A. Ul 

CODn;o FORMS FOR SRC INDEXfNG 

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Microfiche No. 

OTS0001392 

New Doc ID 

84010000001 

Date Produced Date Received 

1 0/0~ /00 

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, RRC•\.JT~J .. ·\TED FL'u\-1E RET/i.FD,\J'<·I·s 

'------·---·--------·-·-- ----·----
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 of Prevention, Pesticides, and Toxic Substances TS-7101 

Environmental Protection Agency 

401 M StrEet, SW 

Room 637, East Tower 

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. 

Chemistry Council'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. 

(\ 

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' ~ ,-;;-

~- tit..'L~ 

Enclosures 

R.r::sponsibJe Care·~ 

CL'1 

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. ...,., 

'--t. 

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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 DA IT 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 examined for 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, 1 7 August !98 9: 0 ECD Princip ies 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 l 0. 1999 

November 17. i 999 

November 23, i999 

December 7, l 999 

March \5 - 2 i, 2000 

April l l -- 1 3 aJ1d l 7. 200\' 

June 14, 200(' 

- I~. UL~~:_- Ma~l ? 

Quality Ass'lu·ance Program Super>;isor 

DATE REPORTED TO 

STUDY DIRECTOR: M""NA G E\-LENT 

November 10, 1999 

l'-Tovember 19. 1999 

November 2.3, 1999 

Decem be• 7, ] 999 

l\:1arch 23 .. 2000 .\larch 2 i. 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'l Test with the Rainbmv Trout 

(Oncorhynchus mybss) 

\V1LDLIFE INTERNATIONAL, LTD_ PROJECT NUN1BER: 439A-lll 

STUDY DLR.ECTOR: 

K,~,~~~ Senior Biologist 

'NlANAGEMENT: 

-

He;;:o.~eger, // ~i:.r~~-- • h_D_ 

Director of Aquatic 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 of Contents ............................................................... . 

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 of Rainbow 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 of Rainbow 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 of HBCD in Edible Fish Tissues of Rainbuw Trout Exposed 

to 0.18 ,Ltg HBCDIL ...................................... ·······--····· ...... . 

Figure 2. Concentrations ofHBCD i..11 Nonedible Fish Tissues of Rahabov; 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 of Rainbow Trout Expose,d 

to 1.8 1-1g HBCD/L ........ . ............................. __ .................... .. 

Figure 5. Concentrations of HBCD 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 of Pesticides, 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 of Wildlife lntemational, Ltd. Project No.: 439A-lll .............. . 

Appendix VI - Temper<.:ture and pH ofWater in the Test Chfuubers ............................................. . 127 

Appendix VTI- Dissolved Oxygen of Water 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\'TRA TION3: 

IvlEAN lvffiASURED TEST 

CONCENTRATIONS: 

TEST DATES: 

- gSUM!\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 l 0 SOL VENT 

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 of this study was to determine ihe bioconceni.ration potential of hexabromocvclododecanc 

(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 uptake phase, the test organisms vvere exposed L."l. one: of three groups: l l A sol>:ent control ml. 

acetone/L); 2) A nominal concentration of 0.34 f..lg HBCDfL, or 3) A norrlli"'la! concenuation of 3 4 f).g HBCD/L 

The 3.4 f..Lg HBCD/L exposure concentration vms chosen by th~ Sponsor based on the '"ater solub:lltY of ilie tesL 

substance. At the start of the 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 of the 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 of these 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 

- l 0-

1\L.\TERIALS AND METHODS 

The study was conducted according to the procedures outh1.ed in the protocol, "HexabromOC):clcdodecane 

(HBCD): A Flow-Through Bioconcentration Test \vita the Rainbow Trout (Oncorhvnciws mykiss)" 

(Appendix I). The protocol was based on procedures outlined in U.S. Environmental Protection Agenc:v Se.ries 

850- Ecological Effects fest Guidelines OPPTS Xurnber 850.1730 (I); ASTJI..-fStandard El022-8.:t Standard 

Practice for Conducting Bioconcentration Tests with Fishes and Saint-mer Bivalve !vfolluscs (2): and OECD 

Guideline for Testing of Chemicals 305, Bioconcentration· Flow-Through Fish Test (3) 

Test Substance 

The test subst<mce consisted of a composite of HBCD samples receivf~d from thr<..;c manufacturers The 

material's identity and date received from each of the 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 ambient conditions A subsan1pie of the composite test substa11cc \VOJS sbpped to Albemarle Corporatlc·n 

for characterization and purity a..r1alyses (Appendix H). The results of the 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 ofT est Sclutions 

·n,c nominal test concentrations >vere 0.34 and 3.4 ~J.g HBCD/L. A primary stock '.vas prepared by 

dissoiving the t..:st substance. in aceturre ;:t a roncentrat: un of 56.7 mg HBCD/L The prin1;:u;· stock wa.s mvcned 

to mix A 3econdary stock wac; prepared at a concentr?.tion of 5.67 mg HBCD 'L by 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. The resul"a:::!t test concentrations were not adjusted for the punt)' of the acti .-e i...>gredi~·nt w test 

substance (90. 0'%). The solvent control 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 HBCD treatment grcLi1

]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 of the recommended 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 of 1.hc SPCClCS 

was verified by the supplier. 

The rainbmv trout were held in Wildlife Intemari -·JaL Ltd. \Nell vvater for l 05 days pnor to ::es:i.ng The 

rainbm.v trout \Vere fed at least once daily during holding. Th:.' fish v.-ere acclimated to test condition~' !~x 49 i1ours 

prior t0 test initiation. During the holdin.; and acclimation penods iJJ.e f\c;h ~;howcd no si~11s •.)f disease c:r s:rcss 

During the 14-ciay holding period preceding t..l)e test. water tcmper<lture:-; fron112.1 tul.2_s·.)c_ ThepHof 

the water ranged 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 of this rc:p{>rt :'\t tc:s\ u1.c 

rainbow trout were collected from 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 daily dw·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, :md the 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 of the test was 72 mm '''lith a range of 64 to 86 mm. The <tverage wet weight (blotted cL.-y) 

was 3.46 grams \\':ith a range of2.33 to 5.52 grams. Loading was defmed as tl1e total wet weight offish per liu 

of test 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 dilution water ii1. the nii.xing chambers prior to deLivery to the test crill.rubus The ±1ow 

of dilution 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 io maix1tain 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. Test chambers were siphoned daily and ciea..r1cd during the test to rerr :excess feed_ 

fecal matter, algae rnd bacterial grmvth. Test chambers 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 remove microorganisms and particles. The results of periodic analyses perfmmed to measure 

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 1 Series model no. A). Temperature vvas also measured in 

all test chambers at LIJ.e begimring and end of the test and at weekly intervals during the test, with a liquid-in-glass 

thermometer. 

