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, OH
i

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 Study 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 EC10 Calculations Performed by the U.S. Fish and Wildlife Service

....................................................................................................................................................... 62

References..................................................................................................................................... 69

Re-Analysis of the Brown Trout Selenium Toxicity Study Presented by Formation 

Environmental and Reviewed by U.S. Fish and Wildlife Service, 2014 Revision -

Addendum ................................................................................................................................... 70

Introduction................................................................................................................................... 71

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

Brown Trout Reproduction Study Summary ................................................................................ 72

Reanalysis of Effects 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 ................. 75
ii

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 Addendum) ............................ 77

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

Calculation of Larval Deformities............................................................................................. 78

Calculation of 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 Deformities 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. Results 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 concentrations 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. ...................................................................................... 84
iii

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 distribution for the four 

data subsets. ...................................................................................................................... 45

Figure 6. Deformities as intermediate fully normal fry, excluding missing fry (Overflow 

Optimistic Case, Deformities Intermediate Case), analyzed by TRAP tolerance 

distribution for the four data subsets................................................................................. 46

Figure 7. Deformities 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, Deformities 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 (Overflow Optimistic Case, Deformities Intermediate Case), analyzed by TRAP 

tolerance distribution for the four data subsets. ................................................................ 52

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 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 deformities, assuming missing 

fry were deformed (Overflow Worst Case, Deformities Intermediate Case), analyzed by 

TRAP tolerance distribution for the four data subsets...................................................... 55
iv

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

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. ..................................................... 61

Figure A1. Summary of issues in plot for calculation of the Review’s deformity EC10 of 6.55 mg 

Se/kg. ................................................................................................................................ 67

Figure A2. Effects of data selection and calculation method on deformity EC10......................... 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 selenium concentrations in eggs, optimistic case. .... 89

Figure AD4. Concentration-response relationship of fully normal brown trout fry 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.......................................................................................................................... 93
v

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 necessitated 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 new 

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 study 

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 treatment 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 recalculations 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 counted 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, which 
vi

calculated deformities as 100% minus the average of the four deformity classes (sum fully 

normal), because U.S. FWS believed it would result in an underestimation 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 normal 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.

Uncertainties regarding the health of fry lost to overflow were incorporated into the deformity 

calculations, by calculating the optimistic and worst-case scenarios for each of the three 

deformity scenarios (maximum- , intermediate- , and minimum-fully normal) 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 possible 

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 

recommended 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 fieldcollected samples and hatchery samples from the Spring Creek Hatchery (SPC). This was the 

dataset recommended for the calculation of the 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 prefertilized, eyed embryos; while excluding fish hatched from eggs from the Saratoga National 
vii

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 to 

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 described 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 EC10

for survival under the worst-case scenario using the dataset consisting of all field-collected and 

hatchery samples. This is lower than the recommended EC20 of 21.63 mg Se/kg dw, and EC10 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 EC10 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 EC10 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 EC10 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 where 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 information contained in the remaining 

data for affected doses, and within the range of known, experimentally-derived EC10s for fish 

exposed to selenium through the diet It was noted that the low U.S. FWS-calculated EC10 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 that 

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 EC10 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 notably 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 (survival 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 effects was eliminated. The reanalysis, 
viii

included here as a 2014 Addendum, includes optimistic and worst case EC10 calculations for 

survival, deformities (fully normal), and combined (survival + fully normal) endpoints. Because 

the final recommended EC10 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 EC10 presented in the 2014 Addendum 

is 15.91 mg Se/kg dw, based on the worst-case scenario for the deformity endpoint, which was 

the most conservative EC10 among the six scenarios that were considered, and is lower than the 

16.76 mg Se/kg dw EC10 included in the Taulbee et al. (2012) report and peer-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, contractorled external peer review in 2012, with the exception of minor grammatical corrections. All data, 

analyses and conclusions are identical to the peer reviewed information. 
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 SiteSpecific Selenium Criterion” by Formation Environmental. 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 (EC10s) 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 methods, 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 EC10 calculations is contained in Appendix A. 

