Acknowledgements This work has been far from a solitary effort. Contributions from mentors, colleagues, friends, and funding organizations have led to the successful completion of this dissertation. Serge Doroshov, my major advisor, has offered his constant support and direction throughout this work. I was fort unate to work with someone with such extensive knowledge and experience in fish physiology and reproduction. I have learned so much from him and will always be grat eful. Dave Hinton has made invaluable contributions to this endeavor by serving on my dissertation committee and by offering his expertise in pathology and ecotoxicology. Sam Luoma has been my long-term mentor and I continually attempt to emulate hi m as a scientist. Sam helped me to form

the thesis of this work while I was a member of his research team at the US Geological Survey. All that I learned from him there, along with his continue d support and advice, has shaped every aspect of this research. The work presented here would not have been possible without the help of Joel

Van Eenennaam and Javier Linares. Their expertise in sturgeon culture, maintenance, sampling and research was critical to this project. I am ex tremely grateful to them for their tireless help and advice th roughout this research. I am also grateful to Paul Lutes and Erik Hallen for their support and a dvice regarding sturgeon maintenance. Ed DePeters has my sincere gratitude fo r allowing me extensive use of his lab. My work there was successful due to Scott Taylor’s constant suppor t and advice. Silas Hung also opened his lab to me, and provided cri tical support with the experimental diets. Silas formulated the juvenile experimental diet and directed its production. He also provided the use of his pelleting equipment fo r production of the adult experimental diet.

- ii -I am very grateful to Xin Deng for his collaboration in the fluorometric analyses, his help with sampling, and for our fruitful discussions on selenium research. The first chapter of this dissertation would not have been possible without David Hinton. His work on the histopathology, as well as his dire ction and training, wa s critical to that research. Similarly, Kevin Kroll’s collabora tion in the isolation and purification of vitellogenin and egg yolk proteins was essential to the research presented here in chapter two. A special thanks to Don Tillitt and Di ana Papoulias of the US Geological Survey for providing training on the microinjection of sturgeon larvae. Javier Linares received that training and then returned to share it with the Doroshov Lab. The microinjection experiment would not have been possible without Javier’s extensive collaboration. I had the fortunate opportunity to wo rk with several unde rgraduate interns including Fiona Sands, Summer Evert, Ma tthew Morales, Karen Kaplan, Rachel Hutchins, Blair Kendal, Carla Rodriguez, Melissa Dyer, Anita Setty, Shinichiro Oku, Michelle Jones, Todd Tolentino, Amanda Cassidy, Dominique Weddle, Angela Sartor,

and Melissa Arevalo. These in dividuals assisted in sampling, chemical analyses, and much more. They worked hard, offered insi ght, and contributed a great deal to this research. I am very grateful to each of them. Obtaining funding for graduate research is becoming increasingly difficult. I am extremely grateful to the following organi zations for their financial support through fellowships, grants and cont racts: the UC Toxic Substa nces Research and Teaching Program, the Ecotoxicology Lead Campus Program at UC Davis, the Graduate Group in Ecology, the US Geological Survey and CALFED.

- iii -During the earlier years of this endea vor, I received valuab le direction and support from several faculty members of the Graduate Group in Ecology. Mike Johnson was especially helpful by continuously providi ng insight and serving as the chair of my qualifying examination committee. Keith Mi les, Barry Wilson, and Joe Cech also contributed to my early studi es and planning. I also l earned much through extensive discussions during seminars and conferences hosted by the Graduate Group in Ecology, its Ecotoxicology AOE, and the Lead Ca mpus Program in Ecotoxicology. Most of all, I am immensely grateful for the support of family and friends throughout this arduous process. My parent s, Jess and Edna Linvi lle, have provided unwavering love and support. My sister and friend, Cynthia Linville, has offered encouragement, advice, and her expertise in the use of the English language. I am grateful to the many people that have bright ened my life so much throughout these past years including Mikaela H untzinger, Jennifer Von Aesc h, Diana Rowan, Anne Salo, Scott Nagy, Marta Sykes, Dena McCann, Darc y Jones, and Hali Rowen. I give my fondest thanks and appreciation to my frie nd and husband, Jed Alexander, who has given his continuous love, sup port and encouragement.

- iv -Abstract Selenium (Se) is a potent toxicant in aquatic ecosystems, threatening higher trophic level species because of its efficient food web transfer. Excess Se consumption in fish has led to reduced growth, significan t histopathology in major organs, reproductive failure, developmental defects and mortality. Se is also an essential nutrient; however, there is a very narrow margin between nutritional and toxic concentrations of this element. White sturgeon ( Acipenser transmontanus ) in San Francisco Bay-Delta consume bivalves containing high levels of Se and exhibit elevated Se concentrations in their muscle, liver and eggs, which may threaten their health and reproduction. Juvenile white sturgeon (47 weeks post-hatc h) were exposed to dietary organic Se (as selenized yeast; ca. 1, 20, 36, and 53 μg/g Se) for six months. The effects of excess dietary Se included the dose-re sponse increase of melanomacropha ge aggregates in liver, the occurrence of cholestasis, a potentially fata l condition, in all treatment groups and inflammation and fused lamellae in gills. No effects were observed on the size or growth

of these individuals. Female white sturgeon, in late stage vite llogenesis, were given dietary Se (as selenized yeast; ca. 1 and 34 μg/g Se) for up to six months. Treatment females

accumulated ca. 11, 10 and 12 Se μ g/g (dw) in muscle, liver and eggs. Transfer of Se to the yolk in developing eggs took place thr ough the yolk protein prec ursor, vitellogenin (VTG). Se uptake by VTG in the liver took place in a dose-dependent manner. Similarly, Se concentrations in eggs showed a dose-dependent rela tionship with Se in plasma VTG. Se in eggs was associated with the yolk proteins, li povitellin, phosvitin and immunoglobulin.

- v -Hormonal treatment was used to induce spaw ning in females. We did not observe an impact on reproduction, as shown by fecundit y, fertilization success and survival to neurulation. Progeny of treatment females (e mbryos and yolk sac larvae) exhibited high Se concentrations (similar to egg concentrations). Yolk utilization in larvae from treatment females led to significant skeletal deformities (lordosis, kyphosis, and scoliosis; ED 25 = 24 μ g/g Se in larvae, dw), edema ( ED 25 = 25 μ g/g Se; Logit, p < 0.0001), and mortality (8% in larvae with ca. 20 μ g/g Se; Kaplan-Meier, p < 0.0001). In addition, yolk sac larvae (from unexposed females) that were microinjected with seleno-L-methionine exhibited developmental defects including skeletal deformities (lordosis, kyphosis, and scoliosis; ED 25 = ca. 17.5 μ g/g Se in larvae, dw) and edema ( ED 25 = ca. 13 μ g/g Se; Logit, p < 0.0001). Mi croinjected yolk sac larvae experienced a greater incidence of mortality (45 - 70% in larvae with ca. 20 μ g/g Se Kaplan-Meier, p < 0.0001) as compared to mate rnally exposed yolk s ac larvae. For both experimental approaches (maternal transfer and microinjection), white sturgeon yolk sac larvae containing Se concentrations above ca. 11 to 15 μg/g demonstrated significant increases of mortality and abnormality rates. A hazard threshold range of ca. 3 to 8 μ g/g Se in developing white sturgeon is suggested for this species. In San Francisco Bay-Delta, j uvenile white sturgeon are susceptible to toxicity at Se concentrations currently observed in some prey items (i.e., bivalves), and accumulate

Se in their tissues at concentrations similar to those observed in our study. This leads us to suspect that juvenile white sturgeon in this area may be experiencing significant liver damage, depending on individual dietary Se exposure. Adult white sturgeon in San Francisco Bay-Delta exhibit Se body burdens similar to those accumulated by treatment

- vi -fish in our adult experiment. This suggests that female white sturgeon in San Francisco Bay-Delta are likely transferring Se to their offspring. Limited data on Se concentrations in white sturgeon eggs and ovaries from th is region supports this conclusion. Our research shows that elevated Se ( ca. 11 – 15 μ g/g Se) in white sturgeon eggs leads to significant developmental defects and mortalit y in developing larvae. Based on the work presented here, we suspect that white sturge on recruitment in San Francisco Bay-Delta is impacted by the accumulation of excess Se in this species. The abundance of this population was recently recorded at a 50-year lo w. Careful management of all processes with potential to increase Se concentrations in the benthic food web is essential to protect sturgeon in San Francisco Bay-Delt a and other high-Se systems.

- vii -Table of Contents

Title page i Acknowledgements ii

Abstract v

Table of contents viii

Table of tables x Table of figures xiii

Introduction Selenium in San Francisco Bay-Delta 1 White sturgeon in San Francisco Bay-Delta 4 Toxicity of selenium 7

Research aims 12

Chapter 1. Toxicity of foodborne se lenium to juvenile white sturgeon, with an emphasis on histopathology of the liver

Introduction 13

Materials and Methods 17

Results 22

Discussion 30 Tables and Figures 41

- viii -Chapter 2. Dose-dependent maternal transport of selenium via vitellogenin in white sturgeon

Introduction 62

Materials and Methods 65

Results 81

Discussion 86 Tables and Figures 94

Chapter 3. Developmental toxicity in white sturgeon larvae exposed to selenium through maternal transport or microinjection

Introduction 126

Materials and Methods 129

Results 141

Discussion 150 Tables and Figures 158

Conclusions 196

References 200

Appendix A. Biliary transport and cholestasis in fish 218

Appendix B. Summary data on spawning induction and success in white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months 227

Tables 228

- ix -Table of Tables

Chapter 1. Toxicity of foodborne selenium to juvenile white sturgeon, with an emphasis on histopathology of the liver

Table 1-1. Selenium content of juvenile experimental diets 41

Table 1-2. Composition of juvenile experimental diet 42

Table 1-3. Selenium in juvenile white sturgeon after six months of dietary selenium exposures 43

Table 1-4. Comparisons of gross morphol ogical characteristics of juvenile white sturgeon followi ng six months of dietary selenium exposures 44

Table 1-5. Gross morphological measuremen ts of juvenile white sturgeon following six months of diet ary selenium exposure 45

Chapter 2. Dose-dependent maternal transp ort of selenium via vitellogenin in white sturgeon

Table 2-1. Selenium content of adult experimental diets 94

Table 2-2. Combined vitellogenin protein samples 95

Table 2-3. Morphological comparisons between exposure groups 96

Table 2-4. Selenium in adult white sturgeon after six months of dietary selenium exposures 97

Table 2-5. Percent moisture of adult white sturgeon tissues 98

Table 2-6. Selenium levels measured in plasma and isolated plasma proteins of white sturgeon after six months of dietary selenium exposures 99

Table 2-7. Selenium concentrations in yol k proteins isolated from eggs of adult white sturgeon followi ng six months of dietary exposures 100

Table 2-8. Selenium in combined vitellogenin samples compared to combined tissue and yolk protein samples 101

- x -Table 2-9. Statistical parameters for the regressions of selenium concentrations in ovarian tissue, eggs, plasma and non-vitellogenin isolated plas ma proteins as a function of selenium concen trations in liver 102

Table 2-10. Pearson correlation coefficients for selenium in plasma non-vitellogenin proteins correla ted to selenium in liver, yolk proteins, and eggs 103

Table 2-11. White sturgeon spawning data 104

Table 2-12. Reproductive performance of st urgeon females in control and treatment groups 105

Chapter 3. Developmental toxicity in whit e sturgeon larvae exposed to selenium through maternal transport or microinjection

Table 3-1. Selenium content of adult experimental diets 158

Table 3-2. Key developmental stages in sturgeon 159

Table 3-3. Size of larvae from control and treatment female white sturgeon 160

Table 3-4. Size of larvae in microinjection study two 161

Table 3-5. Selenium in developing wh ite sturgeon progeny of females following dietary selenium exposur e during late vitellogenesis 162

Table 3-6. Selenium in developing white sturgeon exposed to seleno-L-methionine by microinjection 163

Table 3-7. Occurrence of edema in larvae for each progeny cohort of female white sturgeon exposed to dietary selenium 164

Table 3-8. Occurrence of skeletal deformities in larvae for each progeny cohort of female white sturgeon exposed to dietary selenium 165

Table 3-9. Affected larvae (edema and/or skeletal deformities) in each progeny cohort of female white sturgeon exposed to dietary selenium 166

Table 3-10. Occurrence of developmental defects in larvae from female white sturgeon exposed to dietary selenium 167

- xi -Table 3-11. Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis from microinjection study one 168

Table 3-12. Percentage of larvae with developmental defects for each progeny cohort of microinjection study two 169

Table 3-13. Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis from mi croinjection study two 170

Table 3-14. Percent mortality in larvae from female white sturgeon exposed to dietary selenium 171

Table 3-15. Percent mortality in larvae exposed to selenium via microinjection 172

Table 3-16. Combined mortality in larv ae from maternal exposure and microinjection studies 173

Table 3-17. Probabilities of developmen tal defects in larvae containing elevated selenium levels at the beginning of larval development 174

Table 3-18. Selenium in adult tissues and th eir progeny related to incidence of developmental defects 175

Table 3-19. Potential hazard threshold levels for selenium in developing white sturgeon larvae 176

Appendix B. Summary data on spawning induction and success in white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months.

Table B-1. Detailed spawning data for white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months 228

Table B-2. Summary of spawning induction and success in white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months 232

- xii -Table of Figures

Chapter 1. Toxicity of foodborne selenium to juvenile white sturgeon, with an emphasis on histopathology of the liver Figure 1-1. Selenium levels in liver, blood and muscle after six months of dietary selenium exposures 46 Figure 1-2. Micrograph showing overview of control liver architecture 47 Figure 1-3. Micrograph of control liver showing bile duct 48 Figure 1-4. Micrograph of control liver showing bile retention in a single control liver, with no evidence of biliary stasis 49 Figure 1-5. Micrograph of control liver s howing eosinophilic granular leukocytes 50 Figure 1-6. Micrograph of control liver s howing moderate inflammation 51 Figure 1-7. Micrograph of control liver s howing peribiliary inflammation 52 Figure 1-8. Micrograph showing distended canal icular network in liver of low treatment fish 53 Figure 1-9. Micrograph showing tubuloform swelling in liver of low treatment fish 54 Figure 1-10. Micrograph showing bile duct pro liferation in liver of low treatment fish 55 Figure 1-11. Micrograph showing bile duct pro liferation with inflammation in liver of low treatment fish 56 Figure 1-12. Micrograph showing necrosis and distended canalicular network in liver of medium treatment fish 57 Figure 1-13. Micrograph showing bile duct pr oliferation and peribiliary inflammation in liver of medium treatment fish 58 Figure 1-14. Micrograph showing extensive me lanomacrophage aggregates in liver of high treatment fish 59 Figure 1-15. Micrograph showing distended canalicular network and hepatocyte vacuolation in liver of high treatment fish 60

- xiii -Figure 1-16. Contingency table showing th e occurrences of inflammation and fused lamellae in gills of juvenile white sturgeon exposed to dietary selenium for six months 61

Chapter 2. Dose-dependent maternal transp ort of selenium via vitellogenin in white sturgeon Figure 2-1. Hypothesized mechanism of th e maternal transport of selenium to offspring 106 Figure 2-2. Anion exchange chromatograph of white sturgeon male before and after estrogen treatment 107 Figure 2-3. Anion exchange chromatograph of plasma proteins from vitellogenic white sturgeon female used for this study 108 Figure 2-4. PAGE & Western gels of wh ite sturgeon plasma proteins 109 Figure 2-5. Vitellogenin ELISA standard curve 110 Figure 2-6 . Gel filtration chromatograph of sturgeon egg yolk 111 Figure 2-7 . PAGE and Western gels of crude and purified egg yolk proteins of white sturgeon 112 Figure 2-8. Tissue selenium concentrat ions in adult white sturgeon after ca. six months of dietar y selenium exposures 113 Figure 2-9. Selenium in plasma collected at mid- and end-experiment 114 Figure 2-10. Selenium in vitellogenin isol ated from plasma collected at mid- and end-experiment 115 Figure 2-11. Selenium in non-vitellogenin proteins isolated from plasma collected at mid- and end-experiment 116 Figure 2-12. Selenium concentrations in vitellogenin and non-vitellogenin proteins 117 Figure 2-13. Vitellogenin isolated from plasma collected at mid- and end-experiment 118 Figure 2-14. Selenium in vitellogenin re lated to selenium in liver 119

- xiv -Figure 2-15. Selenium in plasma, ovarian tissue and eggs related to selenium in vitellogenin 120 d) Figure 2-16. Selenium in yolk proteins rela ted to selenium in vitellogenin 121 Figure 2-17. Selenium in isolated yolk prot eins as a function of selenium in eggs 122 Figure 2-18. Selenium in ovary, eggs, pl asma and non-vitellogenin plasma proteins as a function of selenium in liver 123 Figure 2-19. Selenium in non-vitellogenin plasma proteins related to total selenium in plasma 124 Figure 2-20. Selenium in eggs related to selenium in non-vitellogenin plasma proteins 125

Chapter 3. Developmental toxicity in whit e sturgeon larvae exposed to selenium through maternal transport or microinjection Figure 3-1. Selenium concentrations in eggs and developing embryos and larvae from white sturgeon fe males exposed to dietary selenium 177 Figure 3-2. Selenium concentrations in developing white sturgeon larvae following microinjection of seleno-L-methionine 178 Figure 3-3. Developmental defects observed in white sturgeon during larval development 179 Figure 3-4. Percentage of maternally e xposed larvae with edema related to selenium concentr ations in larvae 180 Figure 3-5. Percentage of maternally exposed larvae with skeletal deformities related to selenium concentrations in larvae 181 Figure 3-6. Percentage of maternally exposed larvae with edema and/or skeletal deformities relate d to selenium concentrations in larvae 182 Figure 3-7. Percentage of maternally e xposed larvae with edema and/or skeletal deformities related to selenium concentrations in eggs 183

- xv -Figure 3-8. Percentage of larvae from microinjection study one with edema related to selenium con centrations in larvae at the onset of larval development 184 Figure 3-9. Percentage of larvae from microinjection study one with skeletal deformities related to selenium concentrations in larvae at the onset of larval development 185 Figure 3-10. Percentage of larvae from microinjection study one with edema and/or skeletal deformities related to selenium concentrations in larvae at the onset of larval development 186 Figure 3-11. Percentage of larvae from microinjection study two with edema related to selenium con centrations in larvae at the onset of larval development 187 Figure 3-12. Percentage of larvae from microinjection study two with skeletal deformities related to selenium concentrations in larvae at the onset of larval development 188 Figure 3-13. Percentage of larvae from microinjection study two with edema and/or skeletal deformities related to selenium concentrations in larvae at the onset of larval development 189 Figure 3-14. Percentage of maternally exposed and microinjected larvae with edema related to selenium concentrations in larvae at the onset of larval development 190 Figure 3-15. Percentage of maternally exposed and microinjected larvae with skeletal deformities related to selenium concentrations in larvae at the onset of larval development 191 Figure 3-16. Percentage of maternally exposed and microinjected larvae with edema and/or skeletal de formities related to selenium concentrations in larvae at the onset of larval development 192 Figure 3-17. Probabilities of mortality in larvae from female white sturgeon exposed to dietary selenium 193 Figure 3-18. Probabilities of mortality in larvae following microinjection of seleno-L-methionine 194 Figure 3-19. Probability of mortality in larvae using combined data from selenium maternal transf er and microinjection studies in white sturgeon 195

- xvi -Introduction The overarching goal of this dissertation wa s to elucidate the effects of selenium (Se) bioaccumulation on white sturgeon, Acipenser transmontanus , with an emphasis on the San Francisco Bay population. This introduction outlines the need for the studies presented here and provides additional back ground. Three studies are included in this dissertation. The first investig ates the long-term effects of dietary Se in juvenile white sturgeon. The next study examines the mechan isms of Se maternal transfer in white sturgeon and the impacts of accumulated Se on reproduction. Finally, we investigate the effects of Se on developing white sturgeon embryos and larvae.

Selenium in San Francisco Bay-Delta San Francisco Bay-Delta is the largest es tuary in the western United States. The Delta is formed by the confluence of the Sacr amento and San Joaquin Rivers. Se enters this system in local petroleum refinery wa stewaters and irrigation drainage from the

seleniferous western San Joa quin Valley. The fate and cycling of Se is mediated by chemical speciation, hydrological conditions and water chemistry, among other factors (Luoma and Presser 2000). In the water column , Se is assimilated and transformed into organic Se by phytoplankton (Hu et al. 1997; Vandermeulen and Foda 1988; Wrench 1978). The organic Se is predominately in form of protein-bound selenomethionine and selenocysteine (Fan et al. 2002; Wrench 1978). Organi c Se in phytoplankton is consumed by filter feeders (Luoma et al. 1992) or recycled back to the water column upon decomposition of the plankton (Besser et al. 1994; Gobler et al. 1997; Wang and Guo 2001). In the sediment, Se undergoes micr obial reduction and is transformed intoparticulate elemental and organic Se (Oremland et al. 1990). Se in the sediment can return to the water column via pore waters (Velinsky and Cutter 1991). Se is prevalent throughout northern San Francisco Bay and accumulates in several species found in this region. Risebrough et al. (1977) reported concen trations of 8 to 11

μ g/g Se dry weight (dw) in tr ansplanted bivalves in northern San Francisco Bay. Johns et

al. (1988) reported a mean of 6 μ g/g Se dw in resident bivalve Corbicula spp. sampled near Carquinez Strait. The Selenium Veri fication Study found Se concentrations of 5.13

to 7.90 μ g/g dw in Corbicula spp. from the north Bay between 1987 and 1990 (Urquhart and Regalado 1991; White et al. 1988; White et al. 1989). The filter feeding exotic

bivalve Potamocorbula amurensis contains an average Se level of 15 μ g/g dw (Linville et

al. 2002). This non-native species was introduced into San Francisco Bay in the mid1980’s and has since become the dominant bivalve in the bay (Carlton et al. 1990;

Nichols et al. 1990) and a major food source of bent hic-feeding organisms (Urquhart and Regalado 1991). The high Se le vel and wide distribution of P. amurensis is of great concern because its Se burden significantly ex ceeds the levels shown to cause toxicity in animals consuming bivalves. Prey items containing greater than 10 μ g/g Se have been shown to induce Se toxicity in birds and fish (Adams et al. 1998; Coyle et al. 1993;

Hamilton et al. 1990a; Heinz et al. 1989; Hermanutz et al. 1992; Hoffman et al. 1989;

Woock et al. 1987). Based upon tissue burdens, the fish most vulnerable to Se contamination in San Francisco Bay are be nthic feeders, including white sturgeon (Luoma and Linville 1997; Urquha rt and Regalado 1991; White et al. 1987). White sturgeon sampled from San Francisco Bay-Delta between 1986 and 1990 contained Se at concentrations ranging from 9 to 30 μ g/g dw in liver (n = 52) and 7 to 15μ g/g dw in muscle (n= 99; Urquhart and Regalado 1991; White et al. 1988). Out of six sturgeon females sampled in the San Fran cisco Bay-Delta region in the 1990’s, one contained eggs with 3 μ g/g Se, four had eggs ranging from 8 to 12 μ g/g Se and one female contained eggs with 29 μ g/g Se (Kroll and Doroshov 1991). In 2000, white sturgeon from this region exhibited a mean liver Se concentration of 24 μ g/g, with some samples reaching as high as 40 μ g/g Se (n= 15; Stewart et al. 2004). Linares et al. (2004) reported a mean liver Se concentration of 9.75 μ g/g in 36 sub-adult and adult (age 4 – 18) white sturgeon sampled in San Francisc o Bay between 2002 and 2004. Recently, three white sturgeon captured from San Francisc o Bay-Delta were found to have 7 to 20 μ g/g Se in ovaries containing de veloping eggs (Doroshov Lab, UCD; unpublished data). The Se concentrations found in white sturgeon captured from San Francisco Bay-Delta reach levels previously linked to adverse effect s in other fish (Coughlan and Velte 1989; Gillespie and Baumann 1986; Hermanutz et al. 1992; Lemly 1993b). Although Se levels in white sturgeon from this region are quite high, they are also

variable. Stewart et al. (2004) recently examined this variability and found sampling seasonality and foraging location to be likely explanations. Se levels in P. amurensis , a primary route of Se exposure, are strongly influenced by seasonal variability (Linville et

al. 2002; Luoma and Presser 2000; Stewart et al. 2004). Se is highest in these bivalves after periods of low river outflow. Stewart et al. (2004) used stable isotope ratios to show that foraging location was a primary cause of high variation in wh ite sturgeon tissue Se

burden. To mitigate the Se pollution in San Fr ancisco Bay-Delta, the California San Francisco Bay Regional Water Quality Control Board required local oil refineries toreduce the amount of Se in effluents discharged into the Bay; however, refinery effluent continues to be a sign ificant source of Se in this region (Luoma and Presser 2000). The amount of Se entering the San Fr ancisco Bay-Delta in agricult ural effluents from the San Joaquin Valley may increase in coming year s due to changes in water management (Luoma and Presser 2000). Therefore, Se contamination is a high priority for the management of the San Francisco Bay-Delta ecosystem.

White sturgeon in San Francisco Bay-Delta White sturgeon is one of the two ende mic sturgeons inhabiting the Pacific Northwest. Both white and green ( A. medirostris ) sturgeon have high zoological value and are important to biodiver sity; but white sturgeon has a higher value as a food and game fish. Both species belong to a phylogene tically unique group of ancient ray-finned

fish that has survived for over two hundr ed million years (Sewertzoff 1923). White sturgeon is the largest North American sturge on species with indivi duals recorded at 6 meters in length and over 800 kg in weight (reviewed by Billard and Lecointre 2001; Doroshov 1985). This species is also unus ually long-lived, sometimes surviving 100 years. In San Francisco Bay-Delta, white sturgeon aggregate in northern San Francisco Bay (Suisun and San Pablo Bays) and migrate to spawn in the river systems (Kohlhorst 1980; Kohlhorst et al. 1980). The reproductive strategy of white sturgeon is quite different from that of modern teleosts. Sturgeon exhibit a delayed sexual ma turity, with males typi cally maturing at age 10 to 12 years and females at 15 to 32 years (Doroshov 1985; Doroshov et al. 1997). White sturgeon are long-lived and reproduce repeatedly over thei r lifespan (Doroshov1985). Each reproductive cycle of female white sturgeon includes at least two-years of yolk deposition in the eggs (Doroshov et al. 1997). Yolk deposition is dependent on the yolk precursor, vitellogenin, which is synthesize d in the liver. Vitellogenin is transported by the circulatory system to developing ooc ytes, where it is selectively taken up by receptor-mediated endocytosis (Mommsen a nd Walsh 1988). In the oocyte cytoplasm, the vitellogenin is reprocessed and deposited as yolk. In contrast to other fish, sturgeon store and use their yolk in the form of intr acellular crystal line platelets throughout early development (Dettlaff et al. 1993). Vitellogenin has also b een indicated as a potential vehicle of Se maternal transport in white sturgeon. Kroll and Doroshov (1991) found high, but variable, levels of Se in isolated plasma vitelloge nin and yolk proteins from six sturgeon females caught in the Sacramento River, indicating th e potential risk of developmental Se toxicity in sturgeon of this region. Spawning white sturgeon migrate up river during Marc h through May to broadcast their eggs, which adhere to the substrate until hatching. Sturgeon embryos hatch as underdeveloped (altricial) organisms referred to as yolk sac larvae. During the first 10 days post-hatch, over eigh ty percent of the maternal yolk is rapidly metabolized for growth, development, and for greatly in creased metabolic energy associated with pelagic swimming (Bolker 1993; Dettlaff et al. 1993; Doroshov et al. 1983). White sturgeon exhibit holoblastic development and th eir yolk is digested intracellularly, in contrast to the extraembryoni c yolk of modern teleost fish (Buddington and Doroshov 1986; Dettlaff et al. 1993). Vital organs are different iated during the yolk sac larvae period, such as the gills, liver and kidney.After hatching, the larvae are carried down ri ver with the current. Juvenile white sturgeon may remain in the rivers or delta for a period of years before entering the Bay (Radtke 1966). Juveniles feed on crustaceans and, to a lesser extent, bivalves (McCabe et

al. 1993; Radtke 1966; Schreiber 1962). Adult white sturgeon spend the majority of

theirs lives within San Francisco Ba y (Doroshov 1985; Kohl horst 1980; Kohlhorst et al. 1980). Adults in this area primarily consume mollusks and commonly feed on P.

amurensis (McKechnie and Fenner 1971; Ur quhart and Regalado 1991). Historically, the San Francisco Ba y white sturgeon population decreased dramatically in the early 1900’s due to ove rharvesting (Doroshov 1985; Kohlhorst 1980). This population continues to be impacted by ha bitat destruction, reduc ed river flows, and overharvesting. White sturgeon populations in San Francisco Bay-Delta have been highly variable over the past five decades (Kohlhorst 1980; Kohlhorst et al. 1991; Schaffter and Kohlhorst 1999). Schaffter and Kohlhorst ( 1999) reported a paucity of smaller sturgeon caught in the late 1990’s. Population surveys in 2005 indicated that the San Francisco Bay-Delta white sturgeon popul ation was at a 50-year low, with an estimated 10,000 adult sturgeon (California De partment of Fish and Game Commission 2006).

Observed decreases in the San Franci sco Bay white sturgeon population have

been associated with the occurr ence of dry water years (Kohlhorst et al. 1991). This may be due to several factors, including flow ra te and habitat availabi lity. In addition, Se concentrations in sturgeon food sources si gnificantly increase dur ing low-flow years (Johns and Luoma 1988; Linville et al. 2002; Luoma and Presser 2000). The life history of white sturgeon may render them especia lly vulnerable to Se bioaccumulation andmaternal transport of Se to egg yolk due to a long life span, benthi c feeding habits and a prolonged period of vitellogenesis and yolk depo sition. Se toxicity cannot be discarded as a potential factor affecti ng white sturgeon reproduction and natural recruitment in San Francisco Bay-Delta.

Toxicity of selenium Se is an essential nutrient (0.1 – 1.25 μ g/g diet) for animals, humans and many micro-organisms (Diplock 1976; Gatlin and Wilson 1984a; Hilton et al. 1980; Mayland 1994). Se is also a significant environmen tal toxicant due to its efficient food web transfer (Lemly 1985; Luoma et al. 1992). There is a very narrow margin between

nutritional and toxic co ncentrations of this element. Concentrations as low as 3 μ g/g in diet have led to toxicity in aquatic orga nisms (reviewed by Hamilton 2004). Se toxicity results in reduced growth, significant hist opathology in major organs, alteration of detoxification systems, promotion of oxida tive damage, and mortality (e.g., Hicks et al. 1984; Hoffman et al. 1989; Lemly 1993a; Lemly 1998; Sorensen and Thomas 1988; Stanley et al. 1996; Woock et al. 1987). Se is also a well-documented developmental toxicant to fish and wildlife speci es consuming diets containing 13 μ g/g Se or greater (e.g., Coyle et al. 1993; Gillespie and Baumann 1986; Hamilton et al. 1986; Hermanutz 1992; Hoffman et al. 1988a; Lemly 1993b; Ohlendorf et al. 1986; Woock et al. 1987). Common developmental defects produced by Se toxicity include severe edema (e.g., Gillespie and Baumann 1986), skeletal deformities (scoliosis, lordosis and kyphosis; e.g., Schultz and Hermanutz 1990), and cr aniofacial deformities (e.g., Holm et al. 2005). Each of these defects is likely to reduce the pr obability of survival in developing fishes inthe natural environment. Excess Se has le d to severe teratogenesis, mortality and endangerment or extirpation of fish species residing in areas high in Se (Garrett and Inmann 1984; Hamilton 1999; Lemly 1985; Skor upa 1998). Lemly (2002) has proposed

Se toxic thresholds for freshwater and anadro mous fish as 8 μg/g in muscle, 12 μg/g in liver, and 10 μg/g in ovary or eggs. Se toxicity is well documented in fr eshwater species (reviewed by Hamilton 2004). However, few studies have investigated Se toxicity in euryhaline and marine species. Hamilton et al. (1990a) investigated Se toxicity in chinook salmon ( Oncorhynchus tshawytscha ) and found that 10 μ g/g of dietary Se reduced survival and 5

μ g/g reduced growth. Striped bass ( Morone saxatilis ) fed Se-laden red shiners (39 μ g/g) experienced complete mortality (Coughl an and Velte 1989). In the grass goby ( Zosterisessor ophiocephalus ), a marine fish, the 96hr LD 50 for selenite via ip injection

was 0.29 μ g/g body weight (Tallandini et al. 1996). Very few studies are available on the effects of Se in white sturgeon. Recent work shows that excess Se leads to toxicity in white sturgeon. Dietary exposure of 40 μg/g seleno-L-methionine led to impaired growth in fingerling white sturgeon (25 – 30g) after two weeks (Tashjian et al. 2006). In the same study, the activity level of fingerling wh ite sturgeon decreased with increasing Se exposure. A study investigating the effect s of Se exposure on sa linity tolerance in juvenile white sturgeon indicates a strong inte ractive effect of dietary Se exposure and elevated salinity on survival (Tashjian 2005). Sturgeon mortal ity and time to death were both positively correlated with levels of Se in the diet . To our knowledge, no studies have been published regarding the developmen tal toxicity of Se in white sturgeon.The specific chemical species and ro ute of exposure are critical in the manifestation of Se toxicity. In the natura l environment, Se is found in four oxidation states, selenide (-II; Se -2 ), elemental Se (0, Se 0 ), selenite (IV; SeO 3 -2 ), and selenate (VI;

SeO 4 -2 ). Elemental Se is commonly found in anoxic sediments (reviewed in Frankenberger and Engberg 1998). Selenite and selenate are pr esent as salts in the soil, or as dissolved ions in aquatic systems. Selenide is found in organic forms of Se, such as seleno-amino acids, and as dissolved ions . Organic forms of Se, particularly selenomethionine, have been shown to have greater bioavailability and toxicity than inorganic forms and are often the species to which aquatic organisms are exposed (Hamilton et al. 1990a; Hoffman et al. 1989; Luoma et al. 1992; Reinfelder et al. 1997; Wang and Lovell 1997). Additionally, the diet is the most significant route of exposure leading to Se bioaccumulation and toxicity (e.g., Coyle et al. 1993; Hermanutz 1992;

Luoma et al. 1992). Much of the previous work on Se has utilized waterborne exposures (e.g., Gissel Nielsen and Gi ssel Nielsen 1978; Hodson et al. 1980; Lemly 1982). In addition, the common use of inorganic Se in both waterborne a nd dietary exposures underestimates environmental exposure, uptake and toxicity of Se to aquatic organisms (Fan et al. 2002; Wang and Lovell 1997; Woock et al. 1987). However, the appropriate use of selenomethionine through dietary exposur e in toxicity studies is beginning to be widely recognized. Se is readily adsorbed th rough the gut and metabolized in the liver and kidney through a glutathione-dependent reductive pathway (reviewed by Diplock 1976; Suzuki 2005b). Glutathione (GSH) is a tripeptide nuc leophile consisting of glutamic acid, cysteine and glycine. With the aid of gl utathione-S-transferase enzymes, GSH reducesSe species to the –II oxidation state (selen ide). As a result, GS H becomes oxidized and must be reduced by NADPH-depe ndent glutathione reductase before it can react with another Se compound. The serial reduction of selenate and selenite by GSH produces the intermediary metabolite selenodiglutathione (GSSeSG), which is further reduced to glutathione selenopersulfide (GSSeH). Glutathione selenopersulfide is reduced to hydrogen selenide (H 2 Se), which may undergo autooxidation back to glutathione selenopersulfide (Anundi et al. 1984). Seleno-amino acids are transformed to selenide by methylation (Spallholz et al. 2004) or cleavage of Se through lyase functions (Esaki et al. 1982; Okuno et al. 2005a; Okuno et al. 2005b). Selenide can then undergo various

reactions that could lead to toxicity or excretion. Se excretion is mediated through met hylation by methyltransferase and the cofactor S-adenosylmethionine (SAM); howev er, many methylated metabolites are also toxic (reviewed by Diplock 1976; Francesconi and Pannier 2004). Exposure to excess Se leads to selenosugars and trimethylselenonium ion in the urine (Byard 1969; Kobayashi

et al. 2002). At very high Se levels, the step from di- to tri-methly ation becomes limiting and dimethylselenide is ex creted through respiration (McConnell and Portman 1952). There are several potential modes of action in Se toxicity. Metabolic intermediates of Se are thought to be a significa nt cause of Se toxicit y. Methylselenol, an

intermediate metabolite of bot h selenite and selenomethioni ne, can lead to superoxide production resulting in cellular disruption (Okuno et al. 2005b; Seko et al. 1989; Yan and Spallholz 1991). In fact, the production of reactive oxygen species may be responsible, in part, for the destruction of cancer cells observed with high-Se treatment (reviewed by Drake 2006; Spallholz 1994). The metabolic intermediate selenodiglutathione has beenindicated in the inhibition of protein synthesis (Verni e and Van Leewenhoekhuis 1987),

the reduction of ce llular growth (Wu et al. 1995), and the induction of apoptosis (Lanfear

et al. 1994). The metabolite hydrogen selenide is also extremely toxic (reviewed by Spallholz and Hoffman 2002). Additionally, metabolism of excess Se can alter natural detoxification systems by depleting reduced GSH or the methylat ion co-factor SAM (Anderson and Moxon 1942; Hoffman and Heinz 1988; Hoffman 1977). Reduced GSH, along with glutathione peroxidase, is crucial in det oxifying peroxides and halting lipid peroxidati on. With the aid of glutathione transferas e, reduced GSH actively binds electrophilic toxins and prevents them from binding to macromol ecules (proteins, DNA, enzymes, etc.). Methylation is also a common detoxificati on pathway. Depression of these essential detoxification mechanisms leaves organisms vulnerable to additional toxic insults. Se can also be incorporated into biologi cal molecules in place of sulfur because the two elements have similar chemical prope rties (Diplock 1976; Stadtman 1974). Se is incorporated into proteins as a result of genetic translation (reviewed by Gromer et al. 2005; Suzuki 2005b). A specific genetic c odon prescribes the incorporation of selenocysteine into proteins; however, selenom ethionine is indiscri minately substituted for the methionine codon in genetic translat ion. The amount of selenomethionine that is indiscriminately incorporated into proteins increases with increas ing availability of selenomethionine in tissues (Schrauzer 2000). Similarly, when increased Se is available compared to sulfur, selenocysteine can subs titute for cysteine in protein synthesis (Kramer and Ames 1988; Müller et al. 1994). However, Se and sulfur bonds may have different strength and functi on in biological molecules (R eddy and Massaro 1983), thussome Se-substituted compounds may become unstable or dysfunctional (Lemly 1998; Stadtman 1974).

