Danielle M Cleveland Barnett A Rattner Natalie K Karouna-Renier Julia S Lankton 20230725 Digital tabular data Reston, VA U.S. Geological Survey https://doi.org/10.5066/P9RV1D60 Rebecka Brasso Danielle Cleveland Frank R. Thompson David E. Mosby Kathy Hixson Melissa Roach Barnett A. Rattner Natalie K. Karouna-Renier Julia S. Lankton 2023 publication n/a US Geological Survey https://doi.org/10.3133/sir20235032 This dataset provides results for the following: (1) metals concentrations in soils surrounding breeding song bird nest sites; (2) metals concentrations in invertebrates (earthworm and grub composites) collected near nest sites; (3) metals concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in avian blood samples; and (4) metals concentrations, indicators of oxidative stress and DNA damage, and histopathology in liver and/or kidney tissues collected from breeding song birds. The U.S. Fish and Wildlife Service, along with the U.S. Forest Service, and the Missouri Department of Natural Resources, are pursuing damage assessments under the Natural Resource Damage Assessment and Restoration (NRDAR) program at several locations within the Southeast Missouri Lead Mining District. Lead mining occurred in this region for nearly 300 years and metal residues from smelting, tailings, and chat piles have contaminated soil and water. As part of the ongoing damage assessment work, this study expanded upon an earlier investigation by Beyer and others (2013), which identified adverse effects in ground feeding songbirds from soil-borne lead associated with mining in the Southeast Missouri Lead Mining District. The present study evaluated nesting songbirds to determine whether exposure to lead-contaminated soil adversely affected molecular, cellular, physiological or reproductive function. 2016 2019 ground condition Complete None planned Southeastern Missouri Lead Mining District -92.199999999999 -89.3875 38.2 37.1209 ISO 19115 Topic Category biota environment None Natural Resource Damage Assessment and Restoration nesting songbirds lead-contaminated soil DNA damage oxidative stress delta-aminolevulinic acid dehydratase activity soil invertebrates blood liver kidney histopathology microscopic lesions Data Categories for Marine Planning Mining and Mineral Extraction Alexandria Digital Library Feature Type Thesaurus mine sites industrial sites USGS Thesaurus metal elements metallic mineral resources Marine Realms Information Bank (MRIB) keywords heavy metal pollution USGS Metadata Identifier USGS:64c01955d34e70357a32323f None Southeast Missouri Lead Mining District Common geographic areas Missouri United States USGS Biocomplexity Thesaurus Annelids Birds Insects Integrated Taxonomic Information System (ITIS) 2021 ONLINE_REFERENCE Washington, D.C. Integrated Taxonomic Information System (ITIS) http://itis.gov Danielle M Cleveland U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Chemist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 dcleveland@usgs.gov expert advice All identification of organisms was completed by experts. All birds were identified to species, insects and worms were identified to order. Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata chordates Subphylum Vertebrata vertebrates Infraphylum Gnathostomata Superclass Tetrapoda Class Aves Birds Order Passeriformes Perching Birds Family Turdidae Genus Turdus Robins Species Turdus migratorius American Robin TSN: 179759 Genus Sialia Bluebirds Species Sialia sialis Eastern Bluebird TSN: 179801 Family Icteridae American Blackbirds Orioles New World Blackbirds Genus Molothrus Cowbirds Species Molothrus ater Brown-headed Cowbird TSN: 179112 Family Emberizidae Emberizid Finches American Sparrows Towhees Buntings New World Sparrows Genus Pipilo Towhees Species Pipilo erythrophthalmus Rufous-sided Towhee Eastern Towhee TSN: 179276 Genus Spizella Chipping Sparrows Species Spizella pusilla Field Sparrow TSN: 179443 Family Cardinalidae Cardinals Grosbeaks Saltators Cardinal-Grosbeaks Genus Passerina Varied Buntings Species Passerina cyanea Indigo Bunting TSN: 179150 Genus Cardinalis Cardinals Species Cardinalis cardinalis Northern Cardinal TSN: 179124 Family Parulidae New World Warblers Wood-Warblers Genus Helmitheros Worm-eating Warblers Species Helmitheros vermivorum Worm-eating Warbler TSN: 726195 Order Columbiformes Doves Pigeons Family Columbidae Doves Subfamily Columbinae Genus Zenaida Zenaida Doves Species Zenaida macroura Mourning Dove TSN: 177125 Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Ecdysozoa Phylum Arthropoda Subphylum Hexapoda Class Insecta Subclass Pterygota Infraclass Neoptera Superorder Holometabola Order Coleoptera beetles coléoptères besouro Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Lophozoa Phylum Annelida Class Clitellata Subclass Oligochaetae None. Please see 'Distribution Info' for details. None. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Danielle Cleveland U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Chemist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 dcleveland@usgs.gov Avian, soil, and invertebrate samples were collected in the field by researchers at Southeast Missouri State University (Rebecka Brasso, Kathy Hixson, Melissa Roach), U.S. Forest Service (Frank R. Thompson ), and U.S. Fish and Wildlife Service (David E. Mosby and others). We thank Vanessa Melton, Mike Walther, Alison Hartman, and Sandra Schultz (USGS) for technical assistance with laboratory measurements. W. Nelson Beyer J. Christian Franson John B. French Thomas May Barnett A. Rattner Valerie I. Shearn-Bochsler Sarah E. Warner John Weber David Mosby 20130615 OTHER Archives of Environmental Contamination and Toxicology vol. 65, issue 3 n/a Springer Science and Business Media LLC https://doi.org/10.1007/s00244-013-9923-3 Helen B Burch Abraham L Siegel 19711001 BOOK Clinical Chemistry vol. 17, issue 10 n/a Oxford University Press (OUP) https://doi.org/10.1093/clinchem/17.10.1038 Deborah J. Pain 1987 OTHER United Kingdom University of Oxford 335 p. U.S. Environmental Protection Agency 1996 OTHER Washington, DC U.S. Environmental Protection Agency https://www.epa.gov/homeland-security-research/epa-method-3050b-acid-digestion-sediments-sludges-and-soils U.S. Environmental Protection Agency 2014 OTHER Washington, DC U.S. Environmental Protection Agency https://www.epa.gov/esam/epa-method-6020b-sw-846-inductively-coupled-plasma-mass-spectrometry A. Berlin K.H. Schaller 1974 OTHER Clinical Chemistry and Laboratory Medicine (Z Klin Chem Klim Biochem) vol. 12, issue 8 n/a Z Klin Chem Klim Biochem https://doi.org/10.1515/cclm.1974.12.8.389 J. Fair E. Paul J. Jones 2010 OTHER Washington, DC The Ornithological Council