### FIGURESWYOMINGIDAHO



## SMOKY
CANYON

MINE

Bern Lago Rose

Bench Niter Grace Conda Henry

Smoot Afton

Geneva Sharon Nounan Manson Grover Auburn 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 COUNTYUS HWY 89 STATE HWY 238

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

BY: RCR REV: 0 CHK: SMC SMOKY CANYON MINE

LOCATION OF THE
SMOKY CANYON MINE FIGURE 1 0 5 10

Miles

IDAHO 

### J.R. SIMPLOT COMPANYSage Valley LINCOLN COUNTY - WYOMING CARIBOU COUNTY - IDAHO



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Hoopes Spring HS

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DC-600 CC-350 LSV-2C

CC-150 6800

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PRJ: 009-004.70 DATE: JUN 30, 2010

BY: KSR REV: 0 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 BoundaryFigure 3Whole Body Selenium Concentration Versus Fish LengthREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC2002503003504004505005506000 5 10 15 20 25

Adult Fish Length (mm)Whole Body Selenium inBrown Trout (mg/kg dw)Hatchery FishWild FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 4Whole Body Selenium Concentration Versus Adult Fish Weight0500100015002000250030000 5 10 15 20 25

Adult Fish Weight (grams wet)Whole Body Selenium Concentration (mg/kg dw)Hatchery FishWild FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC051015202530350 50 100 150 200 250 300 350 400 450

Selenium Concentration (mg/kg dw)Length (mm)HoopesSage CkCrow Ck d/sBackgroundAdult BTFigure 5Brown Trout Length Versus Whole Body Selenium ConcentrationREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 6Brown Trout Egg Selenium Concentration by Location051015202530354045 Egg Selenium (mg/kg dw)Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 7Comparison of Adult Fish Size (Length) and the Total Number of Eggs Produced01000200030004000500060000 100 200 300 400 500 600

Total # of eggsAdult Fish Length (mm)NB: Symbols surrounded by a square are SC fishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 8Egg Selenium Concentration Versus Total Egg Abundance01000200030004000500060000 5 10 15 20 25 30 35 40 45

Total # of EggsEgg Selenium (mg/kg dw)Hatchery FishWild FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 9Adult Whole Body Selenium Concentration Versus Total Egg Abundance01000200030004000500060000 5 10 15 20 25

Total # of EggsAdult Whole Body Selenium (mg/kg dw)Hatchery FishWild FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 10Percent Egg Mortality by Location0102030405060708090100

% Egg MortalityLocationNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 11Percent Hatch (or Percent Survival at Hatch) of Alevins0102030405060708090100

% HatchLocationNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 12Relationship Between the Percentage of Eggs that Hatched and thePercentage of Fish that Reached Swim-UpNote: the dashed line indicates a 1:1 agreement.0204060801000 20 40 60 80 100

% Swim-up% HatchSCSPCCC-150CC-350LSV2CThe range of egg seleniumconcentrations for those alevins thatdid not swim up was 26.8 to 40.3mg/kg dw.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 13Egg Selenium Concentration VersusDay to First Hatch and Day to Swim-Up01020304050607080901000 5 10 15 20 25 30 35 40 45

1st hatch & swim-up (day)Egg Se (mg/kg dw)Day to 1st hatchswim-upDid not achieve swim-up (day of test term)SPC hatchery fish (received eyed up)SPC hatchery fish (received eyed up)Egg Selenium (mg/kg dw)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 14Fry Mortality at Swim-Up and Overall Mortality by Location0102030405060708090100 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 LSV2C-006 LSV2C-021 LSV2C-004 LSV2C-010 LSV2C-003

Mortality (%)at swim-upoverallNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 15Percent Mortality from Hatch to Test Termination by Location0102030405060708090100 Mortality (%) Dead upon arrival

None fertilizedNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 16Percent Mortality 15-Day Post Swim-Up Feeding Trial0102030405060708090100