Brown Trout Reproduction Study Summary

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) concentrations 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 

the 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 μg/L, while the surface water concentrations 

in Crow Creek were < 1.5 μg/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 

selenium 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 Crow 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 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 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 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 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) Report 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 Toxicity 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 EC10s 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 EC10 calculations in a 2010 U.S. EPA 
4

evaluation of the Report, Formation Environmental reanalyzed survival in TRAP after 

eliminating the three highest exposure concentrations, and presented the resulting EC10 as 20.8 

mg Se/kg dw.

The FWS (2012) Review of an earlier (2010) draft of the Formation 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 EC10 of 14.9 mg Se/kg dw 

was calculated for survival and an EC10 of 6.55 mg Se/kg dw was calculated for deformities, and 

the Review concluded that the EC10 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 in 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 Overgrowth

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 the 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 from 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 in the wild, and conducted their analysis of 
5

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 after 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 deformities 

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 similarly 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 for 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 in 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 the 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 greater 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 clogged 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 deformity 
6

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 EC10s. 

In this document, we explore the consequences of the 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 EC10s of (a) models where 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 calculation 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., cranialfacial, finfold, spinal, edema) were assessed and analyzed separately 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 as “mean total normal” fish, or the average of the %normal fish for the four deformity 

types, and the EC10s 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 Review 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 proportion 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 deformities 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 

endpoints “intermediate normal” and “minimum normal” in order to assess the possible 
7

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 unaccountedfor 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) 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 (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. 

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 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 previously described, we calculated values for survival from hatch to 15-days post swim up 

differently from both the Formation 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 

did 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 swim 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 fish that died during the 
8

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 the 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, where 

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 survive. This represents the most 

conservative estimate of post swim up survival possible1.

Final counts of estimated surviving fish and total fish for both survival approaches are included 

in Table 1a.

Calculation of Larval Survival (Hatch to 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. Furthermore, 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 analyses, 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 field 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 

recalculation 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 assessment 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.

Intermediate 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 deformity, 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 proportions 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 four 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 

at 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 low 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 total 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 repeated 

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 after 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 deformities 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 individuals 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 different 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 fish. 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 first 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 measurement (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 corresponding 

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 fish 
11

for each of the three ways of measuring deformity (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 deformities 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 combinations 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 Optimistic 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 missing 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 measurements)

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 calculating 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))/H1
12

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 larval 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 

EC10s for deformity calculated in the Formation Environmental (2011) 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 overflow 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 using 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 wildcaught 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

Description 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 

the 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 of 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 from 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., 

EC10) will be overestimated. However, the calculation of an EC10 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 induces effects. The problem with this 

supposition is that the EC10s 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 (Janz 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 hatchery 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 and 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 rates 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 fish assessed for deformities were preserved prior to the post 
14

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 

deformities 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 Review 

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).

Loss 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 EC10 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 

EC10 on which a water quality criterion might hinge.

Nevertheless, we believe the ten field samples that lost individuals 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 available 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 without 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 samples 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 between 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 

concentrations 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 samples 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 lower 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 by 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 Field 

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 poor 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 fish to the abovementioned subset. That is, this set includes all Field, SPC Hatchery, and SC Hatchery fish.

Data Analysis

All analyses were conducted using the U.S. EPA Toxicity Relationship Analysis Program 

(TRAP), version 1.20, except for analyses related 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 log10 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 were 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 larval 

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 intervals, and any model error messages are included 

in Tables 9-12. 
17

Summary of Options for which EC10s Were Calculated

EC10s were calculated for (a) different endpoints, (b) different options for handling 

accidental loss of individuals during the second 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 and other losses (Optimistic Case and Worst Case).

 4 data subsets.

Yields 2 x 4 = 8 EC10s.

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

 4 data subsets.

Yields 4 EC10s.

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 EC10s, 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 produced.

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 EC10s.

The total number of EC10s 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 EC10

(Table 9a; Figure 1). EC10 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 EC10s are 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 EC10 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 EC10 estimates ranging from 16.76 – 17.25 mg Se/kg egg dw (Table 9a; 

Figure 2). The M2 EC10 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 

unaccounted 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 included the two samples with the 

highest selenium concentrations that did not experience complete mortality (17.7 mg Se/kg egg 

dw at station 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 overflow or experience mortality was 

14.0 mg Se/kg egg dw (CC-350-006), the EC10 estimates for larval survival in the three datasets 

that did not exclude samples with overflow were higher than the EC10 estimates for the Overflow 

Optimistic Case obtained after excluding these samples, even after assuming 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 EC10 for larval survival likely falls somewhere between 16.76 mg Se/kg egg dw 

(the lowest EC10 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 used 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 EC10s 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 EC10s, these EC10s 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 corresponding 

EC10 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 EC10s calculated for full test with the Review dataset.