https://www.aaalac.org/pub/?id=E9019213-EE55-98AB-F68E-EF2B10C31360 W.M. Haschek C.G. Rousseaux M.A. Wallig 2013 BOOK n/a Elsevier https://doi.org/10.1016/C2010-1-67850-9 L.G. Luna 1968 OTHER New York, NY McGraw-Hill Scott L. Friedman Rebecka L. Brasso Anne M. Condon 2008 OTHER Journal of Field Ornithology vol. 79, issue 1 n/a Wiley https://doi.org/10.1111/j.1557-9263.2008.00150.x William G. Brumbaugh Christopher J. Schmitt Thomas W. May 20050614 OTHER Archives of Environmental Contamination and Toxicology vol. 49, issue 1 n/a Springer Science and Business Media LLC https://doi.org/10.1007/s00244-004-0172-3 Quality control (QC) measures included the use of NIST-traceable calibration standards, second source and/or laboratory control standards, analysis duplicates and spikes, and calibrated balances and pipets, as appropriate. See the process steps for further QC details for each analytical approach. The data were checked for omissions and duplicates. All values fall within expected ranges. The data set is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details. No formal positional accuracy tests were conducted. No formal positional accuracy tests were conducted. both Songbird tissues were collected utilizing lethal and non-lethal methods, sediment and invertebrates were collected near nesting sites, tissue analysis and metal concentrations were completed utilizing various histological and standard methods that are described in detailed and referenced in the process steps. Soil samples were provided to USGS for total recoverable analyses of cadmium (Cd), cobalt (Co), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn). Samples were sub-aliquots of soil composites collected for ex-situ analyses using portable X-ray fluorescence spectrometry by U.S. Fish and Wildlife Service (USFWS) personnel. Soil composite samples were generally composed of five to six equal aliquots of soil collected at a depth of 5-10 centimeters from a 100 meter square area (totaling approximately one kilogram of soil) within a 100-meter diameter of each nest at the contaminated sites; soil samples collected at the reference sites generally within 20 meters of the location of bird nests. The soils had been air-dried and sieved to less than 2-mm particle size by USFWS. Upon receipt at the USGS laboratory, the soils were lyophilized, homogenized, microwave digested in nitric acid and hydrogen peroxide, using a method similar to USEPA 3050B (Acid digestion: U.S. Environmental Protection Agency. 1996. Acid Digestion of Sediments, Sludges, and Soils, Method 3050B), and then analyzed for total recoverable Cd, Co, Cu, Ni, Pb, and Zn using inductively coupled plasma-mass spectrometry (ICP-MS). Quantification was performed on a dry weight basis (dw). The ICP-MS method was similar to USEPA method 6020B (ICP-MS analysis: U.S. Environmental Protection Agency. 2014. Inductively coupled plasma-mass spectrometry, Method 6020B.) The ICP-MS was equipped with an autosampler/autodilutor with online addition of an internal standard solution. A minimum of four external National Institute of Standards and Technology-traceable calibration standards plus a calibration blank were used to calibrate the instrument response. Established laboratory quality assurance/quality control procedures and sample types, including laboratory spikes, duplicates, and reference materials, were used to verify instrument performance, accuracy, and precision throughout all analyses. Quality control (QC) measures for ICP-MS quantification of Pb in soil samples indicated that the analyses were in-control. Recoveries of Pb in the calibration standards were 90 to 110 percent; and the internal standard recoveries were 60 to 120 percent throughout the analyses. Second source initial (ICVS) and continuing calibration standards (CCVSs) and blanks were analyzed every 10 samples; recoveries of lead from the ICVS and CCVSs were 90 to 110 percent. Lead recoveries from the laboratory control solutions were 90.0 to 102.2 percent. Method and analytical spike recoveries ranged from 94.2 to 109.8 percent, and analytical dilutions had percent differences less than 3 percent. Method triplicates had RSDs of 1.0 to 9.4 percent, and analytical duplicates had RPDs of 0.6 to 3.9 percent. Recoveries of lead from certified reference materials (soils and sediments) ranged from 87.3 to 100 percent of the certified range. The mean blank equivalent concentrations for lead in the procedural blanks was 0.02 mg/kg dw, and the estimated limits of quantification (LOQ) for Pb in the tissues were 0.1 to 0.2 mg/kg dw. Results were censored at the LOQ level. Additional metals were quantified in these samples; results for all analytes were generally in-control, but only the Pb QC has been extensively described (for the purposes of the accompanying publication). 2019 Danielle M Cleveland U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Chemist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 dcleveland@usgs.gov Avian liver, kidney, and blood samples were provided to USGS for total recoverable analyses of cadmium (Cd), copper (Cu), lead (Pb), and/or zinc (Zn). All samples were collected by non-USGS field personnel. Liver and kidney samples were collected following euthanization of captured birds. Blood samples were collected either lethally or non-lethally. Non-lethal blood sampling: Adult Eastern Bluebirds were captured using nest box traps (Friedman and others, 2008) or mist nets, while the adults of all other breeding species were captured in their breeding territory using song playback and mist nets. A 26-gauge, 0.5-inch-long needle was used to puncture the cutaneous ulnar vein and collected the blood in non-heparinized capillary tubes. The volume of blood collected from individuals was no more than one percent of the bird’s body weight in adherence to state and federal guidelines and the recommendations of the Ornithological Council (Fair and others, 2010). Blood from the capillary tubes was immediately transferred into pre-labeled, pre-cleaned glass screw top test tubes; samples were stored on ice in a cooler while in the field. The collection test tubes were left capped until the sample was drawn and were re-capped immediately after filling to reduce or eliminate background contamination. Samples were transferred frozen to the USGS laboratory and subsequently kept frozen (-20 °C) until further processing for analysis. Field method blanks for blood samples, which consisted of opening the collection tubes to the atmosphere (but with no blood collected), were collected in 2016; lead concentrations in the field blanks were below the limits of quantification for all target metals, indicating that zero to negligible external