-, 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 :.

twnL

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~

in the Premanufacture Notice W15ThtO

GENERAL INSTRUCTIONS Ts- /~ N Ht 0 3 I • Yo

not have

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 actual data.

• Before you complete this form, you should read the *Instructions Manual for Premanafadate Notification' (the

Substances Control Ad (TSCA) Instructions Manual is available from the Toxi c

Information Service by calling 202-554-140 4, or faxing 202-55~5603),

• If a user fee has been remitted for this

Remembe

notice (40 CFR 700.45), indicate in the boxes above the TS-userfee identification number you have generated.

P

r, your user fee ID number must also appear on your corresponding fee remittance, which is sent to: EPA, HQ AccountingOperations Branch (p14I-266), .O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSG User Fee.

Part I - GENERAL INFORMATION TEST DATA AND OTHER DATA

You must provide the currentl y correct Chemical Abstracts (CA) Name of the You are required to submit all test data in yourpossession or control newchemical 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 reviewwill not begin until EPA environmental effeds of the manafadqee, processing, distribution receives this information. A letter in supportof your submission should in commerce, use, or disposal of the new chemical sabstaeot: Standard

reference yourTS 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 C3•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 • Otherdata 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 foryou to ~- or control of the ia-bmitter describe pollution prevention and recycling information you may have regarding .

the new substance. A physicaland chemical properties worksheet is located on the-lad page

of this form.

So-called "binding" boxes are included throughout this form for you to indicate

your willingness to be TYPE OF NOTICE (Check Only One) 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 of consent orders

Significant New Use Rules or PMN (Pnemanafadane Notice)

. 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,( py binding on the submitterwhen the Agency odatt approves the exemption application, P~

checking a binding box In this noticed~~ by itself prohibitthe sabmHter •

from

the form

later deviating from

. CD

the info rmation (except chemical identity) reported in ❑ SNUN (Significant New UseNotice) r"l

❑ 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 ExposureExemptiosd 0 723.511(dpJ

as confidential. To assert a claim in an attachment, circle or bracket the ❑

Information you claim as confidential. If you daisforeation is th notl . LVE Modification 11 LOREXModlfaNor

confidential_ you m ust also provide a•..iti ..t vesioe of th tl 9

IIZaChmtptil

fld tial, read the

. For additional Instructions

Instructions Manual

on

.

claiming Information as ISTHIS A CONSOLIDATED PMN7 ~ Yes

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) Replacesprevious 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

8 v~e2utg instructions, w

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

Signature of agent

X

- (if applicable) Date .

FORM EPA 7710-25 (Rev. 5-95) Page 2 •
Part I - GENERAL INFORMATION

Section 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 street)

City, State, ZIP Code

b . Agent (

if Name of authorized offidal 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 street)

City, State, ZIP Code Area Code Number

Telephone

2 . Technical Name Position

Contact

(in US.)

Company

Mailing address (number and str eet)

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 submi

import for the chemical substance

tted a notice of Bona fide intent to

covered by this no

manufacture

tice, enter the d~

or a

~

notice number assigned by EPA. -~

1 ❑ Manufacture 1 import I Type of Noti ce- Mark A) Only Only I

a Binding 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 (CA) 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 appropriate . 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 ec ti 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 4
p. 5 ~

,9NFIDIENTiAL

`C ~
p. 6

cOM~P[t~RiTBAL
r% 7

Part I -- GENERAL INFORMATION -- Continued

Section B - CHEMICAL IDENTITY INFORMATION - Continued

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) belo

absolute molecul ar weight of that composi w 500 and below 1,000 tion.

