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 fortunate 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 grateful. 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 him 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 continued 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 extremely grateful to them for 

their tireless help and advice throughout this research. I am also grateful to Paul Lutes 

and Erik Hallen for their support and advice regarding sturgeon maintenance. 

 Ed DePeters has my sincere gratitude for allowing me extensive use of his lab. 

My work there was successful due to Scott Taylor’s constant support and advice. Silas 

Hung also opened his lab to me, and provided critical support with the experimental diets. 

Silas formulated the juvenile experimental diet and directed its production. He also 

provided the use of his pelleting equipment for 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 direction and training, was critical to that 

research. Similarly, Kevin Kroll’s collaboration 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 Diana 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 work with several undergraduate interns 

including Fiona Sands, Summer Evert, Matthew 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 individuals assisted in sampling, chemical analyses, and 

much more. They worked hard, offered insight, 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 organizations for their financial support through 

fellowships, grants and contracts: the UC Toxic Substances 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 endeavor, I received valuable direction and 

support from several faculty members of the Graduate Group in Ecology. Mike Johnson 

was especially helpful by continuously providing insight and serving as the chair of my 

qualifying examination committee. Keith Miles, Barry Wilson, and Joe Cech also 

contributed to my early studies and planning. I also learned much through extensive 

discussions during seminars and conferences hosted by the Graduate Group in Ecology, 

its Ecotoxicology AOE, and the Lead Campus Program in Ecotoxicology. 

Most of all, I am immensely grateful for the support of family and friends 

throughout this arduous process. My parents, Jess and Edna Linville, 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 brightened my life so much throughout these past 

years including Mikaela Huntzinger, Jennifer Von Aesch, Diana Rowan, Anne Salo, 

Scott Nagy, Marta Sykes, Dena McCann, Darcy Jones, and Hali Rowen. I give my 

fondest thanks and appreciation to my friend and husband, Jed Alexander, who has given 

his continuous love, support 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, significant 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-hatch) 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-response increase of melanomacrophage aggregates in liver, 

the occurrence of cholestasis, a potentially fatal 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 vitellogenesis, 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 μg/g (dw) in muscle, liver and eggs. Transfer of Se to 

the yolk in developing eggs took place through the yolk protein precursor, 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 relationship with Se in 

plasma VTG. Se in eggs was associated with the yolk proteins, lipovitellin, phosvitin and 

immunoglobulin. 

- v - 
Hormonal treatment was used to induce spawning in females. We did not observe 

an impact on reproduction, as shown by fecundity, fertilization success and survival to 

neurulation. Progeny of treatment females (embryos 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; ED25 = 24 μg/g Se in larvae, dw), edema 

(ED25 = 25 μg/g Se; Logit, p < 0.0001), and mortality (8% in larvae with ca. 20 μg/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; ED25 = ca. 17.5 μg/g Se in larvae, 

dw) and edema (ED25 = ca. 13 μg/g Se; Logit, p < 0.0001). Microinjected yolk sac larvae 

experienced a greater incidence of mortality (45 - 70% in larvae with ca. 20 μg/g Se 

Kaplan-Meier, p < 0.0001) as compared to maternally exposed yolk sac 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 μg/g 

Se in developing white sturgeon is suggested for this species. 

In San Francisco Bay-Delta, juvenile 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 this region supports this conclusion. Our 

research shows that elevated Se (ca. 11 – 15 μg/g Se) in white sturgeon eggs leads to 

significant developmental defects and mortality in developing larvae. Based on the work 

presented here, we suspect that white sturgeon 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 low. 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-Delta 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 selenium 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 μg/g Se) 

or treatment (ca. 34 μg/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 morphological characteristics of 

juvenile white sturgeon following six months of dietary 

 selenium exposures 44 

Table 1-5. Gross morphological measurements of juvenile white sturgeon 

following six months of dietary selenium exposure 45 

 

Chapter 2. Dose-dependent maternal transport 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 yolk proteins isolated from eggs 

of adult white sturgeon following 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 plasma proteins as a function 

 of selenium concentrations in liver 102 

Table 2-10. Pearson correlation coefficients for selenium in plasma 

non-vitellogenin proteins correlated to selenium in liver, 

yolk proteins, and eggs 103 

Table 2-11. White sturgeon spawning data 104 

Table 2-12. Reproductive performance of sturgeon females in control 

and treatment groups 105

Chapter 3. Developmental toxicity in white 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 white sturgeon progeny of females 

following dietary selenium exposure 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 microinjection 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 larvae from maternal exposure and 

microinjection studies 173

Table 3-17. Probabilities of developmental defects in larvae containing 

elevated selenium levels at the beginning of larval development 174

Table 3-18. Selenium in adult tissues and their 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 μg/g Se) or treatment (ca. 34 μg/g Se) diets for 

approximately six months. 

Table B-1. Detailed spawning data for white sturgeon fed either control 

(ca. 1.42 μg/g Se) or treatment (ca. 34 μg/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 μg/g Se) or treatment (ca. 34 μg/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 showing eosinophilic granular 

leukocytes 50 

Figure 1-6. Micrograph of control liver showing moderate inflammation 51 

Figure 1-7. Micrograph of control liver showing peribiliary inflammation 52

Figure 1-8. Micrograph showing distended canalicular 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 proliferation in liver of low 

treatment fish 55 

Figure 1-11. Micrograph showing bile duct proliferation 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 proliferation and peribiliary 

inflammation in liver of medium treatment fish 58 

Figure 1-14. Micrograph showing extensive melanomacrophage 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 the 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 transport of selenium via vitellogenin in white 

sturgeon 

Figure 2-1. Hypothesized mechanism of the 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 white 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 concentrations in adult white sturgeon 

after ca. six months of dietary selenium exposures 113 

Figure 2-9. Selenium in plasma collected at mid- and end-experiment 114 

Figure 2-10. Selenium in vitellogenin isolated 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 related to selenium in liver 119 

- xiv - 
Figure 2-15. Selenium in plasma, ovarian tissue and eggs related to 

d) selenium in vitellogenin 120 

Figure 2-16. Selenium in yolk proteins related to selenium in vitellogenin 121 

Figure 2-17. Selenium in isolated yolk proteins as a function of selenium 

in eggs 122 

Figure 2-18. Selenium in ovary, eggs, plasma 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 white 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 females 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 exposed larvae with edema related 

to selenium concentrations 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 related to selenium concentrations 

 in larvae 182 

Figure 3-7. Percentage of maternally exposed 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 concentrations 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 concentrations 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 deformities 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 transfer and microinjection studies 

in white sturgeon 195 

- xvi - 
 

Introduction 

The overarching goal of this dissertation was 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 background. Three studies are included in this 

dissertation. The first investigates the long-term effects of dietary Se in juvenile white 

sturgeon. The next study examines the mechanisms 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 estuary in the western United States. The 

Delta is formed by the confluence of the Sacramento and San Joaquin Rivers. Se enters 

this system in local petroleum refinery wastewaters and irrigation drainage from the 

seleniferous western San Joaquin 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). Organic 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 microbial reduction and is transformed into 

1
 

particulate 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 concentrations of 8 to 11 

μg/g Se dry weight (dw) in transplanted bivalves in northern San Francisco Bay. Johns et 

al. (1988) reported a mean of 6 μg/g Se dw in resident bivalve Corbicula spp. sampled 

near Carquinez Strait. The Selenium Verification Study found Se concentrations of 5.13 

to 7.90 μg/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 μg/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 benthic-feeding organisms (Urquhart and 

Regalado 1991). The high Se level and wide distribution of P. amurensis is of great 

concern because its Se burden significantly exceeds the levels shown to cause toxicity in 

animals consuming bivalves. Prey items containing greater than 10 μg/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 benthic feeders, including white sturgeon 

(Luoma and Linville 1997; Urquhart 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 μg/g dw in liver (n= 52) and 7 to 15 

2
 

μg/g dw in muscle (n= 99; Urquhart and Regalado 1991; White et al. 1988). Out of six 

sturgeon females sampled in the San Francisco Bay-Delta region in the 1990’s, one 

contained eggs with 3 μg/g Se, four had eggs ranging from 8 to 12 μg/g Se and one 

female contained eggs with 29 μg/g Se (Kroll and Doroshov 1991). In 2000, white 

sturgeon from this region exhibited a mean liver Se concentration of 24 μg/g, with some 

samples reaching as high as 40 μg/g Se (n= 15; Stewart et al. 2004). Linares et al. (2004) 

reported a mean liver Se concentration of 9.75 μg/g in 36 sub-adult and adult (age 4 – 18) 

white sturgeon sampled in San Francisco Bay between 2002 and 2004. Recently, three 

white sturgeon captured from San Francisco Bay-Delta were found to have 7 to 20 μg/g 

Se in ovaries containing developing 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 effects 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 white sturgeon tissue Se 

burden. 

To mitigate the Se pollution in San Francisco Bay-Delta, the California San 

Francisco Bay Regional Water Quality Control Board required local oil refineries to 

3
 

reduce the amount of Se in effluents discharged into the Bay; however, refinery effluent 

continues to be a significant source of Se in this region (Luoma and Presser 2000). The 

amount of Se entering the San Francisco Bay-Delta in agricultural effluents from the San 

Joaquin Valley may increase in coming years 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 endemic sturgeons inhabiting the Pacific 

Northwest. Both white and green (A. medirostris) sturgeon have high zoological value 

and are important to biodiversity; but white sturgeon has a higher value as a food and 

game fish. Both species belong to a phylogenetically unique group of ancient ray-finned 

fish that has survived for over two hundred million years (Sewertzoff 1923). White 

sturgeon is the largest North American sturgeon species with individuals recorded at 6 

meters in length and over 800 kg in weight (reviewed by Billard and Lecointre 2001; 

Doroshov 1985). This species is also unusually 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 maturity, with males typically 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 their lifespan (Doroshov 

4
 

1985). 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 synthesized in the liver. Vitellogenin is transported 

by the circulatory system to developing oocytes, where it is selectively taken up by 

receptor-mediated endocytosis (Mommsen and 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 intracellular crystalline platelets throughout early 

development (Dettlaff et al. 1993). Vitellogenin has also been 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 vitellogenin and yolk proteins from six 

sturgeon females caught in the Sacramento River, indicating the potential risk of 

developmental Se toxicity in sturgeon of this region. 

Spawning white sturgeon migrate up river during March 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 eighty percent of the maternal yolk is rapidly metabolized 

for growth, development, and for greatly increased metabolic energy associated with 

pelagic swimming (Bolker 1993; Dettlaff et al. 1993; Doroshov et al. 1983). White 

sturgeon exhibit holoblastic development and their yolk is digested intracellularly, in 

contrast to the extraembryonic yolk of modern teleost fish (Buddington and Doroshov 

1986; Dettlaff et al. 1993). Vital organs are differentiated during the yolk sac larvae 

period, such as the gills, liver and kidney. 

5
 

After hatching, the larvae are carried down river 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 Bay (Doroshov 1985; Kohlhorst 1980; Kohlhorst et al. 

1980). Adults in this area primarily consume mollusks and commonly feed on P. 

amurensis (McKechnie and Fenner 1971; Urquhart and Regalado 1991). 

Historically, the San Francisco Bay white sturgeon population decreased 

dramatically in the early 1900’s due to overharvesting (Doroshov 1985; Kohlhorst 1980). 

This population continues to be impacted by habitat destruction, reduced 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 population was at a 50-year low, with an 

estimated 10,000 adult sturgeon (California Department of Fish and Game Commission 

2006). 

Observed decreases in the San Francisco Bay white sturgeon population have 

been associated with the occurrence of dry water years (Kohlhorst et al. 1991). This may 

be due to several factors, including flow rate and habitat availability. In addition, Se 

concentrations in sturgeon food sources significantly increase during low-flow years 

(Johns and Luoma 1988; Linville et al. 2002; Luoma and Presser 2000). The life history 

of white sturgeon may render them especially vulnerable to Se bioaccumulation and 

6
 

maternal transport of Se to egg yolk due to a long life span, benthic feeding habits and a 

prolonged period of vitellogenesis and yolk deposition. Se toxicity cannot be discarded 

as a potential factor affecting white sturgeon reproduction and natural recruitment in San 

Francisco Bay-Delta. 

 

Toxicity of selenium 

Se is an essential nutrient (0.1 – 1.25 μg/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 environmental toxicant due to its efficient food web 

transfer (Lemly 1985; Luoma et al. 1992). There is a very narrow margin between 

nutritional and toxic concentrations of this element. Concentrations as low as 3 μg/g in 

diet have led to toxicity in aquatic organisms (reviewed by Hamilton 2004). Se toxicity 

results in reduced growth, significant histopathology in major organs, alteration of 

detoxification systems, promotion of oxidative 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 species consuming diets containing 13 μg/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 craniofacial deformities (e.g., Holm et al. 2005). 

Each of these defects is likely to reduce the probability of survival in developing fishes in 

7
 

the natural environment. Excess Se has led 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; Skorupa 1998). Lemly (2002) has proposed 

Se toxic thresholds for freshwater and anadromous 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 freshwater 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 μg/g of dietary Se reduced survival and 5 

μg/g reduced growth. Striped bass (Morone saxatilis) fed Se-laden red shiners (39 μg/g) 

experienced complete mortality (Coughlan and Velte 1989). In the grass goby 

(Zosterisessor ophiocephalus), a marine fish, the 96hr LD50 for selenite via ip injection 

was 0.29 μg/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 white sturgeon decreased with increasing Se 

exposure. A study investigating the effects of Se exposure on salinity tolerance in 

juvenile white sturgeon indicates a strong interactive effect of dietary Se exposure and 

elevated salinity on survival (Tashjian 2005). Sturgeon mortality 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 developmental toxicity of Se in white sturgeon. 

8
 

The specific chemical species and route of exposure are critical in the 

manifestation of Se toxicity. In the natural environment, Se is found in four oxidation 

states, selenide (-II; Se-2), elemental Se (0, Se0), selenite (IV; SeO3

-2), and selenate (VI; 

SeO4

-2). Elemental Se is commonly found in anoxic sediments (reviewed in 

Frankenberger and Engberg 1998). Selenite and selenate are present 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 Gissel Nielsen 1978; Hodson et al. 1980; Lemly 1982). In 

addition, the common use of inorganic Se in both waterborne and 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 exposure in toxicity studies is beginning to be 

widely recognized. 

Se is readily adsorbed through 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 nucleophile consisting of glutamic acid, 

cysteine and glycine. With the aid of glutathione-S-transferase enzymes, GSH reduces 

9
 

Se species to the –II oxidation state (selenide). As a result, GSH becomes oxidized and 

must be reduced by NADPH-dependent 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 (H2Se), 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 methylation by methyltransferase and the 

cofactor S-adenosylmethionine (SAM); however, 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-methlyation becomes limiting 

and dimethylselenide is excreted 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 significant cause of Se toxicity. Methylselenol, an 

intermediate metabolite of both selenite and selenomethionine, 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 been 

10
 

indicated in the inhibition of protein synthesis (Vernie and Van Leewenhoekhuis 1987), 

the reduction of cellular 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 methylation co-factor SAM (Anderson and Moxon 1942; 

Hoffman and Heinz 1988; Hoffman 1977). Reduced GSH, along with glutathione 

peroxidase, is crucial in detoxifying peroxides and halting lipid peroxidation. With the 

aid of glutathione transferase, reduced GSH actively binds electrophilic toxins and 

prevents them from binding to macromolecules (proteins, DNA, enzymes, etc.). 

Methylation is also a common detoxification pathway. Depression of these essential 

detoxification mechanisms leaves organisms vulnerable to additional toxic insults. 

Se can also be incorporated into biological molecules in place of sulfur because 

the two elements have similar chemical properties (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 codon prescribes the incorporation of 

selenocysteine into proteins; however, selenomethionine is indiscriminately substituted 

for the methionine codon in genetic translation. The amount of selenomethionine that is 

indiscriminately incorporated into proteins increases with increasing availability of 

selenomethionine in tissues (Schrauzer 2000). Similarly, when increased Se is available 

compared to sulfur, selenocysteine can substitute for cysteine in protein synthesis 

(Kramer and Ames 1988; Müller et al. 1994). However, Se and sulfur bonds may have 

different strength and function in biological molecules (Reddy and Massaro 1983), thus 

11
 

some 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 relevant to the San Francisco Bay-Delta population. 

Understanding the effects of Se on white sturgeon is a critical link between studying the 

extent of Se contamination in biota and regulating 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 and 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. 

12
 

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 μg/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 quantities (as little as 3 

μg/g Se in diet) this element is a potent environmental toxicant that threatens higher 

trophic level species because of its efficient 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 residing in areas receiving 

wastewaters from the combustion of fossil fuels (Garrett and Inmann 1984; Lemly 1985) 

or drainage from seleniferous land, including both agricultural and natural drainage 

(Hamilton 1999; Presser and Ohlendorf 1987; Skorupa 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 estuary in the western United States. The 

Delta is formed by the confluence of the Sacramento and San Joaquin Rivers. Se enters 

this system in petroleum refinery wastewaters and irrigation drainage from the 

seleniferous western San Joaquin 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 evidenced 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 and eggs show significant levels of Se, 

13
 

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 μg/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 levels of Se, an average of 15 μg/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 sturgeon 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 μg/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 systems, predominately the Sacramento River 

(Kohlhorst 1980; Kohlhorst et al. 1980). Juveniles migrate back to the Bay-Delta, but 

may remain in the lower Sacramento River for several years (Radtke 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 phylogenetically ancient group of ray14
 

finned fish that has survived for over two 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 μg/g Se in muscle (± 9.72 standard deviation; range of 

1.46 – 50 μg/g Se; n= 99) and an average of 26.55 μg/g Se in liver (± 21.76 standard 

deviation; range of 3.24 – 79.42 μg/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 μg/g with some samples reaching as high as 

40 μg/g Se (n = 15; Stewart et al. 2004). These concentrations exceed concentrations 

previously linked to adverse effects in other fish (Coughlan and Velte 1989; Hermanutz

et al. 1992). 

