GREENWICH CHEMICAL CONSULTING, INC. 3300 PGA BOl:LEVARD • SUITE 635

PALM BEACH GARDENS, FL 33410 TELEPHONE 561.578.8110 FACSIMILE 561.578.8120 April 19,2012 .j) 

# .. CONTAINSNO CONFIDENTIAL 

## 'f, BUSINESS INFORMATION By FederalExpress
OPPTDocumentControl Office EPA EastBuilding

# IIIIIIIII/IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~1111111111III/1111 40120000061 Room E6428 U.S. Environmental Protection Agency 1201 ConstitutionAve., NW Washington, DC 20004 Attention: DocketID Number: EPA-HQ-OPPT-2007-0531 40 CFR 799.5087
Re: Submission of Eight Study Plans for Tests Conducted by Univar USA and WEGO Chemical and Mineral under the TSCA Section 4 Test Rule on the 

### Testing ofCertain High Production Volume Chemicals: Second Group of Chemicals Ethanedioic acid CAS RN 144-62-7 (Oxalic acid) DocketIdentificationNo. EPA-HQ-OPPT-2007-0531 40 CFR799.5087
Dear Sir/Madam: On behalf of Univar USA Inc. and WEGO Chemical and Mineral Corporation (the Companies), Greenwich Chemical Consulting, Inc. (Greenwich Chemical Consulting) 

## CONTAINS NO CSICONTAINS NO CONFIDENTIAL BUSINESS INFORMATION U.S. Environmental Protection Agency April 19,2012 Page 2 submits to the U.S. Environmental Protection Agency (EPA or the Agency) the study plans for eight tests to be conducted on the chemical substance, ethanedioic acid [CAS RN 144­ 62-7] which is currently subject to a Toxic Substances Control Act ([SCA) Section 4 test rule. 1 For purposes of this letter, the chemical substance ethanedioic acid is herein referred to as oxalic acid. The Companies flle these study plans in accordance with this test rule and the regulations set forth at40 CFR790.50 and 799.5087. I. General Background Greenwich Chemical Consulting functions and operates as the panel manager for and authorized representative of the Companies for the testing of oxalic acid under this TSCA Section 4 test rule. Therefore, Greenwich Chemical Consulting flIes these study plans for eight of the nine tests on behalf of the Companies. (As discussed below, the chromosome aberration studyplan has already been submitted to EPA.) This test rule addresses 19 high productionvolume (HPV) chemicals that went unsponsored under the Agency's voluntary HPV Chemical Challenge Program. One ofthe 19 chemicals identifled in the rule is oxalic acid. The flnal rule identifles the following nine studies on oxalic acid: • MeltingPoint • BoilingPoint • Vapor Pressure • Water Solubility • Ready Biodegradation • Aquatic ToxicityTesting: Acute toxicity to flsh Acute toxicity to Daphnia Toxicity to plants (algae) • InVitro Mammalian ChromosomeAberrationTest II. Prior Submission of the Chromosome Aberration Test Study Plan and the Current Submission ofthe Remaining EightTests As required under 40 CFR 790.50 and 799.5087, the Companies are required to submit to the Agency study plans prior to the initiation of testing. 2 Because the chromosome aberration test is the most lengthy of the nine studies identifled for oxalic acid, the Companies previously submitted this study plan, and the Agency has responded to this plan.' Atthis time, the Companies herein submit to EPA study plans for theremaining eight tests.

1January 7, 2011; 76 Fed. 1067. 2 It is noted that, unlike other HPV test rules, the rule identifying oxalic acid does not specify a date by which studyplans must be submitted to the Agency. 3 Submission from Greenwich Chemical Consultingto the_\gency dated February 2012.CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION U.S. EnvironmentalProtectionAgency April 19, 2012 Page 3 The Companies have selected CiToxLAB Hungary Ltd. (CiToxLAB) to conduct all nine tests, and the laboratory will conduct these studies at a single location identified in the study plans. Also, Greenwich ChemicalConsultingis serving as the study monitor for this testing. Among other things, all of the study plans identify administrative information, the TSCA Section 4 test rule, the oxalic acid test material, primary laboratory personnel, study objectives, test guidelines, materials and methods, observance of Good Laboratory Practice standards, and otherrelated testing andproceduralinformation. EPA will note that final versions of the four physical/chemical properties study plans are included herein whereas draft versions of the biodegradation and three ecotoxicity study plans are provided. We believe that few, if any, amendments to the physical/chemical properties study plans will be required by EPA. However, based on the Companies' experience with the Agency's review of the chromosome aberration study plan, some changes may be required by EPA. From a procedural point, therefore, CiToxLAB recommended that drafts ofthese four plans be providedinstead offinal versions. III. Use ofOEeDTest Guidelines In the final TSCA Section 4 test rule, EPA specified the use ofASTM, ISO, or CFR test guidelines for the nine studies. In its letter dated November 16, 2011, Univar requested that the corresponding OECD test guidelines be used by CiToxLAB in the conduct of all of these studies. On January 10, 2012, as well as February 23, 2012, the Agency granted this request.4 

### * * * The Companies thank the Agency for its consideration in this matter. For all questions, comments, and determinations concerning this matter, please use the contact information given below. Mr. John V. Massingale Greenwich ChemicalConsulting, Inc. 3300 PGABoulevard, Suite 635 PalmBeach Gardens, FL 33410 Telephone: (561) 578-8110 Facsimile: (561) 578-8120 E-mail: john@grcci.com
.\ Electronic mails from Dr. Paul 

### J. Campanella (EPA) to Mr. John Y . .\Iassingale (Greemllich Chemical Consulting).CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION U.S. EnvironmentalProtection Agency April 19, 2012 Page 4 Sincerely, JohnV. Massingale Principal

cc: Dr. P. Campanella
- EPA Uruvar USA Inc. WEGO ChemicalandMineral Corporation

Enclosures

## CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta



# STUDY PLAN
Determination ofthe Boiling Point ofEthanedioic acid

Study code: 11/339-324AN Study Director: Z501t Sarvari, M.Sc.

03 April 2012 

## CONTAINS NO CBIStudy code: 111339·324AN Study Plan Page 2 of 9

STUDY TITLE:
SPONSORS:

STUDY MONITOR:

TEST FACILITY:

TESTFACILITY MANAGEMENT:

STUDY DIRECTOR:

ASSISTANT SCIENTIST: Determination ofthe Boiling Point ofEthanedioic acid
Univar USA Inc. Address: 17425 NE Union Hill Road, Redmond, W A 98052, USA Phone: +1 425 8893400 Fax: + 14258893760 WEGO Chemical and Mineral Corp. Address: 239 GreatNeck Road, GreatNeck,NY 11021, USA Phone: +15164873510 Fax: +1 5164873794 John V. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410, USA Phone: +} 561 5788110 Fax: +15615788120 E-mail:john@grcci.com CiToxLAB Hungary Ltd. (Fonnerly known as LAB Research Ltd. before Ist September 2011) Address: H-8200 Veszprem, Szabadsagpuszta Phone: +36 88 545 300 Fax: +3688545301 ChristopherBanks, DABT Managing Director Alyson Leyshon, M.Sc. Senior Director ofToxicology Operations Zsolt Sarvari M.Sc. Phone: +36 88 545 361 E-mail: zsolt.sarvari@hu.citoxlab.com Vikt6ria Laky, M.Sc.Study code: 111339-324AN Study Plan Page 3 of 9 LEAD QA*:

RESPONSIBLE PERSONS*:
BASIS OF STUDY:

REPORTING FORMAT:
REPORT COPIES: Szabo1cs Gaty M.Sc. Head ofQuality Assurance Unit Yanda Gyimesi, M.Sc. Deputy Head ofQAU lldik6 Kacz - technical assistant

- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Method A.2, Official Journal of the European Union L 142 of31 May 2008

- Organisation for Economic Co-operation

and Development (OECD), Guidelines for Testing of Chemicals, Guideline No. 103, "Boiling Point", adopted July 27, 1995 Final Report III line with 40 CFR 792 format requirements Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by courier
PROPOSED START OF EXPERIMENT: 04 April 2012 PROPOSED END OF EXPERIMENT: not later than 13 April 2012 DRAFTREPORT: not later than 27 April 2012

* Othertrained, competent personnel could be involved in the study, ifrequired.Study Gode: 11/339-324AN Study Plan Page 4 of 9 STUDYPLAN APRROVAL:

APPROVED BY ON BEHALFOF SPONSOR:

APPROVED BY ON BEHALF OF CiToxLAB:

STUDY DIRECTOR:

QUALITY ASSURANCE: DATE: ChristopherBanks, DABT Managing Director

Zsolt Sarvari, M.Sc.

### 7~-**U~** Yanda Gyimesi, M.Sc. OnbehalfofQAUStudy code: 11/339·324AN Study Plan Page 5 of 9
1. OBJECTIVE OF STUDY Forthe detennination ofthe boiling point, the capillary method will be used. A small amount ofthe test item will be heated in the electronic controlled sample block and the temperature around the sample will be displayed. The sample will be observed visually while raisingthe temperature. A phase transfonnation from liquid to vapour is usually deduced by the fonnation ofbubbles. The test item will be assessed visually for any possible changes in its appearance, consistency and colour. The initial temperature of boiling or other effects observed will be registered with the corresponding heatingtemperature. This study is being perfonned based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087.
2. MATERIALS AND METHODS 2.1 . TEST ITEM Name: Chemical name: Synonym: CAS number: BatchlLotnumber: Molecular formula: Molecularweight: Appearance: Purity: Manufacture date: Expiry date: Storage conditions: 2.1.1. Identification and receipt Ethanedioic acid Ethanedioic acid Oxalic acid 144-62-7 YP2-20110704B C2H20 4x2H20 126.1 (dihydrate form) White crystals 99.71 % 04July 2011 03 July 2012 Controlled room temperature (15-25°C, < 70 RH%)

The test item ofa suitable chemical purity was supplied by the Sponsor. All safety instructions required in the handling and disposal ofthe test item were supplied by the Sponsor as well. Identification of the test item was made by its name, batch, appearance and colourby Central Dispensary Unit ofCiT oxLAB Hungary Ltd.Study code: 11'339-324AN Study Plan Page 6 of 9
2.2. TESTPARAMETERS AND CONDITIONS Test System:

2.3. 2.3.1. Accuracy: STUDY DESIGN Test Item Treatment The test will be carried out using a digital boiling point device (Biichi B540 Melting Point Unit, B540 (No.: 908281») with a temperature range up to 400°C (673 K). 0.5°C

Thetest item will be used in the initial form. 2.3.2. PreliminaryTest The boiling behaviour oftest item will be estimated in a preliminary test. For this, a sample oftest item placed in a glass tube will be heated starting at room temperature up to about 400°C at a rate ofapproximately 10°C/min unless a phase transition orreaction ofthetest item is observed. The sample is observed during the experiment and the temperature will be recorded for each observation documented. 2.3.3. Main test If in the temperature range investigated in the preliminary test, a phase transition ofthe test item is observed, the experiment wiII be repeated with a new test item aliquot in the immediate vicinity ofthe temperature ofthe phase transition observed. For the actual determination of the boiling point, the temperature rise will be adjusted to a maximum of0.5 DC/min. The test conditions and temperature measured by a resistance thermometer located in the block will be documented in the raw data. This experiment will be performed in at least duplicate, ifa boiling is observed. Each day oftesting the atmospheric pressure will be registered.Study code: 11/339·324AN Study Plan Page 7 of 9

2.3.4. Definitions The standard boiling point is defined as the temperature at which the vapour pressure ofa liquid is the same as the standard pressure (101.325 kPa). 2.3.5. Observations Upon approach ofthe boiling temperature bubbles will emerge rapidly from the bottom of the capillary. The boiling temperature is that temperature at which, on momentary cooling, the formation ofbubbles stops and the liquid suddenly rises in the capillary. 2.3.6. Data Handling The measured boilingpoint is depending on the atmospheric pressure. Therefore, the boiling point of a test item is defined as the temperature at which phase transition from the liquid state to vapour takes place at normal atmospheric pressure. This temperature ideally corresponds to the condensation temperature. As the phase transition ofmany test item takes place over a large temperature range, it is often described as the boiling range. Results are given in degrees centigrade and in Kelvin. Conversion ofunits: t [0C] = T [K] - 273.15 where, t Celsiustemperature, degrees Centigrade [0C] T Thermodynamic temperature, Kelvin [K] A correction to normalised pressure can be made. Because the correction factor for the boiling point is a specific parameter for each test item, the correction factor has to be determined. The determination ofthis correction factor is beyond the scope of this study, therefore no correction will be applied to the boilingpoint.Study code: 11/339·324AN Study Plan Page 8 of 9 3. AMENDMENT AND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe study director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of study director's signature. All such amendments will be sent to the sponsor. The study plan amendmentwill be distributed and added to all copies ofthe study plan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect on the outcome ofthe study. 4. GOOD LADORATORYPRACTICE (GLP) AND QUALITY ASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be performed in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. (111.30.) EOM-FVMjoint decree ofthe Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENV/MC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practice regulations specified by EPA 40 CFR 792. The Quality Assurance Unit will conduct inspections of the study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures. 5. ARCHIVES The study documents: study plan and any amendments, all raw data, sample ofthe test item, study report and any amendments, correspondence will be stored according to the Hungarian GLP regulation and to the CiToxLAB Hungary Ltd.'s SOP-s in the archives ofCiT oxLAB Hungary Ltd. 8200 Veszprem, Szabadsagpuszta, Hungary.Study code: 11/339-324AN Study Plan Page 9 of 9 6. STUDY REPORT The study report will include the following information: Test item: chemical information Test method: detailed description ofthe method used Results: all measured and observed data 7. DISTRIBUTION OFTHE STUDY PLAN Archive original Sponsor pdf-file Study Director 1x copy 

### QAU 1x copy

## CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta



# STUDY PLAN Determination ofMelting Point ofEthanedioic acid

Study code: 11/339-344AN Study Director: Zsolt Sarvari, M.Sc.

