Draft Mercury Aquatic Wildlife Benchmarks For The Great Salt Lake Assessment

The U.S. Environmental Protection Agency Region 8 Denver, CO

Date Authors Version Description 6/22/2015 L Guenzel, S Spence, K Jensen 1 External Peer Review Draft 6/23/2016 L Guenzel, K Jensen 2 Revised Draft

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Table of Contents 1. Introduction ...... 2 2. Great Salt Lake Background ...... 2 3. Utility of Mercury Data Available for Assessment ...... 4 4. Identification and Selection of Mercury Effect Benchmarks and Risk Categories ...... 5 5. Decision Criteria ...... 8 6. Benchmark Uncertainties ...... 10 6.1 Linking mercury concentrations in to time spent at GSL ...... 10 6.2 Applicability of literature derived benchmarks and risk thresholds to GSL (freshwater vs. saltwater exposure ...... 12 6.3 Potential differences in species sensitivity for literature derived benchmarks and those species found at GSL ...... 12 6.4 Mercury-selenium interaction considerations ...... 13 6.5 Waterfowl foraging behavior and mercury risk ...... 14 7. Conclusion ...... 15

Attachment 1 ...... 16 A.1 Diet ...... 19 A.2 ...... 30 A.3 Blood ...... 47 A.1 Liver ...... 51 References ...... 56

Tables Table 1. Available mercury data types for the Great Salt Lake assessment ...... 5 Table 2. Proposed aquatic wildlife effect benchmarks for the GSL mercury assessment ...... 6 Table 3. GSL mercury assessment risk categories for blood and liver ...... 7 Table 4. Decision criteria for the GSL Hg assessment ...... 9 Table 5. Potential Hg assessment indicators, availability of benchmarks, and risk thresholds for GSL Hg data ...... 10 Table A1-1: Study design differences in the Heinz (1979) and Heinz et al. (2010a) controlled breeding experiments with mallard ...... 25 Table A1-2. Diet. Evers et al. (2004) Hg in diet risk thresholds for common compared to other diet effect values ...... 27 Table A2-1. Comparison of mercury injected LC50s (Heinz et al. 2009, Kenow et al. 2011, Braune et al. 2012) to egg effect concentrations that were maternally deposited (field and controlled breeding experiments), including no observed adverse effect concentrations (NOAEC) ...... 39 Table A2-2. Egg. Evers et al. (2004) Hg risk thresholds for egg compared to other egg effect values ....44 Table A3. Adult Blood. Evers et al. (2004) Hg risk thresholds for blood compared to other blood effect values ...... 49 Table A4. Adult Liver. The EPA-proposed mercury in liver risk categories for aquatic wildlife ...... 54

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Figures Figure 1. Map of Great Salt Lake features ...... 1 Figure A2.1. The relationship between the number of 1 week old ducklings produced relative to the control (i.e., control normalized) presented in Heinz (1974), Heinz (1979), and Heinz and Hoffman (1998) when A) data are analyzed by generation (N=8) and B) when using an average response from the Heinz (1979) 3 generation study (N=6) ...... 31 Figure A2.2. The relative sensitivity of several wild embryos to injected methylmercury ...... 37 Figure A2.3. All quantified reproductive loss endpoints and the associated THg in egg concentration for all bird species (terrestrial and aquatic) ...... 41 Figure A2.4. The reproductive success and associate THg in egg concentrations for aquatic and aquatic dependent birds ...... 42

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Figure 1. Map of Great Salt Lake features (internet image)

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1. Introduction

This document describes the Environmental Protection Agency (EPA) Region 8’s rationale for selecting aquatic wildlife dietary and tissue mercury benchmarks for use in interpreting available data collected from Great Salt Lake (GSL) and surrounding wetlands. EPA Region 8 became aware of potential human health and/or ecological effects of elevated mercury concentrations in avian tissue and water column measurements in GSL after receiving stakeholder information during the public comment period for Utah’s 2006 statewide water quality report (Integrated Report) and impaired waters list (303(d) list). In addition, more recent monitoring data collected from within GSL confirms mercury concentrations of potential concern. The Utah Division of Water Quality (UDWQ) is working to establish numeric water quality standards to protect the lake's designated uses; however UDWQ has determined this effort may take approximately 10 years and will first focus efforts on criteria that can be derived from traditional water column exposure toxicity tests (i.e. parameters such as copper, arsenic and lead without bioaccumulative concerns). Appendix A-1 of Utah’s 2010 Integrated Report (IR) includes a scoping level assessment of mercury for GSL based on the States’ narrative criterion and identifies the need for peer review of the assessment, the need for expert review of the proposed benchmarks, and potential areas of uncertainty with the benchmarks (UDWQ 2010a). EPA Region 8 conducted a literature review to update and refine the aquatic wildlife dietary and tissue benchmarks for mercury that may be used for data assessment until water quality criteria can be derived. The goal of the literature review was to identify mercury concentrations that are associated with adverse reproductive effects and select benchmarks that would avoid a false positive outcome. The following sections of the document describe how aquatic wildlife dietary and tissue benchmarks have been compiled from existing literature sources and the decision criteria that will be used to evaluate whether GSL and surrounding wetlands meets its designated use for aquatic wildlife.

2. Great Salt Lake Background

GSL is a significant aquatic resource that needs protection, which requires an accurate assessment of condition and ability to support designated uses. It is a globally important migratory bird habitat and a substantial economic resource. Hundreds of millions of dollars are generated each year in association with GSL from , mineral extraction, and brine shrimp (Artemia franciscana ) harvesting.1

GSL is a terminal waterbody that supports unique chemical and biological conditions. Figure 1 provides an overview of the significant features of GSL, including its four main bays: Farmington Bay, Gilbert Bay, Gunnison Bay, and Bear River Bay. Each of the bays are separated by causeways that minimize hydraulic transfer, therefore, the chemistry and ecology of each bay is distinct.

GSL salinity varies with bay and water surface elevation. Salinity typically ranges from 1-6% in Farmington Bay and Bear River Bay to saturation in Gunnison Bay (>= 30%). Salinity in Gilbert Bay can range from 7-15% (HDR, 2014)2. Limited connectivity and density differences between the bays results in bi-directional flow of a deep dense brine overlaid by a less dense brine (UDWQ, 2014a). 3 This stratification is frequently observed in Gilbert Bay and can last for months or even years. The dissolved oxygen concentrations in GSL range from full saturation in the shallow layers to anoxia in the deep

1 Bioeconomics, Inc., 2012. Economic Significance of the Great Salt Lake to the State of Utah . Prepared for State of Utah Great Salt Lake Advisory Council. 2 HDR, 2014. Union Pacific Railroad Great Salt Lake Causeway Final Water and Salt Balance Modeling Report . 3 http://www.deq.utah.gov/locations/G/greatsaltlake/railroadcauseway/index.htm 2 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE brine layer. Due to these extreme and variable conditions, the ecosystems of the various bays are dominated by different types of chemistry and biota, and expected conditions are difficult to predict, as are ecological comparisons between the different bays.

Gilbert Bay is recognized for its prolific populations of brine shrimp and brine flies (Ephydra cinerea ), while Bear River Bay and Farmington Bay are generally too low in salinity to support a significant brine shrimp population. Rather, Bear River Bay and Farmington Bay are dominated by corixids (water boatmen) and, to a lesser extent, by brine flies and midges (chironomids). Fish are absent from the open waters of GSL, except when a combination of low lake levels and high spring runoff contributes to a “freshening” of Farmington Bay and Bear River Bay. During these times, bluegill, carp and other minnows have been noted and several bird species (e.g., pelicans, herons, egrets and cormorants) have been observed actively feeding in these bays.1 The extremely high salinity in Gunnison Bay limits the aquatic life use in this bay to halophilic bacteria and archaea (prokaryotes).

GSL is within a major migratory route for waterfowl and shorebirds in North America 2 (Birdnature, 2003) and offers suitable habitat for millions of birds in the western United States. The abundant food supply of brine shrimp and brine flies, as well as many miles of shoreline, serve as an important habitat for migratory birds, and thousands use the shores of the lake to breed. The second largest population in North America of eared ( nigricollis ) use GSL as a staging area during migration, as well as large numbers of migrating tundra swans ( Cygnus columbianus ), snowy plovers, ( Charadrius alexandrinus ), American avocet ( Recurvirostra americana ), black-necked stilts ( Himantopus mexicanus ), red-necked phalaropes ( Phalaropus lobatus ), marbled godwits ( Limosa fedoa ), white-faced ibis ( Plegadis chihi), and long-billed dowitchers (Limnodromus scolopaceus )3 (Utah Department of Natural Resources, 2000). Up to 80% of the world’s population of Wilson’s phalarope ( Phalaropus tricolor ) use GSL during their fall migration, and the world’s largest documented staging population of the species (600,000 individuals) were observed at GSL in 1991.4

The lake faces many pressures that could affect its ecological structure and function, including: 1) wastewater and stormwater discharges from the Salt Lake City metropolitan area; 2) aerial mercury deposition from regional and global sources; 3) mineral extraction activities occurring on the shores of and discharging to the lake from Kennecott Copper, Great Salt Lake Minerals, and US Magnesium; 4) habitat loss due to expansion of the invasive wetland plant, Phragmites australis (common reed); and 5) the presence of and proposed changes to the railroad causeways that alter circulation patterns, salinity gradients, and aquatic habitats in the lake.

The focus of this document is on the selection of aquatic wildlife dietary and tissue benchmarks for assessing the potential risk of mercury exposure to shorebirds and waterfowl in GSL and surrounding wetlands. UDWQ, United States Geological Survey (USGS), academic and nonprofit researchers, and other local and state agencies have collected mercury (Hg) data from in and around GSL over the last several decades. Elevated pollutant concentrations have been measured in GSL media For example, the USGS reported methylmercury (MeHg) concentrations in the water column of GSL as the highest ever measured in surface waters (33 ng/L) ( Naftz et al. 2009). Additionally, in 2005 and 2006 to present, Utah issued the nation’s first avian tissue consumption advisory for two duck species (common

1 http://en.wikipedia.org/wiki/Great_salt_lake 2 http://www.birdnature.com/flyways.html 3 Great Salt Lake Comprehensive Management Plan Resource Document , 2000. 4 Great Salt Lake Waterbird Survey , 1997-2001.

3 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE goldeneye (Bucephala clangula ) and northern shoveler (Anas clypeata ) in response to mercury concentrations detected in duck muscle from GSL exceeding the EPA’s recommended consumption screening level of 0.3 mg/kg. 1 These advisories remain in place. An extensive survey of mercury in waterbird collected from 33 species of birds breeding within the GSL wetlands during 2010-2012 concluded that mercury concentrations in eggs of birds breeding in GSL are moderate to high when compared to other Hg contaminated ecosystems (Ackerman et al. 2015). Elevated concentrations of mercury have also been identified in sediments, brine shrimp, macroinvertebrates and avian muscle, liver and blood.

3. Utility of Mercury Data Available for Assessment

Under the Clean Water Act (CWA) and Utah’s water quality standards 2, Utah is responsible for protecting aquatic life living within GSL, defined as waterfowl, shore birds and other water-oriented wildlife including their necessary food chain. Therefore, mercury data associated with any of these aquatic life components may be used to evaluate the status of attainment for the aquatic life use under the CWA. Table 1 provides a summary of the types of mercury data that are available for consideration in assessing water quality conditions at GSL.

EPA ranked the available types of mercury data with respect to their scientific certainty, or utility to support a CWA assessment decision, and GSL exposure link to the lake’s designated aquatic life use (waterfowl, shorebirds and their food chain). Without a scientifically defensible benchmark for comparison and a link to GSL, the data are not useful for an aquatic life use assessment. Data utility was assigned as follows: • high utility = effect thresholds or criteria are available from controlled, Hg-only exposure studies that measured reproductive endpoints; • medium utility = effect thresholds for reproduction are derived from field studies (no lab data) or additional uncertainties were identified that limit their use (e.g., total vs. methylmercury concerns in liver, potential Hg-Se interaction, etc.); • low utility = effect thresholds have not been published for reproductive endpoints, results are only qualitative, the existing thresholds provide conflicting information, or the data type is not a component of the aquatic life food web. With respect to ranking the GSL exposure link for each data type, the EPA relied on linkages that have been established in the literature. More information on the GSL exposure link is provided in Section 6.1 (linking mercury concentrations in birds to tine spent at GSL).

1 Utah Waterfowl Advisories – Consumption Advisories 2 GSL Beneficial Uses -- Protected for frequent primary and secondary contact recreation, waterfowl, shore birds and other water-oriented wildlife including their necessary food chain. Utah Administrative Code, Standards of Quality for Waters of the State. http://www.rules.utah.gov/publicat/code/r317/r317-002.htm 4 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE

Table 1. Available mercury data types for the Great Salt Lake assessment Data Type/Indicator Utility for CWA GSL Exposure Comment on Utility, GSL Exposure Link, Assessment Link and Applicability Decision Sediment Low High Translator for water column transfer needed in order to interpret risk. Direct water column data more applicable. Water column Varies with endpoint High Although the utility is high since EPA has and environment: national mercury criteria recommendations for Low – hypersaline freshwater and marine environments and GSL areas; aquatic exposure link is high, the applicability of the wildlife water column criteria to aquatic wildlife is low. Additionally, salinity and aquatic life use of High – aquatic life GSL limits potential use of the EPA’s in saline and nationally recommended freshwater and marine freshwater areas criteria. Shoreline spiders Low High Shoreline spiders are not a component of the aquatic wildlife food web, however, concentrations in tissues can show strong correlations with aquatic insect prey that are a direct link to water column exposures Brine shrimp High High Food chain exposure route – direct link GSL nauplii/cysts water column and avian species use Brine fly adults/larvae High High Food chain exposure route – direct link GSL water column and avian species use Macroinvertebrates High High Food chain exposure route – direct link GSL water column and avian species use Avian adult breast Low Low to Medium Concentration in this tissue may not reflect muscle accumulation during time spent at GSL for migratory species. Avian adult liver Medium Low to Medium Concentration in this tissue may not reflect accumulation during time spent at GSL for migratory species. Avian adult blood Medium Medium Concentration in this tissue likely reflects recent accumulation during time spent at GSL for migratory species. Eggs High Medium Concentration in this tissue likely reflects recent accumulation during time spent at GSL for migratory species. * Low Low to High Concentration in this tissue may not reflect accumulation during time spent at GSL for migratory species. * Hg in adult feathers reflects the exposure that occurred when the feathers were grown. Feathers grown while the bird was utilizing habitats other than GSL have low exposure link. Feathers grown at GSL have high exposure link, including chick or fledgling feathers.

4. Identification and Selection of Mercury Effect Benchmarks and Risk Categories

The EPA reviewed over 390 published scientific papers, mercury assessment reports, and state and federal documents to identify effect benchmarks and risk categories for aquatic wildlife. The EPA focused its review on Hg exposure to avian species that are most relevant to the designated uses of GSL, including freshwater and marine shorebirds and waterfowl. Indicators having medium to high utility and exposure link to GSL were selected to support the GSL assessment.

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Attachment 1 of this report provides details on the types of data that were included in our analysis and summaries of the most relevant toxicity tests and field studies that support the selection of effect benchmarks for avian diet (pgs 19-29) and egg (pgs 30-46) and the mercury risk categories that will be used to assess adult avian blood (pgs 47-50), and adult avian liver (pgs 51-55).

We propose effect benchmarks for indicators that have laboratory data, controlled breeding studies, or quantitative field analyses to support the derivation of scientifically defensible adverse effect concentrations for reproductive endpoints. These data requirements supported the selection of effect benchmarks for mercury in diet and eggs (Table 2). We define an effect benchmark as a mercury concentration that, if exceeded , would likely result in adverse reproductive effects. Lowest observable adverse effect concentrations (LOAEC) have been selected for the effect benchmarks since LOAECs are associated with statistically significant adverse reproductive effects. It is important to note that the LOAEC for a reproductive endpoint is a higher concentration than the No Observed Adverse Effect Concentration (NOAEC) and is a lower concentration than a lethal endpoint.

Table 2. Proposed aquatic wildlife effect benchmarks for the GSL mercury assessment. Indicator Effect benchmark Description Diet 0.5 ppm MeHg dw* LOAEC in diet for reproductive effects in aquatic birds. Value derived from a 3 generation controlled breeding experiment with mallards. Egg 0.8 ppm THg fww; LOAEC in eggs for reproductive effects in aquatic birds. 2.7 ppm THg dw Value derived from a 3 generation controlled breeding experiment with mallards.

Egg concentration at which adverse reproductive effects have been observed in several field studies.

The dw benchmark was derived assuming 70% moisture in mallard eggs. * EPA will utilize a conservative approach where THg = MeHg in a dietary component when only THg data are available (see diet discussion in Attachment 1). Measured MeHg in diet will be given precedence over THg when these data are available. LOAEC = lowest observed adverse effect concentration; dw = dry weight; ww = wet weight; fww = fresh wet weight; THg = total mercury.

