U.S. ENVIRONMENTAL PROTECTION AGENCY 

## PREMANUFACTURE NOTICE
SUBSTANCES Dale

j

## Company Sanitized

• You mustprovide all infonnation requested in this fonn to the extent that it isknown to orreasonably ascertainablebyyou. Makereasonableestimatesifyou do not have actual data. • Beforeyou complete this fonn, you shouldread the "Instructions Manual for PremanufactureNotification" (the InstructionsManual is available from theToxic Substances Control Act (TSCA) Information Servicebycalling202-554-1404, or faxing 202-554-5603). • Ifa user fee hasbeen remitted for this notice (40 CFR700.45), indicate in the boxes above theTS-userfee identification numberyou have generated. Remember, youruser fee ID number must also appearonyour corresponding feeremittance, which is sent to EPA, HQ AccountingOperationsBranch (PM-226), P.O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSCAUser fee.

Part 1- GENERAL INFORMATION You must provide the currentlycorrectChemical Abstracts (CA)Name ofthe new chemical substance, even ifyou claimthe identityas confidential. You mayauthorize anotherperson to submitchemical identity information for you, but your submissionwill notbe complete and thereview will notbegin until EPA receives this information. A letter in support ofyoursubmission should referenceyourTS user fee identification number. You must submitanoriginal andtwocopies ofthis notice includingall testdata. Ifyou claimed any infonnationas confidential, a single sanitizedcopy must alsohe submitted.

Part 11- HUMAN EXPOSURE AND ENVIRONMENTALRELEASE Ifthere are several manufacture, processing, oruse operations to he descnbed in Part II, sections A and B ofthis notice, reprnduce the sections as needed.

Part 111- LIST OF AITACHMENTS Attachadditional shects ifthere is notenoughspace to answera question fully. Label eachcontinuation sheet withthe corresponding section heading. In Part Ill,list these attachments, anytestdata orotherdata and any optional information iocluded inthe notice.

OPTIONALINFORMATION You may include any infonnation that youwant EPA toconsider in evaluatingthe new substance. Onpage II ofthis form, space has heenprovided for you to descnbed pollutionprevention and recycling information you may have regarding the new substance. So-called "binding"boxes are included throughout this form for you to indicateyour willingness to be bound to certainstatements you make inthis section, such as use, productionvolume, protective equipment.

.. This option is intended to reduce delays that routinely accompany the developmentofconsent orders orSignificantNew Use Rules. Exceptin the case ofexemptionapplications (suchasTMEA, LVE, WREX) where certain infonnationprovided in such notification is bindingonthe submitterwhen the Agency approves the exemption application, checking a bindingbox inthis notice by itselfprohibitthe submitter from laterdeviating from the information (except chemical identity) reported in the form.

CONFIDENTIALITY CLAIMS You may claim any information in this notice as confidential. To assert a claim onthe form, mark (X) the confidentialboxnext to the infonnationthat you claim as confidential. To assert a claim in anattachment, circleorbracket the infonnationyou claim as confidential. ICyou claim infonnation in the notices as confidential. you must also provide a sanitjzed version ofthenotice. fincludine: attacbments). Foradditional instructions onclaiming information as confidential, readthe Instructions Manual. TEST DATA AND OTHER DATA You are required to submit all test datain yourpossession orcontrol and toprovide a description ofall otherdataknown to orreasonably ascertainablebyyou, ifthese dataarerelated tothehealth and environmental effects on the manufacture, processing,distribution in commerce,use, ordisposal ofthe new chemical substance. Standard literature citations maybe submitted for datainthe open scientific literature. Complete testdata (written in En!;dishl. not summariesofdata. must besubmitted ifthey do nO! appear in the open literature. You shouldclearly identifY whethertest datais on the substanceor on an analog. Also, the chemical composition ofthe tested material shouldbe characterized. Following are examplesoftest dataand otherdata. Data shouldbe submitted accordingtothe requirements of§nO.50 ofthePremanufactureNotification Rule (40 CFRPart nO).

Test Data (Check Below any included in this notice) Environmental fate data 0 Yes Otherdata 0 Yes Healtheffects data 0 Yes Risk assessments Environmental effects data 181 Yes Structure/activity relationships PbysicaL'Chemical Properties· 181 Yes Testdata not inthepossession orcontrol ofthe submitter

• A physicaland chemical propertiesworksheet is locatedonthe lastpage ofthis fonn. TYPEOFNOTICE (CheckOnly One)**~** 

# e PMN(Premanufacture Notice) 

### i; 0 :z:: .",::0 INTERMEDIATEPMN(submitted in sequencewith filial produD{;:J.) W --1.{TJ 

### ....,..­ SNUN(SignificantNew UseNotice)**~<** ::":rrJ no 

# o TMEA (Test Marketing ExemptionApplication) 

### .r::­ 

# o LVE (Low Volume Exemption) @4OCFR**723.SO(~** 

# o WREX (Low Release/Low Exposure Exemption) @4OCFR723.50(c)(2) 

# o o LVEModification LOREX Modification
IS THIS A CONSOLIDATED PMN? Yes 

# o # ofchemicals (Prenotice Communication # required, enter# on page 3) Mark (x) ifany infonnation in this notice is claimed as confidential.

EPA FORM 7710-25 (Rev. 5-95) Replaces previous editions ofEPA Fonn 77\0-25. 

# /Publicreportingburdcn for this collection ofinfonnation is estimated to average 110 hoursperresponse, including timefor reviewing instructions, searchingexistingdata sources, gathering and maintaining thedata needed, and completing and reviewingthe collection of infonnation. Send commentsregarding the burden estimateorany other aspect ofthis collection ofinfonnation, including suggestions for reducingthis burden, to Chief, Infonnation Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W., Washington, D.C. 20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION
I certifythat to the best ofmyknowledge and belief: 1. The companynamed inPart I, sectionA, subsection 1 a ofthis notice form intends to manufacture orimport for a commercial purpose, otherthan insmallquantities solelyforresearchand development, the substance identified inPartI, Section B. 2. All informationprovided inthis notice is complete and truthful as ofthe date ofsubmission. 3. I am submitting withthis notice all testdatain mypossession orcontrol and a description ofall otherdataknownto or reasonably ascertainable byme as required by §720.50 ofthe PremanufactureNotification Rule.

AdditionalCertification Statements: Ifyou are submitting a PMN,Intermediate PMN, Consolidated PMN, orSNlIN, checkthe following user fee certification statementthat applies: 

### C8J TheCompanynamed in PartI, SectionA has remitted the fee of$2500 specified in40 CFR700.45(b), or 

## D TheCompanynamed in PartI, SectionA has remitted the fee of$1000 for an IntermediatePMN (defmed @40 CFR 700.43) in accordance with40 CFR700.45(b), or 

## D The Companynamed inPart I Section A is a smallbusiness concernunder40 CFR700.43 andhasremitted a fee of$lOO inaccordance with 40 CFR700.45(b). Ifyou are submitting a lowvolume exemption (LVE) application inaccordance with40 CFR723.50( c)(1) ora Lowrelease andlowexposureexemption (LoRex) application inaccordance with40 CFR723.50(c)(2), checkthe following certification statements: 

## D Themanufacturer submitting this notice intends to manufacture orimport the new chemical substance for commercial purposes, otherthan in small quantities solelyforresearch and development, undertheterms of40 CFR723.50. 

# o Themanufactureris familiar withtheterms ofthis section and will comply with those terms; and 

# oThenewchemical substancefor whichthe notice is submitted meets all applicable exemptionconditions. 

# o Ifthis application is for an LVE in accordance with40 CFR723.50(c)(1), the manufacturerintends to commence manufacture ofthe exempted substance forcommercialpurposes within 1year ofthe date ofthe expirationofthe 30day reviewperiod.
Theaccuracy ofthestatements you makeinthis notice sbould reflectyourbestprediction ofthe anticipatedfacts regardinltthecbemical substance described herein. Anyknowllll! andwillful misinternretatlon Is subfecttocriminalnenalty nursuantto 18 USC ]001. Signature and title ofAuthorized Official (Original Signature Required) Dale Confidential

12 December2002 

## x Signatureofagent
- (ifapplicable) Date

EPAFORM 7710-25 (Rev. 5-95) Page2Confidential la. Person Submitting Notice (in U.S.) Company

City, State, ZIPCode Telephone

Ifyou are submitting this notice aspart ofajoint submission, mark (X)thisbox.

# -----------------------3... Position

Telephone

Position b. Agent(if applicable)

c. Joint Submitter(if applicable)

2. Technical Contact (in U.S.) Mailing address (numberand street)
State, ZIP Code

Nameofauthorizedofficial
Company
Mailing address and street)

Name ofauthorized official
Company
Mailingaddress (numberand street)
City, State, ZIP Code

Name ofauthorized official
Company
Mailing street)
City, State, ZIP Code

3. a prenotice (PC) concerning this notice and EPA assigned a PC Number to the notice, enterthe number.

you**~** 4. Ifyou previously submittedan exemptionapplication substance covered bythis notice, enterthe exemption numberassigned by EPA. Ifyoupreviouslysubmitted a PMN for this substance enter the PMN number assignedby EPA (i.e. withdrawnor incomplete).**~** 5. Ifyou have submitted a notice ofBona fide intent to manufactureorimport for the chemical substance coveredbythis notice. enter the notice number assigned by EPA. .. I. Manufacture 6. Type ofNotice

- Mark(X) Only 

## D Binding Option Mark 2. Import Only 

## D Binding Option Mark
EPA FORM 7710-25 (Rev. 5-95) Page 3 Mark(X)**~** 

## D ifnone
Mark (X)**~** ifnone
Mark(X)**~** 

## D ifnone
3. Both 

## DI. Class 1or2 chemical substances (fordefinitions ofclass I andclass 2 substances, see the Instructions Manual) a, Class ofsubstance·Mark(X) I Class I or 2 

### [8;] Class 2 b, Chemical name (Currently correct Chemical Abstracts (CAl Namethat is consistent withTSCA Inventory listings for similar substances, ForClass I substancesa CA Index Name mustbeprovided, ForClass 2 substances eithera CA Index NameorCAPreferred Name must beprovided, which ever is appropriatebased onCA 9C1 nomenclature rules and conventions).

c, Please identify which method you used to develop orobtainthe specified chemical identity infonnation reported in this notice: (check one).**~** Method I (CAS Inventory Expert Service· a copy oftheIdentification**~** Method2 (OtherSource) report obtained from the CAS Inventory Expert Services mustbe submittedasanattachmentto this Molecular fonnula CAS Registry a number already exists the substance)

CAS#

e, Fora class I providea complete and correctchemical structurediagram, For a class 2 substance·(I)List the immediate precursor substances withtheirrespective CAS Registry Numbers, (2) Describe the nature ofthereaction orprocess, (3) Indicatethe rangeofcomposition and the typical composition (where appropriate), (4) Provide a correct representative or partial chemical structure diae:ram. as comolcteascanbeknown. ifonecan bereasonablv ascertained, 

### 1) 

### Precursor substances:

) Nature ofthe reaction process: 3) Range oftypical composition: 4) 

### Representative structure: substance based on

Ifanotherperson will submitchemical identity infonnationfor you (foreither Item I or2), mark(X)thebox at the right. in a 

## x X



### x


## x
X

attach a continuation sheet.

EPA FORM 7710·25 (Rev, 5·95) Page4Confidential a. Indicatethenumber-average weight ofthe lowest molecularweight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflow molecularweight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecularweight ofthatcomposition. Describe the methods ofmeasurement orthebasis for your estimates: GPC

## D Other 

## D: (Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecularweight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. 

## x

b. You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the "Confidential"box next to any item you claim as confidential

c. (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or otherreactant used inthe manufacture of thepolymer. (2) Mark (X) this column ifentry in column (I) is confidential. (3) Indicatethe typical weight percent ofeach monomer or other reactant inthe polymer. (4) Mark (X) the identitycolumn ifyou want a monomer or other reactant used at two weight percent or less to belisted as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in columns (3) and (4) are confidential. (6) Indicate the maximum weight percent ofeach monomeror other reactant that may be present as aresidual in thepolymer as manufactured for commercialpurposes. Mark this column if in column is confidential. Monomer or other reactant and CAS RegiStry Number Confi- Typical ConfiMethod I (CAS Inventory Expert Service - a copy ofthe identification report obtained from CAS Inventory Expert Service must be submitted as as attachment to this % % % % % % %
reported in this notice (checkone). Method 2 (other source) % % % % % % %

d. The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers.

e. Provide a correct representative or partial chemical structure diagram, as complete as can be known, ifonecan bereasonably ascertained.

