# FINAL

Brown Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site-Specific Selenium Criterion

Revised October 2011

Prepared for:


## J.R. Simplot Company

Prepared by:

2500 55 th Street, Suite 200 Boulder, CO 80301Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

i TABLE OF CONTENTS Page

LIST OF TABLES ................................................................................................................ ......... ii

LIST OF FIGURES ............................................................................................................... ....... iii

LIST OF APPENDICES ............................................................................................................ .... v

LIST OF ACRONYMS .............................................................................................................. ... vi

1.0 INTRODUCTION ............................................................................................................... 1 1.1 Background ........................................................................................................... 3 1.2 Objectives .............................................................................................................. 4

2.0 METHODS ....................................................................................................................... . 5 2.1 Wild Fish Collection ............................................................................................... 5 2.1.1 October Fish Collection ............................................................................. 6 2.1.2 November Fish Collection .......................................................................... 7 2.1.3 Hatchery Fish............................................................................................. 8 2.2 Reproduction Study and Laboratory Test Methods ............................................... 9 2.3 Deformity Assessment .......................................................................................... 9 2.3.1 Data Reduction of Deformity Rankings .................................................... 11 2.4 Statistical Analysis ............................................................................................... 11

3.0 RESULTS ....................................................................................................................... 13 3.1 Surface Water Quality ......................................................................................... 13 3.2 Wild Fish .............................................................................................................. 13 3.2.1 Adult Size and Selenium Concentrations -Whole Body and Eggs ........... 14 3.3 Hatchery Fish ...................................................................................................... 15 3.3.1 Adult Size and Selenium Concentrations – Whole Body and Eggs ......... 15 3.4 Reproduction Testing .......................................................................................... 16 3.4.1 Fecundity ................................................................................................. 16 3.4.2 Egg Mortality ............................................................................................ 16 3.4.3 Hatch to Swim Up .................................................................................... 17 3.4.4 Mortality ................................................................................................... 18 3.4.5 Growth ..................................................................................................... 19 3.4.6 Deformities............................................................................................... 19

4.0 ANALYSIS OF RESULTS .............................................................................................. 23 4.1 Surface Water Quality ......................................................................................... 23 4.2 Relationship of Maternal Whole Body to Egg Selenium Concentrations ............. 24 4.3 Relationship of Egg Selenium Concentrations to Specific Effects Endpoints ...... 24 4.3.1 Survival/Mortality and Deformities ........................................................... 26 4.4 Logistic Analyses ................................................................................................. 27 4.4.1 Growth ..................................................................................................... 28 4.4.2 Survival .................................................................................................... 28 4.4.3 Deformities............................................................................................... 29 4.5 Predicted Effects Concentration for Brown Trout – Consistency with Literature . 31 4.6 Extrapolating Selenium Concentrations in Egg Tissue to Whole Body Tissue .... 32 4.7 Data Adequacy .................................................................................................... 33 4.8 Dose Response Analysis Update ........................................................................ 34Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

ii 5.0 SUMMARY AND CONCLUSIONS ................................................................................. 36

6.0 REFERENCES ................................................................................................................ 38

LIST OF TABLES

Table Title

1 Monitoring Locations, Coordinates, and Counts for October 2007 Sampling in Support of the Brown Trout Laboratory Toxicity Studies

2 Summary of Field-Measured Water Quality Parameters Collected in October 2007 for Sampling in Support of the Brown Trout Laboratory Toxicity Studies

3 Water Quality Parameters Measured During the October 2007 Sampling in Support of the Brown Trout Laboratory Toxicity Studies

4 Summary of Total Selenium Concentrations Measured in Surface Waters in Support of the Brown Trout Laboratory Toxicity Studies

5 Monitoring Locations, Coordinates, and Counts for November 2007 Sampling in Support of the Brown Trout Laboratory Toxicity Studies

6 Adult Female Brown Trout Length and Whole-Body Selenium (mg/kg dw) Data

7 Summary of Brown Trout Tissue Selenium Data Collected in Support of the Brown Trout Laboratory Toxicity Studies

8 Effect Concentration (EC x ) Values for Egg Selenium Tissue Concentrations Versus Different Biological Endpoints for Brown Trout

9 Summary of Toxicity Studies that Evaluated Selenium Toxicity to Embryo/Larvae Resulting from Maternal TransferBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

iii LIST OF FIGURES

Figure Title

1 Location of the Smoky Canyon Mine

2 Study Area Boundaries, Monitoring Locations, and Exposure Conditions

3 Adult Fish Length Versus Whole-Body Selenium Concentration

4 Adult Fish Weight Versus Whole Body Selenium Concentration

5 Whole-Body Selenium Concentration Versus Brown Trout Length

6 Brown Trout Egg Selenium Concentration by Location

7 Comparison of Total Number of Eggs Produced and the Adult Fish Size (Length)

8 Total Egg Abundance Versus Egg Selenium Concentration

9 Total Egg Abundance Versus Adult Whole Body Selenium Concentration

10 Percent Egg Mortality by Location

11 Percent Hatch (or Percent Survival at Hatch) of Alevins by Location

12 Relationship between the Percentage of Fish that Reached Swim-Up and the Percentage of Eggs that Hatched

13 Day to First Hatch and Day to Swim-Up Versus Egg Selenium Concentration

14 Fry Mortality at Swim-Up and Overall Mortality by Location

15 Percent Mortality from Hatch to Test Termination by Location

16 Percent Mortality 15-Day Post Swim-Up Feeding Trial by Location

17 Brown Trout Growth based on Larval Fish Length by Location

18 Brown Trout Growth based on Larval Fish Dry Weight by Location

19 Percent Cranio-Facial Deformities for Larval Brown Trout

20 Percent Skeletal Deformities for Larval Brown Trout

21 Percent Fin of Finfold Deformities for Larval Brown Trout

22 Percent Edematous Tissue Frequency for Larval Brown TroutBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

iv LIST OF FIGURES (CONTINUED)

23 Mean GSI Score and Total Number of Fish Evaluated by Location and Sample ID Ranked by Egg Selenium Concentration

24 Surface Water Selenium Concentrations

25 Log Egg Selenium Concentration Versus Log Maternal Whole-Body Selenium Concentration (Wild Fish)

26 Total Egg Abundance from Wild Female Fish Versus Egg Selenium Concentration

27 Percent of Brown Trout Fry that Hatched Versus Egg Selenium Concentration

28 Percent of Trout Achieving Swim-Up Versus Wild Fish Egg Selenium Concentration

29 Larval Fish Weight Versus Egg Selenium Concentration

30 Overall Percent Survival Versus Egg Selenium Concentration

31 Percent Survival (Hatch to Test End) Versus Egg Selenium Concentration

32 Percent Survival in the 15-Day Post Swim-Up Feeding Trial Versus Egg Selenium Concentration

33 Sum Fraction Non-Normal Larvae Versus Egg Selenium Concentration

34 Sum Fraction Normal Larvae Versus Egg Selenium Concentration

35 Mean GSI Score Versus Egg Selenium Concentration

36 TRAP Model Larval Fish Growth Versus Log Egg Selenium Concentration15-Day Post Swim-Up Feeding Trial

37 TRAP Model Survival Versus Log Egg Selenium Concentration15-Day Post Swim-Up Feeding Trial

38 TRAP Model Total Survival Versus Log Egg Selenium Concentration

39 TRAP Model Percent Survival Versus Log Egg Selenium Concentration(Hatch to Test End)

40 TRAP Model Cranio-Facial Deformities (Fraction Normal) Versus Log Egg Selenium ConcentrationBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

v LIST OF FIGURES (CONTINUED)

41 TRAP Model Skeletal Deformities (Fraction Normal) Versus Log Egg Selenium Concentration

42 TRAP Model Finfold Deformities (Fraction Normal) Versus Log Egg Selenium Concentration

43 TRAP Model Edema (Fraction Normal) Versus Log Egg Selenium Concentration

44 TRAP Model Sum Fraction Normal Fish Larvae Versus Log Egg Selenium Concentration

45 TRAP Model Mean Fraction Normal Fish Larvae Versus Log Egg Selenium Concentration

46 Revised Trap Model Percent Survival (Hatch to Test End) Versus Log Egg Selenium Concentration

LIST OF APPENDICES

Appendix Title

A Data Report-Reproductive Success Study with Brown Trout (Salmo trutta) -ENSR

B Detail Figures for Locations where Brown Trout were Collected

C Photographs of example deformities and Summary of the Deformity Rankings for Each Sample-CSU

D Brown Trout Whole Body and Egg Tissue Selenium Analytical Data

E EPA’s TRAP Software and Summary Output Statistics for Each Model Run

F Agency Comments on February 5, 2009 Draft Report

G USEPA Comments on Derivation of Brown Trout EC x Values (December 21, 2010)Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

vi LIST OF ACRONYMS

AOC Administrative Order on Consent
CAS Columbia Analytical Services

CF Cranio Facial

CI Confidence Interval
dw Dry Weight

EC/EC x Effect Concentration

ED Edematous Tissue

FD Finfold
GH-AAS Gaseous Hydride Atomic Absorption
GSI Graduated Severity Index
HS Hoopes Spring
IDEQ Idaho Department of Environmental Quality
ODA Overburden Disposal Area
OLS Ordinary Least Squares
SC Saratoga National Fish Hatchery
SI Site Investigation

SK Skeletal
SPC Spring Creek Trout Hatchery
SSSC Site-Specific Selenium Criterion

TM Technical Memorandum
TRAP Toxicity Relationship Analysis Program

USFS United States Forest Service
USEPA United States Environmental Protection AgencyBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

1 1.0 INTRODUCTION

A chronic site-specific selenium criterion (SSSC) is being developed for Hoopes Spring South Fork Sage Creek (SFSC) and the downstream receiving waters including Sage Creek and Crow Creek upstream of the Idaho and Wyoming State Line. Hoopes Spring is located in Sage Valley near the J.R. Simplot Company (Simplot) Smoky Canyon phosphate mine in Southeastern Idaho (Figure 1). In accordance with the Administrative Order on Consent (AOC) entered into by Simplot, Idaho Department of Environmental Quality (IDEQ), the US Forest Service (USFS), and the US Environmental Protection Agency (U SEPA), a Site Investigation (SI) was conducted at the mine site in 2003 and 2004. Investigations to date have identified elevated concentrations of selenium in surface water being discharged via Hoopes Spring and South Fork Sage Creek Springs, which ultimately discharges to lower Sage Creek. Selenium released from overburden disposal areas (ODAs) at the mine has the potential to migrate vertically downward into the Wells Formation aquifer. Groundwater from the Wells Formation aquifer discharges at Hoopes Spring and South Fork Sage Creek Springs.

Source control actions implemented at the ODAs will limit infiltration to the Wells Formation, but will not immediately reduce selenium concentrations discharged from the Wells Formation via Hoopes Spring and South Fork Sage Creek Springs. In the interim, modification of the selenium surface water quality standard is being investigated.

Field monitoring studies are in progress to ch aracterize the exposure environment, the aquatic community, and the physical habitat. Activities for the field monitoring studies are documented in the April 2007 Work Plan - Field Monitoring St udies for Developing a Site-Specific Selenium Criterion (NewFields 2007a).

This report for the study of brown trout ( Salmo trutta) reproduction is presented as the first of two laboratory studies to evaluate the effects of ambient selenium concentrations in aqueous and dietary media on reproductive success of trout from the site. The second study involves a similar scope, but uses Yellowstone cutthroat trout ( Oncorhynchus clarki ). The laboratory studies were developed to complement information available from literature and an extensive field monitoring program for the study area. These laboratory studies are designed to assess potential effects of selenium accumulated in tissue of wild-caught adult brown trout onreproductive success, especially the development of young fish from fertilization through swimup stages of development. The study design was presented in an October 17, 2007 Technical Memorandum - Methods for Testing Adult Brown Trout Reproductive Success. Laboratory portions of this testing were carried out at ENSR’s environmental toxicology laboratory in Ft. Collins, Colorado by Dr. Rami Naddy. The deformities assessment was performed by Dr. Kevin Bestgen at Colorado State University’s Larval Fish Laboratory.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

2 The approach for the brown trout laboratory reproduction studies was based in part on the following published works:

 Kennedy et al. (2000). The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout ( Oncorhynchus clarki lewisi );

 Holm et al. (2003). An assessment of the development and survival of rainbow trout ( Oncorhynchus mykiss ) and brook trout ( Salvelinus fontinalis ) exposed to elevated selenium in an area of active coal mining;

 Holm et al. (2005). Developmental effects of bioaccumulated selenium in eggs and larvae of two salmonid species; and

 Hardy (2005). Effects of dietary selenium on cutthroat trout ( Oncorhynchus clarki ) growth and reproductive performance.

Based on these and other works, the reproductive success of fish exposed to selenium via diet and water was identified as a highly sensitive endpoint. The approach also reflects the following understanding of the current state of the science regarding selenium toxicity:

 Chronic effects of selenium exposure to fish are due primarily to diet. Chronic toxicity is based on the magnitude and duration of exposure, as well as bio-uptake in the food web. The USEPA (2004) draft criteria document for selenium did not consider or use tests in which aqueous only exposure was tested. EPA states, “[b]ecause diet controls selenium chronic toxicity in the environment and water-only exposures require unrealistic aqueous concentrations in order to elicit a chronic response, only studies in which test organisms were exposed to selenium in their diet alone or in their diet and water were considered in the derivation of a chronic value.”

 Fish appear to be the most sensitive aquatic biota in the area of interest to chronic exposure and toxicity from selenium (Coyle et al. 1993; Hamilton et al. 1990; Hermanutz et al. 1996) (as cited in USEPA 2004).

 Reproductive success is the most sensitive biological end point for assessing selenium toxicity to fish (Lemly 1985a,b, 1992; Gillespie and Baumann 1986; SchuItz and Hermanutz 1990; Coyle et al. 1993) (as cited in Lemly 1993).

 Selenium impacts on reproductive success in fish are strongly correlated to selenium content in eggs (Parametrix 2009) 1 . Selenium in eggs is derived from maternal tissue, and is well correlated with whole body tissue selenium concentrations in maternal adults.

1 Parametrix 2009 is a compilation document that reviews a number of important studies in the sel enium literature. The conclusi ons drawn are based on the scientific evi dence from numerous studies suggesting ovary or egg concentrations are the best tissue to correlate to effects. This position is also supported by USEPA in their revision of the National Selenium Criteria as relayed to the SSSC Work Group by Dr. Charles Delos.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

3  To date, three species of trout (i.e., brook, rainbow, and cutthroat) have been tested for bioaccumulation in adults and effects on development of young (Holm et al. 2003, 2005; Kennedy et al. 2000; Hardy 2005; Rudolph et al. 2008). No published literature has been found that indicates brown trout have undergone such testing to assess potential effects.

