Salinity affects pH and lead availability in two mangrove plant species

Some tropical coastal areas that include mangroves are highly polluted by heavy metals, where lead (Pb) is prevalent. Few studies document how environment affects soil physicochemical characteristics and the availability of heavy metals. This study evaluated how different salt concentrations influenced the accumulation of Pb in plants of Avicennia germinans and Laguncularia racemosa and how salinity modified the substrate pH. Under unsalted conditions, Pb accumulation occurred continuously, mainly in L. racemosa, which was more tolerant to its toxic effects. Salt led to a decreased Pb uptake by the roots and an increase in the substrate pH. In addition, salinity also caused an increase in the translocation of Pb to the leaves. Because L. racemosa was tolerant to Pb, this species could be a better candidate for possible remediation and restoration programs in mangrove areas.


Introduction
Industrial, agricultural and mining activity, in addition to the rise of solid waste and oil spills, have considerably increased the environmental contamination by heavy metals (Du Laing et al 2009. Moreover, because these pollutants cannot be chemically or biologically degraded, they become highly toxic when they accumulate in soil and water, causing poor development and death in living organisms (Páez-Osuna 2005).
Additionally, toxicity of heavy metals depends not only on their concentration but also on their degree of availability, which is determined by the chemical forms in which these elements are found. The speciation of metals depends on changes in the physicochemical parameters of soil and water such as pH, cation exchange capacity, organic matter content, redox conditions, dissolved oxygen concentrations, and lack or excess of nutrients, among others. At the same time, these parameters are influenced by environmental conditions such as hydroperiod, salinity, and temperature (Riba et al 2003, Du Laing et al 2009. Metals with electropositive charges are attracted to surfaces with negative charges given by organic matter or clay, which determines the cation exchange capacity (CEC). A high CEC increases the retention of metal cations in the soil, reducing their metal mobility and availability (Du Laing et al 2009). In addition, pH is an important factor in the availability of metals in the soil. For instance, in acidic soils, where there is a greater release of hydrogen ions (H + ), the competition of the metal cations with these H + can cause the desorption of the metals, which increases their concentration and solubility in the soil solution and their possible absorption by the plant roots (García et al 2002).
In the soil, the fate of heavy metals is given by processes of adsorption/desorption, precipitation/ dissolution, and reincorporation/mineralization, as well as by transport of solutes and extraction by plants (He et al 2005). In saline environments, the concentration of other cations that compete with metal ions for sorption sites increases and metals can then form complexes of high stability and solubility with chloride. Therefore, CEC decreases and metals increase their mobility in the soil. Conversely, metals can also form complexes with other anions (e.g. sulfur), which causes low mobility and availability (Leonard et al 2011). However, the higher the salinity, the more alkaline the soil pH becomes, generating lower metal availability because they precipitate as insoluble hydroxides (Alloway 1995).
Salinity can affect metal ion absorption and transport to the roots. This is because Na + can be absorbed through different channels that compete against other ions (e.g. K + ) for entrance to cells. Conversely, internal transport of heavy metals is more limited and specific, so when competing for entrance to the same channel these are absorbed in lower concentration. This entrance is also influenced by the properties of transport and the concentration of ions in the soil (Li et al 2012, Mei et al 2014. In Mexico, for several decades, the coast of the Gulf of Mexico has been the most impacted by heavy metals, where Pb has a major presence, in the range from 0.28 to 267 μg g −1 dry weight of sediment (Villanueva and Botello 1992, Villanueva and Paéz-Osuna 1996, Botello et al 1999, Medina et al 2004, García 2006, Páez-Osuna and Osuna-Martínez 2011, Vazquez et al 2012, De la Cruz-Landero et al 2013, López 2015. Within these zones are mangroves, which are considered the most productive and diverse natural wetland systems (CONABIO 2013). However, few studies exist on the effect of these metal pollutants on mangrove species, despite that this coastal ecosystem helps to mitigate erosion and stabilize the coast by buffering floods and hurricanes, among other ecosystem services (Ezcurra et al 2009).
Mangrove tree species can accumulate various metals, mainly in their roots (Pulford and Watson 2003, MacFarlane et al 2007, Gabriel and Salmo III 2014, Ochoa et al 2016. However, the influence of environmental conditions on the accumulation capacity of metals for plant species has been marginally studied (Riba et al 2003, Hao et al 2012. A previous study shows that plants of Avicennia germinans and Laguncularia racemosa have a greater capacity for accumulation and tolerance to lead than Rhizophora mangle under low salinity (Cabañas-Mendoza 2014).
Plants of A. germinans and L. racemosa can grow in a large spectrum of salinities, ranging from 0 to 100 ‰ and 0 to 90 ‰, respectively (Wang et al 2011). In addition, these species have specialized structures in the leaves such as glands (A. germinans) or small protuberances on the petioles (L. racemosa) to excrete saline solution, which, when water evaporates, forms crystals on the leaf surface that are wind-dispersed (Sobrado 2001, Francisco et al 2009, Esteban et al 2013. Moreover, leaves accumulate salt as a function of the increase of salinity in the soil Greaves 2000, Sobrado 2004) and, at maturity, they could accumulate salt even in the last stage before their abscission (Cram et al 2002).
The aim of this study was to evaluate how different salinity gradients can cause a change in the pH of the substrate and affect the accumulation of lead in the species A. germinans and L. racemosa. The information obtained can be used as a basis to understand some of the factors that regulate the mobility of heavy metals in saline conditions, such as those where mangroves grow, and identify the responses of the species to lead contamination.

