NaCl Improves Suaeda salsa Aniline Tolerance in Wastewater

: Halophytes have been studied as a model for morphological traits of adaptation to saline environments. However, little information has been given on plant growth, chlorophyll ﬂuorescence responses, and change of ion content in halophytes grown in an aniline–salinity coexistent environment. This study hypothesized that aniline could induce alterations in plant growth, chlorophyll ﬂuorescence, and ion content in Suaeda salsa , but salinity could promote the tolerance of halophytes to aniline. A 6 (aniline) × 3 (NaCl) factorial experiment (for a total of 18 treatments) was conducted to test the above hypothesis. After 30 d of cultivation, roots and shoots were harvested separately to analyze the e ﬀ ects of salinity on the seedling growth under aniline stress. Biomass accumulation was inhibited by aniline treatment, and the inhibition was signiﬁcantly alleviated by 200 mM NaCl. The change in chlorophyll ﬂuorescence in leaves with aniline stress was moderated by the addition of NaCl. The removal e ﬃ ciency of aniline was signiﬁcantly enhanced by moderate salinity. Aniline stress decreased the accumulation of Mg 2 + , but various concentrations of NaCl increased the accumulation of Mg 2 + , especially with 200 mM NaCl in both roots and shoots. Both aniline and salinity decreased the content of Ca 2 + . There was a negative correlation between the K + and NaCl concentrations and between the Cl − and aniline concentrations. Our results indicated that Suaeda salsa may be suitable for the remediation of salinity and aniline-enriched wastewater.


Introduction
Aniline (C 6 H 5 NH 2 ) is an important intermediate in the organic and fine chemical industry [1]. It is typically used for biochemical treatment of azo dye wastewater containing benzidine [2]. This is possibly one of the most important environmental issues [3]. Aniline discharged into water has strong toxicity and inhibits the growth of aquatic animals and plants [4]. It can directly or indirectly harm human health by migration through the food chain [5]. Plants show high sensitivity to aniline according to morphological and physiological aspects [6]. Aniline is also easily adsorbed or oxidized by soil into secondary intermediate products that are more toxic and difficult to degrade [7]. Therefore, aniline is listed as one of the serious organic pollutants that need to be strictly controlled in the environment [8]. Technologies have been proposed to remedy aniline-polluted wastewater [9,10], and plants are effective, convenient, and sensitive, thereby making them ideal indicator organisms for environmental pollutants and gene toxicity effects [11].
However, nearly 10% of dyestuffs in printing and dyeing wastewater are discharged into the environment as effluent after dying and processing [12]. They contain a large amount of salts and ultimately affect the well-being of aquatic ecosystems [13,14]. The damage effected by salt stress on glycophytes is manifested mainly in plant growth [15], limiting photosynthesis [16], ion toxicity, and osmotic stress by changing all aspects of plant physiology [17]. The mineral disturbance induced by the interaction between microelements and essential elements is also one of the causes of toxicity in plants [18]. However, halophytes can grow normally and are able to complete their seed-to-seed cycle at concentrations of 200 mM NaCl or higher [19,20]. Halophyte is a very promising phytoresource with many fundamental and potential uses [21].
Salinity and aniline in printing and dyeing wastewater place further constraints on phytoremediation. In addition to aniline tolerance, the plants used for this purpose must also bear the ion and osmotic stress of high NaCl loads. Suaeda salsa (S. salsa) is an annual halophyte that is widely distributed in China [22]. It was demonstrated that S. salsa can thrive in saline-alkali land [23] and reduce the soil salt content. This suggests that S. salsa may be a viable phytoremediator of saline-alkali land [24]. This species was studied as a model of morphological characteristics adapted to saline environments [25].
The selection of S. salsa was based on the idea that the cross-tolerance of halophytes is relatively straightforward [26]. Cross-tolerance refers to the phenomenon that the resistance of plants to stress causes the resistance to another stress. High concentrations of aniline in printing and dyeing wastewater resources may have a potentially adverse influence on the growth of S. salsa. However, studies indicating the biological physiology of aniline in halophytes have rarely been conducted. In this research, we used S. salsa as a candidate to investigate its cross-tolerance to aniline and salinity. This research provides not only a scientific basis for the evaluation of the toxic effects of aniline on halophytes, but also a technical reference for the adaptive strategy and mechanism of wastewater polluted by aniline and salinity.