Because of the site-specific nature of selenium exposure and toxicity, wild-caught, reproducing fish from the study and reference areas are the best measure of current and potential impacts within this watershed. Brown trout are present in Hoopes Spring, Sage Creek, and Crow Creek. It is one of two predominant trout species (the other being Yellowstone cutthroat trout) found in these creeks and, although introduced, is recreati onally important. Brown trout are by far the most abundant of the two trout species and are known to reproduce throughout the study area and therefore were used as a test species.

1.1 Background

Sage Creek downstream of Hoopes Spring regularly exceeds the chronic water quality standard for selenium. The frequency and magnitude of the exceedences decline downstream with tributary inflows. Only infrequent exceedences have been observed in Crow Creek immediately downstream of Sage Creek. While concentrations of selenium may exceed the surface water standard, it is not an explicit indication that th e aquatic community is impaired. National surface water quality criteria adopted by states as standards do not always take into account the types of species present, nor the site-specific conditions, such as aqueous chemistry which may confound toxicity. Many factors influence the in-stream toxicity of selenium including the bioavailability of the form of selenium, tolerance of resident species (e.g., acclimation), and/or other factors that may enhance or ameliorate toxicity.

The brown trout adult reproduction testing used gravid adult wild fish captured at various locations from the study area (Figure 2), as well as hatchery fish for laboratory controls. Maternal transfer is believed to be one of the key factors influencing reproductive toxicity. Wild pre-spawn brown trout were collected from locations that represent the range of observed surface water selenium concentrations (NewFields 2007b). Aqueous and dietary selenium concentrations translate into a range of exposure conditions resulting in different body-burden loadings for parental fish, specifically adult female trout. It was anticipated that tissue concentrations in parental fish would confirm this. Gametes from the adult wild fish were collected and fertilized to evaluate reproduction. Although young were not exposed to aqueous selenium, they consumed any prot ein-bound organic selenium that was present in the yolk and passed on to the egg via parental exposure. The range and gradient of the selenium exposures, well-defined source area, exceedence of water quality standards and observations of thriving fish populations present a unique situation to examine selenium exposure and
effects.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

4 1.2 Objectives

The objectives of the testing presented herein are as follows:

 Document the range of selenium concentrations in wild parental fish due to in-situ integrated exposure of diet and water;

 Document the selenium concentrations in eggs produced by adults from different locations in the study area;

 Develop a relationship between selenium c oncentrations in maternal whole body tissue and egg tissue; and

 Develop relationships between egg tissue concentrations and measures of reproductive success and viability of young.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

5 2.0 METHODS

The methods for testing adult brown trout reproductive success, including the study design plan and analysis details for the assessment of se lenium exposure, were presented in a SSSC Workgroup – reviewed Technical Memorandum (TM) dated October 17, 2007 (NewFields 2007b). The methods for fish collection, egg collection and fertilization, and laboratory methods are briefly summarized below along with any deviations from the planned methods. ENSR conducted the reproduction testing for brown trout. Columbia Analytical Services (CAS) (Kelso, Washington) conducted the analytical chemistry for selenium concentrations in tissue. Appendix A describes the reproduction studies.

2.1 Wild Fish Collection

Because there is limited information on selenium toxicity to brown trout, there was some uncertainty as to where within the previous ly observed tissue concentration range reproductive effects might occur. To address whether the laboratory study would adequately cover the range of parental tissue concentrations expected in t he system, brown trout tissue data for selenium were compiled for the stream segments of interest to examine the range of variability. The mean and its confidence interval (CI) suggest that the data are somewhat variable (mean (CI) - 13.27±1.995). Since the system is comprised of small streams that have already been sampled numerous times, concern for the system is that over sampling can adversely influence the number of fish locally within the system, especially gravid females. Correspondingly, the goal was to identify the number of fish for collection that would provide a reasonably high probability of spanning the tissue concentration range of interest. The representativeness of the study is ultimately determined by capturing the range of effects and not the total number of fish. Next, the sample size to capture the range of tissue variability was estimated. How many samples are needed to cover the range of population of data (i.e., tissue concentrations), including at least one or more sample(s) that represent the upper 10th percentile? The following formula from Gilbert (1987) was used:



## α = 1-(1-p) n

When rearranged to solve for n, it looks as follows:



## n = ln(1- α )/ln(1-p)Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

6 where:

α = Probability of at least one sample representing the upper p th percentile;

p = target percent in number format; and

n = number of samples.

For this assessment, alpha was set at 0.05 and 0.1, while p was set at a range of percentile values. At a 95 percent confidence level (i.e., alpha = 0.05), 29 samples should ensure that at least one or more samples would represent the upper 10 th percentile (i.e., 90 th ). These 29 samples (i.e., adult females) were to be divided among the four exposure areas within the Crow Creek drainage. Rather than round down to seven fish per location, the number of fish was rounded up to 8 fish per location, which equaled 32 fish for the four locations. Based on Workgroup comments, the recommendation was to include additional fish samples for the reference area, thus an additional eight fish were added as targets for collection from the reference location. In total, for the five locations, it was estimated that collection of about 32 fish from the Crow Creek locations and 8 fish from the reference location (i.e., n=40) should capture the upper 10 th percentile of the data distribution.

The upper 10 th percentile was chosen because there will naturally be extremes in any environmental data, thus attempting to capture the entire range is not practical and over sampling of the spawning-age adults could easily occur in an effort to obtain fish with the highest tissue residues. Use of the 90 th percentile captures a large proportion of the data. Based on the data currently available, the range of concentrations is such that capturing the 90 th percentile, or upper 10 th percentile, will result in capturing fish with 20 mg/kg dry weight (dw) selenium or more. Section 3.2.1 describes the initial data used to derive the upper 10 th percentile and shows how the tissue concentrations from the adults collected for the reproduction study fit within that distribution. Section 4.7 describes the number of fish collected, and that the number of fish was adequate for the intended purpose of the study.

2.1.1 October Fish Collection

From October 22 to October 27, 2007, ripe and running brown trout were collected by electrofishing at eight locations (Appendix B) (Table 1). In Crow Creek and Sage Creek, target
locations included those where brown trout r edds had been previously identified during Fall 2006. Other areas within these locations where brown trout might be expected to congregate and spawn due to favorable conditions, such as water depth, velocity, and substrate were also included (Figure 2).Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

7 Reference locations on Montpelier Creek and Stump Creek were also sampled for spawning brown trout. Snowslide Creek, near its confluence with Montpelier Creek, was evaluated and deemed to have inadequate flow and water depth.

Target age of fish for use in this study was 3+ y ears old. Fish of this age typically range from approximately 230 to 300 mm in length. Fish greater than 230 mm were collected, weighed and measured for length.

Adult brown trout were held instream near the location of capture in flow through “live cars”.
Fish were held until October 30 th , and were fed a commercial fish food pellet daily beginning
October 27 th . From October 27 th to October 30 th , fish were checked daily for signs of ripening. Ultimately, eggs could not be expressed from any of the retained females and all fish were
released.

Water quality data were collected at each of the locations where brown trout were collected and subsequently held for possible spawning. In-situ field parameters, including water temperature, pH, conductivity, and dissolved oxygen were recorded (Table 2). A single water quality grab sample was also collected from each of these locations for analysis of dissolved and total selenium (Table 3 and Table 4).

2.1.2 November Fish Collection

From November 12 to 16, 2007, many of the same reaches sampled in October were sampled again for the presence of ripe and running brown trout (Table 5). Ripe or potentially ripe males and females were again held on-site in flow through “live cars” until they could be checked again for eggs or milt depending up gender. Collection methods were similar to those described above. No additional water quality data for selenium or ambient conditions were collected in
November.

Eggs (from adult female trout) and milt (from adult male trout) were collected in the fieldNovember 13-15, 2007 for conduct of the reproduction tests. Fish were anesthetized using MS222 to loss of equilibrium. Fish weight and length were then measured to the nearest 0.1 g and 1 mm, respectively. Trout were blotted dry, particularly the area around the urogenital opening to remove excess water that might contribute to premature water hardening of the eggs. The milt from several males at each location was expressed using a downward squeezing force, ventrally. Milt from several males was collected into a single plastic bag and stored on ice until added to individual egg batches from all females collected from that location. Eggs from each female were stripped from the vent in a similar fashion as the milt was collected from the males. Eggs from a single female were stripped into a pre-cleaned stainless steel bowl. Any blood, dirt or extraneous material was then removed from the bowl. Approximately 1 ml (depending on relative volume of eggs) of milt was added and then the egg / milt mixture was swirled gently toBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

8 ensure adequate mixing of gametes. The eggs and milt were allowed to sit undisturbed for ~1 minute. Then ~100 ml of local stream water (enough to just cover the eggs) was added to the bowl. The gametes were gently swirled for three minutes. Afterward, an additional 500 ml of stream water was added to water harden the fertilized eggs. The entire contents of the bowl were then poured into a labeled plastic bag and sealed. Each bag was labeled according to the female from which the eggs came, as well as the location. Prior to transport to the ENSR, the bag containing the fertilized gametes was partially inflated with oxygen, placed into a separate bag (double bagged), and returned to storage on ice (@ 4°C). The fertilized gametes were placed in a cooler for storage and transfer to the laboratory to protect them from sunlight and to keep them cold. A transponder that recorded temperature at 1 minute intervals was placed in each cooler prior to shipment to monitor the temperature during transport.

Adult fish were sacrificed for whole body selenium analysis. The adult fish carcasses were packaged in double plastic Ziploc® bags and stored on ice or frozen prior to shipment to ENSR along with the final egg batches. Because egg batches had to be delivered to ENSR within a narrow time window, and because ENSR had a large walk-in cooler/freezer, adult carcasses were initially shipped to ENSR. Once all carcasses were at ENSR and thoroughly frozen, a single shipment which included a subsample of eggs and all the adult fish carcasses for selenium tissue concentration analysis was sent on dry ice to CAS. Total selenium analysis and percent solids content were performed on all the submitted samples according to the methods described in the Work Plan (NewFields 2007a).

2.1.3 Hatchery Fish

Hatchery fish were used as method controls. Two sets of adult brown trout were obtained from the Saratoga National Fish Hatchery, Saratoga, WY (courtesy of Lee Bender) on October 23, 2007. Throughout this study, fish from this hatchery are identified as SC. The initial hatchery fish were obtained approximately three weeks prior to the first field-collected (wild) fish. Eggs from the initial set of hatchery fish were fertilized in the laboratory (transport of eggs and milt separately to the laboratory) according to the methods described above. Maternal fish were sacrificed to obtain whole body selenium tissue concentrations that corresponded to egg clutches from each female, consistent with the methods utilized for the wild fish.

A second set of hatchery fish were obtained in December 2007 from the Spring Creek Trout Hatchery in Lewistown, Montana as an additional method control. Eggs from these fish are referred to as SPC. These gametes were obtained in bulk as eyed eggs (i.e., already fertilized). Parents were unknown and mixed, thus whole body parental tissue concentrations were not obtained. Since the SPC eggs were a batch sample, they were submitted to the analytical laboratory for total selenium analysis as one sample.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

9 The hatchery fish that were obtained served as method controls for the experimental process.. Hatchery fish and the resultant eggs are not subject to the same rigors as wild fish. The additional stresses that wild fish face beyond selenium exposure are not present for hatchery fish. As these non-selenium stressors for wild fish can also affect the test endpoints, including hatchery fish that do not experience these stressors in comparisons between these populations to study endpoints is inappropriate.

While hatchery fish were not used to quantitatively assess effects endpoints, the hatchery fish are important to illustrate the range of method variability that can and does occur in larval fish survival, growth, and deformities when no seleni um exposure has occurred. The data for these hatchery fish and the measured response are included alongside the field-collected fish to illustrate that variability.

2.2 Reproduction Study and Laboratory Test Methods

The reproduction portions of this testing were carried out by ENSR’s environmental toxicology laboratory in Fort Collins, Colorado. The methods are presented in detail in Appendix A. The study plan design was initially developed based on exposure areas and grouping of fish from the same collection areas. However, the study approach was modified to collect trout at several different locations (exposure areas) and raise eggs from each maternal fish as an independent unit (i.e., paired data). The collection of paired data for individual fish is expected to provide better insight on the relationships between tissue concentrations and reproductive success.

Adult trout carcasses and a subsample of eggs were sent to CAS for analysis of total selenium and percent solids. Tissue was analyzed for selenium using Gaseous Hydride Atomic Absorption Spectroscopy (GH-AAS) Method 7742. Percent solids were measured via freeze drying.

2.3 Deformity Assessment

Dr. Kevin Bestgen at Colorado State University’s Larval Fish Laboratory was contracted to conduct the deformity assessment. He evaluated over 10,000 individuals and each individual fish was evaluated for up to four different deformities and four possible levels of deformity (Appendix C). Dr. Bestgen developed a process for assessing deformities which gave specific measurements to each ranking, thereby allowing for some measure of repeatability and accuracy. He received samples essentially as blinds because he did not know what the sample
locations were or their locations relative to selenium concentrations.

The general criteria were adopted from Holm et al. (2003), and included assessments of craniofacial deformities (mostly of the head, eyes, and jaw), vertebral deformities, fin deformities, and edema. The original publication showed pictures of some deformities butBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

10 others, particularly the intermediate categories, were not illustrated or were poorly described. More specific definitions for each of the assessment categories were developed to give better repeatability and consistency across studies, and to aid others in learning the range of deformities possible.

Deformities in each of the categories described above were given a score from 0-3, with 0 being a normal condition and 3 being the most deformed. Some range finding was conducted over the first several samples to find background and severe levels of deformities in each category. Initial samples were rescored as necessary to bring them into compliance with the standards that were used throughout the assessment.

The protocol for assessing damage was to place several fish, head to the left, in a Petri dish and examine them under a dissecting microscope and 10X magnification. The lateral side was examined for spinal deformities (lordosis, kyphosis), appearance of the eye, head and snout shape, edema, and fin deformities. The fish was turned ventrally to look for mouth deformities and further spinal deformities (scoliosis), turned laterally again for the same criteria as the other side, and then dorsally for issues associated with eyes, head size, spinal deformities.

Craniofacial deformities included shortening of the jaw, snout, and missing or poorly developed eye or eyes, and head shape abnormalities. A slightly shortened lower jaw (<= 1 lip width) received a 1, a shortened jaw = 2 lip widths or a slightly shortened and slightly disfigured jaw = 2, and a flat lower jaw or much disfigured (non-functional) jaw = 3. An assessment of fish independent of this study revealed that other brown trout of the same size and developmental state did not have the slight deformity that was assessed as CF =1 for the jaw (J). Thus, the CF = 1 score where the J was concerned were deemed real. A slightly blunted snout (about 50 percent eye diameter, usually is > than that) = 1, ve ry blunt or flat = 2, deformed or bulbous = 3. Eye deformities were scored as one eye blind or poorly pigmented or poorly developed =1, both poorly developed = 2, both blind = 3. Skulls t hat were slightly bulbous (1/3 > normal) = 1, moderately bulbous (2/3 > normal) = 2, and bulbous (1x or > than normal) = 3.

