Marine ecosystems of coastal Antarctica are shaped by the interplay of sea ice dynamics, seasonal light availability, and vertical mixing processes, which together generate spatially heterogeneous and temporally variable habitats for phytoplankton (Bolinesi et al., 2020b; Mangoni et al., 2004, 2017; Moro et al., 2000; Saggiomo et al., 2017, 2021a,b; Massi et al., 2025). In polynya systems such as Terra Nova Bay (TNB, the Ross Sea), strong katabatic winds, and persistent sea ice formation drive brine rejection, leading to the production of high-salinity shelf waters (Bromwich and Kurtz, 1984; Hollands and Dierking, 2016). Conversely, seasonal warming and ice melt inject pulses of low-salinity meltwater into the upper mixed layer. The opposing processes—ice formation and melting—create a highly dynamic and heterogeneous environment. Furthermore, these processes give rise to a wide range of physical and chemical conditions, including the development of brine channels within developing sea ice, the release of freshwater during melt events, and the extrusion of concentrated saline brines during ice growth (Testón-Martínez et al., 2024). The interplay between freezing and melting not only modulates stratification and circulation but also influences biogeochemical fluxes and ecosystem structure across the polynya. Apart from this, these processes also give rise to a mosaic of co-occurring microenvironments—including hypersaline brine channels and pockets embedded within sea ice, the under-ice boundary layer, and surface strata influenced by seasonal meltwater inputs—each defined by steep and dynamic gradients in salinity, temperature, light availability, and nutrient concentrations (Eicken, 2003; Fraser et al., 2023).

The physiological responses and taxonomic composition of phytoplankton communities are highly sensitive to various variables, often exhibiting non-linear and threshold-dependent dynamics (Arrigo et al., 2010; Hoppe et al., 2018; Boyd et al., 2016; Petrou et al., 2016; Hayward et al., 2025). Consequently, spatial and temporal variability in salinity emerges as a key regulator of phytoplankton succession, bloom initiation and termination, and the modulation of biogeochemical fluxes in coastal Antarctic marine systems (Arrigo and McClain, 1994; Arrigo et al., 1998, 1999; Smith et al., 2014).

Diatoms are the principal contributors to primary production in many Antarctic coastal ecosystems, where they play a pivotal role in driving particulate organic carbon (POC) export, regulating silica cycling, and shaping the nutritional quality of food available to higher trophic levels (Arrigo et al., 2008; Smith et al., 2014). Among Antarctic diatoms, the centric genus Chaetoceros is particularly widespread and ecologically influential, dominating both pelagic and sympagic communities in regions such as the Ross Sea (Crosta et al., 1997; Moro et al., 2000; Gogorev and Samsonov, 2016).

Chaetoceros species are well adapted to the extreme variability of polar environments: they colonize brine channels and platelet ice during winter and early spring, act as seed populations for under-ice and surface blooms, and form extensive pelagic blooms under shallow mixed layers and high irradiance conditions (Crosta et al., 1997; Moro et al., 2000; Ligowski et al., 2012; Gogorev and Samsonov, 2016; Saggiomo et al., 2021b; Stuart et al., 2025). Their ecological success is underpinned by a suite of physiological traits, including dynamic regulation of photoprotective pigments, formation of resting spores, synthesis of osmolytes and antifreeze proteins, and plasticity in nutrient allocation strategies (French and Hargraves, 1985; Crosta et al., 1997; Plettner, 2002; Gwak et al., 2010; Van de Poll et al., 2011; Kim et al., 2017).

However, the relative contribution of intraspecific vs. interspecific variability to the physiological responses of Antarctic Chaetoceros remains insufficiently resolved. Empirical data on short-term vs. acclimated responses in Antarctic Chaetoceros taxa are still limited, and different strains within the same species exhibit markedly different tolerances to abrupt salinity shifts. Mixed cultures used in this study may therefore capture a broader and more ecologically realistic range of strain-level responses than single-strain isolates.

