### fn 2- &4[) -, Form Approved. O .M .B. No. 20711-0012 Approval Expires 1Q-31-46. U. S . ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY 

## A PREMANUFACTURE Date of reqFipt '- t :. 

# twn L 

# E PA NOTICE ,
. . ( ~ ~ 35 L/ ' FOR NEW CHEMICAL SUBSTANCES

DOCUMENT CONTROL OFFICER OFFICE OF POLLUTION PREVENTION 

### Company Sanitized AND TOXIC SUBSTANCES, 7407 OF U.S . E.P.A. 401 M STREET, SW WASHINGTON, D .C. 20460 Enter the total number of pages r~ 

# W15ThtO in the Premanufacture Notice 

### GENERAL INSTRUCTIONS 

### Ts- /~ 

# N Ht 0 3 

# I • Yo u must provide all info rmation requested in this form to the extent that it is knows to or reasonably ascertainable by you . Make rdssosable estimates if you do not have actual data. • Before you complete this form, you should read the *Instructions Manual for Premanafadate Notification' (the Instructions Manual is available from the Toxi c Substances Control Ad (TSCA) Inform ation Service by calling 202-554-140 4, or faxing 202-55~5603), • If a user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remembe r, your user fee ID number must also appear on your corresponding fee remittance, which is sent to: EPA, HQ Accounting Operations Branch (p14I-266), P.O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSG User Fee . Part I - GENERAL INFORMATION TEST DATA AND OTHER DAT A You must provide the currentl y correct Chemical Abstracts (CA) Name of the You are required to submit all test data in your possession or control new chemical substance, even If you claim the identity as mnfideatial. You may and to provide a description of all other data known to or reasonably' authorise another person to submit chemical identity Information for you, but ascertainable by you, if theme data an related to the health and your submission will not be complete and the review will not begin until EPA environmental effeds of the manafadqee, processing, distribution receives this information. A letter in support of your submission should in commerce, use, or disposal of the new chemical sabstaeot: Standard reference your TS am fee Identification number. You mad submit an original literature citations may be submitted for data is the open scientific and two copies of this notice including all ted data . If you claimed a n y literatu ie data- information as confidential, a single sanitized copy must also be sobmitted, must be submitted if they do nd aope>< in the omen Lteratonz You should clearly identify whether test data is on the substance or on an analog. Also, Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE the chemical composition of the tested material should be d.araderized. Following are examples of test data and other data. Data should b e If them are several manufacture, processing, or use operations to be described in submitted according to the requirements of 5721L50 of the Pe emanufadnie Part II, sections A and B of this notice, reproduce the sections as needed. Notification Rule (40 C 3•R Part 720). Part III - LIST OF ATTACHMENTS Test Data (Check Below any induded in this notice) Attach additional sheets if them is not enough space to answer a question fully. ❑ Label each continuation sheet with the corresponding section heading. In Part • Environmental fate data ~ yell • Other data yes III, list these attachments, any tea data or other data and any optional information included in the notice. • Health effects data yes Risk assessments .OPTIONAL INFORMATION • Environmental effects data ~Yd Structuffulactivity relationships You may include any Information that you want EPA to consider in evaluating • PhysiealK_heminl Ptopettts •,~1 yes Test data not is t~ie~,~~oa the new substance. On page 11 of this form, space has been provided for you to ~- or control of the ia-bmitter describe pollution prevention and recycling information you may have regarding . the new substance . A physical and chemical properties worksheet is located on the-lad page of this form. So-c alled "binding" boxes are included throughout this form for you to indicate TYPE OF NOTICE (Check Only One) your willingness to be bound to certain statements you make in this notice, such as nse, production volume, protective equipment . . . This option Is intended to reduce delays that routinely accompany the development ofconsent orders or PMN (Pnemanafadane Notice) Significant New Use Rules . Except in the rase of exemption applications (such an TMEA, LYE, LOREX) whore certain Information provided in such notification is p~ RMEDIATE PMN (submitted in sequence with fina,( pyodatt binding on the submitter when the Agency approves the exemption application, P~ checking a binding box In this notice d~~ by itselfprohibit the sabmHter • from later deviating from the info rmation (except chemical identity) reported in ❑ SNUN (Significant New Use Notice) r "l the form . CD ❑ TMEA (Test Marketing Exemption Application) CONFIDENTIALITY CLAIMS ❑ LVE (Low Vol . Exemptio
.) 0 40 CFR 723,50 (c)(1) You may claim any information in this notice as confidential. To assert a claim on the form, mark (J0 the confidential box next to the information that you claim ❑ LOREX (Law Rekaselfow Exposure Exemptiosd 0 723.511(dpJ as confidential. To assert a claim in an attachment, circle or bracket the ❑ Information you claim as confidential. If you daisforeati on is th notl LVE Modification . 11 LOREXModlfaNor confidential_you m ust also provide a•..iti ..t vesioe of th tl 9 IIZaChmtptil. For additional Instructions on claiming Information as IS THIS A CONSOLIDATED PMN7 ~ Yes fld tial, read the Instructions Manual. l)5Mark00 if any information in this notice is claimed as confidential. 1 of chemicals (prenotice Communication / required, enter 1 on page 3 ) EPA FORM 7710-25 (Rev . 5-95) Replaces previous editions of EPA Form 7110-25 .PubLic reporting burden for this collection of information is estimated to average 110 hours per response, indudin time for re w 8 v~e2utg instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information . Send comments regarding the burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M . St., S. W .,, Washington, D.C 20460; and to the Office of Mana ent and Budget, Paperwork Reduction Act (2070-0012), Washington, D .C 20503. 

### CERTIFICATION MMMMMMMM~ I certify that to the best of my knowledge and belief: 1 . The ,company named in Part 1, section A, subsection la of this notice form intends to manufacture or import for a commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, Section B . - 2. All information provided in this notice is complete and truthful as of the date of submission

. 3 . 1 am submitting with this notice all test data in my possession or control and a description of all other data known to or reasonably ascertainable by me as required by §720 .50 of the Premanufacture Notification Rule. Additional Certification Statements : If you are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following user fee certification statem t that applies : The Company named in Part i, Section A has remitted the fee of $2500 specified in 40 CFR 700 .45 (b), o r ❑ The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined 0 40 CFR 700 .43) in accordance with 40 CFR 700.45(b), or ❑ The Company named in Part I, Section A is a small business concern under 40 CFR 700 .43 and has remitted a fee of $100 in accordance with 40 CFR 700 .45 (b) . If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723 .50 (c) (1) or a Low release and low exposure exemption (LoREX) application in accordance with 40 CFR 723.50 (c) (2), check the following certification statements : ❑ The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under the terms of 40 CFR 723.50. ❑ The manufacturer is familiar with the terms of this section and will comply with those terms ; and ❑ The new chemical substance for which the notice is submitted meets all applicable exemption conditions

. ❑ If this application is for an LVE in accordance with 40 CFR 723 .50 (c)(1), the manufacturer intends to commence manufacture of the exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day review period . The accuracy of the statements you make in this notice should reflect your best prediction of the anticipated facts regarding the chemical substance described herein . Any knowing and willful misinterpretation is subject to criminal penalty pursuant to 18 USC 1001 . Confideptial Signature and title of Authorized Official (Original Signature Required) Date
X Signature of agent - (if applicable) Date . FORM EPA 7710-25 (Rev . 5-95) Page 2 •

### Part I - GENERAL INFORMATION

Secti on A - SUBMITTER IDENTIFICATION Confidential Mark (X) the "Confidential" box next to any subsec tion you claim as confidential. Name of authorized official Position la. Person Submitting Notice Company (in U .S.) Mailing address ( number and s treet)

City, State, ZIP Code b . Agent (if Name of authorized of fidal Position applicable) Company

Mailing address (number and street) City, State, ZIP Code Area Code Number Telephone I I c If you are submitting this notice as part of a joint submission, mark (X) this box. Joint Name of authorized official Position Submitter (if applicable) Company

Mailing address ( number and s treet) City, State, ZIP Code Area Code Number Telephone 2 . Technical Name Posi tio n Contact (in US .) Company

Mailing address (number and street) City, State, ZIP Code Area Code Number Telephone . ~ 3. If you have had a prenotice communication (PC) concerning this notice and EPA assigned a PC Number to the notice, enter the number . Mark ()) -~ if none 4. If you previously submitted an exempti on app lication for the chemical substan ce covered by this notice, enter the exemp tion number assigned Mark (
~ by EPA. If you previously submitted a PMN for this substan ce enter the -~ PMN number assigned by EPA (Le withdrawn or incomplete) . if none 5 . If you have submitted a notice of Bona fide intent to manufacture or a import for the chemical substance covered by this notice , enter the d~ ~ notice number assigned by EPA . - ~ 1 ❑ Manufacture 1 import I Type of Noti ce- Mark A) Only Only I a Bindin g Option . ( /- ❑ BindingOption 1 3 . ~ Both Mark (x) Mark (x) FORM EPA 7710-25 (Rev . 5-95) Page 3

### Part I -- GENERAL INFORMATION - Continue d Section B- CHEMICAL IDENTITY INFORMATION : • YO1 °`~~^dt a~+uy correct (C A) nameof the substance t►axd on the nineh Collective Index (90 of CA nomenclature rules and oonventlom. Mark (X) the "Confidential" box next to any item you claim as confidential. Complete either item 1(Class I or 2 substances) or 2(Polymen) as app ropriate . Complete all other items. If another person will submit chemical identity information for you (for either item 1 or 2), mark ()() the box at the right . Confi- Identify the name, company, and address of that person in a continuation sheet . -~ ❑ dential 1 . Class 1 or 2 chemical substances (for definitions of class I and class 2 substances, see the Instructions Manual) a . Class of substance - Mark (X) 1 

### F' 1 Class 1 or 2~(] 

### ss 2 b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. For Class 1 substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA Preferred Name must be provided, whichever is appropriate based on CA 9( :I nomenclature rules and conventions .) .

c. Please identify which method you used to develop or obtain the specified chemical indentity information reported in this notice: (check one). Method 1(CAS Inventory Expert Service - a copy of the Identification aMethad 2 (Other Source) report obtained from the CAS Inventory Expert Service must b e submitted as an attachment to this notice) d. Molecular formula and CAS Registry Number (if a number already exists for the substance)

CAS t

e. For a class 1 substance, provide a complete and correct chemical structure diagram. For a class 2 substance - (1) List the immediate precursor substances with their resp ecti ve CAS Registry Numbers . (2) Describe the nature of the reaction or process. (3) Indicate the range of composition and the typical composition (where appropriate) . (4) Provide a correct representative or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertained.