Skeletal deformities included any deformity of the vertebrae or spines. A slight bend of less than 45 degrees (but > than body width off of straight) or a minor body constriction (e.g., a tight rubber band about the body effect) was given a score of 1, 2 slight bends or constrictions anywhere, or bend of > 45-90 degrees was scored a 2, and multi-directional bends > 90 degrees were given a 3.

Fin deformities included variation in fin or finfol d morphology and a slightly smaller or missing fin (in thin fish, the adipose fin was often absent, indicating fat absorption, not uncommon and scored 1) or one with a bend or incomplete ray development (in older fish) was given a 1, 2 fins damaged or malformed = 2, and > 2 fins malformed or if fins were missing (except adipose) was
= 3.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

11 Edema was detected by an obvious swellin g and fluid buildup, usually abdominally, and ventrally, which often displaced the gut, and was us ually clear fluid that was slightly soft when touched with a blunt probe. Slight edema = 1 was for a fish with up to 1X swelling of the normal body width or depth, up to 2x = 2, and > 2x = 3.

2.3.1 Data Reduction of Deformity Rankings

Individual files, representing scoring sheets, were received for each sample evaluated. All files were combined in Excel to form a master file. Data were summarized using the Pivot table function in Excel to produce counts and percentages of normal fish, deformed fish, and total number of fish evaluated. Similar to the method of Holm et al. (2005), a Graduated Severity Index (GSI) was derived based on the deformity rankings and counts for progeny from each parent. A total score was computed as follows:

[(# fish for CF=1) x (1)] + [(# fish for CF=2) x (2)] + [(# fish for CF=3) x (3)].

This method differs slightly from Holm et al. (2005) as it weights each ranking with more weight given to more severe deformities. Fish scored as 0 (normal) observations did not enter into this calculation of total score. The final GSI score was computed as the sum score/total # fish including those ranked as "0". The total scores were summed and divided by the number of categories of deformities assessed (usually 4) to derive a mean GSI score.

Because the USEPA’s Toxicity Relationship Anal ysis Program (TRAP) version 1.2 (Erickson 2008) logistic functions were designed to deriv e an inverse sigmoidal curve, commonly used to illustrate the dose-response curve of increasing exposure concentration and declining biological observation (e.g., survival, growth, etc.), deformities were evaluated as the sum fraction of normal fish (sum of normal fish/ total number of fish) for each deformity. This approach did not take into account severity of deformity, simply the frequency of deformities which is consistent with USEPA’s (2004) approach to analysis of similar data.

2.4 Statistical Analysis

Multiple test-effects endpoints were measured at different times during the test including: fecundity, fertilization success, hatching success, deformities, length, weight, survival (different times during the study), and tissue concentrations (egg and whole body). These endpoints were consistent with those of Holm et al. (2005), Hardy (2005), and Kennedy et al. (2000). Feeding success was added as a test endpoint to evaluate the change from endogenous to exogenous feeding post swim up.

Scatter plots and best-fit ordinary least squares (OLS) regressions were used as an exploratory tool to evaluate the potential for meaningful relationships. Ordinary least squares regressionBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

12 analysis was used as a preliminary method to assess if relationships existed between individual exposure assessment endpoints (i.e., parental selenium body burdens or egg selenium concentrations) and test-effects endpoints measured in the study. The dose-response relationships for exposure and effects endpoints were evaluated further. Logistic regression analysis was performed using USEPA regressi on-analysis software (TRAP version 1.2; Erickson 2008) for the effects endpoints showing the strongest relationships to the exposure endpoints. USEPA’s TRAP software provides a number of statistical analysis tools, including logistic regression, to evaluate the presence of dose-response relationships. The logistic regression approach is consistent with the met hods utilized by the USEPA in their assessment of dose-response data for the 2004 Draft Criterion. USEPA’s TRAP software also allows for prediction of Effect Concentration (EC) values to estimate thresholds for potential effects for
brown trout. Both EC 10 and EC 20 values were derived for each relevant relationship developed.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

13 3.0 RESULTS

3.1 Surface Water Quality

Water quality data were collected in October during the initial effort to capture spawning trout. Tables 3, 4, and 5 present field measured parameters, conventional, and selenium concentrations in surface waters, respectively. At the upper Crow Creek locations (CC-75, 150, and 350), total selenium ranged from 0.00055 (CC-75) to 0.0015 mg/L (CC-150). In Sage Creek at LSV-2C, the selenium concentration in surface water was 0.0384 mg/L. At the lower Crow Creek locations downstream of Sage Creek, selenium concentrations were 0.0017 mg/L at CC-3A and 0.0028 mg/L at CC-1A. At both the Montpelier and Stump Creek locations, selenium was less than detectable (0.0002 mg/L) (Table 4). No additional water quality data were collected in November, as the time frame between the October and November sampling period was short. The expected exposure for fish from an exposure area is aggregated over a period of time, thus the exposure is best represented by the dietary and surface water selenium concentrations occurring prior to the time of sampling.

Water quality data for selenium have been collected at numerous locations within the study area and outside the study area over a fairly long period of time. Two additional sources of data are
available to evaluate selenium concentrations both temporally and spatially. These datasets
include:

 Fall 2006 to Spring 2008 surface water quality data collected for the SSSC study; and

 Spring and Fall annual effectiveness monitoring program data.

Information from these datasets is presented in Section 4 to help put selenium concentrations in
surface water measured in October 2007 in context.

3.2 Wild Fish

During the October sampling period for spawning brown trout, approximately 300 adult brown trout were captured and 104 were retained as potential spawners. Of these, 36 were males and 68 were suspected females (Table 1). After a brief holding period all of these fish were released at the locations from where they were colle cted, because no eggs could be stripped from the
female fish.

During the second sampling round in November, more than 300 adult brown trout were collected over a five day period. Equivalent numbers of prospective ripe males and femalesBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

14 were retained. Eggs were collected from 26 ripe females from three locations representing a range of ambient selenium concentrations in water, including two locations in the watershed upstream of Sage Creek and one location in Sage Creek. Consistent with the October sampling, the largest number of brown trout was collected in Sage Creek from upstream of South Fork Sage Creek to Hoopes Spring. During October and November, several brown trout were collected at the Meade Peak Ranch (CC-1A and CC-3A). However, no ripe females which would readily express eggs were captured. No ripe fish were collected from the Stump Creek reference area (Table 5).

3.2.1 Adult Size and Selenium Concentrations -Whole Body and Eggs

The initial target range for fish age and size was 3+ years (>230 mm). The trout collected from the field ranged from 265 mm to 391 mm with most of the females being 300 mm or larger. Ripe fish, particularly females, were generally closer to 300 mm in size and often larger than 300 mm. It is assumed that the size range of female fish collected represents the 3 and 4 year old age classes. A narrower range of adult sizes would have been preferred because fish size (i.e., age and maturity) can affect egg production. However, the number of ripe females collected made it necessary to include fish from a wider size range to meet study goals.

The whole-body selenium concentrations measured in the adult female carcasses whose eggs were used in this study are presented in Table 6. Whole-body selenium concentrations measured in field-collected brown trout ranged fr om 4.7 to 22.6 mg/kg dw. Figure 3 shows adult hatchery and wild fish lengths relative to thei r respective whole body selenium concentrations. The hatchery fish are larger and have a lower whole body selenium concentration, while the wild fish have considerable variability in whole body se lenium concentrations reflecting the varying histories of selenium exposure. Figure 4 shows a similar relationship using adult fish weight (wet). The range of hatchery fish weights is larger than that observed for length, however, a similar trend is observed for wild adult fish, exhibiting relatively consistent weights despite the variation in whole body selenium concentration.

Figure 5 shows the size distribution (based on adult length) versus the whole body selenium concentration for the adult female brown trout collected as part of this reproduction study in November (red asterisks), the brown trout tissue concentrations collected as part of the field monitoring for the SSSC studies (Table 7), and any brown trout data collected since 2004 when tissue data were collected for the Smoky Canyon Mine Site Investigation. The range of tissue concentrations illustrated in Figure 5 (excluding the adults for the reproduction study) represents the brown trout data available at the time the adult study was being planned and are the basis for the number of samples derived (Section 2.1) as a target for field collection. It is important to note that previous monitoring and redd evaluations indicated very low numbers of adult fish of spawning size found in the Hoopes Spring channel (HS-3) or Hoopes Spring location (HS). As shown in Figure 5, data for fish tissue concentrations greater than 25 mg/kg dw were only foundBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

15 in fish from Hoopes Spring. The range of tissue concentrations for female trout collected in November spans nearly the entire range of brown trout tissue concentrations collected from the previous field monitoring data.

The concentrations of selenium in egg batches from each female parent were also measured. Selenium concentration in fish eggs collected from CC-150 ranged from 6.2 to 12.8 mg/kg dw. Egg selenium concentrations from CC-350 ranged from 6.9 to 14.0 mg/kg dw, while those from LSV-2C ranged from 11.2 to 40.3 mg/kg dw (Table 6). Figure 6 shows the egg selenium concentrations ordered from lowest to highest from hatchery fish and wild collected fish. Appendix D includes the raw data for selenium concentrations in whole body and egg tissue.

3.3 Hatchery Fish

The Saratoga National Fish hatchery (SC), Saratoga, WY, was the first source of brown trout eggs for the laboratory controls in this study. Because these fish were spawned about 3 weeks ahead of the field-collected (wild) fish, due to spawning time in the field versus spawning time at the hatchery, it was not possible to select smaller spawning females from the hatchery. These eggs were fertilized at ENSR following collection at the fish hatchery. As discussed later, hatch rates were lower than expected and some fungal contamination occurred in the SC egg batches, therefore, a second set of hatchery eggs were obtained. Spawning was complete for SC hatchery fish, thus an alternative source of eggs was necessary.

The Spring Creek Trout Hatchery (SPC), Lewistown, Montana, was the second source of brown trout eggs for the laboratory controls in this study. This second set of eggs was obtained to ensure that the test system was suitable. Because it was late in the season these eggs were only available as eyed–up embryos.

3.3.1 Adult Size and Selenium Concentrations – Whole Body and Eggs

Eight adult trout from the Saratoga hatchery were used, ranging in length from 420 mm to 562mm. Hatchery fish were larger than fish captured at field locations (Table 6). Maternal wholebody selenium concentrations measured in the hat chery trout ranged from 2.5 to 4.3 mg/kg dry wt. Bulk eyed-eggs were received from the SPC, thus no information of specific adult size and whole body maternal tissue concentrations was gathered.

The selenium concentration of the SC hatchery eggs ranged from 0.76 to 1.2 mg/kg dw, while the selenium concentration in the SPC hatchery eggs was 0.73 mg/kg dw (Table 6). As illustrated in Figure 6, egg selenium concentrations in the hatchery fish were considerably lower than egg selenium concentrations in the wild fish.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

16 3.4 Reproduction Testing

Appendix A details all laboratory results of the reproduction study. The following endpoints were measured in the laboratory: fecundity, hatch, growth, survival/mortality, and feeding success (growth) post swim up. Additional measures included day to first hatch, day of swim up, and day of test termination.

3.4.1 Fecundity

Fecundity, measured as the number of eggs/female, was measured for each parent. Section 3.1 of Appendix A details egg quantity per female, number of eggs used in the study, and selenium tissue concentrations. Twenty-six egg samples from wild-collected brown trout were submitted to ENSR for the reproduction study. Of these, one set of eggs was dead upon arrival(LSV-2C-006). The number of eggs from individual field-collected fish ranged from 161 (LSV2C-010) to 1,658 (CC-150-016). The number of eggs collected from the individual SC hatchery fish ranged from 1,248 to 5,448. Figure 7 illustra tes the number of eggs relative to adult size (length). As mentioned, the hatchery fish were larger than the field-collected (wild) fish, and on average had almost two times or more eggs.

Figure 8 shows the relationship of the total number of eggs produced from each female versus the respective concentration of selenium in the eggs. Figure 9 shows the relationship of the total number of eggs produced from each female versus the respective concentration of
selenium in the adult female fish.

3.4.2 Egg Mortality

The goal of the test was to begin each test chamber with 600 fertilized eggs. Not all wild fish produced sufficient egg numbers to do this, so a proportion of eggs was used to begin the test that allowed for adequate biomass of eggs to be left over for selenium concentration analysis. The minimum number of eggs included in a test chamber was 100 (LSV-2C-010). For sample LSV-2C-006, the entire egg batch was declared dead upon delivery to the laboratory and was not included in the test. For sample LSV-2C-007, eggs were initially included in the test, but it was determined, due to high numbers of eggs turning opaque, that these eggs were not fertilized, thus eggs from this sample were also not included in the test. Maternal whole body tissue and an egg subsample were collected for these two samples. Table 3-1 in Appendix A summarizes the number of eggs placed in the study for each sample. Egg mortality was
measured based on the number of fish that hatched subtracted from the number of eggs at test initiation for each sample.

Egg mortality for the SPC parents was low (<3 percent), while egg mortality for SC parents ranged from 6.8 percent to 88 percent. It is not clear if higher egg mortality in SC derived eggsBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

17 was due to incomplete fertilization or due to the fungal problem that arose with these eggs. Egg mortality from parents collected at the background areas ranged from as low as about 3 percent to as high as 85 percent. From the LSV-2C exposure area, egg mortality ranged from 1 percent to 49 percent across each of the conditions: hatchery, background (CC-150), low (CC-350), or high (LSV-2C) exposure, egg mortality was highly variable (Figure 10).

3.4.3 Hatch to Swim Up

Appendix A, Table 3-3 summarizes hatch data for the different samples, including day of first hatch, percent hatched, day of swim up, and percent swim up. Day of first hatch was simply the day when hatching was first observed for eggs in a sample. Percent hatch was the number of eggs that successfully hatched divided by the original number of eggs for that sample times 100. The day of swim up was when greater than 80 percent of the alevins for a specific clutch of eggs reached swim up (i.e., absorbed their yolk and were actively feeding). Percent swim up was the number of alevins that had absorbed their yolk sac and were actively feeding divided by the total number of eggs used to begin the test times 100.

