## 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, DC1 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 fina l 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 Revi ew, 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 201 2 review of 201 0 draft will often be called the Review. This document begins with a summary of the Formation Environmental (2011) Report, followed by the
recommended ten

- percent effect concentrations (EC 10 s) and toxicity endpoints as determined in the Report and as reca lculated 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, an d conclusions of the reassessment of the Report’s brown trout data in light of issues raised in the Review. Finally, a critical
assessment of the Review’s EC 10 calculations is contained in Appendix A.

Brown Trout Reproduction Study Summary

Formation Env ironmental 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 mea sured 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 bro wn 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 a t the time of initial fish collection
was 38.4 μ g/L, while the surface water concentrations in Crow Creek were < 1.5 μ g/L.

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 fertilize d (Formation 2011). The final number of field samples for the reproductive study, defined as the offspring of a female wild

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

- 2C), 32 from the upstream Crow Cree k 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 locati on, 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 contamina tion 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 SP C hatchery and these were split into four sample s 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 fe rtilized 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 sam ple 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 pres erved 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.

E ndpoints 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 recomm ended endpoints, as they produced the best models and were biologically relevant. The
EC 10 s for these endpoints were 17.67 mg Se/kg dw for survival and 19.33 mg Se/kg dw for mean total normal (with respect to deformities) fish. Based on comparisons of mo del fit for alternative EC 10 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 EC 10 as 20.8 mg Se/kg dw.

T he 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 EC 1 0 of 14.9 mg Se/kg dw was calculated for survival and

an EC 10 of 6.55 mg Se/kg dw was calculated for deformities, and the Review concluded that the EC 10 of 6.55 mg Se/kg dw for larval deformity was most appropriate.3 Issues Concerning the Brown Trout Rep roduction Study

A number of experimental and analytical issues associated with the Formation Environmental (2011) brown trout reproduction study ( t he Report) deserve additional attention, as they affected the interpretation of the toxicity data and ultimat ely, 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 Cr ow 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 Deform ity 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 i n 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 n oted 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 al l 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 ov erall deformity rates across the four classes of deformities were analyzed4 differently in the Report, the R eview, and this document. These different analytical methods are further discussed in subsequent sections of this document.

Calculation of Larval S urvival

Larval “hatch to swim up survival” was calculated similarly in both the Formation Environmental (2011) Report and this document. However, the calcul ation 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 R eport, “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 su rvival,” 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 numb er 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 sampl es. 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 , t he FWS (2012) Review
asserted that fish lost to overflow were lost becaus e 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 th e 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, m ore than any other5 factor, the FWS decision to exclude these s amples from their analysis had major consequences in the ir
final modeled EC 10 s.

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 en dpoints, we addressed the assumption that fish lost to overflow were likely dead or deformed by comparing the EC 10 s 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 R eport 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 EC 10 s for both of these endpoints were mathematically equivalent (19.3 mg Se/kg dw; Figure 44

– sum total normal; Figure 45

– mean total normal).

The FWS (2012) Review was critical of the method used by the Report to calculat e defo rmities, 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 R eport. As a result, the Review recalculated deformity rates using the endpoint “maximum fully normal” fish, which was 100% minus the pe rcentage 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 defor mities 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 possib le 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 calcula ting procedure for hatch to 15

- day post s wim 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 swi m 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 i n samples CC

- 150

- 12
and CC

- 150

- 15.

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

- 4 of the Formation (2011) R eport, 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 t o 15

- days post swim up.

As previously d escribed, we calculated values for survival from hatch t o 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 t o 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 uncert ainty 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 healt h.

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 d ata 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 overfl ow, 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 possible 1

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

Calculation of Larval Survival (Hatch t o Swim Up)

Larval survival was separately calculated for the period between hatch and swim up. Because hatch to
swim

- up is a commonly used duration for maternal

- tra nsfer 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
calculati on 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 th e 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 whic h 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 recal culation 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 po st 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 o f 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 ea ch 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 o f 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, t he 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 particula rly, 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 fis h 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 fis h 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, th e number of missing fish

2 For example, if each o f 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 appr oaches 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 o f 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 examine d 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.

F irst, 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 unaccoun ted 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 fo r 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 measurin g 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 asse ssed 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. After10 calculating all six possibl e 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, Surv ival 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 hatch ed 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 no rmal” 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 f or 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

- Wors t Case, Deformed Fish

- Intermediate Case (M2D3) = (H2

- (M2+D3))/H211 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 EC 10 s 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 Environm ental (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 mig ht 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 la boratory (Field and SC) were highly variable, percent hatch of SPC eggs was uniformly high, possibly as a result of differential handling and processing.

3 D escription of fungal infestation and measures taken are described in the Report: ENSR Appendix D
, an appendix within another larger Appendix, also called Appendix D (Formation Environmental 2011)

.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 reca lculation for larval deformity, it will hereafter be referred to as the “Review” subset. The Review contended that all of the egg selenium concentrations of wild

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

- response relationship, but that data from SC hatchery fish should be excluded. The rationale was that when no data are available for fish from an appropriate “background” site (however that is defined), there is the possibility that any point estimate (e.g., EC 10 ) will be overestimated. However, the calculation of an EC 10 does not involve or allow for labeling any sa mple 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 m g/kg, would need to be in the exposure range where selenium induces effects. The problem with this supposition is
that the EC 10 s for other sensitive species (which include other salmonids) fall within a narrow range and
the lowest wild

- caught exposure of 6.2 mg/kg is a factor of 3 below the bottom of that range (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 fer tilized 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 methodolo gical 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 embr yos 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 grou ps.” 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 of fspring 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 conce rns 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 L oss of individuals due to aquaria overflow : During the 15 day post swim up test, 16

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

- caught parents, and 2 of the 8 aquaria containing fish hatched from SC hatchery parents. The Review contended that the lost fish were more likely to have been dead or deformed, and therefore poorer swimmers, and that the Report inappropriately analyzed survival and deformities from these sites in a way that effectively assumed that for a particular aquarium, survival and deformity rates among fish that were los t 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 critic al for defining the EC 10 for other sensitive species (Janz et al. 2010) . Given the configuration of data outside that range, we deem such a data gap to render the study unusable for calculating an EC 10 on which a water quality criterion might hinge.