~ Mark (X) this box if you attach a continua ti on sheet. FORM EPA 7710-25 (Rev . 5-95) Page 4p . 5 ~



## ,9N FIDIENTiAL



# `C ~p . 6


## cOM~P[ t ~RiT B ALr% 7 

### Part I -- GENERAL INFORMATION -- Continue d Section B - CHEMICAL IDENTITY INFORMATION - Continue d 2 Polymers ( For a definition of polymer, see the Instructions Manual.) Confidential a. Indicate the number-average weight of the lowest molecular weight composi tion of the polymer you intend to manufacture . Indicate maximum weight percent of low molecular weight species ( not including residual monomers, reactants, or solvents) below 500 and below 1,000

absolute molecul ar weight of that composition . Describe the methods of measurement or the basis for your estimates : GPC ❑ Other[] :(Spedfy) i) lowest number average molecular weight: fi) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight ❑ Mark ( X) this box if you attach a continuation sheet . b. You must make separate confidentiality claims for monomer or other reactant identity, composition infonztation, and residual information. Mark (X) the "Confidential" box next to any item you claim as confidential . (1) - Provide the specific chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufacture of the polymer. (2) - Mark (X) this column if entry in column (1) is confidential. (3) - Indicate the typical weight percent of each monomer or other reactant in the polymer . (4) - Mark (74 the identi ty column if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer description on the TSCA Chemical Substance Inventory. ( 5) - Mark (X) this column if entries in columns (3) and (4) are confidential. (6) - Indicate the maximum weight percent of each monomer or other reactant that may be present as a residual in the polymer as manufactured for commercial purposes . (7) = Mark (X) this column if entry in column (6) is confidential.

Monomer or other reactant and CAS Registry Number Confi- Typical Identity Confi- Maximum confidential compositi on Mark (JU dential residual dential (1) (2) (3) (4) (5) (6) (7) % /10 % X % % % % % 96 % % % % ❑ Mark (X) this box if you attach a continuation sheet. c . Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice . (ch edc one) . ❑ Method 1 (CAS Inventory Expert Service
- a copy of the identification report ❑ Method 2 ( other source)

obtained from CAS Inventory Expert Service must be submitted as an attachment to this notice) d . The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers . x

e. Provide a correct representative or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertainedMark p4 this box if you attach a continuation sheet. FORM EPA 7710-25 (Rev. 5- 95) Page 5p . 8



# -~. .~.~.~~

5~~. Part I - GENERAL INFORMATION - Continued p . 9

- Section B- CHEMICAL IDENTITY INFORMATION - Continue d I Impu rities (a) - Identify each impurity that may be reasonably anticipated to be present in the chemical substance as saanafadured for mmmercial purposes . Provide the CAS Regist ry Number if available
. If them are unidentified impurities, enter "unidentified ' . (b) -Estimate the maximum weight % of each impuri ty. If there are unidentified impurities, estimate their totai weight % . Impurity and CAS Registry Number Maximum Confi-

(a) percent dential % 

# %

% %

% ❑ Mark (X) this box if you attach a continuation sheet
. 4. Synonyms - Enter any chemical synonyms for the new chemical substance identified in subsection I or 2 . ConfiX ❑ Mark (X) this box if you attach a continuation sheet
. 5 . Trade identi fication - List trade names for the new chemical substance identified in subsection 1 or 2
. ❑ Mark (X) this box if you attach a continuation sheet . 6. Generic chemical name - If you claim chemical identity as confidential, you must provide a generic .cheatical name for your substance that reveals the specific chemical identity of the new chemical substance to the maximum extent possible

. Refer to the TSCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing generic names. 

### L u 0 R . lP b L`( U fL,

❑ Mark (X) this box if you attach a continuati on sheet . 7 . Byproducts - Describe any byproducts resulting from the manufacture, processing, use, or disposal of the new chemical substance. Provide the CAS

Registry Number if available. . Byproduct CAS Registry Number Confi- ( 1) (2) dential

~ ❑ Mark (X) this box if you attach a continuation sheet. FORM EPA 771 0-25 (Rev . 5-95) Page 6P . 10 Part I-- GENERAL INFORMATION -- Continued , Section C - PRODUCTION, IIISPORT, AND USE INFORMATION: Mark pQ the "Con

### fidential" box next to any item you 

### claim as confidential. 1. Production volume
- Estimate the maximum production volume during the first 12 months of production . Also estimate the maximum production volume for any consecutive 12-month period during the first three years of production . Estimates should be on 100% new chemical substance basis . For a Low Volume Exemption application, if you choose to ham your notice reviewed at a Lower production volume than 10,000 kg/y r, specify the volume and m ark ( x) in the binding box. If granted, you are bound to this volume . . Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Con g- Binding dential Option (100% new chemical substance basis) (100% new chemical substance basis) Mark W

~ 

### 2. Use Informa

### tion - You must make

### separate confidentiality claims for the 

### description of the catego

### ry of use, the per

### cent of produc

### tion volume devoted to each categor



### y , the formulation of the new substan

### ce, and other use 

### information. Mark (X) the "Confidential" Box next to any item you 

### claim as confiden

### ti al. a. (1) - 

### Describe each intended 

### category of use of the new 

### chemical substance by 

### function and 

### application- (2) - Mark (X) this column if ent

### ry in column ( 1) is confidential business information (®n . (3) - Indicate your willingness to have the information provided in column (1) binding. (4) - Estimate the percent of total production for the first three years devoted to each category of use. (5) - Mark () ) this column if entry in column (4) is confidential business information (CBn . . (6) - Estimate the percent of the new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category of use. (7) - Mark ()0 this column if entry in column (6) is confidential business information (CBI) . (8) - Indicate % of product volume expected for the listed "use" sectors. Mark more than one box if appropriate . Mark ()() to indicate your willingness to have the use type provided in (8) binding . (9) - Mark () ) this column if ent ry(ies) in column (8) is (are) confidential business information (CBI) .

Category of use (1) CBI B~ ~ Produc- CgI % in I V . of substance expected per use I (by function and application i.e. a dispersive dye for ~k W tion % Formfinishing polyester fibers) ulation Site- Indus- Com. Binding (2) (3) (4) (5) (6) (7) limited sumer trial merdal Option (9)

% yr, X % % % % % % 

### % %

% 

### L % % )c *If you have identified a "consumer" use, please provide on a continuation sheet a detailed description of the use(s) of this chemical substance in consumer products . In addition include estimates of the con centration of the new chemical substance as expected in consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product : ❑ Mark 00 this box if you attach a continuation sheet b. Generic If you claim any category of use description in subsection 2a as confidential, enter a generic description of that use category. Read the Instructions Manual for examples of generic use descriptions. description

❑ Mark ()O this box if you attach a continuation shee t 3. Hazard Information - Indude in the notice a copy ofreasonable facsimile of any hazard warning statement, label, material safety data sheet, 86wi•s - or other 'information wl ► id► willbe provided to any person who is reasonably likely to be exposed to this substance regarding protective a'a0' pment or practices for the safe handling, transport, , or ' of the new substanoe List in part IIi hazard information you include. Mwkt"t _pqui ark 00 this box if You attach hazard informiti FORM EPA 7710-25 (Rev . 5-95) Page 7P. 11 ' Part II
-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Section A- INDUSTRIAL SITES CONTROLLED BY THE SUBIVIITTER Mark pq the'Confidential" box next to any item you claim as confidential. Complete section A for each type of manufacture, p rocessing, or use operation involving the new chemical substance at industrial sites you oontroL Importers do not have to complete this section for operations outside the U .S . ; however, you may stiU have reporting requirements if there are further industrial processing or use operations after import You must describe these operations. See instructions manual. 1 . Operation description C .onfi a . Identity
- Enter the identi ty of the site at which the operation will occur . d endg

Name Site address (number and street) City, County, State, ZIP Code If the same operation will occur at more than one site, enter the number of sites . Identify the 0 0E'r0 ° additional sites on a continuation sheet, and if any of the sites have signi ficantly different 1 ~ production rates or operati ons, include all the information requested in this sec tion for those -~ I sites as attachments. ~ Mark (X) this box if you attach a continuation sheet b . Type - Mark 04 ❑ Manufacturing ❑ Processing ❑ Use c. Amount and Duration - Complete I or 2 as appropriate Maximum kg/batch (loo ~ ..w Hours/batch 1 Batches/year 1 . Batch "'~"°tl

Maximum kg/day (loo ti a.w d,ek ,t Hours/day Days/year 2. Continuou s d. Process description ~ Mark (70 to indicate your willingness to have your process descrip tion binding. (1) Diagram the major unit operation steps and chemical conversions . Include interim storage and transport containers (specify-e.g. 5 gallon pails, 55 gallon drum, rail car, tank truck, etc.). (2) Pro vide the identity, the approximate weight (by kg/day or kg/batch on an 1 0096 new chemical substance basis), and entry point of all starting materials and feedstocks (including reactants, solvents, and catalysts, etc), and of a ll products, recycle streams, and wastes . Include cleaning chemicals (note frequency if not used daily or per batch .). (3) Identify by number the points of release, including small or intermittant releases, to t he environment of the new chemical substance .

