "Daphnia sp., Acute

Immobilisation Test"

• Method C.2 of Commission Regulation (EC) No. 440/20 08

• EPA Ecological Effects Test Guidelines OPPTS 850.10 10 “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 t he Sponsor including the following information if known; description, batch, purity, expiry / rete st date, storage conditions and safety precautions to be taken.

Information as provided by the Sponsor. A Certific ate of Analysis supplied by the Sponsor is given in Appendix 1.

Identification: Sodium Glucoheptonate CAS No. 31138 -65-5
Chemical Name: D-gluco-heptanoic acid, monosodium s alt

CAS Number: 31138-65-5

Purity: 39.94% active ingredient (25.78% beta sodium glucoheptonate + 14.16% alpha sodium glucoheptonate)



### 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 c ontrol 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 co nsidered to be

immobile.

EC 50 : The calculated concentration of test item which r esults in 50% immobilization within the exposure period.

LOEC (Lowest Observed Effect Concentration): The lowest test concentration at which a significan t toxic effect on the test organisms is observed within the exposu re period.

NOEC (No Observed Effect The highest test concentra tion at which no significant toxicHarlan Study Number: 41402586 Study Plan Page 9

Concentration): effect on the test organisms is obs erved within the exposure period.

Treatment: Comprises test item treatments and contr ol (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 te st guideline. A clone of this species was originally supplied by the University of Sheffield/ UK. The clone (defined by the supplier as clone 5) is successfully bred at Harlan Laboratorie s 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 cultu ring records. Culture conditions ensure that reproduction will be by parthenogenesis.

The animals will be single housed in 150 ml glass b eakers containing Elendt M7 medium (see Appendix 2).

To obtain young daphnids for the test which are equ al to or less than 24 hours old, any young daphnids will be removed from the culture one day p rior to initiation of the test such that the young daphnids produced overnight are at the requir ed age.

The daphnids will not be fed during the test.



### 3.3 Test Water

The test water parameters are defined in Appendix 3 . The parental daphnids are cultivated in water of the same quality (in respect to pH, main i ons, 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). The test item contains 39.94% active ingredi ent (ai), all test concentrations will be

corrected for this ai content and the test concentr ations will be expressed in terms of mg ai/L.

Details of the method of preparation including any manipulation of the test item prior to test solution preparation (e.g. boiling to remove crysta ls) will be documented in the data and study

report.Harlan Study Number: 41402586 Study Plan Page 10



### 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 solutions. The size of the test

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

The test vessels will be labelled with the study nu mber and all necessary information to ensure unique identification.



### 3.6 Experimental Conditions

The environmental parameters will be as used to mai ntain the stock organisms.

The test will be performed in a temperature-control led 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 applie d with a 20 – 30 minute transition period.



### 3.7 Study Design

A total of 20 daphnids per treatment will be used a nd 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 te st vessels at initiation of the test.

The test will be started by addition of the test or ganisms 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 arran ged 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 preliminar y range-finding tests or available toxicity data and will be documented in the data prior to th e start of the definitive test. If in the rangefinding test a flat concentration-effect relationsh ip is determined and, thus, a large concentration range will have to be tested, a spacing factor exce eding 2.2 (e.g., 3.2) will be applied.Harlan Study Number: 41402586 Study Plan Page 11

The chosen test concentrations should preferably co ver the range causing 0 to 100% immobility. Concentrations of the test item higher than 100 mg ai/L will usually not be tested.

If no effect is observed in the preliminary range-f inding test at 100 mg ai/L, a Limit Test may be conducted using only this concentration and the cor responding 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.



### 3.8 Evaluations

3.8.1 Test organism observations

The number of immobilised Daphnia in each vessel will be recorded after 24 and 48 hou rs. 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 th e daphnids (sub lethal effects other than immobility) will also be recorded at 24 and 48 hour s.

3.8.2 Water quality criteria

The temperature is recorded daily and pH and dissol ved oxygen measurements at the start and end of the test are made for each replicate vessel. In addition the temperature will be recorded hourly in one of the control vessels using a data l ogger.

The appearance of the test media will be recorded d aily.

3.8.3 Analysis of Test Item Concentrations

3.8.3.1 Sampling

Samples of the test media will be taken at initiati on 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 te st 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 documented by the Stu dy Director in the data.Harlan Study Number: 41402586 Study Plan Page 12

For the purposes of the definitive test, the replic ate test and control vessels will be pooled to give one sample. Details of the pooling of replicate ve ssels (i.e. Replicates R 1 – R 4 pooled) will be given in the study report.

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

3.8.3.2 Storage and sample preparation

All samples will be analyzed immediately after samp ling or stored at approximately -20°C until analysis.

If the test item has been shown to be unstable unde r the storage conditions, the samples will be analyzed immediately after sampling without prior s torage or measures will be taken (e.g. pH adjustment or addition of organic solvents) in orde r 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 con centration 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 concentrati ons 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 p hotospectrometry) will either be provided by the Sponsor and adapted, if necessary, to the analytical conditions at Harlan Laboratories Ltd or developed by Harlan Laboratorie s 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. T he accuracy of the analytical method will be verified by recoveries spanning the concentration r ange to be analyzed within this study.

Analytical pre-experiments may be performed within a different Harlan Laboratories study, a summary of which will be provided to the Sponsor pr ior to test initiation.



### 3.9 Statistical Analysis

The 24 and 48-Hour EC 50 values and associated 95% confidence limits will b e calculated using an appropriate statistical method which will be doc umented in the data. All EC 50 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 o bserved within the exposure period.Harlan Study Number: 41402586 Study Plan Page 13

The Lowest Observed Effect Concentration (LOEC) is the lowest test concentration at which a significant toxic effect on the test organisms is o bserved within the exposure period. If the test has been conducted as a limit test then it is not p ossible to determine a LOEC.



### 3.10 Validity Criteria

For the test to be valid, the following criteria sh ould be fulfilled:

In the control, not more than 10% of the daphnids s hould show immobilization or other signs of disease or stress (e.g., discoloration or unusual b ehavior such as trapping at the surface water).

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

test vessels.Harlan Study Number: 41402586 Study Plan Page 14



## 4 REFERENCES

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



## APPENDICES

Appendix 1 Certificate of AnalysisHarlan Study Number: 41402586 Study Plan Page 16

Appendix 2 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 fol lowing 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 the range o f 7.9 ± 0.3 and will be stored at approximately 21 oC. If necessary the pH may be ad justed 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 17

Appendix 3 Reconstituted Water – Test Water

Prior to the start of the study, the test water (sp ecifications 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 r ange 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, r espectively, 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 puri fied 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 characteristics o f 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.