Research aims The studies presented here were designed to identify potential Se toxicity in white sturgeon, at exposures rele vant to the San Francisc o Bay-Delta population. Understanding the effects of Se on white stur geon is a critical link between studying the

extent of Se contamination in biota and re gulating Se loading in this region. The recruitment success of fishes is not dependent on a singular factor, but rather a synergistic interaction of several factors (Bennett a nd Moyle 1996). Simply addressing high-profile environmental factors, such as habitat loss, may not adequately protect the white sturgeon population of San Francisco Bay-Delta unless the risk of Se toxicity is fully understood and mitigated.Chapter 1. Toxicity of foodborne selenium to juvenile white sturgeon, with an emphasis on histopathology of the liver

Introduction In small quantities (0.1 – 1.25 μ g/g diet) selenium (Se) is an essential nutrient for animals, humans and many micro-organisms (Diplock et al. 1976; Gatlin and Wilson 1984b; Hilton et al. 1980; Mayland 1994). In slightly greater quant ities (as little as 3

μ g/g Se in diet) this element is a potent en vironmental toxicant that threatens higher trophic level species because of its effici ent food web transfer (Hamilton 2004; Lemly 1985; Lemly 2002; Luoma et al. 1992; Stewart et al. 2004). Environmental exposure to Se has led to population-level effects in birds and fish re siding in areas receiving wastewaters from the combustion of fossil fuels (Garrett and Inmann 1984; Lemly 1985) or drainage from selenifer ous land, including both agricu ltural and natural drainage (Hamilton 1999; Presser and Ohlendorf 1987; Skor upa 1998). Se toxicity is well studied in warm, freshwater systems (Hamilton 2004) , but its effects are largely unknown in estuarine systems (Luoma and Presser 2000; Skorupa 1998). San Francisco Bay-Delta is the largest es tuary in the western United States. The Delta is formed by the confluence of the Sacr amento and San Joaquin Rivers. Se enters this system in petroleum refinery wast ewaters and irrigation drainage from the

seleniferous western San Jo aquin Valley. White sturgeon, Acipenser transmontanus , resides in San Francisco Bay (Kohlhorst et al. 1991) and is exposed to high levels of Se through their diet. This is ev idenced by chemical detection of high tissue Se levels in common prey of white sturgeon (Linville et al. 2002; Stewart et al. 2004; White et al. 1988). In addition, sturgeon muscle, liver a nd eggs show significant levels of Se,indicating that diet is a predominant route of exposure to this element (Kroll and Doroshov 1991; Stewart et al. 2004; Urquhart and Regalado 1991; White et al. 1989). Common San Francisco Bay bivalves were shown to contain 3 to 9 μ g/g Se (all values listed as dry weight) in the 1980’s (Johns and Luoma 1988; Urquhart and Regalado 1991; White et al. 1988; White et al. 1989). However an invasive bivalve, Potamocorbula amurensis, introduced into San Francisco Bay in the mid-1980’s accumulates significantly higher leve ls of Se, an average of 15 μ g/g (Linville et al. 2002; Luoma and Presser 2000; Stewart et al. 2004). Since being introduced, P. amurensis has become the dominant bivalve in the bay (Carlton et al. 1990; Nichols et al. 1990) and a major current food source of adult white sturgeon (Stewart et al. 2004; Urquhart and Regalado 1991). Juvenile white sturge on also consume bivalves, although not predominantly (McCabe et al. 1993; Radtke 1966; Schreiber 1962). The high Se level

and wide distribution of P. amurensis is of great concern because consumption of prey containing greater than 10 μ g/g Se induces Se toxicity in birds and fish (Coyle et al. 1993; Hamilton 2004; Hamilton and Buhl 1990; Hamilton et al. 1990b; Hermanutz et al. 1992; Hoffman et al. 1989; Woock et al. 1987). White sturgeon aggregate in north San Francisco Bay (Suisun and San Pablo Bays) and migrate to spawn in the river sy stems, predominately the Sacramento River (Kohlhorst 1980; Kohlhorst et al. 1980). Juveniles migrate back to the Bay-Delta, but may remain in the lower Sacramento Rive r for several years (R adtke 1966). White sturgeon has a very high value as a food and game fish and is important to biodiversity in the Pacific Northwest. Two endemic species of the Pacific Northwest, white and green ( Acipenser medirostris ) sturgeons, belong to a phyloge netically ancient group of rayfinned fish that has survived for over tw o hundred million years, but has experienced rapid decline over the past century (Birstein 1993). White sturgeon sampled from San Francisco Bay between 1986 and 1990 contained an average of 12.57 μ g/g Se in muscle (± 9.72 standard deviation; range of

1.46 – 50 μ g/g Se; n= 99) and an average of 26.55 μ g/g Se in liver (± 21.76 standard deviation; range of 3.24 – 79.42 μ g/g Se; n=54; Urquhart and Regalado 1991; White et

al. 1987; White et al. 1988; White et al. 1989). In 2000, white sturgeon from this region

had a mean liver Se concentration of 24 μ g/g with some samples reaching as high as

40 μ g/g Se (n = 15; Stewart et al. 2004). These concentrations exceed concentrations previously linked to adverse effects in ot her fish (Coughlan and Velte 1989; Hermanutz

et al. 1992). Elevated Se affects growth, condition fact or, hepatosomatic index, and survival in several fish species (Hamilton et al. 1990b; Hilton et al. 1980; Sorensen and Bauer 1984;

Sorensen et al. 1984; Woock et al. 1987). However the current li terature reflects a lack of consistency in these effects, which may be linked to differences between the species, life-stages, and experimental conditions (Hamilton 2004). Exposure to elevated Se concentrations also causes histopathological alterations in several fish species (Coughlan and Velte 1989; Ellis et al. 1937; Hicks et al. 1984; Lemly 1993a; Sorensen 1988;

Sorensen et al. 1984). Se can result in damaged or necr otic liver ce lls; and, thereby alter the structural framework of the organ in survivors (Coughlan and Velte 1989; Ellis et al. 1937; Sorensen et al. 1982a; Sorensen et al. 1983a; Sorensen et al. 1983b; Sorensen et al. 1984). In the kidney, excessive Se concentrations affect glom erular and tubular epithelial cells and can lead to disease states such as proliferative glomerulonephritis,nephrocalcinosis and uremia (Coughlan and Velte 1989; Hicks et al. 1984; Sorensen et

al. 1983a; Sorensen et al. 1982b). Gills of Se-exposed fish exhibit swollen and vacuolated secondary lamellae, which can adversely affect respiration and could potentially lead to death (Lemly 1993a; Sorensen et al. 1982a; Sorensen et al. 1984). Se is also a potent reproductive and developmen tal toxin when transferred from the adult female to the yolk of their eggs (G illespie and Baumann 1986; Lemly 1993b). Due to a paucity of information regardi ng the effects of Se on sturgeon, this study was initiated to determine the susceptibility of juvenile white sturgeon to chronic Se exposure via diet. We reared sturgeon juveniles using three foodborne Se (as selenized yeast) levels and a control diet with the required low Se level for essential nutrition. Selenized yeast, predominately in the form of selenomethionine (Polatajko et al. 2005), was used as a surrogate for organic Se presen t in the aquatic environment. Organic forms of Se, particularly selenomethionine, have b een shown to have grea ter bioavailability and toxicity than inorganic forms and are often the species to which aquatic organisms are exposed (Hamilton et al. 1990b; Hoffman et al. 1989; Luoma et al. 1992; Reinfelder et

al. 1997; Wang and Lovell 1997). Following six m onths of rearing, the juveniles were

sacrificed and tissue Se c oncentrations were measured. In addition, growth, and histopathological and morphologi cal alterations were assess ed. Our hypothesis was that dietary Se at environmentally relevant leve ls would be accumulated in sturgeon tissues, inhibit growth, and induce pathological changes.Materials and Methods Source of Animals White sturgeon juveniles (6 weeks post hatch, mean weight 350 mg) were

obtained from Stolt Sea Farm , California in May 2000. They originated from the same fertilization mixture composed of gametes fr om three female and six male hatchery-bred white sturgeon. Larvae were tr ansported to University of California, Davis (UCD) and reared to 38 weeks post-hatch following standard hatchery protocols (Conte et al. 1988) and monitored for health. During early rearing, a portion of the juveniles were euthanized due to an outbreak of white sturgeon iridovirus (Georgiadis et al. 2001). However, by 25 weeks post-hatch there were no signs of fish illness and the fish exhibited normal growth and survival until 47 weeks post-hatch, prior to initiating the study.

Experimental Design Hatchery reared juveniles (47 weeks old, average weight 575 g) were held in twelve, 5-foot diameter, out door flow-through tanks at the Center for Aquatic Biology and Aquaculture, UCD. The experiment cons isted of four dietary treatments: control (1.04 ± 0.07 μ g/g Se), low (20.05 ± 1.10 μ g/g Se), medium (35.61 ± 3.83 μ g/g Se) and high (52.54 ± 2.32 μ g/g Se; mean ± standard deviati on; Table 1-1). Each dietary treatment was replicated in three randomly assigned tanks, with 10 fish per tank and 30 fish per treatment.Initiation and maintenance At 38 weeks post-hatch, 144 randomly selected fish were anesthetized (200 mg/L MS-222) measured, weighed and randomly distri buted into the experimental tanks. Each tank initially contained 12 fish with mean weight of 465 g, fork length of 39.3 cm and condition factor of 0.76 (100 x W/L 3 ). There were no statistical differences among treatment tanks (ANOVA; p > 0.82). At 46 weeks post hatch, two fish from each tank were sacrificed to provide an initial Se m easurement using techniques described below. Three days later the remaining 120 fish we re gradually transitioned onto respective experimental diets over one week. All juve niles were on full experimental diets by 47 weeks-post hatch. The experiment al tanks received well water at an average flow rate of 168 L/min. The temperature in the tanks averaged 18.6 °C. The dissolved oxygen and percent air saturation averaged 9 mg/L and 95% respectively. Th e fish were observed daily for mortality and morbidity. Mori bund fish were euthanized using MS-222 overdose. The tanks were cleaned weekly, with minimum disturbance to the fish. Following 23 weeks of dietary Se exposure, th e fish were measured for fork length and wet weight, and were necrops ied for tissue analyses.

Experimental Diet Sturgeon were fed a casein-based, purified pelleted diet (Table 1-2), specifically formulated for sturgeon and with a satisfact ory history for reari ng hatchery sturgeon stocks for longer than two years with consistently good growth and free from any abnormal signs (Hung et al. 1987). Optimal feeding rates and nutrient requirements of white sturgeon are known (Hung 1991; Hung and Deng 2002). Dietary ingredients weremixed and then pelleted as described by Hung and Lutes (1987). Four diets were formulated with increasing levels of Se (Table 1-1). Se was added to the diet as selenized yeast (Selenomax®, Ambi Inc.). Sturgeon we re continuously fed the experimental diet, using a 24 hour belt feeder, at a rate of 1.2% body weight per day.

Sampling After 23 weeks of dietary Se exposure, sturgeon were sampled over a three-day period. Fish were stunned by a quick blow to the head, weighed and measured. Blood

was collected from the caudal vein usi ng a 10 ml heparinized Vacutainer® and centrifuged (4000 x g, 10 min) to separate the plasma, which was flash frozen in liquid nitrogen for Se analysis. Fish were then euthanized by MS-222 ove rdose and the spinal cord was cut before proceeding with the ne cropsy. Dissected liver and gonads were weighed for respective somatic indices. Tissue pieces from gill, gonad, liver and posterior kidney were fixed in 10 volumes of 10% phosphate-buffered formalin for histopathological analysis as described below. In addition, samples of liver and muscle were frozen on dry ice and stored at -80 °C for Se analysis.

Selenium Analysis Frozen tissue samples were lyophilized and weighed. Muscle samples were homogenized using a Wiley Mill. Liver sample s were flash-frozen in liquid nitrogen and then pulverized with a mallet. The entire sample of lyophilized plasma was analyzed. Approximately 0.25 g of lyophilized tissue was di gested using a nitric /sulfuric/perchloric acid mixture heated to 230 °C over two days. Se in the digested solution was reduced toselenite by the addition of 6M HCl heated to 90 °C for 1 hour. The reduced samples

were diluted to 25 ml with 4 M HCl and stor ed at 4 °C until spectroscopy analysis (less than 10 days). Total Se was analyzed as selenite us ing Hydride-Generation Atomic Absorption with an EDL Se lamp (PerkinElmer AAnalyst 300 Atomic Adsorption Spectrometer MHS-15). The instrument had a minimum de tection limit of 1 ng Se and was calibrated in the linear region, from 20 to 100 ng Se, usi ng a sodium selenite standard solution. Quality control included spikes, duplicates , certified reference materials (NRC DORM-2, DOLT-2, DOLT-3) and validation by an external laboratory (California Animal Health and Food Safety Laboratory at UCD). All qual ity control measurements agreed with the target concentration within 15 percent. All blanks were less than 1 ng/ml Se. All data is reported as dry weight. Mois ture content was determined as 58.3 ± 6.59 percent in liver and 77.6 ± 1.21 percent in muscle (mean percent moisture ± st andard deviation, n=117). Plasma contained 95.9 ± 0.41 percent moistu re and 42.02 ± 5.7 mg of dry weight per

milliliter.

Gross Morphology

Condition factor was measured as (weight (g)/fork Length 3 (cm)) * 100. Specific growth rate (SGR) was determined for each tank using the formula: SGR = 100 * (lnWeight final – lnWeight initial )/time (days). Organ to body weight relationships were analyzed by regressing log organ weights by log body weights. Body weight to length relationships were analyzed by regressing log body weights against log fork lengths. Log

transformations were used to acc ount for allometric growth.Histological Analyses At necropsy, following euthanasia, the ve ntral abdominal wall was surgically opened and tissues were rapidly removed for pl acement in fixative. Liver (1-3 mm thick piece of the left lateral marg in), kidney (similarly sized pieces from rostral and caudal portions), and gonad were removed. Next, the second gill arch was identified and

removed after transaction at dorsal and ventral midlines. Finally, a wedge of skeletal

muscle from the flank with skin attached was removed and, using a fresh, clean razor blade, a 1-3 mm piece of muscle was trim med from this sample. Tissues were immediately immersed in 10% neutral buffere d saline and stored under refrigeration in individually labeled containers at a ratio of 10 volumes of fi xative to 1 volume of tissue. After completion of fixation, solution was d ecanted into appropriately labeled storage

chambers and each container was filled w ith 0.1 molar phosphate buffer containing 6% sucrose and stored under refrigeration (2 – 4 °C) until time of processing. At the initiation of processing, fixed tissues were trimmed, selec ting the outermost (1-2 mm thick) portions to ensure fixative penetrati on, dehydrated in graded ethanol solutions, cleared, and embedded in paraffin. Sections (6-8 microns thickness) were made using a rotary microtome, and were attached to glass slides coded for each individual and treatment, stained with hematoxylin and eo sin, coverslipped and vi ewed using a Nikon Eclipse E600 microscope with CFI plan fluor objectives. Images were acquired using a Nikon DXM1200 Digital Still Camera and EclipseNet ® Nikon E600 software, individually labeled with orig inal magnification recorded. Images were stored as TIFF files in Adobe Photoshop 6.0.Statistical Analysis Se accumulation was compared across tissues and treatments. Because no suitable transformations to normality could be found for this data, the non-parametric Rank F-Test was used (Neter et al. 1996). To compare mean Se values, a least squares means Students t-test with Bonferroni-adj ustment was applied to the ranked data (experiment-wise α = 0.05; Bonferroni adjusted comparison-wise α = 0.001666). Morphologic measurements were compar ed across treatments using ANOVA and ANCOVA, or Rank F-test when the assumptions of the former models could not be met (Neter et al. 1996). Log 10 transformations were used in regressions of organ to body weight to account for allometr ic growth. Histological surv ey of liver, kidney, and gill

was made and alterations from control anatom y were imaged, ranked as to treatment and recorded. For areal analysis of melanomacrophage centers, random, non-overlapping

fields of liver were selected and the numb er and area of melanomacrophage centers were recorded. Comparisons of the presence of hi stological lesions in gills across treatment levels were made using two-way contingency tables. Statistical software packages JMP and SAS (SAS Institute Inc.) were used for all computations.

Results

Selenium Bioaccumulation Se bioaccumulation was compared across tissues and treatments (Table 1-3). Se

in each tissue increased with in creasing Se in the diet (Ra nk F-Test, p = .0334; adjusted multiple comparisons, α = 0.05; Figure 1-1). In the control treatment ( ca. 1 μ g/g dietary Se) Se accumulated more in blood than in musc le, but Se in liver wa s not different fromeither blood or muscle. There were no differences between Se levels in tissue types in both the low and medium treatments ( ca. 20 & 36 μ g/g dietary Se, resp ectively). In the high treatment ( ca. 53 μ g/g dietary Se), Se accumulated more in liver than in muscle, but

Se in blood was not different from either liver or muscle. Within treatment groups, there were no significant relations hips between Se accumulation and condition factors (Regression; p > 0.05). Before initiating the experiment, 24 juvenile white sturgeon were sacrificed to provide a baseline measurement of Se. Prior to the dietary exposure, the sturgeon had low levels of Se in muscle (1.4 ± 0.20 μ g/g, n=17), liver (1.89 ± 0.69 μ g/g, n=10) and blood (3.66 ± 0.83 μ g/g, n=10; mean Se ± standard deviation).

Morphologic Characteristics Several whole body morphologic characte ristics were compared across Se

treatments. We found no differences between treatments in conditi on factor, fork length, body weight, or specific growth rate (ANOVA, p > 0.075; Tabl es 1-4 & 1-5). Similarly, gonadosomatic, hepatosomatic and weight to le ngth regression slopes were not different between treatments (ANCOVA, p = 0.08 – 0.3; Table 1-4).

Survival Mortality was not significant in this experiment. One mortality occurred in a high treatment tank just prior to initiating di etary Se exposures. A moribund fish was euthanized (MS-222 overdose) in a high tr eatment tank after it was found upside down and motionless during the second month of di etary exposure. One fish from a low

treatment tank was euthanized (MS-222 overdose) in the fourth month of dietaryexposure because it was unable to swim and wa s emaciated. These fish were not sampled for Se or histopathology. No other mortalit ies were observed during the experiment.

Histopathology Pathological changes were observed in liv er, gill arch and posterior kidney of treatment fishes. Histology of gonads revealed mostly adipose tissues with very limited vasculature. No actual gonada l tissue was visible, which is typical in white sturgeon at this age class (Doroshov et al. 1997). The majority of pathological changes were

observed in the liver.

Liver Control liver morphology In control fish ( ca. 1 μ g/g dietary Se), hepatocytes were the major cell type and occupied the most volume in microscopic fields (Figure 1-2). When viewed in longitudinal arrangement, two rows of hepatocytes commonly intervened between adjacent profiles of the microvasculature (si nusoids). This pattern of hepatocyte arrangement is the tubule, common as the ad ult phenotype in lower vertebrates including fishes (Hinton and Couch 1998). Hepatocyte s contained a single nucleus but not all planes of section through hepa tocytes revealed a nuclear prof ile due to the much larger component volume of the cytoplasm (Figure 1-2). With hematoxylin and eosin (H&E) stained preparations of paraffin-embedded liver, the nuclear margins were basophilic (dark gray) and a single nucleolus was seen (Figures 1-2 & 1-3). The cytoplasm stained lightly eosinophilic (pink). Within the cyt oplasm, vacuolar profiles likely denoted lipidthat was removed during tissue processing (a lcoholic dehydration). Other regions of cytoplasm stained more diffusely (gray-pink) but were not compri sed of lipid profiles (Figures 1-2 & 1-3). These were interpreted as areas of glycogen storage. Evidence for hepatocyte polarity was shown (Figures 1-2 & 1-3). Hepatocyte nuclei were located in basal regions of cells (i.e., closer to hepati c sinusoids) while apical cytoplasm was only lightly stained (Figures 1-2 & 1-3) and was directed toward center of he patic tubules (Figures 1-2 – 1-4). The parenchyma was comprised of tubular arrays of hepatocytes and, in certain regions, individual tubules curved and abutte d against adjacent tubules resulting in what appeared as sheets of cells (Figures 1-2 – 1- 4). Sinusoids, the hepatic microvasculature, were easily denoted by the pr esence of erythrocytes whos e cytoplasm stained red and contrasted with the single darkly stained nucleus (Figures 1-2 & 1-3). The luminal

surfaces of sinusoidal endothelial cells were concave and nuclei often showed similar shape. These cells delimited the sinusoids and were separated from adjacent hepatocytes by a perisinusoidal space containing perisi nusoidal macrophages. Within this space, small cells with high nuclear to cytoplasmic ratio and containing a single lipid droplet were seen (not shown in figures). Respectivel y, these cells are the fa t-storing cells of Ito (Hampton et al. 1985; Hinton et al. 1987) which may become activated and assume fibroblastic properties (Hampton et al. 1985). Stromal features included venules (Figures 1-2 & 1-5), arterioles, veins (Figure 1- 3), small muscular arteries, bile ductules and sm all diameter bile ducts. Because all livers were sampled at the left margin, larger va scular and biliary stru ctures more closely associated with the hepatic hilus (near mid line of abdominal cavity) were not included.Biliary epithelial cells frequently accompanied venules (Figure 1-5). The latter were interpreted as branches of the hepatic portal ve in, the afferent venous supply to the liver.

In controls these sites also contained focal aggregates of melanomacrophages (Figure 1- 6). Eosinophilic granular leukoc ytes were frequently seen ne ar small bile ducts (Figure 1-5). Larger diameter veins were often a ssociated with bile ductules and hepatic arterioles (Figure 1-3), resembling mammalia n portal triads (Hinton and Couch 1998). However, foci of mononuclear cell infiltration were common in these sites and melanomacrophage aggregates near large vein s were usually of greater diameter than

those associated with venules. In add ition to accompanying veins, melanomacrophage aggregates were frequently associated with arteries, often surrounding muscular layers and periadventitia of these vessels (Figures 1-5 & 1-6). Eosinophili c granular leukocytes were found near arteries, arteri oles; and in inflammatory fo ci they surrounded small bile ducts (Figures 1-5 & 1-7).

Low Selenium dietary group Mild expansion of number and area of melanomacrophage aggregates was observed in the low Se treatment ( ca. 20 μ g/g dietary Se). There was a 30% incidence of tubuloform hepatocellular swel ling (Figures 1-8 & 1-9). Bile duct proliferation, especially involving transitional biliary epitheli al cells, was seen (Figures 1-10 & 1-11). This occurred in approximately 70% of indivi duals. In addition, apparent biliary stasis

led to marked increase in the volume of the canalicular network. Canaliculi are the initial portions of the intrahepatic biliary system r eceiving bile made by hepatocytes. Normally, in paraffin-embedded material these passageways are not seen ; however, in this group thecondition was seen in each indivi dual. Bile stasis is a hallm ark feature of hepatotoxicity

and criteria for this condition fit thos e of the medical diagnosis (Lee 1996-2005). Limited formation and subsequent excretion of b ile, due to stasis, results in liver necrosis and animal death if prolonged and severe (Beers 2003; Lee 1996- 2005). Perivascular

inflammation was enhanced over control mor phology in 40% of the i ndividuals of this

group.

Medium selenium dietary group Observed changes in the medium treatment ( ca. 36 μ g/g dietary Se) included moderate expansion of melanomacrophage aggr egates in area and in number; increased perivascular inflammation; focal hepatocellu lar necrosis with accumulation of discreet eosinophilic cytoplasmic inclusi ons; biliary canalicular disten sion and biliary epithelial cell (ductular and small ducts) prol iferation (Figures 1-12 & 1-13).

High selenium dietary group Alterations in the high treatment ( ca. 53 μ g/g dietary Se) involved hepatocytes (fatty change, biliary stasis of canalicular network, necrosis, reduction in hepatocyte volume in cells of portal zones, and presen ce of Councilman bodies which are evidence of hepatocellular apoptosis), mononuclear leukocytes (foci of inflammation), macrophages (melanomacrophage aggregation), a nd biliary epithelial cells (proliferation of ductules and small ducts; Figures 1- 14 & 1-15). Maximum melanomacrophage aggregation response occurred in this group (Figure 1-14). There was evidence of areas of parenchyma being divided by septae of pr oliferating bile ductu les. In addition, amosaic of response was seen in some individua ls indicating that two functionally distinct populations of hepatocytes were present. Fi rst, vacuolated hepato cytes (suspected but unconfirmed lipid change) occurre d in distinct zones and th ese zones were lacking in melanomacrophage aggregates. The next z one was one in which hepatocytes were shrunken, stained more intensely, and were likely producing apoptot ic fragments which were then phagocytosed by adjacent cells producing bright red Councilman bodies. These regions contained the melanomacrophage a ggregates. As in other Se concentration groups, the canalicular network was distended; and this, alon g with distension of bile ductular and small ductal lumina, suggests an in ability to transport bile from the liver. This constitutes a major form of toxicity to the liver and may account for some of the cytotoxic effects following Se exposure. That small bile duct proliferation may be seen with other hepatic conditions was suggested by the finding of duct proliferation an d bile stasis in one control fish. The high amplitude swelling of some tubules (seen in on e control and in severa l individuals of the medium and high Se groups) suggests a lack of volume regulation and is a form of acute hepatic tubular necros is. This condition affected groups of individual hepatocytes of

individual tubules. The consistent canalic ular distension, seen only in Se exposed sturgeon constitutes proof that these animals are responding to their dietary exposure.

Gill Gills were analyzed for the occurrences of four prominent lesion types: gill dysplasia, the misshapen secondary lamellae and sometimes bent primary lamellae, which can occur with exposur e of young organisms to Se; fusion of secondary lamellae,which results in reduced surface area for gase ous exchange; inflammation, representing a response to injury; and torsi on of lamellae, where the lame llae are twisted along the long axis. Contingency tables were used to an alyze the presence or absence of each lesion type in control and treatment fish. Significan t differences were found for the presence of the fusion of secondary lamellae and in flammation across treatments (p = 0.024 and 0.041, respectively; Figure 1-16). These effect s were observed more frequently in the lowest treatment group ( ca. 20 μ g/g dietary Se). No statisti cally significant differences were found for the presence of gill dysplasia or torsion of lamellae between treatment groups (p = 0.25 and 0.146, respectively).

Kidney Kidneys of juvenile sturgeon of this study did not appear affected by Se exposure. All kidneys of juvenile groups showed signs of tubulogenesis. However, since the basophilic tubules with numerous mitotic cells and the basophilic glomeruli were seen in the control, low, medium and high Se exposur e groups, this is intere sting but likely not a toxic response. In addition, th e tubulo/glomerulo-genesis app eared to be localized in the periphery of all kidneys, which indicates th at the stem or progenitor cells for this generation are located in the same topographic site in the organ. Eosinophilic material (putative protein) was observed in the glomerular space. This was accompanied by eosinophilic bodies in the cytoplasm of proximal tubular epithelial cells, which was interpreted as epithelial cell protein uptake as an effort to conserve protein. However this change appear ed not only in animals of the experimental groups but also in the control. Despite careful analysis of glomerular space, renalproximal tubules, intertubular structures, a nd generation/regenera tion of tubules and glomeruli, no features of toxic alterations were seen in kidneys of exposed fish. Dietary Se did not appear to cause renal toxicity under the conditi ons of this experiment.

Discussion The pattern of bioaccumulation observed in this experiment concurs with several other studies showing the efficient uptake of Se in fish exposed to increasing dietary concentrations (Cleveland et al. 1993; Crane et al. 1992; Hamilton et al. 1990b; Hamilton

et al. 2002b; Hilton et al. 1980). The greater Se accumulati on in the liver observed in the highest treatment diet ( ca. 53 μ g/g dietary Se) is expected because this organ is very

active in Se metabolism and detoxification (Diplock 1976; Figure 1-1). High variation

was observed in liver Se levels for each of the three treatment groups ( ca. 20 – 53 μ g/g dietary Se). One possible explanation for this variation is that varying liver lipid content between individuals could have biased the qua ntification of Se (expr essed per gram total dried liver tissue). Sturgeon liver has been observed to contain high lipid content, especially in cultured fish (Doroshov 2004; Hung 2004; USACE 2003). Se is not typically found in lipids; a nd, the weight of this component may have biased the Se determination in high lipid samples (Cappon and Smith 1982; Lemly 1982). It is also possible that fish consuming greater amount s of the experimental Se diet would accumulate more Se in their livers compared to those consuming lesser amounts of the same diet. However, there was no relati onship between Se accumulation and condition factor in any treatment group.Se in all tissues of fish receiving low, medium and high experimental diets ( ca. 20

– 53 μ g/g dietary Se) reached levels that have been associated with toxi city in other fish species (Garrett and Inmann 1984; Hermanutz et al. 1992; Hilton et al. 1980; Hodson et

al. 1980; Sorensen and Bauer 1984). Lemly (20 02) proposed thresholds of Se toxic

effects for freshwater and anadromous fish at 8 μ g/g in muscle and 12 μ g/g in liver. Mean liver and muscle Se concentrations surp assed these thresholds in each of the three

treatment levels of the cu rrent study (Figure 1-1). Se-induced toxicity in teleosts has led to reduced juvenile growth, changes in condition factor, changes in organ to body wei ght ratios, tissue damage in gills, liver, kidney, blood, heart and gonads , and mortality (Cleveland et al. 1993; Crane et al. 1992;

Hamilton et al. 1990b; Hermanutz et al. 1992; Hilton et al. 1980; Lemly 1993a; Lemly 1993b; Lemly 1998; Sorensen 1988; Sorensen 1986; Sorensen et al. 1984; Woock et al. 1987). In the current study, we found no signi ficant differences be tween treatments in survival, specific growth rate, condition fact or, fork length, or body weight of juvenile white sturgeon, athough there was an apparent trend of decr ease in length, weight, and growth rate in the high Se treatment (Tab les 1-4 & 1-5). In a recent study, dietary exposure of 40 μ g/g seleno-L-methionine led to impaire d growth in fingerling-stage (0+) white sturgeon (25 – 30g) af ter two weeks (Tashjian et al. 2006). Our older juveniles (1+) may not be as susceptible to Se-induced growth effects and mortality. However, one study found reduced survival and growth in adult bluegills (Lepomis macrochirus) exposed to 10 and 30 μ g/L waterborne Se for the dur ation of one year (Hermanutz et al. 1992). The influence of age on growth impacts in Se-exposed fish remains unclear.Similarly, there were no si gnificant differences in hepatosomatic, gonadosomatic or weight to length regressi ons during the course of this study (ANCOVA, p 0.08 – 0.3; Table 1-4). These findings c ontradict previous work with Se in modern teleosts.

