U.S. ENVIRONMENTAL PROTECTION AGENCY 

PREMANUFACTURE 

NOTICE 

SUBSTANCES 

Dale 

j 

Company Sanitized 

• 	 You must provide all infonnation requested in this fonn to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates ifyou do 

not have actual data. 

• 	 Before you complete this fonn, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from the Toxic 

Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603). 

• 	 Ifa user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remember, 

your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM-226), P.O. 

360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee. 

Part 1-GENERAL INFORMATION 

You must provide the currently correct Chemical Abstracts (CA) Name ofthe new 

chemical substance, even if you claim the identity as confidential. You may authorize 

another person to submit chemical identity information for you, but your submission will 

not be complete and the review will not begin until EPA receives this information. A 

letter in support ofyour submission should reference your TS user fee identification 

number. You must submit an original and two copies ofthis notice including all test data. 

If you claimed any infonnation as confidential, a single sanitized copy must also he 

submitted. 

Part 11- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

Ifthere are several manufacture, processing, or use operations to he descnbed in Part II, 

sections A and B ofthis notice, reprnduce the sections as needed. 

Part 111- LIST OF AITACHMENTS 

Attach additional shects ifthere is not enough space to answer a question fully. Label 

each continuation sheet with the corresponding section heading. In Part Ill, list these 

attachments, any test data or other data and any optional information iocluded in the 

notice. 

OPTIONAL INFORMATION 

You may include any infonnation that you want EPA to consider in evaluating the new 

substance. On page II ofthis form, space has heen provided for you to descnbed 

pollution prevention and recycling information you may have regarding the new 

substance. 

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

willingness to be bound to certain statements you make in this section, such as use, 

production volume, protective equipment. .. This option is intended to reduce delays 

that routinely accompany the development ofconsent orders or Significant New Use 

Rules. Except in the case of exemption applications (such as TMEA, LVE, WREX) 

where certain infonnation provided in such notification is binding on the submitter when 

the Agency approves the exemption application, checking a binding box in this notice 

by itself prohibit the submitter from later deviating 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 on the 

form, mark (X) the confidential box next to the infonnation that you claim as 

confidential. To assert a claim in an attachment, circle or bracket the infonnation you 

claim as confidential. ICyou claim infonnation in the notices as confidential. you must 

also provide a sanitjzed version ofthe notice. fincludine: attacbments). For additional 

instructions on claiming information as confidential, read the Instructions Manual. 

TEST DATA AND OTHER DATA 

You are required to submit all test data in your possession or control and to provide 

a description ofall other data known to or reasonably ascertainable by you, ifthese 

data are related to the health and environmental effects on the manufacture, 

processing, distribution in commerce, use, or disposal ofthe new chemical 

substance. Standard literature citations may be submitted for data in the open 

scientific literature. Complete test data (written in En!;dishl. not summaries ofdata. 

must be submitted ifthey do nO! appear in the open literature. You should clearly 

identifY whether test data is on the substance or on an analog. Also, the chemical 

composition ofthe tested material should be characterized. Following are 

examples oftest data and other data. Data should be submitted according to the 

requirements of §nO.50 ofthe Premanufacture Notification Rule (40 CFR Part 

nO). 

Test Data (Check Below any included in this notice) 

Environmental fate data 0 Yes 	 Other data 0 Yes 

Health effects data 0 Yes 	 Risk assessments 

Environmental effects data 181 Yes 	 Structure/activity relationships 

PbysicaL'Chemical Properties· 181 Yes 	 Test data not in the possession 

or control ofthe submitter 

• A physical and chemical properties worksheet is located on the last page ofthis fonn. 

TYPE OF NOTICE (Check Only One) ~ e 

PMN (Premanufacture Notice) i; 0 

:z:: .",::0 

INTERMEDIATE PMN (submitted in sequence with filial produD{;:J.)

W --1.{TJ

....,..­ SNUN (Significant New Use Notice) ~< 

::":rrJ 

o TMEA (Test Marketing Exemption Application) no

.r::­ o LVE (Low Volume Exemption) @4O CFR 723.SO(~ 

o WREX (Low Release/Low Exposure Exemption) @4OCFR 723.50(c)(2) 

o 	 L VE Modification o LOREX Modification 

IS THIS A CONSOLIDATED PMN? o Yes

# of chemicals 

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

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

/ 

Public reporting burdcn for this collection of infonnation is estimated to average 110 hours per response, including time for reviewing 

instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of 

infonnation. Send comments regarding the burden estimate or any other aspect of this collection ofinfonnation, including suggestions for 

reducing this 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 of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 


I certify that to the best ofmy knowledge and belief: 

1. 	 The company named in Part I, section A, subsection 1 a of this notice form intends to manufacture or import for a 

commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, 

Section B. 