Mortality (%) Dead upon arrival

None fertilizedNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 17Brown Trout Growth based on Larval Fish Length0510152025 average length (mm)Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 18Brown Trout Growth based on Larval Fish Dry Weight0510152025 average dry weight (mg)Note: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 19Percent Cranio-Facial Deformities for Larval Brown Trout0.00%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 LSV2C

Percentage of Samples0(normal)1(slight/few)2(mod/several)3(severe/many)(blank)NAREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 20Percent Skeletal Deformities for Larval Brown Trout0.00%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 LSV2C

Percentage of Samples0(normal)1(slight/few)2(mod/several)3(severe/many)(blank)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 21Percent Fin of Finfold Deformities for Larval Brown Trout0.00%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 LSV2C

Percentage of Sample0(normal)1(slight/few)2(mod/several)3(severe/many)(blank)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 22Percent Edematous Tissue Frequency for Larval Brown Trout0.00%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 LSV2C

Percentage of Sample0(normal)1(slight/few)2(mod/several)3(severe/many)(blank)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 23Mean GSI Score and Total Number of FishEvaluated by Location and Sample IDRanked by Egg selenium Concentration01002003004005006000.00.20.40.60.81.01.21.41.61.82.0 Total Number of Fish in Sample

Mean GSI ScoreGSItotalno.offishNote: The order of locations, as represented on the X axis, is in order of egg selenium concentration.REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 24Surface Water Selenium Concentrations00.010.020.030.040.050.06 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 DateHoopes Spring (HS)00.0050.010.0150.02

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 11/15/2008

Total Selenium (mg/L)Sample DateLower Sage Valley (LSV-4) below HoopesSpring00.0050.010.0150.02 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 11/15/2008

Total Selenium (mg/L)Sample DateCrow Creek - Meade Peak Ranch(CC-1 and CC-1A)Figure 25Log Maternal Whole Body SeleniumConcentration (Wild Fish) VersusLog Egg Selenium ConcentrationsREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = 1.1926x - 0.0071R2 = 0.796600.20.40.60.811.21.41.61.80 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Log Egg Selenium (mg/kg dw)Log Maternal Whole Body Selenium (mg/kg dw)Figure 26Egg Selenium Concentration VersusTotal Egg Abundance from Wild Female FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = -15.989x + 1090.7R2 = 0.1972y = -13.619x + 1044.5R2 = 0.17640200400600800100012001400160018000 5 10 15 20 25 30 35 40 45

Total # of EggsEgg Selenium (mg/kg dw)non normalizednormalizedLinear (non normalized)Linear (normalized)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 27Egg Selenium Concentration VersusPercent of Brown Trout Fry that Hatched0204060801000 5 10 15 20 25 30 35 40 45

% HatchEgg Selenium (mg/kg dw)Wild FishHatchery FishREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCFigure 28Wild Fish Egg Selenium Concentration VersusPercent of Trout Achieving Swim-Upy = -2.6299x + 97.598R2 = 0.48710204060801000 5 10 15 20 25 30 35 40 45

% Swim-upEgg Selenium (mg/kg dw)Wild FishHatchery FishLinear (Wild Fish)Figure 29Egg Selenium Concentration VersusLarval Fish WeightREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = -0.0011x3+ 0.0724x2- 1.4258x + 21.163R2 = 0.6623y = 0.0006x3- 0.0487x2+ 1.0565x + 7.1103R2 = 0.2605101520250 5 10 15 20 25 30 35 40 45

Dry Weight (mg)Egg Selenium (mg/kg dw)Egg Selenium Versus WeightHatchery & Wild FishWild FishPoly. (Hatchery & Wild Fish)Poly. (Wild Fish)Figure 30Egg Selenium Concentration VersusOverall Percent SurvivalREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = 0.0001x3- 0.0075x2+ 0.1372x + 0.0417R2 = 0.5425y = 3E-05x3- 0.0024x2+ 0.035x + 0.6113R2 = 0.30390%10%20%30%40%50%60%70%80%90%100%0 5 10 15 20 25 30 35 40 45

