# 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

. E nvironmental P rotection A gency, 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 Paperi



## 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 Ava ilability 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 Re viewer Comments

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

................................ ................................ ......................... 29 Comment s Submitted by Vince P. Palace, Ph.D.

................................ ................................ .................. 39 Comments Submitted by Daniel Schlenk, Ph.D.

................................ ................................ ................... 471 

## SummarySummary

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

Avail ability and Sufficiency of Data

1. Are the Linville (2006), Linares

- Cas enave 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 expl ain 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 dat a 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 ov erestimate 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 inh erent 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 f or 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, t he 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 experim ents 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 we re unpublished and lacked external peer review. 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

m ethods 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 reviewer s mentioned that micro injection 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 microinje ction. 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 com plex 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

o rganoselenium species.

All three reviewer s concluded that there is too much uncertainty in the Silvestre et al. (2010) and Linares

Cas e nave et al. (2010) studies to be useful in deriving benchmarks. The derivation of toxicity data for green sturgeon is ba sed 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, w ithout more extens ive dosing strateg ies than those conducted in the Silvestre et al. (2010) and Linares

- Cas e nave et al. (2010) studies , it is not possible to carry out extrapolations with any degree of certainty.
Further, one reviewer said that t here is uncertainty in extra polating 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

m ay explain why larval mortality was the most sensitive endpoint in these studies . L arval 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 note d 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 Lin ares

- Cas e nave 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 relat ionship s

. 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 interpretati on 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 f

ish. The third reviewer stat ed 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 t he 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 t hese 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, h e supports the u se of the EC10s because they are more ecologically relevant for protecting populations from impacts. T he 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.



### Avai lability 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 prov ide 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

rec ruitment 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 infor mation that normal regulatory mechanisms may decrease in importance at higher exposures.

 The entire modeled dataset needs more prominent disclosure. Specifically, d ata 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. Projec t 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 impac ts 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 t his report).7 

## Responses to Charge QuestionsResponses to Charge Questions

9 

### Introductory Comments

Reviewer Comments

J an z Preamble :

I am a Professor of Veterinary Biomedical Sciences. I hold a PhD in Pharmacology and Toxicology, a MSc in E n vironmental 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 a nd 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 resp onses 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 experiment al 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 rev iewed 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 use d 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 20 12 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 s elenium 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 da ta would be useful.

Reviewer Comments

J an z 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, s pecies 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

- larv al 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 EC 10 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 endpoin t 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 a mong 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 gree n 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 ecol ogical, 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 seleniu m” (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 speci es

. ” 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 e cology 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. SpeciesResponses to Charge Questions

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

- based guideline. Integr. Environ. Assess. Ma nag . 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. Adam s, 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 Co uncil 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 nich es 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 marine

orie nted)). 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 f or 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 lackResponses 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 ha s 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 t he 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

J an z 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 tox icokinetics 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 belie ved 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 organosele nium 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 missin g 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 tera ta. 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 i s a moreResponses to Charge Questions

13 sensitive endpoint (see page 6, 1 st 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 d iets 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 l arval 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 SeMe t 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” sel enium 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). Anoth er 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 si nce 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 tr ansient, 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 o n 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 unde r the heading White Sturgeon Exposure by Maternal Transfer (additional discussionResponses 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 whit e 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

cons ervative) 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 abov e 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. T his 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 de formities 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 modele d 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 ju st 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 p ublications, 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 inje cted 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 seriousl y 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, 5 th 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 rat io 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 t he 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 toxicit y 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, 2 nd paragraph). In my experience, the variability of toxic responses within progeny of a singl e female are less substantial than the variation that can beResponses 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 gree n 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.