Dissolved oxygen was measured with a Yello·w Springs L1strument Company, l.tl<: }\!fodel5l B 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 of the 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 and end of the 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 of T'Vcter and Wos!e1-rwer· 

Conductivity· vvas measured using a Yellow Springs illst.l.nnent Corcpany, Inc. Model33 SaEnity-C 

Temperature meter. 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 of darkness A 30-rninute transition of low 

lir-hts - went on and off to avoid sudden changes in lwht intensih - ~ .· 
B 03 

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

- 14-

Observations 

All fish were observed once each day to evaluate the number of mortalities and the number of i..<div:id;;als 

exhibiting sigp..s of abnormal behavior. 

Procedures for Exposure ofFish to HBCD 

The test chambers were conditioned by delivering HBCD to the diluter system for approximately 6 days 

before adding the fish. Water samples were collected tv:ice during the pre-test period to con:fum that equilibrium 

concentrations of test substance in the test chambers were achieved prior to adding the fish. Stock solution 

samples were also collected on selected days to confirm the concentration oftest substance in the stock :-:olution..s. 

At the end of the pre-test period, the uptake phase of the test \vas i.Pitiated on November- 2::;. 199(). by 

placing the fish in the test chambers. Rainbow trout were impartially removed from the holding tank in groups of 

1 to 2. The groups of rainbow trout were distributed among t.he test chainbers until each test 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 the rainbow trout were exposed to dilution \vatenvithout f-'J3CD for a penc:d of 

35 days. 

CoHection and Analysis of Water Samples 

Water samples were collected on Days 0 (0 hours and 4 hours), l, 3, 7, 14, 2L 28 &!d 35 oftl1e 

phase. Water sa:11ples were also collected on Days 1, 3, 7, 10, 14, 2 L 28 and 35 of the depurJtion phnsc. Ai. each 

\Vater sampling interval, two water samples were collected from the solvent control and three sarnpks "·ere 

coLlected from each of the tvl..-o HBCD treatment groups. O;;e solvent control sample and two samptcs from eacb 

of the HBCD treatment groups were analyzed for ~IBCD. The remaining 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 of the water samples are nrrwu"iPri in Appendix V. Ali \Vil1er 

stored at approximately 4°C until analysis. 

CoJ!lection and Aiialysis of Tissue 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 sufficient number of fish were collected to provide tvvo replicate samples of sol Yent control 

fish and four replicate samples of each HBCD treatment group. F1sh were impartially removed fwm the tesi 

chambers and euthanized by severing the spinal cord above the opercular region. The fish Vl'ere blotted dry and 

measured for total length and wet weight vvithin approximately 15 minutes of collection, when possible. Each fish 

was then rinsed with dilution water, blotted dry again and dissected into edible arid nonedible tissue fractions 

Dissection -.vas accomplished by making an incision from just posterior to Lfte base of the 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. The remaining tissue was considered the edible tissue Tissue samples ·were tnu"1Sferred to tared 

scintillation vials and weighed. Procedures for extraction and anai:ysis of the tissue s?,nJples are provided i.,, 

Appendix V. All tissue samples were stored at approxi.-nately -14'"'C: until analysis. Tissue samples collected on 

Day 3 arid 7 of the depu;ation phase were not analyzed due to tl1e pattern of depUTation observed 

Tissue Lipid Content and_Q[y~ 

Selected fish were collected to deterrrine hpid content ;:tiJd dry vveig1i: (_Appendi.x 1l1e determination of 

lipid content and dry ·weight provides the potential to express BCF values in temlS oflipidcontent and v,-eight 

Data Analvsjs 

Replicate tissue measurements were averaged to report the concentration of test substance m fsh 1:issue 

Whole fish concentrations •vere calculated based on the sum of the ed.ib le and noned.ible pac-rs. The stead:· -sii:tc 

bioconcentration factor (BCF) values v:ere detemlLi!ed from the tissue concentrations at ""'""'''"'' 

divided by the average water conce:1tration. Tissue concentrations were considered to be at ~n,-,,rpnt 

if three consecutive sets of tissue concentratio~ts ·were not sign.ificarr!Jy different (p > 0.05) using nnah·s1s of 

variance. If apparent steady-state was not achieved, 

concentration on uptake day 35. 

35 BCF values were cakuJate.d using the average tissue 

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) 

BI 0 FA C 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 and ranges of temperature, dissolved m ')'gen, .. pH of the ·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 within the temperature; range of 12± 1 oc established for the test. Dissolved ox·ygen concentrations 

remained 2:8.8 mg!L (81 percent of saturation) throughout the test. Measurements of pH ranged from 7.9 to 8.3. 

Weekly measurements of hardness, alkalinity and conductivity remained consistent throughout tt'1e test (Table l 

and Appendix VIII). 

Observations of Mortality and Clinical Sims 

Observations of mortality and clinical signs are presented in Appendix IX. No mor..alities were ohserved 

during the test. In addition, no treatment-related clinical signs of toxicity 'ivere observed dt.ttit<g tlJ.e test 

Concentrations of HBCD in Water 

Concentrations of .li"BCD in the solvent control were <LOQ ( <0 .0250 !J.g HBCD/L) except on Day 28 &1d 

35 of the depuration phase of the test (Tables 2 and 3). On Days 28 and 35, HBCD was measured. t-bl?ce>-er, it 

is not likely that HBCD was actually present in the so] vent control. It is suspected that the sarnples were e:;,:posed 

to contamination at some point during the analysis. 

