FIGURES
WYOMING

IDAHO

SMOKY

CANYON

MINE

Bern

Lago

Rose

Niter Bench

Grace

Conda

Henry

Smoot

Afton

Geneva Sharon

Nounan

Manson

Auburn Grover

Thayne

Liberty

Giveout

Talmage

Freedom

Bedford

Hellhole

Thatcher

Fairview

Telluride

Alexander

Montpelier

Bennington

Georgetown

Mound Valley

Soda Springs

Wayan (Unincorp)

Osmond Community

CARIBOU COUNTY CARIBOU COUNTY

LINCOLN COUNTY LINCOLN COUNTY

BEAR LAKE COUNTY BEAR LAKE COUNTY

FRANKLIN COUNTY FRANKLIN COUNTY

STATE HWY 238

US HWY 89

US HWY 30

STATE HWY 34

US HWY 89

GOVERNMENT DAM RD

STATE HWY 36

N RESERVOIR RD

STATE HWY 34

US HWY 89

S:\GIS\arcprj2\010109\plt\SSSCP\fig1-1-SCP-region.mxd

Smoky Canyon Mine

PRJ: 0442-004-900.70 DATE: MAY. 05, 2011

REV: 0 BY: RCR CHK: SMC

SMOKY CANYON MINE

LOCATION OF THE

SMOKY CANYON MINE

FIGURE 1

0 5 10

Miles

IDAHO

J.R. SIMPLOT COMPANY
Sage Valley

LINCOLN COUNTY - WYOMING

CARIBOU COUNTY - IDAHO

ge Creek

Deer Creek

Pole Canyon Creek

Crow Creek

Sage Creek

Crow

Creek

Sout h Fork Sage Creek

Manning Creek

g

ee

Creek

North

Fork

Sa

g

e

C

re

ek

Nate Canyon

Books Creek

Warm Creek

Hardmans Hollow

Rock Creek

White Du

Sprin

g

Creek

LINCOLN COUNTY - WYOMING

CARIBOU COUNTY - IDAHO

Hoopes

Spring

HS

HS-3

CC-1A

LSV-4

CC-75

CC-3A

DC-600

CC-350

LSV-2C

CC-150

6800

7000

7200

7400

7600

6600

6400

7800

8000

8200

8400 8600

7000

8200

8200

7200

8400

7000

7400

7200

7400

6800

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8000

7800

8200

7000

6800

6800

8200

7400

7600

7200

8200

8000

6800

7400

6800

7600

7800

7200

7000

7200

7800

8000

8000

8600

7400

8200

7400

7400

7000

7400

7200

7600

7400

7400

7600

7000

7600

7200

8000

7200

8000

7200

7000

8200

7600

7800

6800

6800

7800

6600

8000

8000

8000

7200

7000

8400

8200

7800

8200

6600

8200

7400

8000

8000

8000

8000

7200

7000

7800

8000

7400

8000

8000

7800

7600

7600

8200

7800

8000

8200

7200

7600

8000

7800

8200

8200

8600

8200

7400

6800

7400

7000

6800

7200

8000

7600

7000

6800

8200

8400

7600

7800

8200

7000

7800

8000

8200

7800

7000

6800

7400

8000

8000

7000

7600

8000

7200

7200

7400

7000

8000

8200

S:\GIS\ARCPRJ2\010109\PLT\SSSCP\INTERP_REPT\APPDFIG2_SSCSAMPLOCS.MXD

PRJ: 009-004.70 DATE: JUN 30, 2010

REV: 0 BY: KSR CHK: SMC

J.R. SIMPLOT COMPANY

FIGURE 2

SMOKY CANYON MINE

STUDY AREA BOUNDARIES

AND MONITORING LOCATIONS

Notes: 

Topographic surface reflects 2004 conditions in 

mine disturbance areas.

Legend

Monitoring Locations

Mine Disturbance Areas

0 3,500 7,000

Feet

Study Area Boundary

Site Boundary
Figure 3

Whole Body Selenium Concentration Versus Fish Length

REV: 1

J.R

Site-S

PRJ: 0442-004-

200250300350400450500550600

0 5 10 15 20 

Adult Fish Length (mm)

Whole Body Selenium in Brown Trout (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 4Whole Body Selenium Concentration Versus Adult Fish Weight

0

500

10001500200025003000

0 5 10 15 20 

Adult Fish Weight (grams wet)

Whole Body Selenium Concentration (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

05

101520253035

0 50 100 150 200 250 300 350 

Selenium Concentration (mg/kg dw)

Length (mm)

Hoopes Sage Ck Crow Ck d/s Background Adult BT

Figure 5Brown Trout Length Versus Whole Body Selenium Concentration
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 6Brown Trout Egg Selenium Concentration by Location

05

1015202530354045

Egg Selenium (mg/kg dw)

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

0

100020003000400050006000

0 100 200 300 400 

Total # of eggs

Adult Fish Length (mm)

NB: Symbols surrounded by a square are SC fish
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 8Egg Selenium Concentration Versus Total Egg Abundance

0

100020003000400050006000

0 5 10 15 20 25 30 35 

Total # of Eggs

Egg Selenium (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 9Adult Whole Body Selenium Concentration Versus Total Egg Abundance

0

100020003000400050006000

0 5 10 15 20 

Total # of Eggs

Adult Whole Body Selenium (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 10Percent Egg Mortality by Location

0

102030405060708090

100

% Egg Mortality

Location

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 11Percent Hatch (or Percent Survival at Hatch) of Alevins

0

102030405060708090

100

% Hatch

Location

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

Note: the dashed line indicates a 1:1 agreement. 

0

20406080

100

0 20 40 60 80 

% Swim-up

% Hatch

SCSPCCC-150CC-350LSV2C

The range of egg sconcentrations for did not swim up w

mg/kg dw.
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

0

102030405060708090

100

0 5 10 15 20 25 30 35 40 

1st hatch & swim-up (day)

Egg Se (mg/kg dw)

Day to 1st hatswim-up

Did not achieve swim-up (day of test t

SPC hatchery fish (received eyed up)

SPC hatchery fish (received eyed up)

Egg Selenium (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

0

102030405060708090

100

SPC-001

SPC-002

SPC-003

SPC-004

SPC-005

SPC-006

SC-001

SC-003

SC-004

SC-002

SC-008

SC-007

SC-005

SC-006

CC-150-020

CC-150-017

CC-150-018

CC-350-007

CC-150-016

CC-150-011

CC-150-013

CC-150-012

CC-150-015

CC-350-008

LSV2C-020

LSV2C-019

LSV2C-002

CC-150-009

LSV2C-012

LSV2C-016

CC-350-006

LSV2C-008

LSV2C-007

LSV2C-017

LSV2C-005

Mortality (%) 

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 15Percent Mortality from Hatch to Test Termination by Location

0

102030405060708090

100

Mortality (%)

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 16Percent Mortality 15-Day Post Swim-Up Feeding Trial

0

102030405060708090

100

Mortality (%)

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 17Brown Trout Growth based on Larval Fish Length

05

10152025

average length (mm)

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 18Brown Trout Growth based on Larval Fish Dry Weight

05

10152025

average dry weight (mg)

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 19Percent Cranio-Facial Deformities for Larval Brown Trout

0.00%

10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%

100.00%

SPC SC CC-150 CC-350 

Percentage of Samples

0(normal) 1(slight/few) 2(mod/several) 3(severe/many) (blank) NA
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 20Percent Skeletal Deformities for Larval Brown Trout

0.00%

10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%

100.00%

SPC SC CC-150 CC-350 

Percentage of Samples

0(normal) 1(slight/few) 2(mod/several) 3(severe/many) (blank)
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 21Percent Fin of Finfold Deformities for Larval Brown Trout

0.00%

10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%

100.00%

SPC SC CC-150 CC-350 

Percentage of Sample

0(normal) 1(slight/few) 2(mod/several) 3(severe/many) (blank)
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 22Percent Edematous Tissue Frequency for Larval Brown Trout

0.00%

10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%

100.00%

SPC SC CC-150 CC-350 

Percentage of Sample

0(normal) 1(slight/few) 2(mod/several) 3(severe/many) (blank)
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

0.00.20.40.60.81.01.21.41.61.82.0

Mean GSI Score

GSItotal no. of fish

Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.
REV: 1

J.R

Site-S

PRJ: 0442-004-

Figure 24Surface Water Selenium Concentrations

0

0.010.020.030.040.050.06

5/15/2000

11/15/2000

5/15/2001

11/15/2001

5/15/2002

11/15/2002

5/15/2003

11/15/2003

5/15/2004

11/15/2004

5/15/2005

11/15/2005

5/15/2006

11/15/2006

5/15/2007

11/15/2007

5/15/2008

11/15/2008

Total Selenium (mg/L)

Sample Date

Hoopes Spring (HS)

0

0.005

0.01

0.015

0.02

5/15/2000

11/15/2000

5/15/2001

11/15/2001

5/15/2002

11/15/2002

5/15/2003

11/15/2003

5/15/2004

Total Selenium (mg/L)

Samp

Lower Sage Valley (LS

Sprin

0

0.005

0.01

0.015

0.02

5/15/2000

11/15/2000

5/15/2001

11/15/2001

5/15/2002

11/15/2002

5/15/2003

11/15/2003

5/15/2004

11/15/2004

5/15/2005

11/15/2005

5/15/2006

11/15/2006

5/15/2007

11/15/2007

5/15/2008

Total Selenium (mg/L)