The percent hatch for the SPC hatchery fish was 97.5 to 100 percent. The holding period was significantly shorter for SPC eggs. This difference may contribute to the higher hatch success. The percent hatch for the field collected eggs was 11 to 93 percent. Eggs samples collected from CC-150 ranged from 14.8 to 97.3 percent, with an average of 71.9 percent. Average hatchout for the eggs collected from fish at CC-350 was 56.4 percent. Average hatch out from LSV2C was 83.9 percent (not including LSV-2C-007 whose eggs were not fertilized). The lowest percent hatch from the LSV-2C treatments was 50.6 percent, while the highest was 99.0 percent. The field collected fish eggs (which were fertilized in the field) indicated that the change in fertilization technique (field fertilization vs. laboratory fertilization) resulted in a higher hatch success rate compared with the SC hatchery fish (which were fertilized in the laboratory). Figure 11 illustrates percent hatch for each sample ranked from lowest to highest egg selenium concentration. Figure 12 shows the relationship of percent hatch to swim up. There is for most fish, a 1:1 relationship of hatch to swim up. However, for several LSV-2C samples, while eggs hatched (>50 percent), yolk fry never swam up, despite the extension of the study 15 days past the time of swim up to assess feeding transition. The range of egg selenium concentrations for those alevins that did not swim up was 26.8 to 40.3 mg/kg dw. Except those five samples shown for LSV-2C, eggs that hatched resulted in young that were able to reach swim up. As shown in Figure 13, the eggs from those five samples had the highest selenium concentrations (i.e., greater than 25 mg/kg dw). Figure 13 shows the number of days to first hatch and swim up versus egg selenium concentrations. While hatch rates were similar across the board, the number of days to swim up increased with egg selenium concentrations greater than 25 mg/kg
dw.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

18 3.4.4 Mortality

Mortality was assessed at several different periods during the test, including:

 at swim up ([number of eggs used to begin the test - number of fish surviving to swim up] / total number of eggs at beginning of test] * 100);

 from hatch to test termination ([percent survival at hatch - percent survival at test end]);

 at test termination as overall mortality ([number of eggs used to begin the test - total number of fish at the end of the test] / total number of eggs at beginning of test] *100); and

 at the end of the 15-day post swim up feeding success trial ([number of fish used to begin the post swim up feeding trial, usually n = 100 – number of fish at the end of the feeding trial at 15 days / total number of fish used to begin the test] * 100).

Mortality at swim up ranged from 0 to 6 percent in SPC fish, 7 to 90 percent in SC fish, and about 4 to 92 percent in wild fish (excluding the samples where no eggs were fertilized or eggs died before test initiation). Figure 14 shows mortality at swim up and overall mortality at the end of the test by location ranked by egg selenium concentration. It shows that overall mortality closely tracks mortality at swim up for most samples, except those from LSV-2C where overall mortality is greater than swim-up mortality. No real trends are observable from these data.

Figure 15 shows mortality in a similar fashion for the period, hatch to test termination. Mortality from hatch to the end shows a clear trend of low mortality in most all samples (i.e., <10 percent) and much higher mortality in a number of samples from Sage Creek below Hoopes Spring.

Percent mortality for fish from hatch to test end was substantially greater in the same five LSV2C samples as those that did not achieve swim up (Section 3.4.3) and that had the highest egg selenium concentrations (greater than 25 mg/kg dw). Mortality during this period drops substantially in samples where egg selenium was about 20 mg/kg or less as shown by the 10 percent or less mortality for all remaining samples.

Finally, mortality as a function of the 15-day post swim up feeding trials was evaluated and is shown on Figure 16. The duration during which mortality was measured for this phase of the test was short, extending from swim up to 15 days past swim up to evaluate if fish could transition to active feeding. Similar to mortality from hatch to test end, mortality during thefeeding trial shows highest mortality only occurred in the LSV-2C samples. Isolation of posthatch mortality shows that for fish from most samples, mortality is low and only increases at locations where egg selenium concentrations were highest. Furthermore, the greatest percentage of mortality occurred in the last 15 days of test as shown in the difference between mortality rates in Figures 15 and 16. This is consistent with the prior observations that severalBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

19 of the samples from LSV-2C with the highest egg selenium concentrations did not swim up, therefore, their ability to transition to active exogenous feeding was impaired.

3.4.5 Growth

Growth endpoints measured include length and dry weight. The results of length and dry weight analysis for the target of 20 organisms per chamber at the end of the 15-day post swim up phase of the study are provided in Appendix A, Table 3-5. Average length of fish per sample location is shown in Figure 17. There were no apparent differences in length noted between hatchery fish and wild fish, or among wild fish, following the feeding trial. Growth as measured by dry weight is shown in Figure 18. Similar to the length data, these are presented in a rank order from lowest to highest egg selenium concentrations. There does appear to be an inverse relationship of increasing egg selenium and reduced dry weight. However this difference is not as apparent when the hatchery fish are excluded.

3.4.6 Deformities

Appendix B provides a summary of the counts of deformity rankings for each sample and a series of graphics illustrating sample deformities as a percentage of each sample. Observations made during scoring that resulted in defining a level of severity for a fish being examined are reported below:

 Cranio-Facial Deformities - Usually factors occurred together so a combination of two “1” conditions = 2, three “1” conditions = 3, or a 1 and a 2 = 3, and so on. For example, a deformed jaw and a blind eye = 2, two blind eyes = 2, but a badly deformed jaw (= 2 alone) plus a blind eye (= 1 alone), = 3.

 Skeletal Deformities - Bends caused by skeletal deformities were usually detectable from normal bending of the body during preservation (these fish were usually well preserved, very straight) by presence of a slight or greater bump below the surface of the epidermis on the outside of the bend. However, some fish with SD = 1 had just a very slight bend in the range the deformity described but could be due to preservation or the poor condition of the fish. This was sometimes especially true in larger fish, which may be more muscular and undergo stronger contraction during preservation and thus, bend slightly. A score “CF = 1” was a slight deformity, if at all. The scores of SD = 1 involving kyphosis or lordosis were deemed real because that is an unusual preservation deformity. Also, samples BKD 015 SU (i.e., extra fry from CC-150-015 at swim up), LOW 008 SU (i.e., extra fry from CC-350-008 at swim up), and SC 003 SU (i.e., extra fry from SC-003 at swim up) were re-examined; most fish were very straight so some samples with higher SD scores (e.g., post swim-up samples) were determined accurate. Thin fish were difficult to score, as they often looked emaciated.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

20  Fin and Finfold Deformities - Often fins were malformed associated with vertebral deformities that did not permit proper development. Folded finfolds as a result of preservation were not counted.

 Edema - Edema was not originally scheduled for assessment because it was thought sometimes not a teratogenic effect and may be transitory as fish develop. However, it was assessed because it was common in one early sample and not others, and because it was thought a condition that could affect emergence, mobility, and other factors that may limit survival of fish in the wild. The yolk, which was present in some quantity in some study specimens, also created some swelling but was typically yellowish, opaque, and small, and hard to the touch in preservation.

A sample of 50 fish and a sample of 30 fish were scored twice, the same fish for each batch but not necessarily the same order. This sample was characterized by a low incidence of fin deformities (slow development) and a high incidence of jaw deformities and blindness (SC 003 SU). Those cranio-facial traits are difficult to score because they are additive, and subjective as to severity. Thus, the results may be a conservative view of what score replicability should be like for other traits in other samples that are easier to score.

Replicability of frequency of cranio-facial abnormalities was high among assessments at 50 and 52 percent in the first sample of 50 fish, and identical frequencies of 46.7 percent in each assessment for the sample of 30 fish. The cumulative sums of the scores were also quite close, but reflecting variability in scoring for all three categories of severity in each sample. Replicability of fin ray development assessments for both frequency and the sum of the scores was identical in both samples.

The results of the deformity assessment are visually displayed in Figures 19 through Figure 22. The results of the cranio-facial (CF), skeletal (SK), finfold (FD), and edematous tissue (ED) deformity frequency are depicted separately. Each of these figures is similar for the remaining deformity assessments and present the data on a percent basis for each of the rankings used in
the assessment.

Cranio-Facial Deformities

For CF deformities (Figure 19 and Appendix B Figure 1), hatchery fish, except young from two parents, showed that greater than 80 percent of the young in each sample were ranked as normal. When deformities were present in the hatchery fish, they were ranked as slight. For the two samples that had higher numbers of non-normal fish (SC-003 and 004), craniofacial deformities ranged from slight to severe. For wild fish, from background and low areas (CC-150 and CC-350) from about 65 to 75 percent of all fish were ranked as normal for cranio-facial deformities. CF deformities ranked as slight were observed more often in fish from CC-150. AtBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

21 CC-150, the percentage of normal fish ranged from 52 to 96 percent, whereas at CC-350 it ranged from 59 to 93 percent. For the CC-150 and CC-350 samples, fish not ranked as normal were predominantly ranked as slightly deformed for the CF characteristic. For samples from the LSV-2C area, more than 70 percent of the fish examined ranked as normal. Of the 12 samples submitted from the LSV-2C area, five (LSV-2C 003, 004, 005, 021, and 010) had a higher incidence of slight, moderate, and severe CF deformities in fry examined. For these five samples, fish were more often ranked as moderately deformed than those ranked as slightly deformed for this characteristic. In the remaining 7 samples, from 77 to 97 percent of fish in each sample ranked as normal, similar to the hatchery or background samples (Figure 19).

Skeletal Deformities

For SK deformities, the range of normal fish found in each sample was 60 to 91 percent and collectively, for both sets of hatchery fish was greater than 80 percent for all samples. In fish samples from CC150 and 350, the range of normal fish was 51 to 92 percent. For all sample from CC-150, 80 percent were ranked as normal fish, while at CC-350, only 66 percent of fish were ranked as normal. For fish from the LSV-2C area, the range of normal fish was 0 to 93 percent (Figure 20 and Appendix C Figure 2). Overall, 65 percent of the fish were ranked as normal. For this deformity category, there was a higher frequency of fish in most samples ranked as slight. The frequency of SK deformities in the moderate to severe range for hatchery
fish or fish from CC-150 was low. Moderate to severe SK deformities were observed more frequently in fish from CC-350. Fish from LSV-2C showed a similar trend for SK deformities to those observed for CF deformities. The same five samples had higher percentages of the sample with slight, moderate, and severe SK deformities. For some of the fish in these five samples, larvae had deteriorated and could not accurately be ranked (shown as the blank category Figure 20). As noted in Appendix A, some larvae were clearly dying, but not dead, thus began deteriorating prior to them being preserved.

Fin or Finfold Deformities

For fin or FD deformities, the range of hatchery fish ranked as normal was 81 to 100 percent while overall for both sets of hatchery fish, greater than 90 percent of the fish were ranked as normal. For fish from CC-150, the range was 92 to 100 percent of fish ranked as normal in individual samples, while overall, 98 percent of the CC-150 fish were ranked as normal. The range of fish ranked as normal for CC-350 samples was 73 to 89 percent while overall, 85 percent of the fish from all samples were ranked as normal. Fish ranked as slight moderate, and severe for finfold deformities were observed more often in samples from CC-350 but the percentages were 6 percent or less for each of these categories. Normal fish from the LSV-2C area ranged from 0 to 100 percent. Across all samples, 80 percent of the fish were ranked as normal. Excluding samples LSV-2C 003 and 010, whose fish were too deteriorated to accurately assess fin deformities, the range of normal fish was 16 to 100 percent normal (FigureBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

22 21 and Appendix C Figure 3). The same five samples from LSV-2C either could not be evaluated for this characteristic, due to the condition of the fish in that sample, or had higher frequencies of moderate and severe fin deformities. As stated previously, several samples from LSV-2C experienced mortality before the swim up stage or did not swim up at all. Some of these fish were deteriorated and an accurate assessment of the severity of the deformity for this
condition could not be made.

Edema Deformities

ED was almost non-existent in hatchery fish and fish from CC-150 and CC-350 (Figure 22 and Appendix C Figure 4). Similar to the other three categories of deformities evaluated, edema was present at a higher and more severe level in the same five samples as identified previously for LSV-2C samples.

Graduated Severity Index (GSI)

To capture the severity of each of these deformities into a single unit, a GSI was derived for fry originating from each egg batch as described in Section 2.3.1. The mean GSI score for all treatments ranked from left to right by the lowest to highest egg selenium concentrations is shown in Figure 23. Because a fish can have more than one deformity score, rankings higher than 1 are possible. Mean GSI scores show that for most samples GSI scores are 0.2 or less and a small number of samples ranged from 0.2 to 0.4. Mean GSI scores of 0.4 or less span the range of treatments/sites (hatchery and field). Only a small number of samples, the same five as identified in previous sections, from LSV-2C had mean GSI scores that were substantially greater than 0.4, ranging from 0.87 to 1.7 indicating that these samples had a higher frequency and severity in the level of deformities found. Figure 23 also shows the number of fish assessed from each sample.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

23 4.0 ANALYSIS OF RESULTS

The following section presents preliminary analysis of the brown trout study data. A focus of these analyses is the relationship between maternal tissue concentrations and various reproductive effects identified in literature. Additional analyses will ultimately be conducted in conjunction with the full range of Site-specific information on exposure conditions and the biological community structure. These findings will also be contrasted with the work of others.

4.1 Surface Water Quality

Table 4 shows selenium concentrations from October 2007 surface waters, as well as selenium concentration data collected from pre- and post-October 2007 monitoring events. Both total and dissolved selenium analyses were conducted, and di ssolved selenium is dominant in all surface water samples. At all of the upstream background locations on Crow Creek, the October 2007 total selenium concentrations are within the range of total selenium concentrations measured from 2006 to 2008 during seasonal monitoring. Total selenium concentrations at the Crow Creek locations upstream of Sage Creek (CC-75, CC-150, CC-350) are always below Idaho’s Surface Water Quality selenium standard (0.005 mg/L).

The total selenium concentration at LSV-2C in October 2007 was high (0.0384 mg/L) compared to previous sampling events at this site. Figure 24 shows longer-term monitoring at the Hoopes Spring location immediately upstream of the LSV-2C location and shows that surface water selenium concentrations were increasing in 2007 and 2008.

Longer-term water quality monitoring in Sage Creek near Crow Creek road (LSV-4) provides information on selenium concentrations in Sage Creek before discharge to Crow Creek. Figure 24 shows longer-term selenium concentration data from this location and a gradual increase in selenium concentrations during 2007 and 2008 cons istent with increases observed at Hoopes Spring and South Fork Sage Creek Springs. Concentrations during 2007 and 2008 ranged from 0.008 to 0.011 mg/L total selenium at LSV-4.

Selenium concentrations measured in Crow Creek samples collected downstream of Sage Creek at CC-1A and CC-3A in October 2007 were lower than Idaho’s Surface Water Quality Standard. This is consistent with measurement data before and after this time period for these locations (Table 5). Selenium in Crow Creek downstream of Sage Creek is typically lower than the State Standard as illustrated by data in Figure 24 and Table 4.

Overall, selenium concentrations are highest in Hoopes Spring and Sage Creek immediately below Hoopes Spring (LSV-2C), and decrease in surface water with distance downstream ofBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

24 Hoopes Spring (Figure 24). In Sage Creek upstream of its discharge to Crow Creek, selenium concentrations exceeded the State Standard of 0.005 mg/L, recently by about 2 times. In Crow Creek downstream of Sage Creek, infrequent exceedences of the State Standard have been observed. Regular monitoring data suggested selenium in Crow Creek downstream of Sage Creek is on average about 0.5 times the State Standard (0.005 mg/L). These surface water data set the stage for selenium exposure to fish and their food resources. Exposure is primarily dietary for fish but does include an aqueous component. Whole body maternal tissue concentrations of selenium described below integrate the water and diet exposures to provide a good indicator of exposure and bioaccumulation.

