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, Columbus, OH

and

Charles Delos

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

1

The following is a reassessment of the brown trout reproductive study as presented in the 2011 “Brown 

Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site-Specific 

Selenium Criterion” by Formation 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 stream that regularly exceeds the chronic water quality standard for selenium, and

two locations along Crow Creek downstream of the conflux 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.

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 wildcaught brown trout beginning with their fertilized eggs, was 24; 12 from Sage Creek (station LSV-2C), 3 
2

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 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. 
3

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 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 
4

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 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 
5

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., cranial-facial, 

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 overestimation of normal fry. A detailed description 

of the three calculation methods for deformities is provided in the Methods section of this document.
6

Correction of Errors

In addition to following a calculating procedure for hatch to 15-day post swim up survival that we think 

underestimated mortality during the 15-day post swim up test, as described previously, a careful 

accounting of the data (Appendix F) revealed a number of discrepancies between the reported number 

of fish at the 15-day post swim up test initiation and that the number of fish that could possibly have 

been used in the post swim up study (Column “15d PSU target” in Appendix F). In addition, there were 

four unaccounted-for fish in sample SPC-005, and two unaccounted-for fish each in samples CC-150-12 

and CC-150-15. 

The FWS (2012) Review cited numerous discrepancies between “Survival (%) from Hatch until test 

term,” in Table 3-4 of the Formation (2011) 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 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. 
7

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 any of the four 

 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.
8

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.

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 

 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. 
9

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 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 
10

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

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
11

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 wild-caught fish from stations along Crow Creek or Sage Creek. 

SC Hatchery fish samples were generated in a manner similar to the field samples, as gametes were 

stripped from male and female SC hatchery fish and fertilized in the lab. However, SC Hatchery samples 

were somewhat more affected by the fungal infection than the wild-caught fish, they were the first to 

be added to the hatching test chambers.3

 In contrast to SC and wild-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. 

 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).
12

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 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).
13

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.

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 
14

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 above-mentioned 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.

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.
15

• 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. 
16

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. 
17

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, 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 
18

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, 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 
19

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 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, 
20

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 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 
21

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) and similar 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 
22

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 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%.
23

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 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 
24

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,” 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). 
25

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
26

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
27

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 98a 0.000 0 0.000 0 0.000 0

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

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

LSV2C-008 17.7 194 0.851 165 0.670 130 0.704 137

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

LSV2C-012 13.2 554 0.424 235 0.329 182 0.384 213

LSV2C-016 13.4 530 0.915 485 0.766 406 0.784 415

LSV2C-017 20.5 150 0.813 122 0.593 89 0.647 97

LSV2C-019 12.5 390 0.774 302 0.626 244 0.661 258

LSV2C-020 11.2 296 0.868 257 0.780 231 0.793 235

LSV2C-021 28.1 170a 0.153 26 0.000 0 0.006 1

CC-150-009 12.8 142 0.768 109 0.683 97 0.704 100

CC-150-011 8.4 266 0.429 114 0.229 61 0.343 91

CC-150-012 8.5 282 0.677 191 0.507 143 0.563 159

CC-150-013 8.4 310 0.590 183 0.200 62 0.375 116

CC-150-015 9.1 445 0.519 231 0.404 180 0.461 205

CC-150-016 7.5 23 0.565 13 0.435 10 0.491 11

CC-150-017 6.6 163 0.663 108 0.577 94 0.606 99

CC-150-018 6.9 486 0.593 288 0.311 151 0.427 207

CC-150-020 6.2 558 0.894 499 0.785 438 0.798 445

CC-350-006 14 386 0.513 198 0.000 0 0.252 97

CC-350-007 6.9 131 0.634 83 0.099 13 0.344 45

CC-350-008 9.5 338 0.840 284 0.666 225 0.700 237

a – Maximum number of fry assessed for any given deformity (cranio-facial).
28

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 98a 0.031 3 0.000 0 0.000 0

