Patrick T. Kroboth Benjamin H. Stahlschmidt Duane C. Chapman 20231207 tabular digital data Reston, VA U.S. Geological Survey Authors Open Researcher and Contributor Id (ORCID) are as follows: Travis W. Schaeffer: 0000-0002-8126-8660 https://doi.org/10.5066/P93JXORA A piscicide delivery method was designed to selectively target black carp Mylopharyngodon piceus, an invasive species in North America which possesses specially adapted pharyngeal teeth for crushing mollusk prey. Bait was prepared by attaching a glass vial containing toxicant (antimycin A) to the exterior of Corbicula fluminea clam valve. The vial was designed to break by the force exerted from the fish’s (550 millimeter total length) pharyngeal teeth when the fish attempted to crush and consume the clam. Suitable vial size was tested for encapsulating piscicide and two attachment materials, aquarium epoxy and ultraviolet light (UV) cured attachment material. Toxic baits consisting of antimycin A piscicide were administered in ethanol and acetone carriers to black carp in assembled baits with vials attached to live clam valves at three concentrations (40.0 milligrams per milliliter ethanol and 341.5 and 170.8 milligrams per milliliter acetone). Additionally fathead minnow Pimephales promelas toxicity data from experiments validating the methods of flame sealing glass vials are present in this dataset. Many North American mollusks are imperiled, existing in small, fragmented populations susceptible to predation. A black carp-selective toxic bait deployed appropriately could be used to protect those populations. This data represents several stages in the development of a black carp specific bait and are a culmination of preliminary work. Video footage generated for this project is considered a temporary project record through disposition authority of USGS 1600-01b. As such the video footage is available upon request through 2026 20170228 20180427 ground condition Complete None planned Columbia Environmental Research Center -92.285000 -92.273640 38.914900 38.908060 ISO 19115 Topic Category biota USGS Thesaurus ecotoxicology invasive species National Agricultural Library Thesaurus black carp baits fish poisons USGS Biocomplexity Thesaurus fishes bivalves USGS Metadata Identifier USGS:6495aacfd34ef77fcb01dd07 None Columbia Environmental Research Center USGS Biocomplexity Thesaurus fishes bivalves Integrated Taxonomic Information System (ITIS) 2022 ONLINE_REFERENCE Washington, D.C. Integrated Taxonomic Information System (ITIS) http://itis.gov Patrick T Kroboth U.S. Geological Survey, Columbia Environmental Research Center Fish Biologist mailing and physical

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 pkroboth@usgs.gov expert advice Black carp and fathead minnows from Columbia Environmental Research Center cultured stocks were utilized. Clams were identified by experts. Fish were identified to species. Clams were identified to genus Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Actinopterygii Class Teleostei Superorder Ostariophysi Order Cypriniformes Superfamily Cyprinoidea Family Cyprinidae Genus Mylopharyngodon Species Mylopharyngodon piceus Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Lophozoa Phylum Mollusca Class Bivalvia Subclass Heterodonta Order Veneroida Superfamily Corbiculoidea Family Corbiculidae Genus Corbicula Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Actinopterygii Class Teleostei Superorder Ostariophysi Order Cypriniformes Superfamily Cyprinoidea Family Cyprinidae Genus Pimephales Species Pimephales promelas fathead minnow carpita cabezona tête-de-boule None. Please see 'Distribution Info' for details. It is requested that the authors and the USGS Columbia Environmental Research Center be cited for any subsequent publications that reference this dataset. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Patrick T Kroboth U.S. Geological Survey, Columbia Environmental Research Center Research Fish Biologist mailing address

4200 New Haven Road

Columbia MO 65201 US 573-875-5399 pkroboth@usgs.gov Great Lakes Restoration Initiative Windows 10 version 20H2, build 19042,1348; notepad++ 64-bit x64 version 8.1.9.3; ASTM International 2014 BOOK Unkown ASTM International https://poseidonsciences.com/ASTM_E_729.pdf Brian J. Finlayson Rosalie A. Schnick Richard L. Cailteux Leo DeMong William D. Horton William McClay Charles W. Thompson 200206 OTHER Fisheries vol. 27, issue 6 n/a Wiley Haag, W. R 2012 BOOK New York, NY Cambridge University Press Berger, B. L. Lennon, R. E. Hogan, J. W. 1969 OTHER Investigations in Fish Control Investigations in Fish Control 26 Washington, DC U.S. Fish and Wildlife Service Water quality instruments were calibrated monthly. Analytical instruments ran blanks and spikes to ensure accuracy of results. Data were check following entry for transcription errors and relative logical accuracy by second person associated with the study (not the primary recorder). 