External Peer Review of the

Evaluation of the Toxicity of Selenium to

White and Green Sturgeon

Contract No.: EP-C-07-059

Work Assignment 4-18

Submitted to:

Dave Guiliano

U.S. Environmental Protection Agency, Region 9

75 Hawthorne Street, WTR-2

San Francisco, CA 94105

Submitted by:

Eastern Research Group, Inc.

110 Hartwell Avenue

Lexington, MA 02421-3136

September 21, 2012

Printed on Recycled Paper
i

Contents

Contents...............................................................................................................................................................i

Summary.............................................................................................................................................................1

Introduction ...................................................................................................................................................3

Summary of Reviewer Responses ................................................................................................................3

Availability and Sufficiency of Data.......................................................................................................3

Appropriateness of the Process/Steps of Analysis ..................................................................................5

Reasonableness/Supportability of Assumptions and Conclusions of Analysis.......................................5

Availability of Alternative Methods........................................................................................................5

Key Recommendations..................................................................................................................................6

Additional References...................................................................................................................................6

Responses to Charge Questions........................................................................................................................7

Introductory Comments ...............................................................................................................................9

Availability and Sufficiency of Data ..........................................................................................................10

Appropriateness of the Process/Steps of Analysis ....................................................................................16

Reasonableness/Supportability of Assumptions and Conclusions of Analysis.......................................18

Availability of Alternative Methods...........................................................................................................19

Additional Reviewer Comments.....................................................................................................................21

Appendix A: Individual Reviewer Comments...............................................................................................27

Comments Submitted by David Janz, Ph.D. .........................................................................................29

Comments Submitted by Vince P. Palace, Ph.D. ..................................................................................39

Comments Submitted by Daniel Schlenk, Ph.D....................................................................................47
1

Summary

Summary

3

Introduction

Overall, the reviewers generally agreed with the approach used to derive selenium benchmarks for white and 

green sturgeon. However, they all thought the data were limited—causing much uncertainty—and had issues 

with the microinjection studies. 

Summary of Reviewer Responses

Availability and Sufficiency of Data

1. Are the Linville (2006), Linares-Casenave et al. (2010), Silvestre et al. (2010) and Tashjian et al.

(2006) studies the best available studies that you are aware of, for determining selenium 

benchmark values for mortality and/or abnormalities affecting survival, for green and white 

sturgeon (Acipenser medirostris and Acipenser transmontanus)? Please explain why or why not, and 

if not, identify any other studies and why and how their data would be useful.

None of the reviewers knew of any additional research specific to selenium exposures in white or green 

sturgeon. One reviewer commented that the studies are the most appropriate data sets that are currently 

available on which to model effects in white and green sturgeon. Another reviewer noted that Linville (2006) 

appears to have adequate data for white sturgeon. One reviewer noted that species sensitivity distributions 

that include up to 12 fish species have recently been developed (DeForest et al. 2011; Janz et al. 2010).

These studies indicated a narrow range of benchmarks (EC10s) for developmental toxicity among five fish 

families.

One reviewer replied that the maternal transfer experiment in the Linville (2006) study was the most 

physiologically, ecologically, and toxicologically relevant among the literature. He suggested that the 

derivation of benchmarks rely more heavily on the maternal transfer data from Linville (2006). He supported 

the EC10 of 15.8 μg/g for larval abnormalities in white sturgeon eggs (derived from maternal transfer of 

selenium following dietary exposure of adult female white sturgeon; Linville 2006). He felt the egg EC10 

values overestimate the toxicity. He also noted that the egg EC10 values based on larval mortality in white 

and green sturgeon and larval abnormalities in green sturgeon and are not scientifically sound due to their 

limited data. 

Two reviewers commented on the inherent uncertainty with using data to extrapolate from one species to 

another. One noted that different fish species can have significantly different tissue-to-tissue relationships for 

selenium. He supported the use of regressions established specifically for white sturgeon, but cautioned the 

extrapolation of these regressions to even the closely related green sturgeon. Given that data for the 

threatened species, green sturgeon, are not available for selenium concentrations in diet, muscle, and whole 

body, the reviewer thought that the approach to estimate parameters based on regressions from the more 

readily available data for white sturgeon in conjunction with a correction factor is warranted and valid. 

However, said that the inherent uncertainty should be noted.
Summary

4

2. All three reviewers commented that the data in some of the underlying studies were very limited, 

causing much uncertainty in the derived benchmarks. One reviewer pointed out that the available 

studies for green sturgeon are more like “single experiments without replication.” He said he 

expected to see a more probabilistic species sensitivity distribution method that incorporates 

estimates of uncertainties, given the limited data and uncertainties. Two reviewers pointed out that 

some of the studies were unpublished and lacked external peer review. The underlying studies 

cited above use two different methods to expose sturgeon to selenium: micro-injection of Lselenomethionine into yolk sacs and a diet of selenized yeast to gravid females. Are the two 

methods of exposure and the resulting data comparable? Please explain why or why not. Please 

comment on the Report's use of data from two different methods of exposure.

All three reviewers mentioned that microinjection of L-selenomethionine into yolk sacs is not comparable to 

a diet of selenized yeast to gravid females (i.e., maternal transfer). The toxicokinetics are very different, 

resulting in a more gradual exposure during maternal transfer and a greater sensitivity/worst-case scenario

during microinjection. Two of the reviewers noted that the dietary exposure route is far more relevant than 

microinjection. Neither thought the fact that sturgeon do not feed on selenized yeast presented a problem 

because they consume prey that convert selenium to the complex organoselenides that are present in the 

yeast and responsible for toxicity. Exposure to pure selenomethionine during microinjection may exaggerate 

toxicity (overestimate exposure) because maternal transfer most likely involves exposure to less toxic 

organoselenium species. 

All three reviewers concluded that there is too much uncertainty in the Silvestre et al. (2010) and LinaresCasenave et al. (2010) studies to be useful in deriving benchmarks. The derivation of toxicity data for green 

sturgeon is based on microinjection of progeny from a single female. One of the reviewers expressed concern

that the ratios of toxicity for green sturgeon versus white sturgeon are based on this single data point and that 

this ratio is then used to derive several other benchmark values for green sturgeon.

One reviewer stated that microinjection of L- selenomethionine into developing sturgeon embryos is an 

efficient method of evaluating the effects of this form of selenium to sturgeon larvae. However, without more 

extensive dosing strategies than those conducted in the Silvestre et al. (2010) and Linares-Casenave et al. 

(2010) studies, it is not possible to carry out extrapolations with any degree of certainty.

Further, one reviewer said that there is uncertainty in extrapolating benchmark effect levels from one dataset 

(microinjection) to another (maternal transfer). Mortality was most evident among microinjected white 

sturgeon larvae and deformities were the most prevalent among larvae exposed via maternal transfer. This

suggests the two exposure routes are not physiologically comparable, as evidenced by the higher EC05 and 

EC10 values from the maternal transfer studies relative to the microinjection studies in white sturgeon.

One reviewer had specific concerns about the timing of the microinjection studies. Because the 

selenomethionine was injected into recently hatched larvae, a critical window of exposure between 

fertilization and hatching was missed. This window typically results in developmental abnormalities, which 

may explain why larval mortality was the most sensitive endpoint in these studies. Larval deformities are 

consistently the most sensitive endpoint in the majority of field and laboratory studies of fish exposed to 

selenium via maternal transfer.

One reviewer was critical of the use of “peak incidence of abnormalities” as an endpoint because effects 

were noted to be transient. Classic larval deformities are irreversible. This adds further conservatism into the 

benchmark estimates.

One reviewer noted that the strength of the regression was not provided in Figures 7 through 9. He suspects 

that relatively weak regressions exist due to the small range of selenium concentrations in the limited 

datasets, which seriously limits the strength of the overall model. 
Summary

5

Appropriateness of the Process/Steps of Analysis

3. Is each step in the analysis reasonable and appropriate, and sufficiently explained? For each step, 

briefly explain why or why not. Identify any other steps that you believe should be and/or could be

included to enhance the analysis.

The reviewers felt that the steps in the analysis were reasonable, however, indicated that parts of the analysis 

were limited by insufficient data. One reviewer commented that the data from Silvestre et al. (2010) and 

Linares-Casenave et al. (2010) appear to be insufficient to adequately describe a dose-response relationship. 

However, he concluded that the model should be stable, and wondered whether other published studies could 

be cited to support the dose-response relationships. He also thought that data were sufficient to derive 

relationships among tissues and diets to accurately predict selenium concentrations in the absence of specific 

data to that tissue or diet. Another reviewer agreed with the report’s interpretation that both mortality and

deformity indices are relevant endpoints and said the discussion of this subject is valid and ecologically 

defensible. He supported the fit evaluation and comparability analysis (Tashjian et al. 2006) between adult 

and juvenile fish. The third reviewer stated that the overall concept seems straightforward and can be used

eventually, however, is currently flawed by the lack of data points in critical areas of the regression and 

subsequent extrapolations (specifically in Figures 2, 4, 6, 7, 8, 9, 10, and 11).

Reasonableness/Supportability of Assumptions and Conclusions of Analysis

4. Given the use of the underlying studies cited in the Report, are the assumptions and conclusions in 

each step of the analysis reasonable and supported by the data? Briefly explain why or why not, 

and if not, why not and how it might be changed to enhance the analysis.

One reviewer thought that relying on the microinjection studies resulted in overly conservative estimation of 

benchmarks. He said the maternal transfer experiment from Linville (2006) represents the most 

toxicologically relevant data set to focus this analysis on. The second reviewer noted that several of the 

uncertainties he pointed out are acknowledged by the author, as well as the fact that these are the best data 

available. He commented that the exponential regressions for trophic transfer of selenium from diet to 

maternal tissues and then to larvae from the female fish are appropriate for interpreting the datasets. Further, 

he supports the use of the EC10s because they are more ecologically relevant for protecting populations from 

impacts. The third reviewer concluded that due to the lack of data for extrapolation between tissue, larvae, 

and effects, a larger dataset is needed to conduct the extrapolations.

Availability of Alternative Methods

5. Would you recommend alternative methods or analyses to estimate benchmarks for mortality or 

abnormalities affecting survival, given the available data on green and white sturgeon? If so,

explain what they are and why they might be more appropriate.