Climate forcing is altering the frequency and magnitude of salinity regimes experienced by coastal phytoplankton. In the Ross Sea, episodic reductions in sea ice extent, increased meltwater inputs, and circulation changes have driven regional freshening trends in recent decades (Castagno et al., 2019; Jacobs et al., 2022; Jena et al., 2024), while ice formation and melt processes still produce transient hypersaline plumes (Eicken, 2003; Widell et al., 2006; Peterson, 2018; Porter et al., 2019; Fraser et al., 2023; Miller et al., 2024). Consequently, planktonic populations may experience abrupt salinity spikes or dips lasting seconds to minutes during brine drainage or sustained hypo−/hypersaline periods that persist for days to weeks in stratified layers or isolated brine pockets. Distinguishing physiological outcomes produced by stress severity (moderate vs. extreme), exposure duration, and change direction (hypo vs. hypersalinity) is required to predict phytoplankton responses across future scenarios.

Prior experimental works on salinity impacts have emphasized temperate species or combined stressors; few studies isolate abrupt salinity changes in Antarctic diatoms. Existing polar studies indicate osmoregulation, antioxidant activation, and xanthophyll cycle engagement under stress, but these studies often address gradual acclimation or combined drivers (Gleitz and Thomas, 1992; Petrou et al., 2011; Hernando et al., 2015, 2018; Bozzato et al., 2019; Antoni et al., 2020). Important but unresolved questions include: (1) What are the immediate (hours) physiological responses to an abrupt salinity perturbation akin to brine plumes? (2) Can Antarctic Chaetoceros acclimate during days to weeks after the salinity shift, and what trade-offs arise between population growth, cell size, and pigment composition? (3) Do stress severity (moderate vs. extreme) and direction (hypo vs. hypersalinity) differentially structure responses? and (4) How does salinity modulate pigment: Chl a ratios applied in chemotaxonomy?

We addressed these questions by exposing a mixed Chaetoceros culture (Terra Nova Bay isolate) to a salinity gradient representative of present-day microenvironments and potential future freshening (20‰, 24‰, 34‰, 44‰, and 60‰). We tracked immediate (2 h) and longer-term (4, 8, 12, and 15 days) responses across multiple functional endpoints—maximum quantum efficiency (Fv/Fm), cell density and growth dynamics, cell size and morphology, chlorophyll a content (total and per cell), high-performance liquid chromatography (HPLC) pigment profiles and xanthophyll activity, nitrate and phosphate uptake, and N:P ratios. Our objectives include (1) characterizing short-term vs. acclimation responses to abrupt salinity change, (2) evaluating whether stress severity or direction of salinity change (increase vs. decrease) primarily structures physiological trajectories, (3) identifying trade-offs between growth, size, and pigment investment with ecological consequences, and (4) providing salinity-specific pigment:Chl a reference data for CHEMTAX applications. By combining high-resolution temporal sampling with multiple functional endpoints, this study clarifies mechanistic pathways through which salinity variability may reconfigure phytoplankton performance and community outcomes in a changing Southern Ocean.

Responses clustered more strongly by stress severity (moderate vs. extreme) than by the direction (hypo vs. hypersalinity) of salinity change alone. Moderate treatments (24‰ and 44‰) supported growth relative to the extremes but through contrasting physiological adjustments. At 24‰, Chaetoceros exhibited reduced cell size and lower pigment content per cell, together with a measurable decline in growth rate. These patterns indicate that moderate hyposalinity imposed physiological stress that constrained both biomass production and pigment investment, rather than promoting a shift toward numerical proliferation. The observed decrease in cell size in 24‰ (with more deformation, lysis, and amorphous cells compared with the control) reflects a regulated adjustment under osmotic stress rather than an increase in division rate. Impaired nutrient uptake at 24‰ further supports the conclusion that hyposalinity reduced overall metabolic efficiency.

