### c' ""H 1,...., 1 •.. Li 1 1 

### t.LJ 

### r•t ,,...... 0 r r' 

### I**(',:~~** 1'(.,**~**•• J ri.•**~·** i '.,/ 

### GREENWICH CHEMICAL CONSULTING,

## IJ~fltM>;:~ 16

## I~HIQ: 21 3300 PGA BOULEVARD • SUITE 

### 635

PALM BEACH GARDENS, 

### FL 33410 TELEPHONE 561.578.8110 FACSIMILE 561.578.8120 CBIC Control Number 

# 359537 

# COPY
April 15, 2014

)By CentralDataExchange

OPPT Document Control Office U.S. EnvironmentalProtection Agency EPA EastBuilding RoomE6428 1201 Constitution Ave., NW Washington, DC 20004 Attn.: TSCA Section 4 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

Re: Study Plan Submission and Request for Modification of Test Standards for Testing ConductedbyUnivarUSA underthe TSCASection4 TestRule onthe Testing ofCertain High Production Volume Chemicals: Second Group of Chemicals D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt CAS RN 6381-77-7 (Sodium erythorbate) DocketIdentification No. EPA-HQ-OPPT-2007-0531; 40 CFR799.5087

DearSir/Madam: On behalf of its client, Univar USA Inc. (Univar or the Company), Greenwich Chemical Consulting, Inc. (Greenwich Chemical Consulting) submits to the U.S. Environmental ProtectionAgency (EPA ortheAgency) studyplans and a request for the modification oftest standards conducted on the chemical substance, D-erythro-hex-2-enonic acid, .gamma.- 

# v ""-/**\~**U.S. Environmental Protection Agency April15, 2014 Page 2 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

lactone, monosodium salt [CAS RN 6381-77-7] which is currently subject to a Toxic Substances Control Act (TSCA) Section 4 test rule. 1 Univar ftles this request in accordance with this testrule and the regulations set forth at 40 CFR 790.50 and 790.55. I. General Background As a matter of background, this test rule addresses 19 high production volume (HPV) chemicals that went unsponsored under the Agency's voluntary HPV Chemical Challenge Program. One of the 19 chemicals identified in the rule is D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt which is identified herein as sodium erythorbate. The rule requires manufacturers andimporters ofthese HPVchemicals, including sodium erythorbate, to conduct testing to obtain screening level data for human health effects, environmental effects, chemical fate, andphysical/chemicalproperties. The Agency has issued this test rule under its TSCA Section 4(a)(1)(B) authority based on its determination that HPV compounds, including sodium erythorbate, are "produced in substantialquantities; there is ormay be substantialhuman exposure to them; existing data are insufficient to determine or predict their health and environmental effects; and testing is necessary to develop such data."2 The final rule identifies the following studies and test protocols on sodium erythorbate:
• n-Octanol/WaterPartition Coefficient [40 CFR 799.6755, ASTM E 1147-92, or 40 CFR 799.6756] • Ready Biodegradation3 Aquatic Toxicity Testing4: Acute toxicity to fish [ASTM E 729-96] Acute toxicity to Daphnia [ASTM E 729-96] Toxicity to plants (algae) [ASTM E 1218-04et] Because Univar had notyet begun importing sodium erythorbate with the intent to sell it for TSCA purposes at the time ofthe rule's effective date, the specific dates, as set forth in the fmal rule, for the submission oftheletter ofintentandtestreports were notapplicable. Univar subsequently submitted its letter ofintent to test onJanuary 27, 2014.5 Based on this, EPA stated in its response letter dated February 11, 2014 that study plans would be due April15, 2014 and fmal reports wouldbe due February 11, 2015. 6

1January 7, 2011; 76 Fed. Reg. 1067. 2 Id. at 1069. 3 The test rule identifies six ISO protocols and one ASTM standard for biodegradation testing. The protocol selection depends on the test substance's physical and chemical properties. 4 Because the n-octanol/water partition coefficient is expected to be significantly less than 4.2 based on the compound's high water solubility, Test Group 1is expected to apply. s Upon EPA's receipt ofits January 27, 2014 letter ofintent to test, Univar planned to begin importing this substance for bothTSCA purposes and for continuing exempt FDA purposes. 6 It is noted that since the time this test rule was issued through today, no other companies have come forward indicating their intent to test sodj.um erythorbate under this rule.U.S. Environmental Protection Agency April15, 2014 Page 3

II. Study Plan Submission CONTAINSNO CONFIDENTIAL BUSINESS INFORMATION

As required under 40 CPR 790.50 and 799.5087, the Company is required to submit to the Agency study plans prior to the initiation oftesting. Univar has selected Harlan Laboratories Ltd. (Harlan Laboratories) to conduct all of these tests. The laboratory will conduct these studies at a single location. Also, Greenwich Chemical Consulting is serving as the study monitor for this testing. The study plans included herein identify administrative information, the TSCA Section 4 test rule, the sodium erythorbate test material, primary laboratory personnel, study objectives, test guidelines, materials and methods, observance of Good Laboratory Practice standards, and otherrelated testing and procedural information. III. Request for Modification ofTest Standards for the Use ofOECD Test Guidelines All of the studies require the use of an ASTM, CPR, or ISO standard. Because of the validation. widespreaduse. and broad acceptance ofOrganization for Economic Cooperation and Development (OECD) test guidelines, as well as the near equivalency amongthe OECD. ASTM, CPR, and ISO protocols. Univar requests that EPA permit Univar's lab. Harlan Laboratories, to perform all five studies under the corresponding OECD guidelines. Specifically, we request that the testing be performed under the OECD test guidelines listed below. n-Octanol/WaterPartition Coefficient [OECD 107f • Ready Biodegradation [OECD 301B] 8 • Aquatic ToxicityTesting: Acute toxicity to fish [OECD 20W Acute toxicity to 

### Daphnia [OECD 202r0 Toxicity to plants (algae) [OECD 201]11 One significant benefit in permitting Harlan Laboratories to utilize the OECD protocols for this testing is that it will allow for more straightforward and expedient development of the data. Because it has significant experience and familiarity with the OECD test guidelines, Harlan Laboratories can begin testing immediately. Alternatively, the required use of the ASTM standards would delay the development ofthe test data. 12

7 Harmonizedwith EPA's guidelines under OCSPP 830.7550. 8 Harmonized with EPA's guidelines under OCSPP 835.3110. 9 Harmonized with EPA's guidelines under OCSPP 850.1075. IO Harmonized with EPA's guidelines under OCSPP 850.1010. II Harmonized with EPA's guidelines under OCSPP 850.5400. 12Thelabs contactedby Univar for testingunderanotherTSCA Section 4 testing obligation advised thatbecause ofthe additional time involved, the costs ofperforming tests under the ASTM standards is expected to be 20% to 30% higher.U.S. Environmental Protection Agency April15, 2014 Page 4 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

In addition, Harlan Laboratories was not familiar with the ASTM standards that EPA identifies in this and otherrecentTSCA Section 4 HPV rulemakings. Indeed, the lab was not familiar with orhad experience with theASTM standards andwas surprised tolearn thatEPA was requiring their use in lieu of EPA's own Harmonized Test Guidelines or OECD guidelines. 13 Also, the use offamiliar OECD protocols decreases the chances for errors due toinadvertent deviations from the guidelines. Presumably, the Agency intends to eventually submit these data as part ofdossiers under the international OECD HPV SIDS Program. The development ofdata on sodium erythorbate, under the internationally adopted and accepted OECD guidelines, would facilitate the Agency's efforts atthe OECD level. Itis also notedthat

### all ofthe aboveASTM standardsinternallyreference otherASTM standards which eithermust or should be observed. This adds to the complexity and duration oftesting. Forexample,inthe algae study alone,ASTM E 1218-04e1 references 11 otherASTM standards. Similarly, inthe combined fish andDaphnia standardunderASTM E 729-96, nine otherASTM standards are cited. Conversely, itis notable thatnone oftheASTM standards reference either the OECD guidelines or EPA's HarmonizedTest Guidelines. Eventhoughmostofthe discussion abovepertains totheASTM standards,Univaris formally requesting that EPA also approve the use ofequivalent OECD test guidelines for these tests for which EPA has citedASTM, CPR, or ISO guidelines. 

## * * *
Univar thanks the Agency for its consideration in this matter. For all questions, comments, and determinations concerning this matter, please use the contactinformation given below. Mr.JohnV. Massingale Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 Telephone: (561) 578-8110 Facsimile: (561) 578-8120 E-mail: john@grcci.com

13 See EPA's HarmonizedTest Guidelines at:**http://~'W.epa.gov/ocspp/pubs/frs/home/guidelin.htm.**U.S. Environmental Protection Agency April15, 2014 Page 5 Sincerely,
JohnV. Massingale Principal

cc: Ms. K. Gilje- Univar Ms. S. Lilley

- Univar Mr.

## J. Schaeffer

- EPA CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

# c

STUDY PLAN D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Determination ofPartition Coefficient (n-Octanol/Water)

Study Director: Test Facility:

Sponsor:

StudyMonitor:

Harlan StudyNumber: J A Walker Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE72 2GD 

### UK UnivarUSA Inc. 17425 NE Union Hill Road Redmond WA 98052 UNITED STATES OF AMERICA Greenwich Chemical Consulting, Inc. 3300 PGABoulevard, Suite 635 Palm Beach Gardens FL 33410 UNITED STATES OF AMERICA 41401172

Page 1 of 10Harlan Study Number: 41401172 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 INTRODUCTION AND PURPOSE
.....................................................................................7 1.1 Guidelines I Regulations
..........................................................................................7 2 TEST AND REFERENCE ITEMS
....................................................................................... 8 2.1 Test Item
.................................................................................................................. 8 2.2 Reference Item
......................................................................................................... 8 3 TEST
...................................................................................................................................... 9 3.1 Partition Coefficient (n-OctanoliWater)
.................................................................. 9 3.1.1 Test Specific Guidelines I Regulations
......................................................9 3.1.2 Test System
...............................................................................................9 3.1.3 PrincipleoftheTest.
..................................................................................9 3.1.4 Performance ofthe Test.
............................................................................9 3.1.5 Calculation
............................................................................................... 10Harlan StudyNumber: 41401172


## SIGNATURES
Study Director:

Test Facility Management:

Forthe Sponsor: Study Plan Page 3

J A Walker

Date:

Date:

John V. Massingdale Greenwich Chemical Consulting, Inc.

Date:Harlan Study Number: 41401172 Study Plan Page 4


## GOOD LABORATORYPRACTICE This 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 ofDirectives 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).Harlan StudyNumber: 41401172


## GENERAL INFORMATION Proposed Schedule Experimental StartingDate: Experimental Completion Date: Draft Report Date: 

### Distribution List Study Plan

01 May 2014 17 July 2014 31 July 2014 Page 5

The original study plan will be retained in the study file. Copies ofthe study plan along with any amendments will be distributed as follows: Study Monitor: John V. Massingdale Greenwich Chemical Consulting, Inc. Quality Assurance: Harlan Laboratories Ltd. 

### 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 Study Monitor. Amendments will be distributed to all recipients ofthe 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 Study Monitor for review and comments before issue ofthe final report.Harlan StudyNumber: 41401172 Study Plan Page 6


### 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 ofthe data will be chargeable to the Sponsor. No data will be discarded without contactingthe Sponsorto obtain their written consent.Harlan StudyNumber: 41401172 Study Plan Page 7


## 1 INTRODUCTIONAND PURPOSE The purpose of this study is to evaluate the partition coefficient (n-octanol/water) of the test item. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

### 1.1 Guidelines I 

### Regulations The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
• OECD Guidelines for Testing ofChemicals. Where testing is conducted to satisfy the requirements ofspecific guidelines and/or regulations, this will be documented and reflected in the final report.Harlan StudyNumber: 41401172 Study Plan Page 8


## 2 TEST AND REFERENCE ITEMS 2.1 Test Item The test item will be supplied by or on behalfofthe Sponsor including the following information if known: description, batch, purity, expiry I retest date, storage conditions and safety precautions to be taken. Information as provided by the Sponsor.

Identification:



### 2.2 Reference Item D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]

Reference items, ifused, will be identified inthe individual test sections.Harlan Study Number: 41401172 Study Plan Page 9


## 3 TEST 3.1 Partition Coefficient (n-OctanoVWater) The purpose ofthis test is to evaluate the partition coefficient (Pow) between n-octanol and water for the test item. As the test item is a salt, testing will be performed by the shake-flask method (as indicated by test guidance).
3.1.1 Test Specific Guidelines I Regulations The procedure outlined in this document has been designed to be compatible with the following guidelines and recommendations:
• OECD 107, 27 July 1995
3.1.2 Test System The test system consists ofimmiscible aqueous and n-octanol phases in the same vessel with a defined amount oftest item pre-dissolved in the appropriate phase. Partition of the test item between both phases will be achieved by inversion ofthe mixture. The justification for the selection of the test system and test method ts that they are recommended by international guidelines.
3.1.3 Principle ofthe Test Inprinciple, the log Pow can be determined by the shake flask method (log Pow between -2 and 4). Limitations ofthe method are described in the relevant guidelines.
3.1.4 Performance ofthe Test A preliminary estimate ofthe partition coefficient ofthe test item will be generated. This may be obtained from the ratio ofthe approximate solubilities ofthe test item in n-octanol and water (by visual assessment) or calculated using appropriate modeling software. In order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the equilibrium concentrations ofthe items dissolved in the two phases must be determined.Harlan StudyNumber: 41401172 Study Plan Page 10

Before the partition coefficient can be determined, the two individual phases will be pre-saturated by shaking both n-octanol and water with a sufficient quantity of the opposite phase. A stock solution ofthe test item will be prepared in either n-octanol saturated water or water saturated n-octanol. The actual mass concentration of this stock solution will be determined using a suitable analytical method. Three ratios ofthe two phases will be carried out, each in duplicate, as detailed in the method guidelines. Ratio selection will be based on the characteristics of the test item and analytical methodology, but will contain both twice and half the n-octanol to water ratio of the middle ratio. The test solutions will be partitioned and phase separation performed by centrifugation before analysis to determine the concentration ofthe test item in the initial stock solution and in both phases. The total quantity oftest item present in both phases will be calculated and compared with the quantity ofthe test item originally introduced (derived from the analyzed stock solution concentration).