contamination was introduced into the blood samples during collection or by the pre-cleaned tubes. Field method blanks were therefore not repeated in subsequent study years. Nestlings were hand captured late in the nestling period, approximately one to three days before the expected day of fledging. Site collection blank results for blood samples were converted to the dry weight basis using a nominal sample weight of 0.025 g, and 0.005 L digestate volume. The protocol for nestling blood collection and sampling was the same as for adults; however, a smaller volume of blood was collected from each individual, based on the weight of that individual. Therefore, blood samples from nestlings from the same brood were composited into a single, pooled sample that represented the whole brood. Lethal sampling: Target species (American Robin; Northern Cardinal; Mourning Dove; and Brown-headed Cowbird) were captured in mist nets or funnel traps. Blood samples were collected from each bird via the cutaneous ulnar vein; as before, the drawn blood was immediately transferred from the non-heparinized capillary tubes into pre-labeled, pre-cleaned glass screw top test tubes. Samples were stored on ice in a cooler while in the field, and then frozen (-20 °C) until further processing. Individuals were then euthanized in the field by inhalant carbon dioxide from a gas cylinder (Fair and others, 2010); and the liver and kidneys were immediately dissected using clean, stainless steel dissecting tools. Tissues for lead analyses consisted of one lobe of kidney and an approximately 0.25 g (ww) section of liver, which were placed into two separate, pre-labeled and acid-washed plastic vials (i.e., 1 vial for liver tissue; 1 vial for kidney tissue) and stored on ice in the field. Samples were kept frozen (-20 °C) until transfer to the USGS laboratory and remained frozen until further processing for analysis. Portions for each analysis were taken from consistent locations within each organ to standardize sampling protocols among individuals to the extent possible. Liver and kidney tissues were lyophilized, homogenized, microwave digested in nitric acid and hydrogen peroxide, using a method similar to USEPA 3050B (Acid digestion: U.S. Environmental Protection Agency. 1996. Acid Digestion of Sediments, Sludges, and Soils, Method 3050B), and then analyzed for total recoverable Cd, Cu, Pb, and/or Zn using inductively coupled plasma-mass spectrometry (ICP-MS). Blood samples were digested on a hot block in nitric acid and hydrogen peroxide similar to the method used by Brumbaugh and others, 2005), and the digestates were then analyzed using ICP-MS. Percent moisture was determined gravimetrically by sample weight loss on lyophilization; quantification of all tissue types was performed on a dry weight basis (dw). However, some caution should be used with moisture values for small samples, like nonlethal blood samples. Moisture losses during storage and handling prior to lyophilization are particularly problematic for blood samples due to their small weights and large surface areas. Further, weight measurements of small samples are more susceptible to static, which can add error to balance readouts. The ICP-MS method was similar to USEPA method 6020B (ICP-MS analysis: U.S. Environmental Protection Agency. 2014. Inductively coupled plasma-mass spectrometry, Method 6020B.) The ICP-MS was equipped with an autosampler/autodilutor with online addition of an internal standard solution. A minimum of four external National Institute of Standards and Technology-traceable calibration standards plus a calibration blank were used to calibrate the instrument response. Established laboratory quality assurance/quality control procedures and sample types, including laboratory spikes, duplicates, and reference materials, were used to verify instrument performance, accuracy, and precision throughout all analyses. Quality control (QC) measures for ICP-MS quantification of Pb in blood, liver, and kidney tissues indicated that the analyses were in-control. Recoveries of Pb in the calibration standards were 90 to 110 percent; and the internal standard recoveries were 60 to 120 percent. Second source initial calibration verification standard (ICVS) and continuing calibration verification standards (CCVSs) and blanks were analyzed every 10 samples; recoveries of Pb from the ICVS and CCVSs were 90 to 110 percent. Lead recoveries from laboratory control solutions were 85.8 to 117 percent. Method and analytical spike recoveries ranged from 89.8 to 108 percent, and analytical dilutions had percent differences less than 5 percent. Method triplicates had relative standard deviations (RSDs) of 0.8 to 4.2 percent. Analytical duplicate relative percent differences (RPDs) were 0 to 4.8 percent. Recoveries of Pb from certified reference materials composed of similar tissues (e.g., bovine liver, whole blood) ranged from 80.8 to 114 percent of the certified range, with the exception of one reference material used during liver and kidney analysis, which had a lead recovery of 162 percent for n = 1 of 3 analyses, and one whole blood reference material, which had lead recoveries of 66.2 to 79.4 percent Pb for n = 3 of 11 analyses (i.e., n = 8 of 11 were in the 80.8 to 114 percent recovery range for this material). However, for kidney and liver analyses the weight of reference material used (40 milligrams, mg) was well-below the weight (250 mg) at which the material was test for homogeneity; this discrepancy likely impacted the lead recovery. Similarly, the weight of blood reference material (20 mg) used for digestions was low in comparison to the recommended weight (5 mL of reconstituted material). The mean blank equivalent concentrations for lead in the procedural blanks was 0.01 mg/kg dw. The estimated limits of quantification (LOQs) for Pb in the livers and kidneys were 0.03 to 0.2 mg/kg dw; the estimated LOQs for lead in blood were 0.02 to 0.1 mg/kg dw. Results were censored at the LOQ level. Additional metals were quantified in these samples; results for all analytes were generally in-control, but only the Pb QC has been extensively described (for the purposes of the accompanying publication). The following are potential quality issues for individual samples: Moisture and metals results for samples in vials 013, 021, 027, 048, 059, 060, 064, 065, 067, 068, 076, 122, 125, 128, 129, 136, 137, 154, 157, 158, 163, 197, 201, 204, 208, 211, 213, and 3006 may have additional associated error due to the loss of the Teflon cap liners during field collection. These sample tubes required additional manipulation as follows: (1) insertion of a new liner for digestion, (2) emptying, cleaning, and drying of the tube post-analysis, and (3) removal of the liner to determine the new empty weight for moisture determinations, and calculation of dry sample weight. A droplet of the diluent for this sample (vial 836) was lost during the sample preparation process; results may be slightly biased due to the loss, but the exact extent of the bias cannot be determined. The Teflon cap liner for vial 975 fell out during sample digestion and had to be replaced. It is unknown whether or not any small sample droplets were present on the liner; lost droplets may slightly bias the results, but the presence or exact extent of any bias cannot be determined. A droplet of the digestate for liver sample AMRO-16 was lost during the sample preparation process; results may be slightly biased due to the loss, but the exact extent of the bias cannot be determined. 