4.2 Relationship of Maternal Whole Body to Egg Selenium Concentrations

While more recent studies focus on selenium concentrations in eggs as the best predictor of reproductive effects there is also a substantial amount of information on whole body tissue concentrations in the literature. This initial regression analysis was performed to better understand the relationship between maternal whole body and egg selenium concentrations, and the results of the preliminary analysis confirm a strong relationship. The remainder of the analyses in this study focuses on the effects endpoint relationships to egg selenium concentrations. The relationship between egg selenium and whole body selenium allows for consideration of both measures as the study progresses.

Both selenium concentrations in whole body maternal fish and their eggs were measured. Figure 25 shows the relationship of egg selenium to whole body tissue selenium for wild brown trout collected for this study. This relationship is strong (R 2 = 0.80) with 80 percent of the egg selenium variability in wild fish explained by the selenium content of maternal tissue. It is important that this relationship be strong in order to confidently predict whole body tissue concentrations from egg concentrations. This is consistent with USEPA (2004) methods that developed translators for various tissue types. In this case, effects are related to egg selenium concentration, which, based on the current literature, is the best tissue for measuring a response for effects due to selenium.

4.3 Relationship of Egg Selenium Concentrations to Specific Effects Endpoints

Data for selenium in brown trout egg tissue are evaluated for effects on various endpoints for reproduction. Scatter plots and best-fit ordinary least squares regressions are used as an exploratory tool to further evaluate potential relationships. These regression relationships are not used as a final analysis tool or to determine biological significance of the regression because the distribution of the various effects endpoints (e.g., dependent variable) is typically skewed. Tests for normality were conducted and these data are not normally distributed nor are they log-normally distributed, which is one of the underlying assumptions of a least squaresBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

25 regression analysis. Use of logistic regression approach as an analysis tool is presented in
Section 4.4.

Figure 7 shows total number of eggs per female versus the individual female length. As expected, the smaller, wild fish produced fewer numbers of eggs relative to the larger, hatchery fish. Size may not be the only controlling factor. Overall health of the fish due to environmental and or chemical stressors may also play a role, as discussed previously. As shown in Figures 8 and 9, selenium concentrations in eggs or whole body adults, while varied, suggest relatively similar egg production, although when just examining wild fish egg production, there does appear to be a relationship of decreasing egg abundance with higher parental or egg selenium concentrations. Figure 26 shows wild brown trout egg abundance relative to selenium concentrations in egg tissue. For fish collected as part of this study, the relationship is poor (R 2 = 0.2); only a small percentage of the variability in observed egg production for wild fish is explained by egg selenium concentrations. Because fecundity can be related to the size of the female (i.e., heavier, longer fish, are indicative of older fish potentially in a more robust condition), these data were also normalized to 320 mm by dividing the total number of eggs by the length of the female parent. This value was then multiplied times 320. The red diamonds on Figure 26 illustrate that length-normalized fecundity does not reduce variability nor improve this weak relationship. Based on these data, it is difficult to suggest that fecundity is an important endpoint in assessing selenium effects in trout. The literature and field data will be used to further assess this endpoint through examination of literature-reported fecundity effects and observed population data from the Crow and Sage Creeks.

Figure 27 illustrates the percent hatch of brown trout fry versus egg selenium concentrations for hatchery and wild fish. No apparent relationship was observed. Figure 28 illustrates both hatchery and wild fish egg selenium concentration versus the percent of fry that achieved swim up. The regression analysis indicates a possible relationship (R 2 = 0.49) between the percent of fry that swim up and egg selenium concentration.

Larval fish growth, measured as the dry weight of the organism following the 15-day post swim up feeding trial, was also investigated as a potentially important endpoint. Figure 29 shows dry weights of larval fish versus egg selenium concentrations and two relationships, one including the hatchery fish and one including only the wild fish. Both relationships show decreased dry weight with increasing egg selenium concentrations. However, the strength of the relationship is moderate at best (i.e., its predictive ability) when including the hatchery fish (R 2 = 0.66) data and poor when using the wild fish data (R 2 = 0.26). The growth data provides a strong indication about how inclusion of hatchery fish in the analysis may provide misleading results. Note the
difference in the R 2 values for the two relationships shown in Figure 29. Clearly, hatchery larvae are larger as a group when compared to wild fish. Clearly, egg selenium concentrations in hatchery fish are lower than any wild fish collected. However, many other factors includingBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

26 differences in nutrition, relative physical stresses such as energy needed to forage, and size of wild versus hatchery fish adults may have effects that are independent of selenium exposures.

4.3.1 Survival/Mortality and Deformities

As discussed in Section 3, survival or mortality of eggs, alevins, and swim ups was measured throughout the study. For the purpose of the following analyses and those later presented for the logistic regression analysis, survival will be the endpoint used. The logistic regression analysis software requires that the effects variables be declining in their relationship to the exposure variables, and thus, will only correctly analyze, for example, survival data, not mortality data. To put these data in the same context for those analyses that precede the logistic regression analyses, survival data will be presented.

Survival was evaluated at several key points during the life stage: egg, hatch, swim up, at test termination following a 15-day feeding trial, and as overall survival. Of these, overall survival was initially evaluated as one of the endpoints more obviously correlated to egg selenium concentration. Figure 30 shows a scatter plot of this relationship, including wild and hatchery fish. Both hatchery fish and wild fish survival rates are variable on the lower end of the egg selenium range; however, it is clear from the data presented on Figure 30 that survival drops consistently to 30 percent or less at egg selenium concentrations above about 25 mg/kg dw. A third order polynomial regression provides a best fit model for these data (R 2 = 0.543) with egg selenium explaining about 54 percent of the variability in the overall survival endpoint when only wild fish are considered. The correlation coefficient is lower when hatchery fish are included.

Given the variability of the overall survival/m ortality endpoint, as shown in Figures 14 and 15, and the influence of egg hatch on overall mortalit y/survival, the endpoint of survival/mortality from time of hatch to test end was investigated. Figure 31 illustrates this relationship including wild and hatchery fish. Review of this figure indicates a substantial decrease in survival between 20 and 25 mg/kg dw selenium in eggs. When both wild and hatchery fish are included in the 3rd order polynomial regression, the relationship is strong (R 2 = 0.90) with egg selenium concentrations explaining 90 percent of the variability in survival from hatch to test end. When considering only wild fish, the correlation coefficient R 2 remains essentially the same (0.89).

Figure 32 shows percent survival during the 15 day post swim up feeding trial versus egg
selenium concentrations. When both wild and hatchery fish are included in the 3rd order polynomial regression, the relationship is strong (R 2 = 0.96) with egg selenium concentrations explaining 96 percent of the variability in survival during the post swim up feeding trial. When considering only wild fish, the correlation coefficient R 2 remains essentially the same (0.96). Of the survival terms, survival measured during the post swim up feeding trial presents the strongest relationship to egg selenium concentrations. As discussed later in Section 4.5, theBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

27 strong relationship of post-hatch alevins and fish survival to selenium concentrations in egg or ovary tissue is consistent with the findings of others.

Frequency and severity of deformities, or lack thereof, was also evaluated relative to egg selenium concentrations. Figure 33 shows the sum fraction of non-normal fish larvae relative to egg selenium. The 3rd order polynomial function provided the best fit line (R 2 = 0.85). The increase in non normal fish is evident between 20 to 25 mg/kg egg selenium. Recall that a similar break point was also observed for survival (Figures 31 and 32) which indicates an important break point in two related but different effects endpoints.

Figure 34 shows the sum fraction normal fish versus egg selenium concentration. The 3rd order polynomial function provided the best fit line (R 2 = 0.87) and a slightly stronger relationship than the fraction of non-normal fish. This may result due to the higher numbers of fish that were ranked as normal for each sample as opposed to having some level of deformity. Again, as egg selenium concentration increases, there is a distinct break in the data for the fraction of normal fish, which in this case, decreases substantially at egg selenium concentrations greater than 25 mg/kg dw.

Figure 35 shows the mean GSI score versus egg selenium concentration and again, a 3rd order polynomial provides the best fit line (R 2 = 0.84). The mean GSI score, fraction non-normal fish, and fraction normal fish provide very similar endpoints, albeit based on the same data, the derivation for each is quite different. Fraction non-normal and normal fish are simply frequency values, while the GSI score is frequency and severity of the deformity.

Figure 33 through 35 similarly illustrate an inflection point, similar to the survival endpoints where increased effects are occurring relative to egg selenium concentrations. Due to the strong relationship of the deformity frequency and GSI effects endpoints to egg selenium, additional analyses of the individual frequency for the different deformity categories will also be assessed in the subsequent section.

4.4 Logistic Analyses

Based on the results of the preliminary regression analyses described above, a subset of effects endpoints was selected for logistic-regression analysis 2 to determine dose-response

2 Unlike traditional linear regression models, which assume equality of variance and normal distributions, the logistic regression model does not require nor have the same assumptions which can lead to Type I and Type II errors. Logistic regression has many analogies to OLS regression: logit coefficients correspond to b coefficients in the logistic regression equation, the st andardized logit coefficients correspond to beta weights, and a pseudo R 2 statistic is available to summarize the strength of the relations hip. Logistic regression do es not assume linearity of relationship between the independent variables and the dependent, does not require normally distributed variables, does not assume homoscedasticity, and in general has less stringent requirements. Logistic regression finds the equation that best predicts t he value of the Y variable for each value of the X variable. The Y variable is not directly measured; it is instead the pr obability of obtaining a particular value of a nominal variable.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

28 relationships with egg selenium concentrations. Log-transformed egg selenium tissue concentrations (mg/kg dw) were related to growth ( based on larval fish weight), survival (various stages), and deformities (various measures) (Table 8). USEPA’s TRAP software was used to derive a best fit logistic regression model for each effect endpoint distribution. Summary statistics for each regression run, a graphic of the curve plotting the actual data and predicted curve, and EC x for egg selenium residues based on the endpoint effect distribution are included in Appendix C for each of the models run.

4.4.1 Growth

Growth was measured in the post swim up feeding trial fish at the end of the 15 day period. These fish were carried through the test to the swim up stage. Twenty fish (or fewer if 20 were not available), for each sample, were fed for another 15 days to examine if there might be differences in the ability of swim ups to transition from endogenous to exogenous feeding. Morphological or physiological impairments c ould arise in young fish exposed to elevated selenium that may limit successful growth. Average growth of post feeding swim ups, as measured by dry weight, was related to egg selenium levels. Figure 37 shows this relationship for wild fish. Except for samples LSV-2C- 003 and LSV-2C-010, 20 fish were included in this analysis for every location (Appendix A). The R 2 for this model is 0.208 and, as illustrated in Figure 36, the model fit does not resemble a dose response curve. TRAP software indicates a large standard error for the steepness of the slope. Table 8 shows the EC 10 and EC 20 values for growth along with the predicted confidence intervals. Slope steepness, or lack thereof combined with wide confidence intervals in the predictive ability of the model suggests a poor relationship of growth to egg selenium concentrations. The lack of fit may be due to several
factors.

4.4.2 Survival

Survival was also measured as part of the pos t swim up study. Figure 37 shows the logistic regression curve fitted to survival data in the pos t swim up feeding trial versus log egg selenium
concentrations. The R 2 for this model is high (0.96) and the fit of the predicted data to the observed data is good. Confidence intervals are also narrow for the predicted EC 10 and EC 20 values (Table 8). Recall that percent surviv al measured as part of the 15 day post swim up feeding trial had the best fit polynomial regression to egg selenium concentration as well. While the time frame of this endpoint is narrow, it is based on eggs resulting from maternal transfer of selenium, successful hatch, successful swim up, and survival of the larvae from the alevin endogenous feeding stage to exogenous feeding stage. The dose response curve reflected by this model illustrates a similar breakpoint in effects as previously mentioned.

Total survival through the duration of the test related to log egg selenium concentration is shown in Figure 38. The R 2 for this relationship is 0.31 and the error report of the logistic regressionBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

29 model output from TRAP indicates a large standard error for the slope steepness. As illustrated, the fit of the data between observed and predicted values is low and the width of the
confidence intervals about the EC 10 or EC 20 values is large, encompassing a large range of the curve. While the endpoint is relevant, the variability of the overall survival endpoint is not well suited for the logistic function, and thus its predictive ability for EC x values is low.

Figure 39 shows the logistic regression for log egg selenium concentrations versus percent survival (hatch to test end). As illustrated, the predicted line fits the data well resulting in an R 2 of 0.89. The confidence intervals for the predicted EC 10 and EC 20 values are narrow and no errors (standard error was small and convergence was met) were reported in the TRAP software output (Table 8). Reduced variability of the survival term post-hatch likely strengthens this relationship and removes factors such as incomplete fertilization or egg viability, which could be affected by egg selenium concentrations as well as other factors. The strength of this model suggests it is a good predictor of survival effects post hatch due to selenium
concentrations.

Based on the growth and survival models eval uated, the survival endpoints provide a much stronger measure of effects relative to egg selenium concentrations than does the growth endpoint. EC x values for the three survival endpoints are very similar, although each endpoint represents different stages of development of young fish. Percent survival in the post swim up feeding study and percent survival from hatch to test end both appear to provide data that are strongly related to log egg selenium concentrations in terms of a dose response. Both predict
similar EC x values and narrow confidence intervals about the EC x . Both provide biologically meaningful and relevant measures of effects, although survival during the 15 day post swim up feeding trial is more refined, as the variability of survival pre-swim up is eliminated. Survival from hatch to test end is an inclusive endpoi nt and encompasses the 15 day post swim up survival rate. The similarity of the two endpoints and the effects predicted based on their relationship to egg selenium concentration suggests that either endpoint may provide a suitable measure from which to gage effects.

4.4.3 Deformities

The four primary deformity categories examined were: cranio-facial; skeletal; fin fold; and edema. Initial analyses were conducted to derive fractions or percentages of deformed fish relative to the total number of fish evaluated for an egg clutch. However, the TRAP software is sensitive to a declining effects response versus the exposure variable. For the purposes of fitting within the model framework, these data were structured in terms of the fraction of normal fish (number of normal fish/the total number of fish evaluated for an egg clutch). The GSI data are not structured for use in the TRAP model as GSI scores increase with increasing egg selenium. Summary data and graphics for deformity counts are presented in the earlier results section of this document and in Appendix C.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

30Figure 40 shows the logistic function for log egg selenium versus the fraction normal for craniofacial deformity assessment. Scatter of the observed values relative to the predicted values
reduced the fit of this model as well as the R 2 (0.70). The TRAP software error report of the logistic regression model output indicates a large standard error for the slope steepness. The confidence intervals for the predicted EC 10 and EC 20 values are slightly larger than that found for the total fraction normal endpoint.