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

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

LSV2C-008 17.7 194 0.959 186 0.923 179 0.924 179

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

LSV2C-012 13.2 554 0.977 541 0.968 536 0.968 536

LSV2C-016 13.4 530 0.994 527 0.992 526 0.992 526

LSV2C-017 20.5 150 0.920 138 0.853 128 0.860 129

LSV2C-019 12.5 390 0.977 381 0.967 377 0.967 377

LSV2C-020 11.2 296 0.983 291 0.959 284 0.960 284

LSV2C-021 28.1 170a 0.353 60 0.000 0 0.200 34

CC-150-009 12.8 142 0.965 137 0.908 129 0.911 129

CC-150-011 8.4 266 0.989 263 0.981 261 0.981 261

CC-150-012 8.5 282 0.982 277 0.965 272 0.965 272

CC-150-013 8.4 310 0.690 214 0.623 193 0.644 200

CC-150-015 9.1 445 0.978 435 0.951 423 0.951 423

CC-150-016 7.5 23 0.957 22 0.957 22 0.957 22

CC-150-017 6.6 163 0.988 161 0.982 160 0.982 160

CC-150-018 6.9 486 0.975 474 0.963 468 0.963 468

CC-150-020 6.2 558 0.973 543 0.973 543 0.973 543

CC-350-006 14 386 0.816 315 0.604 233 0.652 252

CC-350-007 6.9 131 0.802 105 0.458 60 0.551 72

CC-350-008 9.5 338 0.956 323 0.893 302 0.897 303

a – Maximum number of fry assessed for any given deformity (cranio-facial).
29

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 98a 0.000 0 0.000 0 0.000 0

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

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

LSV2C-008 17.7 239 0.690 165 0.544 130 0.573 137

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

LSV2C-012 13.2 554 0.424 235 0.329 182 0.384 213

LSV2C-016 13.4 530 0.915 485 0.766 406 0.784 415

LSV2C-017 20.5 169 0.722 122 0.527 89 0.574 97

LSV2C-019 12.5 429 0.704 302 0.569 244 0.601 258

LSV2C-020 11.2 332 0.774 257 0.696 231 0.707 235

LSV2C-021 28.1 170a 0.153 26 0.000 0 0.006 1

CC-150-009 12.8 142 0.768 109 0.683 97 0.704 100

CC-150-011 8.4 266 0.429 114 0.229 61 0.343 91

CC-150-012 8.5 282 0.677 191 0.507 143 0.563 159

CC-150-013 8.4 336 0.545 183 0.185 62 0.346 116

CC-150-015 9.1 445 0.519 231 0.404 180 0.461 205

CC-150-016 7.5 66 0.197 13 0.152 10 0.171 11

CC-150-017 6.6 196 0.551 108 0.480 94 0.504 99

CC-150-018 6.9 486 0.593 288 0.311 151 0.427 207

CC-150-020 6.2 558 0.894 499 0.785 438 0.798 445

CC-350-006 14 386 0.513 198 0.000 0 0.252 97

CC-350-007 6.9 151 0.550 83 0.086 13 0.299 45

CC-350-008 9.5 366 0.776 284 0.615 225 0.646 237

a – Maximum number of fry assessed for any given deformity (cranio-facial).
30

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 98a 0.031 3 0.000 0 0.000 0