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 Lab We prepared a bait by attaching a glass vial containing toxicant (antimycin A) to the exterior of an Corbicula fluminea clam valves. The vial was designed to break by the force of the fish’s teeth. We tested suitable bait sizes for fish [approximately 550 mm total length] and evaluated vial breakage with different attachment materials (epoxy or plastic) and vial sizes. A range test of antimycin A in ethanol (near solubility) was first applied via oral dose to identify a potential lethal volume at a 40.0 mg/mL concentration. Mortality was observed at the 200 microliterL volume. This volume was administered in an assembled bait with host clams and vials attached via epoxy at three concentrations (40.0 mg/mL in ethanol, and 341.5 mg/mL and 170.8 mg/mL, both in acetone.). Antimycin A toxicity was tested via bioassay to compare median lethal concentrations to those previously reported for fathead minnows. Similar methods were applied to compare the effects of flame-sealing glass vials on toxicity of antimycin A solution. Study site and water quality monitoring: All experiments were conducted at the U.S. Geological Survey Columbia Environmental Research Center in Columbia, Missouri. Black carp aquaculture production has been restricted; thus, a limited number of fish were available for experiments. Black carp were procured from Keo Fish Farms (Lonoke, AR) and were kept indoors and fed a diet of pelletized food (Extruded 400 and 450 pellets, Rangen Inc., Buhl, ID). Additional Corbicula fluminea clams were fed to the carp alongside the pellets prior to testing to allow pharyngeal teeth to adapt to a harder, mollusk-based diet prior to experiments. Mean fish total length was 585 millimeters (SD=25) and weight 1.93 kilograms (SD=0.27). Initial tests occurred among nine 400 liter indoor tanks. Flow-through well water with an average hardness 280-300 milligrams per liter maintained water quality at a roughly six-hour water exchange rate. During piscicide experiments this was reduced to five 133 liter tanks to allow for a water exchange rate ranging from five to eight minutes. This increase in water exchange reduced the potential for mortality due to aqueous rather than oral toxicity. Berger et al. (1969) reported that brief (7.5-15 minutes) aqueous treatments in static water produced complete mortality of common carp at 100 micrograms per liters antimycin A despite transfer to freshwater after treatment. We wished to remove the potential effects of aqueous toxicity to replicate field conditions more closely, in which dilution of any piscicide released would be rapid and unlikely to cause mortality. Daily water temperature, dissolved oxygen, and pH measurements were recorded for each tank with a YSI Professional Plus Multiparameter Instrument (YSI Incorporated, Yellow Springs, OH; hereafter YSI). Aeration was maintained with compressed air through an air stone. Water quality measurements showed minimal deviation among treatments in during each experiment. Standard operating procedures for animal care and euthanasia were applied throughout testing in accordance with the Columbia Environmental Research Center’s Institutional Animal Care and Use Committee guidelines and ATSM International (2014). 2018 Attachment and vial sizes: We tested the ability of black carp to break three sizes of borosilicate glass vials filled with a water, flame-sealed, and attached to the exterior of the clam valve by two methods. Vials consisted of a pre-manufactured 1 milliliter vial insert of clear borosilicate glass (5.5 millimeter outside diameter (OD); hereafter large; Wheaton, Millville, NJ), and two hand-drawn colored borosilicate glass vials (OD ranged from 5.08-2.98 millimeter; hereafter medium, and 3.18-2.01 millimeter; hereafter small; Mountain Glass Arts, Asheville, NC). Colored borosilicate glass was chosen to help identify broken glass shards in tanks. A Carlisle pipe ribbon burner (Carlisle Machine Works, Millville, NJ) evenly heated sections of tubing to provide consistent wall thicknesses and outside diameter (OD) as stock tubing was hand-drawn. From initial vial designs we found average wall thicknesses less than 0.3 millimeter produced misshapen and brittle vials, so a target 0.45 millimeter wall thickness was used. Completed unsealed vials were annealed in a Sentry Xpress 4.0 Paragon Kiln (Paragon Industries L. P., Mesquite, TX) oven for 24 hours. Once cooled, vials were filled with a water and food coloring mixture, and flame-sealed with a Smith USA Little Torch (Miller Manufacturing Company, Appleton, WI;). Two materials for exterior attachment of vials to clam valves were tested: aquarium epoxy (Two Little Fishes Inc., Miami Gardens, Florida) and ultraviolet (UV) light cured attachment material (Bondic, Aurora, Ontario). The epoxy was a slow curing material, hardening in 20 minutes after mixing. The UV cured attachment material hardens in seconds of exposure to high intensity UV light. Aquarium epoxy is a two-part material that was mixed and placed on the valve of a blotted and air-dried clam. Vials were then depressed into the epoxy. Epoxy was smoothed along the surface of the valve and around the length of the vial. Epoxy was allowed 30-minutes to cure before tests. Clams were prepared for UV cured attachment material by scouring the exterior of the valve with a coarse file and air-dried. The UV cured attachment