N -- Continued EPA FORM 7710-25 (Rev. 5-95) Page 5Section B
. LHI<:MICAL IDENTITY l'Il'uKMATlON
- Continued 3. Impurities (a) 

### - IdentifY each impurity that may bereasonably anticipated to be present in the chemical substance as manufactured for commercialpurpose. Provide the CAS Registry Number ifavailable. Ifthere are unidentified impurities, enter "unidentified." (b)
- Estimate the maximum weight % ofeach impurity. Ifthere are . :ro. estimatetheir total weight %. Impurity and CAS Registry Number Maximum Confipercent dential

(b) % (a) X

% X

%

%

%

%

% 

### [ 

### ] Mark (X) thisbox ifyou attach a I sheet. 4. Synonyms

- Enter any chemical synonyms for the new chemical identified in subsection I or2.

## ~;~~~ X 

## D Mark (X)this box ifyou attach a continuation sheet. 5. Tradeidentification

- List trade names for the new chemical substance identified in subsection I or2. X 

## D Mark (X)this box ifyou attach a continuation sheet. 6. Generic chemical name - Ifyou claim chemical identifY as confidential, you must provide a generic name for your substancethat reveals the specific chemical identityofthe 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. Fatty acid amide



## D Mark (X) this box ifyou attach a continuation sheet. 7. Byproducts

- Describe

.**h~rn..l"~to** resulting from the manufacture, processing, use, ordisposal ofthenew chemical substance. Provide the CAS Registry Number**if~v~;hhlp R~rn..l"~t**  CAS Registry Number Confi­ -J. (I) (2) dential None



### [ ] Mark (X) this box ifyou attach a cont;n"M;nn**~heet.** Part I -- GENERAL INFORMATION -- Continued
EPA FORM 77\0-25 (Rev. 5-95) Page 6Section C

-- PRODUCTION, IMPORT, AND USE INFORMATION: Mark(X) the "Confidential"box next to any item you claim as confidential. 1. Production volume

­ Estimatethe maximum production volumeduring thefirst 12 months ofproduction. Also estimatethe maximum production volumefor any consecutive l2-monthperiod during the first threeyears ofproduction. Estimates should be on 100% new chemical substancebasis. Fora LowVolume Exemption application, ifyou choosetohaveyournoticereviewed at a lowerproduction volume than 10,000kg/yr, specifythe volumeand mark (x) in thebindingbox. Ifgranted, you arebound to this volume Maximum first 12-monthproduction (kg/yr) Maximum 12-monthproduction (kg/yr) Confi- Binding (100% newchemical substancebasis) (100% newchemical substance basis) dential Option Mark (x) X 2. Use Information

-- You must make separateconfidentialityclaims for thedescription ofthecategoryofuse, thepercent ofproduction volume devoted to each category, the formulation of the new substance, and otheruse information. Mark (X) the "Confidential" Box next to anyitem you claim as confidential. a. (1)

-­ Describeeach intended categoryofuse ofthe new chemical substancebyfunction and application

.. (2) Mark(X) this column ifentrycolumn (1) is confidentialbusiness information (CBI). (3) Indicateyour willingness tohavetheinformation provided in column (1) binding. (4)

-­ Estimatethepercent oftotal production for thefirst three years devoted to each categoryofuse. (5)

-­ Mark (X) this column ifentryin column (4) is confidential business information (CBI). (6)

-­ Estimatethepercent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, orgels as manufactured for commercialpurposes at sitesunderyour control associated with each category ofuse. (7)

-­ Mark (X) thiscolumn ifentry in column (6) is confidential business information (CBI). (8)

-­ Indicate% ofproduct volumeexpected for the listed ''use'' sectors. Markmorethan onebox ifappropriate. Mark (X) to indicate your willingness tohavethe use type providedin (8) binding. (9)

-­ Mark (X) this column ifentrv(ies) in column (8) is (are) confidential business information (CBI). Categoryofuse (I) CBI Binding Produc- CBI %in CBI %ofsubstanceexpectedper use CBI Option tion% Form­ (8) (5) (by function and application i.e. adispersive dye Mart (x) ulation Site­ Con-oj< Indus- Com- Binding for**finishin~** polyesterfibers) (2) (3) (4) (6) (7) limited sumer trial mercial Option (9) X X X X % % % % % % % % % % % % % % oj< Ifyou have identified a "consumer" use, please provideon acontinuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumerproducts.

In addition include estimates oftheconcentration ofthe new chemical substance as expected in consumerproducts and describe the chemical reactions by which this substance loses its identity in the consumerproduct. DMark (X)this box ifyou attach a continuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2a as confidential, entera generic description ofthat category. Read the use InstructionsManual forexamples ofgeneric use descriptions. description Lubricant additive.



# o Mark (X)this box ifyou attach a continuation sheet. 3. Hazard Information

- Include in the notice a copy ofreasonable facsimile ofany hazard warning statement, label, material safety data sheet, orother Binding

information which will beprovidedto any person who is reasonably likely to be exposed to this substance regarding protective equipmentorpractices Option for the safehanding, transport, use, ordisposal ofthe new substance. List in part IIIhazard information you include. Mark(x) 

## r8J Mark (X)this box ifyou attachhazard information. 

### Part 11-- HUMAN EXPOSUREAND ENVIRONMENTAL RELEASE
EPA FORM 7710-25 (Rev. 5-95) Page 7

# x · Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X)the "Confidential"box next to any item you claim as confidential

sites as attachments.
a continuation sheet.
Use Batches/year l. Batch Maximumkglbatch (100"10 new chemic.l Hourslbatch Batches/year subs/once) 2. Continuous d. Process description Mark (X) to indicateyour willingness to haveyour processdescription binding. (1) Diagram themajor unit operation steps andchemical conversions. Include interim storageandtransportcontainers (specifY- e.g. 5 gallon pails, 55 gallon drum, railcar,tanktruck, etc.). (2) Providethc identity,the approximate weight (bykgiday or kgibatch on a 100%new chemical substancebasis), andentrypoint ofall starting materialsand feedstocks (including reactants, solvents, catalysts, etc.), and ofallproducts, recycle streams, and wastes. Includecleaningchemicals (notefrequency ifnot useddaily orperbatch.). (3) IdentifY bynumberthepoints ofrelease, including small or intermittentreleases, to the environmentofthenew chemical substance. Completesection A for each type ofmanufacture, processing, oruse operation involving the new chemical substance at industrial sitesyou controL Importers do nothave to completethis section for operations outside the U.S.; however, you may still havereporting requirements if there arefurther industrial oruse You these See 1. Confiofthe site at which the wi11 occur. dential

Siteaddress (numberand street)
City, County, State,ZIP code
Ifthe same operation will occuratmorethan one site, enterthe numberofsites. IdentifY the additional sites on a continuation sheet, and ifany ofthe sites have significantly different production rates oroperations, include all the information requested in this section for those

Marl<: (X)this box attach a continuation sheet. Part 11-- HUMAN EXPOSUREAND ENVIRONMENTAL RELEASE -- Continued
EPA FORM 7710-25 (Rev. 5-95) Page 8

## I Section A - INDUSTRIAL SITES CONTROLLEDBY THE SUBMITTER

- Continued 2. Occupational Exposure

­ You must make separateconfidentialityclaimsfor thedescription ofworker activity, physical form ofthenew chemical substance, number ofworks exposed, and duration ofactivity. Mark(X) the "Confidential"box nextto any item you claim as confidential. (1)

-- Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling,cleaning, etc.) in which workers may be exposed to the substance. (2)

-- Mark (X) this column ifentry in column (I) is confidential business information (CBI). (3)

-- Describe any protective equipment and engineering controls used to protect workers. (4) and (6) -Indicateyour willingness to have the information provided in column (3) or (5)binding. (5) --Indicatethe physical formes) ofthe new chemical substance (e.g., solid: crystal, granule, powder, or dust)and % new chemical substance(ifpartofamixture) at the time ofexposure. (7)

-- Mark (X)this column ifentry in column (5) is confidential business information (CBI). (8)

-- Estimatethemaximumnumberofworkers involved in each activity for all sites combined. (9)

-- Mark (X) this column ifentry in column (8) is confidential business information (CBI). (10) and (II)

-- Estimate the maximumduration ofthe activity for any worker in hoursperday and days peryear. (12)

-- Mark (X) this column ifentries in columns (10) and (11) are confidential business information CBI). Worker activity CBI Protective Equipment! Binding Physical forms(s) Binding CBI #of CBI duration CBI m (i.e., bagdumping, filling drums) Engineering Controls Option and % new Option Worker Hrs/day Oays/yr Mark (x) substance Mark (x) S (I) (2) (3) (4) (5) (6) (7) Expose (9) (10) (II) (12) d (8)



## D Mark (X) this box ifyou attach a continuation sheet. 3. Environmental Release and Disposal -- You must make separateconfidentialityclaimsfor thereleasenumberand the amount ofthenew chemical substance released and otherrelease and disposal information. Mark (X) the "Confidential"box nextto each itemyou claim as confidential. (I)

-- Enterthe numberofeach releasepointidentified in the processdescription, part II, section A, subsection Id(3). (2)

-- Estimatethe amount ofthenew substance released (a)directly to theenvironment or (b) into control technology (in kg/day orkglbatch). (3)

-- Mark (X) this column ifentries in columns (I) and (2) are confidential business information (CBI). (4) --Identify the media (stack air, fugitive air(optional-see Instruction Manual), surfacewater, on-siteoroff-siteland orincineration, POTW, orother(specify»to which the new substancewill bereleased from thatreleasepoint. (5)

-- a. Describe control technology, ifany, and control efficiency that will beused to limit therelease ofthenew substance tothe enviromnent. Forreleases disposed ofon land, characterizethedisposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for each sitedescribeany additional disposal methods that will be used and whether the waste is subjectto secondaryortertiary on-sitetreatment. b. Estimate the amountreleased to the environment after control technology (in kg/day). (6)

-- Mark (X) this column ifentries in columns (4)and (5) areconfidential business information (CBI). (7) --Identify thedestination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System)numbers for directdischarges or NPDES numbers ofthe POTW (Publici Owned Treatment Works). Mark (X) ifthe POTW name orNPDES # is confidential business information (CBI). Release Amount ofnew substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CBI Number released release

(I) (2a) (2b) e,g.!I1ack air Binding (3) (4) (5a) Mark (X) (5b) (6)

(7)Mark (X)the 

## D POTWprovidename(s) below: CBI 

## D Navigable 

## D Other

- Specify provideNPDES # CBI I--­ destination(s) of waterway releases to water. 

## D Mark (X) this box ifyou attach a continuation sheet.

EPA FOJU.1 7710-25 (Rev. 5-95) Page 9Part11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued SectionB -- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section B for typical processingoruse operations involvingthe newchemical substanceat sites you do not control. Importers do not have to complete this section for operations outside theU.S.; however, you mustreport anyprocessing oruseactivities after import. Seethe Instructions Manual. Complete aseparate section B foreach type of processing, oruse operation involving the newchemicalsubstance. Ifthesameoperation is performed at morethan one sitedescribe the typical operation common tothese sites. Identify additional sites on a continuation sheet. 1. Operation Description To claim information in this section as confidential, circle orbracket the specific information thatyou claim as confidential. (I)

-­ Diagramthe majoronit operation steps and chemical conversions, including interim storage and transport containers (specifY

- e.g. 5 gallon pails. 55 gallon drums, rail cars, tank trucks, etc). On thediagram. identifY by letter and briefly describe each worker activity. (2)

-- Providethe identity, the approximateweight (by kg/day orkglbatch, on an 100% new chemical substance basis). and entl)'point ofall feedstocks (includingreactants. solvents andcatalysts, etc) and all products, recycle streams, and wastes. Include cleaningchemicals (note frequency ifnot used daily orperbatch). (3)

-- IdentifY by numberthepoints ofrelease, ineluding small or intermittent releases, to the environment ofthenew chemical substance. (4) Please enterthe# ofsites (rememberto identi/Y the locationsof these sites on a continuation sheet): ca. 10 # ofsiles



# o Mark (X) thisbox ifyou attach a continuation sheet. 2. Worker Exposure/Environmental Release (I)

--From the diagram above. provide the letter for each worker activity. Complete2-8 for each worker activity described. (2)

-- Estimate thenumberofworkers exposed for all sites combined. (4)

-- Estimatethe typical duration ofexposure per worker in (a) hours per day and (b)daysper year. (6)

-- Describephysical form ofexposure and % new chemical substance (ifin mixture). and any protective equipmentandengineering controls, ifany. used to protectworkers. (7)

-- Estimatethepercentofthenew substanceas formulated when packaged orused as a fmal product. (9)

- From theprocess diagram above, enterthenumberofeach release point. Complete 9-13 for each releasepoint identified. (10) Estimatetheamount ofthe new substancereleased (a) directly to the environment or(b) into control technology to the environment (in kg/day orkglbatch). (12)

-- Describemedia ofrelease i.e. stack air, fugitive air(optional-see Instructions Manual), surface water, on-site oroff-site land orincineration, POTW, or other (specify) andcontrol technology, ifany, that will be used to limit therelease ofthenew substance to the environment. (14)

-­ Identi/ybyproducts which may result from theoperation.