Sorensen et al. (1984) found increased liver weights in green sunfish ( Lepomis cyanellus ) collected from a high Se area compared to t hose collected from a reference area (based on wet weights). Significant effects on body size have also been documented with exposures to elevated Se. Hilton et al. (1980) observed food avoidance and feeding inefficiency (high feed: gain ratio) in rainbow trout ( Salmo gairdneri ) fed a diet containing 15 μ g/g Se as sodium selenite. The absence of mortality and gross morphological effects in the current study may be due to differences in phylogeny (Chondrostei vs. Teleostei lineages), age, Se species, exposure r oute and duration of

exposures. Histopathological analysis of treatment live r revealed significant cholestasis with compensatory changes in biliary epithelium, and expansion of melanomacrophage aggregates in all treatment exposures ( ca. 20, 36, & 53 μ g/g dietary Se). This indicates

that Se interferes with the removal of bile from the liver, which in turn causes the expansion of canaliculi, cholangioles (smallest bile ductules) and pr oliferation of ductules and ducts in the sturgeon liv er. Yellow pigment was seen in the cytoplasm of some hepatocytes, indicating cholestasi s. The disruption of bile flow (cholestasis or bile stasis) is a potentially fatal condition. Normal bile flow depends on a complex transport system. Bile acid synthesis a nd secretion are vital to several critical functions including the elimination of endogenous toxins, xenobiotics and their metabolites, and the assimilation of lipid soluble dietary nutr ients (e.g., vitamin A, K, E and triglycerides; Groothuis andMeijer 1996; Nathanson and Boyer 1991). Bile acids that are accumulated in liver during cholestasis damage mitochondria, generate oxi dative stress, and can lead to cell death (Palmeira and Rolo 2004; Rolo et al. 2004; Sokol et al. 1995). Cholestasis is infrequently reported with Se toxicity and is not typically reported for Se-exposed fish. However, this condition wa s associated with Se toxicity in birds and some mammals. Mallards ( Anas platyrhynchos ) fed a diet containing 32 μ g/g Se for 14 weeks incurred bile duct hyperplasia accompanied by elevated plasma alkaline phosphatase activity and lip id peroxidation (Hoffman et al. 1991). Mallards fed 60 μ g/g Se for 22 to 50 days exhibited bile duct hyperplasia and hepatic necrosis (O'Toole and Raisbeck 1997). Adult birds from Kesterson Reservoir, CA with mean liver Se

concentrations of 94.4 μ g/g (dry weight) showed bile duct hyperplasia and hepatic lesions (Ohlendorf et al. 1988). Buffalo calves ( Bubalus bubalis ) fed Se-enriched wheat straw (8.54 μ g/g Se) for 14 days also showed markedly increased alkaline phosphatase activity levels and enlarged gall bladders (Singh et al. 2002). Pigs exposed to 24.5 to 49 μ g/g Se via diet for up to 80 days showed bile depos ition and necrosis in the liver (Miller and Schoening 1938). While cholestasis is well recognized in r odents and mammals, this condition is only beginning to be under stood in fish species (Cai et al. 2001; Kirby et al. 1995; Lorent

et al. 2004). Cholestasis can resu lt from genetic defects, toxi ns, disease, inflammation and oxidative stress (Trauner et al. 2005). Reduced expression and function of bile transport proteins are important factors in the development of cholestasis (Elferink 2003;

Trauner et al. 1998). Toxins can effect the formati on and secretion of bile, presumably by affecting transport protein ex pression and function (Trauner et al. 1998). Schmitt andcolleagues (2000) demonstrated that oxidative stress could lead to cholestasis by reducing the number of functional transporters in the bile canalicular membrane. These authors concluded that severe glutathione depletion c ould ultimately lead to cholestasis. It is interesting to note that both cholestasis a nd Se toxicity are str ongly associated with oxidative damage. The metabolism of Se can lead to redox cycling and produce superoxides, resulting in oxidative stress and lipid peroxidation (Anundi et al. 1984;

Hoffman et al. 1991; Palace et al. 2004; Seko et al. 1989; Yan and Spallholz 1991). Se can also deplete glutathione, which is crucial in detoxifying the pero xides generated from redox cycling during Se metabolism, as well as other critical cellu lar functions (Hoffman

et al. 1989; Hoffman et al. 1991; Hoffman et al. 1988b; Hoffman 1977). The underlying

mechanisms of Se-induced cholestasis are currently unknown and deserve further investigation. Cholestasi s is explored in more detail in Appendix A. Melanomacrophage aggregates (MMAs) in liv er of treated fish increased in size and frequency with increasing dietary Se. MMA s have also been observed in livers of juvenile Sacramento splittail ( Pogonichthys macrolepidotus ) exposed to dietary Se (Teh

et al. 2004). Several other envir onmental contaminants have also been shown to cause MMAs in the liver and spleen of fish (Akaishi et al. 2004; Blazer et al. 1987; Vethaak et

al. 1996; Wolke et al. 1985). These aggregates are known to increase in number with age and stress, and are commonly found in mature fish (Agius 1985; Blazer et al. 1987; Bonga 1997; Kenedy-Stoskopf 1993; Vethaak and Wester 1996). Cons ideration of the age of experimental fish is necessary when using MMAs as an indication of physiological stress (Rice 2001; Wester et al. 1994). The evaluation of MMAs is appropriate in thisstudy since juveniles were used and all fi sh were exposed to identical holding and sampling stressors.

MMAs have a debated function but are generally suspected to be involved in the defense systems of fish (Bonga 1997; Rice 2001; Wester et al. 1994). Common pigments in these aggregates (lipofuscin, melanin, hemosiderin, and ceroid) are involved in defensive functions such as hemoglobin de gradation, storage of destroyed cellular material, antioxidation, lipid oxidation, and immune function (Rice 2001; Wester et al. 1994). The presence of melanin is particul arly significant in evaluating Se toxicity considering the importance of this pigment in combating oxidative stress. The finding of increased number and area of macrophage aggregations in ju venile sturgeon fed increasing levels of Se suggests that this element produces active oxygen intermediates and that melanin may be used to combat oxidative damage. Dietary Se did not appear to cause re nal toxicity, based on histopathological analysis. Controls and treated fish showed evidence of generation of new glomeruli and tubules. These results differ from prev ious studies in other fishes. Hicks et al. (1984) found kidney lesions in rainbow trout with li ver Se levels similar to those in white sturgeon from this study. Another study f ound significant tubular changes and signs of glomerulonephritis in kidneys of green sunfis h residing in Belews Lake, North Carolina, which is an area of high Se contamination (Sorensen et al. 1984). The green sunfish had liver Se levels similar to juvenile sturgeon in the highest treatment of the present study. In addition, striped bass ( Morone saxatilis ) fed Se-laden fish from Belews Lake, and accumulating muscle Se levels similar to those found in our lowest Se treatment, sustained proximal tubule damage of the ki dneys (Coughlan and Velte 1989). The lackof renal toxicity observed in the present study may relate to differences in phylogeny (Chondrostei vs. Teleostei lineages), Se species, exposure route and duration of

exposures. The occurrence of both fused secondary gill lamellae and inflammation of the gills was highest in the low and medium Se dietary treatments ( ca. 20 & 36 μ g/g dietary Se, respectively). These lesions indicate cellular damage (inflammation) and compromised respiration (fused lamellae) in exposed fish. Effects on the gill are a welldocumented effect in Se-exposed fish, esp ecially the swelling of secondary lamellae (Lemly 1993a; Sorensen et al. 1982a; Sorensen et al. 1984). This study provides further evidence that overexposure to Se leads to impaired morphology and function of gill lamellae. However, reasons for the decreased occurrence of gill effects in the highest treatment ( ca. 53 μ g/g dietary Se) are unclear. There is evidence for several potential mechan isms of Se toxicity in teleosts, birds

and mammals. The metabolism of Se can lead to oxidative damage, which can result in damaged tissues (Anundi et al. 1984; Seko et al. 1989; Yan and Spallholz 1991). Se can also alter natural detoxification system s by depleting glutathione (GSH) or the methylation co-factor S-adenosylmethi onine (Anderson and M oxon 1942; Hoffman and Heinz 1988; Hoffman et al. 2002; Hoffman 1977). Such alterations can leave the organism more vulnerable to both endogenous and exogenous toxicants. Se can also be incorporated into biological molecules in pla ce of sulfur because the two elements have similar chemical properties (Diplock 1976; Stadtman 1974). However, Se and sulfur bonds may have different strength and f unction in biological molecules (Reddy and Massaro 1983), thus some Se-substitu ted compounds may become unstable ordysfunctional (Lemly 1998; Stadtman 1974). In addition, Se can inhib it protein synthesis (Vernie et al. 1974) and react with sulfhydryl groups of proteins or other molecules (Martin 1973). Such cellular disruptions can also lead to tissue damage. Additional

research is needed to understand the mechan ism(s) of Se-induced toxicity in fishes. The present study is novel because it e xposed the actual species of ecological concern to the Se form (organic Se) and expos ure route (dietary) that is predominant in

the natural environment. Mu ch of the previous work on Se has utilized waterborne exposures (e.g., Gissel Nielsen and Gissel Nielsen 1978; Hodson et al. 1980; Lemly 1982). However, the diet is the most si gnificant route of expos ure leading to Se bioaccumulation and toxicity (e.g., Coyle et al. 1993; Hermanutz et al. 1992; Luoma et

al. 1992). In addition, the common use of inorga nic Se in both waterborne and dietary exposures underestimates environmental exposu re, uptake and toxicity of Se to aquatic organisms (Fan et al. 2002; Hoffman et al. 1989; Wang and Lovell 1997). This work incorporates the species of concern rather than extrapolating potential impacts from experimentation with surrogate species. This is rare in ecotoxicol ogy because it requires the ability to culture a speci es of ecological concern. Fo r some phylogenetically ancient fish, such as sturgeon, the use of surrogate species is not possible, or is unreliable, because of the significant anatomical, physio logical and behavioral differences with modern fish. White sturgeon cu lture can be difficult and has taken years of research to become widely successful (e.g., Doroshov 1985; Doroshov et al. 1997). Were there no aquaculture ponds with the ability to produce sturgeon of spawning ma turity, this study would not have been possible.This work shows that juvenile white sturgeon in San Fran cisco Bay-Delta are susceptible to toxicity at Se levels currently observed in their prey, and that environmentally feasible increases of Se in this food web leads to increased toxicity in this species. Each of the three dietary treatment levels has environmental relevance for white sturgeon in San Francisco Bay-Delta. The lowest Se ex perimental diet ( ca. 20 μ g/g Se) is similar to the highes t concentrations found in Potamocorbula amurensis , which is a common food source of San Francisco Bay-Delta white sturgeon (Linville et al. 2002; Luoma and Presser 2000; Stewart et al. 2004). The medium and high experimental diets ( ca. 36 & 53 μ g/g Se, respectively) fall within predicted Se concentrations in bivalves resulting from the proposed in crease of Se-laden agricultura l irrigation discharges into San Francisco Bay-Delta (Luoma and Presser 2000). Previous studies have shown that juve nile white sturgeon from San Francisco Bay-Delta accumulate high Se levels similar to those found in our experiment. Linares et

al. (2004) reported a mean liver Se of 9.75 ± 5.35 μ g/g in 36 sub-adult and adult (age 4 – 18) white sturgeon sampled in San Fr ancisco Bay between 2002 and 2004 (all measurements of variation reported as st andard deviation). Between 1986 and 1990, juvenile white sturgeon captured from this ar ea contained mean Se concentrations of 5.52

± 3.02 μ g/g in muscle and 17.01 ± 15.89 μ g/g in liver (n = 18; Urquhart and Regalado 1991), which is similar to the bioaccumulation levels observed in our experiment. It is clear from this data that juvenile white sturgeon in San Francisco Bay-Delta accumulates high, but variable, Se levels that may not induce short-term mortality but could potentially result in the adverse he alth effects observed in this study.The direct effects of Se on a white st urgeon population are difficult to detect because monitoring is typically accomplished by tracking sub-adults and adults, which are generally at least 9 years of age (S chaffter and Kohlhorst 1999). Thus, poor recruitment is usually not detected until a decade or more after the hatching of a particular year-class. White sturgeon populat ions in San Francisco Bay-Delta have been highly variable over the past five decades (Kohlhorst 1980; Kohlhorst et al. 1991; Schaffter and Kohlhorst 1999). Schaffter and Kohlhorst (1 999) reported a paucity of smaller sturgeon caught in the late 1990’s. Population surveys in 2005 indicated that the San Francisco Bay-Delta white sturgeon population was at a 50-year low, with approximately 10,000 adult sturgeon (Califor nia Department of Fish and Game Commission 2006). It is impor tant to note that this se vere population decrease has occurred approximately a decade after P. amurensis significantly increased the availability of Se in San Francisco Bay (Linville et al. 2002; Luoma and Presser 2000). If the increased availability of Se in the early 1990’s resulted in smaller sturgeon yearclasses, we would expect to observe ev idence of that impact around this time. In conclusion, this work shows that j uvenile white sturgeon experience significant toxicity upon exposure to elevated dietary Se. In San Francisco Bay- Delta, juveniles are susceptible to toxicity at Se levels currently observed in their prey. Relatively small

increases of Se in this food web will lead to increased toxicity to this species. Melanomacrophage aggregates in liver increased in size and number in a dose-response

manner between ca. 20 to 53 μ g/g dietary Se exposures. Chol estasis, a potentially fatal condition, was present in all tr eatment groups. Exposure to ca. 20 and 36 μ g/g dietary Se resulted in significant inflammation and fuse d lamellae in gills, however these effectsoccurred less frequently with exposure to ca. 53 μ g/g dietary Se. T oxicological studies typically utilize a much wider exposure ra nge, with doses differing by orders of magnitude. The narrow exposure range us ed here provides greater ecological significance; however, the lack of large differences between e xposures is not surprising.

Field studies have shown that Se is elevated in several branches of the San Francisco Bay-Delta food web, and that white sturgeon in this region have high tissue Se burdens, similar to that in our experiment. The presen t study indicates that the impacts of Se in this system may be a factor affecting the abundance of white stur geon in San Francisco Bay. Careful management of all processes wi th potential to increase food web Se in San Francisco Bay-Delta is essent ial to sturgeon preservation.Se content of experimental diets

Diet Target Se Content

(μg/g) Measured Total Se (μg/g) Control 1 1.04 ± 0.07 Low 15 20.05 ± 1.10 Medium 30 35.61 ± 3.83 High 45 52.54 ± 2.32

Table 1-1. Se averages ± standard deviation are shown on a dry weight basis (n = 3). Se was added to the diet as selenized yeast (Selenomax®, Ambi Inc.).Composition of experimental diet

Ingredient: % of diet Vitamin Free Casein 31 Wheat gluten 15 Egg White 4 Corn Dextrin 27 Cod Liver Oil 4 Corn Oil 4 Lard 4 Vitamin Premix 1 (w/ cellulose & santoguin) 1 Mineral Premix 2 3 Cellulose 2.5 Choline Chloride 0.5 Yeast Mix 3 4

Table 1-2. Experimental diet composition for juve nile white sturgeon. Increasing levels of selenized yeast were used in the treatment diets. Table 1-1 shows the Se level in each diet.

1 Supplied per 1 Kg diet: 2.69 g Alphacel no n-nutritive bulk; 1.47 g Ascorbic acid; 1.47 mg Biotin; 4.91 g Cellulose; 147.2 mg D Calcium pantothenate; 5.3 mg Folic acid 147.2 mg Inositol; 58.88 mg Menadione; 147.2 mg Niacin; 29.44 mg Pyridoxine HCL; 88.32 mg Riboflavin; 186.46 mg Santoguin; 14.72 mg Thiamine Mononitrate; 2.94 mg Vitamin A acetate (500,000 U/g); 58.88 ug Vita min B-12 crystalline; 98.14 mg Vitamin D3 (400,000 U/g).

2 Mineral premix was purchase from ICN and the com position was given by Bernhart and Tomarelli (1966).

3 Yeast mixture contained selenized yeast and Torula yeast.Se in juvenile white sturgeon aftersix months of dietary Se exposuresData displayed as median, 25th- 75thpercentiles and full rangeMuscle (Se μg/g)Blood (Se μg/g)Liver (Se μg/g)Se dietaryexposures(μg/g)Median25 - 75%RangeMedian25 - 75%RangeMedian25 - 75%Range1.04 ± 0.072.061.61 - 2.491.36 - 2.764.33.85 - 5.012.14 - 6.902.852.14 - 4.491.31 - 7.3520.05 ± 1.1021.5617.40 - 25.9913.18 - 32.4024.0422.23 - 25.8618.75 - 33.0519.4914.20 - 25.927.54 - 53.6535.61 ± 3.8339.7334.55 - 43.5821.28 - 47.5440.9938.19 - 46.2531.01 - 59.2040.231.48 - 45.3217.10 - 90.2452.54 ± 2.3252.9746.67 - 59.2032.06 - 65.5955.3351.33 - 63.1138.52 - 77.9069.8460.29 - 98.3947.02 - 281.64Table 1-3.Sturgeon data displayed as median, 25th- 75thpercentiles, and range of Se (μg/g,dw) levels in liver, blood and muscleafter six months of dietary Se exposures.Dietary Se exposure displayed as mean ± standard deviation Se (μg/g, dw). Data displayedin Figure 1-1.Comparisons of gross morphological characteristics of juvenile white sturgeonfollowing six months of dietary Se exposuresR2Fp-valueModelCondition Factor0.151.740.08ANOVAFork Length0.091.060.40ANOVABody Weight0.101.130.35ANOVASpecific Growth Rate0.361.490.29ANOVAGonadosomatic regression0.592.280.08ANCOVAHepatosomatic regression0.581.950.13ANCOVAWeight to length regression0.841.240.30ANCOVATable 1-4.Parameters from ANOVA and ANCOVA analyses ofgross morphological characteristics betweenjuvenile white sturgeon exposed toca.1, 20, 36, and 53 μg/g dietary Se forsix months. No differences werefound for any of the measured characteristics.Gross morphological measurements ofjuvenile white sturgeon following six months of dietary Se exposuresDietary Se (μg/g)Condition FactorFork Length (cm)Body Weight (kg)Specific Growth Rate1.04 ± 0.077.43x 10-3± 1.7x 10-4(3)60.3 ± 0.86 (3)1.65 ± 0.097 (3)0.58 ± 0.032 (3)20.05 ± 1.107.73x 10-3± 1.7x 10-4(3)59 ± 0.39 (3)1.62 ± 0.051 (3)0.57 ± 0.02 (3)35.61 ± 3.837.96x 10-3± 7.4x 10-5(3)59.5 ± 0.32 (3)1.69 ± 0.039 (3)0.57 ± 0.016 (3)52.54 ± 2.327.86x 10-3± 2.8x 10-4(3)56.4 ± 0.66 (3)1.45 ± 0.103 (3)0.51 ± 0.035 (3)Table 1-5.Measurements of weight, length, condition and growth in juvenile white sturgeon for each dietary exposure level.All data is presented as mean ± standarderror (number of tank replicates). No statistical differences were found betweendietary exposure levels (Table 1-4).Se in juvenile white sturgeon follow ing six months of dietary exposures

Figure 1-1. Se levels in liver, blood and muscle af ter six months of dietary Se exposures. For each tissue type, Se increased with in creasing dietary Se e xposure (non-parametric Rank F-Test, p = 0.03, adjusted multiple comparisons, α = 0.05). In general, muscle accumulated less Se than either liver or blood. Standard box and whiskers plots are used to describe these non-parametric data. The horizontal mid-line represents the median while the upper and lower horiz ontal lines represent the 25 th and 75 th percentiles, respectively. The whiskers show the 10 th and 90 th percentiles. Values labeled with the same letters are not statistically different (non-parametric Rank F-Test, p = 0.03; adjusted multiple comparisons, α = 0.05).Figure 1-2. Section of liver from control group of juvenile sturgeon. Portal venule is at center of field and is associated with a small bile duct (to left of venule). Hepatocyte polarity is shown by localization of nucleus near basal (sinus oidal) plasma membrane in base of cell (horizon tal arrows) and apex directed toward center of hepatic tubule (vertical arrows). Vacuolar profiles (V) in hepatocytes represent sites of lipid while glycogen stores (G) stain more intensely and show no vacuolation. Some hepatocyte profiles do not show a nucleus due to the large difference between cytoplasmic and nuclear volumes.Figure 1-3. Additional micrograph from section of control liver shown in Figure 1-2. Conducting portal vein is shown in center of field and is closely associated with small bile duct (B) and hepatic arteriole (A). Mononuclear leukocytes form cuff around vein. Glycogen storage areas (G) and areas of lipid vacuolation (V) are shown in hepatocytes.Figure 1-4. Section of liver from control group of juvenile sturgeon showing bile retention (arrows) and focus of small bile duct pro liferation. This condition was seen in a single individual. Note absence of eviden ce for biliary stasis involving canalicular network.Figure 1-5. Section of liver from control group of juvenile sturgeon. Eosinophilic granular leukocytes (arrows) are associated with portal venule a nd small bile duct.Figure 1-6. Section of liver from control group of juvenile sturgeon. Comparison of this micrograph with figures 1-2 and 1-3 illustrate rang e of features in livers of control group. A single focus of inflammation involving monon uclear leukocytes is shown in center of field. To left of field, melanomacrophage aggreg ate is larger than those of Figure 1-3 and is associated with inflammation around a small bile duct.Figure 1-7. Section of liver from control group of juvenile sturgeon showing most extreme case of peribiliary inflammation (mononuclear leukocytes). This type infiltration was consistently seen near small bile ducts and often where these were closely associated with arterioles.Figure 1-8. This individual was fed 20.05 ± 1.10 μ g/g Se in diet for 6 months. Red circle in center of tr ansversely sectioned he patic tubule is a canaliculus (small arrow). Longitudinal arrays of canalicular network are shown (CN). These features were not visible in juveniles of contro l group (Figures 1-2 – 1-7).Figure 1-9. This individual was fed 20.05 ± 1.10 μ g/g Se in diet for 6 months. High amplitude swelling of hepatocytes (large whit e cellular arrays) in tubule give rise to tubuloform swelling that was seen at 30% in cidence in this group. This condition was seen in only one of 30 control juvenile st urgeon. Two normal appearing hepatic tubules in transverse section are show n (arrows). Inflammatory cells at center of field surround 8 profiles of small bile ducts (bile duct prol iferation).Figure 1-10. This is an additional view of the i ndividual in Figure 1-8. Continuation of canaliculi into areas of transitional biliary epithelial cells and cholangioles is shown (large arrows). Longitudinal array of bile ductule shows apical margins of cells with staining identical to that of canaliculi. In addition, a site of transitional biliary epithelial cell proliferation is seen (TB).Figure 1-11. This individual was fed 20.05 ± 1.10 μ g/g Se in diet for 6 months. Focus of bile duct proliferation is shown with infl ammatory cells just beneath hepatic capsule.Figure 1-12. This individual was fed 35.61 ± 3.83 μ g/g Se in diet for 6 months. This micrograph shows morphology of portal venul e and adjacent hepatic parenchyma. Hepatocytes show signs of necrosis seen as nuclear lysis (Ly). In addition, cells are swollen and contain discreet particulate mate rial in cytoplasm (arrows). Canalicular network is distended (CN). In addition there are four profiles of tr ansected small bile ducts around the venule.Figure 1-13. This individual was fed 35.61 ± 3.83 μ g/g Se in diet for 6 months. Extensive perivenous (venular lumen is at ri ght of field) and pe ribiliary inflammation involving predominantly mononuclear leukocyte s with occasional eosinophilic granular leukocytes. Hepatocytes appear swollen and a single, bright-red Councilman body is seen in perisinusoidal macrophage at top of field. Biliary ep ithelial cell proliferation is seen (arrow) with cells extending from perivasc ular site into adjacent parenchyma. Also, note the longitudinal arrays of biliary ductu les with red staining in apical plasma membrane region.Figure 1-14. This individual was fed 52.54 ± 2.32 μ g/g Se in diet for 6 months. This field is from liver of individual show ing maximum response of melanomacrophage aggregates. The number of hepatocyte nuclei per area of parenchyma is greatly enhanced and the cytoplasm stains with more intensit y. This indicates that hepatocytes have become smaller in volume. Under this le vel of magnification, a mosaic pattern is evident. Vacuolated hepatocytes occur in zones that are free of melanomacrophage aggregates while hepatocytes, adjacent to macrophage aggregates, are free of vacuolation, smaller (nuclei more abundant), and stain more intensely.Figure 1-15. This higher magnification view of ar ea near that of Figure 1-14 shows features in greater detail. Note vacuolation of hepatocytes in upper half of field. This is consistent with lipid change. Non-vacuolated hepatocytes (lower portion of field) are smaller; and, profiles of hepatic tubules (T ) are more obvious. He patic tubules reveal canalicular network (see red lines in cente r of tubules). Melanomacrophage aggregates of various sizes are prominent. Councilman bodies are seen in four cells in bottom half of field.Effects observed in gill of juvenile white sturgeon followi ng six months of dietary Se exposure

Figure 1-16. Contingency table showing the oc currences of inflammation and fused lamellae in gills of juvenile white sturgeon exposed to dietary Se for six months. The occurrence of inflammation and fused gill is significantly different across treatment levels (p = 0.041 and 0.024, respectively). Th e low and medium tr eatments contribute the most to this difference.Chapter 2. Dose-dependent maternal transp ort of selenium via vitellogenin in white sturgeon

Introduction Selenium (Se) is a well-documented deve lopmental toxicant to fish and wildlife species (Coyle et al. 1993; Gillespie and Baumann 1986; Hamilton 2004; Hamilton et al. 1986; Hermanutz 1992; Hoffman et al. 1988b; Lemly 1993b; Ohlendorf et al. 1986). In small quantities (0.1 – 1.25 μ g/g diet) Se is an essential nutrient for animals, humans and many micro-organisms (Diplock et al. 1976; Gatlin and Wilson 1984b; Hilton et al. 1980; Mayland 1994). However in slightly greater quantities (as little as 13 μ g/g Se in diet) this element can impair fish reproduction and development (Woock et al. 1987). Excess Se has led to severe teratogenesis, mortality and endangerment or extirpation of fish species residing in areas receiving wast ewaters from the combustion of fossil fuels (Garrett and Inmann 1984; Lemly 1985) or dr ainage from seleniferous land (Hamilton 1999; Skorupa 1998). Higher trophic level species are most thr eatened by Se in aquatic ecosystems due

to the efficient food web transfer and biom agnification of this element (Coughlan and Velte 1989; Lemly 1985; Luoma et al. 1992). The predominant pathway of Se transfer in the aquatic food web is through the assimila tion of dissolved Se by phytoplankton, which biotransform inorganic Se into organic Se (Hu et al. 1997; Luoma et al. 1992; Vandermeulen and Foda 1988; Wrench 1978) . Organic Se is accumulated from phytoplankton by filter feeding bivalves (Luoma et al. 1992) and is consequently transferred to secondary consumers, such as fish foraging on bivalves. Several studieshave shown the efficient bioaccumulation of Se in fish exposed to a range of concentrations (Cleveland et al. 1993; Crane et al. 1992; Hamilton et al. 1990b; Hamilton

et al. 2002b; Hilton et al. 1980). Bioaccumulated Se in maturing fish can be passed to the egg yolk, causing teratogenesis of developing embryos a nd complete reproductive failure (Coyle et al. 1993; Gillespie and Baumann 1986; Lemly 1993 b; Schultz and Hermanutz 1990). The maternal transport of contaminants to offspr ing is common in aquatic species. The yolk protein precursor vitellogenin (VTG), synthesi zed in the liver, has b een shown to be the vehicle of maternal transpor t for several pollutants (Fal chuk and Montorzi 2001; Ghosh and Thomas 1995; Monteverdi and Di Giulio 2000). A previous study on white sturgeon has indicated that VTG may also transport Se to the egg yolk (Kroll and Doroshov 1991). Se has chemical properties similar to sulfur and can substitute for sulfur in biological molecules (Diplock 1976; Stadtman 1974). In the case of VTG, Se may be substituted in the sulfur amino acids, methionine, cysteine, and cystine present in the backbone of the VTG molecule (Bidwell and Carlso n 1995; Kroll and Doroshov 1991). Estrogen induces synthesis of VTG in liver , after which VTG is transported by the circulatory system to the growing oocytes (eggs) where it is selectively taken in by receptor-mediated endocytosis (Mommsen a nd Walsh 1988). In the egg cytoplasm the VTG is broken up and accumulated as egg yolk proteins, phosvitin and lipovitellin,

which are stored as nutrients for the embr yo. Kroll and Doroshov (1991) measured Se levels in plasma (5 – 9 μ g/g), VTG (3 – 4 μ g/g), ovulated eggs (3 – 29 μ g/g), phosvitin (3

– 90 μ g/g) and lipovitellin (3 – 11 μ g/g) from six wild-caught white sturgeon females from San Francisco Bay-Delta (dry weight). These elevated, a lthough variable, levelssuggest that VTG is a potential vehicle for the transport of Se to the egg yolk of this valuable endemic species of sturgeon. White sturgeon are especially vulnerable to Se bioaccumulation and maternal transport due to benthic feeding habits, a long life span, and a prolonged (two year) period of vitellogenesis and yol k deposition (Doroshov 1985; Doroshov et al. 1997). White sturgeon is common in San Franci sco Bay-Delta, an area with known Se contamination. San Francisco Bay-Delta recei ves Se through both the refining of fossil fuels and irrigation drainage from the seleni ferous San Joaquin Valley. White sturgeon in this area are exposed to high levels of Se through their diet. This is evidenced by high tissue Se levels in common prey of white sturgeon (Linville et al. 2002; Stewart et al. 2004; White et al. 1988), as well as in sturgeon mu scle, liver and eggs (Kroll and Doroshov 1991; Stewart et al. 2004; Urquhart and Regalado 1991; White et al. 1989). The objectives of this experimental st udy were to verify the mechanism of maternal transport of Se in sturgeon, and to evaluate the bioaccumulation, maternal transport, and reproductive success in white sturgeon exposed to elevated dietary Se. Two groups of eight cultured white sturge on females in the advanced stage of vitellogenesis were fed diets with normal ( ca. 1.42 μ g/g) or elevated ( ca. 34.04 μ g/g) Se (as selenized yeast) for approximately 6 mont hs. Selenized yeast, predominately in the form of selenomethionine (Polatajko et al. 2005), was used as a surrogate for organic Se present in the aquatic environment. Organi c forms of Se, particularly selenomethionine, have been shown to have greater bioavailabil ity and toxicity than inorganic forms and are often the species to which aquati c organisms are exposed (Hamilton et al. 1990b;

Hoffman et al. 1989; Luoma et al. 1992; Reinfelder et al. 1997; Wang and Lovell 1997).Upon acquisition of maturation competence, the fish were induced to spawn and ovulated eggs were fertilized with sper m from non-treated males. Fecundity, fertilization, and neurulation rates were m easured as indicators of reproductive success. Development of embryos and yolk sac larvae was monitored as part of a separate experiment described in Chapter 3 of this di ssertation. Adult females were necropsied for Se analysis. We hypothesized that VTG is the vehicle of maternal tr ansport in fish, as indicated by Kroll and Doroshov (1991; Figure 2-1). We also hypothesized that exposure to elevated dietary Se leads to signifi cant bioaccumulation, maternal transport and reproductive impairment in white sturgeon.

Materials and Methods Source of Animals Sixteen virgin females in mid-vitellogeni c stage of oogenesis were obtained from The Fishery, Inc. (Galt, California) in Se ptember 2001. These sturgeon were from a 1995 year-class and had been reared from birth in circular tanks (with increasing diameter to accommodate growth) with flow-through well water (20 – 21 oC). During the previous year and a half, they had been cultured in 12.2 m diameter concrete tanks, at densities of 60 to 80 kg/m 3 , and fed a commercial high-energy sa lmonid diet (45% pr otein/25% lipid) at approximately 0.25 % body weight/day. The sturgeon were transported to the Pu tah Creek Hatchery Facility (PCHF) at University of California, Davis (UCD) on September 26, 2001 (1-hour travel time). Upon arrival, each fish was anesthetized in 50 mg/L MS-222 until a stage 4 to 5

anesthesia was achieved. A small incision was made in the abdomen to allow insertion ofa catheter for the collection of eggs, whic h were placed into Ringers solution for measurement of egg diameter and weight . Blood was collected from the caudal vasculature, using a 10 ml he parinized Vacutainer®, and we ight and fork length were measured. Each female was PIT tagged in the caudal peduncle re gion under a lateral scute and externally marked with a floy tag. The fish we re randomly assigned to two 6.1 m diameter tanks, with eight in each tank.

Experimental Design Domestic broodstock white sturgeon fema les (mean weight and fork length: 22.71 Kg and 134.59 cm) were held in two 6.1 m diameter outdoor flow-through tanks at PCHF. The experiment consisted of tw o dietary treatments: control (1.42 ± 0.03 μ g/g Se) and treatment (34.04 ± 0.82 μ g/g Se; Table 2-1). Each tank held 8 females and received one of the two experimental diets for a pproximately 6 months. Upon acquisition of maturation competence, the fish were induced to spawn and ovulated e ggs were fertilized with sperm from non-treated males. Fec undity, fertilization and neurulation were measured as indicators of reproductive success. Females were necropsied for Se analysis following spawning or the failure to ovulate.

Initiation and maintenance The sturgeon were maintained on well water (18-19 oC) for approximately 2 months following their arrival at UCD and were slowly transitioned to 100% surface irrigation water in December, 2001, in order to prevent ovarian follicular atresia (LinaresCasenave et al. 2002). The irrigation water source t ypically cools down to 11 to 12 oC inDec to Jan and gradually warms to 15 to 16 oC in April-May. Water flows were approximately 150 liters per minute, and di ssolved oxygen was maintained at > 80% air saturation. Irrigation wate r had a pH of 7.5 to 7.8, hardness of 220 mg/L CaCO 3 and alkalinity of 180 mg/L as CaCO 3 . The fish were acclimated on commercial salmonid diet (Nelson and Sons, Inc., Utah) for approximately one month after their arrival at UCD and were fully transitioned to experimental diet (0.3% body wt/day) by November 17, 2001.

The two 6.1 m diameter tanks were cleaned weekly, with minimum disturbance to the

fish.

Experimental Diet

Two diets were made w ith essential (control; ca. 1 μ g/g) and elevated (treatment;

ca. 34 μ g/g) levels of Se (Table 2-1). Commercial salmonid diet (Nelson and Sons, Inc., Utah) was obtained as mash and pelleted with fish oil (10%) and selenized yeast (2.2%; Selenomax®, Ambi Inc.). Water was added pr ior to pelleting at 14% of the total diet. For the control diet, the selenized yeast mi xture contained 1.3% selenized yeast and 98.7% tortula yeast. Only selenized yeast was added to the treat ment diet. After pelleting, the diet was allowed to air dry on drying racks.

Sampling On February 12, 2002 each female was biopsie d for egg size and maturation. Fish were anesthetized in 50 mg/L MS-222 until a stage 4 to 5 anesthesia was achieved. A

small incision was made in the abdomen to allow insertion of a catheter for the collection of eggs, which were placed into Ringers solution for analyses of egg diameter,polarization index and response to progester one exposure (as desc ribed below). Blood was collected from the caudal vasculature, using a 10 ml heparinized Vacutainer®, and centrifuged (4000 x g, 10 min) to separate the plasma, which was flash frozen in liquid nitrogen for protein isolation and Se analysis. Fish weight and fork length were also measured. Females were sampled repeated ly (approx every 4 weeks) until it was determined that a female was ready for spawning.

Egg Diameter, Polarization Index and Maturation Assay Representative subsamples of vitellogenic eggs (n=15 from each fish) were collected by catheterization a nd the egg diameters were measured using a darkfield dissecting microscope with camera luci da, an image analyzing tablet, and a microcomputer interface (± 0.01 mm). The read iness of a specific female to spawn was determined by examining additional subsampl es of collected eggs to calculate the polarization index (PI), and measure the capac ity to undergo germinal vesicle breakdown (GVBD) in the presence of an in-vitro maturation-inducing steroid, progesterone (Dettlaff et al. 1993; Mims et al. 2002). Eggs were extr acted by a catheter (Conte et al. 1988) and kept in a sturge on Ringer solution (Dettlaff et al. 1993) with penicillin (0.03 g/L) and streptomycin (0.05 g/L). The incision for inserting the cat heter is small (6-9 mm) and is closed using a single suture. Females are returned to the broodstock tank until spawning or further sampling. Domestic white sturgeon females have been sampled in this manner 3 to 5 times, every 4 to 6 w eeks, and have been successfully induced to ovulate, producing eggs with high fertility and hatch rates (Van Eenennaam et al. 2004).Subsamples of 15 eggs per female were used for PI dete rmination. Eggs were boiled for 5 to 6 minutes, chilled on wet ice for 20 minutes, stored in 10% buffered formalin overnight, and bisected along the animal -vegetal axis using a thin, double-edged razor blade. The PI was calculated by meas uring the distance of the germinal vesicle from the inner border of the egg chorion at the animal pole, and dividing this by the animal-vegetal egg di ameter. These measurements were made using a darkfield dissecting microscope w ith camera lucida, an image-analyzing tablet, and a microcomputer interface (± 0.01 mm). The PI alone provides a very good i ndication of female readiness to spawn, but it does not directly measure the capacity of the egg to mature. Thus, the catheterized eggs are also analyzed for their capacity to undergo GVBD in the presence of a maturation-inducing steroid. To determine the capacity of the egg to undergo GVBD, the in vitro egg maturation assay, as described in Dettlaff et

al. (1993), was conducted in sturgeon Ringe r solution at 16 °C for 16 hours. Fifteen eggs were incubated with 5 μg /mL progesterone and 15 were incubated without progesterone to serve as a contro l. Each treatment had two replicates. After incubation, the eggs are boiled, chill ed on wet ice, stored in 10% buffered formalin, bisected and examined for GVBD. The criteria used to select females for induced ovulation were an egg PI of less than 0.10 and 100% GVBD response in the egg maturation assay.Spawning Induction, Egg Fertilization and Embryo Incubation The spawning, fertilization and in cubation protocol followed Conte et al. (1988),

with few modifications. The male s received a single injection of 10 μ g/kg body weight of mammalian GnRHa [D-Ala 6 , Des-Gly 10 ]–LH-RH Ethylamide (Peninsula Laboratories), and the females received a priming injection of 2 μ g/kg GnRHa, followed

in 12 h with resolv ing injection of 18 μ g/kg GnRHa. All injections were intramuscular and given underwater to minimize handling stre ss. Females were held in separate 3.7 m

diameter tanks. Tank bottoms were routin ely checked for released eggs beginning 16 h after the second injection. The ovulating fe male was placed onto a hooded stretcher and ovulated eggs were removed surgically (Conte et al. 1988). Females were anesthetized during surgery using MS-222 solution (100 mg /L), which was pumped across the gills using a submersible pump with a 5 cm diameter vinyl tube and a 100 L ice chest as a

sump.