2018 Danielle M Cleveland U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Chemist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 dcleveland@usgs.gov Target species for lethal sampling (American Robin; Northern Cardinal; Mourning Dove; and Brown-headed Cowbird) were captured in mist nets or funnel traps. Blood samples were collected from each bird via the cutaneous ulnar vein for analyses of microhematocrit and delta-aminolevulinic acid dehydratase (ALAD) activity. For ALAD analysis, blood samples were collected into several heparinized capillary tubes. The contents of the tubes were immediately transferred into a cryovial, frozen in liquid nitrogen in the field, and stored at -80 °C for transfer to the USGS laboratory. One heparinized capillary tube was used to collect a blood sample for microhematocrit; the tube was immediately placed on ice in a cooler while in the field and processed by centrifugation within five hours of collection. ALAD activities in blood samples were determined by the method of Burch and Siegel (1971) as modified by Pain (1987). The ALAD assay was scaled down to a volume of 25 microliters (µL) whole blood for analysis, and all samples were analyzed in duplicate. Samples were incubated pre-assay for 10 minutes at 38 °C. Freshly-prepared aminolevulinic acid substrate (0.01 M ALA, pH 6.65) was then added to each sample tube, and the mixtures were incubated for an additional 60 minutes. The assay was terminated by the addition of 10 percent trichloroacetic acid. The incubation tubes were then centrifuged at 1500×g, and the supernatant was reacted with Erlich’s reagent. The absorbance of the reacted supernatant was measured at 555 nm, background corrected using the absorbance at 630 nm, and pathlength corrected at 90 second intervals, for up to a total of 21 minutes using a FLUOstar® Omega microplate reader (BMG LABTECH Inc., Cary, N.C.). Optimal absorbance occurred at about 15 minutes following the addition of Erlich’s reagent. Activity was calculated two ways; the method of Burch and Siegel (1971) and by the method of Berlin and Schaller (1974) that was used by Pain (1987). Units of ALAD activity correspond to ΔA/mL RBC/h, where A is the background-corrected absorbance at 555 nm, mL RBC is the hematocrit, and h is the time in hours (Burch and Siegel 1971). Hematocrit was measured in the packed red cell fraction on a micro-hematocrit capillary tube reader following centrifugation (IEC International Micro-Capillary Centrifuge, Boston, Mass.) of a plugged capillary tube at 13,460×g for 3 minutes. The packed red cell fraction was measured on a micro-hematocrit capillary tube reader. Note that the hematocrit was measured by non-USGS personnel and is provided here only for the purposes of ALAD determination. Preliminary delta-aminolevulinic acid dehydratase (ALAD) assays using commercially-available heparinized laboratory mouse (Mus musculus) and chicken (Gallus gallus) whole blood (BioIVT, Westbury, N.Y.) were performed to verify assay linearity over incubation time and sample volume, characterize the precision of quadruplicate analyses, and to estimate the limit of detection (LOD). Incubation of varying volumes of mouse and chicken blood (12, 25, 37 and 50 microliters, µL) for 60 minutes yielded comparable blank-corrected absorbances when normalized on a unit volume basis (mean ± standard deviation [SD]; mouse = 0.198 ± 0.017; chicken = 0.157 ± 0.022). A different pool of chicken blood was analyzed on different days (August 9, 2018 and August 13, 2018); both pools yielded comparable mean blank-corrected absorbances when normalized on a unit volume basis (mean absorbance ± SD of 0.072 ± 0.012 and 0.082 ± 0.014). Incubation of 25 µL samples of mouse and chicken blood for varying durations (30, 60 and 90 minutes) yielded comparable mean blank-corrected absorbances (mouse = 0.191 ± 0.014; chicken = 0.161 ± 0.005). Chicken blood assays having varying incubation times were repeated on two additional days and yielded comparable mean blank-corrected absorbances of 0.073 ± 0.003 and 0.086 ± 0.014. Intra-assay precision using quadruplicate determinations of mouse and chicken blood (25 µL) yielded mean blank-corrected absorbances of 0.194 ± 0.009 and 0.164 ± 0.005, respectively. Tests for intra-assay precision were repeated for chicken blood on two additional days and yielded mean blank-corrected absorbances of 0.072 ± 0.005 and 0.081 ± 0.008. An estimated LOD for the ALAD activity assay was determined based on analysis of ten chicken sample replicates without the addition of substrate. Replicate analyses yielded a mean absorbance (± 2 SD) of 0.032; this corresponds to 13.99 units of activity assuming a hematocrit of 40. One Northern Cardinal sample (NOCA-28) was clotted, and therefore not assayed. A Brown-headed Cowbird sample (BHCO-5) was clotted but could be pipetted and was assayed; it is recommended that results for this sample not be in any statistical analyses. A hematocrit value was not obtained for one American Robin sample (AMRO-20), so activity could not be calculated for that sample. The remaining samples (n=52) were assayed in duplicate in three batches (consisting of 6 samples, 18 samples, and 28 samples, respectively); chicken blood was assayed multiple times in each batch as a QC sample. The mean coefficient of variation (CV; mean CV ± sd) for duplicate determinations of the 52 field samples that were assayed was 5.62% ± 3.67. For chicken blood samples, the average intra-batch CV for blank corrected absorbance ranged from 1.93% to 8.64%, and inter-batch CV for blank corrected absorbance was 5.01% ± 3.39. 2018 Barnett A Rattner U.S. Geological Survey, Northeast Region Research Physiologist mailing and physical

308 Center Rd