03 April 2012


## CONTAINS NO C2!Study code: 111339·344AN Study Plan Page 2 of 9
STUDY TITLE: SPONSORS:

STUDY MONITOR:

TESTFACILITY:

TEST FACILITY MANAGEMENT:

STUDY DIRECTOR: Determination ofthe Melting Point ofEthanedioic acid Univar USA Inc. Address: 17425 NE Union Hill Road, Redmond, W A 98052, USA Phone: +I 425 889 3400 Fax: +I 425 889 3760 WEGO Chemical and Mineral Corp. Address: 239 GreatNeck Road, GreatNeck, NY 11021, USA Phone: +1 5164873510 Fax: +1 5164873794 John V. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410, USA Phone: +1 561 5788110 Fax: +1 561 5788120 E·mail:john@grcci.com CiToxLAB Hungary Ltd. (Fonnerly known as LAB Research Ltd. before 1st September 2011) Address: H·8200 Veszprem, Szabadsagpuszta Phone: +3688545300 Fax: +3688545 301 ChristopherBanks, DABT Managing Director Alyson Leyshon, M.Sc. Senior Director ofToxicology Operations Zsolt Sarvari M.Sc. Phone: +36 88 545 361 E-mail: zsolt.sarvari@hu.citoxlab.comStudy code: 11/339-344AN Study Plan Page 3 of 9 ASSISTANT SCIENTIST: Vikt6ria Laky, M.Sc. LEADQA*:

RESPONSIBLE PERSONS*:
BASIS OF STUDY:

REPORTING FORMAT:
REPORT COPIES: Szabolcs Gaty M.Sc. Head ofQuality Assurance Unit YandaGyimesi, M.Sc. Deputy Head ofQAU JIdik6 Kacz - technical assistant

- Commission Regulation (EC)No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Method AI, Official Journal ofthe European Union L 142 of31 May 2008

- Organisation for Economic Co-operation and

Development (OECD), Guidelines for Testing of Chemicals, Guideline No. 102, "Melting Point / Melting Range"; adopted July 27, 1995 Final Report in line with 40 CFR 792 format requirements Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by courier
PROPOSED STARTOF EXPERIMENT: 04 April 2012 PROPOSED END OF EXPERIMENT: not later than 13 April 2012 DRAFT REPORT: not later than 27 April 2012

* Othertrained. competent personnel could be involved in the study, ifrequired.Study code: 111339·344AN Study Plan Page 4 of 9

---..**-.-.~--** STU])YPLANAPRROVAL:

APPROVED BY ON BEHALF OFSPONSOR:

APPROVED BY ON BEHALF OF CiToxLAB:

STUDY DIRECTOR:

QUALITY ASSURANCE:

### O~.~ 

# ..... . ChristopherBanks, DABT ManagingDirector

Zsolt Sfuvari, M.Sc.

## VnA-~'**lJ~** DATE: 

### 0;' 

# 1Jr..( (o.{ L

## ~anda Gyimesi, M.Sc. OnbehalfofQAUStudy code: 111339·344AN Study Plan Page 5 of 9
1. OBJECTIVE OFTHE STUDY For the determination ofthe melting point or melting range, the capillary method will be used. A small amount ofthe test item will be heated in the electronic controlled sample block and the temperature around the sample will be displayed. The sample will be observed visually while raising the temperature. The initial and final melting temperatures will be determined. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth under40 CFR 799.5087.
2. MATERIALSAND METHODS 2.1. TEST ITEM Name: Chemical name: Synonym: CAS number: Batch/Lot number: Molecular formula: Molecularweight: Appearance: Purity: Manufacture date: Expiry date: Storage conditions: 2.1.1. Identification, Receipt Ethanedioic acid Ethanedioic acid Oxalic acid 144-62-7 YP2-20110704B C2H204x2H20 126.1 (dihydrate form) White crystals 99.71 % 04 July 2011 03 July 2012 Controlled room temperature (15-25°C, < 70 RH%)

The test item of a suitable chemical purity was supplied by the Sponsor. All safety instructions required in the handling and disposal ofthe test item were supplied by the Sponsoras well. Identification ofthe test item was made by its name, batch, appearance and colour by Central Dispensary Unit ofCiToxLAB Hungary Ltd.Study code: 11/339·344AN Study Plan Page 6 of 9
2.2. TESTPARAMETERS AND CONDITIONS Test System: Surroundings: Accuracy: 2.3. TESTMETHOD The test will be carried out using a digital melting point device (BUchi B540 Melting Point Unit, (No.: 908281)) with a temperature range up to 400°C (673 K). The test will be performed in air atmosphere at room temperature. < 0.5 

### °c

Before test item analysis, melting point unit will be checked by measuring the melting point of three reference substances: vanillin (mp: 82.3 0c), phenacetin (mp: ]35.3 0c) and caffeine (mp: 236.7 0c). Melting point measurement of test item will be started following the verification ofreference substance's melting point. 2.3.1. Preparation ofthe Glass Tube A small amount oftest item will be charged in a capillary glass tube with a diameter of about 2 mm and packed tightly to obtain a filling level ofapproximately 5 mm. The tube will be heated in the oven ofthe melting point device. 2.3.2. Preliminary Test The melting behaviour oftest item will be estimated in a preliminary test. The test item will be heated in a glass tube from room temperature up to about 400°C with a rate of approximately 10 °Ctmin unless a phase transition orreaction ofthetest item is observed. The sample will be observed during the experiment and the temperature will be read and documented for each observation. 2.3.3. Main Test Ifin the temperature range investigated in the preliminary test, a phase transition oftest item is observed, the experiment will be repeated with a new test item aliquot in the immediate vicinity of the temperature of the phase transition observed. For the actual determination ofthe melting point, the temperature rise will be adjusted to a maximum of 0.5°C/min. This experiment will be performed at least in duplicate.Study code: 11f339-344AN Study Plan Page 7 of 9 2.3.4. Definitions The melting point ofa test item is defined as the temperature at which phase transition from a solid to liquid state occurs at normal atmospheric pressure. 2.3.5. Observations Finely pulverised test items usually show the following stages of melting when a low heating rate is applied: Stage A: Beginningof melting/ wetpoint Fine droplets adhere uniformly on the inside wall ofthe capillary tube. Stage B: Shrinkagepoint A clearance appears between the sample and the inside wall due to shrinkage ofthe melt. Stage C: Col/apsepoint The shrunken sample begins to collapse downwards and liquefies. Stage D: Liquefyingpoint A complete meniscus is formed at the surface, but an appreciable amount ofthe sample remains solid. Stage E: FinalStage of melting There are no solid particles. Duringthe determination ofthe melting temperature, the temperatures are recorded at the beginning ofthe melting and atthe final stage. 2.3.6. DataHandling The melting point ofthe test item ideally corresponds to the solidification or freezing temperature. As the phase transition ofmany test items takes place over a large temperature range, it is often described as the melting range. Ifthe difference between the temperature at the beginning and at the final stage of melting is within the limits ofthe accuracy ofthe method, the temperature at the final stage ofmelting is taken as the melting temperature; otherwise the two temperatures are reported. Results are given in degrees centigrade and in Kelvin. Conversion ofunits: t [0C] =T [K] - 273.15 where t Celsius temperature, degrees Centigrade [0C] T 

### = Thermodynamic temperature, Kelvin [K]Study code: 11/339-344AN Study Plan Page 8 of 9 3. AMENDMENT AND DEVIATIONPROCEDURES Planned changes to the study plan can be made at the discretion ofthe study director and will be agreed with the Sponsor. Detailed descriptions ofall study plan amendments will be effective at the time ofstudy director's signature. All such amendments will be sent to the sponsor. The study plan amendment will be distributed and added to all copies ofthe study plan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect on the outcome ofthe study.
4. GOODLABORATORYPRACTICE (GLP) AND QUALITY ASSURANCE In orderto enablethe results ofthis study to meet legal requirements including regulatory submission and/or safety assessment, it will be performed in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. (1ll.30.) EilM-FVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENVIMC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code ofFederal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practice regulations specified by EPA 40 CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases of the study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures. 5. ARCHIVES The study documents: study plan and any amendments, all raw data, sampleofthe test item, study report and any amendments, correspondence will be stored according to the Hungarian GLP regulation and to the CiToxLAB Hungary Ltd.'s SOP-s in the archives of CiToxLAB Hungary Ltd 8200 Veszprem, Szabadsagpuszta, Hungary.Study code: 11/339·344AN Study Plan Page 9 of 9 6. STUDY REPORT The study report will include the following information: Test item: chemical information Test method: detailed description ofthe method used Results: all measured and observed data 7. DISTRIBUTION OF THE STUDY PLAN Archive original Sponsor pdf-file Study Director 1 x copy 

### QAU ] x copy

## CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta



# STUDY PLAN
Determination ofthe Vapour Pressure ofEthanedioic acid

Study code: 

### 11/339-323AN Study Director: Zsolt Sarvan, M.Sc.

03 April 2012


## CONTAINS NO CB!Study code: 11/339·323AN Study Plan Page 2 of 8 GENERALINFORMATION STUDY TITLE: SPONSORS:

STUDY MONITOR:

TEST FACILITY:

TESTFACILITY MANAGEMENT:

STUDY DIRECTOR:

ASSISTANT SCIENTIST: Determination ofthe Vapour Pressure ofEthanedioic acid Univar USA Inc. Address: 17425 NE Union Hill Road, Redmond, W A98052, USA Phone: +1 425 889 3400 Fax: +1 4258893760 WEGO Chemical and Mineral Corp. Address: 239 Great NeckRoad, GreatNeck, NY 11021, USA Phone: + I 516 487 3510 Fax: +1 5164873794 John V. Massingale Greenwich Chemical Consulting, Inc. Address: 3300PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410, USA Phone: +1 561 5788110 Fax: +1 561 5788120 E-mail:john@grcci.com CiToxLAB Hungary Ltd. (Fonnerly known as LAB Research Ltd. before Ist September20II) Address: H-8200 Veszpn5m, Szabadsagpuszta Phone: +3688545300 Fax: +3688545301 ChristopherBanks, DABT Managing Director Alyson Leyshon, M.Sc. Senior Director ofToxicology Operations ZsoIt Sarvari M.Sc. Phone: +36 88 545 361 E-mail: zsolt.sarvari@hu.citoxlab.com Viktoria Laky, M.Sc.Study code: 111339-323AN Study Plan Page 3 of 8 LEADQA*:

RESPONSIBLE PERSONS*:
BASIS OF STUDY:

REPORTING FORMAT:
REPORT COPIES: Szabolcs Gaty M.Sc. Head ofQuality Assurance Unit Vanda Gyimesi, M.Sc. Deputy Head ofQAU Ildik6 Kacz - technical assistant

- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 ofthe European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Method AA, Official Journal of the European Union L 142 of31 May 2008

- Organisation for Economic Co-operation

and Development (OECD), Guidelines for Testing ofChemicals, Guideline No. 104, "Vapour Pressure", adopted March 23, 2006 Final Report in line with 40 CFR 792 format requirements Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by courier
PROPOSED STARTOF EXPERIMENT: 04 April 2012 PROPOSED END OFEXPERIMENT: not later than 13 April 2012 DRAFTREPORT: not laterthan 27 April 2012


### *Othertrained, competent personnel could be involved in the study, ifrequired,Study code: 11/339-323AN Study Plan Page 4 of 8 STUDY PLAN APIU{OVAL:

APPROVED BY ON BEHALF OF SPONSOR:

APPROVED BY

# ;t.1'~-.. D'? j4.

# ~ ( 

### 2..ID 

### 1'-.. DATE:



### __ ON BEHALF OF CiToxLAB: ChristopherBanks, DABT Managing Director

STUDYDIRECTOR:

### ~1f.V-'**V~** DATE: 0 'J

### ~.( 10Ic QUALITY ASSURANCE: Yanda Gyimesi, M.Sc. OnbehalfofQAU ZsoltSlirvw:i, M.Sc,Study code: 11/339·323AN Study Plan Page 5 of 8

1. OBJECTIVE OF STUDY The measuring principle is equivalent to ASTM 02879-86 (isoteniscope principle) but with only 1ml sample volume. The instrument consists ofa vacuum-sealed measuring chamber ofan overall volume of5 m!. A piezoresistive pressure transducer is integrated in the vacuum chamber. A vacuum valve is installed in the chamber for automatic outgassing of the sample. Cooling and heating is performed with a Peltier element. 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 CFR799.5087. 2. MATERIALSAND METHODS 2.1. TEST ITEM Name: Chemical name: Synonym: CAS number: Batch/Lot number: Molecular formula: Molecularweight: Appearance: Purity: Manufacture date: Expiry date: Storage conditions: Ethanedioic acid Ethanedioic acid Oxalic acid 144-62-7 YP2-20110704B C2H204x2H20 126.1 (dihydrate form) White crystals 99.71 % 04 July 2011 03 July 2012 Controlled room temperature (15-25°C, < 70 RH%) 2.1.1. Identification and Receipt The test item ofa suitable chemical purity was supplied by the Sponsor. All safety instructions required in the handling and disposal ofthe test item were supplied by the Sponsoras well. Identification ofthe test item was made by its name, batch, appearance and colour by Central Dispensary Unit ofCiToxLAB Hungary Ltd.Study code: 11/339·323AN Study Plan Page 6 of 8
2.2. TEST PARAMETERS AND CONDITIONS Test System: MINIVAP VOC, Grabner Instruments, Austria (Serial No.: 08-01-108), DUO 2.5A type two stage Rotary Vane Vacuum Pump, Pfeiffer Vacuum GmbH Limit ofMeasurement: 0.5 kPa 2.3. STUDY DESIGN 2.3.1. Test Item Treatment The test item will be used in the initial form. 2.3.2. Reference Material Water: (prepared by MILLIPORE DIRECT Q3 waterpurification system) The instrument will be checked at the temperatures below. The deviation from the literature data(see below) must be less than 10%. 20°C 2.34 kPa 25°C 3.17 kPa 30°C 4.24 kPa 35°C 5.62 kPa 40°C 7.38 kPa 45°C 9.58 kPa 50°C 12.33 kPa 55°C 15.74kPa 60°C 19.92 kPa 65°C 25.00 kPa 70°C 31.16 kPa 75°C 38.54 kPa 80°C 47.34 kPa 2.3.3. Main Test The sample will be filled into the sample cup and a fully automatic determination of the vapour pressure will be carried out based on the isoteniscope principle. Dissolved air in the sample will be removed by evacuation at 20°C. The vapour pressure as a function oftemperature will be registered in a suitable range in 5 °C steps. The lowest detectable vapourpressure is 0.5 kPa.Studycode: 111339·323AN Study Plan Page 7 of 8 2.3.4. Data Handling The vapour pressure curve (log p versus 1 ff) will be prepared. A line will be fitted to the measured data applying the least squares method with the help of Excel for Windows software. The vapour pressure will be back calculated from the regression line for 20 and 25°c' The mean ofat least two measurements will be reported. 3. AMENDMENT AND DEVIATIONPROCEDURES Planned changes to the study plan can be made at the discretion ofthe study director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of study director's signature. All such amendments will be sent to the sponsor. The study plan amendment will be distributed and added to all copies ofthe study plan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect on the outcome ofthe study. 4. GOODLABORATORYPRACTICE (GLP) AND QUALITYASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be perfonned in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. (111.30.) EiiMFVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENV/MC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practice regulations specified by EPA 40CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard OperatingProcedures.Study code: 11/339·323AN Study Plan Page 8 of 8 5. ARCHIVES The study documents:

