### Re

- Analysis of the Brown Trout Selenium Toxicity Study Presented by

Formation Environmental and Reviewed by U.S. Fish and Wildlife Service

2014 Revision

DRAFT

June 21 , 2014

Office of Water, U.S. Environmental Protection Agency, Washington, DC

Submitted By

Great Lakes Environmental Center, Columbus, OHi TABLE OF CONTENTS

TABLE OF CONTENTS

................................ ................................ ................................ ................. i LIST OF TABLES

................................ ................................ ................................ .......................... ii LIST OF FIGURES

................................ ................................ ................................ ....................... iii Introduction

................................ ................................ ................................ ................................ ..... v Background

................................ ................................ ................................ ................................ . v Re

- Analysis of the Brown Trout Selenium Toxicity Study Presented by Formation Environmental and Reviewed by U.S. Fish and Wildlife Service

................................ ............. 1 Introduction

................................ ................................ ................................ ................................ ..... 2 Brown Trout Reproduction Stud y Summary

................................ ................................ .................. 2 Issues Concerning the Brown Trout Reproduction Study

................................ .............................. 4 Fungal Overgrowth

................................ ................................ ................................ ..................... 4 Use of Alevin in Deformity Analysis and 15 Day Post Swim Up

................................ .............. 4 Calculation of Larval Survival

................................ ................................ ................................ .... 5 Overflow and Loss of Fish during the 15

- Day Post Hatch Survival Feeding Trial

.................... 5 Calculation of Deformity Endpoints

................................ ................................ ........................... 6 Correction of Errors

................................ ................................ ................................ .................... 7 Methods

................................ ................................ ................................ ................................ ........... 7 Calculation of Larval Survival (Full Test)

................................ ................................ .................. 7 Calculation of Larval Survival (Hatch to Swim Up)

................................ ................................ ... 8 Calculation of Deformities

................................ ................................ ................................ .......... 8 Calculation of Mortality Plus Deformity

................................ ................................ .................. 10 Analysis of Subsets of the Data

................................ ................................ ................................ . 12 Data Analysis

................................ ................................ ................................ ............................ 15 Results

................................ ................................ ................................ ................................ ........... 18 Larval Survival (Full Test)

................................ ................................ ................................ ........ 18 Larval Survival for First Portion of Test (Hatch to Swim

- up)

................................ .................. 18 Larval Deformities

................................ ................................ ................................ .................... 19 Larval Major Deformities

................................ ................................ ................................ .......... 22 Larval Mortality Plus Deformity

................................ ................................ ............................... 23 Synthesis

................................ ................................ ................................ ................................ ....... 24 Appendix A: Assessment of EC 10 Calculations Performed by the U.S. Fish and Wildlife Service

................................ ................................ ................................ ................................ ....................... 62 References

................................ ................................ ................................ ................................ ..... 69 Re

- Analysis of the Brown Trout S elenium Toxicity Study Presented by Formation Environmental and Reviewed by U.S. Fish and Wildlife Service, 2014 Revision

- Addendum

................................ ................................ ................................ ................................ ... 70
Intro duction

................................ ................................ ................................ ................................ ... 71 Key Differences between the Original and Updated Reassessment

................................ ......... 71 Brown Trout Reproduction Study Summary

................................ ................................ ................ 72 Reanalysis of E ffects Data

................................ ................................ ................................ ............ 73 Discussion of Issues Addressed in Taulbee et al. 2012 and in 2014 Reanalysis Concerning the Brown Trout Reproduction Study

................................ ................................ ................................ . 75 Use of Alevin in Deformity Analysis and 15 Day Post Swim Up

................................ ............ 75 Overflow and Loss of Fish During the 15

- Day Post Hatch Survival Feeding Trial

................. 75ii

Calculation of Deformity Endpoints

................................ ................................ ......................... 76 Correction of Errors in Previous Documents Related to the Formation Study.

........................ 77 Methods to Address Issues (from Taulbee et al. (2012), and this Addendu m)

............................ 77 Calculation of Larval Survival (Hatch through 15 day post swim up test)

............................... 77 Calculation of Larval Deformities

................................ ................................ ............................. 78 Calculation o f Combined Effects of Mortality and Deformity

................................ ................. 79 Data Analysis

................................ ................................ ................................ ............................ 86 Results

................................ ................................ ................................ ................................ ........... 86 Survival

................................ ................................ ................................ ................................ ..... 86 Deformity

................................ ................................ ................................ ................................ .. 86 Combined Effects of Mortality and Deformity

................................ ................................ ......... 91 Summary

................................ ................................ ................................ ................................ ... 91 2014 Addendum References

................................ ................................ ................................ ......... 94

LIST OF TABLES

Table 1a. Summary of Larval Survival, Full Test.

................................ ................................ ....... 27 Table 1b. Summary of Larval Survival, First Portion of Test (Hatch to Swim Up).

.................... 28 Table 2. Summary of Larval Deformities Excluding Missing Fry.

................................ .............. 29 Table 3. Summary of Major Larval Deformities Excluding Missing Fry.

................................ ... 30 Table 4. Summary of Larval D eformities Incorporating Missing Fry.

................................ ......... 31 Table 5. Summary of Major Larval Deformities Incorporating Missing Fry.

.............................. 32 Table 6a. Summary of Larval Survival plus Deformity endpoint

– lost fish excluded (M1).

...... 33 Table 6b. Summary of Larval Survival plus Deformity endpoint

– lost fish assumed dead (M2).

................................ ................................ ................................ ................................ ........... 34 Table 7. Larval survival for field treatments where fry reached swim up stage.

.......................... 35 Table 8. Larval deformities for field treatments where fry reached swim up stage.

.................... 36 Table 9a. Resu lts of TRAP Analyses for Larval Survival, Full Test

. ................................ ........... 37 Table 9b. Results of TRAP Analyses for Larval Survival, First Portion of Test (Hatch to Swim Up)

. ................................ ................................ ................................ ................................ ... 37 Table 10. Results of TRAP Analyses for Larval Deformities.

................................ ..................... 38 Table 11. Results of TRAP Analyses for Major Larval Deformities.

................................ .......... 39 Table 12. Results of TRAP Analyses for the Combined Effects of Larval Mortality and Deformity.

................................ ................................ ................................ ......................... 40 Table AD1. Brown trout selenium concentrations and survival data from hatch to test end.

...... 81 Table AD2. Brown trout selenium concen trations and deformity data from hatch to test end.

... 83 Table AD3. Brown trout selenium concentrations and survival + deformity data (combined endpoint) from hatch to test end.

................................ ................................ ...................... 84iii LIST OF FIGURES

Figure 1. Survival in full test (hatch through post swim up) excluding missing fry (Method 1: Overflow Optimistic Case) analyzed with TRAP tolerance distribution for the four data subsets.

................................ ................................ ................................ .............................. 41 Figure 2. Survival in full test (hatch through post swim up) including missing fry as dead (Method 2: Overflow Worst Case) analyzed with TRAP tolerance distribution for the four data subsets.

................................ ................................ ................................ ...................... 42 Figure 3. Survival only to swim up (first part of test) analyzed with TRAP tolerance distribution for the four data subsets.

................................ ................................ ................................ ... 43 Figure 4. Deformities as maximum fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................................ ...................... 44 Figure 5. Deformities as minimum fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Worst Case), analyzed by TRAP tolerance distri bution for the four data subsets.

................................ ................................ ................................ ...................... 45 Figure 6. Deformities as intermediate fully normal fry, excluding missing fry (Overflow Opti mistic Case, Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 46 Figure 7. Defo rmities as maximum fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 47 Figure 8. Deformities as minimum fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 48 Figure 9. Deformities as intermediate fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Defor mities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 49 Figure 10. Major deformities as maximum fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 50 Figure 11. Major deformities as minimum fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ ................ 51 Figure 12. Major deformities as intermediate fully free of major deformities, excluding missing fry (Overflo w Optimistic Case, Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ 52 Figure 1 3. Major deformities as maximum fully free of major deformities, assuming missing fry were deformed (Overflow Worst Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ 53 Figure 14. Major deformities as minimum fully free of major deformities, assuming missing fry were deformed (Overflow Worst Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ................................ 54 Figure 15. Major deformities as intermediate fully free of major def ormities, assuming missing fry were deformed (Overflow Worst Case, Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

................................ ..................... 55iv

Figure 16. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on maximum fully free from deformity (Deformity Optimistic Case), analyzed with TRAP for the four data subsets.

................................ ................................ ................ 56 Figure 17. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on minimum fully free from deformity (Deformity Worst Case), analyzed with TRAP for the four data subsets.

................................ ................................ ........................ 57 Figure 18. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on intermediate fully free from deformity (Deformity Intermediate Case), analyzed with TRAP for the four data subsets.

................................ ................................ . 58 Figure 19. Combined effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on maximum fully free from deformity (Deformity Optimistic Case), analyzed with TRAP for the four data subsets.

................................ ................................ . 59 Figure 20. Combined effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on minimum fully free from deformity (Deformity Worst Case), analyzed with TRAP for the four data subsets.

................................ ................................ . 60 Figur e 21. Combi ned effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on intermediate fully free from deformity (Deformity Intermediate Case), analyzed with TRAP for the four data subsets.

................................ ..................... 61 Figure A1. Summary of issues in plot for calculation of the Review’s deformity EC 10 of 6.55 mg Se/kg.

................................ ................................ ................................ ................................ 67 Figure A2. Effects of data selection and calculation method on deformity EC 10

. ........................ 68 Figure AD1. Concentration

- response relationship of brown trout survival from hatch through 15

- days post swim up and selenium concentrations in eggs, optimistic case.

....................... 87 Figure AD2. Concentration

- response relationship of brown trout survival from hatch through 15

- days post swim up and selenium concentrations in eggs, worst case.

.............................. 88 Figure AD3. Concentration

- response relationship of fully normal brown trout fry from hatch through 15

- days post swim up and se lenium concentrations in eggs, optimistic case.

.... 89 Figure AD4. Concentration

- response relationship of fully normal brown trout fr y from hatch through 15

- days post swim up and selenium concentrations in eggs, worst case.

........... 90 Figure AD5. Concentration

- response relationship of fully normal brown trout fry that also survived from hatch through 15

- days post swim up and selenium concentrations in eggs, optimistic case.

................................ ................................ ................................ .................. 92 Figure AD6. Concentration

- response relationship of fully normal brown trout fry that also survived from hatch through 15

- days post swim up and selenium concentrations in eggs,

worst case.

................................ ................................ ................................ ......................... 93v

Introduction This document consists of a reanalysis of the brown trout ( Salmo trutta ) selenium toxicity study presented by Formation Environmental (2011). The original draft reanalysis (Taulbee et al. 2012) completed for the EPA was written in response to a technical review of a draft version of the Formation Environmental toxicity study conducted by the U.S. Fish and Wildlife Service (U.S. FWS 2012). Following a peer

- review of Taulbee et al. (2012), additional information was provided in a quality assurance (QA) report of the Formation Environmental report data by AECOM ( 2012 )

. This nec essitated additional reanalysis, which is included in this document as a 2014 addendum to Taulbee et al. (2012). The 2014 re

- analysis addresses comments made by peer reviewers of the Taulbee et al. (2012) document by providing an revised analysis of the n ew AECOM QA report data

, which reflects counts of fish that were fully free from all deformities,
as well as the number of fish assessed for deformities.

Background Formation Environmental conducted a field

- based brown trout ( Salmo trutta ) reproduction st udy for receiving waters downstream of the Smoky Canyon phosphate mine in Southeastern Idaho in 2007 for the J.R. Simplot Company. A draft report was released in 2010, and the final report was released as Formation Environmental (2011).

The U.S. FWS (2012 ) issued a technical review that identified several issues with the Formation Environmental study. Notable among these issues were the loss of fry during the post swim up portion of the test, fungal growth on numerous egg samples during the study, the tre atment of fry that failed to reach swim up in survival calculations, and the most appropriate model type and measure of effect to use. After addressing the issues with the Formation Environmental study,
the U.S. FWS review included recommended recalculati ons for the survival and deformity endpoints.

The original Taulbee et al. (2012) reanalysis conducted for the EPA examined the issues raised in the U.S. FWS (2012) review through a series of 78 hypothetical scenarios. The loss of fry to
overflow due to the clogging of drains in several treatments during the post swim up portion of the test was addressed by considering an optimistic scenario, where fry lost to overflow were completely excluded from survival calculations (the approach followed in Formation Environmental); and a worst

- case scenario, where all fry lost to overflow were assumed to have been dead (or dying) prior to overflow. Consistent with recommendations in the U.S. FWS review, fry that survived the test but failed to reach swim up were cou nted as having died in the
Taulbee et al. report , based on the assumption that they would not have survived in the wild.

Formation Environmental (2011) presented deformity rates separately for four classes of
deformities: cranial

- facial, skeletal, finfold, and edema. However, no information was provided allowing for the calculation of fish fully free from deformities. The U.S. FWS review was critical of the method used to calculate deformities in the Formation Environmental report, whichvi

calculated deform ities as 100% minus the average of the four deformity classes (sum fully normal)

, because U.S. FWS believed it would result in an underestimat ion of total deformity rates in cases where some deformity rates for specific deformity types were much higher than others. In response to this uncertainty, U.S. FWS calculated deformities as 100% minus the percentage of the most prevalent deformity type ( defined as maximum fully normal). Taulbee et al. (2012) addressed uncertainties regarding the total counts of fry fully free from deformities by considering three scenarios (maximum

- , intermediate

- , and minimum

- fully normal) based on increasingly conservative assumptions of how the different types of deformities overlapped among individuals

. Maximum fully norm al is equal to 1.0 minus the maximum rate of any of the four deformity types. This was the calculation procedure used in the U.S. FWS review. It is the least conservative of the three approaches in that is assumes that the less prevalent deformity
types only occur in individuals already having the most prevalent type. Intermediate fully normal deformities are calculated as the product of “1.0 minus the deformity rate” for each of the four deformity types. For example, if each of the deformity types had a 10% occurrence rate, the intermediate fully normal proportion would be 0.9x0.9x0.9x0.9=0.656. Finally, minimum fully normal is equal to 1.0 minus the sum of the deformity rates for each of the four deformity types.
When the sum is greater than 1.0, the proportion of minimum fully fry is set to 0. This is the most conservative way to estimate deformities, as it assumes that no fish can have more than one type of deformity unless the summed proportion of all deformity rates is greater than 1.

U ncertainties regarding the health of fry lost to overflow were incorporated into the deformity calculations, by calculating the optimist ic and worst

- case scenario s for each of the three deformity scenarios (maximum

- , intermediate

- , and minimum

- fully nor mal) described above

. That is

, the optimistic scenario regarding status of fish lost to overflow reflects an assumption
that fish lost would have been the same as fish that were not lost. For purposes of calculating the deformity endpoint, the worst

- case scenario reflects an assumption that fish lost would have had all four deformity types

. In addition to calculating survival and deformity endpoints, Taulbee et al. (2012) also calculated combined (survival plus deformity) endpoints consisting of all poss ible survival and deformity scenario combinations described above.

Taulbee et al. (2012) examined each individual survival, deformity, and combined endpoint scenario using four data subsets. These data subsets were selected to address the effects of the

r ecommended recalculations performed in the U.S. FWS review to account for the loss of fish to overflow and the presence of fungal growth on eggs in some of the treatments. The first data
subset consisted of all 24 field

- collected samples, and no hatchery samples. This was the dataset analyzed in the Formation Environmental report. The second dataset consisted of 14 field

collected samples and hatchery samples from the Spring Creek Hatchery (SPC). This was the
dataset recommended for the calculation of th e deformity endpoint in the U.S. FWS review. U.S. FWS recommended completely excluding the 10 field

- collected samples where fish were lost to overflow, and recommended including fish from the SPC Hatchery, which arrived as pre

fertilized, eyed embryos; wh ile excluding fish hatched from eggs from the Saratoga Nationalvii

Fish (SC) Hatchery, which were subjected to the fungal overgrowth. The third dataset consisted
of all field

- collected samples plus all fish from the SPC hatchery. This dataset was selected t o isolate the effects of the fungal overgrowth on hatchery fish, while allowing for a comparison between the U.S. FWS recommendation for addressing fish lost to overflow (exclusion of those samples) to optimistic and worst

- case scenario approaches describe d above. The fourth dataset
consisted of all field

- collected samples and all hatchery samples.

Taulbee et al. (2012) provided a final recommendation on 16.76 mg Se/kg dw, based on the EC 10 for survival under the worst

- case scenario using the dataset consi sting of all field

- collected and hatchery samples. This is lower than the recommended EC 20 of 21.63 mg Se/kg dw, and EC 10 of 20.8 mg Se/kg dw for survival, in the 2010 draft and 2011 final Formation Environmental reports, respectively; and higher than the recommended EC 10 of 6.5 mg Se/kg dw for deformities in the 2012 U.S. FWS review. Taulbee et al. (2012) underwent an independent, external peer
review by six expert scientists. The external peer reviewers of Taulbee at al. (2012) all
concurred that an EC 10 of 16.76 mg Se/kg dw based on the scenarios analyzed was a reasonable

worst

- case approach, with the exception of one reviewer who thought the EC 10 derived through
the worst

- case scenario was overly conservative. These peer reviewers also noted that that the U.S. FWS approach of excluding all treatments w h ere fish were lost to overflow unfortunately
excludes all data within the critical range of partial

- effects in the dose

- response data ( i.e., between 0 and 100% effect) , resulting in a loss of key i nformation contained in the remaining data for affected doses, and within the range of known

, experimentally

- derived EC 10 s for fish exposed to selenium through the diet It was noted that the low U.S. FWS

- calculated EC 10 of 6.5

mg Se /kg dw was inconsistent with the observed lack of negative trend in deformities below the

20.5 mg Se /kg dw dataset and was not supported by the data available for consideration

. It was also noted through the Taulbee et al. document peer review that data on the counts of fish th at were fully free from all deformities, as well as the number of fish assessed for deformities, would be especially useful in reducing uncertainty in the calculated EC 10 for the Formation Environmental trout study.

Following the peer

- review of Taulbee et al. (2012), a quality assurance (QA) report of the original raw data from the Formation Environmental brown trout reproductive study was conducted (AECOM 2012). In addition to making numerous minor corrections to the original data, AECOM (2012) also nota bly provided counts of fish that were fully free from all deformities, as well as the number of fish assessed for deformities. This important additional information addressed a major source of uncertainty in the Formation Environmental (2011) study raised in the U.S. FWS (2012) review by providing counts for fry that are fully free from all deformities. These updated deformity data allow for a second reanalysis that greatly reduced the number of hypothetical scenarios for both the deformity and combined (s urvival plus deformity) endpoints that were examined in Taulbee et al. (2012) in order to account for uncertainties in reported deformity counts. That is, the need for analyses to consider maximum

- , intermediate

- , and minimum

- fully normal estimated effect s was eliminated. The reanalysis,viii

included here as a 2014 A ddendum, includes optimistic and worst case EC 10 calculations for survival, deformities (fully normal), and combined (survival + fully normal) endpoints. Because
the final recommended EC 10 in Taulbee et al. (2012) was based on the full (all field

- collected samples plus all hatchery samples), the six scenarios presented in the addendum were only examined using the full dataset. The final recommended EC 10 presented in the 2014 A ddendum
is 15 .91 mg Se/kg dw, based on the worst

- case scenario for the deformity endpoint, which was
the most conservative EC 10 among the six scenarios that were considered , and is lower than the

16.76 mg Se/kg dw EC 10 included in the Taulbee et al. (2012) report and p eer

- reviewed in 2012. The value of 15.91 mg Se/kg dw is the chronic value for brown trout recommended in the External Peer Review Draft Aquatic Life Ambient Water Quality Criterion for Selenium

– Freshwater 2014 (U

. S

. EPA 2014).1



### Re

- Analysis of the Brown Trout Selenium Toxicity Study Presented by Formation Environmental and Reviewed by U.S. Fish and Wildlife Service

DRAFT

August 22, 2012

By

Keith Taulbee Dennis McIntyre Great Lakes Environmental Center, Inc. Columbus, OH

and

Charles Delos Office of Water, U.S. Environmental Protection Agency, Washington, DC

Note: This 2012 Re

- Analysis is identical to the report that underwent independent, contractor

led external peer review in 2012, with the exception of minor grammatical corrections. All data, analyses and conclusions are identical to the peer reviewed inf ormation.2

Introduction The following is a reassessment of the brown trout reproductive study as presented in the 2011 “Brown Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site

Specific Selenium Criterion” by Formation En vironmental. This reassessment is in response to the 2012 “Technical Review: Smoky Canyon Mine Site

- Specific Selenium Criterion Report” by the U.S. Department of the Interior Fish and Wildlife Service (FWS 2012). FWS reviewed Formation’s 2010 draft (not the above 2011 final revision) and addressed several topics unrelated to the brown trout test. However, the current analysis is limited to the sections that discuss the brown trout reproductive study. In particular, it addresses issues raised in Section II of the FWS Review, Recommended Recalculations, and relevant portions of the peer review of the FWS
Review.

Hereafter for simplicity, the Formation Environmental 2011 document (and related 2010 draft report) will often be called the Report, and the FWS 2012 review of 2010 draft will often be called the Review. This document begins with a summary of the Formation Environmental (2011) Report, followed by the recommended ten

- percent effect concentrations (EC 10 s) and toxicity endpoints as determined in the Report and as recalculated in the Review. Next, a description of experimental and analytical issues that affected the recommendations of the Report and the recommended recalculations of the Review are listed. The remainder of the
document includes the m ethods, results, and conclusions of the reassessment of the Report’s brown trout data in light of issues raised in the Review. Finally, a critical assessment of the
Review’s EC 10 calculations is contained in Appendix A.

Brown Trout Reproduction Study Su mmary Formation Environmental was contracted by the J.R. Simplot Company to perform a brown trout ( Salmo trutta ) reproduction study for receiving waters downstream of the Smoky Canyon phosphate mine in Southeastern Idaho. After elevated selenium (Se) conc entrations were measured in receiving waters downstream of the Smoky Canyon mine, this study was commissioned to investigate the possible effects of these elevated Se concentrations on the reproductive fitness of resident brown trout.