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 identity column if you want a monomer or other reactant used at two weight percent or less to be listed as pa rt 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 composition 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 noti ce . (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 5
p. 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

Provide chemical substance as saanafadured for mmmercial purposes.the CAS Registry Number if available. If them are unidentified impurities, enter "unidentified '

. (b) -Estimate the maximum weight % of each impurity. 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 identification - 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 0R. lPb L`( U fL,

❑ Mark (X) this box if you attach a continuation 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 6
P. 10

Part I-- GENERAL INFORMATION -- Continued ,

Section C - PRODUCTION, IIISPORT, AND USE INFORMATION:

Mark pQ the "Con fi dential" 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 mark (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) Cong- 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 category of use, the percent of produc tion volume

devoted to each category , the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you

claim as confidenti al.

a. (1) - Describe each intended category of use of the new chemical substance by function and application-

(2) - Mark (X) this column if entry 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 businessinformation (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 businessinformation (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 genericdescription 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. Mwk_pqui t"t

ark 00 this box if You attach hazard informiti

FORM EPA 7710-25 (Rev. 5-95) Page 7
P. 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 further indust reporting requirements if there are rial processing or use operations after import You must describe these operations. See instructions manual.

1 . Operation description

C.onfi a . Identity - Enter the iden tity 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 opera ti on 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

production rates or operati ons, include all

ficantly different 1 ~

sites as attachments.

the information requested in this sec tion for those -~ I

~ 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. Processdescription ~ Mark (70 to indicate your willingness to have your process description binding.

(1) Diagram the major unit operation steps and chemical conversions

gallon drum, rail car, tank

. Include interim storage and transport containers (specify-e.g. 5 gallon pails, 55 truck, etc.).

(2) P

materials and feedstocks

rovide 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

chemicals (note frequency if not used daily or per batch

(including reactants, solvents, and catalysts, etc), and of a ll products, recycle streams, and wastes. Include cleaning .).

(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 continua tion sheet.

FORM EPA 771 0- 25 (Rev. 5-95) Page 8
p.12

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued

Sec tion 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 substance (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 Protect ive 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 informa tion. 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 Disch arge 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)

k x

(3) (4) (5a) MarkW (5b) (6)

k

(7) Mark (X) the ❑ PO'I1N provide name(s) below: I ❑ Navigable ❑ Other - Specify provide NPDES # 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 9
Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continued

Section B-- INDUSTRIAL SITES CONTROLLED BY OTHER S

Complete section B for typical processing or use

complete

operations involving the new chemical substance at sites you do not control.Importers do not have to

Manual

this section for operations outside the US.; however, you must report any proce ssing or use activities after import See the Instructions

atmore than one site

. Complete a separate section B/o► each type of processing, or use operation inadvrng the new chemicalsubstance. If the same operation is performed describe the typical operation common to these sites. Identify additional sites on a continua tion sheet.

1. Operation Description - To claim information in this section as confidential, circle or bracket the specific informa tion that you daim as mnfidentiaL (1) - Diagram the major unit opera ti on steps and chemical conversions, including interim storage and transport containers (specify- e.g. 5 gallon p

ads 55gallon drums, rail cars, tank trucks, etc) ,

the approximate weight

. On the diagram, identify by letter and briefly describe each worker activity. (2) - Provide the identity, (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

batch) . Indude cleaning chemicals (note frequency if not used daily or per . (3) - Identify by number the points of release, including small or intermittent releases, to the environment of new (4) Please chemical substance. 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 s heet.

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) -

to

Describe

protect

physical form of exposure and % new

workers chemical substance (if in mix ture), and any protecti ve equipment and engineering controls used .

(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 PdIW, or other incineration, (specify) and c o ntrol technology, if any, that will be used to limit the release of the new substance to the environment (14) - Identify byproducts which may result from the operation.

(3), <5). (8), (11), ( 13) and (15) - Mark ()0 this column if any of the proaeeding entries are con fidential business informati on (CBI). Letter # of ®I Duration CBI Protective Equip./ in CBI Release Amount of Of Workers of Engineering Controls/ CBI Media of Release CBI

Form- Number New &cont. Act rol

- 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 10
p. 14

~

IN

~~

*444

~a-
p.15

OPTIONAL POLLUTION PREVENTION INFORMATION

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 manufactured; ( 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 11
16

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 12
p.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 ~~ de

Corifi.

