Emily Karwacki Viviana Mazzei 20221003 spreadsheet Orlando, Florida U.S. Geological Survey https://doi.org/10.5066/P9D52SZ1 https://www.sciencebase.gov/catalog/item/632c61aad34e900e86c5103b An interdisciplinary and multiagency study was conducted in southern Florida to examine harmful algal bloom (HAB) dynamics on the Caloosahatchee River, which drains from Lake Okeechobee to the west into the Gulf of Mexico. Using in-situ mesocosm experiments, ammonium, nitrate, phosphate, and urea were altered from ambient conditions to determine the effects on cyanotoxin biosynthesis gene presence and quantity as measured via quantitative polymerase chain reaction (qPCR). Eight experiments were conducted seasonally from May 2019 through July 2021 at the W.P. Franklin Lock and Dam. The experimental installations consisted of three floating metal cradles each containing four opaque fiberglass chambers filled with local river water. Each chamber was open at the top to allow free flow of air and light but closed off at every other part. This ensured the chambers were in the river but not getting cross-contaminated by exchange with the river water. The twelve chambers were each enriched using one of five treatments depending on the experiment: phosphate (P), nitrate (N), ammonium (A), urea (U) or control (C) – with only four of the five treatments selected, each with three replicates, for every experiment. The P, N, A, and U treatment chambers were enriched by applying a concentrated dosing solution of either dibasic dodecahydrate sodium phosphate, anhydrous sodium nitrate, liquid ammonium hydroxide, or urea, respectively to elevate concentrations above ambient levels. The control chambers were left unenriched. The mesocosms were treated and sampled in approximately 24-hour intervals over a 72-hour period (Time 0, Time 24, Time 48, Time 72). May and July 2021 mesocosms were additionally sampled one week from the initiation of the experiment (T240). Deoxyribonucleic acid (DNA) was extracted from these samples and tested via six qPCR assays to determine the presence and quantity of different cyanotoxin biosynthesis genes. Each qPCR assay tests for a different cyanotoxin biosynthesis gene with one assay testing for cyanobacterial 16S DNA as an internal control. The five toxin biosynthesis genes tested were microcystin (mcyE), nodularin (ndaF), saxitoxin (sxtA), anatoxin (anaC), and cylindrospermopsin (cyrA). These data were collected to examine and document the short-term and independent effects of ammonium, nitrate, phosphate, and urea enrichment on the growth of toxin-producing cyanobacteria and cyanotoxins in the Caloosahatchee River in southern Florida. 20190506 20210805 ground condition Complete None planned Caloosahatchee River -82.0300 -81.2400 26.7900 26.5000 ISO 19115 Topic Category biota Marine Realms Information Bank (MRIB) keywords cyanobacteria ["blue-green algae"] toxin research USGS Thesaurus polymerase chain reaction algal blooms hazards USGS Metadata Identifier USGS:632c61aad34e900e86c5103b Common geographic areas Caloosahatchee W.P. Franklin Lock and Dam 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. Viviana Mazzei U.S. Geological Survey, SOUTHEAST REGION Research Biologist mailing address