(3), Mark(X) this column ifany oftheproceedingentries are confidential business information (CBO. Letter #of Duration CBI ProtectiveEquip. / %in CBl Release Amountof CBI Media ofRelease CBl

of WOlkers of Engineering Controls! Fonn- Number New & Control Act- Exposed Exposure Physical Form and % ulation Substance Technology ivity new substance Released (1) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (lOa) (lOb) (11) (12) (13) A X < X X X X 

# Y 14)

-- Byproducts: 

# o Mark (X) thisbox ifyou attach a continuation sheet, EPA FORM 7710-25 (Rev. 5-95) Page 10

## OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle orbracket the specific information that you claim as confidential. In

### this section you may provide information notreported elsewhere in this form regarding your efforts to reduce orminimizepotential risks associated with activities surrounding manufacturing, processing, use anddisposal ofthe PMN substance. Pleaseinclude new information pertinent to pollutionprevention, including source reduction, recycling activities and saferprocesses orproducts available due to the new chemical substance. Sourcereduction includes the reduction in the amount ortoxicity ofchemical wastes bytechnological modification, process and procedure modification, productreformulation, raw materials substitution, and/or inventory control. Recyclingrefers to the reclamation ofuseful chemical components from wastes that would otherwisebe treated orreleased as air emissions orwaterdischarges, orland disposal. Descriptions ofpollution prevention, sourcereduction and recycling should emphasizepotential risk reduction subsequent to compliance with existingregulatory requirements and canbeeitherquantitative orqualitative. The EPA is interested in the information to assess overall net reductions intoxicityorenvironmental releases and exposures, not the shifting ofrisks to otherenvironmental media ornon-environmental areas (e.g., occupational orconsumerexposure). In addition, information onthe relative cost orperformance characteristics ofthe PMN substanceto potential alternatives maybeprovided. 

### All information provided in

### this section will betakenintoconsideration duringthe review ofthis substance. See InstructionsManual and Pollution Prevention Guidancemanualfor guidance and examples. Describe the expected netbenefits, such as (1) an overall reduction inriskto humanhealthorthe environment; (2) a reduction in the volume manufactured; (3) a reduction in the generation ofwaste materials throughrecycling, source reduction orothermeans; (4) a reduction inpotential toxicity orhumanexposure and/orenvironmental release; (5) anincrease inproductperformance, a decrease in the costofproduction and/or improved operationefficiency ofthe new chemical substance in comparison to existing chemical substances used in similarapplication; or (6) the extentto which the new chemical substance maybe a substitute for an existing substance thatposes a greater overall riskto human health orthe environment.

] Mark(X) this box ifyou attach a continuation sheet.
EPA FOR.rvI 77\0-25 (Rev. 5-95) Page II

### Part III -- LIST OF ATTACHMENTS
Attach continuation sheets for sectionsofthe tonn and test dataand otherdata(includingphysical/chemical properties and structure/activityinfonnation), andoptional infonnation afterthis page. Clearly identify the attachment and the section ofthe fonn to which it relates, ifappropriate. Numberconsecutively the pages ofthe al:tachments. In the column below, enterthe inclusive page numbers ofeach attachment. Mark(X) the "Confidential" box next to any attachment name you claim as confidential. Readthe Instructions Manual tor guidance on how toclaim any infonnation in an attachment as confidential. You must include withthe saniti:liXI copy ofthe notice fonn a sanitized version ofany attachment in which you claim infonnation as confidential. Attachment name Attachment DaI!.Cnumber(s) 

### 14 Material Safetv Data Sheet (MSDS) Physical Chemical PropertiesWorksheet 

### 13 Confidential

X 

### 21 X 

### 24 X 44 X 

### 62 X CAS Inventory Expert Service Reports 

### 82 X

### ~hiS box ifyou attach a continuation sheet. Enterthe attachment name and number.

EPAFORM 7710-25 (Rev. 5-95) Page 12PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical andchemical properties data, pleasecomplete the following worksheet for data you provideand include itin the notice. IdentifY theproperty measured, the pageofthe notice on which theproperty appears, the value oftheproperty, theunits in which theproperty is measured (as necessary), and whetherornot theproperty is claimed as confidential. Thephysical stateofthe neat substance should beprovided. These measured properties should befor the neat (100%pure) chemical substance. Properties that aremeasured for mixtures orformulations should beso noted (% PMN substance in _). You arenot required to submitthis worksheet; however, EPA strongly recommends that you do so, as it will simplifyreview and ensure thatconfidential information is properly protected. You should submitthis worksheet as a supplementto yoursubmission oftest data. This worksheet is not a substitute for submission oftest data.

# ~ Property Value lMeasuredo Confidentml (a) X Physical state ofneat substance X Vaporpressure @ Temperature 20 °C X Density/relative density Solubility @ Temperature °C

Solvent Solubilityinwater Temperature °C Melting temperature Boiling!sublimation temperature{aJ torr Spectra Dissociation constant Particle size distribution X Octanol! waterpartition coefficient Henry's Law constant Volitalization from water Volitalization from soil pH@ concentration Flammability 

# I Explodability Adsorption/ coefficient Other

- Specify (c) 13 (s) (l) (g) 13 <0.1 hPA Torr 13 860 k!!lm3 (25° C)

giL 13 Insoluble giL 48°C >200 °C

13 >3 Estimate Mark (X) (MorE) (d)

E X

M

E E**~**

# R

ATTACHMENT PAGE 13----------



### ------------------------------------------------------------------------------**-------------------------~~!~gI~~=~~~~!i=~~!~=~R~~!---------p-~ge------l-of--7** Revised Replaces As of MSDS ID:

Material Name:

Ingredients OSHA PEL Fatty acid amide Not listed Ingred ents not precisely identified are propr etary or nonhazardous. Values are not product specifications.
3 HAZARDS IDEBTIFICATIOB General: No toxicity information is available on this specific preparation; this health hazard assessment is based on information that is available on the properties of its components. Emergency Overview: Appearance: Cream colored solid, faint odor. MAY CAUSE EYE IRRITATION. INHALATION OR INGESTION MAY CAUSE MUCOUS MEMBRANE IRRITATION. MAY BE HARMFUL IF SWALLOWED. ALL INGREDIENTS NOT CONFIRMED ON EPA TSCA INVENTORY. FOR TSCA-EXEMPT PURPOSES ONLY. Routes of exposure: Eye contactj Skin contact; Inhalation 

# ILL7 Page 2 of Revised Replaces As of

Symptoms of Overexposure (Acute & Chronic) : Eye contact: This material may irritate human eyes following contact. Skin contact: No irritation is likely to develop following contact with human skin. This product will probably not be absorbed through human skin. Inhalation: High concentrations of dusts or particulates may irritate respiratory passages. No toxic effects are known to be associated with inhalation of this material. Ingestion: In humans, irritation of the mouth, pharynx, esophagus and stomach can develop following ingestion of this product. This material may be harmful if swallowed.

### ----------------------------~-~~~~~-~~~-~~~~~~~----------------------------- Eyes:Immediately flush with plenty of water. After initial flushing, remove any contact lenses and continue flushing for at least 15 minutes. Have eyes examined and treated by medical personnel. Skin:Wash material off of the skin with plenty of soap and water. If redness, itching, or a burning sensation develops, get medical attention. Ingestion: DO NOT INDUCE VOMITING. Give one or two glasses of water to drink and refer to medical personnel or take direction from either a physician or a poison control center. Never give anything by mouth to an unconscious person. Inhalation: Remove victim to fresh air. If a cough or other respiratory symptoms develop, consult medical personnel.



# (r

### ------------



### __________________________ ____________________________

_______________________ __________________________

___________________________ _____________________________ 7 Page 3 of Revised Replaces As of MSDS ID:**~_~l~~_~l~~~l~~_~~~~~~~~** Flashpoint and method: 401 deg.F, 205 deg.C Autoignition temperature: No data. Flammable limits (LEL!UEL): No data. Extinguishing media: Water fog, alcohol foam, carbon dioxide, or dry chemical agents. Special fire-fighting protective equipment: Self-contained breathing apparatus with full facepiece and protective clothing. Unusual fire and explosion hazards: None known. Explosion data: Sensitivity to mechanical impact: Not applicable Sensitivity to static discharge: No data**~_~~~l~~~~~~_~~~~~~~_~~~~~~~** Refer to Section 8 for proper personal protective equipment. Steps to be taken in case material is released or spilled: Sweep up and recover or mix material with a moist absorbent and shovel into a chemical waste container. Wash residue from spill area with water containing detergent and flush to a sewer serviced by a permitted wastewater treatment facility.**~_~~~~~l~~_~~_~~~~~~~** Storage requirements: 50-90 deg F in original closed container. Precautions to take during handling and storage: Prevent eye contact. Avoid breathing this material. Do not swallow. Normal chemical handling and storage.-------------

------------------------------------------------------------------------------ 7 Page 4 of Revised Replaces As of MSDS ID:
..**------------------------------------------------------ -------------~---------** 



### __________________**~_~~~~~~~~_~~~~~~~~L~!~~~~~~_~~~!!~! ~~~**  

### _____________________ Exposure limits: No ACGIH TLV or OSHA PEL assigned. Minimize exposure in accordance with good hygiene practice. Engineering controls: Use ventilation adequate to maintain safe levels. Eye protection: Chemical tight goggles. Protective clothing: Impervious gloves. Respiratory protection: Not normally needed if controls are adequate. Other protective equipment: Eyewash station and safety shower in work area.
9 PHYSICAL AND CHEMICAL PROPERTIES Appearance & odor: Cream colored solid, faint odor. Odor threshold: No data Boiling point: >392 deg. F, 200 deg. C Melting point: No data Vapor pressure (mm Hg at 20 deg. C) <0.1 hPa (20 deg. C) Vapor density (air = 1): No data. Solubility in water: Insoluble pH: No data. Specific gravity: No data % Volatile by volume: No data. Cloud point: No data Viscosity: No data Partition Coefficient: No data Coefficient of water/oil distribution: No data Freezing point: No data**.--------------------------------~-------------------- ------------------------** **~~!~~!~~_£~K~!X_Q~!~_~~~~!** Page Revised 5 of 7 Replaces As of MSDS ID: --------......-­**------------------------~~-~~~~~~~~~~~~-~~~~~~~~~~--------------------------** Stability: Stable under normal conditions. Incompatibility: Oxidizing agents. Conditions to avoid: None known. Hazardous decomposition products: Combustion products: Carbon dioxide, carbon monoxide, nitrogen oxides. Hazardous polymerization: Will not occur.

No data available on this material. Carcinogenicity: This material is not listed as a carcinogen by OSHA, IARC, and NTP. Reproductive toxicity/Teratogenicity: No available information. Mutagenicity: No available information. TOxicologically synergistic products: None known.

No data is available on this product.

