Heather L. Walsh Stephanie E. Gordon Adam J. Sperry Michael Kashiwagi John Mullican Vicki S. Blazer 20221123 spreadsheet Reston, VA U.S. Geological Survey https://doi.org/10.5066/P9XZ7UWK Decades of poor reproductive success and young-of-the-year recruitment, in addition to adult mortality, has led to a decline in the smallmouth bass (SMB) population in subwatersheds of the Potomac River. Previous studies have identified numerous biologic and environmental stressors associated with negative effects on SMB health. To better understand the impact of these stressors, the current study was conducted from 2013-2019 to identify temporal changes associated with SMB reproductive health. Grab surface water samples were collected and analyzed for over 300 organic contaminants, including pesticides, phytoestrogens, pharmaceuticals, hormones and total estrogenicity (E2Eq). Adult SMB were collected and sampled for multiple endpoints, including gene transcripts associated with reproduction (molecular), histopathology (cellular), and organosomatic indices (tissue). In males, biomarkers of estrogenic endocrine disruption, including testicular oocytes (TO) and plasma vitellogenin (Vtg) were assessed. The results identified numerous agriculture-related contaminants or land use associated with gene transcripts in males and females and a positive association of pesticides in the immediate catchment with TO severity and E2Eq with plasma Vtg in males. Prevalence of TO and detectable levels of plasma Vtg, liver vitellogenin transcripts (vtg) and testis vtg were high throughout the study. Peaks of complex mixtures of numerous contaminants occurred during the spring/early summer when early development occurs and to a lesser extent in fall/winter during recrudescence. Management practices to reduce exposure during these critical and sensitive periods may enhance reproductive health of economically important sportfishes. Smallmouth bass were from sampled the Potomac River in Dargan, Maryland (located within the Chesapeake Bay drainage) for a long-term, temporal study on reproductive health from 2013-2019 in the spring and fall. Tissues (gonad and liver) and plasma were analyzed for endpoints associated with endocrine disruption, including testicular oocytes (TO) and plasma vitellogenin (Vtg). Gene transcripts were quantified for reproduction-associated genes from the liver from both sexes and from the testis for genes associated with reproduction and immune function. Additionally, the gonadosomatic index for each fish was determined. Correlations between biological endpoints and transcripts were conducted in addition to correlation analyses with highly detected contaminants. 20130329 20190523 observed Complete None planned World -77.80655 -77.69737 39.43487 39.39057 ISO 19115 Topic Category biota None transcript abundance testicular oocytes plasma vitellogenin reproductive health endocrine disruption USGS Metadata Identifier USGS:627a76f3d34e8d45aa6e4e1a None Chesapeake Bay drainage Potomac River Maryland None Smallmouth Bass Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Actinopterygii Class Teleostei Superorder Acanthopterygii Order Perciformes Suborder Percoidei Family Centrarchidae Genus Micropterus Species Micropterus dolomieu TSN: 550562 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. Heather Walsh U.S. Geological Survey, Eastern Ecological Science Center Fish Research Biologist mailing and physical

11649 Leetown Rd

Kearneysville WV 25430 3047244445 hwalsh@usgs.gov Microsoft Excel R Nanostring nSolver 4.0 Softmax Pro 7.1.0 Data are accurate and fall within the acceptable range of values. All analyses were run twice to confirm results. Data is consistent and falls within the expected ranges. Data only represents the reproductive health of smallmouth bass from the Potomac River, Dargan, Maryland from 2013-2019. Smallmouth bass sampling Approximately 20 adult smallmouth bass (≥ 200 mm in length) were collected by boat electroshocking in spring of 2013, 2015 - 2019 and in the fall of 2016 and 2018 near the confluence of Antietam Creek and the Potomac River, near Dargan, Maryland (39.409378, -77.744009). All fish were humanely euthanized with a lethal dose (350 mg/L) of tricaine methanesulfonate (MS-222, Argent