Beltsville MD 20705 US 301-497-5671 301-497-5744 brattner@usgs.gov Target species for lethal sampling (American Robin; Northern Cardinal; Mourning Dove; and Brown-headed Cowbird) were captured in mist nets or funnel traps. Upon completion of blood sampling, individuals were then euthanized in the field by inhalant carbon dioxide from a gas cylinder (Fair and others, 2010); and the livers were immediately dissected using clean, stainless steel dissecting tools. An approximately 125 mg wet weight (ww) section of liver was placed into a cryovial and immediately frozen in liquid nitrogen for analyses of oxidative stress and DNA damage biomarkers. Samples were kept frozen at -80 °C until further processing. All dissecting tools and surfaces were thoroughly cleaned with isopropyl alcohol between dissections. Portions for biomarker analyses were taken from consistent locations within each liver to standardize sampling protocols among individuals to the extent possible. Liver samples (n = 53) were analyzed for biomarkers of oxidative stress, namely total sulfhydryl (TSH), total glutathione (tGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), protein bound sulfhydryl (PBSH), lipid peroxidation (thiobarbituric acid reactive substances; TBARS), and DNA damage (8-hydroxy-2'-deoxyguanosine; 8-OH-dG). We also measured the ratio of GSSG to GSH (GSSG:GSH). For TSH, tGSH, GSH, GSSG, PBSH and TBARS analyses, an approximately 100 mg (ww) piece of frozen liver sample was placed in a 1.5 milliliter (mL) tube on wet ice and mixed with a 0.6 mL volume of 1X phosphate-buffered saline (PBS, pH 7.4; Fisher BioReagents, Waltham, Mass.). Stainless steel beads (0.9 to 2.0 mm blend; Next Advance, Inc., Troy, N.Y.) were added to each tube at 1.3 times the weight of the liver piece; and the samples were homogenized using a Bullet Blender (Next Advance, Inc., Troy, N.Y.). The homogenate was centrifuged at 10,000×g for 10 minutes at 4 °C and the supernatant saved. Two aliquots of each supernatant were diluted in 1X PBS at concentrations of 100 mg/mL and 25 mg/mL, and separately frozen at -80°C until analysis. Concentrations of GSH and tGSH were measured using the DetectX Glutathione Fluorescent Detection Kit (Arbor Assays, Ann Arbor, Mich.) following the manufacturer’s protocol. The 25 mg/mL supernatant was thawed on ice and diluted to 12.5 mg/mL in 1X PBS for the analysis of Brown-headed Cowbird, Northern Cardinal, and Mourning Dove samples; the 25 mg/mL supernatant was similarly thawed on ice but diluted to 6.25 mg/mL in 1X PBS for American Robin samples. TSH was analyzed using the Measure-iT Thiol Assay Kit (Invitrogen Molecular Probes, Eugene, Oreg.) following the manufacturer’s instructions. For this assay, the 25 mg/mL supernatant from Brown-headed Cowbird, Northern Cardinal, and Mourning Dove samples was diluted to 8.33 mg/mL in 1X PBS; the 25 mg/mL American Robin supernatant was diluted to 5.0 mg/mL in 1X PBS. TBARS levels were determined using the QuantiChrom TBARS Assay Kit (Bioassay Systems, Hayward, Calif.) following the manufacturer’s instructions. For TBARS, the 100 mg/mL supernatant was used for all four species, although for some American Robin samples, the supernatant had to be diluted to 12.5 mg/mL in 1X PBS. Concentrations of PBSH were calculated as the difference between TSH and GSH concentrations. Oxidized glutathione (GSSG; i.e., [tGSH-GSH]/2), and the ratio of GSSG:GSH were calculated using the measured endpoints. Total protein concentrations were determined with a 5 µg/µL supernatant (in 1X PBS) using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, Mass). Each sample was assayed in duplicate; and any sample for which the CV of duplicates was greater than 10 percent was re-analyzed. No duplicate CV was greater than 10 percent following re-analysis. Two reference samples were run on each plate to monitor inter-assay variability. Inter-assay coefficients of variance (CVs) for the two reference samples were 9.29 percent and 1.47 percent for TSH, 6.06 percent and 9.39 percent for GSH, and 5.38 percent and 8.44 percent for TBARS. The limit of detection (three times the standard deviation of 10 wells containing assay diluent) was 1.28 micromoles per gram of wet tissue (µmol/g) for TSH, less than 0.001 µmol/g GSH, 0.53 µmol/g tGSH, and 0.03 µmol/g for TBARS (Table 3.1). The reporting limits, which were determined from the lowest calibration standard for which replicates had CVs less than 15 percent, were 6.88 µmol/g for TSH, 0.78 µmol/g for GSH and total glutathione (tGSH), and 0.094 µmol/g for TBARS. DNA was extracted from approximately 5 mg (ww) of liver using the Gentra Puregene Tissue Kit (Qiagen, Gaithersburg, Md.). The concentration and purity of DNA were determined using a NanoDrop 8000 microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, Wilmington, Del.). The samples were normalized to 15 µg DNA/100 µL in hydration solution (10 mM Tris, 1 mM EDTA, pH 7-8) and frozen at -80 °C until analysis. Normalized DNA samples were thawed and denatured by heating for 10 min at 100 °C, cooled on ice for 5 minutes, and microfuged for 5 s. We then added 8.2 µL of 300 mM sodium acetate buffer (pH 5.2; (Fisher, Waltham Mass.), 1.3 µL of 5 mM zinc chloride (Sigma Aldrich, St. Louis, Mo.), and 7 µL of 25 U/mL Nuclease P1 (Sigma-Aldrich) to each sample. The tubes were inverted and microfuged for 5 s, and then incubated at 37 °C for 30 minutes. The samples were pH-adjusted to pH 7.5-8.0 by adding 14 µL 1M Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.5); and then 10.2 µL of 10 U/mL alkaline phosphatase (Thermo Fisher Scientific) was added to each sample. Samples were mixed by inversion, microfuged for 5 s and incubated at 37 °C for 30 minutes. The alkaline phosphatase was inactivated by boiling for 10 minutes at 95 °C, and the samples were then placed on ice. Aliquots (30 µL, 0.075 µg/µL) were stored at -20 °C until analysis. Concentrations of 8-OH-dG were determined by enzyme-linked immunosorbent assay using the DetectX DNA Damage Immunoassay Kit (Arbor Assays, Ann Arbor, Mich.) using a BMG Fluostar Omega microplate reader (BMG Labtech, Cary, N.C.). Standard calibration curves (0.125 to 4 nanograms per milliliter, ng/mL; correlation coefficient, R2 > 0.999), which were used to calibrate the microplate reader response, were fit using a four-parameter model (MARS Data Analysis Software 2.10 R3; BMG Labtech, Cary, N.C.). The LOD was less than 0.001 ng/mL and the reporting limit was 0.25 ng/mL. Inter-assay variability (i.e., percent CV) of the two reference samples were 8.3 percent and 1.1 percent. 2018 Natalie K Karouna U.S. Geological Survey, Northeast Region Research Ecologist mailing and physical

308 Center Rd

