Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Sea levels across the planet are rising, particularly along the eastern coast of the United States. Climate-induced sea level rise can result in the inundation and intrusion of seawater into freshwater drainages. This would alter salinity regimes and lead to the salinization of coastal freshwater ecosystems. Increased salinity levels in freshwater can negatively affect freshwater-dependent species, including native mussels belonging to the order Unionida, which are highly sensitive to changes in water quality. Sea salt is largely made up of sodium and chloride ions, forming sodium chloride, a known toxicant to freshwater mussels. However, sea salt is a mixture that also contains other major ions, including potassium, sulfate, calcium, strontium, and magnesium, among others. Freshwater mussels exposed to sea salt would be exposed to each of the sea salt ions at the same time, resulting in a mixture toxicity effect. The mixture toxicity of these ions on early life stages of freshwater mussels is largely unknown because most research to date has evaluated individual salt ions in relative isolation. Therefore, we conducted acute toxicity tests on early life stages (glochidia and juvenile) of three freshwater mussel species that inhabit Atlantic Slope drainages (nonsalinity-adapted Atlanticoncha ochracea, salinity-adapted A. ochracea, Sagittunio nasutus, and Utterbackiana implicata). Glochidia and juveniles of each species were exposed to a control and six concentrations of Instant Ocean® Sea Salt (IOSS), a synthetic sea salt that closely resembles the ionic composition of natural sea salt. Exposure concentrations were 1 part(s) per thousand (ppt), 2 ppt, 8.5 ppt, 12.5 ppt, 17 ppt, and 34 ppt. We calculated the median effect concentration (EC50) for each of the eight acute toxicity tests and found that glochidia were more sensitive than juveniles to IOSS. At hour 24 EC50s for the glochidia ranged from 0.38 to 3.6 ppt, with the most sensitive freshwater mussel being the nonsalinity-adapted A. ochracea, exhibiting an EC50 of 0.38 ppt (95% confidence interval [CI] 0.33-0.44). Juvenile freshwater mussels exhibited EC50s at hour 96 ranging from 5.0 to 10.4 ppt, with the least sensitive freshwater mussel being the nonsalinity-adapted A. ochracea, exhibiting an EC50 of 10.4 ppt (95% CI 9.1-12.0). Our results show that acute exposure to sea salt adversely affects freshwater mussel viability, particularly glochidia. This information can be used to enhance freshwater mussel conservation strategies in regions that are or will be impacted by climate-induced sea level rise and associated freshwater salinization. Environ Toxicol Chem 2023;42:2478-2489. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Figures 5 and 6 (and their corresponding legends) were inadvertently omitted from the final publication of the original paper [...]

Freshwater mussel taxa are severely imperiled and among the most sensitive to several contaminants, including chloride. Relatively little is known of the toxicity of major ions to glochidia (larvae), particularly for rare species, or the effects of hardness on major ion toxicity to glochidia. Therefore, the primary objectives of this work were to: (1) determine the acute toxicity of major ion salts to glochidia, (2) compare chloride sensitivity of glochidia from common and rare species, and (3) evaluate the relationship between water hardness and chloride toxicity to glochidia. We assessed 24 h EC50s for fatmucket (Lampsilis siliquoidea) glochidia exposed to NaCl, KCl, MgCl2, CaCl2, Na2SO4, MgSO4, CaSO4, and NaHCO3 in moderately hard water. We determined NaCl EC50s for four species and KCl EC50s for glochidia of three species. Toxicity of chloride was generally consistent among the various chloride salts except for KCl, which was more toxic than all others by 1–2 orders of magnitude. Our results suggest that commonly tested species may be generally representative of rare species. Water hardness ameliorated the toxicity of chloride for all species to varying degrees. Results of this work indicate that some existing water quality criteria that do not include mussel toxicity data may not be protective of freshwater mussels.