Two reviewers noted that the authors made the most use of the best available data, although limited. One 

stated that the report provided a thorough analysis. He did not have any suggestions for another approach, but

suggested focusing more on the maternal transfer study (Linville 2006). The other said the methods 

employed for the analysis in the document are appropriate. He noted that the data limitations for the two 

sturgeon species contribute great uncertainty in the application of the model and the derived benchmark 

toxicity threshold values. The third reviewer suggested that a probabilistic approach using data from other 

species would be a better method given the current data restrictions because it would provide a better 

estimate of uncertainty. 
Summary

6

Key Recommendations

 To support the statement that white and green sturgeon are among the most sensitive species to 

adverse effects of selenium, evaluate age distribution data to determine whether there is impaired 

recruitment of the populations in the San Francisco Bay area.

 Rely more heavily on the maternal transfer data from the Linville (2006) study for the derivation of 

benchmarks.

 Modify the discussion about the homeostatic regulation of selenium to include information that

normal regulatory mechanisms may decrease in importance at higher exposures.

 The entire modeled dataset needs more prominent disclosure. Specifically, data to compare the 

model fit against adult females, or males, of reproductive age from other species should be presented 

in Figure 12 or in the appendix for that figure.

 The conclusions based on the model should recognize the serious limitations and uncertainties in the 

existing dataset.

 The limitations of the green sturgeon microinjection studies should be more prominently noted. 

 Include a recommendation for the most urgently needed data that would strengthen the predictive 

capacity of the model.

 Make every attempt to obtain the Kaufmann et al. (2008) data.

 Include selenomethionine embryotoxicity data from other species to add to model.

Additional References

 DeForest, D.K., G. Gilron, S.A. Armstrong and E.L. Robertson. 2011. Species sensitivity 

distribution evaluation for selenium in fish eggs: Considerations for development of a Canadian 

tissue-based guideline. Integr. Environ. Assess. Manag. 7, 1-7.

 GEI Consultants. 2008. Maternal Transfer of Selenium in Fathead Minnows, with Modeling of 

Ovary Tissue-to-Whole Body Concentrations. Project Report # 062790 (PDF attached to this report).

 Holm J, VP Palace, P. Siwik, G Sterling, RE Evans, CL Baron, J Werner, K Wautier. 2005. 

Developmental effects of bioaccumulated selenium in eggs and larvae of two salmonid species. 

Environ. Toxicol. Chem. 24:2373-2381.

 Janz, D.M., D.K. DeForest, M.L. Brooks, P.M. Chapman, G. Gilron, D. Hoff, W.D. Hopkins, D.O. 

McIntyre, C.A. Mebane, V.P. Palace, J.P. Skorupa and M. Wayland. 2010. Selenium toxicity to 

aquatic organisms. Pp. 141-231 In: P.M. Chapman, W.J. Adams, M.L. Brooks, C.G. Delos, S.N. 

Luoma, W.A. Maher, H.M. Ohlendorf, T.S. Presser and D.P. Shaw (Eds.) Ecological Assessment of 

Selenium in the Aquatic Environment. CRC Press, Boca Raton, FL.

 Lemly AD. 1997. A teratogenic deformity index for evaluating impacts of selenium on fish 

populations. Ecotoxicol Environ Saf 37:259–266.

 North American Metals Council. 2008. Selenium Tissue Thresholds. Report compiled by GEI 

Consultants, Golder Associates, Parametrix and the University of Saskatchewan. (PDF attached to 

this report).
7

Responses to Charge Questions

Responses to Charge Questions

9

Introductory Comments

Reviewer Comments

Janz Preamble: 

I am a Professor of Veterinary Biomedical Sciences. I hold a PhD in Pharmacology 

and Toxicology, a MSc in Environmental Resource Studies and a BSc in Ecology. My 

main area of research is aquatic ecotoxicology, with a focus on developmental and 

reproductive toxicities in fish. I have conducted field and laboratory research with over 

20 species of fish over the past 25 years. I have published 82 peer-reviewed scientific 

articles, including 22 on the aquatic toxicology of selenium in fish. My work in this 

area has included field studies investigating ecosystem-level selenium bioaccumulation 

and speciation, and laboratory and field studies determining developmental and 

reproductive toxicities in fishes. These latter studies have included both maternal 

transfer and embryo microinjection of selenomethionine into newly fertilized fish 

embryos. Overall, I feel very well qualified to provide this peer review on the EPA 

manuscript. My goal from the outset is to provide a balanced review based on the 

principles of sound science. In addition, I should state that I have no conflict of interest 

in any way related to this review.

I have carefully read the Beckon (2012) manuscript and the supporting material 

provided. I will say up front that I feel the derived benchmarks are based on a very 

limited data set, much of which remains unpublished, that likely cause significant 

uncertainty in the benchmarks. I also believe the Beckon (2012) manuscript is biased 

towards creating benchmarks that may be overly conservative. In my responses below 

to the charge questions, I will expand on these general comments. I also include 

editorial comments following the responses to charge questions.

Palace I have completed my review of the draft document “Evaluation of the toxicity of 

selenium to white and green sturgeon” by Dr. William N. Beckon of the U.S. Fish and 

Wildlife Service. In the draft document, Dr. Beckon has synthesized the available

experimental evidence pertaining to selenium bioaccumulation, trophic transfer and 

tissue distribution in order to derive benchmark effects concentrations for various 

tissues and larvae of white and green sturgeon. As part of my assessment I have also 

critically reviewed background references which Dr. Beckon has cited and which are 

critical to the analysis provided in the draft document. Specifically, these include 

Linville 2006, Linares-Casenave et al. 2010, Tashjian et al. 2006 and Silvestre et al. 

2010. I am also sure that you are aware of my familiarity with the selenium literature in 

general because of my own research involvement with this specific issue for over 10 

years. With respect to the latter, my review has also considered methodologies and 

approaches used in similar studies with other species in order to fully assess the 

modelled studies in the draft document. I will address each of the Charge Questions in 

your Technical Charge to External Peer Reviewers identified in the Work Assignment 

4-18 of August 2012 in order of their appearance.
Responses to Charge Questions

10

Availability and Sufficiency of Data

1. Are the Linville (2006), Linares-Casenave et al. (2010), Silvestre et al. (2010) and Tashjian et 

al. (2006) studies the best available studies that you are aware of, for determining selenium 

benchmark values for mortality and/or abnormalities affecting survival, for green and white 

sturgeon (Acipenser medirostris and Acipenser transmontanus)? Please explain why or why 

not, and if not, identify any other studies and why and how their data would be useful.

Reviewer Comments

Janz I am extremely familiar with the literature associated with the aquatic ecotoxicology of 

Se in fish and am not aware of any other pertinent studies specifically in white 

sturgeon or green sturgeon that have attempted to develop such benchmarks. 

However, species sensitivity distributions (SSDs) that include up to 12 fish species 

have recently been developed (Janz et al. 2010; DeForest et al. 2011). These SSDs are 

based on the most toxicologically relevant tissue (egg or ovary), are based on both 

embryo-larval mortality and deformities, and indicate that there is a remarkably narrow 

range of benchmarks (EC10s) for developmental toxicity among five fish families, 

with the lowest EC10 values falling between 18-20 μg Se/g egg or ovary. Thus, in my 

opinion the EC10 values for egg Se presented in the Summary Table on page 2 of the 

manuscript are very likely to overestimate the toxicity to both white and green 

sturgeon. The one exception is the EC10 of 15.8 μg/g shown for white sturgeon eggs 

using the effect endpoint of larval abnormalities. In my opinion this EC10, derived 

from maternal transfer of Se following dietary exposure of adult female white sturgeon 

(Linville 2006) is the most physiologically, ecologically and toxicologically relevant 

experiment conducted among the supporting literature for both sturgeon species. I will 

discuss this further in the following charge question. 

Overall, given the weight of evidence, I feel that the egg EC10 values in the Summary 

Table based on larval mortality in white and green sturgeon (6.83 and 3.70 μg/g), and 

on larval abnormalities in green sturgeon (6.59 μg/g, which is derived from relative 

sensitivities to microinjected selenomethionine), are not scientifically sound due to the 

limited data set and concerns about the ecological, physiological and toxicological 

relevance of selenomethionine microinjection (discussed in the following charge 

question). 

On the other hand, white and green sturgeon may very well be “among the most 

sensitive of fish to adverse effects of selenium” (Abstract), but further research would 

be required to substantiate this statement. In contrast, as stated in Tashjian et al.

(2006), “Results of this study suggest that the white sturgeon is relatively less sensitive 

to Se toxicity than other fish species.” It would seem to me that given the life history 

and ecology of sturgeon in the SF Bay area, and the relatively high Se levels in their 

prey, that if they are indeed extremely sensitive they would have been extirpated 

decades ago. Have any population ecology studies been conducted in either of these 

species to build the weight of evidence? Age distribution data would be very useful to 

determine if there is impaired recruitment into these populations.

References cited above, recommended for inclusion in revised manuscript:

DeForest, D.K., G. Gilron, S.A. Armstrong and E.L. Robertson. 2011. Species 
Responses to Charge Questions

11

sensitivity distribution evaluation for selenium in fish eggs: Considerations for 

development of a Canadian tissue-based guideline. Integr. Environ. Assess. 

Manag. 7, 1-7.

Janz, D.M., D.K. DeForest, M.L. Brooks, P.M. Chapman, G. Gilron, D. Hoff, W.D. 

Hopkins, D.O. McIntyre, C.A. Mebane, V.P. Palace, J.P. Skorupa and M. 

Wayland. 2010. Selenium toxicity to aquatic organisms. Pp. 141-231 In: P.M. 

Chapman, W.J. Adams, M.L. Brooks, C.G. Delos, S.N. Luoma, W.A. Maher, 

H.M. Ohlendorf, T.S. Presser and D.P. Shaw (Eds.) Ecological Assessment of 

Selenium in the Aquatic Environment. CRC Press, Boca Raton, FL.