Figure 41 shows the logistic function for log egg selenium versus the fraction normal for skeletal deformity assessment. Similar to the cranio-facial plot, the observed data do not fit the predicted model, although the R 2 value is higher than that of the craniofacial endpoint (R 2 = 0.81). The TRAP software error report of the logistic regression model output indicates a large standard error for the slope steepness, convergence was not reached at the maximum number of model iterations, and the steepness was at a maximum or minimum limit.

Figure 42 shows the logistic function for log egg selenium versus the fraction normal for finfold deformity assessment. The R 2 for this function is low (0.28) probably due to the lack of adequate data at the high end of the egg selenium concentration. Skeletal deformities for some samples could not be accurately assessed. The TRAP software error report of the logistic regression model output indicates a large standard error for the slope steepness, and convergence was not reached at the maximum number of model iterations. The errors associated with this model and poor fit reduce the utility of predicted EC 10 and EC 20 values.

Figure 43 shows the logistic function for log egg selenium versus the fraction normal for edema deformity assessment. The R 2 for this function is high (0.96) and the observed data fit the predicted model well. No errors were reported for this model from the TRAP software output. The predicted EC 10 and EC 20 values and their confidence intervals intersect the predicted dose response curve at the top end of the curve, with no inclusion of higher effects levels at the lower
end of the curve.

Figure 44 illustrates the logistic regression for log egg selenium versus total fraction normal. This endpoint is a summed value proportion of the total number of normal fish per egg clutch to the total number of fish examined for that egg clutch. Because an individual fish could have more than one type of deformity and because it is a summation of fractions, it can be greater than 1 and in fact could be as high as 4. These data show a good fit to the predicted function and the confidence limits for the predicted ECs are narrow. Residual error is small (Appendix E)
and the R 2 is high (0.88). A plot of the EC 10 and EC 20 and their confidence intervals over the predicted curve shows the EC values intersects the predicted line, bisecting the observed data where a clear break in effects has been previously discussed for other endpoints. The confidence limits are tight about the predicted EC 10 and EC 20 values suggesting not only a good fit, but a low variability as well. Figure 45 shows essentially the same relationship, only theBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

31 mean fraction normal was used as the dependent variable. The R 2 is the same as for sum
fraction normal and the EC 10 and EC 20 values are nearly identical.

4.5 Predicted Effects Concentration for Brown Trout – Consistency with Literature

USEPA (2004) opted to use logistic regression analysis to define the dose-response relationship to derive its Draft chronic tissue-based value. The EC 20 was used and defined as a reduction of 20 percent in the response observed at control. As presented above, both EC 20 and EC 10 values were derived using the TRAP software. In its 2004 Draft criterion document, USEPA provides the rationale for selection of the EC 20 as the chronic value. USEPA states that
the EC 20 represents a low level of effect that is gener ally significantly different from the control (U.S. EPA 1999). Smaller reductions in growth, survival, or other endpoints only rarely can be detected statistically. Effect concentrations associated with such small reductions have wide uncertainty bands, making them unreliable for criteria derivation (USEPA 2004). In his work to develop a screening benchmark, Suter (1996) indicates that “the 20 percent figure was chosen because it is a little lower than the mean level of effect on individual response parameters observed at CVs, and it is a minimum detectable difference in population characteristics in the field.” In its revision of the 2004 Draft Selenium Criterion, USEPA is contemplating the use of
EC 10 s for long-term exposure criteria for tissue. Results of the analyses presented as part of this Site-specific laboratory study include both the EC 10 and EC 20 values.

For this study, an analysis consistent with the approach utilized by EPA for the 2004 draft criterion was utilized. For these analyses, “controls” are the response of fish from background locations. Based on the preliminary analysis presented above, the best-suited end points to
calculate EC x for brown trout egg selenium concentrations (dw) would be the sum fraction normal fish (causing less than a some percent reduction in the sum fraction of normal fish/total fish), and survival (causing less than a some percent reduction in survival post hatch). In total, logistic regression models were run for nine di fferent endpoints including growth, survival, and deformities. For almost all (8 out of 9) of the models run, predicted EC 10 or EC 20 values were within 1 to 3 mg/kg dw egg selenium concentra tion of each other, respectively. The two models that appear to have a good overall fit with tight confidence intervals about the EC 10 or EC 20 predictions, and are biologically relevant, are the survival (hatch to test end) and total fraction normal endpoints. EC 10 values for these endpoints are 17.67 and 19.33 mg/kg dw, respectively,
while the EC 20 values for these two endpoints are 21.63 and 21.7 mg/kg dw, respectively.

The endpoint for survival, based on hatch to test end, is consistent with the findings of Rudolph et al. (2008), who found a significant relationship of alevin mortality to egg selenium concentration. It has been suggested that sel enium does not exert its toxic effects until a developing fish absorbs its yolk and accumulated selenium (Lemly 1997 and Holm et al. 2005 as cited in Rudolph et al. 2008). Hatchability of eggs is not affected by elevated selenium evenBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

32 though there may be a high incidence of deformities in resultant larvae and fry, and many may fail to survive (Gillespie and Baumann 1986; Coyle et al. 1983).

Data for brown trout presented in this study showed a highly variable mortality rate prior to hatch, which may have been due several factors, including incomplete fertilization, disease, or reduced egg viability due to elevated selenium concentrations. However, the latter is not consistent with the review by Holm et al. (2005) who reports that although egg selenium is present in the yolk throughout development, it may affect larval development rather than egg development because it is mobilized to a greater degree after hatch.

Deformity frequency, as measured in this study bas ed on fraction normal fish relative to the total number of fish assessed for deformities provides an endpoint that is consistent with the studies of Holm et al. (2005), Kennedy et al. (2000), Hardy (2005), Rudolph et al. (2008), Muscatello et al. (2006), and de Rosemond et al. (2005) in terms of cited developmental effects due to increased egg selenium concentrations. Table 9 shows the range of effects concentrations for the varying endpoints evaluated.

Hodson and Hilton (1983) and Lemly (1997) both suggest that developmental malformations are reliable indicators of chronic selenium toxicity to fish. Lemly (1997) described the sequence of selenium toxicity to larval fish: parental expos ure, maternal deposition of selenium into eggs during vitellogenesis, and subsequent exposure during yolk resorption in developing larvae. Both the literature and the results of this study indicate that survival and developmental malformations of larval fish are clear and supportable endpoints for developing effects concentrations for fish. For brown trout, this study indicates that EC 20 values would range from 21.6 to 21.7 mg/kg egg selenium for alevin surviv al (measured as hatch to test end) and larval deformities (measured as the sum fraction of normal fish), respectively.

4.6 Extrapolating Selenium Concentrations in Egg Tissue to Whole Body Tissue

In Section 4.2, the relationship of maternal whole body selenium concentrations to egg selenium concentrations was derived using wild brown trout collected for this study. The regression relationship has the form:

Log10(y) = 1.1926(Log10x) - 0.0071

The R 2 for whole body maternal selenium concentra tion relationship to selenium concentration in their respective eggs is 0.8. As stated previ ously, this relationship becomes important in this preliminary analysis in order to relate EC x values derived for selenium concentrations in egg tissue back to selenium concentrations in whole body fish. Outside of this specific reproduction study, the larger body of data available for this si te is for whole body tissue. Furthermore, whole body tissue concentration is a more practical endpoint to measure throughout the year than isBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

33 egg tissue. Based on an effects threshold value derived for selenium concentration in eggs and the site-specific relationship developed for whole body to eggs, a whole body tissue concentration of 13.35 mg/kg dw was derived based on the EC 20 for survival and a whole body tissue concentration of 11.27 mg/kg dw was derived based on the EC 10 for survival. Muscatello et al. (2006) reported whole body selenium EC 20 of 15.56 mg/kg dw for northern pike derived from the egg EC 20 . Hardy (2005) reported a whole body NOEC for cutthroat trout of 11.37 mg/kg dw. Conversion of the Holm (2002) and Holm et al. (2003) ovary tissue selenium concentrations presented as chronic values from ovary to whole body using USEPA (2004) equations yields the following values: 19.96 mg/kg dw (rainbow trout), 16.06 mg/kg dw (rainbow trout), and 12.24 mg/kg dw (brook trout). Currently, the Draft National criterion recommends a value of 7.91 mg/kg dw.

These preliminary values of 11.27 or 13.25 mg/kg dw were derived for comparison purposes only to evaluate if the relationships derived for brown trout are consistent with values in the literature from other studies of maternal transfer. Based on the literature reviewed, the brown trout whole body value falls within the range of whole body tissue concentrations reported for other cold water species.

4.7 Data Adequacy

The critical question to be addressed for this study is whether or not the data adequately address the range of tissue concentrations in maternal parents which ultimately affects the offspring produced. Four key points address the adequacy of the data utilized for this study:

1. The goal was to capture adult brown trout with tissue selenium concentrations greater than 20 mg/kd dw, which represented the upper 90 th percentile of the tissue selenium data for brown trout available when this study commenced. That goal was met as shown in the data presented earlier in this document.

2. Studies carried out with the brown trout and eggs collected yielded results that spanned a range of effects, including no or low effects and high and adverse levels of effects. In any toxicity study, being able to define the upper thresholds of effects is a critical component of the study. Effects were well defined for two important endpoints out of several that were evaluated, including survival and deformities for a sensitive life stage of brown trout, which are consistent endpoints defined in the literature for other trout species.

3. The distribution of effects and exposure data indicates that there are no large gaps in the data and that relationships between effects and egg selenium concentrations can be
defined with confidence.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

34 4. Collection of more fish with tissue concentrations higher than those collected for this study will not improve the study because effects are defined near the middle of the egg selenium concentrations which correspond to whole body selenium tissue concentrations that are lower than the upper whole body tissue concentrations (i.e., greater than 20 mg/kg dw).

4.8 Dose Response Analysis Update

Throughout this report, reference has been made to two particular effects thresholds, the EC 10 and EC 20 . The brown trout data presented in the Draft Final Brown Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site-Specific Selenium Criterion (NewFields 2009) were submitted to USEPA for use in their derivation of the National Criterion. The first draft of the Interpretive Report (August 2010) proposed an EC 20 as the SSC for this project based on the brown trout dat a. USEPA provided formal comments (December 21, 2010) on the Draft Interpretive Report and the criterion proposed and suggested that some alternative evaluations may be practical. USEPA’s review of the brown trout data indicates agreement with the selection of the endpoint for survival (hatch to test end). Further, the USEPA has made it clear that they intend to propose an EC 10 in their Draft National Criterion, which includes the brown trout data developed as part of this study. In their comments, EPA stated that, for this project, the EC 10 is a more appropriate endpoint than the proposed EC 20 in developing a proposed SSC for the Smoky Canyon Site. Their primary rationale is that as a bioaccumulative pollutant that accumulates in fish tissue, concentrations in fish tissue are more stable over time than aqueous selenium concentrations. This stability may lead to concentrations that are just below the criterion for extended periods of time.

The TRAP software used to derive the logistic regression conducted throughout this brown trout study also includes two additional non-linear models, threshold sigmoidal and piecewise linear models. USEPA’s comment letter illustrated an investigation of each of these models relative to the logistic model used as part of the brown trout studies presented above, and found that the projected EC x values are likely conservative. As part of the USEPA’s evaluation, another alternative examined exclusion of data points that exceeded 30 mg/kg dw in eggs, due to the fact that effects were already occurring between 15 and 30 mg/kg dw. This approach was investigated as a means of optimizing the model output. By eliminating the three highest data points, the logistic model is able to focus on the region of interest (i.e., between 15 and 30 mg/kg dw egg selenium). Using this approach, the logistic model run using log-transformed exposure data (egg selenium concentrations) versus survival (hatch to test end) results in a
model with a R 2 = 0.99 (Figure 46). This improved model results in an EC 10 equal to 20.8 (95 percent LCL – 19.89, 95 percent UCL – 21.83) mg/kg dw egg selenium and an EC 20 equal to 23.1 (95 percent LCL – 22.37, 95 percent UCL – 23.77) mg/kg dw egg selenium.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

35 Confidence intervals derived for the estimated EC x values are also tight about the estimates and
the standard error of the model is low.

The effects concentration for fraction normal fish was also re-evaluated to assess whether the previously derived EC 10 and EC 20 values were similarly affected. Using the same approach as presented above, the revised EC 10 and EC 20 values for fraction normal fish are 22 and 23.4 mg/kg dw egg selenium, respectively.

Using the brown trout regression model presented in Section 4.6 as a translator between egg selenium and whole body selenium concentrations, the resulting whole body values corresponding to the egg EC 10 and EC 20 values are 12.91 and 14.1 mg/kg dw, respectively.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

36 5.0 SUMMARY AND CONCLUSIONS

The effects of maternal selenium transfer in wild brown trout were evaluated as part of this study. Eggs from wild female brown trout co llected from different locations with varying selenium exposure levels were used to assess a number of reproductive endpoints as part of this study. Initially, the data were plotted and reviewed for any obvious relationships and patterns. In the initial review, a consistent breakpoint was identified where egg selenium concentrations were contrasted with reproduction test endpoints. These observed relationships are consistent with expected dose-response relationships.

Moving forward from these initially-defined relationships, adult whole body and egg selenium concentrations were considered the independent variables in a regression-based analysis approach. The focus of the analysis was narrowed to focus on egg selenium concentration versus growth, survival, and deformity endpoints. Logistic regression was used to develop dose-response relationships and predict egg selenium concentrations related to different effects (EC x ). Post-hatch survival and total deformity frequency (fraction normal) were found to be the most biologically relevant endpoints exhibiting dose response relationships and concurrence of observed data to predicted values. The predicted post-hatch survival EC 20 was 21.63 (95 percent LCL – 17.77, 95 percent UCL – 26.32) mg/kg dw egg selenium, while the EC 10 for this endpoint was 17.68 (95 percent LCL – 13.44, 95 percent UCL – 23.25) mg/kg dw egg selenium. For deformities, the sum fraction normal endpoint, the EC 20 was 21.7 (95 percent LCL – 18.09, 95 percent UCL – 26.02) mg/kg dw egg selenium, while the EC 10 for this endpoint was 19.33 (95 percent LCL – 15.07, 95 percent UCL – 24.79) mg/kg dw egg selenium.

Consistent with comments received from the USEPA relative to derivation of the effect concentrations previously presented, a revised dose-response model was utilized that included using the same logistic regression model, and eliminated egg selenium concentrations above the already defined effects threshold (i.e., >30 mg/kg dw). Using this approach allowed the model to be more focused on the region where effects occur to brown trout. This improved
model results in an EC 20 equal to 23.1 (95 percent LCL – 22.37, 95 percent UCL – 23.77) mg/kg dw egg selenium and an EC 10 equal to 20.8 (95 percent LCL – 19.89, 95 percent UCL – 21.83) mg/kg dw.