Overall Survival (%)Egg Selenium (mg/kg dw)wild fishhatchery fishAllPoly. (wild fish)Poly. (All)Figure 31Egg Selenium Concentration VersusPercent Survival (Hatch to Test End)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = 0.0019x3- 0.1905x2+ 2.9252x + 86.179R2 = 0.8894y = 0.0006x3- 0.0961x2+ 0.9843x + 97.214R2 = 0.89620204060801001200 5 10 15 20 25 30 35 40 45

Survival (%, hatch to test end)Egg Selenium (mg/kg dw)wild fishhatchery fishAll FishPoly. (wild fish)Poly. (All Fish)Figure 32Egg Selenium Concentration VersusPercent Survival in the 15-Day Post Swim-Up Feeding TrialREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy = 0.0018x3- 0.1843x2+ 3.2221x + 84.717R2 = 0.9614y = 0.0002x3- 0.0714x2+ 0.9027x + 97.888R2 = 0.95690204060801001200 5 10 15 20 25 30 35 40 45

Survival (%, 15-Day Post Swim-Up Feeding Trial)Egg Selenium (mg/kg dw)wild fishhatchery fishAll FishPoly. (wild fish)Poly. (All Fish)Figure 33Egg Selenium Concentration VersusSum Fraction Non-Normal LarvaeREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy=‐0.0004x3+0.025x2‐0.4288x+2.5231R2=0.851500.511.522.533.50 5 10 15 20 25 30 35 40 45

Sum Fraction Non-Normal LarvaeEgg Selenium (mg/kg dw)DeformitiesPoly.(Deformities)Figure 34Egg Selenium Concentration VersusSum Fraction Normal LarvaeREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy=0.0002x3‐0.0184x2+0.328x+1.9231R2=0.870500.511.522.533.544.50 5 10 15 20 25 30 35 40 45

Sum Fraction Normal LarvaeEgg Selenium (mg/kg dw)DeformitiesPoly.(Deformities)Figure 35Egg Selenium Concentration VersusMean GSI ScoreREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCy=‐1E‐04x3+0.0079x2‐0.1374x+0.8071R2=0.844900.20.40.60.811.21.41.61.820 5 10 15 20 25 30 35 40 45

Mean GSI ScoreEgg Selenium (mg/kg dw)GSIscorePoly.(GSIscore)Figure 36TRAP ModelEgg Selenium ConcentrationVersus Larval Fish Growth15-Day Post Swim-Up Feeding TrialREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC05101520250.6 0.8 1 1.2 1.4 1.6 1.8 2

Larval Fish Growth (mg dw)LogEggSelenium(mg/kgdw)Actual Datapredict lineEC2095% LCL EC2095% UCL EC20EC1095% LCL EC1095% UCL EC10Figure 37TRAP ModelEgg Selenium Concentration Versus Survival15-Day Post Swim-Up Feeding TrialREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC01020304050607080901001100.6 0.8 1 1.2 1.4 1.6 1.8

Survival 15

‐ Day Post Feeding Trial (%)LogEggSelenium(mg/kgdw)Actual Datapredict lineEC2095% LCL EC2095% UCL EC20EC1095% LCL EC1095% UCL EC10Figure 38TRAP ModelEgg Selenium Concentration Versus Total SurvivalREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC01020304050607080901001100.6 0.8 1 1.2 1.4 1.6 1.8

Total Survival (%)LogEggSelenium(mg/kgdw)Actual Datapredict lineEC2095% LCL EC2095% UCL EC20EC1095% LCL EC1095% UCL EC10Figure 39TRAP ModelEgg Selenium Concentration Versus Percent Survival(Hatch to Test End)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC01020304050607080901001100.6 0.8 1 1.2 1.4 1.6 1.8

% Survival (Hatch to Test End)LogEggSelenium(mg/kgdw)EggSeleniumVersus%Survival(HatchtoTestEnd)Actual Datapredict lineEC2095% LCL EC2095% UCL EC2095% LCL EC1095% UCL EC10EC10Figure 40TRAP ModelEgg Selenium Concentration VersusCranio-Facial Deformities (Fraction Normal)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC00.10.20.30.40.50.60.70.80.911.10.6 0.8 1 1.2 1.4 1.6 1.8

