## U. S. DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE



# Evaluation of the Toxicity of Selenium

to White and Green Sturgeon

Prepared b y Dr. William N. Beckon

Sacramento, California

March 20 12

### ACKNOWLEDGEMENTS

I would like to gratefully acknowledge the assistance and support of the following people in the preparation of this document: Theresa Presser and Sam Luoma both with the U.S. Geological Survey, as well as Joe Skorupa with the U.S. Fish and Wildlife Service , provided insightful

discussions and input over the years on selenium bioaccumulation. Diane Fleck and Eugenia McNaughton both with the U.S. Environmental Protection Agency and Thomas Maurer with the U. S. Fish and Wildlife Service provided kind and pati ent support and guidance throughout the preparation of this report. Robin Stewart with the U.S. Geological Survey provided clam data. Regina Linville, Barbara Osmundson, Javier Linares

- Casenave, Serge Doroshov, and Dennis Lemly provided invaluable review s of early drafts of the report. None of the above is responsible for the final interpretation and conclusions in this report.

Literature citation should read as follows:

U. S. Fish and Wildlife Service. 2012

. Evaluation of the Toxicity of Selenium to White and Green Sturgeon . U.S. Fish and Wildlife Service, Sacramento Fish and Wildlife Office, Environmental Contaminants Division. Sacramento, California. March 20 12

. 48 pp.

## Evaluation of the Toxicity of Selenium to White and Green Sturgeon

Table of Contents

Abstract 1

Summary Table of Sturgeon Selenium Benchmarks 2

Introduction 3

White Sturgeon Exposure by Microinjection 5

White Sturgeon Exposure by Maternal Exposure 1 1

Green Sturgeon Exposure by Microinjection 13

Green Sturgeon Exposure by Maternal Exposure 1 5

Discussion 18

References 23

Appendix I 2 7

Appendix II 41

### BLANK PAGE

### 1 U. S. DEPARTMENT OF THE INTERIOR

FISH AND WILDLIFE SERVICE

Sacramento Fish and Wildlife Office 2800 Cottage Way, Rm W

- 2605 Sacramento, California 95825



## Evaluation of the Toxicity of Selenium to White and Green Sturgeon

Prepared by

Dr. WILLIAM N. BECKON

Environmental Contaminants Division william_beckon@fws.gov

March 2012



## Abstract

Fish of the genus Acipenser (sturgeon) are likely to be among the most vulnerable to selenium exposure in the San Francisco Estuary because these fish feed predominantly on benthic

invertebrates

, including the Asian clam, Corbula amurensis . This clam is an efficient

bioaccumulator o f selenium. The best data available for the most sensitive endpoint for sturgeon come from studies in which the survival of larvae was monitored following micro

- injection of organic selenium (L

- selenomethionine) into the yolk sacs of newly hatched larvae. Benchmark larval selenium concentrations from these studies were translated, by means of regressions, to selenium concentrations in the tissue and diet of adult white and green sturgeon. This analysis indicates that white and green sturgeon are among th e mo st sensitive of fish to adverse effects of selenium, with the listed green sturgeon being the more sensitive of these two species. These levels of sensitivity evidently put sturgeon at substantial risk at current levels of exposure in the

San Francisc o Bay area. Selenium concentrations in food items of sturgeon in the San Francisco Bay area are almost always high enough that they may cause at least 10 percent mortality in hatchling green sturgeon (≥3.58 μg/g), and they are frequently high enough that they may cause at least 10 percent mortality among hatchling white sturgeon (≥10.8 μg/g) as well. Below is a summary of benchmark concentrations of selenium derived here for the diets, whole body, muscle, and eggs of these two sturgeon species.

### 2 Summary Table of Selenium Benchmarks

Concentration of selenium (μg/g dry wt.) corresponding to effect level (proportion affected)

Effect

Level 5% 10% 5% 10% 5% 10% 5% 10%

Species Se in diet

of sturgeon Se in

sturgeon (whole body) Se in

sturgeon
muscle Se in

sturgeon

eggs Effect Form of selenium Exposure Data source

White sturgeon 5.45 10.8 2.55 3.86 3.55 5.56 4.67 6.83 larval mortality L

- selenomethionine microinjection
of larvae Linville 2006

39.3 48.4 8.51 9.65 13.1 15.0 14.0 15.8 larval

abnormalities selenized yeast maternal diet Linville 2006

Green sturgeon 2.51 3.58 1.59 1.97 2.13 2.69 3.03 3.70 larval mortality L

- selenomethionine microinjection
of larvae Linares

- Casenave et al.

2010

8.22 10.1 3.27 3.71 4.65 5.33 5.87 6.59 larval

abnormalities selenized yeast maternal diet* Linares

- Casenave et al. 2010, Linville 2006

* Green sturgeon maternal diet effect levels were calculated from white surgeon maternal dietary exposure larval developmental defects (Linville 2006) adjusted for relative species sensitivity using the ratio of EC10s for peak larval abnormalities from the white and green sturgeon microinjection experiments of Linares

- Casenave et al . (20 10 ).

B enchma rk values listed in this table are shown in boldface type in the following text and graphs where the derivations are explained

and illustrated.

### 3



## Introduction

The San Francisco Bay, San Pablo Bay, and Suisun Bay are listed as impaired by selenium under 303(d) of the Clean Water Act. Of the wildlife that live in, pass through, or are dependent on, these bays, benthic (bottom

- dwelling) fish of the genus Acipenser (sturgeon) are likely to be among the most vulnerable to selenium. This is be cause these large, long

- lived fish feed predominantly on benthic invertebrates (McKechnie and Fenner 1971; Radtke 1966; Ritz 2010)

, and since 1986 the San Francisco Bay benthic community has experienced a massive invasion of an Asian clam, Corbula amurensi s , (Carlton et al. 1990; Nichols et al. 1990) that is a particularly

efficient bioaccumulator of selenium (Linville et al . 2002)

.

Two species of sturgeon are found in the San Francisco Bay estuary: white sturgeon ( Acipenser

transmontanus ) and green sturgeon ( Acipenser medirostris ). White sturgeon in the San Francisco Bay estuary congregate in Suisun and San Pablo Bays where they remain year

- round except for a small fraction of the population that moves up the Sacramento River, and to a lesser extent the San Joaquin River, to spawn in late winter and early spring (Kohlhorst et al . 1991). Thus, many individuals of this species remain year

- round in a region of the San Francisco Bay estuary where they are exposed to dietary items ( Corbula amurensis ) that exceed selenium concentrations of 10 μg/g (dry weight) much of the time ( Linville et al . 2002

, Kleckner et al. 2010). Linares et al

. (2004) found concentrations of selenium as high as 46.7 μg/g in gonads of 39 white sturgeon captured in the San Francisco Bay. Kroll and Doroshov (1991) reported that developing ovaries

of w hite sturgeon from San Francisco Bay contained as much as 71.8 μg/g selenium (dry weight) ; several times the concentration in fish eggs (20 μg/g dry weight) that Lemly (1996) suggested would cause a high level of hazard from reproductive impairment.

Green sturgeon are more marine

- oriented than white sturgeon, but also congregate in coastal bays and estuaries (Adams et al. 2007). Furthermore, green sturgeon in this area are federally listed as threatened under the Endangered Species Act.

Therefore, in view of their status as well as their particular vulnerability to selenium, sturgeons must be pivotal species in establishing regulatory guidelines that would be broadly protective of wildlife in the San Francisco Bay area. The purpose of this report is to provide an analysis of recent sturgeon data that may be relevant to developing such guidelines.

This analysis focuses on the “most sensitive endpoint”, the ecologically relevant a dverse effect that occurs at the lowest concentrations of selenium, relative to the concentrations at which other effects or “endpoints” start to occur. This focus is consistent with the approach used by the USEPA Region 9 Biological Technical Assistance Group for establishing toxicity reference values (CDTSC HERD 2002). It also conforms with prudence and reason; the most sensitive endpoint should trump other effects levels in developing protective guidelines.

Key studies by Linville (2006), Linares

- Ca senave et al. (2010), Silvestre et al. (2010), and Tashjian et al. (2006) provide white and green sturgeon larval and egg toxicity data. Linville (2006) also provides dietary, female muscle and maternal transfer data for white sturgeon while Tashjian et a l. (2006) provides whole body and muscle data for white sturgeon. However, data on green sturgeon dietary, muscle and whole body is not currently available. To the best of our

### 4

knowledge, the best data available for the most sensitive endpoint for sturgeo n come from studies in which the survival of larvae was monitored following micro

- injection of organic selenium (L

- selenomethionine) into the yolk sacs of newly hatched larvae (Linville 2006,

Linares

- Casenave et al. 2010, Silvestre et al. 2010 ). Benchmark larval selenium concentrations from these studies can be translated, by means of regressions, to selenium concentrations in the tissue and diet of adult sturgeon that deposit eggs from which the larvae with those concentrations would hatch. For rarer spe cies, such as green sturgeon, for which there are insufficient data to derive species

- specific regressions, regressions from a surrogate species of the same genus (i.e. white sturgeon) can be used (USEPA 1995). Use of such surrogates and regressions is a protective approach based on best available information. It is an approach that EPA employs extensively in setting water quality standards (e.g. USEPA 2004).