Plant Growth Conditions
The experiment was performed in the greenhouse of Shaanxi University of Science and Technology with temperatures of 26 ± 2 • C/20 ± 2 • C in the day and night, respectively, and a light/dark period of 16 h/8 h. The seeds of the halophyte S. salsa were collected from saline soils in Fukang, Xinjiang (44 • 09 56 N, 87 • 50 55 E). The seeds of S. salsa were randomly selected for cultivation and germinated in an incubator at 25 • C. When the seedlings reached a height of 6-8 cm, seedlings with uniform size were transplanted into clean culture bottles with 1/4 Hoagland's solution (200 mL) for cultivation.

Experimental Design
The stress treatments were started 1 week after the transplantation. The basic irrigation medium was1/4-Hoagland's solution. Salt was supplied with sodium chloride (NaCl) at 0, 100, and 200 mM, the concentration of NaCl solution was increased by 50 mM every day. After 2 weeks of NaCl treatment, 0, 1, 2, 4, 6, and 8 mg·L −1 of aniline (Tianjin Chemical Reagent Factory, Tianjin, China) was added, and then the cultures were incubated for 2 weeks before harvesting. Throughout the process, the exposure solution was replaced daily and completely shielded from light. The experiment was a 3 (NaCl) × 6 (aniline) factorial combination, making a total of 18 treatments, four biological replicates. Each treatment was performed in quadruplicate. The light intensity of the artificial climate chamber (RZX-500C Intelligent Artificial Climate Chamber, Ningbo Jiangnan Instrument Factory) was 12,000 lx.

Plant Growth Measurements
Plants were harvested after washing with deionized water [27]. The plants were divided into root and shoot parts. The dry weight (DW) of the plant was measured after oven-drying the samples at 70 • C for 48 h.

Shoot and Root Elemental Analysis
Dry plant material was ground into fine powder in an agate mortar, and 0.05 g of powder sample was digested using a mixture of HNO 3 :H 2 SO 4 :HClO 4 (10.0:1.0:0.5; v/v/v) for 2.5 h at 110 • C. After cooling, the digestion products were filtered and diluted with distilled water to a total volume of 50 mL. The total concentrations of Na, K, Ca, and Mg were determined by atomic absorption spectrometry (ZEEnit 700P, Analytik Jena AG, Jena, Germany). The concentration of Cl was determined by ionchromatography IC (940 Professional IC Vario, Metrohm, Herisau, Switzerland) [28].
To determine the aniline in water (collected under the concentration of aniline 1, 2, 4, 6, and 8 mg·L −1 ) by HPLC (515, Waters, USA) [29], 900 µL of the sample was withdrawn, immediately filtered with a 0.22 µm filter membrane, and collected in an HPLC vial mixed with 100 µL of acetonitrile to obtain the liquid to be measured. An HPLC 2478 dual-wavelength UV detector was used to determine aniline in the sample. The chromatographic conditions included a wavelength of 240 nm, mobile phase of methanol:water (35:65; v/v), mobile phase pH of 3.0, flow rate of 0.8 mL·min −1 , and injection volume of 10 µL. Each sample was measured three times (Supplemental Figures S1 and S2).

Chlorophyll Fluorescence and Leaf Pigment Content Analysis
Determination of leaf chlorophyll (Chl) fluorescence was made by pulse-amplitude-modulation fluorometer (PAM 2100, H. Walz, Effeltrich, Germany). Before measuring the Chl fluorescence parameters, leaves were placed in a dark-adapted state for 30 min using light exclusion clips. After irradiating the measuring light (1 µmol·m −2 ·S −1 ) and saturating illuminations (3000 µmol·m −2 ·S −1 ), the minimum Chl fluorescence yield in the dark-adapted state (F 0 ) and maximum Chl fluorescence yield in the dark-adapted state (F m ) were measured. After irradiating the light (150 µmol·m −2 ·S −1 ) and saturating illuminations, we measured the minimum Chl fluorescence yield in the light-adapted state (F 0 ), maximum Chl fluorescence yield in the light-adapted state (F m ), and steady-state Chl fluorescence yield in the light-adapted state (F s ). Using these parameters, we calculated the maximum photosystem II (PSII) photochemical efficiency (F v /F m = (F m − F 0 )/F m ) and quantum yield of PSII electron transport (ΦPSII = (F m − F s )/F m ) [31]. The Chl-a, Chl-b, and total Chl were extracted based on the method of Lichtenthaler (1987) [32].

