GREENWICH CHEMICAL CONSULTING, INC. 

3300 PGA BOl:LEVARD • SUITE 635 


PALM BEACH GARDENS, FL 33410 


TELEPHONE 561. 578.811 0 


FACSIMILE 561.578.8120 


April 19,2012 

.j)

.. CONTAINS NO CONFIDENTIAL 'f, 

BUSINESS INFORMATION 

By Federal Express 

OPPT Document Control Office 

EPA East Building IIIIIIIII/IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~ 1111111111 III/ 1111 

40120000061 Room E6428 

U.S. Environmental Protection Agency 

1201 Constitution Ave., NW 

Washington, DC 20004 

Attention: 	 Docket ID 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 of Certain High Production Volume Chemicals: Second Group of 

Chemicals 

Ethanedioic acid 


CAS RN 144-62-7 


(Oxalic acid) 


Docket Identification No. EPA-HQ-OPPT-2007-0531 


40 CFR 799.5087 


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 CSI 

CONTAINS 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 at 40 CFR 790.50 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 study plan has already been submitted to EPA.) 

This test rule addresses 19 high production volume (HPV) chemicals that went unsponsored 

under the Agency's voluntary HPV Chemical Challenge Program. One of the 19 chemicals 

identifled in the rule is oxalic acid. The flnal rule identifles the following nine studies on 

oxalic acid: 

• Melting Point 

• Boiling Point 

• Vapor Pressure 

• Water Solubility 

• Ready Biodegradation 

• 	 Aquatic Toxicity Testing: 


Acute toxicity to flsh 


Acute toxicity to Daphnia 


Toxicity to plants (algae) 


• In Vitro Mammalian Chromosome Aberration Test 

II. 	 Prior Submission of the Chromosome Aberration Test Study Plan and the Current 

Submission of the Remaining Eight Tests 

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.' 

At this time, the Companies herein submit to EPA study plans for the remaining eight tests. 

1 January 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 


study plans must be submitted to the Agency. 


3 Submission from Greenwich Chemical Consulting to the _\gency dated February 2012. 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

April 19, 2012 

Page 3 

The Companies have selected CiT oxLAB 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 Chemical Consulting is 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 other related testing and procedural information. 

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 of these four plans be provided instead of final versions. 

III. Use of OEeD Test Guidelines 

In the final TSCA Section 4 test rule, EPA specified the use of ASTM, 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 Chemical Consulting, Inc. 


3300 PGA Boulevard, Suite 635 


Palm Beach Gardens, FL 33410 


Telephone: (561) 578-8110 


Facsimile: (561) 578-8120 


E-mail: john@grcci.com 


.\ Electronic mails from Dr. Paul J. Campanella (EPA) to Mr. John Y . .\Iassingale (Greemllich Chemical 

Consulting) . 
CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

April 19, 2012 

Page 4 

Sincerely, 

John V. Massingale 

Principal 

cc: 	 Dr. P. Campanella - EPA 

U ruvar USA Inc. 

WEGO Chemical and Mineral Corporation 

Enclosures 
CiToxLAB Hungary Ltd. 

Veszprem - Szabadsagpuszta 

STUDY PLAN 


Determination of the Boiling Point 

of Ethanedioic acid 

Study code: 11/339-324AN 


Study Director: Z501t Sarvari, M.Sc. 


03 April 2012 

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

STUDY TITLE: 

SPONSORS: 

STUDY MONITOR: 

TEST FACILITY: 

TEST FACILITY 

MANAGEMENT: 

STUDY DIRECTOR: 

ASSISTANT SCIENTIST: 

Determination of the Boiling Point of Ethanedioic acid 


Univar USA Inc. 


Address: 17425 NE Union Hill Road, 


Redmond, W A 98052, USA 

Phone: +1 425 8893400 

Fax: + 1 4258893760 

WEGO Chemical and Mineral Corp. 

Address: 239 Great Neck Road, 

Great Neck, 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. 


(F onnerly known as LAB Research Ltd. before I st September 2011 ) 


Address: H-8200 Veszprem, Szabadsagpuszta 


Phone: +36 88 545 300 


Fax: +3688545301 


Christopher Banks, DABT 


Managing Director 


Alyson Leyshon, M.Sc. 


Senior Director of Toxicology 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 of Quality 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 

DRAFT REPORT: not later than 27 April 2012 

* Other trained, competent personnel could be involved in the study, if required. 
Study Gode: 11/339-324AN Study Plan Page 4 of 9 

STUDY PLAN APRROV AL: 


APPROVED BY 


ON BEHALF OF SPONSOR: 


APPROVED BY 


ON BEHALF OF CiToxLAB: 


STUDY DIRECTOR: 


QUALITY ASSURANCE: 


DATE: 

Christopher Banks, DABT 

Managing Director 

Zsolt Sarvari, M.Sc. 

7~- U~ 

Yanda Gyimesi, M.Sc. 

On behalfofQAU 
Study code: 11/339·324AN Study Plan Page 5 of 9 

1. OBJECTIVE OF STUDY 


For the detennination of the boiling point, the capillary method will be used. A 

small amount of the 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. A phase transfonnation 

from liquid to vapour is usually deduced by the fonnation of bubbles. 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 heating temperature. 

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: 

BatchlLot number: 

Molecular formula: 

Molecular weight: 

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 4 x2H20 

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 of the test item were 

supplied by the Sponsor as well. 

Identification of the test item was made by its name, batch, appearance and 

colour by Central Dispensary Unit ofCiT ox LAB Hungary Ltd. 
Study code: 11'339-324AN Study Plan Page 6 of 9 

2.2. TEST PARAMETERS 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 

The test item will be used in the initial form. 

2.3.2. Preliminary Test 

The boiling behaviour of test item will be estimated in a preliminary test. For 

this, a sample of test item placed in a glass tube will be heated starting at 

room temperature up to about 400°C at a rate of approximately 10°C/min 

unless a phase transition or reaction ofthe test 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 of the test item is observed, the experiment wi II be repeated with a 

new test item aliquot in the immediate vicinity of the temperature of the phase 

transition observed. For the actual determination of the boiling point, the 

temperature rise will be adjusted to a maximum of 0.5 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, if a 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 of a liquid is the same as the standard pressure (101.325 kPa). 

2.3.5. Observations 

Upon approach of the 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 of bubbles stops and the liquid 

suddenly rises in the capillary. 

2.3.6. Data Handling 

The measured boiling point 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 of many 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 of units: t [0C] = T [K] - 273.15 

where, 


t Celsius temperature, 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 of this correction 

factor is beyond the scope of this study, therefore no correction will be 

applied to the boiling point. 
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 of the 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 of the 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 of the study. 

4. GOOD LADORA TORY PRACTICE (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-FVM 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 40 CFR 792. 

The Quality Assurance Unit will conduct inspections of the 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, 


sample of the 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 ox LAB 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: detai led description of the method used 

Results: all measured and observed data 

7. DISTRIBUTION OF THE STUDY PLAN 

Archive original 


Sponsor pdf-file 


Study Director 1x copy 


QAU 1x copy 

CiToxLAB Hungary Ltd. 