Mark ()Q this box if you attach a continuation sheet. FORM EPA 771 0- 25 (Rev . 5-95) Page 8p .12

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMTITER

- Continued 2 Occupational Exposure - You must make separate confidentiali ty claims for the description of worker activity, physical form of the new chemical substance, number of workers exposed, and dura tion of activity. Mark (X} the "Confidential" box next to any item you claim as confidential. (1) - Describe the activities (e.g . bag dumping, tote filling, unloading drums, sampling, cleaning, etc) in which workers may be exposed to the substance . (2) - Mark (X) this column if entry in column (1) is confidential business information (CBI). (3) - Describe any protective equipment and engineering controls used to protect workers. (4) and (6) - Indicate you willingness to have the informa tion provided in column (3) or (5) binding . (5) - Indicate the physical form(s) of the new chemical substance (e .g. solid : crystal, granule, powder, or dust) and % new chemical substan ce (if part of a mixture) at the time of exposure . (7) - Mark ()C) this column if entry in column (5) is confiden tial business information (CBI). (8) - Estimate the maximum number of workers involved in each activity for all sites combined . (9) - Mark ( ? ) this column if entry in column (8) is confiden tial business information (CBI) . (10) and (11) - Estimate the maximum duration of the activi ty for any worker in hours per day and days per year. (12) - Mark () C) this column if entries in columns (10) and ( 11) are confiden ti al business information (CBI) . Worker activity ® I Protective Equipment/ lading Physical form(s) lading CBI # of CBI Maxim duration CBI (e .g. bag dumping, filling drums) Engineering Coataota ~) (e.g.sdidpowder) ion Workers and % new W Exposed I irs/day Days/yr substance (8) (10) 1



# x

'77 ❑ Mark ( X) this box if you attach a continuation sheet. 3. Environmental Release and Disposal - You must make separate confidentiality claims for the release number and the amount of the new chemical substance released and other release and disposal information . Mark (X) the "Con fidenti al" box next to each item you claim as confidential. (1) - Enter the number of each release point identi fied in the process description, part II, section A, subsection ld(3 ). (2) - Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology (in kg/day or kg/batch). (3) - Mark 00 this column if entries in columns (1) and (2) are confidential business information (CBI) . (4) - Identify the media of release ie . stack air, fugi tive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, Pt7I1N, or other (please specify) to which the new substance will be released from that release point. (5) - a. Describe control technology, if any, and control efficiency that will be used to limit the release of the new subst ance to the environment. For releases disposed of on land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste . On a continua tion sheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment . b. Estimate the amount released to the environment after control technology (in kg/day). (6) - Mark p4 this column if entries in columns (4) and (5) are confidential business information (CBI). (7) - Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct dischargers or NPDES numbers of the PO'Iti11(Pub lidy Owned Treatment Works) . Mark (X) if the POTW name or NPDES f! is confidential business information (CBI). Release Amount of new substance CBI r Media of elease Control technology and efficiency (you may wish to optionally attach efficiency data) ~i Number , 0 .& *ad air sWding (1) (2a) (2b) (3) (4) (5a) MarkW (5b) (6)



# k x k
(7) Mark (X) the ❑ PO'I1N provide name(s) below : I ❑ Navigable ❑ Other - Specify provide NP DES # CBI destination(s) of waterway releases to water. ❑ Mark (Jq this box if you attach a continuation sheet. FORM EPA 711425 (Rev . 5-95) Page 9Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued Section B-- INDUSTRIAL SITES CONTROLLED BY OTHER S Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control.Importers do not have to complete this section for operations outside the US.; however, you must report any proce ssing or use activities after import See the Instructions Manual. Complete a separate section B / o► each type ofprocessing, or use operation inadvrng the new chemicalsubstance. If the same operation is performed atmore than one site describe the typical operati on common to these sites. Identify addi tional sites on a continua tion sheet. 1. Operation Description - To claim information in this section as confidential, circle or bracket the specific information that you daim as mn fidentiaL (1) - Diagram the major unit opera ti on steps and chemical conversions, including interim storage and transport containers (specify- e.g . 5 gallon pads, 55gallon drums, rail cars, tank trucks, etc) . On the diagram, identify by letter and briefly describe each worker activity. (2) - Provide the identity, the approximate weight (by kg/day or kg/batch, on an 100% new chemical substan ce basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and of a ll products, recycle streams, and wastes . Indude cleaning chemicals (note frequency if not used daily or per batch). (3) - Identify by number the points of release, including small or intermittent releases, to the environment of new chemical substance. (4) Please enter the # of sites ( remember to identify the locations of these sites on a continua tion sheet) : # of sites

Mark () ) this box if you attach a continuation sheet. 2. Worker ExpoaurdEnvironmental Release (1) - From the diagram above, provide the letter for each worker ac tivity. Complete 2-8 for each worker activity described . (2) - Estimate the number of workers exposed for all sites combined. (4) - Estimate the typical duration of exposure per worker in (a) hours per day and (b) days per year. (6) - Describe physical form of exposure and % new chemical substance (if in mixture), and any protective equipment and engineering controls used to protect workers. (7) - Estimate the percent of the new substance as formulated when packaged or used as a final product (9) - From the process diagram above, enter the number of each release point. Complete 9-13 for each release point identified . (10) - Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment ( in kg/day or kg/batch) . (12) - Describe media of release i .e . stack air, fugi ti ve air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, PdIW, or other (specify) and co ntrol technology, if any, that will be used to limit the relea se of the new substance to the environmen t (14) - Identify byproducts which may result from the operation. (3), <5). (8), (11), ( 13) and (15) - Mark ()0 thi s column if any of the proaeeding entries are con fidential business informati on (CBI) . Letter # of ®I Duration CBI Protecti ve Equip. / in CBI Release Amount of CBI Media of Release CBI Of Workers of Engineering Controls/ Form- Number New & cont .rol Act - Exposed Exposure Physical Form and % ula tion Substance Technology ivity new substance Released (1) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) ] Oa) I 10b 11 (12) (13)

.
. _ ~ l

(14) - Byproducts: ❑ Mark (X) this box if you attach a continuation sheet. FORM EPA 7110•25 (Rev . 5-95) Page 10p . 14

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IN ~~ 

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## ~a-p .15

OPTIONAL POLLUTION PREVENTION INFORMATIO N To claim information in this section as confidential circle or bracket the specific information that you claim as con fidential. 

### In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce or minimize potential risks associated with activities surrounding - manufacturing, processing, use and disposal of the PMN substance . Please include new information pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due to the new chemical substance . Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological modification, process and procedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation of useful chemical components from wastes that would otherwise be treated or released as air emissions or water discharges, or land disposal . Descriptions of pollution prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance with existing regulatory requirements and can be either quantitative or qualitative . The EPA is interested in this information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting of risks to-other environmental media or non-environmental areas (e.g ., occupational or consumer exposure) . In addition, information on the relative cost or performance characteristics of the PMN substance to potential alternatives may be provided
. All information provided in this sec tion will be taken into consideration during the review of this substance. See the revised Instructions Manual that includes a Po llution Prevention manual for guidance and . examples . Describe the expected net benefits, such as ( 1) an overall reduction in risk to human health or the environment; (2) a reduction in the volume m anufactured; (3) a reduction in the generation of waste materials through recyc ling, source reduction or other means ; (4) a reduction in potential toxicity or human exposure and/or environmental release ; (5) an increase in product performance, a decrease in the cost of production and/or improved operation efficiency of the new chemical substance in comparison to existing chemical substances used in similar app lications ; or (6) the extent to which the new chemical subst ance may be a substitute for an existing substance that poses a greater overall risk to human health or the environment . 

# Co►~~~D~l~

1 :3 Mark ( X) this box if you attach a continuation sheet . FORM EPA 771425 Qtev . SA 5) Page 1116 

### Part III -- LIST OF 

### ATTACHMENTS Attach continuation sheets for sections of the form and test data and other data (including physical/chemical properties and structure/activity information), and optional information after this page . pearly identify the attachment and the section of the form to which it relates, if appropriate . Number consecutively the pages of the attachments . In the column below, enter the inclusive page numbers of each attachment. .Mark (X) the "Confidential" box next to any attachment name you claim as confidential . Read the instructions Manual for guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy of the notice form a sanitized version of any attachment in which you claim information as confidential. Attachment name Attachment Cong_ e number(s) dentia l 

### Material Safety Data Sheet (MSDS) .

~

X

" • ~ 

# E3 Mark (X) this box if you attach a continuation sheet. Enter the attadunent name and number. FORM EPA 7710-25 (Rev . 5-95) -Page 12p .17 PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and include it in the notice . Identify the property measured, the page of the notice on which the property appears, the value of the property, the units in which the property is measured (as neceaeary), and whether or not the property is claimed as confidential. The physical state of the neat substance should be provided. These measured properties should be for the neat (1 009G pure) chemical substance . Properties that are measured for mixtures or formulations should be so noted (% PMN substance in _).You are not required to submit this worksheet ; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential information is properly protected . You should submit this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission of test data.

Property Mark (X) if Page Value ~~ d e Corifi. ntial (a) provided number ~ ~ ro) (c) Mark (?Q uNort7 (d) Physical state of neat substance (s) ~ W Vapor pressure O C ® Temperature Torr Density/relative density g/cm 3 ~ Solubility ® Temperature 

### Sc Solvent /L Solubility in water 40 Temperature OC OC Melting temperature OC Boiling/sublimation temperature pressure Spectra Dissociation constant Partide size distribution Octanol/water partition eoeffident Henry's Law constant Volitalization from water VoGtalization from soil pH 0 concentration
Explodability Adsorption/coefficient Other - Specify 

# (~p . 1 8



# ~44\ p.79 /



# l5p .20 \p . 21



# ~~p .22 \p .23 /



# Zt4tp . 2 4 ~„• ~F ;~ , ~= Z ~ ~' ~ ~



# REPORT Study Title : ACUTE TOXICITY OF ~ , TO ZEBRA FISH (BRACH NI RER~ O~ ) IN A 96-HOUR STATIC TEST Data Requirements ! Test Guidelines : OECD No . 20 3 EU Commission Directive 92/69/EEC, C .1 Study Director : Dr . Birgit Seyfried Study Completion Date : July 22, 2002 Test Facility : RCC Ltd Environmental Chemistry & Pharmanalytics Division CH-4452 Itingen / Switzerland S sor•

RCC Study Number : ~t Page 1 of 1 8 

# RRCCCp: 25 !~-

-. .~ REPORT Page 2 

# GLP CERTIFICAT E The Swiss GLP Monitoring Authoritie s Swiss Federal Swiss Agency for the Inlercantonal 0111" J office of Environment . Forests e/A~ for the Control of

Public Health and Landscape Medicines 

# Statement of GLP Complianc e It is hereby confirmed that "' .• during the period of August 15 - 17, 2000 August 28 - 29 , 2001 and April 15, 200 2 the following Test Facilities of RCC Ltd 4452 Itingen Switzerland were inspected by the Federal Office of Public Health, the Swiss Agency for th e Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines with respect to the compliance with the Swiss legislation on Good Laboratory Practice . Test Facilities areas of expertise* - Toxicology Division TOX, ACC, MU T - Environmental Chemistry and ACC, ECT, ENF, EMN, Pharmanalytics Division PCT,RES,OTH (Animal metabolism ) - Microbiological Diagnostics by ' Biotechnology & Animal Breeding Division OTH (Microbiology ) The inspection was performed in agreement with the OECD Guidelines for National GLP Inspections and Audits . It was found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813 .016 .5] at the time they were inspected . Federal Office of Public Health The Director Bern, May 2002 Prof
. 