The above hatch to swim up survival results may have the following implications: (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 EC10; and (c) The Review’s editing of the dataset to discard the 10 field samples which 

experienced overflow in the later test has a substantial effect in reducing the EC10 in the portion 

of the test unaffected by the overflow. 

Larval Deformities

Overflow Optimistic Case: For the 12 analyses where fish lost to overflow were not considered, 

EC10 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 was 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 were 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 increase 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 

completely 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 EC10. 

Error-message free EC10 estimates were obtained for all of the data subsets 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 not 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 had 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 combined 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 EC10 estimates using 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 with 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 more likely to have been dead and/or deformed. However, this EC10 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 overflow, 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 effects 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 from 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 (beginning 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 where fish were lost to overflow), the EC10 for the Maximum Fully Normal 
21

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 

EC10 estimate of deformity than the inclusion of the SPC hatchery data, as the EC10 for the 

Field+SPC data subset, which is the Review data set plus the 10 field samples in which fish were 

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 were deformed. The EC10 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 right). In other words, if every fish lost to overflow had been 

deformed, which was the argument for excluding these samples, the EC10 for deformities in 

response to selenium is nearly than three times higher than it was estimated to be in the Review 

as a result of excluding these samples. In all data subsets where no field samples were deleted, 

the EC10 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 

EC10 estimate. The EC10 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 occasionally 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 EC10 was higher because of the decrease in 

intermediate fully normal fish among the SPC hatchery samples. Finally, the EC10 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% fully 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 deformity 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 normal 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 at 
22

concentrations above 20.5 mg/kg that deformity effects in response to selenium were clearly 

apparent. 

For the 9 analyses where fish lost to overflow were assumed to have been deformed, EC10

estimates were lowest for the “Maximum Fully Normal” calculation method for the Field and 

Field+SPC data subsets, highest for 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, EC10 estimates were slightly lower when all missing fish were 

considered deformed 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 considered 

deformed (Table 10; Figure 9). Only the “Maximum Fully Normal” calculation methods 

resulted in error free EC10 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 exhibited 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 EC10 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 EC10 estimate for Minimum FMD was greater than the other two. EC10s estimated 

using the Review data subset were lower than for 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, EC10 estimates 

followed 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, EC10 estimates were similar across the three data subsets (Table 11) 
23

and to the EC10 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 associated 

with the absence of partial responses. With the exception of the “Review” data subset, major 

larval deformities nearly always yielded a lower EC10 than did total larval deformities, regardless 

of whether fish lost to overflow were ignored or considered 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 the 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 Deformity

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 endpoint 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 purposes 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 assessments 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 free 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 performed 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 resulted 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 EC10 estimates were 

fairly close to the highest sample level 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 17.7 
24

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 deformities 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 yield a pronounced underestimation of the combined 

survival and deformities EC10. 

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 great 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 Formation 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 EC10 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 (Se 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 in 
25

reported survival rates and have corrected them; (2 and 3) we are recommending an EC10 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 portion of the test, and are recommending 

a worst-case adjustment in the dataset used in calculation of the EC10; (6) we have examined the 

spectrum of possibilities for calculating the number of normal fish from the available counts of 

deformities. We have found the EC10 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 above 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 survive 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 addressed this latter consideration. 

The other issue that had a meaningful effect on the EC10 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 

herein showed that uncertainty in the health status of the lost fish had comparatively little 

influence on the EC10 estimate, but that discarding the data from the affected samples, as done in 

the Review, introduced so much uncertainty and instability into the EC10 calculation that it 

rendered the dataset unfit for use in national criteria derivation. 

In the progression to determine a representative EC10 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 subset). This application of the data resulted in an EC10 value 

of 16.76 mg Se/kg egg dw. The survival EC10 is not sensitive to the inclusion or exclusion of SC 

and SPC hatchery fish. 