Veszprem - Szabadsagpuszta 

STUDY PLAN 


Determination of Melting Point 

of Ethanedioic 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: 

TEST FACILITY: 

TEST FACILITY 

MANAGEMENT: 

STUDY DIRECTOR: 

Determination of the Melting Point of Ethanedioic 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 Great Neck 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: +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 


Christopher Banks, 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 

Study 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 


Yanda Gyimesi, 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 of the 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 START OF EXPERIMENT: 04 April 2012 

PROPOSED END OF EXPERIMENT: not later than 13 April 2012 

DRAFT REPORT: not later than 27 April 2012 

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

---..-.-.~--

STU])Y PLAN APRROVAL: 

APPROVED BY 


ON BEHALF OF SPONSOR: 


APPROVED BY 


ON BEHALF OF CiToxLAB: 


STUDY DIRECTOR: 


QUALITY ASSURANCE: 

O~.~..... . Christopher Banks, DABT 


Managing Director 


Zsolt Sfuvari, M.Sc. 

VnA-~' lJ~ DATE: 0;' 1Jr..( (o.{ L 

~anda Gyimesi, M.Sc. 

On behalf of QAU 
Study code: 111339·344AN Study Plan Page 5 of 9 

1. OBJECTIVE OF THE STUDY 


For the determination of the melting point or melting range, the capillary method will be 

used. A small amount of the 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 under 40 CFR 799.5087. 

2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: 

Chemical name: 

Synonym: 

CAS number: 

Batch/Lot number: 

Molecular formula: 

Molecular weight: 

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 of the test item were supplied by the 

Sponsor as well. 

Identification of the 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. TEST PARAMETERS AND CONDITIONS 


Test System: 

Surroundings: 

Accuracy: 

2.3. TEST METHOD 

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 of reference substance's melting point. 

2.3.1. Preparation of the Glass Tube 

A small amount of test item will be charged in a capillary glass tube with a diameter of 

about 2 mm and packed tightly to obtain a filling level of approximately 5 mm. The tube 

will be heated in the oven ofthe melting point device. 

2.3.2. Preliminary Test 

The melting behaviour of test 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 or reaction ofthe test 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 

If in the temperature range investigated in the preliminary test, a phase transition of test 

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 of the 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 of a 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: Beginning ofmelting / wet point 


Fine droplets adhere uniformly on the inside wall ofthe capillary tube. 


Stage B: Shrinkage point 


A clearance appears between the sample and the inside wall due to shrinkage ofthe melt. 


Stage C: Col/apse point 


The shrunken sample begins to collapse downwards and liquefies. 


Stage D: Liquefying point 

A complete meniscus is formed at the surface, but an appreciable amount of the sample 

remains solid. 

Stage E: Final Stage ofmelting 


There are no solid particles. 


During the determination of the melting temperature, the temperatures are recorded at the 

beginning ofthe melting and at the final stage. 

2.3.6. Data Handling 

The melting point of the test item ideally corresponds to the solidification or freezing 


temperature. 


As the phase transition of many test items takes place over a large temperature range, it is 


often described as the melting range. If the difference between the temperature at the 


beginning and at the final stage of melting is within the limits of the accuracy of the 


method, the temperature at the final stage of melting is taken as the melting temperature; 


otherwise the two temperatures are reported. 


Results are given in degrees centigrade and in Kelvin. 


Conversion of units: 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 DEVIATION PROCEDURES 


Planned changes to the study plan can be made at the discretion of the 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. GOOD LABORATORY PRACTICE (GLP) AND QUALITY ASSURANCE 

In order to enable the 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 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 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, 


sample of the 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 of the Vapour Pressure 

of Ethanedioic 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 

GENERAL INFORMATION 


STUDY TITLE: 


SPONSORS: 


STUDY MONITOR: 

TEST FACILITY: 

TEST FACILITY 


MANAGEMENT: 


STUDY DIRECTOR: 

ASSISTANT SCIENTIST: 

Determination of the Vapour Pressure of Ethanedioic acid 


Univar USA Inc. 


Address: 17425 NE Union Hill Road, 


Redmond, W A 98052, USA 


Phone: +1 425 889 3400 


Fax: +1 4258893760 


WEGO Chemical and Mineral Corp. 

Address: 239 Great Neck Road, 

Great Neck, NY 11021, USA 


Phone: + I 516 487 3510 


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 I st September 20 II) 


Address: H-8200 Veszpn5m, Szabadsagpuszta 


Phone: +3688545300 


Fax: +3688545301 


Christopher Banks, DABT 


Managing Director 


Alyson Leyshon, M.Sc. 


Senior Director of Toxicology 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 of the 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 of Chemicals, 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 START OF EXPERIMENT: 04 April 2012 

PROPOSED END OF EXPERIMENT: not later than 13 April 2012 

DRAFT REPORT: not later than 27 April 2012 

* Other trained, 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 

ON BEHALF OF CiToxLAB: ;t.1'~-.. DATE: D'? j4. __ ~ ( 2..ID 1'-..

Christopher Banks, DABT 

Managing Director 

STUDY DIRECTOR: 

~1f.V-' V~ DATE: 0 'J ~.( 10Ic QUALITY ASSURANCE: 

Yanda Gyimesi, M.Sc. 

On behalf ofQAU 

Zsolt Slirvw: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 1 ml sample volume. 

The instrument consists of a vacuum-sealed measuring chamber of an overall volume 

of 5 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 CFR 799.5087. 

2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: 

Chemical name: 

Synonym: 

CAS number: 

Batch/Lot number: 

Molecular formula: 

Molecular weight: 

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 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 of the test item was made by its name, batch, appearance and colour by 

Central Dispensary Unit of CiT ox LAB 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 of Measurement: 	 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 water purification 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 of temperature will be registered in a suitable range in 5 °C steps. The lowest 

detectable vapour pressure is 0.5 kPa. 
Study code: 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 of at least two measurements will be reported. 

3. AMENDMENT AND DEVIATION PROCEDURES 

Planned changes to the study plan can be made at the discretion of the 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 of the 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 of the study. 

4. GOOD LABORATORY PRACTICE (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 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 40 CFR 792. 

The Quality Assurance Unit will conduct inspections of the study plan, various phases 

of the study, certain repetitive operations and the report will be audited according to 

applicable Standard Operating Procedures. 
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 of the 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. 

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 I x copy 


QAU 1x copy 

CiToxLAB Hungary Ltd. 

Veszprem - Szabadsagpuszta 

STUDY PLAN 


Determination of the Water Solubility of 


Ethanedioic acid 


Study code: 111339-345AN 


Study Director: Zso1t Sarvari, M.Sc. 


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

STUDY TITLE: 

SPONSORS: 

STUDY MONITOR: 

TEST FACILITY: 

TEST FACILITY 

MANAGEMENT: 

STUDY DIRECTOR: 

ASSISTANT SCIENTIST: 

Detennination of the Water Solubility of Ethanedioic 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 Great Neck 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 Director of Toxicology Operations 


Zsolt Sarvari M.Sc. 


Phone: +36 88 545 361 


E-mail: zsolt.sarvari@hu.citoxlab.com 


Olga Ulbert, M.Sc. 

Study code: 11f339·345AN Study Plan Page 3 of 8 

LEAD QA"': 

RESPONSIBLE PERSONS"': 

BASIS OF STUDY: 

REPORTING FORMAT: 

REPORT COPIES: 

Szabolcs Gcity M.Sc. 


Head of Quality Assurance Unit 


Yanda Gyimesi, 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 of the Council on the Registration, 


Evaluation, Authorisation and Restriction of 


Chemicals (REACH), Method A.6. "Water 


Solubility", Official Journal of the 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 START OF EXPERIMENT: TBD 

PROPOSED END OF EXPERIMENT: TBD 

DRAFT REPORT: TBD 

TBD: to be determined and documented by an amendment 

* Other trained, competent personnel could be involved in the study. if required. 
Study code: 11/339-345AN Study Plan Page 4 of 8 

STUDY PLAN APRROVAL: 


APPROVED BY 


ON BEHALF OF SPONSOR: 


APPROVED BY 


ON BEHALF OF CiToxLAB: 


STUDY DIRECTOR: 


QUALITY ASSURANCE: 

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

\......-r-~ 

Christopher Banks, DABT 

Managing Director 

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

0, <LtA-~: tI~ 

/ Yanda Gyimesi, M.Sc. 


