Effects of Schoenoplectus americanus (Pers.) Volkart ex Schinz & R.Keller and Phragmites australis (Cav.) on the water quality improvement of moderately saline wastewater

Effects of Schoenoplectus americanus (Pers.) Volkart ex Schinz & R.Keller and Phragmites australis (Cav.) on the water quality improvement of moderately saline wastewater | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Schoenoplectus americanus (Pers.) Volkart ex Schinz & R.Keller and Phragmites australis (Cav.) on the water quality improvement of moderately saline wastewater Sarahí Josefina Estrada-Loredo, Rodolfo Cisneros-Almazan, Gerson Alonso Soto-Peña, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5823958/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Wetlands → Version 1 posted 5 You are reading this latest preprint version Abstract Schoenoplectus americanus and Phragmites australis have a great potential for phytoremediation. In this study, the ability of these plants to improve the quality of moderately saline wastewater was tested. Both species were adapted to wastewater using two protocols. In the first, plants were directly exposed to undiluted or diluted wastewater at 12.5%, 25% and 50%. In the second protocol, the plants were gradually acclimated to 12.5%, and then to 25%, 50% diluted and undiluted wastewater for 20 days. Both processes were performed without using substrates. The efficiency of salt removal was assessed by employing plants adapted to undiluted wastewater over a period of 6 months. Direct exposure of S. americanus to wastewater resulted in a 50% reduction in stem height in undiluted wastewater and an arrest of root development in 25%, 50% and 100% wastewater. An exudation of salts was observed in the stem in undiluted wastewater. Shoot formation was not significantly affected. Progressive exposure to wastewater improved stem length by 23% and shoot formation by 13% in 12.5% diluted wastewater. Direct and progressive exposure of Phragmites australis to wastewater did not affect stem development, and increased the number of shoots (24–30%). Root growth reduction was observed during direct exposure to wastewater. Both species improved wastewater quality by reducing 0.8 units pH, as well as the concentration of nitrite (98%), nitrate (50%-90%) and orthophosphate (50%-90%) after 21 days. Therefore, S. americanus and P. australis are a viable option for treating moderately saline wastewater. wastewater stabilization ponds Schoenoplectus americanus Phragmites australis salt removal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction According to the WHO (2023), approximately 771 million people did not have access to safe drinking water, and at least 1.7 billion people worldwide drank water contaminated with fecal matter. This issue is partly due to the fact that 80% of wastewater is discharged without being treated into water bodies (WWAP 2019). The consumption of contaminated water is responsible for one-third of morbidity and mortality in developing countries (Kumar et al. 2021 ). Furthermore, extreme weather events caused by climate change, also affect freshwater ecosystems and consequently their availability for human consumption (Lee et al. 2020 ). Recently, wastewater reuse has become increasingly important and is considered one of the most viable alternatives for addressing climate change and contributing to water resources management (Biswas et al. 2023 ). The contaminants of concern in wastewater include pathogens, and dissolved organic and inorganic matter. To avoid health and the environment risks, the quality of the effluent from the treatment plant must meet specific standards for use in agriculture, horticulture, groundwater recharge, augmentation of riverbeds or natural water bodies, industries and street washing. The most common parameters analyzed in wastewater, besides the pathogen level, include total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN), nitrites and nitrates, chloride and phosphate (Preisner et al. 2020 ). In developing countries, water pollution and drinking water scarcity represent major challenges. These countries have made significant efforts to improve water supply and promote water reuse (Bijekar et al. 2022 ). In India, the range of implemented systems encompasses constructed wetlands and upflow anaerobic sludge blankets. In Africa, duckweed pond systems and septic tanks are employed (Muzioreva et al. 2022 ). Nepal and Indonesia have adopted baffled reactors (Yulistyorini et al. 2019 ), while constructed wetlands are a prevalent wastewater treatment system in South America and Mexico (Ferreira et al. 2021 ). According to Cáñez-Cota ( 2022 ), Mexico had 3,516 municipal wastewater treatment plants in 2018, 72% of which are currently operational. The remaining 980 are inactive due to the high energy costs that have not been covered because of the communities' low or very low marginalization. In addition, most of these wastewater treatment plants are in arid and semi-arid regions characterized by low rainfall and the overexploitation of wells. Macrophytes play an important role in wetland ecosystems because they can remove significant amounts of nutrients from domestic wastewater. Therefore, they represent a technological alternative for rural areas in Mexico or other developing countries. Macrophyte roots act as filters, biodegrading significant amounts of organic matter and promoting nutrient uptake while reducing solar radiation penetration, thereby suppressing algal growth (Huth et al., 2021 ). Two macrophytes with an excellent potential for restoring water bodies are Schoenoplectus americanus (synonym Scirpus americanus ) and Phragmites australis. S. americanus is native to North America, and can grow both in wet or dry environments. It has a very vigorous radical system, and stems from 50 to 220 cm tall. This species can tolerate and remove high concentrations of lead from water (Esquivel-Ramos et al. 2024 ), chromium and total phenols from the tannery industry (Quevedo et al. 2024 ) and is also salt tolerant. Howard and Mendelssohn ( 1999 ) described that biomass and stem density were not affected in plants exposed to 6 and 12 g/L NaCl. Plant height continued to increase in both treatments and did not reach the senescence stage, unlike the oligohaline marshes Panicum hemitomon and Sagittaria lancifolia , which showed reduced root and aerial tissue growth. In addition, S. americanus was approximately twice as salt-tolerant as Typha domingensis . It exhibited a 50% relative growth rate in 9 g/L total dissolved solids (TDS), while T. domingensis did so at 4 g/L TDS (Baeza et al. 2013 ). P. australis is an herbaceous plant that grows in temperate and tropical climates. The plant can reach 4 m in height, is able to remove and accumulate emerging pollutants (Lei et al. 2022 ) and is salt tolerant. Haplotype M has the highest tolerance, showing only a 50% reduction in growth at a concentration of 0.4 M NaCl (Vasquez et al. 2006 ). Salt tolerance has been associated with cation adjustment and water loss. Cation adjustment maintain K + /Na + ratios in leaf laminas, while water loss concentrates solutes in the cell sap, thus reducing the necessity for the synthesis of osmolytes, and the Na + uptake for osmotic adjustment (Lissner et al. 1999 ). Another factor that contributes to the removal of pollutants in constructed wetlands is the substrate used to fill the ponds and provide support for plants. Pumice, perlite, zeolite, and sand have been described as absorbing nitrate, sulfate, and phosphate (Mehrani et al. 2017 ; Ghasemi et al. 2020 ; De Rozari et al. 2020 ). Pollutant removal occurs through physical sedimentation, filtration, sorption in the substrate matrix, ion exchange, gas diffusion, and bio-immobilization in the substrate (Wang et al. 2020 ). In the municipality of Salinas de Hidalgo, in the state of San Luis Potosí, in Mexico (101°42'15.6" west longitude and 22°38'26.4" north latitude) is located the so-called salty valley because of the high salt content in the soil and water. In this area, there is an abandoned stabilization pond (SP) from a water treatment plant. The water in the pond is of low quality, has high salinity, significant organic matter, and possible microbial contamination (Fig. 1 ). The SP receives the city's domestic and industrial wastewater, and given its proximity to the Salinas Lagoon, the SP's wastewater typically has high salt concentrations. The inclusion of artificial wetlands could be a technological alternative for improving the quality of the Salinas wastewater plant. Therefore, this study focused on the ability of S. americanus and P. australis plants to remove salts from wastewater plant. Considering that these emergent macrophytes have more supporting tissues than floating macrophytes, they may have greater potential for storing salt for longer periods of time. We were interested in comparing the response of S. americanus regenerated in vitro , which showed robust growth and had not experienced any stress, with those of P. australis , which had been exposed to environmental salinity conditions. On the other hand, it is known that plant tolerance to salt can be increased by prior exposure to low salt concentrations for a short period of time (Pandolfi et al. 2016 ; Kamanga et al. 2020 ). Therefore, S. americanus and P. australis were directly and progressively exposed to wastewater to analyze if there were differences in the growth and the adaptation process. This study was performed without substrates, in order to specifically understand the role of plants in the removal of salts and major pollutants from moderately saline wastewater. This research should contribute to the development of sustainable and scalable phytoremediation strategies for rural communities in semi-arid regions. 2. Material and methods S. americanus in vitro cultures The S. americanus plants were obtained from in vitro germinated seeds in Murashige and Skoog basal medium, MS (Murashige and Skoog 1962 ) supplemented with 116 µM myo-inositol, 1.2 µM thiamine-HCl, 30 g/L sucrose, and 8.4 mg/L agar, pH 5.7. The in vitro seedlings were sectioned and propagated in MS medium supplemented with 0.5 mg/L benzyladenine (Alfaro-Saldaña et al. 2016 ). The plants were cultivated in solid MS medium until they reached a height greater than 8 cm. Subsequently, they were transferred to 500-ml vessels containing 100 ml of liquid MS medium (without agar) to increase the growth rate, and then underwent constant shaking at 135 rpm, in an orbital shaker. In vitro cultures were maintained within a photoperiod of 16 hours of light and 8 hours of darkness at 25°C in a growth chamber. Establishment of hydroponic cultures of S. americanus and P. australis The in vitro S. americanus plants (15 cm tall) were collected, and their roots were washed with sterile deionized water to remove the medium. The plants were placed in 1-L containers with 400 ml of commercial hydroponic medium (1 g/L) (Hydro Environment, Mexico), pH 5.7. The plants were covered with a plastic bag for 15 days to improve their adaptation to the environment. Subsequently, the plants were transferred to plastic vessels with 10 L of hydroponic medium supplemented with 330 µl of Aquapool algaecide (Aris Industrial, Peru). The surface of the vessels was covered with a styrofoam plate and the hydroponic medium was changed weekly. The plants were maintained under greenhouse conditions until they reached a height of 50 cm. Entire P. australis plants taller than 40 cm and with abundant roots were collected from the banks of the Santiago River (22°10'52.9" N 100°56'09.0" W) in San Luis Potosí. The plants were carefully washed with soap to eliminate soil and some microbials, and then rinsed with water. The plants were placed in 60-L containers with 20 L of commercial hydroponic medium (2 g/L) for 4 weeks to obtain new shoots. New shoots taller than 15 cm, with a diameter greater than 2 cm and defined roots, were cut and transferred to basins containing 10 L of hydroponic medium. The surface of the basin was covered with a styrofoam plate. The plants were maintained in greenhouse conditions with the hydroponic medium being replaced once a week. Physicochemical analysis of the wastewater Water samples were collected from the surface of the stabilization ponds at the Salinas Municipality Water Treatment Plant (22°38'26.4" N, 101°42'15.6" W), placed in glass containers, and stored at 4°C until analysis. The temperature, total suspended solids and electrical conductivity (EC, µs/cm) were measured immediately by using a HI98130 portable conductivity meter (Hanna Instruments, USA). Upon arrival at the laboratory, the pH was measured with the UltraBasic Benchtop pH meter. The TSS value (mg/L) was verified again at 810 nm by using a DR 2800 spectrophotometer. The depth and coordinates of water collection, as well as the date and time of sampling, were also registered. The physicochemical analysis of the wastewater was performed in triplicate. Turbidity measurements were performed using a HACH 2100Q turbidimeter at 850 nm. The resulting values were reported as nephelometric turbidity units (NTU) according to Official Mexican Standard NMX-AA-038-SCFI-2001. The chemical oxygen demand (COD) was analyzed according to Official Mexican Standard NMX-AA-030/1-SCFI-2012 using a thermal reactor (HACH DRB200) with a confidence interval of 15 to 300 mg/L. If the value exceeds 300 mg/L, the sample