TECHNICAL REVIEW OF THE 2004 U.S. EPA DRAFT SELENIUM CRITERIA DOCUMENT

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2 TECHNICAL REVIEW OF THE 2004 U.S. EPA DRAFT SELENIUM CRITERIA DOCUMENT May 2005 Prepared on behalf of: Colorado Wastewater Utility Council Chadwick Ecological Consultants, Inc.

3 TABLE OF CONTENTS EXECUTIVE SUMMARY 4 BACKGROUND 11 1: TECHNICAL REVIEW OF SELENIUM CRITERION 12 Acute Criteria Review Chronic Criterion Review Corrections and Additions to U.S. EPA Chronic Database 15 Recalculation of the Chronic Tissue Criterion Proposed Recalculation 27 Comparison to other Published Thresholds for Fish Tissues 28 Caveats to use of the Whole-Body Tissue-Based Chronic Criterion 29 Specific questions and information requests by the U.S. EPA 2004b) that are relevant to our technical review of the selenium criterion: : IMPLICATION OF THE DRAFT SELENIUM CRITERIA FOR COLORADO WATERS 36 Summary Statistics for Selenium Concentrations in Colorado Spatial Evaluation of Selenium in Colorado Surface Water 42 Fish Tissue 45 3: POTENTIAL IMPLEMENTATION STRATEGIES FOR THE CHRONIC TISSUE- BASED SELENIUM CRITERION 49 Applicability of the draft criterion to Colorado Acclimation/tolerance Implementing tissue-based criteria CONCLUSIONS AND RECOMMENDATIONS 61 Acute Selenium Criteria Chronic Selenium Criteria Caveats to use of the Whole-Body Tissue-Based Chronic Criterion Potential Implementation Strategies for the Chronic Tissue-based Criterion Recommendations LITERATURE CITED 69

4 LIST OF TABLES Table 1. Corrections, deletions, and additions to the 2004 chronic selenium database Table 2. Total selenium concentration of muscle tissue for female carp and male catfish Table 3. Revised chronic tissue-based database for selenium, species mean chronic values SMCV), and genus mean chronic values GMCV) Table 4. Selenium summary statistics for samples collected between 2001 and 2004 in Colorado Table 5. Selenium summary statistics as a function of geological formation in Colorado Table 6. Selenium summary statistics by sub-basin and geology within sub-basins for Colorado LIST OF FIGURES Figure 1. Seliniferous geological formations 37 Figure 2. Cumulative probability distribution of dissolved selenium in surface water in Colorado 39 Figure 3. Comparison of sulfate-normalized acute selenate criteria in Colorado to the current acute WQS of 18.4 µg/l 40 Figure 4. Comparison of water selenium to sulfate-normalized acute selenate criteria in Colorado 41 Figure 5. Cumulative probability distribution of selenium in whole body fish for Colorado 42 Figure 6. Surface water selenium 44 Figure 7. Whole body fish selenium concentrations 47 Figure 8. Whole body selenium concentration for the family centrarchidae vs. concentrations for fish in other families 48 Figure 9. Relationship between Bioaccumulation Factors BAFs) derived from field collected fathead minnows vs. mean dissolved selenium from the Sand Creek, Colorado study sites 58 Figure 10. Examples of site-specific water quality standards resulting from various combinations of co-located water and fish selenium 60

5 TECHNICAL REVIEW OF THE 2004 U.S. EPA DRAFT SELENIUM CRITERIA DOCUMENT EXECUTIVE SUMMARY At the request of the Colorado Wastewater Utility Council, Chadwick Ecological Consultants, Inc. CEC) and Parametrix have reviewed the Draft 2004 Aquatic Life Water Quality Criteria for Selenium proposed by the U.S. Environmental Protection Agency U.S. EPA). The Colorado Wastewater Utility Council is a nonprofit corporation with more than 43 members from municipal and special district wastewater treatment entities seeking to promote Clean Water Act goals through compliance with the regulations implementing those goals. The U.S. Environmental Protection Agency U.S. EPA) issued an updated draft of the Aquatic Life Water Quality Criteria for selenium on December 17, 2004 U.S. EPA 2004a), hereafter referred to as the 2004 Draft document. Both acute and chronic selenium criteria from this document are markedly different from the existing U.S. EPA selenium criteria and their updates. This report includes: 1) a detailed technical review of the new tissue-based chronic criterion derived in the 2004 Draft document, 2) the implications of the draft selenium criteria for Colorado waters, and 3) potential implementation strategies for the chronic tissue-based selenium criterion. We also provide specific input to USEPA s requests for information in the Federal Register Notice announcing the 2004 Draft document last December. Acute Selenium Criteria The proposed acute freshwater criteria in the 2004 Draft document are greatly improved from the previous criterion of 20 µg Se/L criterion U.S. EPA 1987), which was not based on empirical toxicity data. In our review, we note additional toxicity data are available, but not used by the U.S. EPA. Inclusion of these new data expands the acute toxicity databases, but does not change the FAVs or subsequent CMCs. We support and recommend the adoption of the 2004 Draft document proposed acute criteria: Total selenite acute Se +4 = 257 µg/l) Total selenate acute Se +6 = e [lnsulfate)] ) ) The proposed acute criteria for selenite and selenate are scientifically more defensible than the existing acute criterion of 20 µg/l, as the proposed criteria are based on empirical acute toxicity data and better reflect the acute toxicity of selenium to freshwater organisms. 4