Plant material and growth conditions
Black (Avicennia germinans) and white (Laguncularia racemosa) mangrove propagules (about 400 of each species) were collected in the Ria Celestun Biosphere Reserve (the points of collection were 20.857874, −90.383454, and 20.856782, −90.376828; supplementary figure 1). This is a pristine preserve in Yucatan, Mexico, and our group has been working there for about 10 years and has the required collection permits. Propagules were transported in plastic bags with water from the lagoon to a greenhouse. From the pool of propagules, we selected 240 individuals per species, washed them with distilled water, and placed them individually in black plastic bags (20×10 cm) with silica sand 20/30 and agrolite (1:1). Bags with plants were then set into plastic boxes, so they had a shared medium of water and nutrients. Boxes were located in a greenhouse under an average temperature of 20°C at night and 27°C during the day, an average relative humidity ranging from 55% to 84%, and an average total daily photon flux density of 5.4 mol m −2 . The photon flux density was measured with a light meter (Photosynthetic Light PAR, Smart Sensor, S-LIA-M003, Onset, EEUU), air temperature and relative humidity with a Temp Smart Sensor (S-TMB-M003, Onset). All variables were recorded simultaneously every 10 s and 10-min averages were stored with a data acquisition system (HOBO U30-NRC Weather Station Starter Kit, Onset). Plants received water weekly with the commercial fertilizer Miracle-Gro (1 g l −1 , 200 ml of solution per plant).

Acclimatization of plants to salinity
After 4 months, plants were randomly assigned to different saline treatments, with salt concentrations similar to those where species commonly grow under natural conditions. Each week, 80 plants of each species were watered with 0, 7 and 15 mg l −1 of commercial sea salt, during a period of two months; simultaneously, plants were irrigated with commercial fertilizer (Miracle-Gro; 1 g l −1 , 200 ml of solution per plant).

Lead treatments
After the 2-month period, individual plants were assigned to lead treatments within each salt treatment. For each salt treatment, 20 individuals of each species were irrigated with deionized water prepared with 0, 40, 80 and 160 μM of PbNO 3 . During the experiment, plant leaves were fertilized weekly with a nutritive commercial solution (Miracle-Gro; 1 g l −1 ).