material was poured between the valve and vial and cured with a 120-volt UV light. A 1-centimeter wide strip of attachment material was poured over the vial and cured with the same light source. This process was repeated twice to produce two overlapping layers in the middle of the vial leaving the ends exposed. The three vial sizes were attached to the exterior of 22 millimeter maximum shell length clams. This size was large enough to attach a vial without eclipsing the shell. Three baits, one of each vial size, were presented to black carp along with seven similarly sized clams without vials to replicate feeding rates. Tanks were inspected at one, three, and twenty four hours to locate glass shards indicative of broken vials (see Bait_physical_measurements_acceptance.txt). An aerated control tank with flow through well water confirmed retention of attachment material and vials on clams for the duration of tests. Two GoPro Hero 4 cameras monitored the first three hours of feeding, placed at the mouth-level of the fish and just below the water surface providing a wide-angle view of the tank. Video observations of feeding on the three vials sizes and the seven additional clams were combined for each tank (see FishFeedingEventsVideo.txt). 2018 Complete piscicide bait: Toxicity of antimycin A is measured by the cumulative percent composition of its four isomers. For consistency among bait development trails, we applied antimycin A from batch number 096M4064V with reported isomers A1 30.55 percent, A2 18.94 percent, A3 17.99 percent, and A4 18.66 percent (Sigma-Aldrich, St. Louis, Missouri). The total of these percentages represents batch purity; measurements of stock material for bait development were adjusted for batch purity prior to mixing. We confirmed prior to testing that the toxicity of antimycin A batch number 096M4064V did not significantly deviate from expected toxicity via bioassay of fathead minnows Pimephales promelas (Kroboth et al. 2022) according to the methods and results of Burger et al. (1969). Three concentrations of antimycin A were tested at a 200 microliter volume in borosilicate glass vials. The 200 microliter volume represented the maximum that could be encapsulated by the borosilicate glass vial and still broken by the fish. Antimycin A concentrations tested were 1) 40.0 mg/mL antimycin A in ethanol solution, 2) 171 mg/mL antimycin A in acetone solution, and 3) 341.5 mg/mL antimycin A in acetone solution. Later acetone solutions were identified based on the concentrations of the individual concentrate and combined concentrate and diluent from the previously commercially available form of antimycin A, Fintrol. Flammable carriers including ethanol and acetone required a cooling stage between filling and sealing to condense the solution. Vials containing ethanol carriers were stored in a -20 degrees Celsius freezer for a minimum 30 minutes. Later acetone carriers required that the vial be submerged in a dry ice and ethanol slurry to rapidly cool prior to flame-sealing. In preparation for tests, one black carp was stocked into each of five 133-liter tanks. For three days prior to trials, fish were trained by feeding three clams, individually with 0.1 gram of Extruded 400 and 450 fish food pellets (Rangen Inc., Buhl, Idaho) throughout the day. Sealed vials were attached with aquarium epoxy to the valve of clams possessing a surface area large enough to attach the vial (approximately 22 millimeters maximum shell length). Assembled baits were stored in a 15 liter tank for 24 hours to harden before testing. Water quality in this storage tank was maintained via flow-through well water and compressed air. Water temperature was recorded with a YSI ProPlus during this 24-hour period. Once testing commenced, survival was monitored for the first hour then at 3, 6, 24, 48, 72, and 96 hours post treatment. One GoPro Hero 4 camera monitored the first three hours of feeding. Cameras were placed back in tanks at 48 and 72 hours after feeding or breaking of the vial, in the case that initial feeding was delayed. The camera recorded audio and video at the fish’s mouth-level over the bait. This footage was used to evaluate behavior following bait consumption. Broken vials were confirmed by the collection of glass shards at physical checks. During feeding at the 171 mg/mL concentration, fish did not consistently consume baits, thus additional non-bait clams were added to these tanks to stimulate feeding. Tests were terminated after 96-hour observation of individuals following consumption of the bait (see Completed_bait.txt) . 