is diluted until the values fall within the measuring range. The total nitrogen was measured using the Spectroquant® 114763 kit. The confidence interval was 10 to 150 mg/L TN. Nitrite was measured according to the Official Mexican Standard NMX-AA-099-SCFI-2021. The nitrite concentration was calculated from the calibration curve (0.05 mg/L, 0.10 mg/L, 0.15 mg/L, 0.20 mg/L, 0.25 mg/L and 0.30 mg/L) using NaNO 2 as a standard. Nitrates were analyzed according to the protocol described by Gentle et al. ( 2011 ). The nitrate concentration was calculated from the calibration curve constructed with KNO 3 at concentrations of 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, and 7 mg/L. Orthophosphates were determined using the Spectroquant® 114848 test kit with a confidence interval of 0.05 to 5 mg/L. The physicochemical parameters of initial collected water samples were: temperature 29.80°C; pH 7.89; EC 7,520 µS/cm; TSS 3,740 mg/L; COD 2,780 mg/L; TN 71.50 mg/L; 23.10 mg/L nitrites; 30.80 mg/L nitrates; 388.5 NTU of turbidity; and 3.95 mg/L orthophosphates. Direct adaptation of S. americanus and P. australis to wastewater Twenty S. americanus and P. australis plants taller than 50 cm were used for this experiment. The plants were rinsed with water, pruned to 50 cm to ensure that the plants were of uniform size and to assess the extent of increase in stem length over time. The pruning of macrophytes has been reported to improve nutrient uptake (Herzog et al. 2021 ). Four plants of each species were placed in 10 L basins containing 9 L of 12.5%, 25%, 50% or 100% wastewater; dilutions were performed by using deionized water. In addition, two basins containing plants of either species in commercial hydroponic medium were used as controls. The basins were covered by a styrofoam plate and the plants were maintained for 20 days under greenhouse conditions. The water lost by evapotranspiration was replaced by deionized water every 4 days. Stem length, and the number of shoots and leaves were measured every 4 days. Root length was measured at day 1 and 20. Every 4 days, 50 mL of wastewater was collected and the pH, EC and TSS were determined. Progressive adaptation of S. americanus and P. australis to wastewater Twelve plants of S. americanus or P. australis pruned to a height of 50 cm were used. Three plants of each species were placed in four different 10-L basins containing 9 L of 12.5% effluent. Dilutions were performed using deionized water. The plants remained under these conditions for 20 days and were then transferred to basins containing 9 L of 25% wastewater. After 20 days, the plants were exposed to 50% of wastewater for 20 days, and finally to 100% of the wastewater for another 20 days. The water lost by evapotranspiration was replaced with deionized water. Stem growth and the number of new shoots were measured at 4-day intervals, as were water parameters. Root length was measured only at day 0 and 20 to minimize plant disturbance. Effect of S. americanus and P. australis on water quality in stabilization ponds. In this experiment, the plants (n = 12) were cultivated in undiluted wastewater over a period of 6 months. The objective of this experiment was twofold: firstly, to ascertain the ability of the plants to survive in these conditions over an extended period, and secondly, to verify that the mechanisms of tolerance to salt were already active. Four plants of each species (pruned to a 50 cm height) were then placed in three different basins with 9 L of wastewater. Three basins with wastewater and no plants were included as controls. The increase in stem length and root length, and the number of shoots was measured as described in the previous paragraph. For the water quality analysis, 100 mL of wastewater was collected from each tank every 7 days, and the temperature, pH, TSS, COD, NT, nitrites, nitrates, turbidity, EC and orthophosphates were measured. Statistical analysis The height, number of shoots, number of leaves, and water analysis were evaluated using a one-way analysis of variance (ANOVA) to determine whether significant differences existed between groups. Post-hoc comparisons of means were performed using the Tukey test and the significance level was set at 𝑝 < 0.05. Statistical analyses were performed using Statistica 10 software (StatSoft). 3. Results and discussion In vitro regenerated plants of S. americanus successfully adapted to environmental conditions. After 6 months, vigorous and healthy plants with extensive root were obtained in hydroponic media. Variations in development patterns were observed, with 80% of the plants producing between 0 and 10 new shoots, 13.3% between 11 and 20 new shoots, and 6.7% between 21 and 30 new shoots. Regarding stem length, 76.7% of the plants developed stems < 50 cm, 20.0% from 51 to 100 cm and 3.3% from 101 to 150 cm. These differences in development could be due to somaclonal variation induced by in vitro culture (Bairu et al. 2011 ). P. australis required 3 months to obtain vigorous plants in a hydroponic medium. The plants also showed differences in growth patterns. About 50% of the plants produced between 0 and 10 new shoots, 30% between 11 and 20 new shoots, and 20% between 21 and 30 new shoots. The total number of plants had a stem length ≤ 50 cm (data not shown). It has been posited that epigenetic changes, such as DNA methylation, may underlie variations in growth patterns. These variations are more closely associated with microhabitat than with genotype within subspecies (Spens and Douhovnikoff 2016 ) (data not shown). Direct adaptation of S. americanus and P. australis to wastewater S. americanus plants maintained in hydroponic medium (control) and 12.5% wastewater showed an increase in root length of 5 cm and 0.75 cm, respectively. Root loss was observed in 25%, 50%, and 100% wastewater (Fig. 2 a) with no statistical differences (p > 0.05) among treatments. P. australis presented a similar behavior (Fig. 2 b) with an increase in root length of 5.8 and 2.75 cm in the control and 12.5% of residual water, respectively. Furthermore, there was root loss in 25%, 50% and 100% of wastewater, which statistically differed from the control (p > 0.05). These results indicate that direct exposure of S. americanus and P. australis plants to wastewater affected root metabolism, since a significant loss of this tissue was observed in 25%, 50% and 100% wastewater. The inhibition of root growth was also observed in Tartary buckwheat (Zhang et al. 2023 ), tomato, carrot (Jahan et al. 2019 ) and avocado plants (Bernstein et al. 2004 ) exposed to excess salt. The root damage induced by a high level of salinity in S. americanus and P. australis , can be due to ionic stress, osmotic stress, and/or oxidative stress. Ionic stress promotes ion imbalance in the plant. For instance, Na + inhibits the uptake of NH 4 + , K + and PO 4 3− , causing nutrient stress in the plant. In Arabidopsis thaliana , NaCl, KCl, and LiCl reduced root hair density and elongation (Wang et al. 2008 ). Osmotic stress accelerated suberization in the endodermis and exodermis of roots of Zea mays seedlings, significantly decreased hydrostatic hydraulic conductivity, and reduced water and nutrient uptake (Shen et al. 2015 ). The oxidative stress increased mitochondrial ultrastructural damage and reactive oxygen species (ROS) generation in wheat seedling roots (Kononenko et al., 2020 ). Furthermore, it promoted electrolyte leakage, as well as malondialdehyde and hydrogen peroxide production in the stem and roots of Lepidium draba ecotypes (Jamshidi et al. 2020). Regarding the aerial parts, the stem length of S. americanus on day 20 increased between 42 and 60 cm in the control, and in 12.5%, 25% and 50% wastewater, while the increase in 100% wastewater was only 25 cm at the end of the experiment (Fig. 2 c). This difference was statistically significant (p = 0.042). Additionally, salt exudation and stem stiffness were observed in 100% wastewater. The production of shoots ranged between 14 and 17 new shoots in 50% and 100% wastewater, and this number decreased in more diluted wastewater and the control (Fig. 2 e). The data suggest that salts absorbed by the roots of S. americanus were transported and accumulated in the stem. At high salinities, the salt was exuded from this tissue, possibly through excretory glands, as a defense mechanism. To our knowledge, this is the first report describing salt exudation in this species. X-ray microanalysis of maize exposed to 100 mM NaCl showed that salts enter the apoplast, chloroplast, cytosol, and vacuoles almost simultaneously. When vacuoles become saturated, salts are released (Zhao et al. 2010 ). During salt secretion, the ions are transported by small vesicles, which then fuse with the plasma membrane and release the salts. The salt passes through the apical cell wall and cuticle to the environment (Lu et al. 2021 ). P. australis plants exposed to different wastewater dilutions increased stem length (20 to 38 cm) as a function of time, with no significant statistical differences between treatments on day 20 (Fig. 2 d). The lowest growth was observed in the control (15 cm) and was statistically different from 50% wastewater (p < 0.05). P. australis plants produced the highest number of new shoots (27) in 50% wastewater, but in the control and in 12.5%, 25% and 100% wastewater the number of new shoots decreased (14 to 17) (Fig. 2 f). The production of new shoots and the increase in stem length in S. americanus and P. australis could be related to the level of organic matter, as previously described in Pennisetum purpureum , Medicago sativa , Sinapis alba , and Helianthus annuus (Matheyarasu et al. 2016 ). Presumably, the 50% effluent would provide plants with a significant amount of nutrients, but the deleterious effects of salts could be moderate. Conversely, plants exposed to undiluted effluent would have higher levels of organic matter, but would also be exposed to toxic concentrations of salts. In addition, it has been reported that the response to salt may exhibit a bell-shaped dose-response curve. For example, in tomato seedlings, low salt concentrations (25 and 50 mM NaCl) positively affected shoot length, whereas high concentrations (100 to 200 mM NaCl) significantly reduced shoot growth (Rivera et al. 2022 ). Generally, roots were more affected by salinity than aerial parts in both species because they were in direct contact with the wastewater and more exposed to salts. Roots could act as a barrier to reduce salt internalization and decreased its toxicity in aerial parts. After exposure to wastewater, S. americanus plants produced longer stems, while P. australis generated more shoots. This response can be attributed to their intrinsic morphological characteristics. P. australis is a perennial plant that develops an increasing number of shoots during the growing season, whereas S. americanus usually has only a few short leaves, up to about 10 cm long, but has strong and large stems. These results agree with Vasquez et al. ( 2006 ), who reported that salinity conditions induced more shoots per gram of rhizome tissue in P. australis. The analysis of wastewater collected during this experiment is shown in the Online Resource 1. The pH remained between 7 and 8.2, while the water temperature correlated with the maximum temperature. TSS values in undiluted water ranged between 2640 to 3750 ppm. The EC observed in 100% wastewater was 5174 µS/cm- 7470 µS/c, indicating that water has a high concentration of dissolved ions and salt corresponding to moderately saline water (Hillel 2000 ). Adaptation of S. americanus and P. australis using serial dilution S. americanus plants gradually adapted to wastewater exhibited minimal root loss (Fig. 3 a). Stem length increased by 22% in wastewater at 12.5% compared with the control group (p < 0.05), decreasing proportionally with wastewater concentration. This reduction was statistically different from the control (p < 0.05). The number of new shoots ranged between 4.2 and 6.4 with significant differences between the control and 12.