Elevated Se affects growth, condition factor, 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 literature 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 necrotic liver cells; 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 glomerular and tubular epithelial 

cells and can lead to disease states such as proliferative glomerulonephritis, 

15
 

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 developmental toxin when transferred from the adult 

female to the yolk of their eggs (Gillespie and Baumann 1986; Lemly 1993b). 

Due to a paucity of information regarding 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 present in the aquatic environment. 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. 1990b; Hoffman et al. 1989; Luoma et al. 1992; Reinfelder et 

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

sacrificed and tissue Se concentrations were measured. In addition, growth, and 

histopathological and morphological alterations were assessed. Our hypothesis was that 

dietary Se at environmentally relevant levels would be accumulated in sturgeon tissues, 

inhibit growth, and induce pathological changes. 

16
 

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 from three female and six male hatchery-bred 

white sturgeon. Larvae were transported 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, outdoor flow-through tanks at the Center for Aquatic Biology 

and Aquaculture, UCD. The experiment consisted of four dietary treatments: control 

(1.04 ± 0.07 μg/g Se), low (20.05 ± 1.10 μg/g Se), medium (35.61 ± 3.83 μg/g Se) and 

high (52.54 ± 2.32 μg/g Se; mean ± standard deviation; Table 1-1). Each dietary 

treatment was replicated in three randomly assigned tanks, with 10 fish per tank and 30 

fish per treatment. 

17
 

Initiation and maintenance 

At 38 weeks post-hatch, 144 randomly selected fish were anesthetized (200 mg/L 

MS-222) measured, weighed and randomly distributed 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/L3

). 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 measurement using techniques described below. 

Three days later the remaining 120 fish were gradually transitioned onto respective 

experimental diets over one week. All juveniles were on full experimental diets by 47 

weeks-post hatch. The experimental 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. The fish were observed 

daily for mortality and morbidity. Moribund 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, the fish were measured for fork length and 

wet weight, and were necropsied for tissue analyses. 

Experimental Diet 

Sturgeon were fed a casein-based, purified pelleted diet (Table 1-2), specifically 

formulated for sturgeon and with a satisfactory history for rearing 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 were 

18
 

mixed 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 were 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 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 Se analysis. Fish were then euthanized by MS-222 overdose and the spinal 

cord was cut before proceeding with the necropsy. 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 samples 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 digested using a nitric/sulfuric/perchloric 

acid mixture heated to 230 °C over two days. Se in the digested solution was reduced to 

19
 

selenite 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 stored at 4 °C until spectroscopy analysis (less 

than 10 days). 

Total Se was analyzed as selenite using Hydride-Generation Atomic Absorption 

with an EDL Se lamp (PerkinElmer AAnalyst 300 Atomic Adsorption Spectrometer 

MHS-15). The instrument had a minimum detection limit of 1 ng Se and was calibrated 

in the linear region, from 20 to 100 ng Se, using 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 quality 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. Moisture content was determined as 58.3 ± 6.59 percent in liver 

and 77.6 ± 1.21 percent in muscle (mean percent moisture ± standard deviation, n=117). 

Plasma contained 95.9 ± 0.41 percent moisture and 42.02 ± 5.7 mg of dry weight per 

milliliter. 

Gross Morphology 

Condition factor was measured as (weight (g)/fork Length3

 (cm)) * 100. Specific 

growth rate (SGR) was determined for each tank using the formula: SGR = 100 * 

(lnWeightfinal – lnWeightinitial)/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 account for allometric growth. 

20
 

Histological Analyses 

At necropsy, following euthanasia, the ventral abdominal wall was surgically 

opened and tissues were rapidly removed for placement in fixative. Liver (1-3 mm thick 

piece of the left lateral margin), 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 trimmed from this sample. Tissues were 

immediately immersed in 10% neutral buffered saline and stored under refrigeration in 

individually labeled containers at a ratio of 10 volumes of fixative to 1 volume of tissue. 

After completion of fixation, solution was decanted into appropriately labeled storage 

chambers and each container was filled with 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, selecting the outermost (1-2 mm 

thick) portions to ensure fixative penetration, 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 eosin, coverslipped and viewed 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 original magnification recorded. Images were stored as TIFF 

files in Adobe Photoshop 6.0. 

21
 

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-adjustment was applied to the ranked data 

(experiment-wise α = 0.05; Bonferroni adjusted comparison-wise α = 0.001666). 

Morphologic measurements were compared 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). Log10 transformations were used in regressions of organ to body 

weight to account for allometric growth. Histological survey of liver, kidney, and gill 

was made and alterations from control anatomy were imaged, ranked as to treatment and 

recorded. For areal analysis of melanomacrophage centers, random, non-overlapping 

fields of liver were selected and the number and area of melanomacrophage centers were 

recorded. Comparisons of the presence of histological 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 increasing Se in the diet (Rank F-Test, p = .0334; adjusted 

multiple comparisons, α = 0.05; Figure 1-1). In the control treatment (ca. 1 μg/g dietary 

Se) Se accumulated more in blood than in muscle, but Se in liver was not different from 

22
 

either blood or muscle. There were no differences between Se levels in tissue types in 

both the low and medium treatments (ca. 20 & 36 μg/g dietary Se, respectively). In the 

high treatment (ca. 53 μg/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 relationships 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 μg/g, n=17), liver (1.89 ± 0.69 μg/g, 

n=10) and blood (3.66 ± 0.83 μg/g, n=10; mean Se ± standard deviation). 

Morphologic Characteristics 

Several whole body morphologic characteristics were compared across Se 

treatments. We found no differences between treatments in condition factor, fork length, 

body weight, or specific growth rate (ANOVA, p > 0.075; Tables 1-4 & 1-5). Similarly, 

gonadosomatic, hepatosomatic and weight to length 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 dietary Se exposures. A moribund fish was 

euthanized (MS-222 overdose) in a high treatment tank after it was found upside down 

and motionless during the second month of dietary exposure. One fish from a low 

treatment tank was euthanized (MS-222 overdose) in the fourth month of dietary 

23
 

exposure because it was unable to swim and was emaciated. These fish were not sampled 

for Se or histopathology. No other mortalities were observed during the experiment. 

Histopathology 

Pathological changes were observed in liver, gill arch and posterior kidney of 

treatment fishes. Histology of gonads revealed mostly adipose tissues with very limited 

vasculature. No actual gonadal 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 μg/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 (sinusoids). This pattern of hepatocyte 

arrangement is the tubule, common as the adult phenotype in lower vertebrates including 

fishes (Hinton and Couch 1998). Hepatocytes contained a single nucleus but not all 

planes of section through hepatocytes revealed a nuclear profile 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 cytoplasm, vacuolar profiles likely denoted lipid 

24
 

that was removed during tissue processing (alcoholic dehydration). Other regions of 

cytoplasm stained more diffusely (gray-pink) but were not comprised 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 hepatic sinusoids) while apical cytoplasm was only 

lightly stained (Figures 1-2 & 1-3) and was directed toward center of hepatic tubules 

(Figures 1-2 – 1-4). 

 The parenchyma was comprised of tubular arrays of hepatocytes and, in certain 

regions, individual tubules curved and abutted 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 presence of erythrocytes whose 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 perisinusoidal macrophages. Within this space, 

small cells with high nuclear to cytoplasmic ratio and containing a single lipid droplet 

were seen (not shown in figures). Respectively, these cells are the fat-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 small diameter bile ducts. Because all livers 

were sampled at the left margin, larger vascular and biliary structures more closely 

associated with the hepatic hilus (near midline of abdominal cavity) were not included. 

25
 

Biliary epithelial cells frequently accompanied venules (Figure 1-5). The latter were 

interpreted as branches of the hepatic portal vein, the afferent venous supply to the liver. 

In controls these sites also contained focal aggregates of melanomacrophages (Figure 1-

6). Eosinophilic granular leukocytes were frequently seen near small bile ducts (Figure 

1-5). Larger diameter veins were often associated with bile ductules and hepatic 

arterioles (Figure 1-3), resembling mammalian portal triads (Hinton and Couch 1998). 

However, foci of mononuclear cell infiltration were common in these sites and 

melanomacrophage aggregates near large veins were usually of greater diameter than 

those associated with venules. In addition to accompanying veins, melanomacrophage 

aggregates were frequently associated with arteries, often surrounding muscular layers 

and periadventitia of these vessels (Figures 1-5 & 1-6). Eosinophilic granular leukocytes 

were found near arteries, arterioles; and in inflammatory foci 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 μg/g dietary Se). There was a 30% incidence of 

tubuloform hepatocellular swelling (Figures 1-8 & 1-9). Bile duct proliferation, 

especially involving transitional biliary epithelial cells, was seen (Figures 1-10 & 1-11). 

This occurred in approximately 70% of individuals. 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 receiving bile made by hepatocytes. Normally, 

in paraffin-embedded material these passageways are not seen; however, in this group the 

26
 

condition was seen in each individual. Bile stasis is a hallmark feature of hepatotoxicity 

and criteria for this condition fit those of the medical diagnosis (Lee 1996-2005). 

Limited formation and subsequent excretion of bile, 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 morphology in 40% of the individuals of this 

group. 

Medium selenium dietary group

Observed changes in the medium treatment (ca. 36 μg/g dietary Se) included 

moderate expansion of melanomacrophage aggregates in area and in number; increased 

perivascular inflammation; focal hepatocellular necrosis with accumulation of discreet 

eosinophilic cytoplasmic inclusions; biliary canalicular distension and biliary epithelial 

cell (ductular and small ducts) proliferation (Figures 1-12 & 1-13). 

High selenium dietary group

Alterations in the high treatment (ca. 53 μg/g dietary Se) involved hepatocytes 

(fatty change, biliary stasis of canalicular network, necrosis, reduction in hepatocyte 

volume in cells of portal zones, and presence of Councilman bodies which are evidence 

of hepatocellular apoptosis), mononuclear leukocytes (foci of inflammation), 

macrophages (melanomacrophage aggregation), and 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 proliferating bile ductules. In addition, a 

27
 

mosaic of response was seen in some individuals indicating that two functionally distinct 

populations of hepatocytes were present. First, vacuolated hepatocytes (suspected but 

unconfirmed lipid change) occurred in distinct zones and these zones were lacking in 

melanomacrophage aggregates. The next zone was one in which hepatocytes were 

shrunken, stained more intensely, and were likely producing apoptotic fragments which 

were then phagocytosed by adjacent cells producing bright red Councilman bodies. 

These regions contained the melanomacrophage aggregates. As in other Se concentration 

groups, the canalicular network was distended; and this, along with distension of bile 

ductular and small ductal lumina, suggests an inability 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 and bile stasis in one control fish. The high 

amplitude swelling of some tubules (seen in one control and in several individuals of the 

medium and high Se groups) suggests a lack of volume regulation and is a form of acute 

hepatic tubular necrosis. This condition affected groups of individual hepatocytes of 

individual tubules. The consistent canalicular 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 exposure of young organisms to Se; fusion of secondary lamellae, 

28
 

which results in reduced surface area for gaseous exchange; inflammation, representing a 

response to injury; and torsion of lamellae, where the lamellae are twisted along the long 

axis. Contingency tables were used to analyze the presence or absence of each lesion 

type in control and treatment fish. Significant differences were found for the presence of 

the fusion of secondary lamellae and inflammation across treatments (p = 0.024 and 

0.041, respectively; Figure 1-16). These effects were observed more frequently in the 

lowest treatment group (ca. 20 μg/g dietary Se). No statistically 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 exposure groups, this is interesting but likely not a 

toxic response. In addition, the tubulo/glomerulo-genesis appeared to be localized in the 

periphery of all kidneys, which indicates that 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 appeared not only in animals of the experimental 

groups but also in the control. Despite careful analysis of glomerular space, renal 

29
 

proximal tubules, intertubular structures, and generation/regeneration 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 conditions 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 accumulation in the liver observed in the 

highest treatment diet (ca. 53 μg/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 μg/g 

dietary Se). One possible explanation for this variation is that varying liver lipid content 

between individuals could have biased the quantification of Se (expressed 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; and, 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 amounts 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 relationship between Se accumulation and condition 

factor in any treatment group. 

30
 

Se in all tissues of fish receiving low, medium and high experimental diets (ca. 20 

– 53 μg/g dietary Se) reached levels that have been associated with toxicity 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 (2002) proposed thresholds of Se toxic 

effects for freshwater and anadromous fish at 8 μg/g in muscle and 12 μg/g in liver. 

Mean liver and muscle Se concentrations surpassed these thresholds in each of the three 

treatment levels of the current study (Figure 1-1). 

Se-induced toxicity in teleosts has led to reduced juvenile growth, changes in 

condition factor, changes in organ to body weight 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 significant differences between treatments in 

survival, specific growth rate, condition factor, fork length, or body weight of juvenile 

white sturgeon, athough there was an apparent trend of decrease in length, weight, and 

growth rate in the high Se treatment (Tables 1-4 & 1-5). In a recent study, dietary 

exposure of 40 μg/g seleno-L-methionine led to impaired growth in fingerling-stage (0+) 

white sturgeon (25 – 30g) after 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 μg/L waterborne Se for the duration of one year (Hermanutz et al.

1992). The influence of age on growth impacts in Se-exposed fish remains unclear. 

31
 

Similarly, there were no significant differences in hepatosomatic, gonadosomatic 

or weight to length regressions during the course of this study (ANCOVA, p 0.08 – 0.3; 

Table 1-4). These findings contradict 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 those 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 μg/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 route and duration of 

exposures. 

Histopathological analysis of treatment liver revealed significant cholestasis with 

compensatory changes in biliary epithelium, and expansion of melanomacrophage 

aggregates in all treatment exposures (ca. 20, 36, & 53 μg/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 proliferation of ductules 

and ducts in the sturgeon liver. Yellow pigment was seen in the cytoplasm of some 

hepatocytes, indicating cholestasis. 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 and secretion are vital to several critical functions including the 

elimination of endogenous toxins, xenobiotics and their metabolites, and the assimilation 

of lipid soluble dietary nutrients (e.g., vitamin A, K, E and triglycerides; Groothuis and 

32
 

Meijer 1996; Nathanson and Boyer 1991). Bile acids that are accumulated in liver during 

cholestasis damage mitochondria, generate oxidative 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 was associated with Se toxicity in birds and 

some mammals. Mallards (Anas platyrhynchos) fed a diet containing 32 μg/g Se for 14 

weeks incurred bile duct hyperplasia accompanied by elevated plasma alkaline 

phosphatase activity and lipid peroxidation (Hoffman et al. 1991). Mallards fed 60 μg/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 μg/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 μg/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 μg/g Se 

via diet for up to 80 days showed bile deposition and necrosis in the liver (Miller and 

Schoening 1938). 

While cholestasis is well recognized in rodents and mammals, this condition is 

only beginning to be understood in fish species (Cai et al. 2001; Kirby et al. 1995; Lorent

et al. 2004). Cholestasis can result from genetic defects, toxins, 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 formation and secretion of bile, presumably 

by affecting transport protein expression and function (Trauner et al. 1998). Schmitt and 

33
 

colleagues (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 could ultimately lead to cholestasis. It is 

interesting to note that both cholestasis and Se toxicity are strongly 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 peroxides generated from 

redox cycling during Se metabolism, as well as other critical cellular 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. Cholestasis is explored in more detail in Appendix A. 

Melanomacrophage aggregates (MMAs) in liver of treated fish increased in size 

and frequency with increasing dietary Se. MMAs have also been observed in livers of 

juvenile Sacramento splittail (Pogonichthys macrolepidotus) exposed to dietary Se (Teh

et al. 2004). Several other environmental 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). Consideration 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 this 

34
 

study since juveniles were used and all fish 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 degradation, storage of destroyed cellular 

material, antioxidation, lipid oxidation, and immune function (Rice 2001; Wester et al. 

1994). The presence of melanin is particularly 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 juvenile 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 renal toxicity, based on histopathological 

analysis. Controls and treated fish showed evidence of generation of new glomeruli and 

tubules. These results differ from previous studies in other fishes. Hicks et al. (1984) 

found kidney lesions in rainbow trout with liver Se levels similar to those in white 

sturgeon from this study. Another study found significant tubular changes and signs of 

glomerulonephritis in kidneys of green sunfish 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 kidneys (Coughlan and Velte 1989). The lack 

35
 

of 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 μg/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, especially 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 μg/g dietary Se) are unclear. 

There is evidence for several potential mechanisms 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 systems by depleting glutathione (GSH) or the 

methylation co-factor S-adenosylmethionine (Anderson and Moxon 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 place of sulfur because the two elements have 

similar chemical 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 

36
 

dysfunctional (Lemly 1998; Stadtman 1974). In addition, Se can inhibit 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 mechanism(s) of Se-induced toxicity in fishes. 

The present study is novel because it exposed the actual species of ecological 

concern to the Se form (organic Se) and exposure route (dietary) that is predominant in 

the natural environment. Much 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 significant route of exposure 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 inorganic Se in both waterborne and dietary 

exposures underestimates environmental exposure, 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 ecotoxicology because it requires 

the ability to culture a species of ecological concern. For some phylogenetically ancient 

fish, such as sturgeon, the use of surrogate species is not possible, or is unreliable, 

because of the significant anatomical, physiological and behavioral differences with 

modern fish. White sturgeon culture 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 maturity, this study 

would not have been possible. 

37
 

This work shows that juvenile white sturgeon in San Francisco 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 experimental diet (ca. 20 μg/g 

Se) is similar to the highest 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 μg/g Se, respectively) fall within predicted Se concentrations in bivalves 

resulting from the proposed increase of Se-laden agricultural irrigation discharges into 

San Francisco Bay-Delta (Luoma and Presser 2000). 

Previous studies have shown that juvenile 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 μg/g in 36 sub-adult and adult (age 4 – 

18) white sturgeon sampled in San Francisco Bay between 2002 and 2004 (all 

measurements of variation reported as standard deviation). Between 1986 and 1990, 

juvenile white sturgeon captured from this area contained mean Se concentrations of 5.52 

± 3.02 μg/g in muscle and 17.01 ± 15.89 μg/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 health effects observed in this study. 

38
 

The direct effects of Se on a white sturgeon 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 (Schaffter 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 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 population was at a 50-year low, with 

approximately 10,000 adult sturgeon (California Department of Fish and Game 

Commission 2006). It is important to note that this severe 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 evidence of that impact around this time. 