(a) provided number ~~ ntial

ro) (c) Mark (?Q

uNort7 (d)

Physical state of neat substance (s) ~ W

Vapor pressure O

® C Temperature Torr

Density/relative density g/cm3 ~

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 /

l5
p.20

\
p. 21

~~
p.22

\
p.23 /

Zt4t
p. 24

~„• ~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. 203

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

RRCCC
p

: 25

!~-

-. .~

REPORT Page 2

GLP CERTIFICAT E

The Swiss GLP Monitoring Authoritie s

Swiss Federal Swiss Agency for the Inlercantonal 0111"

J officeof 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, 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, MUT

- 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. Zeltner

• 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-61n. 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 performed in compliance with the Swiss Ordinance relating to Good

Laboratory Practice, adopted Februa ry 2"d, 2000 [RS 813 .016.5] . This Ordinance is based

on the OECD Principles of Good Laboratory 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

~~: . . .~~~'Z.. . . . . . . . . . . . . . . . . . . . . . . . . . . . Date: .2 2, .200.2
p. 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 Page5

..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 Seyfried

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 Reports 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, 2002

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 and

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 Ltd

Environmental Chemistry & Pharmanalytics Division

' Zelgliweg1

CH-4452 Itingen / Switzerland

RESPONSIBILITIES

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

ARCHIVING

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~ 7
p. 32

~~.~ i ,r- .,. ._ . . . - .

. . . .:

REPORT Page9

DATA REQUIREMENTS I TEST GUIDELINES

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 Page10

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 PURPOSE

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 DEFINITIONS

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 Observed

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 ITEM

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: Stable

Solubility in water: Miscible

pH in aqueous solution : 3.9 at concentration of 26%

Aggregate state at room temperature : Aqueous dispersion

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 SYSTEM

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).

31
p. 36

~~ rk -

REPORT Page 13

2.4 STUDY DESIGN

2.4.1 Experimental Conditions

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 start 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 test 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 .

127z
p. 37

REPORT Page 14

2.4.2 Dosage and Concentrations

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 EVALUATIONS

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 Criteria

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 DISCUSSION

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 secretion

BA: Distended abdomen SR: Fish lying on side or back on the bo ttom

BO: Fish mainly at the bottom SV: Strong ventilation

of the aquarium TS: Tumbling during 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 abnormalities

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 tions:

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 item

6: test item at the surface

7 : test item lying at the bottom of the aquarium

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 control

Nominal

test item Exposure time

concentration

(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

concentration

(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

concentration

(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 Title

ACUTE TOXICITY OF

TO DAPHNIA MAGNA

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 Seyfried

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 7

RCC
p. 43

~ 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 ~ forthe 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 expertise•

- Toxicology Division TOX, ACC, MUT

- 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 Director

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/~~,1
p. 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

performed 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 Seyfried

~y~~

..,

. .

.~.~

.

~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Date : a2 ,2, eL v02

40
p. 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 UNIT

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

Management

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:! 22 I 2o(3 (z
p. 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
p. 48

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
p. 49

REPORT Page 8

NOW

PREFACE

GENERAL

Study Title : Acute toxicityof~~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

RESPONSIBILITIES

Study Director: Dr. Birgit Seyfried

Deputy Study Director: Dr. Ulrich Memmert

Technical Coordinator: Mrs. Nadja Justrich

Head of RCC Quality Assurance : Mrs. Iris Wuthrich

SCHEDULE

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 Page9

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 PURPOSE

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 DEFINITIONS

Immobilization: those organisms which are not able to swim within 15 seconds

after gentle agitation of the test container are considered to be

" immobile

ECO: the highest test concentration without a significant number of

immobilized test organisms

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

immobile

NOEC (No Observed

Effect Concentration) : the highest test concentration at which no significant toxic effect

on the test organisms is observed
p. 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: Stable

Solubility in water: Miscible

pH in aqueous solution : 3.9 at concentration of 26%

Aggregate state at room temperature : Aqueous dispersion

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 SYSTEM

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 performed 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 : None

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 satisfactory test conditions .

S0
p. 55

REPORT Page 14

2.4 .2 Dosage and Concentrations

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 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 (= start 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 .