2018 As a quality control test 40 fathead minnows (n=280) were stocked among four replicate 10 liter glass test chambers (10 fish in each) consisting of treatment levels: a positive control, negative control, and 5 flame-sealed borosilicate glass vials to test the effect of flame-sealing methods on toxicity of antimycin A. A 160 microliter volume of stock solution was pipetted into each borosilicate glass vial with the pre-pipetted solution cooled. A similar volume of the same solution was pipetted into an unsealed vial (positive control). All piscicide treatments were measured from the same single concentrated solution prepared at the start of testing. The 160 microliter volume from the vials was diluted in a 40 liter carboy with a reconstituted deionized water (hardness 40 parts per million) for each treatment to achieve a treatment aqueous concentration of 0.4 micrograms per liter in each 10 liter glass test chamber. Glass piscicide vials were broken in the 40 liter carboy to facilitate release of the piscicide for dilution. Fathead minnows were additionally stocked at the same rate into 4 replicate test chambers of the same reconstituted deionized water (hardness 40 parts per million; negative control). Water was maintained at a 17 degrees Celsius temperature via partial immersion of test chambers in a water bath. Survival was assessed for adult fathead minnows Pimephales promelas following 24-hour exposure (see Antimycin_A_Toxicity_Fathead_Minnow.txt). During this 24-hour time, adult fathead minnows do not require feeding (ASTM 2014). 2018 Bait_Measurements_FishFeeding_Survival.txt Text (txt) file containing results and measurements from bait feeding trials Attributes include clam dimension, vial dimensions, antimycin A concentration, dates and black carp survival. Producer Defined Tank A numeric identifier for the test chamber tank. Text. Producer Defined A numeric identifier for the test chamber tank. Text. Valid values 4-8. Nom_conc The target concentration of the antimycin A in the test chamber, reported in milligrams per liter. Numeric Producer Defined 40.0 341.5 Milligrams per liter Dose_date A representation of time in which the smallest unit of measure is a day. The value is expressed in YYYYMMDD. It represents the date upon which fish were fed piscicide baits. Numeric. Producer Defined 20170803 20171201 Date Date_broken A representation of time in which the smallest unit of measure is a day. The value is expressed in YYYYMMDD. It represents the date upon which glass shards were observed indicating the vial containing piscicide had been broken. Numeric. Producer Defined . No data Producer defined 20180803 20181205 Date Survived A count of the viable test organisms (black carp) minnows) in the test chamber after 96 hours of exposure. Numeric Producer Defined . No data Producer defined 1 1 Observed individuals Clam_length The maximum shell length measured as the greatest distance between the two values perpendicular to width, reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 21.49 23.27 Millimeters Clam_height The shell height measured as the greatest distance between the two valves reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 18.48 20.16 Millimeters Clam_width The shell width measured as the distance from the hinge to front of the clam beak reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 12.97 14.68 Millimeters Bait_width The bait width measured as the distance from the bottom valve to the top of the attached vial, measured on hinge side of the clam. Reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for Producer Defined 17.5 19.54 Millimeters Diameter The measured outside diameter of the cylindrical vial at a relative midpoint, reported in millimeters. Numeric. Producer Defined 5.07 5.98 Millimeters Wall_width The average wall width (thickness) from four measurements taken at the opening of the vial prior to flame sealing, reported in millimeters. Numeric. Producer Defined 0.39 0.64 Millimeters Antimycin_A_Toxicity_Fathead_Minnow Text (txt) file containing quality control data regarding impact of flame sealing on toxicity of antimycin A to fathead minnows. Producer Defined Treatment A literal value indicating the type of antimycin A used in the exposure. Text Producer Defined None No antimycin A (piscicide) Producer defined Antimycin_A A nominal stock solution of 10 milliliter of 100 milligrams per liter antimycin A in acetone Producer defined Heat_exposed_antimycin_A A nominal stock solution of 10 milliliter of 100 milligram per liter/L antimycin A in acetone and exposed to heat during flame sealing of vials Producer defined Id A shorthand alphanumeric identifier. Assigned in the laboratory. Text Producer Defined A shorthand alphanumeric identifier. Assigned in the laboratory. Text. This identifier can be used to identify the specific heat sealed vials utilized in the exposure. Replicate A shorthand alphanumeric identifier used to distinguish the replicate testing chambers tested under identical conditions. Replicate measurements capture random biological variation. Text Producer Defined A shorthand alphanumeric identifier used to distinguish the replicate testing chambers tested under identical conditions. Replicate measurements capture random biological variation. Text Stocked A count of the test organisms (fathead minnows) placed in the test chamber at the beginning of the exposure. Numeric Producer Defined 10 10 Observed individuals Num_survived A count of the viable test organisms (fathead minnows) in the test chamber after 24 hours of exposure. Numeric Producer Defined 3 10 Observed individuals Bait_Physical_Measurements_Acceptance.txt Text (txt) file containing results and initial measurements from trials of three different vial sizes and two attachment materials