Sch lenk 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 extrapola tions 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 clea rly 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 antagon istically. 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 interaction s 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 

### Appropriatenes s 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 t he analysis.

Reviewer Comments

J an z 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, 3 rd paragraph, I somewhat disagree with the author’s opinion that there is no homeostatic regulation of selenium. As an essential trace element, regu latory 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 leve ls, 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 b ody/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 in terpretation 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 t heir 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 Tashj ian 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 r elative 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. Whil e 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 (Figur e 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 fi sh, 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 reproductiv e 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 event ually 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 uncert ain. 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. Giv en 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 uncertaintie s 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 D r. Beckon suggests (page 18, 3 rd 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 ho meostatic 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 thresho ld 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 mo re appropriate.

Reviewer Comments

J an z 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

sen sitive 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 pr escribed. 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 th e 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 deriv ation 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 CommentsAdditional 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 ph armacological 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 inc lude 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. 20 05. 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 s turgeon, the limited dose response data precludes adequate statistical power to make these determinations.27 

## Appendix A: Individual Reviewer Comments29 

### 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 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 a nd 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 ar e 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 res ponses 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 sur vival, 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

- lar val 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 E C10 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 endpoi nt 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 gr een 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 ec ological, 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 sele nium” (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 whiteDavid Janz, Ph.D.

33 sturgeon is relatively less sensitive to Se toxicity than other fish sp ecies”. 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 populatio n 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 method s 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 du ring 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 fertil ized 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 Sil vestre 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

mos t 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 mos t sensitive endpoint. Why are sturgeons more like birds, where embryo mortality is a more sensitive endpoint (see page 6, 1 st paragraph)? I believe the answer is related to the timing of exposure in these microinjection experiments.

The author argues in d efense 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 abn ormalities 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 separ ate control. Mortality in white sturgeon never exceeded 20%, even at the “high” selenium dose. Importantly, although green sturgeon mortality in the Se treatment appeared toDavid 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 incidenc e 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 fur ther 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 Bec kon 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 stu dies that can be cited to support these relationships?

On page 18, 3 rd 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 involvin g 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 h igher 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 rep orted 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/Supp ortability 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 ex periment 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 a nalyses 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 limi ted 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 materna l 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.

3 7 Ed itorial 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 extre me 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 conclu des 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 shou ld 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 s elenium 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 o ther 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 appear ance.

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 cert ainly 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 fa ctors 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 Meta ls 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. CautionVince 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 marine

oriented)). 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 uncertain ty 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. T wo different methods are used to expose sturgeon to selenium: microinjection of L

selenomethionine 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 relation ship 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 ma ternal 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

ex pected 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 res ults. 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 treatmen t group in which
larvae contained 11.6 μ g/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 fro m 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 μ g/g Se, L

methionine injected larvae contained 3.5 μ g/g Se and the Selenomethionine injected larvae contained 7.3

μ g/g Se). This small range of sele nium 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 d raft 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, 5 th 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 transfe r. 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 mater nal 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 proge ny from a single female in the Linares

Casenave et al. 2010 and Silvestre et al. 2010 studies (page 20, 2 nd 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 nutritionalVince 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 sev eral 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 ste p 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 in terpretation 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 t heir 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 Tashj ian 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 r elative 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. Whil e 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 (Figur e 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 fi sh, 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 reproductiv e 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 Ana lysis

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 en hance 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 exponentia l 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, 3 rd paragraph), the literature contains no support for anything other than an exponential model application for troph ic 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 interpr eting 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 da ta 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 pr escribed. Identific a tion 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 th e 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 deriv ation 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 Con centrations. 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 morta lity 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 rejecte d. 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 pub lished and one wonders what issues prevent the work from being published. Thus, “studies” for green sturgeon were really more like single exp eriments without replication. And while the data may be useful for comparative purposes, the use of the data for

m odeling 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 uncertai nty, 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 s elenized 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 int o 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. Unfortunate ly, 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 Linar es

- Dasenave et al. (2010). It is unclear how the 3 data points in F igure 9 of the draft report were determined. Without a more extensiveDaniel 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 re gard 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

repor t, 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 w ould 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 rep ort 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 yea st 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 i n 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/Supporta bility 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 n ot, 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 throu ghout 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 cu rrent 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,
th e limited dose response data precludes adequate statistical power to make these determinations.