Never theless, 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 indiv iduals 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 mis sing (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 pe riods 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 ach ieved 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 th e 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 subsets14 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 i ncluded 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 experie nced 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

- mentio ned subset. That is, this set includes all Field, SPC Hatchery, and SC Hatchery fish.

Data Analysis

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

A total of 78 TRAP models were analyzed for the different endpoints and calculations methods. This rather comprehensive selection of models represents a full factor ial 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, Fu ll 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 ca lculation 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 ov erflow = 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 EC 10 s 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 EC 10 s.

Endpoint: Survival to swim

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

• 4 data subsets. Yields 4 EC 10 s.

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

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

• 3 sc enarios for combining deformity types (Optimistic, Worst, and Intermediate Cases).

• 4 data subsets. Would yield 2 x 3 x 4 = 24 EC 10 s, but two of the overflow scenarios are identical for 1 of the 4 data subsets, such that only 21 different EC10s are produce d (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 scenari os for combining deformity types (Optimistic, Worst, and Intermediate Cases).

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

The total number of EC 10 s evaluated is thus 78 for all endpoints, scenarios, and data subsets.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 tha t were not lost, the lack of partial responses presents TRAP with certain problems in estimating an EC 10 (Table 9a; Figure 1).

EC 10 estimates ranged from 14.59 for the “Review” dataset, to 20.47

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

- calculated EC 10 s 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 d ata, and provides little basis for expecting the EC 10 to be below 17.7

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

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

– 17.25 mg Se/kg egg dw (Table 9a; Figure 2). The M2 EC 10 estimate for the “Review” dataset of 14.51 mg Se/kg egg dw was qualitati vely 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 include d the two sample s with the highest selenium concentrations that did not experience complete mortality (17.7 mg Se/kg egg dw at stat ion LSV2C

- 008 and 20.5 mg Se/kg egg dw at sta tion 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 EC 10 estimates for larval survival in the three datasets that did not exclude samples with overflow were higher than the EC 10 estimates for the Overflow Optimistic Case obtained after excluding these samples, even after assum ing that every fish lost to overflow had already
died

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

Larval Survival for First Portion of Test (Hatch to Swim

- up)

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

- up is a commonly used duration for maternal

- transfer reproductive tests with trout, the results
can be of interest for criteria der ivation purposes. For the Field, Field+SPC, and All datasets, the EC 10 s fell into a tight range, 20.64

– 20.72 mg Se/kg, in each case (Table 9b; Figure 3). Compared to their corresponding full test EC 10 s, these EC 10 s were around 1% higher than calculated for the Overflow18 Optimistic Case, and 20

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

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

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

- up to 15 days post swim

- up) appears to have had little influenc e on the survival outcome; (b) Inclusion or exclusion of hatchery fish has little influence on the survival EC 10 ; 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 ef fect in reducing the EC 10 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, EC 10 estimates were lowest for the “Maximum Fully Normal” method (combining deformities by Optimistic Case) for three of the four data subsets, highest for the “Minimum Fully Normal” method (combining deformities Worst Case) for all of the data subsets, and intermediate for the “Intermediate Fully
Normal” method for three of the four data subsets (Table 10; Figures 4

- 6). This result was not expected, since the Minimum Fully Normal calculation method was the most conservative, and the Maximum Fully Normal method was the least conservative. However, because of th e 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 d w) 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

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

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

Error

- message free EC 10 estimates were obtained for all of the data 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 internally19 consistent, because deformities were assessed in live fish that had reached swim up for a ll 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, whi ch 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

o ne of the peer reviewers of the Review and will be addressed in a subsequent section within this analysis.

Larval deformity EC 10 estimates using the Maximum Fully Normal calculation method ranged from 6.55 mg Se/kg egg dw for the Review data subset to betw een 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 EC 10 of 6.55 mg Se/kg egg dw (Figure 4

– upper right ) was highly dependent on the combination of the inclusion of SPC hatchery samples, the exclusion of samples where some fish were lost to overflow, and the calculation method for fully normal fish, including the consequence that deformity rates in those sa mples 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 compara ble 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 ga metes 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 platea u, 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 fro m 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 EC 10 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 EC 10 estimate of deformity than the inclus ion of the SPC hatchery data, as the EC 10 for the Field+SPC data subset, which is the Review data set plus the 10 field samples in which fish 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 ov erflow 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 EC 10 for this data subset (Field+ SPC with missing fish included as abnormal data subset, usin g 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 thes e samples, the EC 10 for deformities in response to selenium is nearly than three times higher than it was estimated to be in the Review as a result of excluding these samples. In all data subsets where no field samples were deleted,

t he EC 10 estimates for deformities where missing fish were assumed to have been deformed were similar using the Maximum Fully Normal calculation method, because the larger number of samples from field stations was sufficient to outweigh the effects of the low deformity rates in the SPC samples.

The calculation procedure for the determination of fully normal fish also influenced the Review EC 10 estimate. The EC 10 for the Review dataset using the more conservative Intermediate method of estimating fully normal fish was 8.64 mg Se /kg egg dw (Table 10; Figure 7; Figure A2

– lower left). As previously described, the intermediate calculation method assumed that fish will 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
EC 10 was higher because of the decrease in intermediate fully normal fish among the SPC hatchery samples. Finally, the EC 10 for Maximum Normal fish in t he 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 e gg 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 (Optimisti c 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

con centration 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, EC 10 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, EC 10 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 def ormed; and the Intermediate Normal, All data subset, which was notably (15.81 vs. 21.32 mg Se/kg egg dw) lower21 when all missing fish were considered deformed (Table 10; Figure 9). Only the “Maximum Fully Normal”
calculation methods resulted in error free EC 10 estimates after missing fish were included as deformed.