12703 Research Parkway Suite 200

Orlando FL 32826 US 407-803-5508 407-803-5501 vmazzei@usgs.gov This work was financially supported by the U.S. Army Corps of Engineers' Aquatic Nuisance Species Research Program and the USGS Environmental Health toxins group. Quality of sample DNA was tested using the internal control assay that tested for cyanobacterial 16S DNA. If 16S DNA was not detected in a sample, then that sample was likely either contaminated or did not contain cyanobacterial DNA in sufficient amounts. Additionally, samples were tested at least twice to decrease the likelihood of false positives or negatives, and if a sample had a significantly different gene quantity between the two runs, additional tests were run to determine the gene content. All data were checked for inconsistencies and samples with inconsistent results were run again. This 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 horizontal accuracy tests were conducted. No formal vertical accuracy tests were conducted. The experimental installation consisted of three floating metal cradles each containing four 556-liter fiberglass chambers filled with local river water using a gas-powered pump. The twelve chambers were assigned to one of five treatments, phosphate (P), nitrate (N), Urea (U), or ammonium (A) and a control (C) with no treatment – with only four of the five treatments selected, each with 3 replicates, for every experiment. The P, N, U, and A treatment chambers were enriched by applying a concentrated dosing solution of either dibasic dodecahydrate sodium phosphate (Na2HPO4·12H2O), anhydrous sodium nitrate (NaNO3), crystalline urea (CH4N2O), or liquid ammonium hydroxide (NH4OH), respectively to elevate concentrations above ambient levels. The mesocosms were treated and sampled using a surface water grab at approximately 24-hour intervals over a 72-hour period (Time 0, Time 24, Time 48, Time 72). The May and July 2021 experiments had an additional sampling time one week after the Time 72 sample was collected (Time 240). Samples were collected by kayaking to each chamber, rinsing a 1-liter HPDE bottle with the chamber water, filling the bottle, then sealing and bringing the samples back to shore for processing. 20210805 Water samples were either poured into preserved or unpreserved 50-milliliter (mL) tubes. Preserved samples consisted of 15 mL of sample water, 33 mL of 200 proof ethanol, and 1.5 mL of 3 Molar sodium acetate and were placed in a -20 degrees Celsius freezer until extraction. Unpreserved samples consisted of 20 mL of sample water and were left at room temperature until extraction. 20220601 Unpreserved and preserved samples had differing DNA extraction processes. Preserved samples were centrifuged for 45 minutes at a radial force of 3000 x g at 6 degrees Celsius. The pellet formed was then transferred to a sterile 2-milliliter (mL) tube and a traditional spin-column method was used to extract the DNA using a PureLink Genomic DNA minikit from Invitrogen. Unpreserved samples were centrifuged for 15 minutes at a radial force of 3000 x g at 15 degrees Celsius. The pellet formed was then transferred to a sterile 2-mL tube and a traditional spin-column method was used to extract the DNA using a PureLink Genomic DNA minikit from Invitrogen. The only deviation from the manufacturer's protocol was taken during the incubation stage: preserved samples were incubated for 4 to 5 hours while unpreserved samples were incubated overnight. Finished DNA extracts were stored in a -20 degrees Celsius freezer until analysis. 20220601 Each sample was first tested using the cyanobacteria 16S DNA assay via qPCR to determine whether the DNA the extraction step was successful and whether there was sufficient cyanobacterial DNA present to test for the toxin biosynthesis genes. Samples that were positive for cyanobacterial DNA were then tested for the five cyanotoxin biosynthesis genes via qPCR. Each assay was tested according to its optimal reaction mixture and cycling protocol. Each assay was run on a QuantStudio 5 qPCR cycler. Raw data was processed using the QuantStudio Design and Analysis Software. Quantitative data was recorded for each successful run and the two closest quantities were averaged to provide the final gene quantity. 20220601 mesocosms_year1-3.xlsx Excel (xlsx) file containing the finalized data. Producer Defined SampleUniqueID A unique alphanumeric identifier for each sample. Producer Defined An automatically generated internal sample identifier consisting of 16 characters starting with the letter D followed by the 8-digit date (YYYYMMDD) the sample was analyzed and the letter T followed by the 6 or 4-digit time (Greenwich Mean Time; 24-hour time) the sample file was generated. DateSampled Date on which the sample was collected in the field in month/day/year format (MM/DD/YYYY). Producer Defined 05/06/2019 08/05/2021 TimeSampled Time at which the sample was collected from each chamber. Producer Defined 10:00 14:57 Hour: minute TimeZone Corresponding time zone for the sample collection time. Producer Defined EDT Eastern Daylight Time (Greenwich Mean Time -4). Producer defined Time Time period during the experimental process