Statistical Analyses
Statistical analyses were conducted with analysis of variance (ANOVA) using the statistical software package SPSS 17.0 (SPSS Inc, version 17.0). The least significant difference (LSD) test was applied at 5% probability for the ranking and appraisal of treatment means. The normality and homogeneity of variances were tested before analysis. For the ion content, each treatment group (Aniline alone, Aniline + 100 mM NaCl, and Aniline + 200 mM NaCl) was separately compared with control. Redundancy analysis (RDA) in the CANOCO package was used for evaluation, and the parametric test was replaced by the Monte Carlo test to evaluate the responses of all parameters.

Alleviation of the Effects Caused by Aniline on the Growth of S. salsa by Salinity
S. salsa, as a representative halophyte, was used to investigate the toxicity of aniline. As shown in Figure 1, 1-8 mg·L −1 of aniline significantly (p = 0.003) reduced the DW of shoots, and 4-8 mg·L −1 of aniline reduced the DW of roots; however, 1-2 mg·L −1 of aniline increased the DW of roots. Therefore, the stress of aniline on roots showed the phenomenon of "low promotion and high suppression" (Figure 1). Specifically, the root and shoot DW decrease under 8 mg·L −1 of aniline was about 9% and 25%, compared to the control under non-aniline stress ( Figure 1). Furthermore, 200 mM NaCl increased the root DW by 59% and shoot DW by 27%, compared with those of the control under non-aniline stress. There were significant interactions between the effects of NaCl and aniline on the root (p = 0.008) and shoot (p = 0.05) DW according to the ANOVA (Table S1). The DW of roots under the combined stresses of aniline and NaCl was higher than that under individual aniline stress (p = 0.003), thereby indicating that NaCl alleviated the decrease in root DW caused by aniline (1-8 mg·L −1 ). There were also significant interactions between the effects of NaCl and aniline on shoot DW (p = 0.001) ( Table S1). The results suggested that 200 mM NaCl alleviated the toxicity of aniline. The interactions between aniline and NaCl showed a greater effect on S. salsa shoot and root growth ( Figure 1). S. salsa, as a representative halophyte, was used to investigate the toxicity of aniline. As shown in Figure 1, 1-8 mg·L −1 of aniline significantly (p = 0.003) reduced the DW of shoots, and 4-8 mg·L −1 of aniline reduced the DW of roots; however, 1-2 mg·L −1 of aniline increased the DW of roots. Therefore, the stress of aniline on roots showed the phenomenon of "low promotion and high suppression" (Figure 1). Specifically, the root and shoot DW decrease under 8 mg·L −1 of aniline was about 9% and 25%, compared to the control under non-aniline stress ( Figure 1). Furthermore, 200 mM NaCl increased the root DW by 59% and shoot DW by 27%, compared with those of the control under non-aniline stress. There were significant interactions between the effects of NaCl and aniline on the root (p = 0.008) and shoot (p = 0.05) DW according to the ANOVA (Table S1). The DW of roots under the combined stresses of aniline and NaCl was higher than that under individual aniline stress (p = 0.003), thereby indicating that NaCl alleviated the decrease in root DW caused by aniline (1-8 mg·L −1 ). There were also significant interactions between the effects of NaCl and aniline on shoot DW (p = 0.001) ( Table S1). The results suggested that 200 mM NaCl alleviated the toxicity of aniline. The interactions between aniline and NaCl showed a greater effect on S. salsa shoot and root growth ( Figure 1). Figure 1. The effect of NaCl on the shoot and root dry weight (DW) (mg) of S. salsa under various aniline stress. A, aniline; N, NaCl; * means with different uppercase letters indicate significant difference (p < 0.05) between NaCl treatments at a particular aniline level. ** Means with different lowercase letters are significantly different (p < 0.01) between aniline treatments at a particular salinity level. *** Means denoted by the different letters are significantly different at a p < 0.001 according to the LSD test.