11/15/2008

Sample Date

Crow Creek - Meade Peak Ranch 

(CC-1 and CC-1A)
Figure 25

Log Maternal Whole Body Selenium Concentration (Wild Fish) Versus Log Egg Selenium Concentrations REV: 1

J.R

Site-S

PRJ: 0442-004-

y = 1.1926x - 0.0071

R2 = 0.7966

0

0.20.40.60.8

1

1.21.41.61.8

0 0.2 0.4 0.6 0.8 1 1.2 

Log Egg Selenium (mg/kg dw)

Log Maternal Whole Body Selenium (mg/kg dw)
Figure 26

Egg Selenium Concentration Versus Total Egg Abundance from Wild Female Fish REV: 1

J.R

Site-S

PRJ: 0442-004-

y = -15.989x 

R2 = 0.1

y 

0

200400600800

10001200140016001800

0 5 10 15 20 25 30 35 

Total # of Eggs

Egg Selenium (mg/kg dw)

non normalized normalized Linear (non normalized) Linear (norm
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

0

20406080

100

0 5 10 15 20 25 30 35 

% Hatch

Egg Selenium (mg/kg dw)
REV: 1

J.R

Site-S

PRJ: 0442-004-

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

y = 

0

20406080

100

0 5 10 15 20 25 30 35 

% Swim-up

Egg Selenium (mg/kg dw)
Figure 29

Egg Selenium Concentration VersusLarval Fish Weight REV: 1

J.R

Site-S

PRJ: 0442-004-

y = -0.0011x3 + 0.0724x2 - 1.4258x + 21.16

R2 = 0.6623

y = 0.0006x3 - 0.0487x2 + 1.0565x + 7.1103

R2 = 0.26

05

10152025

0 5 10 15 20 25 30 35 

Dry Weight (mg)

Egg Selenium (mg/kg dw)

Egg Selenium Versus Weight

HatcheWild FiPoly. (HPoly. (W
Figure 30

Egg Selenium Concentration Versus Overall Percent Survival REV: 1

J.R

Site-S

PRJ: 0442-004-

y = 0.0001x3 - 0.0075x2 + 0.1372x + 0.0417

R2 = 0.5425

y = 3E-05x3 - 0.0024x2 + 0.035x + 0.6113

R2 = 0.3039

0%

10%20%30%40%50%60%70%80%90%

100%

0 5 10 15 20 25 30 35 

Overall Survival (%)

Egg Selenium (mg/kg dw)
Figure 31

Egg Selenium Concentration Versus Percent Survival (Hatch to Test End) REV: 1

J.R

Site-S

PRJ: 0442-004-

y = 0.0019x3 - 0.1905x2 + 2.925

R2 = 0.8894 y = 0.0006x3 - 0.0961x2 + 0.9843x + 97.214

R2 = 0.8962

0

20406080

100120

0 5 10 15 20 25 30 35 

Survival (%, hatch to test end)

Egg Selenium (mg/kg dw)
Figure 32

Egg Selenium Concentration Versus Percent Survival in the 15-Day Post Swim-Up Feeding Trial REV: 1

J.R

Site-S

PRJ: 0442-004-

y = 0.0018x3 - 0.1843x2 + 3.22

R2 = 0.9614

y = 0.0002x3 - 0.0714x2 + 0.9027x + 97.888

R2 = 0.9569

0

20406080

100120

0 5 10 15 20 25 30 35 

Survival (%, 15-Day Post Swim-Up Feeding Trial)

Egg Selenium (mg/kg dw)
Figure 33

Egg Selenium Concentration Versus Sum Fraction Non-Normal Larvae REV: 1

J.R

Site-S

PRJ: 0442-004-

y = ‐0.0004x3 + 0.0

R2 = 

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25 30 35 

Sum Fraction Non-Normal Larvae

Egg Selenium (mg/kg dw)

Deformities Poly. (Deformities)
Figure 34

Egg Selenium Concentration Versus Sum Fraction Normal Larvae REV: 1

J.R

Site-S

PRJ: 0442-004-

y = 0.0002x3 ‐ 0.018

R2 = 0

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 5 10 15 20 25 30 35 

Sum Fraction Normal Larvae

Egg Selenium (mg/kg dw)

Deformities Poly. (Deformities)
Figure 35

Egg Selenium Concentration VersusMean GSI Score REV: 1

J.R

Site-S

PRJ: 0442-004-

y = ‐1E‐04x3 + 

0

0.20.40.60.8

1

1.21.41.61.8

2

0 5 10 15 20 25 30 35 

Mean GSI Score

Egg Selenium (mg/kg dw)

GSI score Poly. (GSI score)
Figure 36

TRAP ModelEgg Selenium Concentration Versus Larval Fish Growth15-Day Post Swim-Up Feeding Trial

REV: 1

J.R

Site-S

PRJ: 0442-004-

05

10152025

0.6 0.8 1 1.2 1.4 1.6 1.8 

Larval Fish Growth (mg dw)

Log Egg Selenium (mg/kg dw)
Figure 37

TRAP ModelEgg Selenium Concentration Versus Survival15-Day Post Swim-Up Feeding Trial

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

102030405060708090

100110

0.6 0.8 1 1.2 1.4 1.6 

Survival 15‐Day Post Feeding Trial (%)

Log Egg Selenium (mg/kg dw)
Figure 38

TRAP ModelEgg Selenium Concentration Versus Total Survival REV: 1

J.R

Site-S

PRJ: 0442-004-

0

102030405060708090

100110

0.6 0.8 1 1.2 1.4 1.6 

Total Survival (%)

Log Egg Selenium (mg/kg dw)
Figure 39

TRAP ModelEgg Selenium Concentration Versus Percent Survival (Hatch to Test End)

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

102030405060708090

100110

0.6 0.8 1 1.2 1.4 1.6 

% Survival (Hatch to Test End)

Log Egg Selenium (mg/kg dw)

Egg Selenium Versus % Survival (Hatch to Test End)
Figure 40

TRAP ModelEgg Selenium Concentration VersusCranio-Facial Deformities (Fraction Normal)

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

0.10.20.30.40.50.60.70.80.9

1

1.1

0.6 0.8 1 1.2 1.4 1.6 

Cranio‐Facial Deformities (Fraction Normal)

Log Egg Selenium (mg/kg dw)
Figure 41

TRAP ModelEgg Selenium Concentration Versus Skeletal Deformities (Fraction Normal)

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

0.10.20.30.40.50.60.70.80.9

1

1.1

0.6 0.8 1 1.2 1.4 1.6 

Skeletal Deformities (Fraction Normal)

Log Egg Selenium (mg/kg dw)
Figure 42

TRAP ModelEgg Selenium Concentration Versus Finfold Deformities (Fraction Normal)

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

0.10.20.30.40.50.60.70.80.9

1

1.11.2

0.6 0.8 1 1.2 1.4 1.6 

Finfold Deformities (Fraction Normal)

Log Egg Selenium (mg/kg dw)
Figure 43

REV: 1

J.R

Site-S

PRJ: 0442-004-

TRAP ModelEgg Selenium Concentration Versus Edema (Fraction Normal)

0

0.10.20.30.40.50.60.70.80.9

1

1.11.2

0.6 0.8 1 1.2 1.4 1.6 

Edema (Fraction Normal)

Log Egg Selenium (mg/kg dw)
Figure 44

REV: 1

J.R

Site-S

PRJ: 0442-004-

TRAP ModelEgg Selenium Concentration Versus Sum Fraction Normal Fish Larvae

0.000.501.001.502.002.503.003.504.004.505.00

0.60 0.80 1.00 1.20 1.40 1.60 

Fraction Normal (normal/total)

Log Egg Selenium (mg/kg dw)
Figure 45

TRAP ModelEgg Selenium Concentration Versus Mean Fraction Normal Fish Larvae

REV: 1

J.R

Site-S

PRJ: 0442-004-

0.000.100.200.300.400.500.600.700.800.901.00

0.60 0.80 1.00 1.20 1.40 1.60 

Mean Fraction Normal (normal/total)

Log Egg Selenium (mg/kg dw)
Figure 46

Revised TRAP ModelEgg Selenium Concentration VersusPercent Survival (Hatch to Test End)

REV: 1

J.R

Site-S

PRJ: 0442-004-

0

102030405060708090

100110

0.6 0.8 1 1.2 1.4 1.6 

% Survival (Hatch to Test End)

Log Egg Selenium (mg/kg dw)

Egg Selenium Versus % Survival (Hatch to Test End)
APPENDIX A 

Data Report-Reproductive Success Study with Brown Trout (Salmo trutta)-ENSR 
Prepared for: 

JR Simplot Company and Newfields Company, LLC. 