Cranio

‐ Facial Deformities (Fraction Normal)LogEggSelenium(mg/kgdw)Actual DataEC2095% LCL EC2095% UCL EC20predict lineEC1095% LCL EC1095% UCL EC10Figure 41TRAP ModelEgg Selenium Concentration VersusSkeletal Deformities (Fraction Normal)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC00.10.20.30.40.50.60.70.80.911.10.6 0.8 1 1.2 1.4 1.6 1.8

Skeletal Deformities (Fraction Normal)LogEggSelenium(mg/kgdw)Actual DataEC2095% LCL EC2095% UCL EC20predict lineEC1095% LCL EC1095% UCL EC10Figure 42TRAP ModelEgg Selenium Concentration VersusFinfold Deformities (Fraction Normal)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC00.10.20.30.40.50.60.70.80.911.11.20.6 0.8 1 1.2 1.4 1.6 1.8

Finfold Deformities (Fraction Normal)LogEggSelenium(mg/kgdw)Actual DataEC2095% LCL EC2095% UCL EC20predict lineEC1095% LCL EC1095% UCL EC10Figure 43REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCTRAP ModelEgg Selenium Concentration VersusEdema (Fraction Normal)00.10.20.30.40.50.60.70.80.911.11.20.6 0.8 1 1.2 1.4 1.6 1.8

Edema (Fraction Normal)LogEggSelenium(mg/kgdw)Actual DataEC2095% LCL EC2095% UCL EC20predict lineEC1095% LCL EC1095% UCL EC10Figure 44REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMCTRAP ModelEgg Selenium Concentration VersusSum Fraction Normal Fish Larvae0.000.501.001.502.002.503.003.504.004.505.000.60 0.80 1.00 1.20 1.40 1.60 1.80

Fraction Normal (normal/total)LogEggSelenium(mg/kgdw)Actual Datapredict lineEC2095% LCL EC2095% UCL EC20EC1095% LCL EC1095% UCL EC10Figure 45TRAP ModelEgg Selenium Concentration VersusMean Fraction Normal Fish LarvaeREV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC0.000.100.200.300.400.500.600.700.800.901.000.60 0.80 1.00 1.20 1.40 1.60 1.80

Mean Fraction Normal (normal/total)LogEggSelenium(mg/kgdw)Actual Datapredict lineEC2095% LCL EC2095% UCL EC20EC1095% LCL EC1095% UCL EC10Figure 46Revised TRAP ModelEgg Selenium Concentration VersusPercent Survival (Hatch to Test End)REV: 1J.R. Simplot CompanySite-Specific Selenium CriterionPRJ: 0442-004-900.70BY: SMCDATE: June 2011CHK: SMC01020304050607080901001100.6 0.8 1 1.2 1.4 1.6 1.8

% Survival (Hatch to Test End)LogEggSelenium(mg/kgdw)EggSeleniumVersus%Survival(HatchtoTestEnd)Actual Datapredict lineEC2095% LCL EC2095% UCL EC2095% LCL EC1095% UCL EC10EC10excluded data

### APPENDIX A Data Report-Reproductive Success Study with Brown Trout (Salmo trutta) -ENSRPre pared for:

JR Simplot Company and Newfields Company, LLC. Pocatello, Idaho



# Data Report

Reproductiv e Success Study with Brown Trout ( Salmo trutta )

F inal

Oct ober 2011i Oc tober 2011 Document No. 12699-001-500 

# 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 ................................................................................................................................... 4-1i i October 2011 Document No. 12699-001-500 

# List of Appendices

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

A p pendix 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 studyi ii October 2011 Document No. 12699-001-500 

# List of Tables

Page



### T able 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 ..................... 3-10i v October 2011 Document No. 12699-001-500 

# List of Figures

Page


### F igure 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 .................................................... 3-81 -1 October 2011

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# 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.2 -1 October 2011

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# 2.0 Methods

E NSR 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.2 -2 October 2011

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

i ce 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 CaCO 3 

### ] 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). The2 -3 October 2011

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

o n 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



### A fter 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 at2 -4 October 2011

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

l evel 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 guidancefor 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
N itex