- study plan and any amendments,

- all raw data,

- sample ofthe test item,

- study report and any amendments,

- correspondence will be stored according to the Hungarian GLP regulation and to the CiToxLAB Hungary Ltd.'s SOP-s in the archives ofCiToxLAB Hungary Ltd. 8200 Veszprem, Szabadsagpuszta, Hungary. 6. STUDYREPORT The study reportwill include the following information: Test item: chemical information Test method: detailed description ofthe method used Results: all measured and observed data 7. DISTRIBUTIONOFTHE STUDYPLAN Archive original Sponsor pdf-file Study Director Ix copy QAU 1x copy

## CiToxLAB Hungary 

## Ltd. Veszprem - Szabadsagpuszta



# STUDY PLAN
Determination ofthe Water Solubility of Ethanedioic acid

Study code: 

### 111339-345AN Study Director: Zso1t Sarvari, M.Sc.

17 April 2012Study code: 11f339·345AN Study Plan Page 2 of 8
STUDY TITLE:
SPONSORS:

STUDY MONITOR:

TESTFACILITY:

TESTFACILITY MANAGEMENT:

STUDY DIRECTOR:

ASSISTANT SCIENTIST: Detennination ofthe Water Solubility ofEthanedioic acid
Univar USA Inc. Address: 17425 NE Union Hill Road, Redmond, W A 98052, USA Phone: + I 425 889 3400 Fax: +1 4258893760 WEGO Chemical and Mineral Corp. Address: 239 GreatNeck Road, Great Neck, NY 11021, USA Phone: +1 5164873510 Fax: +1 5164873794 John V. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410, USA Phone: +15615788110 Fax: +1 561 5788120 E-mail: john@grccLcom CiToxLAB Hungary Ltd. (Formerly known as LAB Research Ltd. before 1st September 2011) Address: H-8200 Veszprem, Szabadsagpuszta Phone: +36 88 545 300 Fax: +3688545301 Christopher Banks, DABT Managing Director Alyson Leyshon, M.Sc. Senior DirectorofToxicology Operations Zsolt Sarvari M.Sc. Phone: +36 88 545 361 E-mail: zsolt.sarvari@hu.citoxlab.com OlgaUlbert, M.Sc.Study code: 11f339·345AN Study Plan Page 3 of 8 LEAD QA"':

RESPONSIBLE PERSONS"':
BASIS OF STUDY:

REPORTING FORMAT: REPORTCOPIES: Szabolcs Gcity M.Sc. Head ofQuality Assurance Unit YandaGyimesi, M.Sc. Deputy Head ofQAU I1dik6 Kacz - technical assistant

- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and ofthe Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Method A.6. "Water Solubility", Official Journal ofthe European Union L 1420f31 May

- Organisation for Economic Co-operation and

Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline No. 105, "Water Solubility", adopted July 27, 1995 Final Report in line with 40 CFR 792 format requirements Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by courier
PROPOSED STARTOF EXPERIMENT: TBD PROPOSED END OF EXPERIMENT: TBD DRAFTREPORT: TBD TBD: to be determined and documented by an amendment

* Othertrained, competent personnel could be involved in the study. ifrequired.Study code: 11/339-345AN Study Plan Page 4 of 8 STUDYPLANAPRROVAL:

APPROVED BY ON BEHALF OF SPONSOR:

APPROVED BY ONBEHALF OF CiToxLAB:

STUDY DIRECTOR:

QUALITYASSURANCE: DATE: I**~** /lPP'-{ 201"Z

### \......-r-~ ChristopherBanks, DABT Managing Director

## ~~--...:**~** Zsolt Sarvari, M.Sc.


### 0,**<LtA-~: tI~**  / Yanda Gyimesi, M.Sc. OnbehalfofQAU DATE: \'\

### ~',,,-u.')

# ,'­

DATE: 

### ;f ';t

### ~.( (0) 'LStudy code: 11f339·345AN Study Plan Page 5 of 8
1. OBJECTIVE OF STUDY The purpose of this study is to determine the water solubility of Ethanedioic acid, which is specified by the saturation mass concentration ofthe test item in water at 20 

### ± 1°C. As the expected water solubility is above 10.2gil, the flask method will be applied. 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 40CFR 799.5087. 2. MATERIALSAND METHODS 2.1. TEST ITEM Name: Ethanedioic acid Chemical name: Ethanedioic acid Synonym: Oxalic acid CAS number: 144-62-7 BatchlLot number: YP2-20II0704B Molecular formula: C2H204x2H20 Molecular weight: 126.1 (dihydrate form) Appearance: White crystals Purity: 99.71 % Manufacture date: 04 July 2011 Expiry date: 03 July 2012 Storage conditions: Controlled room temperature (l5-25°C, < 70 RH%) 2.1.1. Identification, Receipt The test item of a suitable chemical purity was supplied by the Sponsor. All safety instructions required in the handling and disposal ofthe test item were supplied by the Sponsor as well. Identification ofthe test item was made by its name, batch, appearance and colour by Central Dispensary Unit ofCiToxLAB Hungary Ltd.Study code: 11/339-345AN Study Plan Page 6 of 8 3. TEST METHOD 3.1. APPARATUS HPLC:

Balances Centrifuge Ultrasonic bath Thermostated Orbital Shaker 3.2. PRINCIPLE OF THE TEST Merck-Hitachi LaChrom HPLC system 0-7000 Interface L-7100 HPLC pump L-nOO Autosampler L-7400 UV Detector

The substance is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved the mixture is cooled and kept at the test temperature, stirring as long as necessary to reach equilibrium. Subsequently, the mass concentration of the substance in the aqueous solution is determined by a suitable analytical method. 3.3. PERFORMANCE OF THE TEST About five times as much Ethanedioic acid will be weighed into a stoppered Erlenmeyer flask as the estimated solubility based on the approximate solubility (to be estimated in a preliminary test). After the addition of the water, the flasks will be agitated at 30°C. Passing 1, 2 and 3 days from each tested solvent, 3 parallel flasks will be removed and re-equilibrated at 20°C for 24 hours. The concentration in the clear solvent phase will be determined by HPLC. The pH ofeach sample will be recorded. The mean ofthree parallel tests will be reported. 3.4. ANALYTICAL METHOD Ethanedioic acid concentration ofthe water samples will be determined by a validated HPLC method.Study code: 11/339-34SAN Study Plan Page 7 of 8 3.6. DATA HANDLING The solubility in water of a substance is specified by the saturation mass concentration ofthe substance in water at a given temperature. The solubility in water will be specified in units ofmass per volume ofsolution. The individual results will be given for each of the three flasks and those results deemed to be constant (repeatability ofless than 15 %)will be averaged and given in units ofmass per volume ofsolution. 4. AMENDMENT AND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe Study Director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of Study Director's signature. All such amendments will be sent to the Sponsor. The study plan amendment will be distributed and added to all copies ofthe studyplan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect onthe outcome ofthe study.
5. GOOD LABORATORYPRACTICE (GLP) AND QUALITYASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be performed in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. (III.30.) EiiMFVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENVIMC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practice regulations specified by EPA 40 CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures.Study code: 11/339·345AN Study Plan Page 6 of 6 6. STUDY REPORT The study report will include the following information: Test item: chemical information Test method: detailed description ofthe method used Results: all measured and observed data 7. ARCHIVES The studydocuments:

- study plan and any amendments,

- all raw data

- sample ofthe test item

- study report and any amendments

- correspondence will be stored in the archives ofCiToxLAB Hungary Ltd., Hungary 8200 Veszprem, Szabadsagpusztaaccordingto the Hungarian GLP and our SOP-so 8. DISTRIBUTION OFTHE STUDY PLAN Archive Sponsor Study Director QAU original pdf-file Ix copy Ix copy

# CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta Hungary



# DRAFT 2 

# STUDYPLAN
Ready Biodegradability ofEthanedioic acid in a Closed Bottle Test

Study code: 1l/339-322AN Study Director: Krisztina Sipos, M.Sc.



## CONTAINS NO CBI 11 April 2012Study code: 11/339-322AN DRAFT Study Plan 2 Page 2 of 15 

### GENERAL INFORMATION STUDY TITLE TEST ITEM SPONSORS

STUDY MONITOR

TEST FACILITY Ready Biodegradability ofEthanedioic acid in a Closed Bottle Test Ethanedioic acid UnivarUSA Inc. Address: 17425 NE Union Hill Road Redmond, W A 98052 USA Telephone: + 14258893400 Facsimile: +14258893760 WEGO Chemical and Mineral Corp. Address: 239 GreatNeck Road GreatNeck, NY 11021 USA Telephone: +1 5164873510 Facsimile: +1 5164873794 Mr. John V. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Telephone: + 1 561 578 8110 Facsimile: + 1 561 578 8120 E-mail:john@grcci.com CiToxLAB Hungary Ltd. (Fonnerly known as LAB Research Ltd. before Ist September 20II) Address: H-8200 Veszprem, Szabadsagpuszta Hungary Phone: 36 88 545-300 Fax: 36 88 545-301Study code: 11/339-322AN DRAFT Study Plan 2 Page 3 of 15

APPROVED BY ON BEHALF OF SPONSOR DATE: John V. Massingale GreenwichChemical Consulting, Inc.
APPROVED BY ON BEHALF OF CiToxLAB DATE: ChristopherBanks, DABT

STUDY DIRECTOR DATE: Krisztina Sipos, M.Sc.
QUALITY ASSURANCE DATE: Szabolcs Gaty, M.Sc.Study code: 11/339-322AN DRAFT Study Plan 2 Page 4 of 15
TECHNICAL STAFF Ecotoxicological staff BASIS OF STUDY OECD Guidelines for Testing ofChemicals, No.: 3010, EPA Guideline, OPPTS 835.3110, Council Regulation (EC)No 440/2008, Method C.4-E REPORTING FORMAT Final Report in line with40 CFR792 format requirements REPORT COPIES Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by courter PRELIMINARYEXPERIMENT: TBD PROPOSED START OF EXPERIMENT: TBD PROPOSEDEND OF EXPERIMENT: TBD DRAFT REPORT: TBD TBO: to be determined and documented by an amendment The detailed study schedule is given in Appendix 1.Study code: 11/339-322AN DRAFT Study Plan 2 Page 5 of 15


## CONTENTS Page 1. 

### OBJECTIVE OFTHE STUDY 

### 6 1.1. PURPOSE 6 1.2. DEFINITIONS 6 1.3. CONDITIONS FOR THE VALIDITY OF THE TEST 6 

### 2. MATERIALSAND METHODS 

### 7 2.1. TEST ITEM 7 2.2. REFERENCE ITEM 7 2.3. IDENTIFICATION, RECEIPT 7 2.4. RECONSTITUTED WATER 8 

### 2.5. TEST SYSTEM 9 2.6. TEST UNITS 9 2.7. TEST CONDITIONS 9 2.8. PREPARATION OF THE TEST SOLUTIONS 10 2.9. COURSE OF THE TEST 11 2.10.MEASUREMENTS 11 2.11.CALCULATION OF RESULTS 12 

### 3. GOOD LABORATORYPRACTICE (GLP) AND QUALITYASSURANCE 13 

### 4. AMENDMENT AND DEVIATION PROCEDURES 12 

### 5. ARCHIVES 13 

### 6. DISTRIBUTION OFTHE STUDY PLAN 

### 14 7. SIGNATURES 

### 14 8. REFERENCES 

### 14 APPENDIX 1 

### 15Study code: 11/339·322AN DRAFT Study Plan 2 Page 6 of 15


### 1. 

### OBJECTIVEOFTHESTUDY 1.1. PURPOSE The purpose ofthis study is to determine the ready biodegradability ofthe test item. The test item is exposedto activated sludge from the aeration tank ofa domestic waste water treatment plant for 28 days. The biodegradation is followed by the oxygen uptake ofthe microorganisms during exposure. As a reference item Sodium benzoate is tested simultaneously under the same conditions as the test item, and functioned as a procedure control. This study is recognised by the test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule andthe regulations setforth at40 CFR 799.5087. 1.2. DEFINITIONS ThODNH4: Theoretical Oxygen Demand, the total amount ofoxygen required to oxidise a chemical completely. It is calculated from the molecular formula, assuming no nitrification occurs, and expressed as mgoxygen required permg test item. BOD: Biochemical Oxygen Demand, the amount ofoxygen consumed by microorganisms when metabolising a test item; also expressed as mg oxygen uptake per mgtest item. 10-day window: the 10 days immediately following the attainment of 10 % biodegradation. The pass level, 60 % ofThOD has to be reached in a 10-day window withinthe 28-d period ofthe test. 1.3. CONDITIONS FORTHE VALIDITY OF THE TEST
• Oxygen depletion in the inoculum control will not exceed 1.5 mg 

### 02/Lafter 28 days. •The residual oxygen concentration in the test flasks will not drop below 0.5 mg 

### 02/L

at any time. •The difference ofduplicate values for the degradation at any time during the test will

not be greaterthan 20 %. •The percentage degradation of the reference item will reach the level for ready

biodegradability (> 60 %)by exposure day 14.Study code: 11/339-322AN DRAFT Study Plan 2 Page 7 of 15


### 2. MATERIALS AND METHODS 2.1. TEST ITEM Name: Chemical name: Synonym: Batch number: CAS number: Molecular Formula: Molecular Weight: Purity: Description ofthetest item: Manufacture date: Retest date: Storage condition: Safety Precautions:

2.2. REFERENCE ITEM Name: Molecular Formula: MolecularWeight [glmoIJ: BatchNumber: Expiry date: Supplier: Storage condition: Safetyprecautions: Ethanedioic acid Ethanedioic acid Oxalic acid YP2-20110704B 144-62-7 C2H20 4 126.1 g/mo

### I(dihydrate form) 99.71% White crystals 04 July 2011 03 July 2012 controlled roomtemperature (15-25°C) Routine safety precautions (lab coat, gloves, safety glasses and face mask) will be applied to assure personnel health and safety.