The US Environmental Protection Agency's short‐term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short‐term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field‐collected effluent in 7‐day exposures. Both mussel species were equally or more sensitive (more than two‐fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7‐day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7‐day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%–52% length increase) when the 1‐ or 2‐week‐old mussels were fed 2 ml twice daily and the 3‐week‐old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314–520 mg Cl/L) among the three age groups, indicating that an age range of 1‐ to 3‐week‐old mussels can be used for a 7‐day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7‐day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392–3409. © 2021 SETAC

Adverse reproductive effects associated with gonadal intersex among freshwater fish could hold considerable implications for population sustainability. Presence of testicular oocytes (TO) is the most common form of intersex and is widespread among centrarchids (sunfishes) of North America and other freshwater teleosts. Placing TO within the toxicological context of adverse outcome pathways (AOPs) to assess ecological risk is a priority for ecotoxicologists due to the association of TO with harmful chemical exposure and adverse reproductive effects in some cases. However, key event relationships between EDC exposure, incidence of TO, and apical outcomes have yet to be fully elucidated - in part due to a lack of knowledge of relationships between intersex gonad physiology and fish health. Understanding the physiological status of intersex fish is critical to assess ecological risk, understand mechanisms of induction, and to establish biomarkers of intersex in fish. In the present study, features of gonad metabolite profiles associated with TO in largemouth bass (LMB, Micropterus salmoides) from an impoundment in Georgia (USA) were determined using GC-MS-based metabolomics. Clinical blood biochemical screens were used to evaluate markers of fish health associated with TO. Results suggest that physiological changes in energy expenditure as well as relatively 'feminized' gonad lipid and protein metabolism may be related to the occurrence of TO in male LMB, and highlight the need to understand relationships between intersex and physical stressors such as elevated temperature and hypoxia. These results provide novel insight to AOPs associated with TO and identify candidate analytes for biomarker discovery.

Growing human populations and increasingly intensive agriculture have resulted in widespread aquatic nitrate pollution. Freshwater mussel populations have been in decline for decades but often are underrepresented in data used for the development of ambient water quality criteria and acute toxicity of nitrate to mussel glochidia has not yet been established. Additionally, toxicity testing with aquatic species often is limited to a few model species; however, relatively little is known about how representative model species are of imperiled species. Therefore, to better define the acute toxicity of nitrate to common and rare aquatic species, we conducted 24-h nitrate acute toxicity tests with glochidia of four species of freshwater mussels, including a federally threatened species (Hamiota altilis) and 7-day tests with larval fish of three species: fathead minnow (Pimephales promelas), tricolor shiner (Cyprinella trichroistia), and tilapia (Oreochromis spp.), across a range of water hardness. Median effective concentrations (EC50s) in freshwater mussel glochidia ranged from 524 to 904 mg/L NO3–N in moderately hard water. In fish, median lethal concentrations (LC50s) ranged from 228 to 1725 mg/L NO3–N and varied with water hardness. Of the species tested, generally sensitivity of the common species was similar to the rare species, although relative sensitivity varied with water hardness. Based on these results, we can conclude that acute lethal effects are unlikely for the fish and mussel species considered here at current environmental levels, but the results of these standardized tests are useful for the development of ambient water quality criteria and other regulatory and management decisions regarding acute nitrate exposures.

