# A. Ul CODn;o FORMS FOR SRC INDEXfNG


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### Aiv1EI;:. CJIEM
Contractor \-VILDUFE INTL LTD·. 

### S

**1-IOPt(_~:~** 

### Document Title LETTER fROM AivJER CHEM

**~lJ!3~\HTTINC1** 

### t:I 

### STUDY OF BR01'v!TNATED FL 

### \Tv1F RETARDAN 

### L W'ATTCI-fl\iiTS & 

### DATED 

### 2S GO

HEXABROt\10CYCI.ODODE('ANF,

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### ..·\TED FL'u\-1E RET/i.FD,\J'<·I·s
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### ---- ·----

# A 03

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# America : Chemistry COURTNEY 

### M. PRICE VIU PRESiDENT CHEMSfAR 

# ~oo RUG 29 Pr\ 3 03 

# Council Goodchenl'strv Makes It Possible

I 

# FYI-00-001392 August 28, 2000

**··~** :} (-)

**~~~n** 

### Dr. SusaD. 

### Wayland Acting 

### Assistant Administrator Office 

### ofPrevention, Pesticides, 

### and Toxic 

### Substances TS-7101 Environmental Protection 

### Agency 401 M StrEet, SW 

### Room 637, EastTower 

### vVashington, DC 20460 Dear Dr. 

### Wayland: Ul 

# Contain NO

The 

### American Chemistry Council 

### makes available 

### to the public and appropiate 

### government agencies final 

### reports of environrnentaL healtl1, and 

### safety research that it 

### manages. In

### keeping •.vith this 

### policy. 

### the following repo.rt(s) tl-cat 

### Lhe Americru.t. ChemistryCouncil's Brominated Flame Retardar1.t bdustry 

### Pa..t.el rece:J.ctly condL:cted 

### are enclosed:

5639. 

### 1) HEXABROMOCYCLODODECAi\.TE (HBCD): A Flow-Tlt.rough Bioconcentration Test with the Rain.bovc' Trout (Oncorhynchu:o. 

### rnykiss!; 

### 2) Broncinated Flame Retard.cm:t

**End-Ls~'r** 

### Su_rve.y-Phase 1. Johns University. These reports do not include con:fidentid information. If

### you have 

### any questions, 

### piease caLl Shcrrna.n of fl1};- 

### staff at 703-?41- Sinc,:en;lv vours.



### (\ 

# L '

**~** 

# ,-;;-

**~- tit..'L~**  

### Enclosures
R.r::sponsibJe

**Care·~** CL'1 0_..,.,


### .

### ...,.,

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### _,

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# A 

# 04

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### HEXABROMOCYCLODODECANE (HBCD): 

### A FLOW-THROUGH BIOCONCENTRATIO?\" TEST 

### W1TH THE RAINBOW TROUT 

### (Oncorhynchus mykiss) FrNAL REPORT WILDLIFE 

### INTERl"\JATIONAL LTD. PROJECT 

### Nlfl\.1BER: 439A-l i 

### l U.S. Environmental Protection Agency· 

### Series 850- Ecological Effects Test Guidelines OPPTS Nmnber 850.1730 and OECD Guideline 305 

### AUTHORS: 

### Kurt R. Drottar 

### Henry 0. Krueger, Ph D.
STUDY TNITIA

### TION DATE: October 

### 19, 1999 

### STUDY COMPLETION DAIT August 10.2000 

### Submitted 

### w Chemicall'v1a..nufacturcrs Association·s 

### Brominated Flame Relardru1t 

### Panel 

### 1300 Wilson Boul.evru·d Adington Virgm.ia 222U9



### 8598 Commerce DriYc EastmL I'vfal}'iand 2160 

### (410) 822-8600

# A 05 WILDLIFE INTERNATION_A_L_,_,_L_T_D_. _____ _ PROJECT

**~0:** 

### 439A-J ll

.. 2- 

### GOOD L-\BORATORY 

### PRt\CfiCE COlVIPLIANCE §TATEM::El'iT 

### SPONSOR: Chemical Manufacturers Assc-ciat10n's Brominate.d Flame Retarcb.nt Industry Panel TITLE: Hexabromocyc!ododecane (HBCD): A Flow-Through Bioconcentration Test

### Vvith tl1e Rai..;>bow Trout 

### (Oncorhvnchus myhss) WILDLIFE 

### INTERNATIONAL, LTD. PROJECT NU1v1BER: 

### 439A-lll STUDY COMPLETION: August 10,2000 This study was 

### conducted in 

### compliance

### \vith Good 

### Laboratory Practice Stzmdard::: as published hv Lhc U.S. Environmental Protection Agency 

### ir1 40 CFR Part 160, 17 August 1989; OECD Prmcip!es of Govd 

### Laboratory Practice (ENV!MC/CHEM (98) 17); and Japan lVlA.FF (59 NohSa.."1, Notification No..•

**~8.:'f'** 

### Agricultural Production Bureau) \vith the 

### foliov ..ing exception: 

### The stability of the test substance tmder 

### storage concEL

**~ns** 

### at the test site ·was not detem1med 

### in accordance 

### with Good 

### Laboratory Practice Stai<dards 

### STIJ1)Y DIRECTOR:



# /~

**Kurt~Drott~~ --~~~---------**  {00 DATe. Senior Biologist 

### SPONSOR APPROVAL
DATE

(! .. •• 

# rS..\Tt:: ....**//~_"-.//_/&_'~_::->**___

# A 06 WILDLIFE INTERNATIONAL, LTD. PROJECT NO 439A-! ll - 3 -

This study\vas examinedfor compliance 

### with Good Laboratmy Practice. StCL'1d<lfds i.lS 

### pubiished 

### by t.hc 

### U.S 2nvironmental Protection Agency 

### in 40 CFR Part 1 60, 17 August !989: 

### 0 ECD Principies of Good Laboratory Practice 

### (ENV/MC/CHEM (98) 17): and Japan 

### MAFF (59 NohSan, Notification 

### >.Jo 3850, Agricultural Protection Bureau). The dates of all inspections and audits ao.d 

### the dates 

### that any 

### findings were reported to the 

### Study Director and Laboratory :tvlanagement were as follows:

ACTIVITY Test Substance Preparacion Test Substance Preparation Test Initiation and \Vater Chemistry Measurements .iv1atri.x Fortification A .

'l.alytical Data and Draft Report Biological Data ar1d Draft Report Final Report 

### DATF CO:NDUCTED November l0. 1999 November 17. i 999 November 23, i999 December 7, l999 March \5 - 2 i, 2000 April l l -- 1 3 aJ1d l7. 200\' June 14, 200('



### -



# I~.**UL~~:_-**

**Ma~l** ? Quality Ass'lu·ance Program Super>;isor DATE REPORTED TO STUDY DIRECTOR: 

### M""NAG E\-LENT November 10, 1999 l'-Tovember 19. 1999 November 2.3, 1999 Decem be• 7, ] 999 l\:1arch 23.. 2000 .\larch 2i. 2

## c;oo Apnl J. 7. 2000

# A 07**~ILDLIFE INTERN~IONA~L~,_L~_T_D_.~~~~--~~~~~~~P_R~O~JE~C~T~N_'0~~-43~9~A~-~11~1**  -4- 

### REPORT \PPROVAL
SPONSOR: 

### Chemical Mar.ufacturers Association's Brominated Flame Retardant IndL!st:r;.- Panel TITLE Hexabromocyclododecane (HBCD)- A Flow-

### Through Biowncentratio'lTestwiththe RainbmvTrout 

### (Oncorhynchus mybss) \V1LDLIFE INTERNATIONAL, LTD_ PROJECT 

## NUN1BER: 439A-lll

STUDY DLR.ECTOR:

# K,~,~~~ Senior Biologist
'NlANAGEMENT:



# - //**~i:.r~~-- He;;:o.~eger,**  • h_D_ Director ofAquatic Toxicology and Non-

### Target Plants 

## 8 ofco DATE

# At\ 

# 08 WILDLIFE INTERNATIONAL, 

## LTD.

**PRGJEC'~ ~0**  

### · 439A-lll

- "i - 

### .fABLE OF CONTEJ\I!S Title/Cover Page..................................................................................... ···········-············...... . Good Laboratory Practice Compliance Statement. Quality Assurance Statement ................................-.................................................................... Report Approval. ......................................................................... Table ofContents ............................................................... . 

### Smnn1ary............................................................................................ Introduction . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . ... . .............................................. 

### Objective ........................................................................ .. Experimental Design............................... Materials and Methods.............................. .. . . . . . . . . . . . .. .. . . .. .. .. . ... . . . . . . . . . . . . . . . ... ....... Results and Discussion. Conclusions .................... References ..................................... TABLES Table 1 - Means and Ranges ofWater Quality Parameters Table 2- Concentrations ofHBCD 

### in Water Samples During tiu:: Uptake Phase. Table 3 - Concentrations ofHBCD in \Vater Samples Dwing the Depuratron Phase. Table 4- HBCD Concentrations 

### in Erliblr, l\onedible and \\'hole Fish Tissues ofRainbow Trout Exposed to 0 18 ,u.g HBCD/L Table 5- Day 35 BCF 

### Estimates for Rainbov.: Trout Exposed to 0 18

**~tg** 

### HBCDiL............. 

### Table 6- BIOFAC Model Estima•es for Rainbow Trout Exposed to 0 18 f.l.g HBCDiL .... 

### .... l 2

. ............. 4 .5



### 8

.. ...... 9



### .... 9

... . 16 18 19

. 2!



### 27

### \NILDUFE INTERNAT!ONAL, 

### LTD. PROjECT NO.· 439A-ll i



## --- -6- 

### TABLE OF COl\TEN'TS


### TABLES (Cont'd.) Table 7- HBCD Concentrations 

### ill Edible, Nonedible and VVhoie Fish 

### Tissues ofRainbow 

### Trout 1.8

**~tg** 

### HBCD,'L............................ 

### Table 8 - Steady-State BCF 

### Values for Rainbm.v Trout Exposed 

### to 1.

### 8 J.lg 

### HBCD/L Table 

### 9- BIOFAC 

### Model Estimates for 

### Rainbovv Trout 

### Exposed 

### io 1.8

**~J.g** 

### HBCD/L ..... 

### FIGlJRES Figure 

### 1. Concentrations ofHBCD 

### in Edible Fish Tissues ofRainbuw Trout Exposed to 

### 0.18 ,Ltg 

### HBCDIL...................................... ·······--····· ....... Figure 

### 2. Concentrations ofHBCD i..11 

### Nonedible Fish Tissues 

### ofRahabov; Trout

**E~<poscd** 

### to 

### 0.18 1-1-g HBCD!L....... . Figure 3. Concentrations ofHBCD in \Vhole Fish

**Ti~sues** 

### ofRainbO'I\ Trout Exposed 

### to 0 18 J-ig 

### HBCD(L. ............ ............ ... .................... __ ·--·--------·----·-··- Figure 4_ 

### Concentrations ofHBCD i.IJ. 

### Edible Fish 

### Tissues ofRainbow Trout Expose,d 

### to 1.8 1-1g 

### HBCD/L ........ .............................. __ ...................... Figure 5. 

### Concentrations ofHBCD in Nonedible Fish Tissues 

### ofRaii1bow Trout Exposed to 1.8 IJ.g 

### HBCD!L............... ···----------------··-··· . .. . _____ ...

**F1~rure** 

### 6. Concentratio,·s ofHBCD in \\'bole Fish Tissues of Rambow Trout Exposed 

### to 1.8 IJ.g 

### HBCD!L....... 

### APPENDICES Appendix 

### I- Protacol, Protocol

**A~mcndmcnt** 

### and Protocol Dc,·iation . Appendix 

### l1- 

### Test_ Substance Characterization ... Appendix I1I- Specif1c Conductance, I-Tardness,

**Aikalinit~,** 

### and 

### of\Vcll \Vater 

### l\llei1sured 

### Duxing the 

### 4-Week Period Immediately Preceding the Test .. 

### 29
34

35

.... 36 37

39 

### 40

41



### 72

### \1'/tLDLIFE INTERNATIONAL, 

### LTD. PROJECT NO· 439A-lll

- 7- 

### TABLE OF CONTE:\'TS

- Continued - APPENDICES (ConCd.) ApiJenc.ix IV- Analyses ofPesticides, Organics, Metals and Other lnorganics in

### Wildlife International, Ltd Well \Vater.. .................................... **~~** ... /_"'' 

### Appendix V- The Analysis ofHBCD Concentrations in Freshwater/Rainbow Trout Tissue 

### in Supp xt ofWildlife 

### lntemational, Ltd. Project No.: 439A-lll ............... Appendix VI - Temper<.:ture and pH ofWater 

### in the Test Chfuubers ............................................. . 

### 127 Appendix VTI- 

### Dissolved Oxygen ofWater in the Test Chambers. ....... 128 Appendix VIII - Hardness,

**A1~alinity,** 

### Conductivity and TOC of\Vater m t(Ie 

### :)oivent Control 1.30 Appendix IX - Apper,dix 

### XCtu.wlativ.o: Percent 

### 1\krtalit:y and Treatment-Related Effect.... ..................... Ul 

### Personnellnvolved in the 

### Study ................................................................. 1 -;-;
...

**l-"~** i

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO. 439A-l I 1

SPONSOR: SPONSOR'S REPRESEl\'TATIVE: LOCA110N OF STUDY, RAW DATA AND A COPY OF TilE FINAL REPORT: WILDLIFE**INTE~'\IATIONAL,** LTD. PROJECT NUMBER: TEST SUBSTAl'\ICE: STIJDY: NO!vllNAL TEST CONCEJ\'TRATION3: IvlEAN lvffiASURED TEST CONCENTRATIONS: TEST DATES: - g

### SUM!\tL-\RY Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel Ms. Wendy SheiTilan Wildlife International, Ltd. Easton, Maryland 21601 

### 439A-lll Hexabromocyclododecane (HBCD) Hexabrom0eydododecane (HBCD): A Flmv-Through Bioconcentration Test 

### \Vith the RainbO\v Trout (Oncorh<.cncims 

### mykiss) ·· Solvent ControL 

### 0.34 and 3.4 1-1-g HBCDfL Solvent Control, 0.18 ar1d l.8 !J.g 

### HBCDfL Experimental Start - November 23, 1999 Bi;)logicaJ 

### Temlination- February 

### L 2000 Experm1ental Termination- February 28, 2000 

# l ii !;**!!:::L=EN=G=T=H=O=F=l=~E=S=T=:==========7=0=D=="ays~I)~'"** 

### Uptake, 35-Dav Depw-at1on) TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGAN1SMS MEASUR.E!v!ENTS OF 

### l0 SOLVENT CONTROL F1SH COLLECTED AT TEST TER!vTINATION: WEIGHT 

### (g): STANDARD LENGTH 

### (mm): RESULTS: 

### (0.18 

### fl.g HBCD/L measured; 0.34 !1-g 

### HBCDIL nominal) 

### Rai;<bo-vv Trout (Onca:-J:ynchus myfciss) 

### ""'->mas Fish Co·nparrv '.B·1X85i - 

### , . ::rsor., t'1ltforru--: 96007



# . ., JUVenue:o:


### Mean= 3.46; Range= 2 33 to 5 52 M.;:an = 

### 72: R<:mge**~** 64 

### to 86 Edible Nonu.iibte DAY35BCF: 

### 6531 20.726 ......... ·-=--====;;;**~** 

### tiote: Steadv-State was not 

### achieved 

### in this**tre<?tment~~""Rr~o~u~p~.**===================="

I LTS: \.'1.8 

### f.lg 

### HBCD/L measured; 3.4**~** 

### HBCDIL nominal) DY-STATE BCF: 

### Edible Nonedibk 

### 4,650 12,866 

### 8.974 11 

### ,, 

## II li :!**---~-----** ----**--~ ·-::-=~o:==·:J** 

### WILDLIFE INTERNATIONAL, 

## LTD. PROJECT NO. 439A- 

### J 11

- 9- 

### INTRGDVCTION A rainbow trout, 

### Uncc hynchus myiass, bioconcentration stJJdy was conducted for the Chen'jcaJ 

### l'vianufacturers Association's Brorrinatec! Flame Retardant Industry Panel at the Wildlife IntematJOn'lL Ltd**aqu~tic** 

### toxicology fa,:.;ility in EastCJ11, 

### Maryland. Them-life phase of

### the tr:st was 

### conducted frcm**~\member** 

### 23. 1999 

### to**~ebmary** 

### 1, 2000. Raw data generated 

### by Wildlife International. Ltd and a copy**oft.~e** 

### fined re-port are filed under 

### Project i'-lumber 

### 439A-lll in archives located on the Wildlife International, Ltd. site 

### OBJECTI\''E The objective ofthis study was to determine ihe bioconceni.ration potential ofhexabromocvclododecanc (HBCD) 

### in rainbow trout. The solubility ofHBCD

### in water at 

### 25°C is 3.4**~~g** 

### HBCD/L The 96-hour LC50 for 

### rainbow trout is >6.8 f..lg HBCD/L. 

### EXPERIMENT,<\_L DESIGN The bioconcentration test consisted of a 

### 35-day uptake phase followed by a 

### 35-day depurat10n 

### phusc During the uptakephase, the test organisms vvere exposed L."l. 

### one: ofthree groups: l l A sol>:entcontrol ml. acetone/L); 2) A nominal concentration of0.34 f..lg 

### HBCDfL, or 3) 

### A norrlli"'la! concenuation of3

### 4 f).g 

### HBCD/L The 3.4 f..Lg 

### HBCD/L exposureconcentration vms chosen 

### by**th~** 

### Sponsorbased on the '"ater solub:lltY ofilie tesL 

### substance. At the start ofthe depuration phase. stock flo\v to the treared groups v;as stopped and the r.1mh.w. 

### trout 

### were exposed to dilution water without 

### HBCD for the remainder ofthe test Each 

### test chamber 

### contained 85 rai.\"1bow trout at test tll..itiJ.tion, <md one replic.:Jte '-Vas tesltod 

### for each treatment and the solvent control. 

### Water samples vvere collected on Day -4 (pre-test),**Da~** 

### -1 (pre-test> on uptake Days 0 (0 and 4 hours), l, 3, 7, 14, 2L 28 ;md 35 and on depuration Days L 

### ?, 7, lO. lei, 2L ?.8 a.nd**~)dunn;;** 

### the test and analyzed for HBCD using liquid chromatography-mass spectrometry also collected at selected V'iater 

### sample collecilon penods dunng the test and analyzni**i~1r** 

### HBCD hy 

### LC"\-IS The results ofthese analyses >vere used to calculate the BCF values, uptoke rates and dcpmation rates in edible tissue. noncdiblc tissue &!d whole fish.