### Th
. 

### Zeltne r • TOX = Toxicology : ACC = Analytical and Clinical Ctxmistry ; ECT = Environmental toxicity on aquatic and terrestria l organisms : ENF = Behaviour in water, soil and air . Bioaccumulation : EMN = Studies on effects on mesocosms and natural

-- . . . . .+ _ , . . .• . .. . • .• or•T - ow . . ,~~w . . . . .~ . . .•i .. . . • orc = a -61 n. ehwii .e • ATH . Other. to be soeeified .p . 26 REPORT Page 3
GOOD LABORATORY PRACTIC E 

# STATEMENT OF COMPLIANCE RCC Study Number: Test Item : Study Director : Dr . Birgit Seyfrie d Study Title : Acute toxicity zebra fish (Brachydanio ren S= our static tes t This study (with the exception of the pre-experiments as mentioned in the report and the first four days of acclimatization of the test fish, which sta rted before finalization of the study plan) has been pe rformed in compliance with the Swiss Ordinance relating to Good Laborato ry Practice, adopted Februa ry 2"d, 2000 [RS 813 .016 .5] . This Ordinance is based on the OECD Principles of Good Laborato ry Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97) 186/Final] . There were no circumstances that may have affected the quality or integrity of the data . Study Director : Dr . Birgit Seyfrie d



# ~~:...~ ~'Z .

# ............................

~ 

### Date : .2 2, .200.2p . 27
REPORT g 

# SIGNATURES

Study Director : Dr . Birgit Seyfried



### e . 

## .... .... .. ... . ... ... ... . .. .. .... .. .. . II _,G Date: .2,2. .,2 vva2

Management : ~ Dr. Uwe Morgenroth I
Date :p . 28 REPORT Page 5

..r_r~ . ; '_ : .. . . t.~`, ._...._ 

# QUALITY ASSURANCE UNI T RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland STATEMEN T Study Number: Test Item : Study Director : Dr . Birgit Seyfrie d Study Title : Acute toxicity o W6-h zebra fi sh (Brachydanio ren n atic test The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management
. Study procedures (with the exception of the pre-experiments as mentioned in the report and the first four days of acclimatization of the test fish, which started before finalization of the study plan) were periodically inspected . The study plan and this report were audited by the Quality Assurance Unit . The dates are given below
.

Dates and Types of QAU Inspections Dates of Repo rts to the Study Director and to the Management June 13, 2002 Study plan June 13, 2002 March 26, 2002 Process based March 28, 2002 July 08/11, 2002 Final report July 11, 200 2 This statement also confirms that this final report reflects the raw data

. Quality Assurance : Mrs . Sabine Gotschi

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

### Date : ~ 

# 22) 200 2,p . 29 f•~'+f--_ r=-~ •_ _ __ - REPORT Page 6 

# CONTENTS
GLP CERTIFICATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 STATEMENT OF COMPLIANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 SIGNATURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 QUALITY ASSURANCE UNIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 CONTENTS CONTINUED (TABLES) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

, . , DATA REQUIREMENTS / TEST GUIDELINES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 SUMMARY OF STUDY PLAN AMENDMENTS
. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1 PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2 MATERIALS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . .: . 11 2 .1 DEFINITIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 2 .2 TEST ITEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 .3 TEST SYSTEM
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 .4 STUDY DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 2 .4 .1 Experimental Conditions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2 .4 .2 Dosage and Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2 .5 EVALUATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2 .5 .1 Determination of the LC50, LCO, LC100, NOEC an d LOEC
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Water Quality Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . 15 3 RESULTS AND DISCUSSION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4 TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

~~-p . 30 t . ., ._ : . ..._ REPORT Page 7 

# CONTENTS CONTINUED (TABLES)

Table 1 : Mortality and visible abnormalities observed at the test fish . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 2 : Appearance of the test medium
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 3 : pH-values in the test medium and the control
. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Table 4 : Oxygen concentrations ( mg/L) in the test medium and th e

control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 18 Table 5 : Temperatures (°C) in the test medium and the control
. . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . 18

C%'~~p . 31 REPORT Page 8 

# PREFAC E GENERAL Study Title : Acute toxicity of zebra fish (Brachydanio rerio) in a 96-hour static test Sponsor : Monitoring Scientist : Test Facility : RCC Lt d Environmental Chemist ry & Pharmanalytics Division ' Zelgliweg 1 CH-4452 Itingen / Switzerland 

## RESPONSIBILITIE S Study Director : Dr . Birgit Seyfried Deputy Study Director : Dr . Ulrich Memme rt Technical Coordinator : Mr. Frederic Levrechon Head of RCC Quality Assurance : Mrs . Iris Wuthrich SCHEDUL E Experimental Sta rting Date : June 14, 2002 Experimental Completion Date : June 21, 2002 Study Completion Date : July 22, 2002 ARCHIVIN G RCC Ltd, CH-4452 Itingen, Switzerland will retain the study plan, raw data, a sample of the test item and the final report of the present study for at least ten years . No data will be discarded without the Sponsor's consent.



# o~ 7p . 32

~~ .~ i ,r- ., . ._ . . . - . . . . . : REPORT Page 9 

# DATA REQUIREMENTS I TEST GUIDELINE S This study followed the procedures indicated by the following internationally accepted guidelines and recommendations : OECD Guideline for Testing of Chemicals, No . 203, Fish, Acute Toxicity Test, 1992 EU Commission Directive 92/69/EEC, Part C .1 : Acute Toxicity for Fish, 199 2 

# SUMMARY OF STUDY PLAN AMENDMENTS First Amendment to Study Pla n Concerning : Alteration : Reason : Header Page 7 Study Number Wrong study number given in the study pla n 2 .3 Test System Test System Wrong test system given in the study plan

C~ ~p,33, REPORT Page 10 

# SUMMARY The acute toxicity of the test item qftto zebra fish (Brachydanio rerio) was determined in a 96-hour static test accor ing to the EU Commission Directive 92/69/EEC, Part C .1 (1992), and the OECD Guideline for Testing of Chemicals No . 203, (1992) . A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of nominal 100 mg/L . Thus the only concentration tested was nominal 100 mg/L and a control
. In the control and at the test concentration of 100 mg/L no mortality or other visible abnormalities were determined during the test period of 96 hours
. Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LCO o to zebra fish were determined to be at least 100 mg/L . The NOEC and the L O- might even be higher than this concentration, but concentrations in ~excess of 100 mg/L have not been tested, according to the guidelines . The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly higher than 100 mg/L . These values could not be quantified due to the absence of toxicity oft the tested concentration
.p' 34 REPORT Page 11 

# 1 PURPOS E ur ose of this 96-hour toxicity test was to evaluate the acute toxicity of the test item aw For this purpose, zebra fish (Brachydanio rerio) were exposed in a static non-renewal test to an aqueous test medium at the concentration of nominal 100 mg/L under defined conditions . This limit test was based on a range-finding test and performed in compliance with the test guidelines to demonstrate that the test item has no toxic effect on fish up to at least this concentration . The test fish were observed for mortality and visible abnormalities
. The used method of application and the tested fish species zebra fish are recommended by the international test guidelines of the OECD and EEC
. 

# 2 MATERIALS AND METHODS 2 .1 DEFINITION S LCO: . The highest test concentration without a significant number of dead test organisms . LC50 : The median lethal concentration, i .e . the calculated concentration of test item which causes death in 50% of the test organisms . LC100 : The lowest test concentration at which all test organisms are dead . NOEC (No Observed " • . Effect Concentration) : The highest test concentration at which no significant toxic effect on the test organisms is observed
. LOEC (Lowest Observe d Effect Concentration) : The lowest test concentration at which a significant toxic effect on the test organisms is observed
.p . 35 REPORT Page 12 

## 2 .2 TEST ITE M The test item and the following information concerning the test item were provided by the sponsor: Identity : •~~~ Batch No . : 4512 O S Expiration date : March, 2004 Purity : >98% Concentration : Dry content 26% Stability in water : Stabl e Solubility in water : Miscibl e pH in aqueous solution : 3 .9 at concentration of 26% Aggregate state at room temperature : Aqueous dispersio n Color : Colorless Vapor pressure : 20 tor r Storage conditions : At room temperature at about 20 °C, away from direct sunlight . Shaken before sampling .


## 2 .3 TEST SYSTE M The study was performed with zebra fish (Brachydanio rerio) . The test fish were obtained from Zoohaus Schaub, CH-4410 Liestal, Switzerland . In accordance with the test guidelines the fish were held in the laboratories of RCC for more than six weeks without any medication . Prior to test start, they were acclimated for one week to the test water and temperature . During holding and acclimatization until one day before the start of the test the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, D-49304 Melle, Germany) . During holding and acclimatization no fish died in the test fish batch and all fish were healthy . From the acclimated test fish batch, 10 fish were measured at the start of the test : The mean body length of the fish was 3 .1 ± 0 .1 cm (Mean ± SD), the mean body wet weight was 0 .28 ± 0 .04 g (Mean ± SD)
.



