An Acad Bras Cienc (2020) 92(Suppl. 2): e20190050 DOI 10.1590/0001-3765202020190050 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal BIOLOGICAL SCIENCES Effect of phosphorus on the toxicity of zinc Running title:PHOSPHORUS to the microalga Raphidocelis subcapitata EFFECT UNDER ZINC TOXICITY FOR ALGAE SUZELEI RODGHER, THAIS M. CONTADOR, GISELI S. ROCHA & Academy Section: BIOLOGICAL EVALDO L.G. ESPINDOLA SCIENCES Abstract: The aim of this study was to evaluate the effect of phosphorus (P) on the toxicity of zinc (Zn) for the alga Raphidocelis subcapitata. P was provided in three concentrations: 2.3 x 10-4 mol L-1, 2.3 x 10-6 mol L−1 and 1.0 x 10-6 mol L−1. Algal cells were acclimated to the e20190050 specifi c P concentrations before the start of the experiment. The chemical equilibrium software MINEQL+ 4.61 was employed to calculate the Zn2+ concentration. After acclimated, the algal cells were inoculated into media containing different Zn concentrations (0.09 92 x 10-6 mol L-1 to 9.08 x 10-6 mol L-1). The study showed that besides the reduction in algal (Suppl. 2) growth rates, phosphorus had an important infl uence on the toxicity of zinc for microalga. 92(Suppl. 2) The inhibitory Zn2+ concentration values for R. subcapitata were 2.74 x 10-6 mol L-1, 0.58 x 10-6 mol L-1 and 0.24 x 10-6 mol L-1 for the microalgae acclimated at P concentrations of 2.3 x 10-4 mol L-1, 2.3 x 10-6 mol L-1 and 1.0 x 10-6 mol L-1, respectively. Ecotoxicological studies should consider the interaction between metal concentrations and varying P values to provide realistic data of what occurs in phytoplankton communities in environments. Key words: Algal density, free zinc, green algae, nutrient. IN TRODUCTION on aquatic environments include changes in the biodiversity, drinking water treatment problems Eutrophication and the introduction of toxic and a reduction in recreational use (Tundisi & materials such as metals are considered major Tundisi 2008, Pereira et al. 2010, Costa et al. 2014). types of degradation of aquatic systems (Wang Some metals, such as zinc, are essential & Dei 2006, Dirszowsky & Wilson 2016, Albano et nutrients for prokaryotic and eukaryotic al. 2018). Ecological concern about phosphorus organisms. Zn is a component of many enzymes is caused by its important role in biological and guarantees biological stability of the genetic metabolism and the low concentrations of material and of biological structures, such as this element in water bodies (Wetzel 2001). the ribosomes and cytoplasmic membranes Phosphorus controls the biological productivity (Eisler 1981). Currently, zinc is employed in of algae in the greater part of the aquatic coating other metals, inorganic medicines, and environment (Grossman 2000, Hessen et al. the manufacture of aesthetic products and 2002, Esteves 2011). However, agro-industrial and non-corrosive alloys interfering (Tsushima et al. domestic sewage discharges may insert excess P 2010). This element is one of the most important into freshwater and marine ecosystems, resulting metals in the economic improvement of China eutrophication (Zeng & Wang 2009, Schindler et (Xueyi et al. 2010), Korea (Jeong & Kim 2018) al. 2016). The potential effects of eutrophication and Poland (Lewicka & Burkowicz 2017). In 2017, An Acad Bras Cienc (2020) 92(Suppl. 2) SUZELEI RODGHER et al. PHOSPHORUS EFFECT UNDER ZINC TOXICITY FOR ALGAE the U.S. Geological Survey (2018) estimated the to green microalgae under limiting phosphate global zinc mining production as 13 million tons, conditions was described by Gao et al. (2016). with 5% increase compared to the previous year. The variable data obtained from research Due to concern about the potential effects of zinc about the interaction between phosphorus and on the biota of aquatic systems, zinc is receiving metal exposure to algae could be related to the attention from the regulatory environmental different procedures adopted in the experiments agencies in European Union and Japan (Bodar (Gao et al. 2016), including the absence of et al. 2005, Van Sprang et al. 2009, Naito et al. metabolic acclimation of algae to changes in 2010, Tsushima et al. 2010). the phosphorus concentration. According to Zinc is an essential element for the literature, excess P in the environment is phytoplankton, being required in both incorporated in the algae as polyphosphate photosynthetic processes and energy storage vesicles, which reduces the toxicity of metallic (Cao et al. 2015). However, values above trace ions by way of detoxifying processes when amounts can be dangerous, modifying cell the ions bind to the polyphosphates (Jensen volumes and inhibiting the growth rate and et al. 1982, Twiss & Nalewajko 1992). Hence, photosynthesis of microalgae (Guanzon Jr et al. previous studies about the interaction between 1994, Mallick & Mohn 2003, Machado et al. 2015). phosphorus and metal exposures have On the other hand, the presence of phosphorus reinforced the need for