Measured concentrations of HBCD during the uptal:.:e phase in the 0. 34 rJ.g HBCD/L treatment. group v,'ere 

extremely variable. The values ranged from <LOQ to 0.231 t'g HBCD/L. Even >N:ith frequ,~ntck.an1I1f of the 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/L which represented 53% of the nominal test concentration Conct'ntrations of 

HBCD during the d~:puration phase were generally <LOQ. 

Measured concentrations ofHBCD during the uptake phase in the 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 5 3'>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 Day 21 l'nd 28 tissue concentrations were not significantly· different (p >0.05). However on uptake Day 

3 5, the tissue concentrations increased unexpectedly. Dunnett's test showed that the Day 3 5 tissue concentrations 

were significantly different from the Day 21 and 28 tissue concentrations (p ::: 0 05). Consequently, steady-state 

was not achieved in this treatment group. The unexpected increase in tissue concentrations on Day 3 5 may have 

been due to the variability in the measured water concentrations in this treatment group. Estimates of h'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:; of the time to reach 90% of .;teady state in edible tissue, nonedible tissue and whole fish ,,·ere 

118, 99 and 101 days, respectively (Table 6 ). DUl-ing Ll;.c depuration phase of the tesi., HBCD -was eiimimted 

slowly with estimates oftime to reach 50% clearance of35, 30 a..'1d 30 days for edible tissue, 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 (Figure 4) An2Jysis of variance 

showed that HBCD concentrations for upta.~e Days 14 - 35 were not significa.ntly different (:_., >0.05). 

Consequently, the BCF value for edible tissue 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 - 3 5. Whole fish concentrations alsc; reached steady·-state on Da:v 21 (Figure 6) and the BCF 'Nas based on the 

average tissue concentrations for days 21 - 35. BIOFAC estimates of the ti.me to reach 90% of steady· 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 of the test, HBCD was eliminated slowly with estimates of time to recch 50% clearance of 19, 

20 and 19 days for edible tissue, nonedible tissue and 'iV~lOk fish, respectively. 

CONCLUSIONS 

Hexabromocyclododecane (HBCD) rapidly bioconcentrllied in the tissues of rainbow trout (Or~corhynclws 

myldss). After only 4 hours of exposure, measurable concentrations of HBCD were present in the fish tissues. 

Rainbow trout exposed to 0.18 j.!g HBCDIL did not reach apparent steady-state tissue concentrations during the 

length of the test Rainbow trout exposed t.o 1. 8 j.!g HBCD/L acru cved steady-state tissue concentrations at Day 

21. In this study, the BCF calculated for the 1.8 j.!g HBCDIL treatment group was considered to be a better 

estimate of the BCF because: 1) Water concentrations were mainta:u1ed more consistent~'); and 2) Sieady-state 

vvas achieved. The BCF values for edible, nonedible and whole fish were 4,650, 12)?.66 and 8,97 4, re::~pecti-,elv 

Clearance ofHBCD from fish tissue during depuration was slow. The BIOF AC estiu:;ates for time to reach 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 Practice for Conducting Bioconcemration Test:; with 

Fishes and Saltwater Bivalve Molluscs. American Society for Testing and l'v1aterials 

3 OECD Guideline for Testing of Chemicals 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 of Water Quality Parameters 

Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel 

HBCD 

Rainbow Trout, Oncorhynchus mykiss 

Well Water 

Temperature' 

(°C) 

12.0 

1!.8- 12.3 

11.9 

1 1.5- 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.kalini ty 

(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 CaC03\ 

137 lOA 

132 - 140 <!- 24.1 

2 At a temperature of l2°C, the dissolved oxygen saturation concentration is 10.8 mg/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 <LOQ1 <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 

1The 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 <LO

0.155 () 1

0.157 () 1

1.34 1.5

1.34 !.5
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 of HBCD in Water D..uing the Depw-ation Phase 

Chemical Manufacturers Association's Brominated Flame Retardant Industry Panel 

HBCD 

Rainbow Trout, Oncorhynchus m}kiss 

Well Water 

Day of Study 

(J-IgHBCDIL) 

3 7 10 14 21 28 

<LOQ1 <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 

1

The Limit ofQuantitation (LOQ) was 0.0250 )J.g HBCD!L 2

Samples presumed to be contarn.L.1ated. 

35 

CC:49~o= 

C.0542 

•.,_ 

0.0648 
Sponsor: 

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 '· Non

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 

(flg HBCD/Kg) 

32.8 

24.8 

24.4 

270 

123 

91.8 

165 

97.4 

382 

310 

366 

456 

733 

1!29 

1199 

583 

937 

91!3 

~(i1 

11 :JO 

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

wt. + noned1ble wt. 

-------------~
- 24" 

Table 4 (Continued) 

HBCD Concentrations in Edible, Noncdiblc and Whole Fish 

Tissues of Rainbow Trout Exposed to 0.18 I li3CD/L 

Sponsor: Chemical Manufachtrcrs Association's Hrominatcd Flame Retardant Industry Panel 

Test Substance: HHCD Test Organism: Rainbow Trout, Oncorhynchus mykiss 

Dilution Water: 

Sample ID 

EG9/N(,l) 

E70!N70 

h71/N71 

E72M72 

EiQ/Nfi2 El\3/NXJ 

E84/NX4 

E85/Nfi5 

Well Water 

Uptake 

Day 

21 

21 

21 

21 

2g 

21\ 

Edible Tissue 

Concentration 

(itg IillCD/Kg) 

66'i 

767 

671 

801 

751 

R81 

727 740 

Edible Tissue Nonedible Tissue 

Weight Concentration 

(g) (!tg liBCD/Kg) 

3.3308 1744 

2 0901 l"'f" 

1.8612 2636 

1 7825 1621 

1.6704 1532 

lli!'i4 2386 

1.4706 2238 

1.4.'109 2426 

Noncdi

W

3.

1.1.

151.

1.

J. 

1.