At 44‰, cells increased in size and pigment per cell but still exhibited slower population growth than the control. This suggests that moderate hypersalinity induced a different allocation pattern—characterized by greater per-cell investment—yet nevertheless resulted in reduced growth. The modest increase in Fv/Fm at 44‰ fell within the range of variability and did not translate into enhanced growth or nutrient assimilation. Fucoxanthin and chlorophyll c concentrations (Figure 7) were highest in control and 44‰ cultures, consistent with increased pigment investment under moderate hypersalinity.

These results indicate that both moderate hypo- and hypersalinity impaired growth, but the magnitude of the effect was asymmetrical: low salinity (24‰) caused stronger reductions in intact cells, growth rate, and phosphate use than the equivalent increase in salinity (44‰).

Extreme salinities (20‰ and 60‰) exceeded effective tolerance ranges for much of the culture. At 20‰, cells exhibited marked deformation and lysis with only a small surviving fraction showing increased size and partial Fv/Fm recovery, reflecting selection for tolerant subpopulations or osmotic swelling artifacts (Gleitz and Thomas, 1992; Hernando et al., 2015). At 60‰, sustained high %DES and elevated photoprotective pigment levels suggest oxidative and photoprotective stress; persistent photoprotection likely consumed energetic reserves and limited growth capacity, contributing to collapse of chlorophyll pools and population density (Petrou et al., 2011; Kholssi et al., 2023). These observations align with previous findings that Chaetoceros spp. tolerate moderate salinity variation but suffer at extremes (Gleitz and Thomas, 1992; Hernando et al., 2018; Liang et al., 2014). Given the near-complete suppression of nutrient assimilation at 60‰, the limited growth observed under hypersaline conditions is unlikely to be sustainable beyond the short term.

Smaller, less pigmented cells promoted by moderate hyposalinity (24‰) reflect reduced per-cell investment and impaired nutrient uptake, consistent with the physiological stress indicated by the decline in growth rate. Increased deformation and lysis under severe hyposalinity (20‰) favor microbial recycling and reduce POC export (Isla et al., 2006). Conversely, larger, more pigmented cells at 44‰ indicate increased per-cell investment under moderate hypersalinity, although a reduced growth rate suggests that this adjustment did not translate into enhanced biomass production.

Pigment:Chl a ratios varied with salinity and time; marker pigments exhibited broader ranges in control and moderate treatments than in extremes. Photoprotective pigments were particularly sensitive to salinity stress (Demers et al., 1991; Lavaud, 2007; Goss and Jakob, 2010; Brunet et al., 2011). The salinity-specific pigment ratios provided in Table 2 can refine CHEMTAX initial matrices for Chaetoceros-dominated samples (Neveux and Panouse, 1987; Mackey et al., 1996; Mantoura and Repeta, 1997; Lazzara et al., 2010; Higgins et al., 2011); however, field application requires caution because pigment ratios are influenced by multiple co-occurring environmental factors. Moreover, the pigment responses observed here represent short-term acclimation patterns; their consistency over longer periods is uncertain, as pigment ratios are likely to vary depending on the duration of salinity exposure and the rate at which salinity fluctuates. These patterns indicate that salinity stress alters nutrient demand and uptake efficiency, with implications for cellular stoichiometry, competitive dynamics, and biogeochemical cycling (see Table 3).

Preliminary comparison with a co-isolated Prorocentrum sp. (exposed to identical hyposaline and hypersaline treatments in a parallel study; unpublished data) demonstrates functional-group specificity: Prorocentrum fully recovered at 20‰ by day 15, whereas Chaetoceros exhibited partial recovery only; while at 60‰, Prorocentrum collapsed entirely, while Chaetoceros persisted with impaired function. Such differences suggest that freshening favors some flagellate groups over diatoms under particular regimes, with community-level implications reminiscent of shifts observed on the Western Antarctic Peninsula (Bolinesi et al., 2020a; Mendes et al., 2013, 2018, 2023; Hamilton et al., 2021).