Two to three males were induced to spermiate at each spawning. Milt was collected from each male with a 60 ml plas tic syringe and a 4 cm long vinyl catheter inserted into the urogenital pore (Conte et al. 1988). Sperm was evaluated for percent initial motility and the time to less th an 10% motility under a compound microscope, using 5 μL of semen diluted immediately with 200 μL of hatchery water. Milt was only used if sperm had over 50% initial motility and duration to 10% motility was at least one minute and thirty seconds. Eggs were ferti lized with 20 ml of pooled milt from 2 to 3 males, diluted 1:200 with hatchery water. Af ter 3 min of fertilization, eggs were gently mixed in a water suspension of Fuller’s Ea rth (100-200 mesh size, Sigma Chemical Co.) for 60 min to prevent egg adhesion. The numbe r of ova collected from each female wasestimated volumetrically (egg count in five 5 ml subsamples). Approximately 1 Liter of fertilized eggs was placed into individual M acDonald hatching jars; and, jars were set-up

to allow hatched larvae from individual fema les to flow into separate 1.2 m diameter circular tanks. Embryo incubation wa s at water temperatures 15 to 16 oC.

Final Necropsy Sampling Females were euthanized and necropsie d following the extraction of ovulated eggs, or when determined to be unable to ovulate (Dettlaff et al. 1993). Euthanasia was performed using an overdose of MS 222 and se vering the spinal cord. Samples of liver, kidney, muscle, spleen, eggs a nd post-ovulatory gonads were fr ozen on dry ice and stored at -80 oC for Se analysis. Prior to euth anasia, blood was collected from the caudal vasculature using a 10 ml heparinized Vacuta iner® and centrifuged (4 000 x g, 10 min) to separate the plasma, which was flash frozen in liquid nitrogen and stored at -80 oC for protein isolation and Se anal ysis. Eggs collected for e gg yolk purification were flash frozen in liquid nitrogen a nd stored at -80 oC. Devel oping embryos were sampled at stage 5 for fertilization success and stage 21 for neurulation success, as described in

Dettlaff et al. (1993).

Vitellogenin Purification and Identification

Plasma Plasma vitellogenin (VTG) was purif ied from estrogenized male white sturgeon plasma using anion exchange chromatogra phy. To verify the identity of estrogeninduced VTG, the chromatographs of the male were compared before and after treatment(Figure 2-2). Purification of VTG was acco mplished as follows: 200 μL of plasma was diluted 1:10 into the running buffer (20 mM bis-tris-propane, 50 mM NaCl, pH 9.0) and then loaded onto the column containing the strong anion exchanger, 20 HQ, and separated using the BIOCAD Perfusion System TM (Perseptive Biosystems). The flow

rate was maintained at 10 mL/min and 1.0 ml fractions were collected. The proteins

absorbance and salt content of the eluent were measured at 280 nm and conductivity in μsiemens. Non-binding protei ns were eluted by washing with 5 column volumes of running buffer (peak P1, Figure 2-3). Sequen tial removal of binding proteins (P2-P4) was accomplished by a linear gradient of salt (50-800 mM). VTG binds the strongest of the plasma proteins at pH 9.0 and is the last protein to elute from the column (Figure 2- 3). Plasma proteins other than VTG, indicat ed as P1-P4 (Figure 2-3), were pooled and indicated as “nonVTG” proteins in PAGE gels and Westerns. After pooling the fractions (VTG or nonVTG peaks), the pH wa s adjusted to 7.0 using 100 mM bis-trispropane (pH 6.0), sodium azide (0.02% v/ v), and protease inhi bitor-Aprotinin (10 KIU/ml) was added. A 1.0 ml aliquot was co llected from each pool, the cryoprotectant - glycerol added (50% v/v) and stored at –20 oC. Freeze/thaw fracture of VTG and yolk proteins is a major contributor to degradation of yolk protei ns and strongly affects ELISA results (personal observation by Kroll/Denslow, Hybridoma lab, University of Florida). Freeze/thaw fracture of yolk proteins is avoide d by the addition of glycerol and storage at –20 oC. At this temperature the solution remain s a liquid. This aliquot would be used for PAGE and Western analysis. The total prot ein content of each pool was determined by Bradford analysis (Coomassie Plus TM , Pierce) using bovine seru m albumin as a standard.VTG levels in the plasma were relatively low because the females used for this study were in late stages of vitellogenesis and were developing pol arized eggs (Doroshov

et al. 1997). Pooling several ion exchange runs was necessary to acquire enough VTG for analysis. In some cases further concen tration using a 10,000 Da size-exclusion spin column (Microcon, Millipore) was necessary for VTG-PAGE analysis.

Plasma PAGE In order to ascertain the purity and identi ty of the purified VTG, the plasma and purified protein pools were separated by elec trophoresis on polyacrylamide gels (PAGE)

and then transblotted to a membrane for We stern analysis. A 4 to 12% gradient gel (NuPAGE TM , Invitrogen) was used to capture all the white sturgeon plasma proteins. The samples run on a gel consisted of the following: 1) male negative control, 2) purified VTG positive control (VTG+, 1.0 μg/lane), 3) th e plasma sample before purification, and 4) the pooled fractions after ion exchange purification (nonV TG proteins pool and VTG). Plasma was diluted 1:50, purified VTG and nonVTG pool was diluted to 0.05 μg/μL in a mixture of Laemmli sample buffer containing dithiothreitol as a reducing agent. The samples were heated to 95 oC for 10 min and then loaded onto the gel. Ten microliters of each sample and a mixture of colored molecular weight markers (See Blue 2 TM , Invitrogen) were added to each lane. The amount 0.5 μg of each purified protein, VTG+, non-VTG and VTG proteins, were added to separa te lanes. The gel was run for 35 min at 200 V. The gel was soaked for 2 to 4 hours in colloidal blue TM stain (Invitrogen) and destained using water. Each gel was sca nned and labeled using the Adobe program. PAGE gels of pooled nonVTG plasma protei ns showed bands at approximately 70, 62and 28 kDa (Figure 2-4). The VTG proteins we re identified using Western analysis, as

described below.

Plasma Western A gel identical to the PAGE gel was used for Western analysis. After running the PAGE gel, the gel was removed and tran sblotted to PVDF membrane (Invitrolon TM , Invitrogen) for 3 hrs at 100 V (4 oC) in buffer (10 mM MES, 10% MeOH, 0.01 % SDS, pH 6). The blot was blocked in blotto, which consisted of 5% instant milk in TBSTZ (20 mM tris, 150 mM NaCl, 0.05% Tween-20, 0.02% azide, pH 7.6) for 2 hrs. A highly specific monoclonal antibody (Mab), 1H 12 (HL 1387) against gulf sturgeon ( Acipenser oncorynchus destoi ) VTG developed by the University of Florida Hybridoma lab (Kroll/ Denslow) was used to identify VTG on the blot s. After blocking the membrane, the blot was incubated overnight in 0.1 ng/ml of the Mab at 4 oC, washed twice with TBSTZ and then incubated for 2 hours in the secondary antibody, anti-mouse IgG (H&L)- alkaline phosphatase diluted 1:1000 in blotto. The blot was rewashed twice, and the color developed by adding the enzyme’s substrate (1-Step TM , Invitrogen). Development was stopped by washing with water. Each blot was dried, scanned, and labeled. The anti-sturgeon VTG Mab is very se nsitive and clearly recognizes white sturgeon purified positive cont rol, gravid female plasma, and purified gravid female plasma VTG (Figure 2-4). It does not reac t with pooled nonVTG plasma proteins. The Mab recognizes 3 different bands of white sturgeon VTG. The VTG proteins are high molecular weight: 180, 75, and 64 kDa (Figure 2-4).Vitellogenin ELISA

Plasma VTG concentrations were dete rmined by sandwich ELISA using a white sturgeon polyclonal antibody, UF131, and gulf sturgeon monoclonal antibody, 1H2, developed by the Doroshov (UCD) and Denslo w (UF) labs, respectively. The ELISA assay was run as follows. A 96-well ELISA plate (Nunc TM , Nalge) was precoated overnight at 4 oC with 5 μg /mL of the Mab (1H2, HL 1387) in PBSZ (10 mM phosphate, 150 mM NaCl, 0.02% azide, pH 7.2). The pl ate was washed four times with PBSTZ (PBSZ with 0.05% tween) using a plate washer (Skan washer, Molecular Devices). The plate was blocked with 1% BSA in TBS TZ (20 mM tris, 150 mM NaCl, 0.05% tween-20, 0.02% azide, pH 7.4) for 2 hours at room temp erature. The plate was re-washed with PBSZ before adding the samples and standards. Unknown samples and controls (positiv e and negative) were diluted 1:100, 1:10,000, 1:100,000, and 1:1,000,000 in PBSZ with th e protease inhibitor- Aprotinin (10 KIU/ml) and loaded onto the ELISA plate. A standard curve using purified white sturgeon VTG (0, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μg/mL), negative controls (nonestrogenized male), and positive controls (vitellogenic female) to be used for intra and inter-assay determination were also added. Each sample

and standard was loaded in tr iplicate (50 μL/well). After in cubating overnight (4 oC) in a humidified Tupperware ® container, the plate was washed as previously described. The polyclonal antibody UF 131, diluted 1:1,000 in blocking buffer (1% BSA in TBSTZ) was added to each well (50 μL) and incubated fo r 2 hours at room temperature. After washing (4X PBSZT), an alkaline phosphata se conjugated detection antibody, goat-antirabbit IgG- F(ab) 2 -AP (Pierce), diluted 1:1000 in blocking buffer was added andincubated for 2 hours at room temperature. The plate was washed and then 1.0 mg/mL of the substrate, para-nitro phenyl phosphate (Sigma), in carbonate buffer (30 mM carbonate, 2 mM MgCl 2 , and pH 9.6) was added to each well (100 μL/well). The plate was scanned at 405 nm using a reader (Spect raMax 384, Molecular Devices) and the data interpreted by the SoftMax Pro program. A number of criteria were employed to maintain high quality data. The coefficient of variation between triplicate absorbance vales was < 10%. Outliers were discarded as long as the CV < 10% otherwise the sample was rerun. Absorbances at 405 nm greater than 1.5 were discarded. Inter and intra-assay variations (CV) were typically < 10% and < 5%, respectively as determined using positive and negative control samples. Figure 2-5 is a typical standard curve using purified white sturgeon VTG and the

sandwich ELISA.

Egg Yolk Purification and Identification Egg processing Ten grams of frozen eggs were homogeni zed in 15.0 ml of gel filtration buffer (25 mM Trizma, 350 mM NaCl, 0.05% azide, pH 8.0) using a glass tissue grinder. The mixture was centrifuged for 30 min at 15,000 x g at 4 oC. This resulted in 3 distinct layers: white fat, yellow aqueous and black pellet on the top, middle, and bottom of the tube, respectively. The middle layer was caref ully removed using a pasture pipette and then recentrifuged to remove any remaining layers. The middle solution was filtered using a glass fiber filter (A/E, Pall) to rem ove any large particles. Azide (0.02%) wasadded to this “crude egg yolk” preparation to prevent bact erial growth, and the solution was frozen (-20 oC). Three milliliters of the crude egg yolk was added to a gel filtration column (1.5 x 90 cm) containing Sepahrose-CL-6B (Pharmacia). This material is designed to separate proteins between 2,000,000 to 1,000 Daltons and ha s been cross-linked to improve flow rate. After loading the egg yolk solution, the flow was maintained at 0.3 mL/min and 6.0 ml fractions were collected every 20 min. The protein absorbance of each fraction was analyzed at 280 nm using UV 96-well plates (Greiner) and a plate reader (SpectraMax 384 Plus, Molecular Devices). After plotting the absorbance of each fraction three main peaks of different molecular weights were evid ent, indicated as E1 through E3 (Figure 2- 6). Fractions within each peak were comb ined and the protease inhibitor-Aprotinin added (10 KIU/ml). The total protein of each pool was determined by Bradford analysis (Coomassie Plus, Pierce) using bovine serum albumin as a sta ndard. A 1.0 ml aliquot of each pool to be used for PAGE and Western an alysis was collected, glycerol added (50% v/v), and proteins were stored at –20 oC.

Egg PAGE & Western Polyacrylamide electrophoresis and West ern analysis of egg yolk proteins (crude and purified) were identical to those descri bed for plasma. Figure 2-7 shows typical gel and western of purified sturge on egg proteins. The anti-s turgeon VTG Mab, 1H2, clearly recognizes the egg yolk proteins : crude preparation before purification, and peak E2. Peak E1 also reacts with the VTG antibody, but to a lesser degree. This may be due to slight contamination from peak E2 due to ove rlap and also the extr emely high sensitivityof the Mab. Peak E2 appears to be lipovitellin due to the cross-react ivity of the anti-VTG antibody. The Mab recognizes smaller sized prot eins than plasma VTG. This may be a result of follicular processing and possible degradation of the proteins despite the addition of protease inhibitor. Peak E3 app ears to be phosvitin sinc e this peak contains small molecular mass proteins which do not bind to Coomassie Blue stain (Wallace and Morgan 1986). Evidently, this fraction is a phosphoprotein and stereotypical of phosvitins and phosvettes. Peak E1 seems to be immunoglobulin, based on its molecular mass (>600 kDa, Kroll 1990) and immunological studies on paddlefish (Acton et al. 1971) and adult white sturgeon (Adkison et al. 1996).

Analysis of Selenium in White Sturgeon Tissues by Hydride Generation ICP-AES White sturgeon tissue samples were homogenized in millipure water using a hand blender and then freeze-dried. Approximately 300 mg of tissue was digested in a mixture of concentrated sulfuric, nitric and perchloric acids with a gradual temperature increase to

330 o C. The samples were then reduced by conc entrated hydrochloric acid heated to 95

o C and measured as selenite by hydrogen ge neration inductively c oupled plasma-atomic emission spectrometry (ICP-AES). The mini mum detection limit was typically 5 ng/ml. Quality control measures included the use ofstandard reference materials (NRC DORM2, DOLT-2, DOLT-3), spiked samples, duplication and blanks with every analytical run. All quality control measurements agreed with th e target concentration within 20 percent.Analysis of Selenium in Plasma an d Isolated Proteins by Fluorometry Plasma and isolated plasma and yolk protei ns were analyzed for total Se using the

method of Fan et al. (1998). VTG samples within the same exposure level were combined for Se analysis in order to obtai n the minimum amount of material required. VTG sample combinations are shown in Table 2-2. Approximately 2 mg of freeze-dried proteins, or 100 μ l plasma, were digested in a mixtur e of nitric and su lfuric acids and gradually heated to 130 o C. The samples were then reduced to selenite in 6 N HCl heated

to 110 °C. The selenite was derivatized to piazselenol by the addition of 0.1% diaminonaphthalene (DAN) reagent (prepa red by sonicating 2,3-diaminonaphthalene hydrochloride, Dojindo Laboratories (Japan), in 0.1 N HCl for 1 h, filtered through 0.45

μ m cellulosic filter, and washed three times with excess cyclohexane) and extracted using 0.5 ml of cyclohexane. This mixture was incu bated at 45 °C in the dark for 30 min, and then shaken to complete the extraction into the cyclohexane layer. Two hundred microliters of the cyclohexane layer was measured for the fluorescence intensity of the piazselenol derivative using a spectrofluor ometer. The minimum detection limit was typically 1 ppb. Quality control measures incl uded the use of spiked samples, duplication and blanks with every analytical run. Qu ality control measurements agreed with the target concentration within 15 percent.

Statistical Analysis Morphologic measurements were comp ared between exposure groups using ANOVA and ANCOVA. Log 10 transformations were used in regressions of body weight to fork length to account for allometric gr owth. The reproductiv e endpoints fecundity,fertilization success and neurulation success were compared between exposure groups by the Wilcoxon Rank Sums Test. Comparisons of mean Se concentrations between two groups were performed using a least s quares means Student’s t-test, ANOVA or

Wilcoxon’s rank sum test. Se concentrations were compared across tissues and exposure groups using two-way ANOVA, nested by indi vidual fish, with a Bonferroni-adjusted Students t-test (experiment-wise α = 0.05; adjusted comparison-wise α = 0.00142). Comparisons of Se concentrations between exposure groups at different sampling time points for plasma and isolated plasma prot eins were performed using two-way ANOVA, nested for repeat measures, with Tuke y’s Honestly Significant Difference (HSD) comparisons test ( α = 0.05). Log 10 transformations were used when necessary to meet the assumptions of parametric models. Nonparametric tests were used when the assumptions of parametric models could not be met. The relationships between Se

concentrations in various tissues were exam ined using linear or nonlinear regressions. Correlation analysis was used when the variab les could not be assumed to be functionally dependent. Weighted least s quares regression was used when the dependent variable did not show constant variance. The weights used included the reci procal of y and the reciprocal of y 2 . The statistical software packag es JMP (SAS Institute Inc.) and SigmaPlot (Systat Software, Inc.) were used for all computations. One fish (treatment fish number 8) in the Se treatment group lost 2.75 kg (~10% body weight), had negative specific growth ra te (- 0.05%), and tissue Se concentrations lower or similar to control group. Apparent ly, this female was not feeding due to avoidance of high Se feed, stress, or inte rnal injury caused by transport to UCD or handling. This fish was excluded from all sta tistical analyses of Se data in this study.Results Gross Morphologic Characteristics

There were no differences in condition factor, fork length, or body weight between exposure groups at either the be ginning or end of the study (ANOVA, p > 0.18; Table 2-3). Body weight to fork length regressions were not significantly different between exposure groups at either the be ginning or end of the study (ANCOVA, p = 0.69 and 0.06 respectively). In addition, the specifi c growth rate (SGR) for the length of the study was not different between the tw o exposure groups (ANOVA, p = 0.94).

Adult Tissue Selenium Comparisons within individual tissues s howed that Se concentrations were significantly higher in Se-tre ated females (Student’s t- test, p < 0.0001; Wilcoxon’s Rank Sums, χ 2 < 0.0017; Table 2-4). Se concentrations were also compared among all tissues and exposure groups (two-way nested ANOVA p < 0.0001; adjusted multiple comparisons, α = 0.05; Figure 2-8). Again, treated females had significantly higher Se concentrations in liver, kidne y, spleen, muscle, ovarian tissue, and eggs compared to control. In control fish, Se concentrations in kidney were greater than in muscle, ovarian tissues and liver, but similar to concentrations in spleen and eggs. Spleen of control fish contained higher levels of Se than did muscle. Se con centrations in ovarian (postspawning) and liver tissues did not differ in control fish. In treatment fish, Se concentrations in kidney were greater than in muscle, ovarian tissues, eggs and liver, but not spleen. Se in treatment fish spleen was gr eater than in liver. The levels of Se in treatment fish muscle, ovarian tissue and eggs were very similar. All Se values reportedabove are based on dry weight analyses. Percen t moisture of each tissue type is listed in

Table 2-5.

Plasma, Isolated Plasma VTG and no nVTG Plasma Protein Selenium Treatment fish had significantly higher le vels of Se in plasma, and isolated plasma proteins, than control fish (St udent’s t-test, p < 0.02; Wilcoxon’s Rank Sums, χ 2 < 0.0017; Table 2-6). Plasma was collected at two time points within the experiment, mid-experiment (88 days dietary exposure) and at spawning or necropsy (126 – 200 days dietary exposure). Se concentrations in plasma, isolated pl asma VTG, or nonVTG plasma proteins did not differ between sa mpling time points within each exposure group, but were significantly different between exposure groups at each sampling time point (two-way nested ANOVAs, p < 0.011; Tukey HSD, α = 0.05; Figures 2-9 – 2-11). In treatment fish, VTG contained higher Se concentrations than those found in nonVTG plasma proteins (two-way ANOVA, p = 0.032; Tukey HSD, α = 0.05; Figure 2-12). Se concentrations of VTG and nonVTG plasma prot eins were similar for control fish. Se values are reported as wet weight for plasma and dry weight for isolated plasma proteins.

Plasma VTG concentrations in cont rol fish were 0.21 ± 0.053 mg/mL (8) and 0.1 ± 0.034 mg/mL (7) at mid- and end-experiment , respectively (ELISA). Treatment fish contained 0.28 ± 0.083 mg/mL (7) and 0.08 ± 0.022 mg/mL (8) plasma VTG at mid- and end-experiment, respectively. Data shown as mean mg VTG/ml plasma ± standard error (number of fish). Plasma VTG concentrati ons were greater during mid-experiment as compared to end-experiment, but were not di fferent between contro l and treatment fish (two-way nested ANOVA, p = 0.048; Tukey HSD, α = 0.05; Figure 2-13). Plasma VTGconcentrations are expected to decrease when sturgeon reach full oocyte maturation, or experience widespread atresia (e.g., fail to ovulate; Doroshov et al. 1997; LinaresCasenave et al. 2002). Due to small sample sizes of isolated pl asma VTG, samples from individual fish from the same exposure group were pooled prior to Se analysis (Table 2-2). Only one pooled VTG sample (n = 4 fish) was availabl e for the final sampling point of treatment fish. One-sample t-tests were calculated betw een mean Se values and the single end data point for treatment VTG (26.29 μ g/g Se; Figure 2-10). Bo th the mid- and endexperiment time points in the control gr oup had mean VTG Se values significantly different from the end-experiment data point in the treatment group (p < 0.02). Within the treatment group, the mid-experiment mean VTG Se concentrati on was not different from the single end-experiment VTG data point (p = 0.32).

Yolk Protein Selenium Se concentrations in isolated yolk protei ns are presented in Table 2-7. Se was highly variable in both egg immunoglobu lin and phosvitin. Only lipovitellin Se

concentrations were found to be statis tically different between exposure groups (Wilcoxon Rank Sums; χ 2 = 0.005; Table 2-7). Phosvitin had higher Se concentrations than lipovitellin within the treatment group (Wilcoxon Rank Sums; χ 2 = 0.005; Table 2- 7). No other significant diffe rences were observed. Se va lues for yolk proteins are reported as dry weight.Relationships of Selenium Distribution in Tissues We hypothesized that Se in the liver w ould become associated with the VTG molecule and transferred through the plasma to the eggs (Figur e 2-1). Se concentrations in isolated plasma VTG increased expone ntially with increasing Se in liver (R 2 = 0.90; p < 0.0001; Figure 2-14; Table 2-8). Se in pl asma, ovarian tissue and eggs increased exponentially with increasing Se concentrations in VTG (R 2 ≥ 0.82; p ≤ 0.0008; Figure 2- 15; Table 2-8). Se in the yolk lipovit ellin, but not phosvitin or immunoglobulin, increased exponentially with increas ing Se concentrations in VTG (R 2 = 0.91; p < 0.0001; Figure 2-16; Table 2-8). Exploring the relationship be tween Se in eggs and in isolated yolk proteins, we f ound that only Se levels in li povitellin could be predicted by Se levels in whole eggs (R 2 = 0.96; p < 0.0001; Figure 2-17). Neither phosvitin nor immunoglobulin Se show significant relationships with whole egg Se. We also explored the possibility of Se in nonVTG plasma proteins contributing to whole egg Se. Se concentra tions in ovarian tissue, eggs , plasma, and nonVTG isolated plasma proteins increased linearly with increas ing Se in liver (Figure 2-18; Table 2-9). Plasma Se showed a significant relationship wi th Se in isolated nonV TG plasma proteins (p < 0.0001; R 2 = 0.82 & 0.92 for mid- and end-experi mental samples, respectively; Figure 2-19). Se concentrations in eggs in creased linearly with Se in nonVTG proteins (R 2 ≥ 0.73; p ≤ 0.0004; Figure 2-20). In the yolk, li povitellin Se concentrations were positively correlated with Se in nonVTG proteins collected mid-experiment ( r = 0.88; p = 0.0007; logtransformed), and end-experiment ( r = 0.66; p < 0.0267; logtransformed; Table 2-10). Se concentrations in phosvitin were positively correlated with Se in nonVTG proteins collected end-experiment ( r = 0.66; p < 0.0267; logtransformed; Table2-10), but not mid-experiment. There was no correlation between Se concentrations in nonVTG proteins and in egg immu noglobulin (Table 2-10). A ll Se values are reported as dry weight, except plasma.

Reproductive Characteristics Five fishes from each exposure group we re hormonally induced to ovulate. The remaining three fishes from each exposure group had either follicular atresia or underdeveloped eggs that did not reach ma turation competence (Appendix B). Three out

of the five control fish ovulat ed (the two that di d not ovulate may have been injected too early; Table 2-11). Four out of the five treatment fish ovulated (although one only partially). The mean PI and percent GVB D was 0.09 and 100% for the ten induced fish,

and 0.15 and 35.4% for the five individuals that were not induced to ovulate (one atretic female died mid-experiment due to technical error). Virgin (first reproductive cycle) females of cultured white sturgeon often do not respond to hormonal treatment as well as iteroparous (second or third cy cle) females. Hence, the ovulation rate after hormone injection cannot serve as an endpoint in Se toxi city trials with a lim ited number of fish. Detailed information on spawning-related para meters is presented in Appendix B. No significant differences were found for fec undity (95 and 72 thousand, in control and treatment), fertilization rate (86 and 88%), and neurula tion survival (60 and 47%) between ovulated fish from the two exposur e groups (Wilcoxon; p > 0.35; Table 2-12). Hatching occurred in all egg ba tches, except of one female (in treatment), but hatching rates could not be properly measured under the condition of “in mass” egg incubation.One fish (treatment fish 3) was excluded from the fecundity analysis due to incomplete

ovulation.

Discussion

The bioaccumulation of Se in adult white sturgeon observed in this experiment concurs with several other studies showing the efficient uptake of Se in fish exposed to increased concentrations (Cleveland et al. 1993; Crane et al. 1992; Hamilton et al. 1990b;

Hamilton et al. 2002b; Hilton et al. 1980). White sturgeon fed treatment diet ( ca. 34 μ g/g Se) had Se concentrations approximately 3 to 9 times greater than those fed control diet ( ca. 1.42 μ g/g Se) in all tissues analyzed, expe ct the yolk proteins phosvitin and egg immunoglobulin (Figures 2-8 – 2-12; Table 2-7) . The kidney and spleen accumulated the greatest amounts of Se, most likely due to th e high metabolic activit ies of these tissues (Diplock 1976; Hilton and Hods on 1983; Sorensen 1986). Relati vely lower levels of Se were observed in liver, which is also highl y metabolic. This may be indicative of the transport of Se from liver to deve loping eggs during vitellogenesis. The average Se in isolated plasma VTG of treatment fish was 8 times higher than that of control fish, and approximated the c oncentration of the trea tment diet (Figure 2- 10). Furthermore, plasma VTG protein cont ained higher Se levels compared to the nonVTG plasma proteins in treatment fish (F igure 2-12). Se concentrations of VTG and nonVTG plasma proteins were similar for cont rol fish. These result s support a tentative conclusion of Kroll and Doroshov (1991), w ho hypothesized that VTG is a potential vehicle for the maternal transport of Se . Kroll and Doroshov (1991) found high, but variable, concentrations of Se in plasma and eggs from six white sturgeon femalescaptured in the Sacramento River. These sturgeon had Se concentrations ranging from

0.19 to 0.33 μ g/ml in plasma, 3.06 to 29.26 μ g/g in eggs, 2.78 to 4.11 μ g/g in VTG, 0.76

to 6.70 μ g/g in egg immunoglobulin, 3.14 to 11.22 μ g/g in lipovitellin, and 3.17 to 90.31

μ g/g in phosvitin (all values based on dry wei ght, except plasma). Se concentrations in ovulated eggs increased linearly with incr easing Se in egg immunoglobulin and phosvitin (R 2 = 0.83 and 0.88, respectively; Kroll and Doroshov 1991). These authors speculated that Se is most likely incorporated into the backbone of the VTG molecules by substituting for sulfur in the sulfur-containing amino acids, methionine, cysteine and cystine (two cystei ne residues linked by a disulfide bond). Se is able to substitute for sulfur in biological molecules because these two elements have similar chemical properties (Diplock 1976; Stadtman 1974). White sturgeon VTG contains 1.49% cysteine a nd 2.92% methionine (based on total moles of amino acids; Bidwell and Carlson 1995). VTG can transpor t toxic levels of cationic metals (Ghosh and Thomas 1995; Lee 1993; Shackley et al. 1981) most likely by binding to phosphate residues. As a lipoglycophosphoprotein, VTG can transport lipophilic toxins such as DDT, dioxin and benzo(a)pyrene (Monte verdi and Di Giulio 2000; Plack et al. 1979; Ungerer and Thomas 1996). The work by Kroll and Doroshov (1991) was the first to indicate VTG as a potential vehicle for the ma ternal transport of Se, and the current study

is the first to examine this e xposure route experimentally. The results of this study clearly indicate that Se accumulated in the liver becomes associated with VTG and is then transported to developing eggs with plasma proteins. As more Se is available in the liver, gr eater amounts of Se are associated with VTG (Figure 2-14). In fact, Se concentrations in VTG increased exponentially with increasingSe concentrations in the livers of fish from this study (R 2 = 0.90; Figure 2-14). Se in plasma, ovarian tissue and eggs showed what appears to be an asymptotic exponential increase with increasing Se concentrations in VTG (R 2 = 0.82 – 0.95; Figure 2-15). This demonstrates the efficient and dose-dependent maternal transport of Se via VTG in white

sturgeon. The relationship between Se concentrations in VTG and in yolk proteins is not as clear (Figure 2-16). Only lipovitellin Se concentrations were found to have a significant relationship to Se in VTG and eggs (R 2 = 0.91 and 0.96, respectively; Figures 2-16 & 2- 17), whereas Se concentrations of phos vitin and egg immunoglobulin were highly variable (CV ranged 0.44 to 1.10 in control) . Kroll and Doroshov (1991) found higher levels of Se in phosvitin than in other yol k proteins. We observed the same trend in treatment fish of this study (Table 2-7); however, there were no st atistical differences between mean Se concentrations in phos vitin and egg immunoglobulin across exposure groups. It is possible that data from a greater number of individuals would have decreased the variation observed in phosvitin and egg immunoglobulin Se concentrations. The impurity of the peak E1 and E3 fractions could also contribute to a high variation of Se concentrations in immunoglobulin a nd phosvitin. Interestingly, Hiramatsu et al. (2002) reported a relatively high proportion of methionine (~2.5%) in lipovitellin purified from yolk proteins of fertile sturgeon hybrid Huso huso x Acipenser ruthenus , but no

detectable level of this amino aci d in the purified phosvitin fraction. Several possible mechanisms exist for the incorporation of Se into the VTG molecule. Se is incorporated into proteins as a result of genetic translation (reviewed by

Gromer et al. 2005; Suzuki 2005a). A specific genetic codon prescribes theincorporation of selenocyste ine (SeCys) into proteins; however, selenomethionine (SeMet) is indiscriminately substituted for the methionine codon in genetic translation. As noted above, white sturgeon VTG consists of 1.49% cysteine and 2.92% methionine (based on total moles of amino acids; Bidwell and Carlson 1995). The amount of SeMet that is indiscriminately incorporated into prot eins increases with increasing availability of SeMet in tissues (Schrauzer 2000). Similarly, when increased Se is available compared to sulfur, SeCys can substitute for cysteine in protein synthesis (Kramer and Ames 1988;

Müller et al. 1994). The results of this study suppor t the hypothesis that as Se becomes more available in the liver, there is an incr ease in the substitution of Se amino acids for sulfur amino acids during protein synthesis.