acclimation processes in the aquatic metabolism is of concern to when the algae are exposed to different supplies ecologists because this element can regulate of phosphorus, to assure that their metabolisms algal productivity, causing eutrophication and express that condition (Lombardi & Maldonado affecting biological diversity. In the natural 2011, Chia et al. 2017, Rocha et al. 2018). aquatic environment, organisms are continually The intensification of eutrophication confronted with simultaneous physical and in freshwater systems justifies the need for chemical perturbations (Chia et al. 2017, Van studies about the interaction between metals Regenmortel et al. 2017, Kong et al. 2018, Roy et al. and phosphorus, due to the influence of these 2018). As a consequence, chemical compounds elements on the physiology of autotrophic interacting with each other in antagonistic and microorganisms (Reynolds 2006, Chia et al. 2013a, synergistic manners can alter their toxic action Gormley-Gallagher et al. 2016). Ecotoxicological to microalgae (Bere et al. 2012, Zhang et al. 2015, tests are helpful tools to estimate the combined Mansano et al. 2017). toxic effects of chemicals in aquatic systems. Of Studies about the interactions between the unicellular algal species, the Chlorophyceae metallic ions and nutrients have highlighted Raphidocelis subcapitata (previously named the influence of nutrients in culture media on Pseudokirchneriella subcapitata) is one of the sensitivity of phytoplankton to metals. The the most suitable for carrying out toxicity toxicities of arsenic (Wang et al. 2013), cadmium tests, due to its high sensitivity to a variety of (Zeng & Wand 2009, Webster et al. 2011), copper contaminants, adequately maintainable in (Serra et al. 2010, Rocha et al. 2016) and chromium laboratory conditions (Wei et al. 2006, Yan et (Qian et al. 2013) to cyanobacterium and green al. 2015, Lewis & Thursby 2018) and for being microalgae have increased in response to the a characteristic species of oligotrophic and reduction of phosphorus in the environment. eutrophic freshwater systems (Blaise & Vasseur To the contrary, a decrease in the toxicity of Zn 2005). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190050 2 | 11 SUZELEI RODGHER et al. PHOSPHORUS EFFECT UNDER ZINC TOXICITY FOR ALGAE The main objective of this study was to in values lower than 1 x 10-6 mol P L-1. Acclimation of evaluate the effects of various phosphorus/ the green algae was carried out by transferring algal zinc combinations on the toxicity of the metal cells in the exponential growth phase with an initial on the green alga Raphidocelis subcapitata. density of 5 x 105 cells mL-1, into semi-continuous The choice of the phosphorus concentrations cultures with specific phosphorus concentrations. tested reflected the natural conditions of The algal cells were maintained for 5 weeks under freshwater aquatic environments, including low these conditions, keeping the algal cell density at 5 and high concentrations of the element (Zeng x 105 cells mL-1. Algal cells were collected every 168 h & Wang 2009, Chia et al. 2017). In this research, and the number of cells in the cultures quantified the microalga R. subcapitata was exposed to using a Neubauer-Improved Chamber to determine three phosphorus concentrations for several the growth rate (Rocha et al. 2016, Chia et al. 2017). The generations up to constant growth rates, to growth rate values were evaluated according to Fogg guarantee that their physiology would reflect (1975). The algal cells were maintained under distinct the nutrient concentrations (Rocha et al. 2016). phosphorus concentrations for 35 days, allowing for Thus, the toxicity tests with zinc were carried out the finding of at least three constant growth rate after acclimation of the microalgae. The results values (statistically similar values, p>0.05). Under this generated in this work represent an important condition, the microalga was considered acclimated contribution to research related to the effect of and its metabolism expressed the P values in the zinc on aquatic systems with different trophic culture medium (Rocha et al. 2016). The experiments states, with emphasis on the physiology of were carried out with 3 replicates per treatment. autotrophic microorganisms. The acclimated algal cells were employed in the toxicity tests with zinc, but the microalgae were not acclimated to the metal. MATERIALS AND METHODS All the laboratory materials used in the Algal culture and acclimation experiments culture and acclimatization experiments were The green alga Raphidocelis subcapitata was washed with 10% HNO3 for 7 days and then obtained from the algal culture collection of the rinsed with deionized water before use. Botany Department of the Federal University of Toxicity tests São Carlos (São Carlos, SP, Brazil). Stock cultures of the R. subcapitata were maintained in L.C. Phosphorus acclimated and exponentially Oligo medium (AFNOR 1980).