The following flow diagram is provided to show the relationship of the larval effect levels an d their translations to selenium in sturgeon diet and tissue.

Flow Diagram of Translation Procedures. White and green sturgeon 5% and 10% larval effect values are determined from microinjection and maternal transfer studies. These are then

translated to selenium concentrations in egg, muscle, whole body, and diet using regressions presented in each of the figures noted.



## Diet

W hole body

Eggs Muscle

Larvae Trophic transfer

Maternal transfer

E ffects: larval survival

and deformities Hatch

Fig. 3

Figs. 1,2,7,8,9,11

Figs. 6,10

Fig. 4 Fig. 5 Key



### Direction of selenium

transfer /partitioning

Direction of

benchmark translation

### 5



## White S turgeon (

Acipenser transmontanus )

At the University of California, Davis, Linville (2006) studied several effects of selenium on white sturgeon, including the toxicity of selenium to larvae after hatching and before the larvae begin to actively feed. During this period of about two weeks, the larval fish obtain nutrition (and toxicants) from a yolk sac. Linville (2006) exposed these larvae to elevated selenium in two ways: (1) by microinjection of L

- selenomethionine into the yolk sacs just after hatch and (2) by exposing mother fish to dietary selenium (selenized yeast) for up to six months before they deposited the eggs, thereby transferring selenium from the mother to the larvae mainly via the yolk sac (“maternal transfer”). We first provide a larval toxicity regression to determine 5% and 10% effect levels for white sturgeon based on the Linville (2006) microinjection study. These effect levels are then translated to egg, muscle, whole body, and diet using other Linville (2006) data and Tashjian et al. (2006). Then we use results from the Linville (2006) maternal transfer study to deter mine 5% and 10% effect levels via maternal transfer and then perform similar translation of those effect levels to egg, muscle, whole body and diet.

White S turgeon Exposure by Microinjection

Linville’s (2006) analysis focused mainly on developmental defec ts in the larvae, but the data she provided show that larval mortality in the microinjection treatments was the most sensitive endpoint of those she investigated (at the 5% and 10% effect levels, mortality occurs at lower

selenium concentrations than defec ts; compare Figures 1 and 2)

.

Figure 1 . Mortality of white sturgeon larvae microinjected with L

- selenomethionine in the yolk sac just after hatching (developmental stage 36). Selenium was measured in a subsample of larvae one day after injection. Mortality is the proportion that died by the time of yolk depletion, when the fish is ready to start eating (stage 45). The data are from Table 3

- 15 in Linville (2006) . Dashed lines indicate 95% confidence boundaries.

LC05 and LC1 0 are the concentrations of selenium at which 5 and 10 percent (respectively) mortality is expected. 

## 0% 50% 100%

1 10 100 Larval mortality



## Selenium in larvae ( μ g/g dry wt.) LC10 = 6.77 μ g/g 

# White Sturgeon

regression: y=1/(1+(20.0/x) 2.02 )



### LC05 = 4.68 μ g/g

### 6

Figure 2

. Incidence of larval edema and/or skeletal deformities among white sturgeon larvae microinjected

with L

- selenomethioni ne in the yolk sac just after hatching . Data are from Tables 3

- 11 and 3

- 13 in Linville (2006)

. EC05 and EC10 are the concentrations of selenium at which 5 and 10 percent (respectively) rates of adverse effects are expected.



### This agrees with the relative sensitivities of bird eggs to selenium, because bird egg hatchability

is a more sensitive endpoint than teratogenesis (embryonic deformities) (Ohlendorf 2003), and bird egg hatchability is the avian equivalent of larval mortal ity (death by the time the yolk is fully depleted and feeding begins) in fish.

White S turgeon Larval Benchmarks

– Microinjection A standard log

- logistic model (Beckon et al. 2008; Ritz 2010) of the larval mortality data yield LC05 and LC10 (the concentra tions of selenium in larvae corresponding to 5% and 10% mortality) of 4.68 μg/g and 6.77 μg/g, respectively (Figure 1). As far as we know, field data on larval concentrations of selenium are not available; therefore, these larval benchmark

concentrations are shown here only for the purpose of anchoring the following calculations of more useful benchmarks in eggs, muscle, whole body, and diet. 

## 0% 50% 100%

1 10 100 Larval developmental defects



## Selenium in larvae ( μ g/g dry wt.) EC10 = 8.65 μ g/g 

# White Sturgeon

regression: y=1/(1+(13.5/x) 4.92 )



### EC05 = 7.43 μ g/g

### 7

White S turgeon Egg Benchmarks

– Microinjection In another experiment, Linville (2006) fed gravid female white stu rgeon selenized yeast for up to six months before they deposited their eggs, and she measured selenium in the eggs and in the larvae that hatched from those eggs. Therefore, the larval benchmark selenium concentrations from Figure 1 (from microinjection e xperiments) can be translated into corresponding egg concentrations (Figure 3 ). This translation indicate s that selenium concentrations of 4.67 μg/g

and 6.83 μg/g in white sturgeon eggs correspond to 5% and 10% mortality rates (respectively) in

the larvae that hatch from those eggs.

Figure 3

. Relationship between selenium in white sturgeon eggs and in the larvae (stage 36) that hatch from the eggs . The data are from Tables 3

- 18 in Linville (2006)

.



# 1 10

1 10 Selenium in eggs ( μ g/g dry wt.)



### Selenium in larvae ( μ g/g dry wt.)



# White Sturgeon

regression: log 10 y=

- 0.019+1.028*log 10 x
R

- squared: 0.997



### LC10 = 6.77 μ g/g LC05 = 4.68 μ g/g 6.83 μ g/g

4.67 μ g/g

### 8

White S turgeon Muscle Benchmarks

– Microinjection The benchmark larval selenium concentrations from Figure 1 translate into maternal muscle

selenium concentrations of 3.55 μg/g ( 5% mortality) and 5.56 μg/g ( 10 % mortality) using a conversion equation based on data from Linvill e’s (2006) work (Figure 4). Evidently, white sturgeon in the San Francisco estuary commonly exceed these benchmarks. In 46 white sturgeon (ages 4

- 18 years) caught in the San Francisco estuary from 2002 to 2004 (Linares et al. 2004),

mean selenium concen tration in muscle was 6.59 μg/g dry wt., above the 10% mortality benchmark. Among these sturgeon, selenium concentrations increased with age; therefore, it is likely that individuals older than 18 years (the age at which reproduction was beginning to occu r in these wildcaught sturgeon) have even higher concentrations of selenium. This suggests that among white sturgeon in the San Fransico estuary, there may be more than 10% risk of mortality

due to selenium.

Figure 4

. Conversion from larval (stage 36) selenium to selenium in muscle tissue of the mother before depositing eggs. The d ata are from Table 3

- 18 in Linville (2006)



# 1 10

1 10 Selenium in maternal muscle ( μ g/g dry wt.)



### Selenium in larvae ( μ g/g dry wt.)



# White sturgeon

regression: log 10 y=

- 0.2644+1.2157*log 10 x

R

- squared: 0.940



### LC10 = 6.77 μ g/g LC05 = 4.68 μ g/g 5.56 μ g/g

3.55 μ g/g

### 9

White S turgeon Whole Body Benchmarks

– Microinjection These muscle concentrations (above) tran slate into maternal whole body selenium concentrations

of 2.55 μg/g (LC05) and 3.86 μg/g (LC10) using a conversion equation based on data from a Tashjian et al. (2006) study (Figure 5). See the Discussion section for more details on this conversion step.

Figure 5

. Relationship between muscle and whole

- body concentrations of selenium in young white sturgeon (initially weighing about 30 g) exposed for 56 days to dietary selenium in the form of L

- selenomethionine

. Data are from Tables 2 and 3 in Tashjian et al

. (2006)

. 

## 1 10 100

1 10 100 Selenium in whole body ( μ g/g dry wt.)



### Selenium in muscle ( μ g/g dry wt.)



## White Sturgeon

regression: y=exp(

- 0.238+0.926*ln(x))

R

- squared: 0.981



### EC10 = 5.56 μ g/g

EC05 = 3.55 μ g/g 3.86 μ g/g

2.55 μ g/g

### 10

White S turgeon Dietary Benchmarks

– Microinjection The larval benchmark selenium concentrations from the microinjection experiment shown in Figure 1 can be translated directly into maternal dietary selenium concentrations by using data from the study by Linville (2006) . In this study, she fed gravid femal e white sturgeon selenized yeast for up to six months before they deposited their eggs. She then measured selenium in the larvae that hatched from those eggs. These data indicate that selenium concentrations of 5.45 μg/g and 10.8 μg/g in the food of adul t female sturgeon correspond to 5% and 10% mortality rates (respectively) in the larvae they produce (Figure 6). See the Discussion section for more information on this translation. Diet items (i.e. Corbula amurensis ) in the San Francisco Bay

estuary ex ceed selenium concentrations of 10.8 μg/g (dry weight) much of the time ( Linville et
al . 2002

, Kleckner et al. 2010) indicating that these selenium benchmarks are environmentally relevant in this system.



