Methodology:
Heterotrophic dinoflagellate species O. marina and Gyrodinium sp. were incubated for 3 hours under two conditions: a control, fed only algal prey I. galbana, and a treatment fed algal prey and microplastic particles. This was done over 15 concentrations of algal prey and plastics. Samples were taken at T0 and Tfinal, with abundances of dinoflagellates, algal prey, and microplastics measured with a Beckman Coulter Counter and verified via microscopy. Ingestion rates of algal prey and of microplastics were measured and compared between treatments to determine the relative proportions of microplastics and prey eaten across a range of particle concentrations. Ingestion rates for all particles types were calculated.

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.

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⁻¹on 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.

To determine if the heterotrophic dinoflagellates were ingesting microplastics and prey at the same rate, or if they displayed a preference for one particle type, short term (3 hrs duration) ingestion experiments were conducted. The two dinoflagellate species were fed only prey or a 1:1 mixture of microplastics and prey. A total of 15 total particle concentrations (prey + microplastics) were used, ranging from 4,000 to 220,000 particles mL⁻¹ (Table 1 of Fulfer & Menden-Deuer, 2021). Each treatment contained 900-1100 cells mL⁻¹ of O. marina or 500 – 800 cells mL⁻¹ of Gyrodinium sp. and was prepared to a final volume of 125 mL. Samples of 10 mL were taken at time 0 and after 3 hours. The initial and final abundances of microplastic particles, prey, and zooplankton were measured with a 100 µm aperture on a Beckman Coulter Multisizer 3 (Beckman Coulter).

microplastic_functional_response.csv