### 31p . 36



## ~~ rk - REPORT Page 13 

## 2 .4 STUDY DESIG N 2 .4 .1 Experimental Condition s One glass aquarium with 5 liters test medium was used for each treatment (the single test concentration and the control) . The test vessels were labeled with the RCC study number and all necessa ry additional information to assure unmistakable identification . At the sta rt of the test 7 fish were introduced into each aquarium in a random order . The loading rate was lower than 1 g fish wet weight per liter test medium . The test media and the control were slightly aerated during the test period . The fish were not fed during the test . A static t est without test medium renewal was chosen since the test item is stable in water ( information given by the sponsor) . Test duration : 96 hours Water temperature : 21-22 °C during the test period (Table 5) Light conditions : A 16-hour light to 8-hour darkness photoperiod, with a 30-minute transition period ( light intensity at light period approximately within the range of 50 to 500 Lux) . Test water : Reconstituted water : analytical grade salts were dissolved in deionized water to obtain the following nominal concentrations : CaCI2 x 2H20 : 2 .0 mmol/L (= 294 mg/L) MgSO4 x 7H20 : 0 .5 mmol/L (= 123 mg/L) NaHCO3 : 0 .75 mmol/L (= 65 mg/L) KCI : 0 .075 mmol/L (= 5 .8 mg/L) Water Hardness : 2 .5 mmol/L (= 250 mg/L) as CaCO3 Alkalinity : 0 .8 mmol/L Ratio of Ca : Mg = 4 : 1 (based on molarity)

Na : K = 10 : 1 (based on molarity) The test water was aerated prior to the preparation of the test media until oxygen saturation was reached .



### 127zp . 37 REPORT Page 14 

### 2 .4 .2 Dosage and Concentration s A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test fish up to the test item concentration of 100 mg/L . Thus, the only test item concentration tested was nominal 100 mg/L . Additionally, a control was tested in parallel (test water without addition of the test item)
. The test medium of nominal 100 mg/L was prepared by dissolving 500 mg of the test item completely in 5 liters test water by ultrasonic treatment for 15 minutes and stirring for 15 minutes at room temperature
. The test medium of the only test concentration of nominal 100 mg/L was freshly prepared just before introduction of the fish (= start of the test) . On request of the sponsor the test concentrations were not analytically verified during the test period . The test concentrations were based on the results of a range-finding test (without GLP)
. However, concentrations in excess of nominal 100 mg/L have not been tested according to the guidelines .The range-finding test and the pre-experiment were not performed in compliance with GLPRegulations and therefore are excluded from the Statement of Compliance . However the raw data, respectively copies of raw data of these tests will be archived under the RCC study number of the present study.

~p . 38 REPORT Page 15 

## 2 .5 EVALUATION S 2 .5 .1 Determination of the LC50, LCO, LC100, NOEC and LOE C The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities . The NOEC and the LCO were determined directly from the raw data . The LOEC, the LC100 and the LC50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested concentration . 

### 2 .5 .2 Water Quality Criteri a The water temperature, pH-values and dissolved oxygen concentrations were measured at the start of the test and once every day during the test in the single test medium concentration and in the control . At the same dates the appearance of the test medium was recorded
.p . 39

M111MORW REPORT Page 16 

# 3 RESULTS AND DISCUSSIO N A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the concentration of nominal 100 mg/L

. The biological results are listed in Table 1 . In the control and in the test medium of 100 mg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish . Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LCO o zebra fish were determined to be at least 100 mg/L . The 96-hour NOEC and the 96-hour LCO might even be higher than this concentration, but concentrations in excess of 100 mg/L have not been tested, according to the guidelines . The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly hi her than 100 mg/L . These values could not be quantified due to the absence of toxicity of~t the tested concentration . No remarkable observations were made concerning the appearance of the test medium . It was a clear solution throughout the entire test duration (Table 2)
. The pH value in the test medium and the control was constantly 7 .9 (Table 3) . The oxygen concentration was always 8 .1 mg/L or higher (Table 4), and thus higher than 60% oxygen saturation . The water temperature ranged from 21 to 22 °C (Table 5) during the test period

.

3_"REPORT Page 17



# 4 TABLES Table 1 : Mortality and visible abnormalities observed at the test fish Abbreviations of visible abnormalities for which fish were observed during the study: AK : Strongly extended gills OB : Fish mainly at the water surface AP : Apathy SA : Mucus secretio n BA : Distended abdomen SR : Fish lying on side or back on the bo ttom BO : Fish mainly at the bottom SV : Strong ventilatio n

of the aquarium TS : Tumbling du ri ng swimming GA : Exophthalmus VF : Changed body colo r KR : Convulsions Numbet of fish tested at the single concentration and the control : 7 Nominal Number of affected fish* ! number of dead fish test item visible abnormalitie s concentration Obse rvation tim e (mg/L) 3 hours 24 hours 48 hours 72 hours 96 hours Control 0/0 0/0 0/0 0/0 0/0 100 0/0 0/0 0/0 0/0 0/0 LC50 > 100 mg/L *number of dead fish plus number of fish with visible abnormalities
Table 2 : Appearance of the test mediu m

Abbrevia ti ons : 0 : no remarkable obse rvations, clear test mediu m 1 : homogeneous dispersion in the water, turbidity obse rvable 2 : noticeable turbidity caused by the test ite m 3 : noticeable coloration caused by the test item 4 : inhomogeneous dispersion of the test item 5 : precipitation of the test ite m 6 : test item at the su rface 7 : test item lying at the bottom of the aqua ri um Nominal test item Exposure time concentratio n (mg/L) 0 h 24 h 48 h 72 h 96 h 100 0 0 0 0 0 

# 3 &p . 41 REPORT Page 18 Table 3 : pH-values in the test medium and the contro l



### Nominal test item Exposure time concentratio n (mg/L) 0 h 24 h 48 h 72 h 96 h Control 7 .9 7 .9 7 .9 7 .9 7 .9 100 7 .9 7 .9 7 .9 7 .9 7 : 9

Table 4 : Oxygen concentrations (mg/L) in the test medium and the control

Nominal test item Exposure time concentratio n (mg/L) 0 h 24 h 48 h 72 h 96 h Control 8 .2 8 .1 8 .3 8 .2 8 .2 100 8 .1 8 .2 8 .4 8 .4 8 .1

Table 5 : Temperatures (°C) in the test medium and the contro l

Nominal test item Exposure time concentratio n (mg/L) 0 h 24 h 48 h 72 h 96 h Control 22 22 21 21 21 100 22 22 21 21 21



# ~~p . 42



## _.=~


# REPORT Study Titl e ACUTE TOXICITY OF TO DAPHNIA MAGN

A IN A 48-HOUR IMMOBILIZATION TEST Data Requirements / Test Guidelines : OECD No . 202, Part I EU Commission Directive 92/69/EEC, C .2 Study Director : Dr . Birgit Seyfrie d Study Completion Date : July 22, 200 2 Test Facility : RCC Ltd Environmental Chemistry & Pharmanalytics Division CH-4452 Itingen / Switzerland

RCC Study Number: Page 1 of 1 7 

# RCCp . 4 3 ~ n ~ tr~r~t. ► .•=t_ r-r"t .~, ~ ., . REPORT Page 2 

# WP`W"* GLP CERTIFICAT E The Swiss GLP Monitoring Authoritie s ^ Swiss Federal Swiss Agency for the Intereantonal Office r~l office of Environment, Forests ~ for the Control of

Public Health and Landscape Medicines 

# Statement of GLP Compliance It is hereby confirmed tha t during the period of August 15 - 17, 2000 August 28 - 29 , 2001 and April 15, 2002 the following Test Facilities of RCC Ltd 4452 Itingen Switzerland were inspected by the Federal Office of Public Health, the Swiss Agency for th e Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines' with respect to the compliance with the Swiss legislation on Good Laboratory Practice . Test Facilities areas of expe rtise• - Toxicology Division TOX, ACC, MU T - Environmental Chemistry and ACC, ECT, ENF, EMN, Pharmanalytics Division PCT,RES,OTH (Animal metabolism) - Microbiological Diagnostics b y Biotechnology & Animal Breeding Division OTH (Microbiology ) The inspection was performed in agreement with the OECD Guidelines for National GLP Inspections and Audits . It was found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813 .016 .51 at the time they were inspected . Federal Office of Public Health The Directo r Bern, May 2002 Prof . Th . Zettner • TOX = Toxicology : ACC = Analytical and Clinical Chemistry ; ECT = Environmental toxicity on aquatic and terrestrial organisms ; ENF = Behaviour in water, soil and air. Bioaccumulalion : EMN = Studies on effects on mesocosms and natural

. . __~ _ . . . . . . . . . . .- . . _ rs__ .~ . .- - .u1 :-- . /1T ►J - /lA- . w A~ e/~~,1p . 44 REPORT Page 3 GOOD LABORATORY PRACTIC E 

# STATEMENT OF COMPLIANC E RCC Study Number : 843571 Test Item : Study Director : Dr . Birgit Seyfried Study Title : Acute toxicity o o Daphnia magna in a 48-hour immobilization test This study (with the exception of the pre-experiments as mentioned in the repo rt) has been pe rformed in compliance with the Swiss Ordinance relating to Good Laborato ry Practice, adopted Februa ry 2"d, 2000 [RS 813 .016 .5] . This Ordinance is based on the OECD Principles of Good Laborato ry Practice, as revised in 1997 and adopted November 261h, 1997 by decision of the OECD Council [C(97) 186/Final] . There were no circumstances that may have affected the quality or integrity of the data . Study Director : Dr . Birgit Seyfrie d

~y~~ . ., . . .~ .~ 

# . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



### Date : 

## a2 ,2

## , eL v0 2



## 40p . 45 REPORT ~~ h~~`Y ~~ Page 4 

# SIGNATURES

Study Director : Dr . Birgit Seyfried



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

Management: h . Dr . Uwe Morgenroth

A- - k~ Date : - 'Lti ~ Z.so2 ._. . .p . 46 :~ ~ REPORT Page 5 n 

# QUALITY ASSURANCE UNI T RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland STATEMEN T RCC Study Number : Test Item : Study Director : Dr. Birgit Seyfried Study Title : Acute toxicity o o Daphnia magna in a 48-hour immobilization tes t The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management
. Study procedures (with the exception of the pre-experiments as mentioned in the report) were periodically inspected . The study plan and this report were audited by the Quality Assurance Unit . The dates are given below.