6 Chronic Selenium Criteria A whole-body tissue-based chronic selenium criterion, such as that proposed by the U.S. EPA, should be the most direct approach to quantify the chronic toxicity of selenium. Selenium is a bioaccumulative contaminant with a dietary exposure route for chronic toxicity. Given that water column selenium is not an accurate measurement of chronic selenium toxicity and bioaccumulation factors BAFs) vary by site, a tissue-based chronic criterion is a reasonable alternative, and a whole body criterion is the most cost effective approach. Following our review of the chronic criterion derivation, we corrected selected data points in the chronic database of the 2004 Draft document and added additional data deemed suitable for inclusion from our review of the literature. This revised database consists of one invertebrate rotifer), 11 fish species from nine genera, and one general family chronic toxicity value Centrarchidae). The U.S. EPA did not use any of their standard approaches for derivation of the chronic freshwater criterion, instead defaulting to the single value from Lemly 1993a) of 7.91 µg/g. While this study is important to the understanding of chronic selenium toxicity to fish, lowering the nation-wide criterion to values derived from this specific study is not appropriate for several reasons. 1. First, and probably most importantly, the experimental design did not follow preferred standard procedures of testing multiple selenium concentrations over multiple temperature regimes to document both the selenium dose response and temperature stress response. 2. Second, this study has not been replicated. National criteria should preferably be derived from multiple, reproducible studies that clearly show differences in whole body selenium concentrations between the fish that were affected and the fish that were not affected. 3. Third, use of the Lemly 1993a) value as the proposed chronic criterion does not follow standard U.S. EPA guidelines for criteria development. However, sufficient data exist for deriving chronic values from all the available chronic fish toxicity data, rather than lowering the final chronic value on the basis of this single study. 4. Furthermore, if winter stress is a significant factor in selenium toxicity, the relationship between selenium toxicity and temperature should be defined and factored in to a final chronic equation, similar to other how water quality parameters have been taken into consideration for many other water quality criteria e.g., sulfate and selenate toxicity). 5

7 Overall, to single out this particular study as the most important and protective value is not scientifically justified, and is not consistent with the rest of the available chronic fish toxicity data. The most appropriate use of the Lemly 1993a) data thus is to simply include the data with other suitable data in the SMCV calculations for Lepomis macrochirus. Data from six families of freshwater animals are present in the chronic selenium tissue database, which eliminates the option of calculating a final chronic value directly since the database does not meet the eight family rule, following U.S. EPA criteria development guidance. Using an acute-chronic ratio is also not appropriate since acute criteria are water column concentrations and the chronic criterion is tissue-based. Therefore, the most appropriate method for chronic criterion calculation is calculation of a 5 th percentile of SMCVs 10.6 µg/g) or use of the lowest SMCV from the final chronic database 10.2 µg/g for the flannelmouth sucker, C. latipinnis). We generally support the 2004 Draft document proposed chronic whole body fish tissue criterion approach. However, we would recommend the 5 th percentile or the lowest SMCV as a chronic effect value: 5 th percentile chronic value = 10.6 µg/g dw wb Lowest SMCV = 10.2 µg/g dw wb These tissue-based values represent a considerable improvement to the existing 5 µg/l water column criterion because they remove much although not all) of the site-specific uncertainties associated with a water-based criterion. Caveats to use of the Whole-Body Tissue-Based Chronic Criterion Although practical and an improvement over a water-based criterion, published whole body selenium concentrations may not yet provide the certainty necessary for a national criterion for a number of reasons. 1. Whole body selenium may not be as ecologically relevant as concentrations measured in reproductive tissue eggs/ovaries), given the considerable variation in effects levels observed for fish with comparable whole body selenium concentrations. 2. Matched whole body selenium and fish population data for Colorado show no measurable impact of elevated tissue levels on fish communities, even with tissue concentrations in excess of the U.S. EPA value of 7.91 µg/g dw wb or our recommended values of µg/g dw wb. 6

8 3. There may not yet be enough scientifically reliable studies for deriving an ecologically relevant and protective national criterion based on whole-body selenium concentrations. Of the 23 chronic values in the database, only five are actually based on measured whole body concentrations and not back-calculated from other tissue concentrations. 4. The accuracy of predicting whole-body concentrations via the individual tissue-to-whole body equations generated by U.S. EPA is unknown. Over 90% of the data points used in regression analyses were for bluegill sunfish. The appropriateness of applying these equations derived primarily from one species to all fish species warm water, cold water, lentic, lotic, etc.) is difficult to support. 5. Whole-body tissue-based criteria will result in the need to collect fish tissue data on a regular basis. Scientific collection permits can be difficult to obtain in watersheds with threatened and endangered species or with stressed populations. Non-destructive muscle biopsy sampling is limited to fish of large size, making the technique of limited use for many streams dominated by smaller, non-game species. In summary, it may still be premature to establish a criterion based on whole-body tissue concentrations. However, the existing chronic criterion of 5 µg/l in the water column is even less scientifically based - so a change is definitely needed. Available data are sufficient to derive a reference tissue-based chronic value or trigger value that will warrant further studies in the watershed. We propose that this would only be used as a monitoring trigger, rather than as default national criterion, because additional data e.g. studies testing multiple concentrations with measured effects and matching whole body selenium) are necessary to derive an ecologically relevant nationwide criterion. Potential Implementation Strategies for the Chronic Tissue-based Criterion Applicability of the draft criterion to Colorado: A significant number over 50%) of whole body selenium concentrations in the available Colorado fish tissue samples exceed the draft criterion of 7.9 µg/g. Like many regions in the western U.S., significant deposits of selenium-rich surface materials naturally elevate selenium concentrations in aquatic ecosystems. However, there is no empirical field evidence that selenium is impacting fish in the Colorado River basin and results from laboratory studies evaluating selenium effects on razorback suckers are conflicting. There are still a number of site-specific issues that need consideration. 7