%, and 12.5% an undiluted wastewater. P. australis showed no root loss and a slight growth of this tissue was observed even at 100% effluent (Fig. 3 b). Stem length was similar at all effluent concentrations tested, but lower in the control treatment (p > 0.05). The number of shoots increased progressively up to 25% effluent and then remained constant at approximately 26 new shoots. In summary, S. americanus mainly developed longer stems while P. australis generated new shoots. The analysis of the effluent collected during this experiment is shown in Online Resource 2. These results suggest that plants exposed to wastewater through a serial adaptation process developed more efficient tolerance mechanisms that allow better growth. The most obvious change was the approximately 2.8-times increase in stem length in S. americanus , and the 3-times increase in new shoot formation in P. australis during progressive exposure to wastewater as compared with direct exposure. It has been documented that prior exposure to low salt concentrations can enhance survival and growth rates. For instance, pre-treatment of Zea mays plants with 25 mM NaCl enhanced the intracellular K/Na ratio, and increased the sequestration of sodium in vacuoles (Pandolfi et al. 2016 ). In olive plants a reduction in salt-induced ultrastructural changes in leaf cells was observed (Pandolfi et al. 2017 ). In pea an improved photosynthetic rate, stomatal conductance, and chlorophyll content were reported (Shaukat et al. 2019 ); in tomato seedlings accumulation of proline, activation of antioxidant enzymes, and a reduction in H 2 O 2 were observed (Kamanga et al. 2020 ). Several mechanisms involved in the physiological adaptation could be participating. P. australis has been classified as a salt excluder, which allows it to reduce Na + transport to shoots and ameliorate salt toxicity. It uses K + for osmotic adjustment in leaves and also accumulates osmoprotectants such as proline and glutamine (Vasquez et al. 2006 ; Hartzendorf and Rolletschek 2001 ; Xie et al. 2020 ). In S. americanus , a 5-fold increase in glycine betaine levels was documented after exposure to 20 ppt salinity (Ewing et al. 1988). Proline and glycine betaine play an important role in scavenging ROS and counteracting NaCl-induced oxidative stress. They also improve hydration and prevent dehydration and protein denaturation. The rapid catabolism of proline provides reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP for recovering from stress and repairing of stress-induced damage (Xie et al. 2020 ). Another observation is that in vitro regenerated S. americanus plants previously unexposed to salt could survive and grow in the direct and progressive adaptation experiments, as well as P. australis derived from plants collected from the environment. It suggests that S. americanus activate their defense systems by retaining and maintaining their stress memory. This is a phenomenon in which information from previous stress is retained to cope with future stress. The regulation of stress memory involves chromatin modifications, DNA methylation, and the involvement of retrotransposons and microRNAs (Sharma et al 2022 ). In Arabidopsis. thaliana during salt stress, the methylation of the gene encoding pyrroline-5-carboxylate synthase was eliminated, leading to the activation of the enzyme and the accumulation of proline. When plants return to normal conditions, the gene remains unmethylated to cope with salt stress when it occurs. Further studies are needed to define the specific mechanism used by S. americanus and P. australis to tolerate the Salinas WWTP. Effect of S. americanus and P. australis on water quality from stabilization ponds. The development of the plants (maintained for 6 months in undiluted wastewater) showed that S. americanus plants exhibited more vigorous root growth as compared with P. australis , but no statistically significant differences (p > 0.05) were observed (Fig. 4 a). S. americanus showed greater stem development (33 ± 5.2) than P. australis (19.1 ± 3.6 cm) (Fig. 4 b), while P. australis generated more new shoots (12.6 ± 1.3) than S. americanus (4.2 ± 0.5) (Fig. 4 c). These differences were statistically different (p < 0.05). Growth parameters were similar to those observed in the progressive adaptation experiment (Fig. 3 ), indicating that both species tolerated prolonged exposure to wastewater. Regarding the physical parameters, the water temperature followed the greenhouse trend temperature and was approximately 6°C lower in the control and in the presence of plants (Fig. 5 a). Turbidity was 4.4 and 4.8 times lower (p < 0.05) on day 7 and 14, respectively, than in the control; nevertheless, on day 21 there were not statistical differences among treatments (Fig. 5 b). In the presence of S. americanus and P. australis on day 7, the EC was 1.5 times lower (3336 µS/cm) than in the control (5020 µS/cm) without differences at the end of the experiment (Fig. 5 c). The content of the TSS in wastewater was extremely high (2640–3750 ppm) but it decreased 4 and 3.5 times on day 7 and 14 in relation to the control; on day 21 the TSS value was similar in all treatments. As expected, the reduction of TSS was associated with a reduction in turbidity. In the presence of S. americanus and P. australis , TSS appears to be retained and absorbed at the root surface. This would explain their rapid decline. It is known that TSS has physical effects beyond salinity stress, such as reduction in water clarity, photosynthetic rate, energy production and consequently plant growth and development (Adjovu et al. 2023 ). The effect of S. americanus and P. australis on chemical water parameters is shown on Fig. 6 . At day 21, the pH of wastewater with plants was close to 7, while in the control group the pH was slightly alkaline (pH = 8) (Fig. 6 a). The lowest COD value was recorded with S. americanus on day 14, but at the end of the experiment there were no statistically significant differences between the control and the presence of the plants (Fig. 6 b). The nitrogen content decreased rapidly to 50% on day 7 with S. americanus and P. australis , but there were no differences between treatments at the end of the experiment (Fig. 6 c). The nitrite decreased to almost zero (0.58) on day 7 in the presence of S. americanus and P. australis (Fig. 6 d), while in the control the concentration was 6 to 10 higher (p < 0.05) on day 21. The concentration of nitrates reduced by about 10 to 15 times with P. australis on day 21, and by 2 times with S. americanus and the control (Fig. 6 e). Additionally, the wastewater was subjected to salinity analysis. The percentage of salinity exhibited a 30% reduction on day 42 (13.63) in the presence of the plants as compared with the initial stage of the experiment and the control (19.5) (data not shown). Orthophosphate values were similar between the control group and with S. americanus but 6 times lower than in P. australis (p < 0.05) at the end of the experiment (Fig. 6 f). The reduction in the physical and chemical parameters at the end of the experiment in the control group is attributed to sunlight, wind, and the participation of microorganisms and algae. Therefore, the control functioned as a microscale SP. In the presence of S. americanus and P. australis the wastewater quality improved approximately 3 times faster as compared with the control without plants. The pH reduction in wastewater could be related to the release of protons during oxidation of NH 3 to nitrates and nitrites. These protons would be responsible for the acidification of the water observed in the presence of the plants (Fig. 6 a). Additionally, the lower pH could be attributed to root exudates. S. americanus exudated aspartic acid, and P. australis released oxalic and citric acids in response to salt (Haviland and Noyce 2024 ; Rocha et al. 2015 ). These exudates could be involved in the elimination of Na + through root exudates as reported in Arabidopsis (Contreras-Cornejo et al. 2014 ). The important reduction in nitrate and TN is attributed to a de-nitrification process, assimilation of NO 3 and the production of nitrogen (Soana et al. 2020 ). Superior P and nitrate removal capacity exhibited by P. australis may be attributable to its higher biomass production and more efficient nutrient extraction from surface water in comparison to S. americanus . During the establishment of hydroponic cultures, it was observed that P. australis demanded high levels of nutrients, as it required twice the concentration of salts (2 g/L) in the hydroponic medium to produce new shoots (data not shown). The results show that S. americanus and P. australis were effective in improving the quality of moderately saline domestic wastewater, since significant reductions in nitrites (97.5%), nitrates (up to 84%), and orthophosphates (91.5%) were observed. Similar results have been described in the literature. Coleman et al. ( 2001 ) observed that Typha, Juncus effuses , and Scirpus validus removed 50–70% of the TSS, BOD, TKN, ammonia, and phosphate from domestic wastewater. Another study, with 12 emergent macrophytes showed a removal efficiency of 68.4% for NH 4 , 70.4% for TN, and 53.7% for COD when plants were exposed to 0.5, 1, and 1.5% salinity (Gao et al. 2015 ). Furthermore, Trema orientalis, Acorus calamus , and Cyperus alternifolius decreased COD, TP, NH 4 + , and NO 3 with efficiencies of 93.5%, 99.6%, 99.5%, and 98.9%, respectively, at low influent concentrations of NaCl (Cui et al. 2025 ). It should be noted, however, that all of these studies were performed using substrates. It is documented that gravel alone improved 6 out of 10 water quality parameters (Coleman et al. 2001 ). Additionally, Omidinia-Anarkoli and Shayannejad ( 2021 ) revealed that an unplanted constructed wetland containing pumice and gravel exhibited the most significant reduction in TSS, biochemical oxygen demand, and COD when compared with a planted wetland. Thus, the higher removal efficiencies reported in those works could also be related to the substrate and not just the plants. In this study, salt removal experiments were conducted without substrates. Consequently, the improvement in wastewater quality is attributable solely to the plants. When incorporating substrates into constructed wetlands, a number of factors must be considered, including the cost, availability, permeability, the potential for reuse, and safety concerns. For instance, the depth of ponds used usually ranged from 45 to 60 cm (Wang et al. 2020 ). Assuming a 1 m 3 of gravel weighs between 1.5 and 1.8 k (due to the characteristics of the material), 0.9 to 1.08 k of substrate are then utilized for each pond. In addition to the inherent costs associated with the materials, their use may also generate waste, if appropriate reuse strategies are not employed. The substrates used after wastewater treatment should not be discharged into the aquatic ecosystem unless such discharge will not have a detrimental effect on the environment. On the contrary, they must be removed and contained, which may require significant surface area (Legal Information Institute, https://www.law.cornell.edu/uscode/text/33/1344 ). The disposal of S. americanus and P. australis plants must be considered as well, as they may accumulate emerging contaminants, such as pharmaceuticals, personal care products and pesticides from domestic wastewater. Nevertheless, these plants can be incinerated for biofuel and biogas production, with the resulting ash being safely contained in a reduced space. Table 1 compares the physicochemical parameters of the effluent when the treatment plant was operating and when it was out of operation, in the absence (control) or presence of S. americanus and P. australis on day 21. The values accepted by the Official Mexican Standard for wastewater discharges into water (NOM-001-SEMARNAT-2021), for public services (NOM-003-ECOL-1997), the WHO (2022) and the Agency for Toxic Substances and Disease Registry (ATSDR 2016) were also included. It is evident that the treatment plant's lack of maintenance substantially increased the pH, TSS, COD, nitrites and nitrates. In the presence of S. americanus and P. australis plants, the values of TN, nitrites, nitrates and orthophosphates decreased, and remained within the limits allowed by Mexican and international regulations. Since S. americanus and P. australis showed a good nitrogen and phosphate removal capacity, they could be used for the phytoremediation of nitrogen-contaminated waters in real-world scenarios. The chemical, agri-food, textile and leather industries produce large amounts of saline wastewater thus the use of plants could be an option for treating the industries´s saline effluents and removing nitrogen and phosphorus. Table 1 Physicochemical analysis of the effluent from the treatment plant, during operation and out of operation, and the comparison with Mexican and international limits for pollutants in effluent discharges Parameter WTF 1 WTO 1 NOM-001 NOM-003 Control P. australis S. americanus Rivers Lakes Agricultural uses Industrial uses °C 18.00 23.33 23.87 24.07 35.00 35.00 TA ± 5.00 pH 7.50 7.90 7.33 7.32 6.50–8.50 SST (mg/L) 100 410 346 340 60 20 20 30 Turbidity (NTU) - 11.51 3.93 7.95 NA NA - EC (µS/cm) 3,630 1,526 1,102 1,250 1000 200 < 700 - DQO (mg/L) 595.60 2,060 1,970 2,030 150 100 20 30 NT (mg/L) 33.30 20.00 10.00 18.67 25.00 15.00 40 Nitrite (mg/L) 0.38 8.26 0.08 0.22 3.00 2 1.00 3 Nitrate (mg/L) 0.92 14.27 4.39 16.90 50.00 2 10.00 3 P0 4 − 3 (mg/L) - 2.31 0.34 2.24 15.00 5.00 20 1 WTF: wastewater treatment plant in function; WTO: wastewater treatment plant out of service 2 World Health Organization (2022) 3 Agency for Toxic Substances and Disease Registry ( ATSDR) Several approaches can be employed to enhance water quality such as, using more S. americanus and P. australis plants or larger plants. In this research, 50-cm plants were employed, but they can reach a height of about 3 m. Therefore, using larger plants could improve the uptake of salts. Another option would be to combine the two species to achieve a synergistic effect and increase the efficiency of phytoremediation, as previously reported (Coleman et al. 2001 ). Conclusion The present study demonstrated that S. americanus and P. australis exhibited a decline in root development when exposed directly to moderately saline domestic wastewater. However, when the plants were exposed to the wastewater in a gradual manner, their growth parameters exhibited an improvement, suggesting that the plants had undergone a physiological adaptation. This adaptation enabled the plants tolerated prolonged exposure to undiluted wastewater (over 6 months). Both species were effective in removing nitrites, nitrates, and orthophosphates, as well as in