### 3.1.5 Calculation The partition coefficient (Pow) is defined as the ratio ofthe equilibrium concentrations (c) ofa dissolved test item in a two-phase system consisting oftwo largely immiscible solvents. The partition coefficient therefore is the quotient oftwo concentrations (no dimension) and is usually given in the form ofits logarithm to base ten (log pow). All observations which may be used to interpret the results ofthe tests will be documented. Where appropriate, mean values and standard deviation will be calculated and linear regression analysis (least square method) will be applied. Calculations performed will be presented in the study report.

# c

STUDY SPECIFIC SUPPLEMENT To General Study Plan: 920-30 D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Assessment ofReady Biodegradability; C02 

## Evolution Test

Study Director: Test Facility:

Sponsor:

StudyMonitor:

Harlan StudyNumber: Proposed Schedule: Experimental StartingDate: Experimental Completion Date: Draft Report Date: NBest 

### Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE722GD 

### UK UnivarUSA Inc. 17425 NE Union Hill Road Redmond WA 98052 UNITED STATES OF AMERICA 

### Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens FL 33410 UNITED STATES OF AMERICA 41401177

31 May 2014 30 June 2014 31 July 2014 Page 1 of4Harlan StudyNumber: 41401177 Study Specific Supplement Page 2



### Test Item Information as provided by the Sponsor.

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

### Special Conditions Study specific parameters:

GSP Section 1.1 Guidelines I Regulations: The study will be conducted according to the following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing ofChemicals (1992) No 301B "Ready Biodegradability: C02

Evolution Test" • Method C.4-C ofCommission Regulation (EC) No. 440/2008 • US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 (Paragraph

(m))
GSP Section 3.2.1 Inoculum: The study will be conducted using aerobic activated sewage sludge from Loughborough sewage treatment works. 

## GENERAL INFORMATION Distribution List The original study specific supplement will be retained in the study file. Copies ofthe study specific supplement and general study plan along with any amendments will be distributed as follows: Forthe Sponsor: John V. Massingale Greenwich Chemical Consulting, Inc. Quality Assurance: Harlan Laboratories Ltd, Shardlow, UKHarlan Study Number: 41401177 Study Specific Supplement Page 3Harlan Study Number: 41401177


## SIGNATURES
Study Director:

Test Facility Management:

Forthe Sponsor: Study Specific Supplement Page4

NBest

Date:

CMead

Date:

John V. Massingale Greenwich Chemical Consulting, Inc.

Date:

Note: These signatures also acknowledge the procedures described in the corresponding General Study Plan.

## GENERAL STUDY PLAN:**920~30** 

# harlan® 

# ...

Assessment ofReady Biodegradability; C02 

## Evolution Test

TestFacility:

Signatures: Author:

TestFacility Management:

Quality Assurance: 

### HarlanLaboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK

N Clarke BSc

# .~.......................... Date: 

## 2

## 9 AUG 

## 2012

J G Riley BSc (Hons) MRQA



# .....**2..~!.~**

# ................... Date: 

## 05 SEP 

## 2012
Page 1 of17General Study Plan: 920-30 Page2



### CONTENTS GOOD LABORATORY PRACTICE
.............................................................................................3 GENERAL INFORMATION
.........................................................................................................4 Study Plan Amendments and Deviations
...............................................................................4 Quality Assurance
..................................................................................................................4 Reporting
...............................................................................................................................4 Archiving
...............................................................................................................................4 1 INTRODUCTION AND PURPOSE
.....................................................................................5 1.1 Guidelines 

### IRegulations
..........................................................................................5 2 TEST AND REFERENCE ITEMS
.......................................................................................6 2.1 TestItem
.................................................................................................................. 6 2.2 Reference Item
.........................................................................................................6 3 MATERIALS AND METHODS
...........................................................................................6 3.1 Definitions and Abbreviations
.................................................................................6 3.2 Test System
..............................................................................................................7 3.2.1 Inoculmn
....................................................................................................7 3.2.2 Justification
................................................................................................7 3.2.3 Preparation ofinoculmn
............................................................................7 3.3 Mineral Medimn
......................................................................................................8 3.4 Dosage
..................................................................................................................... 8 3.4.1 TestItem Preparation
................................................................................ 8 3.4.2 Reference Item Preparation
....................................................................... 8 3.5 Materials
.................................................................................................................. 8 3.6 Experimental Conditions
......................................................................................... 8 3.7 Study Design
............................................................................................................9 3.7.1 TestItem Concentration
............................................................................9 3.7.2 Nmnber ofVessels
.................................................................................. 10 

### 3.7.3 Procedure
................................................................................................. lO 

### 3.8 Evaluations
............................................................................................................ 11 3.8.1 Observations
............................................................................................ 11 3.8.2 IC Analysis
.............................................................................................. 11 3.8.3 IC/TC Ratio
............................................................................................. 11 3.8.4 Analyzers
................................................................................................. 12 3.9 DataEvaluation
..................................................................................................... 12 3.9.1 Percentage Carbon Content
..................................................................... 12 3.9.2 Percentage Biodegradation
...................................................................... 12 3.9.3 Total C02 

### Evolution
................................................................................ 13 3.10 

### Validity Criteria
..................................................................................................... 13 

### 4 REFERENCES
.................................................................................................................... 14 APPENDICES
............................................................................................................................... 15 Appendix I Mineral Medium
............................................................................................................... 15 Appendix 2 Methods ofDissolving!Dispersing TestItem in the Test System
..................................... l6General StudyPlan: 920-30 Page 3


## GOOD LABORATORYPRACTICE 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, therequirements ofDirectives 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).General StudyPlan: 920-30 Page4


## GENERALINFORMATION Study Plan Amendments and Deviations Any intended change to the study plan (general study plan and study specific supplement) 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 ofthe study plan. Deviations (unplanned changes) from the study plan (general study plan and study specific supplement) will be documented 

### and acknowledged by the Study Director. 

### QualityAssurance Quality Assurance will audit and inspect study-related procedures and will report any audit and inspectionresults inwritingtothe Study Director and TestFacilityManagement. 

### Reporting A GLP compliant report will be presented. A draft report will be sent to the Sponsor for review and comments before issue ofthe final report. 

### Archiving Unless instructed otherwise bythe Sponsor, the study plan (general study plan and study specific supplement), all raw data (paper and electronic) and the final reportwill 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 ofthe data will be chargeable to the Sponsor. No datawill be discarded without contacting the Sponsorto obtaintheirwritten consent.General StudyPlan: 920-30 PageS


## 1 INTRODUCTIONAND PURPOSE The purpose of this study is to determine the ready biodegradability of the test item in a biodegradation test by following carbon dioxide (C02 ) evolution ofthe test item by means of inorganic carbon analysis over a period of usually 28 days when exposed to sewage sludge micro-organisms under defined conditions. The results ofthe study are ofvalue in the assessment ofthe persistence ofthe test item in the aquatic environment. 

### 1.1 Guidelines I

### Regulations The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
• OECD Guidelines for Testing ofChemicals (1992) No 301B "ReadyBiodegradability: C02 Evolution Test"
• Method C.4-C ofCommission Regulation (EC) No. 440/2008
• US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m))General StudyPlan: 920-30 Page6


## 2 TESTAND REFERENCE ITEMS 2.1 TestItem The test item will be suppliedby or onbehalfofthe Sponsor including the following information if known; description, batch, purity, expiry 

### I retest date, storage conditions and safety precautions to be taken. Where the test item is administered in a vehicle, and the homogeneity, concentration or stability ofthe test item inthat vehicle is not determined an impact assessment will be made by the Study Director, this will be reflected in the final report and an exception will be included in the GLP compliance statement. 

### 2.2 Reference Item Sodium benzoate, at a concentration of 10 to 20 mg organic carbon/L (equivalent to 17.1 to 34.3 mg/L), will be included inthe study. 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Readily biodegradable:

1

0-Day window: Inorganic carbon (IC):
Total carbon (TC):
Total organic carbon (TOC):
Dissolved organic carbon (DOC): Test items may be considered to be readily biodegradable if**~** 60% biodegradation is attained in a 10-Day window within the 28 days ofthe test. The 10 days immediately following the attainment of 10% biodegradation. Carbon derived from inorganic sources e.g. carbon dioxide, carbonic acid, and carbonate. This is the sum ofthe organic carbon and the inorganic carbon present ina sample. This is the sum of the organic carbon in solution and in suspension.
Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0.45 J.Lm filter or remains in the supernatant after centrifuging at approximately 4000 g for 15 minutes.General StudyPlan: 920-30 Page 7



### 3.2 Test System The test method and test system are consistent with the testing guidelines and should provide a rational basis to assess thereadybiodegradability ofthetest itemunder aerobic conditions. 

### 3.2.1 Inoculum The study will be performed with aerobic activated sewage sludge from the aeration stage ofa local wastewater treatment plant, which treats predominately domestic sewage. Details ofthe source ofinoculum to be used in the study will be specified under the special conditions section ofthe studyplan and documented inthe data.


### 3.2.2 Justification Sewage sludge/treatmentmicro-organisms have been selected following the recommendations of OECD Guideline No 301, Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, andTransformationTest Guidelines OCSPP 835.3110. Pre-adaptation ofthe inoculumto the test item is notpermitted.


### 3.2.3 Preparation ofInoculum The aerobic activated sewage sludge will be washed by centrifugation or settlement and resuspension. The supernatant (liquid phase) will be decanted and the solid material resuspended in tap water or mineral medium. Ifnecessary, this procedure will be repeated but the fmal washwill be in mineral medium. An aliquot ofthe fmal sludge suspension (this maybe homogenized) will beweighed, thereafter dried and the concentration ofsuspended solids willbe calculated. Based on this ratio, calculated amounts ofwet sludge may be suspended in mineral medium to obtain a concentration equivalent to 4 g (±10%) dry material per liter. The inoculum is maintained on aeration priorto use and maybepre-conditioned to experimental conditions for up to 7 days by aeration at test temperature. Prior to use, the sludge may be diluted withmineral medium to a concentration of 1 g dry material perliter. Defined volumes of this diluted activated sludge will be added to each test vessel to obtain a final concentration of 30mg dry material perliter. Preparation and pre-conditioning of the inoculum may be performed with similar concurrent studies. However, the raw data, copies ofthe raw data or transcribed data ofthis work will be included inthe study datafile.General Study Plan: 920-30 Page 8


### 3.3 Mineral Medium The test will be conducted using a defined mineral medium (see Appendix 1). The mineral medium is considered not to contain any contaminant that would affect the integrity or outcome ofthe study. 

### 3.4 Dosage 3.4.1 Test Item Preparation Ifnecessary, pre-experiments will be performed to determine the solubility/dispersibility ofthe test item in the test system. According to the fmdings of these pre-experiments, appropriate procedures will be chosen to prepare the test media (typical methods are summarized in Appendix 2). The test item will be dissolved/dispersed in a defined mineral medium once at the start ofthe test. Ifauxiliary solvents, surfactants orinert carriers are used to aid inthe dispersion, they should not be toxic to bacteria and mustnot be biodegradable or cause foaming underthe test conditions. Details ofthe method ofpreparation will be documented in the data. 

### 3.4.2 Reference Item Preparation The reference item will be dissolved in the mineral medium once at the start ofthe test and addedto the test vessels via a concentrated stock solution. 

### 3.5 Materials Glass test vessels (5 liters) containing 3 liters oftest preparation will be used. The test vessels will be labeled with the study number and all necessary information to ensure unique identification. 

### 3.6 Experimental Conditions Test duration: Normally 28 days. At the discretion ofthe Study Director the test may be terminated before Day 28 if three consecutive determinations confirm complete degradation. With the Sponsor's approval and depending upon the results obtained, the test may be extended for a further 2 to 3 weeks. In such a case the test item could not be classed as readily biodegradable. Any extension would be subject to an additional cost. The test dates will be documented in the data.General StudyPlan: 920-30
Light conditions: Testtemperature:

Aeration:

COz absorption: pH:



### 3.7 Study Design Page9

In darkness. The test will be conducted at a temperature of22 ± 2°C. The room temperature will be recorded throughout the test at least on each working day. Additionally, the temperature may be checked on each sampling occasion in a separate vessel containing water and incubated under the same conditions as the test vessels. COz-free air is bubbled through the solution in each test vessel at a rate of30-100 mL/min per vessel and stirred continuously by magnetic stirrers to prevent settlement ofthe sludge. COzfree air will be produced by passing compressed air through a glass column containing self-indicating soda lime granules**(Carbosorb~** or the air will be passed through a bottle, containing about 750 mL ofa 2 M NaOH solution to trap COz. One empty 500 mL Dreschel bottle is placed in the**com~ressed** air line priorto passage ofthe air through the Carbosorb to act as a moisture trap. The C02 produced by the culture vessels will be collected in 2 x 500 mL Dreschel bottles containing a suitable volume of 0.05 MNaOH. Prior to test start (Day 0), the pH will be measured in each test vessel after the addition of the test or reference item, and, if necessary, adjusted to pH 7.4 ± 0.2 using a diluted hydrochloric acid or sodium hydroxide solution. The pH will be measured again in each vessel at the end ofincubation.