Palace In my opinion, while Dr. Beckon’s model is based on the best available data, that data 

is limited enough that there is serious uncertainty in the model. The studies noted in the 

draft document and in your charge questions are the most appropriate data sets that are 

currently available on which to model effects in white and green sturgeon. I am aware 

of no other studies specific to these 2 species and my recent literature searches also 

yielded no new information specific to these fishes. 

There are certainly more complete datasets to describe trophic transfer and relative 

tissue concentrations from other species of fish. While it could be argued that the more 

complete datasets from other fish species could be used to derive tissue to tissue 

correction factors for application to the sturgeon species in the draft document, in my 

opinion this would be inappropriate. My rationale for this is that different fish species 

can have significantly different tissue-to-tissue relationships for Se (North American 

Metals Council 2008). This makes it imperative that known relationships for a given 

species be used to accurately describe Se concentrations in one tissue using another. In 

fact, even related species (brook trout and rainbow trout) occupying similar ecological 

niches can have significant variability in Se concentrations from one tissue to another 

(Holm et al. 2005). For this reason, I support the use of regressions established 

specifically for white sturgeon but caution the extrapolation of these regressions to 

even the closely related green sturgeon. Caution is especially warranted given that the 

draft document discriminates the ecological niches of white sturgeon (potential year 

round residents of the San Francisco Bay area from the green sturgeon (more marineoriented)). Fish from freshwater and marine environments can have vastly different Se 

kinetics owing to different availability and interactions of the element with sulphur and 

phosphorous, as well with other modifying elements. 

Another area of uncertainty in the model arises from the fact that data for the 

threatened species, green sturgeon, are not available for Se concentrations in diet, 

muscle and whole body. However, these parameters are estimated based on regressions 

from the more readily available data for white sturgeon in conjunction with a 

correction factor for relative toxicity of green sturgeon to white sturgeon that is based 

on microinjection studies using larval fish of both species. For the purposes of 

modelling potential toxicity thresholds, this approach is warranted and valid, but it 

must be noted that there is serious inherent uncertainty in extrapolating relationships 

between tissues of different species. The rationale for this cautionary note is discussed 

in additional detail below. 

Schlenk While each of the studies listed have explored the effects of selenium on green and/or 

white sturgeon, only two (Sivestre et al. (2010) and Tashjian et al. (2006) have 

undergone peer review outside of the University of CA, Davis. Consequently, the lack 
Responses to Charge Questions

12

of external peer review lends some uncertainty to the data. In fact, with essentially an 

N of 1 for the green sturgeon microinjection studies and only 3 data points on the 

concentration-response curve, it is interesting that the Sivestre et al. 2010 study was 

not rejected. In contrast, the thesis of Linville (2006) appears to have adequate data for 

white sturgeon and the 3 committee members represent excellent expertise in the area 

of metal toxicology in fishes. However, it is unfortunate that the thesis has not been 

published and one wonders what issues prevent the work from being published. Thus, 

“studies” for green sturgeon were really more like single experiments without 

replication. And while the data may be useful for comparative purposes, the use of the 

data for modeling exercises to extrapolate from one species to another should be 

considered extremely uncertain. 

Given these uncertainties, one would have expected a more probabilistic species 

sensitivity distribution method that incorporates estimates of uncertainty, rather than 

using single projeny experiments with limited data points for extrapolation.

2. The underlying studies cited above use two different methods to expose sturgeon to selenium: 

micro-injection of L-selenomethionine into yolk sacs and a diet of selenized yeast to gravid 

females. Are the two methods of exposure and the resulting data comparable? Please explain 

why or why not. Please comment on the Report's use of data from two different methods of 

exposure.

Reviewer Comments

Janz In my opinion, injection of selenomethionine (SeMet) into newly hatched larvae is not 

entirely comparable to maternal transfer of Se to eggs by adult females, for the 

following reasons. First, as stated on page 21 (top paragraph) of the manuscript, the 

toxicokinetics of a bolus dose of free L-SeMet will likely be very different than that of 

SeMet incorporated into yolk proteins; the latter would result in more gradual exposure 

during yolk assimilation by the yolksac larvae. Indeed, this is reflected in the greater 

sensitivity of larvae injected with SeMet compared to larvae obtaining their Se dose via 

maternal transfer. (In our preliminary studies injecting SeMet into newly fertilized 

zebrafish embryos (2 hours post-fertilization), we observe similar greater sensitivity 

compared to maternal transfer studies in that species with SeMet-spiked diets). 

Second, maternal transfer of Se will likely involve exposure to organoselenium and 

selenide species other than SeMet, which may be less toxic since SeMet is believed to 

be biotransformed to toxic metabolites that are responsible for developmental toxicities 

(see Janz et al. 2010 and references therein). Thus, exposure to pure SeMet may 

exaggerate toxicity due to the maternal transfer of other, less toxic organoselenium 

species. 

Third, I have some major issues with the microinjection studies described in the 

supporting material. In Linville (2006), Linares-Casanave et al. (2010) and Silvestre et 

al. (2010) SeMet was injected into recently hatched larvae, thus missing a critical 

window of exposure between fertilization and hatch, where developmental 

abnormalities (terata) would be initiated. Thus, it is perhaps not surprising that the 

most sensitive effect observed in these studies is larval mortality rather than terata. It is 

important to note that in the majority of field and laboratory studies of fish exposed to 

selenium via maternal transfer, larval deformities are consistently the most sensitive 

endpoint. Why are sturgeons more like birds, where embryo mortality is a more 
Responses to Charge Questions

13

sensitive endpoint (see page 6, 1st paragraph)? I believe the answer is related to the 

timing of exposure in these microinjection experiments.

The author argues in defense of the microinjection studies that sturgeon do not feed on 

selenized yeast, as was used to feed adult females in Linville (2006). This is true, but 

the dietary exposure route is far more relevant than microinjection. Selenized yeast, 

where selenium is predominantly in the form of SeMet as occurs in natural prey, was 

spiked into diets and in my opinion represents a relevant exposure design. 

I also have some additional comments regarding the four supporting studies. The 

Linville (2006) PhD thesis was the most important study and derivation of benchmarks 

should rely more heavily on these data, especially the maternal transfer experiment. 

However, Linville (2006) is limited by only having a single elevated Se dose. The 

Silvestre et al. (2010) published paper and Linares-Casanave et al. (2010) poster are 

part of the same study, where larval mortality and abnormalities were assessed after 

yolk microinjection. In these studies, there appears to be a confounding factor 

(temperature), where similar larval abnormalities were observed at 26C in the absence 

of SeMet injection compared to SeMet injection alone. In addition, as stated by Beckon 

(2012), these studies were limited by small sample sizes (n=1 green and n=3 white 

sturgeon). In addition, there appeared to be a limited number of larvae evaluated in 

each treatment group (see my comment below regarding Appendix 1). A major flaw 

was that negative controls (“NI”) were not sham-injected; however there was a group 

injected with methionine that served as a separate control. Mortality in white sturgeon 

never exceeded 20%, even at the “high” selenium dose. Importantly, although green 

sturgeon mortality in the Se treatment appeared to be a sensitive endpoint, there was 

only a slight, and not toxicologically relevant, increase in D45 selenium concentration 

in larvae (3.1 vs 3.8 μg/g at 18C). Another surprising result was that most abnormal 

larvae “straightened their bodies and resumed normal swimming”. From this 

observation, I am critical of the use of “peak incidence of abnormalities” as an 

endpoint, which is argued (page 17 top) to be relevant since such “transient 

abnormalities effectively immobilize developing larvae”. My issue here is that classic 

larval deformities arising from excess selenium exposure (skeletal curvatures, 

craniofacial deformities and fin deformities) are irreversible, not transient, effects. The 

only possible transient abnormality is edema. Thus, I find it highly unusual to use 

“peak incidence of abnormalities” as an endpoint. This will add further conservatism 

into estimates of benchmarks.

Overall, in my opinion there is too much uncertainty in the Silvestre et al. (2010) and 

Linares-Casanave et al. (2010) studies that makes their use in deriving benchmarks 

tenuous at best.

Palace The following discussion concerns the data presentation outlined in the Microinjection 

results (pages 5-10 of the draft document). In those sections, the translation of Se 

concentrations in larvae to those expected in eggs of white sturgeon is predicated on 

the relationship being the same for these two compartments in eggs/larvae exposed via 

the maternal diet and those exposed via microinjection. This is because the EC05 and 

EC10 values that were calculated from the data in the larval microinjection studies of 

Linville (2006) are then used by the author to derive corresponding egg concentrations 

from experiments in which the eggs and larvae were “loaded” with Se via the diet of 

the mother. The author acknowledges this uncertainty in the model on page 11 under 

the heading White Sturgeon Exposure by Maternal Transfer (additional discussion 
Responses to Charge Questions

14

of the relative sensitivity via microinjection can be found on page 22 of the draft 

document). In that section Dr. Beckon describes how toxicity thresholds are lower for 

white sturgeon exposed via microinjection versus via maternal transfer. The end result 

of this aspect of uncertainty is that application of the larval toxicity benchmarks from 

the microinjection studies would extrapolate to lower (e.g., more protective or 

conservative) estimates of Se concentrations in eggs from the maternal transfer studies. 

Because the benchmark values from the larval microinjection studies are also used to 

extrapolate muscle, whole body and dietary benchmarks, the same uncertainty noted 

above would apply to these measures. That is, they too may be expected to be 

conservative if microinjected Se is more toxic than maternally transferred Se. 

While a regression equation is provided for Figure 7, the strength of the regression is 

not provided. This is a key determinant of the strength of the modeled results. The 

graphic representation of the model is appropriate, but a further examination of the 

data in Table 3-18 of Linville (2006) reveals that the regression is limited to 

identifying percent deformities in just 2 of the 3 treatment groups (one treatment group 

in which larvae contained 11.6 μg/g of Se yielded 0% deformities). I suspect that a 

relatively weak regression of this limited dataset contributes seriously to the 

uncertainty of the modeled results in the draft document. As similar concern should be 

noted for the regression presented for Figure 8, where mortality is expressed against 

selenium in eggs for just the 3 groups of larvae. Furthermore, Figure 9 presents another 

regression where just 3 data points are presented. These data were derived from Tables 

1 and 2 of the Linares-Casenave et al. (2010) poster and from the data presented in 

Silvestre et al. (2010) in Figure 1 and Table 1. Referring to the original data 

presentations in those publications, it is evident that a relatively small range of 

selenium body burdens is available for analysis of the regression (i.e., non injected 

larvae contained 3.2 μg/g Se, L-methionine injected larvae contained 3.5 μg/g Se and 

the Selenomethionine injected larvae contained 7.3 μg/g Se). This small range of 

selenium concentrations would also provide a relatively weak and non-robust 

regression for the purposes of further data extrapolation. The limited regressions noted 

above would be expected to seriously limit the strength of the overall model presented 

in the draft document. 