On behalfof QAU 


DATE: \'\ ~',,,-u.') ,'­

DATE: ;f ';t ~.( (0) 'L 
Study 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 of the test item in water at 20 


± 1°C. 


As the expected water solubility is above 10.2 gil, 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 40 CFR 799.5087. 


2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: Ethanedioic acid 

Chemical name: Ethanedioic acid 

Synonym: Oxalic acid 

CAS number: 144-62-7 

BatchlLot number: YP2-20 II 0704B 

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 of the test item were supplied by the 

Sponsor as well. 

Identification of the test item was made by its name, batch, appearance and colour by 

Central Dispensary Unit of CiToxLAB 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 of each sample will be recorded. 

The mean of three 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 of mass per volume of solution. 

The individual results will be given for each of the three flasks and those results 

deemed to be constant (repeatability of less than 15 %) will be averaged and given in 

units of mass per volume of solution. 

4. AMENDMENT AND DEVIATION PROCEDURES 

Planned changes to the study plan can be made at the discretion of the 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 of the study plan. 

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 of the 

study. 

5. GOOD LABORATORY PRACTICE (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.) 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 of the study plan, various phases 

of the 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 study documents: 

- study plan and any amendments, 


- all raw data 


- sample ofthe test item 


- study report and any amendments 


- correspondence 


will be stored in the archives of CiToxLAB Hungary Ltd., Hungary 8200 Veszprem, 

Szabadsagpuszta according to the Hungarian GLP and our SOP-so 

8. DISTRIBUTION OF THE STUDY PLAN 

Archive 

Sponsor 

Study Director 

QAU 

original 

pdf-file 

I x copy 

I x copy 
CiToxLAB Hungary Ltd. 

Veszprem - Szabadsagpuszta Hungary 

DRAFT 2 


STUDY PLAN 


Ready Biodegradability of Ethanedioic acid 

in a Closed Bottle Test 

Study code: 1l/339-322AN 

Study Director: Krisztina Sipos, M.Sc. 

CONTAINS NO CBI 

11 April 2012 
Study 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 of Ethanedioic acid 

in a Closed Bottle Test 

Ethanedioic acid 

Univar USA Inc. 

Address: 	 17425 NE Union Hill Road 


Redmond, W A 98052 


USA 


Telephone: + 1 4258893400 


Facsimile: +14258893760 


WEGO Chemical and Mineral Corp. 

Address: 	 239 Great Neck Road 

Great Neck, NY 11 021 

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 I st September 20 II) 


Address: 	 H-8200 Veszprem, Szabadsagpuszta 

Hungary 

Phone: 36 88 545-300 


Fax: 36 88 545-301 

Study code: 11/339-322AN DRAFT Study Plan 2 Page 3 of 15 

APPROVED BY 

ON BEHALF OF SPONSOR DATE: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

APPROVED BY 

ON BEHALF OF CiToxLAB DATE: 

Christopher Banks, 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 with 40 CFR 792 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 

PRELIMINAR Y EXPERIMENT: 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: 11/339-322AN DRAFT Study Plan 2 	 Page 5 of 15 

CONTENTS 

Page 

1. 	 OBJECTIVE OF THE STUDY 6 


1.1. 	 PURPOSE 6 


1.2. 	 DEFINITIONS 6 


1.3. 	 CONDITIONS FOR THE VALIDITY OF THE TEST 6 


2. 	 MATERIALS AND 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 LABORATORY PRACTICE (GLP) AND 


QUALITY ASSURANCE 13 


4. 	 AMENDMENT AND DEVIATION 


PROCEDURES 12 


5. 	 ARCHIVES 13 


6. 	 DISTRIBUTION OF THE STUDY PLAN 14 


7. 	 SIGNATURES 14 


8. 	 REFERENCES 14 


APPENDIX 1 	 15 

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

1. OBJECTIVE OF THE STUDY 

1.1. PURPOSE 

The purpose of this study is to determine the ready biodegradability of the test item. 

The test item is exposed to activated sludge from the aeration tank of a domestic waste 

water treatment plant for 28 days. The biodegradation is followed by the oxygen 

uptake of the 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 and the regulations set forth at 40 CFR 799.5087. 

1.2. DEFINITIONS 

ThODNH4 : 	 Theoretical Oxygen Demand, the total amount of oxygen required 

to oxidise a chemical completely. It is calculated from the 

molecular formula, assuming no nitrification occurs, and expressed 

as mg oxygen required per mg test item. 

BOD: 	 Biochemical Oxygen Demand, the amount of oxygen consumed by 

microorganisms when metabolising a test item; also expressed as 

mg oxygen uptake per mg test 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 within the 28-d period of the test. 

1.3. CONDITIONS FOR THE VALIDITY OF THE TEST 

• Oxygen depletion in the inoculum control will not exceed 1.5 mg 02/L after 28 days. 

• The residual oxygen concentration in the test flasks will not drop below 0.5 mg 02/L 

at any time. 

• The difference of duplicate values for the degradation at any time during the test will 

not be greater than 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 of the test item: 


Manufacture date: 


Retest date: 


Storage condition: 


Safety Precautions: 


2.2. REFERENCE ITEM 

Name: 


Molecular Formula: 


Molecular Weight [glmoIJ: 


Batch Number: 


Expiry date: 


Supplier: 


Storage condition: 


Safety precautions: 


Ethanedioic acid 


Ethanedioic acid 


Oxalic acid 


YP2-20110704B 


144-62-7 


C2H20 4 


126.1 g/moI (dihydrate form) 

99.71% 

White crystals 

04 July 2011 

03 July 2012 

controlled room temperature (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 

C7HsNa02 

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 of a suitable chemical purity was suppJied by the Sponsor. Precautions 


required in the handling and disposal of the test item were supplied by the Sponsor. 


These documents are part of the raw data. 


Identification of the test item was performed in the Central Dispensary of CiToxLAB 


Hungary Ltd. on the basis of the 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 water analytical grade salts* will be added (in the following ratio) to prepare 

the following stock solutions: 

a) Solution: KH2P04 2.l25g 

K2HP04 5.4375g 

Na2HP04 x 12H20 16.795g 

NH4Cl O.l25g 

Deionised water ad 250 mL 

b) Solution: MgS04 X 7 H20 5.625g 

Deionised water ad 250 mL 

c) Solution: CaCb x2 H20 9.10 g 

Deionised water ad 250 mL 

d) Solution**: FeCh x 6 H20 0.25g 

Deionised water ad 1000 mL 

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

the Report 

** If this stock solution will not applied on the day of preparation one drop of concentrated Hel per 

litre will be added 

2.4.2. Ratio of ingredients 

1 mL of the 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 of the final sludge 

suspension will be weighed, dried and the ratio of wet 

sludge to its dry weight will be determined. Based on this 

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 of measurement 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 of test water will be about 9 mg/L at the start of the test (it 

will be measured at the start). 

The pH value of the test water will be checked prior study start and will be adjusted to 

7.4 ± 0.2 with HCI or NaOH if necessary. 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. PREP ARA TION OF THE TEST SOLUTIONS 

Before the preparation of the respective test solutions with test item an aqueous stock 


solution ofthe test item will be prepared and diluted accordingly. 


