Methodology:
Heterotrophic dinoflagellate species O. marina and Gyrodinium sp. were incubated for 5 days under two conditions: a control, fed only algal prey I. galbana, and a treatment fed algal prey and microplastic particles. Samples were taken every 24 hours, with abundances of dinoflagellates, algal prey, and microplastics measured with a Beckman Coulter Counter and verified via microscopy. Growth rates were measured and compared between treatments.

Microplastic Ingestion Experiments:
The possibility and subsequent effects of microplastic ingestion by heterotrophic dinoflagellate species were determined using two treatment conditions: first, a treatment with microplastics, in which heterotrophic dinoflagellates were fed a mixture of algal prey and microplastic particles; and second, an algae-only control, in which heterotrophic dinoflagellates were fed algal prey.

For the grazing experiments, each of the three target heterotrophic dinoflagellate species was incubated with I. galbana and microplastic particles, when applicable, diluted in filtered seawater (FSW) to the target concentrations (Table 1 of Fulfer & Menden-Deuer, 2021). Control treatments of the two prey types in the absence of predators were incubated alongside the grazing experiments. All treatments were prepared in triplicate and in a total volume of 125 mL and incubated in 250 mL polycarbonate bottles on a 12 h: 12 h light-dark cycle at 15°C and a light intensity of 8 – 15 µmol photons m⁻² s⁻¹ on a shaker table at 60 rotations-per-minute (rpm) to reduce settling of microplastic particles.

Fluorescent yellow polystyrene (PS) microplastic particles ranging in diameter from 2.5 to 4.5 µm were used in all microplastic feeding experiments (Spherotech, FP-3052-2). This size range was chosen to mimic the size of the algal prey species. Microplastic particles were rinsed three times in DI water and resuspended in autoclaved, filtered seawater directly before use. For each experiment, prey (IG) control treatments were prepared in triplicate in 125 mL polycarbonate bottles with I. galbana diluted in filtered seawater (FSW) to a final concentration of 70,000 - 100,000 prey cells mL⁻¹.

Microplastic ingestion experiments were run for up to 5 days and sampled daily. Samples of 3 mL were fixed with 10% glutaraldehyde to a final concentration of 0.1% glutaraldehyde. Heterotrophic dinoflagellates were counted via light microscopy. To measure growth rates, subsamples of 10 mL were taken at T0 and every 24 hours for 5 days. Abundances of prey, predators, and microplastic particles were measured with a Beckman Coulter Multisizer 3 (Beckman Coulter) using a 100 µm aperture. Rates were calculated over the entire experimental span and over the exponential growth phase.