In the fall of 2007, pre

- spawn adult brown trout (>230 mm) were collected from three locations downstream of the Smoky Canyon mine. These included one location (station LSV

- 2C) along Sage Creek, a highly impacted site downstream of Hoopes Spring (which drains directly from
th e Smokey Canyon mine) that regularly exceeds the chronic water quality standard for
selenium ; as well as two locations along Crow Creek upstream of the confluence with Sage Creek. Chronic selenium exceedances have occasionally been measured in Crow Creek, but only immediately downstream of Sage Creek. The surface water selenium concentration in Sage
Creek at the time of initial fish collection was 38.4 μ g/L, while the surface water concentrations
in Crow Creek were < 1.5 μ g/L.3

Eggs were collected from 26 adult female fish collected from Sage and Crow Creek in the fall of 2007 and fertilized in the laboratory. A sample of eggs from each female was analyzed for
sele nium content. Eggs from two of the females from Sage Creek were not used; one batch because they were dead upon arrival to the laboratory, and the other because they were not successfully fertilized (Formation 2011). The final number of field samples for the reproductive study, defined as the offspring of a female wild

- caught brown trout beginning with their fertilized eggs, was 24; 12 from Sage Creek (station LSV

- 2C), 3 from the upstream Crow Creek location (station CC

- 350), and 9 from the downstream Cro w Creek location (station CC

- 150). Selenium concentrations in eggs collected from each female ranged from 11.2

- 40.3 mg Se/kg dw at Sage Creek, 6.9

- 14.0 mg Se/kg dw at the upstream Crow Creek location, and 6.2

- 12.8 mg Se/kg dw at the downstream Crow Creek location.

In addition, eggs were collected from 8 ripe females obtained from the Saratoga National Fish Hatchery (SC) to serve as method controls. Similar to wild caught females, they were stripped of eggs and fertilized by milt stripped from adult males from the same hatchery. Selenium concentrations in eggs collected from each SC hatchery female ranged from 0.76

- 1.2 mg Se/kg dw. As a result of lower than expected hatch rates and fungal contamination in some of the SC hatchery samples, additional hatche ry fish were obtained. Because it was late in the hatching season, no fertile females were available from the Spring Creek Trout Hatchery (SPC). Instead, fertilized eyed eggs were sent from the SPC hatchery and these were split into four samples to

serve as a secondary method control. The selenium concentration in eyed eggs obtained from the SPC hatchery samples was 0.73 mg Se/kg dw.

For each sample, the target was to initiate the test with 600 fertilized eggs. Fertilized eggs were monitored through hat ch and swim up (the absorption of the yolk sac by the alevins), and percent hatch, time of hatch, and time to swim up were recorded for all field and hatchery samples. After the fry for a given sample reached swim up, their numbers were culled to a target of 100 individuals / sample for a 15 day post swim up feeding test. If fewer than 100 fish were available for a particular sample, no fish were culled. Culled fish that reached swim up were preserved for subsequent deformity assessment. After 15 days, 20 fish were sacrificed from each sample for length and weight determination, and all remaining fish that survived the 15 day post swim up test were preserved for subsequent deformity assessment.

Endpoints analyzed in the Formation Environmental (2011) Re port included percent hatch of fertile eggs, average time to swim up, survival to swim up, survival to 15

- days post swim up, incidence of deformities, and growth. All endpoints were analyzed using the logistic equation model option in the U.S. EPA Toxicit y Relationship Analysis Program (TRAP). It was determined in the Report that survival from hatch through 15

- day post swim up and total fraction normal (i.e., mean total normal fish) were the recommended endpoints, as they produced the best
models and were biologically relevant. The EC 10 s for these endpoints were 17.67 mg Se/kg dw for survival and 19.33 mg Se/kg dw for mean total normal (with respect to deformities) fish. Based on comparisons of model fit for alternative EC 10 calculations in a 2010 U.S. E PA4

evaluation of the Report, Formation Environmental reanalyzed survival in TRAP after eliminating the three highest exposure concentrations, and presented the resulting EC 10 as 20.8 mg Se/kg dw.

The FWS (2012) Review of an earlier (2010) draft of the Form ation Report, recommended
recalculations for 15

- day post swim up survival and analysis of deformities. Using the triangular distribution of the tolerance distribution analysis type in TRAP, an EC 10 of 14.9 mg Se/kg dw
was calculated for survival and an EC 10 of 6.55 mg Se/kg dw was calculated for deformities, and
the Review concluded that the EC 10 of 6.55 mg Se/kg dw for larval deformity was most appropriate.

Issues Concerning the Brown Trout Reproduction Study A number of experimental and analytical issues associated with the Formation Environmental (2011) brown trout reproduction study (the Report) deserve additional attention, as they affected the interpretation of the toxicity data and ultimately, the effect concentrations derived from these
data i n the Report, the FWS (2012) Review of the Formation Environmental Report, and the current document, which is a reassessment of the Formation Environmental Report in response to the FWS (2012) Review. These issues will be described below.

Fungal Overgrowt h Numerous egg samples were affected by fungal growth during the study. The eggs obtained from the SC hatchery fish were the most affected by the fungus, as they were the first samples added to the test system. A number of steps were taken to minimize th e fungal growth, including salt rinses, formalin rinses, manual scrubbing of the egg cups (but not the eggs), and flushing of the tubing lines connecting the splitter box to individual test chambers with hydrogen peroxide. These steps are detailed within Appendix D of the Formation Environmental (2011) Report. The overall deformity rates of fish hatched from eggs of the SC hatchery samples were similar to deformity rates for fish hatched from the Crow Creek samples, as well as the Sage Creek samples at Se concentrations < 20.5 mg Se/kg dw. The Review cited the fungal growth as the rationale for using SPC hatchery data and not using SC hatchery data in the deformity analysis.

Use of Alevin in Deformity Analysis and 15 Day Post Swim Up No alevin hatched fro m eggs collected at Sage Creek samples with the 5 highest Se exposure concentrations (26.8

- 40.3 mg Se/kg dw) reached swim up. However, alevin from these five exposure concentrations were included in the 15

- day post swim up survival test, and were
included in the final survival calculations in the Formation Environmental (2011) Report. Deformity assessment for these 5 Se exposures was performed on alevin that survived the 15

- day post swim up test as well as preserved alevin that died during the first part of the test. In contrast, deformity and survival assessments were performed using only fish that had reached swim up in all other field samples and in all hatchery samples. The FWS (2012) Review noted alevin that fail to reach swim up would not survive i n the wild, and conducted their analysis of5

survival from hatch through 15

- day post swim up assuming 0% survival for those five high Se samples. We agree with the point raised in the Review, and all survival analyses in this document were also performed a fter setting survival to 0% for those exposure concentrations. Results from the five high Se samples were retained for deformity analysis in both the Review as well as in this document; although overall deformity rates across the four classes of deformiti es were analyzed differently in the Report, the Review, and this document. These different analytical methods are further discussed in subsequent sections of this document.

Calculation of Larval Survival Larval “hatch to swim up survival” was calculated s imilarly in both the Formation Environmental (2011) Report and this document. However, the calculation methods for “hatch

to 15

- day post swim up survival” differed markedly between the Report and this document. In section 3.4.4 (Mortality) and Appendix F of the Report, “hatch to 15

- day post swim up survival” was calculated in such a way that all fish that were preserved prior to the post swim up test were assumed to have survived the entire test, including post swim up. One consequence of this was
that f or some samples, “hatch to 15

- day post swim up survival” was reported as being higher than “swim up to 15

- day post swim up survival,” which is not possible (Table 3

- 4; Appendix F). In contrast, “hatch through 15

- day post swim up survival” was calculated i n this document by first separately calculating “hatch through swim up survival” and “swim up to 15

- day post swim up survival,” and then multiplying the two to calculate “hatch through 15

- days post swim up survival.” Only a portion of the fish surviving t he first part were tested in the second part, and in the majority of cases, this portion was small relative to the total number of fish that survived through swim up. Therefore, the death of a single individual during the post swim up test would have a gr eater effect on overall survival than the death of a single individual prior to the culling. The calculation method used in the Report overestimated 15

- day post hatch survival and subsequent hatch through 15

- day post swim up survival for all samples where culling occurred prior to the post swim up test that experienced mortality during post swim up test.

Overflow and Loss of Fish during the 15

- Day Post Hatch Survival Feeding Trial During the 15

- day post survival test, aquaria overflow resulting from a clo gged drain led to the loss of fish from 10 field samples (5 from Crow Creek and 5 from Sage Creek) and two SC hatchery samples. The field samples lost from Sage Creek included 5 of the 7 samples where fish reached swim up stage, with egg Se concentrations ranging from 11.2

- 20.5 mg Se/kg dw. As a consequence, the highest egg Se concentration for any of the field samples where fish reached swim up stage and did not lose individuals to overflow was 14.0 mg Se/kg dw. The Formation Environmental (2011) Report addressed the overflow issue by analyzing hatch to 15

- day post swim up survival as if fish lost to overflow had never been included in the test. Effectively, this approach assumed mortality of fish lost to overflow would have been identical to mortality of fish not lost to overflow. On the other hand, the FWS (2012) Review asserted that fish lost to overflow were lost because they were most likely either dead or deformed, and used this to justify excluding all samples lost to overflow from their survival and deformity6

analyses. As a result, the highest Se concentration of any sample with fish that reached swim up stage in the Review dataset was 14.0 mg Se/kg dw, whereas the highest concentration of any sample with fish that reached swim up in the Report dataset was 20.5 mg Se/kg dw. As will be further discussed, more than any other factor, the FWS decision to exclude these samples from their analysis had major consequences in their final modeled EC 10 s.

In this document, we explore the consequences of t he decision to exclude samples that lost fish to overflow in for both the survival and the deformity endpoints. For both endpoints, we addressed the assumption that fish lost to overflow were likely dead or deformed by comparing
the EC 10 s of (a) models wh ere all missing fish were assumed to be dead or deformed, as applicable, to (b) models where samples with missing fish were excluded, and to (c) models where missing fish were ignored (as was done in the Report). Full details of these scenarios, and
the c alculation procedures, are described in the Methods section.

Calculation of Deformity Endpoints In the Formation Environmental (2011) Report, each of the four deformity types (i.e., cranial

facial, finfold, spinal, edema) were assessed and analyzed separat ely and in combination. The method used by the Report to calculate the four deformity types in combination was the endpoint “sum total normal” fish. This endpoint, described in section 4.4.3 (Deformities), was calculated by summing the percentage of fish free of each of the four classes of deformity, and could range from 0 (100% deformities, or 0% normal, for each of the four deformity types) to 4 (0% deformities for each of the deformity types). The endpoint “sum total normal” fish was also

calculated a s “mean total normal” fish, or the average of the %normal fish for the four deformity types, and the EC 10 s for both of these endpoints were mathematically equivalent (19.3 mg Se/kg dw; Figure 44

– sum total normal; Figure 45

– mean total normal).

The FWS ( 2012) Review was critical of the method used by the Report to calculate deformities, stating that the endpoint “sum total normal “ fish averages deformities across classes, and will always underestimate the total number of fish with deformities. The Revie w then stated that “fully normal” fish would have been the most appropriate overall measure of deformities. However, the information required to calculate this endpoint was not available in the Report. As a result, the Review recalculated deformity rates using the endpoint “maximum fully normal” fish, which was 100% minus the percentage of the most prevalent deformity type. This endpoint is based on the recognition that the proportion of normal fish cannot have been higher than the lowest normal proporti on of any of the deformity classes.

We agree with the reasoning of the FWS (2012) Review that “fully normal” fish would have been the most appropriate endpoint, and that “maximum fully normal” fish is a more appropriate endpoint for assessing combined de formities than “sum total normal” fish. In this reanalysis, we assess deformities using the endpoint “maximum fully normal” as the most optimistic scenario for deformities, and also assess deformities using two additional, increasingly conservative endpoi nts “intermediate normal” and “minimum normal” in order to assess the possible7

overestimation of normal fry. A detailed description of the three calculation methods for deformities is provided in the Methods section of this document.

Correction of Errors In addition to following a calculating procedure for hatch to 15

- day post swim up survival that we think underestimated mortality during the 15

- day post swim up test, as described previously, a careful accounting of the data (Appendix F) revealed a number of discrepancies between the reported number of fish at the 15

- day post swim up test initiation and that the number of fish that could possibly have been used in the post swim up study (Column “15d PSU target” in Appendix F). In addition, there were four unaccounted

- for fish in sample SPC

- 005, and two unaccounted

for fish each in samples CC

- 150

- 12 and CC

- 150

- 15.

The FWS (2012) Review cited numerous discrepancies between “Survival (%) from Hatch until test term,” in Table 3

- 4 of the Formation (2011) Repor t, and hatch to 15

- days post swim up survival calculated by dividing “total survival” (Table 3

- 4) by “% hatch” (Table 3

- 3). The sources of these discrepancies are a combination of the method Formation (2011) used to calculate “Survival (%) from Hatch unti l test term,” described in the section “Calculation of Larval Survival,” as well as the minor accounting errors described above.

Methods Calculation of Larval Survival (Full Test) The determination of survival involves two sequential parts of the test, (a) hatch to swim up, and (b) swim up to 15

- days post swim up (PSU). Only a portion of the individuals surviving the first part were tested in the second part. Larval survival was e valuated here as the combined endpoints for both parts of the test, thus covering the duration from hatch to 15

- days post swim

up.

As previously described, we calculated values for survival from hatch to 15

- days post swim up differently from both the For mation Environmental (2011) Report and the FWS (2012) Review, which corrected a number of errors regarding the calculation method performed in the Report for
hatch to 15

- day post swim up survival, and assigned a value of 0% survival to those samples that
d id not reach swim up. In addition

, swim up to 15

- days post swim up survival in this document was calculated two different ways, reflecting uncertainty stemming from the loss of some individuals when certain aquaria overflowed due to a clogged drain. One way reflects an optimistic view of the health of those individuals prior to their loss. The other way reflects the
worst case view of their health.

Survival Method M1 (Optimistic Case): The first method of calculating survival during the 15 day post sw im up phase was to divide all fish that survived the 15 day post swim up (post swim up fish assessed for deformities + post swim up fish saved for length and weight measurements) by the sum of all fish that survived the 15 day post swim up test plus all fi sh that died during the8

15 day post swim up test. This method is conceptually identical to how post swim up survival was calculated in the Report, which calculated 15 day post swim up survival as the number of fish at post swim up test initiation minus th e number of fish that died during the post swim up test divided by the number of fish at test initiation. Fish lost to overflow, or to other types of technician error, were subtracted from the number of fish at test initiation

. Survival Method M2 (Worst Case): Survival for Method 2 was calculated following the procedure described in Method 1, with one major exception. Rather than simply discard data from 10 of the 24 field samples as was done in the Review, as well as from 2 of 8 SC hatchery samples, wh ere applicable, 15 day post swim up survival was also calculated by assuming the worst case scenario that every fish that was missing due to overflow, lost to technician error during the initial thinning process, or otherwise unaccounted for did not surviv e. This represents the most conservative estimate of post swim up survival possible 1

.
Final counts of estimated surviving fish and total fish for both survival approaches are included
in Table 1a.

Calculation of Larval Survival (Hatch t o Swim Up) Larval survival was separately calculated for the period between hatch and swim up. Because
hatch to swim

- up is a commonly used duration for maternal

- transfer reproductive tests with trout, these analyses could be of interest for criteria derivation purposes. F urthermore, because there was no loss of fish resulting from overflow or other sources, these analyses involved a single, straightforward calculation procedure, free of any assumptions (e.g., optimistic case or worst case) regarding lost fish. For these a nalyses, survival was calculated as the number of fish that hatched and successfully reached swim up divided by the number of successfully hatching fish (Table 1b). Because they did not reach swim up, 0% survival was assumed for all fish from
the five fie ld samples with selenium concentrations of 26.8 mg Se/kg egg dw or greater.

Calculation of Deformities In order to address the issues raised in the FWS (2012) Review and noted earlier in this document regarding the combined assessment of the four deformity classes, combined deformities were assessed in three different ways in this study. The first method, Maximum Fully Normal, is the method by which deformities were analyzed in the Review. The remaining methods, Intermediate Fully Normal and Minimum Fully Normal, represent two additional increasingly conservative calculation procedures that attempt to address the potential overestimation of fully normal fish.

Maximum Fully Normal (Optimistic Case) : Here deformities were assessed following the
recalculati on performed in the Review. For each sample, it is 1.0 minus the maximum rate of

1 It should be noted that while the number of missing fry from sample LSV2C

- 008 was 46, the fry “mass balance” for that sample revealed that after preserving fry for deformity as sessment following swim up, 100 fry were available for the post swim up test. Of these, 34 were preserved for deformity assessment, 20 were preserved for length and weight measurement, and one died, leaving only 45 fry that could have been missing, which is the number used in this analysis.9

any of the four deformities. It is optimistic in assuming that the less prevalent types of deformities only occur in individuals already having the most prevalent type.

Inte rmediate Fully Normal (Intermediate Case) : Here deformities were calculated following the assumption that the likelihoods of developing a deformity operate independently. When deformities are uncommon, each deformed fish usually has just one type of defo rmity, but the likelihood of a fish having more than one deformity increases as the overall deformity rate
increases. 2

Minimum Fully Normal (Worst Case) : Here the proportions of each of the four types of deformities were summed. If the sum of the proport ions of deformed fish was greater than one, then the minimum fully normal fish was equal to zero. This approach, which assumes that no fish can have more than one type of deformity, is the most conservative way to estimate fully
normal fish.

In each case, the proportions of normal fish were multiplied by the total number of assessed fish
and rounded to the nearest whole number.

It should be noted that number of assessed fish used in these analyses was the maximum number of assessed fish for any of the fou r deformities. This decision was made to facilitate summing and multiplying deformities across endpoints where the number of fish assessed for each of the four endpoints differed. In essence, this approach assumed that for a particular sample, the rate

a t which a particular deformity was observed would have remained the same had more fish been available for analysis. These differences only applied to the samples at the five highest selenium concentrations where no fish reached swim up. Because of the lo w numbers of available fish during the post swim up test, preserved alevin that had died during the pre

- swim up phase of the study were also assessed for deformities, and some of these had degraded to the point where skeletal, edema, and particularly, fin and finfold deformities could not be assessed. This assumption did not affect the proportions; only the counts used in the tolerance distribution analysis, and had no effect on the results of the analysis. Final counts of normal and assessed
fish for the three deformity calculation approaches are included in Table 2.

Major Deformities: The methodology described above was repeated to assess “major” deformities, with major deformities defined as the sum of all level 2 and level 3 deformities. As
with tota l deformities, major deformities were analyzed for the “maximum”, “minimum”, and “intermediate” number of fish free of major deformities” scenario. Again these correspond to Optimistic Case, Worst Case, and Intermediate Case. Final counts of fish free of major deformities (FMD) and assessed fish for the three major deformity calculation approaches are
included in Table 3.

2 For example, if each of the four deformity types is reported to have a 10% frequency in a sample, the probability of an individual not having any of the four deformity types would be 0.9 x 0.9 x 0.9 x 0.9 = 0.656.10

Overflow Loss: As discussed previously, the FWS Review assumed that fish lost due to overflow during the 15 day post swim up test were more prone to death or deformation than fish that were not lost. In order to test this assumption, each of the scenarios described above were also analyzed with the assumption that all fish lost to overflow were deformed. For these analyses, the number of missing fish from the ten field samples and two SC hatchery samples that lost fish were added to the total number of assessed fish and then the maximum, minimum, and intermediate numbers of “fully normal” fish were recalculated. This procedure was repe ated for the analysis of major deformities, but in this case, it was assumed that every fish lost during the post swim up test had a major (level 2 or 3) deformity. Final counts of normal and assessed fish for the three deformity calculation approaches af ter accounting for missing fish are included in Table 4, and final counts of FMD and assessed fish for the three major deformity calculation approaches after accounting for missing fish are included in Table 5.

The end result is that the analysis of all de formities and the analysis of major deformities were each done in six different ways: three ways for combining the different types of deformities (Optimistic Case, Worst Case, and Intermediate Case), multiplied by two ways for handling the
loss of individ uals in the overflow incident (Optimistic Case and Worst Case).

Calculation of Mortality Plus Deformity In a final set of analyses, larval survival and larval deformities were combined into a single endpoint. This combined endpoint was examined six diff erent ways, analogous to the two ways larval survival was examined multiplied by the three ways that types of deformities were considered. The methodologies followed here were not identical for accounting reasons, and are
described as follows.

First, all dead fish were accounted for. In the first approach, dead fish were defined as all recorded dead fish before (alevin stage) or after swim up. In the second approach, dead fish were defined as all recorded dead fish plus all missing and unaccounted for fi sh. For both approaches, every fish from the five samples where fish did not reach swim up was considered to
be dead. The total number of fish was calculated as the total number of fish that hatched minus all missing and unaccounted for fish for the firs t approach, and as the total number of fish that hatched in the second approach. Next, all deformed fish were accounted for. For each sample, a certain number of fish were preserved for either wet weight measurements (at swim up), or length and weight me asurement (following the post swim up test). These fish were not dead at the end of the test, but they were also not assessed for deformities. In order to estimate the number of these fish that were deformed, these totals were multiplied by the correspon ding proportion of deformed fish for each sample, and rounded to the nearest whole fish. This calculation was repeated for each of the three deformity approaches described above (i.e., maximum normal, minimum normal, intermediate normal). These estimated totals were then added to the corresponding number of deformed fish calculated above, resulting in a final count of deformed fish consisting of assessed deformed fish plus estimated deformed unassessed fish11

for each of the three ways of measuring deformit y (i.e., minimum, maximum, intermediate) for each sample.

Because the five samples with fish that failed to reach swim up were all considered dead, and therefore removed from deformity analysis for the combined endpoint, all fish that were assessed
for d eformities for this combined endpoint were alive at the time of preservation. So for purposes of the calculation of this endpoint, double counting was not an issue. Fish were either dead or they were deformed. After calculating all six possible combinat ions of dead plus deformed fish, the number of normal fish was then calculated for each combination by
subtraction.

A description of the factors used to calculate the six possible dead+deformed combinations are as follows:
Dead fish

– M1, Survival Optimi stic Case = Dead alevin plus dead post swim up fish

Dead fish

– M2, Survival Worst Case = Dead alevin plus dead post swim up fish plus missing
and otherwise unaccounted for fish

Hatched fish

– H1, applies to Survival Optimistic Case = Fish hatched minus mi ssing and
otherwise unaccounted for fish.

Hatched fish

– H2, applies to Survival Worst Case = Fish hatched

Deformed fish

– D1, Deformities Optimistic Case = Fish assessed minus “maximum fully normal” fish plus ((1

- proportion of “maximum fully normal” fish) times fish preserved for length and weight measurements)

Deformed fish

– D2, Deformities Worst Case = Fish assessed minus “minimum fully normal” fish plus ((1

- proportion of “minimum fully normal” fish) times fish preserved for length and weight measuremen ts)

Deformed fish

– D3, Deformities Intermediate Case = Fish assessed minus “intermediate fully normal” fish plus ((1

- proportion of “intermediate fully normal” fish) times fish preserved for length and weight measurements)

The final equations for calculat ing the numbers of normal fish and total fish for each of the six combinations are as follows:
Dead Fish

- Optimistic Case, Deformed Fish

- Optimistic Case (M1D1) = (H1

- (M1+D1))/H1

Dead Fish

- Optimistic Case, Deformed Fish

- Worst Case (M1D2) = (H1

- (M1+D2))/H1

Dead Fish

- Optimistic Case, Deformed Fish

- Intermediate Case (M1D3) = (H1

- (M1+D3))/H112

Dead Fish

- Worst Case, Deformed Fish

- Optimistic Case (M2D1) = (H2

- (M2+D1))/H2

Dead Fish

- Worst Case, Deformed Fish

- Worst Case (M2D2) = (H2

- (M2+D2))/H2

Dead Fish

- Worst Case, Deformed Fish

- Intermediate Case (M2D3) = (H2

- (M2+D3))/H2

Final counts of normal and total fish for each of the six dead + deformed combinations are
included in Tables 6a and 6b.