### WJLDLIFE INTERNATIONAL, 

## LTD. PROJECT NO. 4?9A-lll - l0- 

### 1\L.\TERIALS AND METHODS The study was conducted according to the procedures outh1.ed inthe protocol, "HexabromOC):clcdodecane (HBCD): A Flow-Through Bioconcentration Test \vita 

### the Rainbow Trout 

### (Oncorhvnciws mykiss)" (Appendix I). The protocol was based onprocedures outlined 

### in U.S. Environmental Protection Agenc:v Se.ries 

### 850- Ecological Effects fest Guidelines OPPTS Xurnber 850.1730 (I); ASTJI..-fStandardEl022-8.:t Standard 

### Practicefor ConductingBioconcentration Tests with Fishes andSaint-merBivalve!vfolluscs 

### (2): and OECD Guideline for Testing ofChemicals 

### 305, Bioconcentration· 

### Flow-Through Fish Test 

### (3) Test Substance The test subst<mce consisted ofa composite of

### HBCD samples**receivf~d** 

### from thr<..;c manufacturers The material's 

### identity and 

### date received from 

### each ofthe manufacturers is given belmv Wildlife**lntema~ionJl** 

### Lid Manufacturer Lot/Batch 

### Date Received Identification**Nurnb~r** ------------------- 

### Great Lakes Chemical Corporation Not Given June 

### 19, 1998 

### 45L' Eurobrom 

### b.Y. 971201 June25, 

### i998 4520 

### Albemarle Corporation 

### 33449-lSX Ju:1e 29, 1998 The 

### composite

### test substance was 

### assigned Vlildlife 

### International Ltd. identification

### nLrrnber 4615 a..nc1 \.Yas 

### stored 

### under ambientconditions A 

### subsan1pie ofthe 

### composite test substa11cc \VOJS 

### sbpped to Albemarle Corporatlc·n for characterization and purity a..r1alyses (Appendix H). 

### The results ofthe analyses indicated the composi.t:'" tc:st 

### substance was homogeneous and contained the following components 

### Alpha Isomer Beta Isomer Gamma 

### Isomer The conclusion of

### the characterization\vas that 

### the test article wJs 

### HBCD ;.>-ith a The test substance was stored 

### at room 

### temperature.

# A 14 WILDLiFE INTERNATIONAL, 

### LTD. PROJECT

**i~O**

### .. :1.39A-lll

- ll - 

### Preparation ofTest Sclutions ·n,c nominal test concentrations >vere 0.34 and 3.4

**~J.g** 

### HBCD/L. A primary stock '.vas prepared by dissoivingthe 

### t..:st substance. 

### in aceturre ;:t 

### a roncentrat:un of56.7 mg HBCD/L The 

### prin1;:u;· stockwa.s mvcned to mix 

### A 3econdary stock wac; prepared at a concentr?.tion of5.67 mg HBCD 

### 'Lby pr..:Jport]ona: dilution 

### ,,rtlle J., .• ,

### "rry siod· 

### '"itl1

**acec~.ne.** 

### The stock soiu.: Jns wen. :in_1ectcd 

### ir1to the diluter n:ixing

**~hambers** 

### lai. a rate oi 

### 0 n ··. mLiminute) where 

### th;;:y

**Y>en~'** 

### .:;-.;_ed with 

### well water

### fat a mte of

### 5CU mL/miu.'1ttte) t:J 

### adli;:,ethe

### desired test 

### conce: "trat'rms. Theresul"a:::!t test concentrations were not adjusted for the punt)' ofthe acti .-e

**i...>gredi~·nt** 

### w test substance 

### (90.0'%). The solventcontrol was prepared by 

### adding acetone 

### to the 

### r1i.xing chamber for the solvent controL The c0;-;centration ofa:::etone in the solvent contrcl and both 

### HBCDtreatment grcLi 1 

### ]s \\CJS 

### 0.060 

### mLJL Test Organism The rainbow trout, 

### Oncorhynchus myl.:iss, was selected as 

### t.he tesL species 

### for this siud:y. 

### The ra:nbo'' trout is one oftherecommended freshwater fish species for use in bioconcent1auon tests ( 1. :?. ,

**?-~>** 

### Rainbcow 1n_,il! 

### used 

### in the test were obtai!led from Thomas F;:,h Company, /illderson, California. Identification of1.hc SPCClCS 

### was verified 

### by the supplier. The rainbmv trout were held 

### in Wildlife 

### Intemari -·JaL Ltd. \Nell 

### vvater for l05 days pnor to 

### ::es:i.ng The 

### rainbm.v trout\Vere 

### fed at least once daily during holding. Th:.' fish 

### v.-ere acclimatedto test

**condition~' !~x**  

### 49 i1ours 

### prior t0 

### test initiation. During the holdin.; and acclimation penods iJJ.e f\c;h

**~;howcd** 

### no

**si~11s** 

### •.)fdisease c:r 

### s:rcss During

### the 14-ciay holdingperiod 

### precedingt..l)e test. water tcmper<lture:-; fron112.1 

### tul.2_s·.)c_ ThepHof the waterranged from 8.0 to 8 3 and dissolved oxygen nmged from 9.4 

### w l 0.0 mg/L lnstrumentation use-d 

### Co; water measurements 

### are described 

### in the

### Environmemal Condtnons section 

### ofthis 

### rc:p{>rt :'\t 

### tc:s\ u1.c rainbow trout were collectedfrom the acclimation taiJks and 

### i.ndiscrinunole!y dislnbmcd l tu 2

**:;~** J. 

### m:n•:: nne:· rl:c 

### test cbambers until each chamber contained 85 fish. Zeigler Brothers, Inc., Gardners, Pennsy·lvani<l. 

### Rainbo>A trout 'Xt:ft' nci

**\~;.:!** 

### hm.vever, 

### they vver:;., fed dailydw·ing the test. Feeding artd sampling

**sch~~dulcs** 

### v.-crc. ccordi.narcd so the;: f;sh

**'"''"it~** 

### sampled at least four bours after feedi.ng.

# B 01 WILDLIFE 

# !NT!:::RNAT-'-I....::O....::N....::A-'--L_,_,

## _L_T_D_.;.__________ _ PROJECT NO. :t39A-lll

- 12- .1\11 fish used 

### in ;:he test

**w~re** 

### from the same source and year class, :mdthe standard length oftt'1e longest fish was no more than twice the length ofthe shortest. The length and weight offish in the solvent control •v<:re considered tv be representative of aU fish used 

### in the tesL The mean 

### total leng-ch of 10 solvem controi i!sh measured 

### at the end 

### ofthe 

### test was 72 mm '''lith a range 

### of64 to 86mm. The <tverage wet weight (blotted cL.-y) 

### was 3.46 grams \\':ith 

### a range 

### of2.33 to 5.52 

### grams. Loadingwas defmed as 

### tl1e total wetweight offishperliu oftest water that passed tlrrough the test chamber 

### in 7.4 hours, and ;,vas detenuined to be 0 4] 

### g fish/Liday Test Apparatus A continuous-flow diluter 

### was used to deliver each concentration of the test substance 

### aDd a solvent controL Syringe pumps (Harvard Apparatus, South

### Natick, i'vtl\) 

### were used to deli>:er the test 

### substance stock solutions anJ acetone for the solvent control into mixing 

### chambers ass;gned to each treatrnenL 

### The 

### siock solution:;: were mixed 

### witi1 dilutionwater ii1. 

### the nii.xing chambers prior to deLivery 

### to the test 

### crill.rubus The±1ow ofdilution water to the test chambers was controlled by rotameters. The delivery of>.vater irom the ;-ur.,m-:.eters was checked prior to the test and at approximately weekly- intervals th.ereafrer 

### Approximmely 9.0 YOlL!--ne 

### additions of

### test water were delivered to the

**te~'t** 

### chambers every 

### :.A hours. The general operatro;-, oftho:e diL,;,-r 

### was checked at least two tunes a day during the 

### test. Test chambers were 106-L stainless steel 

### aquaria filled 

### with approxilnatdy 80 L 

### of tcs1 :<oluuon 

### The: 

### depth of the test 

### water in a representative chamber 

### v.'as 25 ern. 

### --rest

**charnbe~** 

### s \Ycre indiscriminately positioned 

### in a temperature-controlled v;aier baih deslf,rncd 

### iomaix1tain a cousraru tcmper'l'crrc 

### The water bath w:ts enclosed 

### in a 

### plexiglass ventilation hood 

### m. order to mimmize any PU"A'cu"' 

### for cr:Jsscontamination. Testchambers were siphoneddaily and 

### ciea..r1cd during the test to rerr :excessfeed_ fecal matter, algae rnd

### bacterial grmvth. Testchambers were identified concentration. Dilution \,Vater The water used for holdi,'1g and uru:nediately 

### preceding the test are presen1 

### _ed in 

### the number,

45 rncLc-r:s

# B 02 VJ!LDLIFE INTERNATIONAL, 

### LTD. PROJECT

**~\'0.•** 

### 439A-lll

- 13 - The well water was passed through a sa..'1d filter to remove particles greater than approximate!} 25 

### f..!m, and pumped into a 37,800-L storage tank and aemted with spray nozzles. Prior to use, the -vvater again was frlte.·ed (0.4511J1l) 

### to removemicroorganisms and particles. Theresults ofperiodic analysesperfmmedtomeasure the concentrations of selected contanlln.ants m well

**\'~ater** 

### used

**b~'** 

### Wildlife International, Ltd. are presem<Xl in 

### Appendix 

### IV. Environmental Conditiollii The target temperature range for the test was 12 ± 

### 1°C. Tempcrattrre was recorded 

### continuously 

### in L0.c solventcontrol>v:ith a Fulscope ERIC Recorder (1900 1Seriesmodel no. 

### A). Temperature vvas 

### also measuredin all test chambers atLIJ.e begimring and endofthetest and at weekly intervals during the test, with a liquid-in-glass thermometer. Dissolvedoxygen was measured 

### with a Yello·w Springs L1strument Company, l.tl<: 

### }\!fodel5lB d:ssoh cd ox·ygen meter. Measurements were made daily in each test chamber duri..ng the test. l\:Ieasu.remcnts of were made in eaLh test chamber at the beginning and eu.d ofthe test &.""Jd at V\'eekly intervals during lhe test usmg a Fisher Accumet Model 915 pH meter. Hardness, alkalinil}, conductivity· aDd total organic carbon \TOCi were measured m the 

### sr.:,h·cnt control'" the beginning andend ofthe test, and at 

### ·weekly i_ntei-"·als during ilK: lest. Hardness &.!d all:-:aEcil} 

### were measured by titration based 

### rm procedures 

### in Srandard 

### 1'i1ethods

### for the

**l::~-caminatwn** 

### ofT'Vcter 

### and Wos!e1-rwer· 

### Conductivity· vvas measured 

### using a Yellow Springs illst.l.nnent Corcpany, Inc. Model33 SaEnity-C 

### Temperaturemeter. Total organic "arbon v;as measured using a 

### Sh.L.uadzu model TOC--5000 

### to1 a1 (Jrga'1jc 

### carbon 

### aJ1alyz:er. Ambient room bght was used to illmnir1ate 

### the test. 

### systems. Fluon:sccnt tubes thJI 

### emitted similar to natural sunlight (Colortone® 50) vvcre controlled 

### by an cmtomatic timer to 

### a OJ: 

### 16 hours ofl1ght and 8 hours ofdarkness A 30-rninute transition oflow lir-hts went on and offto avoid sudden changes in lwht intensih



# - -

**~** .·

# B 03 WILDLIFE INTERNATIONAL, LTD. PROJECT NO. 439A-lll



### - 14- Observations All fish were observed once eachday to evaluatethenumberofmortalities and thenumber ofi..<div:id;;als exhibiting sigp..s ofabnormal behavior. Procedures for Exposure ofFish to HBCD The test chambers were conditioned by delivering HBCD tothe diluter system for approximately 6 days before adding the fish. Watersamples were collectedtv:iceduringthe pre-testperiod to con:fum thatequilibrium concentrations oftest substance in the test chambers were achieved prior to adding the fish. Stock solution sampleswere 

### also collected

### on selecteddays to confirmthe concentration oftest substance in thestock :-:olution..s. At the end ofthe pre-test period, the uptake phase ofthe test \vas i.Pitiated 

### on November- 2::;. 

### 199(). by 

### placingthe fish inthe test chambers. Rainbowtroutwere impartially removed from theholdingtank in groups of 

### 1to 2. Thegroups ofrainbowtroutwere distributed among t.he test chainbers until eachtest chamber cc,ntamed 85 fish. The duration of the uptake phase was 35 days. At the end of the uptaJ;\:e phase, stock flow 

### w ibte treatment groups 

### v:as stopped and therainbowtrout were exposedto dilution\vatenvithout f-'J3CD for a penc:dof 35 days. CoHection and Analysis ofWater Samples Water samples were collected on Days 0 (0 hours and 4 hours), l, 3, 7, 14, 2L 28 &!d 35 oftl1e phase. Watersa:11ples were also collected onDays 1, 3, 7, 10, 14, 2 L 28 and 35 ofthedepurJtionphnsc. Ai. each \Vater sampling interval, two water samples were collected from the solvent control and three sarnpks "·ere coLlected from each ofthe tvl..-o 

### HBCDtreatment groups. O;;e solvent control sample and two samptcs from eacb of

### the HBCDtreatment groups were analyzed for

**~IBCD.** 

### Theremaining 

### s&-nples were held in reserve 3S lx1c.h:up 

### samples. All water samples were collected from mjd-depth of each test chamber usL.1g a class A YC·lurnetric 

### pipette. The water samples were analyzed for HBCD 

### by liquid chromatography-mass spectrometn· 

### i!VLS) Procedures for extraction and analysis ofthe water samples are nrrwu"iPri 

### in Appendix V. 

### Ali \Vil1er 

### stored at approximately 4°C until analysis. CoJ!lection and Aiialysis ofTissue Samples Tissue samples were collected on Days 0 (4 hours), L 3, 7, 

### 14.

# n. 28 

### mxd 35 ofilie samples were also collected on Days l, 3, 7, 10, 14, 21,28 ar1d 35 of the depuration ,-\f. 

### each tissue

# H U4 WILDLIFE INTERNATIONAL, LTD. PROJECT NO. 439A-lll

- 15- sampling interval, a sufficientnumber offish were collectedtoprovidetvvo replicate samples ofsolYent 

### control fish and four replicate samples ofeach HBCD treatment group. F1sh were impartially removed fwm the tesi chambers and euthanized by severingthe spinal cord above the opercularregion. The fish Vl'ere blotted dry and measuredfortotallength andwetweightvvithin approximately 

### 15 minutesofcollection, whenpossible. Eachfish was then rinsed with dilution water, blotted dry again and dissected into edible arid nonedible tissue fractions Dissection -.vas accomplished 

### by making an incision fromjust posterior to 

### Lfte base ofthe pectoral fm dorsrdly through the spinal cord. The head, fms and viscera ·were removed from the body and were considered to be nonedib]e tissue. Theremainingtissuewas consideredtheedibletissue Tissue samples ·were tnu"1Sferred totared scintillation vials and weighed. Procedures for extraction and anai:ysis ofthe tissue s?,nJples are provided i.,, 

### Appendix 

### V. All tissue samples were stored at approxi.-nately -14'"'C: until analysis. Tissue samplescollectedon Day 3 arid 7 ofthe depu;ation phase were not analyzed due to tl1e pattern ofdepUTation observed Tissue Lipid Content

**and_Q[y~** 

### Selected fish were collected 

### to deterrrinehpid content ;:tiJd 

### dry

### vveig1i: (_Appendi.x 1l1edetermination of lipid content and dry ·weightprovides thepotential to express BCF values in temlS oflipidcontent and 

### v,-eight Data Analvsjs Replicate tissue measurements were averaged to report the concentration oftest substance m fsh 1:issue Whole fish concentrations •vere calculatedbasedonthe sum ofthe ed.ible andnoned.ible 

### pac-rs. The 

### stead:·-sii:tc bioconcentration factor (BCF) values v:ere detemlLi!ed from the tissue concentrations 

### at ""'""'''"'' dividedbythe average waterconce:1tration. Tissue concentrations were consideredto be at

**~n,-,,rpnt** 

### if three consecutive sets oftissue

**concentratio~ts** 

### ·were not sign.ificarr!Jy different (p > 

### 0.05) using nnah·s1s of variance. Ifapparent steady-statewas not achieved, concentration 

### on uptake day 35. 

### 35 BCF

### values were cakuJate.d using the averagetissue
The kinetic bioconcentratio:n factor (BCFK), edible, nonedible and whole fish using BIOFAC rare and estimation routine vvhich estimates rate constants from a s·et of c:N1 """"' 

### constants are then used to calculate a BCFK (BCFK = 

### K,rK2) 

### BI0 FAC IS 

### a nonLinear hrne-concentration

**datr._~. 1l~esc**  

### rate

### WILDLIFE**!NT~RNATiONAL,** 

### LTD. PROJECT NO.: 439A-l11

- 16- 

### RESULTS A.ND DISCUSSION Water ChemJstr_y Means andranges oftemperature, dissolved m.. ')'gen, pH ofthe ·water in the test chambers are presented in Table l. 

### The individual measurements are given 

### in Appendicies VI and VII. Water temperat-ures m tt'le test chambers were withinthetemperature; rangeof 12± 1 

# oc establishedforthe test. Dissolvedox·ygen concentrations remained 2:8.8 

### mg!L (81 percent

### ofsaturation) throughout

### the test. Measurements ofpHranged from 7.9 to 8.3. Weekly measurements ofhardness, alkalinity andconductivityremainedconsistentthroughouttt'1e test (Table l and Appendix VIII). Observations ofMortality and Clinical Sims Observations ofmortality and clinical signs are presented

### inAppendix IX. Nomor..alities were 

### ohserved during the test. 

### In addition, no treatment-related clinical signs oftoxicity 'ivere observed dt.ttit<g 

### tlJ.e test Concentrations 

### ofHBCD 

### in Water Concentrations of.li"BCD 

### in the solventcontrol were <LOQ (<0.0250 

### !J.g HBCD/L)excepton Day 

### 28 &1d 35 

### ofthe depuration phase ofthe test (Tables 2 

### and 3). On

### Days 28 and 35, HBCDwas measured. t-bl?ce>-er, 

### it 

### is notlikelythatHBCD was actuallypresent

### in the so]ventcontrol. 

### Itis suspected that the sarnples were e:;,:posed to contamination at some point during the analysis. Measuredconcentrations ofHBCD during the uptal:.:e phase 

### inthe 0. 34 

### rJ.g HBCD/Ltreatment.

### group v,'ere 

### extremely variable. The valuesranged from <LOQ to 0.231 

### t'g HBCD/L. Even

### >N:ith**frequ,~ntck.an1I1f** 

### ofthe test chamber to remove potentially adsorptive 

### surfaces, it 

### \Vas 

### difficult to maintain a constant ::cxposurc- <1! 

### this concentration. When concentrations measured during the uptak.e phase were averaged, the mean measured concentration was 0.18 )lg 

### HBCD/Lwhichrepresented 53% ofthe 

### nominal test

### concentration Conct'ntrations of HBCD during the**d~:puration** 

### phase were generally <LOQ. Measured concentrations ofHBCDduring the uptake

### phase inthe 3.4 J.lg HBCD/L treatment 

### group ·were 

### less variable. The values ranged from 0.972 to 2.68 !J.g HBCD/L 

### \:Vl1en concentrations 

### measur:;cd tlJe 

### uptake phase were averaged, the mean measure-.d concentration was 1. 8 p.g 

### I-I.\SCD!L which represeDted 53'>0 of

### WiLDLiFE INTERNATIONAL, 

### LTD. PROJECTNO. 439A-lll



### - 17- the 

### nominal test concentration. 

### Five of the 16 samples 

### analyzed during 

### the depuration 

### phase contaL.11ed measurable HBCD. Concentrations ofHBCD in 

### Fish Tissues Excluding two 

### samples, there ·were I'') 

### quantifiable HBCD concem'rations 

### in the solvent control tissues 

### (Appendix V). The tvvo samples 

### which did contain 

### HBCD appeared 

### to have been conta.."TT.inated 

### The concentrations ofHBCD in 

### tissues offish e:<-lJOSed to 0.18 !J.g 

### HBCDfL are presented 

### in T<Jble 

### 4. HBCD concentrations in edible 

### and nonedible tissues appeared 

### to :reach steady-state at 

### Day 21 (Figures 

### l-3). Uptul:e 

### Day21 l'nd 28 tissue concentrations 

### werenot significantly· different 

### (p >0.05). However on

### uptake Day 35,

### thetissue concentrations increased unexpectedly. Dunnett's test showedthattheDay3 5 

### tissue concentrations 

### were significantly 

### different fromthe 

### Day21 and 28 tissue concentrations 

### (p ::: 0 05). Consequently, steady-state was not achieved 

### inthis

### treatmentgroup. Theunexpectedincrease in tissueconcentrations on Day 35 may have 

### been due to the variability in the measured 

### water concentrations in this treatment group. Estimates ofh'lc 

### BCF values for 

### the 0.18 

### !.lg HBCD/L treatment group \vere 

### based on Day 

### 35 tissue concentrations od:v 

### (Table 5) BIOFAC estimate:; 

### ofthetime to

### reach 90%of.;teady stateinedibletissue, nonedible 

### tissue and whole fish ,,·ere 

### 118, 99 and 101 days, respectively (Table 6). DUl-ing Ll;.c 

### depuration 

### phase ofthe tesi., HBCD -was eiimimted slowly with 

### estimates oftimeto

### reach 50%clearance 

### of35, 30 a..'1d 

### 30 days 

### for edibletissue, nonedibktissue

### ill:d ·whole fish, 

### respectivel:y. The concentrations of HBCD 

### in tissues 

### of fish 

### exposed to 1 3 Ptg B-::BCD/L arc 

### presented in Table 7 

### HBCD concentrations 

### in edible tissues appeared to reach steady-state at Day 14 (Figure4) An2Jysis 

### ofvariance showed 

### that HBCD concentrations for

**upta.~e** 

### Days 14 - 35 were 

### not significa.ntly different (:_., >0.05). 

### Consequently, the 

### BCFvalue for edibletissue was calcillated 

### based on the average tissue concentration 

### forup!ake Days 14-35 (Table 8). Similarly, 

### HBCD concentrations 

### in nonedible tissues reached stead:.-state at. Day 21 

### (Figure 5). 

### Analysis of

### variance shm:ved that HBCD 

### concentrations on uptake 

### 2!, 28 and 35 were not significantly 

### different (p > 

### 0.05) and !:he BCF ·was 

### calculated 

### based on the average tissue concentration 

### for days 21 - 35. Whole fish concentrations alsc; 

### reached steady·-state on Da:v 

### 21 (Figure 6) and the BCF'Nas basedonthe average tissue concentrations for days 21 - 35. BIOFAC estimates ofthe ti.me toreach 90% ofsteady· state in edibie tissue, nonedible tissue and whole fish were 63, 65 ;;md 64 days, respectiYely (Table 9) 

### the

### WILDLIFE INTERNATIONAL. 

### LTD. PROJECT NO.: 439A-11l



### - 18- depuration phase ofthe test, HBCD was eliminated slowly with estimates oftime to recch 

### 50% clearance of 

### 19, 20 and 19 days for edible tissue, nonedible tissue and

**'iV~lOk** 

### fish, respectively. 

### CONCLUSIONS Hexabromocyclododecane (HBCD)rapidlybioconcentrllied inthetissues of rainbowtrout

**(Or~corhynclws** myldss). 

### After 

### only 4 hours ofexposure, measurable concentrations ofHBCD were present 

### in the fish tissues. 

### Rainbowtrout exposedto 0.18 j.!g HBCDIL didnotreach apparentsteady-state tissue concentrations during

### the lengthofthetest Rainbowtroutexposedt.o 

### 1.8 j.!g

### HBCD/L acrucved steady-statetissue concentrations atDay 21. 

### In this study, the BCF calculated for the 1.8 j.!g HBCDIL treatment group was considered to be a better estimate ofthe BCF because: 

### 1) Water concentrations were mainta:u1ed more

**consistent~');** 

### and 2) 

### Sieady-state vvas achieved. TheBCF values for edible,nonedible and whole fish were4,650, 12)?.66 and 8,974,

**re::~pecti-,elv** 

### Clearance ofHBCDfrom fish tissueduring depurationwas slow. The BIOF AC estiu:;ates for timetoreach

**50"--~** 

### clearance were 19, 20 and 19 days for edible, nonedibie and >vhole 

### fish tissue, respectively

### WILDLIFE INTERNATIONAL, LTD. PROJECT**~0.** 

### 439A-lll

- 19- 

### REFERENCES U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelmes 

### (draft), OPPTS ?\umber 850.1730: Fish BL""'F 

### 2 ASTM Standard E1022-84. 1988. 

### Standard Practicefor Conducting Bioconcemration Test:; with Fishes andSaltwaterBivalveMolluscs. American Society for Testing and l'v1aterials 3 OECD Guideline for Testing ofChemicals 305. 