Finquel, Redmond, Washington) according to the USGS Eastern Ecological Science Center’s Institutional Animal Care and Use Committee protocol. Fish were weighed (gm), measured (total length in mm), examined for visible abnormalities and a blood sample was obtained from the caudal vessel using a sterile 3 mL syringe with a 23-gauge needle. Blood was placed into a heparinized Vacutainer tube (Fisher Scientific, Waltham, MA, USA) and stored on wet ice until returned to the laboratory (2–4 h). Blood was centrifuged at 1000× g at 4 °C for 10 min and plasma was aliquoted into cryovials and stored at −80℃. A slit was cut from the anus to the operculum and the gonad and liver were excised and weighed to the nearest 0.01 g. Pieces of liver and gonad tissue were placed into bottles containing Z-Fix® (Anatech Ltd, Battle Creek, Michigan) for histology and small pieces of liver and testes were placed into separate RNALater® (ThermoFisher, Waltham, Massachusetts) tubes for RNA preservation. Samples preserved in RNALater® were kept in the refrigerator for 24 hr prior to freezing at -20℃ until further use. The gonadosomatic index (GSI) was calculated as follows: (gonad weight / body weight) × 100. Histological preparation Pieces of gonad and liver were fixed in Z-Fix® (Anatech, Battle Creek, Michigan) for ≥ 48 hr prior to being processed for histology. Tissue sections were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (Luna 1992). Five to eight cross-sections along the length of one testis were examined for the presence of testicular oocytes and a severity rating was assigned according to Blazer et al. (2007). Testicular oocytes were never found to advance past the primordial follicle stage. A severity score of 1 indicated a single oocyte per field, 2 was two or more oocytes per field not closely associated, 3 was multiple clusters of 2-5 oocytes closely associated, and 4 was zonal groups of more than five closely associated oocytes. A ranking system was also established to classify severity as low, medium, or high. Low ranged from severity scores of 0-0.4, medium ranged from 0.5-1.5, and high was > 1.6. Plasma vitellogenin detection Plasma Vtg concentrations were measured using a direct enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody 3G2 (Caymen Chemical, Ann Arbor, Michigan) as previously described (Denslow et al. 1999; Blazer et al. 2014b). Briefly, plasma samples were diluted as necessary in PBSZ-AP (10 mM phosphate, 150 mM NaCl, 0.02% azide, pH 7.6). SMB plasma Vtg was used as a standard for all plasma analyzed. The plasma Vtg standards were prepared at the University of Florida, Department of Physiological Sciences from plasma of 17β-estradiol exposed male SMB held at the Eastern Ecological Science Center, Leetown Research Laboratory. Optical density was measured on a multiwell plate reader (SpectraMax M4, Molecular Devices Inc., Sunnyvale, California) at 405 nm. Concentrations of the unknowns were determined from the standard curves with the Softmax Pro TM Program version 7.1.0 (Molecular Devices Inc.). Limit of detection was 1 µg/ml. Inter- and intra-assay variability was < 10%. RNA extraction and analyses Liver was collected in RNALater® from all fish used in this study (2013-2019). Testes were also collected in RNALater® in spring of 2015-2017 and fall of 2016 and 2018. Total RNA was extracted with an E.Z.N.A Total RNA Kit I (Omega Bio-Tek, Norcross, Georgia) from approximately 10-15 mg testes and 20-25 mg liver. A DNase treatment step was performed with RNase-free DNAse set I (Omega Bio-Tek) to remove DNA contamination and all samples were eluted in 50 µl nuclease-free water. Purified RNA was quantified with an RNA BR Assay Kit (Agilent, Santa Clara, California) on a Qubit 4 Fluorometer. Transcript abundance analyses were conducted with 50 ng of RNA/sample on the Nanostring nCounter® (Nanostring Technologies, Inc., Seattle, WA) with transcripts from a previously published liver CodeSet (Hahn et al. 2016). Testes transcripts were measured using two custom CodeSets developed from the SMB testes transcriptome (Walsh et al. 2022). A total of 58 (including three housekeeping genes) sequences were used for the smallmouth bass testes immune custom CodeSet (Supplemental Table 1) and an additional 43 (including three housekeeping genes) were included in the smallmouth bass testes reproductive custom CodeSet Supplemental Table 2). Transcript abundance was normalized with internal positive and negative controls and housekeeping transcripts included in each custom CodeSet using nSolver 4.0 (Nanostring Technologies, Inc.). 