### 3.7.1 Test Item Concentration 10 to 20 mg organic carbon/L, in duplicate. The total organic carbon (TOC) content ofthe test item will be calculated based onthe information (molecular formula or carbon content) provided by the Sponsoror specific measurement (e.g. DOC analysis). Lower test concentrations may be employed ifthe test item is toxic to sewage sludge microorganisms. The actual concentration used will be documented in the data. If a repeat test is required due to the test item toxicity, this will be subject to an additional cost. The test item can be assumed to be not inhibitory ifin the toxicity control more than 25% degradation (based on total mg TOC added) occurs within 14 days.General Study Plan: 920-30 Page 10



### 3.7.2 Number ofVessels The following vessels will be prepared:
• 2 test item vessels, containing the test item (10 to 20 mg organic carbon/L) in inoculatedmineralmedium. 

### • 2 inoculumcontrol vessels, containing inoculatedmineral medium alone (blank). • 2 procedure control vessels, containing the reference item, sodium benzoate (10 to 20 mg organic carbon/L), ininoculated mineral medium. • 1 toxicity control vessel, containing inoculated mineral medium, the reference item, sodiumbenzoate (10 to 20 mg organic carbon/L) and the test item (10 to 20 mg organic carbon/1). Experience has shown that an abiotic control is not required. The abiotic control is designed to show chemical degradation ofthe test item and it is considered that C02 would not be produced from such degradation and hence the abiotic control is omitted. Should an abiotic control be required a vessel will be prepared containing the test item in noninoculated mineral media and a sterilizing agent to prevent microbial growth (e.g. 0.1 giL sodium azide or 10 mg/L HgCh). This will only be performed at the request ofthe Sponsor and at an additional cost. Ifauxiliary solvents, surfactarits orinert carriers are used to aid in the dispersion ofthe test item, the control and reference item vessels will be prepared containing an identical concentration of auxiliary solvent, surfactant orinertcarrier as inthe test item vessels. Data from the control and reference group may be shared with similar concurrent studies. However, the raw data, copies ofthe raw data ortranscribed data ofthis workwill be included in the study datafile.


### 3.7.3 Procedure Approximately 24 hours prior to the start ofthe study, to each 5 liter vessel, typically 2400 mL of mineral medium will be added and an appropriate volume of the activated sewage sludge inoculum to give 

### 30 mg ss/1 in the final volume of 

### 3 liters. The test media will be aerated overnightwith co2..:free airto purge the system ofcarbon dioxide. On the following day (Day 0), the test item and/or reference item will be added to the relevant vessels and the volume in all flasks made up to 3 liters by the addition of mineral medium (whichwas overnightpurged with C02-free air). The inoculum control (blank) will be prepared in a similar manner with the omission of test and reference items. Two absorber flasks, containing a suitable volume of0.05 M NaOH which will be documented in the data, will be connectedin series to the exit air line ofeachtest flask.General StudyPlan: 920-30 Page 11

On Day 28 (after pH measurement), 1 mL ofconcentrated HCl will be added to each test flask. Then the flasks will be aerated overnight to drive offany residual/dissolved C02 present in the test suspensions. On Day 29, a sample ofboth absorber flasks will be withdrawn and analyzed for IC to determine residual C02remaining inthe 3 liter test suspensions. Samples ofthe second NaOH absorber flask willbe analyzedto correct for any carryover ofC02. 

### 3.8 Evaluations 3.8.1 Observations The appearance of the test preparations will be observed on Day 0 and then periodically throughout the test and, ifrelevant, changes inthe appearance will be recorded. 

### 3.8.2 IC Analysis Samples (the exact volume will be recorded in the data) are taken from the first C02 absorber vessel for IC analysis on Day 0 and typically every second or third day during the first 14 days (to cover the 10-day window) and on Days 21, 28 and 29 to give a total of nine sampling occasions. Additional sampling days may be included ifrequired for identification ofthe 10-Day window. The volume of sample removed is recorded at each sampling time. On the 29th day the last analysis ofevolved carbon dioxide is made. The second absorbervessel is sampled onDay 0 and Day 29 only inorder to correct for any carryover ofcarbon dioxide. Each sample is subjected to a direct analysis ofthe evolved C02in triplicate. Depending on the degradability of the test item, an additional sample from the second absorber flask may be withdrawn at anearliertime point duringthe exposure period. Samples are stored in glass containers and are either analyzed immediately or stored at approximately -20°C. 

### 3.8.3 IC/TC Ratio For soluble test items, in order to calculate the IC/TC ratio, samples (the exact volume will be recorded in the data) will be removed from the test item and inoculum control vessels after making up to the final volume of3 liters. All samples will be either filtered through a 0.45**~-tm** filter (an initial volume discarded, details to be recorded inthe data) or for those test items which show significant adsorption to glassware, fllters and/or activated sewage sludge, centrifuged (approximately4000 g, 15 min), priorto analysis.General StudyPlan: 920-30 Page 12

In order to enable calculation ofthe IC/TC ratio in the test item preparations for insoluble test items, samples (the exact volume will be recorded in the data) will be removed from the test item prior to dosing and the inoculum control vessels after preparation. All samples will be either filtered through a 0.45 f.Lm filter (an initial volume discarded, details to be recorded inthe data) or centrifuged (approximately 4000 g, 15 minutes), prior to analysis. The nominal TC contribution ofthe test item, e.g. 10 mg C/L, will be added to that obtained from TC analysis for the testitem before calculation ofthe IC/TC ratio.


### 3.8.4 Analyzers All samples will be analyzed using a TOC infrared gas analyzer equipped with an automatic sampler for use ifrequired. For calibration ofthe TOC analyzer, a standard sample is measured at the start or at the end ofeach run to prove conformity with the calibration curve. Details of the analyzerused andthe calibrations performed willbe documented inthe data. 

### 3.9 Data Evaluation 3.9.1 Percentage Carbon Content The theoretical amount ofcarbonpresent inthe test itemis calculated as follows: No ofC atoms x mol wt of C xlOO% mol wt oftest item Where the molecular weight of the test item is not known, the TOC present as test chemical maybe derived from calculationor specific measurement. 

### 3.9.2 Percentage Biodegradation The amount ofC02 produced, expressed as IC (mg), is givenby multiplying the concentration of IC in the NaOH absorbent by the volume of the absorbent (this will be different on each sampling occasion due to theremoval ofsample). The amount of carbon dioxide produced must be corrected for endogenous carbon dioxide production and is, therefore, determined by the difference in carbon dioxide contentbetweenthe experimental and control absorbervessels. The percentage degradationbased on C02 evolution is calculatedfrom: OJ'd d . 

## * mgICfrom testflask- mglCfromCOntrOlflask }00 

### "o egra at10n 

## = . x mgTOC addedas testchemical

• The conversion factor for carbonto carbon dioxide is 3.67General StudyPlan: 920-30 Page 13

The volume obtained on the day of measurement is a cumulative value and no additions are required. The amount ofIC present in the second absorber vessels is also taken into consideration when calculatingthe degradation values for the vessels. 

### 3.9.3 Total C02 

### Evolution The total C02 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for replicates 

### R1and 

### R2 on Day 28 are meaned before substitution into the equation. TotalC02 evolution=mgiC incontrol x 100 x--1

-- %CofC02 test volume

. 100 1 

## =mgiC mcontrol x -- 

### x27.29 3 

### 3.10 Validity Criteria Test items giving a result of**~60%** yield ofC02(within 28 days) should be regarded as readily biodegradable. This level mustbereached within 10 days ofbiodegradation exceeding 10%. The test is considered valid ifthe difference ofthe extremes ofreplicate values ofproduction of C02at the time the plateau is reached, at the end ofthe test or at the end ofthe 10-Day window, as appropriate, is less than 20%. The results of the degradation test are considered valid ifin the same test the reference item**yields~** 60% biodegradation (in a 10-Daywindow) byDay 14. Thetoxicity control should attain**~** 25% degradationbyDay 14 for the test itemto be considered asnon-inhibitory. The total C02 evolution in the control vessels at the end ofthe test should not normally exceed 40 mg/L medium(= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained shouldbe criticallyexamined. The IC content ofthe test item inthe mineral medium atthe beginning ofthetest should be <5% ofthe TC.

# ·.
General StudyPlan: 920-30 Page 14 

# 4 REFERENCES HANDLEY, J. W., MEAD, C., RAUSINA, G. A., WAID, 

### L. J., GEE, J. C. and HERRON, S. J. (2002) The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly Water-soluble Substances. 

### Chemosphere, 48, pp. 529-34. INTERNATIONAL STANDARDS ORGANISATION (ISO) (1995) 

### Standard 10634: Water quality

- Guidance for the preparation and treatment ofpoorly water-soluble organic compounds for the subsequent evaluation oftheir biodegradability in an aqueous medium. Geneva: International Standards Institute.**--"··--~··-··--** ... ------------···--···-------········--···----·-------·-· ··--·-------···-----·····----------·····-·····----·--····· 

### .,
General Study Plan: 920-30 Page 15


## APPENDICES Appendix 1 MineralMedium The mineral medium will be prepared according to the testing guidelines using purified water*. Analytical grade salts will be dissolved inpurified water to obtain the following stock solutions: i) Stock Solutions (a) KH2P04 8.50 giL KzHP04 21.75 g/L NazHP04.2H20 33.40 giL**~Cl** 0.50g/L The pHofthis solution should be 7.4 (b) CaCh or CaC1z.2H20 27.50 giL 36.40 giL 22.50 giL (d) FeCh.6H20 0.25 giL (In orderto avoid having to prepare solution (d) immediately before use add one drop ofconcentrated HCl perliter as a preservative. ii) Preparation To 1liter (fmal volume) ofpurified water* are added the following volumes ofsolutions (a) to (d). 10 ml ofSolution (a) 1ml ofSolution (b) 1ml ofSolution (c) 1ml ofSolution (d) iii) Storage Stock solutions (a) to (d) are stored at approximately 4°C and are stable for a period of 1year.

Ionexchange andreverse osmosis water treatedtap water (ElgaOptima 15+, ElgaPurelab OptionR-15 orElga Purelab OptionR-15 BP)General StudyPlan: 920-30 Page 16
Appendix2 Methods ofDissolving/Dispersing TestItem in the Test System Following the recommendations ofthe International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) typical methods of preparation that may be employed inthe biodegradationtest are summarizedbelow. Readily Water Soluble Items: A concentrated aqueous stock solution ofthe test item will be prepared in reverse osmosis purified deionized water or mineral medium and a suitable aliquot added to inoculated mineral medium to give the required test concentration. In order to prepare an aqueous stock solution, dissolution methods such as ultrasonication, high shearmixing andprolonged stirring/shaking may be used. Low Water Solubility Items: In order to aid dispersion ofthe test item in the test system and increase thepotential for biodegradationto occurthe following methods may be used: Ultrasonication: The required amount oftest item will be dispersed in an aliquot ofmineral medium with the aid ofultrasonication prior to dispersal in inoculated mineral medium to give the required testconcentration. High Shear Mixing: The required amount oftest item will be dispersed in an aliquot ofmineral medium with the aid ofhigh shear mixing prior to dispersal in inoculated mineral medium to give the required test concentration. Adsorption onto Silica Gel: The required amount of test item will be adsorbed onto granular silica gel. The test item/silica gel combination will then be dispersed in an aliquot ofmineral medium prior to dispersal in inoculated mineral medium to give the required test concentration. When dispersing the test item/silica gel combination in the aliquot ofmineral medium, shaking byhand, ultrasonication orhigh shear mixingmay be usedto aid dispersion. Adsorption onto Filter Paper: The required amount oftest item will be adsorbed onto glassfibre filter paper. The test item/filter paper combination will then be added into inoculated mineral mediumto give the required test concentration. When adsorbing the test item onto the glassfibre filter paper efforts should be made to spread the test item across the surface ofthe filter paper in orderto maximize the surface areathat will be exposed to the micro-organisms presentin the test system. Addition onto Glass Slide: The required amount oftest item will be weighed directly onto a glass microscope slide or coverslip. The slide/coverslip carrying the test item will then be added into inoculated minerale medium to give the required test concentration. When weighing the test item onto the slide/coverslip efforts should be made to spread the test item across the surface in orderto maximizethe surface areathat will be exposed to the micro-organisms present in the test system.'·
General StudyPlan: 920-30 Page 17
Preliminary Solution in a Volatile Solvent: A stock solution ofthe test item may be prepared in a suitable solvent e.g. acetone, diethyl ether, chloroform. A suitable aliquot ofthis solvent stock solution will then be dispensed onto a glassfibre filter paper and the solvent allowed to evaporate to dryness prior to addition of the filter paper containing the test item to inoculated mineral medium to give the required test concentration. As with the adsorption onto filter paper method, efforts should be made during addition ofthe solvent stock solution to the filter paper to spread thetest item across the surface ofthe filter paperto maximize its surface area. Care should be taken whenusing this method ofaddition ofthe test item to ensure that all traces ofsolvent are removed prior to addition ofthe filter paper to the test vessel as trace amounts of solvent can cause inhibitory effects or affectpercentage biodegradation values. Alternatively an aliquot ofthe solvent stock solution can be dispensed to an empty test vessel and the solvent allowed to evaporate to dryness prior to addition ofinoculated mineral medium to give the required test concentrations. Again care should be taken to ensure that all traces of solvent areremoved from the test vessel prior to addition ofthe inoculated mineral medium. A solvent stock solution ofthe test item can also be dispensed onto silica gel and the solvent allowed to evaporate to dryness by passing a steady stream ofcompressed air (0.5- 1.0 L /min) over the solvent stock solution/silica gel. In order to ensure the solvent has been removed, the test item coated silica gel can be placed in an oven over night (temperature ofoven to be advised by Study Director). The test item coated silica gel may be stored in a labeled glass container priorto use inthe study. Preliminary Solution in a Non-Volatile, Non-Degradable Solvent: A stock solution ofthe test item may be prepared in a suitable solvent e.g. silicone oil (Polydimethylsiloxane). An aliquot ofthis solvent stock solution will then be dispensed into inoculated mineral medium to give the required test concentration.