Larval Major Deformities

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

- threshold selenium concentration range (6.2

- 20.5 mg Se/kg egg dw), larval major deformities (Levels 2 and 3) were separately analyzed to determine whether they were more likely to
have 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 fro m 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
dis tribution analysis model was problematic for these data (Table 11; Figures 10

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

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

- 16.95 mg Se/kg egg dw), which might be expected given that the Maximum FMD calculation method was less conservative. Counter to what might be expected, for any given dataset the EC 10 estimate for Minimum FMD was greater than the
other two. EC 10 s estimated using the Review data subset were lower than 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 pronounce d 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 cons idered to have had major deformities, EC 10 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 Maxim um FMD calculation method (Table 11; Figure 13

- 15). For any given calculation method, EC 10 estimates were similar across the three data subsets (Table 11) and similar to the EC 10 obtained when excluding missing fish from the analysis. Again, all of the T RAP 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 EC 10 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 somewha t 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 deformi ties (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 than22 estimate larval sensiti vity 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 t he 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 we re 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 th e 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 purpose s 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 t he lack of partial responses that were capable of precisely positioning the downward curve (Table 12; Figures 16

- 21). The EC 10 estimates were fairly close to the highest sample level within the respective data subsets that were not 100% affected by seleni um (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 contai n 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 E C 10 . The 20.5 mg Se/kg egg dw sample represented the highest concentration having response consistent with little or no effect on the combined survival and deformities endpoint. Below this concentration, no matter what assumptions were made about the ove rflow (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 exposur e. 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 discuss ed in the FWS (2012) Review (including its appendix presenting its peer review). A summary of the issues that could affect the use of data to calculate an EC 10 are as follows:

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

- day post swim

- up portion of the test, and are recommending a worst

- case adjustment in the dataset used in calculation of
the EC 10 ; (6) we have examined the spectrum of possibilities for calculating the number of normal fish
from the available counts of deformities. We have found the EC 10 calculations to be relatively insensitive to the how deformities are handled, as long as no Field data are discarded.

In the selection of an effect level considered to be representative of the data, the sample of two of the issues listed above had the gre atest 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 the24 proportion of normal fish as well. Our analysis of the combined effects of larval mortality and deformity
addressed this latte r consideration.

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

- day post swim

- up test. The FWS (2012) Review’s rationale for excluding these samples was based on the uncertainty about whether the lost fish might represent a greater proportion of dead, affected, or abnormal fish. The results of the analyses presented herein showed tha t uncertainty in the health status of the lost fish had comparatively little influence on the EC 10 estimate, but that discarding the data from the affected samples, as done in the Review, introduced so much uncertainty and instability into the EC 10 calcula tion that it rendered the dataset unfit for use in national criteria
derivation.

In the progression to determine a representative EC 10 we treated the five highest selenium samples as having 100% mortality, and retained the ten field and two SC hatchery t reatments 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 a pplication of the data resulted in an EC 10 value of 16.76 mg Se/kg egg dw. The survival EC 10 is not sensitive to the inclusion or exclusion of SC and SPC hatchery fish.

For the endpoints involving deformities, having calculated EC 10 s by 21 ways for all deformities, 21 ways for major deformities, and 24 ways for combined survival and deformities, the weight of evidence indicates that deformities is probably a less sensitive endpoint in this study. Whether evaluated as “maximum fully normal,” “minimum ful ly 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 EC 10 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 th e EC 10 s calculated for survival from hatch to swim up (the first portion of the test) and for combined survival and deformities (in the full test), provided that the dataset is not edited to discard field samples. This final recommended EC 10 of 16.76 mg S e/kg egg is also consistent with egg

- or ovary

- based EC 10 s for other fish species, falling at the lower end of the distribution for seven other species (Janz et al. 2010).25 Table 1 a . Summary of Larval Survival , Full Test

. Number s 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 Survive d Proportion Survived #Fry Survive d
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 27526 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 38727 T able 2

. Summary of Larval Deformities Excluding Missing Fry

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

.

Max. Normal Min. Normal Int. Normal

Station Egg Se (mg Se/kg dw) #Fry Assessed Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm. SC