Dates and Types of QAU Inspections Dates of Reports to the Study Director and to the Managemen t June 13, 2002 Study plan June 13, 2002 April 10, 2002 Process based April 12, 2002 July 08/11, 2002 Final report July 11, 2002 This statement also confirms that this final report reflects the raw data
. Quality Assurance : {Mrs . Sabine Gotsch i

~ .Y. :~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ v 

### Date :! 

# 2 2 I 2

# o(3 (zp . 47 REPORT 

# CONTENTS
GLP CERTIFICATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 STATEMENT OF COMPLIANCE . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . : . 3 SIGNATURES . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 QUALITY ASSURANCE UNIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 CONTENTS CONTINUED (TABLES) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 PREFACE' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 DATA REQUIREMENTS / TEST GUIDELINES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 SUMMARY OF STUDY PLAN AMENDMENTS . . . . . . . . . . . . .: . . . . . . . . . . . . . . . . . . . . . . 9 ~ ~ ~ . . . . . . . . . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1 PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2 MATERIALS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 .1 DEFINITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 2 .2. TEST ITEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 .3 TEST SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 .4 STUDY DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2 .4 .1 Experimental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2 .4 .2 Dosage and Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 2 .5 EVALUATIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 15 2 .5 .1 Determination of the Immobility, NOEC, ECO, EC50 an d EC100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2 .5 .2 Water Quality Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4 TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17p . 4 8 REPOR T 

# AVENWR~! ~<Page7 

# CONTENTS CONTINUED (TABLES )

Table 1 : Influence o on the mobility of Daphnia magna
. . . . . . . . . . . . . . . . . . 17 Table 2 : Appearance of the test medium during the test period
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 3 : Dissolved oxygen concentrations, pH values and temperature i n

the test medium and the control
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17p . 49 REPORT Page 8 NOW 

# PREFACE GENERAL Study Title : Acute toxicity of~~to Daphnia magna in a 48-hour immo ilization test Sponsor: Monitoring Scientist : Nor

Test Facility : RCC Ltd Environmental Chemistry & Pharmanalytics Division Zelgliweg 1 CH-4452 Itingen / Switzerland 

## RESPONSIBILITIE S Study Director : Dr . Birgit Seyfried Deputy Study Director : Dr . Ulrich Memmert Technical Coordinator : Mrs . Nadja Justrich Head of RCC Quality Assurance : Mrs . Iris Wuthrich
SCHEDUL E Experimental Starting Date : June 17, 2002 Experimental Completion Date : June 20, 2002 Study Completion Date : July 22, 2002
ARCHIVIN G RCC Ltd, CH-4452 Itingen, Switzerland will retain the study plan, raw data, a sample of the test item and the final report of the present study for at least ten years . No data will be discarded without the Sponsor's consent
.

L-~p . 50

- REPORT Page 9 

# DATA REQUIREMENTS / TEST GUIDELINE S This study followed the procedures indicated by the following internationally accepted guidelines and recommendations : OECD Guideline for Testing of Chemicals, No . 202, Daphnia sp., Acute Immobilization Test and Reproduction Test, Part I, 1984
. EU Commission Directive 92/69/EEC, C .2, Acute Toxicity for Daphnia, 1992
. 

# SUMMARY OF STUDY PLAN AMENDMENTS There were no amendments to study plan
.p .51 REPORT Page 10 

# SUMMARY The acute toxicity of the test it o Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C .2 (1992), and the OECD Guideline for Testing of Chemicals, No . 202, Part I(1984)
. A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of nominal 100 mg/L . Thus the only concentration tested was nominal 100 mg/L and a control . In the control and at the test item concentration of 100 mg/L no immobilized test organisms were determined during the test period of 48 hours . The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours), and the 48-hour ECO to Daphnia magna were determined to be at least 100 mg/L . These values might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to the EU Commission Directive . The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 m/L . These values could not be quantified due to the absence of toxicity t the test concentration of 100 mg/L .p . 52 ~ REPORT Page 11 ~~am 3,,r~-9s-r ! ! R

~ E~ z ~Y~ .~~ 

# 1 PURPOS E The purpose of the 48-hour toxicity test was to evaluate the influence of the test item ~~n the mobility of Daphnia magna. For this .purpose, juvenile daphnids were exposed in a static test to an aqueous test medium containing the test item at the concentration of nominal 100 mg/L under defined conditions . This limit test was based on a range-finding test and performed in compliance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no .toxic effect on the test organisms up to at least this concentration . The used method of application and the test species Daphnia magna are recommended by the international testing guidelines . 

# 2 MATERIALS AND METHODS 2 .1 DEFINITION S Immobilization : those organisms which are not able to swim within 15 seconds after gentle agitation of the test container are considered to be " immobil e ECO : the highest test concentration without a significant number of immobilized test organism s EC50 : the calculated concentration of test item which results in a 50% immobilization rate EC100 : the lowest test concentration at which all test organisms are immobil e 

### NOEC (No Observed Effect Concentration) : the highest test concentration at which no significant toxic effect on the test organisms is observedp . 53

• ~~ ^ `$ . ; ~ :. .y~~'z,-,. ,,,_, . ,-- T - REPORT Page 12 

## 2 .2 TEST ITE M The test item and the following information concerning the test item were provided by the sponsor: Identity: Batch No . : 4512 OS Expiration date : March, 2004 Purity : >98% Concentration : Dry content 26% Stability in water : Stabl e Solubility in water : Miscibl e pH in aqueous solution : 3 .9 at concentration of 26% Aggregate state at room temperature : Aqueous dispersio n Color : Colorless Vapor pressure : 20 torr Storage conditions : At room temperature at about 20 °C, away from direct sunlight . Shaken before sampling
. 

## 2 .3 TEST SYSTE M The study was performed with young daphnids of a clone of the species Daphnia magna Straus . A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5 . Since this date the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions and total hardness) and under temperature and light conditions identical to those of the tests (see below)
. At the start of the test the used test organisms were 6-24 hours old and were not first brood progeny.p . 54 REPORT Page 13 

## 2 .4 STUDY DESIG N 2 .4 .1 Experimental Condition s The test was pe rformed in 100 mL glass beakers filled with 50 mL test medium . The beakers were covered with glass plates to reduce the loss of water and to avoid the ent ry of dust into the solutions . The test vessels were labeled with the RCC study number and all necessa ry additional information to assure unmistakable identification . At the single test concentration and for the control 20 daphnids were used, divided into two replicates of ten daphnids each . The daphnids were randomly distributed to the test vessels at initiation of the test . The loading rate was lower than one daphnia per 2 mL test solution . A static test - without test medium renewal was chosen since in a pre-experiment (without GLP) the test item concentration in the test medium was constant during the test period of 48 hours . The test was performed in a temperature-controlled room . Water temperature : 20-21 °C during the test period (Table 3) . Light conditions : A 16-hour light to 8-hour darkness photoperiod (with a 30 minute

transition period) . Light intensity at light period between 550 and 685 Lux. Test duration : 48 hours Test water. Reconstituted test water : analytical grade salts were dissolved in purified

water to obtain the following nominal concentrations : CaCl2 x 2H20 : 2 .0 mmol/L (= 294 .0 mg/L) MgSO4 x 7H20 : 0 .5 mmol/L (= 123 .0 mg/L) NaHCO3 : 0 .75 mmol/L (= 65 .0 mg/L) KCI : 0 .075 mmol/L (= 5 .8 mg/L) Water Hardness : 2 .5 mmol/L (= 250 .0 mg/L) as CaCO3 Alkalinity : 0 .8 mmol/L Ratio of Ca : Mg = 4 : 1 (based on molarity) Na : K = 10 : 1 (based on molarity) The test water was aerated prior to the sta rt of the study until oxygen saturation was reached . During the test period the test water was not aerated . 

### Feeding : Non e Positive Control : For evaluation of the quality of the Daphnia magna clone and of

experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfacto 

### ry test conditions . 

# S 0p . 55 REPORT Page 14 

### 2 .4 .2 Dosage and Concentration s The only test item concentration tested was nominal 100 mg/L . Additionally, a control was tested in parallel (test water without addition of the test item) . Thus a limit test was pe rformed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to this concentration . The test medium of the only test concentration of nominal 100 mg/L was prepared by dissolving 29 mg test item completely in 300 mL test water by ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature . The test medium was prepared just before introduction of the daphnids (= sta rt of the test) . On request of the sponsor the test concentrations were not analytically verified during the test period . The test concentration was based on the results of a range-finding test and on results of a pre-experiment to the solubility of the test item (without GLP) . The range-fiqding test and the pre-experiment to the solubility of the test item were not performed in compliance with GLP-Regulations and therefore are excluded from the Statement of Compliance . However, the raw data, respectively copies of raw data of these tests will be archived under the RCC study number of the present study .



# ~lp . 56 REPORT Page 15 

## 2 .5 EVALUATION S 2 .5 .1 Determination of the Immobility, NOEC, ECO, EC50 and EC10 0 The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile) . The EC50 could not be calculated due to the absence of toxicity of the test item
. The NOEC, ECO and EC100 were determined directly from the raw data
. 