2. 	 All information provided in this notice is complete and truthful as of the date of submission. 

3. 	 I am submitting with this notice all test data in my possession or control and a description of all other data known to or 

reasonably ascertainable by me as required by §720.50 of the Premanufacture Notification Rule. 

Additional Certification Statements: 

Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNlIN, check the following user fee certification 

statement that applies: 

C8J 	 The Company named in Part I, Section A has remitted the fee of$2500 specified in 40 CFR 700.45(b), or 

D The Company named in Part I, Section A has remitted the fee of $ 1000 for an Intermediate PMN (defmed @40 CFR 

700.43) in accordance with 40 CFR 700.45(b), or 

D The Company named in Part I Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of$lOO 

in accordance with 40 CFR 700.45(b). 

If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723 .50( c)( 1) or a Low release 

and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check the following certification 

statements: 

D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial 

purposes, other than in small quantities solely for research and development, under the terms of40 CFR 723.50. 

o 	The manufacturer is familiar with the terms ofthis section and will comply with those terms; and 

o 	The new chemical substance for which the notice is submitted meets all applicable exemption conditions. 

o Ifthis application is for an L VE in accordance with 40 CFR 723 .50( c)( 1), the manufacturer intends to commence 

manufacture ofthe exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day 

review period. 

The accuracy of the statements you make in this notice sbould reflect your best prediction of the anticipated facts regardinlt the cbemical substance 

described herein. Any knowllll! and willful misinternretatlon Is subfect to criminal nenalty nursuant to 18 USC ]001. 

Signature and title ofAuthorized Official (Original Signature Required) Dale 

Confidential 

12 December 2002 

x 

Signature ofagent - (ifapplicable) Date 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 2 
Confidential 

la. Person 

Submitting 

Notice (in U.S.) 

Company 

City, State, ZIP Code Telephone 

Ifyou are submitting this notice as part of a joint submission, mark (X) this box. -----------------------3... Position 

Telephone 

Position 

b. Agent(if 

applicable) 

c. 

Joint Submitter (if 

applicable) 

2. 	 Technical 

Contact (in 

U.S.) 

Mailing address (number and street) 

State, ZIP Code 

Name of authorized official 

Company 

Mailing address and street) 

Name of authorized official 

Company 

Mailing address (number and street) 

City, State, ZIP Code 

Name of authorized official 

Company 

Mailing street) 

City, State, ZIP Code 

3. a prenotice (PC) concerning this notice 

and EPA assigned a PC Number to the notice, enter the number. 

you 

~ 

4. 	 Ifyou previously submitted an exemption application 

substance covered by this notice, enter the exemption number assigned by 

EPA. If you previously submitted a PMN for this substance enter the 

PMN number assigned by EPA (i.e. withdrawn or incomplete). ~ 

5. 	 Ifyou have submitted a notice of Bona fide intent to manufacture or import 

for the chemical substance covered by this notice. enter the notice number 

assigned by EPA. .. 

I. Manufacture 

6. Type of Notice - Mark (X) Only

D Binding Option 

Mark 

2. Import 

Only 

D Binding Option 

Mark 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 3 

Mark 

if none 

(X) ~ D

Mark (X) ~ 

if none 


Mark 

if none 

(X) ~ D

3. D Both
I. Class 1 or 2 chemical substances (for definitions of class I and class 2 substances, see the Instructions Manual) 

a, Class of substance· Mark (X) I Class I or 2 [8;] Class 2 

b, Chemical name (Currently correct Chemical Abstracts (CAl Name that is consistent with TSCA Inventory listings for similar substances, 

For Class I substances a CA Index Name must be provided, For Class 2 substances either a CA Index Name or CA Preferred Name must 

be provided, which ever is appropriate based on CA 9C1 nomenclature rules and conventions). 

c, 	 Please identify which method you used to develop or obtain the specified chemical identity infonnation reported in this notice: (check one). 