### 1988 

### Bioconcemratlon. Flmr-Through Fish Test. 4 APHA, AWWA,WPCF. 1985 

### StandardA1ethodsfor the Examination ofWaterand WasteH'arer 16th Edition, American Public**HealL~** 

### Association. American Water Works Association \Vater Pollution Control Federation, New York 5 

### BIOFAC. 1991. September 19, lS91 version. The Dow Chemical Company, Midland, Michiga11.

### WILDLIFE INTERNATIONAL, 

### LTD.

Sponsor: Test Substance: Test Organism: Dilution Water:

Uptake Phase

Nominal Concentration (Jlg 

### HBCDIL) Solvent Control 0.34
3.4 

## -----

- 20- 

### Table 1 Means and Ranges ofWater Quality Parameters Chemical Manufacturers Association's Brominated Flame Retardant IndustryPanel HBCD Rainbow Trout, Oncorhynchus mykiss Well Water

Temperature' (°C) 12.0 1!.8- 12.3 11.9 11.5- 12.0 11.8 11.4- 12.0 

## oo" (mg/L)

9.6 8.8-10.6 9.7 8.8-10.6 9.7 8.8- 10.6 pH 8.1 7.9- 8.3

8.1 7.9- 8.3

8.1 8.0-8.3 Conducti,·ity .f>J.kalinity (umhos/cm) (rng!L as CaC03) 271 178 270- 280 172- 184

1 Temperature measured continuously in the solvent control ranged from appwximately 12.0 to l3.0°C. 

### PROJECT NO.: 439A-lll

TOC Hardness

**~rng** C/L) (mg/L as CaC0 3\ 137 lOA 132 - 140 <!- 24.1

2 At a temperature of l2°C, the dissolved oxygen saturation concentration is 10.8mg/L and 60% saturation ic 6.5 mgil.- 21 - Table 2 Concentrations ofHBCD in Water Samples During the Uptake Phase

Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Concentration **(~tg** HBCD/1 .) Chemical Manufacturers Association's Rrominatcd Flame Retardant Industry Panel

HBCD Raiubow Trout, Oncorhynchus mykiss
Well Water I Day of Study (l!g HHCD/L)

3 7 14 21 ... 21 2

0_ (0 H<:':r2

_ **~** 0 ( 4 Hours)

==--" --- --·-

Solvent Control <LOQ 1 <LOQ <LOQ <LUQ <LOQ <LOQ <LOQ <LOQ

0}1 0.211 0.192 0.169 0.170 0.195 0.252 <LOQ 0.117

0.214

0.211 0.175 () 147 0.172 0.205 <LOQ 0.126

3.•1 2.46 2.16 2.02 2.17 2 <17 2.11 1.22 0.972

2.Ml

2.27 2.17 us 2/15 2 12 0. 982 1.01

1 The Ltmit of

**Qunnttt~tion** (LOQ) was 0.0250

**~tg** I !BCI )/J.

2 **~dditional** samples

**"'::_r~** collected on Day 2 I **d~e** to _low tccovcncs. 28

**~5** <LOQ <LOQ 0.155 () 163 0.157 () 178 1.34 1.56 1.34 !.51 Mean Measured tj • PROJECT NO.: 439A-ld!J Percent of Concentration Nominal **--~** <LOQ 0.!8 51 1.8 53

### U II WILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO.: 439A-lll

Sponsor: Test Substance: Test Organism: Dilution Water: 

### Uptake Phase 

### Mean Measured Concentration

**(~Lg** HBCD!L) Solvent Control 0.18 1.8 

### - 22- Table 3 Concentrations ofHBCD in 

### Water D..uing the Depw-ation Phase Chemical ManufacturersAssociation's Brominated Flame Retardant Industry Panel HBCD Rainbow Trout, Oncorhynchus m}kiss Well Water Day ofStudy (J-IgHBCDIL) 3 7 10 14 21 28 <LOQ 1 <LOQ <LOQ <LOQ <LOQ <LOQ 0.0461: <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0306 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <L00 <LOQ 0.300 <LOQ <LOQ <LOQ <LOQ 0.0412 0.142 <LOQ <LOQ 0.0288 <LOQ <LOQ <LOQ 1The Limit ofQuantitation (LOQ) was 0.0250 )J.g HBCD!L 2Samples presumed to be contarn.L.1ated. 35

**CC:49~o=** C.0542 •.,_ 0.0648Sponsor: Test Substance: Test Organism: Dilution Water: Sample ill E4/N4 ESfi\.15

f-NN(, h7/N7 E17/N17 r.18/NI fl

r.I9/NllJ **~.20iN20** 1}:10/N'lO **1<31/~n** 1 **~J2/r·ll2** f·:nrrn1 L43!r·I4J f.A4/N4tJ E45/N45 f-A!i!N4(, E5(,/N5!i F57!N.'i7 L:'i8!HSS -21- Table 4 I-IBCD Concentrations in Edible, Nonedible and Whole Fish
Tissues of Rainbow Trout Exposed to 0.18 f.!g HBCD/L

Chemical Manutacturers i\ss(Jciation's Brominated flame Retardant Industry Pand

HHCD Rainbow Trout, Oncorhynchus mykiss
Well Water

**---·--···-~** -.--- --- **----~-~~- ~**  _,_

Uptake Day 0 (4 hours)
0 (4 hours)
0 ( 4 hours)

0 (4 hours)

I I l 7 7 7 7 14 14 1''1 II Edible Tissue Edible Tissue · · · ·· · .... '· l'WllCC!lDJC !ISSUe Noneaible Tissue Concentration Weight

(flg HHCD/Kg) (g) 13.9 1.5641 13.9 1.4558
10.0 1.3705 8.38 2/1433 79.9 67 () 90.6 85.4 229

232 213 225 ]](} J(,j

4:1(, 415 4(>2 .'il7 :192 23969 2.2607 2.1009 2.3081 l.3 582 l.2t116 1.7536 1.2130 !.2910 **l.~897**

l.48,\0 2.3696 2.!440

I .')Olf\

I .'JW57
I 5)(,1\ Concentration Weight

(flg HBCD/Kg) (g) 32.8 1.3507 24.8 1.2641 24.4 1.2802 270 2 0041

123 1.9973 91.8 1 7977
165 !.7498 97.4 1.8944
382 10778
310 10750
366 10463
456 0.8662
733 1.1209 1!29 1.2845 1199 1.2958
583 1.9002
937 1.7711
91!3 1.1!699
**~(i1** I ()J49

11 :JO I (,717

' Whole Fish Concentrat:on rgclib !e _\vl.)': **e:_ClL~)<;_~:()l!(U** __ **~_Lil'L!l_0Jj_Q]<;_Iyl** )\_!_!OIIC< 1i blc coJtc. )

wt. + noned1ble wt. 0 PPOJECT NO.: **439A~l ~**  Whole Fish Concentration' (Jlg H8CD/Kg) 22.7 19.0 17.0 16.8 99.5 78.0 121 90.8 297 268 270 321 511 717 792 490 677 748 725

fU8 **-------------~--=:::::=::::::::::::--==--==--~==------:::::===**-24"PROJECTNO.:439A-lu:Table4(Continued)HBCDConcentrationsinEdible,NoncdiblcandWholeFishTissuesofRainbowTroutExposedto0.18Ili3CD/LSponsor:ChemicalManufachtrcrsAssociation'sHrominatcdFlameRetardantIndustryPanelTestSubstance:HHCDTestOrganism:RainbowTrout,OncorhynchusmykissDilutionWater:SampleIDEG9/N(,l)E70!N70h71/N71E72M72EiQ/Nfi2El\3/NXJE84/NX4E85/Nfi5WellWaterUptakeDay212121212g21\EdibleTissueConcentration(itgIillCD/Kg)66'i767671801751R81727740EdibleTissueNonedibleTissueWeightConcentration(g)(!tgliBCD/Kg)3.3308174420901l"'f"1.861226361782516211.67041532lli!'i423861.470622381.4.'1092426NoncdibleTissueWeight(g)3.05091.78541.6679158951.80721.83·'\9J.71931.5337VlholcFishConcentration1(f-lgHHCD/Kg)1181122716001181\1157168115411612E95/N95E96/N%E97/N<J735]CJ]72.'i7263396202•102400351155!.608238901.·1210243835gss1.99.1430511.568718413510421.2609I27421WholehshConcenlr<tlicmcc(cdjbkwt.XedibleGO!ie.wl._,Xnoncdiblccone.)wl.1-nonediblewt.--·:Sponsor: Test Substance: Test Organi srn: Dilutwn

Water: Sample ID E!OG/Nl06 Fl07/N107 i-:1 08/l'll 08
r·: 1 OWN l 09

,., ! 1')/f·JI JC, E140/Nl40 **E14l/~;J4J** J!! 42/1\1142

I 50/f'lilO r--:1

wrm1 F15:'/Nl52 El5J/Nl53 **-~-~~--=~·.--** -==-;--- -25- Table 4 (Continued) HBCD Concentrations in Edible, Nonediblc and Whole Fish
Tissues of Rainbow Trout Exposed to 0.18 !!g H BCD/L

Chemical MnnufRcturcrs Association's Brominaled flame Retardant Industry Panel

HHCD Rainbow Trout, Oncc;rhynchus mykiss
Well Water Depmation Day ]() 10 iO lO ['! H l ,j ., Edible Tissue Concentrntion (ftg IIDCD/Kg)
1 ]()() !()74 1047 14')] nx g74 ]()()2

I Ill :-\15 94\] 101/l **~/()()** Edible Tissue Weight (g) = 2.4333 137M 1.4125

l 0815 1 1499 ()

96i8 10696 () 912[ 1 .3703 1.7160 12102 1.7052 Nonedible Tissue Concentration **(~tg** HECD/Kg) 1769 2161 2334 2021 1"749 4473 3185 4059 2292 2842 3662 3167

fish Conr:-';n(ration

**~** (l;cljblewt),

**r,dlhi~U>'1CJ±_fn.<>l1l:SLtblc** wt. X r.!Qg£diql\;_ con_c;j

wi. rronediblf:! wl. Nonedible Tissue Weight (g) 2.3973 1.5885 1.7169 13982 13073 1 1118 1.2114 1.1301 1.'1 1)2 1.(.5]5 1.2372 uns ec **~** PROJECT NO.:

**439A-~** ·----=- Whole Fish Conccntralion

1 (!lg IIBCD/K..Q__ 1432 1656 1753 ]7()] ]27(i 2804 2161 27.'51 i5G5

I R70 2.155 1884 **-~==~=~;;~~="'--~** - **.~~. ~.** Sponsor: Test Substance: Test Organism: Dilution Water Jl) **C~J** 6i!N 161 **[~162/Nl62**

E1<iJ/N163 E!(,,i/NJ6d El 72/Ni72

EIIJ/1'1173 E J7 eJ/l'J J7 'f El 75,Nl75 L:J83/Nli'.J Ei 4/Nl81 186 - 26- Table 4 (Continued) HBCD Coi'Centrations in Edible, 0lonedible and Whole Fish
Tissues of Rainbow Trout Exposed to 0.18 pg HBCD/L

Chemical **Manul~1cturen· Assoz~iation's**  Brcnninnted Flame Retardant Industry Panel

I ill CD R.o.: 1bow Trout, Oncorhynchus mykiss
Well Water **··=-=~-** -- **=r:~.-----o-~--~-** -- .... **~.::--··,** Edible Tissue Edible Tissue DepurntJon Concentration Weight

Day (flg IIBCD/Kg) (g)

- ··-

21 iR72 1 **.76~3**

21 7 1 ()()09

21 12l'i 2.2.l79

21 ?ns 1 ()()30

JO ,.,_ __ \ 7 1 15 1.iP72

i[l (, 1 () 1.6895

2fl 24(, 24913

28 ·'lri>l L738!

-,) **g()i~** I .4476

5 12() 2.2725

s 707 2.2240

**~i** 741 1041.3 Nonedible Tissue Concentration **(~1g** HBCD/Kg)

!977 [3 1 18 2497 1090 !362 614 1362 1476 1597

330 1049

2

-- Nol' .dihle Tissue Weight (g) 1.7717 I. I 569 I. 8875 !.5778 1.1195 1.5630 22·156 2.09,18 J51jtjJ

198,15 1.99'18 1.0473

L'<.ill.iJle .YJ.Ll( q!!hle cone.) l (noncdibi'C **\'I'L2.C~QiLZcJTI)j';:;~~;j''""x"x=~=~~~-~=~=~~-**

(edibk '-''l + nonedible wt.)

·smnple con1fm1 inn ted td1 concentration r:ould ,Jm he calc•.tlated.

**=~~---;...._-~--"-'""'"'~~~~:"~:·~:::---~.r_'1:~-"--~"-'~:~~;':!:';.~!::e;::;~~-::.::.~:::::::.::..":;".::;-;o:;;==~~ --~'~"===~oo==''···=====**  (j PROJECT NO.: **439A~ll::2** WhokFish Concentratior. 1 (f1g IIDCD/Kg) 1925

0

--

1815 961 1059

612 775 949 1257 221

g(i()

3

# c 02 WILDLIFE 

### iNTERNATIONAL, LTD. PROJECT

**~0** 

### 439A-lll

- 27- 

### Table 5 Da_y 

### 35 BCF Estimates for Rainbmv Trout Exposed tc 0.18 ;..tg HBCD/L Sponsor: Test SHbstance: Test Organism Dilution Water.

Tissue Type Edible Nonedible Whole Fish Chemical Manufacturers

**As~ociation's** BrominatedFlame R:;loLrdant Industrv Panel I-:IBCD Rainbow Trout, Oncorhynchus my/...'iss WeU Water Mean :tvieasured

Mean Measured Day 35 Tissue Test Coneentrdtion Concentration

(J-lgHBCDIL) (ug HBCD/Kr.;) 0.18 1,175.5 0.18 3,730.75 0.18 2,355.25 Day 35 BCF 6,53) 20,726 1:3,085

# c 03 WILDLIFE INTERNATIONAL, LTD. PROJECT NO : 43<:;A-lll

Sponsor: Test Substance: Test Organism: Dilution Water:

Tissue Type Edible Nonedible Whole Fish 

### - 28 - Table 6 

### BIOFAC Model Estimates for Rainbow Trout Exposed to 0.18 !-Lg HBCD/L Chemical Manufacturers Association's Brorninated Flame Retardant Llldustry Panel HBCD Rainbow Trout, 

### Oncorhynchus myhss Well Water

Kinetic Bioconcentration Uptake Rate Factor Constant (BCFK) (k1, L•Kg- 1•Day- 1) 14,039 

### 275 30,242 701 21,940 500 Depuration Rate Constant Ck2, Day- 1 )

0.0196 0.0232 0.0228 Esti.;nate Time to Reach 90'% Steady State (Days)

118 99.3 101 Estimated Time to Reach 50°/o

Clearance

(Doys)

35.4 29.9 30.4- 29- Table 7 HBCD Concentrations in Edible, Nonedible and Whole Fish
Tissues of Rainbow Trout Exposed to 1.8 **~tg** HBCD/L

Sponsor: Chemical Manufacturers Association's Brominatcd Flame Retardant Industry Panel

Test Substance: IillCD Test Organism: Rainbow Trout, Oncorhynchus mykiss **~** PROJECT NO.: **439A~l** lil:

Dilution Water: Well **W~:,a;;;t;;;;er~=========:==:::=:=.:=======::=:':"=:=~=======.:=:===:====================**

wl. + nonedibleSponsor: Test Substancr;: Test Organism: Dilution Water: Sample ID E74/]\!7 4 E75!N75 E7G/N76 E77fN77 E87/N87 E88/N88 Efl9/i\l89 E90!N90 E99/N99 ElOOINJOO r:IOlfNJOI El02/Nl 02 - 30- Table 7 (Continued) HBCD Concentrations in Edible, Nonedible and Whole Fish Tissues of Rainbow Trout Exposed to 1.8 1-1-g HBCD/L

Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel

Hl3CD Rainbow Trout, Oncorhynchus mykiss
Well Water Uptake Day 21 21 21 21 28 28 28 28 35 35 35 35 Edible Tissue Edible Tissue Concentration Weight

**(~tg** HBCD/Kg) (g)

8814 2.6499 9801 8101 12607

1 !205

8905 10061 9759 6550 **~~ms**

10692 9174 J.o7 44 1.6934 1.5397 2.1502 2.0656 1.9036 1.6929 2.3350 1.8894 1.6189 1.3193 Nonedible Tissue Concentration (11g HBCD!Kg) 24197 29737 28478 19567 21523 21455 16465 26294 15931 29848 29841 14563

**~** .. **-~** .. **~~~0~-~** •• **-~~"···--- -::--:-~~-=-=-"~----·-** 

1 \\/hole !'ish Concentration **===-"=:::-~.=;;-=:::=..---:-~:::~::.::z·-** 0ilibJ£::\VL X edibl\! __ (;_onc,_)__± __ **(@ne~liule** wL X nonediblc com; ) (edible wl. +- nonedi'Jle wt.) r PROJECTNO.: **439A~l~** u Nonedible Tissue Whole Fish Weight Concentration 1

(g) (Jlg HBCD!Kg) 2.427H 16169 1.5764 19469 1.6278 18088 **1.~856** 16025 2.1049 12772 2.3595 15597 2.1876 13485 1.7189 18090 2.2231 11126 2.1199 19620 1.9766 21219 1.6753 12189

**~----** ---------------31-PROJECTNO.:439A-IOTable7(Continued)HBCDConcentrationsinEdible,NonedibleandWholeFishTissuesofRainbowTroutExposedto1.8HBCD/LSponsor:ChemicalManufactmersAssociation'sBromi.natcdFlameRetardantlildnstryPanelTestSubstance:HBCDTestOrganism:RainbowTrout,Oncorhynchusmykiss.DilutionEdibleTissueEdibleTissueTissueNonedibleTissueWholeFisha""""mpleIDEl10/Ni!OE111/N111u12/N1l2E!l:l!Ni13E[iUfN143E1t!4/N14,!Fl45/NI45El4G!N146El54!}1154El55!Nl55F:l56/Nl56El57fNI1V!11oleFishConcentrationDepurationWeightConcentrationWeightConccntration1Day(g)(JlllBCD/Kg)._(g)(J.lgHBCDfi<:g)_I1.392715609!.75951290010!0101014141414I1<!43Ui451228681.884117542159981.1291297971.311723414261!67o261099979915354104199916787765751.3459211946H684153251.26531.25251.93460.94611.50733.26491.58362343029!tl6t\966228026280591268317tlJ816952223851.3037185471.2570303652.1985186481.1445177841.6794116122.4738131501.5890124222.J8LI614203U;,ilibk:wl.XedibleconQ,L:i_:.Jl!ont:diblewt.XglllQj(ediblewl.+nonediblewt.)-··-......0\-32-Table7(Conlinued)HBCDConcentrationsinEdible,NonedibleandWholeFishTissuesofRainbowTroutExposedto1.8f.,tgHBCDILSponsor:TestSubstanceTestOrganismDilutionWater:ChemicalManufacturersAssocintion'sBmminatedFlameRetardantIndustryPanelHBCDRainoowTrout,OncorhynchusmykissWellE..;ibleTissueEdibleTissueDepurationConcentrationWeightDay(_g)EIG5/Nl65217195!.6321Ei66/NIGCi2173582.948721El68/Nl6821Ei7(i/NI76E177/NI77El78!N178El79/J\1179E187fNIH7El88/NI811El89/Nl89l1902.29291.35441.28490.86761.67541.90021.46040.9763===1WholeFishConcentmtionwtX__-+:_iJlone_cij_l2lc\Yi_;\'(JlOnccli_blecott_0(edihlewt.+nonecliblcwt.)NonedibleTissueConcentration(ftgHBCD/Kg)134821423618354106131419011546116427177999949990546745NoncdibleTissueWeight1.53622.46232.42141.47492.1302l.5451l.48470.99121.92721.48911.0245PROJECTNO.:O"-"-WholeFishConcentration'._(J.Lg_HBCDI.f!il1024310488135521116173646764203821445681766461065443.....J'"""....-·-·.·---·--·