### 2 .5 .2 Water Quality Criteri a At the start and at the end of the test, the pH values, the dissolved oxygen concentrations and the water temperature were determined in the single test conc6ntration and the control
. The appearance of the test medium was visually recorded at the start of the test and after 24 and 48 hours .:~. REPORT Page 16 

# 3 RESULTS AND DISCUSSION A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Daphnia magna up to the concentration of nominal 100 mg/L. In the control and in the test concentration of 100 mg/L no immobilized test organisms were observed during the test period of 48 hours (Table 1)
. Therefore, the 48-hour NOEC (highest concentration tested v ►i h effects after the exposure period of 48 hours), respectively the 48-hour ECO to Daphnia magna were determined to be at least 100 mg/L . The 48-hour OEC and the 48-hour ECO might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to the EU Commission Directive . The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L . These values could not be quantified due to the absence of toxicity the test concentration of 100 mg/L
. No remarkable observations were made concerning the appearance of the test medium . The test medium was a clear solution throughout the whole test duration (Table 2)
. At the beginning and the end of the test period, the dissolved oxygen concentrations in the test medium and the control were at least 8 .0 mg/L, the pH values ranged from 7 .8 to 7 .9, and the water temperature was between 20 and 21 °C (Table 3)
.p . 5 8
• ;_ - - - REPORT Page 1 7 

# 4 TABLE S Table 1 : Influence the mobility of Daphnia magna

Nominal No . of Immobilized Immobilized test item daphnids daphnids after daphnids afte r concentration tested 24 hours 48 hours (mg/L) No . % No . % ;~ Control 20 0 0 0 0 100 20 0 0 0 0

Table 2 : Appearance'of the test medium during the test perio d

Abbreviations : 0 : no remarkable obse rvations, clear test mediu m 1 : homogeneous dispersion in the water, turbidity observable 2 : noticeable turbidity caused by the test ite m 3 : noticeable coloration caused by the test item 4 : inhomogeneous dispersion of the test item 5 : precipitation of the test ite m 6 : test item at the su rface 7 : test item lying at the bottom of the test beaker Nomina l test item Exposure time concentratio n (mg/L) 0 hours 24 hours 48 hours

. 

## ao0,, 0 0 0

Table 3 : Dissolved oxygen concentrations, pH values and temperature

in the test medium and the contro l Nominal test item Start (0 hours) End (48 hours) concentratio n (mg/L) pH Oxygen Temperature pH Oxygen Temperature

(mg/L) (°C) (mg/L) (°C) Control 7 .9 8 .8 21 7 .9 8 .1 2 0 100 7 .8 8 .3 21 7 .9 8 .0 20p . 5 9
` ~ - 

# REPORT Study Title : TOXICITY O F TO SCENEDE MUS SUBSPICA US IN A 72-HOUR ALGAL GROWTH INHIBITION TEST Data Requirements I Test Guidelines : OECD No . 20 1 EU Commission Directive 92/69/EEC, C .3 Study Director: Dr . Birgit Seyfrie d Study Completion Date : July 22, 2002 Test Facility
. RCC Ltd Environmental Chemistry & Pharmanalytics Division CH-4452 Itingen / Switzerland

. . . , RCC Study Number : Page 1 of 1 9 

# RCCp . 60 REPORT Page 2 

# GLP CERTIFICAT E The Swiss GLP Monitoring Authoritie s Swiss Federal ~ Swiss Agency for the Inlercanlonal Office r~l Office of Environment, Forests for the control Of

Public Health and Landscape Medicine s 

# Statement of GLP Compliance It is hereby confirmed tha t during the period of August 15 - 17, 2000 August 28 - 29, 2001 and April 15, 2002 the following Test Facilities of RCC Ltd 4452 Itingen Switzerland were inspected by the Federal Office of Public Health, the Swiss Agency for th e Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines' with respect to the compliance with the Swiss legislation on Good Laboratory Practice . Test Facilities areas of expertise* - Toxicology Division TOX, ACC, MU T - Environmental Chemistry and ACC, ECT, ENF, EMN, Pharmanalytics Division PCT,RES,OTH (Animal metabolism) - Microbiological Diagnostics b y Biotechnology & Animal Breeding Division OTH ( Microbiology) The inspection was performed in agreement with the OECD Guidelines for National GLP Inspections and Audits . It was found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813 .016 .5) at the time they were inspected . Federal Office of Public Health The Directo r Bern, May 2002 Prof . Th . Zeltne r • TOX - Toxicology : ACC = Analytical and Clinical Chemistry : ECT - Environmental toxicity on aquatic and terrestrial organisms : ENF = Behaviour in water, soil and air . Bioaccumulalion : EMN = Studies on effects on mesocosms and natura l

. . . . . - . . r .nt - n 1 . . . t- -

_ :__, .__ . :__ - - -- . .- - . . .d :-- . P1TU - /b - 6 n iu - -W- A• ''K ~ ~Y~~_<~'?_i ~' : ty F • 4w>Cz• .,~

. . . 1i : .i . . ._ . c . a _,' : REPORT Page 3
~ GOOD LABORATORY PRACTIC E 

# STATEMENT OF COMPLIANCE RCC Study Number: Test Item : Study Director : Dr . Birgit Seyfried Study Title : Toxicity to Scenedesmus subspicatus in a 72-hour algal growth inhibition test This study (with the exception of the p re-experiments as mentioned in the repo rt) has been pe rformed in compliance with the Swiss Ordinance relating to Good Laborato ry Practice, adopted Februa ry 2"d, 2000 [RS 813 .016 .5] . This Ordinance is based on the OECD Principles of Good Laborato ry Practice, as revised in 1997 and adopted November 26th 1997 by decision of the OECD Council [C(97) 186/Final] . There were no circumstances that may have affected the quality or integrity of the data . Study Director : Dr . Birgit Seyfried



# ., .. 03 . .~ ~ ....................................... y ~ ~ Date : AoCy .2.2, .200.L



# ~~p . 62 . ~~~?~~•~~~'-~~~~~ . :,~, i?t:~° .~., REPORT Page 4 

# SIGNATURES

Study Director : Dr . Birgit Seyfrie d

• . 

## . : . 

## . . . : .` . . . . . . . 

## . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Date : Y .2 .2, ~ o oa2 .

Management: Dr . Uwe Morgenrot h

Date : ~ ~ ' ' •



# 5

~9 -_ K REPORT Page 5 

# QUALITY ASSURANCE UNI T RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland STATEMEN T RCC Study Number : Test Item : Study Director : Dr. Birgit Seyfrie d Study Title : Toxicity of Scenedesmus subspicatus in a 72-hour algal growth inhibition test The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management
. Study procedures (with the exception of the pre-experiments as mentioned in the report) were periodically inspected . The study plan and this report were audited by the Quality Assurance Unit . The dates are given below.

Dates and Types of QAU Inspections Dates of Repo rts to the Study Director and to the Managemen t June 13, 2002 Study Plan June 13, 2002 May 21, 2002 Process based May 24, 2002 ° . . July 08/11, 2002 Final Report July 11, 2002

This statement also confirms that this final report reflects the raw data

. Quality Assurance : Mrs . Sabine Gotsch i



# ~• r

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Datej 

# 22, 2~2p . 64 • ~~ REPORT Page 6 

# CONTENTS GLP CERTIFICATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 STATEMENT OF COMPLIANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 SIGNATURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 QUALITY ASSURANCE UNIT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 CONTENTS CONTINUED (TABLES & FIGURES)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 PREFACE . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 DATA REQUIREMENTS / TEST GUIDELINES
. . . . . # . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . .. . . . . . . . . . . . . . . . . . . . .9 . SUMMARY OF STUDY PLAN AMENDMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 1 PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 2 MATERIALS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 1 2 .1 DEFINITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 2 .2 TEST ITEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 2 .3 . TEST SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 2 .4 STUDY DESIGN
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 3 2 .4 .1 Experimental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 2 .4 .2 Dosage and Concentrations
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2 .5 EVALUATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .1 5 2 .5 .1 Counting and Examination of the Algal Cells
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 2 .5 .2 Determination of the Algal,Growth Inhibition and the E C Values
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .15 2 .5 .3 Water Quality Criteria
. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 3 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 4 TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 5 FIGURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

'f'/"p . 65 • ~~~t a'~ ~~ ~,a~`y~~,~~.~' .~ REPORT Page 7 

# CONTENTS CONTINUED (TABLES & FIGURES )
Table 1 : Algal cell densities during the test period of 72 hours
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 2 : Appearance of the test medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Table 3 : pH values in the test medium and in the control
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Table 4 : Water temperature during the test period
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Figure 1 : Growth curves of Scenedesmus subspicatus, incubated for 72 hours
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~p . 66 '~f~ ` VON" REPORT Page 8 

# PREFAC E GENERAL Study Title : Toxicity of to Scenedesmus subspicatus in a 72-hour algal growth inhibition tes t Sponsor: 40%6* Monitoring Scientist : 

# 408ftaftak. Test Facility : RCC Ltd Environmental Chemistry & Pharmanalytics Division Zelgliweg 1 CH-4452 Itingen / Switzerland
RESPONSIBILITIE S Study Director : Dr . Birgit Seyfried Deputy Study Director : Dr . Ulrich Memmert Technical Coordinator : Mrs . Nadja JUstrich Head of RCC Quality Assurance : Mrs . Iris Wuthrich SCHEDUL E Experimental Starting Date : June 14, 2002 E ;:perimental Completion Date : June 21, 2002 Study Completion Date : July 22, 2002 ARCHIVIN G RCC Ltd, CH-4452 Itingen, Switzerland will retain the study plan, raw data, a sample of the test item and the final report of the present study for at least ten years
. No data will be discarded without the Sponsor's consent
.

-Z'p . 67 REPORT Page 9 

# DATA REQUIREMENTS / TEST GUIDELINE S This study followed the procedures indicated by the following internationally accepted guidelines and recommendations : OECD Guideline for Testing of Chemicals, No . 201 : Alga, Growth Inhibition Test, 1984
. EU Commission Directive 92/69/EEC, C .3 : Algal Inhibition Test, 1992
. 

# SUMMARY OF STUDY PLAN AMENDMENTS First Amendment to Study Pla n Concerning : Alteration : Reason : Header Page 7 Study Number Wrong study number given in the study pla n 2 .3 Test System Test System Wrong test system given in the study planp . 68 REPORT Page 10 

# SUMMARY
The influence of the test item W investigate d the growth of the green algal species Scenedesmus subspicatus CH AT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C .3, 1992, and the OECD Guideline No . 201, 1984 . A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Scenedesmus subspicatus to and including the concentration of nominal 100 mg/L . Thus the only concentration tested was nominal 100 mg/L and a control
. The test ite ad clearly no inhibitory effect on the growth of Scenedesmus subspicatus ring the exposure period of 72 hours at the test concentration of 100 mg/L
. The 72-hour NOEC (highest entration tested without toxic effects after the exposure period of 72 hours) of Scenedesmus subspicatus was determined to be at least 100 mg/L . This va ue might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to EU Commission Directive 92/69/EEC
. The 72-hour EC50 and the 72-hour EC100 were clearly higher than 100 m/L . These values could not be quantified due to absence of toxicity o t the tested concentration . -- -• p . 69 

# ANN 9 REPORT Page 11 

# 1 PURPOS E The purpose of this test was to determine the inhibito ry effect of the test ite m on the growth of the freshwater green algal species Scenedesmus subspicatus. Exponentially growing cultures of this algal species were exposed to a test medium containing the test item at the concentration of nominal 100 mg/L under defined conditions . This limit test was based on a range-finding test and pe rformed in compliance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Scenedesmus subspicatus up to at least this concentration . A static, non-renewal exposure system was used . The inhibition of growth in relation to control cultures was determined over a test-period of 72 hours and, thus, over several algal generations . The method of test item application and the test system are recommended by the testing guidelines .

