Laura Christine Claus

Transcription

1 INFLUENCE OF LIFE HISTORY TRAITS ON ACCUMULATION OF POLYBROMINATED DIPHENYL ETHERS AND POLYCHLORINATED BIPHENYLS IN THREE LAKE TROUT POPULATIONS FROM LAKE SUPERIOR By Lur Christine Clus A THESIS Submitted to Michign Stte University In prtil fulfillment of the requirements for the degree of MASTER OF SCIENCE Fisheries nd Wildlife 2011

2 ABSTRACT INFLUENCE OF LIFE HISTORY TRAITS ON ACCUMULATION OF POLYBROMINATED DIPHENYL ETHERS AND POLYCHLORINATED BIPHENYLS IN THREE LAKE TROUT POPULATIONS FROM LAKE SUPERIOR By Lur Christine Clus Bioccumultion of lipophilic contminnts cn be influenced by mny fctors, including life history. Life history trits, such s ge t mturity nd reproductive schedules evolve to optimize the lloction of vilble energy to reproduction nd growth, which my hve implictions for the distribution nd effects of lipophilic contminnts. The gol of this study ws to understnd how life history trits cn influence the distribution of lipophilic contminnts in three distinct lke trout (Slvelinus nmycush) popultions from Lke Superior: siscowet nd len morphotypes from Mrquette nd Michipicoten Islnd lens. The siscowet morphotype is chrcterized by higher body ft content, slower growth nd lower fecundity thn the len. The Michipicoten Islnd len is slow growing len popultion with high reproductive investment t older ges. The concentrtions of PCBs nd PBDEs in the muscle, ovry nd liver were mesured nd compred to popultion relevnt metrics such s fecundity, muscle ft, length nd ge. As predicted, bsed on higher body ft, siscowets hd higher concentrtion of contminnts in the muscle tissue when djusted for exposure. The Michipicoten lens hd reltively higher concentrtions of contminnts in the ovry tissue compred to siscowets of similr ges, which is consistent with their higher fecundities nd gond weights. The ptterns of contminnt bioccumultion between the three popultions suggest tht it is necessry to consider life history trits when monitoring for lipophilic contminnts becuse such ptterns llow for the inference of the effects of contminnts on the generl helth of the fish popultion.

3 ACKNOWLEDGMENTS I begin by thnking Cheryl Murphy who spent countless hours working with me on this project nd providing insight nd guidnce throughout this process. I lso thnk Dn Jones, whose help nd support with the GC/MS nlyses ws invluble. I thnk Mohmed Fsil who provided me with vluble insight to this project. I thnk the Michign Deprtment of Nturl Resources nd Environment for providing necessry equipment nd guidnce with fish smpling nd ge dt, especilly Shwn Sitr. I lso thnk Rick Goetz nd Dn Rosuer for their ssistnce with smpling. I thnk the Ontrio Ministry of the Nturl Resources for smpling ner Michipicoten Islnd. I thnk Beverly Chmberlin t the Michign Stte University Mss Spectrometry Fcility for her endless support with the use of the GC/MS equipment. I thnk Ben Felt for his trips into Cnd nd the U.P. s prt of smpling trips for this reserch nd his work on the fecundity dt. I thnk Ann Boegehold for her work in the lb. I thnk Lindsey Hrtrey for her work with the lipid extrctions. I thnk Ben Schmitt, Dne Lrsen nd the other Murphy lb members who hve ssisted with this reserch. I lso thnk the members of the MIRTH lb who provided support nd friendship throughout this process. Finlly, I thnk my prents, my sister, Jenni nd my fincé, Mtt for their constnt love nd support throughout ll of my eduction. Funding for this reserch ws provided through grnt from the Center for Wter Sciences t Michign Stte University. Additionl funding ws provided by the Center for Integrted Toxicology t Michign Stte University. Funding for trvel nd dditionl nlyses ws provided through the Dr. Howrd A. Tnner Fisheries Excellence Fellowship. iii

4 TABLE OF CONTENTS LIST OF TABLES......v LIST OF FIGURES vi LIST OF SYMBOLS OR ABBREVIATIONS xi INTRODUCTION...1 METHODS.. 9 Field Smpling....9 Contminnt Anlysis Column Construction.13 Contminnt Extrction.14 Qulity Control..16 Contminnt Quntifiction...19 Lipid Extrction...22 Sttisticl Anlysis...24 ANCOVA...24 Multiple Regression...25 RESULTS..29 Life History Results...29 Contminnt Results...42 DISCUSSION Life History Influences...58 Liver Concentrtion Ptterns...61 Muscle Concentrtion Ptterns...65 Ovry Concentrtion Ptterns...66 Hbitt Specific Influences...67 Helth Effect of Contminnts...70 Assumptions, Limittions nd Future directions...72 Implictions for Monitoring...73 Implictions for Mngement: Multiple Stressors...76 Conclusions...79 LITERATURE CITED..81 iv

5 LIST OF TABLES Tble 1. Stndrds nd congeners used for nlysis..18 Tble 2. Amount of contminnts found in blnks nd the verge percent recoveries for the PAR smples...19 Tble 3. Detection Limits nd Limits of Quntifiction 21 Tble 4. List of sttisticl tests used..28 Tble 5. Differences in life history chrcteristics bsed on ANCOVA...32 Tble 6. Best models bsed on AIC regression method 43 Tble 7. Differences in PBDE nd PCB concentrtions in the liver tissue bsed on ANCOVA results Tble 8. Differences in PBDE nd PCB concentrtion in the muscle tissue bsed on ANCOVA results Tble 9. Differences in PBDE nd PCB concentrtions in the ovry tissue bsed on ANCOVA results...54 v