One of the central assumptions of the modelled data is that the “relative sensitivity of 

green and white sturgeon would be the same for naturally transferred selenium as for 

micro-injected selenium” (page 15, 5th line from the bottom). No data or additional 

discussion is provided to support this statement and, as a result, this introduces another 

source of uncertainty in the model’s application. This is especially true since the ratio 

is used to derive many of the benchmark toxicity values for green sturgeon by applying 

it as a correction factor to the white sturgeon benchmark values. However, mortality 

was most evident among microinjected white sturgeon larvae but deformities were the 

most prevalent among larvae exposed via maternal transfer. This suggests that the 2 

exposure routes are not physiologically comparable and that there is uncertainty in 

extrapolating benchmark effects levels from one data set to another. This concern is 

validated by the higher EC05 and EC10 values from the maternal transfer studies 

relative to the microinjection studies in white sturgeon.

Almost certainly, one of the largest contributors to the uncertainty in the current model 

is the derivation of toxicity data for green sturgeon based on microinjection of progeny 

from a single female in the Linares-Casenave et al. 2010 and Silvestre et al. 2010 

studies (page 20, 2nd paragraph). In my experience, the variability of toxic responses 

within progeny of a single female are less substantial than the variation that can be 
Responses to Charge Questions

15

observed among progeny from different females, even at similar concentrations of Se. 

Regardless of whether this variation results from modifying factors such as interactive 

inorganic elements or nutritional differences, uncertainty in either direction 

(underestimated or overestimated toxicity) is introduced into the model. The fact that 

the ratios of toxicity for green versus white sturgeon are based on essentially this single 

data point from green sturgeon, and that this ratio is then used to derive several other 

benchmark values for green sturgeon is concerning. At the very least, this limitation 

should receive earlier and more prominent attention in delineating the model as it is 

outlined. 

Schlenk Microinjection of L-selenomethionine into developing embryos of sturgeon is an 

efficient method of evaluating the effects of this form of selenium to sturgeon larvae 

and with the appropriate replication and dosing strategies could be used to evaluate 

species sensitivity differences. Unfortunately, based on the methods provided in the 

materials, it would appear that only a single dose was provided to embryos in Silvestre 

et al. (2010) and no mention of dose was provided in Linares-Dasenave et al. (2010). It 

is unclear how the 3 data points in Figure 9 of the draft report were determined. 

Without a more extensive dosing strategy such that EC50 values can be used to reduce 

uncertainty in dose-response assessments, it is not possible to carry out extrapolations 

with any degree of certainty. 

With regard to the dietary seleniforous yeast exposures, this is likely a more accurate 

depiction of exposure to selenium in the field. While selenomethione may be one of the 

more toxic forms of selenium, there are clearly other organic forms (selenocysteine 

etc.) that contribute to toxicodynamic, and toxicokinetic differences as well as the 

resulting toxicology of selenium. It is unclear how each of the varied organoselenides 

works synergistically, additively or antagonistically. As discussed in the draft report, 

transport between hepatic and gonadal tissues via vitellogenin and other lipoproteins is 

a critical step in the process. It is unclear how much and when selenium is released 

from the lipoproteins (if at all) and in what chemical form. Microinjection would 

clearly represent a “worst-case” scenario with regard to exposure and likely 

overestimates exposure to selenium. In addition, without the other organoselenide 

species, the exposure oversimplifies the interactions that occur in embryos. While the 

report indeed states that sturgeon do not feed upon yeast, they do consume other 

organisms that convert selenium to the complex organoselenides that are present in the 

yeast and responsible for toxicity.
Responses to Charge Questions

16

Appropriateness of the Process/Steps of Analysis

3. Is each step in the analysis reasonable and appropriate, and sufficiently explained? For each 

step, briefly explain why or why not. Identify any other steps that you believe should be 

and/or could be included to enhance the analysis.

Reviewer Comments

Janz There are instances where there appear to be insufficient data to adequately describe a 

dose-response relationship, specifically data from the Silvestre et al. (2010) and 

Linares-Casanave et al. (2010) studies (Figures 9 and 11 in Beckon 2012). Attempting 

to describe dose-response relationships from such limited data will ignore the 

“steepness” of the dose-response curve and introduce much uncertainty into these 

estimates. In addition, a similar problem arises with the use of two data points to 

describe the relationship between selenium in maternal diet and larvae for white and 

green sturgeon (Figures 6 and 10, respectively). However, these are the available data 

and I agree that the model should be stable. Are there other published studies that can 

be cited to support these relationships?

On page 18, 3rd paragraph, I somewhat disagree with the author’s opinion that there is 

no homeostatic regulation of selenium. As an essential trace element, regulatory 

mechanisms are in place involving absorption, distribution, biotransformation and 

excretion of selenium, which are well documented in the mammalian literature. 

However, these mechanisms are likely only important at normal physiological 

exposure levels, and may decrease in importance at higher exposures. I suggest 

modifying this discussion to take this into account.

I believe there are sufficient data available to derive relationships among tissues (egg, 

muscle and whole body) and diets to accurately predict selenium concentrations in the 

absence of specific data to that tissue or diet. One note is that clams were reported to 

comprise 41% of white sturgeon diet in the wild (Presser and Luoma 2010), and the 

contribution of other prey items to selenium body/tissue burdens is uncertain, as is the 

presumption that green sturgeon diets would be similar.

Palace Notwithstanding the comments noted above, and in general, Dr. Beckon provides a 

reasonable, step wise analysis of the available data to derive benchmark values in 

tissues and whole bodies of both sturgeon species. Specifically, I agree with Dr. 

Beckon’s interpretation that both mortality, or deformity indices are relevant endpoints 

and in my opinion, the discussion of this subject that appears at the top of page 17 is 

valid and ecologically defensible. 

Whole body benchmark values are important because of their use in regulatory 

capacities and for comparisons with existing datasets from other fish species. 

Conversion of muscle to whole body Se concentrations for adult white and green 

sturgeon in Dr. Beckon’s model are based on regressions obtained from Tashjian et al.

(2006). That study exposed juvenile white sturgeon (initial weight ca. 30g) to diets 

amended with selenomethionine for 8 weeks. There are valid reasons to expect that 

juveniles might differ in their muscle to whole body selenium concentrations relative 

to adults. For example, oogenesis can provide a significant depuration mechanism for 

whole body Se in female fish (GEI Consultants 2008, Reference PDF attached to this 

report), especially among those that produce eggs with large yolk reserves. While this, 
Responses to Charge Questions

17

and other physiological differences between adults and juveniles, might be expected to 

introduce uncertainty in the current model, Dr. Beckon has evaluated the fit of the 

juvenile data relative to other empirical data from other fish species (Figure 12) and 

supported the comparability of the data from Tashjian et al. (2006) among these other 

datasets. However, the suggestion that the fit of the current model in comparison to the 

other models with regard to gender and reproductive condition of the fish, requires 

additional substantiation as these data are not specifically presented in Figure 12 or the 

complete dataset supplied in the Appendix for that figure. Most notably, no 

comparisons of the model fit against adult females, or males, of reproductive age from 

other species is possible as the data are presented. Where available, this specific data 

should be presented in either the figure or the Appendix supporting the figure. 

Schlenk The overall concept seems straightforward and the method can eventually be used. 

However, the major flaw is the number of data points used to make the regression and 

subsequent extrapolations. Most plots have essentially data at two ends of the curve 

and a paucity of data in critical areas of the regression (particularly in the middle 

sections of the curve). Such is the case in Figures 2, 4, and especially 6 where the 

entire regression is based on two points! Figures 7 and 8 only have one data point 

above threshold, which suggests the extrapolated points are highly uncertain. Figure 9 

has 3 points (but these are from one spawning event from a single animal). Figure 10 

only has two points. Figure 11 A and B only have one point above threshold. 
Responses to Charge Questions

18

Reasonableness/Supportability of Assumptions and Conclusions of Analysis

4. Given the use of the underlying studies cited in the Report, are the assumptions and

conclusions in each step of the analysis reasonable and supported by the data? Briefly explain 

why or why not, and if not, why not and how it might be changed to enhance the analysis.

Reviewer Comments

Janz Overall, I feel that relying heavily on the SeMet injection experiments has resulted in 

an overly conservative estimation of benchmarks. In my opinion, the maternal transfer 

experiment in chapter 3 of the Linville (2006) thesis, although not without its 

limitations, represents the most toxicologically relevant data set to focus this analysis 

on. 

Palace My comments above note several uncertainties in the model as presented in the draft

document. These uncertainties are generally noted by the author and it is 

acknowledged several times in the document that these are the best data available. 

However, conclusions based on these data should recognize the serious limitations and 

uncertainties in the existing dataset. 

One of the remaining issues which I have not addressed in my comments above 

concerns Dr. Beckon’s use of an exponential model for trophic transfer of Se from diet 

to maternal tissues and then to larvae from the female fish. As Dr. Beckon suggests 

(page 18, 3rd paragraph), the literature contains no support for anything other than an 

exponential model application for trophic transfer of Se from maternal diet to maternal 

tissues and then to larvae (i.e., there is no evidence for homeostatic control of Se 

accumulation and deposition into eggs). As a result, and in my opinion, exponential 

regressions are appropriate for interpreting the datasets. 

I am also supportive of the use of EC10s rather than EC50s to derive sensitivity 

threshold values as the former is more ecologically relevant for protecting populations 

from impacts. Furthermore, the use of this endpoint is supported by modelled 

population level effects in the literature (Lemly 1997). 