Sodium benzoate 

## C

7

## H s Na02 144.10 02225JJ July 2012 Sigma-Aldrich In tightly closed container, under dry conditions at room temperature Routine safety and hygienic procedures
2.3. IDENTIFICATION, RECEIPT The test item ofa suitable chemical purity was suppJied by the Sponsor. Precautions required in the handling and disposal ofthe test item were supplied by the Sponsor. These documents are part oftheraw data. Identification ofthe test item was performed in the Central Dispensary ofCiToxLAB Hungary Ltd. onthe basis ofthe information was supplied by the Sponsor.Study code: 11/339-322AN DRAFT Study Plan 2 Page 8 of 15 

### 2.4. RECONSTITUTED WATER 2.4.1. Stock solutions In deionised wateranalytical grade salts* will be added (in the following ratio) to prepare the following stock solutions: a) Solution: KH2 P04 2.l25g K2 HP04 5.4375g Na2HP04x 12H2 0 16.795g NH4Cl O.l25g Deionised water ad 250 mL b) Solution: MgS04 X 

### 7 H2 0 5.625g Deionised water ad 250 mL c) Solution: CaCb x2 H2 0 9.10 g Deionised water ad 250 mL d) Solution**: FeCh x 6 H2

### 0 0.25g Deionised water ad 1000 mL 

### * Data ofchemicals used for reconstituted water (batch number, supplier, expiry date) will be given in

the Report 

### ** Ifthis stock solution will not applied on the day ofpreparation one drop ofconcentrated Hel per

litre will be added 

### 2.4.2. Ratio ofingredients 1 mL ofthe stock solutions a)
- d) will be combined and filled to a final volume of 1000 mL with deionised water. The test medium will be aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration will be checked.Study code: 11/339·322AN DRAFT Study Plan 2 Page 9 of 15


### 2.5. TEST SYSTEM The inoculum: Activated sludge, microorganisms from a domestic waste water treatment plant. Origin: The activated sludge will be supplied from the sewage plant for domestic sewage in Veszprem, Hungary. Conditioning: The activated sludge used for this study will be centrifuged (for 10 min.). Thereafter the supernatant liquid phase will be decanted. The solid material will be re-suspended in isotonic saline solution and again centrifuged (for 10 min). This washing procedure will be performed three times. An aliquot ofthe final sludge suspension will be weighed, dried and the ratio of wet sludge to its dry weight will be determined. Based onthis ratio, calculated aliquots of washed sludge suspension, corresponding to 3 - 5 g dry material per litre will be mixed with test water (see below) and then aerated until use. The exact amount of inoculum added will be provided in the report. Before use the sludge will be filtered through cotton wool. 2.6. TEST UNITS Type and Size: BOD bottles (300 ml) with special neck and glass stoppers. Identification: Each test flasks will be uniquely identified with at least study code, test group, days ofmeasurement and replicate number. 2.7. TEST CONDITIONS The test will be carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 

## ± 2°C according to guideline. The test flasks will 

### be placed into an incubator and kept at 22 

### ± 2°C, in the dark. Temperature will be measured at least each day of oxygen consumption measurement. The oxygen concentration oftest water will be about 9 mg/L at the start ofthe test (it will be measured at the start). The pH value ofthe test water will be checked prior study start and will be adjusted to 7.4 

### ± 0.2 with HCI or NaOH ifnecessary. The test conditions will be measured with suitable instruments and documented in the raw data.Study code: 11/339·322AN DRAFT Study Plan 2 Page 10 of 15
2.8. PREPARA TION OF THE TEST SOLUTIONS Before the preparation ofthe respective test solutions with test item an aqueous stock solutionofthetest item will beprepared and diluted accordingly. The respective amount ofthe test item will be weighed in directly to reach the required test item concentrationchosen based onthe theoretical oxygendemand (ThODNH4). 1.) Test Item (flasks 1aand 1 b) The chosen test item concentration will be based on the theoretical oxygen demand**(ThO~H4)** ofthe test item and will be given in Amendment to the Study Plan. At the start of the test a suitable amount of Test Item will be thoroughly mixed into the respective amountofaqueous testmedium. 2.) Procedure Control: Sodiumbenzoate (flasks 2a and 2b) Based on the theoretical oxygen demand (ThODNH4) ofSodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate will be mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mglL reference item, respectively a ThODNH4 ofabout 6.012 mg 

### 021L). 

### *the concentration ofthestock solution will be 360 mgIL 3.) Inoculum Control (flasks 3aand 3b) Only filtered inoculum wil1 be added to 3.40 litres ofaqueous test medium. 4.) Toxicity Control (flasks 4aand 4b) Test and reference item stock solutions [suitable amount of Test Item and Sodium benzoate] will be mixed into the respective amount of aqueous test medium corresponding to the investigated test item concentration (chosen based on the ThODNH4) and 3.6

### mg/Lreference item (ThODNH4 of6.012 mg

### 02/L). Microbial inoculum (0.5 mt per litre) will be added to eachpreparation bottIe.Study code: 111339-322AN DRAFT Study Plan 2 Page 11 of 15


### 2.9. COURSE OF THE TEST 2.9.1. Preparation ofTest Flasks A sufficient number ofBOD flasks will be cleaned with 5 - 10 mL ofa wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1% w/v 

### sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid will be poured off, and the bottles will be thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions will be filled into the bottles bubble-free until the bottles will be completely filled. Then they will be tightly stoppered. 2.9.2. The Test Bottles The test bottles will be the followings:

• at least 10 bottles containingthe test item and inoculum • at least 10 bottles containingthe reference item and inoculum (procedure control) • at least 10 bottles containing only inoculum (inoculum control) • at least 10 bottles containing the test item, reference item and inoculum (toxicity control) 2.10. MEASUREMENTS 2.10.1.MeasurementofOxygen The incubation period ofthe closed bottle test is 28 days. The oxygen concentrations will be measured with oxygen meter with a stirring O2 

### electrode. Oxygen measurements will be performed in duplicate at least on days 0, 7, 14,21 and 28. 2.10.2.Temperature Measurement Temperature will be measured at least each day ofoxygen consumption measurement.Study code: 11/339-322AN DRAFT Study Plan 2 Page 12 of 15


### 2.11. CALCULAnON OF RESULTS 2.11.1. Calculation ofBOD The BOD (mg O2 

### per mg test item) expected after each period will be calculated as follows: mg ofT.i.and/or R. i.
-mg ofi.control mg T.i. and/or R.i.lL inflask where: T.i. test item R.i. = 

### reference item, i.control 

### = inoculum control 2.11.2. Calculation ofBiodegradation % 

### The percentage biodegradation of the test item and of the reference item will be calculated as follows: BOD (mg T.i.orR.i.)



### ....**·~·--~~·····~~~~······~~~--~----x100** 

### ThOD NH4INH4 

### (mgO/mgT.i.or R.i.) where: T.i. test item R.i. = 

### reference item, i.control = 

### inoculum controlStudy code: 11f339-322AN DRAFT Study Plan 2 Page 13 of 15


### 3. GOOD LADORATORYPRACTICE (GLP) AND QUALITYASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment it will 

### be performed in compliance with the principles ofGood Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 

### 9/2001. (III.30.) EliM-FVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development whichcorresponds to the OECD GLP, ENV

### IMC/CHEM (98) 17. These principles are consistent with Good Laboratory Practice regulations specified by EPA 40 CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to our Standard Operating Procedures. 4. AMENDMENTAND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe study director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of Study Director's signature. All such amendments will be sent to the Sponsor. The studyplan amendment will be distributed and added to all copies ofthe studyplan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect on the outcome ofthe study. 5. ARCHIVES The study documents and samples:

- study plan and any amendments,

- all raw data,

- sample ofthe test item,

- correspondence,

- studyreport and any amendments will be stored in the archives ofCiToxLAB Hungary Ltd., Hungary 8200 Veszprem, Szabadsagpusztaaccording to the Hungarian GLP and our SOPs.Study code: 11/339-322AN DRAFT Study Plan 2 Page 14 of 15


### 6. DISTRIBUTION OFTHESTUDY PLAN Sponsor: 1 x pdf Archive: ] x original Departments*: 1 x copy each 

### * Study Director, QAU, Central Dispensary 

### 7. SIGNATURES The Study Director, CiToxLAB Hungary Ltd.'s management and QAU will sign the Study Plan before the start ofthe experiment. The signature page with Study Plan will be sent to the Sponsor after the content of the Study Plan has been agreed. If the Sponsor does not sign the Study Plan within 5 working days (or as specified in the contract) then the experiment may be started with the signature ofthe Sponsor being made after experiment initiation. 

### 8. REFERENCES 1. Council Regulation (EC) No 440/2008,Method C.4-E "Closed Bottle Test", Official Journal ofthe European Union L 142 of31 May 2008 2. OECD Guideline for Testing ofChemicals No. 301 D: "Ready Biodegradability: Closed Bottle Test", adoptedJuly 17, 1992 3. EPA Guideline 712-C-98-076: OPPTS 835.3110, "Ready Biodegradability", January 1998 4. Hungarian Good Laboratory Practice Regulations: 9/2001. 

### (III. 30) EtiM-FVMjoint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, 1997 5. OECD Principles of Good Laboratory Practice, adopted by Council on 26th November 1997 [C(97)I86/Final], Environment Directorate, Organisation for Economic Co-operation and Development, ENVIMCICHEM(98)

### 17, Paris 1998 6. Directive 2004/10/EC 

### ofthe European Parliament and ofthe Council of II February 2004 on the harmonisation oflaws, regulations and administrative provisions relating to the application ofthe principles ofgood laboratory practice and the verification of their applications for tests onchemical substances (codified version)Study code: 111339-322AN DRAFT Study Plan 2 Page 15 of 15



## APPENDIXStudy code: 11f339-322AN DRAFT Study Plan 2 Page 16 of 15


### APPENDIX 1 PROPOSED STUDY SCHEDULE PreliminaryExperiment: DD/MM/YY 

### Pre-ExperimentalPeriod Preparation ofReconstituted Waterday day [-1] DD/MM/YY 

### Preparation ofactivated sludge day [0] DD/MM/YY 

### Experimental Period Start ofexperiment day [0] DD/MMNY 

### Preparation oftest flasks day [0] DD/MM/YY 

### Preparationoftest flasks day [0] DD/MM/YY 

### Preparation oftest solutions day [0] DD/MM/YY 

### Incubation period day [0 28] DD/MM- DD/MM/YY 

### Measuring ofoxygen day [0] DD/MMNY 

### Measuring ofoxygen day [7] DD/MM/YY 

### Measuring ofoxygen day [14] DD/MMNY 

### Measuring ofoxygen day [21] DD/MMNY 

### Measuring ofoxygen day [28] DD/MM/YY 

### Measuring oftemperature day [0 28] DD/MM DD/MMNY 

### (at least at each day ofoxygen measurement) End ofexperiment day [28] DD/MM/YY 

### Draft Report not laterthan DD/MM/YY 

### The exact study schedule will 

### be given in an Amendment.

# CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta



# DRAFT 2 

# STUDYPLAN
Acute toxicity testwith Ethanedioic acid on Zebrafish (Brachydanio rerio)

Study code: 111339-009H Study Director: Krisztina Sipos, M.Sc.



## CONTAINS NO CSI
03 April 2012Study code: 111339-009H DRAFT 2 Study Plan Page 2 of 14 

### GENERAL INFORMATION
STUDY TITLE
TESTITEM SPONSORS

STUDY MONITOR

TEST FACILITY Acute toxicity test with Ethanedioic acid on Zebrafish (Brachydanio rerio)
: Ethanedioic acid

: Univar USA Inc.

Address: 17425 NE Union Hill Road Redmond, W A 98052 USA Telephone: + 1425 889 3400 Facsimile: + 1425 889 3760

: WEGO Chemical andMineral Corp.

Address: 239 GreatNeck Road GreatNeck, NY 11021 USA Telephone: +1 5164873510 Facsimile: +1 5164873794

: Mr. John V. Massingale

Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Telephone: + 1561 578 8110 Facsimile: +1 561 5788120 E-mail:john@grcci.com

: CiToxLABHungaryLtd.

(Fonnerly known as LAB Research Ltd. before 1st September 2011.) Address: H-8200 Veszpn:5m, Szabadsagpuszta Phone: 36 88 545-300 Fax: 3688545-301Study code: 11/339-009H DRAFT 2 Study Plan Page 3 of 14

APPROVED BY ON BEHALF OF SPONSOR
APPROVEDBY ON BEHALF OF CiToxLAB

STUDY DIRECTOR
QUALITY ASSURANCE

RESPONSIBLE PERSONFOR ANALYTICALMEASUREMENTS

APPROVED BY ON BEHALF OF IACUC DATE: JohnV. Massingale Greenwich Chemical Consulting. Inc.
DATE: ChristopherBanks, DABT Managing Director
DATE: Krisztina Sipos, M.Sc.
DATE: Szabolcs GMy, M.Sc.

DATE: Zsolt Sarvari, M.Sc.