stabilizing the pH of the wastewater within a relatively short period of time. Since the studies were carried out without substrates, the improvement in water quality can be attributed specifically to the plants. The incorporation of S. americanus and P. australis into outfalls and wastewater treatment plants could reduce operating costs and enhance efficiency. This ecological phytoremediation approach could contribute to the reduction of contaminants in water bodies and provides a viable, low-cost wastewater management alternative for rural communities. Further studies are needed to determine if the plants are able to effectively colonize the SP waterbody, and how their presence would affect the biota of this area. Declarations Ethical Approval No animals or persons were used in this work. Consent to Participate Sarahi Josefina Estrada-Loredo, Rodolfo Cisneros-Almazán, Gerson Alonso Soto-Peña, Alejandro Hernández-Morales and María del Socorro Santos-Díaz give their consent to participate in this paper. Consent to Publish Sarahi Josefina Estrada-Loredo, Rodolfo Cisneros-Almazán, Gerson Alonso Soto-Peña, Alejandro Hernández-Morales and María del Socorro Santos-Díaz give their consent for the publication of this paper. Authors Contribution Sarahi Josefina Estrada-Loredo realized the experimental work, and participated on elaboration of figures and revision of manuscript. Rodolfo Cisneros-Almazán participated in design, revision and discussion of results. Gerson Alonso Soto-Peña and Alejandro Hernández-Morales participated in the experimental design of the study, defining the methodologies, overseeing data acquisition and revision of manuscript. María del Socorro Santos-Díaz is the leader of the group, designed the project and experimental work, participates in revision, discussion, and wrote the paper. Funding No funding was received for conducting this study. Competing Interests The authors declare that they have no known competing interests that could influence the work reported in this paper. Data Availability Statement Data and material are available at the Faculty of Chemistry of the “Universidad Autónoma de San Luis Potosí”. https://repositorioinstitucional.uaslp.mx/xmlui/handle/i/8644 Acknowledgements The authors thank the “Consejo Nacional de Humanidades Ciencia y Tecnología” for the scholarship to SJEL (no. 1181306) and Irvin Rául Ramírez-Morales and Ulises Juárez-Martínez for their technical assistance. References Adjovu GE, Stephen H, James D, Ahmad S (2023) Measurement of total dissolved solids and total suspended solids in water systems: A review of the issues, conventional, and remote sensing techniques. 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Sci Rep 13: 12483. https://doi.org/10.1038/s41598-023-39634-0 Zhao KF, Song J, Fan H, Zhou S, Zhao M (2010) Growth response to ionic and osmotic stress of NaCl in salt‐tolerant and salt‐sensitive maize. J Integr Plant Biol 52: 468-475. https://doi.org/10.1111/j.1744-7909.2010.00947.x Supplementary Files Onlineresource1.pdf Onlineresouce2april.pdf Cite Share Download PDF Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Wetlands → Version 1 posted Editor assigned by journal 27 May, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers invited by journal 10 Apr, 2025 Editor invited by journal 04 Apr, 2025 First submitted to journal 03 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5823958","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441306899,"identity":"0a733c10-3a00-4193-bece-fd97e70235ed","order_by":0,"name":"Sarahí Josefina Estrada-Loredo","email":"","orcid":"","institution":"Universidad Autónoma de San Luis Potosí: Universidad Autonoma de San Luis Potosi","correspondingAuthor":false,"prefix":"","firstName":"Sarahí","middleName":"Josefina","lastName":"Estrada-Loredo","suffix":""},{"id":441306900,"identity":"70628666-aa78-42a0-8377-e7139c73472f","order_by":1,"name":"Rodolfo Cisneros-Almazan","email":"","orcid":"","institution":"Universidad Autónoma de San Luis Potosí: Universidad Autonoma de San Luis Potosi","correspondingAuthor":false,"prefix":"","firstName":"Rodolfo","middleName":"","lastName":"Cisneros-Almazan","suffix":""},{"id":441306901,"identity":"33352972-7ed7-40bd-a59a-cb64026743db","order_by":2,"name":"Gerson Alonso Soto-Peña","email":"","orcid":"","institution":"Universidad Autonoma de San Luis Potosi","correspondingAuthor":false,"prefix":"","firstName":"Gerson","middleName":"Alonso","lastName":"Soto-Peña","suffix":""},{"id":441306902,"identity":"feb30324-47d0-4925-8331-e51389ab2e58","order_by":3,"name":"Alejandro Hernández-Morales","email":"","orcid":"","institution":"Universidad Autónoma de San Luis Potosí: Universidad Autonoma de San Luis Potosi","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Hernández-Morales","suffix":""},{"id":441306903,"identity":"32447928-a153-4f1c-a04d-08252ff028a3","order_by":4,"name":"Maria del Socorro Santos-Díaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBADOSBmPEC8eqBSYyhNgpbEBqK1yPcffvb4Q0Vd+objxx8cLmDYBtaLFxjcSDM3OHDmcO6GMzkGh2cw3DYmaIuBBIOZxMG2A7kbDuQwHOZhuC1HhMOOfwNqqUs3OP/8AUgLD2HPHMgB2cKcYHAjwYA4Wwxu5JRJnDlz2HDmjTdAvxgQ4Regw7ZJVFTUyfOdT3/4uKDiNuEQQwHMDAYkqQdrGQWjYBSMglGABQAAz45DOD/kD98AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5729-196X","institution":"Universidad Autonoma de San Luis Potosi Facultad de Ciencias Quimicas","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"del Socorro","lastName":"Santos-Díaz","suffix":""}],"badges":[],"createdAt":"2025-01-14 04:28:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5823958/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5823958/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13157-025-01965-1","type":"published","date":"2025-07-14T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80367875,"identity":"1d76c851-2b06-4112-a8b2-1baf5aeebc46","added_by":"auto","created_at":"2025-04-11 06:00:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":152856,"visible":true,"origin":"","legend":"\u003cp\u003eAspect of the stabilization pond of the abandoned Salinas wastewater treatment plant. Algae formation and salt accumulation were observed\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/29ea051ca47f5ca84c384a63.png"},{"id":80366332,"identity":"258b2a63-7812-44e4-b7d1-37e723d77a90","added_by":"auto","created_at":"2025-04-11 05:36:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":981563,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of direct exposure to wastewater on growth parameters of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e. Control (●), 12.5% (■), 25% (▲), 50 % (♦), 100 % (×) wastewater. The control was a commercial hydroponic medium. Data with different letters are statistically significant (Tukey test, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/109ec32132568db1ae4768b0.png"},{"id":80366337,"identity":"fee449e4-b913-4b2d-9f6e-0800a29a7c44","added_by":"auto","created_at":"2025-04-11 05:36:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":595667,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of \u003cem\u003eS. americanus \u003c/em\u003e\u0026nbsp;(a) and \u003cem\u003eP. australis \u003c/em\u003e(b) plants in progressively diluted wastewater. Increase in stem length (□), increase in root length (■), new shoots ( ▬). Values with different letters are statistically significant (Tukey test, p\u0026lt;0.05), n =12\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/a01dca4f24a16ddc707a63f9.png"},{"id":80366334,"identity":"08f9fabf-8aa1-4758-bb4c-77485ed4d763","added_by":"auto","created_at":"2025-04-11 05:36:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":409783,"visible":true,"origin":"","legend":"\u003cp\u003eComparison in the development of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants adapted to wastewater for 6 months to wastewater. The figure shows: a) increase in root length at day 21, b) increase of stem length, c) formation of new shoots. \u003cem\u003eS. americanus\u003c/em\u003e (▲ ) and \u003cem\u003eP. australis\u003c/em\u003e (●) plants. Values with different letters are statistically significant (Tukey, p\u0026lt;0.05), n =12\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/07ff1b0ed8637c9706346812.png"},{"id":80366359,"identity":"b1c8080d-9a6b-4d4f-b970-0c29f994a3a2","added_by":"auto","created_at":"2025-04-11 05:36:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":503991,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eS. americanus \u003c/em\u003e\u0026nbsp;and \u003cem\u003eP. australis \u003c/em\u003eplants on physical parameters of wastewater. Control corresponded to water without plants ( ■ ), \u003cem\u003eS. americanus\u003c/em\u003e (▲ ), \u003cem\u003eP. australis\u003c/em\u003e(●), (♦) greenhouse temperature. Data correspond to: a) temperature, b) turbidity, c) electrical conductivity and d) total suspended solids. Values with different letters are statistically significant (Tukey test , p\u0026lt;0.05), n =3\u003c/p\u003e\n\u003cp\u003eOrthophosphate values were similar between the control group and with \u003cem\u003eS. americanus\u003c/em\u003e but 6 times lower than in \u003cem\u003eP. australis\u003c/em\u003e(p\u0026lt;0.05) at the end of the experiment (Figure 6f).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/9b68b6ed3153db4349b4916d.png"},{"id":80367877,"identity":"7b6c77b7-347b-42be-bed1-a24acee30e1e","added_by":"auto","created_at":"2025-04-11 06:00:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":645456,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eS. americanus \u003c/em\u003e\u0026nbsp;and \u003cem\u003eP. australis \u003c/em\u003eon chemical parameters of wastewater. Control corresponded to water without plants ( ■ ), \u003cem\u003eS. americanus\u003c/em\u003e (▲ ) and \u003cem\u003eP. australis\u003c/em\u003e (●) plants. a) pH, b) chemical oxygen demand, c) total nitrogen, d) nitrite, e) nitrate, f) orthophosphate concentration. Values with different letters are statistically significant (Tukey test , p\u0026lt;0.05), n =3\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/680915cf428579517dcc4582.jpeg"},{"id":87219545,"identity":"49d7e21b-5367-4e30-b3be-9e38c757aa1d","added_by":"auto","created_at":"2025-07-21 16:05:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4324764,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/6e98883c-5f21-4201-b89e-b21a5ac0c4df.pdf"},{"id":80366363,"identity":"b77ea82b-7353-4b66-8f86-64e93304cba4","added_by":"auto","created_at":"2025-04-11 05:36:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":145852,"visible":true,"origin":"","legend":"","description":"","filename":"Onlineresource1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/3f2b4f7541e0312c61abbc1e.pdf"},{"id":80366362,"identity":"f6706cf6-8384-4c08-bc74-39474c5e0f91","added_by":"auto","created_at":"2025-04-11 05:36:50","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":146057,"visible":true,"origin":"","legend":"","description":"","filename":"Onlineresouce2april.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5823958/v1/3a442df6a0f8a0b91c40079e.pdf"}],"financialInterests":"","formattedTitle":"Effects of Schoenoplectus americanus (Pers.) Volkart ex Schinz \u0026amp; R.Keller and Phragmites australis (Cav.) on the water quality improvement of moderately saline wastewater","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the WHO (2023), approximately 771\u0026nbsp;million people did not have access to safe drinking water, and at least 1.7\u0026nbsp;billion people worldwide drank water contaminated with fecal matter. This issue is partly due to the fact that 80% of wastewater is discharged without being treated into water bodies (WWAP 2019). The consumption of contaminated water is responsible for one-third of morbidity and mortality in developing countries (Kumar et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, extreme weather events caused by climate change, also affect freshwater ecosystems and consequently their availability for human consumption (Lee et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, wastewater reuse has become increasingly important and is considered one of the most viable alternatives for addressing climate change and contributing to water resources management (Biswas et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe contaminants of concern in wastewater include pathogens, and dissolved organic and inorganic matter. To avoid health and the environment risks, the quality of the effluent from the treatment plant must meet specific standards for use in agriculture, horticulture, groundwater recharge, augmentation of riverbeds or natural water bodies, industries and street washing. The most common parameters analyzed in wastewater, besides the pathogen level, include total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN), nitrites and nitrates, chloride and phosphate (Preisner et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn developing countries, water pollution and drinking water scarcity represent major challenges. These countries have made significant efforts to improve water supply and promote water reuse (Bijekar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In India, the range of implemented systems encompasses constructed wetlands and upflow anaerobic sludge blankets. In Africa, duckweed pond systems and septic tanks are employed (Muzioreva et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nepal and Indonesia have adopted baffled reactors (Yulistyorini et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), while constructed wetlands are a prevalent wastewater treatment system in South America and Mexico (Ferreira et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to C\u0026aacute;\u0026ntilde;ez-Cota (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), Mexico had 3,516 municipal wastewater treatment plants in 2018, 72% of which are currently operational. The remaining 980 are inactive due to the high energy costs that have not been covered because of the communities' low or very low marginalization. In addition, most of these wastewater treatment plants are in arid and semi-arid regions characterized by low rainfall and the overexploitation of wells.