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 55th Street, Suite 200 

Boulder, CO 80301 
Brown Trout 

Laboratory Reproduction Studies for Developing a Site-Specific Selenium Criterion 

Smoky Canyon Mine FINAL October 2011 

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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 ........................................................................ 34
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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 (ECx) 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 Transfer 

 
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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 Trout 
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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 Concentration 
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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 ECx Values (December 21, 

2010)
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LIST OF ACRONYMS 

AOC Administrative Order on Consent 

CAS Columbia Analytical Services 

CF Cranio Facial 

CI Confidence Interval 

dw Dry Weight 

EC/ECx 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 Agency 
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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 (USEPA), 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 characterize 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 Studies 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 on 

reproductive 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. 
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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 selenium literature. The conclusions 

drawn are based on the scientific evidence 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. 
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 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 recreationally 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 the 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 protein-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. 
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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 concentrations in maternal whole body tissue 

and egg tissue; and 

 Develop relationships between egg tissue concentrations and measures of reproductive 

success and viability of young. 
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2.0 METHODS 

The methods for testing adult brown trout reproductive success, including the study design plan 

and analysis details for the assessment of selenium 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 previously 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 the 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) 
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where: 

α = Probability of at least one sample representing the upper pth 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 10th percentile (i.e., 90th). 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 10th percentile of the data distribution. 

The upper 10th 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 90th percentile captures a large proportion of the data. 

Based on the data currently available, the range of concentrations is such that capturing the 90th

percentile, or upper 10th 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 10th

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 redds 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). 
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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+ years 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 30th, and were fed a commercial fish food pellet daily beginning 

October 27th. From October 27th to October 30th, 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 field 

November 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 to 
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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. 
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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 selenium 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 but 
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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, very 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 that 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 finfold 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. 
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Edema was detected by an obvious swelling and fluid buildup, usually abdominally, and 

ventrally, which often displaced the gut, and was usually 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 Analysis Program (TRAP) version 1.2 (Erickson 

2008) logistic functions were designed to derive 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 regression 
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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 regression-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 methods 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 EC10 and EC20 values were derived for each relevant relationship developed. 
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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 collected, 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 females 
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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 from 4.7 to 22.6 mg/kg dw. Figure 3 shows adult 

hatchery and wild fish lengths relative to their 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 selenium 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 found 
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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 562 

mm. Hatchery fish were larger than fish captured at field locations (Table 6). Maternal wholebody selenium concentrations measured in the hatchery 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. 
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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 illustrates 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 eggs 
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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 hatch 

out 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. 
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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 the 

feeding 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 several 
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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. 
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 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. At 
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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 (Figure 
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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. 
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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 dissolved 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 consistent 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 of 
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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 (R2

 = 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 squares 
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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 (R2

= 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 (R2

 = 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 (R2

 = 0.66) data 

and poor when using the wild fish data (R2 = 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 R2

 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 including 
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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 (R2

 = 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/mortality endpoint, as shown in Figures 14 and 15, 

and the influence of egg hatch on overall mortality/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 (R2

 = 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 R2

 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 (R2

 = 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 R2

 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, the 
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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 (R2

 = 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 (R2

 = 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 (R2

 = 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 analysis2

 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 standardized logit coefficients correspond to beta weights, and a pseudo R2

 statistic 

is available to summarize the strength of the relationship. Logistic regression does 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 the value of the Y variable for each value of the X variable. The Y variable is not directly 

measured; it is instead the probability of obtaining a particular value of a nominal variable.
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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 ECx 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 could 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 R2

 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 EC10 and EC20 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 post swim up study. Figure 37 shows the logistic 

regression curve fitted to survival data in the post swim up feeding trial versus log egg selenium 

concentrations. The R2

 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 EC10 and EC20

values (Table 8). Recall that percent survival 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 R2

 for this relationship is 0.31 and the error report of the logistic regression 
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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 EC10 or EC20 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 ECx 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 R2

of 0.89. The confidence intervals for the predicted EC10 and EC20 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 evaluated, the survival endpoints provide a much 

stronger measure of effects relative to egg selenium concentrations than does the growth 

endpoint. ECx 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 ECx values and narrow confidence intervals about the ECx. 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 endpoint 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. 
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Figure 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 R2

 (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 EC10 and EC20 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 R2

 value is higher than that of the craniofacial endpoint (R2 = 

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 R2

 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 EC10 and EC20 values. 