9 Acclimation/tolerance: Fish tissue selenium concentrations in Colorado often exceed the draft or recalculated chronic AWQC, but it is difficult to confirm the presence of reproductive impacts in the field. Natural fish populations in Colorado may have acclimated or are otherwise more tolerant of elevated selenium concentrations than those used to derive the criterion. While there are no studies that explicitly evaluated the ability for aquatic biota to acclimate to selenium, there are studies that provide circumstantial evidence of acclimation or an inherent tolerance to selenium. Relevance of Winter Stress to Colorado Streams: Factoring the winter stress syndrome into a national, state, or site-specific criterion for streams is not appropriate for three reasons. 1. First, the only documented study that specifically addresses winter stress syndrome is the previously discussed Lemly 1993a) study conducted with the bluegill sunfish a species not native to Colorado. 2. Second, regardless of the species present, a defined relationship between temperature and selenium toxicity needs to be established before it can be properly incorporated into a national criterion. 3. Third, the temperature regime tested warm 20 C) vs. cold 4 C) waters, and thus does not accurately mimic natural diurnal and seasonal temperature fluctuation of Colorado streams. Implementation Guidelines: To implement a fish tissue criterion, a guidance document must be written, peer reviewed, and published to standardize the time of collection and type of samples collected and that the fish species sampled are appropriate, analyzed properly, and consistent between studies. This document should be published prior to or in accordance with the tissue criteria document to ensure states will apply the national tissue criterion if accepted) as intended by the U.S. EPA. Proposed fish tissue sampling protocols are provided in this review based on existing U.S. EPA guidance. Site Specific Criteria: Site-specific criteria will likely be an integral component for the implementation of a tissue based chronic selenium criterion due to stream-to-stream, and potentially site-to-site differences in BAFs, resident fish populations, and geology. These relationships may also reveal a more appropriate media than fish tissues for purposes of compliance with the criterion. A performance-based evaluation would specifically utilize present conditions of resident fish populations in comparison to selenium concentrations in tissues and relevant media. 8

10 Linking the Tissue-Based Criterion to a Water-Based Criterion: Measuring selenium in colocated fish and water samples allows one to calculate a site-specific BAF. However, this does not provide a mechanism for deriving the water selenium concentration that would result in a chronic fish selenium criterion. BAFs decrease as the water concentration increases. Further, the slope of the relationship between BAF and exposure concentration for selenium varies depending on site-specific factors. Two basic approaches for estimating the water selenium concentration that would result in the same level of protection afforded by a fish tissue-based selenium criterion are 1) a mechanistic model that would account for site-specific physico-chemical characteristics, such as ph and redox potential, or 2) an empirical model based on co-located fish and water selenium from several regionally related sites. Coupled with a statistical approach, this empirical approach allows one to estimate the water selenium concentration resulting in the fish selenium criterion with a desired level of confidence. Final Recommendations Based on our technical review of the 2004 U.S. EPA draft selenium criteria document, we have the following recommendations: 1. Adopt the acute criteria proposed by U.S. EPA: Total selenite acute Se +4 = 257 µg/l) Total selenate acute Se +6 = e [lnsulfate)] ) ) 2. Adopt the whole body fish tissue chronic criterion approach proposed by U.S. EPA. 3. Remove current chronic criterion of 5 µg/l water column selenium as being inappropriate for this bioaccumulative contaminant. 4. Adopt an updated whole body fish tissue chronic value of µg/g dw wb, following revision of the toxicity database, as a monitoring trigger or screening value rather than a criterion, given the technical uncertainties underlying the value. In other words, the chronic value would be used to screen a waterbody to see if selenium is potentially a problem and warrants further study; but, it would not be directly promulgated as a chronic water quality standard. Exceedence of this monitoring value would trigger additional studies on selenium concentrations in fish tissues, fish population health, and an analysis of the sources of selenium in the waterbody. 5. Consider solicitation of new studies to evaluate the question of whether acclimation or adaptation of natural populations to elevated background selenium concentrations might warrant alternative criteria or approaches for modifying criteria for regions like Colorado. 9

11 The goal would be to derive an alternative chronic criterion that is equally protective of aquatic life, but not be overly conservative in regions where it may be difficult or prohibitively expensive to comply with default national criteria. This would also benefit smaller dischargers that may not have the resources for conducting site-specific studies of the type and detail needed to modify a chronic criterion. 10

