The exposure of plants to salinity of the fertigation solution induces morphophysiological and biochemical responses that occur through molecular mechanisms (Gupta and Huang 2014), which may alter plant physiology and reduce photosynthesis, growth and leaf area values (Anjum et al. 2011). However, plant tolerance or sensitivity varies between species and between cultivars of the same species, and plant growth and yield are the main parameters for assessing the responses of crops to salt stress (Lei et al. 2014). The proportions of the effluent in the fertigation solution did not influence the growth of L. gracilis plants in terms of biomass allocation (Table 3). The results of this study are in line with those found by Oliveira et al. (2019), who worked with the same species irrigated with a solution composed of sodium chloride, dihydrate calcium chloride and hexahydrate magnesium chloride, under greenhouse conditions. With highly saline solutions (9.9 dS m− 1), the authors observed a behavior of lower biomass production in leaves and stems.
These different responses between the present study and that conducted by Oliveira et al. (2019) may be linked to the source of salinity used. The effluent from tilapia cultivation has a significant amount of nutrients originated from the feeds used in the diets of fish and animal excretes; possibly, the nutrients added to the soil reduce the deleterious effects of salinity of the saline nutrient solution, especially the effects of chloride and sodium toxicity on the plants, causing them to have greater tolerance to salinity when fertigation is used as a water and nutritional source in the crops. The absence of change in leaf area among the evaluated treatments may be one of the factors that contributed to the maintenance of growth, since in situations of salt stress, plants reduce their leaf area, characterizing a strategy to avoid high transpiration rates, influencing photosynthesis and consequently limiting biomass production (Silveira et al. 2016; Silva et al. 2011).
The maintenance of growth levels, and consequently of biomass, may have been influenced by the maintenance of adequate K+ levels in the leaves up to T4. Plants often experience reduction of K+ when there is exposure to higher levels of Na+ due to the competition of this ion for the absorption channels of other ions, especially with K+ (Kibria et al. 2017). However, this result was only observed in the treatment with highest salinity, in which there was a reduction in K+ to the detriment of Na+. The Na+/K+ ratio confirms that there was a higher allocation of sodium to the stem in all treatments, except for T5. The mobilization of Na+ ions to the stem and the maintenance of adequate levels of K+ in the leaves allowed greater protection and functionality of the photosynthetic organs, as well as the growth rate of the plant (Miranda et al. 2017). The change of sodium allocation to the leaves in the treatment of highest salinity can be explained as a strategy of defense of the plant, since the leaves expel sodium when they senesce.
Growth is influenced by salinity and by osmotic imbalance and disturbances in the photosynthetic apparatus and osmoregulation (Negrão et al. 2017). Thus, the water retention capacity in leaf tissues, evaluated in this study by the relative water content (RWC) (Table 3), favored the tolerance to salinity, as it kept the cells turgid, providing full functioning of physiological processes, which positively influences the growth process, as shown in the parameters of leaf dry biomass and stem dry biomass (Table 3). The maintenance of RWC levels in L. gracilis subjected to salinity had already been identified by Ragagnin et al. (2014) under greenhouse conditions with different salinity levels.
The maintenance of leaf tissue hydration is a behavior of plants that can adjust osmotically. Thus, some species invest in sugar synthesis or starch breakage in an attempt to mitigate damage caused by stress (Almodares et al. 2008; Santelia and Lawson 2016). In this study, starch contents decreased (Fig. 1B) in treatments with higher salinity levels, indicating that the species was using its polysaccharide reserves to produce sugars for osmoregulation in order to keep leaf tissue hydrated according to its osmotic potential, thus maintaining its metabolism in operation.
In situations of abiotic stress, plants accumulate in the cytosol or vacuoles low-molecular-weight solutes (proline, betaine glycine, sucrose) to maintain water balance and preserve the integrity of membranes, proteins and enzymes (Ashraf et al. 2013; Marijuan and Bosch 2013). However, depending on the intensity and duration of stress, the production of these osmoregulators may or may not be intensified. In this study, it was observed that proline levels decreased at the highest concentrations of the effluent (Fig. 1C). This finding indicates, once again, that sugar production served as the main regulatory mechanism for the maintenance of water potential and leaf tissue turgor. It is important to highlight that in saline treatments with up to 5.5 dS m− 1 the proline levels did not differ from those of the control. It is suggested that this result is probably attributed to the adaptation of L. gracilis to environments with water scarcity and high temperatures. Under these conditions, proline is generally produced to keep the tissues hydrated, avoiding stress by desiccation. Results similar to these were found by Hu et al. (2012) in wheat plants, where proline levels decreased when sugar levels were high because, besides acting on osmoregulation, high values of sugar helped the plant to eliminate ROS (Cuoee et al. 2006).