E95/N95 

E96/N% 

E97/N<J7 

35 ]CJ]7 2.'i726 3396 20

35 1155 !.6082 3890 1.35 gss 1.99.14 3051 135 1042 1.2609 I 

1 Whole hsh Concenlr<tlicm cc (cdjbk wt. X edible GO!ie. wl._,X noncdiblc cone.) 

wl. 1- nonedible wt. 

--·: 
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. 

Noned

W

2

1

1

1

1

1 

1

1

1.

1

1

u
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 __ \ 7115 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 

[3118 

2497 

1090 

!362 

614 

1362 

1476 

1597 

330 

1049 

2 

--

Nol' .dihle Tissu

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==''···===== 
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 Brominated Flame 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 

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

wl. + nonedible 
Sponsor: 

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.) 

None

W
- 31 -

Table 7 (Continued) 

HBCD Concentrations in Edible, Nonedible and Whole Fish 

Tissues of Rainbow Trout Exposed to 1.8 HBCD/L 

Sponsor: Chemical Manufactmers Association's Bromi.natcd Flame Retardant lildnstry Panel 

Test Substance: HBCD Test Organism: Rainbow Trout, Oncorhynchus mykiss 

.Dilution 

Edible Tissue Edible Tissue Tissue Nonedib

a""""mple ID 

El10/Ni!O 

E111/N111 

u 12/N1l2 

E! l:l!Ni 13 

E[iUfN143 

E 1 t!4/N 14,! 

Fl45/NI45 

El4G!N146 

El54!}1154 El55!Nl55 

F:l56/Nl56 

El57fNI 

1 V!11ole Fish Concentration 

Depuration Weight Concentration We

Day (g) (Jl llBCD/Kg) . _(

I 1.3927 15609 !.75

10 

!0 

10 10 14 

14 

14 14 

I 1<!43 Ui451 22868 1.88

15998 1.1291 29797 1.3

261!6 

7o26 

10999 

7991 

5354 

10419 

9916 

7877 

6575 

1.3459 211946 H6

1.2653 

1.2525 

1.9346 

0.9461 

1.5073 

3.2649 

1.5836 

23430 

29!tl6 

t\9662 

28026 28059 

12683 

17tlJ8 

16952 

22385 

1.30

1.25

2.19

1.14

1.67

2.47

1.58

2. J8

U;,ilibk: wl. X edible conQ,L:i_:.Jl!ont:dible wt. X glllQj 

(edible wl. + nonedible wt.) 

-· ·- ...... 
- 32-

Table 7 (Conlinued) 

HBCD Concentrations in Edible, Nonedible and Whole Fish 

Tissues of Rainbow Trout Exposed to 1.8 f.,tg HBCDIL 

Sponsor: 

Test Substance 

Test Organism 

Dilution Water: 

Chemical Manufacturers Associntion 's Bmminated Flame Retardant Industry Panel 

HBCD Rainoow Trout, Oncorhynchus mykiss 

Well 

Edible Tissue E..;ible Tissue 

Depuration Concentration Weight 

Day (_g) 

EIG5/Nl65 21 7195 !.6321 

Ei66/NIGCi 21 7358 2.9487 

21 

El68/Nl68 21 

Ei7(i/NI76 

E177/NI77 El78!N178 El79/J\1179 E187fNIH7 El88/NI811 El89/Nl89 

l 190 

2.2929 

1.3544 

1.2849 

0.8676 

1.6754 

1.9002 

1.4604 

0.9763 

=== 

1 Whole Fish Concentmtion wt X __ -+:_iJlone_cij_l2lc \Yi _;\'(JlOnccli_ble cott_0 

(edihle wt. + nonecliblc wt.) 

Nonedible Tissue 

Concentration 

(ftg HBCD/Kg) 

13482 

14236 

18354 

10613 

14190 

11546 

1164 

2717 

7999 

9499 

9054 

6745 

Noncdib

W

1.5

2.42.4

1.4

2.1

l. 5

l.4

0.9

1.9

1.4

1.0

'""" . ... -·-· .· --- ·- -· 
'\..._.., 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 

BIOF AC 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 of Rainbow 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 

-0 

0.0 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 Trout Exposed 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 I 
VVILULir 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 Exposed to l 8 11g HBCD/L. 

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

0 

0.0 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 of HBCD i11 W"hole fish Tissues of Rainbm": 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 

TEST WITH THE 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 

Brominated Flame Rctardant :Industry Panel 

! 300 Wilson Boulevard 

Arlington, Virginia 22209 

Wildlife 

8598 Commerce Drive 

Easton, Maryland 2160 I 

(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 WITH THE RAINBOW TROUT (Oncorhynchus my kiss) 

SPONSOR: Chemical Manufacturers Association's 

Brominated Flame Retardant Industry Panel 

1300 Wilson 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 of Aquatic Toxicology & Non-Target Plants 

FOR LABORATORY USE ONLY 

Proposed Dates: 

Experimental Experimental 

Start Date: 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. (if applicable): 

PROTOCOL 

~ 

APPR.OV AL 

PROTOCOL NO. 439!100699/RBT-BIO/SUB439 

/6 9/'17 

DATE -----

~ 

Jo /11 /;q 

;ol z /qo7 ~L:::...t_l . ---- DATE 
WILD Ll FE INTER NA_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); 

ASIM Standard El022-84 Standard Practice for 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 at Wildlife International, Ltd. and a copy of the 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 of the test substance in the rainbow trout, Oncorhynchus mykiss. The solubility of 1!-ffiCD in 

water at25°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>f solvent as that used for the tr::atmcnt groups. 

The duration of the 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 

estimated prior to test initiation. During both phases of the 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 (k2), 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 

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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. 

Great Lakes Chemical Corp. 635297G-l October 26, 1995 3462 

Albemarle Corp. 33449-lSX December 20, 1995 3519 

Bromine Compounds Ltd 950303 February 5, 1996 3551 

An equal part (300 g) of each of the 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 of the composite were 

performed on l'vfarch 20, 1996. The results of the analyses indicated the composite test substance 1Nas 

homogeneous and contained the 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 of the study. 