6 LIST OF FIGURES Figure 1. The phenotypic differences between len nd siscowet lke trout (courtesy Shwn Sitr)...7 Figure 2. A) Mp of Lke Superior. Lrge circles indicte smpling loctions (mp courtesy of mspmg.com). B) Loction of two sites of gill net sets off the cost of Mrquette. The numbers on the mp indicte the depth in meters t ech site (mp courtesy of Rlph Tingley III). C) Loctions of gill nets set for lke trout smpling ner Michipicoten Islnd (mp courtesy of the Ontrio Ministry of Nturl Resources).. 10 Figure 3. Loction of the four ftmeter redings tken long right lterl body. Redings were then clibrted with lipid extrction dt to generte muscle ft percentges for lke trout. (Photogrph courtesy of Sr Smith) Figure 4. Histogrm of the ge rnges (yers) of ech popultion. The verticl xis contins the frequency counts for ech ge rnge. Mrquette lens (n=9), Mrquette siscowets (n=12) nd Michipicoten Islnd lens (n=10) Figure 5. The mrginl men fork length (FL) nd FL-t-ge reltionships were compred between the Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). A) Comprison of the mrginl men fork lengths (mm) between lke trout popultions. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different bsed on the results of n ANCOVA. B) Reltionship between fork length (mm) nd ge (yers) of the three lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.02, R 2 =0.88; Mrquette lens: β=0.02, R 2 =0.21nd siscowets: β=0, R 2 =0). Letters indicte popultions tht hve significntly different slopes of the regression line bsed on the results of n ANCOVA Figure 6. The mrginl men body weights nd the reltionships between weight nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). A) Comprison of popultion mrginl men body weights (g) between lke trout popultions. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different bsed on the results of n ANCOVA. B) Reltionship between body weight (1000 g) nd ge (yers) between the Mrquette len, siscowet nd Michipicoten len lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.07, R 2 =0.80; Mrquette lens: β= 0.04, R 2 =0.14 nd siscowets: β=0, R 2 =0). Letters indicte popultions vi

7 tht hve significntly different slopes of the regression line bsed on n ANCOVA. There were no significnt differences between slopes Figure 7. The mrginl men fecundities nd the reltionships between fecundity nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=11). A) Comprison of popultion mrginl men fecundities (number of eggs) between lke trout popultions including dt from Michipicoten lke trout tht ws not included in this study. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Reltionship between totl fecundity (1000 eggs) nd ge (yers) between len, siscowet nd Michipicoten lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.33, R 2 =0.03; Mrquette len: β=0.12, R 2 =0.27; siscowet: β=0, R 2 =0). There were no significnt differences between slopes Figure 8. The mrginl men gond weights nd the reltionships between gond weight nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=11). A) Comprison of popultion mrginl men gond weights (g) between lke trout popultions including dt from Michipicoten lke trout tht ws not included in this study. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Reltionship between gond weight (g) nd ge (yers) between the lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.01, R 2 =0.01; Mrquette lens: β=0.11, R 2 =0.27, siscowets: β= -0.01, R 2 =0.01). There were no significnt differences between slopes Figure 9. The mrginl men fecundities djusted for body weight nd the reltionships between djusted fecundity nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=11). A) Comprison of popultion mrginl men fecundity djusted for body weight (totl eggs/g) between lke trout popultions including dt from Michipicoten lke trout tht ws not included in this study. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Reltionship between fecundity djusted for body weight (totl eggs/g) nd ge (yers) between the lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β= -0.03, R 2 =0.29; Mrquette lens: β=0.08, R 2 =0.26; siscowets: β=0.01, R 2 =0.02) Figure 10. The mrginl men gondosomtic indices (GSI) nd the reltionships between GSI nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=11). A) Comprison of popultion mrginl men GSIs between lke trout popultions including dt from Michipicoten lke trout tht ws not included in this study. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Reltionship between GSI nd ge (yers) between the lke vii

8 trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β= -0.39, R 2 =0.26, Mrquette lens: β=0.75, R 2 =0.28, siscowets: β= -0.09, R 2 =0.06). There were no significnt differences between slopes Figure 11. The mrginl men lipid concentrtions nd the reltionships between lipid concentrtions nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). A) Comprison of popultion mrginl men lipid concentrtions between lke trout popultions. The lipid concentrtions were tken from muscle tissue nd reported s percentge in wet weight. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. Letters indicte popultions tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Reltionship between lipid concentrtion (% lipid wt/w) nd ge (yers) between the lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.07, R 2 =0.01; Mrquette lens: β= -1.26, R 2 =0.67; siscowets: β= -0.39, R 2 =0.04). There were no significnt differences between slopes Figure 12. The mrginl mens of PBDE nd PCB concentrtions in the liver were compred between the popultions long with the reltionship between PBDE nd PCB concentrtions in the liver nd ge. A) Comprison of popultion mrginl men PBDE liver concentrtions (ng/g) between lke trout popultions. B) Comprison of the popultion mrginl men PCB liver concentrtions. Error brs indicte the 95% confidence intervl of the popultion mrginl mens. C) Reltionship between PBDE liver concentrtions (ng/g) nd ge (yers) between the lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.20, R 2 =0.87, n=9; Mrquette lens: β=0.11, R 2 =0.05, n=9; siscowets: β=0, R 2 =0, n=12). D) Reltionship between PCB liver concentrtion (ng/g) nd ge (yers) between the lke trout popultions (Michipicoten lens: β=0.13, R 2 =0.91, n=10; Mrquette lens: β=-0.04, R 2 =0.05, n=9; siscowets: β=0, R 2 =0, n-12). Letters indicte popultions tht hve significntly different slopes of the regression line or popultion mrginl mens bsed on the results of n ANCOVA Figure 13. The mrginl mens of PBDE nd PCB concentrtions in the liver were compred between the popultions long with the reltionship between PBDE nd PCB concentrtions in the liver nd ge t prticulr ge rnges. A) Reltionship between PBDE liver concentrtion (ng/g) nd ge (yers) for ges less thn or equl to 14. Regression lines re included for ech popultion (Michipicoten lens: β=0.20, R 2 =0.39, n=4; Mrquette lens: β=0.25, n=, R 2 =0.17, n=9). B) Reltionship between PCB liver concentrtion (ng/g) nd ge (yers) for ges less thn or equl to ge 14. Regression lines re included for ech popultion (Michipicoten lens: β=0.17, R 2 =0.72, n=9; Mrquette lens: β= -0.08, R , n=4). C) Comprison of the mrginl men PBDE liver concentrtions for lke trout t or bove ge 20: siscowets (n=8), Michipicoten lens (n=5). D) Comprison of the mrginl men PCB liver concentrtions for lke trout t or bove ge 20. Error brs indicte the stndrd errors of the popultion mrginl men. Letters indicte popultions tht hve significntly different slopes of the regression line or popultion mrginl mens bsed on the results of n ANCOVA viii