The level of membrane damage measured by electrolyte leakage as a function of electrical conductivity (Munns and Tester 2008) and the contents of malondialdehyde (MDA) are important indicators of oxidative stress. The results obtained in this study do not point to salt stress, indicating that the salinity of the effluent did not increase the peroxidation of unsaturated fatty acids of the membrane of L. gracilis, confirming the degree of tolerance of the species under study.
Lipid peroxidation patterns can be modulated according to the amount of ROS generated and the defense capacity of the cells. One of the factors that can increase lipid peroxidation is excessive production and non-removal of H2O2. This compound is naturally synthesized by plants and its production occurs mainly through photorespiration or as a result of the dismutation of the superoxide radical, by superoxide dismutase (SOD). H2O2 can react with Fe2+ ions and form hydroxyl radical (OH), a ROS that can peroxide membrane lipids (Sewelam et al. 2016). However, the increase in H2O2 contents in plants will not always lead to increments in lipid peroxidation and oxidative stress, which can result in enzyme inactivation and limitation in plant growth and development (Gil and Turjeta 2010; De Choudhury et al. 2013). Thus, in order to overcome the negative effects caused by ROS, plants have an enzymatic antioxidant defense system, composed mainly of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) (Sewelam et al. 2016). H2O2 generated by SOD triggers CAT and APX enzymes, which are responsible for converting H2O2 into H2O and O2. These enzymes act according to their degrees of specificity; the CAT enzyme acts more significantly under conditions of greater severity (for example, T4), as it requires two molecules of H2O2 for the reaction. On the other hand, the APX enzyme is more sensitive and has the ability to react with only one molecule of H2O2, hence having lower action under the same conditions (for example, T2). This means that, at the salinity level 4, CAT was the most triggered enzyme to remove H2O2 compared to APX (Fig. 4). Increase in CAT activity was observed by Gondim et al. (2012) in a study with corn plants subjected to stress induced by NaCl, where the harmful effects of salinity did not compromise the growth variables. This result is indicated by the authors due to the performance of the antioxidant system, especially CAT.
In stressful situations, the content of pigments can be affected by the decrease in biosynthesis or the acceleration of their degradation (Ashraf and Harris 2013). Thus, in an attempt to adjust itself to the salinity and temperature conditions to which it was exposed, this species invested in the production of photosynthetic pigments, such as carotenoids, which act not only in the photosynthetic apparatus, but also as non-enzymatic components of the antioxidant system in the photoprotection and dissipation of energy, thus avoiding the formation of ROS and assisting in combating oxidative stress, consequently decreasing the harmful effects on the photosynthetic apparatus of the plant. The photoprotective effect of carotenoids assists in the dissipation of energy by exposure to high temperature and luminosity (Domonkos et al. 2013), through proteins present in the lumen of the thylakoids (Li et al. 2000).
As for anatomy, the results indicate that the fish farming effluent favored the development of the vascular system by increasing the number of xylem vessels, which is considered as an anatomical adaptation to salinity, aiming to improve the flow of water, since the frequency of xylem vessels and tracheids are determinant for water conductance (Sánchez-Aguayo et al. 2004). In addition to being responsible for long-distance transport of water and solutes, xylem is related to the mechanical support (Costa et al. 2004), preventing cells from collapsing and preventing the harmful effects of cavitation (Eller et al. 2018).
The presence of calcium oxalate crystals is not a common characteristic to all members of the Verbenaceae family (Souza et al. 2005; Braga et al. 2009); however, the presence of these crystals in leaves of other species of the genus Lippia has been documented, being highlighted as a xeromorphic characteristic (Andersen et al. 2006). On the other hand, the maintenance of the number of auxiliary vessels (NAV) in the leaves of plants subjected to different concentrations of fish farming effluent denotes that in plants subjected to treatment with 100% of fish farming effluent there was no need to invest in the formation of new vascular bundles to optimize the transport of resources.