Considerable amounts of Se were also found to be associated with nonVTG plasma proteins; however, the roles of these proteins in egg development are not well known. We observed a significant linear relationship between Se concentrations in liver and nonVTG plasma proteins (R 2 = 0.65; Figure 2-18; Table 2-9) . The Se concentrations in eggs increased linearly with increasing Se concentrations in nonV TG plasma proteins (R 2 = 0.80 and 0.73 for mid- and end-experiment samples, respectively; Figure 2-20), which may simply reflect the effect of increas ing liver Se. In addi tion, Se concentrations in the yolk proteins lipovitellin and phosvitin were positively correlated with those of nonVTG plasma proteins (Table 2-10). This may suggest that at least some of the nonVTG plasma proteins contribute Se to developing eggs. PAGE gels of the pooled nonVTG plas ma proteins show strong bands at approximately 70, 62 and 28 kDa (Figure 2-4). The identification of these proteins is unknown; however, several known plasma protei ns associated with Se correspond withthese general molecular mass ranges. In part icular, plasma albumin (68 kDa in humans) is a very abundant plasma pr otein that binds with seleni de in the bloodstream (Quinlan et

al. 2005; Shiobara and Suzuki 1998). In addi tion, several selenoproteins have been identified in vertebrates (reviewed by Gromer et al. 2005; Suzuki 2005a). Most importantly, the presence of excess cellular Se can significantly increase the production of selenoproteins (Fletcher et al. 2000; Helmy et al. 2000; Jameson et al. 2002). In humans, the predominant plasma selenoprotein is selenoprotein P (Sel P; 42 kDa), which provides more than half of the total Se in plasma (Mostert 2000; Peri S. et al. 2003). Plasma Sel P is mainly synthesized in the liver and is the only selenoprotein known to incorporate multiple SeCys residues (Hill et al. 1991; Motchnik and Tappel 1990;

Schweizer et al. 2005). Sel P in zebrafish ( Danio rerio ) contains seventeen selenocysteines (~ 50 kDa; Kr yukov and Gladyshev 2000; Tujebajeva et al. 2000). The apparent function of Sel P is to transport Se from the liver to other organs (Burk et al. 1991; Hill et al. 2003; Saito and Takahashi 2002). Add itionally, yolk cells of zebrafish embryos exhibit Sel P gene expression (Thissea et al. 2003; Tujebajeva et al. 2000). Other selenoproteins present in plasma may include the enzymes, glutathione peroxidases (~ 25 kDa), thyroxi de deiodinase (~ 30 kDa), and thioredoxin reductases (~ 55 kDa; Gromer et al. 2005; Kryukov and Gladyshev 2000; Tujebajeva et al. 2000). To date, none of the above selenoproteins have been characterized in white sturgeon. Further study is needed to understand the ro le of these unidentified proteins in Se maternal transport. Several field and laboratory studies have observed Se maternal transport in fish, and consequent developmental toxicity. Gillespie and Baumann (1986) collected andspawned female bluegills ( Lepomis macrochirus ) from a high-Se reservoir with egg Se concentrations of 12 to 55 μg/g. The re sulting larvae exhibited edema, skeletal deformities, and died. Woock et al. (1987) found similar results. Schultz and Hermanutz (1990) experimentally demonstrated the effici ent maternal transport of Se in fathead minnow ( Pimephales promelas ) raised in experimental stre ams. These authors showed that embryos collected from Se-exposed streams contained ca. 19.5 μg/g Se (converted from wet weight using 80% moisture) and deve loped into larvae with edema and skeletal

deformities. Hermanutz et al. , (1992) observed reduced ha tching, edema, skeletal deformities and hemorrhaging in progeny of bluegills held in experimental streams

treated with 10 μ g/L Se. Hamilton et al. (2002a) showed that razorback suckers ( Xyrauchen texanus ) in the seleniferous Colorado Ri ver can transfer significant amounts of Se to their eggs (36 – 60 μ g/g at two sites). These authors determined that a Se burden of 20 μg/g or more in razorback sucker eggs represented a high level of hazard to this endangered species. Holm et al. (2005) found 41.85 and 31.85 μg/g Se (converted from wet weight using 80% moisture) in the eggs of rainbow trout ( Oncorhynchus mykiss ) and brook trout ( Salvelinus fontinalis ) collected from seleniferous areas in Canada. These eggs produced larvae with edema as well as craniofacial and skeletal deformities. In the present study, white st urgeon fed a diet containing ca. 34 μ g/g Se were

found to contain ca. 11, 10 and 12 Se μ g/g (dw) in muscle, liver and eggs, respectively. The treatment diet contained higher levels of Se than are typically found in the San Francisco Bay-Delta food web. The ubiquitous bivalve Potamocorbula amurensis is a primary food source for white sturgeon from th is area and contain an average Se level of

15 μ g/g (dry wt; Linville et al. 2002; Urquhart and Regalado 1991) . However, the aboveSe concentrations in muscle, liver and eggs are similar to those of wild white sturgeon captured from San Francisco Bay-Delta. White sturgeon sampled from this area between

1986 and 1990 contained Se at concen trations ranging from 9 to 30 μ g/g in liver (n= 52)

and 7 to 15 μ g/g in muscle (n= 99; dry wt; Urquhart and Regalado 1991; White et al. 1988). Stewart et al . (2004) reported a mean liver Se of 24 μg/g (n=15; dry wt.) in white sturgeon collected from northern San Franci sco Bay. Out of six sturgeon females sampled in the San Francisco Bay-Delta regi on in the 1990’s, one contained eggs with 3

μ g/g Se, four had eggs ranging from 8 to 12 μ g/g Se and one female contained eggs with

29 μ g/g Se (Kroll and Doroshov 1991). Recentl y, three white sturgeon captured from this region were found to have 7 to 20 μ g/g Se in ovaries cont aining developing eggs (Doroshov Lab, UCD; unpublished data). Th e actual Se concen trations in the experimental animals are more important here than the relevance of the dietary treatment level since we are exploring the transport of accumulated Se from the liver to the developing eggs. Se concentrations in fish receiving treatment diet reached levels that have been associated with reproductive failure in other fish species (Garrett and Inmann 1984; Hermanutz et al. 1992). Lemly (2002) proposed thres holds of Se toxic effects for

freshwater and anadromous fish at 8 μ g/g in muscle, 12 μ g/g in liver, and 10 μg/g in ovary or eggs. Mean muscle and ovary Se concentrations in treatment fish surpassed these thresholds, while Se in liver a pproached the suggested threshold of 12 μ g/g. The reproductive endpoints of fecundity, fertiliza tion rate and neurula tion rate were not

different between control and treatment fish of this study. The lack of impact on fecundity and fertilization is not unusual with Se toxicity (e.g., Coyle et al. 1993; Gillespie and Baumann 1986; Woock et al. 1987), since most organi c Se is likely storedin the yolk. However, as shown in the next chapter, embryos of the treatment fish from this experiment showed signi ficant developmental toxicity. In conclusion, this is the first work to experimentally demonstrate that vitellogenesis provides a signi ficant, dose-dependent, mechanism of Se maternal transport in a fish species. White sturgeon is especially vulnerable to Se maternal transport due to a prolonged (two year) period of vitellogenesis and yolk deposition (Doroshov 1985; Doroshov et al. 1997). In contrast to othe r fish, sturgeon store and use their yolk in the form of in tracellular crystalline platelet s utilized throughout early development, including the embryoni c and larval periods (Dettlaff et al. 1993). The secondary degradation of yolk proteins occurring at egg maturation (Hiramatsu et al. 2006; LaFleur et al. 2005; Romano et al. 2004) has not been studied in sturgeon; however, it appears that incorpor ated Se is conserved in th e eggs through the vitellogenic phase of oogenesis until spawning. The resu lts of our study, along w ith the field data described above, demonstrate that exposure of maturing females to a diet moderately high in Se may present a threat to the repr oduction of white sturgeon residing in the San Francisco Bay-Delta region. This threat b ecomes more prominent as we explore Seinduced developmental toxicity in white sturgeon in the following chapter.Se content of experimental diets

Diet Target Se Content ( μ g/g, dry wt) Measured Total Se ( μ g/g, dry wt) Control 1 1.42 ± 0.03 (6) Treatment 35 34.04 ± 0.82 (5)

Table 2-1. Selenium averages ± standard errors (n) are shown. Selenium was added to the diet as selenized yeast (Selenomax®, Ambi Inc.).Combined VTG protein samples Days of Dietary Exposure Fish Number % of Combined

Sample Combined Sample ID Time Point Control 88 C1 45.2% 88 C2 26.5% 88 C3 28.3% C1 - C3 Mid 88 C4 100.0% C4 Mid 88 C5 27.0% 88 C6 32.1% 88 C7 30.0% 88 C8 10.9% C5 - C8 Mid 126 C1 72.7% 126 C2 27.3% C1 & C2 End 140 C3 35.3% 154 C4 29.4% 168 C5 5.0% 200 C6 15.1% 200 C7 15.1% C3 - C7 End

Treatment 88 T1 50.8% 88 T2 47.2% 88 T3 2.0% T1 - T3 Mid 88 T4 54.7% 88 T5 14.6% 88 T6 30.7% T4 - T6 Mid 88 T6 100.0% T7 Mid 126 T1 58.4% 140 T2 22.2% 154 T3 3.1% 168 T4 16.3% T1 - T4 End

Table 2-2. Sample combinations for analysis of VTG at mid- (88 days) and endexperiment (126 – 200 days). The percent c ontribution from each fish is shown. End sample T5 - T7 was excluded since it included VTG from fish T8, which was not included in Se analyses due to f ood avoidance (as described in the Statistical Analyses section).Morphological comparisons between exposure groups

Start of Study End of Study

ANCOVA F p R 2 F p R 2

Length to Weight Regression (log transformed) 0.17 0.69 0.83 4.36 0.06 0.78

ANOVA F p F p Weight (log transformed) 1.98 0.18 1.49 0.24 Fork Length (log transformed) 1.46 0.25 0.76 0.40

Condition Factor 0.001 0.97 0.03 0.86

SGR 0.006 0.94

Table 2-3. Morphological characteri stics were compared betw een control and treatment groups at both the beginning and end of the st udy. None of the measured characteristics were shown to be statistically different.Se in tissues of adult white sturgeon fed either control or treatment diets for approximately six months

Tissue Control Treatment Liver 1.52 ± 0.23 9.63 ± 0.89 * Kidney 4.64 ± 0.70 33.37 ± 3.04 * Spleen 2.38 ± 0.13 20.57 ± 2.84 † Muscle 1.23 ± 0.15 (6) 11.15 ± 0.78 * Ovarian Tissue 1.41 ± 0.19 9.6 ± 1.42 * Eggs 2.2 ± 0.18 (5) 12.43 ± 1.70 *

Table 2-4. Se levels measured in tissues of white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approxima tely six months. Data displayed as mean Se μ g/g, dw ± standard error. Sample size is 7 unless indicated in parentheses. Data are graphically displayed in Figure 2-8. * Significantly different value compared to control as shown by Students t-test on lognormal data (p < 0.0001). † Significantly different value compared to control as shown by Wilcoxon rank sum test ( χ

2 < 0.0017).Percent Moisture Plasma 94.8 ± 0.4 Kidney 83.7 ± 4.3 Spleen 83.5 ± 2.3 Muscle 75.0 ± 7.3 Ovarian 73.8 ± 13.4 Eggs 63.7 ± 4.1 Liver 53.6 ± 9.0

Table 2-5. Percent moisture of adult white sturgeon tissues (n=7 for plasma, n=3 for all other tissues)

.Se in plasma and isolated plasma proteins of adult white sturgeon fed either control or treatment diets for approximately six months

Tissue Control Treatment

Plasma 1 (μg/mL) 0.21 ± 0.01 (8) 1.28 ± 0.10** Plasma 2 (μg/mL) 0.27 ± 0.05 1.25 ± 0.15** NonVTG 1 Plasma Proteins 7.27 ± 0.30 22.21 ± 3.38 † NonVTG 2 Plasma Proteins 9.1 ± 1.47 24.89 ± 3.43**

VTG §1 4.29 ± 0.61 (3) 37.03 ± 8.18 (3)*

VTG §2 4.31 ± 1.19 (2) 26.29 (1) ‡

Table 2-6. Se levels measured in plasma and is olated plasma proteins of white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months. Data displayed as mean Se μ g/g, dw ± standard erro r, except plasma, which is presented as Se μ g/ml. Sample size is 7 unless i ndicated in parentheses. Data displayed graphically in Figures 2-9 – 2-11.

1 Sampled after 88 days of exposure

2 Sampled at spawning or necropsy, after 126 - 200 days of exposure § Several VTG samples were pooled within treatments to provide enough sample material (Table 2-2) *Significantly different value compared to control as shown by Students t-test on lognormal data (**p < 0.0001 or *p < 0.002) † Significantly different value compared to control as shown by Wilcoxon rank sum test ( χ 2 < 0.0017) ‡ Difference shown by one-sample t-test comparing control mean to the one treatment value (p < 0.02)Se in isolated yolk proteins of adult whit e sturgeon fed either control or treatment diets for approximately six months

Tissue Control Treatment Egg Immunoglobulin 11.95 ± 4.61 (3) 18.43 ± 3.28 (6) Lipovitellin 5.14 ± 0.64 (5) 14.03 ± 1.62 (7) †§

Phosvitin 20.49 ± 9.74 (4) 42.59 ± 17.71 (7) §

Table 2-7. Se concentrations in yolk proteins is olated from eggs of adult white sturgeon following exposure to either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diet for 126 – 200 days. Data displayed as mean Se μ g/g, dw ± standard error (n). † Only Se concentrations in lipovitellin were found to be different between exposure groups (Wilcoxon Rank Sums; χ 2 = 0.005). § Treatment phosvitin was shown to have higher levels of Se than treatment lipovitellin (Wilcoxon Rank Sums; χ 2 = 0.005). No other significant differences were observed.Se in combined VTG samples compared tocombined tissue and yolk protein samplesData presented as mean Seμg/g, dw ± standard error, except plasma, which is presented as Seμg/mlControlFish TimeNumber(s) Point VTG Liver Egg Ovarian Tissue Plasma Phosvitin Lipovitellin Egg ImmunoglobinC1 – C3 Mid 3.57 ± 0.17 (3) 1.13 ± 0.10 (3) 2.23 ± 0.12 (3) 0.99 ± 0.27 (3) 0.19 ± 0.01 (3) 14.18 ± 10.49 (3) 5.96 ± 0.39 (3)17.36 (1)C4 Mid 3.8 ± 0.00 (1) 0.8 (1) 1.61 (1) 1.65 (1) 0.2 (1)4.95 (1) 15.71 (1)C5 - C8 Mid 5.51 ± 0.00 (4) 2.16 ± 0.05 (3) 2.68 (1)1.75 ± 0.19 (3) 0.24 ± 0.01 (4) 39.41 (1) 2.88 (1) 2.79 (1)C1 - C2 End 5.5 ± 0.26 (2) 1.03 ± 0.04 (2) 2.12 ± 0.03 (2)0.74 ± 0.17 (2) 0.21 ± 0.04 (2) 3.70 ± 0.56 (2) 5.84 ± 0.65 (2)C3 - C7 End 3.12 ± 0.23 (5) 1.72 ± 0.28 (5) 2.25 ± 0.33 (3)1.68 ± 0.11 (5) 0.3 ± 0.07 (5) 37.28 ± 2.13 (2) 4.67 ± 0.97 (3) 11.95 ± 4.61 (3)TreatmentFish TimeNumber(s) Point VTG Liver Egg Ovarian Tissue Plasma Phosvitin Lipovitellin Egg ImmunoglobinT1 - T3 Mid 49.34 ± 1.37 (3) 10.44 ± 0.88 (3) 13.04 ± 3.86 (3) 8.49 ± 2.37 (3) 1.39 ± 0.25 (3) 20.86 ± 2.17 (3) 14.79 ± 3.52 (3) 18.72 ± 2.48 (3)T4 - T6 Mid 21.53 ± 0.55 (3) 8.17 ± 1.64 (3) 10.94 ± 1.66 (3)9.11 ± 1.99 (3) 1.19 ± 0.04 (3) 73.34 ± 36.88 (3) 12.07 ± 1.24 (3) 20.15 ± 11.44 (2)T7 Mid 40.21 ± 2.06 (1) 11.6 (1) 15.1 (1)14.4 (1) 1.22 (1) 15.55 (1) 17.63 (1) 14.09 (1)T1 - T4 End 26.29 ± 1.52 (4) 9.77 ± 0.91 (4) 12.4 ± 2.80 (4)7.97 ± 1.75 (4) 1.42 ± 0.20 (4) 23.25 ± 2.84 (4) 14.12 ± 2.58 (4) 16.22 ± 3.05 (4)Table 2-8.Due to small sample sizes, isolated VTGprotein samples were pooled prior to Se analysis (Table 2-2). Here we present Se inpooledVTG samples compared to the corresponding combinations of liver, eggs, ovarian tissue, plasma and isolated egg yolk proteins.Data displayed asmean Seμg/g, dw ± standard error (number of experimental animals contributing to the average), except plasma, which is presented as Seμg/ml.Mid samples were taken after 88 days of dietary exposure. Endsamples were taken after 126 – 200 days of dietary exposure.Variables Model R 2 F stat p value n= Equation Liver v Eggs Linear 0.82 46.25 <0.0001 12 y = 1.21x + 0.71 Liver v Ovarian Linear 0.83 58.00 <0.0001 14 y = 1.00x - 0.08

Liver v Plasma 1 Weighted Linear 0.87 83.10 <0.0001 14 y = 0.12x + 0.05 Liver v Plasma 2 Linear 0.76 39.01 <0.0001 14 y = 0.11x + 0.13 Liver v nonVTG 1 Linear 0.65 20.54 0.0009 13 y = 1.81x + 4.56 Liver v nonVTG 2 Linear 0.65 21.98 0.0005 14 y = 1.88x + 6.49

Table 2-9. Statistical parameters for the regressions of Se concentrations in ovarian tissue, eggs, plasma and nonVTG isolated plasma proteins as a function of Se concentrations in liver (Figure 2-18). 1 Sampled after 88 days of exposure. 2 Sampled at spawning or necropsy, after 126 - 200 days of exposure. Each regression is statistically significant.Se in yolk proteins correlated to Se in nonVTG

nonVTG 1 Proteins nonVTG 2 Proteins Liver 0.86 0.89 Lipovitellin 0.88 0.66

Phosvitin 0.18 0.66 Immunoglobulin 0.62 0.22 Egg 0.91 0.88

Table 2-10. Pearson correlation coefficients for Se in plasma nonVTG proteins correlated to Se in liver, yol k proteins, and eggs (logtransformed data). Significant Pearson coefficients are presented in bold (p < 0.05). Se in nonVTG was positively correlated with Se in liver, lipovite llin, phosvitin, and whole eggs. 1 Collected after 88 days of exposure. 2 Collected after 126 - 200 days of exposure.White sturgeon spawning data

Fish Number Date Spawned/ Necropsied Days of Dietary Exposure Percent Ovulation Estimated Fecundity (number of eggs) Egg Fertilization

Rate (%) Percent Neurulated

Embryos

C1 3/22/2002 126 None C2 3/22/2002 126 None C3 4/5/2002 140 100 73,440 88 36

C4 4/19/2002 154 100 137,243 84 88 C5 5/3/2002 168 100 73,080 86 56

C6 6/4/2002 200 Atretic

C7 6/4/2002 200 Atretic

T1 3/22/2002 126 100 101,238 92 76

T2 4/5/2002 140 100 84,830 88 74

T3 4/19/2002 154 Partial (40-50%) 31,968 94 36

T4 5/3/2002 168 100 66,660 76 0

T5 5/17/2002 182 None

T6 5/17/2002 182 Eggs not polarized

T7 6/4/2002 200 Eggs not polarized

T8 6/4/2002 200 Eggs not polarized *Fish C8 died mid-experiment due to technical error (escaped tank through a rip in the tank covering).

Table 2-11. Results of spawning white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months. More detailed spawning data are presented in Appendix B.Reproductive performance of sturgeon fe males in control and treatment groups

Exposure Estimated Fecundity (number of eggs) Egg Fertilization

Rate (%) Percent Neurulated

Embryos

Control 94,588 ± 21,328 (3) 86 ± 1 (3) 60 ± 15 (3)

Treatment 84,243 ± 9,986 (3) 88 ± 4 (4) 47 ± 18 (4)

Table 2-12. Fecundity, fertilization success and neurulation rates in control and treatment groups. Data presented as mean ± standard error (n). No significant differences were found between control a nd treatment groups for any of the three parameters (Wilcoxon; p > 0.35).Se transport via vitellogenesisBloodFigure 2-1.Hypothesized mechanism of the maternaltransport of Se to offspring. Duringvitellogenesis (yolkdeposition), estrogenis produced in egg follicles and transported to the liver via blood. Estrogen signalsthe liver to produce the yolk protein precursor,vitellogenin (VTG). Se in the liver may beincorporated into the VTG molecule, which is transported to the egg via blood and takenup by the VTG-specific receptor in the egg membrane. Inside theegg, VTG cleaves into the yolkproteins lipovitellin and phosvitin,and Se is stored in the yolk until metabolized during the embryonic and larval stages. Figure modified from Mommsen & Walsh(1988).VTG-SeEstrogen (EEstrogen)Developing EggFollicle CellsVTGReceptorVTG-SePhosvitinLipovitellinSeYolkE (l ble)Liver CellDNARReceptor (R)Vitellogenin-mRNAVitellogeninSeVTG-Seipid soluEBloodFigure 2-2. Anion exchange chromatograph of white sturgeon male before (red line) and after (green line) estrogen treatment.Figure 2-3. Anion exchange chromatograph of pl asma proteins from vitellogenic white sturgeon female used for this study. P1, P2, P3, & P4 were pooled and indicated as “nonVTG” peaks. VTG is the vitellogenin peak.

### PAGE Western

Figure 2-4. PAGE & Western of white sturgeon plas ma proteins. The first lane contains molecular mass markers; the second lane contai ns plasma of nonestrogenized males; the third lane contains purified VTG. The next four groupings are indi vidual females (coded by numbers), with lanes showing plasma, nonVTG pooled proteins , and isolated VTG protein. The PAGE gel of the pooled nonVTG proteins shows a strong band at 62 kDa and two weak bands at 70 and 28 kDa; none of which reacted with anti-VTG antibody in Western. The Western identifies three differe nt bands of white st urgeon VTG: a strong band at 180 kDa and two weak bands at 75and 64 kDa. Fish 6D5C had VTG in plasma but VTG was not detectable in eluted fractions.Mean Value

Figure 2-5. VTG ELISA Standard Curve. Th e x-axis shows VTG concentration ( μ g/ml) while the y-axis shows absorbance at 405 nm.Figure 2-6 . Gel filtration chromatograph of sturgeon egg yolk. Peaks E1, E2, and E3 were identified as egg Immunoglobulin (Ig), Lipovitellin (Lv), and Phosvitin (Pv). Horizontal bars show collected fractions.PAGE Western Figure 2-7 . PAGE and Western gels of crude a nd purified egg yolk proteins of white sturgeon. The first two lanes are molecular markers and purified VTG. The next four lanes are crude yolk protein, and pooled fract ions from gel filtration corresponding to peaks E1, E2, and E3. Peak E3 (Phosvitin) does not have affinity to Coumassie Blue stain.Figure 2-8. Tissue Se concentrations in white sturgeon exposed to ca. 1.5 or 34 μ g/g dietary Se for approximately six months. Bars show mean Se μ g/g, dw, with vertical lines showing standard error of the mean. Sa mple size is 7, except for control muscle and eggs (n = 6 and 5, respectively). Lognor mal data compared by two-way nested ANOVA (p = 0.028) with adjusted mu ltiple comparisons (family α = 0.05). Groups that do not have the same letter in superscript are signifi cantly different. Data are also presented in Table 2-4.Figure 2-9. Se in plasma collected at mid- a nd end-experiment (after 88 and 126 - 200 days of dietary exposure, respectively) compared across exposure groups (two-way nested ANOVA, p < 0.0001). Bars show mean Se μ g/ml with vertical lines showing standard error of the mean. Sample size is 7, except for control plasma (n = 8). Means that do not have the same letter in supers cript are significantly different (Tukey HSD, α = 0.05). Data are also presented in Table 2-6.1 3 2 3

Figure 2-10. Se in VTG isolated from plasma collected at mid- and end-experiment (after 88 and 126 - 200 days of dietary exposur e, respectively) compared across exposure groups (ANOVA; p < 0.011). Bars show mean Se μ g/ml with vertical lines showing standard error of the mean. Due to small sample sizes, isolated VTG protein samples were pooled prior to Se analysis (Table 2-2) . Pooled sample size shown in bars. Means that do not have the same letter in supers cript are significantly different (Tukey HSD, α = 0.05). *Only one data point was available fo r the end time point in the treatment group. One-sample t-tests were calculated between mean Se values and the single end data point for treatment VTG (26.29 μ g/g Se). Both of the time points in the control group had mean Se values significantly different from the end-treatment data point (p < 0.02). The mid-treatment mean Se was not different from the end-treatment data point (p = 0.32). Data are also presented in Table 2-6.Figure 2-11. Se in nonVTG proteins isolated from plasma collected at mid- and endexperiment (after 88 and 126 - 200 days of dietary exposure, respectively) compared across exposure groups (two-way nested ANOVA; p < 0.0001). Bars show mean Se μ g/g, dw, with vertical lines showing standard error of the mean. Sample size is 7. Data not connected by same letter are si gnificantly different (Tukey HSD, α = 0.05). Data are also presented in Table 2-6.Se in VTG and nonVTG from white sturge on fed control or treatment diet for approximately six months

5 4 14 14

Figure 2-12. Se concentrations in VTG and nonVTG proteins. Data from both mid- and end-experiment are combined. Bars show mean Se μ g/g, dw, with vertical lines showing standard error of the mean. Sample size show n in bars (VTG are pooled samples). Data not connected by same letter are significan tly different (two-way ANOVA, p = 0.032; Tukey HSD, α = 0.05).Vitellogenin from adult white sturgeon following approximately six months exposure to dietary Se

Figure 2-13. Vitellogenin isolated from plasma colle cted at mid- and end-experiment (after 88 and 126 - 200 days of dietary exposure, respectively). Plasma VTG concentrations were greater during mid-experi ment as compared to end-experiment, but were not different between control and trea tment fish (two-way nested ANOVA, p = 0.048; Tukey HSD, α = 0.05). Bars show mean VTG mg /g, with vertical lines showing standard error of the mean. Sample size s hown in bars. Data not connected by same letter are significantly different.Figure 2-14. Se in VTG increases exponentially with increasing Se in liver (R 2 = 0.90; p < 0.0001). Data includes Se in VTG isolated from plasma collected at mid- and endexperiment (after 88 and 126 - 200 days of di etary exposure, respectively). All liver samples were collected at the end of the experi ment. Due to small sample sizes, isolated VTG protein samples were pooled prior to Se analysis (Table 2-2). Se in pooled VTG samples is compared to the average Se in the corresponding combina tions of liver data (Table 2-8).Figure 2-15. Se in plasma, ovarian tissue a nd eggs increases exponentially with increasing Se in VTG (weighted regressions used for ovarian and egg). Due to small sample sizes, isolated VTG protein samples were pooled prior to Se analysis (2-2). Se in pooled VTG samples is compared to the aver age Se in the corres ponding combinations of plasma, ovarian tissue and egg data (T able 2-8). Data displayed as Se μ g/g, dw, except plasma, which is presented on the right y-axis as Se μ g/ml. Each regression is statistically significant.

### Se in Vitellogenin Related to Se in Yolk Proteins

Figure 2-16. Se in lipovitellin increases exponen tially with increasing Se in VTG (R 2 = 0.91; p < 0.0001). Se in phosvitin and egg im munoglobulin were not significantly related to Se in VTG (p = 0.74 and 0.32, respectively). Due to small sample sizes, isolated VTG protein samples were pooled prior to Se anal ysis (Table 2-2). Se in pooled VTG samples is compared to the average Se in the corre sponding combinations of yolk protein data (Table 2-8). Data are displayed as Se μ g/g, dw.Figure 2-17. Se in isolated yolk proteins as a func tion of Se in eggs. A significant linear relationship was found between Se in eggs and Se in lipovitellin (R 2 = 0.96; p < 0.0001). Se in phosvitin and egg immunoglobulin were not significantly related to Se in eggs (p = 0.14 and 0.17, respectively).Figure 2-18. Se concentrations in ovary, eggs, plasma and nonVTG isolated plasma proteins as a function of Se concentrations in liver. 1 Sampled after 88 days of exposure. 2 Sampled at spawning or necropsy, after 126 - 20 0 days of exposure. Data displayed as Se μ g/g, dw, except plasma, which is pres ented on the right y-axis as Se μ g/ml. Each regression is statistically signi ficant. Complete regression statistics are presented in Table 2-9.Figure 2-19. Se in nonVTG isolated plasma proteins correlated with Se in plasma. Data presented for samples collected at mid- and end-experiment (after 88 and 126 – 200 days of exposure, respectively). A statistica lly significant linear correlation was found between plasma and nonVTG isolated plasma proteins (p < 0.0001; R 2 = 0.82 and 0.92 for mid- and end-experiment, respectively).Figure 2-20. Se in eggs increase linearly with increasing Se in nonVTG isolated plasma proteins (p < 0.0005). NonVTG proteins were isolated from plasma collected after 1 88 days of exposure and 2 126 - 200 days of exposure. Data displayed as Se μ g/g (dw).Chapter 3. Developmental toxicity in whit e sturgeon larvae exposed to selenium through maternal transport or microinjection

Introduction Selenium (Se) is a well-documented de velopmental toxin to fish and wildlife species (Coyle et al. 1993; Gillespie and Baumann 1986; Hamilton 2004; Hamilton et al. 1986; Hermanutz 1992; Hoffman et al. 1988a; Lemly 1993b; Ohlendorf et al. 1986). Chapter two demonstrated the maternal tran sfer of Se to eggs and embryos during vitellogenesis in white sturgeon. Transfer a nd storage of Se in the egg yolk compromises the development and survival of embryos and yolk sac larvae of several fish species (Gillespie and Baumann 1986; Lemly 1993b; Schultz and Hermanutz 1990; Woock et al. 1987) since they have not fully developed the organs and enzymes necessary for Se detoxification and excretion (e.g., gills, liver and kidney). If white sturgeon larvae are similarly susceptible, then recruitment may be affected in populations residing in high-Se

areas. The direct effects of Se on recruitment ar e difficult to detect in white sturgeon populations because monitoring is typically accomplished by tracking sub-adults and adults, which are generally at least 9 years of age (Schaffter and Kohlhorst 1999). Thus, poor recruitment is usually not detected until a decade or more after the hatching of a particular year-class. Nevertheless, Kohlhorst et al. (1991) have shown that recruitment success is a major determinant of white st urgeon population abundance in San Francisco Bay-Delta. White sturgeon popula tions in this region have b een highly variable over the past five decades (Kohlhorst 1980; Kohlhorst et al. 1991; Schaffter and Kohlhorst 1999).Population surveys in 2005 indicated that the San Francisco Bay-Delta white sturgeon population was at a 50-year low, with an estimated 10,000 adult sturgeon (California Department of Fish and Game Commissi on 2006). White sturgeon only spawn every two or more years (Doroshov et al. 1997), which led the Califor nia Department of Fish and Game to estimate that fewer than 2,000 of these white sturgeon were likely to spawn in 2006 (California Department of Fish a nd Game Commission 2006). Previous studies have hypothesized that the imp act of environmental contamin ants may affect recruitment success of white sturgeon in this area (K ohlhorst 1980; Schaffter and Kohlhorst 1999). The population dynamics of sturgeon make them especially vulnerable to any disruption of recruitment, for example, by Se. San Francisco Bay-Delta receives Se thr ough both the refining of fossil fuels and irrigation drainage from the se leniferous San Joaquin Valley. White sturgeon in this area are exposed to high levels of Se through thei r diet. This is evidenced by high tissue Se levels in common prey of white sturgeon (Linville et al. 2002; Stewart et al. 2004; White

et al. 1988), as well as in sturgeon muscle, liver and eggs (Kroll and Doroshov 1991;

Stewart et al. 2004; Urquhart and Regalado 1991; White et al. 1989). The biology of white sturgeon indicates that this species would be susceptible to Se-induced developmental toxicity. The accu mulation of maternal Se in the egg yolk proteins of white sturgeon was shown in ch apter two of this di ssertation. Sturgeon embryos hatch as underdeveloped (altricial) or ganisms commonly referr ed to as yolk sac larvae. During the first 10 days post-hatch, over eighty percent of th e maternal yolk is rapidly metabolized for growth, development, and for greatly increased metabolic energy associated with constant pelagic swimming of newly emerged white sturgeon larvae(Bolker 1993; Dettlaff et al. 1993; Doroshov et al. 1983). Vital organs such as gills, liver and kidney complete differentiation during this period. Furthermore, in contrast to the extraembryonic yolk of modern teleosts, stur geon store and digest yolk intracellularly (Buddington and Doroshov 1986), and the nutrien ts are transported by a well developed embryonic vascular system comprised of the ducts of Cuvier and yolk veins (Schmalhausen 1991). In fact, the yolk is stored and digested in the endodermal tissue that ultimately differentiates into the stom ach, mid-intestine, liver, and pancreas (Buddington and Doroshov 1986). The intracellu lar digestion and increased metabolism of yolk in sturgeon may exert profound effect s on the development and survival of yolk

sac larvae with elevated Se levels in maternal yolk. The experiments described here were ba sed on the hypothesis that white sturgeon is most sensitive to Se-induced developmen tal defects and morta lities during the rapid yolk utilization and organogenesi s experienced in the first 10 days post-hatch (yolk sac larval phase). The experiments include both maternal exposure and microinjection exposure to Se in post-hatch larvae. In th e maternal exposure experiment, embryos of

females used in the Se maternal transfer e xperiment (described in chapter two) were reared and observed for developmental defect s and mortalities. Se was shown to be incorporated into yolk proteins of these embryos in the previous chapter. In the microinjection exposure experiment, seleno-L-me thionine (Se-L-Met) was injected into the yolk sac of newly hatche d larvae of unexposed females. Microinjection was used because the large size and long ovarian cycl e of white sturgeon make it difficult and expensive to study reproductive toxicity in this species under laboratory conditions. Microinjection mimics maternal transfer of e nvironmental contaminants and is used as animportant tool in ecotoxicology (Black et al. 1985; Metcalfe et al. 1988). Larvae from both experiments were monitored for impacts on size, normal development and survival. Occurrence of edema, skeletal curvatures, a nd mortality were quantified. The maternal exposure experiment provides a causal link between Se in maternal food and developmental effects in progeny. The micr oinjection exposure experiment confirms threshold levels of Se developmental t oxicity in white sturgeon larvae.

Materials and Methods Maternal Exposure Study This study used developing embryos fr om the maternal Se exposure study described in chapter two of th is dissertation. Briefly, sixteen domestic broodstock white sturgeon females ( ca. 22.71 kg and 134.59 cm) were exposed to either control ( ca. 1.4

μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately 6 months (Table 3-1; all Se values reported here are based on dry weight , unless otherwise noted). Upon acquisition of maturation competence, the fish were induced to spawn and ovulated eggs were fertilized with sperm from non-tr eated males. Ferti lized eggs were placed into individual MacDonald hatching jars; and jars were set up to allow hatched la rvae from individual females to flow into separate 1.2 m diameter circular tanks. Embryo incubation was at water temperatures of 15 to 16 oC. After hatching, larvae were acclimated from 16 oC to 18 oC over one day. Larvae

were then moved to an indoor shelter at th e Center for Aquatic Biology and Aquaculture (CABA) at the University of California, Davis (UCD) and placed into flow-through

buckets secured in 1.2 m diameter circular flow-through tanks. Flow-through bucketswere fashioned from 19 liter hi gh-density polyethylene buckets ( ca. 26 cm diameter, 37 cm depth) with screen s covering large windows ( ca. 25 by 30 cm) on each side to provide

a constant cross-flow.For each progeny cohort, 3,000 larvae were randomly distributed into flowthrough buckets, three replicate buckets for stage 40 sampling (n=500) and three replicate buckets for stage 45 sampling (n=500). A pa ir of stage 40 and 45 replicate flow-through buckets was placed in three separate tanks to control for any tank effects. Separate tanks were used for control and treatment group la rvae. Temperature was maintained at 18.56

± 0.13 °C. Flow rate in ta nks ranged from 1.0 to 1.5 L/ min. Dissolved oxygen ranged

between 90 to 95% saturation in all tanks . Natural photoperiod was maintained by artificial light, and approximately 80% of light was blocked by shad e-cloth tank covers. Larvae were observed daily for mortality, morbidity and developmental abnormalities.

Sampling Key sturgeon developmental stages were used to time sample collections for tissue Se and to conduct morphol ogical analysis of larval development (stages 36 – 45, yolk sac larvae). Table 3-2 shows de velopmental staging following Dettlaff et al . (1993) for Russian sturgeon, with specific characte ristics for white sturgeon described by Beer (1981) and Deng (2000). There were slight variations in stag e within samples due to the fast rate of development. Samples were taken for Se analysis at stages 5, 21, 33 (embryos) and 36, 40, and 45 (larvae). Developm ental defects and size were assessed at stages 36, 40 and 45. Sampled larvae were anesthetized in 150 ppm MS-222, overdosed in 500 ppm MS-222, rinsed in millipure water, then either frozen on dry ice and stored at–80 °C (for Se analysis), fixed in 10% buffe red formalin (for developmental assessment), or measured immediately (for size measurement). All post-hatch developing white sturgeon are referred to as larv ae in this chapter. The term larval development is used here to describe the rapid development that takes place between hatching and the initiation of exogenous feeding (stages 36 – 45).

Selenium analysis Approximately 60 – 90 individual embryos or larvae were homogenized in millipure water using a hand blender and then freeze-dried. Approximately 300 mg of tissue was digested in a mixture of concentrated sulfuric, nitric and perchloric acids with a gradual temperature increase to 330 o C. The samples were then reduced by concentrated hydrochloric acid heated to 95 o C and measured as selenite by hydrogen generation inductively coupled plasma-atomi c emission spectrometry (ICP-AES). The minimum detection limit was typically 5 ng/ml. Quality control measures included the use of standard reference materials (N RC DORM-2, DOLT-2, DOLT-3), spiked samples, duplication and blanks with every analytical run. All quality control measurements agreed with the target con centration within 20 percent.