Analysis of Subsets of the Data Each of the approaches for calculating larva l survival, deformity, and survival + deformity described above was analyzed in TRAP using four different subsets of the dataset. The four subsets of the data analyzed were (a) all fish spawned from wild

- caught fish collected from field stations (no fish spawned from hatchery fish or arriving as eyed eggs), (b) fish spawned from
wild

- caught fish collected at field stations excluding those stations where fish were lost to overflow, plus fish arriving as eyed eggs from the Spring Creek Trout (SPC) Hatchery, (c) all fish spawned from wild

- caught fish collected from field stations plus fish from the SPC Hatchery, and (d) all fish. The most important factor behind the large discrepancy between the
EC 10 s for deformity calculated in the Formation Environmental (2 011) Report and the FWS (2012) Review was the subset of data used by each analysis. The four data subsets that will be analyzed in this document are the data subset used in the Formation Environmental (2011) Report (subset a), the data subset used in the FWS (2012) Review for the deformity endpoint (subset b), the data subset that will allow us to both investigate the effects of excluding all field samples where some fish were lost to overflow as well as to test the worst case scenarios that fish
lost to o verflow were likely dead or deformed (subset c), and the full dataset.

Field Data Subset
The “ Field ” data subset consists of the offspring of wild

- caught fish taken from Crow Creek (CC) and Sage Creek (LSV) . It excludes fish spawned in the laboratory usi ng eggs from the Saratoga National Fish (SC) Hatchery and fertilized. It also excludes larval fish hatched from eyed eggs obtained from the Spring Creek Trout (SPC) Hatchery. These might or might not be viewed as appropriate samples to represent the low end of the Se concentration gradient, in that they were not wild

- caught fish collected from the same region at the same time as the wild

caught fish from stations along Crow Creek or Sage Creek.

SC Hatchery fish samples were generated in a manner similar to the field samples, as gametes were stripped from male and female SC hatchery fish and fertilized in the lab. However, SC Hatchery samples were somewhat more affected by the fungal infection than the wild

- caught fish, they were the first to be added to the hatching test chambers. 3 In contrast to SC and wild

-

3 D escription of fungal infestation and measures taken are described in the Report: ENSR Appendix D

, an appendix within another larger Appendix, also called Appendix D (Formation Environmental 2011)

.13

caught samples, eggs of SPC hatchery samples were not fertilized in the laboratory. Rather, SPC samples arrived in the laboratory as pre

- fertilized, partially developed “eyed” embryos. Also, by

th e time the SPC eyed eggs were added to the hatching test chambers, the fungal infestation has been effectively controlled. In addition, while percent hatch of eggs that were fertilized in the laboratory (Field and SC) were highly variable, percent hatch o f SPC eggs was uniformly high, possibly as a result of differential handling and processing.

Field Data Excluding Samples Lost to Overflow Plus SPC Hatchery Data Subset This subset of the data consists of SPC hatchery and field samples, excluding data fr om the 10 field stations where some fish were lost due to overflow during the swim up to 15

- day post swim up portion of the test. As this was the data subset used in the Review recommended recalculation for larval deformity, it will hereafter be referred to as the “Review” subset. The Review contended that all of the egg selenium concentrations of wild

- caught fish were above background. It then contended that data for SPC hatchery fish should be included in its deformities’ analyses (but not its survival analysis) to anchor the low selenium concentration range of the concentration

- response relationship, but that data from SC hatchery fish should be excluded. The rationale was that when no data are available for fish from an appropriate “background” site (however that is defined), there is the possibility that any point estimate (e.g.,
EC 10 ) will be overestimated. However, the calculation of an EC 10 does not involve or allow for labeling any sample as being either background or control. Rather it depends on (mathematical) recognition of a low

- concentration plateau within which responses are unaffected by the toxicant. Consequently, to be a valid argument, the lowest wild

- caught exposure, 6.2 mg/kg, would need to be in the exposure range where selenium in duces effects. The problem with this supposition is that the EC 10 s for other sensitive species (which include other salmonids) fall within a narrow range and the lowest wild

- caught exposure of 6.2 mg/kg is a factor of 3 below the bottom of that range (Jan z et al. 2010). The argument thus postulates that brown trout would have to be far more sensitive than any known species.

As mentioned previously, eggs of field and SC hatchery samples were fertilized similarly in the laboratory, whereas eggs of SPC hatch ery samples arrived as fertilized, eyed eggs. Overall, SPC hatchery fish reached swim up after 34 days, in contrast to 67

- 73 days for fish from SC hatchery and field samples. These methodological differences between the fertilization of the
SPC fish an d remaining fish add additional uncertainty that calls into question their use as samples to anchor the low end of the Se concentration gradient. The Review noted that the advanced age of embryos from the SPC hatchery resulted in a shorter holding time, which may have affected the percent hatch; however these samples were included in the recalculation of the deformity analysis on the justification that these samples “... provide useable estimates of expected background deformity rates because the hatching r ates were all acceptable control values (i.e., >90% hatch) and from hatch to 15

- days post swim

- up fry these controls are strictly comparable in their holding times to the field

- exposed test groups.” This justification is questionable, as the majority of f ish assessed for deformities were preserved prior to the post14

swim up test, such that holding times for fish from SPC hatchery samples were approximately half as long as for fish from all other samples, as well as because there is no evidence that
deformit ies would have more likely developed during the 15

- day post swim up period than during the longer pre swim up (alevin) period (34 days for the SPC offspring and 67

- 73 days for
the wild

- caught offspring that reached swim up). Furthermore, although the Revi ew recommended the inclusion of SPC fish for the recalculation of the deformity analysis, it did not include SPC fish for the recalculation of the larval survival endpoint, even though the concerns of having egg selenium concentrations above background for all wild

- caught fish would also apply (although their inclusion has a minor effect on the final survival estimate).

L oss of individuals due to aquaria overflow : During the 15 day post swim up test, 16

- 45 fish were lost from 10 of the 24 replicate aquaria containing fish hatched from wild

- caught parents, and 2 of the 8 aquaria containing fish hatched from SC hatchery parents. The Review contended that the lost fish were more likely to have been dead or deformed, and therefore poorer swimmers, and that the Report inappropriately analyzed survival and deformities from these sites in a way that effectively assumed that for a particular aquarium, survival and deformity rates among fish that were lost were similar to those that were not lost. As a result, the Review recommended that these samples should be excluded from all analyses. The resulting dataset is sparse, with no exposures in the range between 14 and 27 mg/kg, a range that has been critical for defining the EC 10 for other sensitive species (Janz et al. 2010). Given the configuration of data outside that range, we deem such a data gap to render the study unusable for calculating an
EC 10 on which a water quality criterion might hinge.

Nevertheless, we believe the ten field samples that lost individual s in the overflow still contain considerable amounts of useful information, such that the study need not and should not be discarded. Instead, the uncertainties caused by overflow loss of individuals can be managed by
careful assessment of all the availab le data. The assumption that missing fish were more likely to have been dead or deformed can be investigated by comparing survival during the 15 day post swim up test (Table 7) and deformity rates (Table 8) between stations with missing fish and
those wit hout missing fish. Post swim up test larval survival rates between the two groups were similar; 98.9% where some fish were lost compared to 99.6% where no fish were lost. Deformity rates were highly variable for both groups, but were lower overall for sa mples where fish were missing (24.9%) than where no fish were missing (36.3%). The lower rates of deformities among samples where fish were lost could be the result of lower deformity rates among those samples, or of having a higher proportion of missing fish lost as a result of deformities. Ideally, the Report would have presented deformity rates for fish assessed after swim up separately from fish assessed following the post swim up test. Had this been the case, if the proportions of deformed fish betw een the two collection periods were similar for stations where no fish were lost, but lower among the post swim up test collection period for stations where fish were lost, this would add support to the assumption that the proportions of deformed
fish lost to overflow were greater than for the fish that were not lost.15

For the most part, egg selenium concentrations between the two groups of field samples (missing fish vs. no missing fish) were similar. However, the samples with the two highest selenium

conc entrations where fish achieved swim up, LSV2C

- 008 and LSV2C

- 017 (17.7 and 20.5 mg Se/kg, respectively), also lost fish due to overflow in the post swim up test, and were excluded from the recalculations in the Review. Larval survival in both of those samp les was slightly below the average survival of 98.9% among stations that lost fish (Table 7), suggesting that selenium was having a slight effect on larval survival at those concentrations. Larval deformity
rates for LSV2C

- 008 and LSV2C

- 017 fish were lowe r than the deformity rate of 24.9% for the subset of stations with missing fish as a whole (Table 8), suggesting either a high proportion of deformed fish were lost from these sites, or that deformity, which was highly variable across all but the SPC fish, was actually lower than average at those sites. Given the uncertainty surrounding the health of individuals lost in the overflow, it is appropriate to evaluate data subsets beyond those of the Review, considering the range of possibilities represented b y the previously defined Optimistic Case and Worst Case for the individuals lost in the overflow.

Field Data Plus SPC Hatchery Data Subset The subset of the data consisting of SPC hatchery fish and all wild

- caught stations (hereafter
referred to as the “ Field+SPC” subset) was included to examine the consequences of including or excluding fish from the stations that experienced overflow for the different endpoints and calculation methods. That is, this subset is the Review dataset plus the data from 10 Fi eld samples that experienced aquaria overflow.

The overflow Optimistic Case and Worst Case investigate the consequences of the assumption that dead and/or deformed fish were more likely to have been lost as a result of overflow, either because they were po or swimmers or incapable of swimming. That is, within any particular sample the overflow Optimistic Case assumes that the lost fish had the same likelihood of death or deformity as the fish not lost. The overflow Worst Case counts lost fish among either the dead or the deformed, depending on the endpoint under analysis.

Full Dataset Finally, the full dataset of all wild

- caught and hatchery derived fish (hereafter referred to as the “All” set) was included to examine the effects of adding the SC Hatchery f ish to the above

mentioned subset. That is, this set includes all Field, SPC Hatchery, and SC Hatchery fish.

Data Analysis All analyses w ere conducted using the U.S. EPA Toxicity Relationship Analysis Program (TRAP), version 1.20, except for analyses rela ted to the first portion of the larval survival test,
which were conducted in TRAP version 1.21. Consistent with the recommendations of the FWS (2012) Review, all data were analyzed using the Tolerance Distribution Analysis approach fitted to a Triangular Distribution following log 10 transformation of selenium concentrations. No initial guesses were provided for any of the TRAP model fits, and all initial final estimates were accepted with no additional iterative curve fitting.16

A total of 78 TRAP models w ere analyzed for the different endpoints and calculations methods. This rather comprehensive selection of models represents a full factorial analysis of the various calculation methods for larval survival, larval deformity, larval major deformity, and lar val survival + larval deformity for the four data subsets described above. The breakdown of the
models is as follows:

 Larval Survival, Full Test: 2 calculation methods (overflow Optimistic Case and Worst Case) x 4 data subsets = 8 models.

 Larval Survival , First Portion of Test (Hatch to Swim Up): One method for 4 data subsets
= 4 models.

 Larval Deformity: For overflow Optimistic Case, 3 calculation methods for combining deformity types (Optimistic Case, Worst Case, and Intermediate Case) x 4 data subsets = 12 models. For overflow Worst Case, the same 3 deformity calculation methods x only 3 data subsets after accounting for fish lost to overflow = 9 models. Total 21 models.

 Larval Major Deformity: Just as above, again totaling 21 models.

 Larval Death or Deformity (combined): 2 survival calculation methods x 3 deformity
calculation methods x 4 data subsets = 24 models.

All TRAP results are expressed in mg Se/kg egg dry weight (dw). Results of all TRAP analyses, including EC10 estimates, 95% confidence in tervals, and any model error messages are included
in Tables 9

- 12.17 Summary of Options for which EC 10 s Were Calculated

EC10s were calculated for (a) different endpoints, (b) different options for handling accidental loss of individuals during the sec ond portion of the test, (c) different options for combining deformity types, and (d) different subsets of samples.

Endpoint: Survival for full test (combining hatch to swim

- up, and swim

- up to 15 days post swim

- up). Apply to:

 2 scenarios for overflow an d other losses (Optimistic Case and Worst Case).

 4 data subsets. Yields 2 x 4 = 8 EC 10 s.

Endpoint: Survival to swim

- up (excludes the second portion of the test). Apply to:

 4 data subsets. Yields 4 EC 10 s.

Endpoint: Deformities (for full test since data cannot be disaggregated into the two portions of the test). Apply to:

 2 scenarios for overflow (Optimistic Case and Worst Case).

 3 scenarios for combining deformity types (Optimistic, Worst, and Intermediate Cases).

 4 data subsets. Would yield 2 x 3 x 4 = 24 EC 10 s, but two of the overflow scenarios are identical for 1 of the 4 data subsets, such that only 21 different EC10s are produced (1 x 3 x 4 + 1 x 3 x 3).

Endpoint: Major deformities (full test). As above, apply to:

 2 scenarios for overflow ( Optimistic Case and Worst Case).

 3 scenarios for combining deformity types (Optimistic, Worst, and Intermediate Cases).

 4 data subsets. As with the previous endpoint, because of redundancies among the 24 possible scenarios, only 21 different EC10s are prod uced.

Endpoint: Survival and deformities (full test). Apply to:

 2 scenarios for overflow (Optimistic Case and Worst Case).

 3 scenarios for combining deformity types (Optimistic, Worst, and Intermediate Cases).

 4 data subsets. Yields 2 x 3 x 4 = 24 EC 10 s

.

The total number of EC 10 s evaluated is thus 78 for all endpoints, scenarios, and data subsets.18

Results Larval Survival (Full Test) Overflow Optimistic Case: For larval survival calculated for the Method M1 or Optimistic Case,
which assumed survival of fish lost in the overflow would have been the same as fish that were not lost, the lack of partial responses presents TRAP with certain problems in estimating an EC 10 (Table 9a; Figure 1). EC 10 estimates ranged from 14.59 for the “Review” dataset, to 20.47

- 20.48 for the other datasets; values slightly higher than the highest concentrations in the respective datasets that did not experience complete mortality. Survival was very high across all samples up to 14.0 mg Se/kg egg dw (Review dataset) or 20.5 mg Se/kg egg dw (Field, Field+SPC, and All datasets), and was 0% in the five samples that were 26.8 mg Se/kg egg dw or higher. While
the TRAP

- calculated EC 10 s ar e slightly problematic due to lack of partial responses, visual inspection of the Figure 1 data suggests relatively little uncertainty in how best to fit the data, and provides little basis for expecting the EC 10 to be below 17.7

- 20.5 mg Se/kg, the highest exposure concentrations having good survival.

Overflow Worst Case: When all larvae from overflow tanks were counted as having died (Method M2 or Worst Case), TRAP was able to effectively model larval survival for all but the “Review” dataset, with EC 10 e stimates ranging from 16.76

– 17.25 mg Se/kg egg dw (Table 9a; Figure 2). The M2 EC 10 estimate for the “Review” dataset of 14.51 mg Se/kg egg dw was qualitatively identical to the M1 estimate, with the minor difference occurring as a result of two

unaccou nted for larvae in the CC

- 150

- 12 and CC

- 150

- 15 field samples. Survival results calculated using Method M2 underscore the consequences of excluding the samples that lost fish during the overflow event. Because the excluded samples include d the two sample s with the highest selenium concentrations that did not experience complete mortality (17.7 mg Se/kg egg
dw at stat ion LSV2C

- 008 and 20.5 mg Se/kg egg dw at station LSV2C

- 017), while the highest selenium concentration in samples that did not lose fish to ov erflow or experience mortality was
14.0 mg Se/kg egg dw (CC

- 350

- 006), the EC 10 estimates for larval survival in the three datasets that did not exclude samples with overflow were higher than the EC 10 estimates for the Overflow Optimistic Case obtained afte r excluding these samples, even after assum ing that every fish lost
to overflow had already died

. Since the rationale presented in the Review for excluding these samples was because lost fish were more likely to have been dead or deformed, the M2 results
illustrate that the EC 10 for larval survival likely falls somewhere between 16.76 mg Se/kg egg dw (the lowest EC 10 estimate using the overflow worst case method in which samples were not excluded) and 20.5 mg Se/kg egg dw.

Larval Survival for First Portion of Test (Hatch to Swim

- up) The overflow event and other accidental losses of organisms were not relevant to the first portion of the test, and consequently Optimistic Case and Worst Case perspectives do not apply.
Because hatch to swim

- up is a commonly u sed duration for maternal

- transfer reproductive tests with trout, the results can be of interest for criteria derivation purposes. For the Field,19

Field+SPC, and All datasets, the EC 10 s fell into a tight range, 20.64

– 20.72 mg Se/kg, in each case (Table 9b; Figure 3). Compared to their corresponding full test EC 10 s, these EC 10 s were around 1% higher than calculated for the Overflow Optimistic Case, and 20

– 24% higher than calculated for the Overflow Worst Case. They were around 38% higher than the corr esponding
EC 10 for the Review dataset, 14.96 mg Se/kg (Table 9b; Figure 3), which was itself within 2.5

– 3.1% of the two survival EC 10 s calculated for full test with the Review dataset.

The above hatch to swim up survival results may have the following im plications: (a) The second part of the test (swim

- up to 15 days post swim

- up) appears to have had little influence on the survival outcome; (b) Inclusion or exclusion of hatchery fish has little influence on the
survival EC 10 ; and (c) The Review’s editin g of the dataset to discard the 10 field samples which experienced overflow in the later test has a substantial effect in reducing the EC 10 in the portion of the test unaffected by the overflow.

Larval Deformities Overflow Optimistic Case: For the 12 an alyses where fish lost to overflow were not considered,
EC 10 estimates were lowest for the “Maximum Fully Normal” method (combining deformities by Optimistic Case) for three of the four data subsets, highest for the “Minimum Fully Normal” method (combining deformities Worst Case) for all of the data subsets, and intermediate for the “Intermediate Fully Normal” method for three of the four data subsets (Table 10; Figures 4

- 6). This result was not expected, since the Minimum Fully Normal calculation method w as the most conservative, and the Maximum Fully Normal method was the least conservative. However, because of the lack of partial responses for defining slope, all of the TRAP model fits for deformities calculated using the Minimum Fully Normal method wer e somewhat problematic. The lack of partial responses also resulted in somewhat problematic TRAP model fits for three of the four data subsets where deformities were calculated using the Intermediate Fully Normal method. Inadequate partial responses incr ease the uncertainty in the response slope between those samples with little to no effects (up to 20.5 mg Se/kg dw) and those samples experiencing complete or nearly complete effects (26.8 mg Se/ kg dw and higher). This uncertainty can become problematic when the concentration gap between largely unaffected and completely affected samples increases. In this dataset, however, the concentration gap between the highest sample that was largely unaffected (20.5 mg Se/ kg dw) and the lowest sample that was

comp letely affected (26.8 mg Se/ kg dw) was only 1.3 fold, which is narrower than is typical in
concentration

- response datasets. Consequently, for some of these brown trout data subsets and assessment options, the TRAP models that have inadequate partial (IP) error

- messages might not be particularly problematic. For others, depending on the data configuration, the lack of partials might detract from the reliability of the calculated EC 10

.

Error

- message free EC 10 estimates were obtained for all of the data s ubsets where deformities were calculated using the Maximum Fully Normal method, as a result of partial responses in three of the five samples where fish did not reach swim up. However, it should be noted that because these fish did not reach swim up, they likely would not have survived in the wild. Also,20

only fish that had survived to the point of preservation and had reached swim up were assessed for deformities in all of the other samples. As a result, the comparison of deformities across samples is no t completely internally consistent, because deformities were assessed in live fish that had reached swim up for all of the hatchery samples and for 19 of the 24 field samples, while deformities in the remaining 5 field samples were assessed for fish that h ad not reached swim up and/or had died prior to being preserved. This inconsistency was noted in the Review,
which contended that deformities should have been assessed for both live and dead fish. Alternatively, a new endpoint which considers the combine d effects of mortality and deformity could be assessed. This recommendation of a combined endpoint was made by one of the peer reviewers of the Review and will be addressed in a subsequent section within this analysis.

Larval deformity EC 10 estimates usin g the Maximum Fully Normal calculation method ranged from 6.55 mg Se/kg egg dw for the Review data subset to between 19.73 and 20.13 mg Se/kg egg dw for the three remaining data subsets. The Review contended that the SPC hatchery samples in combination w ith the field samples where no fish were lost was the most appropriate dataset, because none of the egg selenium concentrations among the field samples were representative of background concentrations, and because fish lost from overflowing aquaria

were m ore likely to have been dead and/or deformed. However, this EC 10 of 6.55 mg Se/kg egg dw (Figure 4

– upper right) was highly dependent on the combination of the inclusion of SPC hatchery samples, the exclusion of samples where some fish were lost to overf low, and the calculation method for fully normal fish, including the consequence that deformity rates in those samples where fish did not reach swim up were less than 100%.

While it is true that all of the field sites were above background selenium levels in most parts of the U.S., larval fish from SC hatchery samples were available at selenium concentrations comparable to SPC hatchery samples. Furthermore, it could be argued that the SC hatchery samples provide a more appropriate representation of the low end of the Se concentration gradient, as these fish were hatched from eggs fertilized in the laboratory using gametes stripped from adults, and have variable hatch rates comparable to the hatch rates of fish from field samples. The purpose of adding low concentration samples to a concentration gradient is to provide additional weight to the low

- concentration plateau, thereby providing a more accurate measurement of the factor under investigation, in this case, selenium toxicity, while minimizing
the effe cts of variables not related to the factor under investigation, such as fertilization methods, handling time, etc. The Review contended that the SC hatchery eggs were affected by fungal growths. This is true; however, fungal growth also affected eggs fro m field samples, but to a lesser extent. Based on these methodological differences, it is a reasonable assertion that the lower deformity rates and higher hatch percentage of fish from the SPC hatchery were more a function of methodological issues (beginn ing with eyed eggs that were fertilized in a professional hatchery) than of selenium concentrations. If fish from the SC hatchery samples were included instead of fish from the SPC hatchery samples in the Review data set (excluding the two SC samples wher e fish were lost to overflow), the EC 10 for the Maximum Fully Normal21

calculation method was 13.72 mg Se/kg egg dw, with a 95% confidence interval of 12.69

- 14.83, and was free of any error messages in TRAP (Figure A2

– upper left).