20210620 MS5_Biological and Liver Transcript Data.csv Comma Separated Value (CSV) file containing smallmouth bass biological endpoints and liver transcript abundance data. Producer Defined Sample Smallmouth bass identification number Producer Defined Total number of smallmouth bass collected between 2013-2019. MS5 = (Potomac) main stem site 5. Collection Date Date smallmouth bass were collected. Producer Defined 3/29/2013 March 29, 2013 Producer defined 5/5/2015 May 5, 2015 Producer defined 4/14/2016 April 14, 2016 Producer defined 10/20/2016 October 20, 2016 Producer defined 5/2/2017 May 2, 2017 Producer defined 5/5/2018 May 5, 2018 Producer defined 10/30/2018 October 30, 2018 Producer defined 5/23/2019 May 23, 2019 Producer defined Season Season smallmouth bass were collected. Producer Defined Spring Smallmouth bass collected in the spring. Producer defined Fall Smallmouth bass collected in the fall. Producer defined Sex Sex of each smallmouth bass. Producer Defined M Male Producer defined F Female Producer defined Age Age of each smallmouth bass. Producer Defined 2 9 years 1 Collection Year Year smallmouth bass were collected Producer Defined 2013 2019 Years 1 Year Class Year each smallmouth bass was hatched. Calculated as: collection year - age. Producer Defined 2005 2017 Year 1 Total Length Total length of each smallmouth bass (from snout to tip of caudal fin). Producer Defined 189 456 millimeters 1 Weight Weight of each smallmouth bass. Producer Defined 71 1323 grams Gonad Weight Gonad weight (ovary or testis) of each smallmouth bass. Producer Defined NA No Data Producer defined 0.31 109.77 grams 0.01 Liver Weight Liver weight of each smallmouth bass. Producer Defined ND No Data Producer defined 0.62 29.30 grams 0.01 HSI Hepatosomatic index: liver weight /(body weight – gonad weight)) × 100. Producer Defined ND No Data Producer defined 0.44 4.06 GSI Gonadosomatic index: (gonad weight /body weight) × 100. Producer Defined NA No Data Producer defined 0.0 12.96 Testicular Oocyte Severity Rank Testicular oocyte severity rank assigned according to testicular oocyte severity. Producer Defined NA No Data Producer defined high Testicular oocyte severity > 1.6 Producer defined low Testicular oocyte severity 0-0.5 Producer defined moderate Testicular oocyte severity 0.6-1.5 Producer defined Testicular Oocyte Severity The testicular oocyte severity score of each male collected. Five cross-sections along the length of one testis were examined for the presence of testicular oocytes and a severity rating was assigned. This is the mean severity of the five cross-sections. Each cross section is scored 0-4 with 0 being no testicular oocytes, 1 indicates a single oocyte per field, 2 is two or more oocytes per field not closely associated, 3 is multiple clusters of 2-5 oocytes closely associated, and 4 is zonal groups of more than five closely associated oocytes. Producer Defined NA No Data Producer defined 0.0 3.0 Testicular Oocyte Prevalence Presence or absence of testicular oocytes. Producer Defined NA No Data Producer defined 1 Testicular oocytes present Producer defined 0 Testicular oocytes not present Producer defined Plasma Vitellogenin Concentration of plasma vitellogenin. Producer Defined NA, BD NA = no data BD = below detection Producer defined 0.86 8834.63 microgram/milliliter 1 17BHD 17 beta hydroxysteroid dehydrogenase Producer Defined 48 627 counts 26 ARA Androgen receptor alpha Producer Defined 10 355 counts 26 ARB Androgen receptor beta Producer Defined 23 1417 counts 26 CHG Choriogenin Producer Defined 32 279791 counts 26 EF1a Elongation factor 1 alpha Producer Defined 30280 58731 counts 26 ERA Estrogen receptor alpha Producer Defined 45 1709 counts 26 ERB1 Estrogen receptor beta 1 Producer Defined 23 234 counts 26 ERB2 Estrogen receptor beta 2 Producer Defined 54 667 counts 26 VTG Vitellogenin Producer Defined 5 1023509 counts 26 ETIF3D Eukaryotic translation initiation factor 3D Producer