Disposal method: Discarded product is not a hazardous waste under RCRA, 40 CFR 261. Container disposal: Empty container retains product residue. Observe all hazard precautions. Do not distribute, make available, furnish or reuse empty container except for storage and shipment of original product. Remove all product------------------------------------------------------------------------------

------------------------------------------------------------------------------ 7 Page 6 of Revised Replaces As of MSDS ID:**-------~-----------------------------------------------------------~---------**



### _____________________**!!_~~~~Q~~~_~Q~~!~~~~!~~~_i~~~~~t** 

### _______________________ residue from container and puncture or otherwise destroy empty container before disposal.
14 TRANSPORT IRFOROOATliOg Department of Transportation:

Not regulated. TDG (Transportation of Dangerous Goods)

Not regulated.
1S REGULATORY IRFORRATliOB TSCA (Toxic Substances Control Act) Regulations, 40 CFR 710: All ingredients NOT confirmed on the TSCA Chemical Substance Inventory. FOR TSCA EXEMPT PURPOSES ONLYl DSL (Canadian Domestic Substances List) All ingredients NOT confirmed on the Canadian DSL. OSHA Hazard Communication Standard, 29 CFR 1910.1200, Hazard Summary: Physical Hazards: None. Health Hazards: Irritant (eye) WHMIS Classification: Class D, Division 2 - Toxic. CERCLA and SARA Regulations (40 CFR 355, 370, and 372): This material contains the following chemicals subject to the reporting requirements of SARA 313:   NO SARA 313 CHEMICALS FOR THIS CHEMICAL ** ** SARA 311/312 Hazard Categories: Immediate: YES Delayed: NO Fire: NO Pressure: NO Reactivity: NO State Regulations: None. 

# \9------------------------------------------------------------------------------ 7 Page 7 of Revised Replaces As of

### ~§Q§-!Q~--~ - ----------------------------------------------------------------­



## _____________________**~~_~~~~&~~~~~_~~~~~~~~~~~_i~~~t~L** 

## ________________________**----------------------------~~-~~~~~-~~~~~~~~~-----------------------------** We assigned NFPA ratings and HMIS ratings to this product based on the hazards of its ingredient(s). Because the customer is most aware of the application of the product, he must ensure that the proper personal protective equipment (PPE) is provided consistent with information contained in the product MSDS.* HMIS Information Health 1 Flammability 1 Reactivity 0 NFPA Information Health 1 Flammability 1 Reactivity 0 *This information is intended solely for the use of individuals trained in the particular hazard rating system. This Material Safety Data Sheet was prepared by , Date:
The information herein is given in good faith but no warranty, expressed or implied, is made.



# Z(J**·~** . ' 

# Ecotoxicology StudyReport

.E<;gtoxicolol!v Section 

# 2/

### 2.

<' 

### First Compiled by: 

### Date:
Test Facility

Staff Involved 

### Study Director Deputy Study Director Test Operators
Analytical Supervisor Test Operators Test Starting Date: Test Completion Date: 

### This report contains the summary data relevant to the study but does not include all of the raw data**record~d** 

### during t.he study. The raw data are located in Ecotoxicology Archive file Distribution

### 3.
Contents

Summary 

### 1. Introduction 

### 1.1 Objective 

### 1.2 Description of Test Substance


## 2. Methodology 

### 2.1 Justification of Test System 

### 2.2 Test System 

### 2.3 Basis of the Method 

### 2.4 Preparation of Stock and Test Solutions 

## 3. Results 

### 3.1 Test Conditions 

### 3.2 Test Substance Analysis 

### 3.3 Effect Data


### 4. Conclusions


## 5. References Appendix 1 Test Conditions Appendix 2 Results of Test Substance Analysis Appendix 3 fish wet weights Appendix 4 Test Substance Characterisation Appendix 5 Analytical Method for the Extraction and Gas Chromatography Analysis of Quality Assurance and 

### GlP Compliance Statements 4 

### 5 5 5
5 5 5 5 6 6 6 6 7


### 8
8 

### 10 14 15 

### 16
17 

### 20

### 4. Summary 

### The chronic toxicity of

. to the fresh-water fish Brachydanio rerio 

### (zebra fish) was determined. The response measured was the change in the mean weight of the fish exposed to the test-substance after 14 and 28 days. The method was adapted from the growth-rate test for Oncorhynchus mykiss ( 

### Crossland, N. 0., 1986 [1] and 1 

### [2] ). The test was conducted under flow-through conditions. Analysis indicated that the concentrations of the test solutions were on average '% 

### and

.

### '% of nominal (range 56
- 160 

### %). Effect concentrations are based on mean measured values. There were no inhibitory effects of on the exposed zebra fish at the highest mean measured concentration tested, 

### 1-'9/1. The NOEC could not be determined.

### 5. 1. Introduction 

### 1 .1 Objective To determine the effect of on the growth of juvenile zebra fish (Brachydanio rerio) 

### under flow-through conditions. 1.2 Description of Test**Sub!=:t~n("Q** 

### ; (also known as is an, manufactured

was tested as 

### % commercial product. Further details are given in Appendix 4. 

### 2. Methodology 

### 2.1 Justification of test system Zebra fish (Brachydanio rerio) 

### is a pre-eminent species used in toxicity tests in which it has been shown to be sensitive to toxicants. It is recommended for use in acute toxicity tests by OECD [3] and EEC [4]. 2.2 Test System The parent fish (batch 9Z) spawned on 9f7193 

### and eggs hatched on 13f7193 

### (batch 15Z). The juvenile fish were reared in carbon-filtered tap water and fed on proprietary fish food [5] and Artemia. 

### There were no significant mortalities of the test fish in the week prior to the test. 2.3 Basis of the Method The method is adapted from the growth-rate test for rainbow trout developed by Crossland [1]. In this method the sublethal toxicity of, \to zebra fish was determined.The response measured was the change in the mean weight of the fish exposed to the test substance after 14 and 28 days. [12] The method was a flow-through procedure with approximately 10 volume replacements per day. Continuously flowing test solutions were generated from a constant-head tank containing dilution water and from a series of stocks which were dosed into the test vessels via a peristaltic pump. The water flow-rate was regulated by glass capillaries.

### 6. 2.4 Preparation of Stock and Test Solutions 0.25 g of t"' 

### (N25) was dissolved in 50 ml of methanol using sonication to give as g/I stock (stock A)[6J. 16.7 ml of stock A was diluted 1 in 3 with methanol to produce a 1.67 mg/ml solution (stock B). Two dosing stocks (5 litres of each at 4.2-1.4 mg/I) were prepared by diluting the 5 mg/ml and 1.67 mg/ml stocks with distilled water. Dosing stocks were diluted 1 in 42 to produce nominal concentrations of 33 and 100l-lg/1. A control and a solvent control (containing 0.02 ml methanol/I) were also prepared. Each test solution was dosed at 67 ml/min giving about 10 volume replacements per day. The total flow rates (water + stock) and dosing stock flow rates were determined daily. The pH, dissolved oxygen concentration and temperature were determined daily (except for weekends) in all test concentrations [7,8]. Total hardness was determined daily in the control vessel (except for weekends) [9J. Twenty four hours prior to the start of the test all the fish were starved. The fish were transferred randomly into the test solutions. 16 fish were exposed to each test concentration. The test vessels were placed in the test area according to a randomised distribution. Test vessels were 12 litre all-glass tanks containing 10 litres of test solution with no replicates. Test vessels were aerated during the test. The light intensity was not measured. Normal room lighting was used. The photoperiod was 16 hours light and 8 hours dark. 

### 3. Results 3.1 Test Conditions The experimental means of the water quality are given in Table 1. Table 1 Test Conditions Mean Std dev. Range

Temperature (OC) 23.1 0.2 22.5-23.5 pH 7.5 0.2 7.1-7.8 Dissolved oxygen (mg/I) 8.5 0.5 7.7-10.2 Total hardness (as mg/I CaC03) 

### 100 

### 6 86-108 The pH, DO, total hardness and temperature remained within acceptable limits. 3.2 Test Substance Analysis Determination of the test substance concentrations in the test medium was carried out using GC analysis [10J according to the method described in Appendix 5.

### 7. Analysis indicated that the concentrations of test solutions were on average 

### 96% and 

### 105% of nominal (range 

### 56

- 160 %). Effect concentrations are based on mean measured values. On day 3 a blockage occurred in the capillary dosing into the 

### 100 I1gl1 test vessel. This stopped the dilution water-flow (but allowed some dosing stock through) raising the concentration to 

### 218 

### 11911. This result was not incorporated in the mean concentration overthe test period as it was not representative. The blockage also caused the control vessel to become contaminated 

### (78 I1g/l) as the control and 

### 100 

### 11911 test vessels were supplied with dilution water via the same T-piece. By day 4 concentrations in the control tank had fallen to 

### 0.1 

### 11911. The short exposure to the test substance by the control fish is not thought to have affected the test outcome. The results of the analysis are given in Appendix 2. 3.3 Effect Data The fish mean weights and standard deviations over relevant periods of the test are given in Table 2. The complete data set can be found in Appendix 3 Table 2 Mean fish weights (mg) Treatment Day Control Solvent control 

### 0 87.3 (12.4) 87.5 (10.8) 88.1 (10.3) 88.3 (12.9) 14 106.7 (12.3) 106.0 (11.2) 106.9 (10.5) 107.3 (15.4) 28 129.9 (14.2) 128.8 (15.0) 127.9 (12.7) 131.0 (17.6)**~tanc** 

### ard deViations 

### gIven In 

### oracKets The mean weights were analysed using analysis of variance followed by Dunnett's test 

### [11] to identify any concentrations causing significant inhibitory effects in the zebra fish. None of the analyses indicated a significant ( p > 

### 0.05 ) inhibition in growth. Specific growth rates calculated using mean wet weights are given in table 

### 3. Table 3 Specific growth rates

Growth rate Control Solvent control 

### 0-14 days 

### 1.43 1.37 ·1.38 1.39 0-28 days 

### 1.42 1.38 1.33 1.41 14-28 days 

### 1.41 1.39 1.28 1.43 

# 27

### 8. Analysis of variance and Dunnett's test cannot be applied as only the mean value for each test vessel is available. However the similarity of the r-values suggest no effect at either test concentration. 4. Conclusions There was no effect on the average fish weight or specific growth rates at any of the concentrations tested. The NOEC of to zebra fish ( Brachydanio rerio ) _ 

### determined. There was no inhibitory effect at the highest test concentration of 

### j.1gll. 5. References Note: All SOPs referenced below are Ecotoxicology SOPs. [1] Crossland, N. 

### 0., (1986). A method to evaluate effects of toxic chemicals on fish growth. Chemosphere, 14, 

### (11/12), 1855. {2] ISOIDIS 10229-1. Determination of the Prolonged Toxicity of Substances for Freshwater Fish. Part 1: Methods for the Evaluation of the Effects of Substances on the Growth Rate of Oncorhynchus mykiss Walbaum 

### (Teleostei, Salmonidae). {3] OECD Guidelines for Testing of Chemicals. Section 2. Guideline 203 "Fish­ Acute Toxicity Test" (1984). {4] EEC directive 84/449/EEC 

### 1984. Part C: Methods for the determination of ecotoxicity; C.1 Acute toxicity for fish. [5] SOP 159 01 The Culture of Warm-water Fish: Fathead Minnow (Pimephales promelas) 

### and Zebra Fish (Brachydanio rerio) 

### [6] SOP 128 02 The Use, Maintenance and Calibration of the Mettler AE200 with Printer [7] SOP 038 03 Use of pH Meters: (PW 9418131)
- Philips (290 MK.2)
- Pye Unicam. [8] SOP 040 01 Use of YSI Model 57 Oxygen Meter. [9] SOP 057 03 Determination of Total Hardness by a Titrimetric Method. [10] SOP 177 01 Use of the Perkin Elmer Model 8700 Gas Chromatograph.

### 9. 

### [11] Dunnett, C. W., (1955). A multiple comparison procedure for comparing several treatments with a control. 

### J. Amer. Statist. Assoc., 50, 1096-1112. [12] SOP 047 03 The Use, Maintainence and Calibration of the Mettler HK160 with printer.