The proposed diet and egg effect benchmarks for the GSL mercury assessment are not precedent setting. The Great Lakes Water Quality Initiative (40 CFR § 132, EPA/820/B-95/008 ) and Mercury Study Report to Congress (EPA-452/R-97-008 ) also relied on the Heinz (1979) LOAEC of 0.5 ppm to derive avian wildlife criteria and for Hg risk analyses. 1 Ackerman et al. (2014) recently used a 0.5 ppm dw threshold to assess macroinvertebrate data collected from San Francisco Bay-Delta. Furthermore, the effect threshold of 0.8 ppm fww for eggs (once again based on Heinz (1979)) was used in the Evaluation of the Clean Water Act Section 304(a) Human Health Criterion for Methylmercury: Protectiveness for Threatened and Endangered Wildlife in California (USFWS 2003) and in several mercury assessments

1All reports utilized a diet LOAEC of 0.5 ppm in diet with uncertainty factors and site-specific bioaccumulation factors to derive protective avian wildlife values using methods published in 60 Fed. Reg. 15387 1995. Resulting wildlife criteria range from 530 – 1,300 pg/L total mercury in the water column. The EPA GLI mercury wildlife criterion of 1,300 pg/L has been adopted by Illinois, Indiana, Michigan, Minnesota, Ohio, Pennsylvania, and Wisconsin in state water quality standards. EPA disapproved New York’s wildlife criterion in 2000 [See 65 Fed. Reg. 59732, 59738 (October 6, 2000)] and promulgated the GLI criterion (40 CFR § 132.6(e)). 6 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE and literature reviews that address impacts to bird species in diverse aquatic habitats (e.g., Henny et al. 2002 , Hill et al. 2008 , Thompson 1996 , Shore et al. 2011), including eggs collected from GSL wetlands (USFWS 2009a , 2009b ).

The Great Lakes Initiative (GLI) methodology for the development of wildlife criteria includes the use of uncertainty factors when toxicity data are extrapolated across species and when data for protective endpoints (i.e, NOAECs) are not available (40 CFR § 132). The GLI mercury wildlife criterion is set to the avian wildlife value that takes into consideration the sensitivity of three representative avian species (kingfisher, gull, and eagle). Since a NOAEC was not available for the avian class and the mallard LOAEC is in a different order than representative species, the GLI methodology required the use of two uncertainty factors (UF L (LOAEL-to-NOAEL) and UF A (interspecies extrapolation)).)(EPA/820/B- 95/008 ). This assessment will not include uncertainty factors since an assessment goal is to identify existing mercury risk and avoid a false positive outcome. This results in benchmarks that are less stringent than the GLI wildlife criteria and the wildlife targets derived by U. S. Fish and Wildlife Service (USFWS) for mercury TMDLs that are developed to protect wildlife, such as the San Francisco Bay Mercury TMDL (diet target = 0.03 ppm ww per 3-5 cm fish; egg target <0.5 ppm ww), Guadalupe River watershed TMDL (diet target = 0.05 ppm ww in fish 3-5 cm and 0.1 ppm in fish 15-35 cm) and Clear Lake TMDL (T3 fish diet target = 0.09 ppm ww and T4 fish diet target = 0.19ppm ww).1 In California, Basin Plans and TMDLs are submitted to EPA for review and approval under CWA § 303(c), making the diet targets (aka numeric water quality objectives in fish tissue) applicable for CWA purposes.

For indicators that did not meet the data requirements to identify an effect benchmark (i.e., blood and liver), the EPA proposes use of a risk range to evaluate GSL data, or categorical approach, similar to what was developed by Evers et al. (2004) to assess Hg risk to the (Gavia immer )(Table 3). The risk categories for adult blood and liver will be used by the EPA in a weight of evidence approach for determining risk, and to supplement the assessment conducted with the effect benchmarks. The risk categories for egg and diet discussed in Attachment 1 were only used to validate the appropriateness of the effect benchmarks in Table 2. Use of the effect benchmarks as indicators of high relevance and scientific certainty for egg and diet combined with the risk categories for blood and liver provides a framework of complex toxicological information for a range of stakeholders interested in the condition of GSL and surrounding wetlands.

Table 3. GSL mercury assessment risk categories for blood and liver. See Attachment 1 for additional details on how the thresholds were derived and what each risk category represents. All values are total Hg (THg) ppm ww concentrations. Indicator Low Risk Moderate Risk High Risk Extra High Risk Blood – adult <1.0 1.0 – 3.0 3.0 - 4.0 >4.0 Liver – adult <1.3 1.3 - <5.0 5.0 - <15.0 ≥15.0

1 http://www.swrcb.ca.gov/sanfranciscobay/water_issues/programs/TMDLs/sfbaymercurytmdl.shtml http://www.waterboards.ca.gov/sanfranciscobay/water_issues/programs/TMDLs/guadaluperivermercurytmdl.shtml http://www.waterboards.ca.gov/centralvalley/water_issues/tmdl/central_valley_projects/clear_lake_hg/num_tartget_report.pd f 7 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE

5. Decision Criteria

The designated beneficial uses that apply to GSL and that are protected by the State of Utah’s narrative criteria include:

“…infrequent primary and secondary contact recreation, waterfowl, shore birds and other water- oriented wildlife including their necessary food chain” 1 (Class 5 UAC R317-2)

The designated beneficial uses that apply to wetlands surrounding GSL (i.e. waterfowl management areas) that are also protected by the state narrative include:

“waterfowl shore birds and other water-oriented wildlife not included in Classes 3A, 3B, or 3C, including the necessary aquatic organisms in their food chain” (Class 3D UAC R317-17-2)

This proposed assessment approach is designed to determine if waterfowl, shore birds and other water- oriented wildlife, including their food chain, are at risk from mercury exposure associated with GSL habitat. Based on the severity of risk posed to aquatic wildlife at GSL, the EPA or State of Utah may conclude that the Lake’s beneficial uses are threatened, impaired, or there is insufficient information to make an assessment determination at this time.

Until water quality criteria that are protective of the beneficial uses are developed and approved, the EPA’s analysis will not include a decision as to whether GSL is meeting its beneficial uses. To confirm that the Lake is meeting its beneficial uses and that mercury concentrations are at protective levels would require the use of more conservative benchmarks and conservative statistics for comparison to the benchmarks. Hence, the decision before the Agency is whether or not the Lake is at risk from mercury concentrations, or whether there is not enough information available to make this decision.

The EPA developed decision criteria to support an Agency determination that GSL is threatened, impaired, or that insufficient information is available to make an assessment decision. The EPA proposes use of LOAECs for diet and egg benchmarks to avoid a potential Type I error (concluding there is significant risk when in fact there is not) in an assessment decision. A threatened vs. impaired assessment decision will be determined by the proportion of the data that exceed the diet and egg benchmarks and robustness of the database. The EPA proposes that a threatened determination is appropriate if a small proportion of a dietary component and egg data exceed the effect benchmarks and that Hg concentrations, Hg bioavailability, or benchmark exceedances demonstrate an increasing trend. A dietary component is defined as a taxonomic group that is known to be a food source for GSL aquatic wildlife, grouped by age class when appropriate (e.g., brine flies larvae and adults, brine shrimp cysts, juveniles and adults, and benthic macroinvertebrates). A dietary exceedance does not need to align with the diet of the bird species with elevated Hg in eggs because all “food chain” components of the GSL ecosystem must be able to support aquatic wildlife growth and reproduction and it is unlikely that all food chain components have been targeted for Hg sampling and analysis.

An impairment determination, on the other hand, will be made if the majority of the egg and diet data (50% or more) exceed the proposed effect benchmarks and an additional line of evidence suggests high Hg risk. If neither of these conditions are identified, the Agency will decide there is insufficient

1 Utah Administrative Code, Standards of Quality for Waters of the State . 8 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE information available to make an assessment decision. Table 4 provides a summary of proposed decision criteria.

Table 4. Decision criteria for the GSL Hg assessment. A decision will be based on at least 2 individual years of exceedance during the 12 year period of record with recommended sample sizes. Aquatic wildlife threatened (tiered criteria based on sample size) When the sample size is ≥ 10: 1. Upper percentage (10% or greater) of mercury concentrations in a dietary component and egg in a single species exceed the effect benchmark. and 2. Threatened #3

When the sample size is ≥5 and <10: 1. Upper percentage (10% or greater) of mercury concentrations in a dietary component and in egg in 2 or more species exceed the effect benchmark. and 2. Threatened #3

3. Mercury concentrations, loads to GSL, bioaccumulation, or methylation potential are projected to increase and result in a greater frequency of exceedance of the diet and egg effect benchmarks in the near future. Aquatic wildlife impaired 1. Mean mercury concentration in egg (by species) and a dietary component exceed the effect benchmark (minimum sample size of 20 for diet and egg). and 2. One or more lines of evidence that indicate: o the local environment is the source of the high mercury measured in aquatic wildlife (e.g., mercury in water column, mercury food web studies, and mercury concentrations in avian blood or liver), o mercury in blood or liver indicate high risk to aquatic wildlife, or o mean mercury in egg or a dietary component exceeds the effect benchmark when sample size is <20. Insufficient information available 1. Sample size is <5 in each year during the period of record for diet or egg, or 2. Data do not align with the threatened or impaired statistics as described above.

The ability of the EPA or State of Utah to apply these decision criteria is dependent on the sufficiency and type of available data. All data will be analyzed by species, rather than aggregating data from different species, and results from multiple avian species will be taken into consideration when applying the decision criteria. For example, the Agency may determine GSL is impaired if the mean mercury in black-necked stilt eggs exceeds the egg benchmark, mean mercury in brine shrimp exceed the diet benchmark, and mercury in eared liver indicates high or extra high risk. This type of data analysis will be conducted for all available data for a specific species since all data types may not be available for a single species.

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6. Benchmark Uncertainties

The EPA recognizes there are several areas of uncertainty with the proposed approach (UDWQ 2010b). They include: 1. Linking mercury concentrations in migratory birds to time spent at GSL; 2. Applicability of literature derived benchmarks and risk thresholds to GSL (i.e., how are freshwater exposures different than exposures in the high salinity waters of GSL?); 3. Applicability of literature derived benchmarks for species with different sensitivities than those at GSL; 4. Effects of mercury:selenium antagonism on birds at GSL; and 5. Lack of information regarding avian foraging behavior in the available dataset. 1 Below we summarize how these and other areas of potential uncertainty are addressed in the proposed assessment approach and how proposed decision criteria will be used to determine aquatic wildlife use attainment for the lake. See Attachment 1 for a comprehensive review of all areas of uncertainty associated with the proposed effect benchmarks and risk categories.

6.1 Linking mercury concentrations in birds to time spent at GSL

The EPA evaluated available literature benchmarks for mercury concentrations in the diet, kidney, liver, blood, feathers, brain, and muscle of adult avian species, as well as total mercury concentrations in eggs, chick blood and whole body. With respect to ranking the indicator’s link to time spent at GSL, the EPA relied on linkages that have been established in the literature. Mercury concentrations detected in the avian dietary components represent the most direct exposure link between the water column and adverse effects to GSL waterfowl and shorebirds. Controlled feeding experiments and field studies have shown strong correlations between Hg in diet and adult tissue and egg concentrations. Mercury in resident brine shrimp, brine flies, and other macroinvertebrates provides a measurement of potential exposure to waterfowl through the GSL food web. Therefore, diet is assigned a high exposure link.

Table 5. Potential Hg assessment indicators, availability of benchmarks, and risk thresholds for GSL Hg data. Shaded cells identify high priority indicators, defined by high utility, high or medium exposure link to the lake, and data availability. High priority indicators are given a more significant ranking in the weight of evidence assessment approach. Beneficial Use Direct Indirect Utility of Exposure Link Risk Thresholds GSL Data that must be Indicators Indicators Indicator* to GSL or Effect Available protected Benchmark Identified Support for Waterfowl Hg in diet High High Yes Yes Waterfowl and and/or Shorebirds shorebird including their health food-chain Hg in adult Low Medium No No kidney Hg in adult liver Medium Low/Medium Yes Yes Hg in adult blood Medium Medium Yes Yes Hg in adult Low Low/High** No Limited feathers Hg in adult brain Low Not determined No No

1 Utah 2010 Integrated Report, Chapter 14, Appendix A-2 http://www.deq.utah.gov/ProgramsServices/programs/water/wqmanagement/assessment/docs/2010/11Nov/IR2010/Part2/Ap pxA-2_GSLHgPart2_2010.pdf 10 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE

Beneficial Use Direct Indirect Utility of Exposure Link Risk Thresholds GSL Data that must be Indicators Indicators Indicator* to GSL or Effect Available protected Benchmark Identified Hg in adult Low Low/Medium No Yes muscle Waterfowl Hg in Egg High Medium Yes Yes and/or shorebird reproductive success (hatching, fledgling) Hg in adult diet High High Yes Yes Hg in down Low Medium Yes No feathers Hg in adult liver Medium Low/Medium Yes Yes Hg in adult brain Low Not determined No No Hg in adult blood Medium Medium Yes Yes Hg in chick blood Low Medium Yes No or whole body *Utility was determined by the availability and certainty of effect benchmarks and risk thresholds for the data type. **Hg in adult feathers reflects the exposure that occurred when the feathers were grown. Feathers grown while the bird was utilizing habitats other than GSL have low exposure link. Feathers grown at GSL have high exposure link.

Similar to diet, egg mercury concentrations frequently correlate well with mercury in adult blood, newly hatched chick blood and down feathers in common loon, mallard, and Forster’s (Evers et al. 2004, Heinz et al. 2010b, Ackerman and Eagles-Smith 2009 ). However, correlations of egg mercury concentration with mercury in the local environment (diet and water) can be limited by the amount of time adults spend feeding in the nesting area prior to laying eggs ( Barr 1986 ). This decrease in connection/correlation between exposure and sampling location resulted in the identification of a medium exposure link for mercury in eggs. We acknowledge there will be differences in foraging time at the lake and differences in diets among GSL migratory species, which will influence egg mercury concentrations; however, controlled feeding experiments suggest that mercury in eggs can increase and decrease rapidly if exposure were to change prior to or during the nesting season ( Heinz and Hoffman 2004 ). Therefore, the EPA has determined for purposes of this assessment approach that the mercury in eggs will likely reflect the recent adult exposure that occurred immediately before and during the nesting period.

A medium exposure link was also assigned to indicators that 1) similar to eggs, may reflect foraging in a different habitat or 2) store inorganic mercury, and more accurately represent cumulative exposure to mercury, rather than recent exposure to mercury. These indicators include down feathers, adult blood, liver, muscle and kidney and chick blood or whole body.

Finally, a low exposure link was assigned when mercury in an indirect indicator only reflects exposure during a certain period of time. For example, mercury in feathers reflects exposure that occurred when feathers were grown. This can result in a temporal separation between mercury measured in feathers, internal organs, and the local environment for feathers collected from adult birds (Eagles-Smith et al. 2008 ). Feathers grown while a bird was utilizing habitats other than GSL would have low exposure link, whereas feathers grown at GSL have high exposure link.

Given the variability in utility and exposure linkage, indicators were assigned different levels of significance when establishing the final decision criteria discussed in Section 5. Effect benchmarks were only established for the indictors with high utility and medium or high link to the local environment (i.e., 11 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE mercury in egg and diet) and were given the highest significance. Adverse reproductive effects are more likely to occur when there is an exceedance of an effect benchmark rather than an exceedance of a risk category. Finally, if data was limited or not available for an indicator that meets the utility and exposure linkage requirements, it was not further evaluated for the assessment.

6.2 Applicability of Literature Derived Benchmarks and Risk Thresholds to GSL (Freshwater vs. saltwater exposure) When conducting its literature review, the EPA did not identify papers that address potential differences in sensitivity to mercury when birds are exposed in a freshwater environment versus a saltwater environment. In addition, the benchmarks selected for the assessment address exposures that occur outside of the hypersaline environment and therefore are not influenced by GSL water column chemistry. For example, mercury exposure to the avian embryo occurs in ova and is not influenced by the hypersaline environment in which the mercury originated. Exertion of MeHg toxicity to avian species as a result of accumulation through the diet and measured in the blood, feathers, or liver occurs outside of the environment in which the initial mercury exposure occurred. It is possible that salinity could impact the fate and transport (i.e., methylation rate, uptake, and accumulation by avian species) of mercury in the aquatic habitat but should not alter the toxicity of mercury that ultimately accumulates in the diet or body tissues of avian species. The net accumulation in these tissues is compared to benchmarks and does not depend on an estimate of uptake from the water column. We do not consider the salinity of the environment of origin to be a significant source of uncertainty when the point of exposure occurs outside of that environment and direct measurements of bioaccumulated mercury are compared to benchmarks.