12 TECHNICAL REVIEW OF THE 2004 U.S. EPA DRAFT SELENIUM CRITERIA DOCUMENT BACKGROUND At the request of the Colorado Wastewater Utility Council, Chadwick Ecological Consultants, Inc. CEC) and Parametrix have reviewed the Draft Aquatic Life Water Quality Criteria for Selenium U.S. EPA 2004a), evaluated the implications of the proposed selenium criteria to Colorado watersheds, and derived potential implementation strategies for the new chronic tissuebased selenium criterion. The Colorado Wastewater Utility Council is a nonprofit corporation with more than 43 members from municipal and special district wastewater treatment entities seeking to promote Clean Water Act goals through compliance with the regulations implementing those goals. Selenium Se) is an essential micronutrient required by most aquatic and terrestrial species in order to maintain metabolic function U.S. EPA 2004a). It occurs in virtually all environmental media at least at trace concentrations, including rocks, soils, water, and living organisms. Anthropogenic activities of irrigating seleniferous soils, coal and phosphorous mining, coal-fired power plants, and oil refining have increased selenium beyond background concentrations of many aquatic ecosystems Lemly 1997). Given that selenium is an essential micronutrient, aquatic organisms readily assimilate organic forms of selenium e.g., selenomethionine), yet frequently are not able to excrete selenium at the same rate of consumption at elevated concentrations. This imbalance of intake and excretion can lead to elevated tissue concentrations that can be toxic to the organism. Direct toxic effects have been measured in adults via decreased survival or growth and in young by decreased survival reproductive success), growth, or increased occurrences of larval deformities. Additionally, the margin between required concentrations and those that may become toxic is narrow; perhaps as low as one order of magnitude for some vertebrate species, and highly variable within and between species making toxic thresholds difficult to define. The U.S. Environmental Protection Agency U.S. EPA) issued an updated draft of the Aquatic Life Water Quality Criteria for selenium on December 17, 2004 U.S. EPA 2004a), hereafter referred to as the 2004 Draft document. Both acute and chronic selenium criteria from this document are markedly different from the existing U.S. EPA selenium criteria and their updates. For example, the existing acute value is 20 µg/l water column total selenium. This value was not based on acute toxicity data, but rather was derived applying the acute-to-chronic ratio in reverse from the chronic value of 5 µg/l Canton 1999). The new acute values are derived separately for 11

13 the two common inorganic forms of selenium. The acute selenite selenium IV) value is 515 µg/l, while the acute selenate selenium VI) value is a sulfate-modified equation, where the acute selenium VI = e [lnsulfate)]+3.357). At sulfate concentrations of 100 mg/l this equates to an acute selenate value of 417 µg/l. Clearly, significant changes have been made to acute selenium criteria. The draft chronic selenium criterion also has gone through significant revision. The current EPA criterion is a water column value of 5 µg/l total selenium). However, the 2004 Draft document acknowledges the differential mode of toxicity for chronic selenium, through dietary uptake and bioaccumulation and derives a new chronic standard based on fish tissue concentrations 7.91 µg/g dry wt, with a trigger level of 5.85 µg/g). Mercury is presently the only contaminant with a U.S. EPA approved tissue-based criterion yet that criterion was determined for human health protection). Therefore, the 2004 Draft document proposes the first chronic tissue-based criterion derived to protect aquatic life. Not only is the criterion value significantly different, but data collection and methods used are also entirely different than the existing selenium document. This report summarizes our technical review of the 2004 Draft document, which was conducted in three sections: 1) a technical review of the new tissue-based chronic criterion derived in the 2004 Draft document, 2) the implications of the draft selenium criteria for Colorado waters, and 3) potential implementation strategies for the chronic tissue-based selenium criterion. This review also attempts to address the multiple requests from the U. S. EPA s charge to reviewers U.S. EPA 2004b), when applicable. 1: TECHNICAL REVIEW OF SELENIUM CRITERION Our evaluation of the 2004 Draft document consisted of a thorough investigation of the data used to calculate the updated acute criteria for selenite and selenate, as well as the chronic tissue-based criteria. Data were critically reviewed for toxicological relevance and adherence to U.S. EPA methodology Stephan et al. 1985). Acute Criteria Review The proposed acute freshwater criteria in the 2004 Draft document are greatly improved and generally represent a significant increase from the 1987 criterion of 20 µg Se/L U.S. EPA, 1987). 12

14 Unlike the 20 µg/l criterion, the proposed values were appropriately derived using U.S. EPA s recommended criteria development procedures specified in the 1985 guidance Stephan et al. 1985). One of the substantial improvements to the acute criterion made in the 2004 Draft document is the recognition of the differential toxicity of the two dominant selenium species, selenite and selenate, to aquatic life. The 2004 Draft document proposes separate acute criterion for selenite CMC = 257 µg/l) and selenate CMC = e [lnsulfate)] = 3.357) ), with the selenate criterion corrected with the ambient sulfate concentration - a second major improvement. To derive the proposed criteria, available toxicity data were summarized as Species Mean Acute Values SMAVs), which are the geometric means of available LC 50 s for a given species. The geometric mean of these SMAVs is then estimated to provide a Genus Mean Acute Value GMAV), and the available GMAVs are analyzed to estimate a Final Acute Value FAV). The FAVs are then divided by 2 to extrapolate from an LC 50 to an LC-low) to derive the Criterion Maximum Concentration CMC). In our review of the 2004 Draft document, we noticed missing data that are relevant to consider, yet do not dramatically change the derived acute criterion value. It is essential that criteria are derived from the most up-to-date and comprehensive database, regardless of the significance it has on the final value. Therefore we believe the following study provides acute values that are suitable for use and should be included in the final acute value calculations. Ingersol et al. 1990) conducted a study on the acute and chronic toxicity of selenite as sodium selenite) and selenate as sodium selenate) to D. magna and Chironomus riparius. Acute values from this study for D. magna were included by U.S. EPA in the acute calculations, yet the Chironomus data were ignored. The 2004 Draft document lists the Chironomus values in Table F-1 Other Data on Effects of Selenium on Aquatic Organisms ) without an explanation as to why the values were not used. All tests were 48-hr acute static tests conducted in accordance with the U.S. EPA guidelines Stephan et al. 1985) and appear to be suitable for use. Therefore, we suggest adding the LC 50 s and respective sulfate concentrations for Chironomus listed below to the revised selenite and selenate databases: Sulfate mg/l) Selenite µg/l) Selenate µg/l) 41 7,950 16, ,600 10,500 13