Schlenk As discussed in question 3 above, given the lack of data for extrapolation between 

tissue, larvae and effects, a larger data set is necessary (at least 4-5 data points well 

distributed throughout the curve) to conduct the extrapolations. 
Responses to Charge Questions

19

Availability of Alternative Methods

5. Would you recommend alternative methods or analyses to estimate benchmarks for mortality 

or abnormalities affecting survival, given the available data on green and white sturgeon? If 

so, explain what they are and why they might be more appropriate.

Reviewer Comments

Janz The author has made the most use of limited data and provided a thorough analysis in 

the manuscript. I cannot think of any other approaches that could be used, although as 

stated above I would focus the analysis on the most toxicologically and ecologically

relevant experiment (Linville’s maternal transfer study). In my opinion, an EC10 of 15 

μg Se/g egg or ovary dw (Summary Table page 2, from Linville 2006) would be 

protective of sturgeon in SF Bay, and would still result in these species being the most 

sensitive fish species studied to date.

Palace The methods employed for analysis in this draft document are appropriate and based 

on the best available data at this time. In the case of the 2 sturgeon species in this 

report, the data limitations are serious enough to contribute to great uncertainty in the 

application of the model and the derived benchmark toxicity threshold values. 

Recommendations 

a. More prominent notation of the limitations of the green sturgeon microinjection 

studies, which are based on toxicity thresholds in the larvae obtained from just one 

fish. In fact, more prominent disclosure of the entire dataset that is modelled would 

be prescribed. Identification of these limitations would be useful for guiding 

recommendation “b” noted below. 

b. Include recommendation in the report on the most urgently needed data to 

strengthen the predictive capacity of the model. 

c. Given the importance of the data referenced in the studies of Kaufmann et al.

(2008), and the fact that >4 years have passed since the data were first presented, 

every attempt to gain access to them should be taken. It is anticipated that these 

data would strengthen the model derivation considerably.

Schlenk Based on the limited data in the two species of concern, it would appear that a 

probabilistic approach using data from other species would be a better method given 

the current data restrictions. The probabilistic approach will provide a better estimate 

of uncertainty. 

21

Additional Reviewer Comments

Additional Reviewer Comments

23

Additional Comments Submitted by Dr. David Janz

Editorial comments:

The references cited in the manuscript are inadequate. The manuscript must focus the literature cited on 

original published studies, not “grey literature” such as Beckon (2007), Beckon (2010) and Beckon et al.

(2010). In addition, the reference section is inconsistently formatted, as are the citations in the text.

Page 3 last paragraph: “egg toxicity” should be changed to “embryo toxicity”. Eggs are gametes; embryos 

are organisms that exhibit toxicity.

Page 3 last sentence: change to “data on green sturgeon dietary, muscle and whole body selenium 

concentrations are not available”.

Page 8 last sentence: change to “10% risk of larval mortality”.

Page 22: use of the term “margin of safety” is incorrect. The margin of safety is defined as the LD1/EC99, 

and is used in describing drug safety. 

In this same paragraph on page 22, it is argued that use of a safety factor (e.g., 10) “could push a benchmark 

concentration into the zone of deficiency”. This is another example of extreme conservatism inherent in this 

manuscript. Selenium has a narrow range between essentiality and toxicity, especially for oviparous 

vertebrates such as fish, but this range is still reported as between 10-30 (equivalent to the “therapeutic 

index”, another pharmacological term). If a proposed safety factor pushes the benchmark past essentiality 

into deficiency, then clearly there is no biological realism involved.

The final paragraph leaves me hanging; I suggest a final statement that summarizes and concludes the 

manuscript.

Do a spell check, for example “progeny”, “teratogenic” and “sensitivity” are misspelled in several instances.

The Reference section is full of typos and formatting inconsistencies.

In Appendix 1, the data shown in Figures 1 to 11 should include the sample sizes (n).

In Appendix 2, why show data for Cu, Cd and Zn? I suggest focusing on just Se.
Additional Reviewer Comments

24

Additional Comments Submitted by Dr. Vince Palace

References

GEI Consultants. 2008. Maternal Transfer of Selenium in Fathead Minnows, with Modeling of Ovary 

Tissue-to-Whole Body Concentrations. Project Report # 062790 (PDF attached to this report)

Holm J, VP Palace, P. Siwik, G Sterling, RE Evans, CL Baron, J Werner, K Wautier. 2005. Developmental 

effects of bioaccumulated selenium in eggs and larvae of two salmonid species. Environ. Toxicol. Chem. 

24:2373-2381.

Lemly AD. 1997. A teratogenic deformity index for evaluating impacts of selenium on fish populations. 

Ecotoxicol Environ Saf 37:259–266.

North American Metals Council. 2008. Selenium Tissue Thresholds. Report compiled by GEI Consultants, 

Golder Associates, Parametrix and the University of Saskatchewan. (PDF attached to this report).

I would like to express my thanks to you and the rest of the Eastern Research Group team for giving me the 

opportunity to participate in the review of the draft document. Issues related to selenium bioaccumulation 

and toxicity continue to be an active area of research in my program and I find it personally and 

professionally stimulating to be involved in front-line evaluation of datasets such as the one presented by this 

document. I would certainly welcome similar opportunities to work with your team in the future. 

Please feel free to contact me should you have any additional questions about my review. 
Additional Reviewer Comments

25

Additional Comments Submitted by Dr. Daniel Schlenk

Other comments:

One of the significant statements in the Linville thesis is the lack of reproductive impairment in the white 

sturgeon tested even though concentrations were above benchmark values.

Is there really a difference in species sensitivity between the green and white sturgeon? Kaufman et al. 2008 

is not peer reviewed and while there may be a trend toward enhanced sensitivity in green sturgeon, the 

limited dose response data precludes adequate statistical power to make these determinations. 

27

Appendix A: Individual Reviewer Comments

29

Comments Submitted by

David Janz, Ph.D.

David Janz, Ph.D.

31

Peer review of Beckon, W.N. 2012. Evaluation of the Toxicity of Selenium to 

White and Green Sturgeon.

David Janz, Ph.D.

August 21, 2012

Preamble: 

I am a Professor of Veterinary Biomedical Sciences. I hold a PhD in Pharmacology and Toxicology, a 

MSc in Environmental Resource Studies and a BSc in Ecology. My main area of research is aquatic 

ecotoxicology, with a focus on developmental and reproductive toxicities in fish. I have conducted field 

and laboratory research with over 20 species of fish over the past 25 years. I have published 82 peerreviewed scientific articles, including 22 on the aquatic toxicology of selenium in fish. My work in this 

area has included field studies investigating ecosystem-level selenium bioaccumulation and speciation, 

and laboratory and field studies determining developmental and reproductive toxicities in fishes. These 

latter studies have included both maternal transfer and embryo microinjection of selenomethionine into 

newly fertilized fish embryos. Overall, I feel very well qualified to provide this peer review on the EPA 

manuscript. My goal from the outset is to provide a balanced review based on the principles of sound

science. In addition, I should state that I have no conflict of interest in any way related to this review.

I have carefully read the Beckon (2012) manuscript and the supporting material provided. I will say up 

front that I feel the derived benchmarks are based on a very limited data set, much of which remains 

unpublished, that likely cause significant uncertainty in the benchmarks. I also believe the Beckon (2012) 

manuscript is biased towards creating benchmarks that may be overly conservative. In my responses 

below to the charge questions, I will expand on these general comments. I also include editorial 

comments following the responses to charge questions.
David Janz, Ph.D.

32

CHARGE QUESTIONS

Availability and Sufficiency of Data:

1. Are the Linville (2006), Linares-Casenave et al. (2010), Silvestre et al. (2010) and Tashjian et al.

(2006) studies the best available studies that you are aware of, for determining selenium 

benchmark values for mortality and/or abnormalities affecting survival, for green and white 

sturgeon (Acipenser medirostris and Acipenser transmontanus)? Please explain why or why not, 

and if not, identify any other studies and why and how their data would be useful.

Response:

I am extremely familiar with the literature associated with the aquatic ecotoxicology of Se in fish and am 

not aware of any other pertinent studies specifically in white sturgeon or green sturgeon that have 

attempted to develop such benchmarks. 

However, species sensitivity distributions (SSDs) that include up to 12 fish species have recently been 

developed (Janz et al. 2010; DeForest et al. 2011). These SSDs are based on the most toxicologically 

relevant tissue (egg or ovary), are based on both embryo-larval mortality and deformities, and indicate 

that there is a remarkably narrow range of benchmarks (EC10s) for developmental toxicity among five 

fish families, with the lowest EC10 values falling between 18-20 μg Se/g egg or ovary. Thus, in my 

opinion the EC10 values for egg Se presented in the Summary Table on page 2 of the manuscript are 

very likely to overestimate the toxicity to both white and green sturgeon. The one exception is the EC10 

of 15.8 μg/g shown for white sturgeon eggs using the effect endpoint of larval abnormalities. In my 

opinion this EC10, derived from maternal transfer of Se following dietary exposure of adult female white 

sturgeon (Linville 2006) is the most physiologically, ecologically and toxicologically relevant 

experiment conducted among the supporting literature for both sturgeon species. I will discuss this 

further in the following charge question. 

Overall, given the weight of evidence, I feel that the egg EC10 values in the Summary Table based on 

larval mortality in white and green sturgeon (6.83 and 3.70 μg/g), and on larval abnormalities in green 

sturgeon (6.59 μg/g, which is derived from relative sensitivities to microinjected selenomethionine), are 

not scientifically sound due to the limited data set and concerns about the ecological, physiological and 

toxicological relevance of selenomethionine microinjection (discussed in the following charge question). 

On the other hand, white and green sturgeon may very well be “among the most sensitive of fish to 

adverse effects of selenium” (Abstract), but further research would be required to substantiate this 

statement. In contrast, as stated in Tashjian et al. (2006), “Results of this study suggest that the white 
David Janz, Ph.D.

33

sturgeon is relatively less sensitive to Se toxicity than other fish species”. It would seem to me that given 

the life history and ecology of sturgeon in the SF Bay area, and the relatively high Se levels in their prey, 

that if they are indeed extremely sensitive they would have been extirpated decades ago. Have any 

population ecology studies been conducted in either of these species to build the weight of evidence? 

Age distribution data would be very useful to determine if there is impaired recruitment into these 

populations.