Increase in the Removal Efficiency of Aniline in S. Salsa Seedlings by Salinity
Various initial concentrations (1, 2, 4, 6, and 8 mg·L −1 ) of aniline solution were prepared to investigate the effect of the initial concentration on aniline removal efficiency (RE) under 0, 100, and 200 mM NaCl treatments. As shown in Figure 2, with the treatment of 0 mM NaCl, the RE of aniline gradually decreased with the increase in its initial concentration from the highest efficiency (35.1%) with the 1 mg·L −1 aniline treatment to the lowest efficiency (7.3%) with the 8 mg·L −1 aniline treatment. The effect of NaCl on the shoot and root dry weight (DW) (mg) of S. salsa under various aniline stress. A, aniline; N, NaCl; * means with different uppercase letters indicate significant difference (p < 0.05) between NaCl treatments at a particular aniline level. ** Means with different lowercase letters are significantly different (p < 0.01) between aniline treatments at a particular salinity level. *** Means denoted by the different letters are significantly different at a p < 0.001 according to the LSD test.

Increase in the Removal Efficiency of Aniline in S. Salsa Seedlings by Salinity
Various initial concentrations (1, 2, 4, 6, and 8 mg·L −1 ) of aniline solution were prepared to investigate the effect of the initial concentration on aniline removal efficiency (RE) under 0, 100, Sustainability 2020, 12, 7457 5 of 16 and 200 mM NaCl treatments. As shown in Figure 2, with the treatment of 0 mM NaCl, the RE of aniline gradually decreased with the increase in its initial concentration from the highest efficiency (35.1%) with the 1 mg·L −1 aniline treatment to the lowest efficiency (7.3%) with the 8 mg·L −1 aniline treatment. The regression analysis demonstrated a relationship between the RE (RE%) (y) of plants of treated groups (aniline) and the initial concentration (x) (R 2 = 0.906) ( Figure 2, Figure S3a). At 100 mM NaCl, the RE of 1 mg·L −1 of aniline was promoted by 15.8% compared with that of the non-NaCl treatment (R 2 = 0.982) ( Figure 2, Figure S3a). With the supplementation of 200 mM NaCl, the RE of 1 mg·L −1 of aniline was increased by 32.46% compared with that of the non-NaCl treatment ( Figure 2, Figure S3b) (R 2 = 0.995). The results indicated that moderate NaCl can significantly enhance the RE of aniline in wastewater. The regression analysis demonstrated a relationship between the RE (RE%) (y) of plants of treated groups (aniline) and the initial concentration (x) (R 2 = 0.906) ( Figure 2, Figure S3a). At 100 mM NaCl, the RE of 1 mg·L −1 of aniline was promoted by 15.8% compared with that of the non-NaCl treatment (R 2 = 0.982) ( Figure 2, Figure S3a). With the supplementation of 200 mM NaCl, the RE of 1 mg·L −1 of aniline was increased by 32.46% compared with that of the non-NaCl treatment ( Figure 2, Figure S3b) (R 2 = 0.995). The results indicated that moderate NaCl can significantly enhance the RE of aniline in wastewater.