Concentration of lead in tissues
Root, stem and leaf samples were taken at 0, 2, 7, 15 and 30 days after exposure for the Pb quantification (n=4). Samples were rinsed with distilled water to remove the external metal; only leaves of plants that were grown under salinity conditions were not washed to avoid the loss of the salt crystals. The complete tissue or, when appropriate, one gram of dry tissue was placed in crucibles inside a muffle furnace at 600°C until obtaining white ash.
This ash was then diluted in 4 ml of 3 M hydrochloric acid; each sample was adjusted to 6 ml with 1% nitric acid v/v (Horwitz et al 1970, Perkin-Elmer Corporation 1996 to finally be centrifuged at 4000 rpm for 10 min All samples were measured in an atomic absorption spectrometer (model 55 AA, Agilent Technologies, EE. UU.), using a wavelength of 217 nm and a calibration curve of 0, 1, 5, 25, 50 and 100 ppm of PbNO 3 . Results were expressed based on 1 gram of dry weight; for the concentration of Pb in the plant, the total dry weight of each tissue was used.

Growth parameters
After exposure to Pb and for both species, fresh weight, dry weight, and total length of individuals from all treatment were obtained at 0, 2, 7, 15 and 30 days. Total plant length was measured with a measuring tape from the tip of the longest root to the highest leaf, which was generally above the apical meristem of the stem. Total fresh weight was obtained with an analytical balance; subsequently, tissues were placed in a tumble dryer at 50°C for 3 days to obtain the total dry weight. During the period of exposure to Pb, photographic evidence was also taken to register any physical change in the plants.

Physicochemical parameters
Samples of substrate of each plant were taken at a depth of approximately 5 to 7 cm procuring the area of greatest interaction with the roots; substrate was then dried at room temperature before measurements. Electrical conductivity and pH were measured according to the methodologies proposed by USDA (1999) and by NOM-147-SEMARNAT/SSA1-2004 with some modifications. Fifty grams of substrate were placed into a beaker, 50 ml of distilled water were added and solution was stirred for 5 min; samples were then allowed to stand for one minute for the sedimentation of solids and supernatant was placed in plastic tubes for measurement. Parameter measurements were made using the Ysi Pro model 2030 (Professional Series, YSI Incorporated, EE. UU.) with the sensor calibrated for conductivity with a 10,000 μS cm −1 calibration solution, and SM Titrino 702 (Metrohm AG, Switzerland) calibrated with pH solutions of 7 and 4. Measurements were made every 7 or 15 days, before and during the salt acclimation and at 0, 7, 15 and 30 days during lead treatment.

Statistical analysis
Factorial analyses of variance were carried out to compare the means across the factors Pb treatment, salinity treatment, time of exposure, and tissue of each species for the response variables Pb tissue concentration, total fresh weight, total dry weight, total plant length, and substrate pH. Tukey comparison tests were performed to identify treatment differences. In addition, simple linear regression analyses were applied to explore the relationship between soil physicochemical parameters as well as the relationship of Pb concentration with the other variables analyzed. All analyses were determined at a confidence level of 95% (p<0.05) using the statistical package R version 3.5.1 for Windows.

Concentration of lead in tissues
Tissues of L. racemosa had the highest Pb concentrations in all salt treatment, compared to tissues of A. germinans (p<0.05; tables 1-3, Table S1 is available online at stacks.iop.org/ERC/2/061004/mmedia). For both species, roots were the tissues that accumulated the highest Pb concentrations.
In the absence of salt (table 1), in both A. germinans and L. racemosa Pb uptake by the roots increased significantly with time, as the Pb concentration in the substrate increased. Lead translocated to the leaf was accumulated in proportions similar to those found in the stem, but leaf showed significant increases and the highest Pb concentration at the end of the experiment in both species.
Salinity significantly affected the Pb uptake capacity of both species. Accumulation of Pb by roots decreased and translocation of Pb to the leaves increased (highest values in A. germinans at 7 mg l −1 of salt and in L. racemosa at 15 mg l −1 of salt); the translocation to the stem was similar in all treatments (tables 2 and 3).
During the first 15 days, the Pb concentration in the roots for plants of A. germinans and L. racemosa growing under 7 mg l −1 of salt increased as Pb concentration in the medium increased (table 2); however, at the end of the experiment Table 1. Concentration of endogenous lead (μg g −1 dry weight) found in plants exposed to different concentrations of lead, grown in the absence of salt (0 mg l −1 ) during a 30-day exposure period. Data are means±standard error. Different lowercase letters indicate significant differences among Pb treatments (columns) for each day of exposure, for each tissue and for each species (n = 12). Different capital letters indicate significant differences for comparisons among tissue/species (bold, n = 288), and among exposure days (rows) for each tissue and for each species (roman for A. germinans and italicized for L. racemosa, n = 48). Different lowercase letters indicate significant differences among Pb treatments (columns) for each day of exposure, for each tissue and for each species (n = 12). Different capital letters indicate significant differences for comparisons among tissue/species (bold, n = 288), and among exposure days (rows) for each tissue and for each species (roman for A. germinans and italicized for L. racemosa, n = 48).