## STUDY SPECIFIC SUPPLEMENT To General Study Plan: 902-21 D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Acute Toxicity to Rainbow Trout

Study Director: Test Facility:

Sponsor:

Study Monitor:

Harlan StudyNumber: Proposed Schedule: Experimental Starting Date: Experimental Completion Date: Draft Report Date: D Sacker Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE72 2GD UK Univar USA Inc. 17425 NE Union Hill Road Redmond WA 98052 UNITED STATES OF AMERICA Greenwich Chemical Consulting, Inc. 3300 PGABoulevard, Suite 635 Palm Beach Gardens FL 33410 UNITED STATES OF AMERICA 41401173

30 April 2014 31 May 2014 30 June 2014 Page 1 of4Harlan Study Number: 41401173 Study Specific Supplement Page 2



### TestItem Information as provided by the Sponsor.

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 TestRule and the regulations set forth under 40 CPR 799.5087. 

### Special Conditions Study specific parameters:

GSP Section 1.1 Guidelines 

### IRegulations: The study will be conducted according to the following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing ofChemicals (1992) No 203, "Fish Acute Toxicity Test" GSP Section 3.2 Test System: The study will be conducted using Rainbow Trout (Oncorhynchus my kiss) GSP Section 3.3 Test Water: The study will be conducted using reconstituted water GSP Section 3.7 Study Design: The study will be conducted using 7 fish pertest concentration. Any pH adjustments ofthe exposure solutions will be conducted according to section 14 of OECD 203 details ofwhich will be included in the final test report. Ifmeasured concentrations are ± 20% ofthe nominal concentrations, results will be based on nominal concentrations. Ifmeasured concentrations are not ± 20% ofnominal concentrations, results will be based on geometric mean measured concentrations. The study report will provide a detailed description of the analytical method that will be developed priorto and utilized during the test. All elements described in item 23 ofOECD 203 will be included inthe final test report. Particulate matter concentration, chemical oxygen demand (COD), total organic carbonHarlan Study Number: 41401173 Study Specific Supplement Page 3
(TOC), hardness, alkalinity and conductivity of the dilution water will be determined at the start ofthe study.


## GENERALINFORMATION Distribution List The original study specific supplement will be retained in the study file. Copies ofthe study specific supplement and general study plan along with any amendments will be distributed as follows: Forthe Sponsor: John V. Massingale Greenwich Chemical Consulting, Inc. Quality Assurance: Harlan Laboratories Ltd, Shardlow, UKHarlan StudyNumber: 41401173


## SIGNATURES
Study Director:

Test Facility Management:

Forthe Sponsor: Study Specific Supplement Page 4

D Sacker

Date:

CMead

Date:

John V. Massingale Greenwich Chemical Consulting, Inc.

Date:

Note: These signatures also acknowledge the procedures described in the corresponding General Study Plan.

## GENERAL STUDY PLAN: 902-21
Acute Toxicity to Fish

TestFacility:

Signatures: Author:

TestFacilityManagement:

QualityAssurance: 

### HarlanLaboratories Ltd. ShardlowBusiness Park Shard!ow Derbyshire DE722GD 

### UK

H VryenhoefBSc

# o~~,Lzo,f·········

# ~712

# ........................ Date: 

## 1 ·1· SEP 2012 J G Riley BSc (Hons) MRQA

Date: 

## 1.2 SEP 2012
Page 1 of 16General StudyPlan: 902-21 Page2



### CONTENTS GOOD LABORATORY PRACTICE
.............................................................................................3 GENERAL INFORMATION
.........................................................................................................4 Animal Welfare
......................................................................................................................4 StudyPlan Amendments and Deviations
...............................................................................4 Quality Assurance
..................................................................................................................4 Reporting
...............................................................................................................................4 Archiving
...............................................................................................................................4 1 INTRODUCTION AND PURPOSE
.....................................................................................6 1.1 Guidelines I 

### Regulations
..........................................................................................6 2 TEST AND REFERENCE ITEMS
.......................................................................................7 2.1 Test Item
..................................................................................................................7 2.2 Reference Item
.........................................................................................................7 3 MATERIALS AND METHODS
...........................................................................................7 3.1 Definitions and Abbreviations
.................................................................................7 3.2 Test System
..............................................................................................................7 3.3 Test Water
................................................................................................................8 3.4 Dosage
.....................................................................................................................8 3.5 Materials
..................................................................................................................9 3.6 Experimental Conditions
.........................................................................................9 3.7 Study Design
............................................................................................................9 3.8 Evaluations
............................................................................................................ 11 3.8.1 Test organism observations
..................................................................... 11 3.8.2 Water quality criteria
............................................................................... 11 3.8.3 Analysis ofTest Item Concentrations
..................................................... 12 3.9 Statistical Analysis
................................................................................................ 13 3.10 Validity Criteria
..................................................................................................... 13 4 REFERENCES
.................................................................................................................... 14 APPENDICES
............................................................................................................................... IS Appendix 1 Recommended Fish Species
.............................................................................................. 15 Appendix 2 Test Water
......................................................................................................................... 16General StudyPlan: 902-21 Page3


## GOOD LABORATORYPRACTICE 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 ofDirectives 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), andJapan (MHLW, MAFF and METI).General Study Plan: 902-21 Page4


## GENERALINFORMATION 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 ofthe United Kingdom's Animals (Scientific Procedure) Act 1986. This general study plan is subject to review and the conduct ofthe study may be included as part ofthe Harlan Laboratories Ltd, Shardlow, UK Ethical ReviewProcess. The associated 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. 

### Study Plan Amendments and Deviations Any intended change to the study plan (general study plan and study specific supplement) 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 ofthe study plan. Deviations (unplanned changes) from the study plan (general study plan and study specific supplement) will be documented and acknowledged by the Study Director. 

### QualityAssurance Quality Assurance will audit and inspect study-related procedures and will report any audit and inspectionresults 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 ofthe fmal report. 

### Archiving Unless instructed otherwise by the Sponsor, the studyplan (general study plan and study specific supplement), all raw data (paper and electronic) and the final report will be retained inthe Harlan Laboratories Ltd, Shardlow, UKarchives for five years after which instructions will be sought as.General StudyPlan: 902-21 Page 5**·---~-·----------------------**
to further retention or disposal. Further retention or return ofthe data will be chargeable to the Sponsor. No datawill be discarded without contactingthe Sponsorto obtaintheir written consent.General StudyPlan: 902-21 Page6



### ---------------------------- ..

### --
..

### --..

## ---..

### --.....

## ---- 

## 1 INTRODUCTIONAND PURPOSE The purpose ofthis study is to assess the acute toxicity ofthe test item to freshwater fish. The toxicity will be expressed as the median lethal concentration (LC50) ormedian lethal loading rate (LLso). The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) and the Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) will also be determined ifapplicable. The species used will be a freshwater species representative ofa wide variety ofnatural habitats, and cantherefore be considered as animportant non-target organism in freshwater ecosystems. The dispersal of the test item in the surrounding medium is considered to represent the most probable route ofexposure inthe environment. 

### 1.1 Guidelines I

### 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 ofChemicals (1992) No 203 "Fish, Acute Toxicity Test"
• Method C.1 ofCommissionRegulation (EC) No. 440/2008
• JMAFF Test Guidelines 2-7-1-1 (2000)General Study Plan: 902-21 Page7


## 2 TESTAND REFERENCE ITEMS 2.1 Test Item The test item will be supplied by or on behalfofthe Sponsor including the following information if known; description, batch, purity, expiry I retest date, storage conditions and safety precautionsto be taken. Where the test item is administered in a vehicle, and the homogeneity, concentration or stability ofthe test item in that vehicle is not determined an impact assessment will be made by the Study Director, this will be reflected in the final report and an exception will be included in the GLP compliance statement. 

### 2.2 Reference Item In the interest ofanimal welfare, a positive control using areference item will not be performed. 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations LCsoiLLso:
LOEC (Lowest Observed Effect Concentration) or LOEL (Lowest Observed Effect Loadingrate): NOEC (No Observed Effect Concentration) orNOEL (No Observed Effect Loadingrate): Treatment:


### 3.2 Test System The calculated concentration or loading rate oftest item which results in 50% mortality within the exposure period. The lowest test concentration or loading rate at which a significant toxic effect onthe test organisms is observed within the exposureperiod.
The highest test concentration or loading rate at which no significanttoxic effect on the test organisms is observed within the exposure period. Comprises test item treatments, control (test water only) and solvent control (ifapplicable).

The study will be performed using juvenile fish supplied by an accredited live fish supplier, details ofwhich will be documented inthe data, or from in-house breeding cultures. The species of fish used will be selected depending on the test guideline to be followed and will be documented onthe study specific supplement. Recommended species, along with recommended temperature range and test length range for each species, are given in Appendix 1.General StudyPlan: 902-21 Page 8
Following a 48-Hour settling in period, mortalities are recorded and the following rejection criteria applied.


### > 10% in 7 days: 5-10% in 7 days: < 5%in7 days: rejection ofentirebatch continue acclimatisationfor an additional 7 days acceptance ofbatch The fish will be acclimatised for a minimum period of 12 days at Harlan Laboratories Ltd, with atleast 7 days acclimatisationpriorto test start inwater ofthe quality and temperature to be used inthetest. The fish will be fed commercial fish food, details of which will be documented in the data, regularly on demand. Feeding will be discontinued 24 hours prior to testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome ofthe study. Animals will be maintained in glass or fibreglass tanks at a loadingrate 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 suppliedtoprovide ;;:: 80%Air SaturationValue (ASV). 

### 3.3 Test Water The animals will be maintained in either reconstituted test water, local non chlorinated well water ofdrinking quality ordechlorinated laboratory tap water (see Appendix 2). 

### 3.4 Dosage Ifnecessary, pre-experiments will be performed to determine the solubility and stability ofthe test item in the test water. According to the findings of these pre-experiments, appropriate procedures will be chosento preparethetestmedia. The test item will be dissolved or dispersed in the test water once at the start ofthe test (static test), daily (semi-static test) orcontinuously (dynamic test) depending onthe stability ofthe test item inthetest water. Methods such as prolonged mixing, ultrasonication, high shearforce stirring orthe production of Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the nature ofthe test item. Ifauxiliary solvents or surfactants are used to aid in the dispersion, they will be used at a maximum concentration of 100**~LIL** (fmal volume). Ifthe test item falls into the category of a "difficult substance" as defined by the OECD Guidance Document No.23 (OECD, 2000), the method of preparation of the test media will be based on the recommendations ofthis document.General StudyPlan: 902-21 Page9

Ifthe test item is photodegradable, the preparation ofthe test media will be performed as far as possible inthe dark. Details ofthe method ofpreparationwill be documented 

### inthe data. 

### 3.5 Materials Glass test vessels containing an appropriate amount oftest medium. The test vessels will be covered to reduce evaporation and to avoid entry ofdust into the solutions. Volatile chemicals will be tested in sealed vessels using a volume oftest mediato provide a minimal headspace (i.e. test performed in a closed system). The size ofthe test vessels and the volume oftest solution will depend onthe species used and 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 be supplied to the test vessels via narrow bore glass tubes to give ;:::: 60% Air Saturation Value (ASV) unless evidence ofinstability ofthe test item under aeration is shown. For dynamic, continuous flow tests the diluent water supply only will be aerated. For tests conducted to OPPTS guidelines no auxiliary aeration will be supplied to the test vessels except for static tests where aeration may be used if the oxygen consumption is expected to drop below 60% Air Saturation Value (ASV) due to the chemical characteristics ofthe test item. The use ofaeration willbe documented inthe data. The test will be performed under temperature-controlled conditions. The temperature will depend onthe test species used (see Appendix 1) with a maximum deviation of± 1

# oc during the test. A 16-hour light to 8-hour dark cycle will be applied with a 20- 30 minute transition period. If the test item is light sensitive, the test will be performed as far as possible in the dark or under reduced light conditionsto avoidphotolytic degradation ofthe test item. 

### 3.7 Study Design Sevenfish will be used at eachtest concentration and 

### inthe control(s). The loadingrate should notbe greater

### than 1 g bodyweightperliter oftestmedium for static and semi-statictests. This loading maybeexceeded indynamic, continuous flow tests. 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.General StudyPlan: 902-21 Page 10
Dependent on the test item stability, the test will be conducted with no medium renewal (static test), with daily medium renewal (semi-static test) orcontinuous medium renewal (dynamic test). The choice oftestmediarenewal regimes is dependent upon the known characteristics ofthe test item and the results ofthe pre-test stability analysis. The actual method oftest media renewal will be documented in the data. For static or semi-static tests, the test will be started by addition ofthe test organisms to the test vessels within 30 minutes ofthe test media being added to the test vessels. For tests conducted using continuous medium renewal (dynamic tests), the test will be started after the concentration oftest item has attained a stable plateau. Alternatively the test item may be added to the test vessels at the required concentration prior to starting the dosing apparatus, in such cases the test organisms will be added to the test vessels within 30 minutes ofthe start ofthe dosing apparatus. At least five concentrations ofthe 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 ofthe definitive test. Ifin 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 ofthe 96-Hour LCso (or LLso) and, as far as possible, span the range of 0 to 100% mortality. In accordance with the test guidelines (EC, 2002), concentrations of the test item higher than 100 mg/L (1000 mg/L for agrochemical formulations tested according to JMAFF guidelines), or above the limit of solubility ofthe test item inthe test water (whichever is the lower) will usuallynotbe tested. If no effect is observed in the preliminary range-finding test at the highest concentration recommended in the relevant test guideline, or the limit ofsolubility ofthe test item in the test water (whichever is the lower), a Limit Test may be conducted using only this concentration and the corresponding control(s). In this case, the test will be conducted using a single test vessel together with a control and solvent control vessel, ifappropriate. Where appropriate a threshold test will be used. The lowest ECso/ELso value from an Algal Growth Inhibition test or Acute Toxicity to 

### Daphnia magna study is set as the threshold concentration. A "Limit Test" will then be carried out as detailed previously at this threshold concentration. If no mortalities are observed this indicates that fish is not the most sensitive species and thatthe LC50 is greater than the threshold concentration. Ifone ormore fish from the test group die, the test will be terminated and a test using a range oftest concentrations will be conducted. The control group will be exposed to the test water alone. Inthe interests ofanimal welfare, data from the control group may be shared with similar concurrent studies.General StudyPlan: 902-21 Page 11

Where auxiliary solvents/surfactants are used, a second control will be employed. The test organisms will be exposed to a concentration ofsolvent equal to that employed inthe test series, this solvent concentration will not exceed 100 JlLIL. In the interests of animal welfare, data from the solvent control group may be sharedwith similar concurrent studies. In the interests ofanimal welfare, ifthepH falls outside the range 6-9, the pHwill be adjusted to that ofthe test waterpriorto addition ofthe test organisms to the test preparations. In such cases, the adjustment ofpHwill be discussed withthe Sponsorpriorto proceeding withthe test. 