6.3 Potential differences in species sensitivity for literature derived benchmarks and those species found at GSL The proposed diet and egg effect benchmarks are derived from mallard controlled breeding experiments. The proposed risk thresholds for diet, eggs, and blood are primarily derived from field studies with the common loon. These species do not nest on the shore of hypersaline portions of GSL (Gilbert Bay, Gunnison Bay or associated islands) and are not commonly observed in the open water of GSL. The mallard is, however, commonly observed in freshwater and the saltwater-freshwater interface habitats surrounding the open waters of GSL (Paul and Manning 2002 ). have been observed in the freshwater wetland habitat surrounding the lake, but their occurrence is rare.1

Although the species used to develop the proposed benchmarks and thresholds do not commonly forage in the open water of GSL, the open waters of the lake and shoreline support a diverse assemblage of birds that changes seasonally, including use during winter months, as the hypersaline waters do not freeze. Species that have been observed in the open water and shoreline habitat of GSL include, but are not limited to, American avocet, black-necked stilt, eared grebe, California gull (Larus californicus ), Franklin’s gull (Leucophaeus pipixcan ), Wilson’s phalarope, least sandpiper (Calidris minutilla ), western sandpiper (Calidris mauri ), black-bellied plover (Pluvialis squatarola ), and green-winged teal (Anas carolinensis ) (Paul and Manning 2002). This list is greatly expanded when additional habitats, such as the saltwater-freshwater interface, are taken into consideration (Barber and Cavitt 2012 ). Unfortunately, toxicity data derived from controlled breeding experiments are not available for these

1 Bear River Migratory Bird Refuge Bird List http://www.fws.gov/uploadedFiles/BRR-BirdList.pdf http://wildlife.utah.gov/gsl/waterbirdsurvey/report.htm 12 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE species; therefore, it is necessary to use toxicity data from a surrogate species when deriving appropriate Hg benchmarks for GSL. Results from recent mercury egg injection studies suggest the mallard and common loon may represent more tolerant bird species to mercury exposure (Attachment 1), which suggests these species are not conservative surrogate species for the waterfowl that are expected to use GSL open water and shoreline habitat.

Avian species can vary in their ability to demethylate mercury (Fimreite 1974 , Barr 1986 , Kim et al. 1996 , Scheuhammer et al. 2007 , Wolfe et al. 1998 ). For example, in the order are known to be effective demethylators and tend to have lower percentages of MeHg in organ tissue when compared to other species. Birds that have the ability to efficiently demethylate mercury may have a high body burden without obvious detrimental effects and therefore are considered generally tolerant to mercury. The EPA did not locate any literature studies that suggest the aquatic wildlife that forage and nest on GSL would be efficient demethylators. In the absence of this type of species-specific information, the EPA has determined it appropriate to use toxicity data from a surrogate species that generally utilizes similar habitats. We also evaluated toxicity data for all aquatic birds, but did not use toxicity data from terrestrial birds or birds of prey when identifying the effect benchmarks. This decision is consistent with the approach adopted by the GSL Selenium Science Panel when deriving the selenium tissue criterion for Gilbert Bay, which was based on the sensitivity of the mallard to selenium (UDEQ, 2008 ).

In summary, GSL supports a diverse assemblage of aquatic birds that do not have mercury toxicity data in the scientific literature. Through EPA’s literature review, data was unavailable to suggest GSL waterfowl and shorebird species are more or less tolerant to mercury than other aquatic birds. Although the mallard is the most sensitive species when reviewing controlled breeding experiments, egg injection studies suggest that it is less sensitive than many other aquatic birds; and field studies with the snowy egret and Forester’s tern have documented reproductive loss when mercury in eggs exceeds 0.8 ppm fww. This suggests that benchmarks derived from the mallard are not conservative for GSL species. Risk thresholds derived from common loon field studies validate the diet and egg effect benchmarks (See Attachment 1). Therefore, we determined that the mallard and common loon are reasonable surrogate species for GSL waterfowl and shorebirds.

6.4 Mercury-selenium interaction considerations Results from several field and lab studies suggest that the bioaccumulation of selenium with MeHg can alter mercury toxicity in adult birds and mammals (Cuvin-Aralar and Furness 1991 , Wiener et al. 2003, Shore et al. 2011). Proposed mechanisms of the protective effect of selenium against mercury toxicity include; 1) redistribution of mercury in the presence of Se; 2) competition for binding sites; 3) formation of nontoxic Hg-Se complexes; 4) conversion of toxic forms of Hg into other forms of Hg; and 5) prevention of oxidative damage (Cuvin-Aralar and Furness 1991). The majority of lab studies and literature reviews that present data in support of antagonistic effects have focused on mammals, fish, and adult birds (both aquatic and terrestrial birds). It is less clear if Hg-Se interactions can influence reproductive success or mercury toxicity in embryos or young birds, which represent the most sensitive endpoint for the GSL assessment, and in some situations, synergistic effects have been observed in early life stages.

For example, embryos produced in a controlled breeding experiment where adults were fed a diet containing MeHg chloride and seleno-DL-methionine had a greater frequency and number of deformities than the embryos produced from the adults fed only Se or Hg ( Heinz and Hoffman 1998 ). Results from this study suggest that the occurrence of both Se and Hg may be synergistic to young birds. 13 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE

Additionally, Heinz et al. (2012) and Klimstra et al. (2012) conducted a series of egg injection studies with MeHg and Se separately and in combination to evaluate potential interactions during the embryo development phase. Authors injected commercially produced mallard and chicken eggs and field collected double-crested cormorant (Phalacrocorax auritus ) eggs with different concentrations and mixtures of MeHg chloride and seleno-L-methionine. The type and frequency of deformities and hatching success between the treatments were documented. Both antagonistic and synergistic effects were observed. MeHg appeared to decrease embryo mortality caused by Se, yet increase the number and types of deformities. Authors conclude that additional studies are necessary to understand the biochemical mechanisms underlying these results and to determine if the same interactions occur with maternally deposited Se and Hg.

Results from the limited studies that evaluated Hg-Se interactions during early life stages do not indicate that selenium protects against the adverse effects of Hg on embryo development or reproductive endpoints. Because the EPA’s assessment is focusing on potential reproductive effects due to mercury, it will not be necessary to take selenium concentrations into consideration when evaluating mercury in diet or eggs. It may be more appropriate to consider selenium concentrations when evaluating adult blood or liver data, but these data are not critical to making an assessment decision since blood and liver data are only identified as additional lines of evidence in the decision criteria.

6.5 Waterfowl foraging behavior and mercury risk The EPA’s analysis will use all readily available data as required under the CWA. In an ideal situation, data would have been collected with the objective of making an aquatic wildlife use assessment decision. This approach would include the selection of target bird species known to be sensitive to Hg, an understanding of its foraging behavior, Hg concentrations in all components of its diet, and the associated water column concentrations to support a species-specific ecological risk assessment. The EPA does not anticipate the data will be available to calculate species-specific risk based upon the relative contributions of mercury from different components of diet. Dietary studies for GSL avian species are limited (Barber and Cavitt 2012). However, a large area of the GSL ecosystem (Gilbert Bay) supports a very simple food web (limited diversity), but very abundant prey (brine shrimp and brine flies) for aquatic wildlife. GSL avian studies have documented several taxa that primarily forage in Gilbert Bay (e.g., avocets, grebes, stilts, and gulls) and many more species forage on the lake during winter months when freshwater habitats freeze. As an alternative to the ecological risk assessment approach, this assessment will evaluate the suitability of known avian dietary components provided by GSL that are protected by the GSL designated uses. If mercury in the known avian dietary components supported by GSL and/or surrounding wetlands are expected to adversely affect the avian species and mercury in eggs exceed the benchmarks, beneficial uses are not met, regardless of its contribution to the total diet.

Furthermore, to address questions concerning diet, when possible, data will be evaluated seasonally, spatially and by life stage. Most of the diet data are expected to consist of adult brine shrimp collected during spring, summer, and fall months. We expect that winter brine shrimp data will be limited. If analyses suggest that mercury in diet varies spatially, this information will be taken into consideration when making assessment determinations. For example, listing decisions can be made by habitat type or for specific segments (Bays) of GSL that have Hg concentrations that exceed the effect benchmarks. Additional details on data analyses will be provided in the assessment report.

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7. Conclusion

GSL has been impacted over time by increased urbanization and industrial, agricultural and municipal discharges. The unique hydrology, biogeochemistry and ecology of the lake limits the applicability of numeric criteria applied to other waterbodies in the state. The lack of applicable numeric water quality criteria has precluded an assessment of the impacts of these stressors on the lake. Utah asserts that the lack of published high quality data and scientific uncertainty concerning the fate and transport of mercury in the lake and its associated food web further complicates a potential assessment effort.

Core Component One of Utah’s Great Salt Lake Water Quality Strategy (UDEQ 2014) outlines a proposed approach the State will follow to develop numeric criteria for the lake. Efforts needed to create numeric water quality standards to protect the lake include conducting strategic monitoring and research, development of a wetlands plan, and initiation of significant public outreach and participation. The State believes that numerous existing uncertainties preclude completion of these efforts within the next 10 years.

Since 2007, the EPA Region 8 office has pursued a collaborative approach with Utah to focus and strengthen efforts directed at protecting the beneficial uses of GSL. The EPA proposes an assessment approach to evaluate available environmental data in comparison to aquatic wildlife adverse effects benchmarks to determine whether GSL waterfowl and shorebird’s diets and eggs are being adversely exposed to mercury concentrations. This document presents the EPA’s rationale to support assessment determinations as to whether the aquatic wildlife in GSL and surrounding wetlands is impaired or threatened or whether insufficient information is available to make an assessment determination.

15 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Identification and Selection of Mercury Effect Benchmarks and Risk Categories for Aquatic Wildlife

The scientific literature provides a wide variety of mercury effect benchmarks and thresholds that can be used to establish risk categories for diet and a number of bird tissue types including egg, blood, liver, and feathers. The introduction of Eagles-Smith et al. (2008) provides a summary of the published bird tissue samples that have been used to evaluate mercury effects on birds over the last 40 years. Eagles- Smith et al. (2008) concludes the unique approaches taken by different authors has made it difficult to compare results from different studies. This inconsistency in approaches lends additional complexity when assessing mercury data from a given waterbody; however it also allows the use of multiple lines of evidence when assessing the current condition of the waterbody. The EPA reviewed over 100 published scientific papers, mercury assessment reports, and state and federal documents to identify effect benchmarks and risk categories for aquatic wildlife. Tables 1 and 4 in the main body of this report provide a summary of the potential data types and indirect indicators that were considered by EPA when initiating the assessment process and the relative certainty of benchmarks that have been derived for those data types. Given the availability of data from GSL and the utility of each indicator, we decided to focus our literature review efforts on mercury in diet, eggs, adult blood, and adult liver.

This attachment provides our rationale for the effect benchmarks and risk categories that will be used in the GSL assessment. Here we discuss: • how laboratory-derived, controlled exposure experiments, and field-derived data were used in our analysis; • our rationale for identifying effect benchmarks for egg and diet tissues; • the types of studies that were used to establish the Evers et al. (2004) risk categories; and • the new adult liver tissue risk categories that we developed following Evers’ approach. Although the literature provides effect thresholds for a wide range of avian species, we are only addressing thresholds for species that are most relevant to the designated uses of GSL, which include waterfowl and shorebirds (freshwater and marine), or aquatic wildlife. The presentation of Hg toxicity results from studies conducted with terrestrial, riparian, and predatory birds (e.g., cowbird, grackle, chicken, blackbird, , quail, starling, eagle, kestrel, hawk, and falcon) is limited to data that support discussions on the relative sensitivity of birds to mercury.

The EPA evaluated a wide range of studies and endpoints (direct and indirect indicators) when establishing the mercury effect benchmarks and risk categories for GSL aquatic wildlife. The EPA took the following types of studies into consideration in its analysis of benchmarks and risk thresholds.

• Controlled breeding experiments and laboratory exposures: Controlled breeding experiments expose test organisms to known concentrations of mercury through a controlled diet. Acute and chronic effects are measured in the exposed adults, or embryos and chicks that are produced during the study. EPA gave the highest priority to toxicity data that were derived from controlled, single pollutant exposure studies that measured reproductive endpoints (e.g., hatching success, chick survival, and chick and embryo deformities). • Field studies: Field collected mercury data and analyses have greatly increased the scientific understanding of mercury toxicity to wildlife. The approach and goals of field studies can be highly variable, with some studies utilizing predetermined literature thresholds for assessment and others evaluating population level effects (i.e., direct indicators) to determine site-specific and species-specific risk to mercury. One advantage of field studies that measure direct 16 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

indicators is they can account for modifications in parental nest behavior that reduce reproductive success (e.g., nest abandonment, laying eggs outside of nest, time spent on nest) that have been observed in adults with elevated mercury concentration (Hill et al. 2008, Evers et al. 2008, Eagles-Smith and Ackerman 2010). These nesting behavioral changes are not typically accounted for in laboratory-derived effect benchmarks. Although field studies have an important role in this assessment, they frequently report qualitative observations. Interpretation of results can be limited by the field study design, and some study results may be confounded by other environmental variables (e.g., pH, turbidity, additional pollutants, predation, unnatural water level fluctuation, and drought). Field studies where authors present quantitative results were given a higher ranking than those that present qualitative results. • Literature reviews with proposed benchmarks, thresholds, or risk ranges: Several authors have published mercury literature reviews that include tissue-specific thresholds for a specific species (i.e., common loon) and for birds in general (several species are taken into consideration). Such reviews are presented in this analysis when authors propose thresholds based on their own analysis of the studies. Literature reviews that simply cite thresholds that were originally proposed by a different author were not documented. When authors only report the individual thresholds from the original studies, EPA reviewed the original papers rather than citing the review paper. • Egg injection studies: Egg injection studies evaluate the effect of mercury that has been directly injected into the egg, rather than relying on maternally deposited mercury. Authors of such studies warn that results from these studies do not provide absolute toxicity thresholds given the route of exposure; however results may provide insight on the relative sensitivity of embryos under controlled laboratory conditions. The EPA took the following types of data into consideration:

1. No observed adverse effect concentration (NOAEC): The highest toxicant concentration in which the values for the measured response are not statistically significantly different from those in the control. Typically reported from full or partial-life-cycle toxicity tests. 2. Lowest observed adverse effect concentration (LOAEC): The lowest toxicant concentration in which the values for the measured response are statistically significantly different from those in the control. Typically reported from full or partial-life-cycle toxicity tests. 3. Effective concentration (ECp): Toxicant concentration estimated to cause a specified effect in a designated proportion (p) of test organisms. The effect is species-specific and typically sublethal. The exposure time can also be specified. 4. Lethal concentration (LCp): Toxicant concentration typically as ug/L estimated to produce death in a specified proportion (p) of test organisms. The effect is species-specific. The exposure time is typically specified. 5. Lethal dose (LDp): Toxicant dose expressed as mg/Kg bodyweight estimated to produce death in a specified proportion (p) of test organisms. The effect is species-specific. The exposure time is typically specified. 6. Hazardous concentration (HCp): A concentration that is predicted to be hazardous to a specified proportion (p) of the species for which we have data, derived from a species sensitivity distribution. The results of the literature review identified effect benchmarks and risk categories to be used in the assessment of GSL mercury data. We define an effect benchmark as a concentration that if exceeded, would likely result in adverse reproductive effects. An effect benchmark was proposed for indicators 17 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary that have laboratory data, controlled feeding experiments, or quantitative field analyses to support the derivation of scientifically defensible effect concentrations for reproductive endpoints that include adult behavioral responses, embryotoxic effects, and overall productivity. Examples of adult endpoints include, number of eggs laid per female, nesting success, proportion of eggs laid outside the nest, and incubation success. The embryotoxic endpoints measured in a study will vary with taxa and test duration. We reviewed controlled breeding and laboratory toxicity studies that measured chick deformities, malposition, percent hatch, number of nestlings fledged, and hatchling survival. Data presented in controlled breeding studies with black duck, mallard, and kestrel indicate that hatching success is a less sensitive endpoint than the number of chicks produced and hatchling survival, which incorporates reductions in egg laying, hatching success, and hatchling mortality (Finley and Stendell 1978, Heinz 1979, Albers et al. 2007).

Our literature review also located field studies that quantified reductions in nest survival, egg hatchability, and overall productivity (number of chicks produced per nesting pair). Depew et al. (2012) identifies that productivity arguably is the most ecologically relevant endpoint since it integrates adverse effects of MeHg on adult behavior, egg production and incubation, embryonic development and hatching success, post-hatch parental care, and chick survival. However, productivity measured in a field setting may also be affected by prey availability, predation and anthropogenic disturbances.

Where toxicity studies were not available to identify an effect benchmark for an indicator, we proposed use of a risk range approach as described by Evers et al. (2004) (i.e., low, medium, high, and extra high risk) to interpret blood and liver data as an additional line of evidence to support an assessment determination. Different decision criteria are associated with concentrations that exceed an effect benchmark and the risk categories (see Section 5 of the main body of this report).