Assessment of larval development

Preserved larvae were rinsed with millipure water and visually observed for gross developmental abnormalities. The development was monitored using the morphological criteria described by Dettlaff et al. (1993) for Russian sturgeon, with specific

characteristics for white stur geon described by Beer (1981) and Deng (2000; Table 3-2).The occurrence of edema (overt swelling caused by accumulation of fluid), lordosis (concave dorsoventral curvature of posterior region), kyphosis (dorsoventral curvature of anterior region), and scoliosis (lateral skelet al curvature) was recorded for each sample. Stage 36 samples contained approximately 30 individuals. Samples were collected from each of the three stage 40 and 45 replicate buckets (six buckets per female); each replicate sample contained a pproximately 30 individuals.

Measurement of larval size Body weight and total length were measur ed in approximately 30 larvae of each progeny cohort for three time points. Deform ed larvae were not excluded from these measurements. Total length was measured under a dissecting scope, using a digital image tablet (± 0.01 mm). Larvae were blotte d and weighed individu ally (wet weight) on a microbalance (± 0.0001 g) then placed in ta red whirl packs, lyophilized, and weighed to determined dry weights.

Microinjection Studies Study One Post-neurulated embryos were obtaine d from spawning of domestic stock (Columbia River origin) at Clear Springs F ood Co. (Idaho). Eggs were fertilized on May 19, 2004, and post-neurulated embryos shipped to California in sealed plastic bags with hatchery water (14.5 °C) and oxygen. Upon arri val, the embryos (stage 30-31; Dettlaff et

al. 1993) were acclimated to 15.7 °C for two hours and transferred to cr ystallizing dishessubmerged in tanks. Approximately 90% of post-neurulated embryos received from Idaho survived to hatching and provided viab le yolk sac larvae for this experiment. Newly hatched larvae (stage 36) with normal morphology and behavior (Beer 1981; Dettlaff et al. 1993) were used for microinjectio ns. Microinjections took place on May 28, 2004 following the methods described in detail below. The control larvae underwent the same procedures (anesthesia and microinjection with sterile millipure water). In addition, a second control group of anesthetized but non-injected larvae was used, to account for the physical effect of microinjection. Follo wing injection, larvae

were allowed to recover in fresh water at 15.7 °C before stocking in tanks. The

maintenance of the larval culture is described in detail below.

Se-L-Met was dissolved in autoclaved vi als using sterile millipure water to 7.0, 14.1, and 28.2 mg/mL. Target doses were calcula ted as described below. The injection volume was 25 nL per larva, re sulting in Se dosing of 10, 20 and 30 μg/g (dw). Controls consisted of larvae injected with 25 nL of sterile millipure water (sham-injected) and non-injected larvae. A total of 160 larvae were injected in each treatment and control groups. Within each group the 160 larvae were randomly distributed into four tanks; three replicate survival tanks (n=35) and one sampling tank (n=55). All replication was assigned to tanks by using a random number table. Animals in survival tanks were monitored daily for mortality and morbidity. Sampling tanks were monitored daily for phys ical (e.g., heartbeat, pericardial edema, yolk absorption) and morphological (e.g., skelet al) abnormalities of larvae. Larvae from each treatment were collected from each sampling tank one day post injection and at the endpoint of the experiment (stage 45, yolk depletion) for Se an alysis and morphology.Sampled larvae were anesthetized in 150 ppm MS222, overdosed in 500 ppm MS-222, rinsed in millipure water, then frozen on dr y ice and stored at –80 °C (Se analysis) or fixed in 10% buffered formalin containing 4% sucrose (morphology).

Study Two White sturgeon ova of two females and m ilt (pooled from 4 males) were obtained from a commercial farm (Stolt Sea Farms, CA). The samples of ova (approximately 50 ml) and pooled milt (20 ml) were transported 1 hour to UCD in a temperature-controlled chamber at 15 °C. Upon arrival, sperm were examined for motility. Eggs of two females (referred to here as Female 1 and Female 3) were fertilized (1:100 milt dilution), silted with 5 ppt Fuller’s earth (Sigma) for 1 hour and incubated in tanks in a flow-through water (12.7 °C) re-cir culating system. At start of hatching, temperature was gradually increased to 15.2 °C. Microinjections were initiated after 90% of the larvae had hatched. Microinjection of newly hatched larvae (stage 36) was performed on June 21, 2004 as described belo w. Non-injected control larvae were subjected to anesthesia and similar handling procedures. After injections, larvae were

allowed to recover in fresh water at 15.7 °C before stocking in rearing tanks. The

maintenance of the larval culture is described in detail below.

Se-L-Met was dissolved in sterile vial s using sterile millipure water to 2.11, 6.34, and 19.03 mg/mL. Target doses were calcula ted as described below. The injection volume was 25 nL per larvae, so the target Se doses were 3, 9 and 27 μg/g (dw). Controls were non-injected, sham-injected ( 25 μL millipure water), and L-Met injected larvae (as a control for increased exposure to methionine). Three concentrations of LMet (MW 149.2; Sigma-Aldrich, St. Louis, MO) were used for additional control (2.11, 6.34, and 19.03 mg/g).For each progeny, a total of 445 larvae were used (365 injected and 80 noninjected). Non-injected, sham-injected, a nd low, medium, and high Se-L-Met groups of

80 larvae were each distributed into one surv ival (n=30) and one sampling (n=50) tank. For low, medium, and high L-Met groups, 30 larvae of each group were stocked in a survival tank. All tank assignments were random. Daily monitoring of survival and abnormalities was similar to study one . Larvae were randomly sampled for morphology and Se c ontent at two hours post injection (stage 36), at stage 40 (pyloric sphinc ter formation), and at stage 45 (yolk depletion). Sampled larvae were anesthetized in 150 ppm MS222, overdosed in 500 ppm MS-222, rinsed in millipure water, then frozen on dry ice and stor ed at –80 °C (for Se analysis) or fixed in 10% buffered formalin containing 4% sucrose (for morphology). In addition, each treatment was sample d at the start and endpoint of the experiment to determine live body weight a nd total length. Total lengths of newly

hatched larvae were measured in 10-15 i ndividuals under a dissecting scope, using a digital image tablet (± 0.01 mm). Larvae at the yolk depletion stage (stage 45) were measured with a caliper (± 0.5 mm). Larvae were blotted and weighed individually (wet weight) on a microbalance (± 0.0001 g) then pl aced in tared whirl packs, lyophilized, and weighed to determined dry weights.Larval culture maintenance Microinjection experiments were carried out at CABA at UCD. Indoor flowthrough water recirculation systems with bi ological filter, aeration, YSI thermostat, chiller, and heater were used for all phases of the experiments. Eggs and larvae were held in circular fiberglass ta nks (28 cm diameter, 35 cm depth, flow rate 1.0 – 1.5 L/min). Dissolved oxygen and temperature were recorded daily. A ce rtified calibrated thermometer (National Institute of Standa rds and Technology) was used to adjust incubation temperature to 15.7 ± 0.1 °C. Disso lved oxygen was above 90% saturation in all tanks. Natural photoperiod was maintained by artificial light, and approximately 80% of light was blocked by shade-cloth tank c overs. Ammonia in water ranged from less than 0.003 to 0.014 mg/L NH 3 and pH ranged from 8.2 to 8.3 (DNRA laboratory, UCD).

Se concentration in the water of recirculation systems was be low 1.3 μg/L at the initiation of experiments and 1.2 μg/L at the endpoint of experiments (Flame AA on digested samples, SM3114B and SM303E; Sierra Footh ill laboratory, Inc., Jackson, California; certified for water Se analysis at UCD facilities). Key sturgeon developmental st ages were used to initiate treatments, to collect tissue samples for Se and to conduct morphol ogical analysis at midpoints and endpoints of each experiment. Table 3-2 shows developmental staging following Dettlaff et al

. (1993) for Russian sturgeon, with specific char acteristics for white sturgeon described by Beer (1981) and Deng (2000; Table 3-2). There were slight vari ations in stag e within the sample due to the fast rate of development.Microinjection Newly hatched larvae (stage 36) with normal morphology and behavior (Beer 1981; Dettlaff et al. 1993) were anesthetized in 150 ppm solution of MS-222 at 15.7 °C for approximately one minute. The 100 x 15 Integrid Petri plate (Falcon #1012) was turned upside down and a water saturated Whatman filter paper (90 x 90 cm) placed on top. Using a transfer plastic pipette, ten larvae were lined up in two columns on the paper top for microinjection. The injection site was the upper mid portion of the yolk sac, between the duct of Cuvier and yolk veins, to avoid puncturing the major blood vessels. On average 10 larvae were injected within 2 minutes. After injections, larvae were

allowed to recover in fresh water at 15.7 °C before stocking in tanks. Microinjections were performed using a pressure injector (programmable picoinjector IM 300, Narishige Group, Japa n), a joystick micromanipulator (MN-151, Narishige group, Japan) mounted on a magne tic stand, and aluminosilicate needles (A150-100-10, Sutter Instrument s Co., Novato, CA). Needles were pulled from aluminosilicate tubing (1 mm OD, an d 0.67 mm ID) using a programmable Flaming/Brown micropipette puller (P-97, Sutte r Instruments Co., Novato, CA) to obtain a 10 μm OD and 100 to 150 μm long needle tips. Pulled needles were coated (Sigmacote, Sigma, St. Louis, MO), be veled (EG-44 Microgrinder, Narishige Group, Japan), back filled with approximately 5 μL of injection solution followed by an oil plug

of ca. 10 μL (paraffin oil, #76235, Fluka Production, Germany) using Eppendorf microloaders. Each needle was individually calibrated using a stereoscope (Wild M38) at 400 magnification, equipped with a pre-calib rated eyepiece micrometer. Needle calibration was done by injecting solution into paraffin oil and measuring the dispenseddrop with the eyepiece microm eter. Volume adjustment was done by increasing or decreasing injection time while maintaining th e pressure constant at about 20 psi. The target nanoinjection volume throughout the experiments was 25 ± 2.5 nL (approximately 0.1 % volume of the average size white sturgeon egg). All injections were done under a stereoscope at 160 total magnification (Wild M38), with an X-Y movable stage equipped with a plate holder.

Selenium dosing Se-L-Met (> 98% pure, product # S3132, Si gma-Aldrich, St Louis, MO) was used for the microinjection experiments. Am ount of Se-L-Met (MW 196.1) used for the

different treatments was calculated based on th e target Se dosing per dry weight basis. The newly hatched larvae (stage 36) is 7.35 ± 0.06 and 7.03 ± 0.09 mg (dw) when incubated at 14 and 17 °C respectively (Wang et al. 1985). For calculations of approximate doses of Se, and preparation of stock solution, we assumed the average white sturgeon hatched larv ae to be 7.1 mg (dw) for our temperature 15.7 °C.

Selenium analysis Larvae were frozen on dry ice and stored at -80 o C until preparation for analysis. Each sample (n = 10) was freeze-dried and homogenized using a 0.5 ml mortar and pestle. The homogenates were repeatedly freeze-dried and approxi mately 2 mg of dry material was used for analysis. Total Se concentrations were analyzed by microdigestion followed by fluorometry (Fan et al. 1998). Samples were digested in a mixture

of nitric and sulfuric acid s and gradually heated to 130 o C. The samples were thenreduced to selenite in 6 N HCl heated to 110 °C. The selenite was derivatized to piazselenol by the addition of 0.1% diami nonaphthalene (DAN) reagent (prepared by sonicating 2,3-diaminonaphthalene hydrochlorid e, Dojindo Laboratories (Japan), in 0.1 N HCl for 1 h, filtered through 0.45 μ m cellulosic filter, and washed three times with excess cyclohexane) and extracted using 0.5 ml of cy clohexane. This mixture was incubated at 45 °C in the dark for 30 min, and then shak en to complete the extraction into the cyclohexane layer. Two hundred microliters of the cyclohexane layer was measured for the fluorescence intensity of the piazselenol derivative usi ng a spectrofluorometer. The minimum detection limit was typically 1 μ g/L Se. Quality control measures included the use of spiked samples, duplication and blanks with every analytical run. Quality control measurements agreed with the target concen tration within 15 per cent. Post-injected larvae of microinjection study two were analyzed by hydride generation ICP-AES, following the methods described above for the maternal experiment.

Assessment of larval development Larvae were assessed visually for gross developmental abnormalities throughout the experiment and in preserved larval samp les. The development was monitored using the morphological criter ia described by Dettlaff et al. (1993) for Russian sturgeon, with specific characteristics for white sturgeon described by Beer (1981) and Deng (2000). The occurrence of edema, lordosis, kyphosis, and scoliosis was recorded. Developmental

defects observed in mortalities were in cluded in total occurrence rate.Statistical Analysis Data analysis was performed using JMP-In v. 5.1 (SAS Institute Inc.). Data transformations were used when necessary to meet the assumptions of parametric models. Nonparametric tests were used when the assu mptions of parametric models could not be

met. Measurements of larval size were compared between exposure groups using oneway nested ANOVA (with data transformation), Kruskal-Wall is, or Rank F-Test (Neter

et al. 1996). All hypothesis multiple comparison testing was performe d with either the Tukey-Kramer honestly significant difference (HSD), Dunnets, or least squares means Student’s t-test, at a significance level of α = 0.05. A two-way nested ANOVA model was applied to Se concentrations in developing white sturgeon following logtransformation for data from the maternal experiment and no transformation for microinjection data. All Se values are ba sed on dry weight, unless otherwise noted. Developmental defects were compared using logit analyses. The logit equations were used to estimate median effective doses (ED 50 ) for developmental defects. ANOVA models were also applied to percent deve lopmental defects (two-way nested ANOVA for maternal data, one-way ANOVA for microinjection data). This proportional data was first angular transformed as θ = arcsin 

### √ (Y + 3 / 8 / n + 3 / 4 ), where Y is the observed

number of affected individua ls and n is the sample size, following Anscombe (1948).

This transformation accommodates data with several near-zero values (Zar 1999).

Wilcoxon Rank Sums or Rank F-Tests were used for nonparametric data. For the hypothesis comparisons of developm ental effects in microinjection study two , the control non-injected and sham-injected groups we re not significantly different and were combined into one group. Similarly, the high, medium and low L-methionine sampleswere combined into one group since they were not significantly different from each other. Hypothesis comparisons of developmental e ffects were not possibl e in microinjection study one because only one progeny replicate was us ed. Kaplan-Meier survival analysis was applied to mortality data. The mean and standard deviation of Kaplan-Meier distributions were compared using the least squares means Student’s t-test.

Results Size of Developing White Sturgeon Maternal Exposure No differences were found in weight or length of larvae from female white sturgeon exposed to dietary Se between exposure groups at any of the larval developmental stages (ANOVA or Ra nk F-Test, p > 0.2; Table 3-3).

Microinjection The weights and lengths of larvae from microinjection study one were not measured. At the start of microinjection study two , newly hatched Female 1 larvae had significantly greater weight (Wilcoxon Ra nk Sums; p < 0.0001) but were shorter (ANOVA; p = 0.006) than Female 3 larvae (Table 3-4a). Size comparisons at the end of microinjection study two indicate that larvae from the high Se-L-Met treatment ( ca. 16

μ g/g Se) weigh less than all other groups (nested ANOVA, p < 0.0001; Tukey HSD, α = 0.05; Table 3-4b). However, the high Se-L -Met sample only included larvae from Female 3, which had lower initial weight than larvae from Female 1. This was due to thelimited sample size of this experiment and the prioritization of other analyses. Therefore, the actual affect of high Se-L-Met on larval weight is unclear.

Selenium in Developing White Sturgeon Maternal Exposure Se concentrations in developing white st urgeon were significantly greater in the treatment group for each developmental stag e sampled (two-way nested ANOVA, p < 0.0001; Tukey HSD, α = 0.05; Table 3-5; Figure 3-1) . Comparisons within each exposure level showed no difference in Se concentrations between eggs, embryos and larvae (two-way nested ANOVA, p < 0.0001; Tukey HSD, α = 0.05; Table 3-5). All Se values are based on dry wei ght, unless otherwise noted.

Microinjection Table 3-6 and Figure 3-2 show Se concentr ations in larvae of both microinjection experiments. In microinjection study one Se concentrations are shown for larvae one day after microinjection ( ca. stage 36) and at the experimental endpoint (stage 45). Control groups (endogenous Se) exhibited 2.60 (non-inje cted) and 2.54 (sham-injected) Se (μg/g) at the initiation of the experiment. Post-inj ection Se concentrations in low, med and high treatment groups were 15.8, 21.7 and 46.6 μg/g respectively, approximating target Se doses of 10, 20 and 40 μg/g. At the end of the experiment, Se was measured in the survivors of each group. High (8.99 ± 0.34 μg/g) and medium (10.70 ± 0.34 μg/g) Se treatment groups had lower concentration of Se than at the start of the experiment (Se

shown as mean ± standard error of three re plicate tanks). The lo w treatment group larvaecontained 13.92 ± 1.03 μg/g Se at the experime ntal endpoint. Cont rol non-injected and sham-injected groups exhibited 5.43 ± 0.14 and 5 .19 ± 0.17 μg/g Se, twice as much as the start of the experiment. The discrepancy ma y relate to two different methods of Se analysis and different sample sizes at the st art and end of the experiment (micro-digestion with Fluorometry and n of 5 – 10 at star t; macro-digestion with ICP-AES and n of ca. 45 at end; Table 3-6). There was insufficient data (one progeny rep licate) to perform a statistical comparison of th e above Se concentrations. In microinjection study two Se concentrations are shown for larvae 1 to 2 hours post injection ( ca. stage 36), mid-experiment (stage 40), and end-experiment (stage 45; Table 3-6). Control groups (endogenous Se) exhibited 6.35 ± 0.35 (non-injected) and 5.89 ± 0.77 (sham-injected) Se (mean μg/g ± st andard error of the two progeny cohorts) at the start of the experiment. Post-inject ion Se concentrations in low, med and high treatment groups were 8.74 ± 0.36, 8.96 ± 0.2, and 16.55 ± 1.62 μg/g respectively, which differed from target exposure levels of 3, 9 and 27 μg/g Se. At 1 to 2 hours post injection and at mid-experiment (stage 40), larv ae in the high treatment group contained significantly higher Se than th e other treatment groups and controls (two-way, nested ANOVA, p < 0.0001; Tukey HSD, α = 0.05; Figure 3-2). Additionally, a separate analysis of the mid-experiment data showed that Se concentrations in each of the three treatment groups were significantly higher than in the controls (ANOVA p < 0.002;

Dunnets α = 0.05; Figure 3-2). No other significant differences were observed between Se concentrations between experi mental groups or time points.Developmental Defects in Larvae Maternal Exposure Developmental defects measured included edema and the skeletal deformities, lordosis, kyphosis, and scoliosis (Figure 3-3). Approximately 1,000 larvae from a total of six females were measured for developmental de fects. Logit analysis showed significant associations between the occurrence of edema ( χ 2 = 39.7) and skeletal deformities ( χ 2 = 46.3) and Se concentrations in larvae (p < 0.0001; Tables 3-7 & 3-8; Figures 3-4 & 3-5). Logit probabilities were used to estimate that 10, 25 and 50 percent of the population

would incur edema at ca. 21, 25, and 30 μ g/g Se in larvae, respectively (Figure 3-4). Skeletal deformities would be expected in 10, 25 and 50 percent of the population at ca. 17, 24, and 32 μ g/g Se in larvae, respectively (Figure 3-5). Either of the effects, edema or skeletal deformities, would significantly re duce the chances of survival in the wild. Thus, logit analysis was also applied to the occurrence of edema and/or skeletal

deformities in order to measure the total number of affected larvae. There was a significant association between the total percentage of affected larvae and Se concentrations in both eggs and larvae ( χ 2 = 68.9 and 66.5, respectively, p < 0.0001; Table 3-9; Figures 3-6 & 3-7) . Logit probabilities were us ed to estimate that 10, 25 and 50 percent of the population would become affect ed (edema and/or skeletal deformity) at

ca. 15, 22, and 29 μ g/g Se in larvae, respectively (Figure 3-6). The effective doses are nearly identical for Se in eggs (Figure 3-7). Hypothesis testing of the percentage of developmental defects observed in larvae compared across exposure level and developmental stage revealed some differences (Table 3-10). Overall, the greatest differe nce between control and treatment groups wasin the total percentage of larvae affected (edema and/or skeletal deformities; two-way nested ANOVA following angular tran sformation, p = 0.004; Tukey HSD, α = 0.05). Considered separately, both edema (p = 0.037) and skeletal deformities (p = 0.002) were observed in greater numbers in the treat ment group (two-way nested ANOVA following angular transforma tion; Tukey HSD, α = 0.05). Each type of skeletal deformity was also analyzed separately. Only scoliosis was f ound to appear more often in the treatment group (Wilcoxon Rank Sums, p < 0.025).

Microinjection As with the maternal exposure experi ment, developmental defects measured included edema and the skeletal deformities: lordosis, kyphosis, and scoliosis. For microinjection study one , logit analysis showed significant associations between the occurrence of edema ( χ 2 = 42.7) and skeletal deformities ( χ 2 = 147) and Se concentrations in larvae at the onset of larval developmen t (p < 0.0001; Table 3-11; Figures 3-8 & 3-9). Logit probabilities were used to estimate th at 25 and 50 percent of the population would

incur edema at ca. 13 and 42 μ g/g Se in larvae, respectivel y (Figure 3-8). Skeletal deformities would be expected in 15, 25 and 50 percent of the population at ca. 10, 18,

and 32 μ g/g Se in larvae, respectively (Figure 3-9) . As with the maternal experiment, the total measurement of affected larvae (edema and/or skeletal deformities) was used to reflect the overall impact on each experiment al group. Logit probabilities were used to estimate that 25 and 50 percent of the populati on would become affected (edema and/or skeletal deformity) at ca. 7 and 17 μ g/g Se in larvae, respectively ( χ 2 = 202.8, p < 0.0001;Table 3-11; Figure 3-10). Approximately 500 larvae from one female were measured for developmental defects in study one

. For microinjection study two , logit analysis showed significant associations

between the occurr ence of edema ( χ 2 = 132.3) and skeletal deformities ( χ 2 = 26.4) and Se

concentrations in larvae at the onset of larval devel opment (p < 0.0001; Table 3-12; Figures 3-11 & 3-12). Logit probabilitie s were used to estimate that 15, 25 and 50 percent of the population would incur edema at ca. 11, 13 and 15 μ g/g Se in larvae, respectively (Figure 3-11). Skeletal defo rmities would be expected in 15, 25 and 50 percent of the population at ca. 15, 17, and 21 μ g/g Se in larvae, re spectively (Figure 3- 12). Logit probabilities were used to estimate that 15, 25 and 50 percent of the population would become affected (ede ma and/or skeletal deformity) at ca. 10, 12, and

15 μ g/g Se in larvae, respectively ( χ 2 = 122.7, p < 0.0001; Table 3-12; Figure 3-13). Approximately 500 larvae from a total of two females were measured for developmental

defects in study two

. Hypothesis testing of the percentage of developmental defects observed in yolk sac larvae compared across exposure levels revealed some differences (Table 3-13; study

two ). The high Se treatment showed signi ficantly higher percen tages of edema and lordosis (Rank F-Test, p < 0.005; Tukey HSD, α = 0.05). Greater numbers of larvae with edema were observed in the low Se treatment compared to the medium Se treatment (one-way ANOVA following angular transformation, p ≤ 0.05, Tukey HSD, α = 0.05). A greater percentage of skeletal deformities in general (lordosis, scoliosis, or kyphosis) and of total affected larvae (edema and/or skeletal deformities) was observed in the high treatment group (one-way ANOVA fo llowing angular transformation, p ≤ 0.05, TukeyHSD, α = 0.05). The control non-injected and sham-injected groups were not significantly different and were combined into one group for the above comparisons (Students t-test, p > 0.4). Similarly, the hi gh, medium and low L-methionine data were combined into one group since they were not significantly different from each other (Students t-test, p > 0.4). H ypothesis comparisons of developmental effects were not possible in microinjection study one because only one progeny replicate was used.

Combined Maternal an d Microinjection Data Data from the maternal and microinjecti on experiments were combined to provide a more robust analysis of developmental defects following Se e xposure during larval development. Logit analysis of the comb ined data showed significant associations

between the occurr ence of edema ( χ 2 = 199.8), skeletal deformities ( χ 2 = 340.1) and Se concentrations in larvae at the onset of larval developmen t (p < 0.0001; Tables 3-7 – 3-9, 3-11 & 3-12; Figures 3-14 & 3-15). Logit proba bilities were used to estimate that 15, 25 and 50 percent of the populat ion would incur edema at ca. 18, 26 and 40 μ g/g Se in larvae, respectively (Figure 3-14). Skeletal deformities would be expected in 15, 25 and 50 percent of the population at ca. 18, 23, and 34 μ g/g Se in larvae, respectively (Figure 3-15). Logit probabilities were used to estimate that 15, 25 and 50 percent of the population would become affected (ede ma and/or skeletal deformity) at ca. 11, 15, and

22 μ g/g Se in larvae, respectively ( χ 2 = 543.2, p < 0.0001; Figure 3-16). All Se values are from stage 36 larvae and represent exposures at the onset of larval development.Mortality in Larvae Maternal Exposure Mortality in larvae was analyzed using Kapl an-Meier survival anal ysis. Approximately 14,000 larvae from a total of six females were monitored for survival. Data are categorized in two ways for the analysis: by maternal dietary exposure level and by high ( ca. 21 μ g/g), medium ( ca. 9 μ g/g) and low ( ca. 2 μ g/g) ranges of Se concentrations in eggs. Both approaches revealed significan t differences in the probability of mortality over time (in days post-hatch; Table 3-14; Figure 3-17). When comparing between maternal dietary exposure levels, we find a proba bility of 0.03 (3 percen t) of mortality in treatment larvae by the end of larval de velopment (Kaplan-Meier, p < 0.0001). Comparison of the Kaplan-Meier distribu tions shows that th e treatment group is significantly different from the control gr oup (Student’s t-test, p < 0.0001). However, when the data are categorized by the amount of Se in eggs, we observe a greater difference between groups and a probability of 0.08 (8 percent) of mortality in the high group ( ca. 21 μ g/g) by the end of larval development (Kaplan-Meier, p < 0.0001). Differences between these Kaplan-M eier distributions follow High ( ca. 21 μ g/g Se) >> Low ( ca. 2 μ g/g) > Medium ( ca. 9 μ g/g; Student’s t-test, p < 0.0001).

Microinjection

Incidence of mortalities a nd the percentage of developmental effects observed within mortalities for the microinjection studies are s hown in Table 3-15. Both microinjection studies show a statistically si gnificant difference in the probability of larval mortality between exposure levels, ove r time (Kaplan-Meier, p < 0.0001; Figure 3-18). In microinjection study one , the probability of larval mo rtality in low, medium, and high treatment groups by the end of larv al development were approximately 0.35, 0.45 and 0.65, respectively (Kaplan-Meier, p < 0.0001). Comparison of the Kaplan-Meier

distributions shows th e relationship high ( ca. 46 μ g/g Se) > medium ( ca. 22 μ g/g Se) > low ( ca. 16 μ g/g Se) > control ( ca. 3 μ g/g Se; Student’s t-test, p ≤ 0.008). Approximately

500 larvae from one female were monitored for survival in study one

. In microinjection study two , the low and medium treatment groups are categorized as one group (low) due to indistinguishable Se concentrations. The probability of larval mortality in low and high treatment groups by the end of larval development was approximately 0.05 and 0.70, respectively (Kap lan-Meier, p < 0.0001; Table 3-15; Figure 3-18). Differences between Kaplan -Meier distributions follow high ( ca. 17 μ g/g Se) > low ( ca. 9 μ g/g Se) > control ( ca. 6 μ g/g Se; Student’s t-test, p < 0.001). Approximately

500 larvae from a total of two females were monitored for survival in study two

.

Combined Maternal an d Microinjection Data Table 3-16 summarizes percent mortalit y in white sturgeon larvae following Se exposure using the combined data of the maternal and microinjection studies. The combined data are categorized by Se concentr ations in larvae at the start of larval development ( ca. stage. 36; after hatching in matern al study; several hours post injection in microinjection studies). Ka plan-Meier survival analysis of combined mortality data shows a statistically significant differen ce between exposure categories (p < 0.0001; Figure 3-19). Differences between Kaplan -Meier distributions follow very high ( ca. 46

μ g/g Se) >> high ( ca. 18 – 22 μ g/g Se) = mid ( ca. 12 – 16 μ g/g Se) > low ( ca. 8 – 10μ g/g Se) > control ( ca. 2 – 7 μ g/g Se; student’s t-test, p < 0.05). The probability of mortality by the end of larval developm ent in larvae exposed to 12 to 22 and 46 μ g/g Se were approximately 0.15 to 0.29 and 0.65, re spectively (Kaplan-Meier, p < 0.0001; student’s t-test, p < 0.05).

Discussion This work shows that white sturgeon ar e susceptible to Se-induced developmental toxicity at environmentally relevant levels . The observed effects of Se on larval development included edema, skeletal deformities and mortality. No impact on the size

of larvae was observed. Se maternal transport resulted in signif icant Se burdens persisting in developing larvae (Table 3-5). Se appears to be conserved throughout the yolk utilization period (Figure 3-1). Chapter two of this dissertation revealed that maternally transferred Se was predominantly in the form of yolk proteins. The incorporation of se lenoproteins in larval

tissue and increase of Se in circulation could both contribut e to the conservation of Se during larval development. The excretory mechanisms are limited during the yolk sac phase of larval development in sturgeon, and the efficient mechanisms of gill excretion and glomerular filtration seem to be fully established only at the end of yolk absorption (Dettlaff et al. 1993; Wrobel 2003). The microinjection studies were designed to mimic maternal transfer of Se. Microinjection of Se-L-Met into yolk sac larv ae proved to be a reliable method to study the effects of excess Se on the developmen t of this species. In microinjection study one we successfully targeted the exposure levels of 10, 20 and 40 μg/g Se in larvae (Table 3-6; Figure 3-2). The observed increase of Se in control larvae between the start and end of the experiment was most likely due to differe nt sample sizes (n = 60 and 10, respectively) and different analytical methods empl oyed for the two analyses (ICP-AES and Fluorometry, respectively). In microinjection study two , the actual Se concentrations in microinjected larvae differed from the target exposure levels of 3, 9 and 27 μg/g Se, but essentially provided two le vels of Se exposure ( ca. 9 and 17 μ g/g; Table 3-6; Figure 3-2). In both microinjection studies, we were una ble to accurately obtain target exposures levels that were less than 10 μg/g apart. Small amounts of the compound may have diffused out of the embryo or larvae dire ctly following injection, due to the water solubility of Se-L-Met. Overall, microinj ection of white sturgeon larvae resulted in environmentally relevant levels of Se exposures . Actual exposure levels of Se in larvae ranged from ca. 8 to 47 μg/g (Table 3-6; Figure 3-2) . The loss of Se between the start and end of the microinjection studies contradi cts the findings of the maternal study. This may be due to the fact that Se in the yolk of maternally exposed larvae was protein bound while the microinjected larvae received Se in the form of free amino acids. The observed effects of Se on larval development included edema, skeletal deformities (lordosis, kyphosis and scoliosis), and mortality. Simila r types of deformities were observed in the maternal exposure and microinjection studies. Developmental defects were low in the control groups of all studies (< 4%; Ta bles 3-10, 3-11 & 3-13). Exposure to ca. 10 μ g/g Se led to developmental defects in 0 to 13 percent of maternally exposed larvae (T1 and T3; Table 3-10) and 5 to 13 percent of micr oinjected larvae (low and medium dose of study two ; Table 3-13). Exposure to ca. 16 to 47 μ g/g Se led to very high incidence of developmental defects in microinjected larvae (70 – 84%; Tabl es 3-11 & 3-13).However, maternally exposed larv ae in cohort two (T2) containing ca. 20 μ g/g Se showed considerably less incidence of edema and/or skeletal defects (28% by the end of yolk absorption; Table 3-10), suggesting that seleno-amino acid injection produced more

of an acute effect. In both the maternal exposure and mi croinjection studies, developing larvae showed statistically significant relationships be tween Se concentrations in larvae and the occurrence of edema and skeletal deformities (Logit, p < 0.0001; Figures 3-4 – 3-16). Comparisons of defect occurrence rates be tween exposure groups (Tables 3-10 & 3-13) were less useful than logit analysis, which linked the Se exposure of each progeny cohort directly to the observe d effects. Logit proba bility equations were used to estimate the median effective Se dose (ED 50 ) for each defect (Table 3-17). The term ED X is used here to represent the level of Se content in developing larvae that leads to developmental defects in X percent of the tested population. The probability of developing either edema or skeletal deformities was used to estimate the overall impact on development of larvae exposed to excess Se. In general, the microi njected larvae appeared to be more sensitive to Se; however, many of the ED X values are comparable between studies (Table 3-17). By combining the data of all three studies, it is estimated that larval Se concentrations of