### 10. Appendix 1 Test Conditions pH Day Vessel 

### 1 2 3 4 0 7.6 7.6 7.6 7.5 3 7.4 7.4 7.3 7.3 4 7.6 7.6 7.6 7.6 5 7.7 7.6 7.7 7.6 6 7.6 7.6 7.6 7.6 7 7.5 7.7 7.7 7.7 10 7.3 7.3 7.1 7.3 11 7.2 7.1 7.2 7.1 12 7.4 7.4 7.3 7.3 13 7.3 7.2 7.3 7.3 14 7.2 7.2 7.2 7.2 17 7.4 7.5 7.5 7.5 18 7.3 7.5 7.5 7.5 19 7.5 7.4 7.4 7.5 20 7.5 7.5 7.5 7.5 21 7.5 7.5 7.5 7.6 24 7.5 7.5 7.5 7.5 25 7.6 7.6 7.5 7.5 26 7.5 7.4 7.4 7.4 27 7.5 7.6 7.4 7.5 28 7.8 7.7 7.7 7.7

### 11. Dissolved Oxygen (mg/I) Day Vessel 

### 1 2 3 4 0 8.2 8.1 8.2 8.1 3 8.3 8.3 8.3 8.3 4 8.4 8.1 8.2 8.2 5 8.7 8.4 8.5 8.5 6 8.7 8.6 8.5 8.6 7 7.7 7.8 7.8 7.7 10 8.6 8.3 8.3 8.3 11 8.7 8.9 8.9 8.9 12 8.4 8.3 8.3 8.4 13 8.2 8.2 8.0 8.2 14 8.9 8.7 8.7 8.9 17 8.5 8.5 9.0 9.0 18 9.0 9.0 9.0 9.0 19 8.9 8.8 8.8 8.9 20 8.8 8.6 8.4 8.4 21 8.8 8.6 8.7 8.6 24 8.4 8.3 8.3 8.3 25 8.5 8.2 8.3 8.3 26 8.3 8.1 8.1 8.1 27 10.2 10.0 10.1 10.1 28 8.7 8.6 8.5 8.6



# 3/

### 12.
Total Hardness (as mgtl CaC03) 

### Day Vessel 

### 1 0 100 3 104 4 103 5 104 6 100 7 108 10 102 11 90 12 89 13 90 14 86 17 107 18 100 19 103 20 108 21 99 24 104 25 103 26 104 27 98 28 104

### Temperature eC) Day Vessel 1 2 3 4 0 23.5 23.5 23.5 23.5 1 22.5 23.0 23.0 23.0 2 23.0 23.0 23.0 23.0 3 23.0 23.5 23.0 23.0 4 23.0 23.5 23.0 23.0 5 23.0 23.0 23.0 23.0 6 23.0 23.5 23.0 23.5 7 23.0 23.5 23.0 23.0 8 23.0 23.5 23.0 23.0 9 23.0 23.0 23.0 23.0 10 23.0 23.0 23.0 23.0 11 23.0 23.0 23.0 23.0 12 23.0 23.0 23.0 23.0 13 23.0 23.0 23.0 23.0 14 23.0 23.0 23.0 23.0 15 23.0 23.0 23.0 23.0 16 23.0 23.0 23.0 23.0 17 23.0 23.0 23.5 23.0 18 23.0 23.5 23.0 23.5 19 23.0 23.0 23.0 23.0 20 23.0 23.0 23.0 23.0 21 23.0 23.0 23.0 23.0 22 23.0 23.0 23.0 23.0 23 23.0 23.0 23.0 23.0 24 23.0 23.0 23.0 23.0 25 23.0 23.0 23.0 23.0 26 23.0 23.0 23.0 23.0 27 23.0 23.0 23.0 23.0 28 23.0 23.0 23.0 23.0 (all temperatures quoted in the above table were rounded to the nearest 0.5 °C)

### Appendix 2 Result of Test Substance Analysis Day Nominal concentration 1-1911 control 0 solvent control 0 -1 

# - 1.0 35.2 94.0 0 1.5 < 0.1 42.0 89.0 3 78.1* 1.7 27.4 218* 4 < 0.1 < 0.1 25.1 84.2 11 < 0.1 < 0.1 32.6 123.2 13 2.7 0.0 34.5 112.0 18 < 0.1 0.0 18.6 81.6 20 1.3 2.8 43.5 86.5 25 < 0.1 < 0.1 27.6 118.0 27 

# - 

# - - 159.4 mean < 1.0 < 1.0 31.8±8.0

. 105.3±25.5 * see comment In 

### section 3.2

### Appendix 3 

### Zebra fish wet weights (mg)

Fish Vessell Vessel 2 Vessel 3 Vessel 4 no. Day Day Day Day Day Day Day Day Day Day Day Day 0 14 28 0 14 28 0 14 28 0 14 28 1 73 124 111 106 93 133 87 105 136 100 93 132 2 73 97 122 85 102 137 102 108 123 111 89 138 3 107 118 146 91 91 123 102 99 131 96 109 151 4 83 95 118 103 98 114 80 96 123 112 121 108 5 96 97 120 71 104 134 79 109 132 81 97 143 6 84 103 125 93 110 120 83 87 134 78 111 158 7 106 130 132 98 

### I 123 160 77 104 125 73 115 127 8 100 87 110 88 128 151 80 113 109 73 132 130 9 75 100 146 95 108 119 92 99 147 84 91 107 10 77 95 139 82 96 125 77 118 114 85 109 123 11 85 101 146 97 106 117 80 111 138 75 141 119 12 89 110 117 72 123 107 103 105 102 98 109 169 13 91 103 158 87 94 152 98 117 133 96 111 115 14 106 123 133 77 100 120 82 120 125 90 108 137 15 75 109 121 76 107 131 104 126 147**~** 92 121 16 76 115 134 79 113 117 83 94 

### - 73**~** 118
mean 87.3 106.7 129.9 87.5 106.0 128.8**~.1** 106.9 127.9**~.3** 107.3 131.0 S.D 12.4 12.3 142 10.8 11.2 15.0 10.3 10.5 12.7 12.9 15.4 17.6

### Characterization Sheet Sample Code Name Alternative Name Manufacturer Their Code Supplier Description Information from Certificate of Analysis

, Composition

Carbon Analysis (to GlP} ESl (URlCH) Study Request - . - Carbon :: low/w Hazards Unclassified. Use normal care when handling Storage Room temperature in the dark Expiry Date Not known
Documentation in support of these statements will be found in the fileAppendix 5 

### 17

SAMPLES 

### On each day when extraction and analysis is required the following will be provided (i) > 

### 500mls of carbon-filtered water (ii) > 

### lOOmis of any control, sample or stock solution to be analysed. PREPARATION OF SOLUTIONS 

### STOCK SOLUTION Accuratelv weigh between 4.5 and 6.0 mg of

.Sample labellec:_ into a 100ml glass beaker. Quantitatively transfer it to a 500 ml volumetric flask using at least 3 x 20 ml methanol. Make the solution in the volumetric up to volume with methanol. A fresh solution should be made up on each day of analysis. ALKANE STANDARD SOLUTION Accurately weigh between 20 and 25 mgs of n-docosane (Sigma D-4509) into a 10ml volumetric flask, and make up to volume with chloroform. This solution can be considered to be stable for a month. EXTRACTION PROCEDURE 

### 1) Set up as many 250 ml glass separating flasks in a fume cupboard as required. 2) To each ofthe first four flasks add the followil Flask Name Carbon-filtered water (ml) A 100 

## o B 

### 100 250 C 

### 100 250 x 3 

## =750 D 

### 100 250 x 5 =1250 using a measuring cylinder for the carbon-filtered water, and a 250ul positive displacement pipette for the stock solution. Shake each flask gently for about 10 seconds to allow mixing. 3) To the remaining separating flasks add 100ml (by measuring cylinder) of each sample, control, test concentration, solvent control and diluted test stock solution as required. The stock solution must be diluted down 1 in 10 with carbon-filtered water, before a 100ml portion is taken for analysis required. 4) To each flask add 10 ml chloroform and mix by gentle shaking for about 10 seconds. REMEMEBER TO RELEASE ANY PRESSURE BUILD-UP VIA THE NECK. 5) In turn shake each flask vigorously for 2 minutes. Allow the flask to stand for at least five minutes so that the chloroform and aqueous layers fully separate. Run off the lower chloroform layer into a 25 ml glass vial.18 6) Repeat steps 4) and 5). Run the lower chloroform layer into the appropriate 25ml glass vial which already contains about 10ml of the first chloroform extract from that flask. 7) Put all the 25ml glass vials of chloroform extracts on to a sample concentrator set at 600C and evaporate to dryness under nitrogen. In addition pipette 0.75ml of the stock solution into a 25ml glass vial (Flask X) and this down in the same manner.
MAKING UP SAMPLES The dried down samples should not be dissolved in chloroform until the day of analysis. 250ul ofchloroform should be added with a 250ul positive displacement pipette and then SuI of the alkane standard solution should be added using a SuI Hamilton syringe.

GC ANALYSIS The use of the Perkin Elmer GC 8700 is described in the Ecotoxicology SOP-177. The parameters set up were as follow: Canier gas : Helium at 16 psi Injection: 3 ul of sample via a coolon-column injector Column: 25m CP-Sil 5 CB (id= 0.32mm, film thickness 

## = 0.25um) Oven conditions: 500C for 0.5 minutes and then 300C/min ramping to 3000C. Held at 3000C for 4 minutes Detection: Flame Ionization Detector with temperature = 3500C air pressure 

## = 22 psi hydrogen pressure 

### = 15 psi Procedure A Bleed Calibration will be run and applied prior to any sample analysis, in order to compensate for column bleed during the ramping ofthe oven temperature within the method. 3ul of each of the samples will be injected via the coolon-column injector. The chromatograms obtained should show a peak at 8.5 +/- OS**minute~** corresponding n-docosane, and a peak at 10.5 +/- 0.5 minutes corresponding to ( chromatograms obtained, together with the GC method used, will be printed out. Calculation This method involves the use of an internal alkane standard to correct for minor variations in the small injection volumes used. Data collected from using this method were processed as follows:20 Study Title: The Chronic Toxicity of to Brachydanio Rerio Under Continuous Flow Conditions 28 Day Growth Test
Study Number: Date of Report
GLP COMPLIANCE STATEMENT The study was conducted in compliance with the Laboratory Policy on Good Laboratory Practice, which is based on the Principles of Good Laboratory Practice set forth in the OECD Guidelines for Testing of Chemicals (1982) and in the UK Compliance Programme (1989). Responsibility is accepted for the validity of the data generated in the study. Soil analysis was not carried out to GLP. Signature: Date: Study Director 

# ----------------------------------00000----------------------------------­ QUALITY ASSURANCE STATEMENT Date of Phase Date Reported to Inspection/Audit Study Management 27/08/93 Study Plan Audit 28/08/93 31/08/93 Procedural Inspection 02/09/93 Procedural Inspection 02/09/93 03/09/93 Procedural Inspection 03/09/93 Procedural Inspection 16/09/93 Procedural Inspection 17/09/93 Procedural Inspection 17/09/93 01/10/93 Procedural Inspection 11/03-13/05/94 Study Report Audits 21/03-12/05/94

22/09/93 Facility Inspection 11/10/93 27/07/93 Facility Inspection 11/08/93 (Ecotoxicology- Analytical) The inspections/audits have been conducted by the Quality Assurance Unit in accordance with the Laboratory Policy on Good Laboratory Practice which is based on the OECD Principles of Good Laboratory Practice (1982) set forth in the DoH UK Compliance Programme (1989). The report is considered to be an accurate description of the procedures and practices used for**suc~a** study and an accurate reflection of the raw data. Signature: Date:

## REPORT

- Page 1 of 20

­

# REPORT APPROVAL

STUDY DIRECTOR:

MANAGEMENT:

- Page 2 ­

# CONTENTS

SUMMARY 4 

### PREFACE 5 

### General 5 

### Project staff 5 

### Schedule 5 

### Quality Assurance statement 6 

### Statement of GLP compliance 7 

### Guidelines 8 

### Summary of protocol amendments 8 

### Archiving 8 

### OBJECTIVE 9 

### Purpose 9 

### Definitions 9 

### Rationale 9 

### MATERIALS AND METHODS 10 

### Test system 10 

### Test substance 11 

### Control substance 12 

### Test procedure and conditions 12 

### Preparation of test media 13 

### Determination of C02 14 

### Data evaluation 15 

### Acceptability of the test 15 

### RESULTS· 16 

### Theoretical C02 production 16 

### Biodegradation 16 

### Monitoring of temperature and pH 16 

### Acceptability of the test 16 

### Conclusion 17 

### Table 1 and 2 18 

### Table 3 and 4 19 

### Figure 1 20



### - Page 3 ­

## SUMMARY

was tested for its**read~** 

### biodeQradability in the modified sturm test at nominal concentrations of ,mgtl. The relative degradation values calculated from the measurements performed during the test period of 28 days revealed that at both concentrations

_ .. : was degraded significantly: 28% and 15% for 10 and 20 mgtl respectively. Although substantial biodegradation was recorded after 28 days of incubation,

_ . _ not readily biodegradable under the conditions of the modified Sturm test presently performed. Under the same conditions sodium acetate was degraded by more than 60%.