~ Method I (CAS Inventory Expert Service· a copy of the Identification ~ Method 2 (Other Source) 

report obtained from the CAS Inventory Expert Services must be 

submitted as an attachment to this 

Molecular fonnula CAS Registry a number already exists the substance) 

CAS# 

e, 	 For a class I provide a complete and correct chemical structure diagram, For a class 2 substance· (I) List the immediate 

precursor substances with their respective CAS Registry Numbers, (2) Describe the nature of the reaction or process, (3) Indicate the 

range of composition and the typical composition (where appropriate), (4) Provide a correct representative or partial chemical structure 

diae:ram. as comolcte as can be known. if one can be reasonablv ascertained, 

1) Precursor substances: 

) Nature of the reaction process: 

3) Range of typical composition: 

4) Representative structure: 

substance based on 

If another person will submit chemical identity infonnation for you (for either Item I or 2), mark (X) the box at the right. 

in a 

x 

X 

x 

x 

X 

attach a continuation sheet. 

EPA FORM 7710·25 (Rev, 5·95) 	 Page 4 
Confidential 

a. Indicate the number-average weight of the lowest molecular weight composition of the polymer you intend to manufacture. 

Indicate maximum weight percent of low molecular weight species (not including residual monomers, reactants, or solvents) below 500 and 

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement or the basis for your estimates: GPC D Other D: (Specify) 

i) lowest number average molecular weight: 

ii) maximum weight % below 500 molecular weight: 

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 (if a number exists) of each monomer or other reactant used in the manufacture of 

the polymer. 

(2) Mark (X) this column if entry in column ( I) is confidential. 

(3) Indicate the typical weight percent of each monomer or other reactant in the polymer. 

(4) Mark (X) the identity column if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer 

description on the TSCA Chemical Substance Inventory. 

(5) Mark (X) this column if entries in columns (3) and (4) are confidential. 

(6) Indicate the maximum weight percent of each monomer or other reactant that may be present as a residual in the polymer as manufactured for 

commercial purposes. 

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 of the identification report 

obtained from CAS Inventory Expert Service must be submitted as 

as attachment to this 

% 

% 

% 

% 

% 

% 

% 

reported in this notice (check one). 

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, if one can be reasonably ascertained. 

N -- Continued 

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

3. Impurities 

(a) 	 - IdentifY each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose. Provide the 

CAS Registry Number if available. If there are unidentified impurities, enter "unidentified." 

(b) 	 - Estimate the maximum weight % of each impurity. If there are . :ro. estimate their total weight %. 


Impurity and CAS Registry Number 
 Maximum Confipercent dential 

(b) 

% 

(a) 

X 

% X 

% 

% 

% 

% 

% 

[ ] Mark (X) this box if you attach a I sheet. 

4. Synonyms - Enter any chemical synonyms for the new chemical identified in subsection I or 2. 

~;~~~ X 

D Mark (X) this box if you attach a continuation sheet. 

5. 	 Trade identification - List trade names for the new chemical substance identified in subsection I or 2. 

X 

D Mark (X) this box if you attach a continuation sheet. 

6. 	 Generic chemical name - If you claim chemical identifY as confidential, you must provide a generic name for your substance that reveals 

the specific chemical identity of the new chemical substance to the maximum extent possible. Refer to the 

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

Fatty acid amide 

D Mark (X) this box if you attach a continuation sheet. 

7. 	 Byproducts - Describe .h~rn..l"~to resulting from the manufacture, processing, use, or disposal of the new 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 if you attach a cont;n"M;nn ~heet. 

Part I -- GENERAL INFORMATION -- Continued 

EPA FORM 77\0-25 (Rev. 5-95) 	 Page 6 
Section C -- PRODUCTION, IMPORT, AND USE INFORMATION: 

Mark (X) the "Confidential" box next to any item you claim as confidential. 

1. Production volume ­ Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum 

production volume for any consecutive l2-month period during the first three years of production. Estimates should be on 100% new chemical 

substance basis. For a Low Volume Exemption application, ifyou choose to have your notice reviewed at a lower production volume than 

10,000 kg/yr, specify the volume and mark (x) in the binding box. If granted, you are bound to this volume 

Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confi- Binding 

(100% new chemical substance basis) (100% new chemical substance basis) dential Option 

Mark (x) 

X 

2. Use Information -- You must make separate confidentiality claims for the description ofthe category ofuse, the percent ofproduction volume 

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

you claim as confidential. 