The effect of excluding data from samples with missing fish had an even greater effect on the
EC 10 estimate of deformity than the inclusion of the SPC hatchery data, as the EC 10 for the Field+SPC data subset, which is the Review data set plus the 10 field samples in which fish w ere lost due to overflow, was 19.78 mg Se/kg egg dw (Table 10; Figure 4). The contention that the fish lost to overflow were more likely to have been deformed can be tested by assuming, as was done with the larval survival endpoint, that all missing fish w ere deformed. The EC 10 for this data subset (Field+ SPC with missing fish included as abnormal data subset, using the Maximum Fully Normal calculation method) was 18.47 mg Se/kg egg dw, and was free of any errors (Table 10; Figure 7; Figure A2

– upper rig ht). In other words, if every fish lost to overflow had been deformed, which was the argument for excluding these samples, the EC 10 for deformities in response to selenium is nearly than three times higher than it was estimated to be in the Review

as a re sult of excluding these samples. In all data subsets where no field samples were deleted,
the EC 10 estimates for deformities where missing fish were assumed to have been deformed were similar using the Maximum Fully Normal calculation method, because the larger number of samples from field stations was sufficient to outweigh the effects of the low deformity rates in the SPC samples.

The calculation procedure for the determination of fully normal fish also influenced the Review
EC 10 estimate. The EC 10 for the Review dataset using the more conservative Intermediate method of estimating fully normal fish was 8.64 mg Se/kg egg dw (Table 10; Figure 7; Figure

A2

– lower left). As previously described, the intermediate calculation method assumed that fish
will o ccasionally have more than one deformity, but generally will only have one deformity when deformity rates are low. Although this was more conservative than the Maximum Fully Normal calculation method, the resulting EC 10 was higher because of the decrease in intermediate fully normal fish among the SPC hatchery samples. Finally, the EC 10 for Maximum Normal fish in the Review dataset was 12.97 mg Se/kg egg dw (FigureA2

– lower right) if fish that did not reach swim up were treated as 100% affected (or 0% fu lly normal).

While there were substantial frequencies of deformities in samples ranging from 6.2

- 20.5 mg Se/kg egg dw, the effects were highly variable and had no apparent relationship to selenium concentrations. Six different ways of evaluating the defo rmity data are shown in Figures 3

– 8, corresponding to two ways of addressing the overflow of aquaria (Optimistic Case versus Worst Case), multiplied by three ways of combining deformity types (Optimistic Case, Worst Case, and Intermediate Case). Visual inspection of the upper left “Field” graph of Figures 4

- 9 suggests no relationship between deformities and Se concentration in the 6.2

– 20.5 mg Se/kg range (that is, logs 0.79

– 1.31). Indeed, in all six of these “Field” graphs, the fractions of fully no rmal fish at 17.7 and 20.5 mg Se/kg (the two highest concentration filled circle data points in each graph) were above the median of the Field data points at lower concentrations. It was only at22

concentrations above 20.5 mg/kg that deformity effects in re sponse to selenium were clearly
apparent.

For the 9 analyses where fish lost to overflow were assumed to have been deformed, EC 10 estimates were lowest for the “Maximum Fully Normal” calculation method for the Field and Field+SPC data subsets, highest fo r the “Minimum Fully Normal” method for all of the data subsets, and lowest for the “Intermediate Fully Normal” method for the All data subsets (Table 10; Figures 7

- 9). Overall, EC 10 estimates were slightly lower when all missing fish were
considered defo rmed except for the Intermediate Normal, Field data subset, which was higher after including missing fish as deformed; and the Intermediate Normal, All data subset, which was notably (15.81 vs. 21.32 mg Se/kg egg dw) lower when all missing fish were consid ered deformed (Table 10; Figure 9). Only the “Maximum Fully Normal” calculation methods
resulted in error free EC 10 estimates after missing fish were included as deformed.

Larval Major Deformities Because the majority of deformities were minor (Level 1) , and because deformity rates were highly variable across the pre

- threshold selenium concentration range (6.2

- 20.5 mg Se/kg egg dw), larval major deformities (Levels 2 and 3) were separately analyzed to determine whether they were more likely to have exhib ited a concentration

- response relationship to maternal egg selenium. As with overall larval deformities, the effects of fish lost to overflow were also
considered.

When missing fish were excluded from the analysis, all of the TRAP model fits were free of errors for both the Maximum FMD and Intermediate FMD calculation methods, and all of the TRAP model fits using the Minimum FMD calculation method had multiple errors, indicating that the tolerance distribution analysis model was problematic for these data (Table 11; Figures
10

- 12). For any given data subset, the EC 10 estimate for the Maximum FMD calculation method (16.11

- 18.40 mg Se/kg egg dw) was higher than for the Intermediate FMD calculation method (14.52

- 16.95 mg Se/kg egg dw), which might be expected given that the Maximum FMD calculation method was less conservative. Counter to what might be expected, for any given
dataset the EC 10 estimate for Minimum FMD was greater than the other two. EC 10 s estimated using the Review data subset were lower than f or any of the other data subsets, largely due to the exclusion of the 17.7 and 20.5 mg Se/kg egg dw samples. However, the differences between the Review and the other data subsets were less pronounced than for overall deformities, because the
differences in major deformity rates between SPC hatchery and field samples were less pronounced than the differences in total deformity rates between SPC and field samples.

When fish lost to overflow were considered to have had major deformities, EC 10 estimates
fol lowed the same pattern as when missing fish were excluded from analysis: lowest overall for the Intermediate FMD calculation method, highest for the Minimum FMD method, and intermediate for the Maximum FMD calculation method (Table 11; Figure 13

- 15). For any given calculation method, EC 10 estimates were similar across the three data subsets (Table 11)23

and to the EC 10 obtained when excluding missing fish from the analysis. Again, all of the TRAP models using Maximum FMD and Intermediate FMD calculation methods were error free, while all of the TRAP models using the Minimum FMD method contained multiple errors associat ed with the absence of partial responses. With the exception of the “Review” data subset, major larval deformities nearly always yielded a lower EC 10 than did total larval deformities, regardless of whether fish lost to overflow were ignored or considere d to have had major deformities. Major larval deformities also had a somewhat higher proportion of error free TRAP model fits
that did total deformities.

Overall, major larval deformities appeared to be similar in sensitivity to larval survival. However, inconsistencies in the conditions of the fish that were assessed for deformities (fish that failed to swim up and/or died for five samples compared to live fish that reached swim up for all other samples) cloud any interpretations of the sensitivity of th e deformity or major deformity endpoint. Rather than estimate larval sensitivity using deformities (or major deformities), a less ambiguous approach would be to assess a single endpoint that combines mortality and deformity.

Larval Mortality Plus Deformit y The most comprehensive endpoint would be one that combines the effects of mortality and deformity. One of the peer reviewers of the FWS (2012) Review suggested this endpoint as an alternative to using either survival or deformity. This combined endpoin t also reduces some of the selection bias regarding the relative states of the fish assessed for deformities in the Formation Environmental (2011) Report (e.g., live post swim up vs. live 15

- day post swim up vs. dead), as described previously. For purpose s of the analysis of this endpoint, any fish that failed to reach swim up were considered to have died, as this would have likely occurred in the wild. By removing all fish that failed to reach swim up from the deformity analysis, remaining deformity asse ssments were performed on fish that had reached swim up and were alive at the time of preservation. Eliminating this inconsistency in the condition of fish assessed for deformities allows fish to be classified as either completely unaffected (alive and fr ee of any deformity) or affected (either dead or deformed), with no overlap between the two. As with the other endpoints, calculations can also be made to assume that all fish lost during the overflow event were affected. Operationally, this was performe d by assuming they had died prior to being lost to overflow, but for purposes of the combined endpoint, it does not matter if a fish is “affected” as a result of mortality or deformity.

All 24 possible mortality

- deformity

- data subsetting combinations r esulted in TRAP model fits with one or more error messages, as a result of the lack of partial responses that were capable of precisely positioning the downward curve (Table 12; Figures 16

- 21). The EC 10 estimates were fairly close to the highest sample le vel within the respective data subsets that were not 100% affected by selenium (Table 12). For the “Field,” “Field+SPC,” and “All” data subsets, the 20.5 mg Se/kg egg dw sample was the threshold concentration, while in the “Review” data subset, the 14 mg Se/kg egg dw sample was the threshold concentration, since it did not contain the 17.724

and 20.5 Se/kg egg dw samples. Below these thresholds the field samples showed no apparent relationship between exposure and effect, because survival was high and defor mities were
variable.

The similarities in survival and deformity rates among field samples which lost fish to overflow, combined with subsequent “worst case scenario” analyses demonstrate that simply excluding data from these sites was inappropriate, and y ield a pronounced underestimation of the combined
survival and deformities EC 10

.
The 20.5 mg Se/kg egg dw sample represented the highest concentration having response consistent with little or no effect on the combined survival and deformities endpoint. Below this concentration, no matter what assumptions were made about the overflow (Optimistic Case versus Worst Case) or about combining deformities (Optimistic Case, Worst Case, or Intermediate Case), the variability in the rate of deformities yielded a g reat deal of noise but nothing to suggest a relationship to selenium exposure. In contrast, at the next higher concentration, 26.8 mg Se/kg egg dw, and above, mortality reached 100%.

Synthesis There are several issues regarding the interpretation of the F ormation Environmental (2011) brown trout study, most of which were discussed in the FWS (2012) Review (including its appendix presenting its peer review). A summary of the issues that could affect the use of data
to calculate an EC 10 are as follows:

1. Small inconsistencies in the survival rates for the full test compared with its two component parts (noted by the FWS (2012) Review’s peer reviewers).

2. Poor hatching in the SC hatchery eggs, possibly related to fungal infestation problems,
which to a lesser extent also affected the field sample eggs (from wild

- caught females).

3. Procedural differences in the handling of SPC hatchery eggs compared to field sample eggs and SC hatchery eggs.

4. Failure of fish from the five highest exposure concentration samples (S e egg 26.8 to 40.3 mg Se/kg egg dw) to swim up;

5. The health status of a number of fish that were accidently lost during the 15

- day post
swim

- up test in ten field samples and two SC hatchery samples; and

6. Inability to quantify the total number of normal (not deformed or edematous) fish for each sample.

The analyses presented in this report assessed the impact of all these issues on the determination of an effect level for selenium in brown trout, and each is discussed in the preceding pages. Working down the above list: (1) we have determined the cause of the small inconsistencies in25

reported survival rates and have corrected them; (2 and 3) we are recommending an EC 10 that is insensitive to SC and SPC hatchery issues and fungal issues; (4) we agree with the FWS (2012) Review’s handling of survival in the range 26.8 to 40.3 mg Se/kg, and have therefore equated failure to swim up with failure to survive; (5) we have rigorously considered the uncertainty caused by loss of fish during the 15

- day post swim

- up por tion of the test, and are recommending

a worst

- case adjustment in the dataset used in calculation of the EC 10 ; (6) we have examined the spectrum of possibilities for calculating the number of normal fish from the available counts of
deformities. We have f

ound the EC 10 calculations to be relatively insensitive to the how deformities are handled, as long as no Field data are discarded.

In the selection of an effect level considered to be representative of the data, the sample of two of
the issues listed abov e had the greatest relevance. As discussed in the FWS (2012) Review, no fish in the five field samples with the highest Se concentrations succeeded in reaching swim

- up during the entire testing period. Because this might well translate to a failure to su rvive in the wild, we assigned zero survival to the five highest samples. Furthermore, we think it should be extended to the analysis of the proportion of normal fish as well. Our analysis of the combined effects of larval mortality and deformity address ed this latter consideration.

The other issue that had a meaningful effect on the EC 10 value was the inclusion/exclusion of the ten field samples and two SC hatchery samples that lost fish due to overflow resulting from a clogged drain during the 15

- day post swim

- up test. The FWS (2012) Review’s rationale for excluding these samples was based on the uncertainty about whether the lost fish might represent a greater proportion of dead, affected, or abnormal fish. The results of the analyses presented
here in showed that uncertainty in the health status of the lost fish had comparatively little
influence on the EC 10 estimate, but that discarding the data from the affected samples, as done in the Review, introduced so much uncertainty and instability into the EC 10 calculation that it
rendered the dataset unfit for use in national criteria derivation.

In the progression to determine a representative EC 10 we treated the five highest selenium samples as having 100% mortality, and retained the ten field and two SC hatchery treatments that experienced overflow, while applying the worst case assumption that all lost fish were dead.

The endpoint selected for the brown trout study was larval survival using all field and hatchery samples (designated the “All” data sub set). This application of the data resulted in an EC 10 value of 16.76 mg Se/kg egg dw. The survival EC 10 is not sensitive to the inclusion or exclusion of SC and SPC hatchery fish.

For the endpoints involving deformities, having calculated EC 10 s by 21 ways for all deformities, 21 ways for major deformities, and 24 ways for combined survival and deformities, the weight of evidence indicates that deformities is probably a less sensitive endpoint in this study. Whether evaluated as “maximum fully normal,” “minimum fully normal,” or “intermediate fully normal,”26

the rate of deformities in field samples did not seem to be related to selenium egg concentrations between 6.2 and 20.5 mg Se/kg.

The survival EC 10 of 16.76 mg Se/kg appears to be environmentally co nservative. It is somewhat affected by the worst

- case assumption about the health of fish that were lost when aquaria overflowed during the 15

- day post swim

- up test. It is more restrictive than any of the

EC 10 s calculated for survival from hatch to swim up (the first portion of the test) and for combined survival and deformities (in the full test), provided that the dataset is not edited to discard field samples. This final recommended EC 10 of 16.76 mg Se/kg egg is also consistent with egg

- or ovary

- base d EC 10 s for other fish species, falling at the lower end of the distribution for seven other species (Janz et al. 2010).27

Table 1a. Summary of Larval Survival, Full Test. Numbers of eggs hatched, proportions and numbers of fry surviving from hatch throu gh 15

- day post swim up test for the two calculation methods.

Method 1 Method 2

Station Egg Se

(mg Se/kg dw) #Eggs

Hatched Proportion

Survived #Fry

Survived Proportion

Survived #Fry

Survived
SC

- 001 0.76 143 0.948 136 0.948 136
SC

- 002 0.94 137 0.976 134 0.976 134
SC

- 003 0.83 340 0.971 330 0.874 297
SC

- 004 0.92 187 0.851 159 0.851 159
SC

- 005 1.2 70 0.914 64 0.900 63
SC

- 006 1.2 559 0.986 551 0.986 551
SC

- 007 1.0 183 0.853 156 0.853 156
SC

- 008 0.96 396 0.985 390 0.886 351
SPC

- 001 0.73 596 0.987 588 0.987 588
SPC

- 003 0.73 585 0.966 565 0.966 565
SPC

- 005 0.73 593 0.987 585 0.947 562
SPC

- 006 0.73 598 0.963 576 0.963 576
LSV2C

- 002 12.8 594 0.976 580 0.820 487
LSV2C

- 003 40.3 374 0.000 0 0.000 0
LSV2C

- 004 36 253 0.000 0 0.000 0
LSV2C

- 005 26.8 214 0.000 0 0.000 0
LSV2C

- 008 17.7 265 0.963 255 0.530 140
LSV2C

- 010 38.8 87 0.000 0 0.000 0
LSV2C

- 012 13.2 590 0.973 574 0.973 574
LSV2C

- 016 13.4 570 0.965 550 0.965 550
LSV2C

- 017 20.5 214 0.864 185 0.700 150
LSV2C

- 019 12.5 471 0.953 449 0.582 274
LSV2C

- 020 11.2 357 0.986 352 0.631 225
LSV2C

- 021 28.1 416 0.000 0 0.000 0
CC

- 150

- 009 12.8 171 0.944 161 0.944 161
CC

- 150

- 011 8.4 288 0.993 286 0.993 286
CC

- 150

- 012 8.5 311 0.968 301 0.948 295
CC

- 150

- 013 8.4 400 0.871 348 0.644 258
CC

- 150

- 015 9.1 470 0.994 467 0.974 458
CC

- 150

- 016 7.5 89 0.966 86 0.483 43
CC

- 150

- 017 6.6 223 0.969 216 0.649 145
CC

- 150

- 018 6.9 523 0.967 506 0.967 506
CC

- 150

- 020 6.2 584 0.990 578 0.990 578
CC

- 350

- 006 14 430 0.930 400 0.930 400
CC

- 350

- 007 6.9 178 0.954 170 0.763 136
CC

- 350

- 008 9.5 406 0.940 382 0.677 27528

Table 1b. Summary of Larval Survival, First Portion of Test (Hatch to Swim Up).

Station Egg Se

(mg Se/kg dw) #Eggs

Hatched Proportion

Survived #Fry

Survived
SC

- 001 0.76 143 0.958 137
SC

- 002 0.94 137 0.985 135
SC

- 003 0.83 340 0.982 334
SC

- 004 0.92 187 0.877 164
SC

- 005 1.2 70 0.914 64
SC

- 006 1.2 559 0.996 557
SC

- 007 1.0 183 0.880 161
SC

- 008 0.96 396 0.985 390
SPC

- 001 0.73 596 0.987 588
SPC

- 003 0.73 585 0.966 565
SPC

- 005 0.73 593 0.987 585
SPC

- 006 0.73 598 0.963 576
LSV2C

- 002 12.8 594 0.976 580
LSV2C

- 003 40.3 374 0.000 0
LSV2C

- 004 36 253 0.000 0
LSV2C

- 005 26.8 214 0.000 0
LSV2C

- 008 17.7 265 0.981 260
LSV2C

- 010 38.8 87 0.000 0
LSV2C

- 012 13.2 590 0.973 574
LSV2C

- 016 13.4 570 0.965 550
LSV2C

- 017 20.5 214 0.897 192
LSV2C

- 019 12.5 471 0.953 449
LSV2C

- 020 11.2 357 0.986 352
LSV2C

- 021 28.1 416 0.000 0
CC

- 150

- 009 12.8 171 0.953 163
CC

- 150

- 011 8.4 288 0.993 286
CC

- 150

- 012 8.5 311 0.977 304
CC

- 150

- 013 8.4 400 0.895 358
CC

- 150

- 015 9.1 470 0.994 467
CC

- 150

- 016 7.5 89 0.966 86
CC

- 150

- 017 6.6 223 0.969 216
CC

- 150

- 018 6.9 523 0.967 506
CC

- 150

- 020 6.2 584 0.990 578
CC

- 350

- 006 14 430 0.949 408
CC

- 350

- 007 6.9 178 0.966 172
CC

- 350

- 008 9.5 406 0.953 38729

T able 2

. Summary of Larval Deformities Excluding Missing Fry

. Numbers of fry assessed, proportions and numbers of normal fry for the three calculation
methods.

Max. Normal Min. Normal Int. Normal

Station Egg Se

(mg Se/kg dw) #Fry

Assessed Prop.

Norm. #Fry Norm. Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm. SC

- 001 0.76 115 0.687 79 0.400 46 0.504 58
SC

- 002 0.94 113 0.664 75 0.496 56 0.557 63
SC

- 003 0.83 302 0.576 174 0.364 110 0.460 139
SC

- 004 0.92 140 0.493 69 0.000 0 0.279 39
SC

- 005 1.2 42 0.595 25 0.524 22 0.553 23
SC

- 006 1.2 535 0.908 486 0.813 435 0.824 441
SC

- 007 1 .0 137 0.766 105 0.467 64 0.554 76
SC

- 008 0.96 359 0.811 291 0.710 255 0.731 263
SPC

- 001 0.73 568 0.863 490 0.680 386 0.711 404
SPC

- 003 0.73 545 0.822 448 0.611 333 0.655 357
SPC

- 005 0.73 561 0.848 476 0.647 363 0.685 384
SPC

- 006 0.73 556 0.854 475 0.678 377 0.710 395
LSV2C

- 002 12.8 544 0.917 499 0.858 467 0.864 470
LSV2C

- 003 40.3 98 a 0.000 0 0.000 0 0.000 0
LSV2C

- 004 36 164 a 0.157 26 0.000 0 0.019 3
LSV2C

- 005 26.8 138 a 0.128 18 0.000 0 0.004 1
LSV2C

- 008 17.7 194 0.851 165 0.670 130 0.704 137
LSV2C

- 010 38.8 71 a 0.000 0 0.000 0 0.000 0
LSV2C

- 012 13.2 554 0.424 235 0.329 182 0.384 213
LSV2C

- 016 13.4 530 0.915 485 0.766 406 0.784 415
LSV2C

- 017 20.5 150 0.813 122 0.593 89 0.647 97
LSV2C

- 019 12.5 390 0.774 302 0.626 244 0.661 258
LSV2C

- 020 11.2 296 0.868 257 0.780 231 0.793 235
LSV2C

- 021 28.1 170 a 0.153 26 0.000 0 0.006 1
CC

- 150

- 009 12.8 142 0.768 109 0.683 97 0.704 100
CC

- 150

- 011 8.4 266 0.429 114 0.229 61 0.343 91
CC

- 150

- 012 8.5 282 0.677 191 0.507 143 0.563 159
CC

- 150

- 013 8.4 310 0.590 183 0.200 62 0.375 116
CC

- 150

- 015 9.1 445 0.519 231 0.404 180 0.461 205
CC

- 150

- 016 7.5 23 0.565 13 0.435 10 0.491 11
CC

- 150

- 017 6.6 163 0.663 108 0.577 94 0.606 99
CC

- 150

- 018 6.9 486 0.593 288 0.311 151 0.427 207
CC

- 150

- 020 6.2 558 0.894 499 0.785 438 0.798 445
CC

- 350

- 006 14 386 0.513 198 0.000 0 0.252 97
CC

- 350

- 007 6.9 131 0.634 83 0.099 13 0.344 45
CC

- 350

- 008 9.5 338 0.840 284 0.666 225 0.700 237

a

– Maximum number of fry assessed for any given deformity ( cranio

- facial).30

T able 3

. Summary of Major Larval Deformities Excluding Missing Fry

. Numbers of fry assessed, proportions and numbers of fry free of major (level 2 and 3) deformities (FMD) for the three calculation methods.

Max. FMD Min. FMD Int. FMD

Station Egg Se

(mg Se/kg dw) #Fry

Assessed Prop.