Pocatello, Idaho 

Data Report 

Reproductive Success Study with 

Brown Trout (Salmo trutta) 

Final 

October 2011 
Document No. 12699-001-500 i October 2011

Table of Contents 

1.0 Introduction ................................................................................................................................. 1-1

2.0 Methods ....................................................................................................................................... 2-1

2.1 Spawning of Brown Trout ....................................................................................................... 2-1

2.1.1 Hatchery Trout .......................................................................................................... 2-1

2.2 Laboratory Reproduction Tests ............................................................................................... 2-2

2.3 Dilution Water ........................................................................................................................ 2-7

2.4 Water Chemistry .................................................................................................................... 2-9

2.5 Deformities Assessment ......................................................................................................... 2-9

2.6 Endpoints ............................................................................................................................ 2-10

3.0 Results ......................................................................................................................................... 3-1

3.1 Egg Analyses ......................................................................................................................... 3-1

3.2 Laboratory Study .................................................................................................................... 3-2

3.2.1 Water Chemistry ....................................................................................................... 3-2

3.2.2 Biological Endpoints .................................................................................................. 3-3

3.2.3 Deformity Assessment ............................................................................................... 3-9

4.0 References ................................................................................................................................... 4-1
Document No. 12699-001-500 ii October 2011

List of Appendices 

Appendix A. Select photographs of different phases of the brown trout reproductive study

Appendix B. Wet weight estimation for adult fish from Saratoga hatchery and raw data for adult fish 

Appendix C. Egg counts for Saratoga hatchery fish (SC) and field collected fish 

Appendix D. Summary of fungal treatment methods for SC Hatchery eggs and field collected eggs 

Appendix E. Summary of water quality data selenium and sulfate analysis in water and selenium 

 concentration in eggs 

Appendix F. Number of organisms and survival rates at different stages during the brown trout 

 reproductive study 

Appendix G. Length and dry weight measurements for juvenile brown trout from reproductive study 
Document No. 12699-001-500 iii October 2011

List of Tables 

 Page

Table 2-1 Brown trout treatments and sample identifications for individual lots of fish eggs 

 used in the reproductive study ............................................................................. 2-4 

Table 2-2 Test initiation dates and termination dates for brown trout treatments in the 

 reproduction study ............................................................................................... 2-7 

Table 2-3 Water quality measurements for Horsetooth Reservoir process water 

 (unamended) and Crow Creek Drainage .............................................................. 2-9 

Table 3-1 Estimated number of total brown trout eggs from adult female organisms used 

in the reproductive success study and percent egg mortality............................. 3-2 

Table 3-2 Water hardness (avg ± SD), dissolved oxygen (low and % saturation), pH 

(range),temperature, and conductivity measured in each treatment during 

the reproductive study using brown trout (Salmo trutta) ........................................ 3-3 

Table 3-3 Day of first hatch, percent hatch, day of swim-up, percent swim-up, and 

 percent survival at swim-up for brown trout fry from the reproductive 

 success study ...................................................................................................... 3-4 

Table 3-4 Percent survival in the 15-d post swim-up phase of the study, total survival 

 for the entire study, percent survival from hatch until test termination, and 

 day of test termination for brown trout reproductive 

 study ................................................................................................................... 3-7 

Table 3-5 Standard length and dry weight (avg ± SD) of larval brown trout at test 

 termination .......................................................................................................... 3-9 

Table 3-6 Number of brown trout fry preserved and assessed for deformities ..................... 3-10 
Document No. 12699-001-500 iv October 2011

List of Figures 

 Page

Figure 2-1 Schematic diagram of the test chamber and egg cups for brown trout 

 reproductive study ............................................................................................... 2-4 

Figure 2-2 Location of egg treatments in water baths for brown trout reproductive study ........ 2-5 

Figure 3-1 Relationship between the percentage of organisms that hatched and the 

 percentage of organisms that reached swim-up .................................................... 3-8 
Document No. 12699-001-500 1-1 October 2011 

1.0 Introduction 

A study of brown trout (Salmo trutta) reproduction was conducted by Newfields for the JR Simplot 

Company to evaluate the parental transfer of selenium on the potential effects to offspring. ENSR’s 

Fort Collins Environmental Toxicology Laboratory (FECTL), Fort Collins, CO was retained to 

conduct the laboratory biological exposure portions of this study according to the study design plan 

outlined in the Technical Memorandum – Methods for Testing Adult Brown Trout Reproductive 

Success (Newfields 2007). An assessment of larval trout deformities was performed by Dr. Kevin 

Bestgen at Colorado State University’s Larval Fish Laboratory, which is described in a separate 

document. This report presents the results / data from the laboratory portion of this work. 

 
Document No. 12699-001-500 2-1 October 2011 

2.0 Methods 

ENSR FCETL personnel joined the Newfields team during the November 2007 sampling trip and 

provided assistance with fish collection, egg fertilization, and transport of egg samples to the 

laboratory. The time sensitive nature of transporting fertilized eggs from remote areas to the 

laboratory required integration of laboratory and field staff in this effort and near immediate transport 

of the samples to the laboratory. 

Hatchery fish and gametes were obtained from Saratoga National Fish Hatchery (Saratoga, WY). A 

second set of hatchery fish were obtained in December 2007. This second batch of hatchery fish 

were later obtained (as eyed-eggs) from Spring Creek Trout Hatchery (Lewistown, MT). 

Photographs taken at various points during the study are included in Appendix A. 

2.1 Spawning of Brown Trout 

Fertilization techniques for hatchery fish were slightly different from those of the field collected fish 

because of problems encountered with the SC hatchery eggs (lower than expected survival rates of 

eggs). ENSR engaged various hatchery and fishery personnel for recommendations on the 

fertilization technique. Based on these recommendations, the field methods were altered to fertilize 

eggs in the field instead of bringing the individually collected gametes under oxygen back to the 

laboratory and mixing them to achieve fertilization. 

2.1.1 Hatchery Trout 

Hatchery fish and gametes were obtained from 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. Because hatchery fish were obtained when they were ripe, which occurred prior to 

when fish were spawning in the field, the initial hatchery fish were obtained approximately 3 week 

prior to the first field collected fish. 

Fertilization techniques for the first set of hatchery fish (SC) methods described by Holm et al. 

(2005). Eight adult female and eight male trout were anesthetized using tricaine methanesulfonate 

(MS-222) and stripped by hand (similar to treatment of field fish described below). Eggs from a 

given female were collected directly into a cleaned plastic pan and then transferred into a plastic 

bag. Bags were labeled for that individual. Milt from a single male was collected directly into a small 

plastic bag. Bags were labeled with the individual identifications for each fish and the collection 

location and date. Prior to transport to the laboratory, all bags with gametes were partially filled with 

oxygen, sealed, double bagged, and them placed on ice (~4°C) in a cooler to keep gametes cold 

and out of direct sunlight. A min-max thermometer (Taylor® Digital Wireless Temperature System) 

was placed into the cooler with eggs to monitor temperature during transit to ENSR. The 

temperature range during transport of the SC eggs from Saratoga National Fish Hatchery and the 

FCETL was 6.1 – 9.7°C; with 9.7°C the initial reading when placed into the cooler. 

After stripping, the individual adult females (n=8) from the Saratoga National hatchery were collected 

for determination of total length, weight, percent solids, and whole-body selenium analysis. 
Document No. 12699-001-500 2-2 October 2011 

Sacrificed adult female trout were placed in large plastic bags, double bagged, and then stored on 

ice for transport to ENSR. Total length measurements were taken at ENSR prior to freezing 

samples for shipment to the analytical laboratory for analysis. Wet weight measurements were not 

measured at the laboratory but were estimated using the wet weight data from CAS and the wet 

weight data from the field fish (Appendix B). 

Once received at ENSR’s FCETL (Tuesday, October 23, 2007), eggs were maintained in coolers on 

ice overnight (temperature range overnight was 6-7 °C). The following day, eggs from each 

hatchery female were fertilized using the methods similar to those described by Holm et al. (2005; 

Appendix C). This method entailed placing ‘green’ or unfertilized eggs into a pre-cleaned plastic 

bowl (32 oz Rubbermaid plastic container). Milt (~1 ml, composite from all male fish) was combined 

with the eggs at a rate of 10 ul / 50 ml. The eggs and milt were gently stirred with a glass rod and 

allowed to stand for ~60 seconds. The egg/milt mixture was covered with ~100 ml of laboratory 

process water (i.e., hardness [~50 mg/L as CaCO3] adjusted Horsetooth Reservoir water cooled to 

6°C), swirled for approximately three minutes, after which 500 ml of cooled laboratory water was 

poured over the eggs. The eggs were allowed to water harden for approximately five minutes. After 

water hardening, the fertilized eggs were kept in the Rubbermaid plastic containers with lids loosely 

fastened, placed back into the coolers, and maintained in the dark to gradually warm up to ~10°C 

over the next 24-hours. On Thursday October 25, 2007, 600 eggs were collected from each batch 

of eggs using egg pickers and placed in prepared egg cups. Egg cups were then placed in 

individual test chambers. Remaining eggs not used for the test were then counted and frozen until 

they could be sent to Columbia Analytical Services (CAS), Inc. (Kelso, WA) for total selenium and 

percent solids analysis. Eggs for the SC hatchery fish were estimated using an egg counting 

technique developed for this purpose (Appendix C). Briefly, we counted the number of eggs for a 

given female that would fill a graduated cylinder to a particular volume (either 100 ml or 50 ml). 

Then we poured all the remaining eggs into a graduated cylinder to measure the total egg volume 

for that female. Using the number of eggs in either 50 or 100 ml, we determined the number of eggs 

in the total volume of eggs for that female. Since eggs from different females were of different size, 

this method was completed separately for each egg batch. 

Because of low survival rates associated with the first set of hatchery eggs, a second set of hatchery 

eggs were obtained from Spring Creek Trout Hatchery (Lewistown, MT) on December 4, 2007. 

These eggs were obtained at the eyed-up stage because individual gametes were not available 

from Saratoga National fish hatchery or any other hatchery. These eggs served as method controls 

for the experimental system to ensure that we could expect acceptable survival and growth given 

the design. There were lower than expected survival rates for some treatments from the SC 

hatchery fish, which we believe were related to the fertilization and acclimation methods employed. 