References cited above, recommended for inclusion in revised manuscript:

DeForest, D.K., G. Gilron, S.A. Armstrong and E.L. Robertson. 2011. Species sensitivity distribution 

evaluation for selenium in fish eggs: Considerations for development of a Canadian tissue-based 

guideline. Integr. Environ. Assess. Manag. 7, 1-7.

Janz, D.M., D.K. DeForest, M.L. Brooks, P.M. Chapman, G. Gilron, D. Hoff, W.D. Hopkins, D.O. 

McIntyre, C.A. Mebane, V.P. Palace, J.P. Skorupa and M. Wayland. 2010. Selenium toxicity to aquatic 

organisms. Pp. 141-231 In: P.M. Chapman, W.J. Adams, M.L. Brooks, C.G. Delos, S.N. Luoma, W.A. 

Maher, H.M. Ohlendorf, T.S. Presser and D.P. Shaw (Eds.) Ecological Assessment of Selenium in the 

Aquatic Environment. CRC Press, Boca Raton, FL.

2. The underlying studies cited above use two different methods to expose sturgeon to selenium: 

micro-injection of L-selenomethionine into yolk sacs and a diet of selenized yeast to gravid females. 

Are the two methods of exposure and the resulting data comparable? Please explain why or why 

not. Please comment on the Report's use of data from two different methods of exposure.

Response:

In my opinion, injection of selenomethionine (SeMet) into newly hatched larvae is not entirely 

comparable to maternal transfer of Se to eggs by adult females, for the following reasons. First, as stated 

on page 21 (top paragraph) of the manuscript, the toxicokinetics of a bolus dose of free L-SeMet will 

likely be very different than that of SeMet incorporated into yolk proteins; the latter would result in more 

gradual exposure during yolk assimilation by the yolksac larvae. Indeed, this is reflected in the greater 

sensitivity of larvae injected with SeMet compared to larvae obtaining their Se dose via maternal 

transfer. (In our preliminary studies injecting SeMet into newly fertilized zebrafish embryos (2 hours 

post-fertilization), we observe similar greater sensitivity compared to maternal transfer studies in that 

species with SeMet-spiked diets). 
David Janz, Ph.D.

34

Second, maternal transfer of Se will likely involve exposure to organoselenium and selenide species 

other than SeMet, which may be less toxic since SeMet is believed to be biotransformed to toxic 

metabolites that are responsible for developmental toxicities (see Janz et al. 2010 and references therein). 

Thus, exposure to pure SeMet may exaggerate toxicity due to the maternal transfer of other, less toxic 

organoselenium species. 

Third, I have some major issues with the microinjection studies described in the supporting material. In 

Linville (2006), Linares-Casanave et al. (2010) and Silvestre et al. (2010) SeMet was injected into 

recently hatched larvae, thus missing a critical window of exposure between fertilization and hatch, 

where developmental abnormalities (terata) would be initiated. Thus, it is perhaps not surprising that the 

most sensitive effect observed in these studies is larval mortality rather than terata. It is important to note 

that in the majority of field and laboratory studies of fish exposed to selenium via maternal transfer, 

larval deformities are consistently the most sensitive endpoint. Why are sturgeons more like birds, where 

embryo mortality is a more sensitive endpoint (see page 6, 1st paragraph)? I believe the answer is related 

to the timing of exposure in these microinjection experiments.

The author argues in defense of the microinjection studies that sturgeon do not feed on selenized yeast, 

as was used to feed adult females in Linville (2006). This is true, but the dietary exposure route is far 

more relevant than microinjection. Selenized yeast, where selenium is predominantly in the form of 

SeMet as occurs in natural prey, was spiked into diets and in my opinion represents a relevant exposure 

design. 

I also have some additional comments regarding the four supporting studies. The Linville (2006) PhD 

thesis was the most important study and derivation of benchmarks should rely more heavily on these 

data, especially the maternal transfer experiment. However, Linville (2006) is limited by only having a 

single elevated Se dose. The Silvestre et al. (2010) published paper and Linares-Casanave et al. (2010) 

poster are part of the same study, where larval mortality and abnormalities were assessed after yolk 

microinjection. In these studies, there appears to be a confounding factor (temperature), where similar 

larval abnormalities were observed at 26C in the absence of SeMet injection compared to SeMet 

injection alone. In addition, as stated by Beckon (2012), these studies were limited by small sample sizes 

(n=1 green and n=3 white sturgeon). In addition, there appeared to be a limited number of larvae 

evaluated in each treatment group (see my comment below regarding Appendix 1). A major flaw was 

that negative controls (“NI”) were not sham-injected; however there was a group injected with 

methionine that served as a separate control. Mortality in white sturgeon never exceeded 20%, even at 

the “high” selenium dose. Importantly, although green sturgeon mortality in the Se treatment appeared to 
David Janz, Ph.D.

35

be a sensitive endpoint, there was only a slight, and not toxicologically relevant, increase in D45 

selenium concentration in larvae (3.1 vs 3.8 μg/g at 18C). Another surprising result was that most 

abnormal larvae “straightened their bodies and resumed normal swimming”. From this observation, I am 

critical of the use of “peak incidence of abnormalities” as an endpoint, which is argued (page 17 top) to 

be relevant since such “transient abnormalities effectively immobilize developing larvae”. My issue here 

is that classic larval deformities arising from excess selenium exposure (skeletal curvatures, craniofacial 

deformities and fin deformities) are irreversible, not transient, effects. The only possible transient 

abnormality is edema. Thus, I find it highly unusual to use “peak incidence of abnormalities” as an 

endpoint. This will add further conservatism into estimates of benchmarks.

Overall, in my opinion there is too much uncertainty in the Silvestre et al. (2010) and Linares-Casanave 

et al. (2010) studies that makes their use in deriving benchmarks tenuous at best.

Appropriateness of the Process/Steps of Analysis:

3. Is each step in the analysis reasonable and appropriate, and sufficiently explained?

For each step, briefly explain why or why not. Identify any other steps that you believe should be 

and/or could be included to enhance the analysis.

Response:

There are instances where there appear to be insufficient data to adequately describe a dose-response 

relationship, specifically data from the Silvestre et al. (2010) and Linares-Casanave et al. (2010) studies 

(Figures 9 and 11 in Beckon 2012). Attempting to describe dose-response relationships from such 

limited data will ignore the “steepness” of the dose-response curve and introduce much uncertainty into 

these estimates. In addition, a similar problem arises with the use of two data points to describe the 

relationship between selenium in maternal diet and larvae for white and green sturgeon (Figures 6 and 

10, respectively). However, these are the available data and I agree that the model should be stable. Are 

there other published studies that can be cited to support these relationships?

On page 18, 3rd paragraph, I somewhat disagree with the author’s opinion that there is no homeostatic 

regulation of selenium. As an essential trace element, regulatory mechanisms are in place involving 

absorption, distribution, biotransformation and excretion of selenium, which are well documented in the 

mammalian literature. However, these mechanisms are likely only important at normal physiological 

exposure levels, and may decrease in importance at higher exposures. I suggest modifying this discussion 

to take this into account.
David Janz, Ph.D.

36

I believe there are sufficient data available to derive relationships among tissues (egg, muscle and whole 

body) and diets to accurately predict selenium concentrations in the absence of specific data to that tissue 

or diet. One note is that clams were reported to comprise 41% of white sturgeon diet in the wild (Presser 

and Luoma 2010), and the contribution of other prey items to selenium body/tissue burdens is uncertain, 

as is the presumption that green sturgeon diets would be similar.

Reasonableness/Supportability of Assumptions and Conclusions of Analysis:

4. Given the use of the underlying studies cited in the Report, are the assumptions and conclusions in 

each step of the analysis reasonable and supported by the data? Briefly explain why or why not, 

and if not, why not and how it might be changed to enhance the analysis.

Response:

Overall, I feel that relying heavily on the SeMet injection experiments has resulted in an overly 

conservative estimation of benchmarks. In my opinion, the maternal transfer experiment in chapter 3 of 

the Linville (2006) thesis, although not without its limitations, represents the most toxicologically 

relevant data set to focus this analysis on. 

Availability of Alternative Methods:

5. Would you recommend alternative methods or analyses to estimate benchmarks for mortality or 

abnormalities affecting survival, given the available data on green and white sturgeon? If so, 

explain what they are and why they might be more appropriate.

Response:

The author has made the most use of limited data and provided a thorough analysis in the manuscript. I 

cannot think of any other approaches that could be used, although as stated above I would focus the 

analysis on the most toxicologically and ecologically relevant experiment (Linville’s maternal transfer 

study). In my opinion, an EC10 of 15 μg Se/g egg or ovary dw (Summary Table page 2, from Linville 

2006) would be protective of sturgeon in SF Bay, and would still result in these species being the most 

sensitive fish species studied to date.
David Janz, Ph.D.

37

Editorial comments:

The references cited in the manuscript are inadequate. The manuscript must focus the literature cited on 

original published studies, not “grey literature” such as Beckon (2007), Beckon (2010) and Beckon et al. 

(2010). In addition, the reference section is inconsistently formatted, as are the citations in the text.

Page 3 last paragraph: “egg toxicity” should be changed to “embryo toxicity”. Eggs are gametes; embryos 

are organisms that exhibit toxicity.

Page 3 last sentence: change to “data on green sturgeon dietary, muscle and whole body selenium 

concentrations are not available”.

Page 8 last sentence: change to “10% risk of larval mortality”.

Page 22: use of the term “margin of safety” is incorrect. The margin of safety is defined as the LD1/EC99, 

and is used in describing drug safety. 

In this same paragraph on page 22, it is argued that use of a safety factor (e.g., 10) “could push a benchmark 

concentration into the zone of deficiency”. This is another example of extreme conservatism inherent in this 

manuscript. Selenium has a narrow range between essentiality and toxicity, especially for oviparous 

vertebrates such as fish, but this range is still reported as between 10-30 (equivalent to the “therapeutic 

index”, another pharmacological term). If a proposed safety factor pushes the benchmark past essentiality 

into deficiency, then clearly there is no biological realism involved.

The final paragraph leaves me hanging; I suggest a final statement that summarizes and concludes the 

manuscript.