\u003c/p\u003e \u003cp\u003eMacrophytes play an important role in wetland ecosystems because they can remove significant amounts of nutrients from domestic wastewater. Therefore, they represent a technological alternative for rural areas in Mexico or other developing countries. Macrophyte roots act as filters, biodegrading significant amounts of organic matter and promoting nutrient uptake while reducing solar radiation penetration, thereby suppressing algal growth (Huth et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo macrophytes with an excellent potential for restoring water bodies are \u003cem\u003eSchoenoplectus americanus\u003c/em\u003e (synonym \u003cem\u003eScirpus americanus\u003c/em\u003e) and \u003cem\u003ePhragmites australis. S. americanus\u003c/em\u003e is native to North America, and can grow both in wet or dry environments. It has a very vigorous radical system, and stems from 50 to 220 cm tall. This species can tolerate and remove high concentrations of lead from water (Esquivel-Ramos et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), chromium and total phenols from the tannery industry (Quevedo et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and is also salt tolerant. Howard and Mendelssohn (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) described that biomass and stem density were not affected in plants exposed to 6 and 12 g/L NaCl. Plant height continued to increase in both treatments and did not reach the senescence stage, unlike the oligohaline marshes \u003cem\u003ePanicum hemitomon\u003c/em\u003e and \u003cem\u003eSagittaria lancifolia\u003c/em\u003e, which showed reduced root and aerial tissue growth. In addition, \u003cem\u003eS. americanus\u003c/em\u003e was approximately twice as salt-tolerant as \u003cem\u003eTypha domingensis\u003c/em\u003e. It exhibited a 50% relative growth rate in 9 g/L total dissolved solids (TDS), while \u003cem\u003eT. domingensis\u003c/em\u003e did so at 4 g/L TDS (Baeza et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. australis\u003c/em\u003e is an herbaceous plant that grows in temperate and tropical climates. The plant can reach 4 m in height, is able to remove and accumulate emerging pollutants (Lei et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and is salt tolerant. Haplotype M has the highest tolerance, showing only a 50% reduction in growth at a concentration of 0.4 M NaCl (Vasquez et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Salt tolerance has been associated with cation adjustment and water loss. Cation adjustment maintain K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratios in leaf laminas, while water loss concentrates solutes in the cell sap, thus reducing the necessity for the synthesis of osmolytes, and the Na\u003csup\u003e+\u003c/sup\u003e uptake for osmotic adjustment (Lissner et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother factor that contributes to the removal of pollutants in constructed wetlands is the substrate used to fill the ponds and provide support for plants. Pumice, perlite, zeolite, and sand have been described as absorbing nitrate, sulfate, and phosphate (Mehrani et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ghasemi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; De Rozari et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pollutant removal occurs through physical sedimentation, filtration, sorption in the substrate matrix, ion exchange, gas diffusion, and bio-immobilization in the substrate (Wang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the municipality of Salinas de Hidalgo, in the state of San Luis Potos\u0026iacute;, in Mexico (101\u0026deg;42'15.6\" west longitude and 22\u0026deg;38'26.4\" north latitude) is located the so-called salty valley because of the high salt content in the soil and water. In this area, there is an abandoned stabilization pond (SP) from a water treatment plant. The water in the pond is of low quality, has high salinity, significant organic matter, and possible microbial contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The SP receives the city's domestic and industrial wastewater, and given its proximity to the Salinas Lagoon, the SP's wastewater typically has high salt concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe inclusion of artificial wetlands could be a technological alternative for improving the quality of the Salinas wastewater plant. Therefore, this study focused on the ability of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants to remove salts from wastewater plant. Considering that these emergent macrophytes have more supporting tissues than floating macrophytes, they may have greater potential for storing salt for longer periods of time. We were interested in comparing the response of \u003cem\u003eS. americanus\u003c/em\u003e regenerated \u003cem\u003ein vitro\u003c/em\u003e, which showed robust growth and had not experienced any stress, with those of \u003cem\u003eP. australis\u003c/em\u003e, which had been exposed to environmental salinity conditions. On the other hand, it is known that plant tolerance to salt can be increased by prior exposure to low salt concentrations for a short period of time (Pandolfi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kamanga et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e were directly and progressively exposed to wastewater to analyze if there were differences in the growth and the adaptation process. This study was performed without substrates, in order to specifically understand the role of plants in the removal of salts and major pollutants from moderately saline wastewater. This research should contribute to the development of sustainable and scalable phytoremediation strategies for rural communities in semi-arid regions.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003e \u003cb\u003eS. americanus in vitro\u003c/b\u003e \u003cb\u003ecultures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eS. americanus\u003c/em\u003e plants were obtained from \u003cem\u003ein vitro\u003c/em\u003e germinated seeds in Murashige and Skoog basal medium, MS (Murashige and Skoog \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 116 \u0026micro;M myo-inositol, 1.2 \u0026micro;M thiamine-HCl, 30 g/L sucrose, and 8.4 mg/L agar, pH 5.7. The \u003cem\u003ein vitro\u003c/em\u003e seedlings were sectioned and propagated in MS medium supplemented with 0.5 mg/L benzyladenine (Alfaro-Salda\u0026ntilde;a et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The plants were cultivated in solid MS medium until they reached a height greater than 8 cm. Subsequently, they were transferred to 500-ml vessels containing 100 ml of liquid MS medium (without agar) to increase the growth rate, and then underwent constant shaking at 135 rpm, in an orbital shaker. \u003cem\u003eIn vitro\u003c/em\u003e cultures were maintained within a photoperiod of 16 hours of light and 8 hours of darkness at 25\u0026deg;C in a growth chamber.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of hydroponic cultures of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro S. americanus\u003c/em\u003e plants (15 cm tall) were collected, and their roots were washed with sterile deionized water to remove the medium. The plants were placed in 1-L containers with 400 ml of commercial hydroponic medium (1 g/L) (Hydro Environment, Mexico), pH 5.7. The plants were covered with a plastic bag for 15 days to improve their adaptation to the environment. Subsequently, the plants were transferred to plastic vessels with 10 L of hydroponic medium supplemented with 330 \u0026micro;l of Aquapool algaecide (Aris Industrial, Peru). The surface of the vessels was covered with a styrofoam plate and the hydroponic medium was changed weekly. The plants were maintained under greenhouse conditions until they reached a height of 50 cm.\u003c/p\u003e \u003cp\u003eEntire \u003cem\u003eP. australis\u003c/em\u003e plants taller than 40 cm and with abundant roots were collected from the banks of the Santiago River (22\u0026deg;10'52.9\" N 100\u0026deg;56'09.0\" W) in San Luis Potos\u0026iacute;. The plants were carefully washed with soap to eliminate soil and some microbials, and then rinsed with water. The plants were placed in 60-L containers with 20 L of commercial hydroponic medium (2 g/L) for 4 weeks to obtain new shoots. New shoots taller than 15 cm, with a diameter greater than 2 cm and defined roots, were cut and transferred to basins containing 10 L of hydroponic medium. The surface of the basin was covered with a styrofoam plate. The plants were maintained in greenhouse conditions with the hydroponic medium being replaced once a week.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysicochemical analysis of the wastewater\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWater samples were collected from the surface of the stabilization ponds at the Salinas Municipality Water Treatment Plant (22\u0026deg;38'26.4\" N, 101\u0026deg;42'15.6\" W), placed in glass containers, and stored at 4\u0026deg;C until analysis. The temperature, total suspended solids and electrical conductivity (EC, \u0026micro;s/cm) were measured immediately by using a HI98130 portable conductivity meter (Hanna Instruments, USA). Upon arrival at the laboratory, the pH was measured with the UltraBasic Benchtop pH meter. The TSS value (mg/L) was verified again at 810 nm by using a DR 2800 spectrophotometer. The depth and coordinates of water collection, as well as the date and time of sampling, were also registered.\u003c/p\u003e \u003cp\u003eThe physicochemical analysis of the wastewater was performed in triplicate. Turbidity measurements were performed using a HACH 2100Q turbidimeter at 850 nm. The resulting values were reported as nephelometric turbidity units (NTU) according to Official Mexican Standard NMX-AA-038-SCFI-2001.\u003c/p\u003e \u003cp\u003eThe chemical oxygen demand (COD) was analyzed according to Official Mexican Standard NMX-AA-030/1-SCFI-2012 using a thermal reactor (HACH DRB200) with a confidence interval of 15 to 300 mg/L. If the value exceeds 300 mg/L, the sample is diluted until the values fall within the measuring range.\u003c/p\u003e \u003cp\u003eThe total nitrogen was measured using the Spectroquant\u0026reg; 114763 kit. The confidence interval was 10 to 150 mg/L TN.\u003c/p\u003e \u003cp\u003eNitrite was measured according to the Official Mexican Standard NMX-AA-099-SCFI-2021. The nitrite concentration was calculated from the calibration curve (0.05 mg/L, 0.10 mg/L, 0.15 mg/L, 0.20 mg/L, 0.25 mg/L and 0.30 mg/L) using NaNO\u003csub\u003e2\u003c/sub\u003e as a standard.\u003c/p\u003e \u003cp\u003eNitrates were analyzed according to the protocol described by Gentle et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The nitrate concentration was calculated from the calibration curve constructed with KNO\u003csub\u003e3\u003c/sub\u003e at concentrations of 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, and 7 mg/L.\u003c/p\u003e \u003cp\u003eOrthophosphates were determined using the Spectroquant\u0026reg; 114848 test kit with a confidence interval of 0.05 to 5 mg/L.\u003c/p\u003e \u003cp\u003eThe physicochemical parameters of initial collected water samples were: temperature 29.80\u0026deg;C; pH 7.89; EC 7,520 \u0026micro;S/cm; TSS 3,740 mg/L; COD 2,780 mg/L; TN 71.50 mg/L; 23.10 mg/L nitrites; 30.80 mg/L nitrates; 388.5 NTU of turbidity; and 3.95 mg/L orthophosphates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDirect adaptation of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eto wastewater\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwenty \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants taller than 50 cm were used for this experiment. The plants were rinsed with water, pruned to 50 cm to ensure that the plants were of uniform size and to assess the extent of increase in stem length over time. The pruning of macrophytes has been reported to improve nutrient uptake (Herzog et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Four plants of each species were placed in 10 L basins containing 9 L of 12.5%, 25%, 50% or 100% wastewater; dilutions were performed by using deionized water. In addition, two basins containing plants of either species in commercial hydroponic medium were used as controls. The basins were covered by a styrofoam plate and the plants were maintained for 20 days under greenhouse conditions. The water lost by evapotranspiration was replaced by deionized water every 4 days. Stem length, and the number of shoots and leaves were measured every 4 days. Root length was measured at day 1 and 20. Every 4 days, 50 mL of wastewater was collected and the pH, EC and TSS were determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProgressive adaptation of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eto wastewater\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwelve plants of \u003cem\u003eS. americanus\u003c/em\u003e or \u003cem\u003eP. australis\u003c/em\u003e pruned to a height of 50 cm were used. Three plants of each species were placed in four different 10-L basins containing 9 L of 12.5% effluent. Dilutions were performed using deionized water. The plants remained under these conditions for 20 days and were then transferred to basins containing 9 L of 25% wastewater. After 20 days, the plants were exposed to 50% of wastewater for 20 days, and finally to 100% of the wastewater for another 20 days. The water lost by evapotranspiration was replaced with deionized water. Stem growth and the number of new shoots were measured at 4-day intervals, as were water parameters. Root length was measured only at day 0 and 20 to minimize plant disturbance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eon water quality in stabilization ponds.