Preparation of Test 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 

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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 of the species will be verified by the supplier or by Wildlife International, 

Ltd. personnel using appropriate taxonomic keys, such as Eddy (3). Fish will be gradually acclimated 

to Wildlife International, Ltd. well water, and held for a period of at 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. If mortality exceeds 3% 

during the 48-hour period immediately preceding the test, the entire batch of fish \~ill be rejected or 

held for an additional 14-day period to ensure that they 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 of bias a,-e expected ro affect the results 

of the study. 

The minimum acceptable size of individual test organisms will be determined by the quantity 

of tissue required for measurement of residues. Loading (the total wet weig.!Jt of fish per liter of te..<;t 

solution) will not exceed l.O gram of fish per liter of solution 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 of contaminants 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 of the 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.JJ3vc39 
VVILULit"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 

period preceding the test will be provided in the final report. Analyses will be performed at least once 

annually to determine the concentrations of selected organic and inorganic constituents of the well 

water and results of the most recent GLP analyses will be summarized in the final report. 

Diluter System and Test 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 of dilution water to control and treatment test clwrlbers 

will be regulated by rotameters. A syringe or peristaltic 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 of the 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 of equilibrium concentrations of HBCD in the test chambers before 

introduction of the test organisms. The general operation of the diluter will be checked visually at 

least two times per day during the test. 

Test chambers will be 52-L Teflon"'-lincd stainless steel test chambers containing 

approximately 45 L of test 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 of darkness will be controlled with an automatic timer. 

A 30 minute transition period of low light intensity will be provided when lights go on or off to 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 of Test 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 

chronic toxicity data. The two exposure concentrations of 0.34 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 of the test and at 

weekly intervals during the 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 of pH 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 

PROTOCOL NO. 439/100699/RBT -BIO/SUB439 
E tO 

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WILDLIFE INTERNATIONAL, LID 

- 8 -

at the beginning and end of the test and at weekly intervals during the test. Hardness and alkalinity 

measurements will be made by titration using procedures based on methods in Standard Methods for 

the Examination of Water 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 of behavior 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% of the fish in the trcalrnent 

or control groups. 

Duration of Uptake Phase 

The statistically optimum duration of the uptake phase (u) is near u = l.6/k2, but not mmc than 

3.0/k2, which is equivalent to 95 percent of steady 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 of Depuration Phase 

Two fu:l~;s tt is usually a sufficient time for about 95 per.;:ent removal of the body burden (tv, = 

0.69/k2}, however, the' d~uration phase will continue until the concentratjon ofHBCD residue in the 

organisms attains any one of the following three conditions: 

1. Residues reach less than 10% of steady-state; or 

2. Residues full below the LOQ; or 

3. Sixty (60) days of depuration 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 of the study at the same intervals. l.n addition, wntrol and treated water 

PROTOCOL NO. 4391! 00699/RBT -BIO/SUB439 
VVILULirE... 11'-ll t:..H:i'\IAIIUNAL, Ll U. PKUJECT NU.: 43YA-lll 

E 11 

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'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 of fish used for the study, if possible. Additional control samples of fish 

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 of the test substance and/or reliable 

physico-chemical data from which bioconcentration potential may be estimated, the recommended 

sampling schedule is as follows. 

Phase Sample Interval Water1 Fish2 Action 

Pre-Uptake Start 3 -n~ v 

.F>. - -n2 X - 0 X - Add fish 

Uptake -4 hours 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 Depuration " .r.. X 

3 X v 

.A 

7 X v 

A 

10 X X 

i44 X X 

At least five replicates of water 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 of HBCD. Additional samples v:rill be ch-av;-n as necessa.ry for the a.'U.Iysis 

of dry weight and lipid content. 3 Pre-Uptake water sampling (-n~o -n2} for com'i.rmation of the treatment levels prior to 

addition of fish. 4

Depuration phase sampling \vi!! continue until the concentration ofHBCD residue in 

the organisms attains any one of the conditions outli.rted in the duration of depuration 

phz.se on page 8. 

PROTOCOL NO. 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 of the test substance from which predictions ofbioconcentration rate can be derived and1or 

at the direction of the Sponsor. 

Water Sampling 

At least five replicates of water 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 of a 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 of samples drawn for HBCD analysis will 

be held in reserve and may be analyzed at the direction of the Study Director or Chemistry Principal 

Investigator. The concentration ofHBCD in the v.-ater at each samole interval will be presented as the 

average of the 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 by the Study Director). The remaining replicates of sa.:nples drawn for HBCD a=lysis wiil 

be held in reserve and m.ay be analyzed at the direction of the Study Director or Chemistry Principal 

Investigator. The concentration of HBCD in fish at each sa..-nple iiiterval v,ili be presented as the 

average of the 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 of each fish ·vvill be determined '.~ithin approximately 15 minutes of collection, if 

possible. Each fL~h will be separated into edible a.!'ld noncdiblc tissue. Fish will be stored frozcrr if 

not analyzed immediately. 

PROTOCOL NO. 439/l00699/RBT-BIOiSlJ!3439 
E 13 

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

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WILDLIFE INTERNATIONAL, UD 

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

-]] -

Additional samples will be dravm as necessary for the anal;, is of dry 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 of the intervals noted. The 

lipid content of the 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. 

Data Analysis 

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 signed protocol. 

2. Identification and characterization of the test subst::tnce, if pro-..~ded by the Sponsor. 

3. Dates of initiation and termination of the test. 

4. Test organism holding and acclirP..ation records. 

5. Methods used to prepare stock solutions and dilutions of ilie te:;t substance. 

6. Daily obervations. 

7. Water chemistry calculations (e.g., hardness and aL'<alinity). 

8. Organism weight and length measurements, if 2pplicable 

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/S1JB439 
t'KUJbL 1 NU .. 4.:\':?A-11 i 

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WILDLIFE INTERNATIONAL, LTD 

- 12-

ll. A copy of !he final report. 

FINALF'::_ORT 

A repvrt of !he results of the study will be prepared by Wildlife lmcmational, Ltd. 1bc report 

will include, but not be limited to, the following: 

1. Name and address of the 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, if provided 

by the Sponsor. 

7. Stability and, when relevant to the conduct of the 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 of the meiliods used. 