11 and 22 μ g/g will lead to edema and/or skeletal deformities in 15 and 50 percent of the population, respectively. Mortality increased with increasing larval Se concentrations in both the maternal and microinjection studies (Tables 3-14 & 3- 15; Figures 3-17 & 3- 18). Microinjected larvae showed higher levels of mortality co mpared to larvae in the maternal exposure experiment. In the maternal study, 8 pe rcent mortalities was observed with ca. 20 μ g/gSe exposure, but similar Se exposures were a ssociated with 45 to 70 percent mortalities in the microinjection studies. Mortality was very low or absent in the control groups of all studies (< 2%; Tables 3-14 & 3-15). Wh en considering the combined data from all experiments, the probabilities of mortality by the end of yolk absorption in larvae exposed to 12 to 22 and 46 μ g/g Se were approximately 15 - 29 and 65 percent, respectively (Kaplan-Meier, p < 0.0001; student’s t-te st, p < 0.05; Figure 3-19). There are certainly differences between the microinjection and maternal exposure studies. Mortality was significantly higher in treatment larvae from the microinjection experiment (10 – 70%) compared to the mate rnal exposure experime nt (0 – 8.5%; Tables 3-14 & 3-15). Similarly, edema and/or skeletal deformities occurred at lower Se levels in microinjected larvae (ED 50 = 14.5 – 16.8 μ g/g Se) than in larvae exposed to Se-laden maternal yolk (ED 50 = 28.8 μ g/g Se; Table 3-17). The higher sensitivity of the microinjection larvae is likely due to the effect s of injection stress, fo rm of Se used (free amino acids in microinjection versus protein bound in matern al exposure), and the route of Se exposure (injected versus incorporated into yolk proteins). Microinjected larvae may have absorbed the free Se-L-Met very quickly. Conversely, the maternally exposed larvae absorbed protein-bound Se, as well as vital nutrients, as they digested yolk over

time. This could have resulted in different rates of Se exposure, which may have led to the observed differences between these experi ments. The timing of the production of a critical enzyme in Se detoxification (superoxi de dismutase) has been shown to influence the sensitivity of larvae to Se toxicity (Palace et al. 2004). If greater levels of free Se-LMet were absorbed prior to th e production of key detoxifying enzymes or cofactors, then greater sensitivity would be expected. Th ese suggestions remain speculative untiladditional work elucidates the physiology of larval white sturgeon during exposure to excess Se. Nevertheless, both exposure routes led to adverse effects at Se concentrations exceeding ca. 11 to 15 μg/g in eggs and larvae. The very low control mortality and abnormality rates indicate that white sturgeon la rvae tolerate microinjection well, and that microinjection of yolk sac larvae is a good model for investigating Se-induced toxicity in early life stages. The observed effects on larvae agree with those described in several field and laboratory experiments. The most common types of developmental defects produced by Se toxicity are severe edema and various fo rms of terata, including spinal curvatures (Gillespie and Baumann 1986; Lemly 1993b; Woock et al. 1987). Holm et al. (2005) found 41.85 and 31.85 μg/g Se (converted from we t weight using 80% moisture) in the eggs of rainbow trout ( Oncorhynchus mykiss ) and brook trout ( Salvelinus fontinalis )

collected from seleniferous ar eas in Canada. These eggs pr oduced larvae with edema and

craniofacial and skeletal deformities. G illespie and Baumann (1986) spawned female bluegills ( Lepomis macrochirus) from a high Se reservoir with egg Se concentrations of 12 to 55 μg/g. The resulting larvae exhibited edema, deformities and complete mortality. Other field and laboratory studies have shown similar results in bluegill (Coyle et al. 1993; Hermanutz et al. 1992; Woock et al. 1987). Schultz and Hermanutz (1990) experimentally demonstrated that fathead minnow ( Pimephales promelas ) embryos containing ca. 19.5 μg/g Se (converted from wet wei ght using 80% moisture) resulted in larvae with edema and spinal deformities. Hamilton et al. (2002a) found that eggs from wild razorback suckers ( Xyrauchen texanus ) containing greater than 20 μg/g Se represented a high level of hazar d to this endangered species.Several possible mechanisms of Se toxi city have been described. Palace et al. (2004) demonstrated that selenomethionine expo sure can lead to oxida tive stress in fish larvae, which is known to cause edema and spinal curvatures. They linked superoxide production to methioninase enzyme ac tivity and the ensu ing metabolism of selenomethionine. Superoxide activity aff ects the embryo until significant production of superoxide dismutase provides a detoxificat ion pathway. Individual variation in the expression of either of these enzymes could lead to different timing and frequencies of effects. Additionally, Se can be incorporated into biological molecules in place of sulfur

because the two elements have similar chem ical properties (Diplock 1976; Stadtman 1974). However, Se and sulfur bonds may have different strength and function in biological molecules (Reddy and Massaro 1983), thus some Se-substituted compounds may become unstable or dysfunctional (Lemly 1998; Stadtman 1974). Se can also inhibit protein synthesis (Vernie and Van Leewe nhoekhuis 1987) and reac t with sulfhydryl groups of proteins or other molecules (Mar tin 1973), causing disrupt ions of histogenesis and deformities, which are a common effect of Se toxicity (Lemly 1998). These results indicate that white sturge on populations that bioaccumulate elevated Se may experience decreased recruitment. White sturgeon in San Francisco Bay are exposed to high levels of Se through their diet, evidenced by high Se levels in common prey of white sturgeon (Johns and Luoma 1988; Linville et al. 2002; White et al. 1988), as well as in sturgeon muscle, liver and eggs (Kroll and Doroshov 1991; Urquhart and Regalado 1991; White et al. 1987; White et al. 1988; White et al. 1989). White sturgeon sampled from San Francisco Bay-Delta between 1986 and 1990 contained Se at concentrations ranging from 9 to 30 μ g/g in liver (n= 52) and 7 to 15 μ g/g in muscle (n =99; Urquhart and Regalado 1991; White et al. 1988). Very limited data are available on Se concentrations in the eggs of wild wh ite sturgeon. Out of six sturgeon females sampled in the San Francisco Bay-Delta regi on in the 1990’s, one contained eggs with 3

μ g/g Se, four had eggs ranging from 8 to 12 μ g/g Se and one female contained eggs with

29 μ g/g Se (Kroll and Doroshov 1991). Recentl y, three white sturgeon captured from San Francisco Bay-Delta were found to have 7 to 20 μ g/g Se in ovaries containing developing eggs (Doroshov La b, UCD; unpublished data). In the work described here (chapters 2 and 3) female sturgeon with liver Se

concentrations of 11.0 and 11.6 μ g/g produced larvae with 13 and 28 percent occurrence of developmental defects, respectively (Table 3-18). The remaining treatment female, that was successfully induced to spawn (T1), contained 8.7 μ g/g Se in the liver and produced larvae that were free of developmental defects. In these limited data, maternal brood fish Se muscle concentrations app ear to correspond with the incidence of developmental defects in progeny (Table 3-18). If there is a true relationship between muscle and egg Se levels, then white st urgeon populations could be monitored by analyzing non-lethal core samples of muscle. Se levels in eggs and larvae provide an estimate of the di rect exposure of Se to the developing larvae during yolk uti lization. Such data probably provide the best prediction of Se-toxicity in developing larvae but are very difficult to obtain from wild populations, although naturally spawned eggs can be collect ed using spawning mats. The limited data available suggest that white sturgeon in th e San Francisco Bay-Delta are carrying egg Se burdens that present a significant hazard to their progeny. Table 3-19 shows potential hazard threshold levels for Se in developi ng white sturgeon, based on both the maternaland microinjection exposure expe riments presented here. Larv al Se concentrations of 3.4

and 8.2 μ g/g are predicted to result in edema and/or skeletal deformities in 5 and 10 percent of developing larvae, re spectively. This Se hazard threshold range is based on the observed development of ca. 1,000 maternally exposed larvae and 1,000 microinjected larvae, and the survival of ca. 14,000 maternally exposed larvae and 1,000 microinjected larvae during the course of these experiments. Egg Se levels could potentially be substituted for these larval threshold values, based on the conservation of Se observed throughout the early development in the maternal experiment (dry weight values only; Figure 3-1). Most of the wild sturgeon egg Se data from San Francisco BayDelta fall above this threshold range. This work indicates that Se toxicity is likely contributing to the reduced recruitment obser ved in the white sturgeon population of San Francisco Bay-Delta (California Departme nt of Fish and Game Commission 2006; Schaffter and Kohlhorst 1999). In summary, white sturgeon larvae expos ed to Se by maternal transfer and microinjection demonstrated significant in creases of mortality and abnormality rates (including edema and skeletal deformities) in larvae containing Se concentrations above

ca. 11 to 15 μg/g. The larval Se exposures consisted of environmentally relevant Se levels and revealed defect and mortality ra tes similar to those observed in field and laboratory studies of Se toxicity in other fi sh. This work shows that Se toxicity is expected to cause significant impacts in white sturgeon populations that bioaccumulate elevated levels of Se. A hazard threshold range of ca. 3 to 8 μ g/g Se in developing white sturgeon is suggested for this species.Se content of experimental diets in maternal exposure experiment

Diet Measured Total Selenium ( μ g/g) Control 1.42 ± 0.03 Treatment 34.04 ± 0.82

Table 3-1. Selenium averages ± standard error (dw) are shown. Selenium was added to the diet as selenized yeast (Selenomax®, Ambi Inc.).Key developmental stages in sturgeon Stage 5 2nd cleavage The onset of the first cleavage is indicated by the appearance of a furrow in the animal pole of th e fertilized egg. Due to the holoblastic cleavage and yolky e ggs in sturgeon, the second cleavage furrow appears in the an imal pole resulting in four incompletely divided cells shari ng yolk and cytoplasm material. White sturgeon eggs reach the stage 5 about five hours post fertilization at 15.7 °C (Deng, 2000).

Stage 19 – 23 neurulation The neural folds begin to form around the head region (stage 19), extend to the trunk region (stage 20), and come close together (stage 21-22) to form a neural t ube. By stage 23 the neurulation is completed and a suture-line between the two folds is clearly visible.

Stage 29 S-shaped heart The heart primordium forms as a straight tube, bends as a C-tube, and later as an S-shaped tube (s tage 29). At stage 29 heart beat commences. Stage 36 peak hatch At this stage, hatched yolk sac larvae are 10-11 mm in total length, darkly pigmented, and are relative ly underdeveloped. They lack eye lenses, mouth, functional gills, and have continuous median fanfold.

Stage 40 pyloric sphincter formation At this stage a pyloric sphincter forms between the anterior portion (future stomach) and the posterior po rtion (future intestine) of the yolk sac. The intestinal porti on is void of yolk material, but stomach is not differentiated and is filled with yolk.

Stage 45 yolk depletion At this stage all the fins and fin rays have differentiated. The mouth is well developed and equipped with teeth. Four barbells are located in the ventral side of the snout anterior to the mouth. The gill filaments project from the operculum. Stomach, liver, pancreas, and spleen are differentiated, and some fish had extruded the yolk plug and are ready to start exogenous feeding.

Table 3-2. Developmental staging following Dettlaff et al . (1993) for Russian sturgeon, with specific characteristics for white sturgeon described by Beer (1981) and Deng (2000).Size of larvae from control and treatment female white sturgeon

Weight of Larvae During Development

Control Treatment Mean Weight (mg) Standard Error n Mean Weight (mg) Standard Error n Stage 36 21.86 2.328 3 22.72 3.116 3 Stage 40 24.77 2.066 2 25.82 4.127 3 Stage 45 35.16 4.013 2 32.60 2.472 3

Length of Larvae During Development

Control Treatment Mean Length (mm) Standard Error n Mean Length (mm) Standard Error n Stage 36 11.00 0.186 3 11.55 0.605 3 Stage 40 13.39 0.391 2 14.22 0.338 3 Stage 45 17.27 0.693 2 17.80 0.323 3 n=number of progeny cohorts

Table 3-3. Weight (mg) and length (mm) of larvae at three developmental stages. Larvae are progeny of female white sturgeon exposed to either control ( ca. 1.4 μ g/g) or treatment ( ca. 34 μ g/g) dietary Se for approximately six months during vitellogenesis. Approximately 30 larvae were measured for n number of progeny cohorts. No significant differences were detected between control and treatment larvae for any developmental stage (nested ANOVA or Rank F-Test; p > 0.2).Size of larvae in Microinjection Study Two Weight (mg) Length (mm) Female 1 24.23 ± 1.82 12.48 ± 0.33 Female 3 17.66 ± 2.22 12.85 ± 0.35

Table 3-4a. Initial length and weight (mean ± st andard deviation, n=15) of larvae from Female 1 and Female 3, used for microinjection study two. Newly hatched Female 1 larvae had significantly greater weight (Wilcoxon Rank Sums; p < 0.0001) but were shorter (ANOVA; p = 0.006) than Female 3 larvae.

Weight (mg) Length (mm) Non-injected 42.55 ± 2.96 b 18.45 ± 0.39 ab Sham-injected 42.28 ± 1.74 b 18.60 ± 0.50 a L-Met Low * 45.04 a 18.57 ab L-Met Med 41.76 ± 2.51 b 18.33 ± 0.23 ab L-Met High 41.74 ± 1.00 b 18.09 ± 0.01 bc

Se-L-Met Low 42.49 ± 2.52 b 18.50 ± 0.27 a Se-L-Met Med 42.44 ± 2.45 b 18.67 ± 0.30 a Se-L-Met High § 37.87 c 17.56 c

Table 3-4b. Weight and length (mean ± standard error of the two pr ogeny cohorts; 10 – 15 larvae per cohort) of larvae at stage 45 ( yolk depletion) from treatment and control groups in microinjection study two. Within each measurement type, levels not connected by same letter are significantly diffe rent (nested ANOVA, p < 0.0001; Tukey HSD α = 0.05). *Sample included larvae from Female 1 only, which we re initially higher in wei ght and shorter in length than larvae from Female 3.

§ Sample included larvae from Female 3 only, which were initially lower in weight and longer in length than larvae from Female 1.Selenium in developing white sturgeon progeny from female whitesturgeon exposed to dietary selenium.Se μg/g ± Standard Error (n)TissueControlTreatmentPercent Moisture (Avg ±Standard Deviation)Egg2.25 ± 0.326 (3)12.4 ± 2.801 (4)‡63.7 ± 4.1Stg. 5 Embryo2.32 ± 0.279 (3)11.78 ± 2.711 (4)‡75.7 ± 2.3Stg. 21 Embryo2.21 ± 0.304 (3)8.92 ± 1.173 (3)‡73.8 ± 2.2Stg. 33 Embryo1.95 ± 0.173 (3)12.04 ± 2.98 (3)‡79.5 ± 5.1Stg. 36 Larvae2.26 ± 0.295 (3)12.58 ± 3.113 (3)‡76.5 ± 1.1Stg. 40 Larvae2.34 ± 0.54 (2)13.07 ± 3.875 (3)‡84.5 ± 3.4Stg. 45 Larvae2.28 ± 0.315 (2)13 ± 3.44 (3)‡92.4 ± 1.5Table 3-5.Se in developing white sturgeon progenyof females exposed toeither control (ca.1.4μg/g) or treatment (ca.34μg/g)dietary Se for approximately six months duringvitellogenesis. Data displayed as mean Seμg/g (dw)±standard error (n = progenycohorts; 60 – 90 larvae per cohort).‡Significant difference from the corresponding control value (Student’st-test, p < 0.0001).Selenium in developing white sturgeon exposed to seleno-L-methionine by microinjection Study One Post-injection Se Stage 45 Se Non-injected 2.60 5.43 ± 0.14 Sham-injected 2.54 5.19 ± 0.17 Low 15.8 13.92 ± 1.03 Med 21.7 10.71 ± 0.34 High 46.6 8.99 ± 0.34 a) Study Two Post-injection Se Stage 40 Se Stage 45 Se Non-injected 6.35 ± 0.35 (2) 5.36 ± 0.3 (2) 6.27 ± 1.21 (2) Sham-injected 5.89 ± 0.77 (2) 6.27 ± 0.04 (2) 5.81 ± 0.59 (2) Low 8.74 ± 0.36 (2) 7.89 ± 0.15 (2) 9.46 ± 0.47 (2) Med 8.96 ± 0.2 (2) 9.59 ± 0.34 (2) 8.51 ± 1.85 (2) High 16.55 ± 1.62 (2) 17.61 ± 1.04 (2) 10.13 (1) b)

Table 3-6. Selenium in developing white sturge on exposed to seleno-L-methionine by microinjection. a) Microinjection Study One using progeny of one female white sturgeon. Whole larvae selenium content (μg/g, dw) one day post injection (stage ca. 36; ICP-AES analysis, n = ca. 45 larvae) and at the experi mental endpoint (stage 45; Fluorometric analysis, n = 5 – 10 larvae, data displayed as μ g/g Se ± standard error of sample duplicates). b) Microinjection Study Two using progeny of two female white sturgeon. Whole larvae selenium c ontent one day post injection (stage ca. 36), midexperiment (stage 40) and at the experiment al endpoint (stage 45). All analysis by Fluorometry; data displayed as μ g/g Se ± standard error of progeny replicates (n = progeny cohorts; 5 – 10 larvae per cohort). Stat istical analysis presented in Figure 3-2. Percent moisture in stages 36, 40 and 45 was 69.4 ± 5.6, 84.6 ± 2.3 and 84.3 ± 2.9, respectively (average ± standard deviation).Occurrence of edema in larvae from female white sturgeon exposed to dietary selenium shown by progeny cohort

Control Treatment

Cohort Percent with Edema Larval Se ‡ Cohort Percent with Edema Larval Se ‡ Stage 36 C3 0.00 (1) 2.43 T1 0.00 (1) 11.60 C4 0.00 (1) 1.69 T2 0.00 (1) 18.40 C5 0.00 (1) 2.67 T3 6.67 (1) 7.75 Stage 40 C4 0.00 (3) 1.80 T1 0.00 (3) 11.60 C5 0.00 (3) 2.88 T2 4.44 ± 2.22 (3) 20.40 T3 1.67 ± 1.67 (2) 7.22 Stage 45 C4 0.00 (3) 1.96 T1 0.00 (3) 12.00 C5 0.00 (3) 2.59 T2 15.56 ± 1.11 (3) 19.40 T3 0.00 (2) 7.61

Table 3-7. Percentage of larvae affected by edem a for each progeny cohort of six female white sturgeon exposed to dietary selenium pr esented as average perc entage of replicate samples ± standard error of replicates (num ber of replicate samples, containing 30 larvae each*). Data are shown for each of the larv al developmental stages sampled. Logit analyses of the data is presented in Figure 3-4. ‡ Selenium concentration shown in μ g/g and based on dry weight. *Some replicate samp les contained slightly less than 30 larvae.Occurrence of skeletal deformit ies in larvae from female white sturgeon exposed to dietary selenium shown by progeny cohort

Control Treatment

Cohort Percent with Skeletal Defects Larval Se ‡ Cohort Percent with Skeletal Defects Larval Se ‡ Stage 36 C3 0.00 (1) 2.43 T1 0.00 (1) 11.60 C4 0.00 (1) 1.69 T2 0.00 (1) 18.40 C5 0.00 (1) 2.67 T3 10.00 (1) 7.75 Stage 40 C4 1.11 ± 1.11 (3) 1.80 T1 0.00 (3) 11.60 C5 1.11 ± 1.11 (3) 2.88 T2 14.44 ± 1.11 (3) 20.40 T3 8.33 ± 1.67 (2) 7.22 Stage 45 C4 0.00 (3) 1.96 T1 0.00 (3) 12.00 C5 0.00 (3) 2.59 T2 21.11 ± 1.11 (3) 19.40 T3 13.33 ± 3.33 (2) 7.61

Table 3-8. Percentage of larvae affected by sk eletal deformities (lordosis, kyphosis, or scoliosis) for each progeny cohort of six fe male white sturgeon exposed to dietary selenium. Data shown as average percentage of replicate samples ± standard error of replicates (number of replicate samples, co ntaining 30 larvae each*). Data are given for each of the larval developmental stages sample d. Logit analyses of the data is presented in Figure 3-5. ‡ Selenium concentration shown in μ g/g and based on dry weight. *Some replicate samples contained slightly less than 30 larvae.Affected larvae (edema and/orskeletal deformities) of femalewhite sturgeon exposed to dietary selenium shown by progenycohortControlTreatmentCohortPercentAffected§Egg Se‡Larval Se‡CohortPercentAffected§Egg Se‡Larval Se‡Stage 36C30.00 (1)2.462.43T10.00 (1)11.0011.60C40.00 (1)1.611.69T20.00 (1)20.5018.40C50.00 (1)2.682.67T316.67 (1)7.617.75Stage 40C41.11 ± 1.11 (3)1.611.80T10.00 (3)11.0011.60C51.11 ± 1.11 (3)2.682.88T218.89 ± 1.11 (3)20.5020.40T310.00 ± 0 (2)7.617.22Stage 45C40.00 (3)1.611.96T10.00 (3)11.0012.00C50.00 (3)2.682.59T227.78 ± 2.94 (3)20.5019.40T313.33 ± 3.33 (2)7.617.61Table 3-9.Percentage of larvae affected by edema and/or skeletal deformities for each progeny cohort of six female white sturgeonexposed to dietary selenium. Data are shown as average percentage of replicate samples ± standard error of replicates (numberofreplicate samples, 30 larvae each*). Dataare given for each of the larval developmental stages sampled. Logit analyses of the dataare presented in Figures 3-6 and 3-7.§Larvae counted only once, even if bothedema and skeletal defect occurred.‡Selenium concentration shown inμg/g and based on dry weight.*Some replicate samples contained slightly less than 30 larvae.Occurrence of developmental defects inlarvae from female white sturgeon exposed to dietary seleniumControlTreatmentStageStage364045CombinedStages364045CombinedStagesEdema0 ± 0 (3)0 ± 0 (2)0 ± 0 (2)0 ± 0 (3)2.22 ± 2.22 (3)2.04 ± 1.3 (3)5.19 ± 5.19 (3)3.15 ± 1.75 (3)δLordosis0 ± 0 (3)0 ± 0 (2)0 ± 0 (2)0 ± 0 (3)1.11 ± 1.11 (3)0.37 ± 0.37 (3)0.37 ± 0.37 (3)0.62 ± 0.38 (3)Kyphosis0 ± 0 (3)0.56 ± 0.56 (2)0 ± 0 (2)0.16 ± 0.16 (3)0 ± 0 (3)4.26 ± 3.46 (3)1.85 ± 1.85 (3)2.04 ± 1.29 (3)Scoliosis0 ± 0 (3)0.56 ± 0.56 (2)0 ± 0 (2)0.16 ± 0.16 (3)2.22 ± 2.22 (3)2.96 ± 1.96 (3)8.52 ± 4.27 (3)φ4.57 ± 1.80 (3)φSkeletal‡0 ± 0 (3)1.11 ± 0 (2)0 ± 0 (2)0.32 ± 0.20 (3)3.33 ± 3.33 (3)7.59 ± 4.19 (3)δ11.48 ± 6.16 (3)δ7.47 ± 2.63 (3)δTotalAffected§0 ± 0 (3)1.11 ± 0 (2)0 ± 0 (2)0.32 ± 0.20 (3)5.56 ± 5.56 (3)9.63 ± 5.46 (3)δ13.7 ± 8.02 (3)δ9.63 ± 3.43 (3)δTable 3-10.Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis for progeny of femalewhite sturgeon that wereexposed to dietary selenium. Data shown asaverage percentage of cohort replicates ± standard error of cohort replicates (number ofcohort replicates*). Data are shown for each ofthe larval developmental stages sampled.‡Grouped category of lordosis, kyphosis,and scoliosis.§Larvae with edema and/or skeletal deformities (larvaecounted only once, even if both effects occurred).δSignificantdifference from control value (angular transformed, two-way nested ANOVA, p < 0.05; Tukey HSD,α= 0.05).φSignificant differencefrom control value (Wilcoxon Rank Sums, p < 0.05). *Each cohort replicate represents progeny ofone female. Cohort replicatesforstage 36 contained 25 – 30 larvae each. Cohort replicates for stages 40 and 45 contained 60 – 90 larvae each.Occurrence of developmental defects in larvae exposed to seleno-L-methionineMicroinjection Study OneControl (Non-Injected)(2.6μg/g Se*)Sham-Injected(2.54μg/g Se*)Low Dose(15.8μg/g Se*)Med Dose(21.7μg/g Se*)High Dose(46.6μg/g Se*)Edema0 ± 0 (1)0 ± 0 (1)63.12 ± 0.96 (1)49.98 ± 4.44 (1)35.64 ± 6.18 (1)Lordosis0 ± 0 (1)3.95 ± 2.61 (1)17.48 ± 6.73 (1)30.61 ± 5.3 (1)40.6 ± 5.23 (1)Kyphosis0 ± 0 (1)0 ± 0 (1)9.7 ± 2.56 (1)9.18 ± 1.78 (1)23.76 ± 1.72 (1)Scoliosis0 ± 0 (1)0 ± 0 (1)0 ± 0 (1)8.17 ± 3.67 (1)1.99 ± 0.98 (1)Skeletal‡0 ± 0 (1)3.95 ± 2.61 (1)27.17 ± 7.95 (1)47.97 ± 7.35 (1)66.33 ± 7.72 (1)TotalAffected§0 ± 0 (1)3.95 ± 2.61 (1)76.69 ± 4.86 (1)83.66 ± 3.67 (1)78.21 ± 7.72 (1)Table 3-11.Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis fromMicroinjection Study One. Larvae weremicroinjected with a varying seleno-L-methionine after hatching. Data shown as average percentage ± standard error of threereplicate tanks (number of cohort replicates♦). Data span early larvaldevelopment (stages 36 – 45).‡Grouped category of lordosis, kyphosis, and scoliosis.§Larvae with edema and/or skeletal deformities (larvae counted only once, even if both effects occurred).♦Each cohort replicate represents progeny of one female. Each of the three replicate tanks containedca.35 larvae.*Se exposure at the beginning of larval development (stage 36; Table 3-6).Occurrence of edema in larvae frommicroinjection study twoshown by progeny cohortProgeny F1Progeny F3Seμg/g∗PercentEdemaPercentSkeletal‡PercentTotal Affected§Seμg/g∗PercentEdemaPercentSkeletal‡PercentTotal Affected§Control (non-injected)6.700.003.333.336.000.000.000.00Sham-injected6.660.000.000.005.120.000.000.00Low Se9.108.008.0012.008.3813.333.3313.33Medium Se8.770.003.453.459.160.006.676.67High Se18.1780.0030.0080.0014.9455.1713.7958.62Table 3-12.Percentage of larvae with developmental defects for each progeny cohort ofMicroinjection Study Two, used for logitanalyses (Figures 3-11 – 3-13). Data spanearly larval development (stages 36 – 45)and represent approximately 30 larvae pertreatment.‡Selenium concentration shown inμg/g and based on dry weight.*Selenium concentration represents themicroinjected dose as measured postinjection. Se values are shown inμg/g and based on dry weight.‡Grouped category of lordosis, kyphosis, and scoliosis.§Larvae with edema and/or skeletaldeformities (larvae counted onlyonce, even if both effects occurred). Average data of the two cohorts are shown in Table 3-13.Occurrence of developmental defects in larvae exposed to seleno-L-methionineMicroinjection Study TwoControl♦(6μg/g Se*)L-Met Control•(Se not analyzed)Low Dose(8.7μg/g Se*)Med Dose(9μg/g Se*)High Dose(16.6μg/g Se*)Edema0 ± 0 (2)aδ0 ± 0 (2)aδ10.91 ± 3.64 (2)bδ0 ± 0 (2)aδ67.8 ± 13.56 (2)cδLordosis0 ± 0 (2)aδ0 ± 0 (2)aδ3.64 ± 0 (2)bδ3.39 ± 0 (2)abδ15.25 ± 5.08 (2)bδKyphosis0.85 ± 0.85 (2)1.19 ± 1.19 (2)0 ± 0 (2)0 ± 0 (2)1.69 ± 1.69 (2)Scoliosis0 ± 0 (2)1.19 ± 1.19 (2)1.82 ± 1.82 (2)1.69 ± 1.69 (2)5.08 ± 1.69 (2)Skeletal‡0.85 ± 0.85 (2)a2.38 ± 2.38 (2)ab5.45 ± 1.82 (2)ab5.08 ± 1.69 (2)ab22.03 ± 8.47 (2)bTotal Affected§0.85 ± 0.85 (2)a2.38 ± 2.38 (2)a12.73 ± 1.82 (2)a5.08 ± 1.69 (2)a69.49 ± 11.86 (2)bTable 3-13.Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis fromMicroinjection Study Two. Larvae weremicroinjected with a varying seleno-L-methionine after hatching. Data shown as average percentage of cohort replicates ± standarderror of cohort replicates (number of cohort replicatesθ). Data span early larval development(stages 36 – 45). Within the same row,levels not connected by same letter are significantly different (one-way ANOVA following angular transformation, p≤0.05, TukeyHSD,α= 0.05). Data are not compared between rows.δRank F-Test, p < 0.005; multiple comparisons by Tukey HSD,α= 0.05.θEach cohort replicate represents progeny of one female and contained approximately 30 larvae per treatment.♦Combined control non-injected and sham-injected samples (not significantly different; students t-test, p > 0.4).•Combined L-Met control samples (not significantly different; students t-test, p > 0.4).‡Grouped category of lordosis, kyphosis, and scoliosis.§Larvae with edema and/or skeletal deformities (larvae counted only once, even if both effects occurred).*Se exposure at the beginning of larval development (stage 36; Table 3-6).Mortality in larvae from female white sturgeon exposed to dietary selenium

Stg 40 Stg 45 Egg Se Percent Mortality Percent Mortality Control 2.15 ± 0.54 (2) 0.0 ± 0.0 (2) 0.27 ± 0.0 (2) Treatment 13.04 ± 4.72 (3) 0.04 ± 0.04 (3) 2.95 ± 2.74 (3) a)

Stg 40 Stg 45 Egg Se Percent Mortality Percent Mortality Low 2.15 ± 0.54 (2) 0.0 ± 0.0 (2) 0.27 ± 0.0 (2) Med 9.31 ± 1.70 (2) 0.07 ± 0.07 (2) 0.22 ± 0.12 (2) High 20.5 (1) 0.0 (1) 8.42 (1) b)

Table 3-14. Percent mortality in larvae from fe male white sturgeon exposed to dietary selenium. Mortality data presented as percent mortality ± standard error (number of progeny cohorts; 1,000 – 1,500 larvae per cohort). Egg selenium data are presented as Se μ g/g (dw) ± standard error (number of progeny cohorts). a) Percent mortality by exposure levels. b) The same data but categorized by low, medium and high selenium concentration in eggs. Statistical analyses of these mortality data are presented in Figure 3-17.Effects as Percentage of MortalitiesStudy OneSe (μg/g; dw)PercentMortalityEdemaSpinalCurvatureEdema andSpinalCurvatureEdema and/orSpinalCurvatureControl (non-injected)2.600.0%0.00%0.00%0.00%0.00%Sham-injected2.540.0%0.00%0.00%0.00%0.00%Low Se15.841.0%4.76%13.33%13.33%31.43%Medium Se21.746.7%9.52%27.62%8.57%45.71%High Se46.667.6%4.76%39.05%22.86%66.67%Study TwoControl (non-injected)6.35 ± 0.35 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)Sham-injected5.89 ± 0.77 (2)1.67 ± 1.67 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)L-Met Lownot analyzed0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)L-Met Mednot analyzed0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)0 ± 0 (2)L-Met Highnot analyzed1.67 ± 1.67 (2)0 ± 0 (2)1.67 ± 1.67 (2)0 ± 0 (2)1.67 ± 1.67 (2)Low Se8.74 ± 0.36 (2)10.00 ± 0 (2)6.67 ± 3.33 (2)1.67 ± 1.67 (2)1.67 ± 1.67 (2)10 ± 0 (2)Medium Se8.97 ± 0.2 (2)3.34 ± 3.34 (2)0 ± 0 (2)3.35 ± 3.35 (2)0 ± 0 (2)3.35 ± 3.35 (2)High Se16.56 ± 1.62 (2)70.00 ± 16.67 (2)46.7 ± 3.33 (2)0 ± 0 (2)20 ± 10 (2)66.67 ± 13.33 (2)Table 3-15.Percent mortality in larvae exposed to selenium via microinjection studies. Mortality datapresented as percent mortality±standard error (number of progeny cohorts).Study oneincluded one progeny cohort (n =100 larvae per treatment), whilestudy twoincluded two progeny cohorts (n = 30 larvae per each cohort). Selenium exposure is represented by concentrations in subsamplesoflarvae several hours following microinjection (ca.stage 36). Se data are presented asμg/g Se (dw)±standard error (number ofprogeny cohorts). The percentage of defects observed in mortalities are displayed as percent defect±standard error (number ofprogeny cohorts). Statistical analyses of these mortality data are presented in Figure 3-18.Combined mortality in larvae from mate rnal exposure and microinjection studies

Grouped Exposure Categories Average Percent Mortality Control (~2 – 7 μ g/g Se) 0.44 ± 0.31 (5)

Low (~8 – 10 μ g/g Se) 5.35 ± 2.25 (5) Mid (~12 – 16 μ g/g Se) 31.54 ± 16.01 (3) High (~18 – 22 μ g/g Se) 47.25 ± 22.59 (3) Very High (~46 μ g/g Se) 67.62 (1)

Table 3-16. Percent mortality in larvae from mate rnal and microinjec tion studies of Se exposure in developing white st urgeon. Mortality data presented as percent mortality ± standard error (number of progeny cohorts). Th e combined data are categorized by the Se concentrations in larvae at the start of larval development ( ca. stage. 36; after hatching in maternal study; several hours post injecti on in microinjection st udies; values in dry weight). Statistical analyses of combined mortality data are presented in Figure 3-19.Probabilities of development defects in larvae containing excess SeSkeletalDeformitiesED15ED25ED50LogitEquationMaternal Exposure20.024.431.9P(skeletal deformity) = 1/(1+e(-(-4.64 + 0.145 * (Larval Se ug/g)))Microinjection Study One10.318.332.3P(skeletal deformity) = 1/(1+e(-(-2.54 + 0.08 * (Larval Se ug/g)))Microinjection Study Two14.717.221.4P(skeletal deformity) = 1/(1+e(-(-5.59 + 0.26 * (Larval Se ug/g)))Combined Data17.623.533.7P(skeletal deformity) = 1/(1+e(-(-3.63 + 0.11 * (Larval Se ug/g)))EdemaED15ED25ED50LogitEquationMaternal Exposure22.625.329.9P(edema) = 1/(1+e(-(-7.12 + 0.238 * (Larval Se ug/g)))Microinjection Study One*13.541.8P(edema) = 1/(1+e(-(-1.62 + 0.04 * (Larval Se ug/g)))Microinjection Study Two11.512.814.9P(edema) = 1/(1+e(-(-7.62 + 0.51 * (Larval Se ug/g)))Combined Data17.725.839.8P(edema) = 1/(1+e(-(-3.12 + 0.08 * (Larval Se ug/g)))Total Affected (Edema and/or Skeletal Deformity)ED15ED25ED50LogitEquationMaternal Exposure**17.921.928.8P(affected larvae) = 1/(1+e(-(-4.58 + 0.159 * (Larval Se ug/g)))Microinjection Study One0.56.516.8P(affected larvae) = 1/(1+e(-(-1.79 + 0.11 * (Larval Se ug/g)))Microinjection Study Two10.712.114.5P(affected larvae) = 1/(1+e(-(-6.56 + 0.45 * (Larval Se ug/g)))Combined Data11.115.222.2P(affected larvae) = 1/(1+e(-(-3.48 + 0.16 * (Larval Se ug/g)))Table 3-17.Probabilities of developmental defects in larvae containing elevated Se levels at the beginning of larval development.Probabilities calculated from logit equations (p < 0.0001; Figures 3-4 – 3-16). EDXrepresents the level of Se in larvae (μg/g, dw) expectedto cause a particular effect in X percent of the population. Se concentrations are from larvae at the beginning of larval development (stage36). Defects were measured throughout the period of yolk utilization (ca.first 10 days post-hatch). Data from each experiment is shownboth separately and combined.*Could not be calculated.**Nearly identical to probabilities based on Se in eggs.Se (μg/g; dw) in adult tissues and their progeny related to incidence of developmental defectsExposureFish/ProgenyIDLiverMuscleOvarianEggLarvae‡PercentDevelopmentalDefects*ControlC31.331.281.492.462.430δControlC40.801.221.651.611.690ControlC52.161.481.382.682.670TreatmentT18.729.936.5311.0011.60TreatmentT211.6015.3013.2020.5018.427.78 ± 2.94TreatmentT311.0011.105.747.617.7513.33 ± 3.33Table 3-18.Se concentrations (μg/g; dw) in liver, muscle, ovarian tissue, and eggs from femalewhite sturgeon exposed to eithercontrol (ca.1.4μg/g) or treatment (ca.34μg/g) dietary Se for approximately six months during vitellogenesis (described in Chapter2). Se concentration and occurrence ofdevelopmental defects are shown for the progeny of each female (n = 25 – 30 larvae forstage36; n = 60 - 90 larvae for stages 40 and 45)..‡Newly hatched larvae (stage 36)*Occurrence of edema and/or skeletal deformities at the end of yolk sac developmentδData from stage 36 only, due to very low hatchPotential Hazard Threshold Levels for Se in developing white sturgeon larvae

ED 05 ED 10 Se μ g/g (dry wt) Se μ g/g (wet wt)* Se μ g/g (dry wt) Se μ g/g (wet wt)* Edema 2.2 0.6 11.8 3.2