9 Figure 14. The reltionship between PBDE concentrtions in the muscle tissue nd ge were compred between the popultions: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). Muscle PBDE concentrtions were lso compred between popultions t three ges. Compring three ges re necessry for strong comprison becuse of the differences in the slopes of the regression lines for ech popultion. A) Reltionship between PBDE muscle concentrtion (ng/g) nd ge (yers). Regression lines re included for ech popultion (Michipicoten lens: β=0.15 R 2 =0.76; Mrquette lens: β=0.04, R 2 =0.02; siscowets: β= -0.01, R 2 =0). Letters indicte popultions tht hve significntly different slopes of the regression line to n lph vlue of 0.05 bsed on the results of n ANCOVA. B) Comprison of PBDE muscle concentrtions (ng/g) t ge 17 nd 22 yers. Error brs indicte the stndrd errors of the popultion mrginl men. Comprison of popultion mrginl men PBDE muscle concentrtions (ng/g) between lke trout popultions for ges 12 yers showed no significnt differences between popultions. Letters indicte the significnt difference between mrginl mens. Error brs indicte the stndrd errors of the popultion mrginl men Figure 15. The mrginl men PCB concentrtions in the muscle were compred between the popultions long with the reltionship between PBDE nd PCB concentrtions in the muscle: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). A) Reltionship between PCB muscle concentrtion (ng/g) nd ge (yers). Regression lines re included for ech popultion (Michipicoten lens: β=0.14, R 2 =0.76, Mrquette lens: β=0.02, R 2 =0; siscowets: β=0.04, R 2 =0.11). Letters indicte popultions tht hve significntly different slopes of the regression line bsed on the results of n ANCOVA. B) Comprison of the popultion mrginl mens PCB muscle concentrtions with two outliers removed (ng/g). Error brs indicte the stndrd errors of the popultion mrginl men. Letters indicte popultion mrginl mens cross tht re significntly different to n lph vlue of 0.05 bsed on the results of n ANCOVA Figure 16. The mrginl mens of PBDE nd PCB concentrtions in the muscle djusted for liver concentrtions were compred between the popultions long with the reltionship between PBDE nd PCB djusted muscle concentrtions nd ge: Mrquette lens (n=9), siscowets (n=12) nd Michipicoten lens (n=10). A) Comprison of PBDE muscle concentrtions (ng/g) tht hve been djusted for liver PBDE concentrtions (ng/g). Error brs indicte the 95% confidence intervl of the popultion mrginl mens. B) Reltionship between the PBDE muscle concentrtions (ng/g) djusted for PBDE liver concentrtions (ng/g) nd ge (yers) between the lke trout popultions (Michipicoten lens: β= -0.20, R 2 =0.34; Mrquette lens: β=0.09, R 2 =0; siscowets: β= -0.08, R 2 =0.01). C) Comprison of PCB muscle concentrtions (ng/g) tht hve been djusted for liver PBDE concentrtions (ng/g). Error brs indicte the 95% CIs of the popultion mrginl mens. D) Reltionship between PCB muscle concentrtions (ng/g) djusted for PCB liver concentrtions (ng/g) nd ge (yers) between the lke trout popultions (Michipicoten lens: β=0.04, R 2 =0.11; Mrquette lens: β=0.05, R 2 =0.07; siscowets: β=0.03, R 2 =0.01). Letters indicte popultions tht hve significntly different slopes or popultion mrginl mens bsed on the results of n ANCOVA ix

10 Figure 17. The mrginl mens of PBDE nd PCB concentrtions in the ovry were compred between the popultions long with the reltionship between PBDE nd PCB concentrtions in the ovry nd ge. A) Comprison of popultion mrginl men PBDE ovry concentrtions (ng/g). Error brs indicte the 95% CI of the popultion mrginl men. B) Comprison of popultion mrginl men PCB ovry concentrtions (ng/g). Error brs indicte the 95% CI of the popultion mrginl men. C) Reltionship between PBDE ovry concentrtions (ng/g) nd ge (yers) between the lke trout popultions. Regression lines re included for ech popultion (Michipicoten lens: β=0.12, R 2 =0.91, n=10; Mrquette lens: β=0.13, R 2 =0.61, n=9; siscowets: β=0.02, R 2 =0.02, n=10). D) Reltionship between PCB ovry concentrtion (ng/g) nd ge (yers) between the lke trout popultions (Michipicoten lens: β=0.09, R 2 =0.78, n=10; Mrquette lens: β=0.09, n=9, R 2 =0.24; siscowets: β=0.04, R 2 =0.23, n=12). Letters indicte popultions tht hve significntly different slopes of the regression line or popultion mrginl mens bsed on the results of n ANCOVA Figure 18. The mrginl mens of PBDE nd PCB concentrtions in the ovry were compred between the popultions long with the reltionship between PBDE nd PCB concentrtions in the ovry nd ge: Mrquette siscowets (n=8) nd Michipicoten lens (n=5). A) Reltionship between PBDE ovry concentrtion (ng/g) nd ge (yers) for ges greter thn ge 20 (Michipicoten lens: β= -0.04, R 2 =0.11; siscowets: β=0.07, R 2 =0.24). B) Reltionship between PCB ovry concentrtion (ng/g) nd ge (yers) for ges greter thn 20 (Michipicoten lens: β=0.01, R 2 =0; siscowets: β=0.06, R 2 =0.24). C) Comprison of the mrginl men PBDE ovry concentrtion for ges 20 yers. Error brs indicte the 95% confidence intervl of the popultion mrginl men. D) Comprison of the mrginl men PCB ovry concentrtions for ges 20 yers. Letters indicte popultions tht hve significntly different slopes of the regression line or popultion mrginl mens to n lph vlue of 0.05 bsed on the results of n ANCOVA x

11 KEY TO SYMBOLS OR ABBREVIATIONS AIC Concen. CI GC/MS GSI g Akike informtion criterion concentrtion confidence intervl Gs chromtogrphy / mss spectrometry Gondosomtic index grm µl microliter ml ng/g K ow OMOE PBDE PCB Pop. β TCDD v/v w/w wt/w milliliter nnogrm / grm octnol-wter prtition coefficient Ontrio Ministry of the Environment polybrominted diphenyl ether polychlorinted biphenyl Popultion slope of the regression line 2,3,7,8-tetrchlorodibenzo-p-dioxin volume / volume weight / weight wet weight xi