The respective amount of the test item will be weighed in directly to reach the required 


test item concentration chosen based on the theoretical oxygen demand (ThODNH4). 


1.) Test Item (flasks 1a and 1 b) 

The chosen test item concentration will be based on the theoretical oxygen demand 

(ThO~H4) of the 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 amount of aqueous test medium. 

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b) 

Based on the theoretical oxygen demand (ThODNH4) of Sodium 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 of about 6.012 mg 021L). 

*the concentration ofthe stock solution will be 360 mgIL 

3.) Inoculum Control (flasks 3a and 3b) 

Only filtered inoculum wil1 be added to 3.40 litres of aqueous test medium. 

4.) Toxicity Control (flasks 4a and 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/L reference item (ThODNH4 of6.012 mg 02/L). 

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

2.9. COURSE OF THE TEST 

2.9.1. Preparation of Test Flasks 

A sufficient number of BOD flasks will be cleaned with 5 - 10 mL of a 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 containing the test item and inoculum 

• 	 at least 10 bottles containing the 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. Measurement of Oxygen 

The incubation period of the 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 of oxygen consumption measurement. 
Study code: 11/339-322AN DRAFT Study Plan 2 Page 12 of 15 

2.11. CALCULAnON OF RESULTS 

2.11.1. Calculation of BOD 

The BOD (mg O2 per mg test item) expected after each period will be calculated as 

follows: 

mg of T.i. and/or R. i. - mg of i.control 

mg T.i. and/or R.i.lL in flask 

where: 

T.i. test item 

R.i. = reference item, 


i.control = inoculum control 


2.11.2. Calculation of Biodegradation % 

The percentage biodegradation of the test item and of the reference item will be 

calculated as follows: 

BOD (mg T.i. or R.i.) ....·~·--~~·····~~~~······~~~--~----x100 

ThOD NH4INH4 (mg O/mg T.i. or R.i.) 

where: 

T.i. test item 

R.i. = reference item, 


i.control = inoculum control 

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

3. GOOD LADORATORY PRACTICE (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.) EliM-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. 

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 

of the study, certain repetitive operations and the report will be audited according to 

our Standard Operating Procedures. 

4. AMENDMENT AND DEVIATION PROCEDURES 

Planned changes to the study plan can be made at the discretion of the 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 of the study plan. 

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 of the 

study. 

5. ARCHIVES 

The study documents and samples: 

- study plan and any amendments, 


- all raw data, 


- sample of the test item, 


- correspondence, 


- study report and any amendments 


will be stored in the archives of CiToxLAB Hungary Ltd., Hungary 8200 Veszprem, 

Szabadsagpuszta according to the Hungarian GLP and our SOPs. 
Study code: 11/339-322AN DRAFT Study Plan 2 	 Page 14 of 15 

6. DISTRIBUTION OF THE STUDY 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 of the 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 of the Sponsor being 

made after experiment initiation. 

8. REFERENCES 

1. 	Council Regulation (EC) No 440/2008,Method C.4-E "Closed Bottle Test", Official 

Journal of the European Union L 142 of31 May 2008 

2. 	OECD Guideline for Testing of Chemicals No. 301 D: "Ready Biodegradability: 

Closed Bottle Test", adopted July 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-FVM joint 

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) I 86/Final], Environment Directorate, Organisation for 

Economic Co-operation and Development, ENV IMCICHEM(98)17, Paris 1998 

6. 	Directive 2004/10/EC of the European Parliament and of the Council of II February 

2004 on the harmonisation of laws, regulations and administrative provisions relating 

to the application of the principles of good laboratory practice and the verification of 

their applications for tests on chemical substances (codified version) 
Study code: 111339-322AN DRAFT Study Plan 2 Page 15 of 15 


APPENDIX 

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

APPENDIX 1 


PROPOSED STUDY SCHEDULE 


Preliminary Experiment: DD/MM/YY 

Pre-Experimental Period 

Preparation of Reconstituted Waterday day [-1] DD/MM/YY 

Preparation of activated sludge day [0] DD/MM/YY 

Experimental Period 

Start of experiment day [0] DD/MMNY 

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

Preparation oftest 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 of oxygen day [0] DD/MMNY 

Measuring ofoxygen day [7] DD/MM/YY 

Measuring of oxygen day [14] DD/MMNY 

Measuring of oxygen day [21] DD/MMNY 

Measuring of oxygen day [28] DD/MM/YY 

Measuring of temperature day [0 28] DD/MM DD/MMNY 

(at least at each day of oxygen measurement) 

End of experiment day [28] DD/MM/YY 

Draft Report not later than D D/MM/YY 

The exact study schedule will be given in an Amendment. 
CiToxLAB Hungary Ltd. 

Veszprem - Szabadsagpuszta 

DRAFT 2 


STUDY PLAN 


Acute toxicity test with Ethanedioic acid 

on Zebrafish (Brachydanio rerio) 

Study code: 111339-009H 

Study Director: Krisztina Sipos, M.Sc. 

CONTAINS NO CSI 


03 April 2012 
Study code: 111339-009H DRAFT 2 Study Plan 	 Page 2 of 14 

GENERAL INFORMATION 


STUDY TITLE 

TEST ITEM 

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: + 1 425 889 3400 


Facsimile: + 1 425 889 3760 


: WE GO Chemical and Mineral Corp. 

Address: 	 239 Great Neck Road 


Great Neck, 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 5788120 


E-mail: john@grcci.com 


: CiToxLAB Hungary Ltd. 

(Fonnerly known as LAB Research Ltd. before 1st September 

2011.) 

Address: H-8200 Veszpn:5m, Szabadsagpuszta 

Phone: 36 88 545-300 


Fax: 3688545-301 

Study code: 11/339-009H DRAFT 2 Study Plan Page 3 of 14 

APPROVED BY 

ON BEHALF OF SPONSOR 

APPROVED BY 

ON BEHALF OF CiToxLAB 

STUDY DIRECTOR 

QUALITY ASSURANCE 

RESPONSIBLE PERSON FOR 

ANALYTICAL MEASUREMENTS 

APPROVED BY 

ON BEHALF OF IACUC 

DATE: 

John V. Massingale 

Greenwich Chemical Consulting. Inc. 

DATE: 

Christopher Banks, DABT 

Managing Director 

DATE: 

Krisztina Sipos, M.Sc. 

DATE: 

Szabolcs GMy, M.Sc. 

DATE: 

Zsolt Sarvari, M.Sc. 

DATE: 

Member of IACUC 
Study 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 of Chemicals, No. 203, "Fish, Acute Toxicity Test", 

adopted July 17, 1992 

- EPA Health Effects 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 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACH), Annex Part C, C.l (published in the Official Journal of 

the European Union L 142 of 31 May 2008 

REPORTING FORMAT: 	 Final Report in line with 40 CFR 792 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 

COUrIer 

PRELIMINARY EXPERIMENT: 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 of this study is to evaluate the acute lethal toxicity of the test item on 

Zebrafish (Brachydanio rerio). The test animals will be exposed in a static or semistatic test to aqueous test media containing the test item at different concentrations. 

In case of sufficient effects, the 24-h, 48-h, 72-h and 96-h LCso values will be calculated. 

The recorded effects will include the mortality and symptoms of intoxication of the fish. 

If a limit test is performed the test animals will be exposed at a concentration of 

100 mg/L or the solubility level of the test item to demonstrate that the test item has no 

influence on test animals up to at least this 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 FOR THE VALIDITY OF THE TEST 

- Constant conditions should be maintained in this procedure. 