Beltsville MD 20705 US 301-497-5724 nkarouna@usgs.gov Target species for lethal sampling (American Robin; Northern Cardinal; Mourning Dove; and Brown-headed Cowbird) were captured in mist nets or funnel traps. Upon completion of blood sampling, individuals were then euthanized in the field by inhalant carbon dioxide from a gas cylinder (Fair and others, 2010); and the livers and kidneys were immediately dissected using clean, stainless steel dissecting tools. A liver sample for histopathology consisted of approximately 5x5 mm piece of the liver that was placed into a pre-labeled, plastic sample container containing 1:10 neutral buffered formalin (NBF). One lobe of the kidney was also added to this same sample cup. Portions for each analysis were taken from consistent locations within each organ to standardize sampling protocols among individuals to the extent possible. Histopathology samples were kept submerged in NBF at room temperature until shipping. Immediately prior to shipment to the pathology laboratory, the formalin was drained, and each tissue was wrapped in a formalin-soaked paper towel and sealed back into the labeled container. Upon receipt at the USGS laboratory, the formalin-fixed liver and kidney samples were dehydrated, paraffin-embedded, sectioned at 5µm, and stained with hematoxylin and eosin, Fite’s acid fast, and Ziehl-Neelsen acid fast stains for light microscopic examination (Luna 1968). The tissues were examined for microscopic abnormalities potentially associated with lead toxicosis, including acid-fast intranuclear inclusions in renal tubular epithelial cells, hepatic or renal tubular degeneration and necrosis, hepatic or renal inflammation, hepatic lipidosis, hepatic hemosiderosis, hepatic bile stasis, hepatic biliary hyperplasia, hepatic or renal cyto/karyomegaly, and renal glomerulopathy (Haschek and others, 2013). Abnormalities were scored as present or absent by a single pathologist. 2018 Julia S Lankton U.S. Geological Survey, MIDCONTINENT REGION Veterinary Pathologist mailing and physical

6006 Schroeder Road

Madison WI 53711 US 608-270-2459 608-270-2415 jlankton@usgs.gov Earthworms (Oligochaeta) and a small number of other beetle larvae (i.e., grubs; Coleoptera) representing potential invertebrate prey items of the target songbird species were collected for cadmium (Cd), cobalt (Co), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) analyses. Invertebrates were collected from the top 15-cm of soil by digging with trowels; any invertebrates found were hand-transferred to plastic bags, composited by site, and frozen until processing and analysis. The samples were not depurated or rinsed prior to analyses to approximate incidental soil intake and dietary exposure to songbirds. A total of 23 composite samples (consisting of approximately 90 percent earthworms and 10 percent beetle larvae by weight) were collected. Composite invertebrate sample weights ranged from just over 1 gram to approximately 20 grams wet weight, depending on invertebrate abundance at each site. In general, the Big River floodplain site and areas near human use (i.e., state park campgrounds) had considerably more worms than remote, upland sites such as Magmont Vent, Anschutz Mine, and areas within the reference sites that were far removed from facilities. Upon receipt at the USGS laboratory, the invertebrate composites were lyophilized, homogenized, microwave digested in nitric acid and hydrogen peroxide, using a method similar to USEPA 3050B (Acid digestion: U.S. Environmental Protection Agency. 1996. Acid Digestion of Sediments, Sludges, and Soils, Method 3050B), and then analyzed for total recoverable Pb using inductively coupled plasma-mass spectrometry (ICP-MS). Percent moisture was determined gravimetrically by sample weight loss on lyophilization; quantification was performed on a dry weight basis (dw). The ICP-MS method was similar to USEPA method 6020B (ICP-MS analysis: U.S. Environmental Protection Agency. 2014. Inductively coupled plasma-mass spectrometry, Method 6020B.) The ICP-MS was equipped with an autosampler/autodilutor with online addition of an internal standard solution. A minimum of four external National Institute of Standards and Technology-traceable calibration standards plus a calibration blank were used to calibrate the instrument response. Established laboratory quality assurance/quality control procedures and sample types, including laboratory spikes, duplicates, and reference materials, were used to verify instrument performance, accuracy, and precision throughout all analyses. Quality control (QC) measures for ICP-MS quantification of lead in invertebrates indicated that the analyses were in-control. Recoveries of lead in the calibration standards were 90 to 110 percent; and the internal standard recoveries were 60 to 120 percent throughout the analyses. Second source initial (ICVS) and continuing calibration standards (CCVSs) and blanks were analyzed every 10 samples; recoveries of lead from the ICVS and CCVSs were 90 to 110 percent. Lead recoveries from the laboratory control solutions (LCSs) were 90.3 to 100.8 percent. Method and analytical spike recoveries ranged from 97.6 to 101 percent, and analytical dilutions had percent differences less than 2 percent. A method triplicate for worm tissue had a relative standard deviation (RSD) of 28.3 percent; this was likely due to the presence of soil particulates in the tissue sample. Analytical duplicates had 1.3 to 1.8 relative percent differences (RPDs). Recoveries of lead from certified reference materials composed of invertebrate tissues (e.g., mussels, oysters) ranged from 87 to 100 percent of the certified range. The mean blank equivalent concentrations for lead in the procedural blanks was 0.03 mg/kg dw, and the estimated limits of quantification (LOQ) for lead in the tissues were 0.1 to 0.2 mg/kg dw. Results were censored at the LOQ level. Additional metals were quantified in these samples; results for all analytes were generally in-control, but only the Pb QC has been extensively described (for the purposes of the accompanying publication). 