### 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 ofdeath are absence ofrespiratory movement and the lack ofresponse 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 ofthe LCso value the sacrificed fish will be taken into account as dead fish. The length and weight of a representative batch offish will be determined e.g. fish from the stocktankpriorto the start ofthe test orthe control fish atthe end ofthetest 

### 3.8.2 Water quality criteria Measurements ofpH, dissolved oxygen and temperature in each surviving test vessel (prior to andposttest mediarenewal for semi-static tests) are recorded daily. The appearance ofthe test mediawill be recorded daily.General StudyPlan: 902-21 Page 12



### 3.8.3 Analysis ofTest Item Concentrations 3.8.3.1 Sampling Samples ofthe test media will be taken at least at initiation, termination and an intermediate time point ofthe definitive test for analysis. 

### Inthe range-finding test, samples ofthe 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 (or solvent control where appropriate) and each surviving test group or at the observationpointwhen all fish are dead in a certaintest concentration. Analysis of samples taken from the range-finding test is performed in order to determine the stability ofthe test item under test conditions over the exposure period. The test concentrations provided for analysis will be documented by the Study Directorin the data. Final details ofthe samplesto 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. Ifthe 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 oforganic solvents) in orderto maintain the stability ofthe test item until analysis. It may be necessary for some chemicals which form emulsions or dispersions to filter or centrifuge some samples as part ofthe pre-test validation process to determine the proportion of test item inthe dissolved form. 3.8.3.3 Analysis ofsamples 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, ifnecessary. Stock solutions may also be analyzed, if necessary, for example ifthe test concentrations are belowthe limit ofquantitationofthe analytical method or for reasons ofstability. Ifthe test item is a formulation, test item concentrations will be determined by the measured concentrations ofthe active ingredient(s). WAFs will be characterized by analysis ofthe main testitem component(s)..,
General StudyPlan: 902-21 Page 13

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 ofthe analytical method will not be performed under GLP. Details about the analytical method used will be given in an Appendix ofthe final report. The accuracy ofthe analytical method will be verified byrecoveries spanningthe concentrationrange to be analyzed withinthis study. Analyticalpre-experiments may beperformed within a different Harlan Laboratories study. 

### 3.9 Statistical Analysis The LCso (or LLso) values and associated 95% confidence limits for all appropriate observation times will be calculated using an appropriate statistical method which will be documented inthe data. All LCso (or LLso) values will be roundedto two significantfigures. The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is the highest test concentration (or loading rate) at which no significant toxic effect on the test organisms is observedwithinthe exposure period. The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) isthe lowesttest concentration (or loading rate) at which a significant toxic effectonthe test organisms is observed within the exposure period. Ifthe test has been conducted as a limit test then itis notpossibleto determine a LOEC orLOEL. 

### 3.10 Validity Criteria Forthetest to bevalid, the following criteria shouldbe fulfilled: Inthe (solvent) control, not more than one ofthe fish should die or show signs ofstress during the 96 hours. The dissolved oxygen concentration at the end ofthe test should be**~60%** of Air Saturation Values (ASV) inthe control and test vessels.General StudyPlan: 902-21 Page 14 

# 4 REFERENCES OECD (2002) Guidance Document on the Recognition, Assessmentand Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Series on Testing andAssessment, No. 19. OECD Publishing. OECD (2000) Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. OECD Series onTesting and Assessment, No. 23. OECD Publishing. EUROPEAN COMMISSION (2002) Sanco/3268/2001 rev. 4 (final) Guidance Document on Aquatic Ecotoxicology in the context of Directive 911414/EEC.General Study Plan: 902-21 Page 15


## APPENDICES Appendix 1 Recommended Fish Species The following species are recommended for use according to the OECD, EU, and JMAFF guidelines. Species Recommended Test Recommended Total Temperature Range 

### ec) Length ofTestFish (em) 

### Brachydanio rerio 21-25 OECD 2.0 ± 1.0 Zebrafish EU 3.0±0.5 JMAFF 2.0 ± 1.0 

### Cyprinus carpio 20-24 OECD 3.0 ± 1.0 Common carp EU6.0±2.0 JMAFF 5.0 ± 1.0 

### Lepomis macrochirus 21-25 OECD 2.0 ± 1.0 Bluegill sunfish EU 5.0±2.0 JMAFF 3.0 ± 1.0 

### Oncorhynchus mykiss 13- 17 OECD 5.0 ± 1.0 Rainbow Trout EU 6.0±2.0 JMAFF 5.0 ± 1.0 

### Pimephalespromelas 21-25 OECD 2.0 ± 1.0 Fathead minnow EU 5.0±2.0 JMAFF 2.0 ± 1.0General StudyPlan: 902-21 Page 16
Appendix 2 TestWater Reconstituted Water The reconstituted water will consist ofanalytical grade salts dissolved inpurified water to obtain the following nominal concentrations· Ingredients I Concentration Properties mmoJJL 

## mWL CaC1z.2H20 1.0 147 MgS04.?H20 0.25 61.5 NaHC03 0.38 32.5 KCl 0.038 2.9 WaterHardness 1.25 125 as CaC03 Alkalinity 0.4 

## - The ratio ofCa:Mg andNa:K will be 4:1 and 10:1 respectively based on molarity. The reconstituted water will be aerated until oxygen saturation is reached. The pH ofthe test water after equilibration will be inthe range of7.0to 8.5.
Local Tap Water Local water (non chlorinated well water ofdrinking quality) will be total hardness reduced by ion exchange. The total hardness will be approximately 160 to 250 mg/L as CaC03. The water will be aerated until oxygen saturation is reached. The pH ofthe test water after equilibration will be inthe range of7.8 to 8.8.
Dechlorinated LaboratoryTap Water Laboratory tap water will be dechlorinated by passage through an activated carbon filter (Purite Series 500) and a proportion ofthe 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 CaC03 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.

## STUDY SPECIFIC SUPPLEMENT To General Study Plan: 900-22 

# c

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Daphnia sp., 48-HourAcute Immobilization Test

StudyDirector: TestFacility:

Sponsor:

Study Monitor:

Harlan Study Number: Proposed Schedule: Experimental Starting Date: Experimental Completion Date: Draft Report Date: D Sacker Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE722GD UK UnivarUSA Inc. 17425 NEUnion Hill Road Redmond WA 98052 UNITED STATES OF AMERICA Greenwich Chemical Consulting, Inc. 3300 PGABoulevard, Suite 635 Palm Beach Gardens FL 33410 UNITED STATES OF AMERICA 41401174

30 April 2014 31 May 2014 30 June 2014 Page 1 of4Harlan StudyNumber: 41401174 Study Specific Supplement Page 2



### Test Item Information as provided by the Sponsor.

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

### Special Conditions Study specific parameters:

GSP Section 1.1 Guidelines IRegulations: The study will be conducted according to the following internationally accepted guidelines and recommendations:

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

Immobilisation Test" GSP Section 3.2 Test system Daphnia used in the test should be from a healthy stock (i.e., showing no signs ofstress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discolored animals, etc.). Any pH adjustments ofthe exposure solutions will be conducted according to section 13 of OECD 202 details ofwhich will be included in the final test report. GSP Section 3.8.2 Water quality criteria Temperature, pH and dissolved oxygen measurements will be taken from replicate R1 only at the start and end ofthe test, and prior to and post test medium renewal ifapplicable. Ifmeasured concentrations are ± 20% ofthe nominal concentrations, results will be based on nominal concentrations. If initial measured concentrations are not ± 20% of the nominal concentrations but measured concentrations of the old exposure solution are ± 20% of the initial measured concentrations, results will be based on initial measured concentrations. If measured concentrations are not ± 20% of nominal concentrations, results will be based on geometric mean measured concentrations. The study report will provide a detailed description of the analytical method that will be developed priorto and utilized during the test.Harlan StudyNumber: 41401174 Study Specific Supplement Page 3

All elements described in item 27 ofOECD 202 will be included inthe final test report. 

## GENERALINFORMATION Distribution List The original study specific supplement will be retained in the study file. Copies ofthe study specific supplement and general study plan along with any amendments will be distributed as follows: Forthe Sponsor: John V. Massingale Greenwich Chemical Consulting, Inc. Quality Assurance: Harlan Laboratories Ltd, Shardlow, UKHarlan StudyNumber: 41401174


## SIGNATURES
Study Director:

Test Facility Management:

Forthe Sponsor: Study Specific Supplement Page4

D Sacker

Date:

CMead

Date:

John V. Massingale Greenwich Chemical Consulting, Inc.

Date:

Note: These signatures also acknowledge the procedures described in the corresponding General Study 

### Plan.

## GENERAL STUDY PLAN: 900-22 Daphnia sp., 48-HourAcute Immobilization Test

TestFacility:

Signatures: Author:

TestFacilityManagement:

Quality Assurance: 

### HarlanLaboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE722GD UK

H VryenhoefBSc



### J G Riley BSc (Hons) MRQA

### ~&.~ 

## ........................**~**

# ......**~**

## ................. Date: 

## 12 SEP 

## 2012
Page 1of15General StudyPlan: 900-22 Page2



### CONTENTS GOOD LABORATORY PRACTICE
.............................................................................................3 GENERAL INFORMATION
.........................................................................................................4 StudyPlan Amendments and Deviations
...............................................................................4 QualityAssurance
..................................................................................................................4 Reporting
...............................................................................................................................4 Archiving
...............................................................................................................................4 1 INTRODUCTION AND PURPOSE
.....................................................................................

### 5 1.1 , Guidelines 

### IRegulations
..........................................................................................

### 5 2 TEST AND REFERENCE ITEMS
.......................................................................................6 2.1 Test Item
.................................................................................................................. 6 2.2 Reference Item
.........................................................................................................6 3 MATERIALS AND METHODS
...........................................................................................6 3.1 Definitions and Abbreviations
.................................................................................6 3.2 Test System
..............................................................................................................7 3.3 Test Water
................................................................................................................7 3.4 Dosage
.....................................................................................................................7 3.

### 5 Materials
.................................................................................................................. 8 3.6 Experimental Conditions
.........................................................................................8 3.7 Study design
............................................................................................................8 3.8 Evaluations
............................................................................................................ 10 3.8.1 Test organism observations
..................................................................... 10 3.8.2 Water quality criteria
............................................................................... tO 

### 3.8.3 Analysis ofTestItem Concentrations
..................................................... 10 3.9 StatisticalAnalysis
................................................................................................ 11 3.10 Validity Criteria
..................................................................................................... 12 4 REFERENCES
.................................................................................................................... 13 APPENDICES
............................................................................................................................... 14 Appendix 1 ReconstitutedWater

- ElendtM7 Medium
...................................................................... 14 Appendix 2 Reconstituted Water- TestWater
.................................................................................... 15General StudyPlan: 900-22 Page3


## GOOD LABORATORYPRACTICE 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 ofDirectives 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).General StudyPlan: 900-22 Page4


## GENERALINFORMATION StudyPlan Amendments and Deviations Any intended change to the study plan (general study plan and study specific supplement) 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 ofthe studyplan. Deviations (unplanned changes) from the study plan (general study plan and study specific supplement) will be documented and acknowledgedbythe StudyDirector. 

### Quality Assurance Quality Assurance will audit and inspect study-related procedures and will report any audit and inspectionresults inwritingto the Study Director and Test FacilityManagement. 

### Reporting A GLP compliant report will be presented. A draft report will be sent to the Sponsor for review and comments before issue ofthe fmal report. 

### Archiving Unless instructed otherwise by the Sponsor, the studyplan (general study planand study specific supplement), all raw data (paper and electronic) and the fmal report will be retained inthe Harlan Laboratories Ltd, Shardlow, UKarchives for five years after which instructions will be sought as to further retention or disposal. Further retention or return ofthe data will be chargeable to the Sponsor. No data willbe discarded without contactingthe Sponsorto obtaintheirwritten consent.General StudyPlan: 900-22 PageS


## 1 INTRODUCTIONAND 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 (ECso) or median effectloadingrate (ELso) for immobilization. The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) andthe Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) will also be determined ifapplicable. Daphnia magna is a freshwater invertebrate representative ofa wide variety ofnatural habitats, andcantherefore be consideredas an importantnon-target organismin freshwater ecosystems. The dispersal ofthe test item in the surrounding medium is considered to represent the most probable route ofexposure in the environment. 

### 1.1 Guidelines I

### 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 ImmobilisationTest"
• Method C.2 ofCommission Regulation (EC) No. 440/2008
• JMAFF Test Guidelines 2-7-2-1 (2001)General StudyPlan: 900-22 Page6


## 2 TESTAND REFERENCE ITEMS 2.1 Test Item The test item will be suppliedby or onbehalfofthe Sponsorincluding the following information if known; description, batch, purity, expiry 

### I retest date, storage conditions and safety precautions to be taken. Where the test item is administered in a vehicle, and the homogeneity, concentration or stability ofthe test item in that vehicle is not determined an impact assessment will be made by the Study Director, this will be reflected in the final report and an exception will be included in the GLP compliance statement. 