For the endpoints involving deformities, having calculated EC10s 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 EC10 of 16.76 mg Se/kg appears to be environmentally conservative. 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 

EC10s 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 EC10 of 16.76 mg Se/kg egg is also consistent 

with egg- or ovary-based EC10s 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 through 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 0.677 275
28

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 0.953 387
29

Table 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 98a0.000 0 0.000 0 0.000 0

LSV2C-004 36 164a0.157 26 0.000 0 0.019 3

LSV2C-005 26.8 138a0.128 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 71a0.000 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 170a0.153 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

Table 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 98a0.031 3 0.000 0 0.000 0

LSV2C-004 36 164a0.421 69 0.000 0 0.262 43

LSV2C-005 26.8 138a0.391 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 71a0.225 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 170a0.353 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

Table 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 98a0.000 0 0.000 0 0.000 0

LSV2C-004 36 164a0.157 26 0.000 0 0.019 3

LSV2C-005 26.8 138a0.128 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 71a0.000 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 170a0.153 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 98a0.031 3 0.000 0 0.000 0

LSV2C-004 36 164a0.421 69 0.000 0 0.262 43

LSV2C-005 26.8 138a0.391 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 71a0.225 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 170a0.353 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 6a. 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 0.663 251
34

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 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 (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 0.617 251
35

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 45a98.2

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 45a29 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 282b91 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 EC10. Descriptions of error messages are in the notes below the table. 

Calculation Method Data Subset EC10 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 EC10. Descriptions of error messages are in the notes below the table. 

Calculation Method Data Subset EC10 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 EC10. Descriptions of error messages are in the notes below the table. 

Missing fry not included

Calculation Method Data Subset EC10 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 EC10 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 EC10. Descriptions of error messages are in the notes below the table. 

Missing fry not included

Calculation 

Method

Data Subset EC10 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 EC10 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 TRAP 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 EC10. 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 EC10 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 maximum 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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 9a.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 9a.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Full Test

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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. EC10 values, confidence limits, and any TRAP error messages are listed in 

Table 9b.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Hatch to Swim Up

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Hatch to Swim Up

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Hatch to Swim Up

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Larval Survival, Hatch to Swim Up

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 10.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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 subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 10.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
46

Figure 6. Deformities as intermediate fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities 

Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 10.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
47

Figure 7. Deformities 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.

EC10 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.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10 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.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
49

Figure 9. Deformities as intermediate fully normal fry, assuming missing fry were deformed (Overflow Worst Case, 

Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

EC10 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.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully Normal Fry (Missing as Deformed)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10s, confidence limits, and TRAP messages are in Table 11.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10s, confidence limits, and TRAP messages are in Table 11.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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 subsets. 

EC10s, confidence limits, and TRAP messages in Table 11.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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 distribution for the four data subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 11. Review data subset includes no missing fry but 

is shown here for comparative purposes.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Max. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
54

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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 11. Review data subset includes no missing fry but 

is shown here for comparative purposes.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Minimum Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Min. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
55

Figure 15. Major deformities as intermediate fully free of major deformities, assuming missing fry were deformed (Overflow 

Worst Case, Deformities Intermediate Case), analyzed by TRAP tolerance distribution for the four data subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 11. Review data subset includes no missing fry but 

is shown here for comparative purposes.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully FMD Fry

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Int. Fully FMD Fry (Missing as Major Deformity)

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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 TRAP for the four data subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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), analyzed with TRAP for the four data subsets.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 1 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
59

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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 1

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
60

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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 2

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
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.

EC10 values, confidence limits, and any TRAP error messages are listed in Table 12.

Field

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

Review

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

Field + SPC

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

All

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Mortality 2 + Deformity 3

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - PSU Overflow
62

Appendix A: Assessment of EC10 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 EC10 of 14.9 mg Se/kg and a 

deformity EC10 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 yields a survival EC10 of 14.6 mg Se/kg, while the deformity remains unchanged. 

This Appendix examines the Review’s EC10 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 EC10 of 17.7 mg Se/kg. They also found the EC10s (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 EC10 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 EC10 is so distant from the other species EC10s, 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 

incorporate 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 EC10s 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 for which some organisms 

were lost to overflow, was an essential step for obtaining such a low EC10. The abridged dataset 

was sparse, yielding EC10s that were unstable and sensitive to small nuances in the assumptions 

underlying the calculations, as will be shown below.