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

fish if less than ten are used). 

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

60 % of air saturation value. 

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

throughout the test. If the deviation from the nominal concentration is greater than 

20%, results will be based on the measured concentration. 

2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: 

Chemical name: 

Synonym: 

Batch number: 

CAS number: 

Molecular Formula: 

Molecular Weight: 

Purity: 

Description of the test item: 

Manufacture date: 

Retest date: 

Ethanedioic acid 

Ethanedioic acid 

Oxalic acid 

YP2-20110704B 

144-62-7 

C2H20 4 

126.1 g/mol (dihydrate form) 

99.71% 

White crystals 

04 July 2011 

03 July 2012 
Study code: 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 of the test item were outlined by the 

Sponsor. The test item was identified on the basis of the 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 of addition 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 of the 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 of the solvent in the final test 

medium will be not exceed 100 mg/L (or 0.1 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 of the 

same quality used for holding. 

Light: 12 to 16 hours photoperiod daily. 


Temperature range: 20 - 24°C with a maximum deviation of± 1 °C 


Dissolved oxygen concentration: At least 80 per cent of air saturation 


pH values: 6.0 to 8.5 


Feeding: Feeding daily, until 24 hours before the start 


of the 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 or by a method (e.g. WAF) according to the 

Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures, GEeD No. 23. 

The test solutions will be prepared just before introduction of the fish (start of the 

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 approximate toxicity 

of the 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 

(factor e.g. 10) of the test item 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 

If a 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 vehicle control ifused) will be used in the main test. 

If no 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 if used) will be tested in a limit-test. 

The concentrations in the definitive test will be based on the results of the preliminary 

range-finding test and will be given in Amendment. 

2.5. EXPERIMENTAL ANIMALS 

Species: Zebrafish (Brachydanio rerio) 

Source: "Black Molly" Fish Farm, Veszprem, Hungary 

Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient 

species for acute fish toxicity test. 

Number of animals: at least 7 fish in each concentration and control. 

Body length of animals: 3.0± 0.5 cm 

Acclimatisation: at least 12 days 
Study 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-controlled conditions. 


Circulated, filtered and cooled tap water (same as that 


will be used for the test) 


The fish will not be fed during the test 

The mortalities will be recorded for seven days 

acclimatisation period immediately before the test, the 

following criteria will be applied: 

- mortalities of greater than 10 per cent of 

population in seven days: rejection of entire 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 of the 

experiment. The fish will not be fed during the test. 

The dilution water will be aerated during the test if necessary. 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% of the 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 of 6.0 to 8.5. 

The hardness of the 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 of the 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 of the test. Visible abnormalities in appearance or behaviour will be 

recorded. 

The body weight offish will be recorded on the day 0 (at the start of the main study). 

The loading of the testing liquid in the test-aquaria will be calculated (g fish/litre 

testing liquid) on the basis of the body weight measurements data. 

2.9. ANALYTICAL MEASUREMENTS 

The concentration of the 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 of 

limit test the applied test concentration will be measured). Sample from the control will 

be taken for analysis at least at the start ofthe test. 

Analysis will be performed depending on the results of the analytical method 

validation. 

All samples will be analysed directly after sampling. If analysis 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. 

2.10. STATISTICAL EVALUATION 

In case of sufficient effects the LCso 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 euthanasia by direct depression of the 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 LABORATORY PRACTICE (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 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. AMENDMENT AND DEVIATION PROCEDURES 

Planned changes to the study plan can be made at the discretion of the 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 of the study. 

6. STUDY REPORT 

The study report will include the following information: 


The results of the study will be reported in a detailed final report. 


The original final report, in English will be sent to the sponsor and will include: 


- Name and address ofthe sponsor, the test facility and the study schedule. 


- The names ofthe study director and other scientists and supervisory personnel involved in 

the 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 QA. 
Study code: 111339-009H DRAFT 2 Study Plan Page 11 of 14 

- The statement ofcompliance, signed by the study director. 

- Summary ofthe results ofthe study, including the preliminary test 

The identification of the test item, either by name or code number, concentration, purity, 

composition and properties as far as available. 

- A description ofthe test system, including the materials used, a detailed description ofthe 

formulation ofthe test item. 

A description ofall methods used with references. 

All deviations to the study plan. 

- A description of all circumstances that may have affected the quality or integrity of 

the study. 


A description and assessment for all results including the data calculations and ifnecessary 


statistical analyses. 


- The storage location of all data, study plan, one original version of the [mal report and a 

reference sample ofthe test item and reference item. 

Where appropriate, data will be summarized in tabular form to include mean values 

and a measure of variability. 

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, and as one bound and one unbound paper copies. 

7. ARCHIVES 

The study documents: 

- study plan and any amendments, 


- all raw data, 


- specimen of the 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 of CiToxLAB 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. 
Study code: 11/339-009H DRAFT 2 Study Plan Page 12 of 14 

8. DISTRIBUTION OF THE STUDY PLAN 

Sponsor: 1 x PDF-file, 1 x copy 

Study Director: 1 copy 

QAU: 1 copy 

Archive: 1 original 

Departments*: 1 x copy each 

* Central Dispensary, Scientific Documentation and Analytics 

9. SIGNATURES 

The Study Director, CiToxLAB Hungary Ltd.'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 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 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-FVM joint 

decree of the Minister of Health and the Minister of Agriculture and Regional 

Development which corresponds 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 of the European Parliament 

and the of the Council on the Registration, Evaluation, Authorisation and 

Restrictions of Chemicals (REACh), Annex Part C, C.l (published in the Official 

Journal of the European Union L 142 of 31 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 

on Testing and Assessment No. 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 

Start of experiment day 0 DD/MM/YY 

Preparation of test item solutions day 0 DD/MM/YY 

Analytical sampling day 0 DD/MM/YY 

Measurement of body weight day 0 DDIMM/YY 

Treatment oftest animals day 0 DDIMM/YY 

Measurement oftest conditions day 0 DD/MM/YY 

Observation (3 and 6 h after the treatment) day 0 DD/MM/YY 

Measurement of test conditions day 1 DD/MM/YY 

Observation day 1 DDIMM/YY 

Measurement of test conditions day 2 DD/MM/YY 

Observation day 2 DD/MM/YY 

Measurement of test conditions day 3 DD/MM/YY 

Observation day 3 DD/MM/YY 

Measurement of test conditions day 4 DD/MM/YY 

Observation day 4 DDIMM/YY 

Analytical sampling day 4 DD/MMlYY 

End of experiment day 4 DDIMM/YY 

Draft Report DD/MM/YY 

Remark: 

The exact Study Schedule will be given in Amendment, because the contents of this schedule 

depend on the test item formulation method will be used during the definitive test or on the 

stability of the test item. 
CiToxLAB Hungary Ltd. .It 

Veszprem - Szabadsagpuszta CiTox~B Hungary 

DRAFT 2 


STUDY PLAN 


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

GENERAL INFORMATION 


STUDY TITLE Acute immobilisation test with Ethanedioic acid on 

Daphnia (Daphnia magna) 

TEST ITEM : Ethanedioic acid 

SPONSORS : Univar USA Inc. 

Address: 17425 NE Union Hill Road 

Redmond, W A 98052 

USA 

Telephone: + 1 425 8893400 

Facsimile: + 1 425 889 3760 

: WEGO Chemical and Mineral Corp. 