FMD #Fry
FMD Prop.
FMD #Fry
FMD Prop.
FMD #Fry
FMD SC

- 001 0.76 115 0.930 107 0.835 96 0.844 97
SC

- 002 0.94 113 0.947 107 0.885 100 0.889 100
SC

- 003 0.83 302 0.699 211 0.685 207 0.689 208
SC

- 004 0.92 140 0.679 95 0.464 65 0.541 76
SC

- 005 1.2 42 1.000 42 1.000 42 1.000 42
SC

- 006 1.2 535 0.974 521 0.936 501 0.938 502
SC

- 007 1 137 0.912 125 0.796 109 0.809 111
SC

- 008 0.96 359 0.942 338 0.886 318 0.890 319
SPC

- 001 0.73 568 0.974 553 0.945 537 0.946 537
SPC

- 003 0.73 545 0.956 521 0.917 500 0.920 501
SPC

- 005 0.73 561 0.970 544 0.943 529 0.944 530
SPC

- 006 0.73 556 0.951 529 0.912 507 0.914 508
LSV2C

- 002 12.8 544 0.987 537 0.985 536 0.985 536
LSV2C

- 003 40.3 98 a 0.031 3 0.000 0 0.000 0
LSV2C

- 004 36 164 a 0.421 69 0.000 0 0.262 43
LSV2C

- 005 26.8 138 a 0.391 54 0.000 0 0.079 11
LSV2C

- 008 17.7 194 0.959 186 0.923 179 0.924 179
LSV2C

- 010 38.8 71 a 0.225 16 0.000 0 0.000 0
LSV2C

- 012 13.2 554 0.977 541 0.968 536 0.968 536
LSV2C

- 016 13.4 530 0.994 527 0.992 526 0.992 526
LSV2C

- 017 20.5 150 0.920 138 0.853 128 0.860 129
LSV2C

- 019 12.5 390 0.977 381 0.967 377 0.967 377
LSV2C

- 020 11.2 296 0.983 291 0.959 284 0.960 284
LSV2C

- 021 28.1 170 a 0.353 60 0.000 0 0.200 34
CC

- 150

- 009 12.8 142 0.965 137 0.908 129 0.911 129
CC

- 150

- 011 8.4 266 0.989 263 0.981 261 0.981 261
CC

- 150

- 012 8.5 282 0.982 277 0.965 272 0.965 272
CC

- 150

- 013 8.4 310 0.690 214 0.623 193 0.644 200
CC

- 150

- 015 9.1 445 0.978 435 0.951 423 0.951 423
CC

- 150

- 016 7.5 23 0.957 22 0.957 22 0.957 22
CC

- 150

- 017 6.6 163 0.988 161 0.982 160 0.982 160
CC

- 150

- 018 6.9 486 0.975 474 0.963 468 0.963 468
CC

- 150

- 020 6.2 558 0.973 543 0.973 543 0.973 543
CC

- 350

- 006 14 386 0.816 315 0.604 233 0.652 252
CC

- 350

- 007 6.9 131 0.802 105 0.458 60 0.551 72
CC

- 350

- 008 9.5 338 0.956 323 0.893 302 0.897 303

a

– Maximum number of fry assessed for any given deformity (cranio

- facial).31

T able 4

. Summary of Larval Deformities Incorporating Missing Fry

. Summaries of fry free of deformities after accounting for fry lost during the 15

- day post swim up test. All missing fry were assumed to be deformed for purposes of these calculations.

Max. Normal Min. Normal Int. Normal

Station Egg Se

(mg Se/kg dw) #Fry

Assessed + Missing Prop.

Norm. #Fry Norm. Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm.
SC

- 001 0.76 115 0.687 79 0.400 46 0.504 58
SC

- 002 0.94 113 0.664 75 0.496 56 0.557 63
SC

- 003 0.83 311 0.559 174 0.354 110 0.447 139
SC

- 004 0.92 140 0.493 69 0.000 0 0.279 39
SC

- 005 1.2 42 0.595 25 0.524 22 0.553 23
SC

- 006 1.2 535 0.908 486 0.813 435 0.824 441
SC

- 007 1.0 137 0.766 105 0.467 64 0.554 76
SC

- 008 0.96 369 0.789 291 0.691 255 0.711 263
SPC

- 001 0.73 568 0.863 490 0.680 386 0.711 404
SPC

- 003 0.73 545 0.822 448 0.611 333 0.655 357
SPC

- 005 0.73 561 0.848 476 0.647 363 0.685 384
SPC

- 006 0.73 556 0.854 475 0.678 377 0.710 395
LSV2C

- 002 12.8 560 0.891 499 0.834 467 0.840 470
LSV2C

- 003 40.3 98 a 0.000 0 0.000 0 0.000 0
LSV2C

- 004 36 164 a 0.157 26 0.000 0 0.019 3
LSV2C

- 005 26.8 138 a 0.128 18 0.000 0 0.004 1
LSV2C

- 008 17.7 239 0.690 165 0.544 130 0.573 137
LSV2C

- 010 38.8 71 a 0.000 0 0.000 0 0.000 0
LSV2C

- 012 13.2 554 0.424 235 0.329 182 0.384 213
LSV2C

- 016 13.4 530 0.915 485 0.766 406 0.784 415
LSV2C

- 017 20.5 169 0.722 122 0.527 89 0.574 97
LSV2C

- 019 12.5 429 0.704 302 0.569 244 0.601 258
LSV2C

- 020 11.2 332 0.774 257 0.696 231 0.707 235
LSV2C

- 021 28.1 170 a 0.153 26 0.000 0 0.006 1
CC

- 150

- 009 12.8 142 0.768 109 0.683 97 0.704 100
CC

- 150

- 011 8.4 266 0.429 114 0.229 61 0.343 91
CC

- 150

- 012 8.5 282 0.677 191 0.507 143 0.563 159
CC

- 150

- 013 8.4 336 0.545 183 0.185 62 0.346 116
CC

- 150

- 015 9.1 445 0.519 231 0.404 180 0.461 205
CC

- 150

- 016 7.5 66 0.197 13 0.152 10 0.171 11
CC

- 150

- 017 6.6 196 0.551 108 0.480 94 0.504 99
CC

- 150

- 018 6.9 486 0.593 288 0.311 151 0.427 207
CC

- 150

- 020 6.2 558 0.894 499 0.785 438 0.798 445
CC

- 350

- 006 14 386 0.513 198 0.000 0 0.252 97
CC

- 350

- 007 6.9 151 0.550 83 0.086 13 0.299 45
CC

- 350

- 008 9.5 366 0.776 284 0.615 225 0.646 237

a

– Maximum number of fry assessed for any given deformity (cranio

- facial).32

Table 5. Summary of Major Larval Deformities Incorporating Missing Fry

. Summaries of fry free of major deformities (FMD) after accounting for fry lost during the 15

- day post swim up test. All missing fry were assumed to be deformed for purposes of these
calculations.

Max. FMD Min. FMD Int. FMD

Station Egg Se

(mg Se/kg dw) #Fry

Assessed Prop.

FMD #Fry
FMD Prop. FMD #Fry
FMD Prop. FMD #Fry
FMD
SC

- 001 0.76 115 0.930 107 0.835 96 0.844 97
SC

- 002 0.94 113 0.947 107 0.885 100 0.889 100
SC

- 003 0.83 311 0.678 211 0.666 207 0.669 208
SC

- 004 0.92 140 0.679 95 0.464 65 0.541 76
SC

- 005 1.2 42 1.000 42 1.000 42 1.000 42
SC

- 006 1.2 535 0.974 521 0.936 501 0.938 502
SC

- 007 1.0 137 0.912 125 0.796 109 0.809 111
SC

- 008 0.96 369 0.916 338 0.862 318 0.866 319
SPC

- 001 0.73 568 0.974 553 0.945 537 0.946 537
SPC

- 003 0.73 545 0.956 521 0.917 500 0.920 501
SPC

- 005 0.73 561 0.970 544 0.943 529 0.944 530
SPC

- 006 0.73 556 0.951 529 0.912 507 0.914 508
LSV2C

- 002 12.8 560 0.959 537 0.957 536 0.957 536
LSV2C

- 003 40.3 98 a 0.031 3 0.000 0 0.000 0
LSV2C

- 004 36 164 a 0.421 69 0.000 0 0.262 43
LSV2C

- 005 26.8 138 a 0.391 54 0.000 0 0.079 11
LSV2C

- 008 17.7 239 0.778 186 0.749 179 0.749 179
LSV2C

- 010 38.8 71 a 0.225 16 0.000 0 0.000 0
LSV2C

- 012 13.2 554 0.977 541 0.968 536 0.968 536
LSV2C

- 016 13.4 530 0.994 527 0.992 526 0.992 526
LSV2C

- 017 20.5 169 0.817 138 0.757 128 0.763 129
LSV2C

- 019 12.5 429 0.888 381 0.879 377 0.879 377
LSV2C

- 020 11.2 332 0.877 291 0.855 284 0.856 284
LSV2C

- 021 28.1 170 a 0.353 60 0.000 0 0.200 34
CC

- 150

- 009 12.8 142 0.965 137 0.908 129 0.911 129
CC

- 150

- 011 8.4 266 0.989 263 0.981 261 0.981 261
CC

- 150

- 012 8.5 282 0.982 277 0.965 272 0.965 272
CC

- 150

- 013 8.4 336 0.637 214 0.574 193 0.594 200
CC

- 150

- 015 9.1 445 0.978 435 0.951 423 0.951 423
CC

- 150

- 016 7.5 66 0.333 22 0.333 22 0.333 22
CC

- 150

- 017 6.6 196 0.821 161 0.816 160 0.816 160
CC

- 150

- 018 6.9 486 0.975 474 0.963 468 0.963 468
CC

- 150

- 020 6.2 558 0.973 543 0.973 543 0.973 543
CC

- 350

- 006 14 386 0.816 315 0.604 233 0.652 252
CC

- 350

- 007 6.9 151 0.695 105 0.397 60 0.478 72
CC

- 350

- 008 9.5 366 0.883 323 0.825 302 0.829 303

a

– Maximum number of fry assessed for any given deformity (cranio

- facial).33

Table 6 a. Summary of Larval Survival plus Deformity endpoint

– lost fish excluded (M1). Numbers of total fry (H1) , proportions and numbers of fry surviving and free of deformities for the three calculation methods (D1

- D3) from hatch through 15

- day post swim up (M1)

.

Max. Normal Min. Normal Int. Normal

Station Egg Se

(mg Se/kg dw) #Eggs

Hatched (H1) Prop.

M1D1 #Fry M1D1 Prop. M1D2 #Fry M1D2 Prop. M1D3 #Fry M1D3
SC

- 001 0.76 143 0.653 93 0.380 54 0.480 69
SC

- 002 0.94 137 0.649 89 0.485 66 0.545 75
SC

- 003 0.83 331 0.564 187 0.357 118 0.450 149
SC

- 004 0.92 187 0.424 79 0.000 0 0.240 45
SC

- 005 1.2 70 0.544 38 0.479 34 0.505 35
SC

- 006 1.2 559 0.904 505 0.809 452 0.819 458
SC

- 007 1.0 183 0.662 121 0.403 74 0.478 88
SC

- 008 0.96 386 0.798 308 0.699 270 0.720 278
SPC

- 001 0.73 596 0.851 507 0.670 400 0.701 418
SPC

- 003 0.73 585 0.794 464 0.590 345 0.633 370
SPC

- 005 0.73 585 0.836 489 0.636 372 0.673 394
SPC

- 006 0.73 598 0.823 492 0.653 391 0.683 409
LSV2C

- 002 12.8 578 0.895 517 0.838 484 0.843 487
LSV2C

- 003 40.3 472 0.000 0 0.000 0 0.000 0
LSV2C

- 004 36 353 0.000 0 0.000 0 0.000 0
LSV2C

- 005 26.8 303 0.000 0 0.000 0 0.000 0
LSV2C

- 008 17.7 221 0.828 183 0.653 144 0.686 152
LSV2C

- 010 38.8 158 0.000 0 0.000 0 0.000 0
LSV2C

- 012 13.2 590 0.413 243 0.320 189 0.374 221
LSV2C

- 016 13.4 570 0.883 503 0.739 421 0.756 431
LSV2C

- 017 20.5 195 0.709 138 0.517 101 0.564 110
LSV2C

- 019 12.5 432 0.735 317 0.594 257 0.628 271
LSV2C

- 020 11.2 321 0.855 274 0.768 247 0.781 251
LSV2C

- 021 28.1 517 0.000 0 0.000 0 0.000 0
CC

- 150

- 009 12.8 171 0.727 124 0.647 111 0.667 114
CC

- 150

- 011 8.4 288 0.426 123 0.228 66 0.341 98
CC

- 150

- 012 8.5 309 0.659 204 0.492 152 0.547 169
CC

- 150

- 013 8.4 374 0.521 195 0.176 66 0.331 124
CC

- 150

- 015 9.1 466 0.514 239 0.399 186 0.455 212
CC

- 150

- 016 7.5 46 0.528 24 0.406 19 0.459 21
CC

- 150

- 017 6.6 190 0.638 121 0.555 106 0.584 111
CC

- 150

- 018 6.9 523 0.573 300 0.301 157 0.413 216
CC

- 150

- 020 6.2 584 0.885 517 0.777 454 0.790 461
CC

- 350

- 006 14 430 0.484 208 0.000 0 0.238 102
CC

- 350

- 007 6.9 158 0.606 96 0.095 15 0.329 52
CC

- 350

- 008 9.5 378 0.796 301 0.630 238 0.663 25134

Table 6b

. Summary of Larval Survival plus Deformity endpoint

– lost fish assumed dead (M2). Numbers of total fry (H2) , proportions and numbers of fry surviving and free of deformities for
the three calcu lation methods (D1

- D3) from hatch through 15

- day post swim up (M1)

.

Max. Normal Min. Normal Int. Normal

Station Egg Se

(mg Se/kg dw) #Eggs

Hatched (H2) Prop.

M2D1 #Fry M2D1 Prop. M2D2 #Fry M2D2 Prop. M2D3 #Fry M2D3
SC

- 001 0.76 143 0.653 93 0.380 54 0.480 69
SC

- 002 0.94 137 0.649 89 0.485 66 0.545 75
SC

- 003 0.83 340 0.549 187 0.347 118 0.438 149
SC

- 004 0.92 187 0.424 79 0.000 0 0.240 45
SC

- 005 1.2 70 0.544 38 0.479 34 0.505 35
SC

- 006 1.2 559 0.904 505 0.809 452 0.819 458
SC

- 007 1.0 183 0.662 121 0.403 74 0.478 88
SC

- 008 0.96 396 0.778 308 0.682 270 0.702 278
SPC

- 001 0.73 596 0.851 507 0.670 400 0.701 418
SPC

- 003 0.73 585 0.794 464 0.590 345 0.633 370
SPC

- 005 0.73 589 0.830 489 0.631 372 0.669 394
SPC

- 006 0.73 598 0.823 492 0.653 391 0.683 409
LSV2C

- 002 12.8 594 0.871 517 0.815 484 0.821 487
LSV2C

- 003 40.3 472 0.000 0 0.000 0 0.000 0
LSV2C

- 004 36 353 0.000 0 0.000 0 0.000 0
LSV2C

- 005 26.8 303 0.000 0 0.000 0 0.000 0
LSV2C

- 008 17.7 266 0.688 183 0.543 144 0.570 152
LSV2C

- 010 38.8 158 0.000 0 0.000 0 0.000 0
LSV2C

- 012 13.2 590 0.413 243 0.320 189 0.374 221
LSV2C

- 016 13.4 570 0.883 503 0.739 421 0.756 431
LSV2C

- 017 20.5 214 0.646 138 0.471 101 0.514 110
LSV2C

- 019 12.5 471 0.674 317 0.545 257 0.576 271
LSV2C

- 020 11.2 357 0.769 274 0.691 247 0.702 251
LSV2C

- 021 28.1 517 0.000 0 0.000 0 0.000 0
CC

- 150

- 009 12.8 171 0.727 124 0.647 111 0.667 114
CC

- 150

- 011 8.4 288 0.426 123 0.228 66 0.341 98
CC

- 150

- 012 8.5 310 0.657 204 0.491 152 0.545 169
CC

- 150

- 013 8.4 400 0.487 195 0.165 66 0.309 124
CC

- 150

- 015 9.1 468 0.512 239 0.398 186 0.453 212
CC

- 150

- 016 7.5 89 0.273 24 0.210 19 0.237 21
CC

- 150

- 017 6.6 223 0.544 121 0.473 106 0.497 111
CC

- 150

- 018 6.9 523 0.573 300 0.301 157 0.413 216
CC

- 150

- 020 6.2 584 0.885 517 0.777 454 0.790 461
CC

- 350

- 006 14 430 0.484 208 0.000 0 0.238 102
CC

- 350

- 007 6.9 178 0.537 96 0.084 15 0.292 52
CC

- 350

- 008 9.5 406 0.741 301 0.587 238 0.617 25135

Table 7. Larval survival for field treatments where fry reached swim up stage. Missing fish presumed lost to overflow during 15

- day post swim up test

. Percent survival during
15

- day post swim up test does not include missing fry.

Hatch through swim up 15

- day post swim up test

Station Egg

Se Day of Swim

up Survived Died % Survival Survived Died Missing % Survival Missing Fry
LSV2C

- 002 12.8 68 580 14 97.6 84 0 16 100
LSV2C

- 008 17.7 68 260 5 98.1 54 1 45 a 98.2
LSV2C

- 017 20.5 73 192 22 89.7 78 3 19 96.3
LSV2C

- 019 12.5 73 449 22 95.3 61 0 39 100
LSV2C

- 020 11.2 68 352 5 98.6 64 0 36 100
CC

- 150

- 013 8.4 72 358 42 89.5 72 2 26 97.3
CC

- 150

- 016 7.5 72 86 3 96.6 43 0 43 100
CC

- 150

- 017 6.6 72 216 7 96.9 67 0 33 100
CC

- 350

- 007 6.9 67 172 6 96.6 79 1 20 98.8
CC

- 350

- 008 9.5 67 387 19 95.3 71 1 28 98.6

No Missing Fry
LSV2C

- 012 13.2 73 574 16 97.3 100 0 100
LSV2C

- 016 13.4 73 550 20 96.5 101 0 100
CC

- 150

- 009 12.8 72 163 8 95.3 100 1 99.0
CC

- 150

- 011 8.4 67 286 2 99.3 101 0 100
CC

- 150

- 012 8.5 72 304 7 97.7 97 1 99.0
CC

- 150

- 015 9.1 67 467 3 99.4 98 0 100
CC

- 150

- 018 6.9 72 506 17 96.7 101 0 100
CC

- 150

- 020 6.2 67 578 6 99.0 100 0 100
CC

- 350

- 006 14 72 408 22 94.9 99 2 98.0

a

- listed as 46 missing in the Report. However, a maximum of 45 fish could have been missing for this station after accounting for all other fish that had died or been preserved.36

Table 8. Larval deformities for field treatments where fry reached swim up stage. Deformed fish are “minimum deformed fry” defined as number of fry assessed minus maximum normal fry. Percent deformed does not include missing fry.

Number of Fry Assessed

Station Egg Se Swim Up 15

- day Post Swim Up Total Missing Deformed % Deformed Missing Fry
LSV2C

- 002 12.8 480 64 544 16 45 8.3%
LSV2C

- 008 17.7 160 34 194 45 a 29 14.9%
LSV2C

- 017 20.5 92 58 150 19 28 18.7%
LSV2C

- 019 12.5 349 41 390 39 88 22.6%
LSV2C

- 020 11.2 252 44 296 36 39 13.2%
CC

- 150

- 013 8.4 258 52 310 26 127 41.0%
CC

- 150

- 016 7.5 0 23 23 43 10 43.5%
CC

- 150

- 017 6.6 116 47 163 33 55 33.7%
CC

- 350

- 007 6.9 72 59 131 20 48 36.6%
CC

- 350

- 008 9.5 287 51 338 28 54 16.0%

No Missing Fry
LSV2C

- 012 13.2 474 80 554 319 57.6%
LSV2C

- 016 13.4 449 81 530 45 8.5%
CC

- 150

- 009 12.8 62 80 142 33 23.2%
CC

- 150

- 011 8.4 185 81 266 152 57.1%
CC

- 150

- 012 8.5 204 77 282 b 91 32.3%
CC

- 150

- 015 9.1 367 78 445 214 48.1%
CC

- 150

- 018 6.9 405 81 486 198 40.7%
CC

- 150

- 020 6.2 478 80 558 59 10.6%
CC

- 350

- 006 14 307 79 386 188 48.7%

a

- listed as 46 missing in the Report. However, a maximum of 45 fish could have been missing for this station after accounting for all other fish that had died or been preserved.

b

– 282 fry assessed for deformities, but total number reported as preserved equals 281. Unable to tell from the report when the additional fry was preserved.37

Table 9a . Results of TRAP Analyses for Larval Survival , Full Test

. All values are in mg Se / kg dw egg. LCL and UCL are lower and upper 95% confidence intervals surrounding the EC 10 . Descriptions of error messages are in the notes below the table.

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure
Method 1 Field 20.48 IP, CL 1
Method 2 Field 17.25 16.73 17.79 None 2
Method 1 Review 14.59 13.74 15.49 IP, Conv 1
Method 2 Review 14.51 13.67 15.39 IP 2
Method 1 Field + SPC 20.47 IP, CL 1
Method 2 Field + SPC 16.86 16.32 17.41 None 2
Method 1 All 20.48 IP, CL 1
Method 2 All 16.76 16.22 17.31 None 2 Error Messages: IP

– Inadequate partials, Conv

– Maximum iterations reached without convergence,

CL

– Error estimates cannot be determined, None

– No errors.

Table 9b. Results of TRAP Analyses for Larval Survival, First Portion of Test (Hatch to Swim Up)

. All values are in mg Se / kg dw egg. LCL and UCL are lower and upper 95% confidence intervals surrounding the EC 10 . Descriptions of error messages are in the notes below the table.

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure
Method 1=Method 2* Field 20.72 IP, CL 3
Method 1=Method 2 Review 14.96 14.65 15.29 IP 3
Method 1=Method 2 Field + SPC 20.64 IP, Conv, StLim, CL 3
Method 1=Method 2* All 20.71 IP, CL 3 * For the Field and the All subsets, TRAP version 1.21 replaced version 1.20.38

Table 10 . Results of TRAP Analyses for Larval Deformities. All values are in mg Se / kg dw egg. LCL and UCL are lower and upper 95% confidence intervals surrounding the EC 10 . Descriptions of error messages are in the notes below the table.