In conclusion, this work shows that juvenile 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 μg/g dietary Se exposures. Cholestasis, a potentially fatal 

condition, was present in all treatment groups. Exposure to ca. 20 and 36 μg/g dietary Se 

resulted in significant inflammation and fused lamellae in gills, however these effects 

39
 

occurred less frequently with exposure to ca. 53 μg/g dietary Se. Toxicological studies 

typically utilize a much wider exposure range, with doses differing by orders of 

magnitude. The narrow exposure range used here provides greater ecological 

significance; however, the lack of large differences between exposures 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 present study indicates that the impacts of Se in 

this system may be a factor affecting the abundance of white sturgeon in San Francisco 

Bay. Careful management of all processes with potential to increase food web Se in San 

Francisco Bay-Delta is essential to sturgeon preservation. 

40
 

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.).

41
 

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 Premix1

(w/ cellulose & 

santoguin) 1 

Mineral Premix2 3 

Cellulose 2.5 

Choline Chloride 0.5 

Yeast Mix3 4 

Table 1-2. Experimental diet composition for juvenile 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 non-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 Vitamin B-12 crystalline; 98.14 mg Vitamin D3 (400,000 U/g). 

2

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

3

Yeast mixture contained selenized yeast and Torula yeast. 

42
Se in juvenile white sturgeon after six months of dietary Se exposures 

Data displayed as median, 25th - 75th percentiles and full range 

Muscle (Se μg/g) Blood (Se μg/g) Liver 

Se dietary exposures (μg/g) Median 25 - 75% Range Median 25 - 75% Range Median 25 - 7

1.04 ± 0.07 2.06 1.61 - 2.49 1.36 - 2.76 4.3 3.85 - 5.01 2.14 - 6.90 2.85 2.14 - 

20.05 ± 1.10 21.56 17.40 - 25.99 13.18 - 32.40 24.04 22.23 - 25.86 18.75 - 33.05 19.49 14.20 - 35.61 ± 3.83 39.73 34.55 - 43.58 21.28 - 47.54 40.99 38.19 - 46.25 31.01 - 59.20 40.2 31.48 - 52.54 ± 2.32 52.97 46.67 - 59.20 32.06 - 65.59 55.33 51.33 - 63.11 38.52 - 77.90 69.84 60.29 - 

Table 1-3. Sturgeon data displayed as median, 25th - 75th percentiles, and range of Se (μg/g, dw) levels in liver, after six months of dietary Se exposures. Dietary Se exposure displayed as mean ± standard deviation Se (μg/g, 

in Figure 1-1. 
Comparisons of gross morphological characteristics of juvenile white sturgeon 

following six months of dietary Se exposures 

R2 F p-value Model 

Condition Factor 0.15 1.74 0.08 ANOVA 

Fork Length 0.09 1.06 0.40 ANOVA 

Body Weight 0.10 1.13 0.35 ANOVA 

Specific Growth Rate 0.36 1.49 0.29 ANOVA 

Gonadosomatic regression 0.59 2.28 0.08 ANCOVA

Hepatosomatic regression 0.58 1.95 0.13 ANCOVA

Weight to length regression 0.84 1.24 0.30 ANCOVA

Table 1-4. Parameters from ANOVA and ANCOVA analyses of gross morphological chajuvenile white sturgeon exposed to ca. 1, 20, 36, and 53 μg/g dietary Se for six months. N

found for any of the measured characteristics. 
Gross morphological measurements of juvenile white sturgeon following six months of dietary Se 

Dietary Se (μg/g) Condition Factor Fork Length (cm) Body Weight (kg) Specif

1.04 ± 0.07 7.43 x 10-3 ± 1.7 x 10-4 (3) 60.3 ± 0.86 (3) 1.65 ± 0.097 (3) 0.5

20.05 ± 1.10 7.73 x 10-3 ± 1.7 x 10-4 (3) 59 ± 0.39 (3) 1.62 ± 0.051 (3) 0.35.61 ± 3.83 7.96 x 10-3 ± 7.4 x 10-5 (3) 59.5 ± 0.32 (3) 1.69 ± 0.039 (3) 0.552.54 ± 2.32 7.86 x 10-3 ± 2.8 x 10-4 (3) 56.4 ± 0.66 (3) 1.45 ± 0.103 (3) 0.5

Table 1-5. Measurements of weight, length, condition and growth in juvenile white sturgeon for each d

All data is presented as mean ± standard error (number of tank replicates). No statistical differences wedietary exposure levels (Table 1-4). 
 

Se in juvenile white sturgeon following six months of dietary exposures 

Figure 1-1. Se levels in liver, blood and muscle after six months of dietary Se exposures. 

For each tissue type, Se increased with increasing dietary Se exposure (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 horizontal lines represent the 25th and 75th percentiles, 

respectively. The whiskers show the 10th and 90th 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). 

46
 

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 (sinusoidal) plasma membrane in 

base of cell (horizontal 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. 

47
 

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. 

48
 

Figure 1-4. Section of liver from control group of juvenile sturgeon showing bile 

retention (arrows) and focus of small bile duct proliferation. This condition was seen in a 

single individual. Note absence of evidence for biliary stasis involving canalicular 

network. 

49
 

Figure 1-5. Section of liver from control group of juvenile sturgeon. Eosinophilic 

granular leukocytes (arrows) are associated with portal venule and small bile duct. 

50
 

Figure 1-6. Section of liver from control group of juvenile sturgeon. Comparison of this 

micrograph with figures 1-2 and 1-3 illustrate range of features in livers of control group. 

A single focus of inflammation involving mononuclear leukocytes is shown in center of 

field. To left of field, melanomacrophage aggregate is larger than those of Figure 1-3 and 

is associated with inflammation around a small bile duct. 

51
 

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. 

52
 

Figure 1-8. This individual was fed 20.05 ± 1.10 μg/g Se in diet for 6 months. Red 

circle in center of transversely sectioned hepatic tubule is a canaliculus (small arrow). 

Longitudinal arrays of canalicular network are shown (CN). These features were not 

visible in juveniles of control group (Figures 1-2 – 1-7). 

53
 

Figure 1-9. This individual was fed 20.05 ± 1.10 μg/g Se in diet for 6 months. High 

amplitude swelling of hepatocytes (large white cellular arrays) in tubule give rise to 

tubuloform swelling that was seen at 30% incidence in this group. This condition was 

seen in only one of 30 control juvenile sturgeon. Two normal appearing hepatic tubules 

in transverse section are shown (arrows). Inflammatory cells at center of field surround 8 

profiles of small bile ducts (bile duct proliferation). 

54
 

Figure 1-10. This is an additional view of the individual 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). 

55
 

Figure 1-11. This individual was fed 20.05 ± 1.10 μg/g Se in diet for 6 months. Focus 

of bile duct proliferation is shown with inflammatory cells just beneath hepatic capsule. 

56
 

Figure 1-12. This individual was fed 35.61 ± 3.83 μg/g Se in diet for 6 months. This 

micrograph shows morphology of portal venule and adjacent hepatic parenchyma. 

Hepatocytes show signs of necrosis seen as nuclear lysis (Ly). In addition, cells are 

swollen and contain discreet particulate material in cytoplasm (arrows). Canalicular 

network is distended (CN). In addition there are four profiles of transected small bile 

ducts around the venule. 

57
 

Figure 1-13. This individual was fed 35.61 ± 3.83 μg/g Se in diet for 6 months. 

Extensive perivenous (venular lumen is at right of field) and peribiliary inflammation 

involving predominantly mononuclear leukocytes 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 epithelial cell proliferation is 

seen (arrow) with cells extending from perivascular site into adjacent parenchyma. Also, 

note the longitudinal arrays of biliary ductules with red staining in apical plasma 

membrane region. 

58
 

Figure 1-14. This individual was fed 52.54 ± 2.32 μg/g Se in diet for 6 months. This 

field is from liver of individual showing maximum response of melanomacrophage 

aggregates. The number of hepatocyte nuclei per area of parenchyma is greatly enhanced 

and the cytoplasm stains with more intensity. This indicates that hepatocytes have 

become smaller in volume. Under this level 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. 

59
 

Figure 1-15. This higher magnification view of area 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. Hepatic tubules reveal 

canalicular network (see red lines in center of tubules). Melanomacrophage aggregates 

of various sizes are prominent. Councilman bodies are seen in four cells in bottom half 

of field. 

60
 

Effects observed in gill of juvenile white sturgeon following six months of dietary Se 

exposure 

Figure 1-16. Contingency table showing the occurrences 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). The low and medium treatments contribute 

the most to this difference. 

61
 

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

sturgeon 

Introduction 

Selenium (Se) is a well-documented developmental 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 μg/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 μg/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 wastewaters from the combustion of fossil fuels 

(Garrett and Inmann 1984; Lemly 1985) or drainage from seleniferous land (Hamilton 

1999; Skorupa 1998). 

Higher trophic level species are most threatened by Se in aquatic ecosystems due 

to the efficient food web transfer and biomagnification 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 assimilation 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 studies 

62
 

have 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 and complete reproductive failure (Coyle et al. 

1993; Gillespie and Baumann 1986; Lemly 1993b; Schultz and Hermanutz 1990). The 

maternal transport of contaminants to offspring is common in aquatic species. The yolk 

protein precursor vitellogenin (VTG), synthesized in the liver, has been shown to be the 

vehicle of maternal transport for several pollutants (Falchuk 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 Carlson 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 and 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 embryo. Kroll and Doroshov (1991) measured Se 

levels in plasma (5 – 9 μg/g), VTG (3 – 4 μg/g), ovulated eggs (3 – 29 μg/g), phosvitin (3 

– 90 μg/g) and lipovitellin (3 – 11 μg/g) from six wild-caught white sturgeon females 

from San Francisco Bay-Delta (dry weight). These elevated, although variable, levels 

63
 

suggest 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 yolk deposition (Doroshov 1985; Doroshov et al. 1997). 

White sturgeon is common in San Francisco Bay-Delta, an area with known Se 

contamination. San Francisco Bay-Delta receives Se through both the refining of fossil 

fuels and irrigation drainage from the seleniferous 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 muscle, liver and eggs (Kroll and 

Doroshov 1991; Stewart et al. 2004; Urquhart and Regalado 1991; White et al. 1989). 

The objectives of this experimental study 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 sturgeon females in the advanced stage of 

vitellogenesis were fed diets with normal (ca. 1.42 μg/g) or elevated (ca. 34.04 μg/g) Se 

(as selenized yeast) for approximately 6 months. 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. 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. 1990b; 

Hoffman et al. 1989; Luoma et al. 1992; Reinfelder et al. 1997; Wang and Lovell 1997). 

64
 

Upon acquisition of maturation competence, the fish were induced to spawn and 

ovulated eggs were fertilized with sperm from non-treated males. Fecundity, 

fertilization, and neurulation rates were measured 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 dissertation. Adult females were necropsied for 

Se analysis. We hypothesized that VTG is the vehicle of maternal transport in fish, as 

indicated by Kroll and Doroshov (1991; Figure 2-1). We also hypothesized that exposure 

to elevated dietary Se leads to significant bioaccumulation, maternal transport and 

reproductive impairment in white sturgeon. 

 

Materials and Methods 

Source of Animals 

Sixteen virgin females in mid-vitellogenic stage of oogenesis were obtained from 

The Fishery, Inc. (Galt, California) in September 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/m3

, and fed a commercial high-energy salmonid diet (45% protein/25% lipid) 

at approximately 0.25% body weight/day. 

The sturgeon were transported to the Putah 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 of 

65
 

a catheter for the collection of eggs, which were placed into Ringers solution for 

measurement of egg diameter and weight. Blood was collected from the caudal 

vasculature, using a 10 ml heparinized Vacutainer®, and weight and fork length were 

measured. Each female was PIT tagged in the caudal peduncle region under a lateral 

scute and externally marked with a floy tag. The fish were randomly assigned to two 6.1 

m diameter tanks, with eight in each tank. 

Experimental Design 

Domestic broodstock white sturgeon females (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 two dietary treatments: control (1.42 ± 0.03 μg/g Se) 

and treatment (34.04 ± 0.82 μg/g Se; Table 2-1). Each tank held 8 females and received 

one of the two experimental diets for approximately 6 months. Upon acquisition of 

maturation competence, the fish were induced to spawn and ovulated eggs were fertilized 

with sperm from non-treated males. Fecundity, 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 typically cools down to 11 to 12 oC in 

66
 

Dec to Jan and gradually warms to 15 to 16 oC in April-May. Water flows were 

approximately 150 liters per minute, and dissolved oxygen was maintained at > 80% air 

saturation. Irrigation water had a pH of 7.5 to 7.8, hardness of 220 mg/L CaCO3 and 

alkalinity of 180 mg/L as CaCO3. 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 with essential (control; ca. 1 μg/g) and elevated (treatment; 

ca. 34 μg/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 prior to pelleting at 14% of the total diet. 

For the control diet, the selenized yeast mixture contained 1.3% selenized yeast and 

98.7% tortula yeast. Only selenized yeast was added to the treatment diet. After 

pelleting, the diet was allowed to air dry on drying racks. 

Sampling 

On February 12, 2002 each female was biopsied 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, 

67
 

polarization index and response to progesterone exposure (as described 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 repeatedly (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 and the egg diameters were measured using a darkfield 

dissecting microscope with camera lucida, an image analyzing tablet, and a 

microcomputer interface (± 0.01 mm). The readiness of a specific female to spawn was 

determined by examining additional subsamples of collected eggs to calculate the 

polarization index (PI), and measure the capacity 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 extracted by a catheter (Conte et al.

1988) and kept in a sturgeon Ringer solution (Dettlaff et al. 1993) with penicillin (0.03 

g/L) and streptomycin (0.05 g/L). The incision for inserting the catheter 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 weeks, and have been successfully induced to 

ovulate, producing eggs with high fertility and hatch rates (Van Eenennaam et al. 2004). 

68
 

Subsamples of 15 eggs per female were used for PI determination. 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 measuring 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 diameter. These measurements were made 

using a darkfield dissecting microscope with camera lucida, an image-analyzing 

tablet, and a microcomputer interface (± 0.01 mm). 

The PI alone provides a very good indication 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 Ringer 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 control. Each treatment had two replicates. 

After incubation, the eggs are boiled, chilled 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. 

69
 

Spawning Induction, Egg Fertilization and Embryo Incubation

 The spawning, fertilization and incubation protocol followed Conte et al. (1988), 

with few modifications. The males received a single injection of 10 μg/kg body weight 

of mammalian GnRHa [D-Ala6, Des-Gly10]–LH-RH Ethylamide (Peninsula 

Laboratories), and the females received a priming injection of 2 μg/kg GnRHa, followed 

in 12 h with resolving injection of 18 μg/kg GnRHa. All injections were intramuscular 

and given underwater to minimize handling stress. Females were held in separate 3.7 m 

diameter tanks. Tank bottoms were routinely checked for released eggs beginning 16 h 

after the second injection. The ovulating female 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 plastic 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 than 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 fertilized with 20 ml of pooled milt from 2 to 3 

males, diluted 1:200 with hatchery water. After 3 min of fertilization, eggs were gently 

mixed in a water suspension of Fuller’s Earth (100-200 mesh size, Sigma Chemical Co.) 

for 60 min to prevent egg adhesion. The number of ova collected from each female was 

70
 

estimated volumetrically (egg count in five 5 ml subsamples). Approximately 1 Liter of 

fertilized eggs was placed into individual MacDonald hatching jars; and, jars were set-up 

to allow hatched larvae from individual females to flow into separate 1.2 m diameter 

circular tanks. Embryo incubation was at water temperatures 15 to 16 oC. 

Final Necropsy Sampling

Females were euthanized and necropsied 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 severing the spinal cord. Samples of liver, 

kidney, muscle, spleen, eggs and post-ovulatory gonads were frozen on dry ice and stored 

at -80 oC for Se analysis. Prior to euthanasia, 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 and stored at -80 oC for 

protein isolation and Se analysis. Eggs collected for egg yolk purification were flash 

frozen in liquid nitrogen and stored at -80 oC. Developing 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 purified from estrogenized male white sturgeon 

plasma using anion exchange chromatography. To verify the identity of estrogeninduced VTG, the chromatographs of the male were compared before and after treatment 

71
 

(Figure 2-2). Purification of VTG was accomplished 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 proteins were eluted by washing with 5 column volumes of 

running buffer (peak P1, Figure 2-3). Sequential 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, indicated 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 was adjusted to 7.0 using 100 mM bis-trispropane (pH 6.0), sodium azide (0.02% v/v), and protease inhibitor-Aprotinin (10 

KIU/ml) was added. A 1.0 ml aliquot was collected 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 proteins and strongly affects ELISA 

results (personal observation by Kroll/Denslow, Hybridoma lab, University of Florida). 

Freeze/thaw fracture of yolk proteins is avoided by the addition of glycerol and storage at 

–20 oC. At this temperature the solution remains a liquid. This aliquot would be used for 

PAGE and Western analysis. The total protein content of each pool was determined by 

Bradford analysis (Coomassie PlusTM, Pierce) using bovine serum albumin as a standard. 

72
 

 VTG levels in the plasma were relatively low because the females used for this 

study were in late stages of vitellogenesis and were developing polarized eggs (Doroshov

et al. 1997). Pooling several ion exchange runs was necessary to acquire enough VTG 

for analysis. In some cases further concentration 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 identity of the purified VTG, the plasma and 

purified protein pools were separated by electrophoresis on polyacrylamide gels (PAGE) 

and then transblotted to a membrane for Western analysis. A 4 to 12% gradient gel 

(NuPAGETM, 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) the plasma sample before purification, and 

4) the pooled fractions after ion exchange purification (nonVTG 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 2TM, 

Invitrogen) were added to each lane. The amount 0.5 μg of each purified protein, VTG+, 

non-VTG and VTG proteins, were added to separate lanes. The gel was run for 35 min at 

200 V. The gel was soaked for 2 to 4 hours in colloidal blueTM stain (Invitrogen) and 

destained using water. Each gel was scanned and labeled using the Adobe program. 