Avicennia germinans
the tissue concentration was maintained or even decreased. For both species, the translocation of Pb to the stem was similar among the different treatments and days of exposure. The Pb accumulation in leaves for A. germinans was significantly greater as the time of exposure passed, but it was similar among Pb treatments. At the end of the experiment, in L. racemosa the leaf Pb concentration values increased significantly.
Plants of A. germinans and L. racemosa under 15 mg l −1 of salt had a greater root Pb accumulation than at low salinity (table 3). For L. racemosa, Pb accumulation increased significantly as Pb treatment concentration increased. For A. germinans this also occurred, but after day 15 Pb root concentration decreased. Only L. racemosa had the highest Pb concentrations in the leaf, which was greater than those found in the stem.

Effect of Pb accumulation on plant morphology
Although L. racemosa showed the highest Pb accumulation in all treatments, there were no visible symptoms of toxicity in any of the individuals analyzed even under high concentrations of Pb and at the end of the experiment (figure 1). Only some individuals of A. germinans that grew with or without salt showed leaf yellowing and wilting from day 7, but most damage occurred at 15 and 30 days of exposure, and at the highest Pb concentration (figure 1). In both species, salt crystals in leaves were observed in plants under halophytic conditions because of the mechanism of excretion characteristic of these species.

Effect of Pb and salinity on plant growth
The species with the highest weight and length was A. germinans, but L. racemosa values varied less with salinity than those of A. germinans at the end of the experiment (table 4). In both species, although some plants exposed Table 3. Concentration of endogenous lead (μg g −1 dry weight) found in plants exposed to different concentrations of lead, grown under 15 mg l −1 of salt during a 30-day exposure period. Data are means±standard error. Different lowercase letters indicate significant differences among Pb treatments (columns) for each day of exposure, for each tissue and for each species (n = 12). Different capital letters indicate significant differences for comparisons among tissue/species (bold, n = 288), and among exposure days (rows) for each tissue and for each species (roman for A. germinans and italicized for L. racemosa, n = 48). to 15 mg l −1 of salt had a lower weight and length, there was no significant correlation between the growth variables studied and the electrical conductivity values of the substrate (R 2 <0.1), so that salinity did not influence growth. For A. germinans, the higher the Pb concentration the lower the biomass and length for the plants. In all salt treatments, the ratio of length and dry weight with respect to the total accumulation of Pb in the plant was negative (figures 2 and 3(a)-(f)); but, only in plants that were kept under 7 mg l −1 of salt was the correlation significant. In addition, some decreasing tendencies in the growth values were observed for L. racemosa, but none was significant.

Avicennia germinans
In addition, for the treatments of 0 and 15 mg l −1 of salt in L. racemosa, most of the obtained correlations between Pb tissue concentration and plant biomass or length were positive, but not significant. Larger plants of this species accumulated higher Pb concentrations but toxic effect in this species was not visible (figures 2 and 3). However, in both species, it appears that small-sized plants tend to accumulate lower Pb concentrations (∼10-40 ug g −1 in treatments with 40 and 80 μM of Pb).