Missing fry not included Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure

Maximum Normal Field 20.13 18.74 21.61 None 4
Minimum Normal Field 24.01 IP, StLim, CL 5
Intermediate Normal Field 18.86 17.43 20.40 IP 6 Maximum Normal Review 6.555 5.989 7.174 None 4
Minimum Normal Review 13.50 IP, CL 5
Intermediate Normal Review 8.637 7.998 9.328 None 6 Maximum Normal Field + SPC 19.78 18.50 21.14 None 4
Minimum Normal Field + SPC 23.35 IP, StLim, CL 5
Intermediate Normal Field + SPC 21.20 19.38 23.18 IP, Conv, StLim 6

Maximum Normal All 19.73 18.44 21.10 None 4
Minimum Normal All 23.05 IP, CL 5
Intermediate Normal All 21.32 19.34 23.50 IP, Conv., StLim 6

Missing fry included as abnormal Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure

Maximum Normal Field 19.22 17.83 20.72 None 7 Minimum Normal Field 23.31 IP, StLim, CL 8
Intermediate Normal Field 21.18 19.27 23.29 IP, Conv, StLim 9

Maximum Normal Field + SPC 18.47 17.19 19.84 None 7 Minimum Normal Field + SPC 23.14 IP, StLim, CL 8
Intermediate Normal Field + SPC 20.83 19.39 22.38 IP 9
Maximum Normal All 18.41 17.14 19.77 None 7 Minimum Normal All 23.02 IP, StLim, CL 8
Intermediate Normal All 15.81 14.74 16.95 IP 9 Error Messages: IP

– Inadequate partials, Conv

– Maximum iterations reached without convergence,
StLim

– Steepness at maximum or minimum limit, CL

– Error estimates cannot be determined, None

– No errors.39

Table 11 . Results of TRAP Analyses for Major Larval Deformities. All values are in mg Se / kg dw egg. LCL and UCL are lower and upper 95% confidence intervals surrounding the EC 10 . Descriptions of error messages are in the notes below the table.

Missing fry not included

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure

Maximum FMD Field 18.07 17.11 19.07 None 10
Minimum FMD Field 20.73 IP, CL 11
Intermediate FMD Field 16.63 15.85 17.44 None 12
Maximum FMD Review 16.11 15.50 16.75 None 10
Minimum FMD Review 18.46 IP, StLim, CL 11
Intermediate FMD Review 14.52 14.00 15.05 None 12
Maximum FMD Field + SPC 17.96 17.08 18.89 None 10
Minimum FMD Field + SPC 20.69 IP, CL 11
Intermediate FMD Field + SPC 16.58 15.86 17.33 None 12
Maximum FMD All 18.40 17.36 19.50 None 10
Minimum FMD All 20.77 IP, StLim, CL 11
Intermediate FMD All 16.95 16.11 17.84 None 12

Missing fry included as abnormal

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure

Maximum FMD Field 18.01 16.94 19.15 None 13
Minimum FMD Field 19.78 19.10 20.48 IP, Conv, StLim 14

Intermediate FMD Field 16.70 15.83 17.61 None 15
Maximum FMD Field + SPC 17.68 16.76 18.65 None 13
Minimum FMD Field + SPC 19.37 18.73 20.03 IP, Conv, StLim 14

Intermediate FMD Field + SPC 16.42 15.68 17.20 None 15
Maximum FMD All 17.90 16.91 18.95 None 13
Minimum FMD All 19.70 19.01 20.41 IP, Conv, StLim 14

Intermediate FMD All 16.70 15.88 17.56 None 15 Error Messages: IP

– Inadequate partials, Conv

– Maximum iterations reached without convergence,
StLim

– Steepness at maximum or minimum limit, CL

– Error estimates cannot be determined, None

– No errors.40

Table 12 . Results of T RAP Analyses for the Combined Effects of Larval Mortality and Deformity

. All values are in mg Se / kg dw egg. LCL and UCL are lower and upper 95% confidence intervals surrounding the EC 10 . Descriptions of error messages are in the notes below the table. Abbreviations of mortality

- deformity calculation methods are defined in the “Calculation of Mortality Plus Deformity” subsection of the Methods.

Calculation Method Data

Subset EC 10 LCL UCL Error Messages Figure
M1D1 Field 21.76 0.00149 318,556 IP, X50, St 16
M1D2 Field 23.15 IP, CL 17
M1D3 Field 21.60 16.76 27.83 IP, X50 18
M2D1 Field 21.52 18.58 24.94 IP, St 19
M2D2 Field 21.29 18.88 24.00 IP 20
M2D3 Field 20.94 19.41 22.59 IP, X50, St 21
M1D1 FWS 12.91 12.36 13.49 IP 16
M1D2 FWS 13.50 IP, CL 17
M1D3 FWS 12.89 12.50 13.29 IP, Conv, StLim 18
M2D1 FWS 12.93 12.39 13.49 IP 19
M2D2 FWS 13.50 IP, CL 20
M2D3 FWS 12.89 12.50 13.30 IP, Conv 21
M1D1 Field + SPC 21.39 19.50 23.46 IP, St 16
M1D2 Field + SPC 21.10 IP, CL 17
M1D3 Field + SPC 20.94 IP, CL 18
M2D1 Field + SPC 20.75 19.72 21.83 IP, St 19
M2D2 Field + SPC 20.57 19.25 21.98 IP, X50, StLim 20
M2D3 Field + SPC 20.47 IP, StLim, CL 21
M1D1 All 21.42 19.43 23.61 IP, St 16
M1D2 All 20.78 IP, StLim, CL 17
M1D3 All 20.70 IP, StLim, CL 18
M2D1 All 23.20 IP, StLim, CL 19
M2D2 All 22.77 IP, StLim, CL 20
M2D3 All 21.82 IP, StLim, CL 21 Error Messages: IP

– Inadequate partials, X50

– Large standard error for log(X50) term, St

– Large standard error for steepness term, CL

– Error estimates cannot be determined, Conv

– Maximum iterations reached without convergence, StLim

– Steepness at ma ximum or minimum limit.41

Figure 1. Survival in full test (hatch through post swim up) excluding missing fry (Method 1: Overflow Optimistic Case) analyzed with TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Ta ble 9a.42

Figure 2. Survival in full test (hatch through post swim up) including missing fry as dead (Method 2: Overflow Worst Case) analyzed with TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error mes sages are listed in Table 9a.43

Figure 3. Survival only to swim up (first part of test) analyzed with TRAP tolerance distribution for the four data subsets. Optimistic Case and Worst Case options do not apply. EC 10 values, confidence limits, and any TRAP error messages are listed in
Table 9b.44

Figure 4. Deformities as maximum fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confi dence limits, and any TRAP error messages are listed in Table 10.45

Figure 5. Deformities as minimum fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subse ts. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10.46

Figure 6. Deformities as intermediate fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Intermediate Case), analyzed by TRAP tolerance di stribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10.47

Figure 7. Deformities as maximum fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Deformities Optimistic C ase), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10. Review data subset includes no missing fry but is shown here for comparative purposes.48

Figure 8. Deformities as minimum fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP err or messages are l isted in Table 10. Review data subset includes no missing fry but is shown here for comparative purposes.49

Figure 9. Deformities as intermediate fully normal fry, assuming missing fry were deformed (Overflow Worst Case, Deformities Intermediate Case), an alyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10. Review data subset includes no missing fry but is shown here for comparative purposes.50

Figure 10. Major deformities as maximum fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 s, confidence limits, and TRAP messages are in Table 11.51

Figure 11. Major deformities as minimum fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 s, confidence limits, and TRAP messages are in Table 11.52

Figure 12. Major deformities as intermediate fully free of major deformities, excluding missing fry (Overflow Optimistic Case , Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subset s. EC 10 s, confidence limits, and TRAP messages in Table 11.53

Figure 13. Major deformities as maximum fully free of major deformities, assuming missing fry were deformed (Overflow Worst Case, Deformities Optimistic Case), analyzed by TRAP tolerance distr ibut ion for the four data subsets. EC 10 values, confidence limits, and any TRAP error messa ges are listed in Table 11. Review data subset includes no missing fry but is shown here for comparative purposes.54

Figure 14. Major deformities as minimum fully f ree of major deformities, assuming missing fry were deformed (Overflow Worst Case, Deformities Worst Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 1 1. Review data subset includes no missing fry but is shown here for comparative purposes.55

Figure 15. Major deformities as intermediate fully free of major deformities, assuming missing fry were deformed (Overflow Worst Case, Deformities Intermediate Cas e), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP err or messages are listed in Table 11. Review data subset includes no missing fry but is shown here for comparative purposes.56

Figure 16. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on maximum fully free from deformity (Deformity Optimistic Case), analyzed with T RAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12.57

Figure 17. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on minimum fully free from deformity (Deformity Worst Case), analyzed with TRAP for the four data subsets. EC 10 valu es, confidence limits, and any TRAP error messages are listed in Table 12.58

Figure 18. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on intermediate fully free from deformity (Deformity Intermediate Case), an alyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12.59

Figure 19. Combined effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on maximum fully f ree from deformity (Deformity Optimistic Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12.60

Figure 20. Combined effects, on survival assuming missing fry were dead (Met hod 2: Overflow Worst Case) and on minimum fully free from deformity (Deformity Worst Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12.61

Figure 21. Combined effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on intermediate fully free from deformity (Deformity Intermediate Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP err or messages are listed in Table 12.62

Appendix A : Assessment of EC 10 Calculations Performed by the U.S. Fish and Wildlife Service

The FWS (2012) “Technical Review: Smoky Canyon Mine Site

- Specific Selenium Criterion Report”, herein termed “the Review”, recommended a survival EC 10 of 14.9 mg Se/kg and a deformity EC 10 of 6.55 mg Se/kg for the brown trout maternal

- transfer reproductive study. After making the data corrections described in the main text of this assessment, the approach used by the Review y ields a survival EC 10 of 14.6 mg Se/kg, while the deformity remains unchanged. This Appendix examines the Review’s EC 10 calculation approaches.

Previously Janz et al. (2010), reporting the findings of the 2009 SETAC expert workshop, had
evaluated the same brown trout reproductive data. They judged this species to be among the most sensitive known, with an EC 10 of 17.7 mg Se/kg. They also found the EC 10 s (or comparable NOEC or LOEC values) of the eight species having suitable data to fall within in

narrow band, 17

– 24 mg Se/kg. The Review’s calculation of an EC 10 of 6.55 mg Se/kg for brown trout thus contrasts sharply with the previous understanding of the toxic concentrations of
selenium. Because the Review’s brown trout EC 10 is so distant from the oth er species EC 10 s, and so different from the expert workshop value, it is worthwhile to examine the strength of the evidence underlying its calculation.

The assessment presented in the main text of the current data re

- analysis evaluated 12 ways to incorpora te the Formation Environmental (2011) Report’s survival data, 42 ways to incorporate the deformity data, and 24 ways to incorporate combined survival and deformity data. With two additional options shown in the upper left and bottom right of Figure A2, we have thus
calculated 80 different EC 10 s using the TRAP model’s triangular tolerance distribution, the computational approach used by the FWS (2012) Review. That analysis revealed that the Review’s editing of the dataset, removing information on samples f or which some organisms
were lost to overflow , was an essential step for obtaining such a low EC 10 . The abridged dataset was sparse, yielding EC 10 s that were unstable and sensitive to small nuances in the assumptions underlying the calculations, as will b e shown below.

The Formation Environmental (2011) brown trout test involved two parts, which occur after
fertilization and hatch have been achieved 4 , (a) hatch to swim

- up (mean = 28 days 5 ), and (b)
swim

- up to 15 days post swim

- up. Undertaking the last por tion of the test is unusual; most
selenium maternal

- transfer reproductive tests do not go on that long. That is, they do not extend to an age when the young must be fed to survive. When a drain clogged with food in the last portion of the test, some indi viduals in 10 field samples and 2 SC hatchery samples were lost when aquaria overflowed.

4 Hatching success in such fish species is highly variab le between parents. Because differences are not reliably related to Se exposure, it is customary not to include it in the EC 10 calculation.

5 Hatch to swim

- up duration from Table 3

- 3 of Formation Environmental (2011).63

The FWS (2012) Review responded to this problem by discarding all data from those samples, arguing that the individuals swept out of the tank may have been dead, dyi ng, or deformed; unable to resist the flow over the brim. The effect of removing these data is to leave a concentration gap of 1.9X in the range 14.0

- 26.8 mg Se/kg, a range essential for defining the
EC 10 in all other sensitive species (Janz et al. 2010 ). In addition, having appropriately assigned zero survival to all samples above 26.8 mg Se/kg, due to their failure to swim up (with which we

agree 6 ), the Review dataset lacked exposures displaying partial survival, such that the slope of
the concentrati on

- response curve could not be reliably defined. Recognizing this limitation when discussing its survival EC 10 of 14.9 mg Se/kg, the Review then stated that “the only reasonable information that these data yield is that” the EC 100 “occurs somewhere betwee n 14 and 27” mg Se/kg. In saying that the EC 100 could be as low as 14 mg Se/kg, the Review disregards the fact that at 20.5 mg Se/kg survival was 70%, even after counting fish lost to the overflow among the dead. Despite the uncertainty about the conditi on of the fish lost to overflow, we conclude that samples discarded by the Review actually contain relevant
information.

The data used in calculating the Review’s deformity EC 10 fall in three regions, as shown in Figure A1. At the low end of selenium expo sure are the SPC hatchery fish, obtained because the SC hatchery eggs had developed fungus problems when fertilized and hatched in the lab. The SPC hatchery eggs, in contrast to the eggs spawned from SC hatchery and wild

- caught adults, arrived pre

- fertili zed and substantially older, at the eyed embryo stage. 7 They were held in the lab for only 34 days, as opposed to 67

- 72 days for the eggs spawned from SC and wild

- caught adults. They arrived in the lab after the fungus problems experienced by eggs spawned from SC
and wild

- caught adults had been abated.

In the middle region of the Review dataset are the low

- concentration Field samples (from wild

caught parents), with exposures in the range 6.2

– 14.0 mg Se/kg. Although the Review
describes these as insufficiently low to define the intercept of the respon se curve (the background rate of deformities for the experimental conditions), data for all other sensitive species indicate that these selenium exposures are low enough to reliably define the response intercept, unless
brown trout is far more sensitive th an any known species. But compared to the eyed embryos obtained from the SPC hatchery, the offspring of several but not all of the wild

- caught females were exhibiting elevated deformity rates at concentrations in the range of 6.2

– 14.0 mg Se/kg, as
shown in Figure A1.

Separated by a concentration gap of 1.9X are the high

- end exposures, 26.8

– 40.3 mg Se/kg. When evaluated by the Review’s Maximum Normal approach (an optimistic perspective), not all

6 All survival EC10s that we present in the main text equate failure to swim up with mortality, in accord with the Review’s recommendation.

7 Because the lab fertilization of eggs of salmonids is not a common procedure in toxicity testing labs, it may be recognized that the SPC hatchery would have longer experience in maintaining healthy conditions during fertilization of brown trout eggs.64

of these fish were afflicted with any single type of defo rmity, although it is more likely that essentially all were in fact deformed, as calculated by our Intermediate Normal approach (described in the main text) and shown in the lower left of Figure A2.

Given the Review’s sparse dataset, TRAP defines the respo nse intercept as the SPC hatchery rate, interprets the variable deformity rate in the 6

– 14 mg Se/kg range as having been induced by selenium, and draws a shallow sloping line into the high range. But several factors make this
solution difficult to defen d and unlikely to be valid.

 Given the great difference in the lab handling of SPC eggs, they cannot by themselves define the response intercept. Concerns about the use of these hatchery eggs were expressed in the Review’s peer review.

 With an EC 90 /EC 10 > 5, the Review’s slope is uncharacteristically shallow for selenium. Skorupa (1998, 2005, and 2011) and Lemly and Skorupa (2009) emphasize that selenium
concentration

- response curves are consistently steep. Skorupa (2005) and Skorupa (2011)
indicate a cha racteristic EC 90 /EC 10 = 2. But the EC 10 of 6.55 mg Se/kg hinges on applying a shallow slope.

 In a >2X range above the Review’s EC 10 =6.55 mg Se/kg, the data do not show the expected sharp decline in the fraction normal. Indeed they show no decline whateve r. There is no correlation between selenium concentration and fraction normal in the range 6.2

– 14 mg Se/kg (r 2 =0.005). Lacking any relation between exposure and effect across this 2.3X range, there is no basis for attributing the noisy response in this region to selenium.

 Maximum Fully Normal fish in Field samples in the 6.2

– 14 mg Se/kg range averages a bit higher than SC Hatchery samples in the 0.76

– 1.2 mg Se/kg range, as illustrated in the upper left of Figure A2, again seeming to disaffirm a sele nium

- related effect in this range.

 The lack of any apparent concentration

- response relationship in the 6.2

– 14 mg Se/kg region is not merely a phenomenon shown by these study data. No evidence of a concentration

response relationship within that region can be found in a maternal

- transfer reproductive study in any species

.

 Even after applying the worst case assumption that all fish lost in the overflow event were deformed, the fraction of normal fish at 17.7 and 20.5 mg Se/kg was above the median for
Fiel d samples in 6.2

– 14 mg Se/kg range, thus extending upwards the range within which there is no apparent deformity response to selenium. This holds whether assessed as Maximum Fully Normal, Intermediate Fully Normal, or Minimum Fully Normal. (Figure A2, upper right, shows the Maximum Fully Normal comparison; the data points for 17.7 and 20.5 mg Se/kg are the highest concentration filled circles.)

 The above situation holds for the fraction normal at 17.7 and 20.5 mg Se/kg (counting all missing fish as de formed) even when compared to the median only of the lower concentration Field samples that did not experience overflow. Thus, relative to the random scatter of deformity rates in the 6.2

– 20.5 mg Se/kg range (a 3.3X range), the number of
individuals los t in the overflow event was insufficient to influence the general pattern65

showing no relationship with selenium concentrations in this range (as shown in the upper left of Figures 7, 8, and 9.)

 The Review’s EC 10 calculations hinge on omitting the observati ons in the 17.7 and 20.5 mg Se/kg exposures and the information gap between 14.0 and 26.8 mg Se/kg generated by excluding data in that range from the analyses

. In the main text (pages 17

- 18) we note the influence that removing those observations had on th e survival EC 10 for the first portion of the test (hatch to swim up), which did not experience the overflow event. (The breakout of deformity counts between the first and second portions of the test has not been provided by Formation Environmental, so we could not perform a parallel type of analysis of deformities for the first portion of the test.)

 Working with the data for the full test (hatch to 15 days post swim up) and restoring the samples in which fish were missing (including the data at 17.7 and 20 .5 mg Se/kg) under the

worst

- case assumption that all missing fish were deformed, the solution shifts from the shallow slope curve of Figure A1 (EC 10 of 6.55 mg Se/kg) to the steeper slope curve of Figure A2 upper right (EC 10 18.47 mg Se/kg).

 To a lesser degree, the Review’s 6.55 mg Se/kg calculation depends on a paradoxical interpretation of the health of fish at exposures ≥26.8 mg Se/kg. If the data points above 26.8 mg Se/kg were set to zero to correspond to their failure to swim up, then the slope wo uld be steeper and the EC 10 would increase to 13.0 mg Se/kg. So large an instability, coupled with the difficulty of defending individuals that fail to swim up as being “fully normal”, presents problems.

 Responsive to expectations that EPA will attempt to maintain some degree of consistency in its criteria derivation approaches, we must be concerned about the precedent that would be set by adopting the Review’s Maximum Normal approach. Our Intermediate Normal calculation (see the main text) might be viewe d as a more scientifically reasonable option, while our Minimum Normal calculation represents the worst

- case , most conservative interpretation of the deformity rate. These, however, yield higher rather than lower EC 10 s. This seemingly counterintuitive be havior underscores the instability of the 6.55 mg Se/kg calculation. It is an artifact of using a sparse dataset having a particular configuration, where interpretive nuances making minor changes in the placement of data (such as zeroing the
health of all individuals above 26.8 mg Se/kg) have a strong influence on the EC 10

.

 Egg concentrations in 23 of 24 adult female brown trout taken from the study area (Crow Creek and Lower Sage Creek) exceeded the Review’s EC 10 of 6.55 mg Se/kg (as shown in
Tables 1

- 6). In Lower Sage Creek the median exposure was 19 mg Se/kg, almost 3

- fold
above the Review’s EC 10 . Yet the populations of brown trout did not appear to be manifesting effects commensurate with severe reductions in reproductive success. This may be problem atic to explain if an EC 10 of 6.55 mg Se/kg is in fact the appropriate value.

The FWS (2012) Review has raised important issues, not previously brought to the fore, about the brown trout study. Following up on that Review, this assessment has examined th e issues in detail, calculating the EC 10 in 80 different ways

– 78 ways shown in Tables 9

– 12 and Figures 166

– 21 of the main text of this document, plus two additional ways in the upper left and lower right of Figure A2. Environmentally conservative

, worst

- case assumptions about the response in samples that the Review did not consider indicate that the weight of evidence does not support an

EC 10 as low as either the Review’s deformity EC 10 or its survival EC 10 . Given the issues raised by the Review, we believe that this assessment’s comprehensive examination provides a
defensible basis for a brown trout EC 10 that can be used, together with EC 10 s for other species, as part of the derivation of a national criterion.67

Figure A1. Summary of issu es in plot for calculation of the Review’s deformity EC 10 of 6.55 mg Se/kg. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0.5 1 2 4 8 16 32 64 Maximum Proportion Normal

Egg Selenium (mg/kg) 2.3

- fold range having zero correlation
between Se and effect. As no known species exhibits effects in this range, these more likely define the low

- Se deformity rate for this test.

Eliminating the SC hatchery eggs, which had deformity
rates similar to the 6

- 14 mg/kg wild eggs, the
Review uses the SPC hatchery eggs to represent the low Se background rate, despite the methodological differences in how SPC eggs were handled: (a) not fertilized in the lab, having arrived at a more mature stage, and (b) not experiencing the fungus problems affecting the eggs spawned from SC and wild

caught adults. After arguing against the health of these individuals because they failed to swim up, the Review EC10 (6.5 mg/kg) hinges on the shallow slope defined by treating failure to swim up as being "normal". Were these points set to zero to track their failure to swim up, the Review EC10 would double. 1.9

- fold range within which all other sensitive species

- mean EC10s fall. The Review discarded all data in this range.