Address: 239 Great Neck Road 

Great Neck, NY 11021 

USA 

Telephone: +1 5164873510 

Facsimile: + 1 5164873794 

STUDY MONITOR : Mr. John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Address: 3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

Telephone: + 1 561 5788110 

Facsimile:+15615788120 

E-mail: john@grcci.com 

TEST FACILITY : CiToxLAB Hungary Ltd. 

(Fonnerly known as LAB Research Ltd. before 1 st September 

2011.) 

Address: H-8200 Veszprem, Szabadsagpuszta 

Hungary 

Phone: 36 88 545-300 

Fax: 3688545-301 
Study code: 11/339-023DA DRAFT 2 Study Plan Page 3 of 15 

APPROVED BY 

ON BEHALF OF SPONSOR 

APPROVED BY 

ON BEHALF OF CiToxLAB 

STUDY DIRECTOR 

QUALITY ASSURANCE 

RESPONSIBLE PERSON FOR 

ANAL ¥TICAL MEASUREMENTS 

DATE: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

DATE: 

Christopher Banks, 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 OF EXPERIMENT : (will be given in Amendment) 

DRAFT REPORT : (will be given in Amendment) 

BASIS OF STUDY: 

OECD Guidelines for Testing of Chemicals, No. 202, "Daphnia sp., Acute 

Immobilisation Test," (Adopted: 13 April 2004) OECD: Paris 

EPA Health Effects Test Guidelines, OPPTS 850.1010 

Commission Regulation (EC) No 76112009 of 23 July 2009 laying down test 

methods pursuant to Regulation (EC) No 190712006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACh), Annex Part C, C.2 (published in the Official Journal ofthe 

European Union L 142 of 31 May 2008) 

REPORTING FORMAT: 	 Final Report in line with 40 CFR 792 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 

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 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 of Daphnia 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 of Daphnia being no longer capable of swimming at 

the end ofthe test or being dead will be recorded for each test concentration. 

In case of sufficient effects, the 24-h and 48-h ECso 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 of the test item to demonstrate that 

the test item has no influence on test animals up to at least this test concentration. 

The test method of application 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) Section 4 Test Rule and the regulations set forth at 40 CFR 799.5087. 

1.2. CONDITIONS FOR THE VALIDITY OF THE TEST 

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

10 per cent of the Daphnia should be immobilised or trapped at the surface of the 

water. 

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

should be ~ 3 mg/L of the air saturation value 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 

Molecular Formula: C2H204 

Molecular Weight: 126.1 glmol (dihydrate form) 

Purity: 99.71 % 

Description of the test item: White crystals 

Manufacture date: 04 July 2011 

Retest date: 03 July 2012 

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 
Study 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 of the test item were outlined by the 

Sponsor. The test item was identified on the basis of the 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 of addition 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 of the 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 of the solvent in the final test 

medium will be not exceed 100 mg/L (or 0.1 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 

satisfactory test conditions. 

The details of the 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 of the reconstituted water and the chemicals 

used are given in Appendix 2. 
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2.4. ADMINISTRATION OF THE TEST 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 

Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures, OEeD No. 23. 

The test solutions will be prepared just before introduction of the Daphnia (start of the 

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 of the 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 

If a 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 vehicle control if used) will be used in the main test. 

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 of solubility in the test medium 

whichever is the lower), only this concentration and one control (and one vehicle 

control if used) will be tested in a limit-test. 

The concentrations in the definitive test will be based on the results of the 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 of Public Health 

1097 Budapest Gyali u. 2-6 Hungary 

Breeding The Daphnia are bred in Ecotoxicological Laboratory 

of CiT ox LAB Hungary Ltd. 

Justification of species: Daphnia magna is the standard species of the acute 

immobilisation test. 

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 of animals: There will be 20 animals at each group (concentration 

and untreated control) divided into 4 parallel 

(5 animals!l parallel) 

Age of animals: Less than 24 hours old at the beginning of the test. The 

first breeding of the 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 (water temperature, background colour etc.). 

Holding water: Same water is used as test water (see 2.3.). 

Food and Feeding: The Daphnia are fed with concentrated algal suspension of 

Pseudokirchneriella subcapitata during the holding. 

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 least 4 mL test solutionlanimal) will be used at the test 

concentrations and for the control(s). The Daphnia will not be 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. TEST CONDITIONS 

The vessels will be held for the duration of the 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 of the air saturation value 

at the temperature used. The pH will not vary by more than 1.5 units in anyone test. 

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 case the conditions will be given in an amendment. 

2.8. OBSERV A nONS 

The immobility or mortality of the 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 or behaviour will be recorded. 

The number of immobilised animals and the percentage of immobility will be 

determined at 24 and 48 hours. 

2.9. ANALynCAL MEASUREMENTS 

The concentration of the test item will be measured at all of the 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 start of the test. 

Analysis will be performed depending on the results of the analytical method 

validation. 

All samples will be analysed directly after sampling. If analysis 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 

2.1 O. STATISTICAL EV ALUA TION 

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 LC so calculation may be used if appropriate. 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. 

If a 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 

if necessary. 

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 if necessary. 

3. GOOD LABORATORY PRACTICE (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 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 Practice regulations specified by 

EPA 40 CFR 792. 

The Quality Assurance Unit will conduct inspections of the study plan, various phases 

of the study, certain repetitive operations and the report will be audited according to 

applicable Standard Operating Procedures. 
Study code: 11/339-023DA DRAFT 2 Study Plan Page 11 of 15 

4. AMENDMENT AND DEVIATION PROCEDURES 

Planned changes to the study plan can be made at the discretion of the 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. 

5. STUDY REPORT 

The report will include, but not be limited to the following information: 

The results ofthe study will be reported in a detailed final report. 

The original final report, in English will be sent to the sponsor and will include: 

Name and address of the sponsor, the test facility and the study schedule. 

- The names of the study director and other scientists and supervisory personnel 

involved in the study. 

The signatures of the 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 of the test facility's GLP-Certificate. 

The statement of compliance, signed by the study director. 

- The identification of the test item, either by name or code number, concentration, 

purity, composition and properties as far as available 

- Summary of the preliminary test 

- A description of the test system, including the materials used, a detailed description 

ofthe performance of the test item. 

- A description of all methods used with references. 

A list of all deviations to the Study Plan. 

- A description of all circumstances that may have affected the quality or integrity 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 of all data, study plan, one original version of the 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 of sufficient effect) 

- NOEC, LOEC 

- in case of sufficient effect the calculated dose response curve 

Where appropriate, data will be summarized in tabular form to include mean values 

and a measure of variability. 

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, and as one bound and one unbound paper copies. 

