Among the studied mangroves of Sepetiba Bay, Marambaia is thought to be the most preserved since it lies east of the Coroa Grande and Santa Cruz industrial complex and, hence, the most distant mangrove from anthropic pressure. It is also located in an area under military protection. In 2010 Ferreira et al. found that the concentration of heavy metals in Sepetiba Bay did not exceed the limits recommended in CONAMA Resolution No. 344/2004. However, Cd, Zn and Cu in the water did show unsatisfactory values in some parts of Sepetiba Bay when assessed according to resolution Nº357/2005. One of the many factors that alter the concentration of minerals in Sepetiba Bay is the frequent movement of tidewater and sediment from water entering the bay. In addition, industrial activities have production rates that can alternately reduce and increase pollution. Sepetiba Bay is a coastal area sensitive to regional environmental changes that can create an ecotone where interactions between land and water take place. According to Ribeiro et al. (2013), metal mobility may also change in the short term. The average of dry weight showed a decreasing pattern following the order M> CG> PG (Fig. 4).
Severoglu et al. (2015) suggest that heavy metals constitute one of the main abiotic agents related to growth reduction and alteration in physiological processes. The high SLA value of L. racemosa collected in CG and PG mangroves is consistent with the pollution data based on proximity to the industrial complex. SLA is an important indicator of the impact of pollution in the environment, and it can may be a parameter involved in protection and adaptation of plants (Wuytack et al. 2011).
The amount of Na is three times greater in leaves of Marambaia compared to leaves examined from CG and PG mangroves. This result could be attributed to the degree of salinity in the region where a high hydrodynamism is known to significantly change salinity. Soil salinity can interfere with the uptake and exchange of ions in the soil-plant system, which, in turn, is reflected in the morphology and structure of plants (Bartz et al. 2015).
Leaf structure of L. racemosa was analyzed by Francisco et al. (2009) who reported the presence of leaf glands and trichomes, as well as salt secretory glands. They considered these features to be adaptations to the saline ecosystem. Here we added information about the quantity of salt secretory glands in the leaves of L. racemosa plants growing in different mangroves around Sepetiba Bay and data about the micromorphology of epicuticular wax. Some microscopic views showed the high salt content on leaves and around salt glands (Fig. 5). Evidence suggests that mature leaves of L. racemosa can secrete salt according to its concentration in the soil. That is, if salt increases in the substrate, then salt secretion will be higher in leaves (Sobrado 2004). Through salt glands, salt-tolerant plants also can expel heavy metals (Arrivabene et al. 2016). Leaf structures are particularly relevant to the ecological success of this species, which occupies environments where salinity is high. No leaf samples from mangroves evaluated showed morphological or structural damage.
The plant cuticle consists of nonpolar substances such as cutin, which is the matrix associated with waxes. It is secreted by cells of the epidermis and deposited on an organ of the plant’s surface, such as the leaf. It also seals flowers, stems, and fruits, protecting them from biotic and abiotic stresses (Kunst and Samuels 2009). Intracuticular wax is embedded into the cuticle, and epicuticular wax is found on the cuticle's surface (Koch and Ensikat, 2005). Cuticular waxes are involved in guarding against excessive leaf water loss (residual transpiration) (Hasanuzzaman et al. 2017). The presence of n-alkanes and triterpenes, as the main constituents of epicuticular wax, is recurrent among restinga and mangrove plants that use residual transpiration as a mechanism of tolerance under salinity stress (Oku et al. 2003; Zorat et al. 2011; Hasanuzzaman et al. 2017; Victório et al. 2020). Pentacyclic triterpenoids, such as 𝛽-amyrin and lupeol, which are representative of the oleanane and lupane groups, are widely distributed in mangrove plants associated with features tolerant to salt (Basyuni et al. 2012). Pentacyclic triterpenes were identified as the main components of A. shaueriana epicuticular wax (Victório et al. 2020), as well as the main components of L. racemosa leaves in this study. However, our results are different from those of Rafii et al. (1996) who verified only trace amounts of triterpenes in leaf wax of L. racemosa from Guyana (western Atlantic coast) and 8.5% in wax extract in a population from Gabon (eastern Atlantic coast) that did not present lupeol.
The presence of hydrocarbons in leaf epicuticular wax has proven valuable in chemotaxonomic studies of different botanic families. Alkanes were the most abundant compounds among the hydrocarbons, highlighting hentriacontane (28.01-45.36%) in leaf wax collected in CG. The hydrocarbons hentriacontane and octadecanone are present in leaves of Rhizophora mangle from Africa (Dodd et al. 1995), and hentriacontane is present in leaves of A. shaueriana (Victório et al. 2020). The presence of the hydrocarbon hentriacontane is recorded for the epicuticular waxes of several plant species (Wang et al. 1999), but this was the first evidence of its presence in the Laguncularia genus. As constituents of epicuticular wax, n-alkanes, but not triterpenes, seem to be related to a reduction in cuticular water loss (Buschhaus and Jetter 2012).