Shallow slope not
characteristic of Se.68

Figure A2. Effects of data selection and calculation method on deformity EC 10 . Each figure represents the data subset used for the recalculation of deformities in the FWS Revie w with one component altered. Upper left

– SC hatchery fish vs. SPC hatchery fish . Upper right

– excluded field sample s added, with missing fish assumed to have been deformed. Lower Left

– Intermediate Fully Normal calculation method used. Lower Right

– Fish that failed to reach swim up treated as 0% Normal.69

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Formation Environmental. 2011. Brown t rout l aboratory r eproduction s tudies c onducted in

s upport of d evelopment of a s ite

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.70



### Re

- Analysis of the Brown Trout S elenium Toxicity Study Presented by Formation Environmental and Reviewed by U.S. Fish and Wildlife Service, 2014 Revision

ADDENDUM

May 31, 2014

By

Charles Delos Office of Water, U.S. Environmental Protection Agency, Washington, DC

Submitted By

Great Lakes Environmental Center, Columbus, OH71

Introduction This document is a 2014 Addendum to “Analysis of the brown trout selenium toxicity study presented by Formation Environmental and reviewed by the U.S. Fish and Wildlife Service” (Taulbee et al. 20 12) . This addendum was produced following the availability of additional information from a quality assurance (QA) report of the original raw data from the brown trout reproductive study was conducted ( AECOM 2012 )

. The AECOM (2012) QA report included corrections to the numbers of counts for several replicates, and also most notably provided counts of fish that were fully free from all deformities, as well as counts of fish assessed for deformities. This important additional information addressed a majo r source of uncertainty in the Formation Environmental (2011) study raised in the U.S. FWS (2012) review, in which the
Services stated that counts of fry that were fully free from all deformities were not available. In this update to the 2012 EPA re

- analy sis, theupdated deformity data w ere used in a second reanalysis that greatly reduced the number of hypothetical scenarios for both the deformity and combined (survival plus deformity) endpoints presented in Taulbee et al. (2012) in order to
account for u ncertainties in reported deformity counts.

Th e original draft reassessment performed for EPA ( Taulbee et al. 2012 ), was completed in response to the 2012 “Technical Review: Smoky Canyon Mine Site

- Specific Selenium Criterion Report” by the U.S. Department of the Interior Fish and Wildlife Service ( U.S. FWS 2012). FWS reviewed Formation’s 2010 draft (not the above 2011 final revision) and addressed several topics , including some issues related to the brown trout test. The original reassessment (Taulbee

et al. 2012) addressed issues raised in Section II of the FWS Review, Recommended Recalculations, as well as relevant portions of a peer review conducted on the FWS Technical
Review by calculating effect concentrations for survival and deformities under a w ide range of optimistic and worst case scenarios using different data subsets.

Key Differences between the Original and Update d Reassessment The most important difference between the original draft reassessment (Taulbee et al. 2012) and
this A ddendum is that the additional information provided in the AECOM (2012) QA report removed the uncertainty surrounding total deformity rates. As a result, an EC 10 for total normal fry could be calculated in this A ddendum. This greatly reduced the number of hypothet ical scenarios examined in Taulbee et al. (2012) to estimate fry free from deformities, and by extension, combined survival + fry free from deformities. The second important difference between Taulbee et al. (2012) and this addendum is that every scenario described in Taulbee et al. (2012) was examined separately for four subsets of the data. In contrast, the six scenarios
examined in this A ddendum were only examined using the full (all field

- collected samples plus all hatchery samples) dataset. This dec ision was made because the final recommended EC 10 presented in Taulbee et al. (2012) of 16.76 mg Se/kg dw for the survival endpoint under the

worst

- case scenario was based on the full dataset, and the majority of peer

- reviewers concurred that it was approp riate to use the full dataset.72

This addendum begins with a summary of the Formation Environmental (2011) Report, followed by the recommended EC 10 s and toxicity endpoints as determined in the Formation Environmental (2011) report; and as recalculated in the U.S. FWS (2012) r eview and the U.S. EPA draft reassessment (Taulbee et al. 2012) . Next, experimental and analytical issues pertaining to the specific analyses presented in this A ddendum that affected the

r ecommendations of the F ormation Environmental report, the recommended recalculations of the
U.S. FWS r eview , and the issues addressed by the quality assurance report (AECOM 2012) are described . The remainder of the document includes the methods, results, and conclusions of the updated reassessment using the QA’d brown trout data in light of issues raised in the U.S. FWS

r eview

.

Brown Trout Reproduction Study Summary Formation Environmental was contracted by the J.R. Simplot Company to perform a brown trout ( Salmo trutta ) reprod uction study for receiving waters downstream of the Smoky Canyon phosphate mine in Southeastern Idaho. After elevated selenium (Se) concentrations were measured in receiving waters downstream of the Smoky Canyon mine, this study was
commissioned to invest igate the possible effects of these elevated Se concentrations on the reproductive fitness of resident brown trout.

In the fall of 2007, pre

- spawn adult brown trout (>230 mm) were collected from three locations

near the Smoky Canyon mine. These included o ne location (station LSV

- 2C) along Sage Creek, a highly impacted site downstream of Hoopes Spring (which drains directly from the Smoky Canyon mine) that regularly exceeds the chronic water quality standard for selenium

. In addition, two additional locati ons along Crow Creek upstream of the conflu ence with Sage Creek were selected with the intent of serving as background locations that also contained trout

. Chronic selenium exceedances have occasionally been measured in Crow Creek, but only immediately do wnstream of Sage Creek. The surface water selenium concentration in Sage Creek at the time of initial fish collection was 38.4 μg/L, while the surface water concentrations
in Crow Creek were < 1.5 μg/L.

Eggs were collected from 26 ripe adult female brown trout and fertilized in the field with milt
obtained from males collected at the same site as females. Fertilized eggs were water hardened at the site using stream water, then placed in oxygenated plastic bags and stored on ice in the dark (cooler) for transportation to laboratory. Selenium was measured in adult fish (whole body) and in eggs of field collected females. Eggs from two of the females from Sage Creek were not used; one batch because the eggs were dead upon arrival to the laboratory, and th e other because they were not successfully fertilized (Formation Environmental 2011). The final number of field samples for the reproductive study, defined as the offspring of a female wild

- caught brown trout beginning with their fertilized eggs, was 24; 12 from Sage Creek (station LSV

- 2C), 3 from the73

down stream Crow Creek location (station CC

- 350), and 9 from the up stream Crow Creek location (station CC

- 150).

E ggs were also collected from 8 ripe females obtained from the Saratoga National Fish Hatchery (S C) to serve as method controls. Similar to field

- caught fish, SC hatchery females were stripped of eggs and fertilized by milt from males obtained from the same hatchery. As a result of lower than expected hatch rates and fungal contamination in some of the SC hatchery samples, additional hatchery fish were obtained. Because it was late in the hatching season, no fertile females were available from the Spring Creek Trout Hatchery (SPC). Instead, fertilized eyed eggs were sent from the SPC hatchery in a single batch and these were split into four samples to serve as a secondary method control.

A pproximately 600 fertilized eggs from each female (or 600 eyed embryos for SPC treatments)

were placed in egg cups for hatching and monitoring. Larvae were monito red through hatch and swim up (the absorption of the yolk sac by the alevins), and percent hatch, time of hatch, and time to swim up were recorded for all field and hatchery samples. After swim up, remaining fry were thinned to a target of 100 fry/treatm ent and monitored for an additional 15 day post swim up feeding trial. After 15 days, 20 fish were sacrificed from each sample for length and weight determination, and all remaining fish that survived the 15 day post swim up test were preserved
for subse quent deformity assessment. Fish sacrificed for length and weight determination were
not assessed for deformities. If fewer than 100 fish were available for a particular sample, no fish were culled. Culled fish that reached swim up were preserved for su bsequent deformity

assessment.

Endpoints measured in the Formation Environmental (2011) laboratory study were fecundity, hatch, growth, survival/mortality, and feeding success (growth) post swim up. Larval brown
trout were also evaluated for deformities (craniofacial, vertebral, fin) and edema. For this reanalysis , deformities were combined and assessed as having at least one deformity, or being fully free of deformities (i.e., normal). Test termination ranged from 83 to 88 days after hatch
for all but the Spring Creek Hatchery egg treatments, which occurred 50 days after the arrival of fertilized, eyed embryos from that hatchery.

Reanalysis of Effects Data Se lenium concentrations in eggs collected from the 26 ripe females collected at 3 field locations ranged from 6.2

- 12.8 mg Se/kg dw at CC

- 150, 6.9

- 14.0 mg Se/kg dw at CC

- 350, and 11.2

- 40.3 mg Se/kg dw at LSV2C. Selenium concentrations in Crow Creek (CC) eggs were higher than anticipated, based on water concentrations (Formation Env ironmental 2011). It was suggested by peer

- reviewers of the U.S. FWS (2012) review that the high egg selenium concentrations were likely due to migration of fish from more contaminated to less contaminated waters. Se concentrations in hatchery eggs range d from 0.76

- 1.2 mg Se/kg dw at the SC hatchery, and were74

0.73 mg Se/kg dw at the SPC hatchery. The Se whole body concentration in field collected fish ranged from 7.2

- 22.6 mg/kg dw at LSV03, 4.7

- 8.4 mg/kg dw at CC

- 150, and 5.5

- 9.2 mg/kg dw
at CC

- 350. Se l enium whole body concentrations in SC hatchery fish ranged from 2.5

- 4.3 mg/kg
dw.

Three endpoints (percentage fully free from deformities (% normal), percentage surviving from hatch through 15 day post swim up (% survival), and percentage surviving from h atch through 15 days post swim up AND fully free of deformities (% alive and normal) were analyzed using the updated data from the AECOM (2012) QA report . Selenium concentrations and respective counts are shown in Table 1 (% normal), Table 2 (% survival), and Table 3 (% alive and normal). For all tables, each sample ID represents eggs hatched from a single female fish, with the exception of the 4 SPC samples, which were obtained as eyed eggs.

The chronic measure of effect concentration used in these ana lyses is the EC 10 , which is the concentration of a chemical that is estimated to result in a 10 percent effect in a measured chronic endpoint (e.g., growth, reproduction, survival) (U.S. EPA 2014)

. Plots and EC 10 s of each endpoint for both the optimistic and worst case scenarios are shown in Figure 1. All analyses were performed in TRAP (version 1.21) using tolerance distribution analysis and assuming a triangular data distribution.

Because of concerns raised in a U.S. Fish and Wildlife (2012) review of the Formation Environmental (2011) report, most notably with respect to the consequences of fish lost due to an overflow event during the 15 day post swim up portion of the test, all endpoints were measured according to a both an “optimistic” and a “worst

- case” scenario. The “worst

- case” scenarios

were first introduced in Taulbee et al. (2012) to examine the comment raised in the U.S. FWS (2012) review that fish lost to overflow during the post swim up test were assumed to have been
dead or deformed. As a n alternative to the U.S. FWS proposal that all treatments that lost fish to overflow should be discarded from the study, we examined the effect of those individuals being either dead and/or deformed. Therefore, a total of six EC 10 s (3 endpoints x 2 scenarios/endpoint)
were calculated here.

In all of the analyses described in this Addendum , larvae hatched from eggs from both wild caught fish and hatchery fish were combined in this analysis. This was referred to as the “All”

dataset in Taulbee et al. (2012). The consequences of which subset of data to use were examined in great detail within Taulbee et al. (2012), as each scenario was examined using four different data subsets. The majority of the peer reviews of Taulbee et al. conc urred that the calculation of
an EC 10 from the dataset containing all field and hatchery samples was reasonable. Because the consequences of data subsets were addressed in Taulbee et al. (2012), the recommended EC10 from Taulbee et al. (2012) was calculat ed from the full (All) dataset, and the primary purpose of
the reanalysis in this Addendum is to calculate the “fully free from deformity” endpoint recommended in the U.S. FWS (2012) review using updated data in AECOM (2012), the scenarios presented in thi s Addendum were analyzed using the full (All) dataset.75

Discussion of Issues Addressed in Taulbee et al

. 2012 and in 2014 Reanalysis Concerning the Brown Trout Reproduction Study A number of experimental and analytical issues associated with the Formation E nvironmental (2011) brown trout reproduction study deserve additional attention. Taulbee et al. (2012) includes a description of all of the issues that affected the interpretation of the toxicity data and ultimately, the effect concentrations derived from these data. Those issues that directly affected the analyses performed in this addendum are described below.

Use of Alevin in Deformity Analysis and 15 Day Post Swim Up The U.S. FWS (2012) review noted alevin that fail to reach swim up would not survive in the wild, and conducted their analysis of survival from hatch through 15

- day post swim up assuming 0% survival for those five high Se samples.

For the majority of alevin hatched from field and hatchery collected eggs, only alevin that
reached the swim up stage were included in the 15

- day post swim up survival test, included in the final survival calculations, or assessed for deformities. Alevin hatched from eggs with the 5 highest Se exposure concentrations (26.8

- 40.3 mg Se/kg dw) failed to reach swim up, but were
included in the 15

- day post swim up survival test and in the final survival calculations in the
Formation Environmental study report ( 2011)

. In addition, Formation Environmental (2011) performed d eformity assessment s on alevin from the 5 hig hest Se exposures that survived the 15

- day post swim up test in addition to preserved alevin that died during the first part of the test.

EPA agree s with th e point made by the U.S. FWS that alevin that fail to reach swim up would
not survive in the wild , and thus all survival analyses performed herein were conducted after setting survival for those exposure concentrations where alevin did not reach swim up to 0%

. Consistent with both Formation Environmental (2011) and U.S. FWS (2012), we retained r esult s from the five highest ( > 20.5 mg Se/kg dw) Se samples for deformity analysis

.

Overflow and Loss of Fish During the 15

- Day Post Hatch Survival Feeding Trial During the 15

- day post survival test, aquaria overflow resulting from a clogged drain led to the

p artial loss of fish from 10 field samples (5 from the two Crow Creek stations and 5 from Sage Creek) and two SC hatchery samples. The field samples lost from Sage Creek included 5 of the 7 samples where fish reached swim up stage, with egg Se concentratio ns ranging from 11.2

- 20.5 mg Se/kg dw. Between 9 and 45 (average = 29) larvae were lost from the 12 replicates. As a consequence, the highest egg Se concentration for any of the field samples where fish reached swim up stage and did not lose individuals to overflow was 14.0 mg Se/kg dw. The Formation Environmental (2011) r eport addressed the overflow issue by analyzing hatch to 15

- day post swim up survival as if fish lost to overflow had never been included in the test. Effectively, this approach assume d mortality of fish lost to overflow would have been identical to mortality of76

fish not lost to overflow. On the other hand, the U.S. FWS (2012) r eview asserted that fish lost to overflow were lost because they were most likely either dead or deformed, an d that the only appropriate course of action was to fully exclude all samples lost to overflow from their survival and deformity analyses. As a result, the highest Se concentration of any field sample with fish that reached swim up stage in the U.S. FWS r eview dataset was 14.0 mg Se/kg dw, whereas the highest concentration of any field sample with fish that reached swim up in the Report dataset was 20.5 mg Se/kg dw.

In both Taulbee et al. (2012) and here, w e addressed the assumption that fish lost to ove rflow were likely dead or deformed by comparing the EC 10 s for each endpoint using an optimistic and

worst

- case scenario. In the optimistic scenarios, missing fish were ignored (as was done in Formation Environmental (2011). In the worst

- case assessment s cenario, missing fish were assumed to be dead or deformed, as applicable , which comports with the U.S. FWS (2012)
assertion in their review. The major difference was that EPA retained the treatment groups
whereas the U

. S

. FWS fully exclude d the treatment levels from field samples with missing fish from any further assessment.

Calculation of Deformity Endpoints The U.S. FWS (2012) r eview was critical of the method used by Formation Environmental to calculate deformities, stating that the endpoint “sum total normal“ fish averages deformities across classes, and will always underestimate the total number of fish with deformities. The

FWS r eview then stated that “fully normal” fish would have been the most appropriate overall
measure of deformities. Because the information required to calculate “fully normal” fish was not available in the Formation Environmental report, FWS recalculated deformity rates using the endpoint “maximum fully normal” fish , which was 100% minus the percentage of the most prevalent deformity type. The “maximum fully normal” endpoint is based on the recognition that the proportion of normal fish cannot have been higher than the lowest normal proportion of any of the deformit y classes

, but it still underestimates deformities for all fish exhibiting more than one deformity

.
In the Formation Environmental (2011) r eport, each of the four deformity types (i.e., cranial

facial, finfold, spinal, edema) were assessed and analyzed s eparately and in combination. The method used to calculate the four deformity types in combination (described in section 4.4.3 of Formation Environmental (2011)) was to sum the percentage of fish free of each of the four classes of deformity . This endpoin t (“sum total normal” fish) could range from 0 (100% deformities, or 0% normal, for each of the four deformity types) to 4 (0% deformities for each of the deformity types). The endpoint “mean total normal” fish was also calculated, which was
“sum total no rmal” fish divided by four, the number of deformity types. EC 10 s for both of these endpoints were mathematically equivalent in the Formation Environmental (2011) report (19.3 mg Se/kg dw; Figure 44

– sum total normal; Figure 45

– mean total normal ).77

Taulb ee et al. (2012) agreed with the reasoning of the U.S. FWS (2012) that “fully normal” fish would have been the most appropriate deformity endpoint

, and that “maximum fully normal”

fish was a more appropriate endpoint for assessing combined deformities than “sum total
normal” fish , based on the information available in the Formation Environmental (2011) report. In addition to analyzing deformities using the “maximum fully normal” endpoint, the Taulbee et al. (2012) also investigated numerous additional calcu lation procedures including “minimum fully normal ” and “intermediate fully normal” fish, both for all deformities as well as for major

deformities.

As discussed previously in the introduction, additional data including counts of fish that were fully free from all deformities were made available in a quality assurance report of the brown trout study dataset (AECOM 2012). This occurred after the release and subsequent peer review of the original reassessment, Taulbee et.al. ( 2012 )

. The availability of “ful ly normal” fish data

addressed a concern raised in the U.S. FWS (2012) review, and eliminated the necessity to evaluate the uncertainty regarding the calculation procedures used to estimate total deformities from individual deformity types. As a result, t he deformity endpoint analyzed in this document was “fully normal” fish, which was evaluated for an optimistic and worst

- case scenario addressing the previously discussed assumptions regarding fish lost to overflow during the 15 day post swim up test. Pre vious documents did not have access to the revised count data made available in the AECOM (2012) report.

Correction of Errors in Previous Documents R elated to the Formation Study. The U.S. FWS (2012) r eview cited numerous discrepancies between “Survival (%) from Hatch until test term,” in Table 3

- 4 of the Formation (2011) Report, and hatch to 15

- days post swim up
survival

, calculated by dividing “total survival” (Table 3

- 4) by “% hatch” (Table 3

- 3). The sources of these discrepancies are a combination of the method Formation Environmental (2011) used to calculate “Survival (%) from Hatch until test term,” described in the section “Calculation of Larval Survival,” as well as the minor accounting errors described above.

In addition to following a calculat ing procedure for hatch to 15

- day post swim up survival that we think underestimated mortality during the 15

- day post swim up test, as described in Taulbee et al. (2012) , a number of minor discrepancies and accounting errors were noted in the original

reas sessment.

The QA review conducted by AECOM (2012) corrected these minor errors, and also corrected counts of eggs hatched and numbers of fish assessed by examining the original source data. All analyses performed in this update of the Taulbee et al. (2012 ) reanalysis used corrected data presented in AECOM (2012).

Methods to Address Issues ( from Taulbee et al

. ( 2012 ) , and this Addendum ) Calculation of Larval Survival ( Hatch through 15 day post swim up test )78

The determination of survival involves two sequential parts of the test, (a) hatch to swim up, and (b) swim up to 15

- days post swim up (PSU). Only a portion of the individuals surviving the first part were tested in the second part. Larval survival was evaluated here as the combined endpoints for both parts of the test, thus covering the duration from hatch to 15

- days post swim

up.

As described in Taulbee et al. (2012) , we calculated survival from hatch to 15

- days PSU as the product of % survival from hatch to swim up and % survival from swim up t o 15

- days PSU. For each sample, the resulting % survival from hatch to 15

- days PSU was then multiplied by the number of fry hatched to calculate the estimated number of fry surviving the full test, assuming that no culling had taken place.

S wim up to 15

- d ays post swim up survival was calculated two different ways, reflecting uncertainty stemming from the loss of some individuals when certain aquaria overflowed due to a clogged drain. One way ignores individuals that were lost, as was done in the Formation Environmental (2011) report, and reflects an optimistic view of the health of those individuals prior to their loss. The other way assumes that all individuals lost to overflow were dead or would have died, and reflects the worst case view of their healt h , consistent with assumptions of the U.S. FWS (2012) peer

- reviewers

. Selenium exposure concentrations, counts of eggs hatched, as well as estimated counts and proportions of fry surviving from hatch to swim up, from swim up through 15

- days PSU, from hatc h through 15

- days PSU are included in Table

AD 1.

Calculation of Larval Deformities In the Formation Environmental (2011) report, larvae were evaluated for four types of deformities (cranio

- facial, skeletal, fin and finfold, edema) from a scale of 0 (no de formity) to three (severe deformity) for each of the deformity types. The scoring of deformities followed the widely adopted deformity scoring criteria described in Holm et al. (2003). For example, a slight bend of less than 45 degrees but greater than t he body width of straight, or a minor body constriction (e.g., a tight rubber band about the body) was given a skeletal deformity score of 1; a score of 2 was assigned to a single bend between 45

- 90 degrees or two bends or constrictions anywhere; and a sco re of 3 was assigned to bends greater than 90 degrees. The scoring criteria used by Formation Environmental (2011) are described in detail for all four deformity types within Appendix C of their report. Deformities were reported separately according to d eformity type, and it was not possible to determine which larval fish were free from all deformities.

The availability of fully normal fry data greatly simplified the analysis of the deformity endpoint in this update, compared to the original reassessment. Counts of both fry assessed for deformities, and fully normal fry made prior to the initiation of the 15

- day PSU test and at the
end of the 15

- day PSU test were summed to reflect counts from hatch to test end. Fully normal79

fry were fry that did not have any deformity, regardless of level of severity. All fry assessed for deformities had reached swim up, with the exception of fry from the five samples with the 5 highest Se exposure concentrations (26.8

- 40.3 mg Se/kg dw) . As with survival, the fully norm al fry endpoint was calculated two different ways, reflecting uncertainty stemming from the loss of some individuals when certain aquaria overflowed due to a clogged drain. In the optimistic scenario, fry lost to the overflow event were ignored, and in th e worst case scenario, fry lost to the overflow event were treated as if they were all deformed, by adding the numbers of fry lost to overflow to the total number of fry assessed for deformities. Selenium exposure concentrations, final counts of normal fry, and final counts of fry assessed for both the optimistic and worst case
scenarios are provided in Table AD 2.

Calculation of Combined Effects of Mortality and Deformity The availability of count data of fully normal fry also greatly simplified the analysis of the combined mortality plus deformity endpoint in this update, compared to the or iginal

reassessment

. The total number of fry evaluated for deformities or survival was calculated by summing the number of live fry assessed for deformities with the number of fry that died at some point during the test. As with the survival and deformit y, total surviving normal fry were
calculated in two different ways, reflecting uncertainty stemming from the loss of some individuals when certain aquaria overflowed due to a clogged drain. In the optimistic scenario, fry lost to the overflow event were ignored, and in the worst case scenario, fry lost to the overflow event were treated as if they were either dead or deformed, by adding the numbers of fry lost to overflow to the total number of fry assessed for mortality and deformities. Selenium

exposure concentrations, and all counts necessary to calculate the combined endpoint for both the optimistic and worst case scenarios are provided in Table AD 3.