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this experiment, the plants (n\u0026thinsp;=\u0026thinsp;12) were cultivated in undiluted wastewater over a period of 6 months. The objective of this experiment was twofold: firstly, to ascertain the ability of the plants to survive in these conditions over an extended period, and secondly, to verify that the mechanisms of tolerance to salt were already active. Four plants of each species (pruned to a 50 cm height) were then placed in three different basins with 9 L of wastewater. Three basins with wastewater and no plants were included as controls. The increase in stem length and root length, and the number of shoots was measured as described in the previous paragraph. For the water quality analysis, 100 mL of wastewater was collected from each tank every 7 days, and the temperature, pH, TSS, COD, NT, nitrites, nitrates, turbidity, EC and orthophosphates were measured.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe height, number of shoots, number of leaves, and water analysis were evaluated using a one-way analysis of variance (ANOVA) to determine whether significant differences existed between groups. Post-hoc comparisons of means were performed using the Tukey test and the significance level was set at \u0026#119901; \u0026lt; 0.05. Statistical analyses were performed using Statistica 10 software (StatSoft).\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e regenerated plants of \u003cem\u003eS. americanus\u003c/em\u003e successfully adapted to environmental conditions. After 6 months, vigorous and healthy plants with extensive root were obtained in hydroponic media. Variations in development patterns were observed, with 80% of the plants producing between 0 and 10 new shoots, 13.3% between 11 and 20 new shoots, and 6.7% between 21 and 30 new shoots. Regarding stem length, 76.7% of the plants developed stems \u0026lt; 50 cm, 20.0% from 51 to 100 cm and 3.3% from 101 to 150 cm. These differences in development could be due to somaclonal variation induced by \u003cem\u003ein vitro\u003c/em\u003e culture (Bairu et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. australis\u003c/em\u003e required 3 months to obtain vigorous plants in a hydroponic medium. The plants also showed differences in growth patterns. About 50% of the plants produced between 0 and 10 new shoots, 30% between 11 and 20 new shoots, and 20% between 21 and 30 new shoots. The total number of plants had a stem length ≤ 50 cm (data not shown). It has been posited that epigenetic changes, such as DNA methylation, may underlie variations in growth patterns. These variations are more closely associated with microhabitat than with genotype within subspecies (Spens and Douhovnikoff \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) (data not shown).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDirect adaptation of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eto wastewater\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. americanus\u003c/em\u003e plants maintained in hydroponic medium (control) and 12.5% wastewater showed an increase in root length of 5 cm and 0.75 cm, respectively. Root loss was observed in 25%, 50%, and 100% wastewater (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) with no statistical differences (p \u0026gt; 0.05) among treatments. \u003cem\u003eP. australis\u003c/em\u003e presented a similar behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) with an increase in root length of 5.8 and 2.75 cm in the control and 12.5% of residual water, respectively. Furthermore, there was root loss in 25%, 50% and 100% of wastewater, which statistically differed from the control (p \u0026gt; 0.05). These results indicate that direct exposure of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants to wastewater affected root metabolism, since a significant loss of this tissue was observed in 25%, 50% and 100% wastewater. The inhibition of root growth was also observed in Tartary buckwheat (Zhang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), tomato, carrot (Jahan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and avocado plants (Bernstein et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) exposed to excess salt.\u003c/p\u003e \u003cp\u003eThe root damage induced by a high level of salinity in \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e, can be due to ionic stress, osmotic stress, and/or oxidative stress. Ionic stress promotes ion imbalance in the plant. For instance, Na\u003csup\u003e+\u003c/sup\u003e inhibits the uptake of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e, causing nutrient stress in the plant. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, NaCl, KCl, and LiCl reduced root hair density and elongation (Wang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Osmotic stress accelerated suberization in the endodermis and exodermis of roots of \u003cem\u003eZea mays\u003c/em\u003e seedlings, significantly decreased hydrostatic hydraulic conductivity, and reduced water and nutrient uptake (Shen et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The oxidative stress increased mitochondrial ultrastructural damage and reactive oxygen species (ROS) generation in wheat seedling roots (Kononenko et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, it promoted electrolyte leakage, as well as malondialdehyde and hydrogen peroxide production in the stem and roots of \u003cem\u003eLepidium draba\u003c/em\u003e ecotypes (Jamshidi et al. 2020).\u003c/p\u003e \u003cp\u003eRegarding the aerial parts, the stem length of \u003cem\u003eS. americanus\u003c/em\u003e on day 20 increased between 42 and 60 cm in the control, and in 12.5%, 25% and 50% wastewater, while the increase in 100% wastewater was only 25 cm at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This difference was statistically significant (p = 0.042). Additionally, salt exudation and stem stiffness were observed in 100% wastewater. The production of shoots ranged between 14 and 17 new shoots in 50% and 100% wastewater, and this number decreased in more diluted wastewater and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eThe data suggest that salts absorbed by the roots of \u003cem\u003eS. americanus\u003c/em\u003e were transported and accumulated in the stem. At high salinities, the salt was exuded from this tissue, possibly through excretory glands, as a defense mechanism. To our knowledge, this is the first report describing salt exudation in this species. X-ray microanalysis of maize exposed to 100 mM NaCl showed that salts enter the apoplast, chloroplast, cytosol, and vacuoles almost simultaneously. When vacuoles become saturated, salts are released (Zhao et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). During salt secretion, the ions are transported by small vesicles, which then fuse with the plasma membrane and release the salts. The salt passes through the apical cell wall and cuticle to the environment (Lu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eP. australis\u003c/em\u003e plants exposed to different wastewater dilutions increased stem length (20 to 38 cm) as a function of time, with no significant statistical differences between treatments on day 20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The lowest growth was observed in the control (15 cm) and was statistically different from 50% wastewater (p \u0026lt; 0.05). \u003cem\u003eP. australis\u003c/em\u003e plants produced the highest number of new shoots (27) in 50% wastewater, but in the control and in 12.5%, 25% and 100% wastewater the number of new shoots decreased (14 to 17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eThe production of new shoots and the increase in stem length in \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e could be related to the level of organic matter, as previously described in \u003cem\u003ePennisetum purpureum\u003c/em\u003e, \u003cem\u003eMedicago sativa\u003c/em\u003e, \u003cem\u003eSinapis alba\u003c/em\u003e, and \u003cem\u003eHelianthus annuus\u003c/em\u003e (Matheyarasu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Presumably, the 50% effluent would provide plants with a significant amount of nutrients, but the deleterious effects of salts could be moderate. Conversely, plants exposed to undiluted effluent would have higher levels of organic matter, but would also be exposed to toxic concentrations of salts. In addition, it has been reported that the response to salt may exhibit a bell-shaped dose-response curve. For example, in tomato seedlings, low salt concentrations (25 and 50 mM NaCl) positively affected shoot length, whereas high concentrations (100 to 200 mM NaCl) significantly reduced shoot growth (Rivera et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, roots were more affected by salinity than aerial parts in both species because they were in direct contact with the wastewater and more exposed to salts. Roots could act as a barrier to reduce salt internalization and decreased its toxicity in aerial parts. After exposure to wastewater, \u003cem\u003eS. americanus\u003c/em\u003e plants produced longer stems, while \u003cem\u003eP. australis\u003c/em\u003e generated more shoots. This response can be attributed to their intrinsic morphological characteristics. \u003cem\u003eP. australis\u003c/em\u003e is a perennial plant that develops an increasing number of shoots during the growing season, whereas \u003cem\u003eS. americanus\u003c/em\u003e usually has only a few short leaves, up to about 10 cm long, but has strong and large stems. These results agree with Vasquez et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), who reported that salinity conditions induced more shoots per gram of rhizome tissue in \u003cem\u003eP. australis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe analysis of wastewater collected during this experiment is shown in the Online Resource 1. The pH remained between 7 and 8.2, while the water temperature correlated with the maximum temperature. TSS values in undiluted water ranged between 2640 to 3750 ppm. The EC observed in 100% wastewater was 5174 µS/cm- 7470 µS/c, indicating that water has a high concentration of dissolved ions and salt corresponding to moderately saline water (Hillel \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdaptation of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eusing serial dilution\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. americanus\u003c/em\u003e plants gradually adapted to wastewater exhibited minimal root loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Stem length increased by 22% in wastewater at 12.5% compared with the control group (p \u0026lt; 0.05), decreasing proportionally with wastewater concentration. This reduction was statistically different from the control (p \u0026lt; 0.05). The number of new shoots ranged between 4.2 and 6.4 with significant differences between the control and 12.%, and 12.5% an undiluted wastewater.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. australis\u003c/em\u003e showed no root loss and a slight growth of this tissue was observed even at 100% effluent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Stem length was similar at all effluent concentrations tested, but lower in the control treatment (p \u0026gt; 0.05). The number of shoots increased progressively up to 25% effluent and then remained constant at approximately 26 new shoots. In summary, \u003cem\u003eS. americanus\u003c/em\u003e mainly developed longer stems while \u003cem\u003eP. australis\u003c/em\u003e generated new shoots. The analysis of the effluent collected during this experiment is shown in Online Resource 2.\u003c/p\u003e \u003cp\u003eThese results suggest that plants exposed to wastewater through a serial adaptation process developed more efficient tolerance mechanisms that allow better growth. The most obvious change was the approximately 2.8-times increase in stem length in \u003cem\u003eS. americanus\u003c/em\u003e, and the 3-times increase in new shoot formation in \u003cem\u003eP. australis\u003c/em\u003e during progressive exposure to wastewater as compared with direct exposure.