Salinity Moderation of the Decrease in Leaf Pigment Contents Due to Aniline
Chl, as the important growth indicator, was analyzed with the application of NaCl and aniline. As shown in Figure 3a, the significant differences in Chl-a content under 1 mg·L −1 aniline were caused by 200 mM NaCl, compared with 0 and 100 mM NaCl. As the aniline content increased, the Chl-a content decreased, and NaCl significantly alleviated this effect. With the application of 200 mM NaCl, the content of Chl-a increased by 9.60%, 10.80%, 18.03%, and 34.80%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the aniline control ( Figure 3a). There was no significant effect on the content of Chl-b under 1 and 2 mg·L −1 aniline treatment. The Chl-b content decreased with the increase in aniline, and NaCl also significantly alleviated this decrease under 4-8 mg·L −1 aniline ( Figure 3b). With the application of 200 mM NaCl, the content of Chl-b increased by 2.90%, 6.60%, 9.60%, and 18.13%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the aniline control ( Figure 3b). The ratio of Chl a/b did not significantly decrease with the 1, 2, and 4 mg·L −1 mg·L −1 aniline

Salinity Moderation of the Decrease in Leaf Pigment Contents Due to Aniline
Chl, as the important growth indicator, was analyzed with the application of NaCl and aniline. As shown in Figure 3a, the significant differences in Chl-a content under 1 mg·L −1 aniline were caused by 200 mM NaCl, compared with 0 and 100 mM NaCl. As the aniline content increased, the Chl-a content decreased, and NaCl significantly alleviated this effect. With the application of 200 mM NaCl, the content of Chl-a increased by 9.60%, 10.80%, 18.03%, and 34.80%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the aniline control (Figure 3a). There was no significant effect on the content of Chl-b under 1 and 2 mg·L −1 aniline treatment. The Chl-b content decreased with the increase in aniline, and NaCl also significantly alleviated this decrease under 4-8 mg·L −1 aniline (Figure 3b). With the application of 200 mM NaCl, the content of Chl-b increased by 2.90%, 6.60%, 9.60%, and 18.13%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the aniline control ( Figure 3b). The ratio of Chl a/b did not significantly decrease with the 1, 2, and 4 mg·L −1 mg·L −1 aniline treatments. With the application of 200 mM NaCl, the ratio of Chl a/b increased by 8.2% and 13.4%, under 6 and 8 mg·L −1 , respectively, compared with that of the aniline treatment (Figure 3c). treatments. With the application of 200 mM NaCl, the ratio of Chl a/b increased by 8.2% and 13.4%, under 6 and 8 mg·L −1 , respectively, compared with that of the aniline treatment ( Figure 3c).

Photosystem II Parameters Altered by Aniline and Salinity
To determine whether the NaCl and aniline treatments affected the efficiency of photosynthesis, we measured the maximal efficiency of photochemistry of PSII (Fv/Fm) and ΦPSII. NaCl treatment had very little effect on Fv/Fm (Figure 4a). This suggested that the PSII of S. salsa seedlings was tolerant to NaCl, and NaCl stress had no effect on PSII photochemistry in dark-adapted leaves. However, with the 4, 6, and 8 mg·L −1 aniline treatments, the Fv/Fm of the PSII was significantly reduced by 41.35%, 44.40%, and 47.02%, respectively, compared with that of the control (Figure 4a). Without aniline stress, NaCl showed no effect on Fv/Fm, but under the stress of high concentrations of aniline (4, 6, and 8 mg·L −1 ), 200 mM NaCl displayed a positive promoting effect on Fv/Fm (Figure 4a). The results showed that NaCl counteracted the aniline-induced inhibition of the photochemical activity of PSII. ΦPSII is the product of Fv/Fm, which reflects the actual photochemical reaction efficiency. ΦPSII increased by 5.2% and 12.3% under the 100 mM and 200 mM NaCl treatments (Figure 4b), respectively, which indicated that photosynthetic electron transport was increased under NaCl

Photosystem II Parameters Altered by Aniline and Salinity
To determine whether the NaCl and aniline treatments affected the efficiency of photosynthesis, we measured the maximal efficiency of photochemistry of PSII (F v /F m ) and ΦPSII. NaCl treatment had very little effect on F v /F m (Figure 4a). This suggested that the PSII of S. salsa seedlings was tolerant to NaCl, and NaCl stress had no effect on PSII photochemistry in dark-adapted leaves. However, with the 4, 6, and 8 mg·L −1 aniline treatments, the F v /F m of the PSII was significantly reduced by 41.35%, 44.40%, and 47.02%, respectively, compared with that of the control (Figure 4a). Without aniline stress, NaCl showed no effect on F v /F m , but under the stress of high concentrations of aniline (4, 6, and 8 mg·L −1 ), 200 mM NaCl displayed a positive promoting effect on F v /F m (Figure 4a). The results showed that NaCl counteracted the aniline-induced inhibition of the photochemical activity of PSII. treatments. With the application of 200 mM NaCl, the ratio of Chl a/b increased by 8.2% and 13.4%, under 6 and 8 mg·L −1 , respectively, compared with that of the aniline treatment (Figure 3c).