# 1 10 100

1 10 100 Selenium in larvae (stage 36, μg/g

dry wt.)



## Selenium in maternal diet (μg/g dry wt.) fitted model: log 10 y=0.271+0.542*log 10 x



### LC10 = 6.77 μ g/g

10.8 μ g/g LC05 = 4.68 μ g/g



### 5.45 μ g/g 

# White sturgeon

Figure 6

. Relationship between selenium in the diet (selenized yeast) of gravid female white sturgeon and selenium in the larvae (stage 36) that hatch from the eggs that these females produced. Data for the

fitted model are from Tables 3

- 1 and 3

- 5 in Linville (2006 ). Whiskers indicate standard error bars.

Larval e ffect levels (LC05 and LC10) are from Figure 1.

### 11

White S turgeon Exposure by Maternal Transfer In the experiments of Linville (2006) , adverse effects of selenium on larval white sturgeon occurred at lower concentrations in microinjection treatments than in experiments in which larvae were exposed to elevated selenium solely by “maternal transfer” (selenium deposited in the yolk of deve loping eggs by mother fish that had been exposed to dietary selenium) (compare Figures 2 and 7). However, although the most sensitive known endpoint is the “controlling” endpoint for protection of the species, here we include analysis of the maternal tra nsfer experiment, because this appears to be a more natural mode of exposure (but see Discussion ). In Linville’s (2006) maternal transfer experiment, the most sensitive endpoint for larvae was developmental abnormality rather than mortality (compare Figures 7 and 8). Developmental abnormalities are an equally valid determination of adverse effect or “take” in endangered species management decisions; therefore, in this experiment we focus on these developmental

defects.

White S turgeon Larval Benchmarks

– Maternal Transfer The EC05 and EC10 (the concentrations of selenium in larvae correspondi ng to 5% and 10% abnormalities, that is, edema and/or spinal curvature) are 13.7 μg/g and 15.3 μg/g, respectively (Figure 7). 

## 0%

50% 100%

1 10 100 Developmental defects



## Selenium in larvae ( μ g/g dry wt.) EC10 = 15.3 μ g/g 

# White Sturgeon

regression: y=1/(1+(21.2/x) 6.67 )



### EC05 = 13.7 μ g/g

Figure 7

. Incidence of larval edema and/or skeletal deformities among larvae from eggs of female white sturgeon exposed to dietary selenium in the form of selenized yeast. The data are from Table 3

- 18 in Linville (2006).

### 12

White S turgeon Egg Benchmarks

– Maternal Transfer These larval selenium concentrations translate into egg concentrations of 14.0 μg/g (EC05) and

1 5.8 μg/g (EC10), using the equation of Figure 3.

White S turgeon Muscle Benchmarks

– Maternal Transfer

The above larval concentrations translate into maternal muscle concentrations of 13. 1 μg/g (EC05) and 15.0 μg/g (EC10), using the conversion equation of Figure 4

. Of the 46 white sturgeon (ages 4

- 18 years) caught in the San Francisco estuary from 2002 to 2004 (Linares et al. 2004) some individuals approached, but none exceeded the EC05 benchmark (maximum

selenium concentr ation in muscle: 12.4 μg/g), but see the discussion under White Sturgeon

Muscle Benchmarks

– Microinjection above.

White S turgeon Whole Body Benchmarks

– Maternal Transfer These muscle concentrations translate into whole body selenium benchmark concentr ations of

8.51 μg/g (EC05) and 9.65 μg/g (EC10), using the equation of Figure 5

.

White S turgeon Dietary Benchmarks

– Maternal Transfer

T he larval benchmarks (above) translate into maternal diet selenium concentrations of 39.3 μg/g (EC05) and 48.4 μg/g (EC10) using the equation of Figure 6. These benchmarks are above the maximum concentrations of selenium in diet items ( Corbula amurensis ) collected in San Francisco estuary from 1995 to 2010 (22 μg/g), but see White S turgeon Dietary Benchmarks

– Mi croinjection above

.



## 0% 50% 100%

1 10 100 Larval mortality (stage 45)



## Selenium in egg (μg/g dry wt.) LC10 = 22.8 μ g/g 

# White sturgeon

regression: y=1/(1+(79.0/x) 1.77 ) 

### LC05 = 15.0 μ g/g

Figure 8. Mortality among larvae (Stage 45) from eggs of female white sturgeon exposed to dietary

selenium as selenized yeast

. The d ata are from Table 3

- 1 4 in Linville (2006).

### 13



## Green S turgeon (

Acipenser medirostris )

Since 2006, green sturgeon in the San Francisco Bay

- Delta area have been federally listed under the Endangered Species Act as threatened (71 FR 17757). Therefore, any regulatory benchmark

establ ished by a federal agency, such as the U. S. Environmental Protection Agency, must be reviewed by the National Oceanic and Atmospheric Administration (NOAA) to determine whether that benchmark is protective of the species.

Recently, groups of researchers at the University of California, Davis, have investigated the effects of selenium and methylmercury on both green and white sturgeon in various conditions of salinity and temperature (Kaufman et al. 2008; Linares

- Casenave et al. 2010; Silvestre et al.

2010 ; Walker 2009) . Dietary exposure experiments showed that green sturgeon are “much more sensitive to selenium” than white sturgeon at environmentally relevant concentrations of

selenium (Kaufman et al. 2008) , but data from these experiments are not yet pu blicly available. However, data from selenium micro

- injection experiments are available: in tabular form in the poster of Linares

- Casenave et al. (2010) and in graphical form in the published paper of Silvestre

et al. (2010) . The data from these microinjection experiments confirm that the yolk sac larvae of green sturgeon are more sensitive to selenium than those of white sturgeon (compare Figures 1 and 9)

.



## 0%

50% 100%

1 10 100 Larval mortality



## Selenium in larvae ( μ g/g dry wt.) LC10 = 3.73 μ g/g 

# Green Sturgeon

regression: y=1/(1+(6.57/x) 3.88 )



### LC05 = 3.07 μ g/g

Figure 9. Mortality of green sturgeon larvae microinjected with L

- selenomethionine in the yolk sac just after hatching (developmental stage 36). Selenium was measured one day after injection. Mortality is the proportion that died by the time of yolk depletion, when the fish is ready to start eating (stage 45). The data are from Tables 1 and 2 in Linares

- Casenave et al. (2010) , also shown in Table 1 and Figure 1 in Silvestre et al. (2010)

.

### 14

We first provide a larval toxicity regression to determine 5% and 10% effect levels for green

st urgeon based on the Linares

- Casenave et al. (2010) microinjection study. These effect levels are then translated to egg, muscle, whole body, and diet using the white sturgeon data from Linville (2006) and Tashjian et al. (2006) as a surogate. Then we use results from the Linares

Casenave et al. (2010) green and white sturgeon microinjection study to determine a sensitivity ratio and apply it to the Linville (2006) maternal transfer study to calculate green sturgeon 5%

and 10% effect levels via maternal tr ansfer. As for the white sturgeon we then perform similar translation of those effect levels to egg, muscle, whole body and diet for the green sturgeon.

Green Sturgeon Larval Benchmarks

– Microinjection A standard log

- logistic model of the green sturgeon microinjection data yield larval LC05 and LC10 (the concentrations of selenium in larvae corresponding to 5% and 10% mortality) of 3.07 μg/g and 3.73 μg/g, respectively (Figure 9).

Green Sturgeon Egg Benchmarks

– Microinjection

To the be st of our knowledge, as yet there are no data relating selenium in eggs and larvae for green sturgeon. Therefore, we use the equation for white sturgeon (Figure 3 ) to translate larval benchmark selenium concentrations to corresponding selenium concentrati ons in the eggs from which the larvae hatch. Accordingly, selenium concentrations of 3.03 μg/g and 3.70 μg/g in green sturgeon eggs correspond to 5% and 10% mortality rates (respectively) in the larvae that hatch from those eggs.

Green Sturgeon Muscle Be nchmarks

– Microinjection To the best of our knowledge, no data specific to green sturgeon are yet available to translate

these larval selenium concentrations into concentrations in the muscle of the mother that produced the larvae. Therefore, we use the white sturgeon data referenced above (Figure 4) for the best available approximations of these translations in green sturgeon. Accordingly, the 5%

and 10% larval benchmarks translate into maternal muscle selenium concentrations of approximately 2.13 μg/g and 2.69 μg/g, respectively.