15 Use of these data would change the selenite genus mean acute value GMAV) for Chironomus to 16,715 µg/l and its sensitivity rank would change from 26 th most sensitive to 20 th. When the Ingersoll et al. 1990) data are added to the selenate database, the GMAV for Chironomus would change to 30,969 µg/l and it would rank 11 th most sensitive as adjusted for sulfate). Since these additional data do not affect the sensitivity ranking of the four most sensitive GMAVs for selenite and selenate, the final acute values FAV) and CMCs would not change. Overall, we support the proposed changes to acute selenium criteria, including the derivation of separate criteria for selenite and selenate, as well as incorporating the sulfate correction for the selenate criterion. The proposed values are a considerable improvement to the existing criterion and more accurately represent the acute toxicity of selenium to freshwater organisms. Chronic Criterion Review Exact guidelines for determining data suitability and deriving a tissue-based criterion do not presently exist as they do for water column criteria. However, Stephan et al. 1985) still provides useful guidance on test duration, control survival, and criteria derivation requirements. Data that do not comply with these guidelines should be carefully reviewed prior to inclusion in the database. Some general principles presented in the 1985 Guidelines and used in this analysis, specific to the chronic criterion, include: 1. Life-cycle chronic toxicity tests should begin with embryos, continue through maturation, reproduction, and end not less than 24 days 90 days for salmonids) after hatch. Data should be analyzed on survival and growth of adults and young, maturation of males and females, eggs spawned per female, embryo viability salmonids only), and hatchability. 2. Partial life-cycle and early life-stage chronic tests should begin with immature juveniles and run no less than 24 and days, respectively for fishes, except salmonids, which should run 90 and 60 days, respectively. Data should be analyzed on survival and growth of adults and young, maturation of males and females, eggs spawned per female, embryo viability salmonids only) and hatchability. 3. Data from at least eight families must be available for direct calculation of the acute criteria from the four most sensitive organisms the eight family rule ). These families must include a Salmonid, a second family in the class Osteichthyes, a third family in the phylum Chordata, a planktonic crustacean, a benthic crustacean, an insect, a family in a phylum other than Arthropoda or Chordata, and a family in any order of insect or any phylum not already represented. 14

16 Once again, these guidelines were intended for the derivation of water column criteria. Appropriate, relevant, useable tissue data are generally limited. Therefore, exceptions to many of the standard water exposure toxicity tests guidelines were granted in our analysis to keep useful data in the database. For example, field collected organisms have essentially been used to represent life cycle tests, in which the endpoints measured include reproductive failure or larval deformities. When using field collected organisms, exposure duration and concentration of selenium to adults is unknown and actually not necessary since the bioaccumulative endpoint of adult tissue concentration during reproduction is being used for criteria derivation. Values listed in the 2004 Draft chronic database are expressed as µg/g dry weight dw) whole body wb). To utilize data from studies that report values as concentrations of tissues other than whole body liver, muscle, and ovary) the U.S. EPA developed three quantile regressions to estimate the whole body wb) concentration as a function of the selenium concentration of the selected tissue U.S. EPA 2004a). Equation 1: Selenium wb = exp * ln[se muscle ])) Equation 2: Selenium wb = * [Se ovary ]) Equation 3: Selenium wb = * [Se liver ]) The majority >90%) of data used to derive these equations are based on data for bluegill, yet were used to estimate whole body concentrations for rainbow trout, cutthroat trout, brook trout, fathead minnow, and striped bass. These equations were also used in our review when necessary. Occasionally egg concentration was used in Equation 2 conversion of ovary data to whole body data) when only egg data were available since nearly identical concentrations have been observed between egg and ovary tissue e.g., Coyle et al ). Additionally, to use data from studies that present values in wet weight, moisture content of ovaries/eggs was assumed to be 80%, as did the U.S. EPA in the 2004 Draft document, unless specified otherwise 75.84% for rainbow trout and 85% for fathead minnow). These values were used to convert tissue values from wet weight ww) to dw when moisture content was not reported. Corrections and Additions to U.S. EPA Chronic Database Following standard U.S. EPA methodology, revisions and updates to the existing chronic toxicity database consists of two steps, 1) correcting the existing database, including correcting data that were inappropriately used or reported wrong, as well as deleting data that should not have been in the national database in the first place, and 2) adding any suitable data not presently used to the 15

17 existing database U.S. EPA 1994). Once the database is revised/updated, standard recalculation procedures may proceed following the guidelines outlined by Stephan et al. 1985). Corrections to the Chronic Selenium Fish Tissue Database Corrections to the database include updating data from previously unpublished reports used by U.S. EPA, adding data from new studies not previously available, and removal/deletion of data considered inappropriate following technical review. These changes are noted below. Cutthroat Trout Chronic Values - An updated final report for the Hardy 2002) study used by U.S. EPA, has become available since the release of the 2004 Draft document Hardy 2005). The revised final whole body selenium chronic effects concentrations for cutthroat trout in Hardy 2005) are slightly higher than values reported in Hardy 2002). Final whole body selenium levels at spawning are reported to be 5.87, 9.10, 11.37, and 5.61 µg/g dw in the four highest dietary treatment groups Hardy 2005). No significant effects in growth or reproduction were observed in all treatments. Therefore the chronic value for cutthroat trout from this study is greater than the NOAEC µg/g dw wb). This value should replace the >9.37 µg/g dw reported for this study in the 2004 Draft document Table 1). Brook Trout and Rainbow Trout Chronic Values - Holm 2002) and Holm et al. 2003) present tissue data for the rainbow trout Oncorhynchus mykiss) and brook trout Salvelinus fontinalis). Holm collected gametes from adult rainbow trout from a stream with elevated selenium downstream from active coal mining) and a reference stream in 2000 and Eggs were fertilized in the laboratory and embryos and larvae were reared to the swim-up stage. Fry were evaluated for deformities and other indicators of selenium toxicity, such as edema and finfold. 16