DATE: Member ofIACUCStudy code: 11/339-009H DRAFT 2 Study Plan Page 4 of 14


### TECHNICAL STAFF : Ecotoxicological staff START OF EXPERIMENT : (will be given in Amendment) END OF EXPERIMENT : (will be given in Amendment) DRAFT REPORT : (will be given in Amendment)
BASIS OF STUDY:

- OECD Guideline for Testing ofChemicals, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992

- EPA HealthEffects Test Guidelines, OPPTS 850.1075

- Commission Regulation (EC) No 440/2008 

### of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 

### ofthe European Parliament and ofthe Council on the Registration, Evaluation, Authorisation and Restrictions ofChemicals (REACH), Annex Part C, C.l (published in the Official Journal of the European Union L 142 of31 May 2008
REPORTING FORMAT: Final Report in line with 40 CFR 792 format requirements REPORT COPIES: Original reportto be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by COUrIer 

### PRELIMINARYEXPERIMENT: TBD PROPOSED START OF EXPERIMENT: TBD PROPOSED END OF EXPERIMENT: TBD DRAFT REPORT: TBD TBO: to be determined and documented by an amendment 

### The detailed study schedule is given in Appendix 1.Study code: 11/339-009H DRAFT 2 Study Plan Page 5 of 14 

### 1. OBJECTIVE OF STUDY 1.1. PURPOSE The purpose ofthis study is to evaluate the acute lethal toxicity ofthe test item on Zebrafish (Brachydanio rerio). The test animals will be exposed in a static or semistatic test to aqueous test mediacontaining the test item at different concentrations. In case ofsufficient effects, the 24-h, 48-h, 72-h and 96-hLCsovalues will be calculated. The recorded effects will include the mortality and symptoms ofintoxication ofthe fish. If a limit test is performed the test animals will be exposed at a concentration of 100 mg/L orthe solubility level ofthe test item to demonstrate thatthe test item has no influence ontest animals up to at leastthis test concentration. The test method of application and the test species Zebrafish (Brachydanio rerio) are recommended by the test guidelines. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth at 40 CFR 799.5087.
1.2. CONDITIONS FORTHE VALIDITY OF THE TEST

- Constant conditions should be maintained in this procedure.

- The mortality in the controls should not exceed 10 % at the end ofthe test (or one

fish ifless than ten are used).

- The dissolved oxygen concentration (throughout the test) has to be at least

60 % ofair saturation value.

- The concentration ofthe test item should be at least 80 % ofthe nominal concentration

throughout the test. If the deviation from the nominal concentration is greater than 20%, results will bebasedonthe measured concentration. 

### 2. MATERIALS AND METHODS 2.1. TEST ITEM Name: Chemical name: Synonym: Batch number: CAS number: Molecular Formula: Molecular Weight: Purity: Description ofthe test item: Manufacture date: Retest date: Ethanedioic acid Ethanedioic acid Oxalic acid YP2-20110704B 144-62-7 

### C

2

### H 2

### 04 126.1 g/mol (dihydrate form) 99.71% White crystals 04 July 2011 03 July 2012Studycode: 11/339-009H DRAFT 2 Study Plan Page 6 of 14 

### Storage condition: controlled room temperature (15-25°C) Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) will be applied to assure personnel health and safety. 2.1.1. Identification, Receipt The test item of a suitable chemical purity was provided by the Sponsor. All precautions required in the handling and disposal ofthe test item were outlined by the Sponsor. The test item was identified on the basis ofthe information included in the test item documents, which were supplied by the Sponsor. 2.2. CONTROLS 2.2.1. Untreated Control The dilution water (circulated cooled and filtered) will be used without ofaddition of the test item. 2.2.2. Solvent Control The use of solvents will usually 

### be avoided when possible and used only with the approval ofthe Sponsor. When a solvent substance is used an additional control group will be used which contains the solvent substance at the same concentration as that used in the highest test concentration group. The concentration ofthe solvent in the final test medium will be not exceed 100 mg/L (or0.1 mLIL). 2.2.3. Reference Control Reference controls are not tested in the standard regulatory acute fish tests. 2.3. HOLDING Water: Circulated and filtered water will 

### be used for holding of fish. The fish will 

### be held during the test in water ofthe samequality used for holding. Light: 12 to 16 hours photoperiod daily. Temperature range: 20
- 24°C with a maximum deviation of

### ± 1 

### °C Dissolvedoxygenconcentration: Atleast 80 percent ofair saturation pH values: 6.0 to 8.5 Feeding: Feeding daily, until 24 hours before the start ofthe test.Study code: 11/339-009H DRAFT 2 Study Plan Page 7 of 14
2.4. ADMINISTRATION OF THE TEST ITEM 2.4.1. Formulation The test solutions used in the test will be prepared prior to treatment according to its water solubility: by mechanical dispersion orby a method (e.g. WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, GEeDNo. 23. The test solutions will be prepared just before introduction of the fish (start ofthe experiment). Details will be given in Amendment. 2.4.2. Preliminary Range-Finding Test A non-GLP range-finding test will be conducted to determine the approximatetoxicity ofthe test item so that appropriate test concentrations can be selected for use in the definitive test. At least 3 fish will be exposed to each widely spaced concentration (factore.g. 10) ofthe testitem plus a control, for 96 hours. The results of the preliminary range-finding test will be summarised in an Amendment. 2.4.3. Test item concentrations in the Definitive Test Ifa significant toxic response is observed during the preliminaryrange-finding test, at least five test concentrations in a geometric series (factor not exceeding 2.2) and one control (and one vehicle control ifused)will be used 

### inthe maintest. Ifno significant toxic response is observed in the preliminary range-finding test at a concentration of 

### 100.0 mg test itemlL (or up to its limit of solubility in the test medium whichever is the lower), only this concentration and one control (and one vehicle control ifused) will be tested in a limit-test. The concentrations in the definitive test will be based onthe results ofthe preliminary range-finding test andwill be given in Amendment. 2.5. EXPERIMENTAL ANIMALS Species: Zebrafish (Brachydanio rerio) Source: "BlackMolly" Fish Farm, Veszprem, Hungary Justification ofspecies: Zebrafish (Brachydanio rerio) is one ofthe convenient species for acute fish toxicity test. Number ofanimals: at least 7 fish in each concentration and control. Body length ofanimals: 

### 3.0±0.5 cm Acclimatisation: at least 12 daysStudy code: 11/339-009H DRAFT 2 Study Plan Page 8 of 14
Animal health: Holding water: Food and Feeding: Mortality: Fish are bred in a standard commercial fish farm, under disease- and parasite-controlledconditions. Circulated, filtered and cooled tap water (same as that will beused for thetest) The fish will notbe fed during the test The mortalities will be recorded for seven days acclimatisation period immediately before the test, the following criteriawill be applied:

- mortalities of greater than 10 per cent of population in seven days: rejection ofentire batch.

- mortalities of between 5 and 10 per cent of population: acclimatisation continued for seven additional days.

- mortalities of less than 5 per cent of population: acceptance ofbatch. 2.6. PERFORMANCE OF THE TEST The test duration will be 96 hours. At least 7 fish will be used at the test concentrations and for the control(s). 5 L test solution will be used per aquarium (test group). The maximum loading of 1.0 g fish 

### I L test solution will be at the start ofthe experiment. The fish will not be fed during thetest. The dilution water will be aerated during the test ifnecessary. Each test vessel will be uniquely identified with at least study code and test group.
2.7. TEST CONDITIONS The water temperature, oxygen concentration and pH will be measured at least daily in each test aquarium. The test temperature will be between 20°C and 24°C with a maximum deviation of

### ± 1°C. The oxygen concentration should be 60% ofthe air saturation value at the temperature used. The pH should normally not vary by more than 1.5 units in anyone test, and will be in the range of6.0 to 8.5. The hardness ofthe dilution water will be determined in the National Health Institute before the start of the test. The recommended range for hardness is 10 to 250 mg CaC03/L. The light-dark cycle during the test will be 12
- 16 hours light (artificial illumination) and 8 - 12 hours darkness.Study code: 11/339-009H DRAFT 2 Study Plan Page 9 of 14


### 2.8. OBSERVATIONS Mortality observations will be recorded at 3, 6, 24, 48, 72 and 96 hours. Fish are considered dead iftouching ofthe caudal peduncle produces no reaction. Dead fish are removed when observed and mortalities are recorded. Observations for clinical symptoms will be performed at 3, 6, 24, 48, 72 and 96 hours after the start ofthe test. Visible abnormalities in appearance or behaviour will be recorded. The body weight offish will be recorded on the day 0 (at the start ofthe main study). The loading of the testing liquid in the test-aquaria will be calculated (g fish/litre testing liquid) on the basis ofthe body weight measurements data. 2.9. ANALYTICAL MEASUREMENTS The concentration ofthe test item will be measured, as a minimum, at the highest and the lowesttest concentration, at least atthe beginning and end ofthe test (In the case of limit test the applied test concentration will be measured). Sample from the control will be taken for analysis at least atthe startofthetest. Analysis will be performed depending on the results of the analytical method validation. All samples will be analysed directly after sampling. Ifanalysis directly after sampling is not possible, the samples will be stored in a freezer immediately after sampling, and will be kept stored until analysis. The analytical method will be provided inthe Draft Report. 2.10. STATISTICAL EVALUATION In case ofsufficient effects the LC

### so values will be calculated by Probit analysis with 95 % 

### confidence limits using TOXSTAT or SPSS PC+ software. If a limit test is performed, no statistical analysis will be necessary. The NOEC, LOEC and the LC 100

### will be determined directly from the raw data. 

### 3. ANIMAL WELFARE After the experiment, all fish were humanely killed. Euthanasia for all animals was applied by an overdose of an aqueous solution of 250 mg/L Tricaine Methanesulphonate (MS 222; CAS No.: 886-86-2). This technique is considered to provide a humane euthanasiaby direct depression ofthe brain and vital centers. In case of any mortality during the test the dead fish will be removed as soon as possible.Study code: 11/339-009H DRAFT 2 Study Plan Page 10 of 14 

### 4. GOOD LABORATORYPRACTICE (GLP) AND QUALITY ASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be performed in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 

### 912001. (IIL30.) EUMFVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENV/MC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Registerplus subsequent amendments. These principles are consistent with Good LaboratoryPractice regulations specifiedby EPA40 CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures. 

### 5. AMENDMENT AND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe Study Director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of Study Director's signature. All such amendments will be sent to the Sponsor. The study plan amendment will be distributed and added to all copies ofthe study plan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect onthe outcome ofthe study. 

### 6. STUDY REPORT The study report will include the following information: The results ofthe study will be reported in a detailed final report. The original final report, in English will be sentto the sponsorand will include:

- Nameandaddress ofthe sponsor, thetestfacility andthe study schedule.

- The names ofthe study directorand other scientists and supervisorypersonnel involved in the study. The signaturesofthe studydirectorandthemanagement.

- The signed and dated reports ofeach ofthe individual scientists or other professionals involvedinthe study.

- Thequality assurance statement, signedbythe QA.Study code: 111339-009H DRAFT 2 Study Plan Page 11 of 14

- The statementofcompliance, signedbythe studydirector.

- Summaryoftheresults ofthe study,includingthepreliminarytest The identification ofthe test item, either by name or code number, concentration, purity, compositionandpropertiesas far as available. - A description ofthe test system, including the materials 

### used, a detailed description ofthe formulation ofthetestitem. A description ofall methods usedwithreferences. All deviationstothe studyplan. - A description ofall circumstancesthat may have affected the quality or integrity of the study. A description and assessmentfor all results including the datacalculationsand ifnecessary statisticalanalyses.

- The storage location ofall data, study plan, one original version ofthe [mal report and a reference sampleofthetestitem andreference item. Where appropriate, data will be summarized in tabular form to include mean values and a measure ofvariability. A draft report will be sent to the Sponsor bye-mail for comment and review. The final report will

### be issued as a PDF document, andas onebound and oneunboundpapercopies. 

### 7. ARCHIVES The study documents:

- study plan and any amendments,

- all rawdata,

- specimen ofthe testitem,

- studyreport and any amendments,

- correspondence will be stored according to the Hungarian GLP and to the facility SOP-s in the archives ofCiToxLAB Hungary Ltd., 8200Veszprem, Szabadsagpuszta, Hungary. After the retention time has elapsed all the archived materials listed above would be offered to the Sponsor or retained for a further period if agreed by a contract. Otherwisethe materials will be discarded.Study code: 11/339-009H DRAFT 2 Study Plan Page 12 of 14


### 8. DISTRIBUTION OFTHESTUDY PLAN Sponsor: 1 x PDF-file, 1 x copy Study Director: 1copy QAU: 1copy Archive: 1original Departments*: 1 x copy each 

### *Central Dispensary, Scientific Documentation and Analytics 

### 9. SIGNATURES The StudyDirector, CiToxLAB Hungary Ltd.'s management, QAU will sign the Study Plan before the startofthe experiment. The PDF version ofthe Study Plan will be sent to the Sponsor after the content ofthe Study Planhas been agreed. Ifthe Sponsor does not sign the Study Plan within 5 working days (or as specified in the contract) then the experiment may be started with the signature of the Sponsor being made after experiment initiation.Study code: 111339-009H DRAFT 2 Study Plan Page 13 of 14
10. 

### REFERENCES 26th 

### 1. OECD Principles of Good Laboratory Practice, adopted by Council on November 1997 [C(97)186/Final], Environment Directorate, Organisation for Economic Co-operation and Development, Paris 1998. 2. Hungarian Good Laboratory Practice Regulations: 9/2001. 

### (III. 30.) EUM-FVMjoint decree of the Minister of Health and the Minister of Agriculture and Regional Developmentwhichcorresponds to the OECD GLP, 1997 3. Commission Regulation (EC) No 440/2008 

### of 30 May 2008 laying do\\'Jl test methods pursuant to Regulation (EC) No 1907/2006 

### ofthe European Parliament and the of the Council on the Registration, Evaluation, Authorisation and Restrictions ofChemicals (REACh), Annex Part C, C.l (published in the Official Journal ofthe European Union L 142 of31 May 2008) 4. EPA Guideline 712-C-96-118: OPPTS 850.1075, "Fish Acute Toxicity Test, Freshwater and Marine" April 1996 5. OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992. 6. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series onTesting and AssessmentNo. 23, Paris September 2000.Study code: 11/339-009H DRAFT 2 Study Plan Page 14 of 14 

### APPENDIX 1 STUDY SCHEDULE
Relative date Absolute date Startofexperiment day 0 DD/MM/YY 

### Preparation oftest item solutions day 0 DD/MM/YY 

### Analytical sampling day 0 DD/MM/YY 

### Measurement ofbody weight day 0 DDIMM/YY Treatmentoftest animals day 0 DDIMM/YY Measurement oftest conditions day 0 DD/MM/YY 

### Observation(3 and 6 h afterthe treatment) day 0 DD/MM/YY 

### Measurementoftest conditions day 1 DD/MM/YY 

### Observation day 1 DDIMM/YY Measurement oftest conditions day 2 DD/MM/YY 

### Observation day 2 DD/MM/YY 

### Measurement oftest conditions day 3 DD/MM/YY 

### Observation day 3 DD/MM/YY Measurement oftest conditions day 4 DD/MM/YY 

### Observation day 4 DDIMM/YY Analytical sampling day 4 DD/MMlYY 

### End ofexperiment day 4 DDIMM/YY DraftReport DD/MM/YY 

### Remark: The exact Study Schedule will be given in Amendment, because the contents ofthis schedule depend on the test item formulation method will be used during the definitive test or on the stability ofthe test item.