9. A description of the test system used. Where applicable, the final report she-ill include t.'te 

number of animals used, body weight range, source of supply, speci~s, age, and procedure used 

for identification. 

10. A description of the dosage, dosage regimen, route of administntior~ and duration. 

I 1. A description of all circ.umstanccs that may have affected u.'1c quality or h""ltegrity of u'l<' cbta. 

12. The name of the Study Director, the na..>ncs of oilier 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 of the data, ar.d a statement of the conclusions drawn from the :malys1s 

14. The signed and dated reports of each of the individual scientists or other profe.ssioruJs involved 

in the study, if applicable. 

PROTOCOL NO. 439/l00699/RBT-Bl0/SLJB439 
W!LDUFE INTERi\lATIONAL, LTD. PROJECT NO.: 439A-ll1 

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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. If it 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 of the final report that is being added to or corre.-·red and the reasons for 

the correction or addition. Amendments shall be signed and dated by the Study Director. 

CHA.NG'NG OF PROTOCOL 

Planned changes to the protocol will be in the form of written amendments signed by i.hc Study 

Director and the Sponsor's Representative. Amendments will be c.onsidered as part of the 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 of the 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 of the final rep on 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 

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WILDLIFE INTERNATIONAL, LTC 

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REFERENCES 

US. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test 

Gnidclincs (draft), OPPTS Number 850.1730: Flov:-Through F;<h 7 est. 

2 ASTM Standard E1022-84. 1988. Standard Practice for Conducting Bioconcentration Tests 

with Fishes and Saltwater Bivalve Molluscs. American Society for Testing 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, New York. 

. PROTOCOL NO. 439/i00699/RBT-B!O/SUB439 
WILDLIFE INTERNATIONAL, LTD. 

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WILDLIFE INTERNATIONAL, LTD 

- J 5-

APPEN1)IXI 

IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR 

To be Completed by Sponsor 

PROJECT NO: 439A-lll 

L TestSubstanceidentity(narnetobeusedinthereport): _____ HB"""""'""C""D'----------

Test Substance Sample Code or Batch Number: Wildlife International Ltd. Identification No. 3577 

Test Substance Purity ("/o Active Ingredient): --"--'93,_,_. 6"-'7'--'-'HB~C"'D"'--- Expiration Date: __ _ 

11 Test Substance Olaracrerization 

Have the identity, streogth, purity and composition or other characteristics 

which ~proprialcly defure the test subslaOCe and refi:rence standard been 

determined prior to its use in this study in accordance with GLP Standards? 

Til Test Substance Storage Conditions 

X Yes ___ No 

Please indicate the recommended storage o:mditions at Wildlife lrJ!erP...ational, Ltd. 

Ambient 

Has the stabilitv of the te;;t substance under iliesc storage conditions 

been dcte:rmin&:i in accordance wilh GL'P Standards? __ Yes __ No 

Other pertinent stability information: 

IV. Test Coocentrations: 

V. T oxicit.y Information: 

Adjust test con-centratioo to l 00"/o a.i. ____ baSed 1.1pon !he purity(%) given above. 

Do not adjust test coo=Il:iratioo to l 00% __ _,_x,___.ai. Testfue material AS JS. 

M:unmalian: Ral:LD50 _____ Mou..<eLD50: ------

Aquatic: Invertebrate T CJcicity (EC/LC50) Fish TO'Aicity (I£..50) 

Other Toxicity Information (mcluding findings of chronic and subohroninests): 

VI. Classification of the Compound: 

__ Insecticide 

__ Microbial Agent 

__ Herbicide ___ Fungicide 

__ Economic PoLson 

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 

STUDY TITLE: HEXABROMOCYCLODODECANE (HBCD): A FLOW-THROUGH 

BIOCONCENTRA TION TEST WITH THE 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 Start Date: 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 Substance No.: 3577 

To: Test Substance No.: 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: Test Concentrations: 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. 

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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 

PROJECT NO.: 439A-lll 

Page 2 of3 

To: During the holding and testing periods, the fiSh will be fed at least once daily. Uneaten food and 

feces will be siphoned from the test 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-L Teflon®-lined stain!t:ss steel test chambers containing approximately 

45 L of test solution. 

To: Test chambers will be I 06-L stainless steel test chambers containing approximately 80 L of test 

solution. 

REASON: To correct the size of the test chambers and the volume of test solution. 

AlVIENDMENT: Water Sampling, Page 10 

Change: 

To: 

At least five replicates of water samples will be collected at each interval listed for the analysis 

on-men. 

At each sampling interval, two water samples vvil! be collected from ti1e control and three W3ter 

samples will be collected fr~ :n each of the two HBCD treatment groups. 

REASON: To clarify the 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 of each 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 of dry 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-

WI LDU FE INTERNATIONAL LW 

DEVIATION TO STUDY PROTOCOL 

PROJECT NO.: 439A-lll 

PROJECTNO.: 439A-lll 

Page l of 1 

STUDY TITLE: HEXABROMOCYCLODODECANE (HBCD}: A FLOW -THROUGH 

BIOCONCENTRATION TEST WITH THE RAINBOW TROUf 

(Oncorhynchus mykiss) 

PROTOCOL NO.: 43911 00699/RBT -BIO/SUB439 

SPONSOR: Chemical Manufacturers Association's 

Brominatcd Flame Retardant Industry Panel 

DATE OF DEFACTO 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: The chemist did not realize that dry weights had been amended out of the protoc.ol. It is the best 

judgement of the Study Director that this deviation did not adversely affect fue results of the study. 

STUDYDIRECTOR ~ DATE 

~~L 

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

- 61 -

APPENDIX II 

Test Substance Characterizatio::1 

F 08 
F 09 

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CHEI\llCAL CHARACTERIZATION ANALYTICAL RESULTS 

FOR DETERMJNlNG THE IDENTITY AND PURITY OF 

HEXABROMOCYCLODODECANE (TEST SUBSTA..~CE NOo 

Prepared for. 