Green Sturgeon Whole Body Benchmarks

– Microinjection To the best of our knowledge, no data specific to green sturgeon are yet available to translate the

above muscle selenium concentrations into concentrations in the whole bo dy of the mother that produced the larvae. Therefore, we use the white sturgeon data referenced above (Figure 5) for the best available approximations of these translations in green sturgeon. Accordingly, the

muscle benchmarks translate into maternal who le body (dry weight) selenium concentrations of

about 1. 59 μg/g (LC05) and 1.97 μg/g (LC10).

### 15

Green Sturgeon Dietary Benchmarks

– Microinjection To the best of our knowledge, no trophic transfer data specific to green sturgeon are yet available

to translate larval selenium concentrations or maternal tissue concentrations into concentrations in the diet of the mother that produced the eggs from whic h the larvae hatched. Therefore, we use the white sturgeon data referenced above (Figure 6) for the best available approximation of the appropriate translation. Accordingly, the green sturgeon larval benchmark selenium concentrations from Figure 9 transl ate into selenium concentrations of approximately 2.51 μg/g (EC05) and 3.58 μg/g (EC10) in the food of adult female green sturgeon (Figure 10). Almost all

of the diet items ( Corbula amurensis ) collected in the San Francisco Bay estuary from 1995 to

2010 e xceeded these benchmark concentrations (Kleckner et al. 2010).

Green St urgeon Exposure by Maternal Transfer

– calculated from relative sensitivity As far as we know, for green sturgeon there has been no maternal transfer selenium toxicity experiment comparable to Linville’s (2006) experiment with white sturgeon (Figures 7 and 8). However, if we assume that the relative sensitivity of green and white sturgeon would be the same for naturally transferred selenium as for micro

- injected selenium, then we can calculate a larval selenium sensitivity ratio for green and white sturgeo n, and use this ratio to estimate, for green sturgeon, selenium benchmarks that correspond to those for maternally transferred selenium in white sturgeon. Such a ratio can be obtained from the micro

- injection experiments



# 1 

# 10 100

1 10 100 Selenium in larvae (stage 36, μg/g

dry wt.)



## Selenium in maternal diet (μg/g dry wt.) fitted model: log 10 y=0.271+0.542*log 10 x



### LC10 = 3.73 μ g/g

3.58 μ g/g LC05 = 3.07 μ g/g

2.51 μ g/g

Figure 10. Translation of selenium effect levels for larval green sturgeon (from Figure 9) into corresponding selenium concentrations in maternal diet using trophic and maternal transfer data for white sturgeon are from Linville (2006) (see Figure 6).

### 16

of Linares

- Casenave et al. (2010) on both green and white sturgeon (Figure 11). The ratio of EC10’s ( the sensitivy ratio, white to green) is 10.49/4.49 = 2.34.



## 0% 50% 100%

1 10 100 Peak abnormalities



## Selenium in larvae ( μ g/g dry wt.) LC10 = 10.49 μ g/g 

# White Sturgeon

regression: y=1/(1+(62.06/x) 1.24 )



### LC05 = 5.73 μ g/g



## 0% 50% 100%

1 10 100 Peak abnormalities



## Selenium in larvae ( μ g/g dry wt.) LC10 = 4.49 μ g/g 

# Green Sturgeon

regression: y=1/(1+(8.53/x) 3.42 )



### LC05 = 3.61 μ g/g

Figure 11. Incidence of larval abnormalities among larvae from eggs of (A) white sturgeon and (B)

green sturgeon microinjected with L

- selenomethionine in the yolk sac just after hatching . Data are from Table s 1 and 2 in Linares

- Casenave et al. (2010)

. 

### A

B

### 17

In Figure 11, the effect or “endpoint” used is the peak incidence of abnormalities rather than the incidence of abnormalities at the end of the experiment. This is because transient abnormalities effectively immobilize developing larvae (Serge Doroshov pers.com.). This may cause adverse effects during larval development that are not represented by th e incidence of abnormalities at the end of larval development. In the wild, transient abnormalities resulting in immobilization are likely to result in mortality due to predation or the inability to escape from poor habitat conditions. Even the peak inci dence of abnormalities may not fully represent the full consequences of transient abnormalities, because some individual larvae may be affected by transient abnormalities only at some time other than the time of peak abnormalities in the group

of larvae. Any adverse effects on those larvae are not counted in the analysis presented here. Full accounting of these adverse effects would require tagging and tracking the condition of each

individual larva.

Green Sturgeon Larval Benchmarks

– Maternal Transfer The above sensitivity ratio applied to the white sturgeon maternal transfer benchmark selenium concentrations in larvae (Figure 7) yield the following corresponding green sturgeon benchmark

larval selenium concentrations: larval EC05 = 5.84 μg/g (= 13.7 μg/g /2.34) and EC10= 6.54 μg/g (=15.3 μg/g /2.34).

Green Sturgeon Egg Benchmarks

– Maternal Transfer Corresponding benchmark selenium concentrations for green sturgeon eggs are calculated from the larval concentrations as they were for the injection e xperiment above using Figure 3. The resulting egg benchmarks are EC05 = 5.87 μg/g and the EC10 = 6.59 μg/g.

Green Sturgeon Muscle Benchmarks

– Maternal Transfer Corresponding benchmark selenium concentrations for green sturgeon muscle are calculated

fr om the larval concentrations as they were for the injection experiment above using Figure 4. These translations yield maternal muscle benchmarks of EC05 = 4.65 μg/g and EC10 = 5.33 μg/g.

Green Sturgeon Whole Body Benchmarks

– Maternal Transfer Correspon ding benchmark selenium concentrations for green sturgeon whole body are calculated from the larval concentrations as they were for the injection experiment above using Figure 5. These translations yield maternal whole body benchmarks of EC05 = 3. 27 μg/g and EC10 = 3.71 μg/g.

Green Sturgeon Dietary Benchmarks

– Maternal Transfer Corresponding benchmark selenium concentrations for the diet of green sturgeon are calculated from the larval concentrations as they were for the injection experiment above using Figure 10. This translation produces green sturgeon maternal diet benchmarks of EC05 = 8.22 μg/g and

EC10= 10.1 μg/g.

### 18



## Discussion

The whole

- body LC10 selenium concentration derived here for white sturgeon (3.86 μg/g) is similar to the toxicity threshold of 4 μg/g suggested by Lemly (1996) and used by the U. S. Fish and Wildlife service for warmwater fish (Beckon et al. 2010). Green sturgeon are more susceptible to selenium toxicity; they appear to be about as sensitive to selenium as the more sensitive coldwater fish (Salmonids). The whole

- body LC10 derived here for green sturgeon (1.97 μg/g) is close to the LC10 of 1.84 μg/g for ju venile Chinook salmon, and the EC10 of 2.19 μg/g for juvenile rainbow trout (Beckon et al. 2010). The dietary LC05 and LC10 derived here for green sturgeon (2.51 μg/g and 3.58 μg/g respectively) bracket the concern threshold of 3 μg/g used by the U. S. Fi sh and Wildlife service (Beckon et al. 2010).

Because adult sturgeon are very large, it is difficult to analyze their tissue for selenium on a

whole

- body basis (requiring homogenization of the entire body). Therefore, as far as we know,

data are not avai lable for muscle/whole

- body selenium regressions in adults of these species. The best available data for such a regression (Figure 5) came from only juvenile white sturgeon

data (Tashjian et al . 2006) . However, this regression is in good agreement with a generic

muscle/whole

- body relationship based on a much larger data set comprising several different fish families (Figure 12). Furthermore, the relationship is fairly robust regardless of the gender or reproductive condition of the fish (Figure 13) despi te the suggestion (Osmundson & Skorupa 2011) that selenium is differently partitioned among tissues depending on reproductive status. Therefore, it is likely that the juvenile muscle/whole

- body relationship adequately represents the

muscle/whole

- body rela tionship of adult white and green sturgeon, and that the modest extrapolation of this relationship in this analysis (Figure 5) is reasonable.

Because of the meager data available to translate from larval selenium concentrations to maternal dietary concent rations of selenium (Figures 6 and 10), the choice of model is critical for interpolating between the data points. This translation incorporates two stages: trophic transfer (from maternal diet to maternal tissue) and maternal transfer (from maternal tiss ue to larval tissue). The exponential model (straight line on a log

- log graph) used here for the combined transfer is valid if both transfer stages are adequately represented by exponential models. As far as we know, no one has questioned the applicabil ity of such a model to maternal transfer, but it has been suggested that organisms actively, homeostatically regulate their internal concentrations of selenium (Brix et al. 2005). Such homeostatic regulation would result in trophic transfer

functions that are better represented by “hockey stick” models, which include a plateau region in which internal tissue concentrations are purportedly held constant as exposure increases. If such regulation occurs, it would invalidate the straight

- line exponential mode l, enabling mother sturgeon to consume more selenium without elevating their internal selenium concentrations or those of their eggs and larvae. However, a broad survey of field and laboratory bioaccumulation and trophic transfer data offers no support fo r such homeostatic regulation (for other bioaccumulative metals as well as for selenium). Rather the data strongly support continuous,

non

- homeostatic regulation, with trophic transfer functions that are well described by exponential models (Beckon 2010, Appendix II). Therefore, it is likely that the fitted straight line in Figures 6 and 10 (describing an exponential model) is suitable for translations of concentrations of selenium between maternal diet and larvae. Because only two data points are

availa ble in the published literature for this relationship ( Linville 2006), regression is not

### 19

possible; instead, t he model was fitted manually so as to pass through those two points. The resulting model should be reasonably stable because each of the data poin ts represents the average of three replicates (different cohorts from three different females) of 60 to 90 individual larvae per replicate.