12 Introduction Lipophilic contminnts, such s polybrominted diphenyl ethers (PBDEs) nd polychlorinted biphenyls (PCBs), re usully trnsferred throughout the body of fish by binding to lipoproteins nd triglycerides. The movement nd distribution of triglycerides nd lipoproteins s result of metbolism, growth nd reproduction, constntly reposition the lipophilic contminnts, often leding to uneven ccumultion ptterns in different tissues (Dulfer et l., 1998). Growth nd reproductive processes re influenced hevily by life history (Roff, 2002) nd in this study, I will explore how the life history trits of three different popultions of lke trout (Slvelinus nmycush) cn potentilly influence PBDE nd PCB ccumultion in different tissues. The potentil link between life history trits nd lipophilic contminnt ccumultion ptterns could hve implictions for mngement nd monitoring of lke trout in the Gret Lkes. Environmentl chllenges fced by popultions will dictte birth nd deth rtes nd therefore will influence popultion structure including size t ge (growth), ge structure, ge t mturity, fecundity nd survivl rtes (Roff, 2002). Life history trits re heritble chrcteristics tht re theoreticlly result of evolution nd optimize survivl nd reproduction nd include trits such s ge nd size t mturity, growth rte nd reproductive effort with ge. These trits directly influence popultion structure. Popultions tht fce different environmentl chllenges could select for trits tht llow for different trdeoffs between survivl nd reproduction, nd such trdeoffs could led to different popultion growth trjectories nd reproductive schedules. For exmple, popultion tht experiences high dult mortlity my rech sexul mturity sooner, exhibit incresed growth rtes nd show high reproductive investment t younger ges when compred to longer-lived popultion (Roff, 1

13 2002). These differences in growth rtes nd reproductive outputs between popultions could lso led to different ptterns of lipophilic contminnt bioccumultion. Lipids re n energy source tht cn either be stored or mobilized for use in growth or reproductive processes. In lke trout, the dominnt storge lipids re tricylglyercols (DiGiulio & Hinton, 2008). Stored tricylglycerols provide moderte degree of independence from constnt forge food sources (DiGiulio & Hinton, 2008). Additionlly, lipid storge in some morphotypes compenstes for verticl lift nd drg by reducing n individul s specific grvity (Henderson & Anderson, 2002). Lipids cn be used for egg production nd re trnsferred into the developing egg mss or incorported into vitellogenin, the yolk-precursor protein (Kleinow et l., 2008). Environmentl pressures on popultions will dictte whether lipids re stored, used for growth or reproduction t prticulr ges. Lke trout tht live t greter depth nd store more lipids s muscle ft expend less energy to migrte through the wter column becuse energy is no longer required to use swim bldder (Henderson & Anderson, 2002). Inversely, lke trout tht experience higher rtes of dult mortlity re likely to llocte lipids towrds growth nd reproduction erlier in life. The trde-offs between energy lloctions to growth or reproduction is ment to optimize survivl of the popultion, but hs implictions for the distribution of lipophilic contminnts tht bioccumulte in the body of fish. Lipophilic contminnts re moved through the body by binding with lipoproteins, such s vitellogenin (Ungerer & Thoms, 1996). The movement of lipophilic contminnts within the body begins t the time of exposure. When PCBs nd PBDEs enter the body, they re metbolized by the cytochrome P450 (CYP) enzyme system once they enter the liver (Schlenk et l., 2008). The cytochrome P450 enzyme system is the most dominnt system in the phse I biotrnsformtion oxidtion processes (Schlenk et l., 2008). The CYP enzymes convert 2

14 lipophilic pollutnts into wter-soluble products tht cn be excreted (Schlenk et l., 2008). The prticulr CYP isozyme involved depends on the congener. PCBs with coplnr structures interct with the Ah receptor nd induce CYP1A (Jmes, 2001). CYP2B isozymes re induced by ortho-substituted non-plnr PCBs (Arnold & Feeley, 2002). Oxygen is introduced into the PCB vi the cytochrome P450 enzyme system nd the hydroxylted metbolite my be conjugted with glucuronide or sulfte with UDP-glucuronosyltrnsferse or PAPSsulfotrnsferse s ctlysts (Jmes, 2001). The metbolite is then excreted through the bile; however, when orgnisms re exposed to lipophilic pollutnts t higher rte thn the contminnts re metbolized, the contminnts begin to bioccumulte in the orgnism. Lipophilic contminnts cn bioccumulte disproportiontely throughout the body in pttern relted to the lipid concentrtion of the orgns. Additionlly, the ccumultion efficiencies of tissues vry (Krjlinen et l., 2006). For exmple, when brook trout (Slvelinus fontinlis) were exposed to 2,3,7,8-tetrchlorodibenzo-p-dioxin (TCDD), the totl ssimiltion through dietry exposure for femles ws 84 % (Tietge et l., 1998). The ovries only retined 6% of the totl body burden of TCDD nd 43% of muscle concentrtions(tietge et l., 1998; Wlker et l., 1994). In previous studies, it ws found tht ptterns of lipophilic contminnt ccumultion follows lipid concentrtions throughout the body, including the blood, liver, ovry, muscle nd eggs (Tietge et l., 1998). The ese t which lipophilic contminnts bind to lipoproteins nd triglycerides is process tht is determined by the contminnt s octnol-wter prtition coefficient (K ow ) (Streit et l., 1991). K ow is the rtio of the concentrtion of chemicl in octnol nd wter t equilibrium (United Geologicl Survey, 2010). The octnol is considered surrogte for lipid mtter, nd exmining the K ow llows for n understnding of the fte of chemicls in the 3