Evers et al. (2004) developed MeHg risk categories for diet, egg, adult blood, juvenile blood and feathers for the common loon. The adult blood risk category will be used in our assessment, whereas the risk ranges for diet and eggs are only used to validate the appropriateness of the effect benchmarks. Evers et al. (2004) describes the four risk categories: 1. Low risk = background levels that are minimally impacted by anthropogenic inputs; the upper limit of the low risk category is considered the NOAEL; 2. Moderate risk = elevated levels but the impact levels on the percent of individuals has not yet been determined; 3. High risk = levels with the potential to cause molecular, organism, and/or population effect; lower limit of the high risk category is identified as the LOAEL; 4. Extra high = known impact on loons and other birds. Evers et al. (2004) did not provide risk categories for mercury in liver; however we anticipate adult liver data will be available for the assessment. To utilize these data, the EPA developed adult liver risk categories using effect concentrations identified in the literature review that are equivalent to the Evers approach (see Section A.4).

The results of the literature review are organized by indicator type and are presented in the following order: diet, egg, blood, and liver. When data for several indicators are presented in a single toxicity test or field study, the first indicator presented in this Attachment will contain the detailed review and following sections and tables will only reference the study.

18 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

A.1 Diet

The open waters of GSL and surrounding wetlands provide important foraging and nesting grounds for hundreds of migratory bird species. An assessment of mercury in potential avian diets in the open waters of Gilbert Bay (brine shrimp and brine flies) and surrounding fresher water habitats (macroinvertebrates and fish) provides a direct measurement of the potential exposure of mercury through the GSL food web. Brine shrimp and brine flies are known to constitute a large portion of the diet of several bird species (e.g., American avocet, California gull, common goldeneye, and eared grebe), making brine shrimp and brine flies the focus of our diet assessment of the open waters. Macroinvertebrates and fish will be considered when assessing the fresher water habitats.

Controlled feeding experiments and field studies have shown that mercury exposure through diet can have adverse effects in adult birds, chicks and ultimately the success of bird populations. Acute effects observed in adult birds feeding on a high mercury diet include a wide range of neurological and behavioral effects (e.g., anorexia, leg paralysis) and lethality (Heinz 1979, Spalding et al. 2000). Chronic effects observed in adults and chicks fed a high mercury diet include reduced egg production, reduced egg hatchability, reduced chick survival and growth, reproductive behavioral changes (e.g., laying eggs outside the nest, nest abandonment, decreased rates of key courtship behaviors) liver damage, and immunity suppression (Finley and Stendell 1978 , Heinz 1979 , Bhatnagar et al. 1982, Heinz and Hoffman 1998 , Bouton et al. 1999 , Spalding et al. 2000 , Spalding et al. 2000a , Heinz and Hoffman 2003 , Kenow et al. 2007a , Eagles-Smith and Ackerman 2010, Frederick and Jayasena 2011 ). Here we summarize several controlled breeding and field studies that are frequently cited in literature and that were used to establish the effect benchmarks and risk thresholds that EPA determined were most appropriate for use in the GSL assessment. Table A1-2 presents all the studies that were taken into consideration when selecting a dietary benchmark.

Heinz published several papers in the 1970’s that present results from controlled breeding experiments with the mallard (Heinz 1974 , Heinz 1975a , Heinz 1976a , Heinz 1976b , Heinz 1979 ). Heinz (1979) presents the results of a three generation experiment, summarizing many of the data and results from the earlier publications. In this multigenerational study, adult mallards were exposed to 0.5 and 3.0 ppm Hg as methylmercury dicyandiamide during the first generation and only a single treatment (0.5 ppm) during the second and third generation exposures. Mercury was added to commercial duck breeder mash that contained about 7% water. Early publications described the nominal Hg concentration in diet as a fresh weight (fw) concentration. Later publications ( Heinz et al. 2009 ) describe the nominal diet concentrations as a dry weight (dw) concentration. We agree that the 0.5 ppm Hg exposure is equivalent to a dry weight concentration for the following reasons. If the 7% moisture is taken into consideration, the calculated dw concentration would be 0.54 ppm dw Hg. 1 Additionally, the mean measured THg treatment concentrations for the three generations was 0.53 ±0.006, 0.47 ± 0.021 and 0.43±.037 ppm fw Hg for the 1 st , 2 nd , and 3 rd generations, respectively. Averaging the mean measured diet concentration from the three generations results in an average exposure of 0.48 ppm fw in diet. Taking into account the 7% moisture, this would be equal to a 0.52 ppm dw exposure or 0.5 ppm dw when considering 1 significant digit. Therefore, the nominal concentration of 0.5 ppm dw reasonably characterizes the average dietary exposure that occurred in these experiments. With respect to the 3.0 ppm dw nominal exposure that is presented by Heinz (1974), the mean measured concentration (3.4 ppm dw) was slightly greater than the nominal concentration.

1 [DW] = [WW]/((100-% moisture)/100) 19 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Heinz (1975a) also indicates that the 0.5 ppm dw concentration would be equivalent to a 0.1 ppm ww concentration in a natural duck diet. Although this value can be used to compare Heinz’s results to other studies that measured Hg exposure in a natural diet (e.g., Table A1-2), we are not recommending use of the wet weight data in the assessment in order to avoid the incorporation of uncertainty in the diet benchmark.

Heinz (1979) concluded that the dietary concentration of 0.5 ppm Hg dw, in the form of methylmercury, decreased the reproductive success of mallards and altered the behavior of their young. During the 2 nd generation, and when analyzing the data for all 3 generations together, hens exposed to mercury produced statistically significantly fewer 1-week old ducklings than the control hens (18% and 29% reduction, respectively).1 Although results suggest that mercury adversely affected the 1 st and 3 rd generations, these results were not significant. Given that significantly fewer 1-week old ducklings were produced in the 0.5 ppm exposure, this treatment is determined to be the lowest observed adverse effect concentration (LOAEC).

Finley and Stendell (1978) fed North American black (Anas rubripes ) commercial duck breeder mash dosed with a nominal concentration of 3 ppm Hg as MeHg dicyandiamide for 2 breeding seasons (mean measured Hg in diet = 3.165 ppm dw). Significant adverse effects were observed in the breeders that were fed the mercury diet. The % hatch was reduced on average by 24.5%. The mean number of ducklings that survived to 1 and 4 weeks was significantly reduced in both breeding seasons (79% and 82 % fewer ducklings, respectively).

Burgess and Meyer (2008) utilized quantile regression to analyze field collected loon diet and reproduction data collected from two geographically distant breeding populations of common loons in Wisconsin and Maritime Canada. Loon productivity was measured as the average production for each lake of large chicks (>5-6 weeks old) per territorial loon pair. Data analyses showed that a wide range of productivity is expected when mercury in diet is low, likely in response to other stressors (e.g., lake pH, prey availability, fluctuations in water level). However, loon chick productivity was never high when mercury was high. The authors determined the 90 th quantile regression lines for loon productivity and Hg concentration in both adult female blood and diet (fish prey). The maximum observed loon productivity of 1.4 chick/pair was reduced by 50% when female blood and diet were 4.3 and 0.21 ppm ww, respectively. 2 Additionally, the regression analysis suggests complete reproductive failure when female blood reaches 8.6 ppm ww and diet reaches a concentration of 0.41 p pm ww. The authors recognize that their study contained few sampling locations with Hg greater than 0.3 ppm ww in fish. This results in some uncertainty with the upper threshold for reproductive failure and warrants further study; however, this value is consistent with the Barr (1986) diet threshold for reproductive failure (discussed below).

Shore et al. (2011) provides a literature review of dietary, tissue, and egg Hg concentrations that are associated with adverse effects on reproduction and survival in birds. Their approach differs from other reviews in that effect concentrations were evaluated with a statistical approach to derive proposed indicative values of Hg effects when sufficient data were available, rather than relying on best professional judgment or central tendency. Their approach relied on the following:

1 Mean number of 1-week old ducklings produced in the 2 nd generation control = 50.9 and Hg exposed = 36.3 (p = 0.05). When all three generations are combined the mean number of ducklings in control = 46 and Hg exposed = 37.5 (p = 0.05). 2 The female blood endpoints are later translated to the predicted egg concentrations using the relationship described by Evers et al. (2003) in Section 2 of this Attachment. 20 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

1) Use of the most sensitive effect concentration when multiple effect measures (e.g., chick survival, hatching success) are reported for the same generic endpoint (reproductive success) for a given species; 2) Use of the median species value when multiple studies for the same endpoint are available; 3) Development of species sensitivity distributions (if experimentally derived, author used LOAECs); and 4) When data were available for eight or more species, calculation of the 5 th percentile of the sensitivity distribution (HC5), which approximates a concentration protective of most species (95%). Dietary mercury concentrations associated with adverse reproductive effects were obtained for seven species, including terrestrial birds, ranging from 0.1-10 ppm ww, with a geometric mean of 1.2 ppm ww. The lowest value in the range is the Heinz (1979) LOAEC that was previously described. Insufficient data were available to calculate a HC5, therefore Shore et al. (2011) used an alternative approach to derive the proposed indicative value of >0.25 ppm ww in diet.1

Several publications present the results of a 3 year controlled breeding experiment with white ibises exposed to environmentally relevant MeHg concentrations (Adams and Frederick 2008 , Adams et al. 2009 , Jayasena et al. 2011 , Frederick et al. 2011 , Frederick and Jayasena 2011 ). Test organisms were wild-caught white ibis nestlings raised in a free-flight aviary and fed a food pellet diet sprayed with a solution of MeHg salt dissolved in corn oil. Exposure concentrations included a control, 0.05, 0.1, and 0.3 mg/kg MeHg ww in diet. The authors tested for impacts to foraging behavior and efficiency in juveniles, measured fecal hormones (i.e., estradiol, testosterone, and corticosteroid), monitored key courtship behaviors during the three breeding seasons, reported on the reproductive output, and monitored the survival behavior and survival of the test organisms once the test organisms were released to the wild. Total mercury was measured in feathers and blood.

Adams and Frederick (2008) report on the effect of MeHg exposure on foraging efficiency and behavior during the first 6 weeks of the experiment. Foraging efficiency was significantly different among the feeding groups, however the relationship was nonlinear with the control and highest exposure groups performing similarly. Frederick and Jayasena (2011) present data on the altered pairing and courtship behavior and how this affects the reproductive output. Dosed males showed significant reductions in key courtship behavior, were less likely to be approached by females, and were significantly more unproductive due to male-male pairing. The total number of fledglings produced per female over the 3 breeding seasons was not significantly different among the dosing groups; however, when the unproductive nests resulting from male-male pairing are taken into consideration, the total reproductive output was significantly reduced in all mercury-exposed groups when compared to the control. 2

Depew et al. (2012) derived MeHg dietary screening benchmarks for use in ecological risk assessment for the common loon. The authors reviewed primary and secondary literature for a wide variety of ecological endpoints (i.e., survival, growth, reproduction, behavioral, and subclinical) and included both

1 Shore et al. (2011) used a generic approach recently advocated by the European Commission where the geometric mean value of several species is divided by five. 2 Frederick and Jayasena (2011) report that productivity over the three years was reduced in the 0.05, 0.1, and 0.3 dosed groups by 13.2%, 14.6% and 13.5%, respectively. Male-male pairing was significantly higher in the 0.3 dosed group than the controls in all three years. 21 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary field-based surveys and observational studies in their analysis. Published relationships between loon tissue concentrations and diet were used to convert different measurement matrices (e.g., blood, brain, eggs) to an estimate of dietary MeHg exposure in fish. The authors propose dietary benchmarks for adult behavioral abnormality (0.1 ppm ww), significant reproductive impairment (0.18 ppm ww = geometric mean of productivity and hatch success endpoints), and reproductive failure (0.4 ppm ww), yet acknowledge the adult behavior benchmark provides limited utility at this time due to the high levels of uncertainty in the value.

Risk Categories - Diet

Evers et al. (2004) diet risk categories were primarily derived from results presented by Barr (1986) and supported by additional field studies presented in the report. Barr (1986) presents the results from a field study designed to evaluate the effects of mercury contamination, unnatural water level fluctuations, and turbidity on common loon populations in the drainage basin of Wabigoon-English River system. Study results indicate that loons laid fewer eggs when prey (small forage fish 10-250 g) averaged 0.3-0.4 ppm THg ww and no eggs were laid when prey averaged over 0.4 ppm THg ww. Concentrations greater than 0.3 ppm MeHg ww defines the Evers extra high risk category for diet. It is not clear what endpoint/study was specifically used to establish the low end of the high risk category (0.15 ppm ww); however, this value is just slightly greater than the LOAEL derived from the Heinz studies (when the dw concentration is translated to a fw concentration) and aligns with the low end of the THg fish concentrations associated with depressed reproduction in the Barr (1986) study (Ball Lake perch THg = 0.15 ppm THg ww). Furthermore, when evaluating the reproductive success of the common loon in Maine and New Hampshire with the proposed risk categories, the low risk territorial pairs were 37% more successful in fledging young than the high and extra-high risk territorial pairs (Evers et al. 2004).

Uncertainties Associated with Assessing Hg in Diet

Total vs. methylmercury

Although Evers et al. (2004) states that all of the risk category thresholds are MeHg concentrations, Barr (1986) and Burgess and Meyer (2008) only discuss and present THg ww concentrations in whole body fish and crayfish. The majority of Hg in fish flesh tends to be MeHg, often close to 100% (Scheuhammer 1991, Scheuhammer et al. 1998 ). This was also true for small whole body yellow perch that were sampled from lakes in Kejimkujik National Park and are the typical diet of common loon (mean MeHg levels = 96% of THg; Drysdale et al. 2005). Whole body fish analyses include organs that can vary more in MeHg% than muscle tissue. Data presented by Mason et al. (2000) suggests that MeHg% in whole body samples will vary with fish species and age, with smaller and younger fish having a lower MeHg%.

The MeHg% for aquatic invertebrates is expected to be more variable than fish flesh. Scheuhammer (1991) suggests that invertebrates typically contain < 50% MeHg. Other studies have shown that macroinvertebrate samples collected from freshwater ecosystems can vary from 20-100% MeHg, with predator species containing the greatest MeHg% (Becker and Bigham 1995 , Mason et al. 2000 , Henny et al. 2005 ).

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Saxton et al. (2013) provides useful site-specific data for THg and MeHg in brine shrimp collected from Gilbert Bay of GSL. The data suggests that the MeHg% is consistently high. In this study, brine shrimp were collected on 3 sampling events, transported to the lab in GSL water, transferred to aquaria containing synthetic GSL water, kept for 24 hours without food to allow for gut content purging, and rinsed with high purity water to remove excess salt prior to analysis. MeHg constituted the majority of THg measured in both female brine shrimp (94%±17%) and their eggs (90%±21%). These sampling methods differ slightly from the sampling methods used by Utah Division of Water Quality, which does not provide time for gut content purging (UDWQ 2014b). Without this step, it is possible that the MeHg% in brine shrimp collected from GSL could exhibit greater variability depending on the gut content contribution to the THg body burden and the MeHg% in the diet of the brine shrimp.

Given the wide range of potential MeHg% in invertebrates reported in the literature and the GSL brine shrimp data suggesting that the MeHg% is consistently high, the EPA will utilize a conservative approach and assumed 100% of the mercury in GSL brine shrimp and brine flies is MeHg. All comparisons of GSL data to the Evers diet effect bench mark and risk categories were made with THg ww concentrations.

Mercury in fish vs. invertebrates

Foraging behavior of GSL waterfowl and shorebirds varies among species, with habitat use, and seasonally when preferred habitats are unavailable due to management of impounded GSL wetlands and/or winter freezing (Paul and Manning 2002, Barber and Cavitt 2012). The GSL ecosystem supports a variety of avian foraging guilds including omnivores, piscivores, and species that forage primarily on brine shrimp and flies (e.g., eared grebe). Given this diversity in habitat and food sources supported by GSL, it would be inappropriate to assess the primary food sources (i.e., invertebrates, since fish habitat is limited) with the Evers et al. (2004) risk categories that were derived for piscivorus birds. The Evers’s thresholds are ww concentrations in fish. Fish have a different moisture content and biomagnification potential than the various invertebrate species supported by GSL habitats. To avoid this source of uncertainty in the assessment, all dietary data will be assessed with the effect benchmark of 0.5 ppm as a dry weight concentration (Heinz 1979), rather than the Evers’s dietary risk categories. The risk categories are only presented to allow for a comparison of different studies and the sensitivity of avian species.