Egg tissue concentrations may not be a practical endpoint for routine monitoring because it requires collection of samples during a narrow window of opportunity during autumn of each year. Collection of adult tissue samples is more practical since it can be done with lower effort and with less stringent constraints for the schedule under which sampling must be done. Therefore, the high correlation between selenium in eggs and adult tissue was used to identify an adult tissue concentration that corresponds to the revised EC 20 and EC 10 estimates based onBrown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

37 egg selenium. The resulting adult (whole body) tissue concentration for this Site based on the
revised EC 20 is 14.1 mg/kg dw and based on the revised EC 10 it is 12.9 mg/kg dw.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

38 6.0 REFERENCES

Coyle, J.J., D.R. Buckler, C.G. Ingersoll, J.F. Fairchild, and T.W. May. 1993. Effects of Dietary Selenium on the Reproductive Success of Bluegill Sunfish ( Lepornis macrochirus ). Environ. Toxicol. Chem. 12, 551-565.

de Rosemond SC, Liber K, Rosaasen A. 2005. Relationship between Embryo Selenium Concentrations and Early Life Stage Development in White Sucker ( Catostomus commersoni ) from a Northern Canadian Lake. Bull Environ Contam Toxicol 74: 1134- 1142.

Erickson, R. 2008. Toxicity relationship analysis program (TRAP v.1.20). U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Midcontinent Ecology Division, Duluth, Minnesota, USA.

Gilbert, R.O. 1987. Statistical Methods for Environmental Pollution Monitoring. Van Nostrand Renihold, New York.

Gillespie, R.B. and P.C. Baumann. 1986. Effects of High Tissue Concentrations of Selenium on Reproduction by Bluegills. Trans. Am. Fish. Soc. 115, 208-213.

Hamilton, S.J., K.J. Buhl, N.L. Faerber, R.H. Wiedmeyer, and F.A. Bullard. 1990. Toxicity of Organic Selenium in the Diet to Chinook Salmon. Environmental Toxicology and Chemistry 9:347-358.

Hardy, R.W. 2005. Final Report: Effects of Dietary Selenium on Cutthroat Trout (Oncorhynchus clarki) Growth and Reproductive Performance. Prepared for Montgomery Watson Harza, Bellvue, Washington.

Hermanutz, R.O., K.N. Allen, N.E. Detenbeck, and C.E. Stephan. 1996. Exposure to Bluegill ( Lepomis macrochirus ) to Selenium in Outdoor Experimental Streams. U.S. EPA Report. Mid-Continent Ecology Division. Duluth, MN.

Hodson, P.V. and J.W. Hilton. 1983. The Nutritional Requirements and Toxicity to Fish of Dietary and Waterborne Selenium. Ecol. Bull. 35:335-340.

Holm, J. 2002. Sublethal Effects of Selenium on Rainbow Trout ( Oncorhynchus mykiss ) and Brook Trout ( Salvelinus fontinalis ). Master’s Thesis. Department of Zoology, University of Manitoba, Winnipeg, MB.

Holm, J., V.P. Palace, K. Wautier, R.E. Evans, C.L. Baron, C. Podemski, P. Siwik and G. Sterling. 2003. An Assessment of the Development and Survival of Wild Rainbow Trout ( Oncorhynchus mykiss ) and Brook Trout ( Salvelinus fontinalis ) Exposed to Elevated Selenium in an Area of Active Coal Mining. The Big Fish Bang, H.I. Browman and A.B. Skiftesvik (eds.). Proceedings of the 26th Annual Larval Fish Conference, Bergen, Norway.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

39 Holm, J., V. Palace, P. Siwik, G. Sterling, R. Evans, C. Baron, J. Werner, and K. Wautier. 2005. Developmental Effects of Bioaccumulated Selenium in Eggs and Larvae of Two Salmonid Species. Environmental Toxicology and Chemistry , Vol. 24, No. 9, pp. 2373– 2381.

Kennedy, C.J., L.C. McDonald, R. Loveridge, and M.M. Strosher. 2000. The Effect of Bioaccumulated Selenium on Mortalities and Deformities in the Eggs, Larvae, and Fry of a Wild Population of Cutthroat Trout ( Oncorhynchus clarki lewisi ). Archives of Environmental Contamination and Toxicology 39:46-52.

Lemly, A.D. 1985a. Ecological Basis for Regulating Aquatic Emissions from the Power Industry: The Case with Selenium. Regul. Toxicol. Pharmacol. 5:465-486.

Lemly, A.D. 1985b. Toxicology of Selenium in a Freshwater Reservoir: Implications for Environmental Hazard Evaluation and Safety. Ecotoxicol. Environ. Safety. 10:314-338.

Lemly, A.D. 1993. Teratogenic Effects of Selenium in Natural Populations of Freshwater Fish. Ecotoxicol. Environ. Safety. 26: 181-204.

Lemly, A.D. 1997. A Teratogenic Deformity Index for Evaluating Impacts of Selenium on Fish Populations. Ecotoxicol. Environ. Safety. 37:259-266.

Muscatello, J.R, P.M. Bennett, K.T. Himbeault, A.M. Belknap,and D.M Janz. 2006. Larval Deformities Associated with Selenium Accumulation in Northern Pike (Esox lucius ) Exposed to Metal Mining Effluent. Environ Sci Technol 40:6506–6512.

NewFields. 2007a. Work Plan - Field Monitoring Studies for Developing a Site-Specific Selenium Criterion. Prepared for J.R. Simplot Company. April.

NewFields. 2007b. Technical Memorandum – Methods for Testing Adult Brown Trout Reproductive Success. October 17.

NewFields. 2008. Summary of Approach for Developing a Site-Specific Selenium Criterion Prepared for J.R. Simplot Company. February.

NewFields. 2009b. Draft Final Brown Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site-Specific Selenium Criterion. Prepared for J.R. Simplot Company. June.

Parametrix. 2009. Threshold Development Endpoints – Review of Selenium Tissue Thresholds for Fish: Evaluation of the Appropriate Endpoint, Life Stage, End Effects Level and Recommendations for a Tissue based Criterion. Part II of Selenium Tissue Thresholds: Tissue Selection Criteria, Threshold Development Endpoints, and Potential to Predict Population or Community Effect in the Field. Prepared for the North American Metals Council – Selenium Working Group. Washington, D.C.Brown Trout Laboratory Reproduction Studies for Developi ng a Site-Specific Selenium Criterion Smoky Canyon Mine FINAL October 2011

40 Rudolph, B.L., I. Andreller, and C. Kennedy. 2008. Reproductive Success, Early Life Stage Development, and Survival of Westslope Cutthroat Trout ( Oncorhynchus clarki lewisi ) Exposed to Elevated Selenium in an Area of Active Coal Mining . Environ. Sci. Technol. 2008, 42, 3109-3114.

Schultz, R. and R Hermanutz. 1990. Transfer of Toxic Concentrations of Selenium from Parent to Progeny in the Fathead Minnow ( Pimephales promelas ). Bull. Environ. Contam. Toxicol. 45, 568-573.

Suter, G.W., II. 1996. Toxicological Benchmarks for Screening Contaminants of Potential Concern for Effects on Freshwater Biota. Environmental Toxicology and Chemistry 15:1232-1241.

U.S. Environmental Protection Agency. 1999. 1999 Update of Ambient Water Quality Criteria for Ammonia. U.S. Environmental Protection Agency, EPA/822/R-99-014, Washington, D.C

U.S. Environmental Protection Agency. 2004. Draft Aquatic Life Water Quality Criteria for Selenium-2004. Office of Water, EPA-822-D-04-001.

### TABLES>230<300 >300 males femalesReferenceDownstream 485847 4690589Upstream 485591 4693679Downstream 493711 4738229Upstream 488968 4743637Upstream of Sage CreekDownstream 486291 4710439Upstream 486334 4710260Downstream 487299 4713431Upstream 487204 4713372Downstream 489434 4715598Upstream 489460 4715334Hoopes Spring and Sage CreekDownstream 491398 4720245Upstream 491293 4720575Downstream of Sage CreekDownstream 493433 4719137Upstream 493280 4719057Downstream 494938 4720410Upstream 494648 4720085Totals 128 58 36 68 301Location10/23/2007Table 1Monitoring Locations, Coordinates, and Counts for October 2007 Sampling in Support of the Brown Trout Laboratory Toxicity Studies33MPC-1 Montepelier CreekNorthing Date0ReachTotalBrown Trout# retained# caught and released0Fishing Time(Sec)Stump Creek10/26/2007ReachBoundaryEasting10/22/2007 1008CC-75Crow Creek upstream of WellsCanyonSTUMP-3732105375010 72378 756265904122331 25238441923010618263 12804474142CC-3ACrow Creek downstream of SageCreekLSV-2c10/24/2007CC-150-ReddCC-350Crow Creek downstream of SageCreek and CC-1ACrow Creek downstream of DeerCreekLower Sage Creek downstream ofHoopes SpringCrow Creek upstream of Deer CreekCC-1A10/24/200710/25/200710/22/200738482865142210/22/200810/24/20082034Page 1 of 1Stream Location Date pH (SU) Specific Conductance (umhos/cm) Temp. (°C) Dissolved Oxygen (mg/L) ORP (mV)

Montpelier Creek MC-1 10/23/2007 7.70 437 2.1 12.36 152.1

Stump Creek SC-3 10/26/2007 8.19 610 9.16 11.98 127.2

9/2/2006 7.93 426 7.11 8.22 NM
5/8/2007 8.29 532 7.01 11.59 176.5 8/23/2007 8.04 551 10.32 10.65 131.8 10/22/2007 8.21 572 5.45 11.37 82.5 5/12/2008 8.05 397 6.31 10.55 161.2 9/3/2006 7.58 399 7.94 9.26 NM
5/9/2007 8.34 444 9.42 9.32 204.3
8/24/2007 8.35 437 8.12 11.84 194.3 5/12/2008 8.53 361 10.44 10.00 164.3 8/31/2006 8.89 458 14.8 10.57 NM
5/8/2007 8.47 572 14.27 11.23 104.6
8/23/2007 8.61 643 17.1 11.16 90.1

10/22/2007 8.79 573 6.24 13.06 55.6

5/13/2008 8.44 431 15.04 9.05 202.9

9/8/2006 7.46 461 11.77 5.46 NM
5/14/2007 7.60 503 11.84 5.21 51.9
8/24/2007 7.49 473 11.89 6.32 78.2
5/17/2008 7.33 302 12.02 6.08 201.1
9/6/2006 7.43 489 10.37 7.33 NM
5/12/2007 8.46 484 16.57 7.22 89.5
8/28/2007 8.25 460 17.1 9.41 85.4
5/17/2008 8.38 289 17.05 9.01 152.7
9/6/2006 8.56 478 18.62 6.72 NM
5/12/2007 8.35 498 11.09 8.90 19.7
8/28/2007 8.37 458 14.27 10.68 -37.1
10/25/2007 8.31 462 8.17 11.36 124.4
5/17/2008 8.40 283 18.72 8.92 188.6
9/5/2006 7.81 454 9.49 7.95 NM
5/9/2007 8.50 402 16.54 7.60 125.4

9/1/2006 8.37 590 13.4 9.08 NM
5/10/2007 8.44 591 10.39 9.14 156.5
8/25/2007 8.43 577 10.61 12.62 29.3
10/22/2007 8.31 572 3.06 12.73 129.2
5/14/2008 8.09 358 7.39 9.80 161.2
9/4/2006 7.91 561 11.32 8.94 NM
5/11/2007 8.47 601 9.48 9.28 161.2
8/26/2007 8.20 583 11.26 10.12 19.8
10/24/2007 8.08 616 4.02 11.55 183.7
5/15/2008 8.42 370 13.42 10.49 219.6 Upstream of Sage Creek Table 2

Summary of Field-Measured Water Quality Parameters Collected in October 2007 for Sampling in Support of the Brown Trout Laboratory Toxicity Studies

Reference

CC-3A Crow Creek CC-75

Note: All field monitoring data collected through Spring 2008 is presented on this table. Green shading highlights October 2007 monitoring data. Crow Creek HS

HS-3

Downstream of Sage Creek Sage Creek Hoopes Spring

CC-1A LSV-2C CC-150

CC-350

LSV-4 Hoopes Spring and Sage Creek

Page 1 of 1Water Quality Parameters Measured During the October 2007 Sampling in Support of the Brown Trout Laboratory Toxicity Studies

Stream Location Date Alkalinity (mg/L) Hardness (mg/L as CaCO3) Sulfate, SO4 (mg/L)

Montpelier Creek MPC-1 10/23/2007 95.9J 138J 13.6J

Stump Creek SC-3 10/26/2007 195J 222J 39.2J

9/2/2006 215J 199 24.6 5/8/2007 230J 175 23.5 8/23/2007 214J 152J 25.5 8/23/2007-du p 127J 212J 23.4

10/22/2007 208J 233J 32.6J 5/12/2008 196 208 24.7 5/12/2008-dup 195 205 24.7 9/3/2006 175J 153 11.8 5/9/2007 209 152 14.4 8/24/2007 179J 204J 12.6 5/12/2008 192 199 17.8 CC-150 REDD 10/24/2007 190J 202J 13.1J 8/31/2006 200J 187 16.1 5/8/2007 203J 149 18.4 8/23/2007 134J 197J 17.8 10/22/2007 124J 224J 18.5J 5/13/2008 197 211 23.7

9/8/2006 194 209 47.2 9/8/2006-du p 194 199 46.8

5/14/2007 197 149 48.7 8/24/2007 154J 231J 46.5 5/17/2008 196 232 49.8 9/6/2006 205J 220 39.1 5/12/2007 196 150 44.3 8/28/2007 202J 222J 42.0 5/17/2008 197 223 43.2 9/6/2006 201J 206 38 5/12/2007 209 156 41.0 8/28/2007 205J 223J 40.3 10/25/2007 198J 237J 42.8J 5/17/2008 199 218 38.7 9/5/2006 203J 187 29 5/9/2007 186 129 31.2

9/1/2006 162J 160 24.7 5/10/2007 140 151 22.4 5/10/2007-dup 187 155 28.5

8/25/2007 202J 213J 35.0 10/22/2007 199J 229J 34.9J 5/14/2008 202 221 31.4 9/4/2006 211J 195 17.5 9/4/2006-dup 206J 191 19.3

5/11/2007 206 152 29.9 8/26/2007 195J 212J 35.8 10/24/2007 195J 228J 36.5J 5/15/2008 197 216 32.1

Note: All field monitoring data collected to date is presented on this table. Green shading highlights October 2007 monitoring data. Downstream of Sage Creek HS-3