Mercury (Hg) is a contaminant that enters the environment through natural or anthropogenic means. Ecological risk assessments have examined Hg bioaccumulation and effects in many taxa, but little is known about Hg dynamics in reptiles, or their potential use as bioindicator species for monitoring Hg in aquatic systems. Numerous snake species, like North American watersnakes (Nerodia spp.), are piscivorous and are exposed to Hg through their diet. The purpose of this study was to identify factors associated with Hg accumulation in a common watersnake species and compare Hg concentrations of the snakes to those in fish occupying the same habitats. To this end, we sampled brown watersnakes (Nerodia taxispilota) from the Savannah River, a major river system in the southeastern U.S., and compared N. taxispilota Hg accumulation trends to those of bass (Micropterus salmoides), catfish (Ictalurus and Ameiurus spp.), and panfish (Lepomis and Pomoxis spp.) collected from the same reach. Total Hg (THg) in N. taxispilota tail tips ranged from 0.020 to 0.431 mg/kg (wet weight; mean: 0.104 ± 0.008). Snake tail THg was significantly correlated with blood THg, which ranged from 0.003 to 1.140 mg/kg (0.154 ± 0.019). Snake size and site of capture were significantly associated with tail THg. Snake tail THg increased at sites along and downstream of the area of historic Hg pollution, consistent with fish THg. Snake muscle THg was predicted based on tail THg and ranged from 0.095 to 1.160 (0.352 ± 0.022). To gauge Hg biomagnification in N. taxispilota, we compared predicted snake muscle THg concentrations to THg in fish of consumable size. Average biomagnification factors for THg in N. taxispilota were 3.1 (panfish) and 5.4 (catfish), demonstrating N. taxispilota likely biomagnify Hg through their diet. These results reveal N. taxispilota to be an effective bioindicator species for monitoring Hg in aquatic environments.

We used in situ exposures of juvenile mussels (96-d average, May-September) to investigate the causes of mussel declines. We measured survival and growth of mussels exposed to ambient conditions in 23 streams in Kentucky, USA. Our set of streams included both those that support diverse mussel assemblages (occupied streams) and those that have lost nearly their entire mussel fauna (defaunated streams). We used 2 types of enclosures in each stream: silos, which primarily expose mussels to water, and sediment cages, which provide greater exposure to sediments. We used both enclosure types because some contaminants are more prevalent either in water or sediments. We collected extensive water and sediment chemistry data (163 and 144 analytes, respectively) monthly at each stream and landscape data for each watershed. We found no evidence of acute toxicity. Survival averaged 90% in silos and was only 68% in cages. However, lower survival in cages appeared to be an artifact of the enclosure type (burial), and survival was unrelated to faunal health or water or sediment chemistry in either type of enclosure. In contrast, we found strong evidence that chronic stressors negatively affect growth in defaunated streams. Growth in both enclosure types was uniformly low in all defaunated streams (mean instantaneous growth, as mass = 0.005/d) compared with growth in occupied streams (0.026/d). Variation in water chemistry among streams was described by 2 principal components (PCs). PC2 described a gradient of increasing alkalinity, nutrients, total organic C, and temperature, which corresponded to the underlying geology and physiography in the study region. Growth was positively correlated with PC2, but defaunated streams were conspicuous outliers, suggesting that other factors further limited growth in these streams. PC1 described a gradient of higher row crop agriculture, pesticides, nitrate/nitrite, and karst influence. Mussel growth was negatively correlated with PC1, but agricultural contaminants were not present in all defaunated streams. Variation in sediment chemistry was described by only 1 PC that reflected underlying geology, similar to water chemistry PC2, and there were no consistent patterns of sediment contamination. Overall, lower total organic C and lower water temperature compared with the occupied streams were the main characteristics shared by the defaunated streams. This result suggests that mussel populations in relatively cool, unproductive streams are particularly vulnerable to chronic human impacts that negatively affect growth or other physiological processes.