6. ARCHIVES 

The study documents: 

- study plan and any amendments, 


- all raw data, 


- sample of the 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 of CiT 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 OF THE STUDY PLAN 

Sponsor: 1 x PDF-file, 1 x copy 


Study Director: 1 copy 


QAU: 1 copy 


Archive: 1 original 


Departments* : 1 x copy each 


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

8. SIGNATURES 

The Study Director, CiToxLAB Hungary Ltd.'s management, QAU will sign the Study 

Plan before the start of the experiment. The PDF version of the 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 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-FVM joint 

decree of the Minister of Health and the Minister of Agriculture and Regional 

Development which corresponds to 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 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACh), Annex Part C, C.2 (published in the Official Journal of the 

European Union L 142 of 31 May 2008) 

4. 	EPA Guideline 712-C-96-114: OPPTS 850.1010, "Aquatic Invertebrate Acute 

Toxicity Test, Freshwater Daphnids" April 1996 

5. 	OECD Guideline for Testing of Chemicals, Section 2, No. 202, "Daphnia sp., Acute 

Immobilisation Test", 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 Assessment No. 23, Paris September 2000. 
Study code: 111339-023DA DRAFT 2 Study Plan Page 14 of 15 

APPENDIX 1 

STUDY SCHEDULE 

Relative date Absolute date 

Culturing oftest animals for the testing day [-1] DD/MMIYY 

Start of experiment day 0 DD/MM/YY 

Preparation oftest item solutions day 0 DD/MMIYY 

Analytical sampling day 0 DD/MMIYY 

Measurement of test conditions day 0 DD/MM/YY 

Treatment of test animals day 0 DD/MM/YY 

Measurement oftemperature day 1 DD/MM/YY 

Observation of immobilisation day 1 DDIMM/YY 

Measurement of test conditions day 2 DD/MM/YY 

Observation of immobilisation day 2 DDIMM/YY 

Analytical sampling day 2 DDIMM/YY 

End of experiment 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 OF ISO MEDIUM 


Separate stock solutions of individual trace elements were first prepared in deionised 

water. The medium was prepared of these different stock solutions, so it contained all 

trace elements (combined solution). 

Stock solutions Amount added Stock solutions 

(single substance) to deionised added to deionised water 

waterto prepare media 

(giL) (milL) 

CaCb x2 H2O 11.76 25 

MgS04x7 H2O 4.93 25 

KCI 0.23 25 

NaHC03 2.59 25 
CiToxLAB Hungary Ltd. 


Veszprem - Szabadsagpuszta CiToxlAB .. 
 Hungary 

DRAFT 2 


STUDY PLAN 


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 2012 
Study code: 11/339-022AL DRAFT 2 Study Plan Page 2 of 17 

GENERAL INFORMATION 

STUDY TITLE : Growth inhibition test with Ethanedioic acid 

on Algae (Pseudokirchneriella subcapitala) 

TEST ITEM : Ethanedioic acid 

SPONSORS : Univar USA Inc. 

Address: 17425 NE Union Hill Road 

Redmond, W A 98052 

USA 

Telephone: + 1 425 8893400 

Facsimile: +1 425 8893760 

: WEGO Chemical and Mineral Corp. 

Address: 239 Great Neck Road 

Great Neck, NY 11021 

USA 

Telephone: +1 5164873510 

Facsimile: + 1 516 487 3794 

STUDY MONITOR : Mr. John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Address: 3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

Telephone: +1 561 5788110 

Facsimile: +1 561 5788120 

E-mail: john@grcci.com 

TEST FACILITY : CiToxLAB Hungary Ltd. 

(Fonnerly known as LAB Research Ltd. before] st September 

2011) 

Address: H-8200 VeSZpf(!ID, Szabadsagpuszta 

Hungary 

Phone: 36 88 545-300 

Fax: 3688545-301 
Study 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 

ANALYTICAL MEASUREMENTS 

DATE: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

DATE: 

Christopher Banks, 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 of Chemicals, Section 2, No. 201: "Freshwater Alga 

and Cyanobacteria, Growth Inhibition Test", adopted 23rd 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 of the European Union L 220 of 24 August 2009) 

REPORTING FORMAT: 

REPORT COPIES: 

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 

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 in Appendix 1. 
Study code: 11/339-022AL DRAFT 2 Study Plan Page 5 of 17 

1. OBJECTIVE OF STUDY 

1.1. PURPOSE 

The purpose of the study is to determine the effect of the test item on the growth of the 

unicellular green algal species Pseudokirchneriella subcapitata (formerly known as 

Selenastrum capricornutum). 

Exponentially growing cultures of Pseudokirchneriella subcapitata will be exposed to 

various concentrations of the test item over several generations under defined 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 of the test item to demonstrate that 

the test item has no influence on test 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) Section 4 Test Rule and the regulations set forth at 40 CFR 799.5087. 

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 

growth rate of 0.92 dai 1 

The mean coefficient of variation for section-by-section specific growth rates 

(days 0-1,1-2 and 2-3, for 72-h tests) in the control cultures must not exceed 35%. 

The coefficient of variation of average specific growth rates during the whole test 

period in replicate control cultures must not exceed 7%. 

2. MATERIALS AND METHODS 

2.1. TEST ITEM 

Name: Ethanedioic acid 


Chemical name: Ethanedioic acid 


Synonym: Oxalic acid 


Batch number: YP2-20110704B 


CAS number: 144-62-7 


Molecular Formula: C2H20 4 


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 

Study code: 11/339-022AL DRAFT 2 Study Plan Page 6 of 17 

Storage condition: 


Safety Precautions: 


2.1.1. Identification, Receipt 

controlled room temperature (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 of the test item were outlined by the 

Sponsor. The test item was identified on the basis of the 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 item concentrations. 

2.2.2. Solvent Control 

The use of solvents will usually be avoided when possible and used only with the 

approval of the 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 of the solvent in the final test 

medium will be not exceed 100 mgIL (or 0.1 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 

satisfactory test conditions. 

The results and details of the closest test and range of validity 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. 

The composition of the growth medium and the chemicals used are given in Appendix 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 of Testing 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 prepared just before introduction of the algae (start of the 

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 of the 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 

(factor e.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 

If a 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 if used) will be used in the main test. 

If no significant toxic response is observed in the preliminary range-finding test at a 

concentration of 100 mg test iternlL (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 if used) will be tested in a limit-test. 

The concentrations in the definitive test will be based on the results of the preliminary 

range-finding test and will be given in an Amendment. 
Study code: 11/339-022AL DRAFT 2 Study 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; UTE X 1648; 

ATCC 22662) 

Source: 	 The algae were supplied by the SAG: Collection of Algal 

Cultures, Inst. Plant Physiology, University of Gottingen, 

GERMANY. Cultured under standardised conditions (see 

OECD 201) in the Ecotoxicological Laboratory of 

CiT oxLAB Hungary Ltd. 

Justification of species: 	 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 of about 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 of the 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 thermometer in 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 of the 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 of the 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. ANAL YTICAL 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 

on the aquatic samples generated in the preliminary test. 

In the main study, the concentration of the test item will be measured, as a minimum, 

at the highest and the lowest 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 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. If analysis 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 

Cell Density: 	 the number of cells per mL 

Growth: 	 the increase of cell density over the test period 

Biomass (b): 	 the actual number of cells per volume of medium (cells/mL) 

calculated as the area under the growth curve (A) 

Yield (y): 	 the cell density at the end ofthe test minus the starting cell density 

Average Specific 


Growth Rate (J.1): the increase in cell density per time unit 


the calculated concentration of test item which results in a 50 % 

reduction of biomass (b) relative to the control 

the calculated concentration of test item which results in a 50 % 

reduction of growth rate (J.!) relative to the control 

the calculated concentration of test item which results in a 50 % 

reduction of yield (y) relative to the control 

NOEC: 	 (No Observed Effect Concentration) the highest test concentration 

at which no significant inhibition of growth is observed relative to 

the control 

LOEC: 	 (Lowest Observed Effect Concentration) the lowest test 

concentration at which a significant inhibition of growth is 

observed relative to the control 

2.10.2. Calculation of Average 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 of measured cell density at time tn 

natural logarithm of measured cell density at time to 

time (hour) of the beginning ofthe test 

= time (hour) of nth measurements after the beginning of the test 
Study code: 11/339-022AL DRAFT 2 Study Plan 	 Page 11 of 17 

The percentage inhibition of growth rate (% Ill): 


%1 =~I ·100 % 
 J1 f.1c 

Where %1Il = percent inhibition in average specific growth rate 

mean growth rate ofthe control 

= mean growth rate of test concentration t 

2.10.3. Calculation of Area Under the Growth Curve 

NI - NoNI + N 2 - 2No ( ) Nil I + Nfl - 2No ( ) A = . tl + . t2 - tl + 	 . tn - tn - I 

2 2 	 2 

Where = nominal cell density at time to (start ofthe test) 

mean measured cell density at t] (24 hours) 

mean measured cell density at t2 (48 hours) 

= mean measured cell density at tn 

= time of first measurement after start of the test 

t2 = time of second measurement after start ofthe test 

tn time of nth measurement after start ofthe test 

The percentage inhibition of area (% IA): 

% J = Ac-A, .100 % 

A Ac 

Where percent inhibition in area under the growth curve 

= mean area (biomass) ofthe control 

= mean area (biomass) oftest concentration t 

2.10.4. Calculation ofYield 

Yield is calculated as the biomass at the end ofthe test minus the starting biomass for each 

single vessel of controls and treatments. For each test concentration and control, mean 

yield values were calculated. 