Source of dietary methylmercury

The source and chemical composition of the MeHg used to dose the diet-based toxicity tests has varied over time. Older controlled breeding experiments typically dosed the diet with MeHg dicyandiamide dissolved in propylene glycol, which has been used as a fungicide on seeds. More recent Hg toxicity tests dose avian diets with MeHg chloride dissolved in corn oil. Heinz et al. (2010) identifies the source of the MeHg as an area of uncertainty when comparing Hg toxicity data from different studies and states that they were unaware of any studies that compared the toxicity of the two forms (see additional discussion on this below in Reproducibility of Test Results ). EPA also conducted a literature search in early 2016 and was unable to identify any studies that compare the toxicity of these two forms of MeHg. It is our understanding that the form of selenium used to dose selenium toxicity studies was important when developing the Gilbert Bay selenium criterion. The GSL Selenium Science Panel determined that acceptable tests were limited to those that exposed organisms to the organic form of selenium, selenomethionine, since it is the predominant form of selenium in plant material. Their analysis

23 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary excluded toxicity studies that exposed test organisms to an inorganic form of selenium, sodium selenite. Unlike the selenium toxicity tests where there were clear differences in toxicity resulting from the organic and inorganic form of selenium, both sources of Hg include the most bioavailable form measured in the natural diet of birds – methylmercury. Therefore, until additional data are available that compare the toxicity of the two sources, we have no reason to believe that MeHg dicyandiamide or MeHg chloride would be more or less toxic than natural sources of MeHg.

Reproducibility of Test Results

Heinz et al. (2010a) presents the results from a more recent mallard controlled breeding experiment that nearly replicates the 1st generation 0 and 0.5 ppm diet exposure studies conducted in the 1970’s. The authors did not observe adverse effects in the 0.5 ppm exposure group and instead reported that low levels of Hg in diet can have beneficial effects on mallard reproduction. The mean number of 6 day old ducklings produced in control and 0.5 ppm in diet were 16.8 ± 1.42 and 21.4 ± 1.3, respectively, in the Heinz et al. (2010a). These results are consistent with the 1 st generation results presented in Heinz (1979) where the mean number of 1 week old ducklings produced in the control was also greater than the 0.5 ppm in diet (25.4 and 30.1, respectively). Reproductive loss wasn’t observed until the 2 nd and 3 rd generations, and when the results of all three generations are combined. In both studies, females fed the 0.5 ppm diet consistently laid eggs containing approximately 0.8 ppm Hg ww. The authors and EPA identified the following differences in study design and results as possible explanations for the differences in the study results (Table A1-1).

The most environmentally relevant differences between the 1979 and 2010 studies is the number of generations tested and the duration of the Hg exposure in diet. In both studies, mercury dosing in the 1 st generation began before the breeding season with adult breeders. The 2 nd and 3 rd generation ducks in Heinz (1979), on the other hand, were first exposed to maternally deposited Hg during incubation and then fed a Hg spiked diet for a longer duration (initiated at 9 days old). The multigenerational exposure of Hg to mallards has not been replicated. The long term consistent exposure likely explains the differences in reproductive loss observed in the 2 nd and 3 rd generations, making the results from the 2 nd and 3 rd generations more environmentally relevant to resident bird species. Furthermore, Heinz et al. (2010a) also states that “the most likely explanation for the differences between the earlier studies and our current study is that a diet containing 0.5 ppm Hg as MeHg may fall right at the threshold between causing slight harm to the very most sensitive embryos versus possible benefit to the larger proportion of embryos in the experiment.”

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Table A1-1: Study design differences in the Heinz (1979) and Heinz et al. (2010a) controlled breeding experiments with mallard duck. Variability Source Heinz 1979 Heinz et al. 2010a Exposure duration 3 generations* 1 generation Source of test organism Potentially more sensitive strain Potentially less sensitive strain Mercury exposure MeHg dicyandiamide dissolved MeHg chloride dissolved in in propylene glycol (Heinz corn oil 1974) Hatching success: control 64.9%, 68.8%,63.8%; 57.5% (1 st , 2 nd , 3 rd generation; 65.9% combined) Hatching success : 0.5 in diet 69.3%, 59.8%, 60.6%; 71.8% (1 st , 2 nd , 3 rd generation; 59% combined) Mean number of 1 week-old 25.4, 50.9, 55.9; 16.8 ducklings: control (1 st , 2 nd , 3 rd 46 generation; combined) Mean number of 1 week-old 30.1, 36.3*, 43.5; 21.4* ducklings: 0.5 in diet (1 st , 2 nd , 37.5* 3rd generation; combined) * Note that it is not possible to compare the total number of ducklings produced across generations because the time period of egg collection differed, ranging from 18-21 weeks in Heinz (1979) to 6.5 weeks or45 days in Heinz et al. (2010a).

The EPA took the above variability in the Heinz mallard test results into consideration when evaluating potential effect benchmarks and determining the most appropriate statistical comparisons in the decision criteria. The EPA determined that the 0.5 ppm dw in diet and the associated egg concentration of 0.8 ppm fww are scientifically defensible benchmarks for the assessment given the following: • Although both the 1979 study and the 2010 study observed a slight benefit to reproduction in the first generation, long term effects of the 0.5 ppm exposure in diet resulted in significant reductions in reproduction, which represents an environmentally relevant exposure in a mercury contaminated environment. • Hatching success in the 2010 study was poor, creating uncertainty in the comparison of the 0 and 0.5 ppm exposures. It is possible that different effects between the treatments would have been observed if the hatching success was in the normal range (70-80%). • The authors conclude that a diet containing 0.5 ppm Hg, resulting in 0.8 ppm fww in eggs, likely represents a threshold where adverse effects begin to appear. • Hg in diet and eggs will be one of several lines of evidence the EPA is taking into consideration in its assessment of GSL. • GSL data will be assessed against the dietary and egg effect benchmarks without uncertainty factors that are typically applied when developing protective wildlife criteria to account for interspecies variability in sensitivity (see more on this in Attachment 1 Section A2. Egg)

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Effect Benchmark - Diet

Significant adverse reproductive effects observed in a multigenerational controlled breeding experiment support the use of an effect benchmark of 0.5 ppm MeHg dw in diet for the GSL mercury assessment (Heinz 1979). A mean concentration of Hg greater than 0.5 ppm dw in a known dietary component would be expected to result in egg concentrations associated with adverse reproductive effects. Use of a benchmark that is a dry weight concentration allows for the assessment of dietary data that differ in moisture content. Use of 0.5 ppm dw in the GSL mercury assessment is not precedent setting, for the Heinz (1979) LOAEC of 0.5 ppm was also used to derive the Hg avian wildlife criterion in the Great Lakes Water Quality Initiative (EPA/820/B-95/008) and wildlife risk assessment presented in the Mercury Study Report to Congress (EPA-452/R-97-008).1 To avoid a false positive outcome, this assessment will not include uncertainty factors that are used in the criteria setting process. This decision results in a dietary benchmark that is less stringent than the wildlife criteria adopted by the Great Lakes states and diet targets used in mercury TMDLs, such as the San Francisco Bay Mercury TMDL (diet target = 0.03 ppm ww per 3-5 cm fish).

1All reports utilized a LOAEC of 0.5 ppm with several uncertainty factors and site-specific bioaccumulation factors to derive avian wildlife values using methods published in 60 Fed. Reg. 15387 1995. Resulting wildlife criteria range from 530 – 1,300 pg/L total mercury. 26 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Table A1-2. Diet. Evers et al. (2004) Hg in diet risk thresholds for common loon compared to other diet effect values. Wet weight (ww) concentrations were estimated for studies that report dry weight (dw) concentrations with ww = dw/5. Thresholds are identified as total Hg (THg) or methyl Hg (MeHg) when reported by the author, and blank when the form of Hg is not reported.

Low Risk in Diet <0.05 Moderate Risk in High Risk in Diet MeHg ppm Diet 0.05-0.15 0.15-0.3 Extra High Risk in Diet Reference (ww) MeHg ppm (ww) ppm (ww) >0.3 ppm (ww) Species Study Type Chronic – reproductive effects 0.3-0.4 THg; reduced egg Field: Wabigoon- laying and site >0.4 THg; English River territorial reproductive common system, Ontario, Barr 1986 fidelity failure loon Canada 0.41 THg; common Field: 120 lakes in reproductive loon WI, New failure Brunswick, and Nova Scotia Burgess & Meyer 0.21 THg; (quantile 2008 EC50 5 productivity regression) Depew et al. 2012 0.18 MeHg; 0.4 MeHg; common Literature significant reproductive loon review/data reproductive failure analysis impairment: geometric mean of productivity & hatch success endpoints Heinz 1974 0.7 THg; LOAEC 53.5% reduction Mallard Controlled in 1 week old ducklings (estimated breeding: single from 3.4 dw) generation Heinz 1975, 1979 0.1 THg; LOAEC mallard Controlled 18 % reduction in breeding: 3 1 week old generations ducklings (estimated from 0.5 dw) Heinz & Hoffman 2.0 THg; 85% reduction in 1 week mallard Controlled 1998 old ducklings breeding (estimated from 10 dw)

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Low Risk in Diet <0.05 Moderate Risk in High Risk in Diet MeHg ppm Diet 0.05-0.15 0.15-0.3 Extra High Risk in Diet Reference (ww) MeHg ppm (ww) ppm (ww) >0.3 ppm (ww) Species Study Type Findley & Stendell 0.6 (estimated from 3.1 dw); black duck Controlled 1978 24% reduction in egg hatching & breeding: 2 79% reduction in duckling survival breeding seasons a Frederick & Jayasena 0.05 MeHg; LOAEC 13% white ibis Controlled 2011 reduction in egg productivity breeding: 3 breeding seasons 0.6 (estimated from 3.0 dw) Birds in Literature review Thompson 1996 impaired reproduction general 0.1 THg; lowest 0.25 THg; 1.2 THg; geometric mean of adverse Birds in Literature review b LOAEC estimated HC5 effect concentrations (range 0.1-10) general Shore et al. 2011 (7 species) 0.1 MeHg; Birds in Literature review reproductive general Chan et al. 2003 effects Chronic - other 0.4; immunity common Controlled suppression in loon breeding Kenow et al. 2007a chicks 0.55 MeHg; common Lab – spiked diet LOAEC reduced loon Kenow et al. 2010 motor (chicks) coordination 0.1 MeHg; common Literature midpoint of range loon review/data Depew et al. 2012 for adverse adult analysis behavior 0.5 THg; LOAEC great egret Controlled Behavior, growth, immune function, breeding Spalding et histological changes, biochemical al.2000a/2000b changes 0.4 MeHg; great egret Controlled LOAEC juvenile behavioral breeding c Bouton et al. 1999 changes Adams & Frederick 0.3 MeHg; NOAEC juvenile white ibis Controlled 2008 feeding behavior & efficiency feeding

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Low Risk in Diet <0.05 Moderate Risk in High Risk in Diet MeHg ppm Diet 0.05-0.15 0.15-0.3 Extra High Risk in Diet Reference (ww) MeHg ppm (ww) ppm (ww) >0.3 ppm (ww) Species Study Type Acute Kenow et al. 2003 1.5 THg; common Controlled NOAEC growth loon breeding or survival in chicks Spalding et al. 2000b 5 THg; acute great egret Controlled neurological breeding impacts Frederick et al. 2011 0.3 MeHg ww white ibis Controlled NOAEC adult feeding 3 yrs survival Shore et al. 2011 >6.0 MeHg; 10 MeHg; lowest Birds in Literature review predicted HC5 acute value general (11 species) Thompson 1996 10; Lethal Birds in Literature review general a % reductions = average reduction of the 2 breeding seasons b lowest LOAEC was obtained from Heinz (1979) c Authors report the dose concentration as 0.5 mg methyl HgCl/kg food (fish), which we translated to 0.4 MeHg ppm ww

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A2. Egg Toxicity studies that examine the effect of mercury on reproductive success frequently measure the concentration of mercury in the egg, in addition to mercury in diet and adult tissues. Mercury in egg tissue provides a measurement of the maternally deposited mercury and directly correlates with reproductive endpoints. When embryotoxic endpoints represent the most sensitive endpoint, egg tissue concentrations provide one of the most accurate predictors of potential effects, hence the reason the GSL Gilbert Bay selenium criterion was set to an egg-tissue based criterion.

Reductions in reproductive success and embryotoxic effects have been documented with controlled breeding experiments when adult females are fed MeHg spiked diets. Mercury literature reviews and site assessments frequently cite the Heinz studies that evaluated the effects of maternally deposited MeHg on reproductive success in mallards (Heinz 1974, 1975, 1976a, 1976b, 1979, and Heinz and Hoffman 1998, 2003). Heinz (1979) reported significant reductions in duckling survival to 1 week (18-29%) when eggs contained 0.8 ppm fww (see study details in dietary Section A1). Heinz (1979) identifies the egg concentration as wet weight (ww) concentration, yet given the timing of the sampling and Hg analyses, the 0.8 ww concentration is also equivalent to a fresh wet weight (fww) concentration (see additional details on percent moisture in eggs below).

An effect threshold of 0.8 ppm fww has been used in several mercury assessments and literature reviews that address impacts to bird species in diverse aquatic habitats (e.g., Henny et al. 2002, Hill et al. 2008, Thompson 1996, Shore et al. 2011), including eggs collected from waterfowl areas surrounding GSL (USFWS 2009a, 2009b ). Heinz and Hoffman (2003) later confirmed that the threshold for reproductive impairment in mallards is in the range of 0.8 – 1.0 ppm ww with a study designed to evaluate the lowest Hg concentration in eggs that will harm the most sensitive mallard embryos. In this study, the authors conclude that egg concentrations > 1.0 ppm ww may cause harm, and concentrations >2.0 ppm ww will cause harm to mallard ducklings. Although the egg concentrations in Heinz (1979) and Heinz and Hoffman (2003) are reported as THg, Heinz and Hoffman (2004) later report that 95% of the Hg in the albumen was in the form MeHg and 100% MeHg in the yolk, indicating that 95%-100% of the THg is MeHg.

A significant dose response is observed between the number of 1 week old ducklings and THg in egg when combining data presented in Heinz (1974), Heinz (1979), and Heinz and Hoffman (1998). Data presented in these studies were control normalized and analyzed with the toxicity relationship analysis program (TRAP V1.3a; see Table A.2-2 for model input parameters). The dose response is statistically significant when the individual generations populate the model (Figure A2.1.a; p = 0.03) and when the data from Heinz (1979) is entered as the mean of the 3 generations (Figure A2.1.b; p = 0.05). Although the slope of the toxicity relationship is significant, analyses on pooled datasets integrate within the study variability and between test variability, resulting in greater error and large confidence intervals when the slope is used to estimate low effect concentrations such as an EC10.

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140 A R2 = 0.76

120

100

80

60

40

Reduction in # of20 1wk old ducklings

0 -1.0 -.5 0 .5 1.0 1.5 Log(Egg THg) ppm fww

140 B R2 = 0.87 120

100

80

60

40

Reduction in # of20 1wk old ducklings

0 -1.0 -.5 0 .5 1.0 1.5 Log(Egg THg) ppm fww

Figure A2.1. The relationship between the number of 1 week old ducklings produced relative to the control (i.e., control normalized) presented in Heinz (1974), Heinz (1979), and Heinz and Hoffman (1998) when A) data are analyzed by generation (N=8) and B) when using an average response from the Heinz (1979) 3 generation study (N=6).

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The effect concentrations for common loon derived by Burgess and Meyer (2008) that are previously described in the dietary Section A1 can be translated to an expected egg Hg concentration by using the Hg in female blood and egg relationship for common loon as described by Evers et al. (2003). 1 Translating the common loon female blood EC50 and reproductive failure endpoints to egg concentrations results in an egg EC50 of 2.6 THg fww and reproductive failure when Hg in eggs equals 5.4 THg fww. Both of these values appropriately align with the extra high risk category as described by Evers (2003)(Table A2.2).

Henny et al. (2002), Hill et al. (2008), and Hoffman et al. (2009) present Hg data collected from Lahontan Reservoir located in the Lower Carson River System, Nevada. The Lower Carson River System is impacted by historic gold and silver ore mining and was listed as a Federal Superfund Site in 1990 due to the persistence and degree of Hg contamination in the watershed. Authors collected Hg in blood, histopathological, biochemistry, and dietary data from adults and nestlings, as well as Hg in eggs, and reproductive success data for double-crested cormorants ( Phalacrocorax auritus ), snowy egrets (Egretta thula ) and black-crowned night herons ( Nycticorax nycticorax ). Henny et al. (2002) presents reproductive success data collected in 1997 by comparing the number of young produced per nest when egg Hg concentrations were greater and less than the Heinz (1979) benchmark of 0.8 ppm fww. Snowy egret nests with eggs that ranged from 0.81-1.53 THg ppm fww produced 36% fewer young than nests with THg concentration ≤0.8 ppm, and 53% fewer young than the reference site. Night-heron nests with eggs ≥0.8 ppm fww (ranging from 0.81-1.81 ppm fww), on the other hand, produced a similar number of chicks. Histopathological and biochemistry data collected from adult birds suggest that adverse effects were not observed in adult birds; however, the data collected from young double-crested cormorants, night herons and snowy egrets suggest they experience some degree of neurological and histological damage associated with excessive exposure to dietary mercury.