Effect of salinity on pH and its relation to Pb uptake
In both species, as expected, electrical conductivity (EC) was proportional to the amount of salt in the substrate. Plants grown without salt presented the lowest EC values, which decreased significantly in the last weeks of the experiment (figures 4(a) and (c)). With 7 and 15 mg l −1 of salt, EC increased drastically and gradually; when irrigating with salt and Pb, EC values first decreased and later gradually increased again. In both species and for both treatments with salt, EC values of all weeks of measurement differed statistically.
Substrate pH for plants of A. germinans growing without salt maintained an average range of 4.9 to 5.8, being the lowest among all treatments ( figure 4(b), table S1). For the salt treatments, pH increased gradually and significantly four weeks after starting the salt irrigation and stabilizing at the end of the experiment. Plants of L. racemosa that grew without salt initially had a higher pH (6) than plants of A. germinans, but gradually decreased until the end of the experiment (average range of 5.4 to 6.1, figure 4(d)). Under salinity conditions, the Different lowercase letters indicate significant differences for comparisons among Pb treatments for each parameter within each salinity and each species (n = 80). Different capital letters indicate significant differences for comparison among salinity treatments (underlined for fresh weight, italicized for dry weight and bold for plant length) for each species (n = 240).
former species grew in a less acidic environment compared to A. germinans, since some plants showed point values of pH7 when the plants were acclimated to 15 mg l −1 of salt. In both species, the relationship between EC and pH was significant showing that an increase in the salinity of the substrate can increase the pH; although, the correlation was low (R 2 =0.26 for A. germinans and R 2 =0.21 for L. racemosa; figure 5). Also, EC and pH were related to the roots Pb concentration as well as the EC with the leaf Pb concentration (figures 6 and 7).
For A. germinans, an increase in EC and pH, as well as a longer exposure time to the metal, leads to a lower accumulation of Pb in the roots. Also, L. racemosa had a negative relationship between EC and pH, which increase the first 15 days of exposure. However, correlation values were lower compared to those of A. germinans. Furthermore, for both species, the higher the salinity and longer the time of exposure, the higher accumulation of Pb in the leaf was, especially for L. racemosa at day 30.