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

(2) Mark (X) this column if entry column (1) is confidential business information (CBI). 

(3) Indicate your willingness to have the information provided in column (1) binding. 

(4) -­ Estimate the percent of total production for the first three years devoted to each category of use. 

(5) -­ Mark (X) this column if entry in column (4) is confidential business information (CBI). 

(6) -­ Estimate the percent of the new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for 

commercial purposes at sites under your control associated with each category of use. 

(7) -­ Mark (X) this column if entry in column (6) is confidential business information (CBI). 

(8) -­ Indicate % of product volume expected for the listed ''use'' sectors. Mark more than one box if appropriate. Mark (X) to indicate 

your willingness to have the use type provided in (8) binding. 

(9) -­ Mark (X) this column if entr v(ies) in column (8) is (are) confidential business information (CBI). 

Category of use ( I) CBI Binding Produc- CBI %in CBI % ofsubstance expected per use CBI 

Option tion% Form­ (8) 

(5) 

(by function and application i.e. a dispersive dye Mart (x) ulation Site­ Con-oj< Indus- Com- Binding 

for finishin~ polyester fibers) (2) (3) (4) (6) (7) limited sumer trial mercial Option (9) 

X X X X 

% % 

% % 

% % 

% % 

% % 

% % 

% % 

oj< If you have identified a "consumer" use, please provide on a continuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumer products. 

In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumer products and describe the chemical reactions by which this 

substance loses its identity in the consumer product. 

DMark (X) this box if you attach a continuation sheet. 

b. Generic If you claim any category of use description in subsection 2a as confidential, enter a generic description ofthat category. Read the 

use Instructions Manual for examples of generic use descriptions. 

description Lubricant additive. 

o Mark (X) this box if you attach a continuation sheet. 

3. Hazard Information - Include in the notice a copy ofreasonable facsimile of any hazard warning statement, label, material safety data sheet, or other Binding 

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or practices Option 

for the safe handing, transport, use, or disposal ofthe new substance. List in part III hazard information you include. Mark (x) 

r8J Mark (X) this box if you attach hazard information. 

Part 11-- HUMAN EXPOSURE AND 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 


Maximum kglbatch (100"10 new chemic.l 
 Hourslbatch Batches/year 

subs/once) 

2. Continuous 

d. Process description Mark (X) to indicate your willingness to have your process description binding. 

(1) 	 Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specifY- e.g. 5 gallon pails, 55 gallon 

drum, rail car, tank truck, etc.). 

(2) 	 Provide thc identity, the approximate weight (by kgiday or kgibatch on a 100% new chemical substance basis), and entry point of all starting materials and 

feedstocks (including reactants, solvents, catalysts, etc.), and of all products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not 

used daily or per batch.). 

(3) 	 IdentifY by number the points of release, including small or intermittent releases, to the environment ofthe new chemical substance. 

Complete section A for each type ofmanufacture, processing, or use operation involving the new chemical substance at industrial sites you 

controL Importers do not have to complete this section for operations outside the U.S.; however, you may still have reporting requirements if 

there are further industrial or use You these See 

1. Confiofthe site at which the wi11 occur. dential 

Site address (number and street) 

City, County, State, ZIP code 

Ifthe same operation will occur at more than one site, enter the number of sites. IdentifY the 


additional sites on a continuation sheet, and if any of the sites have significantly different 


production rates or operations, include all the information requested in this section for those 


Marl<: (X) this box attach a continuation sheet. 

Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 8 
Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER - Continued I 

2. Occupational Exposure ­ You must make separate confidentiality claims for the description of worker activity, physical form ofthe new chemical substance, 

number of works exposed, and duration of activity. Mark (X) the "Confidential" box next to 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 if entry in column (I) is confidential business information (CBI). 

(3) -- Describe any protective equipment and engineering controls used to protect workers. 

(4) and (6) -Indicate your willingness to have the information provided in column (3) or (5) binding. 

(5) --Indicate the physical formes) of the new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance (ifpart ofa mixture) 

at the time of exposure. 

(7) -- Mark (X) this column if entry in column (5) is confidential business information (CBI). 

(8) -- Estimate the maximum number of workers involved in each activity for all sites combined. 