Hill et al. (2008) presents 10 years (1997-2006) Hg data for snowy egret and black-crowned night-heron collected form Lahontan Reservoir, NV and an evaluation of the relative role of drought and Hg on their reproductive success. The ten year study period includes data collected under variable flow conditions including drought and flood conditions, whereas the Henny et al. (2002) publication only presents data collected from relatively wet years (1997 & 1998). Hill et al. (2008) presents significant positive relationships between annual water discharge and clutch size and number of young per nest for black- crowned night-heron and positive, yet non-significant, relationships for snowy egret. Snowy egret nesting failed during peak drought years. The relative importance of elevated Hg and drought on the snowy egret reproductive success (i.e., food availability and nest exposure to predators) can be challenging to allocate. Assuming all snowy egret nests with >0.8 ppm THg failed because of THg, Hill et al. (2008) was able to quantify the contribution of THg to the overall percent nest failure for each drought year (% THg loss is in parentheses): 2000-57.1% (13.3%); 2001-93%(13.3%); 2002- 85.7%(60%); 2003-76.5%(20%); and 2004-23.5%(0%). The 5 year average nest failure rate due to THg concentration in eggs was estimated to be 21.3%. Even more dramatic effects are observed when the mean number of young/nest is compared between nests with eggs > or < 0.8 ppm fww. Nests with eggs containing THg > 0.8 ppm fww consistently produced fewer young than nests with THg <0.8 ppm fww, with reduction ranging from 28-100% fewer young produced per nest.

To summarize the Lahontan Reservoir Hg studies, results presented by Henny et al. (2002) and Hill et al. (2008) document reproductive loss in snowy egret when egg THg concentrations exceed 0.8 ppm. Reproductive loss was not observed in black-crowned night herons, which are believed to be less

1 Hg in female blood =(1.5544*egg concentration)+0.2238 32 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary sensitive to Hg. Additionally, the combination of drought and Hg is more stressful than Hg alone and drought may exacerbate the Hg-induced reductions in productivity for sensitive taxa. This conclusion is also supported by young snowy egret blood, organ biochemistry and histopathology data presented by Hoffman et al. (2009) that showed more severe Hg-related adverse effects during drought years.

A field study on mercury in Forster’s tern in San Francisco Bay suggests that are similar in sensitivity to mercury to the mallard (Eagles-Smith and Ackerman 2010). Effects of mercury on Forster’s tern were evaluated by examining the differences in egg mercury among viable eggs and eggs that failed to hatch, successful and unsuccessful nests, and the influence of mercury on the hatching success of an individual egg. Results indicated that 1) eggs collected from abandoned nests had significantly higher Hg concentrations than eggs that failed to hatch or were randomly collected and 2) unsuccessful nests had significantly higher THg concentrations (1.6 ±0.2 ppm fww) than successful nests (0.95±0.08 ppm fww).1 Authors utilized the field data to model the probability of a successful nest and hatching as mercury in eggs increased. These models suggest that a 10% reduction in egg hatchability and 18% reduction in nest survival would be expected with mercury in Forster’s tern eggs equal to 0.88 ppm THg fww. This egg concentration is within the range of effect values (in ww) that have been reported for the mallard.

The literature review conducted by Shore et al. (2011) identified egg mercury concentrations associated with no adverse effects for 15 species that ranged from 0.07-1.6 ppm (geometric mean = 0.4 ppm) and adverse effect concentrations for 10 bird species that ranged from 0.8-5.1 ppm THg ww (geometric mean = 1.9 ppm). The predicted HC5 was determined to be an egg concentration greater than 0.6 ppm.

Risk Categories - Egg

The rationale for the thresholds used to derive the Evers et al. (2004) egg risk categories (Table A1-2) is provided in Evers et al. (2003) . The 2003 study evaluated mercury concentrations in 577 eggs collected from eight states that vary geographically and in their expected egg mercury concentrations (AK, MA, MI, MN, MO, NH, NY, and VT). The authors also considered effect concentrations that have been derived from different field and laboratory analyses. The egg risk categories are described as follows.

• Low risk: 0-0.6 ppm. This range reflects egg concentrations expected in natural environments that are not influenced by enhanced local geological sources, nor from enhanced anthropogenic deposition. The 0.6 ppm ww threshold was selected since Barr (1986) reported reproductive impairment in loons with egg concentrations that exceed 0.6 ppm ww and the Thompson (1996) literature review indicates no adverse effects have been observed at egg concentrations < 0.5 ppm. • Moderate risk: 0.6-1.3 ppm. Eggs containing mercury within this range may be at risk of reproductive impairment for some avian species. • High risk: 1.3 – >2.0 ppm – Adult female loons with blood concentrations >3.0 ppm often exhibit reduced reproductive success. Female adult loons with blood >3.0 ppm are expected to lay eggs with mercury that is > 1.3 ppm. Furthermore, eggs with mercury concentrations >1.3 were significantly smaller than eggs in the low risk range.

1 The authors used a non-destructive microsampling technique when collecting the egg mercury data. All data were reported as fresh egg wet weight (fww), which was determined by dividing the total mass (ww) of the egg content at processing by the predicted fresh egg mass (ww) at laying, and multiplying that value by the Hg concentration (ww). 33 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

• Extra high risk: >2.0 ppm. Thompson (1996) threshold that is considered to have adverse effects in most bird species.

Uncertainties Associated with Assessing Hg in Eggs

Total vs. Methylmercury in Eggs

Similar to the diet risk categories, Evers et al. (2004) identifies the risk threshold concentrations as ppm MeHg ww. Methylmercury data are limited for eggs collected from GSL; however, the literature suggests that Hg in field collected eggs tends to be primarily in the form of MeHg, generally ranging from 75-100% of the THg (Barr 1986, Scheuhammer et al. 2001 , Evers et al. 2003, Heinz and Hoffman 2004, Ackerman et al. 2013 ). Ackerman et al. (2013) presents a comprehensive review of THg and MeHg concentrations in bird eggs. The average percent MeHg for 22 species of birds was 96%. The authors determined the range in percent of THg in the MeHg form was not related to THg concentration in eggs, foraging , nor to a species life history strategy. Given these results, we determined it would be appropriately conservative and reasonable to assume that 100% of the Hg in eggs collected from GSL is in the form of MeHg in this assessment.

Moisture Content in Eggs

Embryo respiration and evaporative water loss reduce egg weight and moisture content as incubation progresses. The total loss of water by the end of incubation can equal 16 % of the initial weight (Drent 1975). Therefore, it is important to understand when the egg was sampled, sample processing, and how the moisture content of the egg was taken into consideration in the results when analyzing egg data and comparing results to effect benchmarks. Authors will report egg Hg concentrations as dry weight (dw), wet weight (ww), and fresh wet weight (fww). Laboratories typically report tissue results as grams of Hg per gram of tissue ww, if the sample was not dried prior to analysis or dw, if the sample is desiccated or freeze dried prior to analysis. Dry weight concentrations can be converted to ww using the % moisture of the sample with the following equation:

dw concentration = ww concentration x______100______(100 - % moisture)

A fww result represents the concentration of a parameter in a freshly laid egg. When Hg analyses are conducted with freshly laid eggs, the ww result is equivalent to a fww result and supports the determination that the Heinz (1979) 0.8 ppm ww LOAEC is equivalent to a fww concentration. Authors have typically used two different methods to determine the fww concentration if there is a time lapse from when the egg was laid, collected, and processed for Hg analysis. First, a fww concentration can be determined at the time of analysis by refilling the air cell in the egg with water prior to determining the egg moisture content and Hg analysis (Hoyt 1979). Alternatively, fww concentrations can be calculated from the total mass (ww) of the egg content at the time of processing and the predicted fresh egg mass (ww) at laying (Stickel et al. 1973, Hoyt 1979, Ackerman et al. 2015). Water loss from an egg will be influenced by relative humidity, egg temperature, and shell porosity (Drent 1975). The moisture content of field collected eggs can typically range from 65-80% (Ohlendorf and Hothem 1995, Ohlendorf and Heinz 2011), with the greatest moisture content observed in freshly laid eggs. Field collected eggs can also include nearly dry eggs if the sampling protocol does not preclude collection of eggs from abandoned nests. Presentation of Hg in egg data as ww can limit data 34 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary analyses since datasets typically include multiple species, incubation stages and years. Unless an egg is sampled immediately after laying, ww concentrations will be biased high when compared to a fww benchmark. Use of Hg in egg data presented as dw and comparing those values to a dw benchmark is another desirable option since weight loss during incubation is almost entirely due to water loss (Drent 1975). To avoid bias due to moisture content of the egg when it was sampled, the mercury assessment should prioritize available GSL egg data as follows: • Fresh wet weight concentrations (including those determined by calculation) will be given priority when available; • Dry weight concentrations will be given second priority when the results are presented as both ww and dw or % moisture data are provided to convert the ww to a dw result; • When only ww concentrations are available (data are not available to estimate a dw or fww concentration), ww concentrations will be treated as equivalent to a fww concentration. To convert the fww egg benchmark to a dw benchmark, the EPA used a 70% moisture content (Stanley et al. 1996, Ohlendorf and Heinz 2011).

Exposure in Migratory Birds

Egg mercury concentrations have been found to correlate well with mercury in adult blood, newly hatched chick blood and down feathers in common loon, mallard, and Forster’s tern (Evers et al. 2004, Heinz et al. 2010b, Ackerman and Eagles-Smith 2009 ). Correlations of egg mercury concentrations with mercury in the local environment (diet and water), on the other hand, can be limited by the amount of time adults spend feeding in the nesting area prior to laying eggs ( Barr 1986 ). There continues to be uncertainty as to how long individual birds have resided at GSL prior to laying eggs. We expect there to be differences in time spent foraging at the lake within and among the migratory species that nest at GSL, however, controlled feeding experiments suggest that mercury in eggs can increase and decrease rapidly if exposure were to change prior to or during the nesting season ( Heinz and Hoffman 2004 ).

Mercury-selenium Interaction

UDEQ’s draft GSL Hg assessment and final selenium report (UDEQ 2008 & 2010) discuss the uncertainty with assessing mercury and selenium (Se) data without recognizing potential antagonistic effects when these toxicants occur together at high concentrations. Frequently, field collected adult animals that have been found with the highest THg concentrations, and no apparent Hg toxicity, also have high Se concentrations and a low % MeHg (Kim et al. 1996, Scheuhammer et al. 2007, Conover and Vest 2009 , Wurtzbaugh et al. 2011 ). Results from field and lab studies indicate that Hg-Se interactions can alter mercury toxicity (Cuvin-Aralar and Furness 1991, Wiener et al. 2003, Shore et al. 2011). Proposed mechanisms of the protective effect of selenium against mercury toxicity include 1) redistribution of Hg in the presence of Se, 2) competition for binding sites, 3) formation of nontoxic Hg- Se complexes, 4) conversion of toxic forms of Hg into other forms of Hg, and 5) prevention of oxidative damage (Cuvin-Aralar and Furness 1991). The majority of publications that present data in support of antagonistic effects have focused on mammals, fish, and adult birds (both aquatic and terrestrial birds). It is less clear if Hg-Se interactions can influence reproductive success or mercury toxicity in embryos or young birds, which represent the most sensitive endpoint for the GSL assessment, and in some situations, synergistic effects have been observed in early life stages.

For example, embryos produced in a controlled breeding experiment where adults were fed a diet containing MeHg chloride and seleno-DL-methionine had a greater frequency and number of 35 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary deformities than the embryos produced from the adults fed only Se or Hg (Heinz and Hoffman 1998). Results from this study suggest that the occurrence of both Se and Hg may be synergistic to young birds.

Additionally, Heinz et al. (2012) and Klimstra et al. (2012) conducted a series of egg injection studies with MeHg and Se alone and in combination to evaluate potential interactions that could occur during the embryo development phase. Authors injected commercially produced mallard and chicken eggs and field collected double-crested cormorant ( Phalacrocorax auritus ) eggs with different combinations of MeHg chloride and seleno-L-methionine. The type and frequency of deformities and hatching success between the treatments were documented. Both antagonistic and synergistic effects were observed. MeHg appeared to decrease embryo mortality caused by Se, yet increase the number and types of deformities. In other words, deformed chicks that would normally die prior to hatching would hatch. Authors conclude that additional studies are necessary to understand the biochemical mechanisms underlying these results and to determine if the same interactions occur with maternally-deposited Se and Hg.

Results from the limited studies that evaluated Hg-Se interactions during early life stages do not indicate that selenium protects against the adverse effects of Hg on embryo development or reproductive endpoints. Given EPA’s assessment is focusing on potential reproductive effects due to mercury, EPA did not determine it necessary to take selenium concentrations into consideration when evaluating Hg in diet or eggs. It may be appropriate to consider selenium data when evaluating adult blood or liver data (see an additional discussion of this in the liver section).

Relative Sensitivity of Aquatic Bird Embryos

To evaluate the relative sensitivity of aquatic wildlife and determine the appropriateness of a mallard Hg toxicity endpoint for the GSL assessment, EPA took into consideration the relative sensitivity of birds observed in egg injection studies and did a meta-analysis of all Hg related effects that are broadly observed in birds. Both of these analyses included Hg toxicity data for all birds, including terrestrial birds.

Egg injection studies have recently been conducted as an alternative to controlled feeding experiments to evaluate the differences in sensitivity of avian embryos to mercury and to validate field-derived toxicity thresholds (Heinz et al. 2009, Kenow et al. 2011, Braune et al. 2012 ). Test species have focused on wild birds that do not perform well in controlled laboratory feeding experiments. The egg injection protocol is described in Heinz et al. (2006) and includes the collection of viable eggs from the field, injecting the eggs with various doses of MeHg dissolved in corn oil, sealing and incubating the eggs, and tracking hatching success (embryos surviving to 90% incubation). Heinz et al. (2009) tested 26 species of birds and had adequate data to calculate median lethal concentrations (LC50s) for 23 species. Kenow et al. (2011) injected common loon eggs with environmentally relevant concentrations of mercury. The Kenow study adapted the Heinz et al. 2006 method for field use (natural incubation with hatching success endpoint) and to minimize potential toxicity from the solvent.1 Braune et al. (2012) also

1 Common loon eggs in the Kenow et al. (2011) experiment were injected at a rate of 0.5 ul/g egg content rather than 1.0 ul/g, as suggested to be acceptable by Heinz et al. (2006). The authors modified the dosing approach in response to a suggestion that the 1.0 µg/L of corn oil/g egg might have some toxicity itself to some embryos (percent survival in the injected control group was 92.9% and 89.3% for the eggs injected with 0.5 ul of corn oil/g versus 1.0 ul corn oil/g, respectively. These differences were not statistically different.

36 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary followed the Heinz et al. (2006) method with a few minor modifications and evaluated the sensitivity of the thick-billed murre (Uria lomvia ) and Arctic tern (Sterna paradisaea ) eggs.1

The results of the egg injection studies are difficult to translate into environmentally relevant egg effect concentrations. Heinz et al. (2009) compared the toxicity of injected Hg to the toxicity of maternally deposited mercury in mallard, chicken and ring-necked and concluded that the injected MeHg is more toxic than maternally deposited MeHg. In other words, one would expect greater LC50s from toxicity tests that rely exclusively on maternally deposited Hg. However, Heinz et al. (2009) also stated that the relative sensitivity, or species sensitivity distribution (Figure A2.2), may provide an environmentally relevant range in the expected avian sensitivity since the mallard, chicken and ring- necked pheasant embryos stay in the same relative order of sensitivity regardless of the mercury origin.

Mercury injected LC50s

10

American avocet

Double-crested cormorant Common loon Mallard

Brown pelican 1 Artic tern Canada Goose LC50 (ppmww) LC50

Embryo Survival Embryo Thick-billed murre Royal tern Herring gull Tri-colored heron Snowy egret White ibis 0.1 0 0.2 0.4 0.6 0.8 1 Percentile

Figure A2.2: The relative sensitivity of several wild bird embryos to injected methylmercury. Aquatic birds relevant to the GSL ecosystem are identified. ♦ = Heinz et al. (2009), ○ = Kenow et al. (2011) and □ = Braune et al. (2012) data. Although the methods used by the authors were slightly different and represent different endpoints, we determined it was still appropriate to compare the LC50s from all studies.