Defined 691 2161 counts 26 RPL8 Ribosomal protein L8 Producer Defined 2179 5040 counts 26 MS5_Testis Immune Transcript Data.csv Comma Separated Value (CSV) file containing smallmouth bass testis transcript abundance data associated with immune function. Producer Defined Sample Smallmouth bass identification number Producer Defined Identification number of male smallmouth bass. Collection Date Date smallmouth bass were collected. Producer Defined 5/5/2015 May 5, 2015 Producer defined 4/14/2016 April 14, 2016 Producer defined 10/20/2016 October 20, 2016 Producer defined 5/2/2017 May 2, 2017 Producer defined 10/30/2018 October 30, 2018 Producer defined Season Season smallmouth bass were collected. Producer Defined Spring Smallmouth bass collected in the spring. Producer defined Fall Smallmouth bass collected in the fall. Producer defined Sex Sex of smallmouth bass. Producer Defined M Male Producer defined 5-HT2BR 5-Hydroxytryptamine receptor 2B Producer Defined 71 2443 counts 23 A-FABP Adipocyte fatty acid binding protein Producer Defined 49 3698 counts 23 C6 Complement component 6 Producer Defined 41 745 counts 23 C7 Complement component 7 Producer Defined 24 2287 counts 23 C8A Complement component 8A Producer Defined 20 2109 counts 23 C9 Complement component 9 Producer Defined 2 2054 counts 23 CXCL11 C-X-C motif chemokine 11 Producer Defined 4 2384 counts 23 CXCL8 C-X-C motif chemokine 8 Producer Defined 20 374 counts 23 CYP3A Cytochrome P450 family 3A Producer Defined 54 860 counts 23 GHR-like Growth hormone receptor-like Producer Defined 6 1886 counts 23 GHR2 Growth hormone receptor 2 Producer Defined 134 1168 counts 23 GPR176 G protein-coupled receptor 176 Producer Defined 17 1851 counts 23 GRTP1 Growth hormone-regulated TBC protein 1 Producer Defined 221 1739 counts 23 HEP1 Hepcidin 1 Producer Defined 8 254 counts 23 HSP71 Heat shock protein 71 Producer Defined 22333 77443 counts 23 IL-15 Interleukin 15 Producer Defined 87 3013 counts 23 IL4I1 Interleukin 4 induced 1 Producer Defined 2 131 counts 23 IRF3 Interferon regulatory factor 3 Producer Defined 90 756 counts 23 ISG15 Interferon-stimulated gene 15 Producer Defined 301 1698 counts 23 LTBP-2 Latent-transforming growth factor beta-binding protein 2 Producer Defined 45 1722 counts 23 LTBP-3 Latent-transforming growth factor beta-binding protein 3 Producer Defined 54 2368 counts 23 LYZC Lysozyme C Producer Defined 4 6386 counts 23 NADPH-P450 Cytochrome P450 reductase Producer Defined 3894 45235 counts 23 NOX1 NADPH oxidase 1 Producer Defined 6 1930 counts 23 PGIS Prostacyclin synthase Producer Defined 84 3695 counts 23 PREB Prolactin regulatory element binding Producer Defined 972 3105 counts 23 PRLR Prolactin receptor Producer Defined 362 2184 counts 23 PTGES Prostaglandin E synthase Producer Defined 512 6164 counts 23 PTGS2 Prostaglandin-endoperoxide synthase 2 Producer Defined 67 2169 counts 23 SAMHD-1 SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 Producer Defined 44 5837 counts 23 SELPLG Selectin P ligand Producer Defined 80 2090 counts 23 SOCS1 Suppressor of cytokine signaling 1 Producer Defined 46 2346 counts 23 SOCS2 Suppressor of cytokine signaling 2 Producer Defined 90 2019 counts 23 SOCS3 Suppressor of cytokine signaling 3 Producer Defined 122 2333 counts 23 SOCS5 Suppressor of cytokine signaling 5 Producer Defined 189 1684 counts 23 SOCS6 Suppressor of cytokine signaling 6 Producer Defined 249 2973 counts 23 SOCS7 Suppressor of cytokine signaling 7 Producer Defined 151 2936 counts 23 TCR T cell receptor Producer Defined 21 2425 counts 23 TGFB1 Transforming growth factor beta-1 Producer Defined 290 2245 counts 23 TGFB1I1 Transforming growth factor beta 1 induced transcript 1 Producer Defined 209 1200 counts 23 TGFB2 Transforming growth factor beta-2 Producer Defined 153 2057 counts 23 TGFB3 Transforming growth factor beta-3 Producer Defined 80 899 counts 23 TGFBR3 Transforming growth factor beta receptor 3 Producer Defined 650 2205 counts 23 TGFBRAP Transforming growth factor-beta receptor-associated protein Producer Defined 28 2174 counts 23 TGFBRAP1 Transforming growth factor-beta receptor-associated protein 1 Producer Defined 235 1535 counts 23 TLR13 Toll-like receptor 13 Producer Defined 101 21049 counts 23 TNFAIP2 Tumor necrosis