In studies with conifer (Pinus sylvestris L.) seedlings, Burkhardt and Pariyar (2014) verified that air pollution degraded (´erosion`) epicuticular waxes that revealed an amorphous appearance resulting in low drought tolerance. These symptoms, which are easily visible through an analysis of the leaf surface by scanning electron microscopy (SEM), are provoked by very diverse chemical environments, such as acid rain or fog, simultaneous exposure to SO2 and NH3 or car exhaust, as pointed out by Viskari et al. (2000). Studies of Arrivabene et al. (2015) detected a higher amount of particulate material, including iron, on the leaf surface of A. schaueriana and L. racemosa, the leaves of which contain salt glands in comparison to Rhizophora mangle. The presence of hydrophobic epicuticular wax covering the leaf blades may be associated with the absorption of chemicals of equal polarity, such as phthalates, pesticides and others. Plasticizers were detected in the leaf epicuticular wax of A. shaueriana and R. mangle from the same mangroves (Victório et al. 2021). Cuticular waxes also act in controlling loss and uptake of polar solutes (Hasanuzzaman et al. 2017).
Cutin is integrated into superimposed waxes; it is an extracellular layer that lines the epidermal cells externally. When plant cells are subjected to stress, the results show up later as alterations in the synthesis of wax, in turn affecting gene expression. Apart from internal processes, biotic and abiotic stresses are involved in the regulation of plant cuticle biosynthesis (Fich et al. 2016; Tafolla-Arellano et al. 2018). Some reports show consistency between the morphology of wax and its composition (Koch and Ensikat 2008). However, variations in wax types caused by changing environmental conditions have been suggested during crystallization, and genes associated with cuticle formation have shown responsiveness to environmental conditions (Koch and Ensikat 2008).
It has been reported that uptake of metals from both soil and leaves influences cuticular wax layer and permeability. A positive correlation between transpiration rate and cadmium (Cd) concentration was reported in Beta vulgaris plants, potentially affecting cuticle biosynthesis and composition. Consequently, changes in chemical composition are reflected in permeability which can then result in high water losses through the cuticle (Greger and Johansson 1992). Evidence suggests that heavy metals alter the cuticle, e.g., the expression of genes involved in Cd tolerance, while Fe deficiency was shown to reduce the amount of cuticular lipids that influence water retention, solute permeability, pathogen infection and disease resistance (Fernández et al. 2008; Tafolla-Arellano et al. 2018).
Mangroves are also exposed to pollution of particulate material through the atmosphere owing to their coastal distribution and proximity to urban centers (Bayen 2012). Particulate pollutants deposited on the leaf surface may be absorbed by leaf tissues and alter the chemical structure of epicuticular waxes, thereby causing morphoanatomical damage, such as chlorosis, necrosis, reduction in photosynthesis and gas exchange (Prasad and Hagemeyer 1999; Naidoo and Chirkoot 2004; Arrivabene et al. 2015). Such absorption may also induce physiological responses of plants and alter production of metabolites. In studies reporting on the epicuticular waxes of Coffea arabica, Lichston and Godoy (2006) verified a decrease in wax content and morphological alteration of wax when leaves were exposed to a copper-based fungicide.
Plants from mangroves, mainly in polluted areas, incorporate some metals in organ tissues. However, results show that metal concentration attained in biota is only high if the plants have accumulated features and are hyperaccumulators (Saenger and McConchie, 2004). Laguncularia racemosa plants accumulate high concentrations of Cr in roots, but low mobility of this element results in correspondingly low content in leaves (Rocha et al. 2009). Saenger and McConchie (2004) also indicate that young leaves accumulate more metals than mature leaves. The metal contents in leaves of mangroves sampled in this study are lower than those observed in normal reference plants grown in uncontaminated soils. Machado et al. (2002) conducted a study with L. racemosa in Guanabara Bay (RJ) and found that heavy metals tend to accumulate in chemical forms that reduce their mobility and, hence, absorption by biota. In addition, a low translocation of heavy metals was observed from the leaves of the litter to other trophic levels, suggesting the low bioavailability of these heavy metals. Leaves may accumulate heavy metals or metabolize them. When leaves fall in the mangrove substrate, a low amount of metals present in the leaf litter is returned to the soil by decomposition and mineralization processes, becoming bioavailable in the environment in a way that culminates in biomagnification processes along the food chain. However, the release of metals from fallen leaves is low (Saenger and McConchie 2004). This ability to keep heavy metals in unavailable forms, together with resistance to these elements in the sediment, may explain the low levels of some heavy metals found in the aerial part of L. racemosa. Still, leaves are important organs in the analysis of heavy metals, essentially because many processes of primary and secondary metabolism occur in leaves. In addition, because of transpiration, the water rises from the root carrying the pollutants dissolved in the water when they are not deposited on the leaf owing to their presence in the atmosphere (Liang et al. 2017).