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

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

LSV2C-008 17.7 239 0.778 186 0.749 179 0.749 179

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

LSV2C-012 13.2 554 0.977 541 0.968 536 0.968 536

LSV2C-016 13.4 530 0.994 527 0.992 526 0.992 526

LSV2C-017 20.5 169 0.817 138 0.757 128 0.763 129

LSV2C-019 12.5 429 0.888 381 0.879 377 0.879 377

LSV2C-020 11.2 332 0.877 291 0.855 284 0.856 284

LSV2C-021 28.1 170a 0.353 60 0.000 0 0.200 34

CC-150-009 12.8 142 0.965 137 0.908 129 0.911 129

CC-150-011 8.4 266 0.989 263 0.981 261 0.981 261

CC-150-012 8.5 282 0.982 277 0.965 272 0.965 272

CC-150-013 8.4 336 0.637 214 0.574 193 0.594 200

CC-150-015 9.1 445 0.978 435 0.951 423 0.951 423

CC-150-016 7.5 66 0.333 22 0.333 22 0.333 22

CC-150-017 6.6 196 0.821 161 0.816 160 0.816 160

CC-150-018 6.9 486 0.975 474 0.963 468 0.963 468

CC-150-020 6.2 558 0.973 543 0.973 543 0.973 543

CC-350-006 14 386 0.816 315 0.604 233 0.652 252

CC-350-007 6.9 151 0.695 105 0.397 60 0.478 72

CC-350-008 9.5 366 0.883 323 0.825 302 0.829 303

a – Maximum number of fry assessed for any given deformity (cranio-facial).
31

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
32

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
33

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 45a 98.2

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

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

LSV2C-020 11.2 68 352 5 98.6 64 0 36 100

CC-150-013 8.4 72 358 42 89.5 72 2 26 97.3

CC-150-016 7.5 72 86 3 96.6 43 0 43 100

CC-150-017 6.6 72 216 7 96.9 67 0 33 100

CC-350-007 6.9 67 172 6 96.6 79 1 20 98.8

CC-350-008 9.5 67 387 19 95.3 71 1 28 98.6

No Missing Fry

LSV2C-012 13.2 73 574 16 97.3 100 0 100

LSV2C-016 13.4 73 550 20 96.5 101 0 100

CC-150-009 12.8 72 163 8 95.3 100 1 99.0

CC-150-011 8.4 67 286 2 99.3 101 0 100

CC-150-012 8.5 72 304 7 97.7 97 1 99.0

CC-150-015 9.1 67 467 3 99.4 98 0 100

CC-150-018 6.9 72 506 17 96.7 101 0 100

CC-150-020 6.2 67 578 6 99.0 100 0 100

CC-350-006 14 72 408 22 94.9 99 2 98.0

a - listed as 46 missing in the Report. However, a maximum of 45 fish could have been missing for this station after 

accounting for all other fish that had died or been preserved.
34

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 45a 29 14.9%

LSV2C-017 20.5 92 58 150 19 28 18.7%

LSV2C-019 12.5 349 41 390 39 88 22.6%

LSV2C-020 11.2 252 44 296 36 39 13.2%

CC-150-013 8.4 258 52 310 26 127 41.0%

CC-150-016 7.5 0 23 23 43 10 43.5%

CC-150-017 6.6 116 47 163 33 55 33.7%

CC-350-007 6.9 72 59 131 20 48 36.6%

CC-350-008 9.5 287 51 338 28 54 16.0%

No Missing Fry

LSV2C-012 13.2 474 80 554 319 57.6%

LSV2C-016 13.4 449 81 530 45 8.5%

CC-150-009 12.8 62 80 142 33 23.2%

CC-150-011 8.4 185 81 266 152 57.1%

CC-150-012 8.5 204 77 282b 91 32.3%

CC-150-015 9.1 367 78 445 214 48.1%

CC-150-018 6.9 405 81 486 198 40.7%

CC-150-020 6.2 478 80 558 59 10.6%

CC-350-006 14 307 79 386 188 48.7%

a - listed as 46 missing in the Report. However, a maximum of 45 fish could have been missing for this station after 

accounting for all other fish that had died or been preserved.

b – 282 fry assessed for deformities, but total number reported as preserved equals 281. Unable to tell from the 

report when the additional fry was preserved.
35

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.
36

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.
37

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.
38

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 mortalitydeformity 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.
39

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
40

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
41

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
42

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
43

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
44

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
45

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
46

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
47

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
48

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
49

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
50

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
51

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
52

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
53

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
54

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
55

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
56

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
57

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
58

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
59

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
60

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 a 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 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, 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 achieved1

, (a) hatch to swim-up (mean = 28 days2), 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. 

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, 

 1 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. 2 Hatch to swim-up duration from Table 3-3 of Formation Environmental (2011).
61

due to their failure to swim up (with which we agree3

), the 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.4

 They were held in the lab for only 34 days, as opposed to 67-72 days 

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

experienced by eggs spawned from SC and wild-caught adults had been abated.

In the middle region of the Review dataset are the low-concentration Field samples (from wild-caught

parents), with exposures in the range 6.2 – 14.0 mg Se/kg. Although the Review portrays 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 fish within any 

one sample were afflicted with that sample's most prevalent type of deformity. However, it is more 

likely that nearly all were in fact affected by at least one type of deformity, 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 call into question the 

appropriateness of this solution.

 3 All survival EC10s that we present in the main text equate failure to swim up with mortality, in accord with the 

Review’s recommendation.

4 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.
62

• 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 concentrationresponse 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. 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 appears to be 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 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, creating an information gap between 14.0 and 26.8 mg Se/kg. 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
63

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”, could be viewed 

as a concern.

• 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 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 appears to be 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 might be problematic to 

explain for an EC10 of 6.55 mg Se/kg.

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 – 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.
64

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.

Having eliminated 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.
65

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
66

References

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