## 1 10 100

1 10 100 Selenium in whole body ( μ g/g dry wt.)



## Selenium in muscle ( μ g/g dry wt.)

white sucker (Osmundson)

green sunfish (Osmundson)

other sp (Osmundson)

regression (all Osmundson)

white sturgeon (Tashjian 2006)

white sturgeon regression

y=exp(0.1004+0.833*ln(x))

R

- squared: 0.91

n=189 y=exp(

- 0.238+0.926*ln(x))

R

- squared: 0.98

n=6

Figure 12

. Relationship between muscle and whole

- body concentrations of selenium in young white
sturgeo n (Tashjian et al . 2006) compared with the relationship between muscle plug and whole

- body concentrations of selenium in representatives of several families of fish caught in the Gunnison and Colorado Rivers and associated tributaries (data from Osmundson et al. 2007, Osmundson & Skorupa 2011).

### 20

For comparisons of the sensitivities of different species and endpoints to selenium, here we use

EC10s rather than the more traditional EC50s. This is because EC10 is an effect level that is closer to levels that may be protective of species. When concentration

- respons e curves have substantially different slopes (as in Figures 1 and 2, and Figure 11), then sensitivity comparisons based on EC50s would poorly represent relative sensitivities at effect levels of EC10 or below.

It should be noted that the green sturgeon ex periment analyzed here (Linares

- Casenave et al.
2010, Silvestre et al. 2010) was performed on the projeny of a single individual (Obtaining multiple individuals for experimentation was not practical because the species is listed as

threatened in this area). Therefore, this analysis may underestimate or overestimate the sensitiviy of the population of green sturgeon from which this individual came. However, these are the best data available for green sturgeon at this time. 

## 1 10 100

1 10 100 Selenium in whole body ( μ g/g dry wt.)



## Selenium in muscle ( μ g/g dry wt.)

white sucker females w/ mature eggs white sucker females w/ immature eggs, small ovaries

white sucker males white sturgeon juveniles (Tashjian 2006)
white sturgeon regression

y=exp(

- 0.238+0.926*ln(x))

R

- squared: 0.98

n=6

Figure 13. Relationship between muscle and whole

- body concentrations of selenium in young white
sturgeon (Tashjian et al . 2006) compared with the relationship between muscle plug and whole

- body

concentrations of selenium in white suckers ( Catastomus commersoni ) segregated by gender and reproductive condition (data from Appendix I in Osmundson & Skorupa 2011).

### 21

In the experiments of Linvi lle (2006) , adverse effects of selenium on larval white sturgeon occurred at lower concentrations in microinjection treatments than in experiments in which larvae were exposed to elevated selenium solely by maternal transfer. If this apparent difference

i s real, it may be related to the fact that in the microinjection experiments, the selenium is introduced suddenly as a free amino acid (L

- selenomethionine) that is readily available for assimilation by the developing larvae, whereas maternally transferred selenium is incorporated into proteins that must be digested by the larvae before assimilation (Linville 2006). Heinz et al. (2011) offered a similar explanation for his observation that methylmercury chloride injected into mallard eggs is more terratogen ic than the same concentration of methylmercury maternally deposited in the egg. However, at the present time, for selenium in white sturgeon larvae, maternal transfer data are more limited (one experiment, two treatments) than microinjection data (multip le experiments, each with several treatments). Additionally, although the maternal transfer mode of exposure may seem to be more natural than injection of selenomethionine into larvae, in the maternal transfer experiments of Linville (2006), the mother fi sh were fed selenium in the form of selenized yeast. Sturgeon do not feed on selenized yeast in the wild, and there are indications that experiments based on exposure to dietary selenized yeast may underestimate selenium assimilation and toxicity (Heinz e t al. 1996). For these reasons, until more data confirm the apparent difference in sensitivity to these two modes of exposure, and clarify reasons for it, prudence and caution impel us to use the data from the microinjection experiments as the basis for p rotective guidelines.

It may be that our analysis of the maternal transfer experiment of Linville (2006) underestimates the potential hazard of trophic and maternal transfer of selenium because of the relative youth of the captive

- reared female sturgeon used in that experiment. White sturgeon captured in the wild from the San Francisco Bay/Estuary region exhibit a clear pattern of increasing selenium bioaccumulation with increasing age (Linares et al. 2004); individuals just beginning to reproduce in th e wild (17 years old) had more than twice the selenium concentrations in muscle than the concentrations in the age class (6 years old) that were used in the Linville (2006) experiment. Still older (>17 years), fully reproductive individuals in the wild ar e likely to accumulate even higher levels of selenium.

It may be that the most sensitive endpoint for selenium effects on sturgeon is neither larval mortality nor larval abnormalities. Possibly these fish are even more sensitive to selenium after

the lar vae have finished yolk sac absorption and begin exogenous feeding, as seems to be the case for at least some salmonids (Beckon 2007, Beckon et al. 2010). To the best of our knowledge, sturgeon at this very sensitive stage have not yet been tested; Tashjia n et al. (2006) tested a later, potentially less sensitive juvenile stage. Fish such as sturgeon could also be very sensitive to selenium effects on behavior, which could be important in the wild for predator avoidance, foraging, or reproductive success. For example, Hopkins et al. (2003) documented a substantial reduction in swimming speed, probably at least partly attributable to selenium, in benthic lake chubsucker ( Erimyzon sucetta ) exposed to coal ash. In green and white sturgeon,

the effects of sel enium on metabolic rates, growth, swimming performance, and avoidance of predators have been studied (Kaufman et al. 2008; Walker 2009) , but the data have not yet been published. Until data on sublethal reproductive and behavioral effects such as these ar e published, to the best of our knowledge, the data used here are the best available for the most sensitive endpoint currently known for these fish.

### 22

The analysis presented here focuses only on the single most sensitive known endpoint. In reality, mortali ty and non

- lethal endpoints are additive. That is, in addition to the mortality of developing embryos directly caused by selenium, a further proportion of surviving individuals

suffers from other adverse effects of selenium: non

- lethal effects (such as s pinal deformities and impaired growth), indirect lethal effects (such as elevated vulnerability to predation due to mobility impairment caused by transitory edema, or other behavioral impairment), and later lethal effects (such as mortality among early sta ge juveniles). For these reasons, the analysis presented here probably underestimates the full extent of adverse effects of selenium on sturgeon. Therefore, benchmarks based on all adverse effects would likely be lower (more stringent) than those derive d here on the basis of the most sensitive endpoint only.

Although uncertainty in data analysis has been viewed as an argument for relaxing protective standards, in reality, uncertainty may require greater stringency. Agencies that implement the Endangere d Species Act are expected to “provide the benefit of the doubt” to listed species (USFWS & NOAA 2004).

A strict interpretation of the “benefit of the doubt” guidance would suggest that, in modeling

dose

- response relationships, rather than a central

- tend ency model (least squares regression: solid line in Figures 1 and 2), some lower (more stringent) confidence bound should be used for deriving a protective guideline. Alternatively, a “safety factor”, “uncertainty factor”, “modifying factor” (USEPA 1993), and/or “margin of safety” could be included in the analysis (“ Margins of safety are essential to any health related environmental standards if a reasonable degree of protection is to be provided against hazards which research has not yet identified.” [Sen ate Committee on Public Works, Report No.91

- 1196 (1970), pp.9

- 10] ). However, in the case of selenium, care must be exercised in implementing any such factor or margin of safety, because selenium is essential at low concentrations, and there is a very narr ow range between essentiality and toxic excess ( e.g ., Figures 3B and 5B in Beckon et al. (2008) ). Larval mortality in sturgeon may be caused by deficiency of selenium as well as by toxic excess of selenium. An excessively large uncertainty factor could push a benchmark concentration into the zone of deficiency. No factor providing any margin of safety is included in the derivation of benchmark selenium concentrations presented here. However, the above analysis, based on the more sensitive microinjectio n route of exposure (rather than the maternal transfer route of exposure), provides conservative benchmarks founded on the best information currently available.