The Formation Environmental (2011) brown trout test involved two parts, which occur after 

fertilization and hatch have been achieved4, (a) hatch to swim-up (mean = 28 days5), and (b) 

swim-up to 15 days post swim-up. Undertaking the last portion 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 individuals in 10 field samples and 2 SC hatchery samples were lost 

when aquaria overflowed. 

 

4 Hatching success in such fish species is highly variable between parents. Because differences are not reliably 

related to Se exposure, it is customary not to include it in the EC10 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, dying, 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 

EC10 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 

agree6), the Review dataset lacked exposures displaying partial survival, such that the slope of 

the concentration-response curve could not be reliably defined. Recognizing this limitation 

when discussing its survival EC10 of 14.9 mg Se/kg, the Review then stated that “the only 

reasonable information that these data yield is that” the EC100 “occurs somewhere between 14 

and 27” mg Se/kg. In saying that the EC100 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 condition 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 EC10 fall in three regions, as shown in 

Figure A1. At the low end of selenium exposure 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-fertilized 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 wildcaught 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 response 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 than 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 deformity, 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 response 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 defend 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 EC90/EC10 > 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 characteristic EC90/EC10 = 2. But the EC10 of 6.55 mg Se/kg hinges on applying a 

shallow slope.

 In a >2X range above the Review’s EC10=6.55 mg Se/kg, the data do not show the expected 

sharp decline in the fraction normal. Indeed they show no decline whatever. There is no 

correlation between selenium concentration and fraction normal in the range 6.2 – 14 mg 

Se/kg (r2=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 selenium-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 concentrationresponse 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 

Field 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 deformed) 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 lost in the overflow event was insufficient to influence the general pattern 
65

showing no relationship with selenium concentrations in this range (as shown in the upper 

left of Figures 7, 8, and 9.)

 The Review’s EC10 calculations hinge on omitting the observations 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 the survival EC10 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 (EC10 of 6.55 mg Se/kg) to the steeper slope curve of 

Figure A2 upper right (EC10 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 would be 

steeper and the EC10 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 viewed 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 EC10s. 

This seemingly counterintuitive behavior 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 EC10.

 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 EC10 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 EC10. Yet the populations of brown trout did not appear to be 

manifesting effects commensurate with severe reductions in reproductive success. This may 

be problematic to explain if an EC10 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 the issues in 

detail, calculating the EC10 in 80 different ways – 78 ways shown in Tables 9 – 12 and Figures 1 
66

– 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 

EC10 as low as either the Review’s deformity EC10 or its survival EC10. Given the issues raised 

by the Review, we believe that this assessment’s comprehensive examination provides a 

defensible basis for a brown trout EC10 that can be used, together with EC10s for other species, as 

part of the derivation of a national criterion. 
67

Figure A1. Summary of issues in plot for calculation of the Review’s deformity EC10 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 wildcaught 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 EC10.

Each figure represents the data subset used for the recalculation of deformities in the FWS Review with one component 

altered. Upper left – SC hatchery fish vs. SPC hatchery fish. Upper right – excluded field samples 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.

Unclogged Field Treatments + Unclogged SC Hatchery

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

All Field + SPC (Missing as Deformed)

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review - Intermediate Fully Normal

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Intermediate Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

Review (fail to swim up as 0% normal)

log10(mg Se/kg egg dw)

0.0 0.5 1.0 1.5 Maximum Fully Normal Fry

0.0

0.2

0.4

0.6

0.8

1.0

SPC Hatchery

SC Hatchery

Field - No Overflow

Field - Fail to swim up

Field - PSU Overflow
69

References

Formation Environmental. 2011. Brown trout laboratory reproduction studies conducted in 

support of development of a site-specific selenium criterion. 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 2011.

Janz, D.M., D.K. DeForest, M.L. Brooks, P.M. Chapman, G. Gilron, D. Hoff, W.A. Hopkins, 

D.O. McIntyre, C.A. Mebane, J.P. Skorupa and M. Wayland. 2010. Selenium toxicity to aquatic 

organisms. In: P.M. Chapman, W.J. Adams, M.L. Brooks, C.G. Delos, S.N. Luoma, W.A. 

