Synthesis-dependent effects of silver nanoparticles on the green freshwater alga Chlorella vulgaris

Silver nanoparticles (AgNPs) synthesized by distinct routes differ in surface chemistry, which may reshape algal photophysiology and redox balance. The effect of biologically produced AgNPs (Agbio) was compared to chemically reduced citrate-capped AgNPs (Agchem), and/or to laser-ablated AgNPs (Agphys) on Chlorella vulgaris as a model organism to understand the nanoparticle effects in aquatic environments. UV–VIS absorption spectroscopy, spectrally resolved laser-scanning confocal microscopy of red chlorophyll fluorescence, chlorophyll fluorescence-lifetime imaging (FLIM), flavin autofluorescence imaging, and time-correlated single-photon counting (TCSPC) of NAD( P )H were combined to resolve their effects, while scanning electron microscopy provided morphological context. The absorption maxima appeared at 407 nm, 410 nm, and 412 nm for Agphys, Agbio, and Agchem, respectively. Agbio produced the strongest suppression of red chlorophyll fluorescence peaking at 680 nm. FLIM recorded with a BP of 700 ± 20 nm, following excitation by a 445 nm ps laser, uncovered specific dynamics over 7 days. Flavin fluorescence, monitored between 483 and 632 nm, rose immediately for Agchem/Agphys but became significant only at Day 7 for Agbio. NAD( P )H fluorescence lifetimes recorded by TCSPC at 420–680 nm (405 nm excitation) revealed an acute drop in NAD( P )H photon counts at Day 1 and a universal increase of Tau1; Tau2 rose persistently for Agbio, increased transiently for Agphys, and showed no sustained elevation for Agchem. The data support a two-phase redox response – early compensation followed by particle-dependent depletion or re-equilibration - and indicate a phototoxicity ranking Agbio > Agchem ≈ Agphys under our exposure conditions. Gathered results show that the synthesis route is a primary determinant of algal responses.