Do a spellcheck, for example “progeny”, “teratogenic” and “sensitivity” are misspelled in several instances.

The Reference section is full of typos and formatting inconsistencies.

In Appendix 1, the data shown in Figures 1 to 11 should include the sample sizes (n).

In Appendix 2, why show data for Cu, Cd and Zn? I suggest focusing on just Se.

39

Comments Submitted by

Vince P. Palace, Ph.D.

Vince P. Palace, Ph.D.

41

I have completed my review of the draft document “Evaluation of the toxicity of selenium to white and green 

sturgeon” by Dr. William N. Beckon of the U.S. Fish and Wildlife Service. In the draft document, Dr. 

Beckon has synthesized the available experimental evidence pertaining to selenium bioaccumulation, trophic 

transfer and tissue distribution in order to derive benchmark effects concentrations for various tissues and 

larvae of white and green sturgeon. As part of my assessment I have also critically reviewed background 

references which Dr. Beckon has cited and which are critical to the analysis provided in the draft document. 

Specifically, these include Linville 2006, Linares-Casenave et al. 2010, Tashjian et al. 2006 and Silvestre et 

al. 2010. I am also sure that you are aware of my familiarity with the selenium literature in general because 

of my own research involvement with this specific issue for over 10 years. With respect to the latter, my 

review has also considered methodologies and approaches used in similar studies with other species in order 

to fully assess the modelled studies in the draft document. I will address each of the Charge Questions in 

your Technical Charge to External Peer Reviewers identified in the Work Assignment 4-18 of August 2012 

in order of their appearance.

Availability and Sufficiency of Data

1. Are the Linville (2006), Linares-Casenave et al. (2010), Silvestre et al. (2010) and Tashjian et al. 

(2006) studies the best available studies.....for determining selenium benchmark values for 

mortality/and or abnormalities affecting green and white sturgeon? 

In my opinion, while Dr. Beckon’s model is based on the best available data, that data is limited enough that 

there is serious uncertainty in the model. The studies noted in the draft document and in your charge 

questions are the most appropriate data sets that are currently available on which to model effects in white 

and green sturgeon. I am aware of no other studies specific to these 2 species and my recent literature 

searches also yielded no new information specific to these fishes. 

There are certainly more complete datasets to describe trophic transfer and relative tissue concentrations 

from other species of fish. While it could be argued that the more complete datasets from other fish species 

could be used to derive tissue to tissue correction factors for application to the sturgeon species in the draft 

document, in my opinion this would be inappropriate. My rationale for this is that different fish species can 

have significantly different tissue-to-tissue relationships for Se (North American Metals Council 2008). This 

makes it imperative that known relationships for a given species be used to accurately describe Se 

concentrations in one tissue using another. In fact, even related species (brook trout and rainbow trout) 

occupying similar ecological niches can have significant variability in Se concentrations from one tissue to 

another (Holm et al. 2005). For this reason, I support the use of regressions established specifically for white 

sturgeon but caution the extrapolation of these regressions to even the closely related green sturgeon. Caution 
Vince P. Palace, Ph.D.

42

is especially warranted given that the draft document discriminates the ecological niches of white sturgeon 

(potential year round residents of the San Francisco Bay area from the green sturgeon (more marineoriented)). Fish from freshwater and marine environments can have vastly different Se kinetics owing to 

different availability and interactions of the element with sulphur and phosphorous, as well with other 

modifying elements. 

Another area of uncertainty in the model arises from the fact that data for the threatened species, green 

sturgeon, are not available for Se concentrations in diet, muscle and whole body. However, these parameters 

are estimated based on regressions from the more readily available data for white sturgeon in conjunction 

with a correction factor for relative toxicity of green sturgeon to white sturgeon that is based on 

microinjection studies using larval fish of both species. For the purposes of modelling potential toxicity 

thresholds, this approach is warranted and valid, but it must be noted that there is serious inherent uncertainty 

in extrapolating relationships between tissues of different species. The rationale for this cautionary note is 

discussed in additional detail below. 

2. Two different methods are used to expose sturgeon to selenium: microinjection of Lselenomethionine into yolk sacs and a diet of selenized yeast to gravid females. Are the two 

different methods of exposure and the resulting data comparable? 

The following discussion concerns the data presentation outlined in the Microinjection results (pages 5-10 of 

the draft document). In those sections, the translation of Se concentrations in larvae to those expected in eggs 

of white sturgeon is predicated on the relationship being the same for these two compartments in eggs/larvae 

exposed via the maternal diet and those exposed via microinjection. This is because the EC05 and EC10 

values that were calculated from the data in the larval microinjection studies of Linville (2006) are then used 

by the author to derive corresponding egg concentrations from experiments in which the eggs and larvae 

were “loaded” with Se via the diet of the mother. The author acknowledges this uncertainty in the model on 

page 11 under the heading White Sturgeon Exposure by Maternal Transfer (additional discussion of the 

relative sensitivity via microinjection can be found on page 22 of the draft document). In that section Dr. 

Beckon describes how toxicity thresholds are lower for white sturgeon exposed via microinjection versus via 

maternal transfer. The end result of this aspect of uncertainty is that application of the larval toxicity 

benchmarks from the microinjection studies would extrapolate to lower (eg. more protective or conservative) 

estimates of Se concentrations in eggs from the maternal transfer studies. Because the benchmark values 

from the larval microinjection studies are also used to extrapolate muscle, whole body and dietary 

benchmarks, the same uncertainty noted above would apply to these measures. That is, they too may be 

expected to be conservative if microinjected Se is more toxic than maternally transferred Se. 
Vince P. Palace, Ph.D.

43

While a regression equation is provided for Figure 7, the strength of the regression is not provided. This is a 

key determinant of the strength of the modeled results. The graphic representation of the model is 

appropriate, but a further examination of the data in Table 3-18 of Linville (2006) reveals that the regression 

is limited to identifying percent deformities in just 2 of the 3 treatment groups (one treatment group in which 

larvae contained 11.6 μg/g of Se yielded 0% deformities). I suspect that a relatively weak regression of this 

limited dataset contributes seriously to the uncertainty of the modeled results in the draft document. As 

similar concern should be noted for the regression presented for Figure 8, where mortality is expressed 

against selenium in eggs for just the 3 groups of larvae. Furthermore, Figure 9 presents another regression 

where just 3 data points are presented. These data were derived from Tables 1 and 2 of the Linares-Casenave 

et al. (2010) poster and from the data presented in Silvestre et al. (2010) in Figure 1 and Table 1. Referring to 

the original data presentations in those publications, it is evident that a relatively small range of selenium 

body burdens is available for analysis of the regression (i.e. non injected larvae contained 3.2 μg/g Se, Lmethionine injected larvae contained 3.5 μg/g Se and the Selenomethionine injected larvae contained 7.3 

μg/g Se). This small range of selenium concentrations would also provide a relatively weak and non-robust 

regression for the purposes of further data extrapolation. The limited regressions noted above would be 

expected to seriously limit the strength of the overall model presented in the draft document. 

One of the central assumptions of the modelled data is that the “relative sensitivity of green and white 

sturgeon would be the same for naturally transferred selenium as for micro-injected selenium” (page 15, 5th

line from the bottom). No data or additional discussion is provided to support this statement and, as a result, 

this introduces another source of uncertainty in the model’s application. This is especially true since the ratio 

is used to derive many of the benchmark toxicity values for green sturgeon by applying it as a correction 

factor to the white sturgeon benchmark values. However, mortality was most evident among microinjected 

white sturgeon larvae but deformities were the most prevalent among larvae exposed via maternal transfer. 

This suggests that the 2 exposure routes are not physiologically comparable and that there is uncertainty in 

extrapolating benchmark effects levels from one data set to another. This concern is validated by the higher 

EC05 and EC10 values from the maternal transfer studies relative to the microinjection studies in white 

sturgeon.

Almost certainly, one of the largest contributors to the uncertainty in the current model is the derivation of 

toxicity data for green sturgeon based on microinjection of progeny from a single female in the LinaresCasenave et al. 2010 and Silvestre et al. 2010 studies (page 20, 2nd paragraph). In my experience, the 

variability of toxic responses within progeny of a single female are less substantial than the variation that can 

be observed among progeny from different females, even at similar concentrations of Se. Regardless of 

whether this variation results from modifying factors such as interactive inorganic elements or nutritional 
Vince P. Palace, Ph.D.

44

differences, uncertainty in either direction (underestimated or overestimated toxicity) is introduced into the 

model. The fact that the ratios of toxicity for green versus white sturgeon are based on essentially this single 

data point from green sturgeon, and that this ratio is then used to derive several other benchmark values for 

green sturgeon is concerning. At the very least, this limitation should receive earlier and more prominent 

attention in delineating the model as it is outlined. 

Appropriateness of the Process/Steps of Analysis

3. Is each step in the analysis reasonable and appropriate and sufficiently explained? 

Notwithstanding the comments noted above, and in general, Dr. Beckon provides a reasonable, step wise 

analysis of the available data to derive benchmark values in tissues and whole bodies of both sturgeon 

species. Specifically, I agree with Dr. Beckon’s interpretation that both mortality or deformity indices are 

relevant endpoints and in my opinion, the discussion of this subject that appears at the top of page 17 is valid 

and ecologically defensible. 

Whole body benchmark values are important because of their use in regulatory capacities and for 

comparisons with existing datasets from other fish species. Conversion of muscle to whole body Se 

concentrations for adult white and green sturgeon in Dr. Beckon’s model are based on regressions obtained 

from Tashjian et al. (2006). That study exposed juvenile white sturgeon (initial weight ca. 30g) to diets 

amended with selenomethionine for 8 weeks. There are valid reasons to expect that juveniles might differ in 

their muscle to whole body selenium concentrations relative to adults. For example, oogenesis can provide a 

significant depuration mechanism for whole body Se in female fish (GEI Consultants 2008, Reference PDF 

attached to this report), especially among those that produce eggs with large yolk reserves. While this, and 

other physiological differences between adults and juveniles, might be expected to introduce uncertainty in 

the current model, Dr. Beckon has evaluated the fit of the juvenile data relative to other empirical data from 

other fish species (Figure 12) and supported the comparability of the data from Tashjian et al. (2006) among 

these other datasets. However, the suggestion that the fit of the current model in comparison to the other 

models with regard to gender and reproductive condition of the fish, requires additional substantiation as 

these data are not specifically presented in Figure 12 or the complete dataset supplied in the Appendix for 

that figure. Most notably, no comparisons of the model fit against adult females, or males, of reproductive 

age from other species is possible as the data are presented. Where available, this specific data should be 

presented in either the figure or the Appendix supporting the figure. 
Vince P. Palace, Ph.D.