- 001 0.76 115 0.687 79 0.400 46 0.504 58
SC

- 002 0.94 113 0.664 75 0.496 56 0.557 63
SC

- 003 0.83 302 0.576 174 0.364 110 0.460 139
SC

- 004 0.92 140 0.493 69 0.000 0 0.279 39
SC

- 005 1.2 42 0.595 25 0.524 22 0.553 23
SC

- 006 1.2 535 0.908 486 0.813 435 0.824 441 SC

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

- 008 0.96 359 0.811 291 0.710 255 0.731 263
SPC

- 001 0.73 568 0.863 490 0.680 386 0.711 404
SPC

- 003 0.73 545 0.822 448 0.611 333 0.655 357
SPC

- 005 0.73 561 0.848 476 0.647 363 0.685 384
SPC

- 006 0.73 556 0.854 475 0.678 377 0.710 395
LSV2C

- 002 12.8 544 0.917 499 0.858 467 0.864 470
LSV2C

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

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

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

- 008 17.7 194 0.851 165 0.670 130 0.704 137
LSV2C

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

- 012 13.2 554 0.424 235 0.329 182 0.384 213
LSV2C

- 016 13.4 530 0.915 485 0.766 406 0.784 415 LSV2C

- 017 20.5 150 0.813 122 0.593 89 0.647 97
LSV2C

- 019 12.5 390 0.774 302 0.626 244 0.661 258
LSV2C

- 020 11.2 296 0.868 257 0.780 231 0.793 235
LSV2C

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

- 150

- 009 12.8 142 0.768 109 0.683 97 0.704 100
CC

- 150

- 011 8.4 266 0.429 114 0.229 61 0.343 91
CC

- 150

- 012 8.5 282 0.677 191 0.507 143 0.563 159
CC

- 150

- 013 8.4 310 0.590 183 0.200 62 0.375 116
CC

- 150

- 015 9.1 445 0.519 231 0.404 180 0.461 205
CC

- 150

- 016 7.5 23 0.565 13 0.435 10 0.491 11
CC

- 150

- 017 6.6 163 0.663 108 0.577 94 0.606 99
CC

- 150

- 018 6.9 486 0.593 288 0.311 151 0.427 207
CC

- 150

- 020 6.2 558 0.894 499 0.785 438 0.798 445
CC

- 350

- 006 14 386 0.513 198 0.000 0 0.252 97
CC

- 350

- 007 6.9 131 0.634 83 0.099 13 0.344 45
CC

- 350

- 008 9.5 338 0.840 284 0.666 225 0.700 237

a

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

- facial).28 T able 3

. Summary of Major Larval Deformities Excluding Missing Fry

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

Max. FMD Min. FMD Int. FMD

Station Egg Se (mg Se/kg dw) #Fry Assessed Prop. FMD #Fry FMD Prop. FMD #Fry FMD Prop. FMD #Fry FMD SC

- 001 0.76 115 0.930 107 0.835 96 0.844 97
SC

- 002 0.94 113 0.947 107 0.885 100 0.889 100
SC

- 003 0.83 302 0.699 211 0.685 207 0.689 208
SC

- 004 0.92 140 0.679 95 0.464 65 0.541 76
SC

- 005 1.2 42 1.000 42 1.000 42 1.000 42
SC

- 006 1.2 535 0.974 521 0.936 501 0.938 502
SC

- 007 1 137 0.912 125 0.796 109 0.809 111 SC

- 008 0.96 359 0.942 338 0.886 318 0.890 319
SPC

- 001 0.73 568 0.974 553 0.945 537 0.946 537
SPC

- 003 0.73 545 0.956 521 0.917 500 0.920 501
SPC

- 005 0.73 561 0.970 544 0.943 529 0.944 530
SPC

- 006 0.73 556 0.951 529 0.912 507 0.914 508
LSV2C

- 002 12.8 544 0.987 537 0.985 536 0.985 536
LSV2C

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

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

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

- 008 17.7 194 0.959 186 0.923 179 0.924 179
LSV2C

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

- 012 13.2 554 0.977 541 0.968 536 0.968 536
LSV2C

- 016 13.4 530 0.994 527 0.992 526 0.992 526
LSV2C

- 017 20.5 150 0.920 138 0.853 128 0.860 129
LSV2C

- 019 12.5 390 0.977 381 0.967 377 0.967 377
LSV2C

- 020 11.2 296 0.983 291 0.959 284 0.960 284
LSV2C

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

- 150

- 009 12.8 142 0.965 137 0.908 129 0.911 129
CC

- 150

- 011 8.4 266 0.989 263 0.981 261 0.981 261
CC

- 150

- 012 8.5 282 0.982 277 0.965 272 0.965 272
CC

- 150

- 013 8.4 310 0.690 214 0.623 193 0.644 200
CC

- 150

- 015 9.1 445 0.978 435 0.951 423 0.951 423
CC

- 150

- 016 7.5 23 0.957 22 0.957 22 0.957 22
CC

- 150

- 017 6.6 163 0.988 161 0.982 160 0.982 160
CC

- 150

- 018 6.9 486 0.975 474 0.963 468 0.963 468
CC

- 150

- 020 6.2 558 0.973 543 0.973 543 0.973 543
CC

- 350

- 006 14 386 0.816 315 0.604 233 0.652 252
CC

- 350

- 007 6.9 131 0.802 105 0.458 60 0.551 72
CC

- 350

- 008 9.5 338 0.956 323 0.893 302 0.897 303

a

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

- facial).29 T able 4

. Summary of Larval Deformities Incorporating Missing Fry

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

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

Max. Normal Min. Normal Int. Normal

Station Egg Se (mg Se/kg dw) #Fry Assessed + Missing Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm. Prop. Norm. #Fry Norm.
SC