~a. 

# 2 MATERIALS AND METHOD S 2 .1 DEFINITION S Cell density : the number of cells per m L 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 (AUC ) Growth rate (r) : the increase of cell density per unit tim e EbCx : the calculated concentration of test item that results in an x% reduction of biomass b relative to the contro l E rCx : the calculated concentration of test item that results in an x% reduction of growth rate r relative to the contro l NOEC ( No Observed Effect Concentration) : the highest test concentration at which no significant inhibition of growth is observed relative to the contro l LOEC ( Lowest Observed Effect Concentration) : the lowest test concentration at which a significant inhibition of growth is observed relative to the contro l



### ~5'~''~~ ; r REPORT Page 1 2 

## 2 .2 TEST ITE M The test item and the following information concerning the test item were provided by the sponsor: Identity: Batch No . : 4512 OS Expiration date : March, 2004 Purity : >98% Concentration : Dry content 26% Stability in water : Stabl e Solubility ih water : Miscibl e pH in aqueous solution : 3 .9 at concentration of 26% Aggregate state at room temperature : Aqueous dispersio n Color : Colorless Vapor pressure : 20 torr Storage conditions : At room temperature at about 20 °C, away from direct sunlight . Shaken before sampling
. 

## 2 .3 TEST SYSTE M The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86 .81 SAG, supplied by the Sammlung von Algenkulturen Gottingen (SAG, Experimentelle Phykologie und Sammiung von Algenkulturen, Albrecht-von-Haller-Institut fur Pflanzenwissenschaften, Universitat Gottingen, D-37073 Gottingen, Germany) . The algae had been grown in the RCC laboratories under standardized condit :ons according to the test guidelines .p 71 - t

. REPORT Page 1 3 

## 2 .4 STUDY DESIG N 2 .4 .1 Experimental Condition s The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines . Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations : Macro-nutrients : NaHCO3 50 .0 mg/L CaCl2 2 H20 18 .0 mg/L NH4CI 15 .0 mg/L MgSO4 x 7 H20 15 .0 mg/L MgCI2 x 6 H20 12 .0 mg/L KH2PO4 1 .6 mg/L Trace elements : Na2EDTA x 2 H20 100 .0 pg/L FeCI3 x 6 H20 80 .0 Ng/L MnCl2 x 4 H20 415 .0 pg/L H3B03 185 .0 pg/L Na2MoO4 x 2 H20 7 .0 pg/L ZnCIz 3 .0 pg/L CoCIZ x 6 H20 1 .5 Ng/L CuClz x 2 H20 0 .01 Ng/L Calculated water hardness of the test water : 0 .24 mmol/L (= 24 mg/L) as CaCO3 . The test was sta rted (0 hours) by inoculation of 10,000 algal cells per mL test medium . These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test under the same conditions as in the test . One day before the sta rt of the test, the pre-culture was diluted threefold to keep the algae in exponential growt h The test design included three replicates per test concentration and six replicates of the control . Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks . The flasks were covered with glass dishes . They were incubated in a temperature controlled water bath at a temperature between 22 and 24 °C (Table 4), and continuously illuminated at a measured light intensity of about 8300 Lux (mean value), range : 7140 to 8980 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the su rface of the test media) . This illumination was achieved by fluorescent tubes ( Philips TLD 36W-1/840), installed above the test flasks . The test vessels were labeled with the RCC study number and all necessa ry additional information to assure unmistakable identification . The test duration was 72 hours . For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control at least once per year to demonstrate satisfacto ry test conditions .

~~p . 72 MrW REPORT Page 14 

### 2 .4 .2 Dosage and Concentration s The only test item concentration tested was nominal 100 mg/L . Additionally, a control was tested in parallel (test water without addition of the test item) . Thus a limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the algae up to this concentration
. The test medium of the only test concentration of nominal 100 mg/L was prepared by dissolving 30 mg test item completely in 300 mL test water by ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature . Additionally, a control was tested in parallel (test water without test item) . The test medium was prepared just before addition of algae (= start of the test ; see Section 2 .5 .1) . On request of the sponsor the test concentrations were not analytically verified during the test period
. The test concentration was based on the results of a range-finding test (without GLP)
. However, concentrations in excess of nominal 100 mg/L have not been tested in compliance with EU Commission Directive 92/69/EEC
.REPORT Page 15



## 2 .5 EVALUATION S 2 .5 .1 Counting and Examination of the Algal Cell s Small volumes of the test media and the control (1 .0-2 .0 ml-) were taken out of all test flasks after 24, 48 and 72 hours exposure, and were not replaced . The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample
. In addition, after 72 hours exposure, a sample was taken from the control and from the single test concentration . The shape of the algal cells was microscopically examined in these samples . 

### 2 .5 .2 Determination of the Algal Growth Inhibition and the EC Value s The inhibition of algal growth was determined from the mean values of counted algal cell densities . The test concentrations corresponding to 10 and 50% inhibition of the algal biomass b(EbC10, EbC50), respectively of the growth rate r(ErC10, E~C50) could not be calculated due to the absence of a toxic effect of the test item
. The LOEC and the NOEC were determined directly from the counted algal cell densities . A statistical evaluation of the algal cell densities was not necessary since the mean algal cell density in the test medium was at all counting dates similar as in the control
. 

### 2 .5 .3 Water Quality Criteri a The pH was measured and recorded in the single test concentration and the control at the start and at the end of the test . The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks . The appearance of the test medium was recorded daily
.

### p 

## z4 REPORT Page 16 

# 3 RESULTS AND DISCUSSIO N A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Scenedesmus subspicatus up to the concentration of nominal 100 mg/L . The mean algal cell densities in the treated test medium of 100 mg/L were identical with those in the parallel control culture throughout the entire test duration (Table 1 and Figure 1)
. Thus, the test item learly had no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at the nominal test concentration of 100 mg/L . This test concentration was therefore determined as the 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours)

. This value might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to EU Commission Directive 92/69/EEC
. The 72-hour LOEC (lowest concentration tested with toxic effects), and the 72-hour EC50 for the algal biomass b and growth rate r could not be quantified due to the absence of a toxic effect of at the tested concentration . However, these parameters were clearly higM than 100 mg/L. The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in the single test concentration and the algal cells in the controls

. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at this nominal concentration
. In the control the cell density increased from nominal N = 1 x 10° cells/ml- at the start of the test (0 hours) to N = 86 x 10" cells/ml- (mean value) after 72 hours (Table 1) . Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled . No remarkable observations were made concerning the appearance of the test media

. All test media were clear solutions throughout the test period (Table 2)
. At the start of the test, the pH values in the test media and the control ranged from 7 .9 to 8 .0 . At the end of the test, in all treatments a pH value of 8 .8 was measured (Table 3) . The water temperature ranged from 22 to 24 °C during the test period (Table 4)
.p . 75 REPORT Page 17 

# 4 TABLE S Table 1 : Algal cell densities during the test period of 72 hours Nominal test item Flask Density of algal cells (cell number x 10,000/mL) after concentration No
. m/L 24 h 48 h 72 h

1 5 .5 5 .6 23 .0 22 .3 99 .5 98 .0 Control 2 5 .9 5 .3 22 .7 21 .6 82 .9 84 .7

3 6 .2 5 .5 24 .3 24 .2 88 .1 88 .5 4 5 .9 6 .0 21 .0 21 .3 80 .5 80 .3 5 5 .5 5 .2 18 .4 17 .7 71 .7 72 .1 6 5 .9 5 .7 23 .2 22 .3 92 .3 92 .6 m 5 .68 21 .83 85 .9 3 s 0 .22 2 .11 9 .43 n 6 6 6 1 3 .6 3 .3 17 .3 16 .8 80 .6 81 . 1 100 2 3 .2 3 .7 21 .4 20 .3 89 .4 88 .7

3 4 .2 4 .0 19 .6 18 .8 88 .6 86 .8 m 3 .67 19 .03 85 .8 7 s 0 .38 1 .91 4 .40 n 3 3 3 m : mean value ; s : standard deviation ; n : number of flasks At the sta rt, 10,000 algal cells/mL were incubated . The tabulated values represent rounded results obtained by calculation using the exact raw data . Table 2 : Appearance of the test mediu m

Abbrevia ti ons : 0 : no remarkable obse rvations, clear test mediu m 1 : homogeneous dispersion in the water, turbidity obse rvable 2 : noticeable turbidity caused by the test ite m 3 : noticeable coloration caused by the test item 4 : non-homogeneous dispersion of the test,item 5 : precipitation of the test ite m 6 : test item at the surface 7 : test item lying at the bo ttom of the flas k

Nominal test item Exposure time concentratio n (mg/L) 0 h 24 h 48 h 72 h 

# F 100 0 0 0 0~~sr'a m : `F . s=•P :" ;. :

4 : b-Z :, :,--I REPORT Page 1 8 Table 3 : pH values in the test medium and in the control

Nominal test item pH values concentratio n (mg/L) Sta rt End Control 7, 9 8 .8 100 8 .0 8 . 8

Table 4 : Water temperature during the test period Temperature (°C) Day 0 (Sta rt) 2 4 Day 1 22 Day 2 22 Day 3 (End) 22p .77 REPORT Page 19 

# 5 FIGUR E Figure 1 : Growth curves of Scenedesmus subspicatus, incubated for 72 hours

100 /.. E o

1 0 C .'/ .

~ .
E

• . ~ 0 24 48 72

test peri od (hours) ~ Control -~ - 100 mg/ L



# ~3