- Page 4 ­

# PREFACE

GENERAL Title Sponsor
Monitoring Scientist Testing Facility

RCC NOTOX Project Test Substance Test System

PROJECT STAFF Study Director Technical Head

SCHEDULE Start of the study 12-02-1991 (Day of addition of inoculum) Completion of the study 13":03-1991 (Last day of titrations)

- Page 5 ­

### QUALITY ASSURANCE STATEMENT

Test Substance Study Director Tit1e
Study procedures were periodica11y inspected and this report was audited by the Quality Assurance Unit. The dates are given be1ow. Dates of QAU Inspections I 

### Audits Dates of Reports to the Study Director and to Management

11-02-1991 20-02-1991 06-03-1991 02-04-1991 11-02-1991 20-02-1991 06-03-1991 02-04-1991



### Manager, Quality Assurance Unit

- Page 6 ­ 

# lf5

### STATEMENT OF GLP COMPLIANCE

Test Substance Study Director Title

To the best of my knowledge and belief, the study described in this report was conducted in compliance with the following Good Laboratory Practice Standards, with the exception that the stability of the test substance in water was unknown:
OECD Principles of Good Laboratory Practice, Paris, Frange. Food and Drug Administration

- Non-clinical laboratory studies; Good Laboratory Practice Regulations, U.S.A. Federal Register, part 58. Environmental Protection Agency

- Pesticide Programs and Toxic Substances Control; Good Laboratory Practice Standards; U.S.A., Federal Register, part 160 and 792 respectively.

Study Director

- Page 7. ­

# ~

### GUIDELINES The study procedures described in this report were based on the following guidelines: Organisation for Economic Co-operation and Development (DECO), DECO guidelines for Testing of Chemicals, Section 3: Degradation and Accumulation, guideline No. 301B: "Ready Biodegradability: Modified Sturm Test" adopted May 12,1981. European Economic Community {EEC),EEC directive 84/449, 

### Part C: Methods for the determination of ecotox;city, Publication No. L251, C5: "Biodegradation: modified Sturm test", adopted September 1984.

SUMMARY 'OF PROTOCOL AMENDMENTS The study protocol**wa~** 

### not amended.

ARCHIVING RCC NOTOX B.V. will archive th& following data for at least 10 years: protocol, report, test substance reference sample, all specimens and raw data.

- Page

# OBJECTIVE

PURPOSE The purpose of the study was to evaluate a non-volatile organic test substance for its ready biodegradability in an aerobic, aqueous medium with microbial activity introduced by inoculation of activated sludge during a test period of 28 

### days.

DEFINITIONS Readily biodegradable: test substances g1vlng a result of greater than 60% 

### yield of C02 

### (within 28 

### days). This level must be reached within 10 

### days of biodegradation exceeding 10%.


### RATIONALE Because of the stringency of this test method a result of less than 60% 

### yield**~f** 

### C02 

### (within 28 

### days) does not necessarily mean that the test substance was not biodegradable under environmental conditions, but indicates that more work would be necessary to establish biodegradability.

- Page 9 ­

# MATERIALS AND METHODS

TEST SYSTEM Source

Treatment

Temperature Reason for selection The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: The activated sludge was aerated for 4 hours. It was then left to settle for at least**~** 

### hour. The supernatant was decanted to provide a sufficiently large volume for a 1% inoculum for each test flask.

This test has been found suitable by the DECO Expert Group Degradationl Accumulation for determining the ready biodegradability of organic chemicals under aerobic conditions.

### TEST SUBSTANCE Identifi

### cation Description Batch Purity Instructions for test sUbstance storage Stability under storage conditions Expiry date Stable for at least 4 hours in vehicles

- Page 11 ­

# 1)

### CONTROL SUBSTANCE Sodium acetate.
TEST PROCEDURE AND CONDITIONS Test duration Test vessels Milli-Q water

Nutrient solutions

Nutrient medium

Barium hydroxide

Test concentrations
Controls 28 days 3-1itre all-glass brown coloured bottles. Water was first purified by reverse osmosis and thereafter passed over carbon and 10nexchange cartridges (Mi11ipore Corp., Bedford, Mass., USA). Stock solutions A) 8.50 9 KH2P04 21.75 9 K2HP04 67.20 9 Na2HP04·12H20 0.50 9 NH4C1 dissolved in 1 1 Mi11i-Q water, pH 7.4+0.2 B) 22.50 9 MgS04.7H20 dissolved in 1 1 Mil1iQwater. C) 36.40 9 CaC12.2H20 dissolved in 1 1 MilliQ water. 0) 0.15 

### 9 FeCl3 dissolved in 1 , Mill;-Q water. E) 40.0 9 (NH4)2S04 dissolved in 1 1 Milli-Q water. The following amounts were added per litre of Milli-Q water for the formulation of the nutrient medium: 2 ml of solution A. 1 ml of solution B, 1 ml of solution C, 4 ml of solution 0, 1 ml of solution E. All nutrients were added to Milli-Q water in such a way that -no precipitation occurred. 4 9 Ba(OH)2.8H20 per litre Mill;-Q water was filtered through filter paper and stored in a sealed vessel to prevent absorption of C02 from the air. The nominal concentrations of the test substance were 10 mg/l (low) and 20 mg/l (high). Positive control: 20 mg/l sodium acetate with inoculum. Blank contrOl to correct for background respiration of the bacteria: contained inoculum but neither test nQr control substance.

- Page 12

­ 

# .:::;/

### Pre-incubation medium
Preparations

PREPARATION OF TEST MEDIA Amixture of oxygen (20%) and nitrogen (80%) was led through a bottle, containing 0.5· - 1 litre 0.025 NBa(OH)2 solution to trap C02 which may be present in small amounts. The C02-free air was sparged through the scrubbing solutions at a constant rate (approximately 1-2 bubbles per second, corresponding to 50-100 ml air/min). 30 ml of the inoculum was added to the medium present in each of the 3-litre test bottles. This mixture was aerated with C02-free air for 24 hours. After the aeration period, three c02-abs'orber bottles were filled with 80 ml 0.025 N Ba(OH)2 and connected in series to the exit air line of each test bottle. Test material was added to two bottles, 10 and 20 mg/l, respectively. The control substance was quantitatively added to one bottle at a concentration of 20 mg/l. The fourth bottle contained onl} inoculum blank. Subsequently, the four bottles were made up to 3 litre with Milli-Q water.

Because the f 

### the test substance in it was quantitatively added to the test media and continuously stirred during the test.

### DETERMINATION OF C02 Experimental C02 production The C02 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of C02 produced was determined by titrating the remaining Ba(OHh wi th 0.05 Nstandardized HCL., Periodically tha C02-absorber nearest to the test bottle was removed for titration. The remaining two absorbers were each moved one

. position in the direction of the test bottle,

and a new absorber filled with 80ml of 0.025 NBa(OH)2 (normalised the same day) was placed at the far end of the series. Measurements Titrations were made when**nec~ssary** 

### (before significant BaC03 precipitate was evident in the second trap); approximately every other day for the first 10 days, and thereafter every fifth day until the 28th day. Phenolphthalein was used as**pH~indicator.** 

### On the 26th day, the pH of the four test bottles was measured, and then 1 ml of concentrated HC' was added to drive off inorganic carbonate. The bottles were aerated overnight, and samples were removed from eaoh bottle to enable the possibility for Dissolved Organic Carbon (DOC) analysis. All samples are stored at -20Q

### C for three months after delivery of this report. The final titration was made on day 28. Theoretical C02 production The theoretical C02 production was calculated from the molecular formula.

- Page 14­ 

# E3

# RESULTS
THEORETICAL C02 PRODUCTION The Theoretical C02 production (ThC02) of (molecular formula: C22H43NO MW=337 g) was calculated to be 2.872 mg C02tm9. The low concentration was 31.9 mg in 3 litre test medium and the high concentration was 60.9 mg in 3 litre test medium. Hence, the theoretical C02**~roduction** 

### following complete degradation was 91.6 mg per 3 litre for the low and 174.9 mg per 3 litre for the high concentration. The positive control contained 58.6 mg sodium acetate (ThC02= 1.073 mg C02tmg) resulting in a theoretical C02 production following complete degradation of 62.9 mg per 3 litre.
BIODEGRADATION The relative degradation values calculated from the measurements performed durina th.e test period revealed signifi·cant degradation of at both concentrations i.e. 28% and 15% for nominal concentrations 10 mgtl and 20 mgtl respectively (see Tables 2 and 3 and Figure 1).
MONITORING OF TEMPERATURE AND pH The temperature of the climate room varied between 18°C and 21.5 °C, with one incidental extreme of 23°C. The pH values of the different test media were: a. blank: control: 4.8;c. (10 mgtl): 5.4; d. mgl1): 5.3. 5.8; b. positive (20

ACCEPTABILITY OF THE TEST The control substance was biodegraded by more than 60% within 20 days and 71% degradation, was reached at the end of the test period (Table 1). The total C02 release in the blank reached a total value of 15 mg C02 (Table 4).



# -**P.~1J**

### CONCLUSION Although substantial biodearadation was recorded after 28 

### days of incubation,_ was not readily biodegradable under the conditions of the modified Sturm test presently performed.

- Page 17 ­

# ~~---- 

### ----------- 

## ----------- ----------- ----------- ----------- ----------- -----



### ---- 

## ----------- 

# ----------- ----------- ----------- ----------- ----------- ----- Table 1 C02-EVOLUTION AND PERCENTAGE OF BIODEGRADATION OF THE CONTROL SUBSTANCE

---- 

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

### Day ml titrant ml titrant Produced CO2 Normality Produced C02 Cumulative Degrad Blank C02 trap in ml Hel HCl mg CO2 C02 mg % 

### 2 37.14 31.72 5.42 0.0471 5.62 5.62 8.9 5 36.34 27.54 8.80 0.0471 9.12 14.74 23.4 7 37.08 33.14 3.94 0.0471 4.08 18.82 29.9 9 37.03 34.43 2.60 0.0471 2.69 21.51 34.2 12 35.00 32.53 2.47 0.0471 2.56 24.07 38.3 16 35.72 27.15 8.57 0.0471 8.88 32.95 52.4 21 36.43 27.63 8.80 0.0471 9.12 42.07 66.9 28 35.34 32.85 2.49 0.0471 2.58 44.65 71.0 28 36.04 37.40 0.00 0.0471 0.00 44.65 71.0 28 34.92 37.40 0.00 0.0471 0.00 44.65 71.0 Theoretical C02 production sodium acetate: 62.9 mg C02/31

Table 2 C02-EVOLUTION AND PERCENTAGE OF BIODEGRADATION OF THE TEST SUBSTANCE LOW

Day ml titrant ml titrant Produced CO2 Normality Produced C02 Cumulative Degrad Blank C02 trap ml HCl N mg mg CO2 % 

### 2 37.14 37.24 0.00 0.0471 0.00 0.00 0.0 5 36.34 35.72 0.62 0.0471 0.64 0.64 0.7 'J 

### 37.08 35.40 1.68 0.0471 1.74 2.38 2.6 9 

### 37.03 35.62 1.41 0.0471 1.46 3.84 4.2 12 35.00 35.46 0.00 0.0471 0.00 3.84 4.2 16 35.72 31.66 4.06 0.0471 4.21 8.05 8.8 21 36.43 29.72 6.71 0.0471 6.95 15.00 16.4 28 35.34 26.71 8.63 0.0471 8.94 23.94 26.1 28 36.04 34.56 1.48 0.0471 1.53 25.47 27.8 28 34.92 35.54 0.00 0.0471 0.00 25.47 27.8



### ---- 

## -----------. ----------- ----------- ----------- ----------- ----------- ----­ Theoretical C02-production test substance: 91. 6 mg C02/3L

- Page 18

­---- 

## ----------- ----------- ----------- ----------- ----------- ----------- -----

----------- 

## ----------- ----------- ----------- ----------- ----------- -----

---- 

## ----------- ----------- ----------- ----------- ----------- -----------

---- 

## ----------- ----------- ----------- ----------- ----------- ----------- Table 3 C02-EVOLUTION AND PERCENTAGE OF BIODEGRADATION OF THE TEST SUBSTANCE HIGH

Day ml titrant ml titrant Produced CO2 Normality Produced C02 Cumulative Degrad Blank C02 trap in ml HCl N 

### mg CO2 C02 mg % 

### 2 37.14 37.44 0.00 0.0471 0.00 0.00 0.0 5 36.34 35.89 0.45 0.0471 0.47 0.47 0.3 7 37.08 36.52 0.56 0.0471 0.58 1.05 0.6 9 37.03 35.78 1.25 0.0471 1.30 2.35 1.3 12 35.00 34.86 0.14 0.0471 0.15 2.50 1.4 16 35.72 27.72 8.00 0.0471 8.29 10.79 6.2 21 36.43 27.70 8.73 0.0471 9.05 19.84 11.3 28 35.34 28.76 6.58 0.0471 6.82 26.66 15.2 28 36.04 36.86 0.00 0.0471 0.00 26.66 15.2 28 34.92 36.34 0.00 0.0471 0.00 26.66 15.2 Theoretical C02 production test sUbstance: 174.9 mg C02/3L