Figure 43 shows the logistic function for log egg selenium versus the fraction normal for edema 

deformity assessment. The R2

 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 EC10 and EC20 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 R2

 is high (0.88). A plot of the EC10 and EC20 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 EC10 and EC20 values suggesting not only a good 

fit, but a low variability as well. Figure 45 shows essentially the same relationship, only the 
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mean fraction normal was used as the dependent variable. The R2 is the same as for sum 

fraction normal and the EC10 and EC20 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 EC20 was used and defined as a 

reduction of 20 percent in the response observed at control. As presented above, both EC20

and EC10 values were derived using the TRAP software. In its 2004 Draft criterion document, 

USEPA provides the rationale for selection of the EC20 as the chronic value. USEPA states that 

the EC20 represents a low level of effect that is generally 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 

EC10s for long-term exposure criteria for tissue. Results of the analyses presented as part of 

this Site-specific laboratory study include both the EC10 and EC20 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 ECx 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 different endpoints including growth, survival, and 

deformities. For almost all (8 out of 9) of the models run, predicted EC10 or EC20 values were 

within 1 to 3 mg/kg dw egg selenium concentration of each other, respectively. The two models 

that appear to have a good overall fit with tight confidence intervals about the EC10 or EC20

predictions, and are biologically relevant, are the survival (hatch to test end) and total fraction 

normal endpoints. EC10 values for these endpoints are 17.67 and 19.33 mg/kg dw, respectively, 

while the EC20 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 selenium 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 even 
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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 based 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 exposure, 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 EC20 values would range from 

21.6 to 21.7 mg/kg egg selenium for alevin survival (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 R2

 for whole body maternal selenium concentration relationship to selenium concentration 

in their respective eggs is 0.8. As stated previously, this relationship becomes important in this 

preliminary analysis in order to relate ECx 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 site is for whole body tissue. Furthermore, whole 

body tissue concentration is a more practical endpoint to measure throughout the year than is 
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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 EC20 for survival and a whole body 

tissue concentration of 11.27 mg/kg dw was derived based on the EC10 for survival. Muscatello 

et al. (2006) reported whole body selenium EC20 of 15.56 mg/kg dw for northern pike derived 

from the egg EC20. 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 90th 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 Developing 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 EC10

and EC20. 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 EC20 as the SSC 

for this project based on the brown trout data. 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 EC10 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 EC10 is a more appropriate endpoint than the proposed EC20 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 ECx 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 R2 = 0.99 (Figure 46). This improved model results in an EC10 equal to 20.8 (95 

percent LCL – 19.89, 95 percent UCL – 21.83) mg/kg dw egg selenium and an EC20 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 Developing a Site-Specific Selenium Criterion 

Smoky Canyon Mine FINAL October 2011 

35 

Confidence intervals derived for the estimated ECx 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 EC10 and EC20 values were similarly affected. Using the same approach as 

presented above, the revised EC10 and EC20 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 EC10 and EC20 values are 12.91 and 14.1 mg/kg dw, respectively. 
Brown Trout 

Laboratory Reproduction Studies for Developing 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 collected 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 

(ECx). 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 EC20 was 21.63 (95 

percent LCL – 17.77, 95 percent UCL – 26.32) mg/kg dw egg selenium, while the EC10 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 EC20 was 21.7 (95 percent LCL – 18.09, 

95 percent UCL – 26.02) mg/kg dw egg selenium, while the EC10 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 EC20 equal to 23.1 (95 percent LCL – 22.37, 95 percent UCL – 23.77) mg/kg 

dw egg selenium and an EC10 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 EC20 and EC10 estimates based on 
Brown Trout 

Laboratory Reproduction Studies for Developing 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 EC20 is 14.1 mg/kg dw and based on the revised EC10 it is 12.9 mg/kg dw. 
Brown Trout 

Laboratory Reproduction Studies for Developing 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 Developing 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 Developing 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 

Reference

Downstream 485847 4690589Upstream 485591 4693679Downstream 493711 4738229Upstream 488968 4743637

Upstream of Sage Creek

Downstream 486291 4710439Upstream 486334 4710260Downstream 487299 4713431Upstream 487204 4713372Downstream 489434 4715598Upstream 489460 4715334

Hoopes Spring and Sage Creek

Downstream 491398 4720245Upstream 491293 4720575

Downstream of Sage Creek

Downstream 493433 4719137Upstream 493280 4719057Downstream 494938 4720410Upstream 494648 4720085

Totals 128 58 36 

Location

10/23/2007

Table 1

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

MPC-1 Montepelier Creek 3 

Northing Date

Reach 

# caught and released # 

0

Fishing Time 

(Sec)

Stump Creek 10/26/2007

Reach 

Boundary Easting

CC-75 10/22/2007 1008 Crow Creek upstream of Wells CanyonSTUMP-3

7

10 

5

3750

1

2378 7 0 7

5626

0

4122

1 3 

0

10

6

18

2

6

3 

0

44

7

CC-3A

Crow Creek downstream of Sage Creek

LSV-2cCC-150-Redd 10/24/2007CC-350

Crow Creek downstream of Sage Creek and CC-1ACrow Creek downstream of Deer CreekLower Sage Creek downstream of Hoopes SpringCrow Creek upstream of Deer Creek

CC-1A

10/24/200710/25/200710/22/2007

384828651422

10/22/200810/24/2008

2034
Stream 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 1
Water 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-dup 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-dup 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 1
Total 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

Stream Location Date

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<300

mm

>300 

mm

males females

Reference

Downstream 493804 4737886Upstream 493711 4738229Downstream 490825 4742505Upstream 490605 4742633

Upstream of Sage Creek

Downstream 487299 4713431Upstream 487178 4712722Downstream 489569 4715684Upstream 489491 4715286

Hoopes Spring and Sage Creek

Downstream 491298 4719723Upstream 491283 4720601

Downstream of Sage Creek

Downstream 493433 4719137Upstream 493136 471904Downstream 494676 4720149Upstream 494036 4719245Downstream 493433 4719137Upstream 493136 471904Downstream 495176 4720411Upstream 494874 4720281

Totals 142 160 34 39 373

Note: 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.