at which the sample was collected. Producer Defined T0 The sample was collected on the first day of the experimental period. Producer defined T24 The sample was collected about 24 hours after the initial (T0) sample was collected. Producer defined T48 The sample was collected about 48 hours after the initial (T0) sample was collected. Producer defined T72 The sample was collected about 72 hours after the initial (T0) sample was collected. Producer defined T240 The sample was collected one week after the last sample (T72) was collected and is labeled as T240. Producer defined Treatment The nutrient treatment applied to the experimental chamber that the sample was collected from. Producer Defined C1 Control treatment, chamber 1 of 3. Producer defined C2 Control treatment, chamber 2 of 3. Producer defined C3 Control treatment, chamber 3 of 3. Producer defined N1 Nitrate treatment, chamber 1 of 3. Producer defined N2 Nitrate treatment, chamber 2 of 3. Producer defined N3 Nitrate treatment, chamber 3 of 3. Producer defined P1 Phosphate treatment, chamber 1 of 3. Producer defined P2 Phosphate treatment, chamber 2 of 3. Producer defined P3 Phosphate treatment, chamber 3 of 3. Producer defined A1 Ammonium treatment, chamber 1 of 3. Producer defined A2 Ammonium treatment, chamber 2 of 3. Producer defined A3 Ammonium treatment, chamber 3 of 3. Producer defined U1 Urea treatment, chamber 1 of 3. Producer defined U2 Urea treatment, chamber 2 of 3. Producer defined U3 Urea treatment, chamber 3 of 3. Producer defined Latitude Latitudinal coordinate of the chamber from which the sample was collected. Producer Defined 26.7211972 The latitude of the sampled chamber during the experimental period. Producer defined Longitude Longitudinal coordinate of the chamber from which the sample was collected. Producer Defined -81.6932056 The longitude of the sampled chamber during the experimental period. Producer defined CollectionMethod The method used to collect the sample in the field. Producer Defined Manual Grab Sample Samples were collected by an individual putting on gloves, manually reaching into the water at the site, and filling a collection bottle with the chamber water. Producer defined 16S_CQ The cycle at which the sample amplified during the cyanobacterial 16S assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 47.35 Cycle number 16S_gene_copies_per_microliter The quantity determined for the sample during the cyanobacterial 16S assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 135000000000.00 Gene copies per microliter mcyE_CQ The cycle at which the sample amplified during the mcyE gene assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 42.67 Cycle number mcyE_gene_copies_per_microliter The quantity determined for the sample during the micorcystin, mcyE, assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 5506035.88 Gene copies per microliter ndaF_CQ The cycle at which the sample amplified during the ndaF gene assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 0.00 Cycle number ndaF_gene_copies_per_microliter The quantity determined for the sample during the nodularin, ndaF, assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 0.00 Gene copies per microliter sxtA_CQ The cycle at which the sample amplified during the sxtA gene assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 42.67 Cycle number sxtA_gene_copies_per_microliter The quantity determined for the sample during the saxitoxin, sxtA, assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 35760.86 Gene copies per microliter anaC_CQ The cycle at which the sample amplified during the anaC gene assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 40.92 Cycle number anaC_gene_copies_per_microliter The quantity determined for the sample during the anatoxin, anaC, assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 2312814.70 Gene copies per microliter cyrA_CQ The cycle at which the sample amplified during the cyrA gene assay run. If there were two positive replicates, the cycle value was averaged. If one replicate was positive and one was zero, the positive cycle value was used. All zero results are true zeroes. Producer Defined 0.00 44.68 Cycle number cyrA_gene_copies_per_microliter The quantity determined for the sample during the cylindrospermopsin, cyrA, assay run in number of gene copies per microliter. If there were two replicates, the value was averaged. If one replicate was positive and one was zero, the positive value was used. Producer Defined 0.00 222407.40 Gene copies per microliter U.S. Geological Survey - ScienceBase mailing and physical

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Denver CO 80225 USA 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 for other purposes, nor on all computer systems, nor shall the act of distribution constitute any such warranty. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Digital Data https://doi.org/10.5066/P9D52SZ1 None 20221003 Viviana Mazzei U.S. Geological Survey, SOUTHEAST REGION Research Biologist mailing address

12703 Research Parkway Suite 200

Orlando FL 32826 US 407-803-5508 407-803-5501 vmazzei@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999