18 Table 1: Corrections, deletions, and additions to the 2004 chronic selenium database. All table values are as µg Se/g dw wb. Species Common Name Existing Chronic Value Updated or Corrected Chronic Value Reference Corrections: Oncorhynchus clarki cutthroat >9.37 >11.37 Hardy 2005; Hardy 2002 Oncorhynchus mykiss rainbow trout Holm 2002; Holm et al Oncorhynchus mykiss rainbow trout 5.79 >16.07* Holm 2002; Holm et al Salvelinus fontinalis brook trout 13.2 >12.24* Holm 2002; Holm et al Salvelinus fontinalis brook trout 12.4 >15.40* Holm 2002; Holm et al Deletions: Pimephales promelas fathead minnow 51.4 Bennett et al Additions: Oncorhynchus mykiss rainbow trout Hilton and Hodson 1983 Xyrauchen texanus razorback sucker >25.37 Hamilton et al. 2002a *see below text for an explanation of the > addition. Data were analyzed for % deformities vs. egg selenium concentration annually and reported separately. The chronic tissue value presented by U.S. EPA for the 2001 rainbow trout data is an EC 20 derived from a clear effect response curve, whereas the value from the 2000 data was derived from the mean tissue concentration of fish sampled from the elevated selenium site. However, both values were originally derived from egg concentrations, back calculated to muscle tissue concentrations using a site-derived regression, and then converted to whole body concentrations using the EPA muscle-to-whole body equation. The ratio of egg-to-muscle selenium is much greater than would be expected, given similar data for other species including the closely related cutthroat trout) it s unclear if this is an observation unique to rainbow trout or anomalous data. Given that larval deformities are expected to be most closely correlated with selenium levels in the egg or ovary, it makes more sense to estimate the whole body selenium concentration directly from eggs for this endpoint using the EPA egg/ovary to whole body equation, especially since the egg concentrations are measured, unlike the muscle concentrations, which were estimated. Furthermore, the additional step of converting egg concentration to a muscle concentration before again converting to whole body increases the potential for error and uncertainty with the end value. Therefore, we propose the 17

19 appropriate conversion for this study is to directly calculate the whole body concentration from egg concentration. This would result in chronic values for rainbow trout of and µg/g dw wb for 2000 and 2001 data, respectively. These values are much closer to chronic values of other closely related species in this genus, including O. tshawytscha and O. clarki. The chronic tissue values presented for the brook trout both 2000 and 2001) and the previously discussed 2000 rainbow trout data are based on the mean tissue concentrations of fish collected from the high selenium streams that had significantly higher % craniofacial deformities in 2000 Luscar Creek) and higher % finfold deformities in 2001 Gregg Creek) for brook trout, and higher % edema Luscar Creek) for rainbow trout compared to the reference stream. For both the brook trout and the 2000 rainbow trout data, regression analyses of these % deformities as a function of egg selenium concentration were inconclusive meaning no EC 20 could be calculated. In fact, there was no relationship between selenium concentrations and % deformities for brook trout. Eggs with selenium concentrations near the chronic values suggested by U.S. EPA for brook trout resulted in deformities from 0% to approximately 21% for finfold deformities and 0% to approximately 40% for craniofacial deformities. Several brook trout produced eggs with higher selenium concentrations than the proposed chronic values and had <5% deformities in all categories, including one with an egg concentration of approximately 14.5 µg/g ww which converts to µg/g dw wb). Additionally, in 2001 all % deformities for brook trout from the stream with the highest mean selenium values Luscar Creek, 8.02±0.77 µg/g ww egg) were not significantly greater than % deformities of the reference site. Furthermore, the author states relationships between selenium and deformities require additional study in brook trout Holm 2002). No other brook trout tissue values exist for comparison. We support the use of the mean tissue concentration for brook trout from the site with elevated selenium for However, like the rainbow trout, the whole body concentration should be calculated directly from the egg concentration 6.37 µg/g ww egg or 12.4 µg/g dw wb) rather than muscle concentration 3.79 µg/g ww muscle or 13.2 µg/g dw wb). We do not support the use of the specific site-mean concentration used by the U.S. EPA for the 2001 data i.e., the site-mean concentration from the site with intermediate concentrations). Use of this site is unnecessary since no effects were observed at the site with highest selenium concentration, either. In fact, the site with higher selenium concentrations had a lower % deformities value and in both cases no apparent negative relationship was observed between tissue selenium values and % deformities of larvae for any of the brook trout data. Therefore, the whole body chronic effect concentration for brook trout from 18