Crow Creek CC-1A

CC-3A Hoopes Spring and Sage Creek

LSV-2C

LSV-4 Hoopes Spring HS

Sage Creek Table 3

Upstream of Sage Creek Reference

Crow Creek CC-75

CC-150

CC-350

Page 1 of 1Total Selenium (mg/L) Dissolved Selenium (mg/L)
Reference
10/23/2007 0.0002UJ 0.0002UJ 10/23/2007-dup 0.0002UJ 0.0002UJ Stump Creek SC-3 10/26/2007 0.0002UJ 0.0002R

9/2/2006 0.00053 0.00057 5/8/2007 0.00047J 0.00046J 8/23/2007 0.00033J 0.00033J8/23/2007-dup 0.00079J 0.0004J10/22/2007 0.00055J- 0.0002UJ 5/12/2008 0.0012 0.0012 5/12/2008-dup 0.0012 0.0011

9/3/2006 0.00062 0.00067 5/9/2007 0.00083J 0.00092J 8/24/2007 0.00059J 0.00068J5/12/2008 0.0018 0.0014 5/12/2008-dup NM NM CC-150 REDD 10/24/2007 0.0015J- 0.0011J9/1/2006 0.00083 0.00082
5/8/2007 0.00084J 0.0011J
8/23/2007 0.0002UJ 0.00026J10/22/2007 0.0003J- 0.0002UJ
5/13/2008 0.001 0.00089

9/8/2006 0.0174 0.0174 9/8/2006-dup 0.0174 0.0168

5/14/2007 0.0301J 0.0205J
8/24/2007 0.0242J 0.0214J5/17/2008 0.0296 0.0273
9/6/2006 0.0108 0.0092
5/12/2007 0.0198J 0.018J
8/28/2007 0.0158J 0.0161J5/17/2008 0.0223 0.026
9/6/2006 0.0095 0.0093
5/12/2007 0.0135J 0.0135J
8/28/2007 0.0144J 0.0143J10/25/2007 0.0384J- 0.0279J5/17/2008 0.0145 0.0141

9/1/2006 0.0029 0.0027
5/10/2007 0.0016J 0.0012J 5/10/2007-dup 0.0025J 0.002J

8/25/2007 0.0014J 0.0022J10/22/2007 0.0028 0.0013J5/14/2008 0.0032 0.0029
9/4/2006 0.003 0.0029 9/4/2006-dup 0.0029 0.0027

5/11/2007 0.0013J 0.0014J 5/11/2007-dup NM NM

8/26/2007 0.0011J 0.0018J8/26/2007-dup NM NM

10/24/2007 0.0017J- 0.001J5/15/2008 0.0036 0.0026 Upstream of Sage Creek Montpelier Creek MPC-1 Table 4 Summary of Total Selenium Concentrations Measured in Surface Waters in Support of the Brown Trout Laboratory Toxicity Studies

Selenium Concentrations
Date Location Stream

CC-350 CC-150

Hoopes Spring and Sage Creek

J - Estimated, NM-Not measured, Bold concentrations are those currently exceeding the State standard for total selenium (0.005 mg/L). Sage Creek LSV-2C

Downstream of Sage Creek

Crow Creek CC-1A

CC-3A

Note: All field monitoring data collected through Spring 2008 is presented on this table. Green shading highlights October 2007 monitoring data. HS-3 Crow Creek

Hoopes Spring HS CC-75

Page 1 of 1>230<300mm>300mmmales femalesReferenceDownstream 493804 4737886Upstream 493711 4738229Downstream 490825 4742505Upstream 490605 4742633Upstream of Sage CreekDownstream 487299 4713431Upstream 487178 4712722Downstream 489569 4715684Upstream 489491 4715286Hoopes Spring and Sage CreekDownstream 491298 4719723Upstream 491283 4720601Downstream of Sage CreekDownstream 493433 4719137Upstream 493136 471904Downstream 494676 4720149Upstream 494036 4719245Downstream 493433 4719137Upstream 493136 471904Downstream 495176 4720411Upstream 494874 4720281Totals 142 160 34 39 373Note: Note all trout collected or retained were spawned. Some females which appeared initially to be ripe, based on external features or even an initial expulsion of eggs did not provide eggs when stripping was conducted.14330312109 out of 13 females expressed eggs8111/13/200712143Stopped counting at ~40, too manyfish. Estimate >300 fish >230mmcaught7,1291769005102CC-3A and downstreamBetween CC-3A and CC-1A,upstream of CC-1ACrow Creek downstream of SageCreekCrow Creek downstream of SageCreek and CC-1A3,184CC-350Crow Creek downstream of DeerCreekCC-1ACrow Creek downstream of SageCreekLSV-2cLower Sage Creek downstream ofHoopes SpringCC-15011/12/200711/15/2007Stump Creek11/16/2007Nate property to CC-150 andupstream of CC-150Stump-1 and 3Stump Creek0323Date261843NC8,3472,594427,090 4128372511/14/200711/16/200711/13/20073,44111/15/2007captured, checked for ripeness,released >300 mm, 2 femalesappeared ripe, no eggs expressed# Caught Retained RipeTable 5Monitoring Locations, Coordinates, and Counts for November 2007 Sampling in Support of the Brown Trout Laboratory Toxicity StudiesReachLocationReachBoundaryFishingTime(Sec)NotesTotalBrown TroutTotal #EggSamplesSubmittedEasting Northing920130287Page 1 of 1Saratoga National Fish Hatchery (SC-001) 498 1,855 a 3.6 0.76 Saratoga National Fish Hatchery (SC-002) 420 1,089 a 4.1 0.94 Saratoga National Fish Hatchery (SC-003) 520 2,072 a 3.7 0.83 Saratoga National Fish Hatchery (SC-004) 562 3,350 a 4.3 0.92 Saratoga National Fish Hatchery (SC-005) 558 2,927 a 3 1.2 Saratoga National Fish Hatchery (SC-006) 439 1,111 a 3.1 1.2 Saratoga National Fish Hatchery (SC-007) 449 1,561 a 2.7 1 Saratoga National Fish Hatchery (SC-008) 494 1,927 a 2.5 0.96

Spring Creek Fish Hatchery (SPC-001) Spring Creek Fish Hatchery (SPC-002) Spring Creek Fish Hatchery (SPC-003) Spring Creek Fish Hatchery (SPC-004) Spring Creek Fish Hatchery (SPC-005) Spring Creek Fish Hatchery (SPC-006) Crow Creek (CC-150-009) 324 315 8.4 12.8 Crow Creek (CC-150-011) 342 351 5.6 8.4 Crow Creek (CC-150-012) 317 269 6.7 8.5 Crow Creek (CC-150-013) 332 376 5.9 8.4 Crow Creek (CC-150-015) 313 281 6 9.1 Crow Creek (CC-150-016) 391 621 7 7.5 Crow Creek (CC-150-017) 265 178 5.6 6.6 Crow Creek (CC-150-018) 308 279 4.7 6.9 Crow Creek (CC-150-020) 310 318 7.2 6.2 Crow Creek (CC-350-006) 370 475 9.2 14 Crow Creek (CC-350-007) 350 416 5.5 6.9 Crow Creek (CC-350-008) 335 341 8.5 9.5 Lower Sage Creek (LSV2C-002) 304 280 8.9 12.8 Lower Sage Creek (LSV2C-003) 300 260 13.8 40.3 Lower Sage Creek (LSV2C-004) 290 260 17.9 36 Lower Sage Creek (LSV2C-005) 294 250 13.6 26.8 Lower Sage Creek (LSV2C-006) 346 420 17.2 26.9 Lower Sage Creek (LSV2C-007) 315 290 6.7 18.6 Lower Sage Creek (LSV2C-008) 296 230 9.6 17.7 Lower Sage Creek (LSV2C-010) 311 314 22.6 38.8 Lower Sage Creek (LSV2C-012) 360 434a 7.2 13.2 Lower Sage Creek (LSV2C-016) 300 260 9.2 13.4 Lower Sage Creek (LSV2C-017) 341 310 13.2 20.5 Lower Sage Creek (LSV2C-019) 330 364 8.6 12.5 Lower Sage Creek (LSV2C-020) 280 241 11.3 11.2 Lower Sage Creek (LSV2C-021) 307 317 20 28.1

a Data not found or measured. Weights were estimated from the relationship between the wet weights of field collected fish and wet weights measured by CAS (r 2 = 0.963). Hatchery Fish

Wild Fish Table 6 Adult Female Brown Trout Length and Whole-Body Selenium (mg/kg dw) Data

Location Sample ID Total length (mm) Wet weight (g) Egg Selenium Concentration

(mg/kg dw) Whole-body

Selenium Concentration

(mg/kg dw)

Hatchery Fish (Second Set) 0.73

Page 1 of 1Count Mean Min Max

9/3/06 4 5.83 4.75 7.71
5/9/07 3 8.67 8.00 10.00
8/24/07 2 5.20 4.30 6.10
10/30/07 1 5.40 5.40 5.40
11/15/07 9 6.34 4.70 8.40
5/13/08 3 9.82 8.61 11.80
8/31/06 1 7.40 7.40 7.40
5/8/07 0 na na na
8/23/07 3 5.43 4.60 6.00
11/15/07 3 7.73 5.50 9.20
5/13/08 0 na na na

9/6/06 6 19.45 16.00 22.82
5/12/07 4 12.78 8.50 22.20
8/28/07 9 22.67 10.80 33.30
11/14/07 20 14.03 6.20 23.80
5/16/08 6 20.25 11.40 29.60

9/1/06 3 9.76 8.15 11.86
5/10/07 2 9.05 7.40 10.70
8/25/07 11 9.95 6.30 14.80
10/30/07 1 8.70 8.70 8.70
5/14/08 5 17.54 16.40 18.30
9/4/06 3 11.15 9.14 14.34
5/11/07 4 9.20 7.50 12.70 8/27/07 13 11.25 7.80 15.60
10/24/07 4 6.50 5.30 8.00 5/14/08 4 15.38 15.00 15.80

na-Not Applicable. Crow Creek CC-150

CC-350 Upstream of Sage Creek

Crow Creek CC-1A

CC-3A Table 7 Summary of Brown Trout Tissue Selenium Data Collected in Support of the Brown Trout Laboratory Toxicity Studies

Stream Location Date Brown Trout (mg/kg dw)

Sage Creek

Sage Creek LSV-2C

Downstream of Sage Creek

Note: All field monitoring data collected through Spring 2008 is presented on this table. Green shading highlights October/November 2007 monitoring data.

Page 1 of 150 20 10

Growth 46.23 33.79 28.13 95% LCL 27.05 22.84 13.09 95% UCL 79.01 50.00 60.44 15-Day Post Survival 34.73 24.52 20.00 95% LCL 33.11 22.26 17.37 95% UCL 36.42 26.99 23.02 Total Survival 24.83 21.43 19.66 95% LCL 19.27 13.60 10.75 95% UCL 32.00 33.77 35.98 Survival Hatch -Test End 30.52 21.63 17.68 95% LCL 27.58 17.77 13.44 95% UCL 33.78 26.32 23.25

Fraction normal 26.43 21.70 19.33 95% LCL 23.94 18.09 15.07 95% UCL 29.19 26.02 24.79 CF fraction normal 26.04 22.31 20.37 95% LCL 22.06 15.91 12.79 95% UCL 30.75 31.27 32.47 SD fraction normal 25.13 23.30 22.29 95% LCL 19.89 15.01 12.68 95% UCL 31.76 36.18 39.20 FD fraction normal 27.65 23.22 20.96 95% LCL 24.27 17.85 14.30 95% UCL 31.49 30.19 30.73 ED fraction normal 26.98 21.23 18.45 95% LCL 25.31 18.96 15.82 95% UCL 28.76 23.77 21.52

CF-Cranio-facial deformity SD-Skeletal deformity FD-Fin deformity ED-Edematous Tissue deformity 0.96 Effect Concentration (EC x ) Values for Egg Selenium Tissue Residues Versus Different Biological Endpoints for Brown Trout

Deformities 0.21

0.96

0.31

0.89

0.28 Table 8

0.88

0.68

0.81 Growth and Survival R 2 Biological Endpoints Effect Concentration (EC x )

Page 1 of 1NOEC LOECEC10EC20Bryson et al. 1984FieldLarval mortalityOvary -- <49 -- --FieldHatchability/swim-up Ovary >9.1 ---- --FieldHatchability/swim-up Ovary - <30 -- --Field Hatchability/swim-up Ovary >14.8 ---- --FieldHatchability/swim-up Ovary >9.2 ---- --Gillespie and Baumann 1986 FieldLarval edemaOvary -- <38.6 -- --LabLarval edemaOvary 3.94 21.1 15 17LabLarval edemaEgg 8.55 25.81 21 23LabLarval mortalityWB 7168 8.5LabLarval mortalityOvary 20 3524 27LabLarval mortalityEgg 22.5 41.3 22 26MesocosmLarval edemaWB 4.4 21.8 -- --MesocosmLarval edemaOvary 17.3 69-- --LabReproductionWB >7.5 ---- --LabReproductionOvary >10.92 ---- --Schultz and Hermanutz 1990 Mesocosm Larval edema/lordosis Ovary - <39.3 --Field Larval deformities/edema WB ----33 --Field Larval deformities/edema Ovary ----45aBrook troutHolm et al. 2005 Field Larval deformities Egg >20 - 20 (EC06) -Kennedy et al. 2000Field Larval deformities/ mortality Egg >21 ---Lab Larval deformities/ mortality WB >11.37 ---Lab Larval deformities/ mortality Egg >16.04 ---FieldLarval deformitiesEgg 20.6 46.8 --FieldAlevin mortalityEgg --17 23Rainbow troutHolm et al. 2005FieldLarval deformitiesEgg 17 2526 29Northern pikeMuscatello et al. 2006FieldLarval deformitiesEgg 3.8 31.28 20.4 33.55White suckerde Rosemond et al. 2005FieldLarval deformitiesEgg -- 26 (EC13) -GEI ConsultantsaAn ovary-based EC10of 44.6502μg/g was estimated from a whole-body EC10of 33.07μg/g based on the whole-body-ovary selenium relationship for fathead minnows (FHM) presented inGEI Consultants (2008): FHM [Se] dw WB = 0.75826*(FHM ovary [Se] dw) - 0.78645.Original table Source: Selenium Tissue thresholds - Tissue Selection Criteria, Threhsold Development Endpoints, and Potential to Predict Population or Community Effects in the Field(NAMC 2009).Hardy 2005Rudolph et al. 2008Fathead minnowTable 9BluegillSummary of Toxicity Studies that Evaluated Selenium Toxicity to Embyo/Larvae Resulting from Maternal TransferCutthroat troutSpeciesReferenceAdultExposureHermanutz et al. 1996Ogle and Knight 1989Coyle et al. 1993Bryson et al. 1985aBryson et al. 1985bDoroshov et al. 1992Selenium Concentration(μg/g dry weight)Endpoint TissuePage 1 of 1