The fish farming effluent did not interfere in the water balance of the cells in such a way to hamper the transport of water, so the mesophyll thickness was similar in all treatments evaluated (Table 5; Fig. 5), indicating that there was water maintenance in the cells, since water availability is one of the factors that first affect the maintenance of cell turgor (Santos and Carlesso 1998), which may therefore affect the mesophyll thickness.
Essential oils are components of the secondary metabolism of plants that are extracted from several parts (Oussalah et al. 2007). These compounds guarantee some advantages, acting for example as antioxidants and in the fight against microbial agents (Gutierrez et al. 2008). However, the chemical composition and content of the essential oil of a same species are associated with a variety of factors. According to Morais (2009), genetic traits, age and some climatic and environmental factors can lead to significant changes in the production of secondary metabolites by plants. Depending on the environment in which the plant is located, the salt concentration of the medium and the time of exposure, its metabolic route can be redirected, causing biosynthesis of different compounds and alteration of the content and chemical composition of essential oils (Neffati and Marzouk 2008). The conditions of the growing environment did not alter the chemical composition and content of oil under the conditions evaluated in this study. Similar results for the levels of thymol and carvacrol were found by Albuquerque et al. (2012) and Ragagnin (2014) in plants of the same species.
According to literature reports, conditions of low and high salinity of irrigation water do not interfere with the yield of the essential oil of L. gracilis species (Oliveira et al. 2019). Based on the published data, the results obtained showed coherence because the most frequent components to appear in the essential oils of many species of Lippia are limonene, β-caryophyllene, p-cymene, camphor, linalool, α-pinene and thymol (Pascoal et al. 2001).
Chemical composition of L. gracilis essential oils obtained from other states in northeastern Brazil showed that: in Ceará, the essential oil was characterized by thymol (30.6%) and p-cymene (10.7%) as major components; in Piauí, there was carvacrol (47.7%) and p-cymene (19.2%); and in Sergipe, carvacrol (23.52%), p-cymene (4) (15.82%), γ-terpinene (14.17%) and menthol (10.97%) (Gomes et al. 2011). The quantitative variations observed in the chemical composition of the essential oils of L. gracilis are probably due to genetic factors such as different forms of the trichomes present in different species of Lippia and edaphoclimatic conditions such as climate, relief, lithology, temperature, air humidity, radiation, soil type, wind, atmospheric composition and rainfall location, as well as conditions under which the plant is grown (Santos et al. 2004).
The essential oil from L. gracilis, with different saline treatments, showed bacteriostatic and bactericidal activity against Gram-positive and Gram-negative bacteria. The antimicrobial activity obtained in this study may be explained mainly by the phenylpropanoids carvacrol and thymol, compounds that are usually found in Lippia species and have shown action against bacteria and fungi. The essential oil from L. origanoides showed activity against bacteria such as methicillin-resistant Staphylococcus aureus, and the essential oil from L. menosides showed activity against Escherichia coli, Enterococcus faecalis, Salmonella enteritidis, Serratia marcescens, Candida albicans and Mycobacterium smegmatis. The antimicrobial activity was associated with the presence of the phenolic monoterpenes, carvacrol (41.77%) and thymol (10.13%) (Girón et al. 1991; Lacoste et al. 1986; Oliveira et al. 1990).
Essential oils are compounds that, for being hydrophobic, are easily diffused through the cell wall of microorganisms and cause damage to the membrane, especially with regard to fluidity and permeability (Millezi et al. 2012). Gram-negative bacteria, in general, have greater resistance to the action of essential oils, due to the greater complexity of their plasma membrane, which acts as a barrier to the diffusion of hydrophobic components of essential oils (Naik et al. 2010).
It is known that fish farming effluent has high levels of excrement and nutrients that can modify the characteristics of the fertigation solution in such a way to make its use unfeasible in other activities, such as in agriculture when using salinity-sensitive species (Mercante et al. 2004; Mainardes Pinto and Mercante 2003). The results of this study are relevant to the planning and management for the use of fish farming effluent, which is currently disposed of inadequately and without any utilization. In this context, the set of results obtained support the responses observed in the growth, anatomical and biochemical parameters, consolidating the indication of fish farming effluent used in this experiment for reuse in the irrigation of L. gracilis, an endemic species of the Caatinga and with pharmacological potential.