3 

31 

21

0

81 

11/13/2007

121

43

7,129

17

0 69 

10 5

2

CC-3A and downstreamBetween CC-3A and CC-1A, upstream of CC-1A

Crow Creek downstream of Sage CreekCrow Creek downstream of Sage Creek and CC-1A 3,184

CC-350 Crow Creek downstream of Deer CreekCC-1A Crow Creek downstream of Sage CreekLSV-2c Lower Sage Creek downstream of Hoopes SpringCC-150 11/12/2007 11/15/2007

Stump Creek

11/16/2007

Nate property to CC-150 and upstream of CC-150

Stump-1 and 3

Stump Creek

0

32

3

Date

26

43 18

NC

8,3472,5947,090 41 42

2837

25

11/14/200711/16/200711/13/2007

11/15/2007 3,441

# Caught Retained Ripe

Table 5

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

Reach Location Reach Boundary Fishing Time (Sec) 

Total Brown Trout 

Easting Northing

0 2

13

28 

7
Saratoga National Fish Hatchery (SC-001) 498 1,855a 3.6 0.76

Saratoga National Fish Hatchery (SC-002) 420 1,089a 4.1 0.94

Saratoga National Fish Hatchery (SC-003) 520 2,072a 3.7 0.83

Saratoga National Fish Hatchery (SC-004) 562 3,350a 4.3 0.92

Saratoga National Fish Hatchery (SC-005) 558 2,927a 3 1.2

Saratoga National Fish Hatchery (SC-006) 439 1,111a 3.1 1.2

Saratoga National Fish Hatchery (SC-007) 449 1,561a 2.7 1

Saratoga National Fish Hatchery (SC-008) 494 1,927a 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 (r2

 = 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 1
Count 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 1
50 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 (ECx) 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

R2 Biological Endpoints

Effect Concentration

(ECx)

Page 1 of 1
NOEC LOEC 

Bryson et al. 1984 Field Larval mortality Ovary -- <49 

Field Hatchability/swim-up Ovary >9.1 -- 

Field Hatchability/swim-up Ovary - <30 

Field Hatchability/swim-up Ovary >14.8 -- 

Field Hatchability/swim-up Ovary >9.2 -- 

Gillespie and Baumann 1986 Field Larval edema Ovary -- <38.6 

Lab Larval edema Ovary 3.94 21.1 

Lab Larval edema Egg 8.55 25.81 

Lab Larval mortality WB 7 16 

Lab Larval mortality Ovary 20 35 

Lab Larval mortality Egg 22.5 41.3 

Mesocosm Larval edema WB 4.4 21.8 Mesocosm Larval edema Ovary 17.3 69 

Lab Reproduction WB >7.5 -- Lab Reproduction Ovary >10.92 -- 

Schultz and Hermanutz 1990 Mesocosm Larval edema/lordosis Ovary - <39.3 

Field Larval deformities/edema WB -- -- 

Field Larval deformities/edema Ovary -- -- 

Brook trout Holm et al. 2005 Field Larval deformities Egg >20 - 

Kennedy et al. 2000 Field Larval deformities/ mortality Egg >21 - 

Lab Larval deformities/ mortality WB >11.37 - 

Lab Larval deformities/ mortality Egg >16.04 - 

Field Larval deformities Egg 20.6 46.8 Field Alevin mortality Egg - - 

Rainbow trout Holm et al. 2005 Field Larval deformities Egg 17 25 

Northern pike Muscatello et al. 2006 Field Larval deformities Egg 3.8 31.28 

White sucker de Rosemond et al. 2005 Field Larval deformities Egg - - 

GEI Consultants 

a An ovary-based EC10 of 44.6502 μg/g was estimated from a whole-body EC10 of 33.07 μg/g based on the whole-body-ovary selenium relationship for fathead minn

GEI 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 Communit(NAMC 2009).

Hardy 2005 Rudolph et al. 2008 

Fathead minnow 

Table 9

Bluegill 

Summary of Toxicity Studies that Evaluated Selenium Toxicity to Embyo/Larvae Resulting from Mater

Cutthroat trout 

Species Reference Adult Exposure 

Hermanutz et al. 1996 Ogle and Knight 1989 Coyle et al. 1993 Bryson et al. 1985a Bryson et al. 1985b Doroshov et al. 1992 

Selenium Co

Endpoint Tissue (μg/g dry