# '\..._.., uo WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll

- 33- 

### Table 8 Steady-State BCF Values for Rainbow Trout Exposed to 1.8

**~g** 

### HBCD/L Sponsor: Test Substance: Test Organism: Dilution Water:

Tissue Type Edible Nonedible Whole Fish Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel HBCD Rainbow Trout, 

### Oncorhynchus mykiss Well Water Mean Measured Test Concentration

(J.l.gHBCDIL) 1.8 

### 1.8 1.8 Uptake Days at Steady-State 

### 14,21,28,35

21,28,35 

### 21, 28,35 Mean Measured Steady-State Tissue Concentration (J..lg HBCD/Kg) 

### 8,370 

### 23,158 

### 16,154 Steady-State BCF 

### 4650 

### !2866 8974

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll

Sponsor: Test Substance: Test Organism: Dilution Water:
Tissue Type Edible Nonedible \}/hole Fish 

### - 34- Table 9 

### BIOFAC Model Estimates for Rainbow Trout Exposed to 1.8 J..Lg 

### HBCD/L Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel HBCD Rainbow Trout, Oncorhynchus myl..iss Well Water Kinetic Bioconcentration Uptake Rate Depuration Rate Factor Constant Constant (BCFK) 

### Ckt, L•Kg- 1•Day- 1 ) 

### Ck2, Day- 1 )

9,826 362 0.0369 

### 23,303 821 0.0352 16,450 591 0.0359 Esti...'11.ate Time Estimated Time to Reach 90% to Reach 50% Steady State Clearance (Days) (Days)

62.5 18.8 65.4 19.7 64.1 19.3

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-111
Figure l. -35- 

### Concentrations ofHBCD in

### Edible Fish Tissues ofRainbow Trout Exposed to 0 18 1-Lg 

### HBCD/L

:I'D 439H-111 

### 0.18 ug/L Edible T1esue

0 D

### 0.0 10.0 20.0 30.0 

### Kl 27l!:.5Sll t\2 0.01956 KB 

## = 14039.50

0

140.0 50.0 60.0 70.0 60.0 I



# ~------------------D-c_y ___________________________ 

# j

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.· 439A-lll
Figure 2. - 36- 

### Concentrations of HBCD in 

### Nonedible Fish Tissues of Rainbow Trout Exposed to 

### 0 18 fl.g HBCD!L.

::1'0 1.139R-111 

### a.18 ug/L Nonecl!ble T1sE<U8

to

m @ (!) 0

# ~ ro (') Kl 701.265 K2 0.02319 l'\B 

### = 302l11. 96

OJ='**~** ...... OJ :;) N



### "' 0 ro ID

N 

# - 00.0 10.0 20.0 

### 30.0 LJ,O.O 

### Doy 50.0 60.0 70.0 

### BO.O

### WILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO.· 439A-lll

- 37- 

### Figure 3. Concentrations ofHBCD in

### Whole Fish Tissues ofRainbm.v TroutExposed to 0.18 f.l.g 

### HBCD/L.

m 0

..... D 439R-111 0.18 uo/L 

### Whole Flsh ,, r\1 soo.227 tr? ::: D. 02260 f\B ::: 21939. 55

**~------~----~------~-----L------~-----+------~----~** 0.0 10.0 20.0 30.0 1.!0 .. 0 

### Dey 50.0 60.0 70.0 

### BO.D

**'-----------------------------------------------------------------··---~·** ··- 

# I IVVILULir t::... IN I t::...t-<NAIIUNAL, L I U. 

### PROJECT NO.: 439A.-lll
Figure 4.



# 13 - 38- 

### Concentrations ofHBCD 

### in Edible Fish Tis<>ues of !:\.ainbow Trout Exposedto l 8 11g HBCD/L.

U>o l!39R-111 1.8 u9/L Edible. Tissue

00.0 

### 10.0 20.0 

### 30.0 t.lO.O Dc\j 50.0 

### Kl 362.308 t\2 

## = 0. 03657 1'\B 

### = 9525.35

61]. 0 70.0

----- --··---··--------

### WILDLIFE 

### INTERNATIONAL, 

### LTD. PROJECT NO.: 439A-lll
Figure 5. -39- 

### Concentrations of HBCD in Nonedible Fish Tissues of Rainbmv Trout Exposed to 1 8 )-lg HBCD/L.

Ulo !!39R-111 1.8 '--'9/L Nonedibie Tissue t\ 1 620.601 

### r\2 

## = o. o:::s22

**-+------1-----l----l-----+---+-----1---~:----.** 0.0 10.0 20.0 30.0 liD. 0 Oo!:J 60.0

**~0.0** BO.D



### ------------------------------ ------·····--

## Ul VVILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO. 439A-l1l figure 6. -40- 

### Concentrations ofHBCD i11 

### W"hole fish Tissues ofRainbm": Trout Exposed 

### to 18 fl.g HBCD/L

'"' 0 LJ39R-!1l 1.8 ug/L

**~~hole** Fish r.! (1) 

### KB 59L !52 0.0359[, 15450.27

-----1-----1--- **~--~----~-----+----~----4** 0.0 10.0 20.0 30.0 10.0 50.J 

### co. 0 60.0



### -------------------·--·-- ----------

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll -41- 

### APPENDIX I Protocol, Protocol Amendment and Proto..:ol Deviation

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll -42-

PROTOCOL HEXABROMOCYCLODODECANE (HBCD): A FLOW-TIIR.OUGH BIOCONCENTRATION TESTWITHTHE RAINBOW TROUT (Oncorhynchus mykiss) U.S. Environmental Protection Agency Series 850 -Ecological Effects Test Guidelines OPPTS Number 850.1730 anrl OECD Guideline 305

Submitted to Chemical Manufacturers Association's BrominatedFlame Rctardant :Industry Panel !300Wilson Boulevard Arlington, Virginia 22209 

# Wildlife 8598 Commerce Drive Easton, Maryland 2160I (4!0) 822-8600
October 6, 1999 

# Ltd.

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll -43-
WILDLIFE INTERNATIONAL, LTD -2- HEX.ti.BROMOCYCLODODECANE {HBCD): A FLOW-THROUGH BIOCONC!Th'TRATION TEST WITHTHE RAINBOW TROUT (Oncorhynchus my kiss) SPONSOR: Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel 1300Wilson Boulevard Arlington., Virginia 22209 SPONSOR'S REPRESENTATIVf: Ms. Wendy Sherman TESTING FACILITY: Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STIIDY DIRECT(JR: Kurt Drottar, Senior Aquatic Biologist L4.BORATORY MANAGEMENT: Henry 0. Krueger, Ph.D. Director ofAquatic Toxicology & Non-Target Plants 

### FOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental StartDate: Termination Date: ___ _ ProjcctNo.: 

### 4'6q)l. - li I Test Concentrations:__ -"0-"'.3"-'4-"an"-"d"'-.
**"'-3.""4-"J..tg""-'a"'.~iJL'-"'----------------** Test Substance No.: __]5.If..__ Reference Substance No. (ifapplicable): PROTOCOL APPR.OV AL



# ~

PROTOCOLNO. 439!100699/RBT-BIO/SUB439 

### /6 

# 9/'17 DATE ----- Jo

# /11 /;q



## ~ ;ol z /qo7

**~L:::...t_l** . ---- DATE

### WILDLl FE INTERNA_T_! O_N_A_L_.:...,_L_T_D_.____________ P_R....:..O_JE_C_T_N_O_._:_4_39_A_-_ll_l



# E 05 -44-
WILDLIFE INTERNATIONAL, LID - 3 - INTRODUCTION Wildlife International, Ltd. v.ill conduct a bioconcentration test with the rainbow trout (Oncorhynchus my/.:iss) for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 (l); ASIMStandard El022-84 StandardPracticefor Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (2); and OECD Guideline for Testing of Chemicals 305, Bioaccumulation: Flow-Through Fish Tesc (3). Raw data for all work performed atWildlife International, Ltd. and a copy ofthe final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative

**loca•~on** to be specified in the final report. PURPOSE The pwpose of this study is to obtain laboratory data characterizing the bioconcentration potential ofthe test substance inthe rainbowtrout, Oncorhynchus mykiss. The solubility of1!-ffiCD in waterat25°C is 3.4 J.lg!L (ppb). The acute 96-hour LC50 for rainbow trout is >6.8 ;tg/L. EXPERIMENTAL DESIGN Rainbow trout will be exposed to two test concentrations (x and lOx) and a solvent (dimethylformamide, DlviF) control. Each group will consist of one test chamber. The test

**v;~ll** be diuided into two phases: uptake and depuration. During the uptake phase, fish in the treatment group; will be exposed to sublethal concentrations ofiillCD, while fish i.rJ. the control group w'Jl be mcposed to dilution water containing the same amount C>fsolvent as thatused for thetr::atmcnt groups. The duration ofthe uptake phase (3 hours to 28 days) and the depuration phase (6 hours to 60 days) may vary according to the time required to reach stea.Gy-state. Th;; time to steady-state will be estimatedpriorto test initiation. During both phases ofthe test, ttost organisms ;md water samples are collected and analyzed for HBCD content. These values will be used to determine the steady-state concentration, the uptake rate constant (k,), the depuration rate wnstant (k 2), the kinetic bioconcentration fuctor (BCF'£) and the steady sta.tc bioconcentration fuetor (BCF) for whole body tissues, cdibic tissue (skin on) and nonedible tissue.

PROTOCOL NO. 439/l006991RBT-BIO/SUB439

# ~ 

# 06 -45-
WILDLIFE INTERNATIONAL, LTD -4- MATERIALS Al\'D METHODS Test Substance The test substance consists of a composite of hexabromocyciododecane (HBCD) samples received from three manufacturers. The materials' identity and date received from each of the manufacturers is give n below: Manufacturer Lot/Batch Date Received Wildlife International Ltd. IDNo. GreatLakes Chemical Corp. 635297G-l October26, 1995 3462 Albemarle Corp. 33449-lSX December 20, 1995 3519 Bromine Compounds Ltd 950303 February 5, 1996 3551 An equal part (300 g) ofeach ofthe manufacturer's HBCD material vvas placed in a 2-L, high density polyethylene (HDPE) bottle. The bottle was placed on a reciprocating shaker for two hours. The composite test substance was assigned Wildlife International, Ltd. identification number 3577 and is being stored under ambient conditions. Initial analyses of subsamples ofthe composite were performed on l'vfarch 20, 1996. The results ofthe analyses indicatedthe composite test substance 1Nas homogeneous and containedthe folloVI.ffig components: HBCD - beta isomer HBCD - alpha isomer HBCD - gamma isomer TotalHBCD 8.5% 6.0% 79.1% 93.6% Another subsample of the composite te..'1 substance will be shipped to Ailbcmarle Corp. for characterization and homogeneity analyses to demonstrate st2.bility and confirm its activitv for t.hc purpose ofthe study. Preparation ofTest Concentrations A primary stock solution ofHBCD will be prepared by transferring the HBCD to a volumetric flask and diluting to an appropriate volume with DMF. The solvent concentration in the solvc.nt control and the HBCD treatment groups will not exceed 0.1 mL!L.
PROTOCOL NO. 439/100699/RBT-BIO/SUB439

### WILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO.: 439A-I 11 -46-
WILDLIFE INTERNATIONAL, LTD

- 5 - Test Organism The test organism used in this study will be the rainbow trout, Oncorhynchus mykiss. Rainbow trout represent an important group of aquatic vertebrates, and have been selected for use in the test based upon past use history in the laboratory. Fish will be obtained from a commercial supplier or hatchery, and the identity ofthe species will be verified by the supplier or by Wildlife International, Ltd. personnel using appropriate taxonomic keys, suchas Eddy (3). Fish will be gradually acclimated to Wildlife International, Ltd. well water, and held for a period ofat least 14 days prior to the test. .f the fish are cultured at water temperatures that differ from the test temperature, they >vill be brought to test temperature at a rate not exceeding 3 "C per 72 hours during ho!ding. Ifmortality exceeds 3% during the 48-hour period immediately preceding the test, the entire batch offish

**\~ill** be rejected or held for an additional 14-day period to ensure thatthey are healthy. Fish will be handled as little as possible, but when handling is necessary, it will be done carefully, gently, and q•.J.ddy. To control bias, fish will be removed from holding tanks ·with nets and impartially distnl->uted to the test chambers. No other forms ofbias a,-e expected ro affect the results ofthe study. The minimum acceptable size ofindividual test organisms will be determined by the quantity oftissue required for measurement ofresidues. Loading (the total wet weig.!Jt offish per liter ofte..<;t solution) will not exceed l.O gram offish per liter ofsolution that passes through a test chamber in 24 hours. During the holding period the test fish will be fed at least once daily. The diet will consist of live or frozen brine shrimp nauplii (Artemia sp.), and/or commercial food during holding and testing. Specifications for acceptable levels ofcontaminants in fish diets have not been established. However, there are no known levels of contaminants reasonably expected to be present in L'lc die< tint arc considered to interfere with the purpose or conduct ofthe test. Dilution Water Water used for the holding and testing of rainbow trout INill be obtained from a well approximately 40 meters deep located on the Wildlife Interr.ation.al, Ltd. site. The v,;ater wiU be passed through a sand filter and pumped into a 37,800-L storage tan.k where the ''vatcr

**"~lt** be aerated PROTOCOL NO. 439/i00699/RBT-B!O/Sl.JJ3vc39VVILULit"t:... IN I !::...1-<:NAIIUNAL, Ll 

### U. 

### PROJECTNO.: 439A-lll



# E 08 -47-
WILDLIFE INTERNATIONAL, LID -6- with spray nozzles. Prior to use the water will be filtered to 0.45 11m in order to remove fine particles. Water used for holding and testing is characterized as moderately hard. Typical values for hardness, alkalinity, pH and specific conductance are approximately: Hardness, mg/L as CaC03 Alkalinity, mg!L as CaC03 pH Specific Conductance, ).tillhos/cm 145 190

8.1 330 Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the consistency of the well water. Means and ranges of the measured parameters for the four-week periodpreceding thetest will be provided in the final report. Analyses willbe performed at least once annually to determine the concentrations of selected organic and inorganic constituents of the well water and results ofthe most recent GLP analyses will be summarized inthe final report. Diluter System andTest Conditions A continuous-flow diluter will be used to provide two concentrations of the: test substance (0.34 and 3.4 !J.g!L) and a control. The flow ofdilution water to control and treatment test clwrlbers will be regulated by rotameters. A syringe orperistaltic pump will be used to inject the test substance worki!!g stock solution into a mixing chamber where ililution to the test r.onccntration occurs. Solvent without the test substance will be introduced to the control test ch.aml. · .1 an identical manner. Flow rates will be adjusted so that each test chamber receives at least six volume additions of water every 24 hours. Delivery ofthe test substance worki.ng stock solution wiill be initiated at least 43 hours prior to the test. Duplicate water samples from each test chamber will be collected at least twice to verify the establislunent ofequilibrium concentrations ofHBCD in the test chambers before introduction ofthe test organisms. The general operation of the diluter will be checked visually at leasttwo times perday during the test. Test chambers will be 52-L Teflon"'-lincd stainless steel test chambers containing approximately 45 L oftest soluti.on. Test chambers will be placed in a water bad; to maintain 2. test temperature of 12±1 °C. The test chambers and water bath will be enclosed in a piexig!ass hood in PROTOCOL NO. 439/l00699!RBT-B!O/SUB439

# E 09 - 48-
WILDLIFE INTERNATIONAL, LTD -7- order to minimize pokntial cross-contamination. Ambient light during testing will be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g. Colortone® 50 or equivalent). A photoperiod of 16 hours of

**ligh~** and 8 hours ofdarkness will be controlled with an automatic timer. A 30 minute transition period oflow light intensity will be provided when lights go on or offto avoid sudden changes in light intensity. Test chambers will be identified by the project number, replicate and test concentration. PROCEDURES The test will be divided into two phases: uptake and depuration. Organisms in the solvent control group

**v.~ll** be exposed to well v•ater in the uptake and depuration phases. Organisms in the treatment groups will be exposed to the test substance in the uptake phase and well water in the depuration phase. Selection ofTest Concentrations The test concentrations selected will not stress, irritate, or otherwise adversely affect the organisms. The test concentrations were selected in conjunction I'<Tih the Spo!lSor based on acute or chronictoxicity data. Thetwo exposure concentrations of0.34 and 3.4 f.tg/L differ by a factor of 10. Chemic.ai!Physica1 Measurements Temperature will be monitored and recorded r.ontinuously during the entire test in the solvent control test chamber using a Fulscope ERIC Recorder (1900 J Series Model A), or equivalent. Temperature also will be measured in every test chamber at the beginning and end ofthe test and at weekly intervals duringthe test using a liquid-in-glass thermometer. Dissolved oxygen will be measured daily in every test cbainber during the test using a Yellow Spring Instruments Model 51B dissolved oxygen meter, or equivalent. l.n the event that dissolved oxygen levels fall below 60% saturation, appropriate actions "vili be taken after consultation with the Spons0r. Measurements ofpH will be made in every test chamber at the beginning and end of the test ar.d at weekly intervals during the test using a Fisher Accumet Model 915 pH meter, or equivalent. Hardness, alkalinity, conductivity and total organic carbon will be mcao;ured in the control treatment PROTOCOLNO. 439/100699/RBT-BIO/SUB439

# E tO -49-

WILDLIFE INTERNATIONAL, LID - 8 - at the beginning and end ofthe test and at weekly intervals during the test. Hardness and alkalinity measurements will be made by titration using procedures based on methods in StandardMethodsfor the Examination ofWater and Wastewater (3). Conductivity will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. Total organic carbon will be measured using a Shimadzu model TOC-5000 total organic carbon analyzer, or equivalent. 3iological Measurements Observations ofbehavior and mortality will be made daily. For a test to be considered valid., mortality and abnormal behavior should not be apparent in more than 10% ofthe fish inthe trcalrnent or control groups. Duration ofUptake Phase The statistically optimum duration ofthe uptake phase (u) is near u 

### = l.6/k2, but not mmc than 3.0/k 2, which is equivalent to 95 percent ofsteady state. A pre-test estimate of k2 will be obtained from a test ,vith a similar material, water solubility data, or the n-octanol/water partition coefficient. The uptake phase will continue until the concentration of HBCD residues in body tissues reaches steady-state, but not for more than 30 days. 'The criterion for attainment of steady-state is that three conse...--utive HBCD residue concentrations in fish arc not significantly different from each other. Duration ofDepuration Phase Two

**fu:l~;s** tt is usually a sufficient time for about 95 per.;:ent removal ofthe body burden (tv, = 0.69/k 2 }, however, the'

**d~uration** phase will continue until the concentratjon ofHBCD residue in the organisms attains any one ofthe following three conditions: 1. Residues reach less than 10% ofsteady-state; or 2. Residues full below the LOQ; or 3. Sixty (60) days ofdepuration have elapsed. Sample Intervals for Water and Fish Collection Fish and water 

### "''ill be sampled no less than five times during the upta.l.;e phase and four times during the depuration phase ofthe study at the same intervals. l.n addition, wntrol and treated water PROTOCOLNO. 4391!00699/RBT-BIO/SUB439VVILULirE... 11'-ll t:..H:i'\IAIIUNAL, Ll U. PKUJECT NU.: 43YA-lll



# E 11 -50-
'v\/lLDLIFE INTERNATIONAL, LTD -9- will be sampled no less than twice prior to addition of fish to the chambers to confmn the concentrations desired. Control fish for the preparation of analytical quality control samples will be drawn from the same batch offish used for the study, ifpossible. Additional control samples offish and water may be collected and prepared as stability samples at least one week prior to test initiation. ln the absence of information on the bioconcentration potential ofthe test substance and/or reliable physico-chemical data from which bioconcentration potential may be estimated, the recommended sampling schedule is as follows. Phase SampleInterval Water1 Fish2 Action Start Pre-Uptake3

**-n~** v 

## - .F>. -n2 X 

# - 0 X - Add fish Uptake -4hours X X l day X X 3 day X 

# I X

7 X X 14 X X 21 X X 28 v y,;: A Transfer 1 

### " X .r.. Depuration 3 X v .A 7 X v A 10 X X i44 X X At least five replicates ofwater samples will be collected at each interval listed for the analysis of HBCD. Additionall samples 11'1i.l.l be drawn as necessary for the determination of pH, hardness, alkalinity, specific conductance, and total mganic carbon {TOC). 2 Su:fficiem fish will be collected to allow the preparation of fuur replicate samples

from each treatment group and duplicate samples from the control group for t.'le analysis ofHBCD. Additional samples v:rill be ch-av;-n as necessa.ry for the a.'U.Iysis ofdry weight and lipid content. 3 Pre-Uptake water sampling

**(-n~o** -n2} for com'i.rmation ofthe treatment levels prior to

addition offish. 4Depuration phase sampling \vi!! continue until the concentration ofHBCD residue in

the organisms attains any one ofthe conditions outli.rted in the duration ofdepuration phz.se on page 8.