Diffusive gradients in thin films (DGT) were assessed for their predictive capability of fathead minnow and yellow lampmussel bioaccumulation in copper (Cu) and lead (Pb) mixed metal exposures. Nine treatments with a matrix of 3 Cu and 3 Pb concentrations were utilized. Exposures were coupled, with organisms and DGT exposed in tanks for 6 days. The Cu measured in fish, mussel, and DGT was found not to be influenced by Pb treatment, whereas Pb accumulation was impacted by the interaction of Cu and Pb treatment. The Pb accumulation increased with increasing Cu concentration, which was attributed to the different speciation of Cu and Pb in the water where Cu binds preferentially to ligands, decreasing its bioavailability and concomitantly displacing Pb from complexing sites. The DGT values were significantly correlated with accumulated Cu and Pb in the fish, but not with Pb in the mussel. In addition, DGT was determined to better predict aquatic organism bioaccumulation of Cu than the inorganic Cu fraction calculated by the speciation model, because DGT accumulated not only inorganic metal fractions but also complexes of metal and organic matter. The present study provides insights into metal speciation in polluted environments, extends the understanding of using DGT as a tool for estimating metal bioavailability, and provides implications for the selection of geochemical modeling, biological sampling, and passive sampling techniques for monitoring trace metal contamination. Environ Toxicol Chem 2019;38:61-70. © 2018 SETAC.

Skipjack Herring (Alosa chrysochloris) has long been proposed as the sole host for Reginaia ebenus (Ebonyshell) and Elliptio crassidens (Elephantear), but these relationships were unconfirmed because of difficulties with maintaining this fish species in captivity. We confirmed the suitability of Skipjack Herring as host for both mussel species, and we also showed that Alabama Shad (Alosa alabamae) is an additional suitable host for E. crassidens; both fish species produced large numbers of juvenile mussels. No other fish species tested (n = 12) were suitable hosts for either mussel species. Our results, combined with results from other studies, suggest these mussel species are specialists on genus Alosa. Traditional methods for host identification were problematic for herrings because of their sensitivity to handling and the large volumes of water required to maintain them in captivity. In addition to traditional methods, we confirmed the suitability of these fishes as hosts using a novel technique in which fish gills infected with glochidia were excised from sacrificed fishes and held in recirculating holding tanks with flow until metamorphosis was complete. Completion of metamorphosis on excised gills required glochidia spend at least 11–17 d encapsulated on live fishes before gill excision. This technique may be useful for other large or sensitive fishes that do not lend themselves well to traditional methods for host identification. Confirmation of Alosa spp. as primary hosts for R. ebenus and E. crassidens supports the idea that dams and other river modifications that disrupt migrations of these fishes are key factors in the range restrictions of these mussel species.

Using a coupled method of diffusive gradients in thin films (DGT) exposure with aquatic organism bioassays, we assessed the use of DGT as a tool for estimating copper (Cu) bioavailability in contaminated waters. The DGT-accumulated Cu fraction could possibly be used as a surrogate for other assessments of metal bioavailability. The Cu concentrations in fathead minnow (Pimephales promelas) and yellow lampmussel (Lampsilis cariosa) soft tissue were compared with DGT-accumulated Cu after 2, 4, and 6 d of exposure to a Cu concentration series in static, water-only assays. The DGT-accumulated Cu was found to include free Cu ions, labile inorganic Cu complexes, and labile dissolved organic matter Cu complexes, compared with Cu speciation output from the biotic ligand model. Regressions of Cu concentrations between DGT and fathead minnow at 4 and 6d of exposure demonstrated linear relationships. The Cu bioaccumulated in yellow lampmussel was overpredicted by DGT at Cu concentrations greater than 10 mu gL(-1), which may be caused by internal regulation of Cu. The speciation component of the biotic ligand model predicted relationships between inorganic Cu and animal-accumulated Cu that were similar to predicted relationships between DGT-indicated Cu and animal-accumulated Cu at all deployment durations. Environ Toxicol Chem 2018;37:1535-1544. (c) 2018 SETAC

