Calcium‑Mediated Exopolymeric Substance Release in Marine Phytoplankton Exposed to Engineered Nanoparticles

Abstract

Engineered nanoparticles (ENPs) are rapidly incorporated into consumer products, yet their impact on marine ecosystems remains poorly understood. We examined the response of four diatom species—Odontella mobiliensis, Skeletonema grethae, Phaeodactylum tricornutum, and Thalassiosira pseudonana—and one green alga, Dunaliella tertiolecta, to model ENPs: 25 nm TiO₂, 10–20 nm SiO₂, and 15–30 nm CeO₂. SiO₂ ENPs amplified extracellular polymeric substance (EPS) secretion by 200–800 %, whereas TiO₂ induced the lowest EPS release. Concurrently, intracellular Ca²⁺ concentrations rose significantly upon ENP exposure, indicating a Ca²⁺-dependent secretion pathway. Elucidating this mechanism offers a foundation for developing strategies to mitigate potential ecological risks posed by ENPs in marine environments.

Background

Engineered nanoparticles (ENPs), ranging from 1 to 100 nm, are integral to products such as inks, paints, cosmetics, and pharmaceuticals. In 2017, U.S. nanotechnology funding reached $1.4 billion, reflecting the sector’s growth [1–3]. As ENPs inevitably enter aquatic systems, their high surface reactivity raises concerns about reactive oxygen species (ROS) generation and other toxicological effects [5–6]. Marine phytoplankton, the ocean’s primary producers, have demonstrated sensitivity to ENPs, with documented detrimental effects on growth and viability [7–11]. Yet, the cellular responses of phytoplankton to diverse ENPs are not fully defined [12–21]. Phytoplankton routinely produce exopolymeric substances (EPS), a complex array of polysaccharides and proteins that mediate biofilm formation, metal scavenging, and protection against environmental stressors [22–27]. EPS secretion is often a stress response, and calcium (Ca²⁺) signaling has been implicated in regulating EPS release in diatoms [28–31]. While ENPs can perturb intracellular Ca²⁺ pathways [29,32–34], no systematic assessment of Ca²⁺-mediated EPS secretion in response to TiO₂, SiO₂, or CeO₂ has been reported.

Results and Discussions

ENP Characterization

Dynamic light scattering (DLS) revealed TiO₂ particles predominantly at 25 nm, SiO₂ at 10–20 nm, and CeO₂ at 15–30 nm (Fig. 1). Minor subpopulations up to 70 nm likely result from aggregation. These measurements confirmed manufacturer specifications and ensured consistency across experiments.

Figure 1. DLS size distribution of TiO₂ (a), SiO₂ (b), and CeO₂ (c) in L1 medium after sonication. Concentration: 1 µg/mL.

ENPs Induce Intracellular Ca²⁺ Elevation

Fluo‑4AM imaging demonstrated that 1 mg/mL SiO₂ increased intracellular Ca²⁺ by 50–300 %, CeO₂ by 150–200 %, and TiO₂ by ~40 % across all tested species (Fig. 2). TiO₂ induced only modest Ca²⁺ changes, likely reflecting its pronounced cytotoxicity and associated cell death [37–39]. These results establish that distinct ENPs elicit species‑specific Ca²⁺ signaling, a prerequisite for downstream EPS secretion.

Figure 2. Intracellular Ca²⁺ responses in Dunaliella tertiolecta (a), Thalassiosira pseudonana (b), Skeletonema grethae (c), Phaeodactylum tricornutum (d), and Odontella mobiliensis (e) following 1 mg/mL exposure to TiO₂ (green), SiO₂ (red), CeO₂ (purple), and control (blue). Data represent averages of 20 cells.

ENP‑Induced EPS Release

Using an enzyme‑linked lectin assay (ELLA), we quantified EPS secretion normalized to total genomic DNA. SiO₂ stimulated EPS release up to 1,000 % in Skeletonema grethae and 900 % in Phaeodactylum tricornutum, with 500–800 % increases in the remaining species (Fig. 3). TiO₂ triggered significant EPS secretion only in Skeletonema and Phaeodactylum, mirroring its limited Ca²⁺ response. CeO₂ had modest effects across taxa. The magnitude of EPS release correlated strongly with the observed Ca²⁺ elevations, reinforcing the Ca²⁺-dependent secretion model.

Figure 3. EPS release from each phytoplankton species treated with TiO₂ (circles), SiO₂ (triangles), and CeO₂ (squares) at concentrations ranging from 10 µg/mL to 5 mg/mL. Values represent mean ± SD (n = 3).

Conclusions

Our study demonstrates that engineered nanoparticles, particularly SiO₂, can provoke substantial, Ca²⁺-mediated EPS secretion in marine phytoplankton. This response varies among species and nanoparticle types, highlighting the complexity of nanomaterial–biota interactions. Future research should extend these findings to natural seawater matrices and assess ecological consequences of altered EPS dynamics, such as biofilm formation and pollutant sequestration.

Methods

Phytoplankton cultures (Odontella mobiliensis, Dunaliella tertiolecta, Skeletonema grethae, Phaeodactylum tricornutum, Thalassiosira pseudonana) were grown in L1 medium at 24 °C under a 14:10 h light:dark cycle. ENPs (TiO₂, SiO₂, CeO₂) were sonicated in water, reconstituted in filtered L1, and characterized by DLS (Brookhaven BI‑200SM). Cells were exposed to ENPs (10 µg/mL–5 mg/mL) for 48 h in 96‑well plates; EPS was harvested from supernatants and quantified by ELLA using Concanavalin A–HRP conjugate. Intracellular Ca²⁺ was measured with Fluo‑4AM dye on a Nikon Eclipse TE2000‑U. DNA content was determined by NanoDrop after extraction with ZR‑96 Quick‑gDNA kit. Statistical analysis employed GraphPad Prism, reporting means ± SD from at least three independent replicates.