- 001 0.76 115 0.687 79 0.400 46 0.504 58
SC

- 002 0.94 113 0.664 75 0.496 56 0.557 63
SC

- 003 0.83 311 0.559 174 0.354 110 0.447 139
SC

- 004 0.92 140 0.493 69 0.000 0 0.279 39
SC

- 005 1.2 42 0.595 25 0.524 22 0.553 23
SC

- 006 1.2 535 0.908 486 0.813 435 0.824 441
SC

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

- 008 0.96 369 0.789 291 0.691 255 0.711 263
SPC

- 001 0.73 568 0.863 490 0.680 386 0.711 404
SPC

- 003 0.73 545 0.822 448 0.611 333 0.655 357
SPC

- 005 0.73 561 0.848 476 0.647 363 0.685 384
SPC

- 006 0.73 556 0.854 475 0.678 377 0.710 395
LSV2C

- 002 12.8 560 0.891 499 0.834 467 0.840 470
LSV2C

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

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

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

- 008 17.7 2 39 0. 690 165 0.544 130 0.573 137
LSV2C

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

- 012 13.2 554 0.424 235 0.329 182 0.384 213
LSV2C

- 016 13.4 530 0.915 485 0.766 406 0.784 415
LSV2C

- 017 20.5 169 0.722 122 0.527 89 0.574 97
LSV2C

- 019 12.5 429 0.704 302 0.569 244 0.601 258
LSV2C

- 020 11.2 332 0.774 257 0.696 231 0.707 235
LSV2C

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

- 150

- 009 12.8 142 0.768 109 0.683 97 0.704 100
CC

- 150

- 011 8.4 266 0.429 114 0.229 61 0.343 91
CC

- 150

- 012 8.5 282 0.677 191 0.507 143 0.563 159
CC

- 150

- 013 8.4 336 0.545 183 0.185 62 0.346 116
CC

- 150

- 015 9.1 445 0.519 231 0.404 180 0.461 205
CC

- 150

- 016 7.5 66 0.197 13 0.152 10 0.171 11
CC

- 150

- 017 6.6 196 0.551 108 0.480 94 0.504 99
CC

- 150

- 018 6.9 486 0.593 288 0.311 151 0.427 207
CC

- 150

- 020 6.2 558 0.894 499 0.785 438 0.798 445
CC

- 350

- 006 14 386 0.513 198 0.000 0 0.252 97
CC

- 350

- 007 6.9 151 0.550 83 0.086 13 0.299 45
CC

- 350

- 008 9.5 366 0.776 284 0.615 225 0.646 237

a

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

- facial).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 u p test. All missing fry were assumed to be deformed for purposes of these calculations.

Max. FMD Min. FMD Int. FMD

Station Egg Se (mg Se/kg dw) #Fry Assessed Prop. FMD #Fry FMD Prop. FMD #Fry FMD Prop. FMD #Fry FMD
SC

- 001 0.76 115 0.930 107 0.835 96 0.844 97
SC

- 002 0.94 113 0.947 107 0.885 100 0.889 100
SC

- 003 0.83 311 0.678 211 0.666 207 0.669 208
SC

- 004 0.92 140 0.679 95 0.464 65 0.541 76
SC

- 005 1.2 42 1.000 42 1.000 42 1.000 42
SC

- 006 1.2 535 0.974 521 0.936 501 0.938 502
SC

- 007 1 .0 137 0.912 125 0.796 109 0.809 111
SC

- 008 0.96 369 0.916 338 0.862 318 0.866 319
SPC

- 001 0.73 568 0.974 553 0.945 537 0.946 537
SPC

- 003 0.73 545 0.956 521 0.917 500 0.920 501
SPC

- 005 0.73 561 0.970 544 0.943 529 0.944 530
SPC

- 006 0.73 556 0.951 529 0.912 507 0.914 508
LSV2C

- 002 12.8 560 0.959 537 0.957 536 0.957 536
LSV2C

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

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

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

- 008 17.7 239 0.778 186 0.749 179 0.749 179
LSV2C

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

- 012 13.2 554 0.977 541 0.968 536 0.968 536
LSV2C

- 016 13.4 530 0.994 527 0.992 526 0.992 526
LSV2C

- 017 20.5 169 0.817 138 0.757 128 0.763 129
LSV2C

- 019 12.5 429 0.888 381 0.879 377 0.879 377
LSV2C

- 020 11.2 332 0.877 291 0.855 284 0.856 284
LSV2C

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

- 150

- 009 12.8 142 0.965 137 0.908 129 0.911 129
CC

- 150

- 011 8.4 266 0.989 263 0.981 261 0.981 261
CC

- 150

- 012 8.5 282 0.982 277 0.965 272 0.965 272
CC

- 150

- 013 8.4 336 0.637 214 0.574 193 0.594 200
CC

- 150

- 015 9.1 445 0.978 435 0.951 423 0.951 423
CC

- 150

- 016 7.5 66 0.333 22 0.333 22 0.333 22
CC

- 150

- 017 6.6 196 0.821 161 0.816 160 0.816 160
CC

- 150

- 018 6.9 486 0.975 474 0.963 468 0.963 468
CC

- 150

- 020 6.2 558 0.973 543 0.973 543 0.973 543
CC

- 350

- 006 14 386 0.816 315 0.604 233 0.652 252
CC

- 350

- 007 6.9 151 0.695 105 0.397 60 0.478 72
CC

- 350

- 008 9.5 366 0.883 323 0.825 302 0.829 303

a

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

- facial).31 Table 6 a. Summary of Larval Survival plus Deformity endpoint

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

- D3) from hatch through 15

- day post swim up (M1)

.

Max. Normal Min. Normal Int. Normal

Station Egg Se (mg Se/kg dw) #Eggs Hatched (H1) Prop. M1 D1 #Fry M1 D1 Prop. M1 D2 #Fry M1 D2 Prop. M1 D3 #Fry M1 D3
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 25132 Table 6 b

. 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. M2 D 1 #Fry M2 D1 Prop. M2 D2 #Fry M2 D2 Prop. M2 D3 #Fry M2 D3
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 25133 Tabl e 7 . Larval survival for field treatments where fry reached swim up stage

. Missing fish presumed
lost to overflow during 15

- day post swim up test

. Percent survival during 15

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

Hatch through swim up 15

- day post swim up test

Station Egg Se Day of Swim up Survived Died % Survival Survived Died Missing % Survival Missing Fry
LSV2C

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

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

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

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

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

- 150

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

- 150

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

- 150

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

- 350

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

- 350

- 008 9.5 67 387 19 95.3 71 1 28 98.6

No Missing Fry
LSV2C

- 012 13.2 73 574 16 97

. 3 100 0 100
LSV2C

- 016 13.4 73 550 20 96

. 5 101 0 100
CC

- 150

- 009 12.8 72 163 8 95

. 3 100 1 99.0
CC

- 150

- 011 8.4 67 286 2 99

. 3 101 0 100
CC

- 150

- 012 8.5 72 304 7 97

. 7 97 1 99.0
CC

- 150

- 015 9.1 67 467 3 99

. 4 98 0 100
CC

- 150

- 018 6.9 72 506 17 96

. 7 101 0 100
CC

- 150

- 020 6.2 67 578 6 99

. 0 100 0 100
CC

- 350

- 006 14 72 408 22 94

. 9 99 2 98.0

a

- listed as 46 missing in the Report. However, a maximum of 45 fish could have been missing for this station after accounting for all other fish that had died or been preserved.34 Table 8 . Larval deformities for field treatments where fry reached swim up stage

. Deformed fish are
“minim um deformed fry” defined as number of fry assessed minus maxim um normal fry

. Percent deformed does not include missing fry.