Percent inhibition in yield (% Iy): 

I =-=-----=- .100 % y 

where: 	 Yc = mean value for yield in the control group 


Yi = mean value 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 2 Study 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 of the 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 of sufficient effects the ErCso, EbCSO and EyCso values of the test item and their 

confidence limits will be calculated using preferably Probit-analysis by TOXSTAT 

SPSS PC+ software. Other models for LCso calculation may be used if appropriate. 

Statistical comparisons of biomass, average specific growth rates and yield in control 

and in treated groups will be carried out using analysis of variance (ANOV A) and 

Bonferroni t-Test (a 0.05) by TOXSTAT or SPSS PC+ software. 

If a 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 

TOXST A T software to compare means. 

For the determination of the LOEC and NOEC, the calculated mean biomass, growth 

rates and yield at the test concentrations will be tested on significant differences to the 

control values by Bonferroni t-Test. 

3. GOOD LADORA TORY PRACTICE (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, ENV IMC/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 

of the 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 of the 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 

distri buted and added to all copies of the 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 of the study. 

5. STUDY REPORT 

The report will include, but not be limited to the following information: 

- Name and address ofthe sponsor, the test facility and the study schedule. 

- The names ofthe study director and other scientists and supervisory personnel involved in 

the 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, signed by the study director. 

The identification of the test item, either by name or code number, concentration, purity, 

composition and properties as far as available. 

- A description ofthe test system, including the materials used, a detailed description ofthe 

formulation ofthe test item. 

- A description ofall method'i used with references. 

- All deviations to the study plan. 

- A description of all circumstances that may have affected the quality or integrity of the 

study. 

- A description and assessment for all results including the data calculations and ifnecessary 

statistical analyses. 

- The storage location of all data, study plan, one original version and one certified copy of 

the final report and a reference sample ofthe test item and reference 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 of cell concentrations 

- growth curves 

- graphical presentation ofthe concentration effect relationship 

- EC values and method of calculation 

- NOEC, LOEC 

- other 0 bserved effects 

- results of analytical measurements 

Where appropriate, data will be summarized in tabular form to incl ude mean values 

and a measure of variability. 


A draft report will be sent to the Sponsor by 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 paper copies. 


6. ARCHIVES 

The study documents: 

- study plan and any amendments, 


- all raw data, 


- sample of the 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 of CiT ox LAB 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 OF THE STUDY PLAN 

Sponsor: 1 x PDF-file, 1 x copy 


Study Director: 1 copy 


QAU: 1 copy 


Archive: 1 original 


Departments*: 1 x copy each 


>I< Central Dispensary, Scientific Documentation 
Study 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 of the experiment. The PDF version of the 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 of the Sponsor being made after 

experiment initiation. 

9. REFERENCES 

1. 	 OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga 

and Cyanobacteria, Growth Inhibition Test", 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 L 220 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 Assessment No. 23, Paris September 2000. 
Study code: 11/339-022AL DRAFT 2 Study Plan Page 16 of 17 

Start of experiment 

Preparation oftest item solutions 

Analytical sampling 

Measurement oftest conditions 

Treatment (Inoculation of alga cells) 

Measurement oftemperature 

Measurement of cell concentration 

Measurement oftemperature 

Measurement of cell concentration 

Measurement of test conditions 

Measurement of cell concentration 

Analytical sampling 

End of experiment 

Draft Report 

APPENDIX I 


STUDY SCHEDULE 

Relative date 

day 0 

day 0 

day 0 

day 0 

day 0 

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 of this 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 OEeD MEDIUM 


Separate stock solutions will be first prepared in deionised water. The growth medium 

will be prepared by adding an appropriate volume of these different stock solutions to 

deionised water in order to 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 

Stock solution 1 

(macro nutrients) CaCh x 2 H2O 18.0 mglL 

MgS04 x 7 H2O 15.0 mglL 

KH 2P04 1.6 mglL 

FeCb x 6 H2O 64.0 f..lglL Stock solution 2 

(iron) Na2EDTA x 2H 2O 100.0 f..lg/L 

H3 B03 185.0 f..lg/L 

MnCh x 4 H2O 415.0 f..lglL 

Stock solution 3 

(trace elements) 

ZnCI 2 

CoClz x 6 H2O 

3.0 f.tg/L 

1.5 f..lglL 

CuClz x 2 H2O 0.01 f..lglL 

Na2Mo04 x 2 H2O 7.0 f.tglL 

Stock solution 4 

(bicarbonate) NaHCO) 50.0 mglL 
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foo: ~atmr.~ by dw! l'NI'IIbcl"I~( ~5 II> the m.pmes. For sh,~ lendorr.d fOl" F~£. Gn-Id ,...,..''''C pl~..,t I>\f<f¢l'lN 11:>< IppI;;;Aldc Scr>1.:. Otoilk I.J..M!!,.... Not IN ANY EVE",T. SUBJECT m TIlE SERVICE CONDITIONS ON THIS AIR W A rBll.L UR TItE APPLICABLE SERVfC£ OUIOE. 

TRA'NSPORTAnoN AOR£EMENT. liTANDARD CONDITIONS. TARIfF OR CONTRACT OF CAARIAGE. F£DEX WON'T Bf. UABLE FOR ANY OMIAOES. WltEmER DIRECT. INDIRECT. INC'tnE,,"AL SPf.CJ.~L OR CONSEQlIE.N"I1N.•• IN EXCF_<;S OF mE.DE.Cl.-\RED V;,tUE 1)&$1(() OQ 

US$9 01 PERLB (lJSSlOJI f>ER KltLil (OR TIlE EQUIVALENTlN UX'ALCllRRf.NCY) WHICHEVf.R 15 GREATf.R (OR l"l CANADA IN EXCF.sSOf TII£ DECLARED \;"..\lJ.'F. FOR CARRlAG£i(!NCWI)fNG BLT"lOT LlM1TEDTOLQSSOf INCOME OR I'ROFJTS) WHE11iER OR NOT 

FEDEX K~£W Oi! SHOULD HAV£ KNOWN TIlAT S\;CH DAMAOts MIUUT BE rNCURR.ED INC!.l!OING ooT NOT UMITf.D TO LOSS OD INCOME OR PROfITS F",jE~ ""OIl', be 1,.toI~ f<lf")'_ ...1& «_l'_, ""lI<:ludmg INlI'lOl hmlUd III 1nt."<>!'f"t<.1 <l«urailclI of cnr. '"If''apcr'' ;",o.dT"acni 

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