2016 Danielle M Cleveland U.S. Geological Survey, Columbia Environmental Research Center Supervisory Research Chemist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 dcleveland@usgs.gov The Southeast Missouri Lead Mining District is defined in History of Mining in the Southeast Missouri Lead District and Description of Mine Processes, Regulatory Controls, Environmental Effects, and Mine Facilities in the Viburnum Trend Subdistrict By Cheryl M. Seeger. Chapter 1 of Hydrologic Investigations Concerning Lead Mining Issues in Southeastern Missouri Edited by Michael J. Kleeschulte https://pubs.usgs.gov/sir/2008/5140/pdf/Chapter1.pdf Elements_avian_tissue_soil_invertebrates These results are total recoverable concentrations of elements (cadmium, cobalt, copper, lead, nickel, zinc) in avian blood, liver and kidney; soil; and invertebrate samples measured by USGS in the laboratory Producer Defined Sample_ID A unique identifier assigned to the sample Producer Defined A unique alphanumeric identifier assigned to the sample. Taxon The taxonomic identification of the sample organism. Producer Defined The taxonomic identification of the sample organism. NA The attribute is not applicable Producer defined Unknown The value of the attribute is unknown Producer defined Sex The identified sex of the sample organism Producer Defined Female The organism was determined to be female Producer defined Male The organism was determined to be male Producer defined Unknown The value of the attribute is unknown Producer defined NA The attribute is not applicable Producer defined Age_class The determined age class of the sample organism Producer Defined NA The attribute is not applicable Producer defined AHY A bird known to have hatched before the calendar year of banding; year of hatch otherwise unknown. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. HY A bird capable of sustained flight and known to have hatched during the calendar year in which it was banded. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. L A nestling or young bird incapable of sustained flight. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. U A bird that cannot be placed in any of the other age classes. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. Unknown The value for the attribute is unknown Producer defined Matrix The matrix from which the sample was collected for analysis Producer Defined Blank The matrix sampled was a method blank Producer defined Blood The matrix sampled was blood. Producer defined Kidney The matrix sampled was kidney. Producer defined Liver The matrix sampled was liver. Producer defined Soil The matrix sampled was soil Producer defined Invertebrate The matrix sampled was beetles, worms or grubs Producer defined Site A literal value representing the common term used to identify the sampling location Producer Defined A literal value representing the common term used to identify the sampling location, See entity Sampling_sites for more detail Year An indicator of the year in which the sample was collected. The value is expressed in YYYY. Producer Defined 2016 2018 Moisture The percent moisture in the sample, measured as weight loss on lyophilization. Producer Defined NA The attribute is not applicable Producer defined Unknown The value for the attribute is unknown Producer defined 23.1 93.3 percent Element An identifier for the element analyzed in the sample. Producer Defined Cd Cadmium, CASRN 7440-43-9 Producer defined Co Cobalt, CASRN 7440-48-4 Producer defined Cu Copper, CASRN 7440-50-8 Producer defined Ni Nickel, CASRN 7440-02-0 Producer defined Pb Lead, CASRN 7439-92-1 Producer defined Zn Zinc, CASRN 7440-66-6 Producer defined Result The analytical result (concentration) of the ELEMENT in the sample as milligrams per kilogram on a dry weight basis. Producer Defined LT # Indicates that the measured concentration of the ELEMENT was less than (LT) the limit of quantification of (#) milligrams per kilogram on a dry weight basis Producer defined 0.02 13600 Milligrams per kilograms ALAD_in_blood These results are delta-aminolevulinic acid dehydratase (ALAD) activities in avian blood samples using the method of Burch and Siegel (1971) as modified by Pain (1987). All blood samples that were collected for ALAD analyses were from adult birds (age class AHY) that were lethally sampled in 2018. Producer Defined Sample_ID A unique identifier assigned to the sample Producer Defined A unique alphanumeric identifier assigned to the sample Taxon The taxonomic identification of the sample organism. Producer Defined The taxonomic identification of the sample organism. NA The attribute is not applicable Producer defined Sex The identified sex of the sample organism Producer Defined Female The sample organism was determined to be female Producer defined Male The sample organism was determined to be male Producer defined Site A literal value representing the common term used to identify the sampling location Producer Defined A literal value representing the common term used to identify the sampling location, See entity Sampling_sites for more detail Year An indicator of the year in which the sample was collected. The value is expressed in YYYY. Producer Defined 2018 2018 Hematocrit The volume occupied by the erythrocytes expressed as a percentage of the total volume of blood, as measured via centrifugation by non-USGS field personnel within 5 hours of blood sample collection. Producer Defined 21 58 percent ALAD_Activ The delta-aminolevulinic acid dehydratase (ALAD) activity where units of activity correspond to the change in absorbance at 555 nanometer per milliliter of red blood cells per hour. Producer Defined 10.00 312.38 activity units Biomarkers_in_livers These results are concentrations of biochemical markers of oxidative stress in avian liver samples. All liver samples collected were from adult birds (age class AHY) that were lethally sampled in 2018. Producer Defined Sample_ID A unique identifier assigned to the sample Producer Defined A unique alphanumeric identifier assigned to the sample Taxon The taxonomic identification of the sample organism. Producer Defined The taxonomic identification of the sample organism. NA The attribute is not applicable Producer defined Sex The identified sex of the sample organism Producer Defined Female The sample organism was determined to be female Producer defined Male The sample organism was determined to be male Producer defined Site A literal value representing the common term used to identify the sampling location Producer Defined A literal value representing the common term used to identify the sampling location, See entity Sampling_sites for more detail Year An indicator of the year in which the sample was collected. The value is expressed in YYYY. Producer Defined 2018 2018 Biomarker An identifier for the biomarker analyzed in the liver sample on a wet tissue basis. Producer Defined TBARS Thiobarbituric acid reactive substances, measured as micromoles of analyte per gram of wet sample. Producer defined GSH Reduced glutathione, measured as micromoles of analyte per gram of wet sample. Producer defined tGSH Total glutathione, measured as micromoles of analyte per gram of wet sample. Producer defined GSSG Oxidized glutathione, measured as micromoles of analyte per gram of wet sample. Producer defined GSSG:GSH The ratio of GSSG to GSH Producer defined TSH Total sulfhydryl, measured as micromoles