Number of Fry Assessed

Station Egg Se Swim Up 15

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

- 002 12.8 480 64 544 16 45 8.3%
LSV2C

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

- 017 20.5 92 58 150 19 28 18.7%
LSV2C

- 019 12.5 349 41 390 39 88 22.6%
LSV2C

- 020 11.2 252 44 296 36 39 13.2%
CC

- 150

- 013 8.4 258 52 310 26 127 41.0%
CC

- 150

- 016 7.5 0 23 23 43 10 43.5%
CC

- 150

- 017 6.6 116 47 163 33 55 33.7%
CC

- 350

- 007 6.9 72 59 131 20 48 36.6%
CC

- 350

- 008 9.5 287 51 338 28 54 16.0%

No Missing Fry
LSV2C

- 012 13.2 474 80 554 319 57.6%
LSV2C

- 016 13.4 449 81 530 45 8.5%
CC

- 150

- 009 12.8 62 80 142 33 23.2%
CC

- 150

- 011 8.4 185 81 266 152 57.1%
CC

- 150

- 012 8.5 204 77 282 b 91 32.3%
CC

- 150

- 015 9.1 367 78 445 214 48.1%
CC

- 150

- 018 6.9 405 81 486 198 40.7%
CC

- 150

- 020 6.2 478 80 558 59 10.6%
CC

- 350

- 006 14 307 79 386 188 48.7%

a

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

b

– 282 fry assessed for deformities, but total number reported as preserved equals 28 1. Unable to tell from the report when the additional fry was preserved.35 Table 9 a . Results of TRAP Analyses for Larval Survival , Full Test

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

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

– Inadequate partials, Conv

– Maximum ite rations reached without converge nce, CL

– Error estimates cannot be determined , None

– No errors

.

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

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

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure Method 1=Method 2 * Field 20. 72 IP, CL 3 Method 1=Method 2 Review 14.96 14.65 15.29 IP 3 Method 1=Method 2 Field + SPC 20.64 IP, Conv , StLim, CL 3 Method 1=Method 2 * All 20.71 IP , CL 3 * For the Field and the All subsets, TRAP version 1.21 replaced version 1.2 0.36 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 EC 10 . Descriptions of error messages
are in the notes below the table.

Missing fry not i ncluded Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure Maximum Normal Field 20.13 18.74 21.61 None 4 Minimum Normal Field 24.01 IP, StLim, CL 5 Intermediate Normal Field 18.86 17.43 20.40 IP 6 Maximum Normal Review 6.555 5.989 7.174 None 4 Minimum Normal Review 13.50 IP, CL 5 Intermediate Normal Review 8.637 7.998 9.328 None 6 Maximum Normal Field + SPC 19.78 18.50 21.14 None 4 Minimum Normal Field + SPC 23.35 IP, StLim, CL 5 Intermediate Normal Field + SPC 21.20 19.38 23.18 IP, Conv, StLim 6 Maximum Normal All 19.73 18.44 21.10 None 4 Minimum Normal All 23.05 IP, CL 5 Intermediate Normal All 21.32 19.34 23.50 IP, Conv., StLim 6

Missing fry included as abnormal Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure Maximum Normal Field 19.2 2 17.8 3 20. 7 2 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 18.4 7 17.1 9 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 17.1 4 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 ite rations reached without converge nce, 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 EC 10 . Descriptions of error messages are in the notes below the table.

Missing fry not included

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

Missing fry included as abnormal

Calculation Method Data Subset EC 10 LCL UCL Error Messages Figure
Maximum FMD Field 18.01 16.94 19.1 5 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 1 8.65 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 T RAP Analyses for the Combined Effects of Larval Mortality and Deformity

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

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

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

– Inadequate partials, X50

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

– Large standard error for steepness term, CL

– Error estimates cannot be determined, Conv

– Maximum iterations reached without convergence, StLim

– Steepness at 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 T RAP
tolerance distribution for the four data subsets . EC 10 values, confidence limits, and any TRAP error message s are listed in Table 9 a

. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow40 Figure 2. Survival in full test (hatch through post swim up) including missing fry as dead (Method 2: Overflow Worst Case) analyzed wit h TRAP
tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 9a. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Full Test

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow41 Figure 3. Survival only to swim up (first part of test) analyzed with TRAP tolerance distribution for the four data subsets. Optimisti c Case and Worst Case options do not apply. EC 10 values, confidence l imits, and any TRAP error messages are listed in Table 9b. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Hatch to Swim Up

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Hatch to Swim Up

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval Survival, Hatch to Swim Up

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Larval 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 Overflow42 Figure 4

. Deformities as m aximum fully normal fry , excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by

TRAP tolerance distribution for the four data subset s . EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow43 Figure 5

. Deformities as minimum fully normal fry, excluding missing fry (Overflow Optimistic Case, Deformities Worst Case), analyzed b y TRAP
tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow44 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. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 10. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow45 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. EC 10 values, confidence limits, and any TRAP error messages are listed in
Table 10. Review data subset includes no missing fry but is shown here for c omparative purposes. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. 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 Overflow46 Figure 8