for potential piscicide bait development. Attributes include clam dimension, vial dimensions and categorical descriptor, vial attachment material, presentation and acceptance dates. Producer Defined Tank A numeric identifier for the test chamber tank. Text. Producer Defined A numeric identifier for the test chamber tank. Text. Valid values 1-9. Material A literal value representing the type of material used to attach vials to the clam valve. Text Producer Defined Epoxy Epoxy attachment material, AquaStik aquarium putty Producer defined UV Ultraviolet-light cured attachment material, Bondic UV-cured plastic Producer defined Size A categorical alpha variable indicating the type and size class of vial tested. Text Producer Defined L A pre-manufactured 1 mL vial insert of clear borosilicate glass (5.5 mm outside diameter) Producer defined M A hand-drawn colored borosilicate glass vial (outside diameter ranged from 5.08-2.98 mm) Producer defined S A hand-drawn colored borosilicate glass vial (outside diameter ranged from 3.18-2.01 mm) Producer defined Clam_length The maximum shell length measured as the greatest distance between the two values perpendicular to width, reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 20.06 25.38 Millimeters Clam_height The shell height measured as the greatest distance between the two valves reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 18.13 22.35 Millimeters Clam_width The shell width measured as the distance from the hinge to front of the clam beak reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for clarification. Producer Defined 13.17 16.15 Millimeters Bait_width The bait width measured as the distance from the bottom valve to the top of the attached vial, measured on hinge side of the clam. Reported in millimeters. Numeric. See Bait_dimension_diagram.pdf for Producer Defined 15.01 20.57 Millimeters Vial_length The measured maximum length of an attached sealed vial, this includes any points or tips created by flame sealing, reported in millimeters. Numeric. Producer Defined 22.16 31.06 Millimeters Diameter The measured outside diameter of the cylindrical vial at a relative midpoint, reported in millimeters. Numeric. Producer Defined 2.01 5.66 Millimeters Hour A count of the 60 minute hours that have elapsed since the test began when data were collected. Reported in hours. Numeric. Producer Defined 1 24 Hours Vial_status A categorical indicator of the status of a vial after black carp foraging. Text. Producer Defined 0 Vial was not broken during feeding Producer defined 1 Vial was broken during feeding Producer defined Fish_Feeding_Events_Video.txt Text (txt) file containing tabular data from video footage of fish feeding events. Producer Defined Tank A numeric identifier for the test chamber tank. Text. Producer Defined A numeric identifier for the test chamber tank. Text. Valid values 1-9. Material A literal value representing the type of material used to attach vials to the clam valve. Text Producer Defined Epoxy Epoxy attachment material, AquaStik aquarium putty Producer defined UV Ultraviolet-light cured attachment material, Bondic UV-cured plastic Producer defined Bait_Ingested A categorical alpha identifier for the bait items ingested by black carp. Text Producer Defined W Clam without a vial attached. Producer defined S Clam with a vial of 3.18-2.01 millimeter outside diameter attached Producer defined L Clam with a vial of 5.5 millimeter outside diameter attached Producer defined M Clam with a vial of 5.08-2.98 millimeter outside diameter attached Producer defined N No feeding observed Producer defined Event A numeric identifier for the observed feeding event. Text Producer Defined A numeric identifier for the observed feeding event. Text. Valid values 1-168 We prepared a bait by attaching a glass vial containing toxicant (antimycin A) to the exterior of a clam valve. The vial was designed to break by the force of the fish’s teeth. We tested suitable bait sizes for fish [~550 mm total length] and evaluated vial breakage with different attachment materials (epoxy or plastic) and vial sizes. A range test of antimycin A in ethanol (near solubility) was first applied via oral dose to identify a potential lethal volume at a 40.0 mg/mL concentration. Mortality was observed at the 200 µL volume. This volume was administered in an assembled bait with host clams and vials attached via epoxy at three concentrations (40.0 mg/mL ethanol, and 341.5 mg/mL and 170.8 mg/mL acetone.). Antimycin A toxicity was tested via bioassay to compare median lethal concentrations to those previously reported for fathead minnows. Similar methods were applied to compare the effects of flame sealing glass vials on toxicity of antimycin A solution. Q GS ScienceBase U.S. Geological Survey mailing and physical

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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. 20231207 CERC Data Manager U.S. Geological Survey, Columbia Environmental Research Center Natural Resource Data Manager mailing and physical

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Columbia MO 65201 United States 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