(9) -- Mark (X) this column if entry in column (8) is confidential business information (CBI). 

(10) and (II) -- Estimate the maximum duration ofthe activity for any worker in hours per day and days per year. 

(12) -- Mark (X) this column if entries 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., bag dumping, 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 if you attach a continuation sheet. 

3. Environmental Release and Disposal -- You must make separate confidentiality claims for the release number and the amount of the new chemical substance 

released and other release and disposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. 

(I) -- Enter the number of each release point identified in the process description, part II, section A, subsection I d(3). 

(2) -- Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology (in kg/day or kglbatch). 

(3) -- Mark (X) this column if entries in columns (I) and (2) are confidential business information (CBI). 

(4) --Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, POTW, or other (specify» to 

which the new substance will be released from that release point. 

(5) -- a. Describe control technology, if any, and control efficiency that will be used to limit the release ofthe new substance to the enviromnent. For releases 

disposed of on land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet, for 

each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate 

the amount released to the environment after control technology (in kg/day). 

(6) -- Mark (X) this column if entries in columns (4) and (5) are confidential business information (CBI). 

(7) --Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or 

NPDES numbers of the POTW (Publici Owned Treatment Works). Mark (X) if the POTW name or NPDES # is confidential business information (CBI). 

Release Amount of new 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 POTW provide name(s) below: CBI D Navigable D Other - Specify provide NPDES # 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 9 
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers do not have to complete this 

section for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate 

section B for each type ofprocessing, or use operation involving the new chemical substance. If the same operation is performed at more than one site describe the 

typical operation common to these sites. Identify additional sites on a continuation sheet. 

1. Operation Description To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. 

(I) -­ Diagram the major onit 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 the diagram. identifY by letter and briefly describe each worker activity. (2) -- Provide the identity, the approximate weight 

(by kg/day or kglbatch, on an 100% new chemical substance basis). and entl)' point of all feedstocks (including reactants. solvents and catalysts, etc) and all 

products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not used daily or per batch). (3) -- IdentifY by number the points ofrelease, 

ineluding small or intermittent releases, to the environment ofthe new chemical substance. (4) Please enter the # of sites (remember to identi/Y the locations of 

these sites on a continuation sheet): 

ca. 10 

# ofsiles 

o Mark (X) this box if you attach a continuation sheet. 

2. Worker Exposure/Environmental Release 

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

(2) -- Estimate the number of workers exposed for all sites combined. 

(4) -- Estimate the typical duration of exposure per worker in (a) hours per day and (b) days per year. 

(6) -- Describe physical form of exposure and % new chemical substance (if in mixture). and any protective equipment and engineering controls, if any. used to 

protect workers. 

(7) -- Estimate the percent ofthe new substance as formulated when packaged or used as a fmal product. 

(9) - From the process diagram above, enter the number of each release point. Complete 9-13 for each release point identified. 

(10) Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kglbatch). 

(12) -- Describe media of release i.e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, or other 

(specify) and control technology, if any, that will be used to limit the release of the new substance to the environment. 

(14) -­ Identi/ybyproducts which may result from the operation. 

(3), Mark (X) this column ifany of the proceeding entries are confidential business information (CBO. 

Letter #of Duration CBI Protective Equip. / %in CBl Release Amount of 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) this box if you 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 or bracket the specific information that you claim as confidential. 

In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce 

or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal 

ofthe PMN substance. Please include new information pertinent to pollution prevention, including source 

reduction, recycling activities and safer processes or products available due to the new chemical substance. 

Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological 

modification, process and procedure modification, product reformulation, raw materials substitution, and/or 

inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would 

otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions of pollution 

prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance 

with existing regulatory requirements and can be either quantitative or qualitative. The EPA is interested in the 

information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting 

ofrisks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In 

addition, information on the relative cost or performance characteristics ofthe PMN substance to potential 

alternatives may be provided. 

All information provided in this section will be taken into consideration during the review of this 

substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and 

examples. 

Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in the 

volume manufactured; (3) a reduction in the generation ofwaste materials through recycling, source reduction or other means; (4) a 

reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in 

the cost ofproduction and/or improved operation efficiency ofthe new chemical substance in comparison to existing chemical 

substances used in similar application; or (6) the extent to which the new chemical substance may be a substitute for an existing 

substance that poses a greater overall risk to human health or the 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 sections ofthe tonn and test data and other data (including physical/chemical properties and 

structure/activity infonnation), and optional infonnation after this page. Clearly identify the attachment and the section of the fonn to 

which it relates, if appropriate. Number consecutively the pages ofthe al:tachments. In the column below, enter the inclusive page 

numbers of each attachment. 

Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual tor guidance on 

how to claim any infonnation in an attachment as confidential. You must include with the saniti:liXI copy ofthe notice fonn a sanitized 

version of any attachment in which you claim infonnation as confidential. 

Attachment name Attachment 

DaI!.C number(s) 

14 

Material Safetv Data Sheet (MSDS) 

Physical Chemical Properties Worksheet 13 

Confidential 

X 

21 X 

24 X 

44 X 

62 X 

CAS Inventory Expert Service Reports 82 X 

~hiSbox ifyou attach a continuation sheet. Enter the attachment name and number. 

EPA FORM 7710-25 (Rev. 5-95) Page 12 
PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

To assist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and 

include it in the notice. IdentifY the property measured, the page of the notice on which the property appears, the value of the property, 

the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical 

state ofthe neat substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. 

Properties that are measured for mixtures or formulations should be so noted (% PMN substance in _). You are not required to 

submit this worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential 

information is properly protected. You should submit this worksheet as a supplement to your submission of test data. This worksheet 

is not a substitute for submission oftest data. 

Property 

~ Value lMeasuredo Confidentml 

(a) 

X 

Physical state of neat substance 

X 

Vapor pressure 

@ Temperature 20 °C 

X 

Density/relative density 

Solubility 

@ Temperature °C 

Solvent 

Solubility in water Temperature °C 

Melting temperature 

Boiling! sublimation temperature{aJ torr 

Spectra 

Dissociation constant 

Particle size distribution 

X 

Octanol! water partition coefficient 

Henry's Law constant 

Volitalization from water 

Volitalization from soil 

pH@ concentration 

Flammability 

Explodability I

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 

----------

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-------------------------~~!~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 

ILL 

Page 2 of 7 

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 

------------

__________________________ ___________________________ _ 

_______________________ _________________________ _ 

___________________________ ____________________________ _ 

Page 3 of 7 

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. 
-------------

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Page 4 of 7 

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 
------------------------------------------------------------------------------

------------------------------------------------------------------------------

Page 6 of 7 

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 

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Page 7 of 7 

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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 Study Report 


. 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 lit res 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 file 
Appendix 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 of the 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 of chloroform 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 of the 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 Mil1iQ water. 

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 

A mixture of oxygen (20%) and nitrogen (80%) 


was led through a bottle, containing 0.5· - 1 


litre 0.025 N Ba(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 N standardi zed 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 

N Ba(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 Cumul ati 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 

.:..~ --+ 

~ 

--~-

--­ o ), =*-~-TI~ I 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 


61 

4 


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 as that 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 test vessels 

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 

of the 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 1 Oml 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 

of an 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 of the 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 of the 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 that there 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 a ppeared 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 

1-19ures 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 77.7 
APPENDIX 3 Preparation of M7 Elendt Medium 

Substance Conc. of Stock (mgll) Volume added to 20 I 

Distilled Water (ml) 

H3B03 57190 2.5 

MnCI2.4H2O 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 

KH2P04 1430 2.0 

K2HP04 1840 2.0 

An Fe·EDTA Solution is prepared by adding 1.991 gil FeS04.7H20 and 5.0 gil Na2EDTA.2H20 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 

Cyanocobalamine (B12) 10 2.0

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 of tnese.,statements will be found in the fife 
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). 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 
Inventory Expert Service 


A division of the American Chemical Society Phone: 800-631-1884. 614-+17-3600 (ext. 2308) 

fax: 614-447-3747 

E-mail:answers@c:as.org 

INVENTORY EXPE}tT SER.'lICE REPOR'r 

lES-ORDER NUMBER: 


REGISTRY NUMBER: 


CA INDEX NAME 

," 

Please print the above CA Index Name on the appropriate page of your "PMN. 

i If this box is checked, CAS has made correction(s) "marked in red to 

~ . . your IES ord.r. pl•••• make the ..... correction(s) to your _ b.fore 

" 

submitting it to the EP~. 

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