PAGE gels of pooled nonVTG plasma proteins showed bands at approximately 70, 62 

73
 

and 28 kDa (Figure 2-4). The VTG proteins were 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 transblotted to PVDF membrane (InvitrolonTM, 

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), 1H12 (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 blots. 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-StepTM, Invitrogen). Development was 

stopped by washing with water. Each blot was dried, scanned, and labeled. 

The anti-sturgeon VTG Mab is very sensitive and clearly recognizes white 

sturgeon purified positive control, gravid female plasma, and purified gravid female 

plasma VTG (Figure 2-4). It does not react 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). 

74
 

Vitellogenin ELISA 

 Plasma VTG concentrations were determined by sandwich ELISA using a white 

sturgeon polyclonal antibody, UF131, and gulf sturgeon monoclonal antibody, 1H2, 

developed by the Doroshov (UCD) and Denslow (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 plate 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 TBSTZ (20 mM tris, 150 mM NaCl, 0.05% tween-20, 

0.02% azide, pH 7.4) for 2 hours at room temperature. The plate was re-washed with 

PBSZ before adding the samples and standards. 

Unknown samples and controls (positive and negative) were diluted 1:100, 

1:10,000, 1:100,000, and 1:1,000,000 in PBSZ with the 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 triplicate (50 μL/well). After incubating 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 for 2 hours at room temperature. After 

washing (4X PBSZT), an alkaline phosphatase conjugated detection antibody, goat-antirabbit IgG- F(ab)2-AP (Pierce), diluted 1:1000 in blocking buffer was added and 

75
 

incubated 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 MgCl2, and pH 9.6) was added to each well (100 μL/well). The plate 

was scanned at 405 nm using a reader (SpectraMax 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 homogenized 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 carefully 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 remove any large particles. Azide (0.02%) was 

76
 

added to this “crude egg yolk” preparation to prevent bacterial 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 has 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 evident, indicated as E1 through E3 (Figure 2-

6). Fractions within each peak were combined 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 standard. A 1.0 ml aliquot of 

each pool to be used for PAGE and Western analysis was collected, glycerol added (50% 

v/v), and proteins were stored at –20 oC. 

Egg PAGE & Western

 Polyacrylamide electrophoresis and Western analysis of egg yolk proteins (crude 

and purified) were identical to those described for plasma. Figure 2-7 shows typical gel 

and western of purified sturgeon egg proteins. The anti-sturgeon 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 overlap and also the extremely high sensitivity 

77
 

of the Mab. Peak E2 appears to be lipovitellin due to the cross-reactivity of the anti-VTG 

antibody. The Mab recognizes smaller sized proteins 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 appears to be phosvitin since 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 oC. The samples were then reduced by concentrated hydrochloric acid heated to 95 

o

C and measured as selenite by hydrogen generation inductively coupled plasma-atomic 

emission spectrometry (ICP-AES). The minimum detection limit was typically 5 ng/ml. 

Quality control measures included the use of standard reference materials (NRC DORM2, DOLT-2, DOLT-3), spiked samples, duplication and blanks with every analytical run. 

All quality control measurements agreed with the target concentration within 20 percent. 

78
 

Analysis of Selenium in Plasma and Isolated Proteins by Fluorometry 

Plasma and isolated plasma and yolk proteins 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 obtain the minimum amount of material required. 

VTG sample combinations are shown in Table 2-2. Approximately 2 mg of freeze-dried 

proteins, or 100 μl plasma, were digested in a mixture of nitric and sulfuric acids and 

gradually heated to 130 oC. 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 (prepared by sonicating 2,3-diaminonaphthalene 

hydrochloride, Dojindo Laboratories (Japan), in 0.1 N HCl for 1 h, filtered through 0.45 

μm cellulosic filter, and washed three times with excess cyclohexane) and extracted using 

0.5 ml of cyclohexane. This mixture was incubated 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 spectrofluorometer. The minimum detection limit was 

typically 1 ppb. Quality control measures included the use of spiked samples, duplication 

and blanks with every analytical run. Quality control measurements agreed with the 

target concentration within 15 percent. 

Statistical Analysis 

Morphologic measurements were compared between exposure groups using 

ANOVA and ANCOVA. Log10 transformations were used in regressions of body weight 

to fork length to account for allometric growth. The reproductive endpoints fecundity, 

79
 

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 squares 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 individual 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 proteins were performed using two-way ANOVA, 

nested for repeat measures, with Tukey’s Honestly Significant Difference (HSD) 

comparisons test (α = 0.05). Log10 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 examined using linear or nonlinear regressions. 

Correlation analysis was used when the variables could not be assumed to be functionally 

dependent. Weighted least squares regression was used when the dependent variable did 

not show constant variance. The weights used included the reciprocal of y and the 

reciprocal of y2

. The statistical software packages 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 rate (- 0.05%), and tissue Se concentrations 

lower or similar to control group. Apparently, this female was not feeding due to 

avoidance of high Se feed, stress, or internal injury caused by transport to UCD or 

handling. This fish was excluded from all statistical analyses of Se data in this study. 

80
 

Results 

Gross Morphologic Characteristics 

There were no differences in condition factor, fork length, or body weight 

between exposure groups at either the beginning 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 beginning or end of the study (ANCOVA, p = 0.69 

and 0.06 respectively). In addition, the specific growth rate (SGR) for the length of the 

study was not different between the two exposure groups (ANOVA, p = 0.94). 

Adult Tissue Selenium 

Comparisons within individual tissues showed that Se concentrations were 

significantly higher in Se-treated females (Student’s t-test, p < 0.0001; Wilcoxon’s Rank 

Sums, χ2 < 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, kidney, 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 concentrations 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 greater than in liver. The levels of Se in 

treatment fish muscle, ovarian tissue and eggs were very similar. All Se values reported 

81
 

above are based on dry weight analyses. Percent moisture of each tissue type is listed in 

Table 2-5. 

 

Plasma, Isolated Plasma VTG and nonVTG Plasma Protein Selenium 

Treatment fish had significantly higher levels of Se in plasma, and isolated 

plasma proteins, than control fish (Student’s t-test, p < 0.02; Wilcoxon’s Rank Sums, χ2 

< 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 plasma VTG, or nonVTG 

plasma proteins did not differ between sampling 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 proteins 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 control 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 concentrations were greater during mid-experiment as 

compared to end-experiment, but were not different between control and treatment fish 

(two-way nested ANOVA, p = 0.048; Tukey HSD, α = 0.05; Figure 2-13). Plasma VTG 

82
 

concentrations 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 plasma 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 available for the final sampling point of treatment 

fish. One-sample t-tests were calculated between mean Se values and the single end data 

point for treatment VTG (26.29 μg/g Se; Figure 2-10). Both the mid- and endexperiment time points in the control group 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 concentration was not different 

from the single end-experiment VTG data point (p = 0.32). 

 

Yolk Protein Selenium 

Se concentrations in isolated yolk proteins are presented in Table 2-7. Se was 

highly variable in both egg immunoglobulin and phosvitin. Only lipovitellin Se 

concentrations were found to be statistically 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 differences were observed. Se values for yolk proteins are 

reported as dry weight. 

83
 

Relationships of Selenium Distribution in Tissues 

We hypothesized that Se in the liver would become associated with the VTG 

molecule and transferred through the plasma to the eggs (Figure 2-1). Se concentrations 

in isolated plasma VTG increased exponentially with increasing Se in liver (R2 = 0.90; p 

< 0.0001; Figure 2-14; Table 2-8). Se in plasma, ovarian tissue and eggs increased 

exponentially with increasing Se concentrations in VTG (R2 ≥ 0.82; p ≤ 0.0008; Figure 2-

15; Table 2-8). Se in the yolk lipovitellin, but not phosvitin or immunoglobulin, 

increased exponentially with increasing Se concentrations in VTG (R2 = 0.91; p < 

0.0001; Figure 2-16; Table 2-8). Exploring the relationship between Se in eggs and in 

isolated yolk proteins, we found that only Se levels in lipovitellin could be predicted by 

Se levels in whole eggs (R2

 = 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 concentrations in ovarian tissue, eggs, plasma, and nonVTG isolated 

plasma proteins increased linearly with increasing Se in liver (Figure 2-18; Table 2-9). 

Plasma Se showed a significant relationship with Se in isolated nonVTG plasma proteins 

(p < 0.0001; R2 = 0.82 & 0.92 for mid- and end-experimental samples, respectively; 

Figure 2-19). Se concentrations in eggs increased linearly with Se in nonVTG proteins 

(R2 ≥ 0.73; p ≤ 0.0004; Figure 2-20). In the yolk, lipovitellin 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; Table 

84
 

2-10), but not mid-experiment. There was no correlation between Se concentrations in 

nonVTG proteins and in egg immunoglobulin (Table 2-10). All Se values are reported as 

dry weight, except plasma. 

Reproductive Characteristics 

Five fishes from each exposure group were hormonally induced to ovulate. The 

remaining three fishes from each exposure group had either follicular atresia or 

underdeveloped eggs that did not reach maturation competence (Appendix B). Three out 

of the five control fish ovulated (the two that did 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 GVBD 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 cycle) females. Hence, the ovulation rate after hormone 

injection cannot serve as an endpoint in Se toxicity trials with a limited number of fish. 

Detailed information on spawning-related parameters is presented in Appendix B. No 

significant differences were found for fecundity (95 and 72 thousand, in control and 

treatment), fertilization rate (86 and 88%), and neurulation survival (60 and 47%) 

between ovulated fish from the two exposure groups (Wilcoxon; p > 0.35; Table 2-12). 

Hatching occurred in all egg batches, except of one female (in treatment), but hatching 

rates could not be properly measured under the condition of “in mass” egg incubation. 

85
 

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 μg/g 

Se) had Se concentrations approximately 3 to 9 times greater than those fed control diet 

(ca. 1.42 μg/g Se) in all tissues analyzed, expect 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 the high metabolic activities of these tissues 

(Diplock 1976; Hilton and Hodson 1983; Sorensen 1986). Relatively lower levels of Se 

were observed in liver, which is also highly metabolic. This may be indicative of the 

transport of Se from liver to developing 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 concentration of the treatment diet (Figure 2-

10). Furthermore, plasma VTG protein contained higher Se levels compared to the 

nonVTG plasma proteins in treatment fish (Figure 2-12). Se concentrations of VTG and 

nonVTG plasma proteins were similar for control fish. These results support a tentative 

conclusion of Kroll and Doroshov (1991), who 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 females 

86
 

captured in the Sacramento River. These sturgeon had Se concentrations ranging from 

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

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

μg/g in phosvitin (all values based on dry weight, except plasma). Se concentrations in 

ovulated eggs increased linearly with increasing Se in egg immunoglobulin and phosvitin 

(R2 = 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 cysteine 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 and 2.92% methionine (based on total moles of amino acids; 

Bidwell and Carlson 1995). VTG can transport 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 (Monteverdi 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 maternal transport of Se, and the current study 

is the first to examine this exposure 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, greater amounts of Se are associated with VTG 

(Figure 2-14). In fact, Se concentrations in VTG increased exponentially with increasing 

87
 

Se concentrations in the livers of fish from this study (R2 = 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 (R2 = 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 (R2 = 0.91 and 0.96, respectively; Figures 2-16 & 2-

17), whereas Se concentrations of phosvitin 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 yolk proteins. We observed the same trend in 

treatment fish of this study (Table 2-7); however, there were no statistical differences 

between mean Se concentrations in phosvitin 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 and 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 acid 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 the 

88
 

incorporation of selenocysteine (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 proteins 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 support the hypothesis that as Se becomes 

more available in the liver, there is an increase 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 (R2 = 0.65; Figure 2-18; Table 2-9). The Se concentrations 

in eggs increased linearly with increasing Se concentrations in nonVTG plasma proteins 

(R2

 = 0.80 and 0.73 for mid- and end-experiment samples, respectively; Figure 2-20), 

which may simply reflect the effect of increasing liver Se. In addition, 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 plasma 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 proteins associated with Se correspond with 

89
 

these general molecular mass ranges. In particular, plasma albumin (68 kDa in humans) 

is a very abundant plasma protein that binds with selenide in the bloodstream (Quinlan et 

al. 2005; Shiobara and Suzuki 1998). In addition, 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; Kryukov 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). Additionally, 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), thyroxide 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 role 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 and 

90
 

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, skeletal 

deformities, and died. Woock et al. (1987) found similar results. Schultz and Hermanutz 

(1990) experimentally demonstrated the efficient maternal transport of Se in fathead 

minnow (Pimephales promelas) raised in experimental streams. 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 developed into larvae with edema and skeletal 

deformities. Hermanutz et al., (1992) observed reduced hatching, edema, skeletal 

deformities and hemorrhaging in progeny of bluegills held in experimental streams 

treated with 10 μg/L Se. Hamilton et al. (2002a) showed that razorback suckers 

(Xyrauchen texanus) in the seleniferous Colorado River can transfer significant amounts 

of Se to their eggs (36 – 60 μg/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 sturgeon fed a diet containing ca. 34 μg/g Se were 

found to contain ca. 11, 10 and 12 Se μg/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 this area and contain an average Se level of 

15 μg/g (dry wt; Linville et al. 2002; Urquhart and Regalado 1991). However, the above 

91
 

Se 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 concentrations ranging from 9 to 30 μg/g in liver (n= 52) 

and 7 to 15 μg/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 Francisco Bay. Out of six sturgeon females 

sampled in the San Francisco Bay-Delta region in the 1990’s, one contained eggs with 3 

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

29 μg/g Se (Kroll and Doroshov 1991). Recently, three white sturgeon captured from 

this region were found to have 7 to 20 μg/g Se in ovaries containing developing eggs 

(Doroshov Lab, UCD; unpublished data). The actual Se concentrations 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 thresholds of Se toxic effects for 

freshwater and anadromous fish at 8 μg/g in muscle, 12 μg/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 approached the suggested threshold of 12 μg/g. The 

reproductive endpoints of fecundity, fertilization rate and neurulation 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 organic Se is likely stored 

92
 

in the yolk. However, as shown in the next chapter, embryos of the treatment fish from 

this experiment showed significant developmental toxicity. 

In conclusion, this is the first work to experimentally demonstrate that 

vitellogenesis provides a significant, 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 other fish, sturgeon store and use 

their yolk in the form of intracellular crystalline platelets utilized throughout early 

development, including the embryonic 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 incorporated Se is conserved in the eggs through the vitellogenic 

phase of oogenesis until spawning. The results of our study, along with the field data 

described above, demonstrate that exposure of maturing females to a diet moderately 

high in Se may present a threat to the reproduction of white sturgeon residing in the San 

Francisco Bay-Delta region. This threat becomes more prominent as we explore Seinduced developmental toxicity in white sturgeon in the following chapter. 

93
 

Se content of experimental diets 

Diet

Target Se Content 

(μg/g, dry wt) 

Measured Total Se 

(μg/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.).

94
 

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 contribution 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 food avoidance (as described in the Statistical Analyses

section). 

95
 

Morphological comparisons between exposure groups 

Start of Study End of Study 

ANCOVA F p R2 F p R2

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 characteristics were compared between control and treatment 

groups at both the beginning and end of the study. None of the measured characteristics 

were shown to be statistically different. 

96
 

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 

μg/g Se) or treatment (ca. 34 μg/g Se) diets for approximately six months. Data 

displayed as mean Se μg/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 

(χ2 < 0.0017). 

97
 

 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). 

98
 

Se in plasma and isolated plasma proteins of adult white sturgeon fed either control 

or treatment diets for approximately six months 

Tissue Control Treatment 

Plasma1 (μg/mL) 0.21 ± 0.01 (8) 1.28 ± 0.10** 

Plasma2 (μg/mL) 0.27 ± 0.05 1.25 ± 0.15** 

NonVTG1 

Plasma Proteins 7.27 ± 0.30 22.21 ± 3.38†

 

NonVTG2 

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 isolated plasma proteins of white sturgeon 

fed either control (ca. 1.42 μg/g Se) or treatment (ca. 34 μg/g Se) diets for approximately 

six months. Data displayed as mean Se μg/g, dw ± standard error, except plasma, which 

is presented as Se μg/ml. Sample size is 7 unless indicated 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 (χ2 < 0.0017) 

‡

Difference shown by one-sample t-test comparing control mean to the one treatment value (p < 0.02) 

99
 

Se in isolated yolk proteins of adult white 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 isolated from eggs of adult white sturgeon 

following exposure to either control (ca. 1.42 μg/g Se) or treatment (ca. 34 μg/g Se) diet 

for 126 – 200 days. Data displayed as mean Se μg/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. 

100
Se in combined VTG samples compared to combined tissue and yolk protein samples 

Data presented as mean Se μg/g, dw ± standard error, except plasma, which is presented as Se μg

Control  Fish Time Number(s) Point VTG Liver Egg Ovarian Tissue Plasma Phosvitin Lipov C1 – 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 C4 Mid 3.8 ± 0.00 (1) 0.8 (1) 1.61 (1) 1.65 (1) 0.2 (1) 4.95  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  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 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 ± 0Treatment  Fish Time Number(s) Point VTG Liver Egg Ovarian Tissue Plasma Phosvitin Lipov T1 - 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 ±  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 ±  T7 Mid 40.21 ± 2.06 (1) 11.6 (1) 15.1 (1) 14.4 (1) 1.22 (1) 15.55 (1) 17.63  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 ± Table 2-8. Due to small sample sizes, isolated VTG protein samples were pooled prior to Se analysis (Table 2-2). Here wVTG samples compared to the corresponding combinations of liver, eggs, ovarian tissue, plasma and isolated egg yolk protmean Se μg/g, dw ± standard error (number of experimental animals contributing to the average), except plasma, which is p

Mid samples were taken after 88 days of dietary exposure. End samples were taken after 126 – 200 days of dietary exposu
 

Variables Model R2 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 Plasma1

Weighted 

Linear 0.87 83.10 <0.0001 14 y = 0.12x + 0.05 

Liver v Plasma2 Linear 0.76 39.01 <0.0001 14 y = 0.11x + 0.13 

Liver v nonVTG1 Linear 0.65 20.54 0.0009 13 y = 1.81x + 4.56 

Liver v nonVTG2 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. 2Sampled at spawning or necropsy, after 126 - 200 

days of exposure. Each regression is statistically significant. 