Egg counts were not performed for the SPC eggs, as these were not obtained from individual 

female fish and thus not comparable with other treatments. 

 

2.2 Laboratory Reproduction Tests 

The temperature range for the cooler (#1) that held the trout from LSV2C (received at ENSR on 

November 15, 2007) was 3.4 – 8.7°C (excluding the first 10 min acclimation period). The 
Document No. 12699-001-500 2-3 October 2011 

temperature range for the second cooler that contained the remaining field eggs (received at ENSR 

on November 16, 2007) was 5.1 – 10.3 °C. Once back at the laboratory, eggs were allowed to 

slowly warm up over a 24-hour period to ~10°C (range of 6.2 – 8.4°C for cooler #1; range of 7.9 – 

8.9 °C for cooler #2) prior to introduction into egg cups and test chambers. All remaining eggs were 

counted and frozen until they could be sent to the analytical laboratory for analysis of total selenium 

and solids. A list of the different locations from which fish were collected (i.e., treatments) and the 

individual identifications for each are provided in the table below (Table 2-1). 

Table 2-1. Brown trout treatments and sample identifications for individual lots of fish eggs used in 

the reproductive study. 

Saratoga NF 

Hatchery 

(SC) 

Spring Creek 

Hatchery 

(SPC) 

Background Se 

Field Location 

(CC-150) 

Low Se Field 

Location 

(CC-350) 

High Se Field 

Location 

(LSV2C) 

SC-001 SPC-001 CC-150-009 CC-350-006 LSV2C-002 

SC-002 SPC-002 CC-150-011 CC-350-007 LSV2C-003 

SC-003 SPC-003 CC-150-012 CC-350-008 LSV2C-004 

SC-004 SPC-004 CC-150-013 LSV2C-005 

SC-005 SPC-005 CC-150-015 LSV2C-006 

SC-006 SPC-006 CC-150-016 LSV2C-007 

SC-007 CC-150-017 LSV2C-008 

SC-008 CC-150-018 LSV2C-010 

 CC-150-020 LSV2C-012 

LSV2C-016 

LSV2C-017 

LSV2C-019 

LSV2C-020 

LSV2C-021 

 Note: CC – Crow Creek; LSV – Lower Sage Creek 

After the 24-h temperature acclimation period, a target of 600 eggs were transferred (in low ambient 

light) from each batch of field collected eggs using egg pickers, and placed in prepared egg cups. 

Egg cups were then placed in individual test chambers in the water bath. The remaining eggs for 

each treatment not used for the test were then counted by hand (Appendix C) and frozen until they 

could be sent to Columbia Analytical Services (CAS), Inc. (Kelso, WA) for total selenium and 

percent solids analysis. 

Eggs were transferred from collection bags to egg cups in low ambient light using egg pickers. The 

egg cups were submersed in ~10°C water. Egg cups were constructed of polyvinyl chloride (PVC) 

schedule 40 pipe (approximately 5 cm ID and 3.8 cm depth) with a nitex screen bottom. Ten 

individual units were attached in a 2 x 5 layout design using silicon, so that each egg cup consisted 

of 10 individual cells (Figure 2-1). Eggs were evenly distributed into all 10 of the cells of the egg 

cups. For instance, the treatments initiated with 600 eggs had 60 eggs placed into each egg cup 

cell. While the original intent was to maintain the ten replicates for a given fish throughout the study, 

this was not feasible due to the water demands and space limitations. Therefore, organisms from all 

replicates were combined in the test chamber at hatch out. 

Egg cups were hung with clips and fishing line in Sterilite® plastic test chambers (11.4 L). Each 

chamber was aerated for the duration of the test to maintain the dissolved oxygen concentration at 
Document No. 12699-001-500 2-4 October 2011 

sufficient levels (>60%). The volume in each test chamber was approximately 5 L maintained at the 

level of the top of the drain pipe which consisted of a piece of 5-mm ID glass tubing inserted through 

a silicone stopper which is pressed into a small hole drilled in the side of the chamber. Chambers 

and water volume were of sufficient size to maintain a loading rate of < 5 g of fish per L of water in 

each test chamber. Spent water overflowed out of the glass standpipes and into the water bath 

before being discharged directly to a conduit connecting both baths. This water was treated with an 

ultraviolet light disinfection unit prior to discharge into the laboratory waste water. After swim-up 

occurred, the drain openings were covered with a small piece of nylon mesh to prevent loss of 

organisms. In general, methods employed for this study followed ASTM (2006) standard guidance 

for conducting early life stage tests with fish, although modifications were made to account for studyspecific hypotheses and test design criteria (e.g., number of eggs). 

Figure 2-1. Schematic diagram of the test chamber and egg cups for brown trout reproductive study. Inset 

shows individual cells of egg cups (n = 10) within a chamber. Aeration tube not shown. 

With the test solution volume of ~5 L and a flow rate of 20 ml/min, each test vessel received ~ 5.7 

volume additions per day. Test chambers were held in two separate water baths with the 

temperature of the water baths controlled by chillers. Chambers were randomly placed in one of the 

two water baths so that each bath had an equal number of chambers (or as close as possible) from 

each treatment. Chambers within a given bath were then randomly placed into different locations 

within the water bath (Figure 2-2). 

Egg cups with 

Nitex 

Screen 

60 eggs / cup 

Solution Level

Inflowing solution

Outflow with Nitex 

E D C B A

I J H G F 
Document No. 12699-001-500 2-5 October 2011 

Figure 2-2. Location of egg treatments in water baths for brown trout reproductive study. 

Bath #1 Bath #2

HIGH002 BK013 SC004 HIGH004 

LOW008 SC005 HIGH019 SC003 

BKD020 HIGH010 HIGH008 BK012 

HIGH016 HIGH005 

HIGH021 SPC004* SPC006 

SPC005 BK017 SC006 

 HIGH017 BK016 

LOW006 BK015 BK009 

SC007 BK011 LOW007 SC002 

SPC001 BK018 

SC008 HIGH012 SPC003 SPC002* 

SC001 HIGH003 HIGH020 HIGH007 

*denotes smaller test chamber (~2.5 L) 

Note: BKD = CC-150, LOW = CC-350, and HIGH = LSV2C 

Two smaller glass chambers (2.5 L) used previously in ELS studies were also initiated with eggs 

from SPC (20 eggs per egg cup / chamber, 20 ml/min flow). These two chambers were included as 

an additional set of performance controls to compare with SPC eggs initiated using the regular 

design (i.e., 600 eggs per egg cup per chamber) 

The exposure chambers were housed in temperature-controlled water baths. Target temperatures 

in the test chambers were 10 ± 2°C. Dissolved oxygen concentrations were maintained at ≥ 60 

percent of saturation (5.6 mg/L at 5,200 feet elevation and 10 °C). Embryos and fry were 

maintained under dim lighting (approximately 0.8 foot-candles) until swim-up occurred, after which 

they were held in ambient lighting (approximately 16 ft-c) with 16 hours of light per 24-hour period. 

Egg cups were maintained submerged in each test chamber until all eggs hatched or were noted as 

dead. Originally, dead eggs were not going to be removed until after all live eggs eyed up. 

However, because of the observance of fungus on some of the dead eggs, dead eggs were 

carefully removed from the egg cups prior to the eyed up stage in some instances. The number of 

dead eggs removed each day was recorded for each test chamber. Eggs or embryos were 

considered dead if they appeared opaque and/or developed visible fungal infections. As hatching 

occurred, the numbers of live alevins, as well as the dead alevins (or eggs) that were removed, were 

recorded on a daily basis. However, because of the movement of the alevins due to their own 

swimming ability as well as from the movement of the water from aeration it was difficult to get an 

accurate count on the number of newly hatched alevins on a daily basis. Since this measurement 

was unreliable, it was discontinued and counts were largely based on the number of dead 

organisms removed from a particular chamber. When eggs hatched, alevins were gently removed 

to the bottom of the surrounding test chamber using a large bore glass pipette and the remaining 

egg shell was removed. Organisms that died as eggs or while hatching were recorded and 

preserved in Stockard’s solution. Eggs that had the amniotic fluid (e.g., yolk) leak out just about the 

time of hatching were termed, ‘dead while hatching’ (DWH). Any organisms that were not found 

during the test were considered dead, except during the 15-d swim-up study. For this phase of the 
Document No. 12699-001-500 2-6 October 2011 

study, any missing organisms were considered missing and were excluded from survival analysis. 

Fish were considered dead if no gill movement or visible response was observed in response to 

gentle prodding. Egg cups were removed after all living eggs hatched. Test initiation and 

termination dates for each treatment are provided below (Table 2-2). 

Table 2-2. Test initiation dates and termination dates for brown trout treatments in the reproduction 

study. 

Fish Treatment 

/ Location Test Initiation Date Test Termination Date(s) 

SC Oct. 25, 2007 Jan. 17, 2008 

SPC Dec. 4, 2007 Jan. 22, 2008 

CC-150 (BKD) Nov. 17, 2007 Feb. 7 & 12, 2008 

CC-350 (LOW) Nov. 17, 2007 Feb. 7 & 12, 2008 

LSV2C (HIGH) Nov. 16, 2007 Feb. 7 & 12, 2008 

Eggs (primarily SC treatments) were treated with salt (NaCl) and, later, formalin in an attempt to 

reduce fungal growth. Days and type of treatment are located in Appendix D. Fungus appeared to 

affect the SC treatments more than later field-collected (i.e., wild) eggs and the second set of 

hatchery treatments as additional UV disinfection systems were incorporated at other locations prior 

to initiation of these other treatments. 