Water quality and contaminants have been frequently identified as critical stressors for freshwater mussels, many species of which are highly imperiled throughout North America and the world. Nutrient pollution, specifically nitrate, has become one of the most prevalent causes of water quality degradation globally, with increasing anthropogenic input from suburban and agricultural runoff, municipal wastewater, and industrial waste. Nitrate acute toxicity is generally low for aquatic species, but the potential effects of nitrate exposure are largely unknown for freshwater mussels, particularly during the parasitic stage of their complex lifecycle. Therefore, this study was designed to determine the effects of short-term nitrate exposure at environmentally relevant concentrations on juvenile production in two freshwater mussel species. Lampsilis siliquoidea and L. fasciola glochidia were exposed to nitrate (0, 11, or 56 mg NO3-N/L) for 24 h before inoculation on a primary host, Largemouth Bass (Micropterus salmoides). Glochidia attachment, metamorphosis success, and total number of juveniles produced were monitored on individual fish. Exposure of L. siliquoidea glochidia to 56 mg NO3-N/L nitrate resulted in a significant (p = 0.02) 35% reduction of total juveniles produced, a combined result of moderate decreases in both glochidia attachment and metamorphosis success. A similar trend (28% reduction; p = 0.06) was evident with 11 mg NO3-N/L. No effects were apparent for L. fasciola, suggesting species-specific differences in responses even among closely related species. These results are the first to suggest that glochidia exposure to nitrate may adversely affect juvenile recruitment in some species. Findings from these studies are important for improving characterization of the hazards of nitrate pollution to aquatic life and this work will help better define the role of water quality in assessing habitat suitability for mussel conservation efforts.

The Atlantic Tripletail Lobotes surinamensis is a popular sport fish for which age and growth data are scarce in general and nonexistent for Georgia (GA), USA, waters. These data are necessary to ensure that management regulations are adequate to protect this species, especially given its popularity as a sport fish. We evaluated whether otoliths and spines were suitable for determining the estimated age (hereafter, age) and growth rates of Atlantic Tripletails, and we ascertained whether one method was more accurate than the other. Atlantic Tripletails were sampled by angling and trawling during March 30-August 10, 2009, and March 14-August 6, 2010, in nearshore GA waters of the Atlantic Ocean. During the study, 243 Atlantic Tripletails were captured and sampled for aging structures. Sagittal otoliths and the first dorsal spine were removed from each fish and used to estimate the age and growth rate. Mean differences in TL at age for spine and otolith data were evaluated with ANOVA. Estimated ages for males and females ranged from 1 to 5years based on otoliths and spines. Both otolith and spine mean TLs at ages 1 and 2 were significantly different from each other as well as all other age-classes, whereas mean TLs for ages 3-5 were not significantly different. Differences in Atlantic Tripletail TL among the otolith- and spine-derived age-classes were not significant. Each method used to age Atlantic Tripletails had advantages and disadvantages. Otoliths had higher initial reader agreement than spines, although agreement between the structures was 84.1%. However, otoliths require sacrifice of the fish, whereas a spine can be taken without sacrificing the fish. The lack of concrete life history data and population estimates suggests that when feasible, nonlethal aging methods would be preferred over lethal methods to ensure the survival of Atlantic Tripletail populations.

Hatchery propagation plays an essential role in restoration or augmentation of freshwater mussel populations, which are among the most critically imperiled of all taxa. A unique feature of freshwater mussel life history is an obligate parasitic relationship with fish in which the larval mussels (glochidia) must attach to host fish and metamorphose to the juvenile stage. Hatcheries can produce large quantities of juvenile mussels for restoration and research purposes; however, in cases where suitable host fish species have not been identified, options for propagation are severely limited. Although specific biochemical mechanisms mediating metamorphosis remain largely unknown, previous research suggests that the plasma cortisol level in the host is an important factor. Manipulation of cortisol to induce or increase metamorphosis in otherwise marginal or non-host fish may provide a valuable tool in culturing mussel species for which the primary host(s) are unknown or prohibitively difficult to rear in a captive environment. The objective of the present study was to further investigate the potential for exogenous cortisol treatment to increase attachment and metamorphosis on marginal or non-host fish. Cortisol treatment (intraperitoneal injection) of known host species (Largemouth Bass Micropterus salmoides and Bluegill Lepomis macrochirus) did not affect attachment or metamorphosis; however, in non-hosts (Channel Catfish Ictalurus punctatus and Goldfish Carassius auratus) and marginal hosts (Blue Tilapia Oreochromis aureusxNile Tilapia Oreochromis niloticus hybrids), attachment generally increased with increasing cortisol, though only significantly so in Goldfish. Metamorphosis was significantly increased on the marginal hosts but was not induced on the non-hosts. This study provides clear evidence that cortisol manipulation of marginal hosts can increase juvenile mussel production and may be an important tool to maximize propagation efforts, particularly for imperiled species.