15 environment. A chemicl with high K ow, rnging between 2 nd 7, will bioccumulte t greter rtes thn chemicl with lower K ow (Streets et l., 2006). The lipophilic contminnts of interest in this study include two industril persistent contminnts with high K ow, PCBs nd PBDEs. PCBs, in the pst, were used in vriety of commercil nd industril pplictions including fluorescent light bllsts, electricl trnsformers, plsticizers in plstic nd rubber, nd cble insultion. The mnufcture nd use of PCBs ws prohibited in 1976, however the relese of PCBs into the environment continues due to existing equipment nd processes (United Ntions Environment Progrmme, 2002). PCBs re highly lipid soluble; the logrithms of K ow vlues for tetr, pent, hex nd oct PCBs rnge from 5.46 to 7.67 (Hwker & Connell, 1988). PBDEs re clss of brominted flme retrdnts used in items such s furniture fom, plstics nd drperies. PBDEs re lso highly lipid soluble; the logrithmic K ow vlues for pentbde-99 rnge from 6.5 to 8.5 (US EPA, 2008b). Production of these compounds re not bnned in the United Sttes, however the mnufcture of pentbde nd octbde cesed strting in 2005 (US EPA, 2011). Both PCBs nd PBDEs cn be trnsported tmosphericlly from hevily polluted res nd deposited in more remote res (Dtt et l., 1999 nd Schmide et l., 2007). The tmospheric deposition of these contminnts complictes mngement of these contminnts in the Gret Lkes ecosystem. PCBs nd PBDEs re ubiquitous in the Gret Lkes nd in fish becuse of their widespred usge nd chemicl composition nd my contribute to dverse helth effects within qutic ecosystems. As top predtors in the Gret Lkes ecosystem, lke trout hve historiclly been known to bioccumulte lipophilic contminnts. PCB levels in whole lke trout from the Gret Lkes hve declined since the 1970s, lthough recently the levels hve stbilized nd 4

16 remin bove levels requiring consumption wrnings in some loctions (US EPA & Environment Cnd, 2009). Concentrtions of PBDEs in lke trout eggs from the Gret Lkes hve incresed in the lst 20 yers (Vlters et l., 2005). The PBDE levels in whole lke trout from ll five of the Gret Lkes ws incresing through the erly 2000s, but more recently we re beginning to see decline (US EPA & Environment Cnd, 2009). Concentrtions of these contminnts in prticulr tissues hve helth implictions for humns tht consume lke trout (Devult et l., 1996) nd the helth of the fish popultions (Mule et l., 2005; Olsson et l., 1999). PBDEs nd PCBs induce sublethl toxic effects, which cn led to stress on humn nd fish popultions. PCBs hve been found to ffect the immune system in fish nd cn be embyotoxic (Mule et l., 2005; Olsson et l., 1999). PCBs re endocrine disruptors tht reduce the uptke of vitellogenin by oocytes nd reduce fecundity (Hlden et l., 2010; Rose et l., 2003). Additionlly, the plnr PCB congeners produce dioxin-like effects (Tillitt et l., 2008). PBDEs, s endocrine disruptors, show mny structurl similrities to dioxins, which suggest tht they my hve toxic properties tht re similr to dioxins (US EPA, 2010b). The structurl similrities between PBDEs nd thyroid hormones suggest disruption of the immune system (Luthe et l., 2008; Birchmeier et l., 2005). The siscowet nd len morphotypes from Lke Superior long with the Michipicoten Islnd len popultion were chosen for this study becuse of the unique life history trits exhibited for ech popultion. Popultions of lke trout from Lke Superior cn be grouped into two ecologiclly distinct morphotypes (Figure 1). Len nd siscowet lke trout morphotypes re estimted to hve diverged from ech other round the time of the Wisconsin Glcil Episode, lthough debte exists s to whether the morphotypes diverged before or fter recoloniztion 5

17 (Pge et l., 2004; Eshenroder, 2008). The bthymetric distribution of siscowets nd lens result in key life history differences, most noticeble is the reltive lipid content (Sitr et l., 2008). Popultions of siscowets re found in lrge bundnces t depths greter thn 100 meters nd re robust with higher body ft content, wheres len lke trout popultions re most bundnt in depths less thn 50 meters nd re slender with low body ft content (Eschmeyer & Phillips, 1965; Hnsen et l., 1995). The higher lipid content in siscowets is hypothesized to overcome drg nd reduce energy expenditures during verticl migrtion (Henderson & Anderson, 2002). Whether these morphologicl differences result from phenotypic plsticity or genetic differences is still under investigtion, but studies indicte genetic bsis for differences in lipid levels between lens nd siscowets (Goetz et l., 2010, Eschmeyer nd Phillips, 1965). In ddition to differences in lipid ccumultion nd distribution ptterns, siscowets nd lens lso show differences in growth nd fecundity, which my hve implictions for contminnt distribution. Len lke trout grow fster thn siscowets nd hve higher reltive fecundity (Hnsen et l., 1995). The Michipicoten Islnd lke trout is believed to be seprte genetic strin of len lke trout (Meredith, 2008). Michipicoten lens grow slower thn the Mrquette lens nd live in north-estern Lke Superior where they re best ble to persist becuse fishing pressure nd se lmprey (Petromyzon mrinus) prsitism rtes re low (Bronte et l., 2007; Ebener, 1998). The growth rte of the individul is importnt to the ccumultion of lipophilic contminnts becuse of the effect of biodilution: fster growing individul will hve lower concentrtions of lipophilic contminnts t specific length compred to slower growing individul (Simoneu et l., 2005). However, this theory of biodilution ssumes individuls with different growth rtes experienced similr contminnt exposure rtes. 6

18 Figure1. The phenotypic differences between len nd siscowet lke trout (courtesy Shwn Sitr). The interction of life history trits of the popultions nd lipophilic contminnts like PBDEs nd PCBs cuses unique concerns for humn helth nd lke trout popultion mngement. Accumultion of lipophilic contminnts into the muscle tissue of lke trout tht re being consumed by humns cretes humn helth concerns. Siscowets re being considered for use s high-grde fish oil dietry supplements becuse of their high popultion numbers nd lipid content. Humn consumption of fish oils hs been ssocited with mny humn helth benefits including prevention of crdiovsculr diseses (Mrik & Vron, 2009) nd siscowet lke trout hve been found to contin very high levels of omeg-3 ftty cids (Wng et l., 1990). Fish oil extrcted from siscowets could be used s nutritionl supplements nd used to produce high-vlue fish mel. However, if siscowets store lrge mounts of lipids, nd thus lipophilic contminnts, the benefits of consumption of omeg-3 ftty cids could be compromised by incresed exposure to hlogented contminnts. Life history trits cn vry within species nd I suspect tht life history trits should be tken into considertion when using lke trout dt for public helth recommendtions nd when considering the response of popultion to stressor such s PBDEs nd PCBs. 7