45

Reasonableness/Supportability of Assumptions and Conclusions of the Analysis

4. Given the use of the underlying studies cited in the Report, are the assumptions and conclusions in 

each step of the analysis reasonable and supported by the data? Briefly explain why or why not, 

and if not, why not and how it might be changed to enhance the analysis.

My comments above note several uncertainties in the model as presented in the draft document. These 

uncertainties are generally noted by the author and it is acknowledged several times in the document that 

these are the best data available. However, conclusions based on these data should recognize the serious 

limitations and uncertainties in the existing dataset. 

One of the remaining issues which I have not addressed in my comments above concerns Dr. Beckon’s use 

of an exponential model for trophic transfer of Se from diet to maternal tissues and then to larvae from the 

female fish. As Dr. Beckon suggests (page 18, 3rd paragraph), the literature contains no support for anything 

other than an exponential model application for trophic transfer of Se from maternal diet to maternal tissues 

and then to larvae (i.e. there is no evidence for homeostatic control of Se accumulation and deposition into 

eggs). As a result, and in my opinion, exponential regressions are appropriate for interpreting the datasets. 

I am also supportive of the use of EC10s rather than EC50s to derive sensitivity threshold values as the 

former is more ecologically relevant for protecting populations from impacts. Furthermore, the use of this 

endpoint is supported by modelled population level effects in the literature (Lemly 1997). 

Availability of Alternative Methods

5. Would you recommend alternative methods or analyses to estimate benchmarks for mortality or 

abnormalities affecting survival, given the available data on green and white sturgeon? If so, 

explain what they are and why they might be more appropriate.

The methods employed for analysis in this draft document are appropriate and based on the best available 

data at this time. In the case of the 2 sturgeon species in this report, the data limitations are serious enough to 

contribute to great uncertainty in the application of the model and the derived benchmark toxicity threshold 

values. 
Vince P. Palace, Ph.D.

46

Recommendations 

a) More prominent notation of the limitations of the green sturgeon microinjection studies, which are based 

on toxicity thresholds in the larvae obtained from just one fish. In fact, more prominent disclosure of the 

entire dataset that is modelled would be prescribed. Identification of these limitations would be useful for 

guiding recommendation “b” noted below. 

b) Include recommendation in the report on the most urgently needed data to strengthen the predictive 

capacity of the model. 

c) Given the importance of the data referenced in the studies of Kaufmann et al. (2008), and the fact that >4 

years have passed since the data were first presented, every attempt to gain access to them should be 

taken. It is anticipated that these data would strengthen the model derivation considerably.

I would like to express my thanks to you and the rest of the Eastern Research Group team for giving me the 

opportunity to participate in the review of the draft document. Issues related to selenium bioaccumulation 

and toxicity continue to be an active area of research in my program and I find it personally and 

professionally stimulating to be involved in front-line evaluation of datasets such as the one presented by this 

document. I would certainly welcome similar opportunities to work with your team in the future. 

Please feel free to contact me should you have any additional questions about my review. 

References

GEI Consultants. 2008. Maternal Transfer of Selenium in Fathead Minnows, with Modeling of Ovary 

Tissue-to-Whole Body Concentrations. Project Report # 062790 (PDF attached to this report)

Holm J, VP Palace, P. Siwik, G Sterling, RE Evans, CL Baron, J Werner, K Wautier. 2005. Developmental 

effects of bioaccumulated selenium in eggs and larvae of two salmonid species. Environ. Toxicol. Chem. 

24:2373-2381.

Lemly AD. 1997. A teratogenic deformity index for evaluating impacts of selenium on fish populations. 

Ecotoxicol Environ Saf 37:259–266.

North American Metals Council. 2008. Selenium Tissue Thresholds. Report compiled by 

GEI Consultants, Golder Associates,, Parametrix and the University of Saskatchewan. (PDF attached to this 

report).
47

Comments Submitted by

Daniel Schlenk, Ph.D.

Daniel Schlenk, Ph.D.

49

Charge Questions:

Availability and Sufficiency of Data:

1. Are the Linville (2006), Linares-Casenave et al. (2010), Silvestre et al. (2010) and Tashjian et al.

(2006) studies the best available studies that you are aware of, for determining selenium 

benchmark values for mortality and/or abnormalities affecting survival, for green and white 

sturgeon (Acipenser medirostris and Acipenser transmontanus)? Please explain why or why not, and 

if not, identify any other studies and why and how their data would be useful.

While each of the studies listed have explored the effects of selenium on green and/or white sturgeon, 

only two (Sivestre et al. (2010) and Tashjian et al. (2006) have undergone peer review outside of the 

University of CA, Davis. Consequently, the lack of external peer review lends some uncertainty to the 

data. In fact, with essentially an N of 1 for the green sturgeon microinjection studies and only 3 data 

points on the concentration-response curve, it is interesting that the Sivestre et al. 2010 study was not 

rejected. In contrast, the thesis of Linville (2006) appears to have adequate data for white sturgeon and 

the 3 committee members represent excellent expertise in the area of metal toxicology in fishes. 

However, it is unfortunate that the thesis has not been published and one wonders what issues prevent the 

work from being published. Thus, “studies” for green sturgeon were really more like single experiments 

without replication. And while the data may be useful for comparative purposes, the use of the data for 

modeling exercises to extrapolate from one species to another should be considered extremely uncertain. 

Given these uncertainties, one would have expected a more probabilistic species sensitivity distribution 

method that incorporates estimates of uncertainty, rather than using single projeny experiments with 

limited data points for extrapolation.

2. The underlying studies cited above use two different methods to expose sturgeon to selenium: 

micro-injection of L-selenomethionine into yolk sacs and a diet of selenized yeast to gravid females. 

Are the two methods of exposure and the resulting data comparable? Please explain why or why 

not. Please comment on the Report's use of data from two different methods of exposure.

Microinjection of L-selenomethionine into developing embryos of sturgeon is an efficient method of 

evaluating the effects of this form of selenium to sturgeon larvae and with the appropriate replication and 

dosing strategies could be used to evaluate species sensitivity differences. Unfortunately, based on the 

methods provided in the materials, it would appear that only a single dose was provided to embryos in 

Silvestre et al. (2010) and no mention of dose was provided in Linares-Dasenave et al. (2010). It is 

unclear how the 3 data points in Figure 9 of the draft report were determined. Without a more extensive 
Daniel Schlenk, Ph.D.

50

dosing strategy such that EC50 values can used to reduce uncertainty in dose-response assessments, it is 

not possible to carry out extrapolations with any degree of certainty. 

With regard to the dietary seleniforous yeast exposures, this is likely a more accurate depiction of 

exposure to selenium in the field. While selenomethione may be one of the more toxic forms of 

selenium, there are clearly other organic forms (selenocysteine etc.) that contribute to toxicodynamic, 

and toxicokinetic differences as well as the resulting toxicology of selenium. It is unclear how each of 

the varied organoselenides work synergistically, additively or antagonistically. As discussed in the draft 

report, transport between hepatic and gonadal tissues via vitellogenin and other lipoproteins is a critical 

step in the process. It is unclear how much and when selenium is released from the lipoproteins (if at all) 

and in what chemical form. Microinjection would clearly represent a “worst-case” scenario with regard 

to exposure and likely overestimates exposure to selenium. In addition, without the other organoselenide 

species, the exposure oversimplifies the interactions that occur in embryos. While the report indeed 

states that sturgeon do not feed upon yeast, they do consume other organisms that convert selenium to 

the complex organoselenides that are present in the yeast and responsible for toxicity.

Appropriateness of the Process/Steps of Analysis:

3. Is each step in the analysis reasonable and appropriate, and sufficiently explained? For each step, 

briefly explain why or why not. Identify any other steps that you believe should be and/or could be 

included to enhance the analysis.

The overall concept seems straightforward and the method can eventually be used. However, the major 

flaw is the number of data points used to make the regression and subsequent extrapolations. Most plots 

have essentially data at two ends of the curve and a paucity of data in critical areas of the regression 

(particularly in the middle sections of the curve). Such is the case in Figures 2, 4, and especially 6 where 

the entire regression is based on two points!! Figures 7 and 8 only have one data point above threshold, 

which suggests the extrapolated points are highly uncertain. Figure 9 has 3 points (but these are from 

one spawning event from a single animal). Figure 10 only has two points. Figure 11 A and B only have 

one point above threshold. 
Daniel Schlenk, Ph.D.

51

Reasonableness/Supportability of Assumptions and Conclusions of Analysis:

4. Given the use of the underlying studies cited in the Report, are the assumptions and conclusions 

in each step of the analysis reasonable and supported by the data? Briefly explain why or why 

not, and if not, why not and how it might be changed to enhance the analysis.

As discussed in question 3 above, given the lack of data for extrapolation between tissue, larvae and 

effects, a larger data set is necessary (at least 4-5 data points well distributed throughout the curve) to 

conduct the extrapolations. 

Availability of Alternative Methods:

5. Would you recommend alternative methods or analyses to estimate benchmarks for mortality or 

abnormalities affecting survival, given the available data on green and white sturgeon? If so, 

explain what they are and why they might be more appropriate.

Based on the limited data in the two species of concern, it would appear that a probabilistic approach 

using data from other species would be a better method given the current data restrictions. The 

probabilistic approach will provide a better estimate of uncertainty. 

Other comments:

One of the significant statements in the Linville thesis is the lack of reproductive impairment in the white 

sturgeon tested even though concentrations were above benchmark values

Is there really a difference in species sensitivity between the green and white sturgeon?? Kaufman et al 

2008 is not peer reviewed and while there may be a trend toward enhanced sensitivity in green sturgeon, 

the limited dose response data precludes adequate statistical power to make these determinations.