### 2.2 Reference Item For evaluation ofthe quality of the Daphnia magna clone and the experimental conditions, potassium dichromate will be tested as a positive control twice a yearto demonstrate satisfactory conditions ofthe test. The reference item will be supplied by a commercial supplier and will be stored as specified by the supplier. 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Immobilization:

ECso/ELso:
LOEC (Lowest Observed Effect Concentration) or LOEL (Lowest Observed Effect Loading rate): NOEC (No Observed Effect Concentration) or NOEL (No ObservedEffectLoadingrate): Treatment: Those organisms which are not able to swim within 15 seconds after gentle agitation ofthe test container are considered to be immobile. The calculated concentration or loading rate oftest item which results in 50% immobilization withinthe exposure period. The lowest test concentration or loading rate at which a significant toxic effect onthe test organisms is observed within the exposureperiod. The highest test concentration or loading rate at which no significanttoxic effect onthe test organisms is observed within the exposureperiod. Comprises test item treatments, control (test water only) and solvent control (ifapplicable).General Study Plan: 900-22 Page7


### 3.2 Test System The study will be performed with young daphnids ofthe 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 clone 5) is successfully bred at Harlan Laboratories Ltd under temperature and light conditions identical tothose ofthetest. Duringthe culturingperiod, 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 byparthenogenesis. 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 ofthe test such that the young daphnids produced overnight are atthe required age. The daphnids will not befed during the test. 

### 3.3 Test Water The test water parameters are defined in Appendix 2. The parental daphnids are cultivated in water ofthe same quality (inrespectto pH, main ions, and total hardness). · 

### 3.4 Dosage Ifnecessary, pre-experiments will be performed to determine the solubility and stability ofthe test item in the test water. According to the findings of these pre-experiments, appropriate procedures will be chosento preparethetest media. The test item will be dissolved or dispersed in the test water once at the start ofthe test (static test), daily (semi-static test) or continuously (dynamic test) depending onthe stability ofthe test item inthetest water. Methods such as prolonged mixing, ultrasonication, high shear force stirring orthe production of Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the nature ofthe test item. Ifauxiliary solvents or surfactants are used to aid in the dispersion, they will be used at a maximum concentration of 100 J..I.LIL (final volume). Ifthe test item falls into the category of a "difficult substance" as defined by the OECD Guidance Document (OECD, 2000), the method ofpreparation ofthetest mediawill be based onthe recommendations ofthis document.General StudyPlan: 900-22 Page 8

Ifthe test item is photodegradable, the preparation ofthe test media will be performed as far as possible 

### inthe dark. Details ofthe method ofpreparation will be documented inthe data. 

### 3.5 Materials Glass test vessels containing at least 50 mL oftest medium will be used. The test vessels will be covered to reduce evaporation and to avoid entry ofdust into the solutions. Volatile chemicals will be tested in completely filled and stoppered test vessels (i.e. test performed in a closed system). The size ofthe test vessels and the volume oftest 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. 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

# oc during the test. A 16-hourlight to 8-hour dark cycle will be applied with a 20

- 30 minute transition period. The light intensity during the light period will be between 200 and 1200 Lux and will be documented in the raw data. Ifthe test item is light sensitive, the test will be performed as far as possible in the dark orunderreduced light conditionsto avoidphotolytic degradation ofthe test item. 

### 3.7 Study design A total of20 daphnids per treatment will be used and divided into four replicates. The following conditions will be met: Total volume perreplicate Volume 

### I daphnid Loadingrate 

### IL 2::10 mL 2::2m.L :::;500 daphnids The daphnids willberandomly distributed to the test vessels at initiation ofthe test. For static or semi-static tests, the test will be started by addition ofthe test organisms to the test vessels within 30 minutes ofthe test media being added to the test vessels. For tests conducted using continuous mediumrenewal (dynamic tests), the test will be started after the concentration oftest item has attained a stable plateau. Alternatively the test item may be added to the testGeneral StudyPlan: 900-22 Page9

vessels atthe required concentration prior to starting the dosing apparatus, in such cases the test organisms will be added to the testvessels within 30 minutes ofthe start ofthe dosing apparatus. The duration ofthe testwill be 48 hours. Dependent on the test item stability, the test will be conducted with no medium renewal (static test), with daily medium renewal (semi-static test) or continuous medium renewal (dynamic test). In a semi-statictest, the Daphnia will be transferredto the fresh mediaby wide-bore pipette. The choice oftest mediarenewal regimes is dependent upon the known characteristics ofthe test item and the results ofthe pre-test stability analysis. The actual method oftest media renewal will be documented inthe data. At least five concentrations ofthe 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-fmding tests or available toxicity data and will be documented in the data prior to the start ofthe definitive test. Ifin 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. 

### In accordance with the test guidelines (EC, 2002), concentrations ofthe test item higher than 100 mg/L (1000 mg/L for agrochemical formulations tested according to JMAFF guidelines), or above the limit ofsolubility ofthe testitem inthe test water (whichever is the lower) will usually notbetested. If no effect is observed in the preliminary range-finding test at the highest concentration recommended in the relevant test guideline, or the limit ofsolubility ofthe test item in the test water (whichever is the lower), a Limit Testmay be conducted using onlythis concentration and the corresponding control(s). The control group will be exposed to the test water alone. Data from the control group may be shared with similar concurrent studies. Where auxiliary solvents/surfactants are used, a second control will be employed. The test organisms will be exposed to a concentration of solvent equal to that employed in the highest concentration in the test series, this solvent concentration will not exceed 100 J..tLIL. Data from the solventcontrol group may be shared with similar concurrent studies. The test will be carried out without adjustment ofpH. Ifthe pH falls outside the range 6-9, the Sponsor will be contacted for advice on whether a second test should be performed with adjustment ofpHto that ofthe test water. However, pH adjustment will not be carried out ifthe test solutions are changed to any significant extent and ifa chemical reaction or precipitation is caused.General StudyPlan: 900-22 Page 10


### 3.8 Evaluations 3.8.1 Test organism observations The nwnber 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 ofthe testbeaker will be consideredto be immobile. Any abnormal behaviour or abnormal appearance ofthe daphnids (sub lethal effects other than immobility) will also be recorded at 24 and 48 hours.


### 3.8.2 Water quality criteria For static and dynamic flow tests the temperature is recorded daily and pH and dissolved oxygen measurements are made for each test vessel at the start and end ofthe test. For semi-static tests, pH, dissolved oxygen and temperature are measured in each test vessel at the start ofthe test, priorto andposttest mediwnrenewal and atthe end ofthe test. The appearance ofthe test mediawill be recorded daily.


### 3.8.3 Analysis ofTest Item Concentrations 3.8.3.1 Sampling Samples ofthe test media will be taken at initiation, before and after the test mediwnrenewal (if applicable), and termination ofboth the range-finding and definitive tests for analysis. Where possible duplicate samples will be taken at each occasion, these samples may also be analyzed shouldthe need arise. Analysis of samples taken from the range-finding test is performed in order to determine the stability ofthe test item under test conditions over the exposure period. The test concentrations provided for analysis will be docwnented bythe Study Director in the data. For the purposes ofthe definitive test, the replicate test and control (or solvent control) vessels will be pooledto give one sample. Ifthe test medium volwne from the actual test is too small to perform the required analyses, requisite volwnes of the freshly prepared test media will be incubated separately (without daphnids), butunder identical conditions as inthe actual test. Final details ofthe samples to be taken and the sample size, etc, will be docwnented inthe data.General StudyPlan: 900-22 Page 11

3.8.3.2 Storage and samplepreparation All samples will be analyzed immediately after sampling or stored at approximately -20°C until analysis. Ifthe 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 oforganic solvents) inorderto maintainthe stability ofthe test item until analysis. It may be necessary for some chemicals which form emulsions or dispersions to filter or centrifuge some samples as part ofthe pre-test validation process to determine the proportion of testitem inthe dissolvedform. 3.8.3.3 Analysis ofsamples The test item formulations will be analyzed for concentration and stability by Harlan Laboratories Ltd, Shardlow, UKAnalytical 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 normallybe analyzed. Theremaining samples may be analyzed, ifnecessary. Stock solutions may also be analyzed, ifnecessary, for example ifthe test concentrations are belowthe limitofquantitation ofthe analytical method orfor reasons ofstability. If the test item is a formulation, test item concentrations will be determined by the measured concentrations ofthe active ingredient(s). WAFs will be characterized by analysis ofthe main test item component(s). 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 ofthe final report. The accuracy ofthe analytical method will be verified byrecoveries spanningthe concentrationrange tobe analyzed withinthis study. Analyticalpre-experiments may be performedwithin a different HarlanLaboratories study. 

### 3.9 Statistical Analysis The 24 and 48-Hour EC50 (or EL50) values and associated 95% confidence limits will be calculated using anappropriate statistical method which will be documented inthe data. All ECso (or EL50) values willberoundedto two significantfigures.General StudyPlan: 900-22 Page 12

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is the highest test concentration (or loading rate) at which no significant toxic effect on the test organisms is observed within the exposure period. The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) is the lowesttest concentration (or loading rate) at which a significanttoxic effect onthe test organisms is observed within the exposure period. Ifthe test has been conducted as a limit testthen 

### it is notpossibleto determine a LOEC orLOEL. 

### 3.10 Validity Criteria Forthe testto bevalid, the following criteria shouldbe fulfilled: Inthe (solvent) control, notmore than 10% ofthe daphnids should show immobilization or other signs ofdisease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). The dissolved oxygen concentration at the end ofthe test should be ?:3 mg/L in the control and test vessels.'•
General StudyPlan: 900-22 Page 13 

# 4 REFERENCES EUROPEAN COMMISSION (2002) Sanco/3268/2001 rev. 4 (final) Guidance Document on Aquatic Ecotoxicology in the contextof Directive 91/414/EEC. 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. OECD (2000) Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. OECD Series onTesting and Assessment, No. 23. OECD Publishing.General StudyPlan: 900-22 Page 14


## APPENDICES Appendix 1 Reconstituted Water- ElendtM7Medium Elendt M7 medium will be prepared by dissolving the following analytical grade salts and additives indeionised orpurified water atthe following concentrations: Main Compounds TraceElements• mg/L CaCh.2H20 MgS04.1HzO KC1 NaHC03 Na2Si03.9H20 NaN03 KH2P04 KzHP04

Water Hardness: Alkalinity:**.~** 294 B H3B03 123 Fe FeS04.7H20 2 5.8 Mn MnChAH20 65 Li LiC1 10 Rb RbC1 0.27 Sr SrCh.6H20 0.14 Br NaBr 0.18 Mo Na2Mo04.2H20 Co CuC}z.2H20 Zn ZnCh Co CoCh.6H20 I KI Se Na2Se03 

## v**~vo3** EDTA Na2EDTA.2H20 2.5 mmol/L (= 250 mg/L as CaC03) 0.9mmol/L Vitamins p.g/L 125 Thiamine HC1 75 50 Cyanocoba1amine (B 12) 1.0 25 Biotin (B6) 0.75 12.5 12.5 12.5 3.1 6.3 1.6 6.3 2.5 2.5 1.0 0.3 625

The pH of the prepared media will be in the range of 7.9 ± 0.3 and will be stored at approximately 21 °C. Ifnecessarythe pHmay be adjusted withNaOH or HCl.

1 Concentrations ofthe targetelements in bold; the source compounds are given inthe next column 2 Stock solutions ofFeS04.7H2 0 and Na2 EDTA.2H2 0 are combined and autoclaved immediatelyGeneral StudyPlan: 900-22 Page 15


### Appendix 2 Reconstituted Water- Test Water
Prior to the start ofthe 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 of7.6- 8.0. Ifnecessary the pH may be adjusted withNaOH orHCl. The pH will normally not vary bymore than 1.5 during the test. The ratio ofCa:MgandNa:K will be 4:1 and 10:1, respectively, based onmolarity. Reconstituted test water according to ISO 6341 (ISO, 1996) will be used in the study. It will consist ofanalytical grade salts dissolved in purified water with a conductivity of< 5J.tS cm-1 to obtainthe following nominal concentrations: Ingredients 

### I Concentration Properties mmoVL mg/L CaCh.2H2 0 2.0 294 MgS04.1H20 0.5 123 NaHC03 0.75 65 KCl 0.075 5.8 Water Hardness 2.5 250 as CaC03 Alkalinity 0.8 

# -

## STUDY SPECIFIC SUPPLEMENT To General Study Plan: 908-25 D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Algal Growth Inhibition Test Study Director:
TestFacility:

Sponsor:

Study Monitor:

Harlan StudyNumber: Proposed Schedule: Experimental Starting Date: Experimental Completion Date: Draft Report Date: H Vryenhoef


### Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE722GD UK 

### UnivarUSA Inc. 17425 NEUnion Hill Road Redmond WA 98052 UNITED STATES OF AMERICA 

### Greenwich Chemical Consulting, Inc. 3300 PGA Boulevard, Suite 635 Palm Beach Gardens FL 33410 UNITED STATES OF AMERICA 41401175

30 April2014 31 May 2014 30 June 2014

Page 1 of4Harlan StudyNumber: 41401175 Study Specific Supplement Page 2



### Test Item Information as provided by the Sponsor.

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 TestRule and the regulations set forth under 40 CPR799.5087. 