Maher, H.M. Ohlendorf, T.S. Presser and D.P. Shaw (Eds.) (1st Ed., pp. 141-231). CRC Press, 

Pensacola, FL.

Lemly, A.D. and J.P. Skorupa. 2007. Technical issues affecting the implementation of US 

Environmental Protection Agency's proposed fish tissue-based aquatic criterion for selenium.

Integ. Environ. Assess. Manag.3(4): 552-558.

Skorupa, J.P. 1998. Selenium poisoning of fish and wildlife in nature: Lessons from twelve real

world examples. (pp. 315-354). In: W.T. Frankenberger and R.A. Engberg (Eds.), Environmental 

Chemistry of Selenium. Marcel Dekker, New York, NY.

Skorupa, JP. 2005. Hazards of Selenium for Aquatic Life. 

http://wvmdtaskforce.com/proceedings/05/pptskorupa.pdf. (Accessed 2012-03-19).

Skorupa, J. 2011. Soil and water sciences winter seminar. Univ. California, Riverside. 

http://envisci.ucr.edu/seminars/regular/skorupa.pdf. (Accessed 2012-03-19).

USFWS (United States Fish and Wildlife Service). 2012. Technical review: Smoky Canyon 

Mine site-specific selenium criterion report. Division of Environmental Quality. Environmental

Contaminants Program. Available online at: 

http://www.fws.gov/contaminants/pdf/ReviewSmokyCanyonMineSeleniumReport.pdf.
70

Re-Analysis of the Brown Trout Selenium 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, OH
71

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. 2012). 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 major 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-analysis, theupdated deformity data were 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 uncertainties in reported deformity counts. 

The 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 wide range of 

optimistic and worst case scenarios using different data subsets. 

Key Differences between the Original and Updated Reassessment

The most important difference between the original draft reassessment (Taulbee et al. 2012) and 

this Addendum is that the additional information provided in the AECOM (2012) QA report 

removed the uncertainty surrounding total deformity rates. As a result, an EC10 for total normal 

fry could be calculated in this Addendum. This greatly reduced the number of hypothetical 

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 Addendum were only examined using the full (all field-collected samples plus 

all hatchery samples) dataset. This decision was made because the final recommended EC10

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 appropriate to use the full dataset.
72

This addendum begins with a summary of the Formation Environmental (2011) Report, followed 

by the recommended EC10s and toxicity endpoints as determined in the Formation

Environmental (2011) report; and as recalculated in the U.S. FWS (2012) review and the U.S. 

EPA draft reassessment (Taulbee et al. 2012). Next, experimental and analytical issues 

pertaining to the specific analyses presented in this Addendum that affected the

recommendations of the Formation Environmental report, the recommended recalculations of the 

U.S. FWS review, 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 

review.

Brown Trout Reproduction Study Summary

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) concentrations 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 

near 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 the Smoky 

Canyon mine) that regularly exceeds the chronic water quality standard for selenium. In 

addition, two additional locations along Crow Creek upstream of the confluence 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 downstream 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 the 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 the 
73

downstream Crow Creek location (station CC-350), and 9 from the upstream Crow Creek 

location (station CC-150).

Eggs were also collected from 8 ripe females obtained from the Saratoga National Fish Hatchery 

(SC) 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.

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

were placed in egg cups for hatching and monitoring. Larvae were monitored 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/treatment 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 subsequent 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 subsequent 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

Selenium 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 Environmental 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 ranged from 0.76-1.2 mg Se/kg dw at the SC hatchery, and were 
74

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. Selenium 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 hatch 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 analyses is the EC10, 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 EC10s 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 an 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 EC10s (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. concurred that the calculation of 

an EC10 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 calculated 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 this 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 Environmental 

(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 deformity assessments on alevin from the 5 highest 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 agrees with the 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 results 

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 

partial 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 concentrations 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) 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 
76

fish not lost to overflow. On the other hand, the U.S. FWS (2012) review asserted that fish lost 

to overflow were lost because they were most likely either dead or deformed, and 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 review 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, we addressed the assumption that fish lost to overflow 

were likely dead or deformed by comparing the EC10s 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 scenario, 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 excluded the treatment levels from field samples with missing fish 

from any further assessment.