# CiToxLAB Hungary Ltd. .It Veszprem - Szabadsagpuszta

# CiTox~B Hungary



# DRAFT 2 

# STUDYPLAN
Acute immobilisation test with Ethanedioic acid on Daphnia 

# (Daphnia magna)
Study code: 111339-023DA Study Director: Krisztina Sipos, M.Sc.



## CONTAINS NO eel

03 April 2012Study code: 11/339-023DA DRAFT 2 Study Plan Page 2 of 15 

### GENERAL INFORMATION STUDYTITLE Acute immobilisationtest with Ethanedioic acid on Daphnia (Daphnia magna) 

### TESTITEM : Ethanedioic acid SPONSORS : UnivarUSA Inc. Address: 17425 NE Union Hill Road Redmond, W A 98052 USA Telephone: + 1425 8893400 Facsimile: + 1425 889 3760
: WEGO Chemical and Mineral Corp.

Address: 239 GreatNeckRoad Great Neck, NY 11021 USA Telephone: +1 5164873510 Facsimile: + 15164873794 STUDY MONITOR : Mr. JohnV. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGA Boulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Telephone: + 1561 5788110 Facsimile:+15615788120 E-mail:john@grcci.com TESTFACILITY : CiToxLAB Hungary Ltd. (Fonnerlyknown as LAB Research Ltd. before 1 st September 2011.) 

### Address: H-8200 Veszprem, Szabadsagpuszta Hungary Phone: 36 88 545-300 Fax: 3688545-301Study code: 11/339-023DA DRAFT 2Study Plan Page 3 of 15

APPROVED BY ON BEHALF OF SPONSOR

APPROVED BY ON BEHALF OF CiToxLAB

STUDY DIRECTOR
QUALITYASSURANCE

RESPONSIBLEPERSONFOR ANAL ¥TICALMEASUREMENTS DATE: John V. Massingale Greenwich Chemical Consulting, Inc.

DATE: ChristopherBanks, DABT Managing Director
DATE: Krisztina Sipos, M.Sc.
DATE: Szabolcs Gaty, M.Sc.

DATE: Zsolt Sarvari M.Sc.Study code: 11/339-023DA DRAFT 2 Study Plan Page 4 of 15



### TECHNICAL STAFF : Ecotoxicological staff START OF EXPERIMENT : (will be given in Amendment) END OFEXPERIMENT : (will be given in Amendment) DRAFT REPORT : (will be given in Amendment)
BASIS OFSTUDY: OECD Guidelines for Testing of Chemicals, No. 202, 

### "Daphnia sp., Acute Immobilisation Test," (Adopted: 13 April 2004) OECD: Paris EPA Health EffectsTest Guidelines, OPPTS 850.1010 Commission Regulation (EC) No 76112009 of 23 July 2009 laying down test methods pursuant to Regulation (EC) No 190712006 

### ofthe European Parliament and ofthe Council on the Registration, Evaluation, Authorisation and Restrictions ofChemicals (REACh), Annex Part C, C.2 (published in the Official Journal ofthe European Union L 142 of31 May 2008)

REPORTING FORMAT: Final Report in line with 40 CFR792 format requirements REPORT COPIES: Original report to be archived One PDF copy will be sentby email One bound and one unbound hard copy will be sentby couner PRELIMINARYEXPERIMENT: TBD PROPOSED START OF EXPERIMENT: TBD PROPOSED END OF EXPERIMENT: TBD DRAFT REPORT: TBD TBD: to be determined and documented by an amendment 

### The detailed study schedule is given in Appendix 1.Study code: 11/339-023DA DRAFT 2 Study Plan Page 5 of 15
1. 

### OBJECTIVE OF STUDY 1.1. PURPOSE The purpose of this study is to evaluate the influence of the test item on the mobility/survival ofDaphnia magna. 

### Young Daphnia 

### will be exposed in a static or semi-static test to aqueous test media containing the test item at different concentrations. The percentage ofDaphnia 

### being no longer capable ofswimming at the end ofthe test orbeing dead will be recorded for eachtest concentration. In case ofsufficienteffects, the 24-h and 48-hEC

### so values will be calculated. If a limit test is performed the test animals will be exposed at a concentration of 100 mg/L or the maximum water solubility level ofthe test item to demonstrate that the test item has no influence ontest animals up to at least this test concentration. The test method ofapplication and the test species Daphnia magna 

### are recommended by the test guidelines. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section4 TestRule and the regulations set forth at 40 CFR799.5087. 1.2. CONDITIONS FORTHE VALIDITY OF THE TEST

- in the control, including any control containing the solubilising agent, not more than

10 per cent ofthe Daphnia 

### should be immobilised or trapped at the surface ofthe water.

- the dissolved oxygen concentration at the end ofthe test in control and test vessels

should be**~** 

### 3mg/L ofthe air saturationvalue at the temperature used. 

### 2. MATERIALS AND METHODS 2.1. TEST ITEM Name: Chemical name: Synonym: Batch number: CAS number: Ethanedioic acid Ethanedioic acid Oxalic acid YP2-20110704B 144-62-7 MolecularFormula: C2H204 Molecular Weight: 126.1 glmol (dihydrate form) Purity: 99.71% 

### Description ofthe test item: White crystals Manufacture date: 04 July 2011 Retest date: 03 July 2012 Storage condition: controlledroom temperature (15-25°C) Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) will be applied to assure personnel healthStudy code: 11/339-023DA DRAFT 2 Study Plan Page 6 of 15


### 2.1.1. Identification, Receipt The test item of a suitable chemical purity was provided by the Sponsor. All precautions required in the handling and disposal ofthetest item were outlined by the Sponsor. The test item was identified on the basis ofthe information included in the test item documents, which were supplied by the Sponsor. 2.2. CONTROLS 2.2.1. Untreated Control The dilution water (ISO-medium) will be used without ofaddition ofthe test item. 2.2.2. Solvent Control The use of solvents will usually be avoided when possible and used only with the approval ofthe Sponsor. When a solvent substance is used an additional control group will be used which contains the solvent substance at the same concentration as that used in the highest test concentration group. The concentration ofthe solvent in the final test medium will be notexceed 100 mg/L (or0.1 mUL). 2.2.3. Reference Control For the evaluation of the quality of the Daphnia 

### clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactorytest conditions. The details ofthe last study will be given in the reports. 2.3. DILUTION WATER Reconstituted water (ISO medium, according to OECD 202) will be used as dilution water in the experiment. The composition ofthe reconstituted water and the chemicals used are given in Appendix 2.Study code: 11/339-023DA DRAFT 2 Study Plan Page 7 of 15


### 2.4. ADMINISTRATION OF THETEST ITEM 2.4.1. Fonnulation The test solutions used in the test will be prepared prior to treatment according to its water solubility: by mechanical dispersion or by a method according to the Guidance DocumentonAquatic ToxicityTestingofDifficultSubstancesand Mixtures, OEeDNo. 23. The test solutions will be preparedjustbefore introduction ofthe 

### Daphnia (start ofthe experiment). Details will be given by Amendment. 2.4.2. Preliminary range-Finding Test A preliminary range-finding test will be conducted to detennine the approximate toxicity ofthe test item so that appropriate test concentrations can be selected for use in the definitive test. At least 5 daphnids will be exposed for 48 hours. The results of the preliminary range-finding test will be summarised in an Amendment. 2.4.3. Test item concentrations in the Definitive Test Ifa significant toxic response is observed during the preliminary range-finding test, at least five test concentrations in a geometric series (factor not exceeding 2.2) and one control (and one vehiclecontrol ifused) will be used in

### themaintest. If no significant toxic response is observed in the preliminary range-finding test at a concentration of 100 mg test itemJL (or up to its limit ofsolubility in the test medium whichever is the lower), only this concentration and one control (and one vehicle control ifused) will be tested in a limit-test. The concentrations in the definitive test will be based on the results ofthe preliminary range-finding test and will be given in an Amendment.Study code: 11/339-023DA DRAFT 2 Study Plan Page 8 of 15


### 2.5. EXPERIMENTAL ANIMALS Species and strain: 

### Daphnia magna (Straus) Source: National Institute ofPublic Health 1097 Budapest Gyali u. 2-6 Hungary Breeding The 

### Daphnia are bred in Ecotoxicological Laboratory ofCiToxLAB Hungary Ltd. Justification ofspecies: 

### Daphnia magna is the standard species of the acute immobilisationtest. Previous generation Daphnia of the previous generation should be reared with conditions similarly to the test conditions as possible. Test daphnia from stage with high reproduction activity (normally 2-4 weeks old) are preferred. Number ofanimals: There will be 20 animals at each group (concentration and untreated control) divided into 4 parallel (5 animals!l parallel) Age ofanimals: Less than 24 hours old at the beginning ofthe test. The first breeding ofthe previous generation should not be used. Acclimatization: To avoid the necessity of adaptation prior to the test, the water used in the test will be similar to the culture conditions (watertemperature, background colouretc.). Holding water: Same water is used as test water (see 2.3.). Food and Feeding: The 

### Daphnia are fed with concentrated algal suspension of 

### Pseudokirchneriellasubcapitataduring theholding. 2.6. PERFORMANCE OF THE TEST The test duration will be 

### 48 hours. Twenty animals, divided into four groups (glass beaker) offive animals each (at least4 mL test solutionlanimal) will be used at the test concentrations and for the control(s). The

### Daphnia will notbe fed during the test. The dilution water may be aerated prior to the addition of the test item and test animals. Each test vessel will be uniquely identified with at least study code, test group and replicate. 2.7. TESTCONDITIONS The vessels will be held for the duration ofthe study in a climate chamber, the test temperature will be between 18°C and 22°C with a maximum deviation of

### ± 1°C. The water temperature will be 

### determined at least daily in one control test vessel and continuously with a minimax thermometer in the climate chamber.Study code: 11/339-023DA DRAFT 2 Study Plan Page 9 of 15 

### The oxygen concentration and pH will be measured at least in the control(s) and in all of the test concentration test vessels at least at the start and at the end of the experiment. The oxygen concentration should be 2 

### 3 mglL ofthe air saturation value at the temperature used. The pH will not vary by more than 1.5 units in anyonetest. The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mglL (as CaC03). 

### The light-dark cycle during the test will be 16 hours light (artificial i1lumination) and 8 hours darkness; complete darkness will be used if the test item is unstable in light, in this casethe conditions will be given in an amendment. 2.8. OBSERVAnONS The immobility or mortality ofthe Daphnia 

### will be determined by visual observation after 24 and 48 hours. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. Also, other visible abnormalities in appearance orbehaviour will be recorded. The number of immobilised animals and the percentage of immobility will be determined at 24 and 48 hours. 2.9. ANALynCALMEASUREMENTS The concentration ofthe test item will be measured at all ofthe test concentration, at least at the beginning and end of the test (In 

### the case of limit test the applied test concentration will be measured). Sample from the control will be taken for analysis at least at the startofthetest. Analysis will be performed depending on the results of the analytical method validation. All samples will be analysed directly after sampling. Ifanalysis directly after sampling is not possible, the samples will be stored in a freezer immediately after sampling, and will be kept stored until analysis. The analytical method will be provided in the Draft Report.Study code: 11/339-023DA DRAFT 2 Study Plan Page 10 of 15


### 2.1O. STATISTICAL EVALUATION In case of sufficient effects the ECso values will be calculated preferably by Probit analysis with 95 % confidence limits using TOXSTAT or SPSS PC+ software. Other models for LCso calculation may be used ifappropriate. If

### a limit test is performed, no statistical analysis will be necessary. Statistical analysis on immobilization may be assessed by analysis of variance (ANOVA) and Bonferroni t-Test (a 

### 0.05) by TOXSTAT or SPSS PC+ software if necessary. Ifa limit test will be perfonned, immobilization in control and in treated group may be analysed using 2 Sample t-Test (a

### = 0.05) by TOXSTAT software to compare means ifnecessary. For the detennination of the LOEC and NOEC, the immobilization at the test concentrations may be tested on significant differences to the control values by Bonferroni t-Test ifnecessary. 3. GOOD LABORATORYPRACTICE (GLP)AND QUALITYASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be perfonned in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. 

### (IIL30.) EiiMFVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENV/MCICHEM (98) 

### 17, which is consistent to the United States Food and Drug Administration Title 

### 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 

### 22 December 

### 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practiceregulations specified by EPA40 CFR 

### 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures.Study code: 11/339-023DA DRAFT 2Study Plan Page 11 of 15


### 4. AMENDMENT AND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe Study Director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of Study Director's signature. All such amendments will be sent to the Sponsor. The study plan amendment will be distributed and added to all copies ofthe studyplan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect on the outcome ofthe study. 5. STUDY REPORT 

### The report will include, but notbe limited to the following information: Theresults ofthe study will be reported in a detailed final report. The original final report, in English will be sentto the sponsor and will include: Name and address ofthe sponsor, the test facility and the study schedule.

- The names of the study director and other scientists and supervisory personnel involved inthe study. The signatures ofthe study director and the management.

- The signed and dated reports of each of the individual scientists or other professionals involved in the study.

- The quality assurance statement, signed by the QAU. - A copy ofthe test facility's GLP-Certificate. The statement ofcompliance, signedbythe study director.

- The identification ofthe test item, either by name or code number, concentration, purity, composition and properties as far as available

- Summaryofthe

### preliminarytest - A description ofthe test system, including the materials used, a detailed description ofthe performance ofthe test item. - A description ofall methods used with references. A list ofall deviations to the Study Plan. - A description ofall circumstances that may have affected the quality orintegrity of the study.Study code: 11f339·023DA DRAFT 2 Study Plan Page 12 of 15


### - A description and assessment for all results including the data calculations and if necessary statistical analyses.