Alevins (recently hatched young with yolk sacs) were monitored daily for mortality. Dead organisms 

were removed and placed in Davidson’s solution. As alevins approached swim-up, trout chow was 

offered to the organisms to determine if they were actively feeding. The swim-up date was set 

based on when at least 80% of the alevins had absorbed their yolk sac and were actively feeding. 

At the swimup stage, organisms were thinned down to a target of 100 organisms per test chamber, 

preserving all the extra organisms in Davidson’s solution for the deformities assessment. If there 

were less than 100 organisms in the test chamber then organisms were counted and left in the test 

chamber; however, no organisms were preserved at this stage for deformities analysis. All living 

larval fish were then maintained for the 15-d post swim-up stage of the study. Dead organisms were 

counted and removed daily, saved by placing them in Davidson’s solution. Swim-up trout were 

started on a 4% body weight ration of salmon starter #1 (purchased from Aquatic Biosystems, Fort 

Collins, CO) over three feedings daily (i.e., morning, noon, evening) during the week, and at least 

two feedings daily on weekends. Weight of a swim-up fry was determined by sacrificing one fish out 

of seven hatchery treatments (SC) and determining a wet weight. The wet weight for these seven 

fish averaged 0.105 g; therefore, daily feeding was ~0.4 g trout chow assuming 100 fry. 

At initiation of the swim-up stage the flow rate into each chamber (except the 2.5L chambers) was 

altered to 40 ml/min and taller stand pipes were added to adjust the total volume to ~9 L to account 

for loading requirements based on the anticipated growth of the organisms. Loading for the 

hatchery fish was < 2.5 g/L (assumes a wet wt of 0.2 g for 100 fish in 8 L of water). Once feeding 
Document No. 12699-001-500 2-7 October 2011 

started, test chambers were siphoned daily (in p.m. prior to feeding) to remove remaining food and 

fecal material. 

At the end of the 15-d post swim-up study, all remaining organisms were sacrificed via immersion in 

isopropyl alcohol. A sub-set of 20 organisms was rinsed with deionized water, blotted dry and 

measured for standard length (tip of snout to caudal peduncle). All remaining organisms were 

preserved in Davidson’s solution for deformities assessment. 

Length measurements were taken on the day of test termination for all organisms except the SC001 through SC-007 fish. Length measurements for these organisms were taken after a few days in 

isopropyl alcohol because they could not all be measured on the day of test termination. Length and 

wet weight measurements on 10 fish from SC-008 chamber were taken prior to storage and after 

storage to see if storage in alcohol altered the length measurement statistically (pre-isopropyl 

storage avg = 22.6 mm, post-isopropyl storage avg. = 21.2 mm, p =0.0055). Because of the 

difference in lengths pre- and post-preservation, all remaining organisms were measured for length 

prior to preservation. Following length measurements, organisms were preserved in isopropyl 

alcohol until dry weight could be determined. For dry weight analysis, each fish was transferred to a 

tared weight boat and dried at 100 °C for at least 48 hours. After removal from the drying oven, the 

weigh boats were placed in a dessicator to prevent absorption of moisture from the air, until weighed 

(dry weight) to the nearest 0.01 mg. 

2.3 Dilution Water 

The dilution/control water used in this study was FCETL process water obtained from Horsetooth 

Reservoir. The ambient incoming water is coarse-filtered (through a sand filter and polypropylene 

core filters [10 and 1 micron]) to remove indigenous organisms, particulate matter, and 

contaminants. Water then passes through an ultraviolet light disinfection system before being 

stored in large holding tanks. This water is periodically analyzed for contaminants. Horsetooth 

Reservoir process water is very soft to soft water according to USEPA (2002), with both hardness 

and alkalinity typically 20 - 30 mg/L as CaCO3 (Table 2-3). Background sulfate levels in unaltered 

Horsetooth water are ~5.0 mg/L. Ambient (unheated) laboratory Horsetooth reservoir water was 

metered into a large holding vessel (~100 gallons) and chilled with a counter-current cooling process 

to help maintain the target water temperature in the test chambers of 10°C. Water from the holding 

vessel was adjusted to increase the hardness and sulfate so that it would be higher than ambient 

levels and more similar to the field conditions (Table 2-3). Given the soft water conditions of the 

laboratory Horsetooth water and the volume of water used on a daily basis, it was impractical to 

match the water quality characteristics of the site. 
Document No. 12699-001-500 2-8 October 2011 

Table 2-3. Water quality measurements for Horsetooth Reservoir process water (unamended) and 

Crow Creek Drainage. 

Horsetooth Reservoir1 Crow Creek2

Parameter Average Range Average Range 

Hardness (mg/L) 33 26.5 – 41.8 171 129 – 220 

Alkalinity (mg/L) 28.8 25 – 33 197 140 – 231 

Sodium (mg/L) 3.5 2.7 – 5.5 3.3 1.0 – 6.5 

Potassium (mg/L) <1 --- <1 <1 – 1.8 

Sulfate (mg/L) 5.6 3.4 – 10 27 7.5 – 48.7 

Chloride (mg/L) 1.9 0.5 – 3.6 7.3 0.2 – 89 

DOC (mg/L) 2.4 2.1 – 2.9 1.0 0.34 – 2.18 

1Horsetooth Reservoir laboratory process water (Fort Collins, CO) from 2000 to 2004 measured at ENSR’s 

FCETL. 

2Crow Creek drainage as characterized by surface water from Crow Creek, Sage Creek, and Hoopes Spring 

surface water quality data (Newfield 2007) 

Calcium sulfate (CaSO4.2H2O; Ben Franklin Aquacal, Plaster City, CA) and magnesium sulfate 

(MgSO4.7H2O; The PQ Corporation, Valley Forge, PA) were added at a ratio of 1.82:1 calcium: 

magnesium (molar basis) to deionized water to prepare a super hardness stock solution of ~2,000 

mg/L as CaSO3. This super stock was metered into the holding vessel to achieve a target hardness 

of ~50 mg/L as CaCO3 and sulfate concentration of ~20 mg/L. Water hardness was measured daily 

during the study, while sulfate concentration was monitored periodically. 

The super stock solution of extremely hard Horsetooth water (~2,000 mg/L hardness) was metered 

from two Mariotte bottles (5-gallon glass bottle) into a head tank which then was metered into the 

large holding vessel along with dilution water inflow. The inflow of the ambient laboratory 

Horsetooth water was approximately 1,400 ml/min. The super hardness stock solution was 

delivered into the holding vessel at a target rate of 14 ml/min. Batches of the super hard stock 

solution were prepared every three days and the Mariotte bottles were filled daily throughout the 

study. Flows on the main dilution water (unadjusted Horsetooth water) and the drip flowing from the 

head tank (Mariotte bottles fed this tank) were measured at least once daily throughout the study. 

Water from the holding vessel, now adjusted to a water hardness of ~50 mg/L and slightly cooled, 

was then pumped over to a head tank set up above the dilutor panels and water baths holding all 

test chambers. Flows into the head boxes above each dilutor panel feeding the test chambers for 

that bath were maintained by providing a constant head pressure using a submersible pump from 

the holding vessel and an over-flow recirculating system (i.e., excess flow was returned from the 

head tank above the test chambers back to the 100 gal holding vessel). 

Water from the head tank flowed into diluter panels constructed out of glass, silicone adhesive, and 

silicone stoppers. Adjusted Horsetooth process water was delivered to the test chambers through 

(3/8 I.D. x 1⁄2 O.D. x 1/16 thickness, inch) polyethylene tubing. The dilutor panel delivered modified 

Horsetooth water to up to 24 test chambers per water bath. Flow rate into each chamber was 
Document No. 12699-001-500 2-9 October 2011 

adjusted in the splitter box to deliver a target rate of 20 ml of test solution per minute to each 

chamber. After swim-up had occurred and the 15-d post swim-up study was underway, the flow 

rates were adjusted to a target of 40 ml of test solution per minute per chamber. 

2.4 Water Chemistry

Temperature (°C), pH (s.u.), dissolved oxygen (mg/L) concentrations, and conductivity (μS/cm) were 

measured and recorded in one chamber for each test treatment daily. Hardness (as mg/L CaCO3) 

was measured from the dilutor panel or from a test chamber daily during the study. Total ammonia 

(mg/L as N) was measured in selected test chambers (LSV2C-010, CC-150-016, and CC-350-006) 

once feeding was initiated. Sulfate concentration was measured from water collected from the 

dilutor panel or from test chambers. Determinations of waterborne sulfate concentrations were 

made at Paragon Analytics, Inc. (Fort Collins, Colorado, USA) using ion chromatography (EPA 

Method 300.0). 

Water samples for total recoverable and “dissolved” selenium analyses were collected, prepared, 

and preserved from selected test chambers during the course of the study. Briefly, approximately 

50 to 250 ml of test solution was collected for analysis of either dissolved or total selenium analysis. 

Aqueous analytical samples were analyzed at either Paragon Analytics Inc. or ACZ Laboratories 

(Steamboat Springs, CO). Dissolved selenium samples were filtered through 0.45 μm filters (GHP 

Acrodisc Syringe Filters, Pall Gelman Scientific, Ann Arbor, MI, USA) prior to placing in the 

polypropylene sample containers and preserved with 1% nitric acid. Samples were analyzed at 

ACZ using an ICP-MS (EPA Method 200.8) or at Paragon Analytics, Inc. using method SW3005A 

(ICP-OES). 