Table 4 C02-EVOLUTION IN THE BLANK

Day stand.Ba(OH) ml titrant Produced CO2 Normal 

### ity Produced C02 Cumulati 

### ve . ml HCl 

### * 4 Blank ml HCl N 

### mg C02 mg

---- 

## ----------- 

# ----------- ----------- ----------- --------r-- ----------­

2 9.71 37.14 1.70 0.0471 1.76 loB 5 9.50 36.34 1.66 0.0471 1.72 3.5 7 9.51 37.08 0.96 0.0471 0.99 4.5 9 9.49 37.03 0.93 0.0471 0.96 5.5 12 9.49 35.00 2.96 0.0471 3.07 B.6 16 9.42 35.72 1.96 0.0471 ·2.03 10.6 21 9.23 36.43 0.49 0.0471 0.51 11.1 28 9.14 35.34 1.22 0.0471 1.26 12.4 28 9.14 36.04 0.52 0.0471 0.54 12.9 28 9.14 34.92 1.64 0.0471 1.70 14.6

- Page 19

­

### Figure 1: Biodegradation of in the modified Sturm test: [J: 

### Sodium acetate; * : Low (10 

### mg/l); + : 

### High (20 mg/l).



# - ­ -- -**-~** 

## b 

## i 

# o d 

## e g 

# r a d 

## a t i 

# o n**.:..~** 

## --+

# ~**--~-** 

# --­**=*-~-TI~** I 

# o ), I i 

# o 5 10 15 20 25 

# COURSE OF BIODEGRADATION IN TIME**-p~-**

# , ,

, 

### 2 First Compiled By : Date : 05/05/94 Sponsor: Test Facility:

Staff Involved : Study Director Deputy Study Director Test Operator

Analytical Supervisor Analytical Supervisor (Deputy) Analytical Operator 

### Study Starting Date : 08/09/93 Study Completion Date : 29/09/93
This study report contains all the summary data but it does not contain all the raw data Le.cmde.cL.durina thp.**~tudy.** 

### The raw data are located in Ecotoxicology Archive file


### Distribution

Further distribution and / or copying of this report is not permitted. Copy No. 

# 5:

### 3 

### Contents Page Summary 

### 4
1.1ntroduction 

### 5 1

.1 Objective 

### 5 1.2 Test Substance Description 

### 5 1.3 Test System 

### 5
2. Methodology 

### 5 2.1 Basis of the Method 

### 5 2.2 Preparation of Stock and Test Solutions 6
3. Results 6 3.1 Test Conditions 6 3.2 Test Substance Concentrations 

### 7 3.3 Effect Data 

### 8
4. Effect Concentrations 

### 8
'5. References 

### 9 Appendix 1 Test Conditions 11 Appendix 2 Effect Data 

### 14 Appendix 3 Preparation of

_ Medium 16 

### Appendix 4 Characterisation Data 

### 17 Quality Assurance and GLP Compliance Statements 

### 18


# 614 

### Summary The chronic toxicity of 'to the freshwater cladoceran Daphnia magna 

### was determined according to a stanaard method conforming with OECD Guideline 202 [1]. However, "Clone 5" Daphnia 

### were used instead of the usual "URPSL Clone". The Daphnia 

### were exposed to two concentrations and two controls (including a solvent control). Elendt medium was usea {recIpe appended) as this has shown to support adequate Daphnia 

### reproduction without the need to add additives, such as organic seaweed-derived extract, to boost production of neonates. The test was conducted under static-renewal conditions with daily renewal of the test solutions. Daphnia 

### were fed daily on 1.7 x 105 

### cells/ml of "algal feed for Daphnia". 

### Analysis of ther test solution concentrations indicated that concentrations of fresh solutions were on average 83.5% of nominal and of old solutions were on average 65.2% of nominal values. Statistical analysis of the effect data using an Analysis of Variance followed by a comparison of treatment means using Dunnett's Test [11] indicated that there was no significant effect on Daphnia 

### from either of the concentrations tested. Chronic inhibitory effects caused by must, therefore, be greater than 80 

### 1J9/1.

### 5 1. Introduction 1 .1 Objective To determine the effect of on the reproduction of Daphnia magna. 

### 1.2 Test**~llhstance** 

### Descriotion

>was tested as a 

### Yo commercial substance. FlfrfneTaetails are given in Appendix 5. 1.3 -Test System Daphnia magna 

### (Crustacea : Cladocera) is a planktonic, filter-feeding invertebrate, widely distributed in the freshwaters of temperate regions around the world. It is widely considered to be sensitive to changes in water quality. The Daphnia 

### used in this test were "Clone, 5", originally obtained from the Department of Animal**~nd** 

### Plant Sciences, University of Sheffield. Daphnia magna

### were cultured in the laboratory according to the procedures described in SOP 019 [2]. Cultures were maintained in a synthetic medium with the same chemical composition and similar pH and dissolved oxygen concentration asthat used to prepare testsoiutions. Culture medium consists of 800 mi.. VI7 medium. The cultures are fed daily with cells of the green algae Chlorella vulgaris. 

### Cultures are reset using 10 neonates, when they become gravid their numbers are reduced from 10 to 5 and the production of neonates results in their daily removal. 2. Methodology 2.1 Basis of the Method The test method is described in SOP 046 [3]. On the day of the test, ,Daphnia 

### neonates were removed from the cultures. These neonates were placed in a 200 ml crystallising dish containing synthetic Elendt M7 medium (within 2°C of that of the culture vessels) until used in the test.

, .' , 

### One Daphnia 

### neonate was placed in each of the test vessels, 4 oz glass bottles, containing 100 ml of test solution. There were 1 0**n~nli('.at.e.s** 

### for each treatment : control, solvent control (methanol), 33**~g!l** 

### and 1 00**~g/r** 

### The testvessels were placed in the test area according to a randomiseo Olstrlouuon [4] and in the temperature range 20.0
- 21.5°C and under normal laboratory light intensity with a 16 hour light, 8 hour dark photoperiod. The Daphnia 

### were fed daily on 1.7 x 105 

### cells/ml of Chlorella vulgaris. 

### Each day, all vessels were assessed for adult survival and numbers of neonates. Neonates were removed daily. 

# j

### 6 

### 2.2 Preparation of Stock and Test Solutions The stock solution was prepared by dispersing 

### 0.05 g 

### N25 in 

### 50 ml of methanol, using a sonic bath, to make a 

### 1.0 gil stock (Stock A) [5]. 

### 16.7 ml of Stock A was then made up to 

### 50 ml with methanol to form a 

### 0.33 gil stock (Stock 8). Test solutions were prepared by adding known volumes of these stocks to 

### 1.5 litres Elendt medium [6]. The solvent control was prepared by adding 

### 0.15 ml methanol to 

### 1.5 litres of Elendt. Test solutions were renewed daily. The pH, dissolved oxygen concentration and total hardness (as 

### mg/l CaC03) 

### of selected test solutions were measured on Days 0, 

### 1, every Tuesday and Friday and Day 

### 21 using the appropriate methods 

### [7,8,9)



### 3. Results 

### 3.1 Test Conditions The results of the analysis of test conditions are shown in Table 1. Deviations from the ran.ge specified for temperature were recorded. These were mostly due to failure ofthe temperature control system in the testing room. This is not considered to have had a significant influence on the outcome of the study. Table 1 Test Conditions Mean Standard Deviation Maximum Value Minimum Value Test Solution Temperature (OC) 

### 19.7 1.6 25.0· 14.5 pH 

### 7.6 0.2 8.1 7.2 Dissolved Oxygen (mg/l) 

### 8.2 0.9 9.3 4.5 Total Hardness (mg/l as CaC03) 

### 245 5 256 234 Further details of the test conditions are given in Appendix 1.

### 7 CT/N25/02 

### 3.2 Test Substance ConcAntrations Concentrations of 

### were determined by the following method with the results given in**table'~'2:** 

### An analytical stock solution of: ,in methanol was prepared, and various aliquots 

### « 2ml) were used t6spike 100mi Elendt medium in 2S0ml separating flasks to obtain standards covering the range concentrations present in the test samples. 100mi of each test sample, or dilution thereof, was also measured into 2S0ml separating flasks. 10ml chloroform was then added to each flask, and the flask shaken for 2 minutes, and then the chloroform layer run off into a 2Sml glass vial. A further 1Oml chloroform was then added to each flask and a second extraction carried out, with the resulting chloroform layer being combined with that from the first extraction. The chloroform extracts were then dried down on a sample concentrator at 60°C under nitrogen, and made up in 2S0ul chloroform, prior to the addition of Sui ofan alkane internal standard (about 2000ma n-docosane/l chloroform). In addition an aliquot of the analytical stock solution of _ •chloroform, equivalent to one that was spiked into one ofthe flasks, was directly dried down and made up as above. The purpose was to allow a single recovery to be determined on each day of analYSis. GC analysis, with column bleed compensation, was carried out on the Perkin Elmer GC 8700 [101 using the following conditions:
Carrier: Gas:Helium at 16 psi Injection: 3ul of sample via a cool on-colun:m injector Column: .' ,,' 3 CB (id=0.32mm, film hickness=0.2Sum) Oven: SO°C for 0.5min then 30°C/min ramp to 300°C, and held for 4min Detector: Flame Ionization Detector with: temperature = ,.' _'C air =, psi hydrogen 

## = psi

### 8 Table 2 Concentrations of Test Substance


### DAY Nominal Concentration (lJg/l) 0.0 0.0 (Solvent) 0 < 0.1 < 0.1 22.3 63.7 new < 0.1 < 0.1 22.1 69.0 1 old 

# - - 13.0 55.9 new < 0.1 < 0.1 33.0 101.8 5 old 

# - - 28.4 87.5 new < 0.1 < 0.1 24.0 77.7 9 old 

# - 

# - 24.1 43.3 new 16.7 

### # < 0.1 27.6 106.5 12 old 

# - - 21.6 67.0 new < 0.1 < 0.1 20.1 85.3 16 old 

# - - 19.6 64.9 new < 0.1 < 0.1 26.2 134.1 19 old 

# - 

# - 20.0· 79.2 Mean 23.2 79.7 Std dev 5.0 24.0 

### # sample re-analysed 24 hours later showed no present suggesting initial result may have been due to contamination during the extraction procedure. Note:values calculated using an average ofthe calibration plots generated during the test to minimise underestimates of concentrations during the early period of the test caused by inadequate conditioning. The mean measured concentrations (new and old test solutions) were· '% and ,% of the nominal 

### 1-19/1 and 1-1

### gIl concentrations respectively. The average fall in concentration before renewal of the test solutions was much greater at the higher concentration (approximately '%), than at the lower concentration (approximately

,,%).

### 9 3.3 Effect Data A summary of the effect data are reported in Table 3. Further details are given in Appendix 2. Table 3 Summary of Effect Data Mean Measured

Concentration (lJg/l) Mean Cummulative Number of Neonates at Day 21 Mean Time to First Brood (days) Mean Size of First Brood Percentage Adult Mortality at Day 21 0.0 61.9 11.2 13.5 0 78.3 11.4 19.5 0 70.4 11.5 16.1 0 0.0 Solvent 77.7 10.2 15.3 20


### 4. Effect Concentrations Statistical analysis of the effect data using an Analysis of Variance followed by a comparison of treatment means using Dunnett's Test [11] indicated thatthere was no

.significant effect on**Daphni~** 

### from either of the concentrations tested. Chronic

inhibitory effects caused by \11ust, therefore, be greater than' I-Igli.

### 10 5. References [1] OECD Guidelines for Testing of Chemicals. Section 2. Guideline 202 

### "Daphnia sp.,Acute Immobilisation Test and Reproduction Test." [2] Ecotoxicology SOP \' [3) Ecotoxicology SOP 

### [4] Ecotoxicology SOF [5] Ecotoxicology SOP , [6) Ecotoxicology SOP [7] Ecotoxicology SOP [8] Ecotoxicology SOP [9] Ecotoxicology SOP, [10 Ecotoxicology SOP The Laboratory Culture of 

### Daphnia magna. Daphnia magna Semi Static Reproduction Test. Random.BAS Computer Program. The Use, Maintenance and Calibration of the Mettler AE 200 with Printer. Maintenance and Operation of Rainin EDP Plus Electronic Pipette.