- The storage location ofall data, study plan, one original version ofthe final report and a reference sample ofthe test item. Results:
- tabular presentation of the immobilization rate for the individual test concentrations and the control together with the exposure period and test substance concentrations

- the 48h ECso, 

### including confidence intervals (in case ofsufficienteffect)

- NOEC, LOEC

- in case ofsufficienteffect the calculated dose response curve Where appropriate, data will be summarized in tabular form to include mean values and a measure ofvariability. A draft report will 

### be sent to the Sponsor bye-mail for comment and review. The final reportwill be issued as aPDF document, and as onebound andone unboundpapercopies. 

### 6. ARCHIVES The study documents:

- study plan and any amendments,

- all raw data,

- sample ofthe test item,

- study report and any amendments,

- correspondence will be stored according to the Hungarian GLP and to the facility SOP-s in the archives ofCiT ox LAB Hungary Ltd., 8200 Veszprem, Szabadsagpuszta, Hungary. After the retention time has elapsed all the archived materials listed above would be offered to the Sponsor or retained for a further period if agreed by a contract. Otherwise the materials will be discarded. 

### 7. DISTRIBUTION OFTHESTUDY PLAN Sponsor: 1 x PDF-file, 1 x copy Study Director: 1copy QAU: 1copy Archive: 1original Departments

## * : 1 x copy each 

### * Central Dispensary, Scientific Documentation and AnalyticsStudy code: 11/339-023DA DRAFT 2 Study Plan Page 13 of 15


### 8. SIGNATURES The Study Director, CiToxLAB Hungary Ltd.'s management, QAU will signthe Study Plan before the start ofthe experiment. The PDF version ofthe Study Plan will be sent to the Sponsor afterthe content ofthe Study Plan has been agreed. Ifthe Sponsor does not sign the Study Plan within 5 working days (or as specified in the contract) then the experiment may be started with the signature of the Sponsor being made after experiment initiation. 

### 9. REFERENCES 26th 

### 1. OECD Principles of Good Laboratory Practice, adopted by Council on November 1997 [C(97)186IFinal], Environment Directorate, Organisation for Economic Co-operation and Development, Paris 1998. 2. Hungarian Good Laboratory Practice Regulations: 9/2001 

### (III. 30) EUM-FVMjoint decree of the Minister of Health and the Minister of Agriculture and Regional Development which correspondsto the OECD GLP, 1997 3. Commission Regulation (EC) No 76112009 

### of 23 July 2009 laying down test methods pursuant to Regulation (EC) No 1907/2006 

### ofthe European Parliament and ofthe Council on the Registration, Evaluation, Authorisation and Restrictions ofChemicals (REACh), Annex Part C, C.2 (published in the Official Journal ofthe European Union L 142 of31 May 2008) 4. EPA Guideline 712-C-96-114: OPPTS 850.1010, "Aquatic Invertebrate Acute Toxicity Test, FreshwaterDaphnids" April 1996 5. OECD Guideline for Testing ofChemicals, Section 2, No. 202, 

### "Daphnia sp., Acute ImmobilisationTest", updated 13th 

### April 2004. 6. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and AssessmentNo. 23, Paris September2000.Study code: 111339-023DA DRAFT 2 Study Plan Page 14 of 15


### APPENDIX 1 STUDY SCHEDULE Relative date Absolute date Culturing oftestanimals for the testing day [-1] DD/MMIYY 

### Start ofexperiment day 0 DD/MM/YY 

### Preparation oftestitem solutions day 0 DD/MMIYY 

### Analytical sampling day 0 DD/MMIYY 

### Measurement oftest conditions day 0 DD/MM/YY 

### Treatmentoftest animals day 0 DD/MM/YY 

### Measurementoftemperature day 1 DD/MM/YY 

### Observation ofimmobilisation day 1 DDIMM/YY 

### Measurement oftest conditions day 2 DD/MM/YY 

### Observation ofimmobilisation day 2 DDIMM/YY 

### Analytical sampling day 2 DDIMM/YY 

### End ofexperiment day 2 DDIMM/YY


### Draft Report DDIMM/YY 

### Remark: The exact Study Schedule will 

### be given in Amendment.Study code: 11/339-023DA DRAFT 2 Study Plan Page 15 of 15 APPENDIX 

### 2 COMPOSITION OFISO MEDIUM
Separate stock solutions ofindividual trace elements were first prepared in deionised water. The medium was prepared ofthese different stock solutions, so it contained all trace elements (combined solution). Stock solutions Amount added Stock solutions (single substance) to deionised addedto deionised water waterto prepare media (giL) (milL) CaCbx2 H2O 11.76 25 MgS04x7 H2 O 4.93 25 KCI 0.23 25 NaHC03 2.59 25

# CiToxLAB Hungary Ltd. Veszprem - Szabadsagpuszta 

# CiToxlAB .. Hungary



# DRAFT 2 

# STUDYPLAN
Growth inhibition Test with Ethanedioic acid on Algae 

# (Pseudokirchneriella subcapitata)
Study code: 111339-022AL Study Director: Krisztina Sipos, M.Sc.



## CONTAINS NO CBI 03 April 2012Study code: 11/339-022AL DRAFT 2Study Plan Page 2 of 17


### GENERAL INFORMATION STUDYTITLE : Growth inhibitiontest with Ethanedioic acid on Algae 

### (Pseudokirchneriella subcapitala) TEST ITEM : Ethanedioic acid SPONSORS : UnivarUSAInc. Address: 17425 NE UnionHill Road Redmond, W A 98052 USA Telephone: + 1425 8893400 Facsimile: +1 425 8893760

: WEGO Chemical andMineral Corp.

Address: 239 GreatNeckRoad Great Neck, NY 11021 USA Telephone: +1 5164873510 Facsimile: + 1 516487 3794 STUDYMONITOR : Mr. JohnV. Massingale Greenwich Chemical Consulting, Inc. Address: 3300 PGABoulevard, Suite 635 Palm Beach Gardens, FL 33410 USA Telephone: +1 561 5788110 Facsimile: +1 561 5788120 E-mail:john@grcci.com TESTFACILITY : CiToxLAB Hungary Ltd. (Fonnerlyknown as LAB Research Ltd. before]st September 2011) Address: H-8200 VeSZpf(!ID, Szabadsagpuszta Hungary Phone: 36 88 545-300 Fax: 3688545-301Study code: 11/339-022AL DRAFT 2 Study Plan Page 3 of 17

APPROVED BY ON BEHALF OF SPONSOR

APPROVED BY ON BEHALF OF CiToxLAB

STUDY DIRECTOR

QUALITY ASSURANCE

RESPONSIBLE PERSON FOR ANALYTICALMEASUREMENTS DATE: John V. Massingale Greenwich Chemical Consulting, Inc.

DATE: ChristopherBanks, DABT Managing Director

DATE: Krisztina Sipos, M.Sc.

DATE: Szabolcs Gaty, M.Sc.

DATE: Zsolt Sarvari, M.Sc.Study code: 11/339-022AL DRAFT 2 Study Plan Page 4 of 17


### TECHNICAL STAFF Ecotoxicological staff START OF EXPERIMENT (will be given in Amendment) END OF EXPERIMENT (will be given in Amendment) DRAFT REPORT (will be given in Amendment) BASIS OF STUDY:

- OECD Guideline for Testing ofChemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 23 rd 

### March, 2006.

- EPA Ecological Effects Test Guidelines, OPPTS 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996

- Commission Regulation (EC) No 761/2009 

### of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 

### laying down test methods pursuant to Regulation (EC) No 1907/2006 

### of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal ofthe European Union L 220 of24 August 2009)
REPORTING FORMAT: REPORT COPIES: Final Report in line with 40 CFR792 format requirements Original report to be archived One PDF copy will be sent by email One bound and one unbound hard copy will be sent by couner PRELIMINARY EXPERIMENT: TBD PROPOSED START OF EXPERIMENT: TBD PROPOSED END OF EXPERIMENT: TBD DRAFT REPORT: TBD TBD: to be determined and documented by an amendment 

### The detailed study schedule is given inAppendix 1.Study code: 11/339-022AL DRAFT 2 Study Plan Page 5 of 17 

### 1. OBJECTIVEOFSTUDY 1.1. PURPOSE The purpose ofthe study is to determine the effect ofthe test item onthe growth ofthe unicellular green algal species Pseudokirchneriella subcapitata 

### (formerly known as Selenastrum capricornutum). 

### Exponentially growing cultures of Pseudokirchneriella subcapitata 

### will 

### be exposed to various concentrationsofthe test item over several generationsunderdefined conditions. If a limit test is performed the test species will be exposed at a concentration of 100 mg/L or the maximum water solubility level ofthe test item to demonstrate that the test item has no influence ontest species up to at least this test concentration. The algal growth in relation to a control culture is determined over a fixed period of 72 hours. The method of application and the test species Pseudokirchneriella subcapitata

### is recommended by the test guidelines. In the first instance, only the preliminary range-finding test will be performed. In parallel, feasibility work for analytical will be performed. When a suitable analytical method is established for the appropriate concentration range, the main study will be conducted, following a Study Plan amendment. This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section4 Test Rule and the regulations setforth at 40 CFR799.5087. 1.2. VALIDITY CRITERIA OF THE STUDY The cell concentration in the untreated control cultures should have increased by a factor of at least 16 within three days (72 hours), corresponding to a specific growthrate of0.92

## dai 1 

### The mean coefficient of variation for section-by-section specific growth rates (days 0-1,1-2and 2-3, for 72-h tests) inthe control cultures must not exceed 35%. The coefficient ofvariation ofaverage specific growth rates during the whole test period inreplicate control cultures must not exceed 7%. 

### 2. MATERIALS AND METHODS 2.1. TESTITEM Name: Ethanedioic acid Chemical name: Ethanedioic acid Synonym: Oxalic acid Batch number: YP2-20110704B CAS number: 144-62-7 Molecular Formula: 

### C 2

### H 2

### 04 

### Molecular Weight: 126.1 glmol (dihydrate form) Purity: 99.71% Descriptionofthe test item: White crystals Manufacture date: 04 July 2011 Retest date: 03 July 2012Study code: 11/339-022AL DRAFT 2 Study Plan Page 6 of 17 

### Storage condition: Safety Precautions:

2.1.1. Identification, Receipt controlled roomtemperature (15-25°C) Routine safety precautions (lab coat, gloves, safety glasses and face mask) will be applied to assure personnel health and safety.

The test item of a suitable chemical purity was provided by the Sponsor. All precautions required in the handling and disposal ofthe test item were outlined by the Sponsor. The test item was identified on the basis ofthe information included in the test item documents, which were supplied by the Sponsor. 2.2. CONTROLS 2.2.1. Untreated Control Algal growth medium will be inoculated with algal cells (without test item) and examined in parallel to the test itemconcentrations. 2.2.2. Solvent Control The use of solvents will usually be avoided when possible and used only with the approval ofthe Sponsor. When a solvent substance is used an additional control group will be used which contains the solvent substance at the same concentration as that used in the highest test concentration group. The concentration ofthe solvent in the final test mediumwill be notexceed 100 mgIL (or0.1 mUL). 2.2.3. Reference Control For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactorytest conditions. The results and details of the closest test and range ofvalidity for this test will be given in the report. 2.3. DILUTION WATER Reconstituted algal growth medium (OECD medium, according to OECD 201) will be used as dilution water in the experiment. Thecomposition ofthe growth medium andthe chemicalsused are giveninAppendix 2.Study code: 11/339-022AL DRAFT 2 Study Plan Page 7 of 17


### 2.4. ADMINISTRATION OF THE TEST ITEM 2.4.1. Dilution and Preparation ofTesting Solutions The test solutions used in the test will be prepared prior to treatment according to its solubility in the test medium: by mechanical dispersion or by a method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. The test solutions will be preparedjust before introduction ofthe algae (start ofthe experiment). Details will be given in Amendment. 2.4.2. Preliminary range-Finding Test A preliminary range-finding test will be conducted to determine the approximate toxicity ofthe test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells will be exposed to each widely spaced concentration (factore.g. 10) ofthe test item plus a control, for 72 hours. The results of the preliminary range-finding test will be summarised In 

### an Amendment. 2.4.3. Test item concentrations in the Definitive Test Ifa significant toxic response is observed during the preliminary range-finding test, at least five test concentrations in a geometric series (factor not exceeding 3.2) and one control (and one vehicle control ifused) will be used in

### themaintest. Ifno significant toxic response is observed in the preliminary range-finding test at a concentration of 100 mg test iternlL (or up to its limit ofsolubility in the test medium whichever is the lower), only this concentration and one control (and one vehicle control ifused) will be tested in a limit-test. The concentrations in the definitive test will be based on the results ofthe preliminary range-finding test and will be given in an Amendment.Study code: 11/339-022AL DRAFT 2Study Plan Page 8 of 17


### 2.5. EXPERIMENTAL ORGANISMS Species: Pseudokirchneriella subcapitata 

### (formerly known as Selenastrum capricornutum) 

### Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662) Source: The algae were supplied by the SAG: Collection ofAlgal Cultures, Inst. Plant Physiology, University of Gottingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. Justification ofspecies: The species of Pseudokirchneriella subcapitata 

### used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines. Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regUlarly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The preculture will be prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period ofabout three days. When the algal cultures contain deformed or abnormal cells, they will be discarded. 2.6. PERFORMANCE OF THE TEST The exposure time will be 72 hours. The test will be started (0 hours) by inoculation of a biomass of approximately 104 

### algal cells per mL test medium. The number of cells added to the test medium will be given in the Final Report. The test will be performed with at least three replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate, in Erlenmeyer flasks, will be continuously shaken by a laboratory orbital shaker. The flasks will be covered with air-permeable stoppers. Each test vessel will be uniquely identified with at least study code, test group and replicate.Study code: 11/339-022AL DRAFT 2 Study Plan Page 9 of 17 

### 2.7. TEST CONDITIONS The vessels will be held for the duration ofthe study in a climate chamber, the test temperature will be between 21°C and 24°C with a maximum deviation of