PROTOCOLNO. 439/100699/RBT-BIO/S\.JB439

# E 12 J:'KVJtL 1 NV.:

**4~~A-lll** 

### -51 -
WiLDLIFE INTERNATIONAL, LTD

- !0- The number and frequency of sample intervals may be changed based on physico-chemical properties ofthe test substance from which predictions ofbioconcentration rate can be derived and1or atthe direction ofthe Sponsor. Water Sampling Atleast five replicates ofwater samples will be collected at each interval listed for the analysis ofHBCD. The HBCD concentration will be determined for a minimum of one rep!icate ofa control and two replicates at each treatment level for each interval in which samples are analyzed (as specified by the Study Director). The remaining repiicates ofsamples drawn for HBCD analysis will be held in reserve and may be analyzed at the direction ofthe Study Director or Chemistry Principal Investigator. The concentration ofHBCD in the v.-ater at each samole interval will be presented as the average ofthe replicate measurements, as applicable. Additional samples will

**b~** dravm as necessary for the determination of pH., hardness. alkalinity, spe-cific conductance, and total organic carbon (TOC). Fish Sampling To assess the HBCD content in fish tissues, sufficient fish will be collected to allow the preparation of four replicate samples from each tre.mnent group and duplicate samples from the control group. The HBCD concentration will be determined for a minimum of one replicate of a control and fotrr replicates at each treatment level for each intervcl in which samples are analy-.xd {as specified bythe Study Director). The remaining replicates ofsa.:nples drawn for HBCD a=lysis wiil be held in reserve and m.ay be analyzed at the direction ofthe Study Director or Chemistry Principal Investigator. The concentration of HBCD in fish at each sa..-nple iiiterval v,ili be presented as the average ofthe replicate measurements. Fish sampled for detenrjnation of HBCD content vvili be imparti2l!y removed from the test chambers, rinsed ·with dilution water, blotted dry, and sacrific.e.d. The weight (wet weight, blotted dry) and totaJ length ofeach fish ·vvill be determined

**'.~ithin** approximately 15 minutes ofcollection, if possible. Each

**fL~h** will be separated into edible a.!'ld noncdiblc tissue. Fish will be stored frozcrr if not analyzed immediately. PROTOCOLNO. 439/l00699/RBT-BIOiSlJ!3439

# E 13 __________ PKUJcCI NU.: 4.:i'-JA-1.!1 

### -52 ..
WILDLIFE INTERNATIONAL, UD



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



## - ]] 

### - Additional samples will be dravm as necessary for the anal;,is ofdry weight ;md lipid conterrt__ Analytical Method Water and tissue samples will be analyzed for HBCD using liquid chromatography-mass spectrometry. The analytical methodology used will be described in-the final report_ Tissue Lipid Content At a minimum, fish v.1.ll be sampled from the control aquarium at Day 0 (upon transfer of fish to aquaria), at an interval where the HBCD concentration j__,-,_ fish bas reached steady state (or following 28 days of exposure), and at the end of the depuration period. Fish will be sampled to generate four samples from the control and two treated aquaria at each ofthe intervals noted. The lipid content ofthe tissue homogenates will be determined by chlorofonn!mefr..anol**extr~ction_** 1hc tissue percent solids will also be determined to allow quantitation on a dry-weight basis. DataAnalysis Results of tissue analyses will be prcscntc<:l on a wet weight basis. The steady-state bioconcentration factor for !he test substance (BCF), kinetic bioconcentration factor (BCFK) uptake rate (k1), and depuration rate (k2) will be expressed for edible tissue, nonedible tissue and whole fish_ RECORDS TO JBE 1\i.I\INTA!i\-lED Records to be maintained for data generated at \Vlldlife International, Ltd. vvill include bm not be limited to:

l. A copy cfu'le signedprotocol. 2. Identification and characterization ofthe test subst::tnce, if**pro-..~ded** by the Sponsor. 3. Dates ofinitiation and termination ofthe test. 4. Test organism holding and acclirP..ation records. 5. Methods used to prepare stock solutions and dilutions ofilie te:;t substance. 6. Daily obervations. 7. Water chemistry calculations (e.g., hardness and aL'<alinity). 8. Organism weight and length measurements, if2pplicable 9. The meiliods used to analyze test substance concentrations and the results oi" a::nl;-1.icat measurements. 10. Statistical calculations. PROTOCOL N0. 439/l00699/RBT-Bi0/S1JB439t'KUJbL 1 NU.. 4.:\':?A-11 i 

### -53 -
WILDLIFE INTERNATIONAL, LTD

- 12- ll. A copy of!he final report. FINALF'::_ORT A repvrt of !he results ofthe study will be prepared by Wildlife lmcmational, Ltd. 1bc report will include, butnot be limited to, the following: 1. Name andaddress ofthe fucility performing the study. 2. Dates on which the study was initiated and completed. It is the responsibility ot the Sponsor to provide the final date that data are recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories. 3. A state:nent of compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures

**~tated** in the approved protocol, including ao.""ly changes m the original protocol. 5. Statistical methods employed for analyzing ilie daa. 6. The test, control and reference substances identified by mune, chemical abstracts number or code number, strength, purity, and composition or other appropn'l.te characteristics, ifprovided bythe Sponsor. 7. Stability and, when relevant to the conduct ofthe study, the solubility of ilie test, control and reference substan= under the conditions of administration, if provided by the Spons•)r or contracted to Wildlife International Ltd. 8. A description ofthe meiliods used. 9. A description of the test system used. Where applicable, the final report she-ill include t.'te number ofanimals used, body weight range, source ofsupply,

**speci~s,** age, and procedure used for identification. 10. A description ofthe dosage, dosage regimen, route of

**administntior~** and duration. I 1. A description ofall circ.umstanccs that may have affected u.'1c quality or h""ltegrity ofu'l<' cbta. 12. The name ofthe Study Director, the na..>ncs ofoilier scientists or professionals, and the names cf all supervisory personnel, involved in the study. 13. A description of the transformations, calculations, or operations performed on u'1e. d.a\D, a summary anct analysis ofthe data, ar.d a statement ofthe conclusions drawn from the :malys1s 14. The signed and dated reports ofeach ofthe individual scientists or other profe.ssioruJs involved in the study, ifapplicable. PROTOCOL NO. 439/l00699/RBT-Bl0/SLJB439

### W!LDUFE INTERi\lATIONAL, 

### LTD. PROJECT NO.: 439A-ll1 -54-
WILDLIFE iNTERNAT{ONAL, LTD

. l3 - 15. The location where all specimens, raw data, and the fmal report are to be stored. 16 A statement prepared by'the Quality Assurance Unit listing the dates that study inspections and audits were made and the d:rtes of any findings reported to the Study Director and Manage·

**men~.** !7. Ifit is necessary to make corrections or additions to a final report after it has been acceptc..i, such changes shall be in 'the form ofamend.rncnt by the Study Director. The amendment sho!.!ld clearly identiiy the part ofthe final report that is being added to or corre.-·red and the reasons for the correction or addition. Amendments shall be signed and dated bythe Study Director. CHA.NG'NG OF PROTOCOL Planned changes to the protocol will be in the form ofwritten amendments signed by i.hc Study Director and the Sponsor's Representative. Amendments will be c.onsidered as part ofthe protocol and will be attache<! to the final protocol. Any other changes will be in the form of writteP, deviations signed by the Study Direcror and filed with the raw data. .IIJi changes to the protocol will be indicated in the firL:J.l report. GOOD LABORATORY PR.I\CTICES This study will be conducted in accordance v,;th Good Labrratory Practice Staud.ards for EPA (t10 CFR Part 160); OECD Principles of Good J...,aboratcry Practice {El\'V/MCiCEEM (98) l.7); and Japan N.'AFF (59 NohSan, Notification No. 3850, A_gricuH.ural PmdJcticn Bureau). Each study conrlucted by \Vildlife International Ltd. is routinely cxa..rni.ned by the Wildlife l.ntemational Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Sta.ndard Operati.nt; Procedures and the specified protocol. A statement of compliance 'lvith Good Laboratory Pmctic.."'S will be prepared for all portions ofthe study conducted by \Vildiife Interm;tional Ltd. Tile Spouse; will be responsible for compliance with Good L-'<boratory Practices tor procedures performed by othec laboratories (e.g., residue analyses or pathology). Raw data for an \':ork performed at \Vildlife international Ltd. and a copy ofthe final repon v:il! be filed by project number in archives Jo.:.2.tcd oo the Wildlife International Ltd. site, or at an alternative loeation lo be specified in the final report

PROTOCOL NO. 4391!00699/RBT-BIO/S1.JB,B9

### WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll -55-
WILDLIFE INTERNATIONAL, LTC

- 14- REFERENCES US. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Gnidclincs (draft), OPPTS Number 850.1730: Flov:-Through F;<h 7est. 2 ASTM Standard E1022-84. 1988. StandardPracticefor Conducting Bioconcentration Tests with Fishes andSaltwater BivalveMolluscs. American Society forTesting and Materials. 3 OECD Guideline fo;· Testing of Chemicals 305. !996. Bioconcentration: Flow-Through Fish Test. 4 APHA, AWWA, WPCF. 1995. Star.dard Methods for the Examination of Water and Wastewater. 17th Edition, American Pt.blic Health Association. American Water Works Association. \Vater Pollution Control Federation, NewYork.

. PROTOCOL NO. 439/i00699/RBT-B!O/SUB439

### WILDLIFE INTERNATIONAL, LTD. -56-
WILDLIFE INTERNATIONAL, LTD

- J5- 

### APPEN1)IXI IDENTIFICATION OFTESTSUBSTANCE BY SPONSOR TobeCompleted by Sponsor 

### PROJECT NO: 439A-lll

L TestSubstanceidentity(narnetobeusedinthereport): _____ HB"""""'""C""D'---------- TestSubstance SampleCodeor BatchNumber: Wildlife International Ltd. IdentificationNo. 3577 TestSubstance Purity ("/oActive Ingredient): --"--'93,_,_.

**6"-'7'--'-'HB~C"'D"'---** ExpirationDate: __ _ 11 TestSubstanceOlaracrerization Havethe identity, streogth,purityand compositionorothercharacteristics which

**~proprialcly**defure thetestsubslaOCeand refi:rence standardbeen determinedpriortoits use inthis studyin accordancewithGLP Standards? Til TestSubstance Storage Conditions X Yes ___ No Pleaseindicatethe recommended storage o:mditions atWildlife lrJ!erP...ational, Ltd. Ambient Hasthe stabilitv ofthete;;tsubstanceunderiliesc storageconditions beendcte:rmin&:i in accordance wilhGL'P Standards? __ Yes __ No Otherpertinentstabilityinformation: IV. TestCoocentrations:
V. Toxicit.yInformation: Adjusttestcon-centratioo to l00"/o a.i.

____ baSed1.1pon !hepurity(%) givenabove. Donotadjusttestcoo=Il:iratiooto l00%

__ _,_x,___.ai. Testfue material ASJS. M:unmalian: Ral:LD50 _____ Mou..<eLD50: ------ Aquatic: Invertebrate TCJcicity (EC/LC50) FishTO'Aicity (I£..50)
OtherToxicity Information (mcluding findings ofchronic andsubohroninests): VI. Classificationof the Compound:

__ Insecticide

__ Microbial Agent __ Herbicide ___ Fungicide

__ EconomicPoLson Oilicr.__________________________ ___
PROTOCOL NO. 4391l00699/RBT-BIO/SUB4?9

### WILDLIFE INTERNATIONAL, LTD. -57-

WILDLIFE INTERNATIONAL LTD" AMENDMENTTOSTUDYPROTOCOL 

### PROJECT NO.: 439A-lll

PROJECT NO.: 439A-l1 I Page 1 of3

STUDYTITLE: HEXABROMOCYCLODODECANE (HBCD): A FLOW-THROUGH BIOCONCENTRA TION TESTWITHTHE RAINBOW TROUT (Oncorhynchus mykiss) PROTOCOL NO.: 439/!00699/RBT-Bl0/SUB439 SPONSOR: Chemical Manufacturers ksociation's Brominated Flame Retardant Industry Panel EFFECTIVE DATE: November 10, 1999 AMENDMENT: Page 2 Add: Experimental StartDate: ll/17/99 Experimental Termination Date: 12129/99 At'\fi:NDJVIENT NO.: ! PROJECT NO.: 439A-l11

REASON: The above information was not known when the protocol was signed by the Study Director. AMENDMENT: Page 2 Change: Test SubstanceNo.: 3577 To: Test SubstanceNo.: 4615 REASON: The Sponsor requested that Wildlife use the most recent composite sample. AMENDMENT: Page 2 Change: Test Concentrations: 0.34 and 3.4 J.Lg a.i.IL. To: TestConcentrations: 0.34 and 3.4 J.Lg HBCD/L REASON: The te:.--t will be performed on an AS IS ba,;s.

**AMEND~1ENT:** Test Substance, Page 4 Delete: Entire Section REASON: The section pertains to test substance No. 3577 which will not be used.

KR£);\439AII/Ipro I



### ~v\-<_,....>••d 

### \f'( 

### <Qfl

**~MC.** l1-

**~v-4<)**

### WILDLIFE INTERNATIONAL, LTD. -58-

WILDLIFE INJERNATlQNAL LTD" AMENDMENT: Test Organism, Page 5 Change: During the holding period the test fish will be fed at least once daily. 

### PROJECT NO.: 439A-lll

PROJECTNO.: 439A-lll Page 2 of3

To: Duringthe holding and testing periods, thefiSh will be fed at least oncedaily. Uneaten food and feces will be siphoned from thetest chambers shortly after feeding (30 minutes to 1 hour). REASON: To clarify feeding during the test and to allow for siphoning. AMENDMENT: Diluter System and Test Conditions, Page 6 Change: Test chambers will be 52-LTeflon®-lined stain!t:ss steel test chambers containing approximately 45 L oftest solution. To: Test chambers will be I06-L stainless steel test chambers containing approximately 80 L oftest solution. REASON: To correct the size ofthe test chambers and the volume oftest solution. AlVIENDMENT: Water Sampling, Page 10 Change: To: At least five replicates ofwater samples will be collected at each interval listed for the analysis 

## on-men. Ateach sampling interval, two water samples vvil! becollected from ti1e control and three W3ter samples will be collected

**fr~** :n each ofthe two HBCDtreatment groups. REASON: To clarifythe number ofwater samples which will be collected. AMENDMENT: Fish Sampling. Page 10 Change: To: Each fish will be separated into edible and nonedible tissue. Each fiSh will be separated into edible ami nonedible tissue and the wet weight ofeach tissue fraction (i.e., edible and nonedible) wil! be recorded. REASON: Edible and nonedible tissue weights are needed calculate whole body concentrations.

KFD:\439Afll\!Jra I

### WILDLIFE INTERNATIONAL, LTD.

WILDLIFE INTERNATIONAL L · AMENDJV£ENT: Fish Sampling, Page ll 

### -59- 

### PROJECT NO.: 439A-lll

IPROJECTNO.: 439A-lll Page 3 of3

Delete: Additional samples will be drawn as necessary for the analysis ofdry weight and lipid

**~c:>ntenL** REASON: Dry weight determinations will nut be made_ 

## 1/{IOffl DATE

SPONSOR'S PRESENTATIVE DATE' '

KRD:\43\'AIIIVJro I

### WiLDLIFE INTERNATIONAL, LTD. -60-

WILDU FE INTERNATIONAL LW DEVIATION TO STUDY PROTOCOL 

### PROJECT NO.: 439A-lll

PROJECTNO.: 439A-lll Page l of 1

STUDYTITLE: HEXABROMOCYCLODODECANE (HBCD}: A FLOW-THROUGH BIOCONCENTRATION TEST WITH THERAINBOW TROUf (Oncorhynchus mykiss) PROTOCOL NO.: 4391100699/RBT-BIO/SUB439 SPONSOR: Chemical Manufacturers Association's Brominatcd Flame Retardant Industry Panel DATE OFDEFACTO DEVIATION: February 25,2000 DEVIATION NO.: l PROJECf NO.: 439A-lll

DEVIATION: Fish dry weights were determined. Dry weight measurements were amended out of the protocol in Amendment No. 1. REASON: Thechemist did not realize that dry weights hadbeen amended out oftheprotoc.ol. It is the best judgementoftheStudyDirectorthat this deviation didnot adversely affectfue results of the study.

STUDYDIRECTOR



### ~ DATE



# ~~L

KRD:\·139AI I /lprc/1VVILDLIFE INTERNATIONAL, LTD. PROJECT NO. 439A-lll



### - 61 - 

### APPENDIX II Test Substance Characterizatio::1



# F 08

# F 09 -62- 

### CHEI\llCAL CHARACTERIZATION ANALYTICALRESULTS FORDETERMJNlNGTHE IDENTITY AND PURITY OF HEXABROMOCYCLODODECANE (TEST**SUBSTA..~CE** 

### NOo

Prepared for.

Prepared by: 

### Dr. 

### MarciaL.Barny, Study

### Monitor Health and EnvironmentDepartment 

### Albemarle Corporation 451 Florida Street Baton Rouge, La. '70801 George M. :Ricks, Smdy Chemist Toxicology and Industrial Laboratory Albemarle Corporation 8000 G.S.RL Road 

### BatonRouge, La. 70820



# 19411'111-L.JLirr::.. H'lll 1::..1""(1'\IA.JIUI'\IA.L, Ll U. 

### PROJECT NO.: 439A-lll



# F 10 - 63-


### ALBEMARLE CORPORATION HEALTH AND E.t'NIRONMENT DEPARTMEJ'\.'T CHEMICAL CHARACTERIZATIONANALYTICAL RESULTS FOR

**DETE~"'NG** 

### THE IDENTITY AND PURITY OF HEXABROMOCYCLODODECANE (TEST 

### SUBSTA..N"CE NO. 4615)
1 Reference ProtocolNo.: II. Sponsor:

III. Performing Laboratory:

IV. Dates ofPerfunnance: V. Test Article:

V1 Object:ive/II..1ethodo!ogy: HBCD-002 Albemarle Corporation Health andEnvironment Department 451 Florida Street BatonRouge, La. 70801 StudyMonitor: MarciaL. Hardy, D.V.M., .!>h.D_ Albemarle Corporation Albemarle Technical. Center 8000 G.S.Rl Aveooe BatonRouge, La. 7082.0 Study Chemist: GeorgeM Ricks, 1\.i.S., CLJ.i. 09/04/98 (Test Article received from Wildlife futer:w...rional, LtdL) 09/29/98 {FmalReport Issued) Hexabmmocyclododecane., (Test SubstanceNo. 4615) Thetest article employed in !Pis study was a composite sample ofB:BCD from. tll:iree companies. The sample w-e;.;; mixed at Wildlife lnte:mational, l.tdL, 8598 Commerce Drive, Easton, Macyland 21602, and forwarded to the AlbemarleTeclmica! Centerfor analysis. The identity ofone sample., taken !from the center ofthe bulk sample mlxrure, was confumed using Fourier Transform Infrared analysis using SOPNo. 1\RS-284-R4. fu this procedure, the sample spectrum was compared

**~o** an Aldrich standard reference spectrum ofHBCD In addition, seven separnte samples, taken !from various locations in the bulk sample mixture (top left, top

**ri~..ht,** middle !e..."t, middle riglrt, bottom left. and bottom right as we!.l as a samplle taken from the center ofthe

**~-nu-e)** was analyzed using 

# 195

# F 11 vn. Data/Record Retention: rl'-.V.J.C.\......1 l'lV .. '-t:J7.M...-ll!. 

### -64-

ProtonNuclearMagnetic Resvnance SOP No. ARS-l7l-RJ to determine the isomer distribution and purity ofthe mixture. Chain ofcustodyand sample hanillm.g

**pro~m:-es** werefullowed as per SOP No. ARS-OD1-R6. TableI contains the an.a!ytical !reSUlts and corrciu.siou from the above analyses. The study conformed to ilie requirements ofEPA TSCA GLP's listed tmde.r "'-{) CFR Part 792. There were no ciK=stan= obs-•Tved that mav have affectedthe quality orintegrity ofilie data.
- All completed logbooks, spectra, original data and reports shall bekeptfiled inthe custody ofthe Study Chemist until theToxicology Studies are completed, afterwhichtime they will be furwarded to the GU"' Cooroinator and stored in the designated H&E archives at the address listed under Section II RSponsor.