### Special Conditions Study specific parameters:

GSP Section 1.1 Guidelines I Regulations: The study will be conducted according to the following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing of Chemicals (2006) No 201 * "Freshwater Alga and

Cyanobacteria, Growth Inhibition Test" • Method C.3 ofCommission Regulation (EC) No. 761/2009 GSP Section 3.2 Test System: The study will be conducted usingPseudokirchneriella subcapitata GSP Section 3.6 Experimental Conditions: Incubation ofthe test vessels will be in an INFORS Multitron® Version 2 incubator Ifmeasured concentrations are ± 20% ofthe nominal concentrations, results will be based on nominal concentrations. Ifmeasured concentrations are not ± 20% ofnominal concentrations, results will be based on geometric mean measured concentrations. The study report will provide a detailed description of the analytical method that will be developed priorto and utilized during the test. All elements described in item 61 ofOECD 201 will be included in the final test report.

• Annex 5 corrected 28 July 2011Harlan Study Number: 41401175 Study Specific Supplement Page 3


## GENERALINFORMATION Distribution List The original study specific supplement will be retained in the study file. Copies ofthe study specific supplement and general study plan along with any amendments will be distributed as follows: Forthe Sponsor: John V. Massingale Greenwich Chemical Consulting, Inc. Quality Assurance: Harlan Laboratories Ltd, Shardlow, UKHarlan StudyNumber: 41401175


## SIGNATURES
Study Director:

Test Facility Management:

Forthe Sponsor: Study Specific Supplement Page 4

H Vryenhoef

Date:

CMead

Date:

John V. Massingale Greenwich Chemical Consulting, Inc.

Date:

Note: These signatures also acknowledge the procedures described in the corresponding General Study Plan.

## GENERAL STUDY PLAN: 908-25 

## Algal Growth Inhibition Test

TestFacility:

Signatures: Author:

Test FacilityManagement:

QualityAssurance: 

### HarlanLaboratoriesLtd. ShardlowBusiness Park Shardlow Derbyshire DE722GD UK

H VryenhoefBSc

Date: 

## 1 1SEP 2012 J G Riley BSc (Hons) MRQA

•••••••••••••••••••••**~** •••• 10
........**~** ••••••••••••••• Date: 

## 1

# 2 SEP 201!
Page 1of19General Study Plan: 908-25 Page2



### CONTENTS GOOD LABORATORY PRACTICE
.............................................................................................3 GENERAL INFORMATION
.........................................................................................................4 StudyPlanAmendments and Deviations
...............................................................................4 Quality Assurance
..................................................................................................................4 Reporting
...............................................................................................................................4 Archiving
...............................................................................................................................4 1 INTRODUCTION AND PURPOSE
.....................................................................................5 1.1 Guidelines 

### IRegulations
..........................................................................................5 2 TEST AND REFERENCE ITEMS
.......................................................................................6 2.1 Test Item
..................................................................................................................6 2.2 Reference Item
.........................................................................................................6 3 MATERIALS AND METHODS
...........................................................................................6 3.1 Definitions and Abbreviations
.................................................................................6 3.2 Test System
..............................................................................................................7 3.3 TestWater
................................................................................................................7 3.4 Dosage
.....................................................................................................................7 3.5 Materials
..................................................................................................................8 3.6 Experimental Conditions
.........................................................................................8 3.7 Study Design
............................................................................................................ 8 3.8 Re-growth Experiment
.......................................................................................... 10 3.9 Colored Test Items
................................................................................................. 10 3.10 Evaluations
............................................................................................................ 11 3.10.1 Test Organism Observations
................................................................... 11 3.10.2 Water Quality Criteria
............................................................................. 11 3.10.3 Analysis ofTest Item Concentrations
..................................................... 11 3.11 Data Analysis
......................................................................................................... 13 3.11.1 Plotting Growth Curves
........................................................................... 13 3.11.2 Calculation ofGrowthRates
................................................................... 13 3.11.3 Yield
........................................................................................................ 14 3.11.4 Plotting Concentration-Response Curves
................................................ 15 3.11.5 Statistical Analysis
.................................................................................. 15 3.12 Validity Criteria
..................................................................................................... 15 4 REFERENCES
.................................................................................................................... 17 APPENDICES
............................................................................................................................... 18 Appendix 1 CultureMedium
................................................................................................................ 18 Appendix 2 Testing ofVolatile Test ltems
........................................................................................... l9General Study Plan: 908-25 Page3


## GOOD LABORATORYPRACTICE 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 ofDirectives 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·MET!).General Study Plan: 908-25 Page4


## GENERALINFORMATION Study Plan Amendments and Deviations Any intended change to the study plan (general study plan and study specific supplement) 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 ofthe studyplan. Deviations (unplanned changes) from the study plan (general study plan and study specific supplement) will be documented and acknowledgedbythe Study Director. 

### Quality Assurance Quality Assurance will audit and inspect study-related procedures and will report any audit and inspectionresults inwritingto the Study Director and TestFacilityManagement. 

### Reporting A GLP compliant report will be presented. A draft report will be sent to the Sponsor for review and comments before issue ofthe fmal report. 

### Archiving Unless instructed otherwise bythe Sponsor, the studyplan (general study plan and study specific supplement), all rawdata (paper and electronic) andthe final report will be retained inthe 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 ofthe data will be chargeable to the Sponsor. No datawillbediscarded without contactingthe Sponsorto obtaintheirwritten consent.General StudyPlan: 908-25 Page 5


## 1 INTRODUCTIONAND PURPOSE The purpose ofthis study is to assess the acute toxicity ofthe test item to a freshwater alga. The test endpoint is inhibition of growth, expressed as the logarithmic increase in algal biomass (average growth rate) during the exposure period. The toxicity will be expressed as the concentration (or loading rate) bringing about a specified x% inhibition ofgrowth (e.g. 50%) and expressed as the ErCx (orErLx). The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL), and the No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) will also be determined ifapplicable. An additional response variable recommended by the Test Guidelines is yield, which may be required to fulfil specific regulatory requirements in some countries. Results may therefore also be expressed interms ofinhibition ofyield (EyCx orEyLx). The study will use one ofthe following species offreshwater alga which are representative ofa wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems: 

### • Pseudokirchneriella subcapitata • Desmodesmus subspicatus (formerly known as 

### Scenedesmus subspicatus) • Anabaena jlos-aquae • Naviculapelliculosa The dispersal ofthe test item in the surrounding medium is considered to represent the most probable route ofexposure inthe environment. 

### 1.1 Guidelines I

### Regulations The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
• OECD Guidelines for Testing ofChemicals (2006) No 201 "FreshwaterAlga and Cyanobacteria, Growth InhibitionTest" 1

• Method C.3 ofCommission Regulation (EC) No. 761/2009
• JMAFF Test Guidelines 2-7-7

1Annex5 corrected: 28 July 2011General StudyPlan: 908-25 Page6


## 2 TESTAND REFERENCE ITEMS 2.1 Test Item The testitemwill be supplied by or onbehalfofthe Sponsor including the following information if known; description, batch, purity, expiry 

### I retest date, storage conditions and safety precautions to be taken. Where the test item is administered 

### in a vehicle, and the homogeneity, concentration or stability ofthe test item in that vehicle is not determined an impact assessment will be made bythe Study Director, this 

### will be reflected in the fmal report and an exception 

### will be included in the GLP compliance statement. 

### 2.2 Reference Item For evaluation ofthe quality of the algal culture and the experimental conditions, potassium dichromate will be tested as a positive control twice a year to demonstrate satisfactory conditions ofthe test. The reference item will be supplied by a commercial supplier and 

### will be stored as specified by the supplier. 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Biomass:

Growth: Growth Rate (Average Specific Growth Rate):
Yield: Measurement variable (fluorescence or algal cell density) as surrogate measure for algal biomass. The calculated concentration or loading rate oftest item that results in an x% reduction ofthe respective growth parameter relative to the control (or solvent control, ifapplicable) within a stated exposure period. The increase 

### inbiomass overthe test period.
The logarithmic increase in biomass during the exposure period. The value of the measurement variable at the end of the exposure period minus the measurement variable's value at the start of the exposure period, calculated to express biomass increase during the test.General StudyPlan: 908-25

LOEC (Lowest Observed Effect Concentration) orLOEL (Lowest Observed Effect Loading rate): NOEC (No ObservedEffect Concentration) orNOEL (No Observed Effect Loadingrate): Treatment: Section-by-Section Growth Rate:



### 3.2 Test System Page7

The lowest test concentration or loading rate at which a statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable). The highest test concentration or loading rate at which no statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable). Comprises test item treatments, control (test medium only) and solvent control (ifapplicable). Specific growth rate for each day during the course ofthetest (days 0-1, 1-2 and 2-3).

The study will be performed using either Pseudokirchneriella subcapitata, Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus), Anabaenajlos-aquae or Navicula pelliculosa. These freshwater alga species are recommended by the relevant test guideline. The test species used will be identified inthe study-specific supplement ofthe corresponding study. An inoculum culture ofalgae will be set up three to four days before the start ofthe test. The algae will be cultivated under the test conditions and will be kept in the exponential growth phase until inoculation ofthe test solutions. 

### 3.3 Test Water Culture medium as defined inAppendix 1. 

### 3.4 Dosage Ifnecessary, pre-experiments will be performed to determine the solubility and stability ofthe test item in the test water. According to the findings of these pre-experiments, appropriate procedures will be chosento preparethe test media. Thetest item willbe dissolved or dispersed inthe testwater once at the start ofthe test. Methods such as prolonged mixing, ultrasonication, high shear force stirring orthe production of Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the nature ofthe test item. Ifauxiliary solvents or surfactants are used to aid in the dispersion, theyGeneral Study Plan: 908-25 Page 8

will be used at a maximum concentration of 100 J!L/L (final volume). Ifthe test item falls into the category of a "difficult substance" as defined by the OECD Guidance Document (OECD, 2000), the method ofpreparation ofthe test media will be based on the recommendations ofthis document. Ifthe test item is photodegradable, the preparation ofthe test media will be performed as far as possible inthe dark. Details ofthe method ofpreparation will be documented inthe data. 

### 3.5 Materials Glass test vessels containing at least 15 mL test medium will be used. The test vessels will be covered/plugged to reduce evaporation and to avoid entry of dust into the solutions. Volatile chemicals will be tested in completely filled and stoppered test vessels (i.e. test performed in a closed system). The size ofthe test vessels and the volume oftest solution will be documented inthe data. The test vessels will be labeled with the study number and all necessary information to ensure unique identification. 

### 3.6 Experimental Conditions The environmental parameters will be as usedto maintain the stock organisms. The test flasks will be incubated under one ofthe following conditions:
• INFORS Multitron® Version 2 incubator under constant illumination (between4440 and 8880 lux) provided by warm white lighting (380

- 730 nm) at 24 ± 1 

# oc and agitated by orbital shaker (approximately 150 rpm). Temperature, light intensity and oscillation rate ofthe incubation chamberwill berecorded daily throughoutthe test. • Temperature-controlled water bath under constant illumination (between 4440 and 8880 lux) provided by fluorescent tubes installed above the test flasks. The light intensity will notvary by more than ±15% from the average light intensity overthe incubation area. The temperature will be maintained at between 21 and 24°C and maintained at± 2°C. Thetest solutions will be continuously stirredbymagnetic stirrers. 

### 3.7 Study Design The test design will include three replicates per test concentration, six replicates ofthe control and, ifappropriate, sixreplicate solvent control flasks. Ifthe presence ofparticles in the test media could interfere with the determination ofthe algal biomass, eg ifusing a Coulter® Particle Counter, one additional replicate per test concentrationGeneral Study Plan: 908-25 Page9**---------------------------··~·············--····----** containing the test media without algae will be prepared inthe definitive test as particle controls. These additional replicates will be incubated under the same conditions as the test media with algae and will be used for the correction of the algal biomass determination. In cases where preliminary work indicates no counts due to dispersed test item, the blank will consist ofculture medium alone. Depending on the specific requirements ofthe relevant test guidelines, the following conditions will be met:
Initial cell density (cells/mL) Testduration (hours) A static test design will be applied. OECD/EC 2-5 X 103 72 

### JMAFF 5 X 

### 1 03

- 

### 1 X 

### 1 0

4 72

At least five concentrations ofthe 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 ofthe definitive test. Ifin the rangefinding test a flat concentration-effect relationship is determined and, thus, a large concentration range will have to betested, a spacing factor exceeding 2.2 (e.g. 3.2) will be applied. The lowest test concentration should have no significant effect on the growth of algae. The concentration series should preferably cover the range causing 5 to 75% inhibition of algal growth rate. In accordance with the test guidelines (EC, 2002), concentrations ofthe test item higher than 100 mg/L (1000 mg/L for agrochemical formulations tested according to JMAFF guidelines), or above the limit ofsolubility ofthe test item in the test water (whichever is the lower) will usually notbetested. If no effect is observed in the preliminary range-finding test at the highest concentration recommended in the relevant test guideline, or the limit ofsolubility ofthe test item in the test water (whichever is the lower), a Limit Test may be conducted using only this concentration and the corresponding control(s). In this case, the number of test concentration replicates will be doubled to six. The control group will be exposed to the test water alone. Data from the control group may be shared with similarconcurrent studies. Where auxiliary solvents/surfactants are used, a second control 

### will be employed. The test organisms will be exposed to a concentration ofsolvent equal to that employed inthe test series, this solvent concentration will not exceed 100 JJLIL. Data from the solvent control group may be shared with similarconcurrent studies. The test will be carried out without adjustment ofpH. Ifthe pH falls outside the range 6-9, the Sponsor will be contacted for advice on whether a second test should be performed withGeneral StudyPlan: 908-25 Page 10
adjustment ofpHto that ofthe test water. However, pH adjustment will not be carried out ifthe test solutions are changed to any significant extent and ifa chemical reaction or precipitation is caused. 

### 3.8 Re-growth Experiment For JMAFF requirements, a re-growth experiment will be conducted as above using the control (or solvent control) cultures and a single test group that 

### has shown significant inhibition of growth. The samples will be incubated for a further 10 days (maximum) under test conditions with samples of the cultures being taken at 0 hours and periodically thereafter for the measurement ofalgal biomass to determine when/ifre-growth occurs. There-growth experiment will not be carried outifa "limittest" has been conducted. 