~l
p. 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 Criteria

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. 58

•

;_ - - -

REPORT Page 17

4 TABLES

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 medium

1 : homogeneous dispersion in the water, turbidity observable

2 : noticeable turbidity caused by the test item

3 : noticeable coloration caused by the test item

4: inhomogeneous dispersion of the test item

5: precipitation of the test item

6: test item at the surface

7: test item lying at the bottom of the test beaker

Nominal

test item Exposure time

concentration

(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 control

Nominal

test item Start (0 hours) End (48 hours)

concentration

(mg/L) pH Oxygen Temperature pH Oxygen Temperature

(mg/L) (°C) (mg/L) (°C)

Control 7.9 8.8 21 7.9 8.1 20

100 7.8 8.3 21 7.9 8.0 20
p. 59

` ~ -

REPORT

Study Title :

TOXICITY OF

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 Seyfried

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

RCC
p. 60

REPORT Page 2

GLP CERTIFICATE

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, MUT

- 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. Zeltner

• 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 - n1 . . . t- -_:__, .__ . :__ - - --. .- -. . .d :-- . P1TU - /b- 6n iu --W-A
• ''K ~ ~Y~~_<~'?_i ~'

: ty • 4w>Cz•

F .,~

.

.

. 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 pre-experiments as mentioned in the repo rt) has been

performed in compliance with the Swiss Ordinance relating to Good Laborato ry Practice,

adopted February 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 Seyfried

• . . : . . . .: .` . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Date: Y .2.2, ~ o oa2.

Management: Dr. Uwe Morgenroth

Date: ~ ~ ' ' •

5 ~
9 -_ K

REPORT Page 5

QUALITY ASSURANCE UNIT

RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland

STATEMEN T

RCC Study Number:

Test Item:

Study Director: Dr. Birgit Seyfried

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 Reports to the

Study Director and to the

Management

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 Gotschi

~• r

~

:

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

Datej 22, 2~2
p. 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 Page7

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 . . . . . . . . 19

~
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

RESPONSIBILITIES

Study Director: Dr. Birgit Seyfried

Deputy Study Director: Dr. Ulrich Memmert

Technical Coordinator: Mrs. Nadja JUstrich

Head of RCC Quality Assurance : Mrs. Iris Wuthrich

SCHEDULE

Experimental Starting Date : June 14, 2002

E;:perimental Completion Date : June 21, 2002

Study Completion Date : July 22, 2002

ARCHIVING

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 GUIDELINES

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 plan
p. 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 PURPOSE

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 METHODS

2.1 DEFINITIONS

Cell density: the number of cells per mL

Growth: the increase of cell density over the test period

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

calculated as the area under the growth curve (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

ErCx: 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 12

2 .2 TEST ITEM

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: Stable

Solubility ih water: Miscible

pH in aqueous solution : 3.9 at concentration of 26%

Aggregate state at room temperature : Aqueous dispersion

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 13

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 surface 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 EVALUATIONS

2.5.1 Counting and Examination of the Algal Cells

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 Criteria

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 TABLES

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

Control 2 5

1 5.5 5.6 23.0 22.3 99.5 98.0

.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

m 5

6 5.9 5.7 23.2 22.3 92.3 92.6

s 0

.68 21.83 85.93

n 6 6 6

.22 2.11 9.43

100 2 3

1 3.6 3.3 17.3 16 .8 80.6 81. 1

.2 3.7 21.4 20.3 89.4 88.7

m 3

3 4.2 4.0 19.6 18 .8 88.6 86.8

.67 19.03 85.87

s 0

n 3 3 3

.38 1.91 4.40

m: mean value; s: standard deviation; n : number of flasks

At the start, 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 medium

Abbrevia tions:

0 : no remarkable observations, clear test medium

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 flask

Nominal test

item Exposure time concentration

(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 18

Table 3 : pH values in the test medium and in the control

Nominal test item pH values

concentration

(mg/L) Start End

Control 7,9 8.8

100 8.0 8.8

Table 4: Water temperature during the test period

Temperature (°C)

Day 0 (Start) 24

Day 1 22

Day 2 22

Day 3 (End) 22
p.77

REPORT Page 19

5 FIGURE

Figure 1 : Growth curves of Scenedesmus subspicatus, incubated for 72 hours

100

/..

E

o

1 0

C .'/ .

~.

E

• . ~

0 24 48 72

test period (hours)

~ Control -~ - 100 mg/L

~3