We conducted an exposure experiment with Diffusive Gradients in Thin- Films (DGT), fathead minnow (Pimephales promelas), and yellow lampmussel (Lampsilis cariosa) to estimate bioavailability and bioaccumulation of Cu. We hypothesized that Cu concentrations measured by DGT can be used to predict Cu accumulation in aquatic animals and alterations of water chemistry can affect DGT's predict ability. Three water chemistries (control soft water, hard water, and addition of natural organic matter (NOM)) and three Cu concentrations (0, 30, and 60 μg/L) were selected, so nine Cu-water chemistry combinations were used. NOM addition treatments resulted in decreased concentrations of DGT-measured Cu and free Cu ion predicted by Biotic Ligand Model (BLM). Both hard water and NOM addition treatments had reduced concentrations of Cu ion and Cu-dissolved organic matter complexes compared to other treatments. DGT-measured Cu concentrations were linearly correlated to fish accumulated Cu, but not to mussel accumulated Cu. Concentrations of bioavailable Cu predicted by BLM, the species complexed with biotic ligands of aquatic organisms and, was highly correlated to DGT-measured Cu. In general, DGT-measured Cu fit Cu accumulations in fish, and this passive sampling technique is acceptable at predicting Cu concentrations in fish in waters with low NOM concentrations.

To inform Dracunculus medinensis (Guinea worm) eradication efforts, we evaluated the role of fish as transport hosts for Dracunculus worms. Ferrets fed fish that had ingested infected copepods became infected, highlighting the importance of recommendations to cook fish, bury entrails, and prevent dogs from consuming raw fish and entrails.

Various anthropogenic activities have resulted in concentration of heavy metals and contamination of surrounding environments. Historically, heavy metal contamination at the Savannah River Site (SRS) in South Carolina has resulted from accidental releases of stored waste generated from nuclear weapon production in the early 1950s. Songbirds inhabiting and using resources from these areas have the potential to bioaccumulate metals but there is limited information on metal concentration levels in areas suspected of contamination as well as uncontaminated sites. Nonlethal tissues samples from avian blood and feathers provide a reliable approach for determining the bioavailability of these pollutants (As, Cd, Cr, Cu, Hg, Ni, Pb, Se, and Zn). The objectives of this study were to survey terrestrial heavy metal contamination at the SRS on potentially bioavailable contaminated (PBC) sites through blood and feather samples from resident Northern Cardinals (Cardinalis cardinalis) and migratory Great Crested Flycatchers (Myiarchus crinitus) and quantify sex-specific concentrations within species. Samples were collected in April to June of 2016. Cardinals had lower blood concentrations of Hg (β=-0.17, 85% CL=-0.26, -0.09) and Se (β=-0.33, 85% CL=-0.50, -0.16) than flycatchers. Cr feather concentrations were less in cardinals (β=-1.46, 85% CL=-2.44, -0.49) and all feathers of both species from reference locations had significantly less Zn (β=-67.92, 85% CL=-128.71, -7.14). Results indicate flycatchers were exposed to differing heavy metal levels during feather formation on their wintering grounds as compared to their recent exposure (through bloods samples) on their breeding grounds. Sex of individuals did not have a significant impact on bioaccumulation in either species. Overall, metal concentration levels in both species indicate minimal risk for acute toxicity; however, there is limited research on wild passerine populations with similar concentration levels. Therefore, further research on reproductive success of these birds should be explored.