If selenium deficiency can occur in larval sturgeon, then data spanning a full range of

concen trations from deficiency to toxic excess would best be represented by a biphasic model (Beckon et al . 2008) . We know of no data as yet available relating larval survival of sturgeon to selenium in the zone of tissue concentrations that are likely to cause deficiency

— probably < 1 μg/g (whole body dry weight), based on experiments with juveniles of other species (Beckon et
al. 2010). Therefore, for the available data, the simple log

- logistic model used here (Figures 1, 2, 7, 8, 9 and 11) should be adequate.

### 23

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Beckon, W. 2007. Selenium risk to salmonids with particular reference to the Central Valley of California. Poster presented at the American Fisheries Society, September 2007, San

Francisco CA.

Beckon, W. 20 10. Trophic t ransfer of selenium and metals: does regulation o ccur in food

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### 27 

## Appendix I

Data and Methods

Log

- logistic m odels were fitted to dose

- response relationships by least squares non

- linear regression, using the “nls” and “nls2” function s in the “R” statistical program. Confidence (95%) bounds were determined for non linear models using the “nls2”, “predict”, and “ as.lm.nls ”

function s in “R” . Linear models for translations of tissue concentrations were determined using

the “lm” function in “R” (except for Figures 11 an d 12, as noted below).



## White sturgeon (

Acipenser transmontanus )

Figure 1

Selenium effect on mortality of microinjected white sturgeon larvae. Data source: Table 3

- 15 in Linville (2006).

Treatment Selenium

in larvae (μg/g dry wt.) Larval

mortality
Study 1 non inj 2.6 0 Study 1 sham inj 2.54 0 Study 1 low 15.8 0.41 Study 1 medium 21.7 0.467 Study 1 high 46.6 0.676 Study 2 non inj 6.36 0 Study 2 sham inj 5.89 0.0167 Study 2 low 8.74 0.1 Study 2 medium 8.97 0.0334 Study 2 high 16.56 0.7

Formula: y ~ 1/(1 + (e/x)^b)

Parameters: Estimate Std. Error t value Pr(>|t|)

e 20.0490 3.0848 6.499 0.000188 ***

b 2.0233 0.6224 3.251 0.011683 *

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.1392 on 8 degrees of freedom

Number of iterations to convergence: 9 Achieved convergence tolerance: 4.346e

- 06

### 28

Figure 2

Selenium effect on defects of microinjected white sturgeon larvae. Data source: Tables 3

- 11 and

3

- 13 in Linville (2006).

Treatment Selenium

in larvae (μg/g dry wt.) Edema

and/or spinal

curvature Study 1 non inj 2.6 0 Study 1 sham inj 2.54 0.0395 Study 1 low 15.8 0.7669 Study 1 medium 21.7 0.8366 Study 1 high 46.6 0.7821 Study 2 low 8.74 0.1273 Study 2 medium 8.97 0.0508 Study 2 high 16.56 0.6949 Study 2 non/sham inj 6 0.0085

Formula: y ~ 1/(1 + (e/x)^b)

Parameters: Estimate Std. Error t value Pr(>|t|)

e 13.5260 0.8507 15.900 9.44e

- 07 ***

b 4.9174 1.1663 4.216 0.00395 **

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.09781 on 7 degrees of freedom

Number of iterations to convergence: 9 Achieved convergence tolerance: 7.008e

- 06



### Fig ure 3

Selenium in white sturgeon eggs and larvae. Data source: Table 3

- 18 in Linville (2006).

Call: lm(formula = log10(egg) ~ log10(larva), data = WhiteSturgeonLinvile3_18)

Residuals:

1 2 3 4 5 6

0.013976

- 0.008056 0.009141

- 0.033171 0.031295

- 0.013184

### 29

Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept)

- 0.01930 0.02187

- 0.882 0.427 log10(larva) 1.02763 0.02711 37.909 2.89e

- 06 ***

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.02548 on 4 degrees of freedom Multiple R

- squared: 0.9972, Adjusted R

- squared: 0.9965

F

- statistic: 1437 on 1 and 4 DF, p

- value: 2.892e

- 06



### Figure 4

Maternal transfer of s eleni um in white sturgeon. Data source: Tables 3

- 18 in Linville (2006)

.

Treatment Selenium

in

maternal

muscle ( μ g/g dry wt.) Selenium

in larvae ( μ g/g dry wt.)

Control C3 1.28 2.43

Control C4 1.22 1.69

Control C5 1.48 2.67

Treatment T1 9.93 11.6

Treatment T2 15.3 18.4

Treatment T3 11.1 7.75

Call: lm(formula = log10(muscSe) ~ log10(larvaeSe), data = WhiteSturgeonLinville3_18 )

Residuals:

1 2 3 4 5 6

- 0.09717 0.07372

- 0.08385

- 0.03273

- 0.08856 0.22859

Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept)

- 0.2644 0.1237

- 2.137 0.09937 . log10(larvaeSe) 1.2157 0.1533 7.930 0.00137 **

--- Signif. codes: 0 '***' 0.001 ' **' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.1441 on 4 degrees of freedom Multiple R

- squared: 0.9402, Adjusted R

- squared: 0.9252

F

- statistic: 62.88 on 1 and 4 DF, p

- value: 0.001369

### 30

Figure 5

Selenium in muscle and whole body in young whi te sturgeon. Data source: Tables 2 and 3 in Tashjian et al. (2006).

Selenium

in

muscle (μg/g dry wt.) Selenium

in whole body (μg/g dry wt.)

8.2 5.2

17.2 11.8

22.9 14.7

36.8 22.5

52.9 34.4

54.8 27.5

Call: lm(formula = log(WB) ~ log(musc ), data = WhiteSturgeonTTF)

Residuals:

1 2 3 4 5 6

- 0.06139 0.07235 0.02715 0.01373 0.10234

- 0.15419

Coefficients:

Estimate Std. Error t value Pr(>|t|) (Intercept)

- 0.23765 0.21398

- 1.111 0.328997 log(musc) 0.92565 0.06399 14.467 0.000133 ***

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.1051 on 4 degrees of freedom Multiple R

- squared: 0.9812, Adjusted R

- squared: 0.9766

F

- s tatistic: 209.3 on 1 and 4 DF, p

- value: 0.0001327

### 31

Figure 6

Trophic and maternal transfer in white sturgeon. Data source: Tables 3

- 1 and 3

- 5 in Linville (2006).

Selenium

in

maternal
diet (μg/g dry wt.) Selenium

in larvae (stage 36, μg/g dry wt.)

1.42 2.26

34.04 12.58



### Figure 7

Developmental defects in white sturgeon larvae exposed to selenium via maternal diet. Data

source: Table 3

- 18 in Linville (2006).

Treatment Selenium

in larvae ( μ g/g dry wt.) Developmental

defects

Control C3 2.43 0

Control C4 1.69 0

Control C5 2.67 0

Treatment T1 11.6 0

Treatment T2 18.4 0.2778

Treatment T3 7.75 0.1333

Formula: defects ~ 1/(1 + (e/larvaeSe)^b)

Parameters: Estimate Std. Error t value Pr(>|t|)

e 21.246 4.084 5.202 0.00651 **

b 6.666 8.410 0.793 0.47230

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.06662 on 4 degrees of freedom

Number of iterations to convergence: 65 Achieved convergence tolerance: 9.34e

- 06

### 32

Figure 8

Mortality in white sturgeon larvae exposed to selenium via maternal diet. Data source: Table 3

- 14 in Linville (2006).

Grouped

exposure category Selenium in egg

(μg/g dry wt.) Larval mortality

(stage 4 5)

low 2.15 0.0027

med 9.31 0.022 high 20.5 0.0842

Formula: Mortality ~ 1/(1 + (e/SeEggs)^b)

Parameters: Estimate Std. Error t value Pr(>|t|)

e 78.98147 3.86639 20.43 0.0311 *

b 1.76984 0.06086 29.08 0.0219 *

--- Signif. codes : 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.00103 on 1 degrees of freedom

Number of iterations to convergence: 3 Achieved convergence tolerance: 7.521e

- 06

### 33



## Green sturgeon (

Acipenser medirostris )

Figure 9

Selenium ef

fect on mortality of microinjected green sturgeon larvae. Data source: Javier

Linares

- Casenave, et al. poster ( 2010 ).

Treatment Selenium in

larvae (μg/g dry wt.) Larval

mortality

Se

- L

- Met 7.3 0.6

L

- Met 3.5 0.12

Non

- Inj 3.2 0.01

Formula: y ~ 1/(1 + (e/x)^b)

Parameters: Estimate Std. Error t value Pr(>|t|)

e 6.5661 0.4116 15.952 0.0399 *

b 3.8791 0.9478 4.093 0.1526

--- Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 0.06239 on 1 deg rees of freedom

Number of iterations to convergence: 5 Achieved convergence tolerance: 6.219e

- 06



### Figure 10

Data and fitted model are the same as in Figure 5

Figure 11A

Selenium effect on peak abnormalities of microinjected green sturgeon larvae. Da ta are from

Tables 1 and 2 in Linares

- Casenave et al. 2010

.