of analyte per gram of wet sample. Producer defined PBSH Protein bound sulfhydryl, measured as micromoles of analyte per gram of wet sample. Producer defined 8OHdG 8-hydroxy-2'-deoxyguanosine, measured as nanograms of analyte per milliliter of sample. Producer defined Result Analytical result (concentration) of the BIOMARKER in the liver tissue sample. Producer Defined NA The attribute is not applicable Producer defined 0.0 4645.9 See Units Units Units of measure for the analytical result Producer Defined Units of measure for the analytical result NA The attribute is not applicable Producer defined Ratio The ratio of biomarker GSSG to GSH Producer Defined NC The ratio of GSSG to GSH could not be calculated because the result for GSSG was 0 micromoles per gram. Producer defined 0.15 11.70 unitless NA The attribute is not applicable Producer defined Histopathology_livers_kidneys Results of histopathological examinations of individual avian liver and kidney tissues for lesions associated with exposure to lead and other metals. Producer Defined Sample_ID A unique identifier assigned to the sample Producer Defined A unique alphanumeric identifier assigned to the sample Taxon The taxonomic identification of the sample organism. Producer Defined The taxonomic identification of the sample organism. Sex The identified sex of the sample organism Producer Defined Female The sample organism was determined to be female Producer defined Male The sample organism was determined to be male Producer defined Unknown The value for the attribute is unknown Producer defined Age_class The determined age class of the sample organism Producer Defined AHY A bird known to have hatched before the calendar year of banding; year of hatch otherwise unknown. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. HY A bird capable of sustained flight and known to have hatched during the calendar year in which it was banded. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. U A bird that cannot be placed in any of the other age classes. https://www.pwrc.usgs.gov/bbl/manual/age.cfm#:~:text=A%20nestling%20or%20young%20bird,age%20HY%20until%20December%2031. Unknown The value for the attribute it unknown Producer defined Tissue The type of tissue that was examined for microscopic lesions. Producer Defined Kidney The tissue examined was kidney. Producer defined Liver The tissue examined was liver. Producer defined Site A literal value representing the common term used to identify the sampling location Producer Defined A literal value representing the common term used to identify the sampling location, see entity Sampling_sites for details Year An indicator of the year in which the sample was collected. The value is expressed in YYYY. Producer Defined 2017 2018 Lesion An identifier of the microscopic lesion for which the tissue was examined by a single pathologist. Producer Defined AFI The lesion was acid fast inclusions, defined as discrete acid-fast positive intranuclear inclusions consistent with lead inclusions in renal tubular epithelium (Ziehl-Neelsen and Fite's acid fast stains). Producer defined BHY The lesion was biliary hyperplasia, defined as increased numbers of bile duct profiles in portal triads. Producer defined BS The lesion was bile stasis, defined as increased abounts of yellow-green pigment in bile caniliculi or hepatocytes. Producer defined GLOM The lesion was glomerulopathy, defined as any glomerular abnormality. Producer defined HEMO The lesion was hemosiderosis, defined as increased amounts of golden-green pigment in Kupffer cells or hepatocytes. Producer defined KINF The lesion was inflammation in the kidney tissues, defined as interstitial or tubular inflammation of any type, including lymphoplasmacytic inflammation, granulocytic inflammation, and granulomas. Producer defined LINF The lesion was inflammation of any type and location in the liver tissues, including granulomas, granulocytic inflammation, and Kupffer cell hyperplasia. Producer defined LIP The lesion was lipidosis; defined as discrete clear vacuoles within hepatocytes. Producer defined MGY The lesion was cytomegaly/karyomegaly, defined as enlargement of cells or nuclei of the renal tubular epithelium. Producer defined KNEC The lesion was renal tubular degeneration or necrosis. Producer defined LNEC The lesion was hepatocellular degeneration or necrosis. Producer defined Occurrence An indicator of the presence or absence of the microscopic LESION within the TISSUE being examined. Producer Defined Absent The lesion was absent. Producer defined Present The lesion was present. Producer defined Sampling_sites A general description of the sites where samples were collected by name, type (reference or contaminated), and a central global positioning system (GPS) point within the collection site, indicating the general location of the site within southeastern Missouri Producer Defined Site A literal value representing the common term used to identify the sampling location Producer Defined A literal value representing the common term used to identify the sampling location Site_Type An indicator of the environmental conditions of the sampling site based on proximity to known mining and tailing locations. Producer Defined Contaminated A site where mining, milling, and smelting processes released lead into the environment; there may be ongoing releases of lead, and soil lead concentrations exceeded 38 parts-per-million as measured by X-ray fluorescence spectrometry. Producer defined Reference A site that has no history of lead releases from mining, milling, or smelting processes; soil lead concentrations were less than 38 parts-per-million as measured by X-ray fluorescence spectrometry. Producer defined Latitude Geographic coordinate that specifies the north–south position of a point on the Earth's surface. Reported in decimal degrees, WGS84 datum. The point is centrally located at the SITE Producer Defined 37.314183 38.109464 decimal degree Longitude Geographic coordinate that specifies the east-west position of a point on the Earth's surface. Reported in decimal degrees, WGS84 datum. The point is centrally located at the SITE Producer Defined -91.120926 -89.54655 decimal degree GS ScienceBase U.S. Geological Survey mailing and physical

Denver Federal Center, Building 810, Mail Stop 302

Denver CO 80225 United States 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. 20230814 CERC Data Managers U.S. Geological Survey, Columbia Environmental Research CenterSOUTHEAST REGION Natural Resources Data Management Specialist (Data Steward) mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 gs-mw-cerc_data_manager@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999