1 Thick-billed murre and Arctic tern eggs were also injected at a rate of 0.5 ul/g egg content. Braune et al. (2012) deviated from the Heinz method by using safflower oil as a solvent rather than corn oil and AirPore Tape instead of a hot glue gun to seal the injection hole. 37 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

We expanded the Heinz et al. (2009) analysis of the egg injection species sensitivity distribution to include new studies and field data to inform our decision on the magnitude of the egg effect benchmark. This analysis was needed since 1) benchmarks and risk thresholds for this assessment are derived from mallard and common loon studies and 2) the injection studies suggest that the mallard and common loon exhibit relatively low sensitivity to injected mercury compared to other birds that are expected to occur at GSL or could represent birds expected to occur at GSL (Figure A2.2). Table A2-1 presents the egg injection derived LC50s with the summary of the maternally deposited studies presented in Heinz et al. (2009), the NOAECs presented in Shore et al. (2011; Table 18.1), and new studies not presented in the previous two publications. We did not include the adverse effect concentrations presented by Shore et al. (2011) since the magnitude of the effect associated with the egg concentration varied, making it challenging to do inter-species comparisons. Our analysis included species beyond aquatic wildlife since inclusion of these species will inform the environmental relevance of the sensitivity distribution. Comparisons were made at the species level when possible, however data were also compared at the genus level for tern and pelican, which assumes that species in the same genus would have similar sensitivity to Hg.

A comparison of NOAECs suggests that birds with low injected LC50s also had some of the lowest NOAECs; however, this provides little information on the relative sensitivity of these species since NOAECs are either a product of the exposure study design or the uncontrolled exposure from field conditions, with the latter being more likely since most of the results are field studies. Comparisons of maternally deposited egg concentrations that result in adverse effects is also less than ideal given the differences in study design, endpoints measured, and level of effects that are observed; however, we attempted to compare endpoints with similar environmental relevance. We were especially interested in studies conducted with birds that represent the most sensitive species in the species sensitivity distribution and that nest in the GSL ecosystem, such as the white ibis. We were unable to locate mercury embryo toxicity studies for the white ibis. Rather, existing mercury toxicity studies focus on adult behavioral effects that reduce nesting success and productivity. A controlled breeding experiment that tested embryo sensitivity is however available for the American kestrel (Albers et al. 2007), which exhibits the same sensitivity to injected mercury as the white ibis. Albers et al. (2007) reported a 16% reduction in egg hatchability and 41% reduction in the number of nestlings fledged when mercury in egg was 2 ppm ww. The 2 ppm egg concentration was associated with the lowest mercury dose in the experiment (exposure in diet = 0, 0.7, 2.0, 3.3, 4.6, 5.9 ppm dw). The next most sensitive species with endpoints derived from a controlled feeding experiment is the ring-necked pheasant, which reports a 46 % decrease in hatching success when mercury in eggs is 1.5 ppm ww. Comparison of the results from kestrel and pheasant studies where the mercury was maternally deposited suggests that pheasants may be more sensitive to mercury than kestrels (16% hatch reduction when egg = 2.0 vs. 46 % hatch reduction when egg = 1.5 for kestrel and ring-necked pheasant, respectively).

38 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Table A2-1. Comparison of mercury injected LC50s (Heinz et al. 2009, Kenow et al. 2011, Braune et al. 2012 ) to egg effect concentrations that were maternally deposited (field and controlled breeding experiments), including no observed adverse effect concentrations (NOAEC). Several birds with injection LC50s studies are not presented because we were unable to locate egg effect thresholds when the Hg was maternally deposited. Common Genus Injected Maternally Maternally deposited threshold Reference Name LC50s deposited endpoint (ppm ww) threshold (ppm) Ibis Plegadis 0.12 0.2 ww NOAEC Henny and Herron 1989, as cited in Shore et al. 2011 Kestrel Falco 0.12 0.08 ww NOAEC Albers et al. 2007 2 ww Controlled exposure: 16% Albers et al. 2007 reduction in # eggs hatched and 41% reduction in nestlings fledged Egret Egretta 0.15 >0.8 fww Field: 21% nest failure rate and 27- Hill et al. 2008 100% reduction in # young/nest during drought Egret Egretta 0.15 0.8- fww Field: 36-51% reduction in # Henny et al. 2002 1.53 young/nest Herring Larus 0.28 0.51 ww NOAEC Gilman et al. 1977, as gull cited in Shore et al. 2011 Tern Sterna 0.4-0.95 0.54 fww Field: 10% reduction in Forster's Eagles-Smith and tern nest survival Ackerman 2010 0.9 fww Field: 10% reduction in Forster's Eagles-Smith and tern egg hatchability; 18% Ackerman 2010 reduction in nest survival Ring- Phasianus 0.44 1.5 ww Controlled exposure: 46% decrease Fimreite 1971, as cited necked in hatching success in Heinz et al.2009 pheasant Common Sterna 0.87 1 ww NOAEC Eisler 2004, Fimreite Tern 1974, as cited in Shore et al. 2011 2.4 ww Field: 73% hatch reduction Fimreite 1974 Pelican Pelecanus 0.89 0.47 ww NOAEC for white pelican Wiemeyer et al. 2007 Chicken Gallus 1.5* 5 ww Controlled exposure: 72% decrease Tejning 1967, as cited in in hatching success Heinz et al. 2009 10 ww Controlled exposure: 83% decrease Tejning 1967, as cited in in hatching success Heinz et al. 2009 Mallard Anas 1.79 0.8 fww Controlled exposure: 18% Heinz 1979 reduction in 1-week old ducklings over 3 generations 5.5 ww Controlled exposure: 55% Heinz et al. 1974, as reduction in egg hatching cited in Heinz et al. 2009 16 ww Controlled exposure: 75% Heinz and Hoffman reduction in hatching success 1998, as cited in Heinz et al. 2009 Common Gavia 1.78 1 ww NOAEC (field) Barr 1986, as cited in loon Shore et al. 2011 2-3 ww Field: reduction in egg laying Barr 1986 2.6 fww Field: 50% reduction in chick Burgess and Meyer productivity 2008; Evers et al. 2003 *Injection LC50 estimated from data presented in Figure 1 in Heinz et al. (2009) since the LC50 appears to contain a typo. Estimated value is further supported by data presented in the text, which states a 68% decrease in hatching was observed when chicken eggs were injected with 1.6 ppm Hg.

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It is interesting to note that the injected LC50s for terns (Royal tern, common tern and Arctic tern) are in the same range as the egg concentrations that resulted in reduced egg hatchability and nest survival observed with the Forster’s tern (Eagles-Smith and Ackerman 2010). The magnitude of the effect observed in the field (10-18% reduction) is less than the 50% reduction modeled by the LC50, however it is unlikely that laboratory derived effect concentrations will translate to the exact levels of effect that will be observed in a natural nesting environment.

In summary, it is clear that the environmental relevance of the injection studies is still to be determined. At this time we are unable to determine if the species sensitivity distribution resulting from the injection studies is useful for estimating the relative ranking of embryo sensitivity of wild birds that do not perform well in controlled laboratory feeding experiments. Additional controlled feeding and field studies that measure the effect of maternally deposited mercury on embryos are needed to determine the true range of avian mercury sensitivity. Heinz et al. (2009) concludes that “using the threshold value for impaired reproduction of 0.8-1.0 ppm mercury, derived from controlled breeding studies with game farm mallards might not provide complete protection for many species of wild birds…however, because only a very small percentage of mallard embryos were actually harmed when eggs contained 0.8-1.0 ppm mercury, this range (Heinz and Hoffman 2003), if used as a default to protect the embryos of a wild bird, might provide protection against a high percentage of mortality among embryos, even though it might not protect the very most sensitive individual embryos.”

The EPA also evaluated the avian toxicity dataset by expanding the maternally deposited Hg reproductive effect dataset in Table A.2.1 to include all measured reproductive effects and the associated egg concentrations. The analysis included toxicity data with quantified effects for all bird species (including terrestrial birds and those without injection LC50s), exposures, and reproductive endpoints. If a field study identified an ambient concentration as NOAEC, we assumed the reproductive success or chick productivity was equal to 100%. The data were sorted and graphed the following ways: all data (Figure A2.3.); data with similar endpoints (e.g., nesting success and hatchability vs. nestlings/chicks produced per nest/female; results are not presented); and only aquatic birds (Figure A2.4). Data that measured reproductive loss with a threshold (e.g., snowy egret data in Henny et al (2002) and Hill et al. (2009)) were not included in the analysis since we were unable to calculate the mean Hg in egg concentration associated with the increased reproductive loss. We are not presenting the regression analyses calculated in TRAP since the dataset contains sources of variability (i.e., inter-species variability in X 50 – 50%reduction in the effect variable) that violates the underlying assumption of the model that there is a single true X50 .

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140 black duck 130 black-crowned night -heron 120 chicken 110 100 common loon 90 common tern 80 Forester's tern 70 herring gull 60 ibis 50 40 kestrel Reproductive Success Reproductive 30 mallard 20 ring-necked pheasant 10 white pelican 0 0.01 0.1 1 10 Egg THg ppm ww

Figure A2.3. All quantified reproductive loss endpoints and the associated THg in egg concentration for all bird species (terrestrial and aquatic). Field derived NOAECs were assumed to have 100% successful reproduction. Values that are >100% identify the mallard exposures that performed better than the controls in controlled breeding experiments.

The data presented in Figure A2.3 clearly shows that adverse reproductive effects are observed when egg concentrations exceed 1.0 ppm ww. NOAECs were not observed at egg concentrations that exceed 1.0 ppm ww. Reductions in reproductive success are first observed at 0.5 ppm ww in the Forster’s tern. Reductions in productivity are observed when the terrestrial species are removed from the dataset, and for multiple endpoints that vary in their sensitivity (Figure A2.4).

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140 130 A 120 110 black duck 100 90 black-crowned night-heron 80 common loon 70 common tern 60 herring gull 50 40 ibis 30 mallard

% chick productivitychick % 20 white pelican 10 0 0.01 0.1 1 10 Egg THg ppm ww 110 B 100 90 black duck 80 black-crowned night-heron 70 common loon 60 common tern 50 Forester's tern 40 herring gull 30 ibis % egg% hatchability

% nest survival and/or and/or survival % nest 20 mallard 10 white pelican 0 0.01 0.1 1 10 Egg THg ppm ww Figure A2.4. The reproductive success and associate THg in egg concentrations for aquatic and aquatic dependent birds. Figure A presents chick productivity measured at the number of young produced per nest/breeding pair (field observations) or per female (controlled breeding exposures) compared to a control. Figure B presents reductions in nest survival and hatchability with the associated THg concentration. The field derived NOAECs for the black-crowned night-heron, common loon and common tern overlap at 1.0 ppm ww. The Heinz (1979) data were entered as individual generations rather than the mean of 3 generations.

Effect Benchmark - Egg

Controlled breeding experiments, laboratory data and reductions in reproductive success observed in field studies support use of an egg effect benchmark of 0.8 ppm THg fww in the GSL mercury assessment. A bird population where the majority of the eggs exceed 0.8 ppm is likely to experience adverse reproductive effects that would impair the GSL waterfowl designated use. To avoid a false positive outcome, this assessment will not include uncertainty factors that are used in the criteria setting 42 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary process. To avoid bias due to moisture content of the egg when it was sampled, the mercury assessment should prioritize available egg data as follows: • Fresh wet weight concentrations (including those determined by calculation) will be given priority when available; • Dry weight concentrations will be given second priority when the results are presented as both ww and dw or % moisture data are provided to convert the ww to a dw result; • When only ww concentrations are available (data are not available to estimate a dw or fww concentration), ww concentrations will be treated as equivalent to a fww concentration.

43 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Table A2-2. Egg. Evers et al. (2004) Hg risk thresholds for egg compared to other egg effect values. Values are identified as methylmercury (MeHg) or total mercury (THg), when reported by the authors, and blank when author does not report the form of Hg. All values are wet weight (ww), unless identified as fresh weight (fw) or fresh wet weight (fww). Low Risk in Eggs <0.5 Moderate Risk in Eggs High Risk in Eggs Extra High Risk in Eggs MeHg 0.5 – 1.3 MeHg 1.3 -2.0 MeHg >2.0 MeHg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type Chronic – reproductive effects Barr 1986 common loon Field: Wabigoon- 2.0-3.0 MeHg; reduced egg English River laying and site territorial fidelity system, Ontario, Canada Burgess and 2.6 THg fww: 5.4 THg fww; common loon Field: 120 lakes in Meyer 2008; 50% reduction reproductive WI, New Evers et al. in productivity failure Brunswick and 2003 Nova Scotia (quantile regression)e Eagles- 0.54 THg fww; 0.9 THg fww; Forster's tern Field: San Smith and 10% reduction 10% reduction Francisco Bay, Ackerman in nest survival in egg CA 2010 hatchability and 18% reduction in nest survival Fimreite 2.4 MeHg (3.65 THg);73% common tern Field: 1974 hatch reduction Northwestern Ontario d Heinz 1974; 5.5 THg; 53.5% reduction in 1 mallard Controlled Heinz et al. week old ducklings breeding 2009 Heinz 1979 0.83 THg; mallard Controlled LOAEC 18% breeding: average reduction in 1 of 3 generations a week old ducklings Heinz and 16 THg; 85% reduction in 1 mallard Controlled Hoffman week old ducklings breeding 1998

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Low Risk in Eggs <0.5 Moderate Risk in Eggs High Risk in Eggs Extra High Risk in Eggs MeHg 0.5 – 1.3 MeHg 1.3 -2.0 MeHg >2.0 MeHg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type Finley & 5.11; 24% reduction in egg black duck Controlled Stendell hatching and 79% reduction in feeding: average 1978 duckling survival of 2 breeding seasons b Wiemeyer et 0.18 – 0.47; American Field: Pyramid al. 2007 no signs of white pelican Lake, NV impact Henny et al. 0.81-1.53 THg; 36-51% reduction snowy egret Field: Lahontan 2002 in # young/nest Reservoir, NV, 1997&1998 Hill et al. >0.8 THg; 21.3% average nest snowy egret Field: Lahontan 2008 failure rate during drought Reservoir, NV, 10 (range = 0-60%), 28-100% year analysis reduction in # young/nest Hill et al. >0.8 THg; black-crowned Field: Lahontan 2008 NOAEC normal night-heron Reservoir, NV, 10 chick year analysis productivity Eisler 1987 <0.9 to 2.0; fw Birds in Literature review adverse effects/proposed criteria general Thompson < 0.5; little > 2.0; some Birds in Literature review 1996 detrimental detrimental general effect effects Shore et al. >0.6 THg; 0.8 THg; lowest 1.9 THg; Birds in Literature Review 2011 predicted HC5 adverse effect geometric general concentration mean of (10 species) adverse effect concentrations (range=0.8- 5.1) Chronic - other Evers et al. 1.3-2.0 THg; common loon Multi field study 2003 significantly analysis/ literature decreased egg review volume

45 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

Low Risk in Eggs <0.5 Moderate Risk in Eggs High Risk in Eggs Extra High Risk in Eggs MeHg 0.5 – 1.3 MeHg 1.3 -2.0 MeHg >2.0 MeHg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type Ackerman et 0.65 fww; American Field: San al. 2014 demethylation avocet, black- Francisco Bay, threshold necked stilt, CA c Caspian tern, Forster’s tern Heinz and 1.0 THg; lowest concentration >2.0 THg; likely neurological mallard Controlled Hoffman associated w/ deformed embryo effects in ducklings feeding 2003 Shore et al. 0.4 THg; geometric mean of Birds in Literature review 2011 NOAECs (range=0.07-1.6) general (15 species) a % reduction = average of 3 generations. The mean number of 1 week old ducklings produced in the control groups for the 1 st , 2 nd and 3 rd generation was 25.4, 50.9, and 55.9, respectively, whereas the Hg dosed group produced 31.1, 36.3 and 43.5 ducklings. When all three generations are combined, the average number of ducklings produced in the control and mercury treatments was 46.0 and 37.5, respectively. b % reductions = average reduction of the 2 breeding seasons. The mean hatchability in the control for the 1 st and 2 nd breeding season was 72% and 57% whereas the % hatchability in the mercury treatments was 44 and 52%, respectively. The mean number of ducklings surviving to 1 week in the controls was 39 and 34 and 5 and 11 in the mercury groups, respectively. c the concentration where demethylation is initiated, translated from the liver demethylation threshold presented by Eagles-Smith et al. (2009b) d The 73% reduction in hatching assumes the potential success rate is 100%. Fimreite determined a 27% hatching success (30 of 111 eggs) and 10-12 percent fledging success rate in Ball Lake. Normal productivity was observed in the control lake, Wabigoon Lake, which had a mean THg and MeHg concentration of 1.0 and 0.82 ppm ww, respectively. e The Burgess and Meyer (2008) quantile regression derived effect concentrations for female blood translated to an egg concentration using the relationship described in Evers et al. (2003).

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A.3 Blood - Adult

Blood is frequently sampled to evaluate recent dietary exposure of newly hatched chicks and adult birds to Hg. Blood is a desirable matrix to sample since it can be sampled with non-lethal sampling techniques, reducing the impacts to the bird population of interest. Mercury in blood typically correlates well with the concentration of mercury in the local diet, other internal tissues, and eggs (Kahle and Becker 1999 , Heinz et al. 2010b), making it a good indicator of potential reproductive success and a meaningful indicator for the GSL assessment.