Treatment Selenium

in larvae (μg/g dry wt.) Peak

abnormalities

Se

- L

- Met 8.6 0.08

L

- Met 2.1 0.02

Non

- Inj 2.1 0.01

### 34

S

- PLUS 6 analysis:

Formula: Abnorm ~ 1/(1 + (e/SeLarvae)^b)

Parameters:

Value Std. Error t value

e 62.06060 26.492800 2.34255

b 1.23579 0.249522 4.95262

Residual standard error: 0.00707107 on 1 degrees of

freedom

Correlation of Parameter Estimates:

e

b

- 0.985



### Figure 11B

Selenium effect on peak abnormalities of microinjected white sturgeon larvae. Data are from

Tables 1 and 2 in Linares

- Casenave et al. 2010

.

Treatment Selenium

in larvae (μg/g dry wt.) Peak

abnormalities

Se

- L

- Met 7.3 0.37

L

- Met 3.5 0.04

Non

- Inj 3.2 0.04



### S

- PLUS 6 analysis:

Formula: Abnorm ~ 1/(1 + (e/SeLarvae)^b)

Parameters:

Value Std. Error t value

e 8.52964 0.130246 65.4887

### 35

b 3.42169 0.202209 16.9216

Residual standard error: 0.00820243 on 1 degrees of freedom

Correlation of Parameter Estimates:

e

b

- 0.755



### Figure 12

Selenium in muscle and whole body in representatives of several families of fish caught in the

Gunnison and Colorado Rivers and associated tributaries. Data sources: Barbara Osmundson

pers. com., Osmundson et al. ( 2007 ) , Osmundson & Skorupa ( 201 1 ) . For comparison, the white sturgeon data and regression from Figure 5 are also shown (orange color).

ID Species Selenium in

whole body (μg/g dry wt.) Selenium in muscle (μg/g dry wt.)

