Dual Fluorescence-Lipid Endpoints Resolve Species- and Metal-Specific Toxicity Patterns in Marine Diatoms

Trace metals are persistent stressors in coastal ecosystems, yet most marine algal toxicity assessments still rely on freshwater model species and growth-based endpoints that provide limited mechanistic resolution. Here, we quantified the sensitivity of two ecologically contrasting marine diatoms-the benthic Cylindrotheca closterium and the planktonic Thalassiosira weissflogii-to ten environmentally relevant metals using a dual-endpoint approach that integrates chlorophyll fluorescence (photosystem function) and Nile Red-based lipid-body fluorescence (metabolic reallocation). Fluorescence-based EC₁₀ values revealed distinct species- and metal-specific patterns, with C. closterium consistently responding at lower concentrations and Hg producing the strongest inhibition in both species (EC₁₀ ≈ 0.04-0.06 mg L^-1). Lipid-body accumulation detected earlier metabolic disturbance for several metals, particularly Hg, As, Cr(VI), and Cd, and frequently occurred at concentrations where fluorescence remained minimally affected. These sequential thresholds indicate that pigment impairment and metabolic reallocation represent mechanistically distinct stages of the cellular stress response that differ among metals and between diatom guilds. Comparison with published toxicity data shows that the dual-endpoint sensitivities observed here fall within, or slightly above, the upper range of reported microalgal responses, underscoring the pronounced susceptibility of benthic diatoms to redox-active and thiol-reactive metals. The strong agreement between fluorescence-based EC values and traditional growth-derived benchmarks for key metals further supports fluorescence as an operationally efficient endpoint suitable for integration into emerging ISO marine algal bioassays. Overall, this study demonstrates that pairing a rapid functional marker with a mechanistically informative metabolic biomarker enables metal-specific toxicity fingerprinting and provides an ecologically grounded basis for incorporating benthic diatoms into coastal metal risk assessment frameworks.