Calculation of Deformity Endpoints

The U.S. FWS (2012) review 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 review 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 deformity classes, but it still underestimates deformities for all fish exhibiting more 

than one deformity. 

In the Formation Environmental (2011) report, each of the four deformity types (i.e., cranialfacial, finfold, spinal, edema) were assessed and analyzed separately 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 endpoint (“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 normal” fish divided by four, the number of deformity types. EC10s 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

Taulbee 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 calculation 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 “fully 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, the 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. Previous documents did not have access to the revised count data made 

available in the AECOM (2012) report.

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

The U.S. FWS (2012) review 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 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 in Taulbee 

et al. (2012), a number of minor discrepancies and accounting errors were noted in the original 

reassessment.

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 to 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.

Swim up to 15-days 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 health, 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 hatch through 15-days PSU are included in Table 

AD1.

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 deformity) 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 the 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 score 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 deformity 

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 normal 
79

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 normal 

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 the 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 AD2.

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 original 

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 deformity, 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 AD3. 

A total of six EC10s were calculated in this 2014 Addendum. These include an optimistic and a 

worst case scenario for the survival, deformity, and combined (survival plus deformity) 

endpoints. The six scenarios are listed in the text box below.
80

EC10s Calculated in this Addendum

1. Larval Survival – Optimistic Case Scenario 

 Est. # survived hatch 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 Scenario

 # 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 EC10 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 AD1. Brown trout selenium concentrations and survival 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 failed to 

survive.

Sample IDa

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-001c0.73 598 0.987 1.000 1.000 0.987 0.987 590 590

SPC-002c0.73 585 0.966 1.000 1.000 0.966 0.966 565 565

SPC-005c0.73 589 0.986 1.000 1.000 0.986 0.986 581 581

SPC-006c0.73 593 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 0.534 255 140
82

Sample IDa

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 AD2. 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 IDa

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-001c0.73 427 570 570

SPC-002c0.73 371 545 545

SPC-005c0.73 400 561 561

SPC-006c0.73 427 556 556

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 AD3. 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 IDa

whole body

Se, mg/kg 

dw

Egg Se

mg/kg 

dw

# 

Normal

# Normal 

that were 

dead at 

assessment

# Normal 

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 post 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-001c0.73 427 427 570 8 578 578

SPC-002c0.73 371 371 545 20 565 565

SPC-005c0.73 400 400 561 8 569 569

SPC-006c0.73 427 427 556 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 0 267 267 267
85

Sample IDa

whole body

Se, mg/kg 

dw

Egg Se

mg/kg 

dw

# 

Normal

# Normal 

that were 

dead at 

assessment

# Normal 

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 post 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 1.21. Consistent with the recommendations of the U.S.FWS (2012) review, all 

data were analyzed using the Tolerance Distribution Analysis approach fitted to a Triangular 

Distribution following log10 transformation 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 AD1; Figure AD1). At exposure 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 EC10 for the optimistic case full test survival endpoint is 20.40 mg 

Se/kg dw (Figure AD1). 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 EC10 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 hatching. 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), 

survival 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 AD1; Figure AD2). The resulting EC10 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 

AD2). This result is very similar to, but very slightly higher than, the EC10 of 16.76 mg Se/kg 

dw for the optimistic case full test survival endpoint 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 EC10 for fry fully free from deformities is 18.36 mg Se/kg dw, with 95% confidence intervals 

of 16.91-19.94 mg Se/kg dw (Figure AD3). When fry lost to overflow during the 15-day PSU 

test (full test) are assumed to have been deformed (worst case), the EC10 for fry fully free from 

deformities is 15.91 mg Se/kg dw, with 95% confidence intervals of 14.77-17.13 mg Se/kg dw 

(Figure AD4). 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 the 
87

optimistic and the worst-case scenarios. Because deformities are calculated differently in the 

updated reassessment, direct comparisons to the original assessment cannot be made. However, 

EC10s for the maximum number of fry fully free from deformities endpoint provides the most 

similar comparisons, and in the original reassessment, the EC10s for the optimistic and worstcase scenarios were 19.73 mg Se/kg dw and 18.41 mg Se/kg dw, respectively (Taulbee et al. 