20 this study should be derived from the mean egg concentration from the Luscar Creek site 8.02 µg/g ww egg or 15.4 µg/g dw wb). This value better represents the NOAEC for brook trout from the 2001 data, as opposed to the concentration used by U.S. EPA. Additionally, since both of the derived values likely represent NOAECs, we propose to add a > to the chronic values derived from site means to denote the true chronic tissue level is more than likely greater than the table values. We also propose adding a > to the revised µg/g dw value reported for the 2000 rainbow trout data due to the error associated with the toxicological endpoint with the greatest % deformities 30.8±27.4 edema), which was extremely influenced by fry produced from a single female. All other females tested with varying selenium concentrations, including one with a much higher selenium tissue concentration than the proposed toxic value, were close to 100% normal. Fathead Minnow Chronic Values - Bennett et al. 1986) evaluated food web dynamics of dietary selenium exposure. Selenium exposed algae were fed to rotifers, which were then fed to fathead minnow larvae Pimephales promelas) under three different feeding regimes. A significant decrease in body weight was observed in larvae exposed to selenium compared to controls for two of the three experiments. The geometric mean of whole body selenium concentrations of exposed fathead minnows from all three experiments was included in the 2004 Draft chronic tissue database. However, these data were determined not suitable for use in chronic toxicity calculations due to the variation in test duration and feeding regimes. The larvae were fed a control diet for 2-8 days, the selenium enriched diet for 6-8 days at very high concentrations), and then followed with the control diet for the remaining 2-19 days. Total experiment duration ranged from 9-30 days. Exposure to the selenium enriched diet was very short - certainly too short to be considered a true chronic toxicity test for Pimephales. Additionally, the one test that ran for 30 days was actually fed a control diet for the last 19 days before the test was terminated and tissues were collected. Since exposure duration does not comply with guidelines for chronic toxicity tests with fish Stephan et al. 1985), we suggest removal of this value from the chronic database Table 1). This is the only data point we suggest for removal from the 2004 chronic tissue-based database. 19

21 Additions to the Chronic Selenium Fish Tissue Database Rainbow Trout Chronic Values - Hilton and Hodson 1983) evaluated the effect of increased dietary carbohydrate on the selenium metabolism in rainbow trout Oncorhynchus mykiss) and the toxicity of selenium to O. mykiss. Two diets of high and low carbohydrate content were supplemented with 0, 5, and 10 µg selenium/g dw and fed to juvenile rainbow trout for 16 weeks. Significant reductions in growth were observed in test organisms fed both diets in the highest selenium concentration tested. The 2004 Draft document presented chronic values calculated from the NOAEC and LOAEC selenium concentrations of liver dissected immediately following completion of the experiment. The resulting chronic values are and µg/g dw liver for the low and high carbohydrate diet tests, respectively. Although both values are presented, only the chronic value calculated from the high carbohydrate treatment µg/g dw) was included in the 2004 Draft chronic database. Both tests produced approximately the same reduction in weight 55% reduction in the low carbohydrate treatment and 52% reduction in the high carbohydrate treatment), and no other obvious factors exist for omission of the low carbohydrate chronic value. Therefore, both chronic values should be included in the chronic database for rainbow trout. Using equation III, the estimated whole body selenium concentration from µg/g dw liver is µg/g dw. This value was added to the revised chronic database Table 1). Razorback Sucker Chronic Value: Hamilton et al. 2002a) conducted a field experiment with the endangered razorback sucker Xyrauchen texanus). Adult fish were stocked at three off stream sites of the Colorado River near Grand Junction, with historically low, medium and high selenium concentrations. Test organisms were previously used in a reproductive study Hamilton et al. 2001) and originally obtained from the Wahweap Fish Facility, Big Water, Utah. An additional reference test was run with hatchery brood stock adults at the low selenium site. The test was initiated in July 1996 and run through April 1997, when fish were collected for laboratory spawning experiments. Total selenium concentrations at sites ranged from 1.8 µg/l to 148 µg/l in water, 4.4 to 48.1 µg/g dw in invertebrates, and 0.42 to µg/g dw in surface sediment during the exposure period. Spawning experiments were conducted in a laboratory and incubated in site water and reference water. Data were analyzed for percent survival and hatch as well as number of deformities by site and source Wahweap fish vs. hatchery brood stock). Mean selenium egg concentrations were 6.0±0.2 µg/g dw for the low selenium site, 6.9±0.2 µg/g dw for the brood stock, 40.1±1.0 µg/g dw for the intermediate selenium site, and 54.7±1.1 µg/g dw for the high selenium site. No significant differences were found in % survival and % hatch 20

22 among the three egg sources. The greatest number of deformities was observed in the brood stock larvae, which also had relatively low egg selenium concentrations. Additionally, the number of deformities was consistently greater for tests using the reference water than those using site water. Based on the lack of effects of selenium concentrations on % deformities, survival, and hatch for any treatment, the NOAEC would be equal to the highest measured mean egg concentration 54.7 µg/g dw). After conversion using the U.S. EPA equation, this results in a whole body chronic value > µg/g dw. This value is within the range of existing chronic values for the razorback sucker and thus should be added to the revised chronic tissue database. Reference Chronic Values for Carp and Catfish - Fan et al. 2002) collected microphytes, macroinvertebrates, fish, and bird embryos from two agricultural drainage basins located within the Tulare Lake Drainage District and San Luis Drain Canal, CA. Three fish species catfish, carp, and mosquito fish) were collected, sectioned into liver, gonad and muscle tissues, and analyzes for total selenium. Gonad and liver tissues from carp Cyprinus carpio) and catfish Ictalurus sp.) were analyzed for histopathologic alterations and compared to the relevant selenium concentrations. Severity of gonad alterations in carp tended to increase with selenium concentration, with moderate to severe inflammation of gonads and atresia degeneration) occurring in carp sampled with the highest selenium concentrations Table 2). We calculated a chronic value of 8.17 µg/g dw wb via the geometric mean of the NOAEC, LOAEC, and equation I. All catfish gonads appeared normal regardless of selenium concentrations, therefore the chronic value for catfish is greater than the geometric mean of all measured concentrations >5.95 µg/g dw wb). Values for both species were added to the revised chronic database as reference values for carp and catfish, but not used in criteria derivation since the effects of cellular alteration on survival, growth, or reproduction have not been quantified. 21