Aqueous water samples were also collected at the hatchery sites (SC and SPC) for analysis of total 

and dissolved selenium so background levels of selenium at the hatcheries could be compared with 

selenium levels from other fish areas. These samples were collected in April 2008 (SC) or October 

2008 (SPC) and sent to ACZ for analysis. Total recoverable and dissolved Se concentrations in the 

water from the Saratoga National Fish Hatchery was 1.7 μg/L, while total recoverable and dissolved 

Se concentrations in the water from Spring Creek hatchery was <0.1 μg/L (Appendix E). 

2.5 Deformities Assessment 

Extra fry (excluding the target of 100 fry kept for the post swim-up phase of the study) were removed 

and preserved in Davidson’s Solution at swim-up for deformity examination. Any deformed fry were 

removed at this point and preserved as part of the extra fry. Upon test termination, an additional 

batch of fish (per treatment) were preserved similarly and saved for deformity assessment. Of the 

100 organisms included in the 15-d post swim-up phase of the study, the target was to save 80 of 

these fry for deformity assessment (the other 20 were for length and dry weight analysis). 
Document No. 12699-001-500 2-10 October 2011 

Dead fish and alevins were removed during the study and preserved for deformity assessment as 

well. However, many of these organisms did not preserve well because they were in various states 

of decay. Because of the poor tissue condition of these dead organisms they were not originally 

evaluated (i.e., necrotic tissue conditions and/or presence of fungus made analysis and 

observations difficult). A subsequent analysis was conducted on approximately 100 of these fish 

(per batch) on five samples in which there was no or little data. These additional samples were from 

LSV2C -003, -004, -005, -010, and -021. All samples for deformity analysis were sent to Dr. Kevin 

Bestgen at CSU. Data from these samples were incorporated in the deformity assessment 

performed. 

2.6 Endpoints 

Multiple test endpoints were utilized for this test at different times during the test. Fecundity, hatch, 

deformities, length, weight, survival (different times during the study), tissue concentrations (egg and 

whole body), and feeding success were proposed test endpoints. These endpoints were similar with 

those of Holm et al. 2005, Hardy 2005, and Kennedy et al. 2000 on which the test described herein 

was based. 

Total egg production for each female was counted as a measure of fecundity. Survival was 

determined based on the number of surviving fish at hatch, swim-up, and at test termination 

compared to the number of eggs at test initiation. Percent hatch was determined as the number of 

live fish and alevins at day of first hatch compared to the number of eggs at test initiation. Other 

endpoints included day of swim-up, day of test termination, and measurements on survival larval fry 

at test termination (length and dry weight). 
Document No. 12699-001-500 3-1 October 2011 

3.0 Results 

3.1 Egg Analyses 

The number of eggs used from a given female depended on the total number of eggs provided by 

that female. While the target was 600 eggs per female, certain organisms did not provide that 

many total eggs. Therefore, we attempted to maximize the number of eggs used in the reproduction 

study while leaving enough for selenium analysis. For treatments with fewer eggs (e.g., LSV2C010), eggs were added to each replicate of the egg cup in small numbers (10 at a time) to ensure 

equal numbers in each replicate. Once that target number was added to each replicate egg cup 

cell, the number of eggs remaining was evaluated to see whether more eggs could be added to the 

egg cup. This process was repeated until no fewer than 61 eggs remained for Se analysis. For the 

field collected eggs, the most that remained was 732 eggs; however, the average number of eggs 

saved for Se analysis was 329. The number of eggs used in the study from a particular female, the 

total number of eggs the female produced, and the percent egg mortality are presented (Table 3-1). 

One set of eggs were completely dead upon arrival (LSV2C-006). Because there were no viable 

eggs upon inspection, these eggs were not used in the reproductive study but were retained for 

selenium analysis. From 100 to 600 eggs were used to initiate the reproductive studies for the 

remaining fish sampled (Table 3-1). As mentioned, this corresponded to the total number of eggs 

produced by a particular female. The total number of eggs from field collected organisms ranged 

from 161 (LSV2C-010) to 1,658 (CC-150-016). By contrast, the range of eggs collected from the SC 

hatchery fish ranged from 1,248 to 5,448. The total number of eggs could not be counted 

individually for the SPC hatchery fish as they were previously composited from multiple females and 

delivered as eyed-up eggs. 
Document No. 12699-001-500 3-2 October 2011 

Table 3-1. Estimated number of total brown trout eggs from adult female organisms used in the 

reproductive success study. 

Location Sample ID #Eggs placed in 

study 

Total # of eggs 

from fish 

Egg 

Mortality (%) 

SC-001 600 4,173 76.2 

SC-002 600 4,005 77.2 

SC-003 600 5,120 43.3 

Hatchery Fish SC-004 600 1,248 68.9 

SC-005 600 5,448 88.3 

SC-006 600 3,176 6.8 

SC-007 600 3,224 69.5 

SC-008 600 4,005 34 

SPC-001 600 -- 0.7 

Second set of SPC-002 20 -- 0 

SPC-003 600 -- 2.5 

Hatchery Fish SPC-004 21 -- 0 

SPC-005 600 -- 1.2 

SPC-006 600 -- 0.3 

CC-150-009 600 1,215 71.5 

CC-150-011 300 488 4.0 

CC-150-012 350 556 11.2 

CC-150-013 600 1,234 33.3 

CC-150-015 600 1,003 21.7 

CC-150-016 600 1,658 85.2 

CC-150-017 250 414 10.8 

Wild CC-150-018 600 959 12.8 

CC-150-020 600 1,332 2.7 

CC-350-006 600 1,154 28.3 

CC-350-007 600 1,174 70.3 

CC-350-008 600 922 32.3 

LSV2C-002 600 1,096 1.0 

Fish LSV2C-003 400 474 6.5 

LSV2C-004 500 766 49.4 

LSV2C-005 300 476 28.7 

LSV2C-006 -- -- 100 

LSV2C-007 500 773 100 

LSV2C-008 300 372 11.7 

LSV2C-010 100 161 13 

LSV2C-012 600 1,031 1.7 

LSV2C-016 600 826 5.0 

LSV2C-017 300 447 28.7 

LSV2C-019 500 693 5.8 

LSV2C-020 400 525 10.8 

LSV2C-021 600 1,208 30.7 

3.2 Laboratory Study 

3.2.1 Water Chemistry 

The water quality parameters (pH, temperature and dissolved oxygen [DO]) monitored daily during 

the study were within acceptable ranges for the survival of brown trout (Table 3-2). 
Document No. 12699-001-500 3-3 October 2011 

Table 3-2. Water hardness (avg ± SD), dissolved oxygen (low and % saturation), pH (range), 

temperature, and conductivity measured in each treatment during the reproductive study using 

brown trout (Salmo trutta). 

Fish Treatment 

Water 

Hardness 

(mg/L) 

Minimum 

Dissolved 

Oxygen 

(mg/L) & % 

Saturation pH (s.u.) 

Avg ± SD 

Temp (°C) 

Temp. 

Range (°C) 

Conductivity 

(μS/cm) 

SC 49.3 ± 3.2 7.8 / 83 7.4 – 7.9 10.1 ± 0.7 8.5 – 11.9 104 – 196 

SPC 48.6 ± 3.6 7.5 / 80 7.4 – 7.9 10.5 ± 0.7 8.6 – 11.9 106 – 146 

CC-150 (BKD) 48.4 ± 3.5 7.5 / 80 7.4 – 7.9 10.5 ± 0.8 8.7 – 12.5 107 – 175 

CC-350 (LOW) 48.4 ± 3.5 7.4 / 79 7.5 – 7.9 10.6 ± 0.8 8.8 – 12.2 104 – 167 

LSV2C (HIGH) 48.4 ± 3.5 7.1 / 76 7.3 – 7.8 10.6 ± 0.8 8.7 – 12.5 107 – 161 

Note: At 5,200 feet elevation and 10 °C, 60% dissolved oxygen saturation is 5.63 mg/L 

Alkalinity was measured at least weekly in the laboratory Horsetooth dilution water and it averaged 

28.2 ± 1.4 mg/L (as CaCO3) between October 25, 2007 and February 12, 2008. Ammonia was 

measured in select treatments (LSV2C-010, CC-150-016, and CC-350-006) during the 15-d postswim-up feeding portion of the study and was <1.0 mg/L in all test chambers. Sulfate, measured 12 

times over the course of the study, averaged 22.9 (range 17 – 27) mg/L (Appendix E). Water 

temperature measured in the chambers for each water bath and water hardness are presented over 

the course of the study (Appendix E). 

Aqueous selenium measured in the hardness adjusted Horsetooth water or in specific test 

chambers from October 23, 2007 to February 12, 2008 were < 5 μg/L (12 total measurements; 

Appendix E). The only time that selenium was detected was on January 22, 2008 for a collected 

sample from the SPC-001 chamber (result was 9.9 μg/L). There was insufficient sample to have 

this value re-verified by a separate laboratory with lower detection limits. 