### Screen

60 eggs / cup

Solution Level Inflowing solution

Outflow with Nitex 

### A B C D E F G H I J2 -5 October 2011

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### 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 the2 -6 October 2011

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

F ish 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 T est Initiation Date Test Termination Date(s)

SC O ct. 25, 2007 Jan. 17, 2008

SPC D ec. 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



### E ggs (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 feeding2 -7 October 2011

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

f ecal 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 CaCO 3 

### (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.2 -8 October 2011

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

C row Creek Drainage.

Horsetooth Reservoir 1 Crow Creek 2

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

So dium (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

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

FCETL.

2 Crow Creek drainage as characterized by surface water from Crow Creek, Sage Creek, and Hoopes Spring surface water quality data (Newfield 2007)



### Calcium sulfate (CaSO 4 

### .2H 2 

### O; Ben Franklin  

### Aquacal  , Plaster City, CA) and magnesium sulfate (MgSO 4 

### .7H 2 

### O; 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 CaSO 3 

### . This super stock was metered into the holding vessel to achieve a target hardness of ~50 mg/L as CaCO 3 

### 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 was2 -9 October 2011

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

ch amber. 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 ( μ S/cm) were measured and recorded in one chamber for each test treatment daily. Hardness (as mg/L CaCO 3 

### ) 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 μ m 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).2 -10 October 2011

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

w ell. 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).3 -1 October 2011

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# 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 reproductionstudy 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.3 -2 October 2011

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### 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 E gg

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 6 00 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 2 0 -- 0 SPC-003 600 -- 2.5 Hatchery Fish SPC-004 2 1 -- 0 SPC-005 600 -- 1.2 SPC-006 600 -- 0.3
CC-150-009 600 1,215 71.5 C C-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).3 -3 October 2011

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### 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

( μ S/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

C C-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

N ote: 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 CaCO 3 

### ) between October 25, 2007 and February 12, 2008. Ammonia wasmeasured 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.3 -4 October 2011

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### 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 1 st

hatch % hatch a Day of swim-up % Swimup Survival

(%) at
S wim-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
C C-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 0 b 8.0 LSV2C-004 40 50.6 88 0 b 30.2 LSV2C-005 40 71.3 88 0 b 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 0 b 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 0 b 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% (average3 -5 October 2011

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### 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 notabsorb 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 were3 -6 October 2011

Document No. 12699-001-500 

### 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.3 -7 October 2011

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### 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

s wim-up stage Total

Survival (%) Survival (%)

from Hatch

u ntil 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 1 00 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 4 9 SPC-003 100 94.2 96.7 49 Hatchery Fish SPC-004 100 100 100 4 9 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 C C-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-up3 -8 October 2011

Document No. 12699-001-500 

### (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

LSV2C3 -9 October 2011

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### 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 2 3.246 ± 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 2 0.852 ± 4.1 SPC-003 20 23.3 ± 2.1 21.807 ± 5.4 Hatchery Fish SPC-004 20 23.4 ± 2.0 2 1.564 ± 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 C C-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.3-1 0 October 2011

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### 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

w e r e preserved

at test termination Number of fish

assessed that had died during the study

SC -0 01 38 77 -- SC -0 02 34 79 -- SC-003 233 69 -- Hatchery Fish SC-004 60 80 --

SC -0 05 0 42 -- SC-006 457 78 -- SC-007 61 76 -- SC-008 289 70 -- SPC-001 488 80 -- Second set of SP C -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 -- C C -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 ~100 a

L S V2C-004 0 64 ~100 a

LS V2C-005 0 49 ~100 a

LS V2C-008 160 34 -- LSV2C-010 0 0 ~84 a

LS V2C-012 474 80 -- LSV2C-016 449 81 -- LSV2C-017 92 58 -- LSV2C-019 349 41 -- LSV2C-020 252 44 -- LSV2C-021 0 69 ~100

a W h ile 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 o f the main Brown Trout report.4 -1 October 2011

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# 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.