EDS analysis evidenced high concentrations of calcium (Ca), magnesium (Mg), potassium (K) and sodium (Na) (Fig. 4), indicating the high salinity of the mangrove environment surrounding Sepetiba Bay where the marine influence is greater (Lacerda et al. 1985). The ability to tolerate high concentrations of salt may be associated with tolerance to heavy metals since both depend, in part, on common physiological mechanisms (Manousaki and Kalogerakis 2011). Ca, K, and Mg will work by inhibiting the absorption of heavy metals, and their levels will increase whenever stress is caused by heavy metals. Potassium will act to restore osmotic pressure, and the plant will protect itself by increasing the levels of Ca and Mg to a threshold, after which they start to decrease (Severeglu et al. 2015). Using EDS, Zn was detected only in leaves from CG (0.39 ATOM%). Its absence in other mangroves may be associated with experimental conditions or optimization of the analysis by EDS since the presence of Zn for all mangroves was verified in analyses by ICP-OES. Comparing these methods, a large number of minerals were identified through ICP, revealing that this type of analysis is more accurate than that performed with EDS. However, the use of standards is required in EDS analysis to better indicate the concentration of minerals.
Considering the analysis by ICP-OES, it follows that CG, as the mangrove area nearest the industrial complex, would have high values of heavy metals, as verified by both spectroscopy and spectrometry. The high concentrations of Zn (0.335 mg/L) and Cd (0.016 m/L) for PG samples in relation to other mangroves also suggest the influence of industrial pollutants in this area, in contrast to Marambaia, which would be the most distant from industrial pollution. Through the analysis by ICP-OES, only high concentrations of Zn were confirmed in CG. Zn is a metal present in the lithosphere, but it also indicates anthropogenic pollution. Zn is a naturally occurring metal, but it is often associated with anthropogenic sources, and it can be considered a key indicator of polluted areas, such as contaminated urban areas (Alharbi et al. 2019). Also, Zn is probably associated with the waste from the metallurgical plants in the West Zone of Rio de Janeiro and Coroa Grande (Itaguai City). The higher concentration of Zn (<50 ppm) was also observed in leaves of L. racemosa in experiments carried out by Bernini et al. (2010) from leaves collected in an estuarine mangrove of the São Mateus River, Espírito Santo, Brazil.
According to Ramos and Silva (2006), mangrove forests are important biochemical filters of heavy metals to coastal areas since these elements would otherwise be accumulated in the organs of perennials, such as branches and leaves, with high renewal rate. Heavy metals like Cd, Cr, Pb and Zn present high toxicity to environment, and their significant bioaccumulation causes many problems in ecosystems because they are not biologically degraded, but rather accumulate in biota and in abiotic environments as trapped particulate in mangrove sediments (Mathivanan and Rajaram 2013).
Mineral resources correspond to an important material base for socioeconomic progress. Besides Zn and Cd, other metals, such as Ba, Si, Sb, Ti and V, are employed in different industries, in particular, those in the Sepetiba Bay area, and thus also found in leaves of L. racemosa. For example, Ti, Sb and V are used in the production of metal alloys. Because of the lower values from some minerals, it is possible that they do not originate from industrial sources, but, instead, are the result of biogeochemical cycling.
Barium (Ba) is a toxic chemical used in various industries, such as oil well drilling, production of rubber and paper, fireworks, manufacture of glass, paints and pigments, composition of batteries, and the composition of fluorescent lamps, and it causes many disturbances in plant development (Sleimi et al. 2021). Silicon (Si) has important applications in computers and the production of silicone polymers. Antimony (Sb) is employed mainly in metal alloys, and some fire-resistant compounds are also used in paintings, ceramics, enamels, rubber vulcanization and fireworks. However, Sb is potentially toxic and has no role in biological functions. Sb in plants can lead to toxicity, so low to moderate concentrations can result in damage to plant growth and development, including photosynthesis, lipid peroxidation and oxidative stress (Natasha et al. 2019). The maximum value reported in leaves is 1.5 mg/kg, but in most samples, like our data (0.2 mg/kg), the concentrations were below 0.5 mg/kg (Pérez-Sirvent et al. 2011). These chemical elements are indicators of industrial development and were detected in samples of L. racemosa leaves, but not in concentrations considered toxic.