A total of six EC 10 s were calculated in this 2014 Addendum . These include an optimistic and a

worst c ase scenario for the survival, deformity, and combined (survival plus deformity) endpoints. The six scenarios are listed in the text box below.80 

### EC 10 

### s Calculated in this Addendum

1. Larval Survival

– Optimistic Case Scenario

Est. # survived hat ch to end “optimistic case” divided by # Eggs hatched (Table

AD1)

2. Larval Survival

– Worst Case Scenario

Est. # survived hatch to end “worst case” divided by # Eggs hatched (Table AD1).

3. Larval Deformities (fully normal)

– Optimistic Case Scenar io

# Normal divided by # Assessed for deformities “optimistic case” (Table AD2)

4. Larval Deformities (fully normal)

– Worst Case Scenario

# Normal divided by # Assessed for deformities “worst case” (Table AD2)

Scenario used for the EC 10 calculation used by EPA in the criteria document.

5. Combined Endpoint (surviving and fully normal)

– Optimistic Case Scenario

# Normal divided by # Live fish assessed + # died during test “optimistic case”

(Table AD3)

6. Combined Endpoint (surviving and fully normal)

– Worst Case Scenario

# Normal divided by # Live fish assessed + # died during test + # lost during post

swim up (Table AD3)

Optimistic case scenarios ignore larval fish lost to overflow during post swim up portion of the test. Worst case scenarios assume all larval fish lost to overflow were
either dead or deformed.81

Table AD 1. Brown trout selenium concentrations and survival data from hatch to test end. Worst case counts assumed that all fish lost to th e overflow event during the post swim up portion of the study would have failed to survive.

Sample ID a whole body

Se, mg/kg dw Egg Se

mg/kg dw # Eggs Hatched Prop.

Survival. Hatch to swim up Prop survival.

Post swim up. “Optimistic Case” Prop survival.

Post swim up. “Worst Case” Prop survival.

Hatch to end. “Optimistic case” Prop survival.

Hatch to end. “Worst case” Est. # survived.

Hatch to end. “Optimistic case” Est. #

survived. Hatch to end. “Worst case”

SC

- 001 3.6 0.76 144 0.951 0.990 0.990 0.942 0.942 136 136

SC

- 002 4.1 0.94 138 0.978 0.990 0.990 0.968 0.968 134 134

SC

- 003 3.7 0.83 340 0.982 0.989 0.890 0.971 0.874 330 297

SC

- 004 4.3 0.92 189 0.868 0.971 0.971 0.842 0.842 159 159

SC

- 005 3 1.2 70 0.914 1.000 0.984 0.914 0.900 64 63

SC

- 006 3.1 1.2 564 0.988 0.990 0.990 0.978 0.978 551 551

SC

- 007 2.7 1 188 0.856 0.970 0.970 0.830 0.830 156 156

SC

- 008 2.5 0.96 396 0.985 1.000 0.900 0.985 0.886 390 351

SPC

- 001 c 0.73 598 0.987 1.000 1.000 0.987 0.987 590 590

SPC

- 002 c 0.73 585 0.966 1.000 1.000 0.966 0.966 565 565

SPC

- 005 c 0.73 589 0.986 1.000 1.000 0.986 0.986 581 581

SPC

- 006 c 0.73 593 0.971 1.000 1.000 0.971 0.971 576 576

CC

- 150

- 009 8.4 12.8 173 0.942 0.990 0.990 0.933 0.933 161 161

CC

- 150

- 011 5.6 8.4 288 0.993 1.000 1.000 0.993 0.993 286 286

CC

- 150

- 012 6.7 8.5 314 0.965 0.990 0.990 0.955 0.955 300 300

CC

- 150

- 013 5.9 8.4 402 0.891 0.973 0.720 0.866 0.641 348 258

CC

- 150

- 015 6 9.1 479 0.971 1.000 1.000 0.971 0.971 465 465

CC

- 150

- 016 7 7.5 89 0.966 1.000 0.500 0.966 0.483 86 43

CC

- 150

- 017 5.6 6.6 223 0.969 1.000 0.670 0.969 0.649 216 145

CC

- 150

- 018 4.7 6.9 522 0.969 1.000 1.000 0.969 0.969 506 506

CC

- 150

- 020 7.2 6.2 584 0.990 1.000 1.000 0.990 0.990 578 578

CC

- 350

- 006 9.2 14 432 0.944 0.980 0.980 0.926 0.926 400 400

CC

- 350

- 007 5.5 6.9 181 0.950 0.988 0.790 0.938 0.751 170 136

CC

- 350

- 008 8.5 9.5 407 0.951 0.986 0.710 0.938 0.675 382 275

LSV2C

- 002 8.9 12.8 584 0.993 1.000 0.840 0.993 0.834 580 487

LSV2C

- 003 13.8 40.3 404 0.000 0.000 0.000 0.000 0.000 0 0

LSV2C

- 004 17.9 36 309 0.000 0.000 0.000 0.000 0.000 0 0

LSV2C

- 005 13.6 26.8 287 0.000 0.000 0.000 0.000 0.000 0 0

LSV2C

- 008 9.6 17.7 263 0.989 0.982 0.540 0.971 0.534 255 14082

Sample ID a whole body

Se, mg/kg dw Egg Se

mg/kg dw # Eggs Hatched Prop.

Survival. Hatch to swim up Prop survival.

Post swim up. “Optimistic Case” Prop survival.

Post swim up. “Worst Case” Prop survival.

Hatch to end. “Optimistic case” Prop survival.

Hatch to end. “Worst case” Est. # survived.

Hatch to end. “Optimistic case” Est. #

survived. Hatch to end. “Worst case”

LSV2C

- 010 22.6 38.8 108 0.000 0.000 0.000 0.000 0.000 0 0

LSV2C

- 012 7.2 13.2 591 0.971 1.000 1.000 0.971 0.971 574 574

LSV2C

- 016 9.2 13.4 570 0.965 1.000 1.000 0.965 0.965 550 550

LSV2C

- 017 13.2 20.5 217 0.885 0.963 0.780 0.852 0.690 185 150

LSV2C

- 019 8.6 12.5 471 0.953 1.000 0.610 0.953 0.582 449 274

LSV2C

- 020 11.3 11.2 357 0.986 1.000 0.640 0.986 0.631 352 225

LSV2C

- 021 20 28.1 424 0.000 0.000 0.000 0.000 0.000 0 0

a SC

– Saratoga National Fish Hatchery; SPC

– Spring Creek Trout Hatchery; CC

– Crow Creek; LSV

– Sage Creek

b Test end was 15 days after swim up.

c Arrived as fertilized, eyed

- eggs. No whole body Se measurement possible.83

Table AD 2. Brown trout selenium concentrations and deformity data from hatch to test end. Worst case counts assumed that all fish lost to the overflow event during the post swim up portion of the study would have been deformed.

Sample ID a whole body

Se, mg/kg dw Egg Se

mg/kg dw # Normal # Assessed for deformities. “Optimistic Case” # Lost to

overflow during post swim up test # Assessed for

deformities plus # lost. “Worst Case”

SC

- 001 3.6 0.76 63 115 115

SC

- 002 4.1 0.94 72 113 113

SC

- 003 3.7 0.83 131 302 9 311

SC

- 004 4.3 0.92 46 140 140

SC

- 005 3 1.2 23 42 42

SC

- 006 3.1 1.2 457 535 535

SC

- 007 2.7 1 93 137 137

SC

- 008 2.5 0.96 283 359 10 369

SPC

- 001 c 0.73 427 570 570

SPC

- 002 c 0.73 371 545 545

SPC

- 005 c 0.73 400 561 561

SPC

- 006 c 0.73 427 556 556

CC

- 150

- 009 8.4 12.8 106 142 142

CC

- 150

- 011 5.6 8.4 87 266 266

CC

- 150

- 012 6.7 8.5 156 282 282

CC

- 150

- 013 5.9 8.4 137 310 26 336

CC

- 150

- 015 6 9.1 210 445 445

CC

- 150

- 016 7 7.5 13 23 43 66

CC

- 150

- 017 5.6 6.6 99 163 33 196

CC

- 150

- 018 4.7 6.9 195 486 486

CC

- 150

- 020 7.2 6.2 453 558 558

CC

- 350

- 006 9.2 14 120 386 386

CC

- 350

- 007 5.5 6.9 68 131 20 151

CC

- 350

- 008 8.5 9.5 269 338 28 366

LSV2C

- 002 8.9 12.8 483 544 16 560

LSV2C

- 003 13.8 40.3 2 100 100

LSV2C

- 004 17.9 36 16 142 142

LSV2C

- 005 13.6 26.8 8 149 149

LSV2C

- 008 9.6 17.7 147 194 45 239

LSV2C

- 010 22.6 38.8 5 80 80

LSV2C

- 012 7.2 13.2 217 554 554

LSV2C

- 016 9.2 13.4 440 530 530

LSV2C

- 017 13.2 20.5 110 150 19 169

LSV2C

- 019 8.6 12.5 267 390 39 429

LSV2C

- 020 11.3 11.2 240 296 36 332

LSV2C

- 021 20 28.1 8 172 172

a SC

– Saratoga National Fish Hatchery; SPC

– Spring Creek Trout Hatchery; CC

– Crow Creek; LSV

– Sage Creek

b Test end was 15 days after swim up.

c Arrived as fertilized, eyed

- eggs. No whole body Se measurement possible.84

Table AD 3. Brown trout selenium concentrations and survival + deformity data (combined endpoint) from hatch to test end.
Worst case counts assumed that all fish lost to the overflow event during the post swim up portion of the study would have failed to survive or have been deformed.

Sample ID a whole body

Se, mg/kg dw Egg Se

mg/kg dw # Normal # Normal

that were dead at

assessment # Nor mal

and alive # Live fish

assessed for deformities # Fish died

during test # Fish lost to

overflow during post swim up test # Live fish

assessed + # died during test. “Optimistic case” # Live fish

assessed + # died during test + # lost during po st swim up. “Worst case”

SC

- 001 3.6 0.76 63 63 115 8 123 123

SC

- 002 4.1 0.94 72 72 113 4 117 117

SC

- 003 3.7 0.83 131 131 302 7 9 309 318

SC

- 004 4.3 0.92 46 46 140 28 168 168

SC

- 005 3 1.2 23 23 42 6 48 48

SC

- 006 3.1 1.2 457 457 535 8 543 543

SC

- 007 2.7 1 93 93 137 30 167 167

SC

- 008 2.5 0.96 283 283 359 6 10 365 375

SPC

- 001 c 0.73 427 427 570 8 578 578

SPC

- 002 c 0.73 371 371 545 20 565 565

SPC

- 005 c 0.73 400 400 561 8 569 569

SPC

- 006 c 0.73 427 427 556 17 573 573

CC

- 150

- 009 8.4 12.8 106 106 142 11 153 153

CC

- 150

- 011 5.6 8.4 87 87 266 2 268 268

CC

- 150

- 012 6.7 8.5 156 156 282 12 294 294

CC

- 150

- 013 5.9 8.4 137 137 310 46 26 356 382

CC

- 150

- 015 6 9.1 210 210 445 14 459 459

CC

- 150

- 016 7 7.5 13 13 23 3 43 26 69

CC

- 150

- 017 5.6 6.6 99 99 163 7 33 170 203

CC

- 150

- 018 4.7 6.9 195 195 486 16 502 502

CC

- 150

- 020 7.2 6.2 453 453 558 6 564 564

CC

- 350

- 006 9.2 14 120 120 386 26 412 412

CC

- 350

- 007 5.5 6.9 68 68 131 10 20 141 161

CC

- 350

- 008 8.5 9.5 269 269 338 21 28 359 387

LSV2C

- 002 8.9 12.8 483 483 544 4 16 548 564

LSV2C

- 003 13.8 40.3 2 2 0 0 395 395 395

LSV2C

- 004 17.9 36 16 16 0 0 289 289 289

LSV2C

- 005 13.6 26.8 8 8 0 0 267 267 26785

Sample ID a whole body

Se, mg/kg dw Egg Se

mg/kg dw # Normal # Normal

that were dead at

assessment # Nor mal

and alive # Live fish

assessed for deformities # Fish died

during test # Fish lost to

overflow during post swim up test # Live fish

assessed + # died during test. “Optimistic case” # Live fish

assessed + # died during test + # lost during po st swim up. “Worst case”

LSV2C

- 008 9.6 17.7 147 147 194 4 45 198 243

LSV2C

- 010 22.6 38.8 5 5 0 0 97 97 97

LSV2C

- 012 7.2 13.2 217 217 554 17 571 571

LSV2C

- 016 9.2 13.4 440 440 530 20 550 550

LSV2C

- 017 13.2 20.5 110 110 150 28 19 178 197

LSV2C

- 019 8.6 12.5 267 267 390 22 39 412 451

LSV2C

- 020 11.3 11.2 240 240 296 5 36 301 337

LSV2C

- 021 20 28.1 8 8 0 0 404 404 404

a SC

– Saratoga National Fish Hatchery; SPC

– Spring Creek Trout Hatchery; CC

– Crow Creek; LSV

– Sage Creek

b Test end was 15 days after swim up.

c Arrived as fertilized, eyed

- eggs. No whole body Se measurement possible.86

Data Analysis All analyses were conducted using the U.S. EPA Toxicity Relationship Analysis Program (TRAP), version 1.2 1. Consistent with the recommendations of the U.S

. FWS (2012) r eview, all data were analyzed using the Tolerance Distribution Analysis approach fitted to a Triangular
Distribution following log 10 transformati on of selenium concentrations.

Results Survival When fry lost to overflow during the 15

- day PSU test (full test) are not counted (optimistic case), survival is high (83

- 99%) for fry in hatchery and field collected samples through the 20.5 mg/kg exposure concentration (Table AD 1; Figure AD 1). At exposur e concentrations greater than 20.5 mg/kg there is complete mortality, as none of the fry from those concentrations reached the
swim

- up stage. The resulting EC 10 for the optimistic case full test survival endpoint is 20.40 mg Se/kg dw (Figure AD 1). Because of the steep mortality threshold between the 20.5 mg/kg and 26.8 mg/kg exposure concentrations, confidence intervals could not be calculated. This result is

very similar to, but very slightly lower than, the EC 10 of 20.48 mg Se/kg dw for the optimistic case full test survival endpoint calculated in the original reassessment (Taulbee et al. 2012). The hatch through swim up portion of the test was longer than the post swim up test, as fry on average reached swim up between 67

- 73 days after hatc hing. The exceptions were fry obtained from the Spring Creek Fish Hatchery (SPC), which arrived as eyed embryos and reached swim up after an average of 34 days.

When fry lost to overflow during the full test are assumed to have been dead (worst case),

s urvival is more variable among the field stations where fry reached swim up, ranging from 48.3% (station CC

- 150

- 016

- 7.5 mg Se/kg dw) to 99.3% (station CC

- 150

- 011

– 8.4 mg Se/kg dw) (Table AD 1; Figure AD 2). The resulting EC 10 for the worst case full test survival endpoint is 16.79 mg Se/kg dw, with 95% confidence intervals of 16.27

- 17.33 mg Se/kg dw (Figure

AD 2). This result is very similar to, but very slightly higher than, the EC 10 of 16.76 mg Se/kg dw for the optimistic case full test survival endpoi nt calculated in the original reassessment (Taulbee et al. 2012).

Deformity When fry lost to overflow during the 15

- day PSU test (full test) are not counted (optimistic case),
the EC 10 for fry fully free from deformities is 18.36 mg Se/kg dw, with 95% con fidence intervals
of 16.91

- 19.94 mg Se/kg dw (Figure AD 3). When fry lost to overflow during the 15

- day PSU test (full test) are assumed to have been deformed (worst case), the EC 10 for fry fully free from deformities is 15.91 mg Se/kg dw, with 95% confide nce intervals of 14.77

- 17.13 mg Se/kg dw (Figure AD 4). Compared to survival, proportions of larvae with at least one deformity were more variable throughout the Se concentration range through 20.5 mg Se/kg dw, for both the87

optimistic and the worst

- case sc enarios. Because deformities are calculated differently in the updated reassessment, direct comparisons to the original assessment cannot be made. However,
EC 10 s for the maximum number of fry fully free from deformities endpoint provides the most
similar comparisons, and in the original reassessment, the EC 10 s for the optimistic and worst

case scenarios were 19.73 mg Se/kg dw and 18.41 mg Se/kg dw, respectively (Taulbee et al. 2012).

Figure AD 1. Concentration

- response relationship of brown trout surviv al from hatch through 15

- days post swim up and selenium concentrations in eggs, optimistic case. EC 10 = 20.40 mg Se/kg dw. Confidence intervals could not be calculated.88

Figure AD 2. Concentration

- response relationship of brown trout survival from hatch through 15

- days post swim up and selenium concentrations in eggs, worst case. EC 10 = 16.79 mg Se/kg dw, with 95% confidence intervals of 16.27

- 17.33 mg Se/kg/dw.89

Figure AD 3. Concentration

- response relationship of fully normal brown trout fry from hat ch through 15

- days post swim up and selenium concentrations in eggs, optimistic case. EC 10 = 18.36 mg Se/kg dw, with 95% confidence intervals of 16.91

- 19.94 mg Se/kg/dw.90

Figure AD 4. Concentration

- response relationship of fully normal brown trout fry from hatch through 15

- days post swim up and selenium concentrations in eggs, worst case. EC 10 = 15.91 mg Se/kg dw, with 95% confidence intervals of 14.77

- 17.13 mg Se/kg/dw.91

Combined Effects of Mortality and Deformity When fry lost to overflow during the 15

- day PSU test (full test) are not counted (optimistic case),
the EC 10 for fry that both survived the full test and were fully free from deformities is 21.58 mg Se/kg dw (Figure AD 5). When fry lost to overflow during the 15

- day PSU test (full test) are
assumed to have been dead or deformed (worst case), the EC 10 for fry that both survived the full test and were fully free from deformities is 21.18 mg Se/kg dw (Figure AD 6). Confidence intervals could not be calculated for either endpoint, as proportions of surviving fry free from deformities were highly variable throughout the Se exposure range up to 20.5 mg Se/kg dw, followed by a steep threshold at all Se exposure concentrations greater than 20.5 mg Se/kg dw, where 100% of fry were either dead or deform ed (Table AD 3; Figures AD 5

- AD 6). Because deformities are calculated differently in the updated reassessment, direct comparisons to the original assessment cannot be made. However, EC 10 s for the maximum number of surviving fry fully free from deformities endpoint provides the most similar comparisons, and in the original reassessment, the EC 10 s for the optimistic and worst

- case scenarios were 21.42 mg Se/kg dw and 23.20 mg Se/kg dw, respectively (Taulbee et al. 2012).

Summary The availability of counts of fry fully free from deformities from the AECOM (2012) QA report removed much of the uncertainty surrounding the evaluation of the deformity endpoint for the Simplot brown trout reproductive study. The recommended EC 10 of 15.91 mg Se/kg dw for fry fully f ree from deformities for the worst case scenario provides a conservative measure of deformity rates that accounts for the possibility that fry lost to overflow during the 15

- day PSU portion of the test were lost because they were already deformed, and ther efore less able to swim against the current of the overflowing water. The EC 10 for the deformity endpoint evaluated using the worst case scenario for the full dataset in this update of the reassessment is lower than
and replaces the EC 10 of 16.76 mg Se/kg dw survival endpoint evaluated using the worst case scenario for the full dataset in original reassessment (Taulbee et al. 2012), as deformity has been shown to be the most conservative endpoint when data for full normal fry were made available.
The EC 10 of 15.91 mg Se/kg dw supersedes the EC 10 of 16.76 mg Se/kg dw because of the additional information made available by the AECOM (2012) QA report , and is the chronic value for brown trout recommended in the External Peer Review Draft Aquatic Life Ambient Water Quality Criterion for Selenium

– Freshwater 2014 (U.S. EPA 2014).92

Figure AD 5. Concentration

- response relationship of fully normal bro wn trout fry that also survived from hatch through 15

- days post swim up and selenium concentrations in eggs, optimistic case. EC 10 = 21.58 mg Se/kg dw. Confidence intervals could not be calculated.93

Figure AD 6. Concentration

- response relationship of fu lly normal brown trout fry that also survived from hatch through 15

- days post swim up and selenium concentrations in eggs,
worst case. EC 10 = 21.18 mg Se/kg dw. Confidence intervals could not be calculated.94

2014 Addendum References

AECOM

. 2012 . Data q uality a ssurance r eport, r eproductive s uccess s tudy with b rown t rout ( Salmo trutta ). Final Report to JR Simplot Company and Newfields Company, LLC. December 2012

.

Formation Environmental. 2011. Brown t rout l aboratory r eproduction s tudies c onducted in

s upport of d evelopment of a s ite

- s pecific s elenium c riterion. Appendix D in Technical Support Document: Proposed Site

- Specific Selenium Criterion, Sage and Crow Creeks, Idaho. Prepared for J.R. Simplot Company by Formation Environmental. Revised October 201 1.

Holm, J., V.P. Palace, K. Wautier, R.E. Evans, C.L. Baron, C. Podemski, P. Siwik and G. Sterling. 2003. An assessment of the development and survival of wild rainbow trout ( Oncorhynchus mykiss ) and brook trout ( Salvelinus fontinalis ) exposed to elevated selenium in an area of active coal mining. In : The Big Fish Bang, H.I. Bowman and A.B. Skiftesvik ( E ds. ) Proceedings of the 26 th Annual Larval Fish Conference, Bergen, Norway.

Taulbee, W. K., D. O. McIntyre and C. Delos. 2012. Analysis of the b rown t rout s elenium t oxicity

s tudy p resented by Formation Environmental and r eviewed by U.S. Fish and Wildlife Service. Draft Report to U.S. EPA Health and Ecological Criteria Division

, Contract No. EP

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U.S. EPA (United States Environmental Protection Agency). 2014. External p eer r eview d raft

a quatic l ife a mbient w ater q uality c riterion for s elenium

– f reshwater 2014. U.S. Environmental Protection Agency

, Office of Water

, Washington

, D.C. EPA 822

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U.S. FWS ( U nited S tates Fish and Wildlife Service ) . 2012. Technical r eview: Smoky Canyon

m ine s ite

- s pecific s elenium c riterion r eport. Division of Environmental Quality

, Environmental Contaminants Program. Available online at: http://www.fws.gov/contaminants/pd f/ReviewSmokyCanyonMineSeleniumReport.pdf

.