**~** The ana.lytical samples were placed in the GLP Sample

**Arch..~e.**

GeOrge Ricks, MS.., C.l.H. STUDY CHEMIST



# 196**~ ~** 

N I-'

ill

''-..l TABLE I CHEMICAL CHARAt."TERIZATION ANALYTICAL RESULTS FOR DETERMINING THE IDENTITY AND PURITY OF IIEXABROMOCYCLODODECANE (TEST SUBSTANCE NO. 4615) Reference Study No. HBCD-001 CiiEMICAIL NAME: He:ubromocyclododeune
C.A.S. No.: 3194-55-6

MOLECULAR JFORMUJLA: C 41 IR 10 !1Jr 4

**-~** PHVSICAJl.lli'ORM: White Powder CWSMICA!L STRUCT'UJRE: "'

"' "' "'

;· ANAJLVSKS RESULTS ANALYSIS i ANALYST

FT-ffi liM.TE .

The sample FT -llR spectrum matched !hat of the Aldrich reference spec!mm. All spectra l 09/2! /98

are 011 file with I he original data. WdgM% W.T. CoiJb II W.T. Cobb I

**To~** !Lel'i Top JRigh! Mlti!Lelft Mhl Wglli li.Mm lldi l'l!m RlgM C¢nler **Avcr~geW!.%**

illNMB

_Alpha Isomer 6.4 6.5 6.6 6.7 16.3 5.9 6.4 6.4 I 09/JHB/98

/ Beta Isomer 41.6 4.1 4.8 4.6 4.6 4.3 4.2 4.5

**~emma** Isomer 79.2 79.4 78.2 78.5 79.5 79.9 78.7 79.1 1 I

le!rnbromo- 0.6 0.9 0.9 0.6 0.9 0.5 0.7 0.7 I cydododecane I 1

Oiber **U•lli.now~~** 9 I 9.0 9,4 9.5 ll.5 9.3 9.9 9.2 I

isobulanol 0.1 0.1 0.1 0.1 0.2 0.1 0.1 **O~IJ!** j I l **[~~~~,L~~l;! ;Lrtic!~~eE,~Lttei~s**  **I~** CD. Tl:e **pu'it>'~~~~~y~~;~;o:~:_:=** 0"1 '-'' < < r

0

r ,

fT1 z

-1

fT1

ll z **~** 0 z

)>

r **~** 0 '"0 **~** ...... m

n

.....,

z

0 ..,.

w

1.0

>- 1 ,_.

,_.

.......

# F 13 -66-

TOXICOLOGY AND INDUSTRIAL HYGlliNE L.4.BOR<%.TOR'\! CHEI\llCAL CHARACI'ERIZATION ANALYTICAL FOR DETERlYrL1\"fl\iG THE IDENTITY AND PURITY OF

**HEXABROMO~~CLODODECf~E** ALBEMARLE QUALITY ASSURA1\'CE UNIT STATEl.Vi!ENT Inspections were conducted under rhe applicable Good LaboratoryPractice regulations in accord with the requirements ofEP A, TSCA GLP's listed under 40 CFR_

**P~_rt** 792_ Findings were reported to management on the following dates: TYPE AUDIT INSPECTION DATE REPORT 

# I DATE-JREPORT

# l DATE ISSUED TO ISSl!£DTO 

### i STUDY DIRECTOR 

### I lVI:ANAG:&'VIENT 

### i STUDY MOJ'<TIOR 

# L ____J 1---- Phase 09/17/98 09/29/98 1 09/29/98 1 DraftReport 09/29/98 09/29/98 \ o9/29/98 

# I 

# i{Hjqf Dace 

# co 198

# F 1WILDLIFE INTERNATIONAL, LTD.

- 67-



### . ANALYTICALRESULTS FORDETERMJ:ININ"G

HEXABROMOCYCLODODECANERT

**DEVELO:P~AL** 

## TOXICITY BEXABROMOCYCLODODECANE (HBCO) W1L 

### Jl.esetJ:Feh Lrl.bomibrle:s

**~(nent;e** Stmi'yNlil.

**}~1**J©iHJ

**~~**



### Dr.Ma:rels L lli.Jrll""y, Stud:,_·

**l\1©1i:t.H:~r**

**II~Uh ~l'lld**  

### En

### viroJUment

**.Al~~le** 

### Corpor-.ati<n.l. 

### 451

**:Fl>:~tida S~d** 

**B~t©tm** 

### Rcmge.

**G!."';org~** 

### M.

**Ril~;:~,** 

## Smey

**Ch~lt'll.iil.t**

**To::ac~)lo~or ~J~Ad**  

### b.d:i!:urtrial

**""~>"""'"·'~""""'**



## 2 PROJECT NO.: 439A-lll

### WILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO.: 439A-lll -68-



### ALBEMARLE CORPORAYION HEALTHAND Em'"lRONMENT DEP 

### ANALYTICALRESULTS

**FORDE~G** 

### THE STitlBILITY

**Ol~'** 

## BEXABR01',10CYCLODODECANE INSUPPORT ""A P:E!ENATAL 

### DE,VELOP:MENT.ALTOXICITYSTUDY

**BE"'\ABROMOCYCLODODEC~** 

## (HBCD) INR.A.:l'S" W1L

**R~eurr..h** 

### LtilJon:dt.JrmRtifertmct! Study

**ll~J.** WJL.186tlfl9

Alben:m:de Co:!poll<!tion

**~lillrl~D~~t**

**4.51lF1~**Street

**B~ Rm:.g~:;**  La. 70801 .Brody

**~..mrl:roro ~** 

### t. &r:iy,D.V

**~1Vt.,** Ph.D.

**A1~e** C©XF«<."C$.J.:n1

**JUh~:WTeclmiew C~rer**  

### 8!JDO G.SJU Av;;:mw Baton

**R.a1J~e,** 

### l-1!. ?0820 Stwiiy Cl,;;mist:

**Q~** tlL 

### Ricks, lVi.S"> Cll-I. 

### 01101199 (Prom:ool A:PP!ovedJ iH!l5!S9 tF.'IrillJ.

**~rt**h"iUed)

**Eeor~:J.oc~'ll)d,Jrl=ri_e T:~**  **Sub,4l:l:r:~** 1-:"lD. 4615) A

### pre-study Silltl

**~'fif-sttlliy ~1~**  oftil;; ru:at testiilriide waspr,;pa;md 

### :at

### V.TfL

**R~-e;a,;~;h Lahon1.tori~**  **In~.,** 1407

**G~Kge** 

### Rm:ld,

**A1<1:!J.End~** OH44805--9281 and

**iol'"''J&'T~::l**

### w the

**.AJ.b~ru:'l~**Tee:®icd D:1lte:r foe ;.tnhili.ry

**clr::~.mlinru:ion..** 

### Tlw :rtRbilit)• offue

**;n,e~t** regt 

### BJ:tk:l;;; 'Wa3 

### c"'i:ru::.n:illl<:odby

**rorop~g** fhe purity

**(isom..~**disttibtlrion) of1:he

**pr~Ett-1-d.y** sru-:c:ple to

**tl~** c;f

**'!.h~** FTc,to:c. Nuc!.e2.r

**iMt~eti.~:: Rcc:so:o.an;~**  (SOP Nc.

**,<>~J:S-111.;l_D)** w2iS

**~>loyd** to d"t;;;:ew.Jne the bom,oJf dis!J:ihution oHbe '""'""-'''',;"· Cl:uliTI, 1!2llrl Sa:mple · wa:> wndnebed

- w

**t~!:iJ.bliS:hed** st1mdar;}

**uv;::J.~,;.w"'** m>C;.cedm:es. 

# 201VVILULII-1::... IN!t:..I-<NAIIUNAL, LIU.



## vn. Results: 

### PROJECT NO. 439A-lll -69-

Table 

### l eru:mri.ns the mmlytica! rew..db a.nd. crmciu..sim::l fromthl:: above

**m:W~.** 

### No

**::ltn.!hti~cl** Df"l.":le dllm

**'~.ras**

**perf~** 

### AdditiomllJy, :UQ

**c.L~tac._~ V!EJ1~**  ODst:.r\IG.'d. tJ:J£\J:

**might~~**fu© qwility ·Df ;:hi:: da:t;_;" 

### 'Th!: study coll:lfi:n:moo m 1\fu:_

**requlrt::i:n~; ~:>f**  El?A TSCA (40 

### CFR.Plm '792) alldEPAFIFRA CFR.Pllrt 160) Good

**La~~Re~!md** theOECD [C(91)U61FUW]

**Good~zy ~~**  **R~~gulii!tioll!i.** 

### All. origirullnnv

**~ ~**  **~..ll be~** 

### file.tintbJ:l

**c~dy** 

### oftbl

**Study~llldll**

### the

**~gy ~**  **~**

**camp~** 

### aftr..r

**whi~ timt~** they

## \'\iill be

**~.tded**

### ro the 

### GLP

**-~ arui~** 

# w.

**~ desigr;l~**  H£E nmu'\1!:!-3<rtthe

**~lisred mW"~:S~** 

## n:

**"S~."**



# ~lr/.Jj_ DATE



## 2020 w 1\.)

0

(.).) TADLEI ANALYTICAL RJB:SVLTS FOR DETERMINJ!NG TilE STABILlTY OF HEXABROMOCYCLODODJECANE IN

SUPPORT OF MA PRENATAL DEVEWPMENTAL TOXICITY STUDY OFmb.uuJROMOCYCLODODECANE

. .

(l!mCD) IN RATS" {Fm ReseMCh IA6orotorlu 1Ufmne4! Siw:(p Nlll'. 'WJL..186009} CHEMICAL NAME: **li!Rll!hrollilloeyd!idod~!!.le** I C.A.S. NUMBER: :U!U-55-6 !:!2!!CUIL.AR **__]Q~LA~ ~~8~**  j I'UYSICAL FORM: Wlilte J!>ovvde1· **~** NMR.RESUJ..,TS (Wt. %) ANALYSIS .ANALYST
SAMJP'LEID DATE

**Dt~te** B!m!pnoo ISO.Mlll'.R§ **·r~!:l-1!b~!l-> /;lpli~**  **~111** Gmlillll!!fll **~\ltt~Une !irollmi~~>PI**  Umill!ll'lntll

..

lfmCJ'» . U/J-il/9® II .iii 5-.li'J 713 u 0..1 !ll.'Hl **IJNfiJ~** W.1'.CGJb&

'

**w:i'.~abb** J

**~ p./2!!1'9~**  6.'1 5.'2 7i.7 0.6 D.l 1.1 **Olffi~/99**

. **~~~~~~~";;imcnwas~bi:T~the©Olarsooft~~=~::~** --.) 0

# G 04 - 71 - 

### Quality Assurance Unit Statement Albemarle Corporntion 451 Florida Street Baton Rouge, LA 70801 Study; Analytical Results for DeteTmining the Stability

**oflHicxab~ro:;mo:H:yclodlodlccane** in Support of" A PrenatalDevelopmentalTo:rlcity Study of

**Hexab~romocydml!odccane** (I!!BCD) in Rats" WilResearch Laborntories Reference Study No. '\VJ!L-.186009 This studywas inspected by the Albemarle QualityAssurance Unit ancl the findings were reported to manage}llent andthe studydirectoronthe dates listed below: Study Phase Inspected Date ofPhas<: Inspection **~ofReport** to StudyDirector DraftFinal Report A.udit January 14, 1999 January 14, 1999



# ~~ B. Kristm Hoover GLP Consultant Date: January 14, 1999



# 204 Dale ofReport So

**Manag~** January 14, 1999VVILUL!t'"t:.. I!'! I t:...Ml'lAIIUI'!AI..., II-i!...'.



# G 05 -72- 

### APPENDIX III Specific Conductance, Hardness, Alkafutity and pH ofWell Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: 

### Test Substance: Test Organism: Dilution Water: Specific Conduc\.ance

**(~os/cm)** 

### Hardness (mg!L as CaC03) 

### Alkalinity (mg/L as CaC03) pH Chemical Manufacturers Association's 

### Brominated Flame Retardant HBCD 

### Rainbow Trout, 

### Oncorhynchus mykiss 

### Well Water Mean 

### 311(N=4) 128 (N = 

### 4) 17l(N=4) 8.l(N=4) Range 310-315 124- 132 170- 172 

### 8.1-8.2 Pane!

# G 06 -73- 

### APPENDIXN Analyses ofPesticides, Organics, Metals and Other lnorganics 

### in Wildlife International, Ltd. Well Water 1 ANALYSIS MEASURED CONCENTRAlOON l'vlisce!Janeons Measurements Total Dissolved Solids 286 Arrunonia Nitrogen < 0.050 Total Organic Carbon2 < l.O Total Cyanide < !0.0 Organochlorines and PCBs Aldrin < 0.005 AlphaBHC < 0.005 Beta BHC 

### '· 0.005 De!taBHC < 

### o.oor Ganuna BHC (Lindane) < O.OC. Chlordane < •J.02.'i DDD,pp' <**~-~"** DDE,pp' < 0**~** DDT,pp' < 1.\.l(h Dieldrin < u.005 Endosulfan, A < 0.005 Endosulfan, B < 0.005 Endosulfan Sulfate < O.oJS Endrin < 0.010 Endrin Aldehyde < 0.005 Heptachlor < 0.005 Methoxychlor < 0.007 Heplachlor Epoxide < 0.005 Toxaphene < 0.500 PCB-1016 < 0.260 PCB-1221 < 0.260 PCB-!232 < 0.260 PCB-1242 < 0.720 PCB-1248 < 0.720 PCB-1254 < 0.720 PCB-1260 < 0.720 Metals and OtherInorgani.cs Aluminum' < 100 Arsenic' < 25.0 Beryllium' < 0.50 Cadmium' < 1.0 Calcium' 35.0 Chromium' < 2.0 Cobalt' < 1.0 Copper' < 20.0 Iron < 100 Lead' < lO.O Magnesium' 13.5 Manganese' < 1.0 Mercury < 0.20 Molybdenum' < 2.0 Nickel' < 2.0 Potassium' 6.62 Selenium' < 25.0 Silver' < 1.0 Sodium' 213 Zinc' < 20.0 1 Analyses performed by QST EnvironmentaL Gainesville, Florida for samples coliected on November 3 through :'-im,ember !. 199'. 2 Analyses performed by Wildlife International Ltd. for the sample collected on 1\ovembcc 5, 1997. 3 Analyses performed by Wildlife International Ltd. for samples collected on J'\ovcmbcr 5 through 7, 1997.**~ng/L** mg/L mg/L J.Lg/L**f.l!f1~** f.lliL ).ig/L }lg/L ).1£/L ,U.g/L }L&'L ).lg!L .u.g/L J.Lg<'L .u.g!L J-Lg<L jl.g,'L J.Lg;L ).lg;'L J.l.g;'L flg;L )J.giL**~tg/L** !J.g/L f.l&'L J.Lg!L p..g/L fl.&L J-Lg.'L fl.&'L vg/L lw/L f;_g:L ;.t&L mgiL J..!.&'L fl&'L f.lg/L i-L&'L i-L&'L mg:'L IJ-giL flg.'L ,t.-tg/L t-tg/L mg/L ).lgiL t-tgt"L rng/L J.l.g>'L

### WILDLIFE 

### INTERNATIONAL, LTD. 

### PROJECT NO.: 

### 439A-lll -74-


### APPEI\rniX V
THE Al'\l'ALYSIS OF HEX.I\BROMOCYCLODODECA.l\JE (HBCD) CONCENTRA TWNS 

### IN FRESHWATER/RAINBOW TROUT TISSUE 

### IN Sl3PPORT OF 

### WILDLIFE INTERNATIONAL, 

### LTD. PROJECT NO.: 439A-lll



# G 07Y V 1 L..L..J L..l r

**~;;;..** I I'<I I i;;;..r\.1'<1.1'-'\ I I ....... 1'1.1'-'\.L.. '.-.:.::L_I

**U~.**----------------..:,.t'..:,JK..:.;U.:.;J;_:C:_'L:..·.:_l

**_.:.1'\I~U:;..:.:..** :__

q...:,:.:).:.;':J;_:A..:.-_:1_:1..:_1



# G 08 -75- 

### REPORT APPROVAL
SPONSOR: Chemical Manufacturers Association's Brominated Flame

**RF'<:~rdant** 

### Panel TITLE: Hexabromocyclododecane (HBCD): A Flow-Through Bioconcentration Test with the Rainbow Trout 

### (Oncorhynchus mykiss) WILDLIFE INTERNATIONAL, LTD. PROJECT 

### NO.: 439A-lll

PRINCIPAL fl\l\/ESTIGATOR:

DATE
MANAGEMENT:


### Willaffi



# f~<~ DATE Manager, Analytical Chemistry

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-lll



# G 09 - 76- Introduction Freshwater samples and tissue samples were collected from a flo\v-tlrrough aquatic test to determine the bioconcentration potential of hexabromocyclododecill1e ir1 the rainbmv trout 

### (Oncorhynchus mykiss). The study was conducted by \Vildlife International, Ltd aild identified as Project Number 439A-Ill. The analyses of freshwater and tissue samples were pertorrned at '0/ikllife International, Ltd. by high performance liquid chromatography Vi!.ith 

### mass spectrometry (HPLC/1\iS). Water samples were ex-tracted and analyzed by HPLC/MS. Tissue samples were homogenized, extracted and analyzed by HPLC/MS. Freshwater samples were collected and analyzed from November 19, 1999 

### to February 

### 1, 7000. Tissue samples were collected from November 

### 23, 1999 to February 

### l, 2000 and analyzed from November 29, 1999 to February 

### 8, 2000. Occasionaliy, stock samples were collected wit.l-t freshwater samples to confirm diluter performance 

### and stock stability during the uptake phase 

### ·:)fthe test. Diluter stocks were diluted 

### and analyzed by HPLC/MS. Diluter stocks \vere collected 

### and analyzed 

### on November 

### 19, 22, 30 and December 

### 7, 1999. Additional tissue samples were collected an.d 

### a.IL:'1lyzed gravimetrically for lipid content. Samples for lipid content were collected on November 19, 1999, December 28, 1999 and February 

### 1, 2000 and a..>alyzed bet\veen February 

### 9 and 

### ll, 2000. Test Substance The test substance, HBCD, was used to 

### prepare calibration standards and 

### matrix fortification samples and was identified as Wildlife International, Ltd. identification number 4615. Analytical Method The analytical methodology for the analysis of HBCD in 

### stock, f:resh1.vater, tissue a..1d stability samples is described below. The methodology also includes proce.iures used in 

### the preparation of tissue quality control samples and the analysis oftissue samples for lipid content. Stock Samples Stock samples were co11ected in 20-rnL glass vials. An 

### aliquot ofeach stock sample was initially diluted in 

### acetonitrile (ACN) followed 

## b:· a secondary dilution in 

### 75% ACN 25% H 2

### 0 Ahquots of diluted stock samples were transferred to autosampler vials and submitted for analysisVV I LULl 

### r 1::.. H'l I 1::..1""<1"\IA I IUNAL, LTD. .I:'KU.ItC 1 NU.:**4J~A-i** 11



# G 10 -77- 

### Freshwater Samples A method flow chart for the analysis ofHBCD in freshwater is presented in 

### Figure 1. Freshwater samples (50-mL 

### aliquots) were volumetrically collected in 125-mL 

### separator:v fmttrels 

### that contained 25 

### mL of dichloromethane (DCM). The separatory furmds were shaken for one minute. After t"he aqueous and organic phases separated, the organic phase (lm.ver of each was 

### drained into a 125-mL round-bottom flask. The extraction procedure was repeated \vith a second 25-mL aliquot ofDCM. The organic extracts from the second ex-traction were combi..."IJ!ed with the extracts from the first e,._iraction in the appropriate flasks. The extracts were rotary evaporated under vacuum in 

### \'Vater baths maintained at 

### approximately 

### 40°C until 

### 1-2 mL ofeach extract remained. 

### The remaining 

### DCM ,,,.as evaporated to dryness under a gentb stream of nitrogen and the residues were reconstituted v1.ith the 

### requisite volume of 75% ACN: 25% H 2

### 0. Aliquots of the reconstituted extracts were transferred to 

### autosampler vials and submitted for analysis. Freshwater quality control (QC) samples (matrix blanks and fortifications) -vv-ere processed in the same marmer as the test samples, except each QC sample was prepared by first adding 50 mL of freshwater to a 125-mL 

### separatory :funnel, fortifying the 

### water with the appropriate JHBCD 

### stock solution using a gas-tight syringe (for matrix fortification samples) and then adding the initial 25-ml. aliquot of DCM. Matrix blank samples \vere not fortified with t.he test substance. Back-up freshwater samples were collected at each sa:rnrling interval in 

### glass French-square bottles that contained 25 mL of DCM. 

### Back-up fresh\vater samples w·ere stored refrigerated. If 

### necessary, the back-up freshwater samples were removed from the 

### walk-in cooler, traruferred to 

### 125-mL separatOI}' funnels, and processed using the same procedures deser:ibed above. Tissue QC 

### Samples Edible and non-edible fish tissue QC 

### samples were prepared from the same lot of culrurc fish 

### as used in the test (lot # 

### 99-19 RAI). 

### Fish from that lot Vi'e1e collected and separated into bulk edible and non-edible tissue samples. The ·weight of each type ofbulk fish tissue s3.!-nple was recorded and the bull< fish tissue samples were stored frozen until homogenization. Each bulk fish tissue type vvas 

### removed from the freezer and quantitatively transfen·ed to a Waring st..-1.i.-rl1ess steel blender for initial homogenization. Based upon the total weight ofbulk fish tissue received, a 

### nom.in..al volume of saturated

# G 11 -78- sodium chloride (NaCl) was measured in a graduated cylinder to yield a target homogenate density of 0.25 g of fish tissue/mL. The tissue was blended with 

### small amounts of saturated NaCl until well mixed. The mixture was transferred to a second graduated cylinder. The remaining

**sai'LlraJ~ed** I\

### aCl v;;as used to rinse the blender and poured into the second graduated cylinder. TI1e 

### total volur:1e 

### in the graduated cylinder was recorded. The mixture was transferred back to the origi_.rJ.al sample storage container and :finally blended 

### 'With a Janke 

### & Kunkle, U1tra-Turra'X, Model T25 homogenizer. A Teflon° coated stir bar was placed in the sample container and the container was placed on a magnetic sti.r plate. 