Prepared by: 

Dr. MarciaL. Barny, Study Monitor 

Health and Environment Department 

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 

Baton Rouge, La. 70820 

194 
11'111-L.JLirr::.. H'lll 1::..1""(1'\IA.JIUI'\IA.L, Ll U. PROJECT NO.: 439A-lll 

F 10 

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ALBEMARLE CORPORATION 

HEALTH AND E.t'NIRONMENT DEP ARTMEJ'\.'T 

CHEMICAL CHARACTERIZATION ANALYTICAL RESULTS FOR 

DETE~"'NG THE IDENTITY AND PURITY OF 

HEXABROMOCYCLODODECANE (TEST SUBSTA..N"CE NO. 4615) 

1 Reference Protocol No.: 

II. Sponsor: 

III. Performing Laboratory: 

IV. Dates ofPerfunnance: 

V. Test Article: 

V1 Object:ive/II..1ethodo!ogy: 

HBCD-002 

Albemarle Corporation 

Health and Environment Department 

451 Florida Street 

Baton Rouge, La. 70801 

Study Monitor: MarciaL. Hardy, D.V.M., .!>h.D_ 

Albemarle Corporation 

Albemarle Technical. Center 

8000 G.S.Rl Aveooe 

Baton Rouge, La. 7082.0 

Study Chemist: George M Ricks, 1\.i.S., CLJ.i. 

09/04/98 (Test Article received from Wildlife futer:w...rional, LtdL) 

09/29/98 {Fmal Report Issued) 

Hexabmmocyclododecane., (Test Substance No. 4615) 

The test 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 

Albemarle T eclmica! Center for analysis. 

The identity of one sample., taken !from the center of the 

bulk sample mlxrure, was confumed using Fourier 

Transform Infrared analysis using SOP No. 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 of the ~-nu-e) was analyzed using 

195 
F 11 

vn. Data/Record Retention: 

rl'-.V.J.C.\......1 l'lV .. '-t:J7.M...-ll!. 

-64-

Proton Nuclear Magnetic Resvnance SOP No. ARS-l7l-RJ 

to determine the isomer distribution and purity of the 

mixture. Chain of custody and sample hanillm.g pro~m:-es 

were fullowed as per SOP No. ARS-OD1-R6. 

Table I contains the an.a!ytical !reSUlts and corrciu.siou 

from the above analyses. The study conformed to ilie 

requirements ofEP A TSCA GLP's listed tmde.r "'-{) CFR 

Part 792. There were no ciK=stan= obs-•Tved that mav 

have affected the quality or integrity of ilie data. -

All completed logbooks, spectra, original data and reports 

shall be kept filed in the custody of the Study Chemist until 

the Toxicology Studies are completed, after which time 

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 RESU

DETERMINING THE IDENTITY AND PURITY O

IIEXABROMOCYCLODODECANE (TEST SUBSTANCE N

Reference Study No. HBCD-001 

CiiEMICAIL NAME: He:ubromocyclododeune 

C.A.S. No.: 3194-55-6 

MOLECULAR JFORMUJLA: C41IR10!1Jr4 -~ PHVSICAJl.lli'ORM: White Powder 

CWSMICA!L STRUCT'UJRE: 

"' 

"' 

"' "' 

;· ANAJLVSKS RESULTS 

FT-ffi 

The sample FT -llR spectrum matched !hat of the Aldrich reference spec!mm. All sp

are 011 file with I he original data. 

WdgM% 

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 

/

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 

le!rnbromo- 0.6 0.9 0.9 0.6 0.9 0.5 0.7 0.7 

cydododecane 

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

isobulanol 0.1 0.1 0.1 0.1 0.2 0.1 0.1 O~I

[~~~~,L~~l;! 

;Lrtic!~~eE,~Lttei~s 

I~ CD. Tl:e pu'i
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 Laboratory Practice 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 

Draft Report 09/29/98 09/29/98 \ o9/29/98 I 

i{Hjqf 

Dace 

co 

198 
F 1 

WILDLIFE INTERNATIONAL, LTD. 

- 67-

. ANALYTICAL RESULTS FOR DETERMJ:ININ"G 

HEXABROMOCYCLODODECANE RT 

DEVELO:P~AL TOXICITY 

BEXABROMOCYCLODODECANE (HBCO) 

W1L Jl.esetJ:Feh Lrl.bomibrle:s ~(nent;e Stmi'y Nlil. }~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 

HEALTH AND Em'"lRONMENT DEP 

ANALYTICAL RESULTS FORDE~G THE STitlBILITY Ol~' 

BEXABR01',10CYCLODODECANE IN SUPPORT ""A P:E!ENATAL 

DE,VELOP:MENT.AL TOXICITY STUDY 

BE"'\ABROMOCYCLODODEC~ (HBCD) IN R.A.:l'S" 

W1L R~eurr..h LtilJon:dt.Jrm Rtifertmct! 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~ of til;; ru:at test iilriide 

was pr,;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<:od by 

rorop~g fhe purity (isom..~ disttibtlrion) of 1: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. 

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

vn. Results: 

PROJECT NO. 439A-lll 

-69-

Table l eru:mri.ns the mmlytica! rew..db a.nd. crmc iu..sim::l 

from thl:: 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) alld EPA FIFRA CFR.Pllrt 160) Good 

La~~Re~!md theOECD 

[C(91)U61FUW] Good~zy ~~ R~~gulii!tioll!i. 

All. origirullnnv ~ ~ ~..ll be~ file.tin tbJ: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 <rt the 

~lisred mW"~:S~ n: "S~." 

~lr/.Jj_ 

DATE 

202 
0 

w 

1\.) 

0 

(.).) 

TADLEI 

ANALYTICAL RJB:SVLTS FOR DETERMINJ!NG TilE STABILlTY OF HEXABROM

SUPPORT OF MA PRENATAL DEVEWPMENTAL TOXICITY STUDY OFmb.uuJR

. . 