\u003c/p\u003e \u003cp\u003eIt has been documented that prior exposure to low salt concentrations can enhance survival and growth rates. For instance, pre-treatment of \u003cem\u003eZea mays\u003c/em\u003e plants with 25 mM NaCl enhanced the intracellular K/Na ratio, and increased the sequestration of sodium in vacuoles (Pandolfi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In olive plants a reduction in salt-induced ultrastructural changes in leaf cells was observed (Pandolfi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In pea an improved photosynthetic rate, stomatal conductance, and chlorophyll content were reported (Shaukat et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); in tomato seedlings accumulation of proline, activation of antioxidant enzymes, and a reduction in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were observed (Kamanga et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral mechanisms involved in the physiological adaptation could be participating. \u003cem\u003eP. australis\u003c/em\u003e has been classified as a salt excluder, which allows it to reduce Na\u003csup\u003e+\u003c/sup\u003e transport to shoots and ameliorate salt toxicity. It uses K\u003csup\u003e+\u003c/sup\u003e for osmotic adjustment in leaves and also accumulates osmoprotectants such as proline and glutamine (Vasquez et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hartzendorf and Rolletschek \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eS. americanus\u003c/em\u003e, a 5-fold increase in glycine betaine levels was documented after exposure to 20 ppt salinity (Ewing et al. 1988). Proline and glycine betaine play an important role in scavenging ROS and counteracting NaCl-induced oxidative stress. They also improve hydration and prevent dehydration and protein denaturation. The rapid catabolism of proline provides reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP for recovering from stress and repairing of stress-induced damage (Xie et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother observation is that \u003cem\u003ein vitro\u003c/em\u003e regenerated \u003cem\u003eS. americanus\u003c/em\u003e plants previously unexposed to salt could survive and grow in the direct and progressive adaptation experiments, as well as \u003cem\u003eP. australis\u003c/em\u003e derived from plants collected from the environment. It suggests that \u003cem\u003eS. americanus\u003c/em\u003e activate their defense systems by retaining and maintaining their stress memory. This is a phenomenon in which information from previous stress is retained to cope with future stress. The regulation of stress memory involves chromatin modifications, DNA methylation, and the involvement of retrotransposons and microRNAs (Sharma et al \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eArabidopsis. thaliana\u003c/em\u003e during salt stress, the methylation of the gene encoding pyrroline-5-carboxylate synthase was eliminated, leading to the activation of the enzyme and the accumulation of proline. When plants return to normal conditions, the gene remains unmethylated to cope with salt stress when it occurs. Further studies are needed to define the specific mechanism used by \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e to tolerate the Salinas WWTP.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eS. americanus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eP. australis\u003c/b\u003e \u003cb\u003eon water quality from stabilization ponds.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe development of the plants (maintained for 6 months in undiluted wastewater) showed that \u003cem\u003eS. americanus\u003c/em\u003e plants exhibited more vigorous root growth as compared with \u003cem\u003eP. australis\u003c/em\u003e, but no statistically significant differences (p \u0026gt; 0.05) were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). \u003cem\u003eS. americanus\u003c/em\u003e showed greater stem development (33 ± 5.2) than \u003cem\u003eP. australis\u003c/em\u003e (19.1 ± 3.6 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), while \u003cem\u003eP. australis\u003c/em\u003e generated more new shoots (12.6 ± 1.3) than \u003cem\u003eS. americanus\u003c/em\u003e (4.2 ± 0.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These differences were statistically different (p \u0026lt; 0.05). Growth parameters were similar to those observed in the progressive adaptation experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that both species tolerated prolonged exposure to wastewater.\u003c/p\u003e \u003cp\u003eRegarding the physical parameters, the water temperature followed the greenhouse trend temperature and was approximately 6°C lower in the control and in the presence of plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Turbidity was 4.4 and 4.8 times lower (p \u0026lt; 0.05) on day 7 and 14, respectively, than in the control; nevertheless, on day 21 there were not statistical differences among treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn the presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e on day 7, the EC was 1.5 times lower (3336 µS/cm) than in the control (5020 µS/cm) without differences at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The content of the TSS in wastewater was extremely high (2640–3750 ppm) but it decreased 4 and 3.5 times on day 7 and 14 in relation to the control; on day 21 the TSS value was similar in all treatments. As expected, the reduction of TSS was associated with a reduction in turbidity. In the presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e, TSS appears to be retained and absorbed at the root surface. This would explain their rapid decline. It is known that TSS has physical effects beyond salinity stress, such as reduction in water clarity, photosynthetic rate, energy production and consequently plant growth and development (Adjovu et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effect of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e on chemical water parameters is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. At day 21, the pH of wastewater with plants was close to 7, while in the control group the pH was slightly alkaline (pH = 8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The lowest COD value was recorded with \u003cem\u003eS. americanus\u003c/em\u003e on day 14, but at the end of the experiment there were no statistically significant differences between the control and the presence of the plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe nitrogen content decreased rapidly to 50% on day 7 with \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e, but there were no differences between treatments at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The nitrite decreased to almost zero (0.58) on day 7 in the presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), while in the control the concentration was 6 to 10 higher (p \u0026lt; 0.05) on day 21. The concentration of nitrates reduced by about 10 to 15 times with \u003cem\u003eP. australis\u003c/em\u003e on day 21, and by 2 times with \u003cem\u003eS. americanus\u003c/em\u003e and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eAdditionally, the wastewater was subjected to salinity analysis. The percentage of salinity exhibited a 30% reduction on day 42 (13.63) in the presence of the plants as compared with the initial stage of the experiment and the control (19.5) (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOrthophosphate values were similar between the control group and with \u003cem\u003eS. americanus\u003c/em\u003e but 6 times lower than in \u003cem\u003eP. australis\u003c/em\u003e (p \u0026lt; 0.05) at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reduction in the physical and chemical parameters at the end of the experiment in the control group is attributed to sunlight, wind, and the participation of microorganisms and algae. Therefore, the control functioned as a microscale SP.\u003c/p\u003e \u003cp\u003eIn the presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e the wastewater quality improved approximately 3 times faster as compared with the control without plants. The pH reduction in wastewater could be related to the release of protons during oxidation of NH\u003csub\u003e3\u003c/sub\u003e to nitrates and nitrites. These protons would be responsible for the acidification of the water observed in the presence of the plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Additionally, the lower pH could be attributed to root exudates. \u003cem\u003eS. americanus\u003c/em\u003e exudated aspartic acid, and \u003cem\u003eP. australis\u003c/em\u003e released oxalic and citric acids in response to salt (Haviland and Noyce \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rocha et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These exudates could be involved in the elimination of Na\u003csup\u003e+\u003c/sup\u003e through root exudates as reported in \u003cem\u003eArabidopsis\u003c/em\u003e (Contreras-Cornejo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe important reduction in nitrate and TN is attributed to a de-nitrification process, assimilation of NO\u003csub\u003e3\u003c/sub\u003e and the production of nitrogen (Soana et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Superior P and nitrate removal capacity exhibited by \u003cem\u003eP. australis\u003c/em\u003e may be attributable to its higher biomass production and more efficient nutrient extraction from surface water in comparison to \u003cem\u003eS. americanus\u003c/em\u003e. During the establishment of hydroponic cultures, it was observed that \u003cem\u003eP. australis\u003c/em\u003e demanded high levels of nutrients, as it required twice the concentration of salts (2 g/L) in the hydroponic medium to produce new shoots (data not shown).\u003c/p\u003e \u003cp\u003eThe results show that \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e were effective in improving the quality of moderately saline domestic wastewater, since significant reductions in nitrites (97.5%), nitrates (up to 84%), and orthophosphates (91.5%) were observed. Similar results have been described in the literature. Coleman et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) observed that \u003cem\u003eTypha, Juncus effuses\u003c/em\u003e, and \u003cem\u003eScirpus validus\u003c/em\u003e removed 50–70% of the TSS, BOD, TKN, ammonia, and phosphate from domestic wastewater. Another study, with 12 emergent macrophytes showed a removal efficiency of 68.4% for NH\u003csub\u003e4\u003c/sub\u003e, 70.4% for TN, and 53.7% for COD when plants were exposed to 0.5, 1, and 1.5% salinity (Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, \u003cem\u003eTrema orientalis, Acorus calamus\u003c/em\u003e, and \u003cem\u003eCyperus alternifolius\u003c/em\u003e decreased COD, TP, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, and NO\u003csub\u003e3\u003c/sub\u003e with efficiencies of 93.5%, 99.6%, 99.5%, and 98.9%, respectively, at low influent concentrations of NaCl (Cui et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It should be noted, however, that all of these studies were performed using substrates. It is documented that gravel alone improved 6 out of 10 water quality parameters (Coleman et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Additionally, Omidinia-Anarkoli and Shayannejad (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) revealed that an unplanted constructed wetland containing pumice and gravel exhibited the most significant reduction in TSS, biochemical oxygen demand, and COD when compared with a planted wetland. Thus, the higher removal efficiencies reported in those works could also be related to the substrate and not just the plants. In this study, salt removal experiments were conducted without substrates. Consequently, the improvement in wastewater quality is attributable solely to the plants.\u003c/p\u003e \u003cp\u003eWhen incorporating substrates into constructed wetlands, a number of factors must be considered, including the cost, availability, permeability, the potential for reuse, and safety concerns. For instance, the depth of ponds used usually ranged from 45 to 60 cm (Wang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Assuming a 1 m\u003csup\u003e3\u003c/sup\u003e of gravel weighs between 1.5 and 1.8 k (due to the characteristics of the material), 0.9 to 1.08 k of substrate are then utilized for each pond. In addition to the inherent costs associated with the materials, their use may also generate waste, if appropriate reuse strategies are not employed. The substrates used after wastewater treatment should not be discharged into the aquatic ecosystem unless such discharge will not have a detrimental effect on the environment. On the contrary, they must be removed and contained, which may require significant surface area (Legal Information Institute, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.law.cornell.edu/uscode/text/33/1344\u003c/span\u003e\u003cspan address=\"https://www.law.cornell.edu/uscode/text/33/1344\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The disposal of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants must be considered as well, as they may accumulate emerging contaminants, such as pharmaceuticals, personal care products and pesticides from domestic wastewater. Nevertheless, these plants can be incinerated for biofuel and biogas production, with the resulting ash being safely contained in a reduced space.