, Use of Philips pH Meters (PW 9410/30).

Use of YSI Model 57 Oxygen Meter. Determination of Total Hardness. Use of the Perkin Elmer Model 8700 Gas Chromatograph. [11] Dunnett C W (1955) A Multiple Comparison Procedure for Comparing Several Treatment Means with a Control. 

### J Amer. Statist. Assoc. 50 p096.11 APPENDIX 1 Test Conditions Test Solution Temperature
Vessel 1 

### Vessel 11 Day old new old new



### 0 

# - 24.0
- 

### 24.5 1 21.0 21.0 21.0 21.0 2 19.5 20.0 19.0 20.0 3 19.0 21.0 19.0 21.0 4 18.0 18.0 18.0 18.0 5 20.0 20.5 20.0 20.5 6 19.0 20.5 19.0 20.5 7 19.0 20.5 20.5 20.5 8 18.0' 20.0 19.5 20.0 9 17.5 18.5 17.0 19.0 10 16.0 19.0 16.0 19.0 11 15.0 17.0 14.5 17.0 12 17.0 19.0 17.0 19.0 13 19.0 20.5 18.5 20.5 14 19.5 20.5 19.0 20.5 15 20.0 20.5 19.0 20.5 16 20.0 21.0 19.0 21.5 17 19.5 20.5 19.0 21.0 18 19.5 20.5 ·18.5 20.5 19 20.0 20.0 20.0 20.0 20 20.5 21.0 20.0 21.0 21 20.0
- 

### 20.0 

## - Vessel 21 Vessel 22 old new old new



# - 25.0 

## - 24.0 20.5 21.0 21.0 21.0 19.0 20.0 18.5 20.0 19.5 21.0 20.0 21.0 19.0 18.0 19.0 18.0 20.5 20.5 20.0 20.5 19.5 20.5 20.5 20.5 19.5 20.5 20.0 20.5 18.0 20.0 19.5 20.5 17.5 19.0 18.0 19.0 16.0 19.0 16.5 19.0 14.5 17.0 15.0 17.0 17.5 19.0 18.5 19.0 18.5 20.5 18.5 20.5 19.0 20.5 19.0 20.5 20.0 21.0 21.0 21.0 19.5 21.5 20.0 21.5 20.5 20.5 20.0 20.5 20.0 20.0 20.5 20.5 20.0 20.0 19.5 20.0 20.5 21.0 21.5 21.0 21.0 - 

### 21.0 

## - Vessel 31 Vessel 40 old new old new



## - 24.5 

# - 24.5 21.0 21.0 21.0 21.0 19.0 20.0 19.0 20.0 19.5 21.0 20.0 21.0 19.5 18.0 19.0 18.0 20.5 20.5 20.5 20.5 19.5 20.5 

# - 

# - 19.5 20.5 

# - - 19.5 20.5 

# - - 17.5 19.0 

# - - 16.0 19.0 

# - - 14.5 17.0 

## - 

# - 17.5 19.0 

## - 

# - 19.0 20.5 

# - - 

### 19.5 20.5 

# - 

# - 20.0 21.0 

# - - 19.5 21.5 

## - 

# - 19.5 20.5 

## - 

# - 19.5 20.5 

## - 

# - 20.0 20.0 

# - 

# - 20.5 21.0 

# - - 20.5 

# - - - NB. Readings ceased in vessel 

### 40 due to the death of the Daphnia.

### 12 APPENDIX 1 Test Conditions (cont.) Total Hardness (mgtl CaC03) 

### Day Test Concentration (1-I9/1) 

### 0.0 

### 0 239 238 236 

## old 234 240 242 2 new 

### 234 240 239 

## old 254 248 251 6 new 

### 245 245 246 

## old 251 240 249 9 new 

### 242 236 240 

## old 254 248 245 13 new' 

### 248 243 243 

## old 250 250 244 16 new 

### 248 248 247 

## old 247 243 246 20 new 

### 249 248 248 21 

## old 247 256 248 0.0 Solvent 

### 234 238 240 255 246 249 237 245 244 247 248 242 249 249



# 7D

### 13 APPENDIX 1 Test Conditions (cont.) 

### pH Mean Test Concentrations (pgll) Day Control Solvent Control old new old new old new old new 

### 0


# - 7.7 

# - 7.9


# - 7.9 

# - 7.9 1 7.5 7.7 7.6 7.8 7.6 7.9 7.6 7.9 2 7.6 7.8 7.5 7.9 7.7 7.9 8.1 7.7 6 7.5 7.5 7.3 7.4 7.2 7.4 7.4 7.4 9 7.3 7.6 7.3 7.7 7.3 7.7 7.4 7.7 13 7.6 8.0 7.8 7.9 7.4 7.9 7.3 <8.0 16 7.4 * 

### 7.2 7.6 7.2 7.6 7.3 7.6

, 

### 20 7.5 7.6 7.5 7.6 7.4 '7.6 7.6 7.6 21 7.5


# - 7.2


# - 7.3


# - 7.3 

# -
Dissolved Oxygen Concentration (mg/l)


### Day Mean Test Concentrations (pg/l) Control Solvent Control old new old new old new old new 

### 0 

# - 8.6
- 

### 8.5


# - 8.3 

# - 8.6 1 8.7 8.8 9.0 8.8 8.5 8.9 9.2 8.8 2 

### 8.1 8.1 7.8 8.0 8.2 8.1 8.8 8.4 6 9.2 8.8 8.3 8.4 8.5 8.5 9.1 8.7 9 8.2 8.4 7.5 8.5 7.5 8.5 8.5 8.6 13 9.0 8.2 7.6 9.3 8.0 9.1 8.0 9.2 16 8.5 * 6.5 8.7 6.0 8.7 7.9 8.6 20 8.4 8.7 5.2 8.6 4.5 8.8 6.7 8.6 21 8.0 - 

### 6.1
- 

### 6.7 

# - 7.2 

# - * indicates the solution was discarded in error.

### 14 

### APPENDIX 2 Effect Data Mean Time to Release of First Brood (Days) Replicate Mean Measured Concentration**(~g/l)** 

### 0.0 1 

### 11 13 11 2 13 12 10 3 10 11 12 4 10 12 15' 5 13 10 11 6 11 12 11 7 11 11 11 8 12 11 1·2 9 12 11 12 10 9 11 10 Mean 

### 11.2 11.4 11.5

SO 

### 1.3 0.8 1.4 0.0 Solvent 

### 10 11 10 10 10 9 10 13 9·

- 

### 10.2 1.2 9rowth of adult appeared to be inhibited. Where no data are given adult died before release of first brood. 

### Total Number of Neonates per Surviving Adult Replicate Mean Measured Concentration**(~g/l)** 

### 0.0 0.0 Solvent 

### 1 67 67 85 84 2 48 56 87 88 3 61 (1) 97 75 77 4 54 51 28 88 5 46 82 62


# - 6 62 65 82 73 7 84 81 73 65 8 81 95 62 (1) 69 9 46 94 (2) 52 (1) 

# - 10 69 93 96


# - Mean 

### 61.9 78.3 70.4 77.7

SO 

### . 14 17 20 9 1-1

### 9ures in brackets refer to numbers of dead neonates. Where no data are given the adult did not survive the test. 

# ,1"

### 15 APPENDIX 2 Effect Data Size of First Brood Replicate 0.0 Mean Measured Concentration (lJglI) 0.0 Solvent 

### 1 17 17 21 15 2 16 22 12 19 3 12 20 12 14 4 11 19 12 11 5 8 20 19 16· 6 14 19 16 '15 7 11 13 18 12 8 18 23 15 20 9 16 21 18 16· 10 12 21 18 

# - Mean 

### 13.5 19.5 16.1 15.3

SO 

### 3.2 2.8 3.2 2.9 Adults died on Oay 

### 17. Mean Cumulative Number of Neonates from Adults Surviving to Day 21 

### Day Mean Measured Concentration (1J9/1) 0.0 0.0 Solvent i 

### 9 1.2 

# - 

# - 2.1 I 

### 10 3.5 2.0 3.0 9.6 11 7.7 11.8 10.4 12.3 12 11.1 17.8 14.9 12.3 13 18.1 19.5 17.4 21.9 14 22.0 33.5 25.9 28.1 15 27.4 38.9 35.9 32.0 16 34.1 41.5 38.7 39.9 17 38.1 54.5 45.5 53.0 18 44.9 58.4 53.6 57.7 19 49.5 64.8 59.5 63.3 20 52.9 71.1 61.7 70.9 21 61.9 78.3 70.4 77.7APPENDIX 3 Preparation of M7 Elendt Medium Substance Conc. of Stock (mgll) Volume added to 20 I Distilled Water (ml) H3B03 57190 2.5 MnCI2.4H 2O 7210 0.25 LiCI 6120 0.25 RbCI 1420 0.25 SrCI2·6H2O 3040 0.25 NaBr 320 0.25 Na2Mo04 1260 0.25 CuCI2·2H2O 335 0.25 ZnCI 260 1.0 CoCI2·6H2O 200 1.0 KI 65 . 1.0 Na2S20 3 ·43.8 1.0 NH"V03 11.5 "­ 1.0 CaCI2·2H2O 29380 200 MgSO".7H2O 246600 10.0 KCI 58000 2.0 NaHC03 64800 20.0 Na2Si03·5H2O 3730 40 NaN03 2740 2.0 KH 2P04 1430 2.0 K

2HP04 1840 2.0 An Fe·EDTA Solution is prepared by adding 1.991 gil FeS04.7H20 and 5.0 gil Na2EDTA.2H 20 and autoclaving immediately. 5.0ml of this solution is needed to prepare 20 litres Elendt medium. As well as the above, a vitamin stock solution is prepared by adding the following to 1 litre deionized water:

. Substance Cone. of Stock (mgtl) Volume added to 20 I Distilled Water (ml) Thiamine Hydrochloride 750 2.0 Cyanocobalamine (B 12) 10 Biotine 7.5
• The vitamin stock is stored frozen in small aliquots. Vitamins are added to the media shortly before use.APPENDIX 4 Characterisation Data



### Sample Code Name Alternative Name Manufacturer Their Code Supplier Description Information from Certificate of Analysis

Carbon Analysis (to GLP) ESL (URLCH) Study Request. Hazards Storage Expiry Date 

## Characterization Sheet

Carbon 

# = . Yow/w Unclassified. Use nonnal care when handling Room temperature in the dark Not known
Documentation in support oftnese.,statements will be found in the fifeStudy Number: Date of Report
GLP COMPLIANCE STATEMENT
The study was conducted in compliance with the Laboratory Policy on Good Laboratory Practice, which is based on the Principles of Good Laboratory Practice set forth in the OECD Guidelines for Testing of Chemicals (1982) and in the UK Compliance Programme (1989). Some of the data in support of characterisation of the test substance were not generated to Good Laboratory Practice. Responsibility is accepted for the validity of the data generated in the study. Signature: Date: Study Director 

# -------------------------------------------------,---00000---------------------------------------------------­ QUALITY ASSURANCE STATEMENT Date of Phase Date Reported to InspectionlAudit Study Management 03/09/93 Study Plan Audits 03/09/93 09/09/93 Procedural Insp (Analytical) 09/09/93 27/07/93 Facility Insp (Analytical) 11108193 22/09/93 ,Facility Insp (Biology) 11110/93 02/08/94-07/10/96 Study Report Audits 05/08/94-07/10/96 Studies of the type described in this report are conducted in a manner which involves frequent repetition of identical or similar procedures. At the time of this study, Process based inspections were made by the Quality Assurance Unit of critical phases relevant to this type of study. Inspections of any given critical phases were conducted on studies selected,on a periodic basis. Any adverse findings from such inspections were reported promptly to the relevant Study Director and to Line Management. Date of Process Ph,ase Date Reported to Inspection Study Management 08/09/93 Study Conduct 08/09/93 Study Conduct 08/09/93 22/09/93 Study Conduct 22/09/93 The inspections/audits have been conducted by the Quality Assurance Unit in accordance with the Laboratory Policy on Good Laboratory Practice which is based on the OECD Principles of Good Laboratory Practice (1982) set forth in the DoH UK Compliance Programme (1989). The report is considered to be an accurate description of the procedures and practices used for such a study and an accurate reJlectiAA of the raw data. Signature: Date: Quality Assurance Manager

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