3.2.2 Biological Endpoints 

The day of first hatch for the SC hatchery fish ranged from 40 to 47 days, although all but two had 

started hatching by day 42 (Table 3-3). The second set of hatchery fish (received at the eyed up 

stage) hatched from nine to 13 days after receipt, while the treatment fish hatched from 36 to 43 

days. The slightly lower temperatures for the SC hatchery fish likely explain the slightly longer day 

to first hatch. 
Document No. 12699-001-500 3-4 October 2011 

Table 3-3. Day of first hatch, percent hatch, day of swim-up, percent swim-up, and percent survival 

at swim-up for brown trout fry from the reproductive success study. Eggs from the second set of 

hatchery fish were obtained at the eye-up stage. 

Location Sample ID Day of 1st

hatch % hatcha Day of swim-up 

% Swimup 

Survival 

(%) at 

Swim-up 

Stage 

SC-001 42 23.8 69 22.8 22.8 

SC-002 41 22.8 69 22.5 22.5 

SC-003 40 56.7 69 55.7 55.7 

Hatchery Fish SC-004 40 31.1 69 27.3 27.3 

SC-005 42 11.7 69 10.7 10.7 

SC-006 40 93.2 69 92.8 92.8 

SC-007 47 30.5 69 26.8 26.8 

SC-008 46 66.0 69 65.0 65.0 

SPC-001 11 99.3 34 98.0 98.0 

Second set of SPC-002 13 100 34 100 100 

SPC-003 11 97.5 34 94.2 94.2 

Hatchery Fish SPC-004 13 100 34 100 100 

SPC-005 11 98.8 34 97.5 97.5 

SPC-006 9 99.7 34 96.0 96.0 

CC-150-009 39 28.5 72 27.2 27.2 

CC-150-011 42 96.0 67 95.3 95.3 

CC-150-012 40 88.8 72 86.8 86.8 

CC-150-013 39 66.7 72 59.7 59.7 

CC-150-015 40 78.3 67 77.8 77.8 

CC-150-016 43 14.8 72 14.3 14.3 

CC-150-017 42 89.2 72 86.4 86.4 

Wild CC-150-018 41 87.2 72 84.3 84.3 

CC-150-020 38 97.3 67 96.3 96.3 

CC-350-006 41 71.7 72 68.0 68.0 

CC-350-007 38 29.7 67 28.7 28.7 

CC-350-008 37 67.7 67 64.5 64.5 

LSV2C-002 36 99.0 68 96.7 96.7 

Fish LSV2C-003 41 93.5 88 0b

 8.0 

LSV2C-004 40 50.6 88 0b

 30.2 

LSV2C-005 40 71.3 88 0b

 37 

LSV2C-006 NA 0.0 --- --- --- 

LSV2C-007 NA 0.0 --- 0 0 

LSV2C-008 39 88.3 68 86.7 86.7 

LSV2C-010 42 87.0 88 0b

 25 

LSV2C-012 42 98.3 73 95.7 95.7 

LSV2C-016 39 95.0 73 91.7 91.7 

LSV2C-017 41 71.3 73 64.0 64.0 

LSV2C-019 38 94.2 73 89.8 89.8 

LSV2C-020 40 89.2 68 88.0 88.0 

LSV2C-021 38 69.3 88 0b

 21.7 

 

a Percent hatch and percent survival at hatch were synonymous endpoints. 

 

b Did not achieve swim-up stage before test was terminated; remaining alevins still had yolk sacs (test Day 88). 

Percent hatch and percent survival at hatch were synonymous endpoints for all treatments. The 

percent hatch of the SC treatments was lower than anticipated, ranging from 11.7 – 93.2% (average 
Document No. 12699-001-500 3-5 October 2011 

of 42%), although this was likely due to the different fertilization technique used for the SC hatchery 

fish and some fungal problems. The percent hatch for the SPC hatchery fish was 97.5 – 100%, 

although the holding period was significantly shorter because they were received as eyed eggs. 

The percent hatch for the field collected eggs was typically better than the SC hatchery eggs. Eggs 

collected from CC-150 treatments ranged from 14.8 to 97.3%, with an average of 71.9%. Average 

hatch out for the eggs collected from fish at CC-350 was 56.4%, while that for the eggs from LSV2C 

was 83.9% (not including LSV2C-006 or -007 whose eggs were either dead upon arrival or were not 

fertilized). The lowest percent hatch from the LSV2C treatments was 50.6%, while the highest was 

99.0%. The field organisms indicated that the change in fertilization technique resulted in a higher 

hatch success rate compared with the SC hatchery fish. 

The day of swim-up for the SC hatchery fish was at 69 days and was 34 days for the SPC hatchery 

fish (Table 3-3). For the majority of the field treatments, the day of swim-up was between 67 to 73 

days, regardless of the collection location. There were five treatments where the alevins did not 

absorb their yolk sac and reach the swim-up stage. These treatments were: LSV2C-003, LSV2C004, LSV2C-005, LSV2C-010, and LSV2C-021. These organisms were maintained for the duration 

of the study and taken down on test day 88 with the last treatments that had finished the 15-d post 

swim-up portion of the study (Table 3-3). 

The next two endpoints were very similar, the percentage of organisms that reached the swim-up 

stage and percent survival at the swim-up stage (i.e., on the day of swim-up). Because the fry on 

the day of swim-up had already absorbed their yolk sac, these values were the same for most 

treatments (Table 3-3). The only treatments for which these values were different were for the five 

in which the alevins did not reach swim-up (listed above). Survival at this point was determined for 

the remaining alevins and ranged between 8.0 and 37%. 

The last phase of the studies consisted of the 15-d post swim-up study. The first three endpoints for 

this phase of the study consisted of survival in the 15-d study, total survival for the entire study, and 

day of test termination. For this phase, each treatment was initiated with a target of 100 of the 

surviving fry and maintained for 15-d to monitor growth to assess whether there were any latent 

effects post swim-up. All treatments were initiated with 100 fry per chamber except the following 

listed below. The number of organisms at initiation of this phase is listed in parenthesis: 

• SC-005 (62) 

• SPC-002 (20) & SPC-004 (21) 

• CC-150-016 (86) 

• CC-350-006 (101) 

• LSV2C-003 (32), LSV2C-004 (151), LSV2C-005 (111), LSV2C-010 (25), LSV2C-016 (101), 

& LSV2C-021 (130) 

For some of the treatments, i.e., SC-005, CC-150-016, LSV2C-003, LSV2C-004, LSV2C-005, 

LSV2C-010, and LSV2C-021, only this many fish (or alevins) were left alive at this point in the study. 

Since some of these were below the target of 100 or had not yet reached swim-up stage, they were 
Document No. 12699-001-500 3-6 October 2011 

not thinned out, but maintained all the remaining live organisms through the duration of the study. 

The remaining treatments mentioned were either initiated with fewer eggs (i.e., SPC-002 and SPC004) or had an extra organism above the target number. For some of the test chambers, fry were 

lost during this stage of the study due to food clogging the drain pipe, resulting in an overflow of the 

test chamber. In calculating survival during the 15-d study and overall survival, these organisms 

were excluded as a technician error. Other technician errors also occurred in the study, for example 

during thinning when two individual fish were killed (SC-003 and SC-005). A list of the treatments 

that lost organisms due to an overflow of the chamber and the number lost (i.e., not recovered at the 

end of the study) are listed below. 

• SC-003 (9) & SC-008 (10) 

• CC-150-013 (26), CC-150-016 (43), CC-150-017 (33) 

• CC-350-007 (20) & CC-350-008 (28) 

• LSV2C-002 (16), LSV2C-008 (46), LSV2C-017 (19), LSV2C-019 (39), & LSV2C-020 (36) 

Survival during the 15-d post swim-up stage was relatively high (Table 3-4). Excluding the five 

LSV2C treatments that had poor survival at swim-up (-003, -004, -005, -010, and -021), all other 

treatments had survival above 96.0%. Survival for the five mentioned LSV2C treatments ranged 

from 28.1% to 68.5%, indicating that only these treatments had substantial mortality at this phase of 

the study (Appendix F). 

Day of test termination for all but the SPC treatments ranged from 82 to 88 days (Table 3-4). The 

SC hatchery fish were terminated on day 84, while the SPC fish were terminated on day 49. Total 

survival for the entire study is presented in Table 3-4. Total survival for the SC hatchery fish ranged 

from 10.7% to 92.7%, and ranged from 94.2% to 100% for the SPC hatchery fish. 
Document No. 12699-001-500 3-7 October 2011 

Table 3-4. Percent survival in the 15-d post swim-up phase of the study, total survival for the entire 

study, and day of test termination for brown trout reproductive study. 

Location Sample ID 

Survival (%) 

in 15-d Post 

swim-up 

stage 

Total 

Survival 

(%) 

Survival (%) 

from Hatch 

until test 

term. 