### ±2°C. The water temperature will be determined at least daily in one vessel filled with water only and continuously with a minimax thermometerin the climate chamber. The pH will be measured in the control(s) and in the used test concentration in each test vessel at the start and at the end ofthe experiment. The pH should normally not vary by more than 1.5 units in anyone test. Illumination: fluorescent lamps will be used with a spectral range of 400-700 nm, continuous lighting with light intensity of 4440-8880 lux (mean value+I-15% variation) at the liquid surface. 2.8. OBSERVATIONS The cell concentration will be determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Any morphological deviations ofthe algal cells noted will be reported. The behaviour of the test item in test medium will be determined daily in all test concentrations. 2.9. ANALYTICAL MEASUREMENTS The analytical method will be developed to match the concentration range identified in the preliminary range-finding test. The analytical feasibility work will be carried out onthe aquatic samples generated in the preliminarytest. In the main study, the concentration ofthe test item will be measured, as a minimum, at the highest and the lowest test concentration, at least at the beginning and end ofthe test (In the case oflimit test the applied test concentration will be measured). Sample from the control will be taken for analysis at leastat the start ofthe test. For volatile, unstable or strongly adsorbing test substances, additional samplings for analysis at 24 hour intervals during the exposure period will be used in order to better define loss of the test substance. In all cases, determination of test substance concentrations need only be performed on one replicate vessel at each test concentration. Analysis will be performed depending on the results of the analytical method validation. All samples will be analysed directly after sampling. Ifanalysis directly after sampling is not possible, the samples will be stored in a freezer immediately after sampling, and will be kept stored until analysis. The analytical method will be provided in the study report.Study code: 11/339-022AL DRAFT 2 Study Plan Page 10 of 17
2.10. RESULTS EVALUATION 2.10.1.Definitions CellDensity: the number ofcells per mL Growth: the increase ofcell density overthe test period Biomass (b): the actual number of cells per volume of medium (cells/mL) calculated as the area underthe growth curve (A) Yield (y): the cell density at the end ofthe test minus the starting cell density AverageSpecific GrowthRate (J.1): the increase incell density pertime unit the calculated concentration of test item which results in a 50 % reduction ofbiomass (b) relative to the control the calculated concentration oftest item which results in a 50 % reductionofgrowthrate (J.!) relative to the control the calculated concentration oftest item which results in a 50 % reduction ofyield (y) relative to the control NOEC: (No Observed Effect Concentration) the highest test concentration at which no significant inhibition ofgrowth is observed relative to the control LOEC: (Lowest Observed Effect Concentration) the lowest test concentration at which a significant inhibition of growth is observedrelative to the control 2.10.2.Calculation ofAverage Specific Growth Rate Concentration-effect relationship was calculated by comparing growth rates in control and test cultures in the following way. The average specific growth rate (J.!) for individual cultures are calculated from the following relationship: In(Nn)-ln(NO ) J.!= ..**-.~.~~-** 

### tn-to Where natural logarithm ofmeasured cell density at time tn natural logarithmofmeasured cell density attimeto time (hour) ofthe beginning ofthetest 

### = time (hour) ofnth measurements afterthe beginning ofthe testStudy code: 11/339-022AL DRAFT 2 Study Plan Page 11 of 17 Thepercentage inhibition ofgrowth rate (% Ill): %1

### =~I ·100 % J1 f.1c Where %1 Il 

### = percent inhibition in average specific growth rate mean growthrate ofthe control = mean growthrate oftest concentrationt 2.10.3.Calculation ofAreaUnderthe GrowthCurve NI - NoNI 

### + N 2 - 

### 2No ( ) Nil I

### + Nfl - 

### 2No ( ) A = . tl 

### + . t2 - tl 

### + . tn - tn - I 2 2 2 Where = nominal cell density attime to (start ofthe test) mean measured cell density at t] (24 hours) meanmeasured cell density at t2 (48 hours) = meanmeasured cell density attn = time offirst measurement after start ofthe test t2 = time ofsecond measurement after startofthe test tn time ofnth measurement after start ofthe test The percentage inhibition ofarea (% IA): 

### % J = Ac-A, .100 % A Ac Where percent inhibition in areaunder the growth curve = mean area (biomass) ofthe control 

### = meanarea (biomass) oftest concentrationt 2.10.4.CalculationofYield Yield is calculated as the biomass atthe end ofthe test minus the startingbiomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated. 

### Percentinhibitioninyield (% 

### Iy): I 

## =-=-----=- . 100 %

y where: 

### Yc = meanvalue foryield inthe control group 

### Yi = meanvalue for yield for the test concentration Area under the growth curve, average specific growth rate and yield will be calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.Study code: 11/339-022AL DRAFT 2Study Plan Page 12 of 17


### 2.10.5. Statistical evaluation The section-by-section specific growth rates in the control cultures will be assessed (calculated as the specific growth rates for each day during the course ofthe test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period. The inhibition of alga growth will be determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations will be calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co.lOne Microsoft Way/Redmond, WA 98052-6399). In case ofsufficient effects the ErCso, EbCSO and EyCso values ofthe test item and their confidence limits will be calculated using preferably Probit-analysis by TOXSTAT SPSS PC+ software. Othermodels for LCsocalculationmay be used ifappropriate. Statistical comparisons ofbiomass, average specific growth rates and yield in control and in treated groups will be carried out using analysis ofvariance (ANOVA) and Bonferroni t-Test (a 

### 0.05) by TOXSTAT or SPSS PC+ software. Ifa limit test will be performed, biomass, average specific growth rates and yield in control and in treated group will be analysed using 2 Sample t-Test (a 

### 0.05) by TOXSTA T software to compare means. For the determination ofthe LOEC and NOEC, the calculated mean biomass, growth rates and yield atthe test concentrations will be tested on significant differences to the control values by Bonferroni t-Test.


### 3. GOODLADORATORYPRACTICE (GLP)AND QUALITY ASSURANCE In order to enable the results of this study to meet legal requirements including regulatory submission and/or safety assessment, it will be performed in compliance with the principles of Good Laboratory Practice Regulations as requested by the Sponsor and specified in national Hungarian GLP Regulations: 9/2001. (III.30.) EtiMFVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, ENVIMC/CHEM (98) 17, which is consistent to the United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments. These principles are consistent with Good Laboratory Practiceregulations specified by EPA 40 CFR 792. The Quality Assurance Unit will conduct inspections ofthe study plan, various phases ofthe study, certain repetitive operations and the report will be audited according to applicable Standard Operating Procedures.Study code: 11/339·022AL DRAFT 2 Study Plan Page 13 of 17


### 4. AMENDMENT AND DEVIATION PROCEDURES Planned changes to the study plan can be made at the discretion ofthe Study Director and will be agreed with the Sponsor. Detailed descriptions of all study plan amendments will be effective at the time of Study Director's signature. All such amendments will be sent to the Sponsor. The study plan amendment will be distributed and added to all copies ofthe study plan. Unplanned changes to the study plan will be documented in the raw data and communicated to the Sponsor. The chapter "Deviations to the Study Plan" in the final report will reflect every study plan deviation, the reason for the study plan deviation and its anticipated effect onthe outcome ofthe study. 

### 5. STUDY REPORT The report will include, but notbe limited to the following information:

- Nameand addressofthe sponsor,thetest facility andthe study schedule.

- The names ofthe study directorand other scientists and supervisorypersonnel involved in the study. The signatures ofthe studydirectorandthemanagement. The signed and dated reports ofeach ofthe individual scientists or other professionals involved inthe study. Thequality assurance statement, signedbythe QAU. - A copyofthetestfacility's GLP-Certificate. The statementofcompliance, signedbythe studydirector. The identification ofthe test item, either by name orcode number, concentration, purity, compositionandproperties as far asavailable. - A description ofthe test system, including the materials used, a detailed description ofthe formulation ofthetest item. - A descriptionofallmethod'i usedwithreferences.

- All deviations to the studyplan. - A description ofall circumstances 

### that may have affected the quality or integrity ofthe study. - A descriptionandassessment for all results includingthedatacalculations andifnecessary statistical analyses.

- The storage location ofall data, study plan, oneoriginal version and one certified copy of thefinal reportandareference sampleofthetestitemandreference item.Study code: 111339-022AL DRAFT 2 Study Plan Page 14 of 17 

### Results:
- cell concentration for each flask at each measuring point and method for measuring cell concentration

- mean values ofcell concentrations

- growth curves

- graphical presentation ofthe concentration effect relationship

- EC values and method ofcalculation

- NOEC, LOEC

- other 0

### bserved effects

- results ofanalytical measurements Where appropriate, data will be summarized in tabular form to include mean values and a measure ofvariability. A draft report will be sent to the Sponsorby email for comment and review. The final report will be issued as a PDF document and a Word document and as one bound and one unbound papercopies. 

### 6. ARCHIVES The study documents:

- study plan and any amendments,

- all rawdata,

- sample ofthe test item,

- studyreport and any amendments,

- correspondence will be stored according to the Hungarian GLP and to the facility SOP-s in the archives ofCiToxLAB Hungary Ltd., 8200 Veszprem, Szabadsagpuszta. After the retention time has elapsed all the archived materials listed above would be offered to the Sponsor or retained for a further period if agreed by a contract. Otherwise the materials will be discarded. 

### 7. DISTRIBUTION OFTHE STUDY PLAN Sponsor: 1 x PDF-file, 1 x copy Study Director: 1copy QAU: 1copy Archive: 1original Departments*: 1 x copy each
>I< Central Dispensary, Scientific DocumentationStudy code: 11/339-022AL DRAFT 2 Study Plan Page 15 of 17


### 8. SIGNATURES The Study Director, CiToxLAB Hungary Ltdo's management, QAU will sign the Study Plan before the start ofthe experiment. The PDF version ofthe Study Plan will be sent to the Sponsor after the content ofthe Study Plan has been agreed. Ifthe Sponsor does not sign the Study Plan within 5 working days (or as specified in the contract) then the experiment may be started with the signature of the Sponsor being made after experiment initiation. 

### 9. REFERENCES 1. OECD Guideline for Testing ofChemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth InhibitionTest", adopted 23rd 

### March, 2006. 2. Commission Regulation (EC) No 76112009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 

### laying down test methods pursuant to Regulation (EC) No 1907/2006 

### of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal ofthe European Union L220 of24 August 2009) 3. EPA Ecological Effects Test Guidelines, OPPTS 850.5400, Algal Toxicity, Tiers I 

### and II,

### EPA 712-C-96-164, Adopted April 1996 4. Directive 200411

### O/EC of the European Parliament and of the Council of 11 

### February 2004 5. OECD Principles of Good Laboratory Practice, adopted by Council on 26th November 1997 [C(97)186/Final], Environment Directorate, Organisation for Economic Cooperation and Development, ENV

### IMC/CHEM(98)17, Paris 1998 6. Hungarian Good Laboratory Practice Regulations: 9/2001 

### (III. 30) EUM-FVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP ENV

### IMC/CHEM (98) 17 7. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and AssessmentNo. 23, Paris September 2000.Study code: 11/339-022AL DRAFT 2 Study Plan Page 16 of 17

Startofexperiment Preparationoftest item solutions Analytical sampling Measurement oftest conditions Treatment (Inoculationofalgacells) Measurementoftemperature Measurement ofcell concentration Measurementoftemperature Measurement ofcell concentration Measurement oftest conditions Measurementofcell concentration Analytical sampling End ofexperiment
Draft Report APPENDIX I 

### STUDY SCHEDULE
Relative date day 0 day 0 day 0 day 0 day0 day 1 day 1 day 2 day 2 day 3 day 3 day 3 day 3 Absolute date DD/MM/YY DD/MM/YY DD/MM/YY DDIMM/YY DD/MM/YY DD/MM/YY DD/MM/YY DD/MM/YY DD/MM/YY DDIMM/YY DD/MM/YY DD/MMIYY DDIMM/YY
DD/MMIYY Remark: The exact Study Schedule will be given in Amendment, because the contents ofthis schedule depend on the test item formulation method will be used during the definitive test or on the stability ofthe test item.Study code: 111339'()22AL DRAFT 2 Study Plan Page 17 of 17 

### APPENDIX 2 

### COMPOSITION OF OEeDMEDIUM Separate stock solutions will be first prepared in deionised water. The growth medium will be prepared by adding an appropriate volume ofthese different stock solutions to deionised waterin orderto achieve the final concentrations.
Final concentration in the Stock solution Substance prepared growth medium NH4Cl 15.0 mglL MgCI2 x 6 H2O 12.0 mglL Stocksolution 1 (macro nutrients) CaCh x 2 H 2 O 18.0 mglL MgS04 x 7 H2O 15.0 mglL KH2P04 1.6 mglL FeCb x 6 H2O 64.0 f..lglL Stocksolution 2 (iron) Na2EDTA x 2H2O 100.0 f..lg/L H3B03 185.0 f..lg/L MnCh x 4 H2O 415.0 f..lglL Stocksolution 3 (trace elements) ZnCI2 CoClzx 6 H2O 3.0 f.tg/L 1.5 f..lglL CuClz x 2 H2O 0.01 f..lglL Na2Mo04 x 2 H2 O 7.0 f.tglL Stock solution 4

(bicarbonate) NaHCO) 50.0 mglLFedEx Ship Manager API Label Page Page 1of 1
From: (561) 578-8110 Origin 10: PBlA JOHN V. MASSINGALE GREENWICH CHEMICAL CONSULTING, INC. 3300 PGA BLVD SUITE 635 PALM BEACH GARDENS, FL33410 J121011l219022:5 Ship Date: 19APR12 ActNgt 1.0LB CAD: 10209719JM1SXI2600 Delivery Address BarCode 1111111111111111111111111111111111111111111 Ref# Invoice # PO# Dept# RELEASE#3785346 SHIP TO: (202) 564·8930 OPPT Document Control Office U.S. Environmental Protection Agenc Room E6428 1201 Constitution Ave., NW Washington, DC 20004
lRK# 7983 0288 4677 10201 FRI- 20 APR A1 STANDARD OVERNIGHT 

# XC RDVA 20004 DC·US 

### DCA

5t2GtlCUDJA278
This AWB label shouldprint in full on one page Ifthe label does not print cOlUplelely on one page use the ,;shrink to filI.I or t Iwhole pageU option on your page settings. Of change all the pagemargins to O.7cm. Please can your local office for further assistance ifrequired WARNING: USE ONLYTHE PRINTED ORIGINAL LABELS fOR SHIPPING TO ENSURETHETIMELY lJELIVERYOf YOUR PACKAGE.

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