The half-life of mercury in blood varies with species and life stage and can range from a few days in chick blood (e.g., 3-6 days) to months for juveniles and adults ( Fournier et al. 2002 ). For chicks, mercury concentrations in blood are expected to vary over a short period of time, with highest concentrations expected immediately after hatching due to maternally deposited Hg into the egg, which is then reduced by mass dilution and Hg depuration into feathers ( Ackerman et al. 2011 ). Furthermore, nearly 100% of the Hg in blood is typically MeHg (Fournier et al. 2002, Henny et al. 2002 ), the most bioavailable and toxic form of mercury, making it a good media for evaluating potential effects.

Compared to the diet and egg indicators, we located relatively few studies that report the adult blood Hg concentration with adverse reproductive effects (Table A3). The three year white ibis study is the only controlled breeding experiment that presents the Hg in adult blood (see additional study details in A.1 Diet). Heinz et al (2010b) presents mallard data that suggests a one-to-one ratio would be expected in adult female blood and Hg in eggs. Two field studies on the common loon also report Hg in adult blood with adverse effect concentrations.

The Burgess and Meyer (2008) quantile regression analysis modeled a 50% reduction in chick productivity and reproductive failure when female concentrations are equal to 4.3 and 8.6 ppm ww, respectively. These results confirm that loons with Hg concentrations in the Evers et al. (2004) extra high risk blood category would be expected to have significantly reduced chick productivity (see discussion in Section A1-1 for more details on the Burgess and Meyer analyses). An additional study by Evers et al. (2008) reported a 41% reduction in reproduction when blood mercury is >3.0 ppm, suggesting that significant reductions in productivity would also be expected for loons with mercury in the high risk category.

Risk Categories – Adult Blood

Evers et al. (2004) present risk thresholds for adult and juvenile blood. Although mercury in chick blood correlates well with reproductive success, chick blood data have not been collected from GSL nesting species. Therefore, we did not do an extensive review of the chick blood risk categories, nor did we attempt to summarize literature effect concentrations for mercury in juvenile blood. For the adult risk categories, Evers et al. (2004) derived the thresholds from blood hormone analyses and confirmed the threshold with field observations (Table A3). Significantly greater mean corticosterone levels were observed in the moderate, high and extra high risk group categories when compared to the low risk category. 1 The adult blood risk categories are described as follows. • Low risk: 0 - < 1.0 ppm. Reference condition. • Moderate risk: 1.0 - <3.0 ppm. Adult loons with 1-2 and 2-3 ppm THg in blood had significantly greater corticosterone levels compared to the 0-1 ppm category.

1 Corticosterone hormones are released during periods of stress. 47 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary

• High risk: 3.0 - <4.0 ppm. Adult loons with 3-4 and >4.0 ppm THg in blood had significantly greater corticosterone levels than the lower mercury categories. Furthermore, adult female loons with blood concentrations >3.0 ppm often exhibited reduced reproductive success. • Extra high risk: >4.0 ppm. Known impacts on loons and other birds. Uncertainties Associated with an Assessment of Adult Blood

Several field studies have documented gender differences in blood mercury concentrations of birds collected from the same habitats, with males typically having greater concentrations of mercury than females ( Evers et al. 1998 , Meyer et al. 1998 , Eagles-Smith et al. 2009a , Evers et al. 2004, Tsao et al. 2009, Robinson et al. 2012 ). Proposed reasons for these observed differences in concentration include 1) female’s ability to depurate Hg into eggs, 2) sexual dimorphism (i.e., larger males are able to consume larger prey with higher mercury concentrations) and 3) foraging and guild adaptation (i.e., greater detoxification potential in females to protect embryos)(Robinson et al. 2012). Evers et al. (2004) does not clarify if the risk thresholds are gender specific, therefore our review and analysis did not differentiate gender when analyzing GSL blood data.

Effect Benchmark – Adult Blood

EPA determined insufficient data are available to develop a mercury effect benchmark for adult blood. Rather, blood data will be analyzed with the risk ranges.

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Table A3. Adult Blood. Evers et al. (2004) Hg risk thresholds for blood compared to other blood effect values. Values are identified as methylmercury (MeHg) or total mercury (THg), when reported by the authors, and blank when author does not report the form of Hg. All values are wet weight (ww) unless identified otherwise. Moderate Risk Low Risk in Blood in Blood High Risk in Blood Extra High Risk in Blood <1.0 MeHg 1.0 – 3.0 MeHg 3.0 – 4.0 MeHg >4.0 MeHg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type Chronic - reproduction Field: North >3.0 THg; 41% American <1.0 THg; control reduced breeding Evers et al. 2008 group reproduction common loon loon dataset Field: 120 lakes in WI, New Brunswick and Nova 4.3 THg; 8.6 THg; Scotia Burgess and Meyer EC50 chick failed (quantile 2008 productivity reproduction common loon regression) 0.73 THg; LOAEC significant reduction in egg productivity (13.2%), reduced male Controlled courtship behaviors and breeding: 3 Frederick & Jayasena increases in male-male breeding 2011 pairing white ibis seasons 0.8 THg LOAEC; Hg Heinz 1979 in female blood when Lab – spiked Heinz et al. 2010b egg = 0.8 THg mallard diet Chronic - other 0.66; LOAEC reduced motor common loon Lab – spiked Kenow et al. 2010 coordination (chicks) diet American avocet, 1.3; black-necked Field: San demethylation stilt, Caspian tern, Francisco Ackerman et al. 2014 threshold Forster’s tern Bay, CA*

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12 THg; LOAEC behavior, growth, immune function, Spalding et histological changes, Controlled al.2000a/2000b biochemical changes great white egret breeding Acute > 3.77 THg; 85-90% adult survival and Meyer et al. 1998 return rate common loon Field: WI 3.95 THg fw; NOAEC adult Controlled Frederick et al. 2011 survival white ibis feeding 3 yrs *the concentration when demethylation is initiated, value was translated from a liver concentration of 8.5 ppm dw (Eagles-Smith et al. 2009)

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A.4. Adult Liver

Total mercury concentrations tend to be highest in the liver, kidney, and spleen when compared to other avian tissues that are commonly monitored in mercury assessments (Fimreite 1974, Bhatnagar et al. 1982, Henny et al. 2002 , Eisler 1987 ). Liver is monitored more frequently than kidney and spleen, and therefore was the focus of our review. Concentrations are high in liver given its essential role in mercury detoxification via demethylation and excretion. Avian species vary in their ability to demethylate Hg, and therefore also vary in the percent MeHg of the THg in the liver (Fimreite 1974, Barr 1986, Scheuhammer et al. 2008). For example, seabirds in the order Procellariiformes are known to be effective demethylators and tend to have lower percentages of MeHg in organ tissue when compared to other species (Kim et al. 1996, Scheuhammer et al. 2008, Wolfe et al. 1998).

The ability of an individual bird to demethylate mercury has also been shown to be age-dependent, with adult and juvenile birds showing greater demethylation potential than new born chicks (Henny et al. 2002, Kenow et al. 2007b, Eagles-Smith et al. 2009b). Greater demethylation potential as the chick ages and in adults may provide additional protection against mercury toxicity. This has been identified as one of the potential explanations for why reproduction endpoints are more sensitive than adult toxicity endpoints. Given these species-specific and age-dependent differences in an individual bird’s ability to demethylate mercury, some authors have warned that correct interpretation of liver data requires characterization of the various species of Hg and that total Hg concentration in liver alone is not sufficiently informative to make confident toxicological judgments (Wolfe et al. 1998, Wiener et al. 2003, Rattner et al. 2011 ). Furthermore, large changes in liver mass due to the natural history of the species of concern (e.g., seasonal variation, fasting, or deposition of mass in preparation for migration), may confound the evaluation of contaminants in liver ( Rattner and Jehl 1997 ).

Risk Categories – Adult Liver

Evers et al. (2004) does not provide risk categories for mercury in liver; however our literature review documented several studies that have reported adult liver concentrations associated with adverse effect concentrations. Given the volume of available studies and suggested adverse effect thresholds, the EPA developed risk categories for liver using an approach similar to Evers et al. (2004)(Table A4). The liver categories are described as follows. • Low risk: 0 - <1.3 ppm. The upper threshold of the low risk category recognizes the liver concentration of the female ducks fed the 0.5 ppm dw treatment in the Heinz (1979) controlled breeding experiments. The 0.5 ppm in diet treatment was the LOAEC for reproductive success. Heinz (1979) reports the liver concentration as a THg concentration; however, since the exposure diet was 100% MeHg, we would expect the majority of the THg to be MeHg. We did not locate any studies where adverse effects were observed below 1.3 ppm in liver. • Moderate risk: 1.3 - <5.0 ppm. The upper threshold of the moderate risk category recognizes the conservative liver threshold proposed by Zillioux et al. (1993) and mercury concentrations that correlate with adverse effects in great white heron. Although the upper threshold is greater than the lowest adverse effect concentration reported by Shore et al. (2011), the low liver value reported by Shore is from a pheasant study and therefore is less applicable to the GSL waterfowl designated use. • High risk: 5.0 - <15.0 ppm. The upper threshold of the high risk category recognizes the concentration in liver that has been associated with adverse behavioral effects in great egret

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(Spalding et al. 2000a & 2000b), yet is lower than the lowest acute adverse effect concentration reported by Shore et al. (2011). • Extra high risk: ≥ 15.0 ppm. Concentrations greater than or equal to 15 ppm in liver have been associated with known reproductive effects and acute effects in birds. Uncertainties Associated with an Adult Liver Assessment

Total vs. Methylmercury

Very few of the studies that were used to develop the liver risk categories report the % MeHg. We acknowledge that the % MeHg in each of the studies is likely variable. Scheuhammer et al. (2007) suggests that MeHg in liver generally predominated when THg concentrations are less than approximately 10 ppm ww. Although it would be preferential to base the liver risk categories on MeHg concentrations, data are not available for such an analysis. Therefore the liver risk categories are based on THg concentrations and should be assessed with adult THg concentrations.

Gender Differences

Similar to blood, differences between male and female liver concentrations of mercury are expected. An assessment that exclusively evaluated female liver concentrations would provide the most direct measurement of potential reproduction risk and would most closely correlate with the reproductive effect thresholds derived from female ducks in controlled breeding experiments. However, in the field, gender differences in liver concentration can vary by location, time of sampling (pre-breeding vs. breeding) and species ( Gochfeld and Burger 1987 , Eagles-Smith et al. 2009a). Therefore, gender was not taken into consideration in the derivation of the liver risk categories when using liver data .

Mercury-selenium Interaction

UDEQ’s draft GSL Hg assessment and final selenium report (UDEQ 2008 & 2010) discuss the uncertainty with assessing mercury and selenium (Se) data, without recognizing potential antagonistic effects when these toxicants occur together at high concentrations. Frequently, field collected animals that have been found with the highest THg concentrations, and no apparent Hg toxicity, also have high Se concentrations and a low % MeHg (Kim et al. 1996, Scheuhammer et al. 2007, Conover and Vest 2009 , Wurtzbaugh et al. 2011 ). Results from field and lab studies indicate that Hg-Se interactions can alter mercury toxicity (Cuvin-Aralar and Furness 1991, Wiener et al. 2003, Shore et al. 2011). Proposed mechanisms of the protective effect of selenium against mercury toxicity include 1) redistribution of Hg in the presence of Se, 2) competition for binding sites, 3) formation of nontoxic Hg-Se complexes, 4) conversion of toxic forms of Hg into other forms of Hg, and 5) prevention of oxidative damage (Cuvin- Aralar and Furness 1991).

The majority of lab studies and literature reviews that present data in support of antagonistic effects have focused on mammals, fish, and adult birds (both aquatic and terrestrial birds). Although we determined egg Hg data will be analyzed independent of the selenium concentration since synergistic effects have been observed in early life stages (see Attachment 1, Section 2), results from Heinz and Hoffman (1998) suggest that the interaction of Hg and Se can be opposite for adults and young. The controlled breeding experiment fed adults a diet containing MeHg chloride, seleno-DL-methionine, and Hg + Se. Embryos from the adults fed the Hg + Se diet had a greater frequency and number of deformities than the

52 DRAFT – DELIBERATIVE – DO NOT QUOTE OR CITE Attachment 1 – Literature Benchmark and Risk Threshold Summary embryos produced from the adults feed only Se or Hg (Heinz and Hoffman 1998). Results from this study suggest that the occurrence of both Se and Hg may be synergistic to young birds.

Additionally, Heinz et al. (2012) and Klimstra et al. (2012) conducted a series of egg injection studies with MeHg and Se alone and in combination to evaluate potential interactions that could occur during the embryo development phase. Authors injected commercially produced mallard duck and chicken eggs and field collected double-crested cormorant eggs with different combinations of MeHg chloride and seleno-L-methionine. The type and frequently of deformities and hatching success between the treatments were documented. Both antagonistic and synergistic effects were observed. MeHg appeared to decrease embryo mortality caused by Se, yet increase the number and types of deformities. Authors conclude that additional studies are necessary to understand the biochemical mechanisms underlying these results and to determine if the same interactions occur with maternally deposited Se and Hg.

Results from the limited studies that evaluated Hg-Se interactions during early life stages do not indicate that selenium protects against the adverse effects of Hg on embryo development or reproductive endpoints. Given EPA’s assessment is focusing on potential reproductive effects due to mercury, EPA did not determine it necessary to take selenium concentrations into consideration when evaluating Hg in diet or eggs. It may be appropriate to consider selenium concentrations when evaluating adult blood or liver data, but is not necessary for our analysis since assessment decisions will not rely exclusively on these indirect indicators.

Uncertainty Summary

Taking the above uncertainties with the adult liver effect thresholds into consideration, the THg in adult liver data will only be taken into consideration with other lines of evidence when making assessment decisions. No assessment decisions will be determined from adult liver data alone.

Effect Benchmark – Adult Liver

EPA determined insufficient data are available to develop an effect benchmark for adult liver. Rather, liver data will be analyzed with the proposed risk ranges. The Hg:Se molar ratio will also be taken into consideration when concentrations are available for both parameters.

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Table A4. Adult Liver. The EPA-proposed mercury in liver risk categories for aquatic wildlife. All values are total mercury (THg) ppm wet weight (ww), unless otherwise noted. The Hg:Se molar ratio will also be taken into consideration when concentrations are available for both parameters. Low Risk in Moderate Risk in Liver Liver High Risk in Liver Extra High Risk in Liver <1.3 THg 1.3-<5.0 THg 5.0 <15.0 THg ≥15 THg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type Chronic-reproduction 1.33; THg LOAEL Controlled 18% reduction in 1 breeding: week old ducklings average of 3 Heinz 1979 mallard generations Heinz and 22 THg; 85% reduction in mallard Controlled Hoffman 1998 1 week old ducklings (females) breeding 23.07; 24% reduction in hatchability & 79% Controlled Finley and reduction in duckling breeding: 2 Stendell 1978 survival black duck seasons*** 1.53 MeHg; reduced nesting 29.7 THg; reduced Barr 1986 success nesting success common loon Field study 20.7 THg; 73% hatch Fimreite 1974 reduction common tern Field study >2.0 THg; LOAEC 8.7 THg; geometric (pheasant) mean of adverse Birds in reproduction effects general Literature Shore et al. 2011 (range 2-52) (5 species) review Chronic-other Spalding et al. >5.0; increased disease great white 1991 and emaciation heron Field study* >6.0 THg; correlated Spalding et al. with mortality from great white 1994 chronic disease heron Field study** 15 THg; LOAEC; behavior, growth, Spalding et al. immune function, histological changes, Controlled 2000a/2000b biochemical changes great egret breeding 5.0 THg; Conservative Zillioux et al. threshold for significant aquatic birds Literature 1993 toxic effects in general review

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Low Risk in Moderate Risk in Liver Liver High Risk in Liver Extra High Risk in Liver <1.3 THg 1.3-<5.0 THg 5.0 <15.0 THg ≥15 THg Reference ppm (ww) ppm (ww) ppm (ww) ppm (ww) Species Study Type 2.9 THg (8.5 THg American dw); demethylation avocet, black- threshold necked stilt, Eagles-Smith et Caspian tern, Field al. 2009b Forster’s tern Study**** Acute 71 THg (Se = 19 ppm): frank neurological effects Heinz and 65 THg (Se = 114 ppm): mallard Controlled Hoffman 1998 NOAEC (males) breeding Scheuhammer 30; frank neurological Birds in Literature 1991 effects general review 20 – 30; range of significant toxic effects Non-marine Literature Thompson 1996 and death birds review 18.4 THg; Birds in lowest acute >20 THg; predicted HC5 general Literature Shore et al. 2011 value (5 species) review *analyzed dead carcasses, multiple age classes evaluated (2.5 months - adult) **analyzed dead carcasses, most birds were juveniles *** % reductions = average reduction of the 2 breeding seasons **** the concentration when demethylation is initiated

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