### \'v'h.ile the mixture was gently stirring, 5-mL aliquots of the homogenate were removed -vvith a calibrated Oxford pipetor and transferred to 20-mL glass EPA vials. Vials containing edibie fish tissue samples \Vere labeled QC-1 through QC-30 and vials containing non-edible fish tissue samples were labeled QC-31 through QC-63. Tne tissue QC samples were stored frozen until t..hey were used 

### in the preparation of matrix blank and matrix fortification samples or storage stability sat-nples. The actual density for each type offish tissue homogenate was

**~0.2** 

### g/mL, calculated as bulk fish tissue weight divided by 

### total homogenate volume (saturated NaCl volume 

### + tissue vohm1e) 

### By 

### transferring 5 mL ofhomogenate, each tissue QC sample

**contained~** 

### 1.0 g oftissue (5 mL x 0.2 g/mL). Since both the uptake and depuration phases of the test were e2>.iended, an insufficient number of tissue QC samples remained for the final edible fish tissue sample analysis (depuration Day 35), edible fish Iissue sample reanalysis (depuration Day 21) 

### and sampk storage stability analysis. Consequently, the back-up edible fish tissue sample collected from the solvl."nt control at uptake Day 0 was used in the preparation oftissue QC samples for the depuration Day 35 ;:malysis,

**zw-:~d** 

### extra solvent control edible and non-edible fish tissue homogenates from uptake Days 0 and 1 

### were used i.e 

### preparation of tissue QC 

### samples for tl:c depuration Day 21 

### reanalysis and storable stability analysis. Tissue San1ples A method flow chart for the analysis ofHBCD in fish tissues is presented in 

### Figure 2. 

### Ecible <md non-edible fish tissue samples v•lere collected from the test in 20-mL glass EPA vials ;wd stored frozen, 

### if necessary, until analysis. Upon analysis, tissue samples ;,vere removed from the freezer, 

### ifne{;essary, and batched by 

### sarn.pling ii1terval and tissue type. At that ti.il1e, two ofthe ap!Jropriate tissue QC 

### samples dso were removed from the freezer. Test and QC 

### tissue samples vvere ailov,:ed to thavv. One tissue QC

# G 12 -79- sample was designated as the matrix blank sample. The other tissue QC sample was designated as the matrix fortification sample and fortified \'llith the appropriate HBCD stock solutior. using a gas-tight syringe. Tissue samples received during the test varied in \'Veight (

**~** 

### l-3 g). 

### sample preparation, it ·was decided to use the same tissue sample quantity in the methodology rather tl1an variable tissue sample quantities. To keep tissue sample quantities consistent, the volume of saturated NaCl added to each sample during sample homogenization was adjusted such that a 5-.mL aiiquot of the homogenate cont.1.ined approximately one gram oftissue. The folloV1i.11g equation was v.sed to calculate the saturated NaCI volume required for each tissue sample: TissueWeight(g) ..!..:Q_g_ SatnratedNaCl Volume (mL) 

### +TissueVohune (mL) 5 mL 1.0 g x (Saturated

**~aCl** Volume (mL) +Tissue Volume (mL)) = 5 mL x Tissue Weight (g) 1.0 g x (Saturated NaCl Volume (mL) +Tissue Volume (rnL)) 1.0 g (5 mL x Tissue Weight (g)) 1.0 g Saturated NaC! Volume (mL) +Tissue Volume (mL) =



## 2._~ x Tissue Weight (g) c Saturated NaCl Volume (mL) 

### = 5



## ~li.. x Tissue Weight (g)- Tissue Volu.tnc; (rro.L) b 5 mL . Tissue Weight(r;Q_ Saturated NaCl Volume (rnL) 

### =-g- x T1ssuc Weig,'1t (g)- Tissue Density (g/IDL) Tis3ue weight was the weight ofthe edible or non-edible test tissue sample received for analysis. Tissue density was determined during tissue QC sample preparation and v.:as calculated as bulk fish tissue weight divided by tissue volume (total homogenate volume·· saturated NaCl volume). Edible fish tissue density was 1.328 g/mL ar1d non-edible fish tissue density ·was l.l 86 gin:J, 

### The appropriate volume of saturated NaCl \vas added to each test tissue sample using a graduated glass pipet or equivalent. The test tissue samples were homogenized :tor approx.irnately one minute usiiJ.g a hand-held tissue shredder. If the total homogenate volume vvas >5 

### m.L, 5 mL of tile sample '.vas transferred to a second labeled 20-.mL glass EPA vial using a calibrated Oxford 

### pipetor.

**Th•~** 

### remaining homogenate in the original vial Vi/as stored frozen in 

### case it \vas neede-,d for reanalysis.VV I L...UI-1 

### r £:.. II'\! l t:..M:I'\!A l !UI'IAL..., LTD. .t"KVJtL-1 l'IU.: .:t.:l':fA.-111



# G 13 - 80- Test and QC tissue samples were eh.'tracted by adding 10 mL ofethyl acetate to each vial, capping the vials, shaking them well and centrifuging the vials at 2000 rpm for 

### approximate!-:,: 5 mi..11utes. 

### The organic phase (top layer) of each sample was transferred to a 50-mL pear shaped flask The e;;.--rraction procedure was repeated with a 

### second 10-mL aliquot of ethyl acetate. 1lle 

### e>;:tracts from the 

### second eh.'traction were combined with the e>..1:racts from the first eA.'traction i.n 

### the solvent was removed from the samples with rotary evaporation. flasks. The**Ea~h** 

### sample was reconstituted vvi.th 1-2 

### mL of DCM and transferred to a deactivated florisi!:s packed column. The columns used for this step ofthe method vvere 

### Kimax·:s>, ll mm x 30G mm (internal diameter x length) chromatographic columns. They were equipped vvith removable PTFE stopcocks and replaceable glass tips. Columns were prepared as follows: a plug of Teflon<Z· wool was i.'1Serted at**"t.~e** 

### base of each column. With the column valves closed, the columns were partially filled vvith DCM.**An.~ydrous** 

### sodium sulfate, Florisil® and additional anhydrous sodium sulfate were sequentially added to each colunm at a height of approximately 1, 6 and 2 em, respectively, using a :!iumeL When a coiumn wac; 

### ready to use, the DCM was drained and the sample wa<> added. The so.•nple was allowed to drip through the column into a clean 15-mL graduated centrifuge tube. The pear sLaped flask vvas r,J1Se,d 

### vvi.th a 2-mL aliquot of DCM and the rinse was transfetTed to**th~** 

### colllllliJ. ensuring to rinse**t,~e** 

### sides of the colurrm during the transter. The rinse was collected into the centrifuge tube as 

### it dripped tt'1rough the 

### colunm. The rinse procedure was repeated v.'ith a 2-mL aliquot of DCM foHmved 

## t:v· a 6-mL aliquot of DCM. The samples**(~12** 

### mL) were evaporated to dryness under nitrogen. Each sample was reconstituted with 10 mL of ACN and transferred to a 125-mL separaLOl)' funnel that contained 15 

### mL of hexane. Each centrifuge tube was rinsed "l?iTi.ti'1 

### 15 

### mL of ACN and the rinse was transferred to the respective separatory funnel. Tne separatOl)' funnels Yvere 

### shaken 3.J."1d the iower ACN phase from each sample was collected 

### in a 125-mL pear shaped or round-bottom tla.sk ll<e e:>..1:raction procedure was repeated "l.vith a st::cond 25-m.L aliquot of ACN atid the ex"tr;Jcts we:c<e combined in the appropriate flasks. The samples were rotary evaporated to dr;mess Each sample '-\'as reconstituted with the requisite volUJ.-ne of 75% ACN:**25~ o**  

### H"O using Class A gbssvvare. Additional dilutions •sere performed, as necessary, using Class A volumetric flasks, gas-tight syringes and/or Class A volumerric pipets to bring expected sample concentrations within the range ofthe calibration standards. of

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECTNO.: 439A.-l11



# G 1 - 81 - 

### reconstituted samples and/or subsequent dilutions thereof were tr::..nsferred to autosaJnp!er vials and submitted for analysis. 

### If necessary, the method was split into two ;,vorking days. Duri11g 

### the: first 

### ,.,·ere homogenized with 

### saturated NaCl, extracted \Vith ethyl acetate. rotary , samples recoDstn:uted 

### >.vith 

### DCM and stored refrigerated. 

### On the second worl<.ffig day, 

### the samples \Vere 

### :·emoved from the 

### 1valk-m cooler, transferred to the deactivated Florisil® packed colum.11s and the r::st of

### tJ1e method was 

### completed. Back-up tissue samples were coilected 

### and stored frozen. 

### If necessa;y·, the back-up tissue samples removed from the freezer, allowed to thaw 

### and processed using the same 

### procedures described above. 

### If it was necessary t0 

### reanalyze remai..Aing stored homogenates, 1iwse samples \Vere remo'-'ed from the freezer, 

### allm:v to thaw and processed using the same procedures 

### described above 

### from the 

### initi;;:J ethyl 

### acetate addition step forward. Tissue Storage Stability Samples Stability· samples were 

### prepared at 

### test initiation to estabbsh test substance stability in fisb tis::;ues 

### stored frozen 

### during the study. Two tissue 

### QC samples 

### of each fish tissue 

### tyJ::<e**"~-Vere** 

### remov;;:'.Cl from the freezer and allowed to 

### thaw. The edible and non-edible 

### fish tissues 

### v>ere eacb 

### fortified at 100 and 

### l000**~tg** 

### HBCD/Kg using the appropriate .H:BCD stock solution and a gas-tight syr'u1ge The 

### stabi1it-. samples were returned to the freezer. Follmving 76 days of frozen storage, t.'-le stability samples ,,·ere removed from the freezer and analyze<"l. Fxtra sclvent control homogenate \VZ!.S 

### used to prepare nvo ne\o.· edible and two new non-'='dib.!e fish tissue fortification samples at the same concentrations as 

### u'le samples since none of the original tissue QC samples remained The samples (nch'iy fortifieJ ;md 

### 76 days old) were processed usmg the same procedures described for tissue**Ti~JJe** 

### Samples for Lipid Content A method flowchart for the analysis of !ipicl 

### content in i1sh 

### tissue is pre.oeni.cd. m ' Edible 

### and non-edible fish tissue samples for lipid content v:ere collected from u'1e test m 20--ml. 

### glass EP/\ \1.1ls 

### and stored frozen until analysis. Sample weights were recorded at the time of coiicction tissue samples designated for lipid content detem1ination \Vere removed frc1m 

### d1e freeze1 

### and a.lknved 

### to thaw. 

### For each sample, l0 mL of

### \J 

## UJ. WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-lll

. 82- 

### sample was homogenized for approximately one minute uswg a h3Jlt1-hclct homogenate was transferred to a 250-mL separatory fi.mnel that contained 2c: 

### 50 mL ofmethanol. Each sample vial v·as rinsed with an additional 

### 10 mL 

### ot shredder l::ach


### rinse V\'as poured into the 

### respective separatory funnel. The 

### separato0· t'l;nnels vJere .sbaken \V:tb 

### ventiug for approxi....'llately 

### one minute. Fifty milliliters of chlorofonn 

### foUov;:ed 

### 50 no.L of saturated 

### N2Cl \Va;, 

### added 

### to each separatory funnel. The separatOl)' finmels were 

### briet1y S\vided 

### vvit.'1 ventii1g. The pha.ses \vere allow·ed 

### to separate. For each sample, the chloroform layer was drai,'1ed 

### 1hrough a po,·der fLmnel packed with Teflon® 

### wool and anhydrous sodilm1 

### sulfate 

### ir.:w a 250-mL round-butwm 

### flask. An additional 50-mL aliquot of chloroform vvas added to each separator;..· funnel and the e;-.i:raction a.nc 

### draining procedures were repeated. 1l1e 

### C:\.'tracts were rotary evaporated in a water bath maintained at approximately 40°C to near dryness. Each eA.'tract was transferred to a pre-v;eighed labeled scmtillatiOn

. 1 Via•. 

### Each 250-mL 

### rmmd-bottom flask 

### was rinsed -;,.vith a 

### sma.ll volurt1e 

### of chloroform 

### and t.':le rinse \vas transferred to the respective scintillation via1. The remaining solvem in each vial ,,vas evaporated under 2. 

### gentle stream 

### of nitrogen or clean dry air. The vials were re\veighed and the yveights \vere recorded Lipid contenc 

### \\'as calculated for each s&...-n.pk- as the lipid weight (rng) cuvided by 

### the fish weight The back-up edible and non-edibl fish tissue samples collected for lipid content \\'ere usee to determine tissue percent 

### solids on a dry->\'( 

### gtt basis 1l:1e 

### tissue in 

### uncapped 20-rnL. gl2ss E_P_. '\ ,,ials, 

### were weighed before and after drying for L'uee 

### days in a '-'acuum uven mai.'1tai.."1ed at

**.54~58°(** 

### and approximately 30 inches of Hg. Tissue percent solids v:ere calculated for each sample as the fi.sh -zissue weight (g) 

### minus the \Veight 

### change <g) 

### divided by 

### the fish tissue >-veight (g) times one

**hundre.c~** 

### chan.f:;e \Vas 

### calculated as wet

**>~:eight** 

### (g) before ch"'ying dry weight (g\ :--fter Concentrations of HBCD \'-:ere determined by 

### high pcr±orma;.J.cc liquid 

### Hewlett-Packard Model 1100 

### :-tigh Performance Liquid Chrom?Jograph 

### Elmer i\PI 

### lOOLC l\Aass

**Spectrome~er** 

### ChnJm<'ltograp:jc. 

### :'rism analytical colunm (150 mm

**~.-** 

### 2 mm, summarized i.t! 

### T:ble l.

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-lll



### - 83- fortification/Calibration Stocks and Sta.'1dards Three primary .:tock solutions of 

### HBCD were prepared for use i11 

### rhe accurately \veighing the test substance and volumetrically dissolving 

### it in solvnt. Dctvl \Vas 

### used for che first and third stock preparation and tetrahydrofuran (THF) was used for the second stock preparation The primary stock solutions contained 1.00 mg HBCD/mL and were diluted as appropriate with THF yielding secondary stock solutions that ranged from 1.00 

### to 100 ).lg 

### HBCD/rnL. The secondary stock solutions were then diluted as appropriate 

### vvi.th THF 

### yielding tertiary stock solutions 

### that ranged from 

### 0.01 00 to 1.00 

### }lg HBCD/mL The <>econdary and 

### tertiary stock solutions were prepared at approximately weekl:;.· intervals and used in 

### the preparation offortification samples. The first primary stock solution prepared in 

### DCM ,,.·as also diluted in 

### ACN yielding
- 1.

### 00 jJ.g 

### HBCD/mL stock solution. This ACN stock solution was used 

### iJ1 t..he preparation of calibration standards 

### for freslnvater (includes diluter

**sto~ks** 

### when

**app!i~able}** 

### and tissue sample set analyses. l'Liiquots 

### of ilie ACN stock solution \Vere diluted in 

### 75% ACN: 

### 25%,

**NA~·JOpure®** 

### water as 

### follows: Stock Stock Final Standard 

### Concentration 

### Aliquot Diluted Volume Concentration Dilution Solvent (u.g/mL) (mL) (mL) (••g/L)

1.00 0.0500 

### 200 0.250 75% ACN: 25'% HcO 1.00 0.0500 

### 100 0.500 75% ACN: 25S-·o H20 1.00 0.100 

### 100 1.00 75";;, ACN: 25% H:O I.OO 

### 0.250 100 2.50 75'% ACN: 25% H20 1.00 0.500 100 5.00 75% ACN: 25':·'0 H:O 1.00 0.750 100 7.50 75%ACN 250,0 H 20 1.00 1.00 100 10.0 75%ACN 

### 25%H:O 

### Two additional calibration standards were prepared from a secondary Till stock solution that was prepared from the first primary 1.00 ).lg 

### HBCD/mL DCM stock 

### solution. Aliqouts of the LOO

**~·g** 

### :HBCD/mL THF stock solution ;.vere

**diluted~''!** 

### 75% ACN: 25% NA.i"\l'Opure"' \.Vater 

### as follows: ! Stock 

# I Concentration Stock Final Standard Aliquot Diluted Volume Concentration Dilution Solvent

_____jJ:tg/mL) (mL) (mL) (!:J:g/L) 

## I 1.00

**2.~0** 100 25.0 75'}(, ACN: 25% 

### I 1.00 5.00 100 50.0

**75~/D** i\CN:

**25~·D** I

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-ll1 

### -84- All stocks and standards were prepared usmg a 

### combination of Class A volumetric flasks, volumetric pipets, and/or gas tight syringes. Each stock solut!on was assigned a 

### code, which was recorded on a stock preparation log sheet. Calibration Curve and Limit ofQuantitation identification

Calibration standards ofHBCD, ranging in concentration from 0.250 

### to 5.00 

### f.!g/L, were analyzed 

### with 

### each freshwater sample set. Calibration standards ofHBCD, ranging in concentration from 1.00 

### to 50.0 

### J.Lg/L, were analyzed with 

### each tissue sample set except uptake Day 0. The uptake Day 0 edible 

### and non-edible tissue sample sets were a.."lalyzed with standards ranging in 

### concentration from 0.250 to 5.00 

### J.Lg/L. Sometimes, partial or complete tissue sample sets were reanalyzed vvith freshwater sample sets ifthe initial analysis showed that the tissue samples were diluted outside the calibration range. For each analysis, a set of five or seven calibration standards was injected at the beginning and 

### end of 

### the analytical run. 

### In addition, a minimum 

### ofone standard was injected foUo-vv:ing ever:r· five test samples. A 

### calibration curve was derived from regression analysis using the 

### in.stwmental responses ofthe 

### calibration standards. Line::tr regression equations were generate::l. using the peak area responses versus the respective concentrations of the calibration standards. A 

### ty-pical calibration 

### curve is presented in 

### Figure 4. 

### The concentration of HBCD in the fresh<.Nater and 

### sto..:;k samples was deter.nined by 

### substituting the peak area responses into the linear regression equation as follows: HBCD 

### in sample (!J.g/L) 

### =[(peak area- y..

### intercept)/slope]*diiution factor
0 

### R measured HBCD concenLration

**(~** x 

### 100 Yo 

### ecovery = . .. . 

### nominal 

### 'HBCD 

### concentrauon

**(J.Lg/T~)** 

### The concentration of HBCD 

### in the tissue and stability

**sam~**

### Jes was determined by substimting the peak area responses into the linear regression equation as follows: HBCD in sample (f.Lg/Kg) 

### =[(peak area- y-intercept)/slope] *dilution factor Dilution factor for tissue samples= 

### primary dilution x secondary dilution 

### Primary dilution= [final 

### volume (mL)/initial weight (g)]. and 

### Secondary dilution= [final 

### volwne (mL)/initia! voiume (mL)J 

### where: 

### Representative 

### ion chromatograms of low and high calibration 

### stan.dards are presented in Figures 

### 5 

### and 6, 

### respeetively.

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-lll 

### -85- The method limit of quantitation (LOQ) 

### for freshwater samples ;vas 0.0250

**~lg** 

### HBCDIL, calculated as the product ofthe lowest calibration standard (0.250 ]J.g :HBCD/L) a..11d 

### tbe dilutior1 

### factor of the matrix blank sample (0.1

### 00) analyzed concurrently vvith 

### the test samples. The LOQ 

### for edible and non-edible fish tissue samples was l.OO !J.g HBCDFKg, for uptake Day 0 sample sets, 25.0 1-Lg HBCD/Kg, for uptake Days 1, 

### 3 and 7 and depuration Day 35 sample sets, lli!d 125 

### f.lg HBCD/Kg, for uptake Days 14, 21, 

### 28 and 35 and depuration Days 1, 10, 14, 

### 21 and 28 sample sets. The LOQ 

### was calculated for each analysis as the product ofthe lowest calibration standard (1.00 r..tg 

### HBCD!L) and the dilution factor ofthe matrix blank sample analyzed concurrently with 

### the test samples. The dilution factor of the matrix blank sample was dependent on the concentration ofJHBCD present in the test samples. Matrix blank dilution factors were 1.00, 

### 25.0 or 125 

### w.L/g duringthe study. Freshwater Matrix Blank and Fortification Samples Along with the actual freshwater sample analyses, 19 freshwater matrix blank samples ;,vere analyzed to determine possible interterence. Two freshwater matrix blaT1k 

### samples (439A-H l-:MJ..\B-17 

### and 439A-lll-MAB-19) were above the LOQ during the test resulting from the introduction of suspect contamination in the laboratory (Table 2). A representative ion chromatogram of a fresh..,,-ater matrix blank sample is presented in Figure 7. Freshwater samples were fortified at 0.0500 and 4.00 

### f.Lg/L 

### (for the uptake Days 0, 1, 

### 3 and 7 sample sets) or 0.100 

### and 4.00 f..!.g/L 

### (for aJJ other sample sets) using a stock solution ofHBCD prepared in 

### 1HF. Freshwater m2. 'Tix fortifications were prepared &,J1d 

### analyzed concurrently \:vith each smnple set and the analytical results were not corrected for mean procedural recovery. Recoveries ranged from 71.2 to 116% 

### ofnominal concentrations (Table 2). A representative ion chromatogram of a freshwater matrix fortification is presented in 

### Figure 8. Tissue Matrix Blank and Fortification Samples .t\lvng with the actual tissue sample analyses, 15 

### edible and 15 

### non-edible fish tissue matrix blank 

### samples were analyzed to determine possible interference (Tables 3 and 4, respectively). No interferences were observed at or above tl1e applicable limits of quantitation during the test. Representative ion

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT 

### NO.: 439A-lll -86- 

### cL-ornatograms of edible and non-edible fish tissue matrix blw..k samples are presented in Figures 9 

### and 10, 

### respectively. Edible and non-edible fish tissue homogenates 'i-vere 

### fortified at 0 1 

### 00 r.he uptake Day 0 sample set), 0.500 

### (for the uptake Days 1 

### and 3 

### sample sets) or 10.0 j.lg 

### (±or all other sample 

### sets) using a stock solution of HBCD prepared in THF. Tissue matrix fortifications vvere prepared and analyzed concurrently with each sample set and the analytical results 

### were not corrected for mean 

### procedural recovery. Edible fish tissue recoveries ranged from 

### 5l.5 to 93.5% of nominal concentrations (Table 

### 3). Non-edible fish tissue recoveries ranged from 

### 51.3 to 91.1% (Tables 

### 4}. Representative ion chromatograms of edible and non-edible fish tissue matrix fortification samples are presented in Figures ll 

### and 12, 

### respectively. Example Calculations The analytical result and percent recovery for freshwater sample 439A-lll-4, 

### from the 0.3cJ. )1-g/L 

### nominal HBCDtreatment group, was calculated using the following equations:

. (peak area- y-intercept) . 

