Salinity tolerance of macroalgae Gracilaria birdiae

: This study aimed to determining the manner in which salinity influenced the growth of the macroalgae Gracilaria birdiae, with the objective of identifying its tolerance limits to this abiotic variable. The specimens were submitted to nutrient-enriched water of varying salinities (0, 10, 20, 30, 40, 50, and 60 ppt) for a 30-day period. Initially, under extreme salinity conditions (0 and 60 ppt) the growth of the G. birdiae suffered a negative impact. The macroalgae biomass exposed to 0 and 10 ppt salinities showed a reduction from day six until the experiment was completed. The macroalgae biomass exposed to salinities 20, 30, 40, and 50 ppt showed an increase, with no significant differences between the four treatments. This suggested that this salinity range was comfortable for this species to develop. We concluded that salinity is a crucial parameter which controls the growth of the G. birdiae. This seaweed was negatively influenced when exposed to values equal to or below 10 ppt and equal to 60 ppt, demonstrating good tolerance to salinities of 20, 30, 40 and 50 ppt.


INTRODUCTION
In general, macroalgae cultivation shows high productivity, with genus Gracilaria (red algae) ranking among the most cultivated of the marine vegetables. In fact, the compounds and extracts find use in several economic applications (STENGEL et al., 2015;FAO, 2018;TORRES et al., 2019). The macroalgal biomass is used in phycolloid industries as a potential agar source, while its processed extracts have rheological or bioactive characteristics which are beneficial as dietary supplements, in cosmetics and for animal feed (PEREIRA & YARISH, 2008;LERAT et al., 2018). Macroalgae are also extensively employed in agriculture, enhancing the productivity of the crop and raising their tolerance levels to overcome biotic and abiotic stresses (BATTACHARYYA, 2015). They are also used in aquaculture, as they have bioremediation efficiency, particularly in terms of nutrient removal from water (MARINHO -SORIANO, 2008).
Living beings exhibit a variety of tolerance limits to the different abiotic variables that they are exposed to in order to survive, grow and reproduce. Salinity ranks high among these variables and is considered one of the most significant, which can influence the metabolism and reproductive capacity of aquatic organisms (WHARTON, 2002;PEREIRA et al., 2017;FATH, 2018). Salinity can also control Alves et al. several ecological and biological aspects of the benthic marine algae, such as a species of red algae, Gracilaria birdiae, which flourishes along coastal regions, where the salinity values reveal a high variation range due to the wind, precipitation and tide actions (SCHMIDT et al., 2015;PEREIRA et al., 2017).
A good understanding of the tolerance limits of the abiotic variables, including salinity, is of practical importance to feasibly cultivate many macroalgae species (KALIAPERUMAL et al., 2001;KALIAPERUMAL et al., 1993). In fact, CHOI et al., (2010), in their evaluation of the ways in which the salinity affects the growth, photosynthetic activity and the internal nutrient composition of Ulva pertusa, reported salinity to exert greater influence, more than the other environmental factors, like light and temperature. In their evaluation, PEREIRA et al., (2017) studied the effects of salinity on the physiology of the macroalgae Acanthophora spicifera, and reported that extreme salinities lowered the growth rate in this species. From the data given above, our goal in this study was to estimate how salinity affected the G. birdiae macroalgal growth. As this alga is widely distributed along the coastal regions of northeastern Brazil, this study was done to determine its tolerance limits in order to provide information that can facilitate its cultivation because there is a limited number of studies on the effect of salinity on its growth.