### 3.9 Colored Test Items Colored test items may absorb biologically relevant wavelengths and thereby may impair algal growth due to a reduction in the light available for photosynthetic growth. Using the standard algal test method it is not possible to differentiate between reduced algal growth by light absorption ofthe colored test solutions or an inherenttoxic effect ofthe test item. The absorption spectra ofchlorophyll a, the principal photoreceptor in the chloroplasts ofgreen plants, shows a double peak (Bidwell, 1979) with maximum absorption occurring at approximately 460 nm and 665 nm. As such, ifcolored solutions formed by the test item show significant absorbance at either ofthese wavelengths it would reduce the light intensity available for photosynthetic growth to occur. In cases where the**t~st** solutions are shown to have distinct absorbance at460 nm or 665 nm, the range-fmding test and the definitive test will therefore be carried out according to the modification to the standard methodology currently recommended by various competent authorities. This includes reducing the test volume in the range of5- 25 mL in order to shorten the light path through the test medium and increasing the light intensity to approximately 8880 lux (±15%) in order to reduce as much as possible the light absorbance effect ofthe colored test solutions. If, after conducting a range-finding test using the modified method, areduction in algal growth is observed, current regulatory advice is to change the test method to a 

### Lemna Growth Inhibition Test (OECD, 2006) as 

### Lemna are a floating plant species and hence colouration of the test solutions does not affect photosynthetic growth.General StudyPlan: 908-25 Page 11

----------------------------·····--···-············-- 

### 3.10 Evaluations 3.10.1 Test Organism Observations A small volume ofthe algal suspension will be withdrawn from each test flask daily for the measurement ofalgal biomass, and will not be replaced. The algal concentration ofeach sample is determined by direct counting using a Coulter® Particle Counter, haemocytometer and light microscope orby fluorescence measurement. Three determinations are made for each sample. Observations will be made onthe test itempreparations daily during the exposure period. To determine a potential effect ofthe test item on the appearance ofalgal cells, a sample will be takenfrom the test and control cultures atthe end ofthe test. The shape and size ofthe algal cells will be inspectedmicroscopically and any abnormalities recorded.


### 3.10.2 Water Quality Criteria The pH ofthe test media will be measured in all treatments at the start and end ofthe test. The pH ofthe controls should not normally deviate by more than 1.5 pH units during the test. An explanation will be provided in the report ifpH deviations in excess of1.5 units are observed. The water temperature will be measured daily in an Erlenmeyer flask filled with water and incubatedunderthe same conditions as the test flasks. The appearance ofthetest mediawill be recorded daily.


### 3.10.3 Analysis ofTest Item Concentrations 3.10.3.1 Sampling Samples ofthe test media will be taken at initiation and termination ofboth the range-finding and definitive tests for analysis. Where possible duplicate samples will be taken at each occasion, these samplesmay also be analyzed should the need arise. Analysis of samples taken from the range-finding test is performed in order to determine the stability ofthe test item undertest conditions overthe exposure period. The replicate testvessels at each concentration will be pooled to give one sample. The test concentrations provided for analysis will be documentedbythe Study Directorinthe data. For the purposes ofthe definitive test, the replicate test and control (or solvent control) vessels will be pooled to give one sample. Should the analysis ofthe range-finding test samples show a decline in measured test concentration over the test duration then additional test samples may be prepared at test initiation with the omission ofalgal cells and incubated alongside the test from which samples willbetaken for analysis attesttermination.General StudyPlan: 908-25 Page 12

Ifrequired, in order to investigate possible losses oftest item due to volatility when the test item vessels are opened on a daily basis, a fourth replicate vessel may be prepared at 0 hours and incubated alongside the other exposure vessels. This additional replicate will remain unopened until termination ofthe test when itwill be analyzed for test item concentration. Ifthe test medium volume from the actual test is too small to perform the required analyses, requisite volumes of the freshly prepared test media will be incubated separately, but under identical conditions as inthe actual test. Final details ofthe samples to be taken andthe sample size, etc, will be documented inthe data. 3.10.3.2 Storage and samplepreparation All samples will be analyzed immediately after sampling or stored at approximately -20°C until analysis. Ifthe 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 oforganic solvents) in order to maintain the stability ofthe test item until analysis. It may be necessary for some chemicals which form emulsions or dispersions to filter or centrifuge some samples as part ofthe pre-test validation process to determine the proportion of testitem inthe dissolved form. 3.10.3.3 Analysis ofsamples 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 72-Hour NOEC will not normally be analyzed. The remaining samples may be analyzed, ifnecessary. Stock solutions may also be analyzed, ifnecessary, for example ifthe test concentrations are belowthe limit ofquantitation ofthe analytical method or for reasons ofstability. Ifthe test item is a formulation, test item concentrations will be determined by the measured concentrations ofthe active ingredient(s). WAFs will be characterized by analysis ofthe main testitem component(s). 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 ofthe analytical method will not be performed under GLP. Details about the analytical method usedGeneral StudyPlan: 908-25 Page 13

will be given in an Appendix ofthe final report. The accuracy ofthe analytical method will be verifiedbyrecoveries spanning the concentration range to be analyzed withinthis study. Analytical pre-experimentsmaybe performed within a different HarlanLaboratories study. 

### 3.11 Data Analysis 3.11.1 Plotting Growth Curves The cell density in the control and test cultures together with the test item concentrations and times ofmeasurement, recorded to the nearest whole hour, are tabulated and growth curves are constructed byplotting cell density (logarithmic axis) againsttime (arithmetic axis). All data points are critically examined and if it is obvious that procedural mistakes can be identified and/or considered highly likely, the specific data point is marked as an outlier and not included in subsequent statistical analyses. Reasons for rejection ofa data point as an outlier will be clearly stated in the final report. Acceptable reasons are only procedural mistakes and notjustbad precision. Outliers (marked as such) will be retained amongst the datapoints shown inthe final report. 

### 3.11.2 Calculation ofGrowth Rates The average specific growth rate for a specified period is calculated as the logarithmic increase inbiomass from the equation:

where:


## =
= = average specific growthrate from time t1 to tn. cell concentration att1. cell concentration at tn. time offirst measurement. time ofnthmeasurement. The average specific growth rate for each replicate control and test item vessel is calculated for the period 0 - 72 hours using the nominally inoculated cell concentration as the starting value ratherthanthe measured starting value inorderto increase the precision ofthe calculation. In additionthe section-by-section growth rate (0

- 

### 24, 24-48 and 

### 48

- 72 hours) is calculated for the control cultures and the results examined in order to determine whether the growth rate remains constant.General StudyPlan: 908-25 Page 14
Percentage inhibition of growth rate for each replicate test item vessel is calculated using the following equation:
I 

## =J.lc- J.lt X 100

r J.lc where:


## = = percentage inhibition ofaverage specific growthrate. mean average specific growth rate for the control cultures. average specific growthrate for the test culture. Where auxiliary solvents/surfactants are used, percentage inhibition in the test cultures is calculated by comparisonto the solvent control cultures. 

### 3.11.3 Yield Yield is calculated as the increase in biomass over a specified period using the following equation:

where:


## = Yield. cell concentration atthe beginning ofthe specifiedperiod. cell concentration atthe end ofthe end ofthe specified period. For each test concentration and control, the mean value for yield, along with the standard deviation is calculated for the period 0

- 72 hours. Percentage inhibition ofyield is calculated using the following equation:

Where


## = = = percentage inhibition ofyield mean value for yield inthe control group meanvalue for yield for the treatment groupGeneral StudyPlan: 908-25 Page 15



### 3.11.4 Plotting Concentration-Response Curves For each individual test vessel, percentage inhibition (arithmetic axis) is plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation e.g. Xlfit or ToxRat. Any data points identified as outliers in the first phase of data evaluation are disregarded when fitting the concentration-response curve. The EC10, EC2o, ECso (or ELto, EL2o, ELso) values are read from the graph. Where appropriate 95% confidence limits for the EC5o(or ELso) will be calculated, using either the simplified method ofevaluating dose-effect experiments ofLitchfield and Wilcoxon (1949) or by Fieller's Theorem (1944). All EC50 (or EL50) values and 95% confidence limits will be rounded to two significant figures.


### 3.11.5 StatisticalAnalysis The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) is the lowest concentration (or loading rate) tested at which significant (P 

### < 0.05) inhibition ofgrowth relative to the control is observed within a given exposure time. However, all test concentrations above the LOEC (or LOEL) must have an effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation ofhowthe LOEC hasbeen selected will be given inthe testreport. The LOEC (or LOEL) for growth rate and yield at 72 hours will be determined using analysis of variance (ANOVA) incorporating a statistical test for homogeneity ofvariance and an adequate posthoc test (e.g. Dunnetttest). The No Observed Effect Concentration (NOEC) orNo Observed Effect Loading rate (NOEL) is the test concentration (or loading rate) immediately below the LOEC (or LOEL) which, when compared to the control, has no statistically significant effect (P**~** 0.05), within a given exposure time. Where a limit test has been conducted, it is not possible to determine a LOEC (or LOEL), and the NOEC (orNOEL) will be the limittest concentration. 

### 3.12 Validity Criteria Forthe testto be valid, the following performance criteria shouldbemet: The cell concentration of the control cultures must increase by a factor of at least 16 after 

### 72 hours. The mean ofthe coefficients ofvariation ofthe daily section-by-section specific growth rate in the control cultures after 

### 72 hours 

### (0- 24, 24-48 and

### 48 -72hours) must not exceed 35%.

## ., General StudyPlan: 908-25 Page 16
------------------·········-··--·-··-··-·-·· 

### ·-------------

The coefficient variation ofthe average specific growth rate 

### in replicate control cultures after 72 hours mustnot exceed 7%.'I General StudyPlan: 908-25 Page 17 

# 4 REFERENCES BIDWELL, R. G. S. (1979) PlantPhysiology. 2nd ed. London: CollierMacmillan. FIELLER, E. C. (1944) A fundamental formula in the statistics ofbiological assay, and some applications. QuarterlyJournal of Pharmacy andPharmacology, 17, pp. 117-123. HERMAN, D. C., INNISS, W. E. and MAYFIELD, C. I. (1990) Impact of volatile aromatic hydrocarbons, alone and in combination, on growth of the freshwater alga Selenastrum capricomutum.Aquatic Toxicology, 18, pp. 87-100. INTERNATIONAL STANDARDS ORGANISATION (ISO) (1999) Standard 14442: Water quality

- Guidelinesfor algal growth inhibition tests with poorly soluble materials, volatile compounds, metals andwaste water. Geneva: International Standards Institute. LITCHFIELD, 

### J. T. and WILCOXON, F. (1949) A Simplified Method of Evaluating DoseEffectExperiments. J. Pharmacol. Exp. Ther., 96, pp. 99-113. MAYER, P., NYHOLM, N., VERBRUGGEN, E. M. 

### J., HERMENS, 

### J. L. M. and TOLLS, J. (2000) Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. Environmental Toxicology andChemistry, 19, pp. 2551-2556. OECD (2000) Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. OECD Series onTesting and Assessment, No. 23. OECD Publishing. OECD (2006) Test No. 221: Lemna sp. Growth Inhibition Test. OECD Guidelines for the Testing ofChemicals, Section 2: Effects on Biotic Systems. OECD Publishing. EUROPEAN COMMISSION (2002) Sanco/3268/2001 rev. 4 (final) Guidance Document on AquaticEcotoxicology in the contextof Directive 91/414/EEC. ToxRatProfessional, ToxRat® Solutions GmbH Xlfit, ID Business Solutions Ltd.General StudyPlan: 908-25 Page 18


## APPENDICES Appendix 1 Culture Medium Reconstituted test water (AAP/ASTM medium) prepared according to OECD Test Guideline No 201 will be used for algal cultivation and testing. Analytical grade salts will be dissolved in purified waterto obtainthe following final concentrations: Ingredients Concentration Macro-nutrients NaHC03 15.0mg/L K2HP04 1.044 mg/L MgS04.1H20 14.6 mg/L MgCh.6HzO 12.16 mg/L CaCh.2HzO 4.41 m£"!11 NaN03 25.5 mg/L Trace elements H3B03 186.0 ug/L MnCbAH20 415.0 ug/L ZnCh 3.27 ug/L CoClz.6H20 1.43 ug!L CuCb.2H20 O.Ol2ug/L Na2Mo04.2H20 7.26 ug/L FeCh.6H20 160.0 ug/L Na2EDTA.2HzO 300.0 uwL The pH will be adjusted to 7.5. The water hardness (calculated) of the test water will be 0.15 mmol/L (= 15 mg/L as CaC03). In tests conducted using Navicula pelliculosa the culture medium will be supplemented with Na 2

Si03.9H20 ata final concentration of 1.4 mg Si/LGeneral Study Plan: 908-25 Page 19
Appendix 2 TestingofVolatile TestItems
In cases where the test item is known (or suspected) to be volatile, the following amendments to the study design may be made in order to minimize possible losses oftest item over the test period:
• The test vessels used will be glass conical flasks completely filled with test preparation and sealed with ground glass stoppers.
• Additional sodium bicarbonate (up to 500 mg/L final concentration) may be added to the prepared culture medium as defined by Herman 

### et al (1990) and Mayer 

### et al (2000) to counter act the change in pH due to algal growth. The pH ofthe culture medium will be adjusted to 7.5 ± 0.1 with 

### O.lN NaOH orHCl priorto use.
• HEPES buffer (1430 mg/L) may be added to the prepared culture medium to counter act .the change inpHdue to algal growth (ISO, 1999)
• In order to investigate possible losses oftest item due to volatility when the test vessels are opened on a daily basis a fourth replicate at each concentration will be prepared at 0 hours and incubated alongside the other exposure vessels. This fourth replicate will remain unopened until termination of the test when it will be analyzed for test item concentration. Details ofthese amendments to the study designwill be documented in the data.