### HBCD m sample (ug!L) = ,

**~<** dilution factor · s.ope Peak area = 4722 

### Y-intercept = 

### 268.05856 Slope= 

### 2078.28491 Initial volume (V;) = 50.0 mL Final 

### volume (Vf) 

## = 5.00 

### rnL Dilution factor (V tf\1;) = 

### 0.100

. . (4722- 268.05856) 

### HBCD m 

### sample

**~!J..g/L)** = 

### 2078_ 28491 x 

### O.lOO HBCD in 

### sample ()-lg/L) = 

### 0.2143 )-lg/L

0 ,/ 

### R . l measured HBCD concentration (J-tg/L2 

### x 1c>c> ;o 

### ecovery m samp e = . . . · • · · 

### nomm.al HBCD concentratiOn lJ.tg./U 0.2143 t:::gtL 'OO % Recover<; m sample = -

**~'"'** x 1 • 0 ..:;4 f.l.gtL %Recovery in sample= 63.0%**.I.!~V.JL\.......-1 l~V**  .. '"'t.J71A.-1.l!



### - 87- The analytical result for non-edible fish tissue sample 439A-lll-N-84, 

### from the 0.34 flg/L nominal 

### HBCD treatment group, was calculated using the tollmving equation: 

# teeak area - v-intercept) . 

### HBCD in sample (f.Lg/Kg) = 

### 1 x d1iution 

### faccor s ope Peak Area= 33491 

### Y-intercept = 

### 367.91983 Slope= 1850.16565 

### Primary Dilution: Initial 

### Weight (W;) 

## = 1.0 

### g Final Volume (V£) 

## = 25.0 mL 

### Secondary Dilution: Initial Volume (V;) 

### = 1.00 

### mL Final Volume (Vr) 

## = 5.00 

### mL 

### Dilution Factor (Vr./W; x 

### V{V,) 

## = 125 mL!g



### . . - (33491- 367.91983) 

### HBCD at 

### mstrument (f..i.g/L)- 

### 1850_ 16565 

### HBCD 

### in sample (f..l.g/Kg) 

## =HBCD 

### at instrument (f.Lg/L) x dilution factor 

### x unit 

### conv-ersions
• r..r _ 

### 17.9 f..l.g 

### 125 mL . L 

### HBCD 

### m sample ()l.g,Kg)- L x g x 

### 1000 mL 1000 g X 

### Kg 

### HBCD in 

### sample (f..l.g/Kg) = 

### 2238 f.!g/Kg 

### RESULTS 

### Freshwater Sample Analvsis Freshwater samples were 

### collected and analyzed 

### for 

### I-IBCD concentrations on Days -4 

### and -l 

### during the pre-uptake phase ofthe test and Days 0 (0 and 4 hours), l, 3, 7, 14, 21, 28 aTld 

### 35 duri-.ng the uptake phase ofthe test. Uptake was suspended on Day 35 ofthe test and depuration began. During u'le 

### depuration phase ofthe test, freshwater samples were collected and analyzed for 

### HBCD concentrations on Days 1, 3, 7, 10, 14, 21, 28 and 35. Measured concentrations of"HBCD 

### in the pre-test diluter verification (pre-uptake phase) 

### san1ples ranged 

### from <LOQ to 

### 0.0274 1--ig 

### HBCDrL in t:"'le solvent 

### control, 54 6 to 

### 99.8% of the nominal concer>tration in 

### the 0.34

**~g** 

### HBCD/L treatment group and 77 0 to

**82.8~'o** 

### ofthe nomina! concentration in 

### the 3 ...:

**~g** 

### HBCDrL treatment group (TabJ.e 

### 5) DuriiJ.g the uptake phase ofthe test, measured concentrmiom of HBCD in the solvent con1.Tol freshwater samples were <LOQ. 

### Freshwater samples collected fror.1 

### the 0.34 and 3.4 !J.g 

### t-111CD;L 

### treatment groups ranged fTom <LOQ 

### to

### WILDLIFE INTERNATIONAL, L_To. 

### PROJECT NO.: 439A-lll

- 88- 74.2% 

### of the nominal concentration and 28.6 

### to 78.8% of 

### tht> 

### nominal concentration, respectively (Table 6). During the depuration phase ofthe test, measured co:.centratioils d I-IBCD in 

### the 

### freshwater samples were typically <LOQ. On depuration Days 28 Jlld 35, the 0.34 and 3 4

**~tg** 

### treatment groups had at least one freshwater sample 

### "''ith a 

### measured concentration of 

### Jt-<IBCD that \vas :S:2.6x 

### the LOQ. Additionally, the 3.4 IJ.g 

### HBCD/L treatment group had a measured concentration ofHBCD in 

### one freshv•.;ater sample that was 6x, 12x 

### and slightly above the LOQ 

### on depuration Days 1, 3 ;md lO, 

### respectively (1able 6). A 

### representative ion chromatogram of 

### a freshwater sample is presented in 

### Figure 13. 

### Stock Sample Analvsis Stock samples were collected on pre-test Days -4 

### and -1 

### and uptake Days 7 

### and 14 to confirm 

### diluter performanee and stock stability. Me2sured concentrations ofHBCD ranged from 89.2 

### to 99.9% 

### of nominal and 93.5 to 103% of nominal 

### in the 5670 !-18 HBCDIL and 56700

**~tg** 

### H.BCDIL stock sampies, respectively (Table 7). Tissue Sample Analysis Tissue samples were collected on uptake Days 0 (4 hours), 1, 3, 7, 14, 21, 28 and 

### 35 and 

### depuration Days 1, 3, 7, 10, 14, 21, 28 and 35 ofthe test. At the request ofthe Sponsor, tissue sarnples collected on depuration Days 3 

### and 7 

### vvere not analyzed. Measured concentrations ofHBCD in 

### all but one 

### solvent control edible fish tissue sample were <LOQ (Table 

### 8). The results ofanalyses 

### ofedible fish tissue samples collected from the 0.34 !J.g 

### HBCDIL and 3.4 !J.g 

### HBCD.'L treatment groups are presented in Table 8. 

### Measured concentrations of HBCD in 

### all 

### but one sclvent control non-edible fish tissue sam.ple were <LOQ (Table 9). 

### The results of analyses of non-edible fish tissue samples coilected from 

### the 0.34 

### !.tg 

### HBCD/L and 3.4 

### IJ.g HBCDIL treatment groups 

### axe

**pres~nted** 

### in 

### Table 9. A 

### representative ]on 

### chromatogram of

### a non-edible fish tissue sample is presented in 

### Figure 14. 

### Stabilitv Sample Analvsis Stability samples 

### were prepared at test 

### initiation (uptake Day 0) aJld stored 

### frozen 

### The results of stability sample analyses after 76 days offreezer storage are presented in Table l0.

### WILDLIFE INTERNATIONAL, LTD. 

### PROJECT NO.: 439A-lll

- 89- Tissue Sample Analysis for Lipid Content and Tissue Percent Solids Tissue samples were collected on uptake Days 0 (0 hour) and 35 a11d 

### depurat]on Day 35 to determine lipid content 

### in fish tissues on a wet-weight basis. TI1e results of in edibk and non-edible fish tissues are presented in Tables 

### 11 and 

### 12, r;::spectively. T!tK: 

### tissue samples collected for determination of lipid content were dried to detennine tissue percent solids. Tbe results of tissue percent solids determinations in 

### edible and non-edible fish tissues are presented i11 

### T<.bles 13 2nd 

### 14, respectively.VVILUUFE IN II::.HNA I jUNAL, LTD. 

### PROJECT NO.: 439A-lll 

### -90- Table 

### 1 

### Typical HPLC/MS Operational Parameters INSTRUMENT: SOURCE: 

### ANALYTICAL COLUMN: OVENTEMPERATIJRE: STOP TIME: 

### FLOW RATE: MOBJIT.,E PHASE: INJECTION VOLUME: HBCD 

### PEAK RETE:N"'TION TIME: HBCD MONITORED MASS: fie\vleil-Packilld 

### 1v1odel 1100 High 

### Pcrforrrlance with 

### a Perkin-Elmer API IOOLC 

### Mass ...,n,,rw.·,,m 

### Ctcmm<rtograph Perkm-Elmer Heat·ed 

### Nebulizer Operated in 

### Selective Ion Jvloni.toring (SIM) 

### Mode Keystone Prism (150 mm x 

### 2 mm, 5-f-Lm 

### partide size) 

### 6.00 min 0.250 mL/min 

### 15% NANOpure Water® 

### \v:ith 0.1% Formic Acid: 85% Acetonitrile 100 

## IlL Approximately 3.7 minutes 640.5 

### amu

### VVILDLIFE INTERNATIONAL, LTD. PKUJECT NU.: 43':-JA-111



### - 91 - Table 2 M:rtrix Blanks and Fortifications Analyzed Concurrently During Freshwater Sample Analyses Sampie 1-iumber

(439A-l 11-) PT-M.t-.!3-1 PT-!vfAS-1 PT-MAS-2 PT-MAB-2 ?T-MAS-3 PT-MAS-4

MAB-1 MAS-I MAS2 MAB-2 MAS-3 MAS-4

**~1AB-3** MAS-5 MAS-6 Mlill-4 MAS-7 MAS-8 MAB-5 MAS-9 MAS-10 M.?J3.-6 MAS-ll MAS-i'.2 :VfAB-7 MA.S-13 MA.S-14 MAB-8 :'viAS-15 :VfAS-16 Concentration ofHexabromocyclododecane (HBCD) (>tgiL) Fortified Measured'

0.00 <LOQ 0.0500 0.0463 4.00 4.41 0.00 <LOQ 0.0500 0.0446 4.00 3.58 0.00 <LOQ 0.0500 0.0418

4.00 3.55 0.00 <LOQ 0.0500 0.0439

4.00 3.64 0.00 <LOQ 0.0500 0.0435 4.00 3.64 0.00 <LOQ 0.0500 0.0403

4.00 i.l6 0.00

**<~OQ** 0.0500 

### ef356

4.00 4.03 0.00 <LOQ 0.100 0.100 4.00 4.04 0.00 <LOQ 0.100 0.0909 4.00 4.08 0.00 <LOQ 0.100 0.0)43 4.00 3.83 9:0:.5 110 89.3 89.5 83.6 88.8 87.8 91.1 87.C• 9l.l 80.6 28.93 71.2 101 100 J.O!

**90.~~** !02 94.3 95.7 1The limit of quantitation (LOQ) was 0.0250 )J.g/L, calcubted as the product of the JowcSI calibration st:mdard <:0.250 c,g'L) :md the dilution factor ofthe matrixblank sample (0.!00). 2 Results were generated using MacQuan, version 1.5 and 1.6 sofuvare. M:mual calculmions may differ slightly. 3 Result was low because stock used for fortification had started to degrad·c. 4 Re;ult was high because sample was exposed to suspect contamination.

### WILDLIFE INTERNATIONAL, LTD.



### - 92- Table 2 (continued) Matrix Blanks and Fortifications Analyzed Concurrently During Freshwater Sample l\umber Concentration ofHexabromocyclododecane (HBCD) (.ug/L)

(439A-ll1-) Fortified Measurcd 1

MAB-9 0.00 <LOQ MAS-17 0.100 0.0796 MAS-18 4.00 3.67 MAB-10 0.00 <LOQ MAS-19 0.100 0.116 MAS-20 4.00 4.Jl MAB-11 0.00 <LOQ 'vfAS-21 0.100 0.0856 M!.S-22 4.00 3.67 MAB-12 0.00 <LOQ :Y!AS-23 0.100 0.0972 MAS-24 4.00 3.95 MAB-13 0.00 <LOQ MAS-25 0.100 0.101 MAS-2, 4.00 3.84 MAB-14 0.00 <LOQ l\-L<\S-27 0.100 0.0929 M.<\S-28 4.00 3.78 MAB-15 0.00 <LOQ M.<\S-29 0.100 0.0864 \,fAS-30 4.00 3.91 MAB-16 0.00 <LOQ**~{AS-31** 0.100 0.0955**~--AS-32** 4.00 4.39 l\'AB-17 0.00 0.0461" !viAS-33 0.100 0.115 MAS-34 4.00 3.98 MP 'l-18 0.00 <LOQ M.A.S-35 0.100 0.219 MAB-19 0.00 0.0432" MAS-36 0.100 0.0953 MAS-37 4.00 3.63 

### PROJECTNO.: 439A-lll

79.6 91.9 []6 !03 85.6 91.6 97.2 98.7 101 96.1 9'0 Q 94.5 86.4 97.7 95.5 liO 115 99.6

95.3 90.9 1 The limit of quantitation (LOQ) was 0.0250 f,LgiL, calculated as the product of the lowest calibralion standard (0.250 !J.giL) and the dilmion factor ofthe matrix blank sample (0.100).**~Results w<~re** **ge<~erated** using MacQuan, version 1.5 and 1.6 software. lvlanual CBiculations way differ siightly.
, Result was lowbeoau.:c 'ltock used for fortification had s1arted to degrac\e. 4 Result was highbecause samplewas exposed to suspect contamination.

### WILDLIFE INTERNATIONAL, Lm. 

### PROJECT NO.: 439A-lll -93- 

### Table 3 Matrix Blanks and Fortifications Analyzed Concurrently during Edible Fish Tissue Sa.cuple 

### Analyses S:unple Number Concentration ofHexabromocyclododecan<o (HBCD)

**(~tg!K~)** Perc • .:-nl (439A-ll1·) Fortified Measured

E-MAB-1 0.00 < l.OO E-MAS-2 100 82.1 82.1 E-MAB-2 0.00 < 25.0 E-MAS-3 500 434 86.8 E-MAB-3 0.00 < 25.0 E-MAS-4 500 4!7 83.5 E-MAB-4 0.00 < 25.0 E-M.-\.8-5 1000 720 72.0 E-MAB-5 0.00 < 125 E-MA.<:-6 10000 6906 69.1 E-lviAB-6 0.00 < 125

**E-~fAS-7** iOOOO 8929 89.3 E-MAB-7 0.00 < 125 E-MAS-8 IOOOO 7501 75.0 E-MAB-8 O.CD < 125 E-MA.S-9 10000 51% 51.5

**E-~fA.B-9** 0.00 < 125 E-MAS-10 10000 7930 79.3 E-MAB-10 0.00 < l25 E-M.bcS-11 !0000 6908 69.1 E-MAB-11 0.00 < 125 E-MAS-12 10000 8431 84.3 E-MAB-12 0.00 <. 125 E-M. .:\S-!3 10008 9185 91.9

**E-~fAB-i3** 0.00 < 125 E-MAS-14 10000 SOlo 60.2 E-.MAB-14 0.00 < 25.0 E-MAS-15 10000 9353 93.5 E-MAB-15 0.00 <25.0 E-MAS-16 100 87.7 87.7 E-MAS-17 1000 631 63.1 1 Less than values conrespond to limit ofquantitation

**(T~OQ).** for each

**anal~'sis**.. the LOQ was calcubted as the prodncl ofthe lowest c;;lohr:nion standard (1.00 !J.g/L) and the dilution factor of!he matrix blank sa.-np!e (1.00. 25.0 or 125 m.Ug). 2 Results were generated using MacQuan, version 1.5 and 1.6 softw::>re. Manual calculations may difier slightly.VVILULll""" t:... IN I t:...h:NA IIUNAL, LTD. 

### PROJECT NU.: 439A-li 1 

### -94- 

### Table 4 Matri..x Blanks and Fortifications Analyzed Cwcurrently during Non-Edible Fish Tissue Sa.1nple A.nalyses Sample Number Concentration ofHexabromocyclododecane (HBCD) (;tg/l(g) ?c-;n.:;,;m

(439A-Ill-) Forc.ified lvleasured'

**-.,.,=---~--·-·<=---** "J-MAB-1 0.00 < 1.00 N-MAS-1 100 78.5 78.5 N-MAB-2 0.00 < 25.0 N-MAS-2 500 415 32.9 N-MAB-3 0.00 < 25.0 N-MAS-3 500 3423 6SA' N-MAB-4 0.00 <25.0 N-l:vfAS-4 !000 830 83.0

**~-MAB-5** 0.00 <. !25 N·l:vL<\.S-5 10000 7155 71.5 N-MAB-6 0.00 < 125 :-\-:vfAS-6 10000 9109 91.1 N-MAB-7 0.00 < 125 ::--1-MAS-7 10000 7290 72.9 N-MAB-8 0.00 < 125 N-MAS-8 10000 8735 87.4 N-MAB-9 0.00 < !25 N-MAS-9 10000 7417 74.2 N-MAB-10 0.00 < 125 N·MAS-10 10000 8581 $5.8 N-MAB·ll 0.00 < 125

**~-MAS-11** 10000 6939

**69.~1** N-MAB-12 0.00 < 125 N-MAS-12 10000 6998 7(L0 N-l:vlAB-13 O.CO < 125 N-MAS-13 lOOOO 5.)13 55.1 N-MAB-14 0.00 < 25.0 N-MAS-14 !0000 5484 54.8 N-l:v1AB-15 0.00 < 25.0 N-MAS-15 100 51.3 

### su N-M.:..S-16 !000 690 69.0

.

**-~- ----~--=~--~-=-.,**  ' Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product oftlw lowes! CJ.librc!;cn standard ( 1.00 J.LgiL) and the dilution factor ofthe matrix blank sample(! 00, 25.0 or 125 mLig). 2 Results were generated using MacQuan, version 1.5 and 1.6

**sofiwar~.** :'vianua] calculations may differ sfigh(]y_ 3 Mean result (341 anJ 342 Jl.g/Kg. 68.1 and 68.6cy-O) of duplicate reamputation and

**reinj~f:'tion** of original samp!e. First

**Jn~!~ysis** :-tct

**rc.~pcnc~;**

because recovery was low.

### 4 Southwest 

# I 5 Southeast 6 Midwest 

# == 7 Southeast 

### 8 Midwest 

# t 9 Northeast Polypropylene resin compounding Dry blending and extrusion Compounding Dry blending and extrusion 

### Plastics Dry blending and extrusion Water-based Wet blending dispersions Tnermop!astic Dry blending 

# I compounding Wire cable 1 

### Dry

**b!endi~g** 

# Ijacketing 

## m and extrus1on



### 5 )

# I Not given Not given Not given ) I I 

# li 200 10 5 I I i 

## I Not given Not given J',iot given 

## I 6 •!2 

### "!2 

# II ----4 96 

### 40

**~t5** i 

## i l -j 

### Il Not given Not given Not given 

## ! _______!