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

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. 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 Overflow47 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. EC 10 values, confidence limits, and any TRAP error messages are listed
in Table 10. Review data subset includes no missing fry but is shown here for compa rative purposes. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully Normal Fry (Missing as Deformed)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. 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 Overflow48 Figure 10

. Major deformities as maximum fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformities Optimistic Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 s , confidence limits, and TRAP messages are in Table 1 1

. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow49 Figure 11

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

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow50 Figure 12

. Major deformities as intermediate fully free of major deformities, excluding missing fry (Overflow Optimistic Case, Deformiti es Intermediate Case), analyzed by TRA P tolerance distribution for the four data subsets. EC 10 s, confidence limits, and TRAP messages in Table 11. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. 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 Overflow51 Figure 13

. Major deformities as maximum fully free of major deformities, assuming missing fry were deformed (Overflow Worst Case,
Deformities O ptimistic Case), analyzed by TRAP tolerance distribution for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 11. Review data subset includes no missing fry but is shown here for comparative purposes. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Max. 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 Overflow52 Figure 14

. Major deformities as m inimum 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. EC 10 values, confidence limit s, 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

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Minimum Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Min. 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 Overflow53 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. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 11. Review data subset includes no missing f ry but is shown here for comparative purposes. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully FMD Fry

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. Fully FMD Fry (Missing as Major Deformity)

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Int. 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 Overflow54 Figure 16

. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on maximum fully free from defor mity (Deformity Optimistic Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12

. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow55 Figure 17

. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on minimum fully free from defor mity (Deformity Worst Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in
Table 12. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow56 Figure 18

. Combined effects, on survival excluding missing fry (Method 1: Overflow Optimistic Case) and on intermed iate fully free from deformity (Deformity Intermediate Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 1 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow57 Figure 19

. Combined effects, on survival assuming missing fr y 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. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 1

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow58 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. EC 10 values, confidence limits, and any TRAP err or messages are listed in Table 12. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 2

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow59 Figure 21

. Combined effects, on survival assuming missing fry were dead (Method 2: Overflow Worst Case) and on intermediate fully free f rom deformity (Deformity Intermediate Case), analyzed with TRAP for the four data subsets. EC 10 values, confidence limits, and any TRAP error messages are listed in Table 12. Field

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 Review

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0

Field + SPC

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 All

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Mortality 2 + Deformity 3

0.0 0.2 0.4 0.6 0.8 1.0 SPC Hatchery SC Hatchery Field - No Overflow Field - PSU Overflow60 

### Appendix A
Assessment of EC 10 

### Calculations Performed by the U.S. Fish and Wildlife Service

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

- Specific Selenium Criterion Report”, herein
termed “the Review ”, recommended a survival EC 10 of 14.9 mg Se/kg and a deformity EC 10 of 6.5 5 mg Se/kg for the brown trout maternal

- transfer reproductive study . After making the data corrections described in the main text of this assessment, t he approach used by the Review yields a survival EC 10 of

14 .6 mg Se/kg, while the deformity remains unchanged. This Appendix examines the Review’s EC 10 calculation approach es

.
Previously Janz et al. (2010)

, reporting the findings of the 2009 SETAC expert workshop, had evaluated
the same brown trout reproductive data

. They judged this species to be among the most sensitive
known , with an EC 10 of 17.7 mg Se/kg . T hey also found the EC 10 s (or comparable NOEC or LOEC values)
of the eight species having suitable data to fall within a narrow band, 17

– 24 mg Se/kg. T he Review’s
calculation of an EC 10 of 6.5 5 mg Se/kg for brown trout thus contrasts sharply with the previous understanding of the toxic concentrations of selenium. Because the Review’s brown trout EC 10 is so
distant from the other species EC 10 s , and so di fferent 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 option s shown in the upper left
and bottom right of Figure A2, we have thus calculated 80 different EC 10 s using the TRAP model’s triangular tolerance distribution, the computationa l approach used by the FWS (2012) Review. Tha t analysis revealed that the Review’s editing of the dataset, removing information , was an essential step for obtaining such a low EC 10 . The abridged data set was sparse , yielding EC 10 s 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 achieved 1 , ( a ) hatch to swim

- up ( mean = 28 days 2 ), and ( b ) swim

- up to 15 days post swim

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

- tran s fer 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 sample s and 2 SC hatchery sample s were lost when aquaria overflowed.

The FWS (2012) Review responded to this problem by discarding all data from those sample s, 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 EC 10 in all other sensitive species (Janz et al. 2010)

. In addition, having appropriately assigned zero survival to all sample s 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 EC 10 calculation.

2 Hatch to swim

- up duration from Table 3

- 3 of Formation Envi ronmental (2011).61 due to their failure to swim up (with which we agree 3 ) , 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 EC 10 of 14.9 mg Se/kg, t h e Review then stated
that “the only reasonable information that these data yield is that ” the EC 100 “occurs somewhere
between 14 and 27 ” mg Se/kg. In saying that the EC 100 could be as low as 14 mg Se/kg, the Review disregards the fact that at 2 0 .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 sample s discarded by the Review actually contain relevant information.

The data used in calculating the Review’s deformity EC 10 fall in three regions, as shown in Figure A1. At the low end of selenium exposure are the SPC hatchery fish , obtained because the SC hatchery eg gs 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 reg ion 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 th ese selenium exposures

are low enough to reliably define the response intercept , unless brown trout is far more sensitive than any k nown species . But compared to the eyed embryos obtained from the SPC hatchery, the offspring
of s everal 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 Figu re 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

.

Give n 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 t he Review’s peer review.

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

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

• In a >2X range above the Review’s EC 10 =6.55 mg Se/kg, the data do not show the expected sharp decline in the fract ion 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 (r 2 =0.0 05 ). 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 H atchery samples in the 0.76

– 1.2 mg Se/kg range, as illustrated in the upper l eft 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 . N o evidence of a concentration

response relationship with in 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, Inter mediate 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 norma l 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 sample s that did not experience overflow

. Thus, r elative 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
seleni um concentrations in this range (as shown in the upper left of Figures 7, 8, and 9.)