Multivariate patterns indicate that salinity acts as a strong ecological filter shaping phytoplankton functional traits. The consistent clustering of samples along the PCA axes, together with the PERMANOVA outcomes, demonstrates that salinity and time independently structure physiological trajectories. However, the physiological adjustments documented reflect short-term responses to a constant salinity shift; in natural settings, fluctuating or transient salinity changes elicit different or more variable pigment and growth responses.

These results highlight the central role of salinity in modulating trait expression and ecological function in polar phytoplankton communities. This study imposed a single salinity shift and maintained it throughout the exposure period; however, natural polar environments are characterized by rapid, transient brine pulses, and cyclic salinity fluctuations driven by ice formation and melt dynamics (Eicken, 2003; Peterson, 2018). To better simulate in situ conditions, future studies must incorporate pulsed and oscillatory salinity regimes, alongside additional environmental drivers such as temperature, irradiance, and nutrient availability (Dong et al., 2016; Hernando et al., 2018). Molecular identification of species within the culture is also essential to resolve intra-genus variability and clarify species-specific responses (Gogorev and Samsonov, 2016; Mock and Junge, 2007). Automated cell enumeration may help reduce observer bias, while direct measurements of reactive oxygen species and osmolyte accumulation may clarify underlying stress mechanisms (Plettner, 2002; Janknegt et al., 2008).

Although species composition within the mixed Chaetoceros culture remains to be molecularly resolved, its use reflects the ecological complexity of Antarctic coastal phytoplankton communities, where multiple strains and closely related species co-occur and interact within highly variable microenvironments (Mangoni et al., 2009; Saggiomo et al., 2017; Saggiomo et al., 2021a). Mixed cultures thus offer a more realistic framework for assessing community-level responses to salinity stress, while capturing potential compensatory or synergistic dynamics that may be masked in clonal systems. While species-level attribution is inherently limited, the functional endpoints evaluated—growth dynamics, pigment regulation, and nutrient uptake—are broadly representative of Chaetoceros-dominated assemblages (Crosta et al., 1997; Pelusi et al., 2020). The consistent and interpretable patterns observed across various treatments suggest that the responses captured are biologically meaningful and ecologically relevant. Nonetheless, future studies integrating clonal isolates and molecular tools may be critical to disentangle intra- and inter-specific mechanisms and refine trait-based predictions under evolving polar climate scenarios (Hamilton et al., 2021; Mendes et al., 2023; Stuart et al., 2025).

Chaetoceros spp. from Terra Nova Bay exhibited marked physiological plasticity across the salinity gradient, with responses strongly dependent on stress severity. Moderate deviations from ambient salinity supported growth of these diatoms but altered cell size, pigment allocation, and nutrient uptake, while extreme salinities led to structural damage, pigment loss, and impaired population performance. Salinity-driven pigment shifts also affect chemotaxonomic resolution by altering pigment:chlorophyll a ratios used as markers in CHEMTAX, potentially changing the detectability and relative contribution of diatoms and other functional groups in a changing Southern Ocean. The observed acclimation patterns further indicate that salinity acts as an important ecological filter in Antarctic coastal systems.

Within the limits of the measured parameters, our results show that salinity modulates growth, pigment investment, and nutrient assimilation. These physiological adjustments influence cellular resource use and contribute to shaping community responses under climate-driven freshening. Although broader implications for energy transfer and biogeochemical fluxes cannot be directly assessed from this dataset, the contrasting responses observed under moderate vs. extreme salinity shifts suggest potential consequences for ecosystem functioning.

Overall, these findings highlight salinity as a central driver of phytoplankton physiological responses in polar environments, with possible downstream effects on community resilience, trophic interactions, and the functioning of Southern Ocean ecosystems under ongoing climate change.