(l!mCD) IN RATS" {Fm ReseMCh IA6orotorlu 1Ufmne4! Siw:(p Nlll'. 'WJ

CHEMICAL NAME: li!Rll!hrollilloeyd!idod~!!.le I C.A.S. NUMBER: :U!U-5

!:!2!!CUIL.AR __]Q~LA~ ~~8~ j I'UYSICAL FORM: Wli

NMR.RESUJ..,TS (Wt. %) 

SAMJP'LEID 

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 

' 

~ 

p./2!!1'9~ 

6.'1 5.'2 7i.7 0.6 D.l 1.1 

. 

~~~~~~~";;imcnwas~bi:T~the©Olar
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 Prenatal Developmental To:rlcity Study of Hexab~romocydml!odccane 

(I!!BCD) in Rats" Wil Research Laborntories Reference Study No. '\VJ!L-.186009 

This study was inspected by the Albemarle Quality Assurance Unit ancl the findings were 

reported to manage}llent and the study director on the dates listed below: 

Study Phase 

Inspected 

Date ofPhas<: 

Inspection 

~ofReport 

to Study Director 

Draft Final Report 

A.udit 

January 14, 1999 January 14, 1999 

~~ B. Kristm Hoover 

GLP Consultant 

Date: January 14, 1999 

204 

Dale of Report So 

Manag~ 

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

G 05 

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APPENDIX III 

Specific Conductance, Hardness, Alkafutity and pH of Well 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 

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APPENDIXN 

Analyses of Pesticides, 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 Other Inorgani.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'AL YSIS OF HEX.I\BROMOCYCLODODECA.l\JE (HBCD) CON CENTRA TWNS 

IN FRESHWATER/RAINBOW TROUT TISSUE 

IN Sl3PPORT OF 

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

G 07 
Y 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 ·:)f the 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 of tissue samples for lipid content. 

Stock Samples 

Stock samples were co11ected in 20-rnL glass vials. An aliquot of each stock sample was initially 

diluted in acetonitrile (ACN) followed b:· a secondary dilution in 75% ACN 25% H20 Ahquots of 

diluted stock samples were transferred to autosampler vials and submitted for analysis 
VV I LULl r 1::.. H'l I 1::..1""<1"\IA I IUNAL, LTD. .I:'KU.ItC 1 NU.: 4J~A-i 11 

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Freshwater Samples 

A method flow chart for the analysis of HBCD 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 of DCM. 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 of each 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% H20. 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 of bulk 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 of bulk 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 of fish 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 of tissue (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 of tissue 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 of HBCD 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, if ne{;essary, and 

batched by sarn.pling ii1terval and tissue type. At that ti.il1e, two of the 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 of tissue. The folloV1i.11g equation was v.sed to calculate the saturated 

NaCI volume required for each tissue sample: 

Tissue Weight (g) ..!..:Q_g_ 

Satnrated NaCl 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 of the 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 of ethyl 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 of the 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 of the 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 

l 000 ~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, l 0 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 2 c: 

50 mL of methanol. 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 l OOLC 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 of fortification 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 H20 

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 

I 1.00 5.00 100 50.0 75~/D i\CN: 25~·D 
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-ll1 

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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 of Quantitation 

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 of HBCD, 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 if the 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 of one standard was injected foUo-vv:ing ever:r· five test samples. A 

calibration curve was derived from regression analysis using the in.stwmental responses of the 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 Yo ecovery = . 100 .. . 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 

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The method limit of quantitation (LOQ) for freshwater samples ;vas 0.0250 ~lg HBCDIL, 

calculated as the product of the 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 of the lowest calibration standard (1.00 r..tg 

HBCD!L) and the dilution factor of the matrix blank sample analyzed concurrently with the test samples. 

The dilution factor of the matrix blank sample was dependent on the concentration of JHBCD present in 

the test samples. Matrix blank dilution factors were 1.00, 25.0 or 125 w.L/g during the 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 of HBCD 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% of nominal 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 

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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 HBCD treatment group, was calculated using the following equations: 

. (peak area- y-intercept) . HBCD m sample (ug!L) = , ~< dilution factor · s.ope 

Peak area = 4 722 

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 1 c>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 of the test and depuration began. During u'le 

depuration phase of the 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 of the 

nomina! concentration in the 3 . ..: ~g HBCDrL treatment group (TabJ.e 5) DuriiJ.g the uptake phase of the 

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 

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74.2% of the nominal concentration and 28.6 to 78.8% of tht> nominal concentration, respectively 

(Table 6). During the depuration phase of the 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 of HBCD 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 of the test. At the request of the Sponsor, tissue sarnples 

collected on depuration Days 3 and 7 vvere not analyzed. Measured concentrations of HBCD in all but 

one solvent control edible fish tissue sample were <LOQ (Table 8). The results of analyses of edible 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 7 6 days of freezer storage are presented in Table l 0. 
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 439A-lll 

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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 

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Table 1 

Typical HPLC/MS Operational Parameters 

INSTRUMENT: 

SOURCE: 

ANALYTICAL COLUMN: 

OVEN TEMPERATIJRE: 

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 

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Table 2 

M:rtrix Blanks and Fortifications Analyzed Concurrently During Freshwater Sample Analyses 

Sampie 1-iumber 

(439A-l 1 1-) 

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 e f356 

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 

1 The 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 of the matrix blank 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. 

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Table 2 (continued) 

Matrix Blanks and Fortifications Analyzed Concurrently During Freshwater 

Sample l\umber Concentration ofHexabromocyclododecane (HBCD) (.ug/L) 

(439A-ll1-) Fortified Measurcd1 

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 of the matrix blank sample (0.1 00). 

~Results w<~re ge<~erated using MacQuan, version 1.5 and 1.6 software. lvlanual CBiculations way differ siightly. 

, Result was low beoau.:c 'ltock used for fortification had s1arted to degrac\e. 4 Result was high because sample was exposed to suspect contamination. 
WILDLIFE INTERNATIONAL, Lm. PROJECT NO.: 439A-lll 

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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 of quantitation (T~OQ). for each anal~'sis .. the LOQ was calcubted as the prodncl of the 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 

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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 of tlw lowes! CJ.librc!;cn 

standard ( 1.00 J.LgiL) and the dilution factor of the 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 

compounding I 

Wire cable 1 Dry b!endi~g I jacketing m and extrus1on 

5 

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Not given I Not given Not given ) 

I 

I 

200 li 10 5 I I i 

Not I given Not given J',iot given I 

6 •!2 "!2 II 

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96 40 ~t5 i 

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_______!