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e compares the physicochemical parameters of the effluent when the treatment plant was operating and when it was out of operation, in the absence (control) or presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e on day 21. The values accepted by the Official Mexican Standard for wastewater discharges into water (NOM-001-SEMARNAT-2021), for public services (NOM-003-ECOL-1997), the WHO (2022) and the Agency for Toxic Substances and Disease Registry (ATSDR 2016) were also included. It is evident that the treatment plant's lack of maintenance substantially increased the pH, TSS, COD, nitrites and nitrates. In the presence of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants, the values of TN, nitrites, nitrates and orthophosphates decreased, and remained within the limits allowed by Mexican and international regulations. Since \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e showed a good nitrogen and phosphate removal capacity, they could be used for the phytoremediation of nitrogen-contaminated waters in real-world scenarios. The chemical, agri-food, textile and leather industries produce large amounts of saline wastewater thus the use of plants could be an option for treating the industries´s saline effluents and removing nitrogen and phosphorus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical analysis of the effluent from the treatment plant, during operation and out of operation, and the comparison with Mexican and international limits for pollutants in effluent discharges\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWTF\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eWTO\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eNOM-001\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eNOM-003\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP. australis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. americanus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRivers\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLakes\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAgricultural uses\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eIndustrial uses\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e°C\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.87\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e35.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eTA ± 5.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003e6.50–8.50\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSST\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e346\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e340\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTurbidity\u003c/p\u003e \u003cp\u003e(NTU)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.51\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.93\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003cp\u003e(µS/cm)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,630\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,526\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,102\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,250\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026lt; 700\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e -\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDQO\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e595.60\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,060\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,970\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,030\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.67\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrite\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e3.00\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e1.00\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e50.00\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e10.00\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP0\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e− 3\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e1\u003c/sup\u003eWTF: wastewater treatment plant in function; WTO: wastewater treatment plant out of service\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e2\u003c/sup\u003e World Health Organization (2022)\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003csup\u003e3\u003c/sup\u003e Agency for Toxic Substances and Disease Registry \u003csup\u003e(\u003c/sup\u003eATSDR)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eSeveral approaches can be employed to enhance water quality such as, using more \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e plants or larger plants. In this research, 50-cm plants were employed, but they can reach a height of about 3 m. Therefore, using larger plants could improve the uptake of salts. Another option would be to combine the two species to achieve a synergistic effect and increase the efficiency of phytoremediation, as previously reported (Coleman et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe present study demonstrated that \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e exhibited a decline in root development when exposed directly to moderately saline domestic wastewater. However, when the plants were exposed to the wastewater in a gradual manner, their growth parameters exhibited an improvement, suggesting that the plants had undergone a physiological adaptation. This adaptation enabled the plants tolerated prolonged exposure to undiluted wastewater (over 6 months). Both species were effective in removing nitrites, nitrates, and orthophosphates, as well as in stabilizing the pH of the wastewater within a relatively short period of time. Since the studies were carried out without substrates, the improvement in water quality can be attributed specifically to the plants. The incorporation of \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e into outfalls and wastewater treatment plants could reduce operating costs and enhance efficiency. This ecological phytoremediation approach could contribute to the reduction of contaminants in water bodies and provides a viable, low-cost wastewater management alternative for rural communities. Further studies are needed to determine if the plants are able to effectively colonize the SP waterbody, and how their presence would affect the biota of this area.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo animals or persons were used in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSarahi Josefina Estrada-Loredo, Rodolfo Cisneros-Almaz\u0026aacute;n, Gerson Alonso Soto-Pe\u0026ntilde;a, Alejandro Hern\u0026aacute;ndez-Morales and Mar\u0026iacute;a del Socorro Santos-D\u0026iacute;az\u003csup\u003e\u0026nbsp;\u003c/sup\u003egive their consent to participate in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSarahi Josefina Estrada-Loredo, Rodolfo Cisneros-Almaz\u0026aacute;n, Gerson Alonso Soto-Pe\u0026ntilde;a, Alejandro Hern\u0026aacute;ndez-Morales and Mar\u0026iacute;a del Socorro Santos-D\u0026iacute;az give their consent for the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSarahi Josefina Estrada-Loredo realized the experimental work, and participated on elaboration of figures and revision of manuscript. Rodolfo Cisneros-Almaz\u0026aacute;n participated in design, revision and discussion of results. Gerson Alonso Soto-Pe\u0026ntilde;a and Alejandro Hern\u0026aacute;ndez-Morales participated in the experimental design of the study, defining the methodologies, overseeing data acquisition and revision of manuscript. Mar\u0026iacute;a del Socorro Santos-D\u0026iacute;az\u003csup\u003e\u0026nbsp;\u003c/sup\u003eis the leader of the group, designed the project and experimental work, participates in revision, discussion, and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing interests that could influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and material are available at the Faculty of Chemistry of the \u0026ldquo;Universidad Aut\u0026oacute;noma de San Luis Potos\u0026iacute;\u0026rdquo;. https://repositorioinstitucional.uaslp.mx/xmlui/handle/i/8644\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the \u0026ldquo;Consejo Nacional de Humanidades Ciencia y Tecnolog\u0026iacute;a\u0026rdquo; for the scholarship to SJEL (no. 1181306) and Irvin R\u0026aacute;ul Ram\u0026iacute;rez-Morales and Ulises Ju\u0026aacute;rez-Mart\u0026iacute;nez for their technical assistance.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdjovu GE, Stephen H, James D, Ahmad S (2023) Measurement of total dissolved solids and total suspended solids in water systems: A review of the issues, conventional, and remote sensing techniques. Remote Sens 15: 3534. https://doi.org/10.3390/rs15143534\u003c/li\u003e\n\u003cli\u003eAlfaro-Salda\u0026ntilde;a EF, P\u0026eacute;rez-Molphe-Balch E, Santos-D\u0026iacute;az MS (2016) Generation of transformed roots of \u003cem\u003eScirpus americanus\u003c/em\u003e Pers. and study of their potential to remove Pb\u003csup\u003e2+\u003c/sup\u003e and Cr\u003csup\u003e3+.\u003c/sup\u003e Plant Cell Tiss Organ Cult 127: 15-24. https://doi.org/10.1007/s11240-016-1025-2\u003c/li\u003e\n\u003cli\u003eAgency for Toxic Substances and Disease ATSDR, may 6, 2016. https://www.atsdr.cdc.gov/es/phs/es_phs204.html#:~:text=condici%C3%B3n%20de%20salud.-,%C2%BFQu%\u003cbr\u003eC3%A9%20son%20el%20nitrato%20y%20el%20nitrito?,manufactura%20de%20municiones%20y%20explosivos.\u003c/li\u003e\n\u003cli\u003eBaeza K, Lopez-Hoffman L, Glenn E P, Flessa K, Garcia-Hernandez J (2013) Salinity limits of vegetation in Cienega de Santa Clara, an oligotrophic marsh in the delta of the Colorado River, Mexico: implications for an increase in salinity. 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J Integr Plant Biol 52: 468-475. https://doi.org/10.1111/j.1744-7909.2010.00947.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"wastewater, stabilization ponds, Schoenoplectus americanus, Phragmites australis, salt removal","lastPublishedDoi":"10.21203/rs.3.rs-5823958/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5823958/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eSchoenoplectus americanus\u003c/em\u003e and \u003cem\u003ePhragmites australis\u003c/em\u003e have a great potential for phytoremediation. In this study, the ability of these plants to improve the quality of moderately saline wastewater was tested. Both species were adapted to wastewater using two protocols. In the first, plants were directly exposed to undiluted or diluted wastewater at 12.5%, 25% and 50%. In the second protocol, the plants were gradually acclimated to 12.5%, and then to 25%, 50% diluted and undiluted wastewater for 20 days. Both processes were performed without using substrates. The efficiency of salt removal was assessed by employing plants adapted to undiluted wastewater over a period of 6 months. Direct exposure of \u003cem\u003eS. americanus\u003c/em\u003e to wastewater resulted in a 50% reduction in stem height in undiluted wastewater and an arrest of root development in 25%, 50% and 100% wastewater. An exudation of salts was observed in the stem in undiluted wastewater. Shoot formation was not significantly affected. Progressive exposure to wastewater improved stem length by 23% and shoot formation by 13% in 12.5% diluted wastewater. Direct and progressive exposure of \u003cem\u003ePhragmites australis\u003c/em\u003e to wastewater did not affect stem development, and increased the number of shoots (24\u0026ndash;30%). Root growth reduction was observed during direct exposure to wastewater. Both species improved wastewater quality by reducing 0.8 units pH, as well as the concentration of nitrite (98%), nitrate (50%-90%) and orthophosphate (50%-90%) after 21 days. Therefore, \u003cem\u003eS. americanus\u003c/em\u003e and \u003cem\u003eP. australis\u003c/em\u003e are a viable option for treating moderately saline wastewater.\u003c/p\u003e","manuscriptTitle":"Effects of Schoenoplectus americanus (Pers.) Volkart ex Schinz \u0026amp; R.Keller and Phragmites australis (Cav.) on the water quality improvement of moderately saline wastewater","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 05:36:42","doi":"10.21203/rs.3.rs-5823958/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2025-05-27T08:08:43+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-10T18:42:38+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T17:42:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Wetlands","date":"2025-04-04T04:30:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Wetlands","date":"2025-04-03T19:36:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99a522ce-0c25-4261-8cb2-2fe246a8094a","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T16:04:21+00:00","versionOfRecord":{"articleIdentity":"rs-5823958","link":"https://doi.org/10.1007/s13157-025-01965-1","journal":{"identity":"wetlands","isVorOnly":false,"title":"Wetlands"},"publishedOn":"2025-07-14 15:57:31","publishedOnDateReadable":"July 14th, 2025"},"versionCreatedAt":"2025-04-11 05:36:42","video":"","vorDoi":"10.1007/s13157-025-01965-1","vorDoiUrl":"https://doi.org/10.1007/s13157-025-01965-1","workflowStages":[]},"version":"v1","identity":"rs-5823958","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5823958","identity":"rs-5823958","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}