BSW

- WS3 WS 3.81 2.88

BSW

- WS4 WS 4.21 4.83

BSW

- WS7 WS 3.33 3.7

BSW

- WS8 WS 4.51 3.7

2PW

- WS1 WS 6.32 8.42

2PW

- WS2 WS 6.77 9.36

2PW

- WS3 WS 10.99 15.5

2PW

- WS4 WS 12.65 23.6

MWB

- WS2 WS 5.72 9.37

MWB

- WS1 WS 8.32 11.5

2MW

- WS1 WS 3.92 6.07

2MW

- WS2 WS 3.79 4.56

2FL

- WS1 WS 9.92 12.3

2FL

- WS3 WS 5.28 9.23

WS

- FL

- 1 WS 10.72 9.44

WS

- FL

- 2 WS 5.92 9.44

WS

- FL

- 3 WS 7.03 10.5

WS

- FL

- 4 WS 6.4 11.4

WS

- FL

- 5 WS 6.27 9.57

### 36 WS

- FL

- 6 WS 5.33 9.29

WS

- FL

- 8 WS 6.15 9.75

WS

- FL

- 9 WS 5.59 10.5

AC

- WSWB1 WS 8.8 11.1

AC

- WSWB2 WS 8.71 12.1

MW

- WS2 WS 11.37 12.8

MW

- WS6 WS 10.7 16

MW

- WS7 WS 8.44 12.1

PS2

- WS1 WS 6.95 8.99

PS2

- WS3 WS 7.49 10.6

PS2

- WS4 WS 10.28 12.6

PS2

- WS5 WS 6.73 11.6

GV1

- WS1 WS 2.09 2.81

GV1

- WS4 WS 1.78 2.53

GV1

- WS6 WS 3.21 4.31

GV1

- WS8 WS 2.27 3.52

GV1

- WS9 WS 3.08 4.27

GV1

- WS10 WS 3.04 3.14

GV1

- WS13 WS 2.79 3.58

GV1

- WS14 WS 2.51 2.95

GV1WS15 WS 3.43 4.09

GV1WS16 WS 2.83 3.59

WS

- FL

- 7 WS 3.1 5.58

WS

- FL

- 10 WS 5.51 6.29

2FL

- WS2 WS 7.02 9.13

2FL

- WS4 WS 7.3 8.47

2FL

- WS5 WS 2.41 3.04

BSW

- WS1 WS 2.72 4.39

BSW

- WS6 WS 2.7 3.23

BSW

- WS10 WS 2.55 1.63

2HW

- WS1 WS 19.6 28.1

AC

- WS2 WS 9.8 12.1

MW

- WS1 WS 8.69 11.8

MW

- WS4 WS 8.73 12.6

MW

- WS5 WS 9.08 12.3

AC

- WSWB3 WS 13.4 18

GV1

- WS2 WS 3.12 2.81

GV1

- WS5 WS 2.43 3.15

GV1

- WS7 WS 2.14 3.14

GV1WS11 WS 3.18 4.32

GV1WS12 WS 2.75 3.4

AC

- GS1 GS 10.8 14.7

ACOGS1 GS 22.8 28.1

ACO

- GS3 GS 8.79 12.9

RW

- GS1 GS 15.37 21.9

LW

- GS2 GS 4.75 4.95

LW

- GS4 GS 5.74 6.11

LW

- GS6 GS 4.43 5.19

2MW

- GS2 GS 3.75 5.14

BSW

- GS2 GS 11.9 15.7

### 37

2PW

- GS1 GS 6.43 10.1

2HW

- GS1 GS 9.51 11.5

FL

- GS

- 1 GS 9.13 10.5

GS

- FL

- 8 GS 6.24 7.2

GS

- FL

- 9 GS 7.04 9.26

GS

- FL10 GS 7.72 7.65

FL

- GS11 GS 6.19 5.99

FL

- GS12 GS 10.2 12

FL

- GS14 GS 9.71 12.1

FL

- GS16 GS 9.88 12.5

SW

- GS2 GS 7.18 7.49

MW

- GSF5 GS 8.99 11.3

MW

- GSF6 GS 9.7 13.6

MW

- GSF7 GS 8.89 13.2

MW

- GSF8 GS 9.81 12.4

MW

- GSF9 GS 9.87 12.5

HW

- GS1 GS 10.27 8.59

PS2

- GS1 GS 5.34 5.34

PS2

- GS2 GS 10.14 11.9

PS2

- GS3 GS 11.83 13.6

GV1GS1 GS 3.26 3.79

GV1GS2 GS 3.95 4.22

GV1GS3 GS 4.33 4.08

GV2GS1 GS 3.65 4.23

GV2GS2 GS 6.22 5.74

GV2GS3 GS 3.46 4.41

GV3

- GS1 GS 4.38 3.52

GV3

- GS2 GS 5.59 5.45

GV3

- GS6 GS 4.87 4.95

GV3

- GS9 GS 4.44 4.32

BSW

- GS1 GS 7.96 10.1

ACO

- GS2 GS 7.87 11.9

MWB

- GS1 GS 6.36 11.1

MWB

- GS2 GS 8.67 11.8

MWB

- GS3 GS 8.34 11

GS

- FL

- 1 GS 6.08 7.08

GS

- FL

- 2 GS 5.62 6.65

GS

- FL

- 3 GS 18.1 26.4

FL

- GS

- 5 GS 9.4 9.62

GS

- FL

- 6 GS 12.2 16.7

SW

- GS1 GS 5.29 8.12

SW

- GS3 GS 7.3 10.6

MW

- GSF1 GS 9.28 14.2

MW

- GSF2 GS 6.82 11.3

MW

- GSF4 GS 7.5 12.8

SC

- FMS1 FMS 3.1 4.09

SC

- FMS2 FMS 2.63 3.79

FL

- FMS1 FMS 4.48 7.28

FL

- FMS2 FMS 3.5 5.23

FL

- FMS3 FMS 2.95 3.56

### 38

FL

- FMS4 FMS 4.42 6.15

FL

- FMS5 FMS 3.12 4.63

32

- FMS1 FMS 2.19 4.23

32

- FMS5 FMS 1.95 4.28

UCS

- FMS1 FMS 2.76 3.57

RP

- FM3 FMS 4.22 5.72

RP

- FM4 FMS 4.55 5.6

FL

- BHS3 BHS 1.97 2.3

FL

- BHS4 BHS 1.3 1.47

FL

- BHS5 BHS 2.42 3.07

PW3

- BHS1 BHS 5.62 8.57

FL

- BHS11 BHS 3.91 5.16

FL

- BHS1 BHS 2.11 2.52

FL

- BHS2 BHS 2.18 2.72

RP

- BH1 BHS 2.11 2.76

RP

- BH2 BHS 2.43 3.04

UCS

- BHS1 BHS 2.13 3.64

PW3

- CP1 CCP 11.7 20

PW3

- CP2 CCP 4.78 8.24

PW3

- CP3 CCP 4.1 6.56

PW3

- CP4 CCP 6.29 7.81

CL

- CP1 CCP 23.12 24.2

RP

- CC1 CCP 6.32 6.1

32

- C

- 1 CCP 4.01 5.06

FL

- CP

- 1 CCP 5.91 10.2

FL

- CP

- 2 CCP 9.02 11.5

FL

- BT2 BT 5.02 4.01

RFL

- BT1 BT 4.55 3.17

FL

- BT1 BT 5.52 6.27

FL

- BT11 BT 4.3 3.64

CR

- RTC5 RTC 6.44 6.22

CR

- RTC6 RTC 6.83 6.87

CR

- RTC7 RTC 8.4 9.84

CR

- RTC9 RTC 4.08 4.34

CRRTC10 RTC 5.27 5

FL

- RTC1 RTC 6.55 7.29

JARVRTC RTC 5.51 6.96

RP

- RT1 RTC 7.23 7.42

UCS

- RTC3 RTC 3.84 5.74

UCS

- RTC4 RTC 3.63 5.56

CR

- BG

- 1 8.78 12.9

FL

- BH3 BBH 7.31 7.49

FL

- BH4 BBH 4.83 3.94

FL

- BH1 BBH 5.47 4.26

FL

- BH2 BBH 7.64 7.42

FL

- BH8 BBH 8.59 7.82

FL

- BH9 BBH 9.61 5.7

RFL

- BH1 BBH 6.61 7.77

RFL

- BH2 BBH 2.03 9.22

HW

- BH1 BBH 4.92 4.67

### 39 HW

- BH2 BBH 5.3 3.35

LCBH

- 1 BBH 2.9 2.04

BH

- FL2 BBH 3.94 4.63

BH

- FL

- 3 BBH 4.57 3.91

2PW

- BH1 BBH 4.99 4.36

FL

- CCF1 CCF 3.97 5.31

PW3

- CC1 CCF 3.34 3.58

FL

- CCF2 CCF 3.41 3.41

RFL

- CF1 CCF 2.63 3.67

UCS

- CC1 CCF 2.04 3.95

? CCF 1.88 1.95

CRCF

- 1 CCF 3.35 3.7

32 CCF 2.35 1.49

CL

- LMB1 7.03 8.45

CL

- SMB1 5.42 6.93

CL

- SMB2 4.19 3.67

GVICSMB3 5.07 5.48

GVICSMB5 4.9 7.7

CRRSMB1 5.51 6.45

CRRSMB7 7.82 11



### Linear least squares regression performed on natural

- log

- transformed data using Microsoft

Excel:

SUMMARY OUTPUT

muscle to WB Regression Statistics
Multiple R 0.908382 R Square 0.8251578 Adjusted R Square 0.8242229

Standard Error 0.2307081

Observations 189

ANOVA

df SS MS F Significance

F
Regression 1 46.97407909 46.97408 882.5361 9.86504E

- 73

Residual 187 9.953306565 0.053226

Total 188 56.92738565

Coefficients Standard

Error t Stat P

- value Lower 95% Upper

95%

Intercept 0.1003801 0.055508758 1.808365 0.072156 - 0.009123739 0.209884

X Variable 1 0.8326865 0.028029496 29.70751 9.87E

- 73 0.77739187 0.887981

### 40

Figure 13

Selenium in muscle and whole body in white sucker caught in the Gunnison and Colorado Rivers

and associated tributaries. Data source: Osmundson & Skorupa ( 201 1 ) . The white sucker data are the same as in Figure 12, but here separated by gender and reproductive condition of females. For comparison, the white sturgeon data and regression from Figure 5 are also shown (orange color).

### 41 

## Appendix II

It has been suggested that homeostatic regulation of selenium in food chains can be discerned in bioaccumulation across several steps of trophic transfer (Brix et al. 2005), requiring that at least one trophic transfer exhibit a “hockey stick” function. I f such homeostatic regulation occurs, it would invalidate the exponential model (straight line on a log

- log graph) used in Figures 6 and 10. Below are five examples of the best species

- specific and location

- specific bioaccumulation relationships currently available. All these examples show field data from Beckon et al. (2010), incorporating estimates of lag time; all are well characterized by exponential models, and exhibit no evidence of homeostatic regulation at any link in the food chain. These resul ts are consistent with the model used in Figures 6 and 10.



# 0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in water (μg/L)



# Largemouth bass

### 42



# 0.1 

# 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in water (μg/L)



# Fathead minnow

0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in water (μg/L)



# Red shiner

### 43



# 0.1 1 10 

# 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in water (μg/L)



# Waterboatman

0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in water (μg/L)



# Siberian freshwater shrimp

### 44

The following four graphs show the best available data from laboratory studies of trophic transfer of selenium across a single link in the food chain. All support the conclusion that trophic transfer of selenium does not involve homeostatic regulation but is well represented by exponential functions (straight lines on log

- log graphs), consistent with the model used in Figures 6 and 10.



# 0.1

1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



# Selenium in diet ( μg/g dry wt)



# White sturgeon
Tashjian et al. 2006



# 0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



## Selenium in diet ( μg/g dry wt)



# Bluegill

Cleveland et al. 1993

### 45 

# 0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



## Selenium in diet ( μg/g dry wt) 

# Fathead minnow

Ogle and Knight 1989



# 0.1 1 10 100

0.1 1 10 100 Selenium in tissue (μg/g whole body dry wt)



## Selenium in diet ( μg/g dry wt) 

# Daphnia magna

Besser et al. 1993

### 46

The following four graphs show the best available data on trophic transfer functions for metals, both essential (copper and zinc) and nonessential (cadmium). These data suggest that the above

conclusion for selenium is generalizable to trophic transfer of metals: there is no evidence of homeostatic regulation of these elements anywhere in the food chain; trophic transfer functions are well represented by exponential models.



# 1 10 100 1000

10 100 1000 10000 Copper in tissue (μg/g whole body dry wt)



# Copper in diet (μg/g dry wt)



# Rainbow trout

Mount et al. 1994



# 10 100 1000 10000

1 10 100 1000 Zn in tissue (μg/g dry wt)



# Zn in water nominal (μg/L)

Algae
Chlamydomonas reinhardtii 

## Guan & Wang. 2004

### 47 

# 0.01 0.1 1 10 100

0.01 0.1 1 10 Cd in tissue (μg/g whole body dry wt)



# Cd in diet (μg/g dry wt)

Mayfly Centroptilum triangulifer

Xie et al. 2009

Aug Nov Jan



# 0.01 0.1 1 10

0.1 1 10 100 1000 Cd in tissue (μg/g whole body dry wt)



# Cd in water (ng/L)

Periphyton

Xie et al. 2009

Aug

Nov
Jan

### 48

References (Appendix II only )

Beckon WN, Gordus A, Eacock MCS. 2010. Biological effects. Ch. 7 in Grassland Bypass Project.

Besser JM, Canfield TJ, La Point TW. 1993. Bioaccumulation of organic and inorganic selenium in a laboratory food chain. Environ Toxicol Chem 12:57

– 72.

Brix KV, Toll JE, Tear LM,. DeForest DK, Adams WJ. 2005. Setting site

- specific water quality standards by using tissue residue thresholds and bioaccumulation data. Part 2. Calculating

site

- specific selenium water quality standards for protecting fish and birds. Environ

Toxicol Chem 24:231

– 237.

Cleveland L, Little EE, Buckler DR, Weidmeyer RH. 1993. Toxicity and bioaccumulation of waterborne and dietary selenium in juvenile bluegill ( Lepomis macrochirus ). Aquatic Toxicology 27:265

- 280.

Guan R, Wang WX. 2004. Dietary assimilation and elimination of Cd, Se, and Zn by Daphnia

magna at different metal concentrations. Environ Toxicol Chem 23:2689

– 2698.

Kleckner, A.E., Stewart, A.R., Elrick, K., and Luoma, S.N., 2010, Selenium and stable is otopes of carbon and nitrogen in the benthic clam Corbula amurensis from Northern San Francisco Bay, California: May 1995

– February 2010: U.S. Geological Survey Open

- File Report 2010

- 1252, 34 p.

Mount DR, Barth AK, Garrison TD, Barten KA, Hockett JR. 19 94. Dietary and waterborne exposure of rainbow trout ( Oncorhynchus mykiss ) to copper, cadmium, lead and zinc using a live diet. Environ Toxicol Chem 13(12): 2031

- 2041.

Ogle RS, Knight AW. 1989. Effects of elevated foodborne selenium on growth and repro duction of the fathead minnow ( Pimephales promelas ). Arch Environ Contam Toxicol 18:795

– 803.

Tashjian DH, Teh SJ, Sogomonyan A, Hung SSO. 2006. Bioaccumulation and chronic toxicity of dietary L

- selenomethionine in juvenile white sturgeon ( Acipenser tran smontanus ). Aquatic Toxicology 79:401

- 409.

Xie L, Funk DH, Buchwalter DB. 2009. Trophic transfer of Cd from natural periphyton to the grazing mayfly Centroptilum triangulifer in a life cycle test.