2012).

Figure AD1. Concentration-response relationship of brown trout survival from hatch 

through 15-days post swim up and selenium concentrations in eggs, optimistic case.

EC10 = 20.40 mg Se/kg dw. Confidence intervals could not be calculated. 

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Surviving

0.0

0.2

0.4

0.6

0.8

1.0
88

Figure AD2. Concentration-response relationship of brown trout survival from hatch

through 15-days post swim up and selenium concentrations in eggs, worst case.

EC10 = 16.79 mg Se/kg dw, with 95% confidence intervals of 16.27-17.33 mg Se/kg/dw.

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Surviving

0.0

0.2

0.4

0.6

0.8

1.0
89

Figure AD3. Concentration-response relationship of fully normal brown trout fry from 

hatch through 15-days post swim up and selenium concentrations in eggs, optimistic case.

EC10 = 18.36 mg Se/kg dw, with 95% confidence intervals of 16.91-19.94 mg Se/kg/dw.

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Fully Normal

0.0

0.2

0.4

0.6

0.8

1.0
90

Figure AD4. Concentration-response relationship of fully normal brown trout fry from 

hatch through 15-days post swim up and selenium concentrations in eggs, worst case.

EC10 = 15.91 mg Se/kg dw, with 95% confidence intervals of 14.77-17.13 mg Se/kg/dw.

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Fully Normal

0.0

0.2

0.4

0.6

0.8

1.0
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 EC10 for fry that both survived the full test and were fully free from deformities is 21.58 mg 

Se/kg dw (Figure AD5). When fry lost to overflow during the 15-day PSU test (full test) are 

assumed to have been dead or deformed (worst case), the EC10 for fry that both survived the full 

test and were fully free from deformities is 21.18 mg Se/kg dw (Figure AD6). 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 deformed (Table AD3; Figures AD5-AD6). Because 

deformities are calculated differently in the updated reassessment, direct comparisons to the 

original assessment cannot be made. However, EC10s for the maximum number of surviving fry 

fully free from deformities endpoint provides the most similar comparisons, and in the original 

reassessment, the EC10s 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 EC10 of 15.91 mg Se/kg dw for fry 

fully free 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 therefore less able to swim 

against the current of the overflowing water. The EC10 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 EC10 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 EC10 of 15.91 mg Se/kg dw supersedes the EC10 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 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.

EC10 = 21.58 mg Se/kg dw. Confidence intervals could not be calculated. 

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Surviving and Fully Normal

0.0

0.2

0.4

0.6

0.8

1.0
93

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.

EC10 = 21.18 mg Se/kg dw. Confidence intervals could not be calculated. 

log (mg Se/Kg egg dw)

0.0 0.5 1.0 1.5

Prop. Surviving and Fully Normal

0.0

0.2

0.4

0.6

0.8

1.0
94

2014 Addendum References

AECOM. 2012. Data quality assurance report, reproductive success study with brown trout 

(Salmo trutta). Final Report to JR Simplot Company and Newfields Company, LLC. December 

2012.

Formation Environmental. 2011. Brown trout laboratory reproduction studies conducted in 

support of development of a site-specific selenium criterion. 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 2011.

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 (Eds.)

Proceedings of the 26th Annual Larval Fish Conference, Bergen, Norway.

Taulbee, W.K., D.O. McIntyre and C. Delos. 2012. Analysis of the brown trout selenium toxicity 

study presented by Formation Environmental and reviewed by U.S. Fish and Wildlife Service. 

Draft Report to U.S. EPA Health and Ecological Criteria Division, Contract No. EP-C-09-001, 

August 22, 2012.

U.S. EPA (United States Environmental Protection Agency). 2014. External peer review draft 

aquatic life ambient water quality criterion for selenium – freshwater 2014. U.S. Environmental 

Protection Agency, Office of Water, Washington, D.C. EPA 822-P-14-001.

U.S. FWS (United States Fish and Wildlife Service). 2012. Technical review: Smoky Canyon 

mine site-specific selenium criterion report. Division of Environmental Quality, Environmental 

Contaminants Program. Available online at: 

http://www.fws.gov/contaminants/pdf/ReviewSmokyCanyonMineSeleniumReport.pdf.