102
 

Se in yolk proteins correlated to Se in nonVTG

nonVTG1

Proteins 

nonVTG2

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, yolk 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, lipovitellin, phosvitin, and whole eggs. 1

Collected after 88 

days of exposure. 2

Collected after 126 - 200 days of exposure. 

103
 

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 μg/g Se) or 

treatment (ca. 34 μg/g Se) diets for approximately six months. More detailed spawning 

data are presented in Appendix B. 

104
 

Reproductive performance of sturgeon females 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 and treatment groups for any of the three 

parameters (Wilcoxon; p > 0.35). 

105
Se transport via vitellogenesis 

Blood 

Figure 2-1. Hypothesized mechanism of the maternal transport of Se to offspring. During vitellogenesis (yolk d

is produced in egg follicles and transported to the liver via blood. Estrogen signals the liver to produce the yolk p

vitellogenin (VTG). Se in the liver may be incorporated into the VTG molecule, which is transported to the egg up by the VTG-specific receptor in the egg membrane. Inside the egg, VTG cleaves into the yolk proteins lipovi

and Se is stored in the yolk until metabolized during the embryonic and larval stages. Figure modified from Mom

(1988). 

VTG-Se

Estrogen (E

Estrogen

)

Developing Egg 

Follicle Cells 

VTG 

Receptor 

VTG-Se 

Phosvitin 

Lipovitellin 

Se 

Yolk 

E (l ble)

Liv

DNA

R

Receptor (R

Vit

VT

ipid solu

EBlood 
 

Figure 2-2. Anion exchange chromatograph of white sturgeon male before (red line) and 

after (green line) estrogen treatment. 

107
 

Figure 2-3. Anion exchange chromatograph of plasma 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. 

108
 

 PAGE Western 

Figure 2-4. PAGE & Western of white sturgeon plasma proteins. The first lane contains 

molecular mass markers; the second lane contains plasma of nonestrogenized males; the 

third lane contains purified VTG. The next four groupings are individual 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 different bands of white sturgeon 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. 

109
 Mean Value 

Figure 2-5. VTG ELISA Standard Curve. The x-axis shows VTG concentration (μg/ml) 

while the y-axis shows absorbance at 405 nm. 

110
 

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. 

111
 

 

 PAGE Western 

Figure 2-7. PAGE and Western gels of crude and 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 fractions from gel filtration corresponding to 

peaks E1, E2, and E3. Peak E3 (Phosvitin) does not have affinity to Coumassie Blue 

stain. 

112
 

Figure 2-8. Tissue Se concentrations in white sturgeon exposed to ca. 1.5 or 34 μg/g 

dietary Se for approximately six months. Bars show mean Se μg/g, dw, with vertical 

lines showing standard error of the mean. Sample size is 7, except for control muscle and 

eggs (n = 6 and 5, respectively). Lognormal data compared by two-way nested ANOVA 

(p = 0.028) with adjusted multiple comparisons (family α = 0.05). Groups that do not 

have the same letter in superscript are significantly different. Data are also presented in 

Table 2-4. 

113
 

Figure 2-9. Se in plasma collected at mid- and 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 μg/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 superscript are significantly different (Tukey HSD, α = 

0.05). Data are also presented in Table 2-6. 

114
 

3 2 3 1

Figure 2-10. Se in VTG isolated from plasma collected at mid- and end-experiment 

(after 88 and 126 - 200 days of dietary exposure, respectively) compared across exposure 

groups (ANOVA; p < 0.011). Bars show mean Se μg/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 superscript are significantly different (Tukey HSD, α = 

0.05). *Only one data point was available for 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 μg/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. 

115
 

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 

μg/g, dw, with vertical lines showing standard error of the mean. Sample size is 7. Data 

not connected by same letter are significantly different (Tukey HSD, α = 0.05). Data are 

also presented in Table 2-6. 

116
 

Se in VTG and nonVTG from white sturgeon 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 μg/g, dw, with vertical lines showing 

standard error of the mean. Sample size shown in bars (VTG are pooled samples). Data 

not connected by same letter are significantly different (two-way ANOVA, p = 0.032; 

Tukey HSD, α = 0.05). 

117
 

Vitellogenin from adult white sturgeon following approximately six months 

exposure to dietary Se 

Figure 2-13. Vitellogenin isolated from plasma collected at mid- and end-experiment 

(after 88 and 126 - 200 days of dietary exposure, respectively). Plasma VTG 

concentrations were greater during mid-experiment as compared to end-experiment, but 

were not different between control and treatment 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 shown in bars. Data not connected by same 

letter are significantly different. 

 

118
 

Figure 2-14. Se in VTG increases exponentially with increasing Se in liver (R2 = 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 dietary exposure, respectively). All liver 

samples were collected at the end of the experiment. 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 combinations of liver data 

(Table 2-8). 

119
 

Figure 2-15. Se in plasma, ovarian tissue and 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 average Se in the corresponding combinations of 

plasma, ovarian tissue and egg data (Table 2-8). Data displayed as Se μg/g, dw, except 

plasma, which is presented on the right y-axis as Se μg/ml. Each regression is 

statistically significant. 

120
 

Se in Vitellogenin Related to Se in Yolk Proteins 

Figure 2-16. Se in lipovitellin increases exponentially with increasing Se in VTG (R2 = 

0.91; p < 0.0001). Se in phosvitin and egg immunoglobulin 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 analysis (Table 2-2). Se in pooled VTG samples 

is compared to the average Se in the corresponding combinations of yolk protein data 

(Table 2-8). Data are displayed as Se μg/g, dw. 

121
 

Figure 2-17. Se in isolated yolk proteins as a function of Se in eggs. A significant linear 

relationship was found between Se in eggs and Se in lipovitellin (R2 = 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). 

122
 

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 - 200 days of exposure. Data displayed as 

Se μg/g, dw, except plasma, which is presented on the right y-axis as Se μg/ml. Each 

regression is statistically significant. Complete regression statistics are presented in 

Table 2-9. 

123
 

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 statistically significant linear correlation was found 

between plasma and nonVTG isolated plasma proteins (p < 0.0001; R2 = 0.82 and 0.92 

for mid- and end-experiment, respectively). 

124
 

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 μg/g (dw). 

125
 

Chapter 3. Developmental toxicity in white sturgeon larvae exposed to selenium 

through maternal transport or microinjection 

Introduction 

Selenium (Se) is a well-documented developmental 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 transfer of Se to eggs and embryos during 

vitellogenesis in white sturgeon. Transfer and 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 are 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 sturgeon population abundance in San Francisco 

Bay-Delta. White sturgeon populations in this region have been highly variable over the 

past five decades (Kohlhorst 1980; Kohlhorst et al. 1991; Schaffter and Kohlhorst 1999). 

126
 

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 Commission 2006). White sturgeon only spawn every 

two or more years (Doroshov et al. 1997), which led the California 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 and Game Commission 2006). Previous studies 

have hypothesized that the impact of environmental contaminants may affect recruitment 

success of white sturgeon in this area (Kohlhorst 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 through both the refining of fossil fuels and 

irrigation drainage from the seleniferous 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 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 accumulation of maternal Se in the egg yolk 

proteins of white sturgeon was shown in chapter two of this dissertation. Sturgeon 

embryos hatch as underdeveloped (altricial) organisms commonly referred to as yolk sac 

larvae. During the first 10 days post-hatch, over eighty percent of the 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 

127
 

(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, sturgeon store and digest yolk intracellularly 

(Buddington and Doroshov 1986), and the nutrients 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 stomach, mid-intestine, liver, and pancreas 

(Buddington and Doroshov 1986). The intracellular digestion and increased metabolism 

of yolk in sturgeon may exert profound effects on the development and survival of yolk 

sac larvae with elevated Se levels in maternal yolk. 

The experiments described here were based on the hypothesis that white sturgeon 

is most sensitive to Se-induced developmental defects and mortalities during the rapid 

yolk utilization and organogenesis 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 the maternal exposure experiment, embryos of 

females used in the Se maternal transfer experiment (described in chapter two) were 

reared and observed for developmental defects 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-methionine (Se-L-Met) was injected into 

the yolk sac of newly hatched larvae of unexposed females. Microinjection was used 

because the large size and long ovarian cycle of white sturgeon make it difficult and 

expensive to study reproductive toxicity in this species under laboratory conditions. 

Microinjection mimics maternal transfer of environmental contaminants and is used as an 

128
 

important 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, and mortality were quantified. The maternal 

exposure experiment provides a causal link between Se in maternal food and 

developmental effects in progeny. The microinjection exposure experiment confirms 

threshold levels of Se developmental toxicity in white sturgeon larvae. 

Materials and Methods 

Maternal Exposure Study 

This study used developing embryos from the maternal Se exposure study 

described in chapter two of this dissertation. Briefly, sixteen domestic broodstock white 

sturgeon females (ca. 22.71 kg and 134.59 cm) were exposed to either control (ca. 1.4 

μg/g Se) or treatment (ca. 34 μg/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-treated males. Fertilized eggs were placed into individual 

MacDonald hatching jars; and jars were set up to allow hatched larvae 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 the 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 buckets 

129
 

were fashioned from 19 liter high-density polyethylene buckets (ca. 26 cm diameter, 37 

cm depth) with screens 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 pair 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 larvae. Temperature was maintained at 18.56 

± 0.13 °C. Flow rate in tanks 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 shade-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 morphological analysis of larval development (stages 36 – 45, 

yolk sac larvae). Table 3-2 shows developmental staging following Dettlaff et al. (1993) 

for Russian sturgeon, with specific characteristics for white sturgeon described by Beer 

(1981) and Deng (2000). There were slight variations in stage 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). Developmental 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 

130
 

–80 °C (for Se analysis), fixed in 10% buffered formalin (for developmental assessment), 

or measured immediately (for size measurement). All post-hatch developing white 

sturgeon are referred to as larvae 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 oC and measured as selenite by hydrogen 

generation inductively coupled plasma-atomic emission spectrometry (ICP-AES). The 

minimum detection limit was typically 5 ng/ml. Quality control measures included the 

use of standard reference materials (NRC DORM-2, DOLT-2, DOLT-3), spiked samples, 

duplication and blanks with every analytical run. All quality control measurements 

agreed with the target concentration 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 sturgeon described by Beer (1981) and Deng (2000; Table 3-2). 

131
 

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 skeletal 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 approximately 30 individuals. 

Measurement of larval size

Body weight and total length were measured in approximately 30 larvae of each 

progeny cohort for three time points. Deformed 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 blotted and weighed individually (wet weight) on 

a microbalance (± 0.0001 g) then placed in tared whirl packs, lyophilized, and weighed to 

determined dry weights. 

Microinjection Studies 

Study One

Post-neurulated embryos were obtained from spawning of domestic stock 

(Columbia River origin) at Clear Springs Food 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 arrival, the embryos (stage 30-31; Dettlaff et 

al. 1993) were acclimated to 15.7 °C for two hours and transferred to crystallizing dishes 

132
 

submerged in tanks. Approximately 90% of post-neurulated embryos received from 

Idaho survived to hatching and provided viable 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 microinjections. 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. Following 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 vials using sterile millipure water to 7.0, 

14.1, and 28.2 mg/mL. Target doses were calculated as described below. The injection 

volume was 25 nL per larva, resulting 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 physical (e.g., heartbeat, pericardial edema, 

yolk absorption) and morphological (e.g., skeletal) 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 analysis and morphology. 

133
 

Sampled larvae were anesthetized in 150 ppm MS222, overdosed in 500 ppm MS-222, 

rinsed in millipure water, then frozen on dry 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 milt (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-circulating 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 below. 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 vials using sterile millipure water to 2.11, 6.34, 

and 19.03 mg/mL. Target doses were calculated 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 L134
 

Met (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, and low, medium, and high Se-L-Met groups of 

80 larvae were each distributed into one survival (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 content at two hours post injection (stage 

36), at stage 40 (pyloric sphincter 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 stored at –80 °C (for Se analysis) or fixed in 

10% buffered formalin containing 4% sucrose (for morphology). 

In addition, each treatment was sampled at the start and endpoint of the 

experiment to determine live body weight and total length. Total lengths of newly 

hatched larvae were measured in 10-15 individuals 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 placed in tared whirl packs, lyophilized, and 

weighed to determined dry weights. 

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Larval culture maintenance 

Microinjection experiments were carried out at CABA at UCD. Indoor flowthrough water recirculation systems with biological filter, aeration, YSI thermostat, 

chiller, and heater were used for all phases of the experiments. Eggs and larvae were 

held in circular fiberglass tanks (28 cm diameter, 35 cm depth, flow rate 1.0 – 1.5 L/min). 

Dissolved oxygen and temperature were recorded daily. A certified calibrated 

thermometer (National Institute of Standards and Technology) was used to adjust 

incubation temperature to 15.7 ± 0.1 °C. Dissolved 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 covers. Ammonia in water ranged from less 

than 0.003 to 0.014 mg/L NH3 and pH ranged from 8.2 to 8.3 (DNRA laboratory, UCD). 

Se concentration in the water of recirculation systems was below 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 Foothill laboratory, Inc., Jackson, California; 

certified for water Se analysis at UCD facilities). 

Key sturgeon developmental stages were used to initiate treatments, to collect 

tissue samples for Se and to conduct morphological analysis at midpoints and endpoints 

of each experiment. Table 3-2 shows developmental staging following Dettlaff et al. 

(1993) for Russian sturgeon, with specific characteristics for white sturgeon described by 

Beer (1981) and Deng (2000; Table 3-2). There were slight variations in stage within the 

sample due to the fast rate of development. 

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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, Japan), a joystick micromanipulator (MN-151, 

Narishige group, Japan) mounted on a magnetic stand, and aluminosilicate needles 

(A150-100-10, Sutter Instruments Co., Novato, CA). Needles were pulled from 

aluminosilicate tubing (1 mm OD, and 0.67 mm ID) using a programmable 

Flaming/Brown micropipette puller (P-97, Sutter 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), beveled (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-calibrated eyepiece micrometer. Needle 

calibration was done by injecting solution into paraffin oil and measuring the dispensed 

137
 

drop with the eyepiece micrometer. Volume adjustment was done by increasing or 

decreasing injection time while maintaining the 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, Sigma-Aldrich, St Louis, MO) was used 

for the microinjection experiments. Amount of Se-L-Met (MW 196.1) used for the 

different treatments was calculated based on the 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 larvae to be 7.1 mg (dw) for our temperature 15.7 °C. 

Selenium analysis

Larvae were frozen on dry ice and stored at -80o

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 approximately 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 acids and gradually heated to 130 oC. The samples were then 

138
 

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 (prepared by 

sonicating 2,3-diaminonaphthalene hydrochloride, Dojindo Laboratories (Japan), in 0.1 N 

HCl for 1 h, filtered through 0.45 μm cellulosic filter, and washed three times with excess 

cyclohexane) and extracted using 0.5 ml of cyclohexane. This mixture was incubated 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 spectrofluorometer. The 

minimum detection limit was typically 1 μg/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 concentration within 15 percent. 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 samples. The development was monitored using 

the morphological criteria 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 included in total occurrence rate. 

139
 

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 assumptions of parametric models could not be 

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

et al. 1996). All hypothesis multiple comparison testing was performed 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 based on dry weight, unless otherwise noted. 

Developmental defects were compared using logit analyses. The logit equations were 

used to estimate median effective doses (ED50) for developmental defects. ANOVA 

models were also applied to percent developmental 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 individuals 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 developmental effects in microinjection study two, the control 

non-injected and sham-injected groups were not significantly different and were 

combined into one group. Similarly, the high, medium and low L-methionine samples 

140
 

were combined into one group since they were not significantly different from each other. 

Hypothesis comparisons of developmental effects were not possible in microinjection 

study one because only one progeny replicate was used. 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 Rank 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 Rank 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 

μg/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 the 

141
 

limited 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 sturgeon were significantly greater in the 

treatment group for each developmental stage 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 weight, unless otherwise noted. 

Microinjection

Table 3-6 and Figure 3-2 show Se concentrations 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-injected) and 2.54 (sham-injected) Se (μg/g) 

at the initiation of the experiment. Post-injection 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 replicate tanks). The low treatment group larvae 

142
 

contained 13.92 ± 1.03 μg/g Se at the experimental endpoint. Control 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 may relate to two different methods of Se 

analysis and different sample sizes at the start and end of the experiment (micro-digestion 

with Fluorometry and n of 5 – 10 at start; macro-digestion with ICP-AES and n of ca. 45 

at end; Table 3-6). There was insufficient data (one progeny replicate) to perform a 

statistical comparison of the 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 ± standard error of the two progeny cohorts) 

at the start of the experiment. Post-injection 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), larvae in the high treatment group contained 

significantly higher Se than the 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 experimental groups or time points. 

143
 

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 defects. Logit analysis showed significant 

associations between the occurrence of edema (χ

2 = 39.7) and skeletal deformities (χ2 

= 

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 μg/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 μg/g Se in larvae, respectively (Figure 3-5). Either of the effects, edema 

or skeletal deformities, would significantly reduce 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 used to estimate that 10, 25 and 

50 percent of the population would become affected (edema and/or skeletal deformity) at 

ca. 15, 22, and 29 μg/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 difference between control and treatment groups was 

144
 

in the total percentage of larvae affected (edema and/or skeletal deformities; two-way 

nested ANOVA following angular transformation, 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 treatment group (two-way nested ANOVA following 

angular transformation; Tukey HSD, α = 0.05). Each type of skeletal deformity was also 

analyzed separately. Only scoliosis was found to appear more often in the treatment 

group (Wilcoxon Rank Sums, p < 0.025). 

Microinjection

As with the maternal exposure experiment, 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 (χ2 = 147) and Se concentrations 

in larvae at the onset of larval development (p < 0.0001; Table 3-11; Figures 3-8 & 3-9). 