factor alpha induced protein 2 Producer Defined 65 896 counts 23 TNFAIP3 Tumor necrosis factor alpha induced protein 3 Producer Defined 134 2255 counts 23 TOLLIP Toll-interacting protein Producer Defined 721 3758 counts 23 TRAF2 Tumor necrosis factor receptor associated factor 2 Producer Defined 23 399 counts 23 TRAF3 Tumor necrosis factor receptor associated factor 3 Producer Defined 165 958 counts 23 TRAF5 Tumor necrosis factor receptor associated factor 5 Producer Defined 30 132 counts 23 TRAF6 Tumor necrosis factor receptor associated factor 6 Producer Defined 148 551 counts 23 VPS39 Vacuolar protein sorting 39 Producer Defined 395 2145 counts 23 ZBPA33 Z-DNA binding protein A33 Producer Defined 184 1082 counts 23 ACTB Beta actin Producer Defined 21435 85730 counts 23 EIF3D Elongation translation initiation factor 3D Producer Defined 2392 5439 counts 23 40Srps18 40S ribosomal protein S18 Producer Defined 4826 10007 counts 23 MS5_Testis Reproductive Transcript Data.csv Comma Separated Value (CSV) file containing smallmouth bass testis transcript abundance data associated with reproduction. Producer Defined Sample Smallmouth bass identification number. Producer Defined Identification number of male smallmouth bass. Collection Date Date smallmouth bass were collected. Producer Defined 5/5/2015 May 5, 2015 Producer defined 4/14/2016 April 14, 2016 Producer defined 10/20/2016 October 20, 2016 Producer defined 5/2/2017 May 2, 2017 Producer defined 10/30/2018 October 30, 2018 Producer defined Season Season smallmouth bass were collected. Producer Defined Spring Smallmouth bass collected in the spring. Producer defined Fall Smallmouth bass collected in the fall. Producer defined Sex Sex of smallmouth bass. Producer Defined M Male Producer defined 17BHD 17 beta hydroxysteroid dehydrogenase Producer Defined 228 2706 counts 18 3BHD 3 beta hydroxysteroid dehydrogenase Producer Defined 1165 4447 counts 18 42sp43 P43 5S RNA-binding protein Producer Defined 13 3978 counts 18 ARA Androgen receptor alpha Producer Defined 279 973 counts 18 ARB Androgen receptor beta Producer Defined 246 1193 counts 18 BACTIN Beta actin Producer Defined 20864 91845 counts 18 CYPP45011B Cytochrome P450 Family 11 subfamily B Producer Defined 115 1714 counts 18 AROMATASE Estrogen synthetase Producer Defined 5 924 counts 18 DMRT1 Doublesex and mab-3 related transcription factor 1 Producer Defined 1523 12866 counts 18 DMRT2 Doublesex and mab-3 related transcription factor 2 Producer Defined 98 1877 counts 18 DMRT3 Doublesex and mab-3 related transcription factor 3 Producer Defined 39 805 counts 18 ERA Estrogen receptor alpha Producer Defined 25 139 counts 18 ERB1 Estrogen receptor beta 1 Producer Defined 258 1557 counts 18 ERB2 Estrogen receptor beta 2 Producer Defined 478 2216 counts 18 SOX11 SRY-box transcription factor 11 Producer Defined 29 664 counts 18 SOX7 SRY-box transcription factor 7 Producer Defined 87 1382 counts 18 SOX9 SRY-box transcription factor 9 Producer Defined 565 6059 counts 18 STAR Steroidogenic acute regulatory protein Producer Defined 55 2081 counts 18 VTG Vitellogenin Producer Defined 13 1500 counts 18 VTGC Vitellogenin C Producer Defined 12 1938 counts 18 WNT5B Wingless-type MMTV integration site family, member 5B Producer Defined 20 522 counts 18 ZAR1 Zygote arrest 1 Producer Defined 6 1812 counts 18 DRSDRFM11 Dehydrogenase/reductase SDR family member 11 Producer Defined 95 2968 counts 18 FST Follistatin Producer Defined 1173 5203 counts 18 FST3 Follistatin 3 Producer Defined 301 2039 counts 18 GRHR Growth hormone releasing hormone receptor Producer Defined 24 512 counts 18 NOBOX Newborn ovary homeobox-encoding gene Producer Defined 12 1828 counts 18 INHA Inhibin alpha Producer Defined 3272 12575 counts 18 INHBB Inhibin beta B Producer Defined 131 1543 counts 18 IGFBP1 Insulin-like growth factor binding protein 1 Producer Defined 73 2311 counts 18 IGFBP2A Insulin-like growth factor binding protein 2A Producer Defined 57 1456 counts 18 IGFBP3 Insulin-like growth factor binding protein 3 Producer Defined 33 1391 counts 18 IGFBP5 Insulin-like growth factor binding protein 5 Producer Defined 57 1250 counts 18 NANOS Nanos homolog Producer Defined 12 934 counts 18 RR2 Relaxin receptor 2 Producer Defined 68 1363 counts 18 UP1 Uncharacterized protein 1 Producer Defined 6 1790 counts 18 UP2 Uncharacterized protein 2 Producer Defined 6 4044 counts 18 ZP3 Zona pellucida 3 Producer Defined 39 2482 counts 18 ZP3IX1 Zona pellucida 3ix1 Producer Defined 4 1630 counts 18 ZP4 Zona pellucida 4 Producer Defined 30 1076 counts 18 ZPAX Zona pellucida AX Producer Defined 15 1744 counts 18 40Srps18 40S ribosomal protein S18 Producer Defined 6147 13855 counts 18 ETIF3D Elongation translation initiation factor 3D Producer Defined 1838 8043 counts 18 Total E2Eq.