19 Additionlly, mobiliztion of lipophilic contminnts through the body llows for the mternl trnsfer of contminnts into the ovries. Accumultion of endocrine disrupting contminnts in the ovries, nd thus the eggs, presents potentil helth concerns for the developing embryo (Miller et l., 1993). The dditionl stress of lipophilic contminnts dds to the multitude of stressors fced by lke trout in the Gret Lkes nd contributes to the decline of nturlly reproducing len lke trout popultions. My study investigtes the role of life history trits on ptterns of lipophilic contminnt ccumultion in three lke trout popultions. The energy trde-offs between growth nd reproduction for lens nd siscowets hve been reported in previous studies (Eschmeyer nd Philips, 1965; Hnsen et l., 1995, Miller et l., 2000). My hypothesis is tht the distribution of PCBs nd PBDEs will be unique to ech popultion nd will reflect life history trde-offs. I predict tht len popultions with greter reproductive investment will hve higher concentrtions of PCBs nd PBDEs in the ovry tissue thn siscowets. The siscowets, due to lrger stores of muscle ft re predicted to hve higher concentrtions of PCBs nd PBDEs in the muscle tissue. My reserch investigted this hypothesis by smpling these three lke trout popultions from Lke Superior nd collecting life history dt, such s ge, fecundity, muscle lipid concentrtion nd weight. Anlyses of muscle, ovry nd liver tissue smples for selected PCB nd PBDE congener concentrtions were performed using gs chromtogrphy/mss spectrometry methods. The life history nd contminnt dt were then nlyzed in tndem to understnd the interction between life history of popultion nd contminnt concentrtions. My study will improve our understnding of the reltionship between life history nd lipophilic contminnts; reltionship tht cn hve implictions for the mngement nd consumption of lke trout. 8

20 Methods Field Smpling Sexully mture femle lke trout were collected during September nd October of 2008 nd 2009 off the cost of Mrquette, USA nd Michipicoten Islnd, Cnd; both in Lke Superior (Figure 2A). Len nd siscowet lke trout were collected offshore of Mrquette s prt of the Michign Deprtment of Nturl Resources project smpling. The Mrquette smpling used bottom gill nets with individul nylon, mutifilment pnels (91.4 m x 1.8 m) nd mesh sizes including 10.2, 11.4, 12.7 nd 14.0 cm. The nets were set t two sites for twenty-four hours before lifting. The first site, tht trgeted siscowets, ws between 375 meters nd 385 meters deep (ltitude nd longitude ). The second site ws selected to cpture lens, nd ws between 109 nd 145 meters (ltitude nd longitude ) (Figure 2B). The mesh sizes were selected in order to trget mture lke trout (greter thn 550 mm). The minimum length requirement ws bsed on current knowledge of verge mture lke trout lengths (Goetz et l., in review). The Mrquette smpling resulted in the cpture of thirtyfour femle lke trout, but only nine lens nd twelve siscowets were mture femles. The lke trout smpled from Michipicoten Islnd were prt of the Ontrio Ministry of Nturl Resource s effort to stock their htchery progrms (Felt, unpublished). Lke trout were cught by gill nets with 50 nd 100 foot pnels of 2 nd 2.5 inch monofilment. The nets were set for between 3 nd 70 hours before lifting t seven min sites (Figure 2C). The smpling trip occurred over the course of bout 20 dys in both 2008 nd A totl of 73 lke trout were cught in 2008, but only twenty mture femles were cught ner Michipicoten Islnd between 2008 nd Ten 9

21 lke trout from the Michipicoten popultion were included in this study. A. Michipicoten Islnd Mrquette B North N Mrquette Munising C. Michipicoten Islnd N Figure 2. A) Mp of Lke Superior. Lrge circles indicte smpling loctions (mp courtesy of mspmg.com). B) Loction of two sites of gill net sets off the cost of Mrquette. The numbers on the mp indicte the depth in meters t ech site (mp courtesy of Rlph Tingley III). C) Loctions of gill nets set for lke trout smpling ner Michipicoten Islnd (mp courtesy of the Ontrio Ministry of Nturl Resources). 10

22 On site, vrious mesurements were mde including morphology metrics, length nd fork length, weight nd ftmeter redings. Len nd siscowet lke trout were identified bsed on their morphometry tht included fin size, eye loction, eye size nd viscerl ft (Bronte & Moore, 2007; Eschmeyer nd Philips, 1965). Length nd fork length were mesured onsite to the nerest milimeter. Weight ws mesured to the nerest grm nd ovry nd liver mss ws tken to 0.1 grm. Muscle ft mesurements were tken long the outer body cvity with fish ftmeter (Model FM-692, Distell Inc.,West Lothin, Scotlnd). Fish were then filleted with the skin on nd the muscle tissue, liver nd ovries were removed. The tissue smples were stored seprtely in luminum foil nd frozen t -80 C until nlysis. The ftmeter redings were tken t consistent loctions long the right side of the outer body (Figure 3). The ftmeter is noninvsive microwve sensor tht emits low frequency microwve bem (2 GHz ±20 MHz) tht efficiently hets wter dielectriclly nd provides reding of wter content of the tissue. Ftmeter redings were converted to lipid percentges bsed on reltionship of lipid to wter content in fish, clibrted using lipid extrction dt (see Lipid Extrction section). Figure 3. Loction of the four ftmeter redings tken long right lterl body. Redings were then clibrted with lipid extrction dt to generte muscle ft percentges for lke trout. (Photogrph courtesy of Sr Smith) 11

23 Additionl informtion ws collected offsite fter collections. Ages were determined by counting the nnuli on the otoliths. Fecundity ws lter determined by extrcting four fresh ovry smples pproximtely 15 grms, which were weighed nd ll eggs were counted in ech smple. The number of eggs per grm of ech smple ws verged nd multiplied by gond mss to determine fecundity. Photos of pproximtely 25 eggs from ech fish were tken on Leic MZ6 dissecting scope. The pictures were then imported into Axiovision Rel. 4.7 where the numbers of pixels long the dimeter of the egg were mesured. The pixel number of known length ws used to convert the pixel mesurements into egg dimeter mesurements. 12