Logit probabilities were used to estimate that 25 and 50 percent of the population would 

incur edema at ca. 13 and 42 μg/g Se in larvae, respectively (Figure 3-8). Skeletal 

deformities would be expected in 15, 25 and 50 percent of the population at ca.10, 18, 

and 32 μg/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 experimental group. Logit probabilities were used to 

estimate that 25 and 50 percent of the population would become affected (edema and/or 

skeletal deformity) at ca. 7 and 17 μg/g Se in larvae, respectively (χ

2 = 202.8, p < 0.0001; 

145
 

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 occurrence of edema (χ

2 = 132.3) and skeletal deformities (χ2 = 26.4) and Se 

concentrations in larvae at the onset of larval development (p < 0.0001; Table 3-12; 

Figures 3-11 & 3-12). Logit probabilities were used to estimate that 15, 25 and 50 

percent of the population would incur edema at ca. 11, 13 and 15 μg/g Se in larvae, 

respectively (Figure 3-11). Skeletal deformities would be expected in 15, 25 and 50 

percent of the population at ca. 15, 17, and 21 μg/g Se in larvae, respectively (Figure 3-

12). Logit probabilities were used to estimate that 15, 25 and 50 percent of the 

population would become affected (edema and/or skeletal deformity) at ca. 10, 12, and 

15 μg/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 significantly higher percentages 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 following angular transformation, p ≤ 0.05, Tukey 

146
 

HSD, 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 high, 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). Hypothesis comparisons of developmental effects were not 

possible in microinjection study one because only one progeny replicate was used. 

Combined Maternal and Microinjection Data

Data from the maternal and microinjection experiments were combined to provide 

a more robust analysis of developmental defects following Se exposure during larval 

development. Logit analysis of the combined data showed significant associations 

between the occurrence of edema (χ

2 = 199.8), skeletal deformities (χ2 = 340.1) and Se 

concentrations in larvae at the onset of larval development (p < 0.0001; Tables 3-7 – 3-9, 

3-11 & 3-12; Figures 3-14 & 3-15). Logit probabilities were used to estimate that 15, 25 

and 50 percent of the population would incur edema at ca. 18, 26 and 40 μg/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 μg/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 (edema and/or skeletal deformity) at ca. 11, 15, and 

22 μg/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. 

147
 

Mortality in Larvae 

Maternal Exposure

Mortality in larvae was analyzed using Kaplan-Meier survival analysis. 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 μg/g), medium (ca. 9 μg/g) and low (ca. 2 μg/g) ranges of Se concentrations in 

eggs. Both approaches revealed significant 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 probability of 0.03 (3 percent) of mortality in 

treatment larvae by the end of larval development (Kaplan-Meier, p < 0.0001). 

Comparison of the Kaplan-Meier distributions shows that the treatment group is 

significantly different from the control group (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 μg/g) by the end of larval development (Kaplan-Meier, p < 0.0001). 

Differences between these Kaplan-Meier distributions follow High (ca. 21 μg/g Se) >> 

Low (ca. 2 μg/g) > Medium (ca. 9 μg/g; Student’s t-test, p < 0.0001). 

Microinjection

Incidence of mortalities and the percentage of developmental effects observed 

within mortalities for the microinjection studies are shown in Table 3-15. Both 

microinjection studies show a statistically significant difference in the probability of 

larval mortality between exposure levels, over time (Kaplan-Meier, p < 0.0001; Figure 3-

148
 

18). In microinjection study one, the probability of larval mortality in low, medium, and 

high treatment groups by the end of larval development were approximately 0.35, 0.45 

and 0.65, respectively (Kaplan-Meier, p < 0.0001). Comparison of the Kaplan-Meier 

distributions shows the relationship high (ca. 46 μg/g Se) > medium (ca. 22 μg/g Se) > 

low (ca. 16 μg/g Se) > control (ca. 3 μg/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 (Kaplan-Meier, p < 0.0001; Table 3-15; Figure 

3-18). Differences between Kaplan-Meier distributions follow high (ca. 17 μg/g Se) > 

low (ca. 9 μg/g Se) > control (ca. 6 μg/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 and Microinjection Data

Table 3-16 summarizes percent mortality in white sturgeon larvae following Se 

exposure using the combined data of the maternal and microinjection studies. The 

combined data are categorized by Se concentrations in larvae at the start of larval 

development (ca. stage. 36; after hatching in maternal study; several hours post injection 

in microinjection studies). Kaplan-Meier survival analysis of combined mortality data 

shows a statistically significant difference between exposure categories (p < 0.0001; 

Figure 3-19). Differences between Kaplan-Meier distributions follow very high (ca. 46 

μg/g Se) >> high (ca. 18 – 22 μg/g Se) = mid (ca. 12 – 16 μg/g Se) > low (ca. 8 – 10 

149
 

μg/g Se) > control (ca. 2 – 7 μg/g Se; student’s t-test, p < 0.05). The probability of 

mortality by the end of larval development in larvae exposed to 12 to 22 and 46 μg/g Se 

were approximately 0.15 to 0.29 and 0.65, respectively (Kaplan-Meier, p < 0.0001; 

student’s t-test, p < 0.05). 

Discussion 

This work shows that white sturgeon are 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 significant 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 selenoproteins in larval 

tissue and increase of Se in circulation could both contribute 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 larvae proved to be a reliable method to study 

the effects of excess Se on the development 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-

150
 

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 different sample sizes (n = 60 and 10, respectively) 

and different analytical methods employed 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 levels of Se exposure (ca. 9 and 17 μg/g; Table 3-6; Figure 3-2). 

In both microinjection studies, we were unable 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 directly following injection, due to the water 

solubility of Se-L-Met. Overall, microinjection 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 contradicts 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. Similar types of deformities were observed in the 

maternal exposure and microinjection studies. Developmental defects were low in the 

control groups of all studies (< 4%; Tables 3-10, 3-11 & 3-13). Exposure to ca. 10 μg/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 microinjected larvae (low and medium dose of 

study two; Table 3-13). Exposure to ca. 16 to 47 μg/g Se led to very high incidence of 

developmental defects in microinjected larvae (70 – 84%; Tables 3-11 & 3-13). 

151
 

However, maternally exposed larvae in cohort two (T2) containing ca. 20 μg/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 microinjection studies, developing larvae 

showed statistically significant relationships between 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 between 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 observed effects. Logit probability equations were used to estimate the 

median effective Se dose (ED50) for each defect (Table 3-17). The term EDX 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 microinjected larvae appeared to be more sensitive 

to Se; however, many of the EDX 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 μg/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 compared to larvae in the maternal exposure 

experiment. In the maternal study, 8 percent mortalities was observed with ca. 20 μg/g 

152
 

Se exposure, but similar Se exposures were associated 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). When 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 μg/g Se were approximately 15 - 29 and 65 percent, 

respectively (Kaplan-Meier, p < 0.0001; student’s t-test, 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 maternal exposure experiment (0 – 8.5%; Tables 

3-14 & 3-15). Similarly, edema and/or skeletal deformities occurred at lower Se levels in 

microinjected larvae (ED50 = 14.5 – 16.8 μg/g Se) than in larvae exposed to Se-laden 

maternal yolk (ED50 = 28.8 μg/g Se; Table 3-17). The higher sensitivity of the 

microinjection larvae is likely due to the effects of injection stress, form of Se used (free 

amino acids in microinjection versus protein bound in maternal 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 experiments. The timing of the production of a 

critical enzyme in Se detoxification (superoxide 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 the production of key detoxifying enzymes or cofactors, then 

greater sensitivity would be expected. These suggestions remain speculative until 

153
 

additional 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 larvae 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 forms 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 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 and 

craniofacial and skeletal deformities. Gillespie 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 weight 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 hazard to this endangered species. 

154
 

Several possible mechanisms of Se toxicity have been described. Palace et al.

(2004) demonstrated that selenomethionine exposure can lead to oxidative stress in fish 

larvae, which is known to cause edema and spinal curvatures. They linked superoxide 

production to methioninase enzyme activity and the ensuing metabolism of 

selenomethionine. Superoxide activity affects the embryo until significant production of 

superoxide dismutase provides a detoxification 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 chemical 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 Leewenhoekhuis 1987) and react with sulfhydryl 

groups of proteins or other molecules (Martin 1973), causing disruptions of histogenesis 

and deformities, which are a common effect of Se toxicity (Lemly 1998). 

These results indicate that white sturgeon 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 μg/g in liver (n= 52) and 7 to 15 μg/g in muscle (n = 

155
 

99; Urquhart and Regalado 1991; White et al. 1988). Very limited data are available on 

Se concentrations in the eggs of wild white sturgeon. Out of six sturgeon females 

sampled in the San Francisco Bay-Delta region in the 1990’s, one contained eggs with 3 

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

29 μg/g Se (Kroll and Doroshov 1991). Recently, three white sturgeon captured from 

San Francisco Bay-Delta were found to have 7 to 20 μg/g Se in ovaries containing 

developing eggs (Doroshov Lab, UCD; unpublished data). 

In the work described here (chapters 2 and 3) female sturgeon with liver Se 

concentrations of 11.0 and 11.6 μg/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 μg/g Se in the liver and 

produced larvae that were free of developmental defects. In these limited data, maternal 

brood fish Se muscle concentrations appear 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 sturgeon populations could be monitored by 

analyzing non-lethal core samples of muscle. 

Se levels in eggs and larvae provide an estimate of the direct exposure of Se to the 

developing larvae during yolk utilization. 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 collected using spawning mats. The limited data 

available suggest that white sturgeon in the 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 developing white sturgeon, based on both the maternal 

156
 

and microinjection exposure experiments presented here. Larval Se concentrations of 3.4 

and 8.2 μg/g are predicted to result in edema and/or skeletal deformities in 5 and 10 

percent of developing larvae, respectively. 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 observed in the white sturgeon population of San 

Francisco Bay-Delta (California Department of Fish and Game Commission 2006; 

Schaffter and Kohlhorst 1999). 

In summary, white sturgeon larvae exposed to Se by maternal transfer and 

microinjection demonstrated significant increases 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 rates similar to those observed in field and 

laboratory studies of Se toxicity in other fish. 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 μg/g Se in developing white 

sturgeon is suggested for this species. 

157
 

Se content of experimental diets in maternal exposure experiment 

Diet

Measured Total 

Selenium (μg/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.).

158
 

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 the fertilized egg. Due to the 

holoblastic cleavage and yolky eggs in sturgeon, the second 

cleavage furrow appears in the animal pole resulting in four 

incompletely divided cells sharing 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 tube. 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 (stage 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 relatively 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 portion (future intestine) of the 

yolk sac. The intestinal portion 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). 

159
 

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 μg/g) or 

treatment (ca. 34 μg/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). 

160
 

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 ± standard 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 ± 2.96b 18.45 ± 0.39ab

Sham-injected 42.28 ± 1.74b 18.60 ± 0.50a

L-Met Low * 45.04a 18.57ab

L-Met Med 41.76 ± 2.51b 18.33 ± 0.23ab

L-Met High 41.74 ± 1.00b 18.09 ± 0.01bc

Se-L-Met Low 42.49 ± 2.52b 18.50 ± 0.27a

Se-L-Met Med 42.44 ± 2.45b 18.67 ± 0.30a

Se-L-Met High § 37.87c 17.56c

Table 3-4b. Weight and length (mean ± standard error of the two progeny 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 different (nested ANOVA, p < 0.0001; Tukey HSD α = 

0.05). 

*Sample included larvae from Female 1 only, which were initially higher in weight 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. 

161
Selenium in developing white sturgeon progeny from female white sturgeon exposed to dietary 

Se μg/g ± Standard Error (n) 

Tissue Control Treatment

Percent Moisture (Avg ± 

Standard Deviation)

Egg 2.25 ± 0.326 (3) 12.4 ± 2.801 (4)‡ 63.7 ± 4.1 

Stg. 5 Embryo 2.32 ± 0.279 (3) 11.78 ± 2.711 (4)‡ 75.7 ± 2.3 

Stg. 21 Embryo 2.21 ± 0.304 (3) 8.92 ± 1.173 (3)‡ 73.8 ± 2.2 Stg. 33 Embryo 1.95 ± 0.173 (3) 12.04 ± 2.98 (3)‡ 79.5 ± 5.1 

Stg. 36 Larvae 2.26 ± 0.295 (3) 12.58 ± 3.113 (3)‡ 76.5 ± 1.1 Stg. 40 Larvae 2.34 ± 0.54 (2) 13.07 ± 3.875 (3)‡ 84.5 ± 3.4 Stg. 45 Larvae 2.28 ± 0.315 (2) 13 ± 3.44 (3)‡ 92.4 ± 1.5 

Table 3-5. Se in developing white sturgeon progeny of females exposed to either control (ca. 1.4 μg/g) or treatm

dietary Se for approximately six months during vitellogenesis. Data displayed as mean Se μg/g (dw) ± standard 

cohorts; 60 – 90 larvae per cohort). ‡Significant difference from the corresponding control value (Student’s t-tes
 

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 sturgeon 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 experimental endpoint (stage 45; 

Fluorometric analysis, n = 5 – 10 larvae, data displayed as μg/g Se ± standard error of 

sample duplicates). b) Microinjection Study Two using progeny of two female white 

sturgeon. Whole larvae selenium content one day post injection (stage ca. 36), midexperiment (stage 40) and at the experimental endpoint (stage 45). All analysis by 

Fluorometry; data displayed as μg/g Se ± standard error of progeny replicates (n = 

progeny cohorts; 5 – 10 larvae per cohort). Statistical 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). 

163
 

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 edema for each progeny cohort of six female 

white sturgeon exposed to dietary selenium presented as average percentage of replicate 

samples ± standard error of replicates (number of replicate samples, containing 30 larvae 

each*). Data are shown for each of the larval developmental stages sampled. Logit 

analyses of the data is presented in Figure 3-4. ‡

Selenium concentration shown in μg/g 

and based on dry weight. *Some replicate samples contained slightly less than 30 larvae. 

164
 

Occurrence of skeletal deformities 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 skeletal deformities (lordosis, kyphosis, or 

scoliosis) for each progeny cohort of six female white sturgeon exposed to dietary 

selenium. Data shown as average percentage of replicate samples ± standard error of 

replicates (number of replicate samples, containing 30 larvae each*). Data are given for 

each of the larval developmental stages sampled. Logit analyses of the data is presented 

in Figure 3-5. ‡Selenium concentration shown in μg/g and based on dry weight. *Some 

replicate samples contained slightly less than 30 larvae. 

165
Affected larvae (edema and/or skeletal deformities) of female white sturgeon exposed to dietary selenium s

cohort 

Control Treatment

Cohort

Percent 

Affected§ Egg Se‡ Larval Se‡ Cohort

Percent 

Affected§ Egg Se‡ Larval Se‡

Stage 36 C3 0.00 (1) 2.46 2.43 T1 0.00 (1) 11.00 11.60 

C4 0.00 (1) 1.61 1.69 T2 0.00 (1) 20.50 18.40 

C5 0.00 (1) 2.68 2.67 T3 16.67 (1) 7.61 7.75 

Stage 40 C4 1.11 ± 1.11 (3) 1.61 1.80 T1 0.00 (3) 11.00 11.60 

C5 1.11 ± 1.11 (3) 2.68 2.88 T2 18.89 ± 1.11 (3) 20.50 20.40 

T3 10.00 ± 0 (2) 7.61 7.22 

Stage 45 C4 0.00 (3) 1.61 1.96 T1 0.00 (3) 11.00 12.00 

C5 0.00 (3) 2.68 2.59 T2 27.78 ± 2.94 (3) 20.50 19.40 

T3 13.33 ± 3.33 (2) 7.61 7.61 

Table 3-9. Percentage of larvae affected by edema and/or skeletal deformities for each progeny cohort of six femexposed to dietary selenium. Data are shown as average percentage of replicate samples ± standard error of repli

replicate samples, 30 larvae each*). Data are given for each of the larval developmental stages sampled. Logit a

are presented in Figures 3-6 and 3-7. 

§Larvae counted only once, even if both edema and skeletal defect occurred. ‡Selenium concentration shown in μg/g and based on dry weight. 

*Some replicate samples contained slightly less than 30 larvae. 
Occurrence of developmental defects in larvae from female white sturgeon exposed to dietary selenium 

Control Treatment

Stage Stage 

36 40 45 Combined Stages 36 40 45 

Edema 0 ± 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) 

Lordosis 0 ± 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) 

Kyphosis 0 ± 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) 

Scoliosis 0 ± 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)φ 

Skeletal ‡ 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)δ 11.48 ± 6.16 (3)δ 

Total 

Affected § 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)δ 13.7 ± 8.02 (3)δ 

Table 3-10. Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis for progeny of female whiexposed to dietary selenium. Data shown as average percentage of cohort replicates ± standard error of cohort re

cohort replicates*). Data are shown for each of the larval developmental stages sampled. ‡Grouped category of 

and scoliosis. §Larvae with edema and/or skeletal deformities (larvae counted only once, even if both effects occdifference from control value (angular transformed, two-way nested ANOVA, p < 0.05; Tukey HSD, α = 0.05). φfrom control value (Wilcoxon Rank Sums, p < 0.05). *Each cohort replicate represents progeny of one female. stage 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-methionine 

Microinjection Study One 

Control (Non-Injected) 

(2.6 

μg/g Se*) 

Sham-Injected (2.54 

μg/g Se*) 

Low Dose 

(15.8 

μg/g Se*) 

Med Dose 

(21.7 

μg/g Se*) 

Hig

(46.6 

Edema 0 ± 0 (1) 0 ± 0 (1) 63.12 ± 0.96 (1) 49.98 ± 4.44 (1) 35.64 

Lordosis 0 ± 0 (1) 3.95 ± 2.61 (1) 17.48 ± 6.73 (1) 30.61 ± 5.3 (1) 40.6 

Kyphosis 0 ± 0 (1) 0 ± 0 (1) 9.7 ± 2.56 (1) 9.18 ± 1.78 (1) 23.76 

Scoliosis 0 ± 0 (1) 0 ± 0 (1) 0 ± 0 (1) 8.17 ± 3.67 (1) 1.99 

Skeletal ‡ 0 ± 0 (1) 3.95 ± 2.61 (1) 27.17 ± 7.95 (1) 47.97 ± 7.35 (1) 66.33 

Total 

Affected § 0 ± 0 (1) 3.95 ± 2.61 (1) 76.69 ± 4.86 (1) 83.66 ± 3.67 (1) 78.21 

Table 3-11. Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis from Microinjection Study microinjected with a varying seleno-L-methionine after hatching. Data shown as average percentage ± standard replicate tanks (number of cohort replicates♦). Data span early larval development (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 contained ca. 35 larvae. 