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample Date Date grab water sample was collected. Producer Defined Date is defined as month/day/year E2Eq Total estrogenicity (E2Eq) was quantified from water samples with a bioluminescent yeast-based estrogen screen (BLYES) and reported relative to 17β-estradiol equivalents. Producer Defined BD Below detection Producer defined 0.131 BD ng/L 0.002 Hormones.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample Date Date grab water sample was collected Producer Defined Date is defined as month/day/year 3-beta-Coprostanol Hormone analzyed in filtered water samples. Producer Defined BD Below detection Producer defined 239 BD ng/L 200 Cholesterol Hormone analzyed in filtered water samples. Producer Defined BD Below detection Producer defined 1050 BD ng/L 200 cis-Androsterone Hormone analzyed in filtered water samples. Producer Defined BD Below detection Producer defined BD 3.84 ng/L 0.4 trans-Diethylstilbestrol Hormone analzyed in filtered water samples. Producer Defined BD Below detection Producer defined BD 1.53 ng/L 0.4 Testosterone Hormone analzyed in filtered water samples. Producer Defined BD Below detection Producer defined BD 23.4 ng/L 0.8 Pesticides.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample Date Date grab water sample was collected Producer Defined Date is defined as month/day/year Lab-Analysis either from California Lab (CA) or National Water Quality Laboratory (NWQL) Indication of whether the lab-Analysis was performed at the USGS Organic Chemistry Research Laboratory, Sacramento, CA (CA) or USGS National Water Quality Laboratory (NWQL) Producer Defined CA USGS Organic Chemistry Research Laboratory, Sacramento, CA Producer defined NWQL USGS National Water Quality Laboratory Producer defined Atrazine Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.4 ng/L 2.3 Carbaryl Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 23.2 ng/L 6.5 Diazinon Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 1.4 ng/L 0.9 Metolachlor Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.4 ng/L 1.5 Pendimethalin Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 8.5 ng/L 2.3 Simazine Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.1 ng/L 5 3,4-Dichloroaniline Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.2 ng/L 8.3 Azoxystrobin Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 114.0 ng/L 3.1 Fipronil Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.0 ng/L 2.9 Fipronil sulfone Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 4.0 ng/L 3.5 Myclobutanil Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 31.3 ng/L 6 Tebuconazole Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 316.0 ng/L 3.7 3,5-Dichloroaniline Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 9.5 ng/L 7.6 Boscalid Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 10.4 ng/L 2.8 Fenhexamid Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 104.0 ng/L 7.6 Prometon Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 19.2 ng/L 2.5 Metalaxyl Pesticide in filtered water samples using both CA method and NWQL method Producer Defined BD Below detection Producer defined BD 108.0 ng/L 5.1 Pharmaceuticals.