24 Contminnt Anlysis PBDEs nd PCBs were quntified using contminnt extrction procedure nd quntified using gs chromtogrphy/mss spectrometry (GC/MS) methods. This section detils the column construction, contminnt extrction, qulity control nd contminnt quntifiction. Column Construction Two chromtogrphy columns were constructed using mixtures of pcking mterils nd used for contminnt extrction (see Contminnt Extrction). Before the chromtogrphy columns were constructed, pcking mterils were prepred. First, nhydrous sodium sulfte nd silic gel with mesh of 70/230 were ctivted by plcing in n oven t 215 C for t lest 70 hours before use. Once the silic gel ws ctivted, it ws mixed with vriety of chemicls in order to crete pcking mterils used in the column construction. The 44% (weight/weight) sulfuric cid/silic pcking mteril ws mde by mixing 56 grms of ctivted silic with 25 ml of concentrted sulfuric cid until the pcking ws free flowing. The 10% (w/w) silver nitrte/silic pcking mteril ws mde by dissolving 6 grms of silver nitrte in 23 ml of HPLC grde wter. The silver nitrte/wter mixture ws then combined with 53 grms of ctivted silic nd shken until the pcking ws free flowing. The 33% (w/w) 1 M sodium hydroxide/silic pcking mteril ws prepred by weighing 1.3 grms of sodium hydroxide nd dissolving it into 33 ml of HPLC grde wter. The wter/sodium hydroxide mixture ws combined with 67 grms of ctivted silic nd shken until the pcking ws free flowing. All pcking mterils were stored in desicctors until use. The extrction column ws prepred by plugging 250 ml cylindricl funnel first with glss wool nd then with 25 g of 44% sulfuric cid/silic nd finlly with 8 g of nhydrous 13

25 sodium sulfte. The mount of 44% sulfuric-cid/silic in the extrction column ws incresed for more smples with higher concentrtions of lipids, which ws indicted by the color chnge of the silic gel mixtures. The cid-bse column ws prepred by plugging chromtogrphy column (14 x 3.5 cm i.d.) with glss wool nd dding, in order from bottom to top: 1.5 g 10% silver nitrte/silic pcking, 1.0 g ctivted silic pcking, 2.0 g 33% sodium hydroxide/silic pcking, 1.0 g ctivted silic pcking, 4.0 g 44% sulfuric cid/silic pcking, 2.0 g ctivted silic pcking nd 2.0 g nhydrous sodium sulfte. All glsswre ws rinsed three times with cetone, three times with toluene nd twice with dichloromethne between ech smple. Contminnt Extrction All digestion, extrction nd GC/MS procedures were modified from the Ontrio Ministry of the Environment s method (OMOE, 2004). Approximtely 5 grms of muscle, ovry nd liver tissue ws homogenized before contminnt extrction. Muscle tissue ws homogenized with dry ice in commercil grde blender. Ovry tissue nd liver tissue ws thwed nd mnully homogenized using shrp blde. Ech smple ws digested overnight in 50 ml of concentrted hydrochloric cid. The hydrochloric cid ws pre-extrcted before contct with the smple using 3:1:1 rtio of hydrochloric cid, dichloromethne nd hexne to prevent contmintion of the smple. The first column used during the extrction process ws the extrction column (see Column Construction). The extrction column ws pre-rinsed with 50 ml of hexne nd the elute ws discrded. The digested tissue solution (lso referred to s the cid-lyer) ws extrcted with 40 14

26 ml of hexne by mixing in seprtory funnel for 2 minutes. The hexne nd cid lyers were then llowed to seprte nd then the hexne extrct ws run through the extrction column to remove wter, excess lipids nd other polr compounds. The cid-lyer ws returned to the originl flsk nd the extrction ws repeted twice nd dded the hexne from ech extrction to the extrction column nd t the end, discrded the remining cid-lyer. To ensure mximiztion of extrction, the originl flsk nd the seprtory funnel were rinsed twice with 20 ml of hexne, which ws dded to the extrction column. Elute from extrctions ws collected in round bottom flsk nd plced on rotry-evportor t 45 C until only bout 2 ml of the elute remined. In between individul smple extrctions, the rotry evportor ws rinsed with ml of hexne. Ech smple ws subjected to chromtogrphic clenup with the cid-bse column, which ws mde with silic gel mixtures (see Column Construction). The cid-bse column ws pre-rinsed with 50 ml of hexne nd the elute ws discrded. Then ech smple extrct ws dded to the cid-bse column. The originl round-bottom flsk contining the smple ws rinsed three times with 2 ml hexne nd dded to the cid-bse column. Then, 100 ml of hexne ws dded to the cid-bse column followed by 80 ml of 50:50 dichloromethne nd hexne mixture. The cid-bse column ws llowed to drin to bed level between ech ddition. Once the column ws well drined, the round bottom flsk contining the elute ws plced on the rotry evportor until pproximtely 1 ml ws remining in the flsk. The concentrted smple extrct ws then trnsferred to clen glss vil nd the round bottom flsk rinsed twice with hexne nd then twice with dichloromethne with ech rinse ws dded to the glss vil. The glss vil ws stored t -80 C until evported using nitrogen evportor. The smple ws 15

27 not completely evported nd the remining smple ws trnsferred into n mber glss utosmpler vil with 100 µl insert. Qulity Control The ntive stndrds were purchsed from Wellington Lbortories (Guelph, Ontrio) nd were spiked in the precision nd recovery (PAR) smple between every 20 tissue smples in order to understnd recovery of ntive PBDEs. The ntive stndrd includes BDE 28, 47, 66, 99, 100, 153 nd 154 (Tble 1). The internl stndrd [ 13 C 12 ]- 2,2,4,4 -TeBDE (47L) ws lso purchsed from Wellington Lbortories (Guelph, Ontrio). A spiked procedure blnk (SPB) ws nlyzed between every 10 smples nd included known concentrtion of internl stndrd nd bout 50mL of pre-extrcted hydrochloric cid (extrcted s described below). The internl stndrd ws dded to ever 12 y smple t known concentrtion in order to determine recovery rtes, but concentrtions were not corrected bsed on recovery. Clibrtion stndrds were purchsed from Wellington Lbortories (Guelph, Ontrio) nd included [ 13 C 12 ]- 2,2,4,4 -TeBDE (47L), 2,4,4 -TrBDE (28), 2,2,4,4 -TeBDE (47), 2,3,4,4 -TeBDE (66), 2,2,4,4,5-PeBDE (99), 2,2,4,4,6-PeBDE (100), 2,2,4,4,5,5 -HxBDE (153) nd 2,2,4,4,5,6 -HxBDE (154) (Tble 1). This set of clibrtion stndrds generted clibrtion curve, which included dt from 4 different concentrtions. The R 2 vlues of the clibrtion curves were ll greter thn 0.90 suggesting strong reltionship between concentrtion nd re under the curve of the pek generted by the GC/MS. 16