Discussion
We found that Avicennia germinans and Laguncularia racemosa accumulated Pb, mainly in the roots, and translocate it in low concentrations towards the aerial parts (phyto-stabilization), which has already been reported for other mangrove species (Nirmal Kumar et al 2011, Wang et al 2013, Cabañas-Mendoza 2014Pérez-Sirvent et al 2017, Qiu and Qiu 2017. This mechanism is common in tree species because it allows them to be more tolerant to the toxic effects (Diez 2008, Trejo-Calzada et al 2015, Salas-Luévano et al 2017. Under no salinity conditions, Pb accumulation tends to be higher when the level of contamination and the time of exposure to the Pb is higher.
In all Pb treatments, L. racemosa not only had the highest accumulation capacity but also the highest tolerance without showing any visible damage; both biomass and length did not change for this species, even though some plants accumulated high Pb concentrations. Our results showed that A. germinans could have a greater vulnerability to the toxic effects of Pb as time of exposure passed.
We corroborated that pH increased in the substrate at higher salinities, but the relationship between these two variables was low. Few studies have shown the influence of salinity on pH (Saraswat et al 2015). More experiments with longer residence times and higher salt concentrations in the soil are required to show an evident relationship. Besides, other environmental variables can also influence changes in pH, such as temperature which, when increases, causes a decrease in pH (Dotro et al 1994).
At higher salt concentrations there is an increase in the uptake of Cd, Zn and Pb in some plants, due to competition with other cations (Na + , K + , Ca 2+ , Mg 2+ ) for sorption sites. This could increase the formation of complexes with chloride anions, causing desorption of metals and their mobility (Fritioff et al 2005, Gharaibeh et al 2015. Additionally, some studies on sediments and different plant species show that higher salinity leads to a decrease in the mobilization and accumulation of Ni, Cu, Zn, Cd and Pb in plant tissues (Smolders and McLaughlin 1996, Riba et al 2003, Fritioff et al 2005, Leblebici et al 2011. These last studies support our results, because we found that salinity caused a lower accumulation of Pb in the root. In addition, in this study, the greater the exposure period, a decrease in Pb availability of the metal occurred, mainly in A. germinans. This can occur because metals can also bind to other anions forming less soluble complexes that can limit the absorption of the metal through coagulation, precipitation and flocculation (Smolders and McLaughlin 1996). Few studies mention the influence of environmental variables on the uptake of metals for mangrove species (Dai et al 2017, Pittarello et al 2018. For instance, Cheng et al (2012) reported that an increase in salinity reduces the loss of radial oxygen, which stimulates the lignification of the exodermis, and alters the permeability of the root, influencing a lesser accumulation of metals (Pb, Zn and Cu) at low salinity. Our results agreed with these findings, since at low salinity in the substrate there was the lowest Pb tissue accumulation.  Nevertheless, some studies show that the transformation of free ion metals to complexes, and their low availability, is not the only reason why metal accumulation in plants decreases in the presence of salt. Mei et al (2014) show that a blockage in the Ca 2+ channels significantly decreases the absorption of Na + , K + , Ca 2+ , Mg 2+ and Cd 2+ by the root of amaranth plants grown under different concentrations of NaCl. This study also reports that a blockage of the K + channels diminishes the uptake of Na + , and K + but not of Ca 2+ , Mg 2+ , and Cd 2+ , which suggests competition between the Na + and Cd 2+ for passage through the channels of Ca 2+ . This affinity of Na + for the sites of sorption in the walls of the root cells displaces other ions and, consequently, results in a low accumulation of the metal in the roots. These findings can help explain the decrease of metal in mangrove roots, which can lead to more studies on the specificity of the transporters in the accumulation of metals in mangroves.
Our results showed that the accumulation of Pb in roots was associated with pH changes; and that the decrease in the availability of Pb in the roots is more evident at longer time of exposure to the metal. A study also reports that the accumulation of metals in plants is more effective when the pH is low, since acidification releases metals associated with sediments (Riba et al 2003). But further studies are needed to know how pH increase of the substrate affects Pb availability. An interesting finding in our study was that salinity also influences a greater translocation of Pb to the leaves. This can occur because these species excrete salt through their leaves. Burchett (1999, 2000) show that the mangrove species Avicennia marina, treated with Zn and Cu, has an increase in these metals in the leaf and in the salt crystals on leaf surfaces, suggesting a strategy of the plant to deal with excess micronutrients by eliminating them through the glandular trichomes of the epidermis. This could help explain the lowest root Pb concentration values at the end of the experiment for mangrove plants in the low salinity treatment.
Soils in mangrove ecosystems, in addition to having a wide salinity gradient, are also flooded either temporarily or permanently and are rich in organic matter, which influences changes in pH and other physicochemical variables, such as processes of oxide reduction (Lugo andMedina 2014, Naidoo 2016). Plants of A. germinans and L. racemosa can co-exist in basins or semi-flooded areas, but when salinity is high, A. germinans dominates, whereas when salinity is low the dominant species can be L. racemosa (Reese 2009, Zaldivar-Jiménez et al 2010. Both species are distributed within bands or plots along the Gulf of Mexico and the Pacific coast (López-Portillo and Ezcurra 2002). Based on our findings, L. racemosa has several advantages that would allow it to be a better candidate for the remediation of Pb in these ecosystems. However, more studies under higher salinities need to be done to show the potential of A. germinans. Figure 7. Relationships between tissue Pb concentration (root and leaves of Laguncularia racemosa) and substrate electrical conductivity (EC) and pH, for the three salinity treatments at 2, 7, 15 and 30 d for Pb treatments of 40 μM ( ), 80 μM ( ) y 160 μM ( ).

Conclusion
Salinity, and the changes caused in pH, influenced the availability, accumulation and translocation of Pb in A. germinans and L. racemosa, which was more evident under prolonged periods of time. Nevertheless, L. racemosa not only had a greater capacity for Pb accumulation and tolerance, but this species also occurs in conditions where the availability of Pb discharges may be greater, which makes it more efficient for phytoremediation. Additionally, A. germinans can be more efficient for phytoremediation in places with low pollution. The contamination by heavy metals in coastal lagoons of Mexico is worrisome so it is urgent to carry out more studies on how these species, and other mangrove species, can coexist and cope with these pollutants and if this could somehow help heavy metal pollution remediation.