Dataset: Prey Engulfment as the Dominant Pathway of Methylmercury Uptake in a Heterotrophic Dinoflagellate Experiment

Methods are detailed in Myer et al. (2025).

All materials and supplies that were in contact with plankton cultures were sterilized, and all glassware was acid-washed and combusted. Experiments utilized both filtered and artificial seawater. Natural seawater was collected from Long Island Sound, filtered, autoclaved, and refrigerated at 18°C. Artificial seawater (ASW) was prepared using MilliQ water with added salts. ASW was filtered and also refrigerated at 18°C. Plankton culture methods are detailed in Myer et al. (2025).

Eight-hour plankton (Oxyrrhis marina (urn:lsid:marinespecies.org:taxname:109902)) exposure experiments were carried out in triplicate vessels (Pyrex glass bottles) for each treatment. Experimental variables included temperature, salinity, and organic matter type, with variations in dissolved organic carbon (DOC) and prey (Isochrysis galbana (urn:lsid:marinespecies.org:taxname:573884)). One additional treatment involved using heat-killed O. marina, following modified methods from Zhong & Wang (2009).

Temperature experiments were conducted at 12, 15, and 22 °C. Salinity was manipulated through dilutions of ASW with MilliQ water (11 and 17) from a stock solution (34). The low DOC treatment was prepared using ASW with a final concentration of 130 µM DOC, and the high DOC treatment was prepared from filtered natural seawater with a final concentration of 210 µM DOC.

The experiment that tested the control of organic matter on MeHg uptake used higher MeHg spike concentration (5 ng/L) and O. marina cell concentration (5000 cells/L), than the consecutive salinity and temperature experiments (MeHg: 1.7 ng/L; O. marina: 3000 cells/L).

MeHg was spiked before the addition of O. marina to allow for equilibration. In the experimental treatments utilizing I. galbana as prey, these cells were added at the same time as the MeHg spike. Experimental flasks were inoculated with O. marina, starting the experiment. Cells and water were collected immediately (t₀ = 0 hours), and then again at 4 and 8 hours. At these time points, 85 mL was filtered sequentially using an acid-washed vacuum filtration tower using 10, then 3, and lastly 0.2 µm MilliporeSigma Isopore Polycarbonate Membrane Filters. All filters were placed into new 15 mL Falcon tubes and stored briefly in a dark cooler, prior to freezing (-20 °C), and until acid digestion and MeHg analysis. 1mL of unfiltered water was sampled from each bottle and preserved with Lugol’s solution for cell counting. For DOC analysis, 30 mL of 0.2 µm filtered seawater was collected and acidified with hydrochloric acid, then refrigerated at 18°C. For dissolved MeHg analysis, filtrate was collected into 125 mL PETE bottles and acidified with trace metal HCl. For the DOC experiment, bulk solution was collected for analysis and dissolved MeHg was calculated.

MeHg analyses were performed using the Tekran 2700 Automated Methylmercury Analysis System. Particulate MeHg analysis followed previously established procedures modified from the EPA Method 1630 (Hammerschmidt & Fitzgerald, 2005). Briefly, filters were digested with nitric acid overnight at a 60 °C. The subsequent digest was diluted with MQ water, neutralized with potassium hydroxide (KOH), and buffered to a pH in a range of 4.0-4.5 with 2 M acetate. 30 µL of sodium tetraethylborate (NaBET4) was used to volatilize the MeHg within the sample into methylethylmercury (MeEtHg). Calibration was prepared based on a five-point standard curve using MeHgCl standard solution (Alfa Aesar). The detection limit was 0.012 ng/L. The quality of measurements was determined based on routinely analyzed standard solutions and sample replication. The RSD for sample replicates was 20% (n=2-4 per sample; n=20 total).

Seawater was processed following previously established methods (Munson et al., 2014). The day before analysis, seawater was spiked with sulfuric acid (H2SO4, TraceMetal Grade, Fisher Chemical; final concentration: 1% vol./vol.) for overnight digestion. A small volume of cold (4 °C) 2.5% L-ascorbic acid was added to the solution, which was buffered using 8N KOH and 2N acetate, prior to ethylation with NaBET4. The detection limit for the seawater analytical runs was 0.007 ng/L, and the RSD was 9% for sample replicates (n=3 per sample, n=15 total). MeHg in water samples was validated using standard addition to experimental samples in triplicate. The recovery of the standard spike to samples was 109 ± 15% (n=2-3 per sample; n=11 total).

Cells were enumerated using a stereo microscope (Fisher brand, 5.0x magnification) and a Sedgwick-Rafter cell counting chamber. For each 1 mL sample of O. marina, three columns were chosen randomly, counted, and averaged. I. galbana cells were enumerated using the Multisizer Coulter Counter II. Cells were counted in duplicate, and if counts differed by ≥10%, a third count was taken and averaged.