*Se exposure at the beginning of larval development (stage 36; Table 3-6). 
Occurrence of edema in larvae from microinjection study two shown by progeny cohort 

Progeny F1 Progeny F3 

Se μg/g∗

Percent 

Edema

Percent 

Skeletal‡

Percent 

Total Affected§ Se μg/g∗

Percent 

Edema

Percent 

Skeletal‡

Control (non-injected) 6.70 0.00 3.33 3.33 6.00 0.00 0.00 

 Sham-injected 6.66 0.00 0.00 0.00 5.12 0.00 0.00 

Low Se 9.10 8.00 8.00 12.00 8.38 13.33 3.33 

Medium Se 8.77 0.00 3.45 3.45 9.16 0.00 6.67 

High Se 18.17 80.00 30.00 80.00 14.94 55.17 13.79 

Table 3-12. Percentage of larvae with developmental defects for each progeny cohort of Microinjection Study T

analyses (Figures 3-11 – 3-13). Data span early larval development (stages 36 – 45) and represent approximately 

treatment. 

‡Selenium concentration shown in μg/g and based on dry weight. 

*Selenium concentration represents the microinjected dose as measured post injection. Se values are shown in μg/g and based on dry w

‡ Grouped category of lordosis, kyphosis, and scoliosis. §Larvae with edema and/or skeletal deformities (larvae counted only once, even if both effects occurred). Average data of the two coho

13. 
Occurrence of developmental defects in larvae exposed to seleno-L-methionine 

Microinjection Study Two 

Control♦

(6 

μg/g Se*) 

L-Met Control•

(Se not analyzed) 

Low Dose 

(8.7 

μg/g Se*) 

Med Dose 

(9 

μg/g Se*) 

High 

(16.6 

μg

Edema 0 ± 0 (2)aδ 0 ± 0 (2)aδ 10.91 ± 3.64 (2)bδ 0 ± 0 (2)aδ 67.8 ± 13

Lordosis 0 ± 0 (2)aδ 0 ± 0 (2)aδ 3.64 ± 0 (2)bδ 3.39 ± 0 (2)abδ 15.25 ± 5

Kyphosis 0.85 ± 0.85 (2) 1.19 ± 1.19 (2) 0 ± 0 (2) 0 ± 0 (2) 1.69 ± 1

Scoliosis 0 ± 0 (2) 1.19 ± 1.19 (2) 1.82 ± 1.82 (2) 1.69 ± 1.69 (2) 5.08 ± 1

Skeletal ‡ 0.85 ± 0.85 (2)a 2.38 ± 2.38 (2)ab 5.45 ± 1.82 (2)ab 5.08 ± 1.69 (2)ab 22.03 ± 8

Total Affected § 0.85 ± 0.85 (2)a 2.38 ± 2.38 (2)a 12.73 ± 1.82 (2)a 5.08 ± 1.69 (2)a 69.49 ± 1

Table 3-13. Percentage of larvae affected by edema, lordosis, kyphosis, and scoliosis from Microinjection Study 

microinjected with a varying seleno-L-methionine after hatching. Data shown as average percentage of cohort re

error of cohort replicates (number of cohort replicatesθ). Data span early larval development (stages 36 – 45). W

levels not connected by same letter are significantly different (one-way ANOVA following angular transformatioHSD, 

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 female 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 

μg/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. 

171
Effects as Percentage of Mortalities 

Study One Se (μg/g; dw)

Percent Mortality Edema

Spinal Curvature

Edema and Spinal Curvature

Control (non-injected) 2.60 0.0% 0.00% 0.00% 0.00% 

Sham-injected 2.54 0.0% 0.00% 0.00% 0.00% 

Low Se 15.8 41.0% 4.76% 13.33% 13.33% 

Medium Se 21.7 46.7% 9.52% 27.62% 8.57% 

High Se 46.6 67.6% 4.76% 39.05% 22.86% 

Study Two

Control (non-injected) 6.35 ± 0.35 (2) 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 

Sham-injected 5.89 ± 0.77 (2) 1.67 ± 1.67 (2) 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 

L-Met Low not analyzed 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 

L-Met Med not analyzed 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 0 ± 0 (2) 

L-Met High not analyzed 1.67 ± 1.67 (2) 0 ± 0 (2) 1.67 ± 1.67 (2) 0 ± 0 (2) 

Low Se 8.74 ± 0.36 (2) 10.00 ± 0 (2) 6.67 ± 3.33 (2) 1.67 ± 1.67 (2) 1.67 ± 1.67 (2) 

Medium Se 8.97 ± 0.2 (2) 3.34 ± 3.34 (2) 0 ± 0 (2) 3.35 ± 3.35 (2) 0 ± 0 (2) 

High Se 16.56 ± 1.62 (2) 70.00 ± 16.67 (2) 46.7 ± 3.33 (2) 0 ± 0 (2) 20 ± 10 (2) 

Table 3-15. Percent mortality in larvae exposed to selenium via microinjection studies. Mortality data presented ± standard error (number of progeny cohorts). Study one included one progeny cohort (n = 100 larvae per treatme

included two progeny cohorts (n = 30 larvae per each cohort). Selenium exposure is represented by concentratiolarvae several hours following microinjection (ca. stage 36). Se data are presented as μg/g Se (dw) ± standard er

progeny cohorts). The percentage of defects observed in mortalities are displayed as percent defect ± standard er

progeny cohorts). Statistical analyses of these mortality data are presented in Figure 3-18. 
 

Combined mortality in larvae from maternal exposure and microinjection studies 

Grouped Exposure 

Categories

Average Percent 

Mortality

Control (~2 – 7 μg/g Se) 0.44 ± 0.31 (5) 

Low (~8 – 10 μg/g Se) 5.35 ± 2.25 (5) 

Mid (~12 – 16 μg/g Se) 31.54 ± 16.01 (3) 

High (~18 – 22 μg/g Se) 47.25 ± 22.59 (3) 

Very High (~46 μg/g Se) 67.62 (1) 

Table 3-16. Percent mortality in larvae from maternal and microinjection studies of Se 

exposure in developing white sturgeon. Mortality data presented as percent mortality ±

standard error (number of progeny cohorts). The 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 injection in microinjection studies; values in dry 

weight). Statistical analyses of combined mortality data are presented in Figure 3-19. 

173
Probabilities of development defects in larvae containing excess Se 

Skeletal Deformities 

ED15 ED25 ED50 Logit Equation 

Maternal Exposure 20.0 24.4 31.9 P(skeletal deformity) = 1/(1+e(-(-4.64 + 0.145 * (Larval Se ug/g))) 

Microinjection Study One 10.3 18.3 32.3 P(skeletal deformity) = 1/(1+e(-(-2.54 + 0.08 * (Larval Se ug/g))) 

Microinjection Study Two 14.7 17.2 21.4 P(skeletal deformity) = 1/(1+e(-(-5.59 + 0.26 * (Larval Se ug/g))) 

Combined Data 17.6 23.5 33.7 P(skeletal deformity) = 1/(1+e(-(-3.63 + 0.11 * (Larval Se ug/g))) 

Edema 

ED15 ED25 ED50 Logit Equation 

Maternal Exposure 22.6 25.3 29.9 P(edema) = 1/(1+e(-(-7.12 + 0.238 * (Larval Se ug/g))) 

Microinjection Study One * 13.5 41.8 P(edema) = 1/(1+e(-(-1.62 + 0.04 * (Larval Se ug/g))) 

Microinjection Study Two 11.5 12.8 14.9 P(edema) = 1/(1+e(-(-7.62 + 0.51 * (Larval Se ug/g))) 

Combined Data 17.7 25.8 39.8 P(edema) = 1/(1+e(-(-3.12 + 0.08 * (Larval Se ug/g))) 

Total Affected (Edema and/or Skeletal Deformity) 

ED15 ED25 ED50 Logit Equation 

Maternal Exposure** 17.9 21.9 28.8 P(affected larvae) = 1/(1+e(-(-4.58 + 0.159 * (Larval Se ug/g))) 

Microinjection Study One 0.5 6.5 16.8 P(affected larvae) = 1/(1+e(-(-1.79 + 0.11 * (Larval Se ug/g))) 

Microinjection Study Two 10.7 12.1 14.5 P(affected larvae) = 1/(1+e(-(-6.56 + 0.45 * (Larval Se ug/g))) 

Combined Data 11.1 15.2 22.2 P(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 dProbabilities calculated from logit equations (p < 0.0001; Figures 3-4 – 3-16). ED

X represents the level of Se in larva

to cause a particular effect in X percent of the population. Se concentrations are from larvae at the beginning of larva36). Defects were measured throughout the period of yolk utilization (ca. first 10 days post-hatch). Data from each eboth 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 defects 

Exposure 

Fish/ 

Progeny 

ID Liver Muscle Ovarian Egg Larvae‡

Percent 

Developmental 

Defects*

Control C3 1.33 1.28 1.49 2.46 2.43 0δControl C4 0.80 1.22 1.65 1.61 1.69 0 Control C5 2.16 1.48 1.38 2.68 2.67 0 

Treatment T1 8.72 9.93 6.53 11.00 11.6 0 Treatment T2 11.60 15.30 13.20 20.50 18.4 27.78 ± 2.94 Treatment T3 11.00 11.10 5.74 7.61 7.75 13.33 ± 3.33 

Table 3-18. Se concentrations (μg/g; dw) in liver, muscle, ovarian tissue, and eggs from female white sturgeon econtrol (ca. 1.4 μg/g) or treatment (ca. 34 μg/g) dietary Se for approximately six months during vitellogenesis (d2). Se concentration and occurrence of developmental defects are shown for the progeny of each female (n = 25 36; 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 development δData from stage 36 only, due to very low hatch 
 

Potential Hazard Threshold Levels for Se in developing white sturgeon larvae 

ED05 ED10 

Se μg/g 

(dry wt) 

Se μg/g 

(wet wt)* 

Se μg/g 

(dry wt) 

Se μg/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 developmental defects in larvae from both 

maternal and microinjection experiments. EDX represents the level of Se in stage 36 

larvae expected to cause a particular effect 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 sturgeon 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 (average ± standard deviation). 

 

176
 

Selenium in developing progeny of female white sturgeon exposed to dietary 

selenium 

Figure 3-1. Selenium concentrations in eggs and developing embryos and larvae from 

white sturgeon females exposed to dietary selenium. Selenium is presented as mean Se 

μg/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. 

 

177
Selenium in developing white sturgeon exposed to seleno-L-methionine by microinjectio

a) Study One b) Study Two Figure 3-2. Selenium concentrations in developing white sturgeon larvae following microinjection of seleno-L-

Microinjection Study One using progeny of one female white sturgeon. Whole larvae selenium content at the stinjection, stage ca. 36; ICP-AES analysis; n = ca. 45 larvae) and end of the experiment (stage 45, Fluorometry anlarvae, data displayed as μg/g Se ± standard error of sample duplicates). b) Microinjection Study Two using prowhite sturgeon. Whole larvae selenium content at the start (one day post injection, stage ca. 36), middle (stage 4

experimental endpoint (stage 45). All analysis by Fluorometry; n = 5 – 10 larvae; data displayed as μg/g Se ± sta

progeny replicates. All values from Study Two include two progeny replicates except the end, High treatment. G

by the same letter are significantly different (two-way, nested ANOVA, p < 0.0001; Tukey HSD, α = 0.05). *Vadifferent from control (ANOVA p < 0.002; Dunnets α = 0.05). Data are also presented in Table 3- 6. 
d)

a) b)

d) e) c) 

Figure 3-3. Developmental defects observed in white sturgeon during larval development. a) A normal larvae. 

edema (overt swelling due to fluid accumulation). c) Larvae showing kyphosis (dorsoventral curvature of anteri

showing lordosis (concave dorsoventral curvature of posterior region). e) Larvae with scoliosis (lateral skeletal 

edema. 
Progeny of selenium-exposed white sturgeon 

larvae 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 pr

sturgeon females exposed to dietary selenium. Data includes three progeny cohorts from the control group and three group. Data for three larval development stages are included (stages 36, 40 and 45). Logit analysis shows a significaselenium concentrations in larvae and the occurrence of defects (χ2 = 39.7, p < 0.0001; prob(edema) = 1/(1+e(-(-7.12 + 0.2

The right y-axis shows the actual percentage of affected larvae in samples containing 30 – 90 larvae each. The left y-probability of larvae to develop edema at a given larval selenium concentration. (b) ED50 (29.90 μg/g), ED25 (25.28 μ

μg/g) values were calculated from the logit equation. 
Progeny of selenium-exposed white sturgeon 

larvae with skeletal deformities related to larval selenium 

(a) (b) Figure 3-5. Percentage of larvae with skeletal deformities related to selenium concentrations in larvae (μg/g; dw

progeny of adult white sturgeon females exposed to dietary selenium. Data includes three progeny cohorts from 

three from the treatment group. Data for three larval development stages are included (stages 36, 40 and 45). Lo

significant association between selenium concentrations in larvae and the occurrence of defects (χ2 = 46.3, p < 0.

deformity) = 1/(1+e(-(-4.64 + 0.145 * (Se in Larvae ug/g))). (a) The right y-axis shows the actual percentage of larvae with sk

samples containing 30 – 90 larvae each. The left y-axis shows the probability of larvae to develop skeletal defor

larval selenium concentration. (b) ED50 (31.92 μg/g), ED25 (24.37 μg/g) and ED10 (16.81 μg/g) values were calcequation. 
Progeny of selenium-exposed white sturgeon 

larvae with edema and/or skeletal defects related to larval selenium 

(a) (b) Figure 3-6. Percentage of larvae with edema and/or skeletal deformities related to selenium concentrations in la

Larvae were progeny of adult white sturgeon females exposed to dietary selenium. Data includes three progeny control group and three from the treatment group. Data for three larval development stages are included (stages 

analysis shows a significant association between selenium concentrations in larvae and the occurrence of defects 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 affsamples containing 30 – 90 larvae each. The left y-axis shows the probability of larvae to develop edema and/or 

at a given larval selenium concentration. (b) ED50 (28.83 μg/g), ED25 (21.92 μg/g) and ED10 (15.01 μg/g) values 

the logit equation. 
Progeny of selenium-exposed white sturgeon 

larvae with edema and/or skeletal defects related to egg selenium 

(a) (b) Figure 3-7. Percentage of larvae with edema and/or skeletal deformities related to selenium concentrations in egLarvae were progeny of adult white sturgeon females exposed to dietary selenium. Data includes three progeny control group and three from the treatment group. Data for three larval development stages are included (stages analysis shows a significant association between selenium concentrations in larvae and the occurrence of defects 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 affeccontaining 30 – 90 larvae each. The left y-axis shows the probability of larvae to develop edema and/or skeletal selenium concentration in eggs. (b) ED50 (29.81 μg/g), ED25 (22.56 μg/g) and ED10 (15.32 μg/g) values were calequation.
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 related to selenium concentrations in larvae 

(μg/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 (χ2 = 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 analysis. The right y-axis shows the actual 

percentage of larvae with edema in samples containing ca. 100 larvae each. ED50 (41.8 

μg/g) and ED25 (13.5 μg/g) values were calculated from the logit equation. 

184
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 (μg/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 (χ2 = 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 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. 100 larvae each. ED50 (32.3 μg/g), ED25 (18.3 μg/g) and ED15

(10.3 μg/g) values were calculated from the logit equation. 

185
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 and/or skeletal deformities related to 

selenium concentrations in larvae (μg/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 (χ2 = 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 probability 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. ED50 (16.8 μg/g) and ED25 (6.5 μg/g) values were 

calculated from the logit equation. 

186
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 related to selenium concentrations in 

larvae (μg/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 association between selenium concentrations in larvae and the 

occurrence of edema (χ2 = 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 logit analysis. The right y-axis shows the 

actual percentage of larvae with edema in samples containing ca. 30 larvae each. ED50

(14.9 μg/g), ED25 (12.8 μg/g) and ED15 (11.5 μg/g) values were calculated from the logit 

equation. 

187
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 skeletal deformities related to selenium 

concentrations in larvae (μg/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 association between selenium concentrations in larvae 

and the occurrence of skeletal deformities (χ2 = 26.4, p < 0.0001; prob(skeletal 

deformity) = 1/(1+e(-(-5.59 + 0.26 * (Se in Larvae ug/g))). The left y-axis shows 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. ED50 (21.4 μg/g), ED25

(17.2 μg/g) and ED15 (14.7 μg/g) values were calculated from the logit equation. 

188
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 and/or skeletal deformities related to 

selenium concentrations in larvae (μg/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 association between selenium concentrations in 

larvae and the occurrence of defects (χ2 = 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 probability 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. ED50 (14.5 μg/g), ED25 (12.1 μg/g) and ED15 (10.7 μg/g) 

values were calculated from the logit equation. 

189
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 related to selenium concentrations in 

larvae (μg/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 (χ2 = 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 analysis. The right y-axis shows the actual 

percentage of larvae with edema in samples containing ca. 30 - 200 larvae each. ED50

(39.8 μg/g), ED25 (25.8 μg/g) and ED15 (17.7 μg/g) values were calculated from the logit 

equation. 

190
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 skeletal deformities related to selenium 

concentrations in larvae (μg/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 skeletal deformities (χ2 = 340.1, p < 0.0001; prob(skeletal 

deformity) = 1/(1+e(-(-3.63 + 0.11 * (Se in Larvae ug/g))). The left y-axis shows 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. ED50 (33.7 μg/g), 

ED25 (23.5 μg/g) and ED15 (17.6 μg/g) values were calculated from the logit equation. 

191
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 and/or skeletal deformities related to 

selenium concentrations in larvae (μg/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 defects (χ2 = 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 probability 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. ED50 (22.2 μg/g), ED25 (15.2 μg/g) and ED15 (11.1 

μg/g) values were calculated from the logit equation. 