Day of test 

termination 

SC-001 97.0 22.7 98.9 84 

SC-002 99.0 22.3 99.5 84 

SC-003 98.9* 54.7 98.0 84 

Hatchery Fish SC-004 100 27.3 96.2 84 

SC-005 100 10.7 99.0 84 

SC-006 98.0 92.7 99.5 84 

SC-007 96.0 26.3 95.8 84 

SC-008 100* 65.0 99.0 84 

SPC-001 100 98.0 98.7 49 

Second set of SPC-002 100 100 100 49 

SPC-003 100 94.2 96.7 49 

Hatchery Fish SPC-004 100 100 100 49 

SPC-005 96 96.8 98.0 49 

SPC-006 100 96.0 96.3 49 

CC-150-009 99 27.0 98.3 87 

CC-150-011 100 95.3 98.7 82 

CC-150-012 97 86.0 98.1 87 

CC-150-013 97.3* 57.5 93.0 87 

CC-150-015 98 77.5 98.9 82 

CC-150-016 100* 15.4 99.7 87 

CC-150-017 100* 84.3 97.2 82 

Wild CC-150-018 100 84.3 96.5 87 

CC-150-020 100 96.3 98.9 82 

CC-350-006 98.0 67.7 96.3 87 

CC-350-007 98.8* 26.0 98.6 82 

CC-350-008 98.6* 62.6 97.1 82 

LSV2C-002 100* 96.6 97.6 83 

Fish LSV2C-003 28.1 2.25 8.8 88 

LSV2C-004 55.6 16.8 66.2 88 

LSV2C-005 62.2 23.0 51.7 88 

LSV2C-008 98.2* 84.2 95.9 83 

LSV2C-010 44.0 11.0 24.0 88 

LSV2C-012 100 95.7 97.4 88 

LSV2C-016 100 91.7 96.7 88 

LSV2C-017 96.3* 60.5 89.2 88 

LSV2C-019 100* 88.9 94.7 88 

LSV2C-020 100* 86.8 97.6 83 

LSV2C-021 68.5 14.8 45.5 88 

* - missing organisms at test termination were not included in calculation. 

The range of total survival for the CC-150 treatments was 15.4% to 96.3%, that for the CC-150 

treatments was 26.0% to 67.7%, and for the LSV2C treatments was 2.25% to 96.6% (Table 3-4). 

For most of these treatments, there was not a substantial difference between the survival rate at 

swim-up and the number of organisms that hatched (Figure 3-1). For most of the treatments, the 

number of organisms that hatched reached the swim-up stage. One group did not hatch or swim-up 
Document No. 12699-001-500 3-8 October 2011 

(LSV2C-007), while five groups had substantial hatch (>50%) but did not reach swim-up (as 

mentioned previously). Percent survival, from hatch until test termination was included in Table 3-4. 

Figure 3-1. Relationship between the percentage of organisms that hatched and the percentage of organisms 

that reached swim-up. Note, the dashed line indicates a 1:1 agreement. 

 The results of length and dry weight analysis for the target of 20 organisms at the end of the 15-d 

post swim-up phase of the study are provided below (Table 3-5). Raw data are in Appendix G. 

0

20

40

60

80

100

0 20 40 60 80 100

% Hatch

% swim-up

SC

SPC

CC-150

CC-350

LSV2C
Document No. 12699-001-500 3-9 October 2011 

Table 3-5. Standard length and dry weight (avg ± SD) of larval brown trout at test termination. The 

number of larval fish measured for each treatment is included (n). 

Location Sample ID n 

Average Standard 

Length (mm) 

Average Dry Weight 

(mg) 

SC-001 20 21.4 ± 1.5 18.338 ± 4.3 

SC-002 20 22.2 ± 0.93 18.470 ± 3.3 

SC-003 20 22.8 ± 1.3 22.602 ± 4.8 

Hatchery Fish SC-004 20 22.2 ± 1.9 23.246 ± 7.5 

SC-005 20 21.8 ± 1.2 19.386 ± 4.0 

SC-006 20 21.8 ± 0.83 18.240 ± 3.3 

SC-007 20 21.0 ± 1.5 18.567 ± 3.9 

SC-008 20 21.2 ± 1.1 15.118 ± 3.3 

SPC-001 20 24.4 ± 2.1 22.564 ± 6.1 

Second set of SPC-002 20 22.8 ± 1.5 20.852 ± 4.1 

SPC-003 20 23.3 ± 2.1 21.807 ± 5.4 

Hatchery Fish SPC-004 20 23.4 ± 2.0 21.564 ± 5.3 

SPC-005 20 23.0 ± 1.9 20.792 ± 5.8 

SPC-006 20 23.2 ± 1.6 22.386 ± 6.1 

CC-150-009 20 21.8 ± 1.3 13.188 ± 3.2 

CC-150-011 20 20.1 ± 0.85 9.248 ± 2.3 

CC-150-012 20 21.4 ± 0.89 12.840 ± 2.8 

CC-150-013 20 22.6 ± 1.3 15.582 ± 3.4 

CC-150-015 20 22.6 ± 1.2 14.486 ± 3.2 

CC-150-016 20 22.8 ± 1.2 16.736 ± 4.0 

CC-150-017 20 21.0 ± 0.83 10.652 ± 2.4 

Wild CC-150-018 20 21.3 ± 1.0 13.244 ± 3.3 

CC-150-020 20 20.2 ± 1.4 11.248 ± 3.6 

CC-350-006 20 21.4 ± 1.1 12.342 ± 3.1 

CC-350-007 20 21.3 ± 1.3 11.009 ± 3.7 

CC-350-008 20 20.6 ± 0.94 12.089 ± 3.1 

LSV2C-002 20 20.1 ± 1.2 11.070 ± 3.2 

Fish LSV2C-003 9 21.3 ± 0.71 9.710 ± 2.4 

LSV2C-004 20 21.2 ± 0.89 9.979 ± 2.1 

LSV2C-005 20 20.2 ± 1.1 10.967 ± 2.7 

LSV2C-008 20 20.4 ± 0.51 9.342 ± 1.1 

LSV2C-010 11 19.9 ± 0.83 8.092 ± 2.5 

LSV2C-012 20 22.0 ± 0.46 13.256 ± 0.8 

LSV2C-016 20 21.8 ± 1.5 15.836 ± 5.0 

LSV2C-017 20 23.7 ± 1.0 18.878 ± 4.0 

LSV2C-019 20 23.6 ± 1.4 19.320 ± 5.8 

LSV2C-020 20 21.8 ± 1.0 11.574 ± 2.7 

LSV2C-021 20 20.2 ± 1.5 10.656 ± 3.9 

3.2.3 Deformity Assessment 

Below is a list of the number of specimens preserved and analyzed at either swim-up or test 

termination for deformities (Table 3-6). The majority of fish that had died during the test were 

preserved but were not evaluated because of the poor state that they were end by the time death 

had occurred. As mentioned, a subset of these dead organisms were evaluated for deformities and 

included with the results of the assessment conducted on organisms that were alive when 

preserved. 
Document No. 12699-001-500 3-10 October 2011 

Table 3-6. Number of brown trout fry preserved and assessed for deformities. Samples were 

preserved at swim-up, at test termination, or upon death. All organisms preserved at swim-up and 

test termination were assessed for deformities; however, only select samples from organisms that 

died during the study were evaluated. 

Location Field 

Sample ID 

Number of fish 

assessed that 

were preserved 

at swim-up 

Number of fish 

assessed that 

were preserved 

at test 

termination 

Number of fish 

assessed that 

had died during 

the study 

SC-001 38 77 -- 

SC-002 34 79 -- 

SC-003 233 69 -- 

Hatchery Fish SC-004 60 80 -- 

SC-005 0 42 -- 

SC-006 457 78 -- 

SC-007 61 76 -- 

SC-008 289 70 -- 

SPC-001 488 80 -- 

Second set of SPC-002 0 0 -- 

SPC-003 465 80 -- 

Hatchery Fish SPC-004 0 0 -- 

SPC-005 485 76 -- 

SPC-006 476 80 -- 

CC-150-009 62 80 -- 

CC-150-011 185 81 -- 

CC-150-012 204 77 -- 

CC-150-013 258 52 -- 

CC-150-015 367 78 -- 

CC-150-016 0 23 -- 

CC-150-017 116 47 -- 

Wild CC-150-018 405 81 -- 

CC-150-020 478 80 -- 

CC-350-006 307 79 -- 

CC-350-007 72 59 -- 

CC-350-008 287 51 -- 

LSV2C-002 480 64 -- 

Fish LSV2C-003 0 0 ~100a

LSV2C-004 0 64 ~100a

LSV2C-005 0 49 ~100a

LSV2C-008 160 34 -- 

LSV2C-010 0 0 ~84a

LSV2C-012 474 80 -- 

LSV2C-016 449 81 -- 

LSV2C-017 92 58 -- 

LSV2C-019 349 41 -- 

LSV2C-020 252 44 -- 

LSV2C-021 0 69 ~100 

aWhile a subset of ~100 organisms were evaluated, scoring criteria were not always possible due to the poor physical 

state at preservation. 

For this assessment, the scoring criteria results of the fry preserved at swim-up, the fry preserved at 

test termination, and the fry preserved upon death (select samples) were combined. A summary of 

the raw data can be found in Appendix D of the main Brown Trout report. 
Document No. 12699-001-500 4-1 October 2011 

4.0 References 

American Society of Testing and Materials. 2006. Standard Guide for Conducting Early Life-stage 

 Tests with Fishes. E1241-05, Philadelphia, PA. 

Hardy, Ronald W. 2005. Final Report: Effects of Dietary Selenium on Cutthroat Trout 

(Oncorhynchus clarki) Growth and Reproductive Performance. Prepared for Montgomery 

Watson Harza. December 14. 

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, 24(9): 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. 

Newfields, LLC. 2007. Technical Memorandum. Methods for Testing Adult Brown Trout 

 Reproductive Success. Letter from S. Covington and S. Werner to D. Essig, IDEQ. Prepared 

 for J.R. Simplot. 

U.S. Environmental Protection Agency. 2002. Short-term methods for estimating the chronic toxicity 

 of effluents and receiving waters to freshwater organisms. Fourth Edition. EPA/821/R- 

 02/013.