Vanadium (V) is a metal widely present in the environment and distributed in leaf organs of plants in low concentrations. Compared to other species, the leaves of L. racemosa accumulated it at very low concentration (0.05 mg/kg). The lowest registered to leaves was found in smilograss - Piptatherum miliaceum (0.1 mg/kg of V) (Aihemaiti et al. 2020). Studies indicated that some phosphate fertilizers present high concentrations of V (90-180 mg/kg) as a contaminant (Vachirapatama et al. 2002), suggesting that this element is widespread in soils, water and vegetables through phosphorus fertilization. For plants, depending on concentration, V can be harmful to development in high concentrations by disrupting energy metabolism and matter cycling. It can also inhibit some enzymes, protein synthesis, and ion transport, as well as reduce growth rate, cause root and shoot abnormalities, or even death of plants. In low levels, the results may be positive by elevating plant height, root length, and biomass production associated with increased chlorophyll content, seed germination, essential mineral uptake, and assimilation of nitrogen and its utilization (Aihemaiti et al. 2020).
From an anatomical point of view, the L. racemosa leaf presented tissue organization similar to that described for the family and genus mentioned in the classical literature (Metcalfe and Chalk 1950). The anatomical pattern observed in the samples of L. racemosa differed slightly from that described by Silva et al. (2010), who evaluated the same species in a mangrove in the state of São Paulo by comparing a highly impacted area with a non-impacted one. In the epidermis, for example, the authors observed only epicuticular wax in granules. Baker (1980) suggests that epicuticular wax in plates are produced by primary alcohols, such as triterpenoids, resulting in amorphous morphology, as we observed here.
Salt-secreting structures in the epidermis seem to be an unusual feature. It has been suggested that plants evolutionarily tend to adapt to the saline condition rather than having salt glands. Only a few orders of Angiosperms such as Poales and Myrtales, including Combretaceae, Caryophyllales, Lamiales, and Solanales have salt glands (Flowers et al. 2010), indicating an independent evolutive origin. The secretory glands actively eliminate salts, keeping them within certain limits (Larcher 1995). In our study, the number of salt glands varied (2-3 glands/mm2), different from what was observed by Silva et al. (2010) who found less than 1 gland/mm2. This reduction may be related to the levels of salinity found in each collection site, especially from the proximity of large rivers that flow into the site investigated by Silva et al. (2010) and which would have reduced the salinity of the soil. The very high content of Na in leaves from M, compared to that found in leaf samples from CG and PG mangroves, is simply suggestive of high salinity as a characteristic of the local ecosystem. This study did not verify any changes in the patterns of salt gland distribution for L. racemosa. The anatomical characteristics of the salt glands, which are located at the bottom of an epidermal cavity, corroborate the description provided by Francisco et al. (2009) and Dassanayake and Larkin (2017) regarding cell organization and composition.
In the mesophyll of L. racemosa herein evaluated, we observed that the thickness varied from 397.94 to 430.13 µm, values similar to those found in leaves collected in northern Brazil (Lucena et al. 2011) in a place not affected by industrial pollution, suggesting that the leaves of the species evaluated here did not present significant alterations in the organization of the palisade and spongy parenchyma. In leaves investigated here, we observed an isolateral structure in the three sites selected, even as we acknowledge the dorsiventral mesophyll cited by Lima et al. (2013) and Lucena et al. (2011). Isolateral leaves are found in plants living in sites where incident light is received from upper and lower orientations, possibly improving the photosynthetic process in an otherwise growth-limiting environment. As part of the mesophyll, the spongy parenchyma, similar to an aquiferous tissue, is a common, but no less remarkable, tissue in halophytes and has a fundamental role in the dilution of salts that are absorbed together with water and that can accumulate in levels toxic to the plant (Silva et al. 2020; Larcher 1996).
In the epidermis and mesophyll, we observed an intense reaction pointing to the presence of phenolic compounds in all collection areas. The occurrence of phenolic compounds, including flavonoids and derived phenolic acids, in the epidermal cells and in the palisade parenchyma close to the adaxial and abaxial surfaces may be related to photoprotection of the underlying tissues, guaranteeing their integrity, even under conditions of intense luminosity, as they relieve the photo-oxidative stress and limit the formation of reactive oxygen species (ROS) in chloroplasts or reduce their formation (Zhang et al. 2018).
Histochemical analyses revealed the presence of Zn associated with druses in the parenchymatic tissue of the leaves. According to Silva et al. (2010), most vascular plants store some type of mineralized material, with druses being the most common form. One of the functions of the deposition of calcium oxalate in the leaves, the main substance of the composition of druses, is to maintain a low concentration of Ca in the vicinity of the cells of the stoma. Ca is engaged during the opening of the stomata, and the greater number of druses may be related to several changes in metabolism (Silva et al. 2010), including those caused by heavy metal stress, which explains the large number of druses in histological analyses.