23 Table 2: Total selenium concentration of muscle tissue for female carp and male catfish as presented in Table 2 of Fan et al. 2002). Chronic value for carp = 8.17 µg/g dw geometric mean of the NOAEC and LOAEC) and >5.95 µg/g dw for catfish geometric mean of all values). Replicate Sex Total Se µg/g dw Gonad status Whole body a Se µg/g dw Geometric mean carp-4 F 8.3 normal NOAEC) carp-5 F 7.5 mild carp-7 F 9.8 moderate/severe LOAEC) carp-3 F 9.9 moderate/severe 8.86 carp-1 F 14.3 Severe catfish-1 M 6.3 normal catfish-2 M 6.5 normal 6.09 catfish-3 M 7.4 normal 6.83 catfish-4 M 5.5 normal 5.24 catfish-5 M 6.5 normal 6.09 catfish-6 M 6 normal 5.67 a estimated using U.S. EPA equation I Recalculation of the Chronic Tissue Criterion After correcting the chronic database of the 2004 Draft document and adding data deemed suitable for inclusion from our literature review, a revised chronic tissue-based database for selenium was compiled Table 3). The revised database consists of 1 invertebrate rotifer), 10 fish species from 8 genera, and one general family chronic toxicity value Centrarchidae). This database is the basis for the subsequent chronic tissue-based criterion calculations, with species by species discussions of calculations of species mean and genus mean chronic values and the data used or not used) for the calculations provided below. Species Brachionus calyciflorus Oncorhynchus tshawytscha Oncorhynchus mykiss Table 3: Revised chronic tissue-based database for selenium U.S. EPA Table 4 of the 2004 Draft document), species mean chronic values SMCV), and genus mean chronic values GMCV). All toxicity values presented are expressed as µg/g dry weight whole body. Common Chronic Name Toxicological Endpoint Value SMCV GMCV Reference rotifer EC 20 for rotifer dry weight chinook salmon rainbow trout after 4d Dobbs et al EC 20 for juvenile growth >12.82 Hamilton et al EC 20 for juvenile growth Hamilton et al MATC for juvenile growth Hilton and Hodson 1983: e >14.73 Hicks et al MATC for juvenile growth Hilton and Hodson 1983: a,e Hicks et al MATC for juvenile survival and growth e Hilton et al

24 Species Common Name Toxicological Endpoint Chronic Value SMCV GMCV Reference Chronic value for larval deformities > b,d Holm 2000; Holm et al EC 20 for craniofacial deformities b,d Holm 2000; Holm et al Oncorhynchus clarki cutthroat trout NOAEC for embryo/larval deformities and mortality > c >11.14 Kennedy et al NOAEC for embryo/larval deformities > b Hardy 2005 Salvelinus fontinalis brook trout Chronic value for craniofacial deformities > b,c >13.73 >13.73 Holm 2002, Holm et al Chronic value for finfold deformities > b,c Holm 2002, Holm et al Cyprinus carpio carp MATC for histopathological alteration of ovaries 8.17 a * Fan et al Ictalurus sp. catfish Histopathological alteration of ovaries > 5.95 a >5.95 >5.95* Fan et al Pimephales promelas Catostomus latipinnis Xyrauchen texanus Lepomis macrochirus fathead minnow flannelmo uth sucker razorback sucker bluegill MATC for pre-spawning adult growth 5.96* <18.21 <18.21 Ogle and Knight 1989 LOAEC for larval fish dry weight after 8d < 73.00* Dobbs et al LOAEC for larval edema and lordosis < d Schultz and Hermanutz 1990 NOAEC for survival and growth > >10.20 >10.20 Beyers and Sodegren 2001a NOAEC for survival and growth > >23.95 >23.95 Beyers and Sodegren 2001a NOAEC for survival and Beyers and Sodegren growth > b NOAEC for larval survival and hatch > a,d Hamilton et al. 2002a LOAEC for larval mortality < c * Bryson et al Chronic value for swim-up larvae < c * Bryson et al. 1985a Chronic value for swim-up larvae > c * Bryson et al. 1985a NOAEC for swim-up larvae > 5.45 c * Bryson et al. 1985b Chronic value for larval survival < 28.20* Gillespie and Baumann 1986 Chronic value for larval survival < d * Gillespie and Baumann 1986 EC 20 for larval survival 8.95 Coyle et al LOAEC for juvenile mortality at 4 C < 7.91 Lemly 1993a NOAEC for juvenile mortality at 20 C > 6.00* Lemly 1993a MATC for larval survival, edema, lordosis and hemorrhaging Study II Hermanutz et al NOAEC for juvenile growth > 3.74* Bryson et al. 1985b NOAEC for juvenile survival > Cleveland et al NOAEC for larval survival, edema, lordosis and hemorrhaging Study III > Hermanutz et al