192
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 analysis of time to larval mortality shows a 

statistically significant difference between control and treatment groups (p < 0.0001). b) 

When data are categorized by selenium concentrations in the eggs, a larger difference is 

detected in time to larval mortality (Kaplan-Meier; p < 0.0001). Differences between 

Kaplan-Meier distributions follow a) High >> Low > Medium, and b) Treatment > 

Control (Student’s t-test, p < 0.0001). 

193
Mortality in larvae following microinjection of seleno-L-methionine 

a) Study One b) Study Two Figure 3-18. Probabilities of mortality in larvae following microinjection of seleno-L-methionine in a) Microinj

and b) Microinjection Study Two. Data are categorized by Se in larvae at the onset of larval development (ca. st

microinjection study two, the low and medium treatment groups were combined into one Se exposure group (low

3- 15). Control non-injected and sham-injected data were combined for both studies. Both studies show a statist

difference in the probability of larval mortality between exposure levels (Kaplan-Meier, p < 0.0001). Difference

Meier distributions follow a) High > Medium > Low > Control (Student’s t-test, p ≤ 0.008), and b) High > Low > t-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 microinjection studies in white sturgeon. 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.

 

195
 

Conclusions 

This work shows that white sturgeon experience Se-induced toxicity at 

environmentally relevant dietary concentrations. Juveniles experienced cholestasis, a 

potentially fatal liver condition, when exposed to diets containing ca. 20 to 53 μg/g Se as 

selenized yeast (predominately selenomethionine). Adult females exposed to ca. 34 μg/g 

dietary Se transferred toxic levels of Se to their young through vitellogenesis. Our 

maternal transfer work is the first to experimentally demonstrate the incorporation of Se 

into the vitellogenin molecule, which supports 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 μg/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 effects of excess Se on the development of this species. 

Microinjection was used to mimic maternal transfer because the large size and long 

ovarian cycle of white sturgeon make it difficult and expensive to study reproductive 

toxicity in this species under laboratory conditions. White sturgeon larvae tolerate 

 

196
 

microinjection 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 route (dietary) that is 

predominant in the natural environment. Avoidance of using a surrogate species is 

especially important when studying sturgeon, since this phylogenetically ancient fish is 

significantly different from modern fish in anatomical and physiological characteristics. 

In addition, the narrow exposure range used here provides greater ecological significance 

by demonstrating the effects of relatively small increases in exposure. 

In San Francisco Bay-Delta, juvenile 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 their tissues at concentrations similar to those 

observed in our study. This leads us to suspect that juvenile white sturgeon in San 

Francisco Bay-Delta may be experiencing cholestasis, depending on individual dietary Se 

exposure. This potentially fatal liver condition 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 sturgeon in San Francisco Bay-Delta, typical Se 

exposure levels in the benthic food web (ca. 15 μg/g) are below the dietary Se level to 

which reproductive females were exposed in these experiments (ca. 34 μg/g). However, 

the experimental sturgeon accumulated Se concentrations in liver, muscle and eggs (ca.

10 – 12 μg/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 were 

 

197
 

exploring the transport of accumulated Se from the liver to the developing eggs. Our 

research indicates that female white sturgeon 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 sampled in the San Francisco Bay-Delta region 

show 3 to 29 μg/g Se (dw) in eggs or egg-bearing ovary (Kroll and Doroshov 1991; 

Doroshov Lab, UCD, unpublished data). Eight of those ten sturgeon exhibited egg Se 

levels above our proposed hazard threshold range of 3 to 8 μg/g Se in larvae (dw). Our 

maternal transport study showed strong conservation of Se between the egg and larval 

stages, indicating that egg Se concentrations may be interchangeable with larval 

concentrations (when both are 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 affecting the recruitment of white sturgeon in this region. We 

observed severe edema and skeletal deformities in larvae containing Se concentrations 

above ca. 11 to 15 Se μg/g. These defects are expected to significantly decrease the 

probability of survival in white sturgeon larvae. The effected larvae have limited 

mobility and can be consumed by predators or may fail to start exogenous feeding. In 

this way, Se-induced developmental defects are an unseen factor in white sturgeon 

recruitment. White sturgeon eggs and gravid ovaries should be monitored for Se 

concentrations to determine the magnitude of potential impact on reproduction. 

Additionally, Se concentrations in the benthic food web should be routinely monitored 

since relatively small increases of Se in the food web can lead to increased toxicity to this 

species. Careful management of all processes with potential to increase Se 

 

198
 

concentrations in the benthic food web is essential to protect sturgeon in San Francisco 

Bay-Delta and other high-Se systems. 

 

199
 

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Appendix A. Biliary transport and cholestasis in fish 

Hepato-biliary transport of biliary constituents and their precursors from blood to 

bile includes three major steps. First, the uptake of substances from blood plasma across 

the sinusoidal membrane and into the hepatocytes must occur. This is followed by 

intracellular transport and metabolism. Finally, the transport and excretion of the parent 

substance or metabolites across hepatocyte membranes into biliary passageways occurs 

(Groothuis and Meijer 1996; Nathanson and Boyer 1991). Bile flow is primarily 

controlled by the energy dependent (ATP-dependent) secretion of biliary constituents into 

bile canaliculi and the resulting osmotic flow of water (Elferink 2003). 

 In the well-studied mammalian model, bile salts and organic ions are removed 

from sinusoidal blood by Na+-dependent Na+/taurocholate cotransporters (NTCP) and 

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 are secreted from several different transport 

proteins in hepatocyte canalicular membranes, 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), which are related to P-glycoprotein (Arrese 

and Ananthanarayanan 2004; Trauner and Boyer 2003). These ATP-dependent transport 

proteins remove bile salts, phospholipids, cholesterol, organic ions, phase II conjugates, 

toxins, and other bile constituents from hepatocytes into biliary canaliculi (Arrese and 

Trauner 2003; Ferenci et al. 2002; Trauner and Boyer 2003). ATP-independent chloridebicarbonate anion exchanger protein (AE2), found in hepatocyte and bile duct epithelial 

 

218
 

cell membranes, secretes bicarbonate 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 ducts and is transported to the 

gall bladder and the intestinal lumen by the larger cystic and common bile ducts. 

The architecture of fish liver differs significantly from that of mammals (Hinton 

and Couch 1998). Teleosts livers exhibit tubular architecture, characteristic of the lower 

vertebrates, with canaliculi formed by basolateral plasma membranes of adjacent 

hepatocytes and transitional biliary passageways, the tubular lumena, lined by apical 

plasma membrane of hepatocytes and by transitional 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. Biliary 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 primitive 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 enterohepatic circulation for a 

 

219
 

bile alcohol sulfate in fish liver, indicating specific hepato-biliary transport mechanisms 

for this class of compounds (Fricker et al. 1997). Bile alcohols do not appear to share the 

transport mechanisms identified for 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 membrane of skate liver that 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 defects, toxins, disease, inflammation and 

oxidative stress (Trauner M 2005). Reduced expression and function of bile transport 

proteins are important factors in the development of cholestasis (Elferink 2003; Trauner

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

can also contribute to cholestasis. Toxins can effect the formation and secretion of bile, 

presumably by affecting transport protein expression and function (Trauner et al. 1998). 

Schmitt et al. (2000) demonstrated that oxidative stress can lead to cholestasis by 

reducing the number of functional transporters in the canalicular membrane. These 

authors concluded that severe glutathione depletion induces cholestasis with a retrieval of 

MRP2 from the canalicular membrane. The impact of oxidative stress on hepatic 

 

220
 

glutathione are reviewed by Lu et al. (Lu 1999). Hepatocytes under oxidative stress have 

a reduced ability to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH), 

which is essential for several cellular functions. Cellular GSH may become depleted 

under high levels of oxidative stress. Inflamation 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, generate 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 several compensatory changes aimed at 

establishing alternative elimination pathways for biliary constituents (Trauner M 2005). 

A commonly observed compensatory change is the development of additional hepatic 

biliary ducts (bile duct hyperplasia), 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 nuclear 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 include increased expression 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 during chronic cholestasis (Trauner M 2005; Wagner

et al. 2004). 

 

221
 

 While cholestasis and related disease states, 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 cholestasis should be further investigated. 

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Arrese M, Trauner M. 2003. Molecular aspects of bile formation and cholestasis. Trends 

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polluted areas of Lake Ontario. Carcinogenesis 16(12):2923-2929. 

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Marine Environmental Research 58(2-5):199-204. 

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Mrp2 gene encoded conjugate export pump from the canalicular membrane 

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to Hydrophobic Bile Acids. Gastroenterology 109(4):1249-1256. 

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activity of P-glycoprotein in cultured hepatocytes from Oncorhynchus mykiss. 

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Gastroenterology & Hepatology 14(10):946-959. 

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Guo G, Marschall H, Denk H and others. 2004. Role of adaptive hepatobiliary 

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226
 

Appendix B. Summary data on spawning induction and success in white sturgeon 

fed either control (ca. 1.42 μg/g Se) or treatment (ca. 34 μg/g Se) diets for 

approximately six months. 

 

 

227
Table B-1. Detailed spawning data for white sturgeon fed either control (ca. 1.42 

μg/g Se) or treatment (ca

for approximately six months. 

Date Fish #1

Pit 

Tag # Tank #

Spawn 

Hold 

Tank #

Cont

/ Trt

FL 

(cm)

BW 

(kg)2 K

Egg 

Diameter PI

% GVBD

Prog

% GVB

Contr

9/26/2001 140A 203 Trt 137.5 22.5 0.87 2.66 2/12/2002 140A 203 Trt 140.5 25.2 0.91 3.10 0.13 100 0 3/13/2002 140A 203 Trt 140.5 25.2 0.91 3.10 0.09 100 0 3/22/2002 T1 123 ovulated and good fertility about 50% hatch 

9/26/2001 772A 204 Cont 136 23.65 0.94 2.85 2/12/2002 772A 204 Cont 138.5 24.4 0.92 3.23 0.11 0 0 

3/13/2002 772A 204 Cont 138.5 24.4 0.92 3.21 0.09 100 0 3/22/2002 C1 125 no ovulation (in hindsight yolk not well polarized) 

9/26/2001 594D 204 Cont 138 24.4 0.93 2.88 2/12/2002 594D 204 Cont 139.5 24.8 0.91 3.28 0.12 57 0 

3/13/2002 594D 204 Cont 139.5 24.8 0.91 3.21 0.10 100 0 3/22/2002 C2 126 no ovulation (in hindsight yolk not well polarized) 

9/26/2001 6F06 203 Trt 135.5 23.9 0.96 2.53 2/12/2002 6F06 203 Trt 139 24.6 0.92 3.15 0.17 90 0 

3/28/2002 6F06 203 Trt 139 24.6 0.92 3.19 0.08 100 0 

4/5/2002 T2 126 ovulated and good fertility about 50% hatch 
Date Fish #1

Pit 

Tag # Tank #

Spawn 

Hold 

Tank #

Cont

/ Trt

FL 

(cm)

BW 

(kg)2 K

Egg 

Diameter PI

% GVBD

Prog

% GV

Cont

9/26/2001 4F7F 204 Cont 121 19.55 1.10 2.73 2/12/2002 4F7F 204 Cont 122.5 20.5 1.12 3.21 0.15 67 0 

3/28/2002 4F7F 204 Cont 122.5 20.5 1.12 3.19 0.08 100 0 

4/5/2002 C3 125 ovulated and good fertility very poor hatch (<1%) 

9/26/2001 0005 204 Cont 140 25.15 0.92 2.75 

2/12/2002 0005 204 Cont 142 25.8 0.90 3.20 0.15 100 0 

3/13/2002 0005 204 Cont 142 25.8 0.90 3.10 0.12 100 0 

4/9/2002 0005 204 Cont 142 25.8 0.90 3.19 0.09 100 77 

4/19/2002 C4 125 ovulated 

9/26/2001 6D5C 203 Trt 136 22.9 0.91 2.92 2/12/2002 6D5C 203 Trt 138 24.1 0.92 3.14 0.13 100 0 

3/13/2002 6D5C 203 Trt 138 24.1 0.92 3.11 0.12 100 0 

4/9/2002 6D5C 203 Trt 138 24.1 0.92 3.21 0.09 100 0 

4/19/2002 T3 126 partial ovulation 

9/26/2001 4304 203 Trt 133 24.05 1.02 2.71 2/12/2002 4304 203 Trt 136.5 25.9 1.02 3.09 0.15 0 0 

3/13/2002 4304 203 Trt 136.5 25.9 1.02 3.08 0.09 0 0 3/28/2002 4304 203 Trt 136.5 25.9 1.02 3.14 0.10 0 0 4/25/2002 4304 203 Trt 136.5 25.9 1.02 3.24 0.07 100 0 

5/3/2002 T4 125 ovulated, hatch almost nothing 
Date Fish #1

Pit 

Tag # Tank #

Spawn 

Hold 

Tank #

Cont

/ Trt

FL 

(cm)

BW 

(kg)2 K

Egg 

Diameter PI

% GVBD

Prog

% GVB

Contr

9/26/2001 6F36 204 Cont 127.5 19.6 0.95 2.74 2/12/2002 6F36 204 Cont 130 20.8 0.95 3.12 0.16 0 0 3/28/2002 6F36 204 Cont 130 20.8 0.95 3.12 0.13 77 0 4/25/2002 6F36 204 Cont 130 20.8 0.95 3.14 0.08 100 0 

5/3/2002 C5 126 ovulated, hatch about 40% 

9/26/2001 3A67 204 Cont 129 17.85 0.83 2.90 2/12/2002 3A67 204 Cont 131 21.2 0.94 3.14 0.16 0 0 

3/28/2002 3A67 204 Cont 131 21.2 0.94 3.10 0.16 0 0 4/25/2002 3A67 204 Cont 131 21.2 0.94 atretic 

6/4/2002 C6 necropsied 

9/26/2001 6D74 204 Cont 140.5 24.05 0.87 2.86 2/12/2002 6D74 204 Cont 142 25.8 0.90 3.17 0.13 0 0 

3/13/2002 6D74 204 Cont 142 25.8 0.90 3.12 0.11 100 0 

4/9/2002 6D74 204 Cont 142 25.8 0.90 3.10 0.12 100 0 5/8/2002 atretic 6/4/2002 C7 necropsied 

9/26/2001 1D75 203 Trt 147 27.4 0.86 2.75 2/12/2002 1D75 203 Trt 149 28.8 0.87 3.09 0.13 4 0 

3/13/2002 1D75 203 Trt 149 28.8 0.87 3.09 0.12 0 0 

4/9/2002 1D75 203 Trt 149 28.8 0.87 3.10 0.11 77 0 5/8/2002 1D75 203 Trt 149 28.8 0.87 3.10 0.15 77 0 

5/17/2002 T6 necropsied 
Date Fish #1

Pit 

Tag # Tank #

Spawn 

Hold 

Tank #

Cont

/ Trt

FL 

(cm)

BW 

(kg)2 K

Egg 

Diameter PI

% GVBD

Prog

% GVB

Contr

9/26/2001 192E 203 Trt 129 21 0.98 2.53 2/12/2002 192E 203 Trt 134.5 22.7 0.93 3.17 0.18 18 0 

4/9/2002 192E 203 Trt 134.5 22.7 0.93 3.23 0.15 100 0 5/8/2002 192E 203 Trt 134.5 22.7 0.93 3.22 0.15 100 0 

5/17/2002 T5 125 injected, no ovulation, necropsied 9/26/2001 6D0E 203 Trt 148.5 30.4 0.93 2.88 2/12/2002 6D0E 203 Trt 148.5 30.4 0.93 3.25 0.19 0 0 

4/9/2002 6D0E 203 Trt 148.5 30.4 0.93 3.20 0.17 0 0 5/8/2002 6D0E 203 Trt 148.5 30.4 0.93 3.26 0.17 0 0 

5/29/2002 6D0E 203 Trt 148.5 30.4 0.93 3.23 0.17 0 0 

6/4/2002 T8 necropsied 

9/26/2001 293B 203 Trt 128 18.9 0.90 2.35 2/12/2002 293B 203 Trt 128 18.9 0.90 3.20 0.22 0 0 

4/9/2002 293B 203 Trt 128 18.9 0.90 3.25 0.18 0 0 5/8/2002 293B 203 Trt 128 18.9 0.90 3.27 0.17 0 0 

5/29/2002 293B 203 Trt 128 18.9 0.90 3.25 0.17 0 0 

6/4/2002 T7 necropsied 

1Control female number C8 (PIT Tag 4519) was atretic on 2/12/02 and later died due to technical error (escaped 

the tank covering). 

2Body weight for 2/12/02 estimated from fork length. 
Table B-2. Summary of spawning induction and success in white sturgeon fed either control (ca. 1.42 μg/g 

(ca. 34 μg/g Se) diets for approximately six months. 

T1 C1 C2 T2 C3 C4 T3 T4 

PIT Tag# 140A 772A 594D 6F06 4F7F 0005 6D5C 4304 1st Inj Date 3/20/2002 3/20/2002 3/20/2002 4/3/2002 4/3/2002 4/17/2002 4/17/2002 5/1/2002 1st Inj Time 10:15pm 10:20pm 10:25pm 9:50pm 9:50pm 10pm 10pm 10pm 2nd Inj Date 3/21/2002 3/21/2002 3/21/2002 4/4/2002 4/4/2002 4/18/2002 4/18/2002 5/2/2002 2nd Inj Time 9:57am 10:00am 10:05am 10am 10am 10am 10am 10am 1st Inj Temp (°C) 14.8 15.0 13.0 14.6 14.6 14.5 14.8 14.7 2nd Inj Temp (°C) 15.3 15.2 14.0 14.7 14.7 15.1 15.3 15.3 Ovul Temp (°C) 15.0 15.4 15.3 15.3 15.4 15.5 Latency (h) 19.0 21.0 21.0 23.0 25.5 24.5 Ovulation % 100 no ovul no ovul 100 100 100 partial (40-50%) 100 Volume Eggs (mls) 3250 

(sampled 

follicles) 

(sampled 

follicles) 3400 2550 4350 950 2200 

# eggs/ml 31.15 

(100% 

gvbd) 

(20% 

gvbd) 24.95 28.8 31.55 33.65 30.3 

# Eggs Collected (est fecundity) 101238 84830 73440 137243 31968 66660 Used pooled milt for fertilization from # of males 2 3 3 4 4 4