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample Date Date grab water sample was collected Producer Defined Date is defined as month/day/year Hexamethylenetetramine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD, ND Below detection, No data Producer defined BD 328 ng/L Guanylurea Pharmaceutical and other compounds in filtered water samples. Producer Defined BD, ND Below detection, No data Producer defined BD 1130 ng/L Gabapentin Pharmaceutical and other compounds in filtered water samples. Producer Defined ND, BD No data, Below detection Producer defined BD 155 ng/L Piperonyl butoxide Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 1.03 ng/L 1.53 Acetaminophen Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 12.6 ng/L 3.56 Bupropion Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 13.8 ng/L 3.57 Caffeine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 40 ng/L 18.1 Carbamazepine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 56.6 ng/L 0.84 Cimetidine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 21.7 ng/L 5.56 Cotinine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 8.51 ng/L 1.27 Diphenhydramine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 6.78 ng/L 2.9 Ranitidine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 56.3 ng/L 38.3 Sulfamethoxazole Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 187 ng/L 20.8 Trimethoprim Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 16.5 ng/L 3.8 Morphine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 28.4 ng/L 2.81 Lidocaine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 71.6 ng/L 3.05 Meprobamate Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 29.1 ng/L 17.2 Dextromethorphan Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 6 ng/L 1.64 Triamterene Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 6.46 ng/L 2.63 Acyclovir Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 70.9 ng/L 4.44 Metformin Pharmaceutical and other compounds in filtered water samples. Producer Defined ND No data Producer defined BD 459 ng/L 6.56 Nicotine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD 106 ng/L 28.9 Oxycodone Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 30.8 ng/L 4.97 Carisoprodol Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 39.4 ng/L 2.5 Methadone Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 8.54 ng/L 3.81 Methocarbamol Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 148 ng/L 4.36 Atenolol Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 9.54 ng/L 2.66 Citalopram Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 6.14 ng/L 1.32 Hydrocodone Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 2.64 ng/L 2.1 Fexofenadine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD 218.0 ng/L 3.98 methyl-1H-benzotriazole Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 765 ng/L 28.2 Tramadol Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 102 ng/L 3.02 Diltiazem Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 85.3 ng/L 5.08 Metoprolol Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 41.7 ng/L 13.8 Sitagliptin Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 67 ng/L 19.5 Venlafaxine Pharmaceutical and other compounds in filtered water samples. Producer Defined 3.52 62.3 ng/L 0.9 Famotidine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD 9.53 ng/L 2.13 Lamivudine Pharmaceutical and other compounds in filtered water samples. Producer Defined BD Below detection Producer defined BD 5.19 ng/L 3.22 Desvenlafaxine Pharmaceutical and other compounds in filtered water samples. Producer Defined 16.7 184.0 ng/L 3.75 Phytoestrogens.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample Date Date grab water sample was collected Producer Defined Date is defined as month/day/year Biochanin A Phytoestrogen in filtered water samples. Producer Defined BD Below detection Producer defined BD 38 ng/L 0.5 Daidzein Phytoestrogen in filtered water samples. Producer Defined BD Below detection Producer defined BD 130 ng/L 0.5 Equol Phytoestrogen in filtered water samples. Producer Defined BD Below detection Producer defined BD 110 ng/L 0.5 Formonentin Phytoestrogen in filtered water samples. Producer Defined BD Below detection Producer defined BD 54 ng/L 0.5 Genestein Phytoestrogen in filtered water samples. Producer Defined BD Below detection Producer defined BD 12 ng/L 0.5 GS ScienceBase U.S. Geological Survey mailing address

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. Digital Data https://doi.org/10.5066/P9XZ7UWK None 20221123 Heather Walsh U.S. Geological Survey, Eastern Ecological Science Center Fish Research Biologist mailing and physical

11649 Leetown Rd

Kearneysville WV 25430 3047244445 hwalsh@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999