Skeletal Deformities 6.4 1.7 13.3 3.6

Edema and/or Skeletal Deformities 3.4 0.9 8.2 2.2

Table 3-19. Estimated probabilities of developm ental defects in larvae from both maternal and microinjec tion experiments. ED X represents the leve l of Se in stage 36 larvae expected to cause a particular eff ect in X percent of the population (Logit, p < 0.0001; Table 3-17). These effect levels are presented as potential hazard threshold levels for Se in newly hatched white sturge on larvae. Defects were measured throughout the period of yolk utilization ( ca. first 10 days post-hatch). Egg Se levels could potentially be substituted for the stage 36 Se thresholds suggested here since maternally transferred Se was conserved throughout early development in the maternal experiment (dry wt; Figure 3-1). *Estimated using the average percent moisture of stage 36 larvae from both studies, 73% ± 5.3 (ave rage ± standard deviation).Selenium in developing pr ogeny of female white sturgeon exposed to dietary selenium

Figure 3-1. Selenium concentrations in eggs an d developing embryos and larvae from white sturgeon females exposed to dietary sele nium. Selenium is presented as mean Se μ g/g (dw) ± standard error. The number of cohorts from individual maternal sturgeon are presented in each bar. Each cohort sample contained approximately 30 individuals. Groups not connected by the same letter are significantly different (lognormal, two-way nested ANOVA, p < 0.0001; multiple comparisons with Tukey HSD, α = 0.05). Data are also presented in Table 3- 5.Selenium in developing whitesturgeon exposed to seleno-L-methionine by microinjectiona) Study Oneb) Study TwoFigure 3-2.Selenium concentrations in developing white sturgeonlarvae following microinjection of seleno-L-methionine.a)Microinjection Study Oneusing progeny of one female whitesturgeon. Whole larvae seleniumcontent at the start (one day postinjection, stageca.36; ICP-AES analysis; n =ca.45 larvae) and end of the experiment (stage 45, Fluorometry analysis, n = 5 – 10larvae, data displayed asμg/g Se ± standard error of sample duplicates).b)Microinjection Study Twousing progeny of two femalewhite sturgeon. Whole larvae selenium contentat the start (one day post injection, stageca.36), middle (stage 40) and at theexperimental endpoint (stage 45). All analysis by Fluorometry; n = 5 – 10 larvae; data displayed asμg/g Se ± standard error ofprogeny replicates. All values from Study Two include two progeny replicates except the end, High treatment. Groups not connectedby the same letter are significantly different(two-way, nested ANOVA,p < 0.0001; Tukey HSD,α= 0.05). *Value is significantlydifferent from control(ANOVA p < 0.002; Dunnetsα= 0.05). Data are also presented in Table 3- 6.d)b)a)e)d)c)Figure 3-3.Developmental defects observed in white sturgeon during larval development. a)A normal larvae. b) Larvae withedema (overt swelling due to fluid accumulation). c) Larvaeshowing kyphosis (dorsoventral curvature of anterior region). d)Larvaeshowing lordosis (concave dorsoventral curvature of posterior region). e) Larvae withscoliosis (lateral skeletal curvature)andedema.Progeny of selenium-exposed white sturgeonlarvae with edema related to larval selenium(a) (b)Figure 3-4.Percentage of larvae with edema related to selenium concentrations in larvae (μg/g; dw). Larvae were progeny of adult whitesturgeon females exposed to dietary selenium. Data includesthree progeny cohorts from the control group and three from the treatmentgroup. Data for three larval development stages are included(stages 36, 40 and 45). Logit analysis shows a significant association betweenselenium concentrations in larvae and the occurrence of defects (χ2= 39.7, p < 0.0001; prob(edema)= 1/(1+e(-(-7.12 + 0.238 * (Se in Larvae ug/g))). (a)The right y-axis shows the actual percentage of affected larvae in samples containing 30 – 90 larvae each. The left y-axis shows theprobability of larvae to develop edema at a given larval selenium concentration. (b) ED50(29.90μg/g), ED25(25.28μg/g) and ED10(20.67μg/g) values were calculated from the logit equation.Progeny of selenium-exposed white sturgeonlarvae with skeletal deformities related to larval selenium(a) (b)Figure 3-5.Percentage of larvae with skeletal deformitiesrelated to selenium concentrations in larvae (μg/g; dw). Larvae wereprogeny of adult white sturgeon females exposedto dietary selenium. Data includes three progeny cohorts from the control group andthree from the treatment group. Data for three larval development stages are included (stages36, 40 and 45). Logit analysisshows asignificant association between selenium concentrations in larvae and the occurrence of defects (χ2= 46.3, p < 0.0001; prob(skeletaldeformity)= 1/(1+e(-(-4.64 + 0.145 * (Se in Larvae ug/g))). (a) The right y-axis shows the actual percentage of larvae with skeletal deformities insamples containing 30 – 90 larvae each. The left y-axis shows the probability of larvae to develop skeletal deformities at a givenlarval selenium concentration. (b) ED50(31.92μg/g), ED25(24.37μg/g) and ED10(16.81μg/g) values were calculated from the logitequation.Progeny of selenium-exposed white sturgeonlarvae with edema and/or skeletaldefects related to larval selenium(a) (b)Figure 3-6.Percentage of larvae with edema and/or skeletal deformities related to selenium concentrations in larvae (μg/g; dw).Larvae were progeny of adult white sturgeon females exposed to dietary selenium. Data includesthree progeny cohorts from thecontrol group and three from the treatment group. Data for three larval development stages are included (stages 36, 40 and 45). Logitanalysis shows a significant association between selenium concentrations inlarvae and the occurrence of defects (χ2= 66.5, p <0.0001; prob(defects)= 1/(1+e(-(-4.58 + 0.159 * (Se in Larvae ug/g))). (a) The right y-axis shows the actual percentage of affected larvae insamples containing 30 – 90 larvae each. The left y-axis shows the probability of larvaeto develop edema and/or skeletal deformitiesat a given larval selenium concentration. (b) ED50(28.83μg/g), ED25(21.92μg/g) and ED10(15.01μg/g) values were calculated fromthe logit equation.Progeny of selenium-exposed white sturgeonlarvae with edema and/or skeletaldefects related to egg selenium(a) (b)Figure 3-7.Percentage of larvae with edema and/or skeletal deformities related to selenium concentrations in eggs (μg/g; dw).Larvae were progeny of adult white sturgeon females exposed to dietary selenium. Data includesthree progeny cohorts from thecontrol group and three from the treatment group. Data for three larval development stages are included (stages 36, 40 and 45). Logitanalysis shows a significant association between selenium concentrations inlarvae and the occurrence of defects (χ2= 68.9, p <0.0001; prob(defects)= 1/(1+e(-(-4.52 + 0.152 * (Se in Eggs ug/g))). (a) The right y-axis shows the actual percentage of affected larvae in samplescontaining 30 – 90 larvae each. The left y-axis shows the probability of larvae to develop edema and/or skeletal deformities at a givenselenium concentration in eggs. (b) ED50(29.81μg/g), ED25(22.56μg/g) and ED10(15.32μg/g) values were calculated from the logitequation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of edema related to larval selenium exposure Microinjection Study One

Figure 3-8. Percentage of larvae with edema relate d to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected with seleno-L-methionine in microinjection study one . Data includes one progeny cohort and spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between selenium concentrations in larvae and the occurrence of defects ( χ 2 = 42.7, p < 0.0001; prob( edema ) = 1/(1+e (-(-1.62 + 0.04 * (Se in Larvae ug/g)) ). The left y-axis shows the probability of larvae to develop edema at a given larval selenium concentration, as determined by logit analys is. The right y-axis shows the actual percentage of larvae with edema in samples containing ca. 100 larvae each. ED 50 (41.8 μ g/g) and ED 25 (13.5 μ g/g) values were calculat ed from the logit equation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of skeletal deformities related to larval selenium exposure Microinjection Study One

Figure 3-9. Percentage of larvae with skeletal deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected w ith seleno-L-methionine in microinjection study one . Data includes one progeny cohort and span s early larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between selenium concentrations in larvae and the occurrence of defects ( χ 2 = 147, p < 0.0001; prob( skeletal deformity ) = 1/(1+e (-(- 2.54 + 0.08 * (Se in Larvae ug/g)) ). The left y-axis shows the pr obability of larvae to develop skeletal deformities at a given larval sele nium concentration, as determined by logit analysis. The right y-axis s hows the actual percentage of la rvae with skeletal deformities in samples containing ca. 100 larvae each. ED 50 (32.3 μ g/g), ED 25 (18.3 μ g/g) and ED 15 (10.3 μ g/g) values were calcula ted from the logit equation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of skeletal deformities and/or edema related to larval selenium exposure Microinjection Study One

Figure 3-10. Percentage of larvae with edema a nd/or skeletal deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected with seleno-L-methionine in microinjection study one

. Data includes one progeny cohort and spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant asso ciation between selenium concentrations in larvae and the occurrence of defects ( χ 2 = 202.8, p < 0.0001; prob( defects ) = 1/(1+e (-(-1.79 + 0.11 * (Se in Larvae ug/g)) ). The left y-axis shows the proba bility of larvae to develop edema and/or skeletal deformities at a given larval selenium concentration, as determined by logit analysis. The right y-axis shows the actual percentage of affected larvae in samples containing ca. 100 larvae each. ED 50 (16.8 μ g/g) and ED 25 (6.5 μ g/g) values were calculated from the logit equation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of edema related to larval selenium exposure Microinjection Study Two

Figure 3-11. Percentage of larvae with edema re lated to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected with seleno-L-methionine in microinjection study two . Data includes two progeny cohorts and spans ear ly larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between se lenium concentrations in larvae and the occurrence of edema ( χ 2 = 132.3, p < 0.0001; prob( edema ) = 1/(1+e (-(-7.62 + 0.51 * (Se in Larvae ug/g)) ). The left y-axis shows the probability of larvae to develop edema at a given larval selenium concentration, as determined by l ogit analysis. The ri ght y-axis shows the actual percentage of larvae with edema in samples containing ca. 30 larvae each. ED 50 (14.9 μ g/g), ED 25 (12.8 μ g/g) and ED 15 (11.5 μ g/g) values were calculated from the logit equation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of skeletal deformities related to larval selenium exposure Microinjection Study Two

Figure 3-12. Percentage of larvae with skelet al deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected w ith seleno-L-methionine in microinjection study two . Data includes two progeny cohorts and sp ans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between selenium concentrations in larvae and the occurrence of skeletal deformities ( χ 2 = 26.4, p < 0.0001; prob( skeletal deformity ) = 1/(1+e (-(-5.59 + 0.26 * (Se in Larvae ug/g)) ). The left y-axis s hows the probability of larvae to develop skeletal deformities at a given larval selenium concentration, as determined by logit analysis. The right y-axis shows the actual percentage of larvae with skeletal deformities in samples containing ca. 30 larvae each. ED 50 (21.4 μ g/g), ED 25 (17.2 μ g/g) and ED 15 (14.7 μ g/g) values were calculat ed from the logit equation.

### White sturgeon larvae following microinjection of seleno-L-methionine Occurrence of skeletal deformities and/or edema related to larval selenium exposure Microinjection Study Two

Figure 3-13. Percentage of larvae with edema a nd/or skeletal deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Larvae were microinjected with seleno-L-methionine in microinjection study two

. Data includes two progeny cohorts and spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant asso ciation between selenium concentrations in larvae and the occurrence of defects ( χ 2 = 122.7, p < 0.0001; prob( defects ) = 1/(1+e (-(-6.56 + 0.45 * (Se in Larvae ug/g)) ). The left y-axis shows the proba bility of larvae to develop edema and/or skeletal deformities at a given larval selenium concentration, as determined by logit analysis. The right y-axis shows the actual percentage of affected larvae in samples containing ca. 30 larvae each. ED 50 (14.5 μ g/g), ED 25 (12.1 μ g/g) and ED 15 (10.7 μ g/g) values were calculated from the logit equation.

### White sturgeon larvae following selenium exposure Occurrence of edema related to larval selenium exposure Combined Data from Maternal and Microinjection Experiments

Figure 3-14. Percentage of larvae with edema re lated to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Plot includes all data from the maternal exposure and microinjection experiments. Data includes nine progeny cohorts and spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between selenium concentrations in larvae and the occurrence of edema ( χ 2 = 199.8, p < 0.0001; prob( edema ) = 1/(1+e (-(-3.12 + 0.08 * (Se in Larvae ug/g)) ). The left y-axis shows the probability of larvae to develop edema at a given larval selenium concentration, as determined by logit analys is. The right y-axis shows the actual percentage of larvae with edema in samples containing ca. 30 - 200 larvae each. ED 50 (39.8 μ g/g), ED 25 (25.8 μ g/g) and ED 15 (17.7 μ g/g) values were calculated from the logit equation.

### White sturgeon larvae following selenium exposure Occurrence of skeletal deformities related to larval selenium exposure Combined Data from Maternal and Microinjection Experiments

Figure 3-15. Percentage of larvae with skelet al deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Plot includes all data from the maternal exposur e and microinjection experiments. Data includes nine progeny cohorts and spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant association between selenium concentrations in larvae and the occurrence of skeletal deformities ( χ 2 = 340.1, p < 0.0001; prob( skeletal deformity ) = 1/(1+e (-(-3.63 + 0.11 * (Se in Larvae ug/g)) ). The left y-axis s hows the probability of larvae to develop skeletal deformities at a given larval selenium concentration, as determined by logit analysis. The right y-axis shows the actual percentage of larvae with skeletal deformities in samples containing ca. 30 - 200 larvae each. ED 50 (33.7 μ g/g), ED 25 (23.5 μ g/g) and ED 15 (17.6 μ g/g) values were calculat ed from the logit equation.

### White sturgeon larvae following selenium exposure Occurrence of skeletal deformities and/or edema related to larval selenium exposure Combined Data from Maternal and Microinjection Experiments

Figure 3-16. Percentage of larvae with edema a nd/or skeletal deformities related to selenium concentrations in larvae ( μ g/g; dw) at the start of larval development ( ca. stage 36). Plot includes all data from the matern al exposure and microinjection experiments. Data includes nine progeny cohorts a nd spans early larval development ( ca. stages 36 – 45). Logit analysis shows a significant asso ciation between selenium concentrations in larvae and the occurrence of defects ( χ 2 = 543.2, p < 0.0001; prob( defects ) = 1/(1+e (-(-3.48 + 0.16 * (Se in Larvae ug/g)) ). The left y-axis shows the proba bility of larvae to develop edema and/or skeletal deformities at a given larval selenium concentration, as determined by logit analysis. The right y-axis shows the actual percentage of affected larvae in samples containing ca. 30 - 200 larvae each. ED 50 (22.2 μ g/g), ED 25 (15.2 μ g/g) and ED 15 (11.1 μ g/g) values were calculated from the logit equation.

### Mortality in larvae from female white sturgeon exposed to dietary selenium

a)

b) Figure 3-17. Probabilities of mortality in larvae from female white sturgeon exposed to dietary selenium. a) Kaplan-Meier analys is of time to larval mortality shows a statistically significant differe nce between control and treat ment groups (p < 0.0001). b) When data are categorized by selenium concentr ations in the eggs, a larger difference is detected in time to larval mortality (Kap lan-Meier; p < 0.0001). Differences between Kaplan-Meier distributions follow a) Hi gh >> Low > Medium, and b) Treatment > Control (Student’s t-test, p < 0.0001).Mortality in larvae following microinjection of seleno-L-methioninea) Study One b) Study TwoFigure 3-18.Probabilities of mortality in larvae following microinjection of seleno-L-methionine ina) Microinjection Study Oneandb) Microinjection Study Two. Data are categorized bySe in larvae at the onsetof larval development (ca.stage 36). Inmicroinjectionstudy two, the low and medium treatment groups werecombined into one Se exposure group (low,ca.9μg/g Se; Table3- 15). Control non-injected and sham-injected data were combined for both studies.Both studies show astatistically significantdifference in the probability of larval mortality between exposure levels (Kaplan-Meier, p < 0.0001). Differences between KaplanMeier distributions follow a) High > Medium> Low > Control (Student’s t-test, p≤0.008), and b) High > Low > Control (Student’st-test, p < 0.001).

### Mortality in larvae from selenium maternal transfer and microinjection studies.

Figure 3-19. Probability of mortality in larvae using combined data from selenium maternal transfer and microinj ection studies in white sturgeo n. Kaplan-Meier analysis of time to larval mortality shows a statistically significant difference between exposure categories (p < 0.0001). Differences between Kaplan-Meier distributions follow Very High >> High = Mid > Low > Control (student ’s t-test, p < 0.05). Percent mortality presented in Table 3- 16.Conclusions This work shows that white sturgeon experience Se-induced toxicity at environmentally relevant dietary concentratio ns. Juveniles experi enced cholestasis, a potentially fatal liver condition, wh en exposed to diets containing ca. 20 to 53 μ g/g Se as selenized yeast (predominately selenometh ionine). Adult females exposed to ca. 34 μ g/g dietary Se transferred toxic levels of Se to their young through vitellogenesis. Our maternal transfer work is the first to experi mentally demonstrate th e incorporation of Se into the vitellogenin molecule, which suppor ts the earlier hypothesis of Kroll and Doroshov (1991). Most importantly, we have shown that Se uptake into VTG, and ultimately the developing eggs, takes place in a dose-responsive manner. This indicates that even moderate increases, or decreases, in the accumulation of Se by reproductive females will affect the exposure level to their offspring. White sturgeon larvae containing Se concentrations above ca. 11 to 15 Se μg/g, following Se maternal transfer or microinjection, demonstrated significant increases of mortality and abnormality rates (including edema and skeletal deformities). Based on the work presented here, the hazard threshold for Se in developing white sturgeon should be set somewhere between 3

and 8 μ g/g Se in yolk sac larvae (dw). The microinjection of seleno-L-methionine into yolk sac larvae proved to be a reliable method to study the e ffects of excess Se on the deve lopment of this species. Microinjection was used to mimic maternal transfer because the large size and long ovarian cycle of white sturgeon make it di fficult and expensive to study reproductive toxicity in this species un der laboratory conditions. Wh ite sturgeon larvae toleratemicroinjection well, as indicated by absent or low mortality and defects in the shaminjected groups. This model may be useful in future studies. The work presented here is novel because it exposed the actual species of ecological concern to the Se form (organic Se) and exposure rout e (dietary) that is predominant in the natural environment. Avoidance of using a surrogate species is especially important when st udying sturgeon, since this phylogen etically ancient fish is significantly different from modern fish in an atomical and physiological characteristics. In addition, the narrow exposure range used he re provides greater ec ological significance by demonstrating the effects of rela tively small increases in exposure. In San Francisco Bay-Delta, j uvenile white sturgeon are susceptible to toxicity at Se concentrations currently observed in some prey items (i.e., bivalves). In addition, juveniles in this region accumulate Se in thei r tissues at concentrations similar to those observed in our study. This leads us to su spect that juvenile white sturgeon in San Francisco Bay-Delta may be experiencing chol estasis, depending on individual dietary Se exposure. This potentially fatal liver conditi on disrupts the flow of bile, which prevents the excretion of many toxicants and can lead to direct toxicity by damaging liver tissue. The extent of cholestasis in juvenile white sturgeon from San Francisco Bay-Delta should be investigated. For adult white sturge on in San Francisco Bay-Delta, typical Se exposure levels in the benthic food web ( ca. 15 μ g/g) are below the di etary Se level to which reproductive females were exposed in these experiments ( ca. 34 μ g/g). However, the experimental sturgeon accumulated Se conc entrations in liver, muscle and eggs ( ca.

10 – 12 μ g/g Se) that are similar to body burdens of sturgeon in the San Francisco Bay population. The extent of accumulation in liver is most important here since we wereexploring the transport of accumulated Se from the liver to the developing eggs. Our

research indicates that female white stur geon in San Francisco Bay-Delta are likely transferring Se to their offspring. Limited data from this region supports this conclusion. Data from ten white sturgeon females sample d in the San Francisco Bay-Delta region

show 3 to 29 μ g/g Se (dw) in eggs or egg-be aring ovary (Kroll and Doroshov 1991; Doroshov Lab, UCD, unpublished data). Eigh t of those ten sturgeon exhibited egg Se levels above our proposed hazard threshold range of 3 to 8 μ g/g Se in larvae (dw). Our maternal transport study showed strong cons ervation of Se between the egg and larval stages, indicating that egg Se concentrati ons may be interchangeable with larval concentrations (when both ar e calculated on a dry weight basis). Based on this information, it is likely that the accumulation of excess Se by female sturgeon in San Francisco Bay-Delta is affec ting the recruitment of white sturgeon in this region. We observed severe edema and skeletal deformities in larvae containing Se concentrations

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Na + -independent organic anion transporting proteins (OATP) in hepatocyte basolateral membranes (Ferenci et al. 2002; Trauner and Boyer 2003). Following metabolism and intracellular transport, bile constituents ar e secreted from several different transport proteins in hepatocyte canalicular memb ranes, including both adenosine triphosphate (ATP)-dependent and –independent transport systems. The ATP-dependent transport proteins include multi-drug resistance proteins (MDR), multidrug-associated proteins (MRP) and bile salt export pumps (BSEP), wh ich are related to P-glycoprotein (Arrese and Ananthanarayanan 2004; Trauner and B oyer 2003). These ATP-dependent transport proteins remove bile salts, phospholipids, chol esterol, organic ions , phase II conjugates, toxins, and other bile constituents from hepa tocytes into biliary canaliculi (Arrese and Trauner 2003; Ferenci et al. 2002; Trauner and Boyer 2003). ATP-independent chloridebicarbonate anion exchanger prot ein (AE2), found in hepatocy te and bile duct epithelialcell membranes, secretes bicarbon ate into the bile flow (Kanno et al. 2001; Trauner and Boyer 2003). Bile collected in canaliculi, which are formed by the spaces between hepatocytes, flows into intrahepatic bile ductules and ducts, which are formed by biliary epithelial cells. Bile exits the liver through the hepatic bile du cts and is transported to the gall bladder and the intestinal lumen by th e larger cystic and common bile ducts.

The architecture of fish liver differs si gnificantly from that of mammals (Hinton and Couch 1998). Teleosts livers exhibit tubular architecture, characteristic of the lower vertebrates, with canalicu li formed by basolateral pl asma membranes of adjacent hepatocytes and transitional biliary passage ways, the tubular lumena, lined by apical plasma membrane of hepatocytes and by transi tional biliary epithelial cells, the bile preductular epithelial cells (Hampton et al. 1988). White sturgeon also has a tubular liver, as demonstrated in the first chapter of this dissertation. B iliary transport is only beginning to be understood in fish, although it has become clear that some transport mechanisms may be conserved in vertebrate evolution (Boyer et al. 1976; Cai et al. 2000;

Cai et al. 2001; Cai et al. 2002). The uptake of bile acids in fish has been shown to involve a Na + -independent carrier system (Fricker et al. 1994; Fricker et al. 1987; Rabergh et al. 1994). Several studies have identified and functionally characterized the uptake of organic ions by OATPs in skate ( Raja erinacea ) liver (Cai et al. 2002; Jacquemin et al. 1995; Wang et al. 2001). These studies suggested that this prim itive Skate OATP is most closely related to human OATP. Sulfated bile alcohols, rather than bile acids, are commonly the major constituent of fish bile (Karlaganis et al. 1989; Kihira et al. 1984; Tammar 1974).

Fricker et al. (Fricker et al. 1997) demonstrated efficient en terohepatic circulation for abile alcohol sulfate in fish liver, indicating specific hepato-b iliary transport mechanisms for this class of compounds (Fricker et al. 1997). Bile alcohols do not appear to share the transport mechanisms identified fo r bile acids in fish (Fricker et al. 1994). Transport systems involved in the secretion of bile into canaliculi also appear to resemble mammalian systems. MDR transport proteins have been identified in the livers of several fish species (Albertus and Laine 2001; Cai et al. 2003; Doi et al. 2001; Sturm

et al. 2001; Tutundjian et al. 2002). Several studies indicate that MRPs are also involved in the secretion of fish bile (Cai et al. 2003; Rebbeor et al. 2000; Sauerborn et al. 2004).

Ballatori et al. (Ballatori et al. 2000) provided functional and structural evidence for a BSEP protein analogue in the canalicular memb rane of skate liver th at transports both bile salts and bile alcohols into biliary canaliculi (Ballatori et al. 2000). Rabergh et al. (Rabergh et al. 1992) demonstrated that toxicants can disrupt hepatobiliary transport processes in Rainbow Trout. Cholestasis can result from genetic defect s, toxins, disease, inflammation and oxidative stress (Trauner M 2005). Reduced expression and function of bile transport proteins are important factors in the devel opment of cholestasis (Elferink 2003; Trauner

et al. 1998). Structural or functi onal damage to the hepatocyte and biliary epithelial cells

can also contribute to cholesta sis. Toxins can effect the formation and secretion of bile, presumably by affecting transport pr otein expression and function (Trauner et al. 1998).

Schmitt et al. (2000) demonstrated that oxidative stress can lead to cholestasis by reducing the number of functi onal transporters in the canalicular membrane. These

authors concluded that severe glutathione depletion induces ch olestasis with a retrieval of

MRP2 from the canalicular membrane. The impact of oxidative stress on hepaticglutathione are reviewed by Lu et al. (Lu 1999). Hepatocytes under oxidative stress have a reduced ability to reduce oxi dized glutathione (GSSG) to reduced glutathione (GSH),

which is essential for several cellular func tions. Cellular GSH may become depleted under high levels of oxidative stress. Inflam ation can also result in the inhibition of biliary transport proteins due to proinflammatory cytokines in the liver, which also leads to oxidative stress (Spirli et al. 2001; Trauner et al. 1999). Bile acids that are accumulated in liver during cholestasis damage mitochondria, genera te oxidative stress,

and can lead to cell death (Palmeira and Rolo 2004; Rolo et al. 2004; Sokol et al. 1995).

Cholestatis has been shown to induce se veral compensatory changes aimed at establishing alternative elimination pathways for biliary constitu ents (Trauner M 2005). A commonly observed compensatory change is the development of additional hepatic biliary ducts (bile duct hype rplasia), which can be induced by several conditions associated with cholestasis (reviewed by LeSage et al. 2001). The expression of biliary transport proteins is regulated by various nuc lear receptors, which can also initiate compensatory changes in biliary transport systems (Karpen 2002). Such adaptive changes can provide alternative elimination pathways for bile constituents through increased expression of transport proteins and induction of alternative transport proteins in the liver and kidney. Compensatory changes also incl ude increased expressi on of phase I and II detoxifying enzymes (Trauner M 2005). The adaptations discussed here may help to limit the build-up of toxic biliary constituents, however recent work suggests that they are not sufficient to prevent liver injury duri ng chronic cholestasis (Trauner M 2005; Wagner

et al. 2004).While cholestasis and related disease stat es, such as primary biliary cirrhosis and primary sclerosing cholangitis, are well recognized in rodents and mammals, hepatobiliary biliary related disease states are only beginning to be understood in fish species (Cai et al. 2001; Kirby et al. 1995; Lorent et al. 2004). The underlying mechanisms of toxicologically-induced chol estasis should be further investigated.

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Tammar AR. 1974. Bile Salts in Fishes. Florki n, Marcel and Bradley T. Scheer (Ed.). Chemical Zoology, Vol. 8. Deuterostomians, Cyclostomes and Fishes. Xxii+682p. Illus. Academic Press 3, Inc.: New York, N.Y., U.S.a.; London, England. 1974. 595-612. Trauner M, Boyer JL. 2003. Bile salt trans porters: Molecular characterization, function, and regulation. Physiological Reviews 83(2):633-671. Trauner M, Fickert P, Stauber RE. 1999. Infl ammation-induced cholestasis. Journal of Gastroenterology & Hepatology 14(10):946-959. Trauner M, Meier PJ, Boyer JL. 1998. Mol ecular pathogenesis of cholestasis. New England Journal of Medicine 339(17):1217-1227. Trauner M WM, Fickert P, Zollner G. 2005. Molecular regulation of hepatobiliary transport systems: clinical implications for understanding and treating cholestasis. J Clin Gastroenterol. 39(4 Suppl 2):S111-24. Tutundjian R, Cachot J, Leboulenger F, Minier C. 2002. Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot ( Scophthalmus maximus ). Comparative Biochemist ry & Physiology. Part B, Biochemistry & Molecular Biology 132B(2):463-471.Wagner M, Fickert P, Zollner G, Fuchsbichler A, Gumhold J, Silbert D, Zatloukal K, Guo G, Marschall H, Denk H and others . 2004. Role of adaptive hepatobiliary transporter expression and re gulation of bile acid synt hetic enzymes in MDR2-/- Mice: A model for chronic cholestatic live r disease due to sclerosing cholangitis Journal of Hepatology 40 (Supplement 1):163 Wang W, Seward DJ, Li L, Boyer JL, Balla tori N. 2001. Expression cloning of two genes that together mediate organic solute and st eroid transport in the liver of a marine vertebrate. Proceedings of the National A cademy of Sciences of the United States of America 98(16):9431-9436.Appendix B. Summary data on spawning induction and success in white sturgeon fed either control ( ca. 1.42 μ g/g Se) or treatment ( ca. 34 μ g/g Se) diets for approximately six months.Table B-1. Detailed spawning data for white sturgeon fed either control (ca.1.42μg/g Se) or treatment (ca.34μg/g Se) dietsfor approximately six months.DateFish #1PitTag #Tank #SpawnHoldTank #Cont/ TrtFL(cm)BW(kg)2KEggDiameterPI% GVBDProg% GVBDControlComments9/26/2001140A203Trt137.522.50.872.662/12/2002140A203Trt140.525.20.913.100.1310003/13/2002140A203Trt140.525.20.913.100.091000well polar3/22/2002T1123ovulated and good fertility about 50% hatch9/26/2001772A204Cont13623.650.942.852/12/2002772A204Cont138.524.40.923.230.1100no pectorals,gonads a littlefatty3/13/2002772A204Cont138.524.40.923.210.0910003/22/2002C1125no ovulation (in hindsight yolk not well polarized)9/26/2001594D204Cont13824.40.932.882/12/2002594D204Cont139.524.80.913.280.125703/13/2002594D204Cont139.524.80.913.210.101000some fat ingonads3/22/2002C2126no ovulation (in hindsight yolk not well polarized)9/26/20016F06203Trt135.523.90.962.532/12/20026F06203Trt13924.60.923.150.179003/28/20026F06203Trt13924.60.923.190.081000well polarized4/5/2002T2126ovulated and good fertilityabout 50% hatchDateFish #1PitTag #Tank #SpawnHoldTank #Cont/ TrtFL(cm)BW(kg)2KEggDiameterPI% GVBDProg% GVBDControlComments9/26/20014F7F204Cont12119.551.102.732/12/20024F7F204Cont122.520.51.123.210.156703/28/20024F7F204Cont122.520.51.123.190.081000well polarized4/5/2002C3125ovulated and good fertilityvery poor hatch (<1%)9/26/20010005204Cont14025.150.922.752/12/20020005204Cont14225.80.903.200.1510003/13/20020005204Cont14225.80.903.100.1210004/9/20020005204Cont14225.80.903.190.0910077well polarized4/19/2002C4125ovulated9/26/20016D5C203Trt13622.90.912.922/12/20026D5C203Trt13824.10.923.140.131000no left pectoral3/13/20026D5C203Trt13824.10.923.110.121000no left pectoral4/9/20026D5C203Trt13824.10.923.210.091000well polarized4/19/2002T3126partial ovulation9/26/20014304203Trt13324.051.022.712/12/20024304203Trt136.525.91.023.090.15003/13/20024304203Trt136.525.91.023.080.0900all prog at PI = 0.03/28/20024304203Trt136.525.91.023.140.1000all prog at PI = 0.0,med polar.4/25/20024304203Trt136.525.91.023.240.071000well polarized5/3/2002T4125ovulated, hatch almost nothingDateFish #1PitTag #Tank #SpawnHoldTank #Cont/ TrtFL(cm)BW(kg)2KEggDiameterPI% GVBDProg% GVBDControlComments9/26/20016F36204Cont127.519.60.952.742/12/20026F36204Cont13020.80.953.120.16003/28/20026F36204Cont13020.80.953.120.13770med polarization4/25/20026F36204Cont13020.80.953.140.081000well polarized5/3/2002C5126ovulated, hatch about 40%9/26/20013A67204Cont12917.850.832.902/12/20023A67204Cont13121.20.943.140.16003/28/20023A67204Cont13121.20.943.100.1600all prog at PI = 0.04/25/20023A67204Cont13121.20.94atretic6/4/2002C6necropsied9/26/20016D74204Cont140.524.050.872.862/12/20026D74204Cont14225.80.903.170.13003/13/20026D74204Cont14225.80.903.120.1110004/9/20026D74204Cont14225.80.903.100.121000low-med polarization5/8/2002atretic6/4/2002C7necropsied9/26/20011D75203Trt14727.40.862.752/12/20021D75203Trt14928.80.873.090.1340left pectoral curled3/13/20021D75203Trt14928.80.873.090.1200very fatty gonad4/9/20021D75203Trt14928.80.873.100.11770low-med polarization5/8/20021D75203Trt14928.80.873.100.15770low-med polarization5/17/2002T6necropsiedDateFish #1PitTag#Tank #SpawnHoldTank #Cont/ TrtFL(cm)BW(kg)2KEggDiameterPI% GVBDProg% GVBDControlComments9/26/2001192E203Trt129210.982.532/12/2002192E203Trt134.522.70.933.170.181804/9/2002192E203Trt134.522.70.933.230.1510005/8/2002192E203Trt134.522.70.933.220.1510005/17/2002T5125injected, no ovulation, necropsied9/26/20016D0E203Trt148.530.40.932.882/12/20026D0E203Trt148.530.40.933.250.19004/9/20026D0E203Trt148.530.40.933.200.17005/8/20026D0E203Trt148.530.40.933.260.17005/29/20026D0E203Trt148.530.40.933.230.17006/4/2002T8necropsied9/26/2001293B203Trt12818.90.902.352/12/2002293B203Trt12818.90.903.200.22004/9/2002293B203Trt12818.90.903.250.18005/8/2002293B203Trt12818.90.903.270.17005/29/2002293B203Trt12818.90.903.250.17006/4/2002T7necropsied1Control female number C8 (PIT Tag 4519)was atretic on 2/12/02 and later died due to technical error (escaped tank through a rip inthe tank covering).2Body weight for 2/12/02 estimated from fork length.Table B-2. Summary of spawning induction andsuccess in white sturgeon fed either control (ca.1.42μg/g Se) or treatment(ca.34μg/g Se) diets for approximately six months.T1C1C2T2C3C4T3T4C5T5PIT Tag#140A772A594D6F064F7F00056D5C43046F36192E1st Inj Date3/20/20023/20/20023/20/20024/3/20024/3/20024/17/20024/17/20025/1/20025/1/20025/15/20021st Inj Time10:15pm10:20pm10:25pm9:50pm9:50pm10pm10pm10pm10pm9pm2nd Inj Date3/21/20023/21/20023/21/20024/4/20024/4/20024/18/20024/18/20025/2/20025/2/20025/16/20022nd Inj Time9:57am10:00am10:05am10am10am10am10am10am10am9am1st Inj Temp (°C)14.815.013.014.614.614.514.814.714.615.12nd Inj Temp (°C)15.315.214.014.714.715.115.315.315.315.6Ovul Temp (°C)15.015.415.315.315.415.515.5Latency (h)19.021.021.023.025.524.524.0Ovulation %100no ovulno ovul100100100partial (40-50%)100100no ovulVolume Eggs (mls)3250(sampledfollicles)(sampledfollicles)34002550435095022002400(sampledfollicles)# eggs/ml31.15(100%gvbd)(20%gvbd)24.9528.831.5533.6530.330.45(no gvbd)# Eggs Collected(est fecundity)1012388483073440137243319686666073080Used pooled milt forfertilization from # ofmales2334444