MATERIALS AND METHODS
Individual samples were drawn from the macroalgae Gracilaria birdiae along the coastal region of the state of Ceará -Brazil (3° 13'06.1" S and 39° 15',51.4" W). The manual collection was done during the low tide, and only those individuals showing good physiological condition, with no signs of depigmentation were selected. The plant material was then packed in isothermal boxes with water drawn from the habitat itself and transported to the Aquaculture Sector of the Federal Rural University of the Semi-Arid Region, where this experiment was performed.
A completely randomized experimental design was adopted. The treatments included several salinities (0, 10, 20, 30, 40, 50 and 60 ppt) with three replicates each, to account for a total of 21 experimental units, each with 40 liters, using the reference value (control) of 30 ppt (meaning, the identical salinity of the seawater from where the macroalgae were collected). Salinities of 10 and 20 ppt were achieved by adding fresh water to the seawater, while salinities above 30 (40, 50 and 60 ppt) were obtained by adding hypersaline water from saline. Freshwater was used to achieve zero ppm salinity. All the experimental units were enriched by the addition of von Stosch (VS) solution (Table 1), prepared according to EDWARDS (1970), including the modifications, according to YOKOYA (1996). Constant aeration was provided throughout the 30-day experiment.
In each experimental unit, the macroalgae had initial fresh biomass of 40 g, which corresponded to 6.5 g of dry mass. In this experiment, the dry mass of the macroalgae used was estimated from the linear regression equation between the fresh mass (MF) and dry mass (MS) of the macroalgae collected from the same place as the individuals used in the experiment (MS = 0.0018 + 0, 1644 * MF (r2 = 0.9819; n = 28)).
The macroalgae were weighed (fresh mass) once in every three days and then replaced  Table 2). The data referring to the dry biomass were analyzed employing the Shapiro-Wilk and Bartlett tests to determine the normality and homoscedasticity, respectively. For variables showing normal distribution and homogeneous variance, the analysis of variance (one-way ANOVA) was applied and a posteriori the Tukey test, to identify the significant differences (P<0.05) between the treatments.

RESULTS AND DISCUSSION
The G. birdiae had initial average biomass of 34.3 gMS.m -2 , which showed no significant difference between treatments (P = 0.8437) ( Figure  1). On day three of the cultivation, the biomass significantly reduced at the extreme salinity conditions, that is, at 0 and 60 ppt. However, under the conditions of salinity 60 ppt, the G. birdiae again increased in biomass on day six (Figure 1), and remained constant until day twenty-one, after which it once again decreased until the experiment was completed, achieving final average biomass of 28.8 gMS.m -2 (Figure 2). The biomasses of the macroalgae cultivated at salinities 0 and 10 ppt showed a reduction from day six until the experiment ended. In the treatment with salinity 0 ppt, the G. birdiae entered senescence totally on day twentyseven, while on day thirty, this species submitted to salinity 10 ppt showed a significantly decreased average biomass compared to the other treatments (1.1 gMS.m -2 ) (Figure 2). The negative influence on the G. birdiae growth, when subjected to the extremes of salinity, was probably caused by a disturbance in the cellular mechanism, which inhibited the reaction centers of photosystems I and II. KIRST (1990) reported that higher or lower salinity levels induced this response, causing either the decrease or interruption of photosynthesis and the subsequent reduced growth. The growth of the G. corticata species also showed significant negative responses when exposed to extreme salinity ranges (15 and 55) (KUMAR et al., 2010).
Although the species exhibited significant differences when exposed to the salinity extremes, it also showed wide tolerance of this parameter in the intermediate salinities. The macroalgae revealed a biomass increase when subjected to salinities of 20, 30, 40 and 50 ppt, during which they remained significantly unchanged throughout the experiment and achieved a final average dry biomass of 40.37, 39.20, 35.07 and 36.31 gMS.m -2 , respectively (Figures 1 and 2). This lack of significant differences in the G. birdiae growth in this interval indicates its ability to tolerate a wide variation in salinity, probably connected to its eurialin characteristic. In a study performed by exposing the red macroalgae Grateloupia doryphora to different salinities, it was reported that they too exhibited higher tolerance to salinities of 22 to 42 ppt (SIMON et al., 1999), a range very similar to that observed in the present study for G. birdiae. The wide interval of salinity tolerated by the species in the present study may be linked to its ability to adapt as it inhabits the regions between the tides, which are subject to constant salinity changes.

CONCLUSION
We concluded that the salinity controls the G.birdiae growth, with the interval between 20 and 50 ppt being the most favorable for this seaweed species to flourish. Conversely, the G. birdiae growth is adversely affected by salinities of 0 and 10ppt. Results from this study supply data that may be relevant to the cultivation of this macroalgae in a monoculture or a multitrophic system.