28 The PCB clibrtion stndrds were purchsed from Cmbridge Lbortories (Andover, MA). The clibrtion stndrds generted clibrtion curve with dt t 6 different concentrtions nd included 2,3,4,4,5-PeCB (123), 2,3,3,4,4,5-HxCB (156), nd 2,3,4,4,5,5 -HxCB (167) (Tble 1). The internl stndrd BDE 47L ws dded to ech clibrtion stndrd t known concentrtion. The R 2 vlues of the clibrtion curves were ll bove

29 Tble 1. Stndrds nd congeners used for nlysis Type of Stndrd Precision nd Recovery (PAR) Internl Stndrd PBDE Clibrtion Stndrd PBDE Clibrtion Stndrd (continued) PCB Clibrtion Stndrd Source purchsed Wellington Lbortories (Guelph, Ontrio) Wellington Lbortories (Guelph, Ontrio) Wellington Lbortories (Guelph, Ontrio) Wellington Lbortories (Guelph, Ontrio) Cmbridge Lbortories (Andover, MA) Congeners included in Stndrd BDE 28 2,4,4 -TrBDE BDE 47 2,2,4,4 -TeBDE BDE 66 2,3,4,4 -TeBDE BDE 99 2,3,4,4 -TeBDE BDE 100 2,2,4,4,6-PeBDE BDE 153 2,2,4,4,5,5 -HxBDE BDE 154 2,2,4,4,5,6 -HxBDE BDE 47L [ 13 C 12 ]- 2,2,4,4 -TeBDE BDE 47L [ 13 C 12 ]- 2,2,4,4 -TeBDE BDE 28 2,4,4 -TrBDE BDE 47 2,2,4,4 -TeBDE BDE 66 2,3,4,4 -TeBDE BDE 99 2,3,4,4 -TeBDE BDE 100 2,2,4,4,6-PeBDE BDE 153 2,2,4,4,5,5 -HxBDE BDE 154 2,2,4,4,5,6 -HxBDE PCB 123 2,3,4,4,5-PeCB PCB 156 2,3,3,4,4,5-HxCB PCB 167 2,3,4,4,5,5 -HxCB BDE 47L [ 13 C 12 ]- 2,2,4,4 -TeBDE 18

30 All solvents were either nlyticl regent grde or HPLC grde. All smples were nlyzed in duplicte. The verge percent recoveries for the PAR smples were reported long with the mount of ech contminnt found in the blnks (Tble 2). Tble 2. Amount of contminnts found in blnks nd the verge percent recoveries for the PAR smples Congener number Gretest mount found in blnk (ng) Averge % recovery for the Precision nd Recovery Smples BDE BDE BDE BDE BDE BDE BDE PCB tetr unknown PCB stndrds were not included PCB in PAR PCB pent unknown # PCB pent unknown # PCB PCB PCB hex unknown # PCB hex unknown # Contminnt Quntifiction The smples were nlyzed using n Agilent 6890N gs chromtogrph coupled to n Agilent 5973 mss spectrometer with helium s the crrier gs (Michign Stte University Mss Spectrometry Fcility). The smples were injected s 1 µl in splitless mode with 2 minute purge time nd the injection port ws held t 270 C. The GC column used ws 30 m x 0.25 mm (i.d.) cpillry tube coted with DB-5MS nd 0.25 µm film thickness (J&W Scientific, Folsom CA). The GC temperture progrm ws s follows: 110 C for 1 min., 45 C/min to 19

31 200 C, 6 C/min to 240 C, 4 C/min to 325 C nd held t 325 C for 5 min. The MS trnsfer line ws held t 280 C. Selected ion monitoring ws used to detect two msses for the tri-, pent- nd hex-bdes nd the tri-, pent- nd hex-pcbs. The selected ion monitoring reported only the two ion msses determined to correspond to the moleculr ion of the selected congeners of PBDEs nd PCBs. Smples with low signl-to-noise rtios were reinjected with 2 µl of extrct. A signl to noise rtio less thn 10 ws considered not quntifible nd less thn 3 ws considered not detectble. The detection limits nd limits of quntifiction were used to replce contminnt redings with low signl to noise rtios with rndom number (Tble 3). A smple with signl to noise rtio less thn 3 for prticulr congener ws ssigned rndom number between the zero nd the detection limit. A smple with signl to noise rtio less thn 10 for prticulr congener ws ssigned rndom number between the detection limit nd the limit of quntifiction. The use of rndom numbers ws bsed on my ssumption tht the overll concentrtions for given tissue ginst the frequency of fish with tht concentrtion would form bell-curve. However, the limits detection of the GC/MS mchine would truncte this curve t the detection limit nd limit of quntifiction. Therefore, smples with low signl-to-noise rtios were ssigned rndom numbers in plce of zeros in order to provide more norml distribution of PBDE nd PCB concentrtions. 20

32 Tble 3. Detection Limits nd Limits of Quntifiction Congener number Detection limit (ng) Limit of Quntifiction (ng) BDE BDE n/ BDE BDE BDE BDE BDE PCB tetr unknown PCB PCB pent unknown # PCB pent unknown # PCB PCB PCB hex unknown # PCB hex unknown #2 n/ n/ Clibrtion curves for both PBDEs nd PCBs were generted in order to relte the output of the GC/MS dt to output from known concentrtions. A clibrtion curve for PBDEs ws generted before nd fter every set of smples run through the GC/MS. The PCB clibrtion curve ws generted once for ll smples injected t 1 µl nd once for smples injected t 2 µl. The isotopic rtio between primry nd secondry ion msses ws within ±10% of theoreticl vlues. The detection of second ion mss llowed me to correctly determine tht the compound ws PCB or PBDE; dditionlly the use of the secondry ion mss llowed for the inclusion of severl PCB congeners tht were not identified in the stndrd. One unknown tetr- PCB, two unknown pent-pcbs nd two unknown hex-pcbs were detected with enough frequency throughout the smples to dd to the nlysis. These unknown PCB congeners were identified bsed the known moleculr mss of primry nd secondry ion nd n isotopic rtio within ±10% of theoreticl vlues. The concentrtions were determined using MssLynx V4.1 nd QunLynx softwre (Wters Corp.), fter conversion of the ChemSttion dt files to MssLynx file formt. 21