Metals may cause damage to the biota of contaminated environments. Moreover, using multiple endpoints in ecotoxicological studies is useful to better elucidate the mechanisms of toxicity of these compounds. Therefore, this study aimed to evaluate the effects of cadmium (Cd) and cobalt (Co) on growth, biochemical and photosynthetic parameters of the microalgae Raphidocelis subcapitata, through quantification of lipid classes composition, chlorophyll a (Chl a) content, maximum (Φ_M) and effective (Φ'_M) quantum yields and efficiency of the oxygen-evolving complex (OEC). Both metals affected the algal population growth, with an IC_50-96h of 0.67 and 1.53 μM of Cd and Co, respectively. Moreover, the metals led to an increase in the total lipid content and reduced efficiency of OEC and Φ_M. Cell density was the most sensitive endpoint to detect Cd toxicity after 96 h of treatment. Regarding Co, the photosynthetic parameters were the most affected and the total lipid content was the most sensitive endpoint as it was altered by the exposure to this metal in all concentrations. Cd led to increased contents of the lipid class wax esters (0.89 μM) and phospholipids (PL - at 0.89 and 1.11 μM) and decreased values of triglycerides (at 0.22 μM) and acetone-mobile polar lipids (AMPL - at 0.44 and 1.11 μM). The percentage of free fatty acids (FFA) and PL of microalgae exposed to Co increased, whereas AMPL decreased in all concentrations tested. We were able to detect differences between the toxicity mechanisms of each metal, especially how Co interferes in the microalgae at a biochemical level. Furthermore, to the best of our knowledge, this is the first study reporting Co effects in lipid classes of a freshwater Chlorophyceae. The damage caused by Cd and Co may reach higher trophic levels, causing potential damage to the aquatic communities as microalgae are primary producers and the base of the food chain.

In the environment, algae are exposed to several stressors such as limitation of essential nutrients and excess of toxic substances. It is well known the importance of phosphorus (P) supply for healthy metabolism of algae and impacts at this level can affect the whole aquatic trophic chain. Aluminum (Al) is the most abundant metal on Earth and it is toxic to different trophic levels. Processes related to P and Al assimilation still need to be clarified and little is known about the responses of microalgae exposed to the two stressors simultaneously. We evaluated the effects of environmental concentrations of Al and P limitation, isolated and in combination, on growth, pigment production and photosynthesis of the freshwater microalga Raphidocelis subcapitata. Both stressors affected cell density, chlorophyll a, carotenoids, and maximum quantum yield. Al did not affect any other evaluated parameter, while P limitation affected parameters related to the dissipation of heat by algae and the maximum electron transport rate, decreasing the saturation irradiance. In the combination of both stressors, all parameters evaluated were affected in a synergistic way, i.e., the results were more harmful than expected considering the responses to isolated stressors. Our results indicate that photoprotection mechanisms of algae were efficient in the presence of both stressors, avoiding damages to the photosynthetic apparatus. In addition, our data highlight the higher susceptibility of R. subcapitata to Al in P-limited conditions.

Phosphorus (P; macronutrient) and cobalt (Co; micronutrient) are essential for algal healthy metabolism. While P provides energy, Co is a co-factor of several enzymes and component of B12 vitamin. However, in concentrations higher or lower than required, P and Co alter algal metabolism, impacting physiological processes (e.g., growth and photosynthesis), usually in a harmful way. In the environment, algae are exposed to multiple stressors simultaneously and studies evaluating the algal response to the combination of macronutrient limitation and micronutrient excess are still scarce. We assessed the effects of P limitation and Co excess, isolated and combined, in Raphidocelis subcapitata (Chlorophyceae), in terms of growth, pigments production, and photosynthetic parameters. Except for the photochemical quenching (qP) and the efficiency in light capture (α) under P limitation, all parameters were affected by both stressors, isolated and combined. Under P limitation, chlorophyll a was the most sensitive parameter; while excess of Co affected most the photoprotective mechanisms of algae, altering the non-photochemical quenchings qN and NPQ, influencing the light use and dissipation of heat by algae. The combination of two stressors resulted in a significant decrease in algal growth, with synergistic responses in growth and pigments production, and antagonism in the photosynthetic parameters. We suggest that algal metabolism was altered during P limitation acclimation and the excess of Co was used in a beneficial way by P-limited algae in photosynthesis, resulting in the well-functioning of the photosynthetic apparatus in the combination of both stressors. However, more studies are needed to understand which mechanisms are involved in this adaptation which resulted in antagonism in photosynthetic processes and synergism in growth and pigments production.