• The Review’s EC 10 calculation s hinge on omitting the observations in the 17.7 and 20.5 mg Se/kg

exposures , creating an information g ap between 14 .0 and 26.8 mg Se/kg

. In the main text (pages 17

- 1 8) we note the influence that removing those observations had on the survival EC 10 for the first portion of the test (hatch to swim up), which did not experience the over flow event. ( The breakout of deformity counts between the first and second portions of the test ha s not been provided by Formatio n Environmental, so we could not perform a parallel type of analysis of deformities for the first portion of the test. )

• W orking with the data for the full test (hatch to 15 days post swim up) and restoring the sample s in which fish were missing (including the data at 17.7 and 20.5 mg Se/kg ) under the63 worst

- case assumption that all missing fish were deformed, the solution shifts from the shallow slope curve of Figure A 1 (EC 10 of 6.55 mg Se/kg) to the steep er slope curve of Figure A 2 upper right (EC 10 18 .47 mg Se/kg)

.

• To a lesser degree, the Review’s 6.55 mg Se/kg calculation depends on a paradoxical interpretation of the health of fish at exposures ≥26.8 mg Se/kg. If the data points above 2 6.8 mg Se/kg were set to zero to correspond to their failure to swim up, then the slope would be
steeper and the EC 10 would increase to 13.0 mg Se/kg . So large an instability, coupled with the difficulty of defending individuals that fail to swim up as being “ fully normal ”

, could be view ed

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 R eview’s Maximum Normal approach . Ou r 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 EC 10 s. 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 EC 10 .

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

- 6)

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

- fold abo ve the Review’s
EC 10 . Yet the populations of brown trout did not appear to be manifesting effect s commensurate with severe reductions in reproductive success

. This m ight be problematic to explain for an EC 10 of 6.55 mg Se/kg.

The FWS (2012) Review has raised important issues, not previously broug ht to the fore, about the brown trout study. Following up on th at Review, this assessment has examined the issues in detail , calculating
the EC 10 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 u pper left and lower right of Figure A2. Environmentally
conservative worst

- case assumptions about the response in sample s that the Review did not consider indicate that the weight of evidence does not support an EC 10 as low as either the Review’s deformit y EC 10 or its survival EC 10 . Given the issues raised by the Review, we believe that this assessment’s comprehensive examination provides a defensible basis for a brown trout EC 10 that can be used

, together with EC 10 s for other species, as part of the deri vation of a national criterion

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

1

0.5 1 2 4 8 16 32 64Maximum 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 wild

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

- fold range within which all other sensitive species

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

Shallow slope not characteristic of Se.65 Figure A2. Effects of data selection and calculation method on deformity EC 10 . Each figure represents the data subset used for the recalculation of deformities in the FWS Review with one component altered. Upper left

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

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

– Intermediate Fully Normal calculation method used. Lower Right

– Fish that failed to reach swim up treated as 0% Normal. Unclogged Field Treatments + Unclogged SC Hatchery

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 All Field + SPC (Missing as Deformed)

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0

Review - Intermediate Fully Normal

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Intermediate Fully Normal Fry

0.0 0.2 0.4 0.6 0.8 1.0 Review (fail to swim up as 0% normal)

log 10 (mg Se/kg egg dw) 0.0 0.5 1.0 1.5Maximum 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 Overflow66 

### Re ferences

Formation Environmental. 2011. Brown Trout Laboratory Reproduction Studies Conducted in Support of Development of a Site

- Specific Selenium Criterion. Appendix D in Technical Support Document: Proposed Site

- Specific Selenium Criterion, Sage and Crow Creeks, Idaho. Prepared for J.R. Simplot Company by Formation Environmental. Revised October 2011.

Janz, D.M., D.K. DeForest, M.L. Brooks, P.M. Chapman, G. Gilron, D. Hoff, W.A. Hopkins, D.O. McIntyre, C.A. Mebane, J.P. Skorupa, M. Wayland. 2010. S elenium Toxicity to Aquatic Organisms. In P.M. chapman, W.J. Adams, M.L. Brooks, C.G. Delos, S.N. Luoma, W.A. Maher, H.M. Ohlendorf, T.S. Presser, and D.P. Shaw, Eds. (1 st ed., pp. 141

- 231). Pensacola, FL. CRC Press.

Lemly , AD, and JP Skorupa . 2007. Tech nical issues affecting the implementation of US Environmental Protection Agency's proposed fish tissue

- based aquatic criterion for selenium. Integrated Environmental Assessment and Management , 3 (4): 552

– 558.

Skorupa, J.P. 1998. Selenium poisoning of fish and wildlife in nature: Lessons from twelve real world examples. Pp. 315

- 354 in: W.T. Frankenberger and R.A. Engberg (eds.), Environmental Chemistry of
Selenium . Marcel Dekker, New York, New York.

Skorupa, JP. 2005. Hazard s of Selenium for Aquatic Life. http://wvmdtaskforce.com/proceedings/05/pptskorupa.pdf (Accessed 2012

- 03

- 19)

Skorupa, J. 2011. Soil and Water Sciences Winter Seminar. University of California, Riverside. http://envisci.ucr.edu/seminars/regular/skorupa.pdf (Accessed 2012

- 03

- 19)

U.S. Fish and Wildlife Service. 2012. Technical Review: Smoky Canyon Mine Site

- Specific Selenium Criterion Report. Division of E nvironmental Quality. Environmental Contaminants Program. Available
online at: http://www.fws.gov/contaminants/pdf/ReviewSmokyCanyonMineSeleniumReport.pdf