Photosystem II Parameters Altered by Aniline and Salinity
To determine whether the NaCl and aniline treatments affected the efficiency of photosynthesis, we measured the maximal efficiency of photochemistry of PSII (Fv/Fm) and ΦPSII. NaCl treatment had very little effect on Fv/Fm (Figure 4a). This suggested that the PSII of S. salsa seedlings was tolerant to NaCl, and NaCl stress had no effect on PSII photochemistry in dark-adapted leaves. However, with the 4, 6, and 8 mg·L −1 aniline treatments, the Fv/Fm of the PSII was significantly reduced by 41.35%, 44.40%, and 47.02%, respectively, compared with that of the control (Figure 4a). Without aniline stress, NaCl showed no effect on Fv/Fm, but under the stress of high concentrations of aniline (4, 6, and 8 mg·L −1 ), 200 mM NaCl displayed a positive promoting effect on Fv/Fm (Figure 4a). The results showed that NaCl counteracted the aniline-induced inhibition of the photochemical activity of PSII. ΦPSII is the product of Fv/Fm, which reflects the actual photochemical reaction efficiency. ΦPSII increased by 5.2% and 12.3% under the 100 mM and 200 mM NaCl treatments (Figure 4b), respectively, which indicated that photosynthetic electron transport was increased under NaCl ΦPSII is the product of F v /F m , which reflects the actual photochemical reaction efficiency. ΦPSII increased by 5.2% and 12.3% under the 100 mM and 200 mM NaCl treatments (Figure 4b), respectively, which indicated that photosynthetic electron transport was increased under NaCl treatment. ΦPSII Sustainability 2020, 12, 7457 7 of 16 was significantly reduced under aniline stress in a dose-dependent manner. ΦPSII was the lowest under 0 mM NaCl treatment and 8 mg·L −1 aniline stress. The decrease in the ΦPSII value caused by 8 mg·L −1 of aniline was 55.20%, but after the supplementation of 200 mM NaCl, the observed decrease was only approximately 20.73%. These results indicated NaCl alleviated the decrease in ΦPSII due to aniline stress.
The photochemical quenching coefficient (qP) measures the proportion of PSII reaction centers capable of photochemistry [33]. There was no significant effect on qP under the 1 mg·L −1 aniline treatment. The qP decreased with the increase in aniline, and 200 mM NaCl significantly alleviated the decrease by 8.90%, 6.40%, 16.80%, and 22.51%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the control ( Figure S4). These results indicated that the application of NaCl increased the quantum yield and protected the photosynthetic system from aniline damage.

Cl − Concentration in S. salsa Tissues
Both the root and shoot Cl − concentrations were increased by the individual effects of NaCl (Figure 5c, Figure 6c). Specifically, 200 mM NaCl increased the root and shoot Cl − concentrations by 82.05% and 80.80%, respectively, and 100 mM NaCl increased the root and shoot Cl − concentrations by 66.91% and 61.56%, respectively. Furthermore, 4-8 mg·L −1 of aniline significantly decreased the shoot Cl − concentrations under 100 mM NaCl, but in the roots, 2, 4, 6, and 8 mg·L −1 of aniline significantly decreased the Cl − concentrations under the 100 mM NaCl treatment (p = 0.001). In the treatment with 200 mM NaCl, aniline significantly decreased the shoot Cl − concentrations, and decreased the shoot Cl − concentrations under 8 mg·L −1 aniline (Figure 5c, Figure 6c). These results indicated that aniline can inhibit the accumulation of Cl − in S. salsa seedlings.
Sustainability 2020, 12, x FOR PEER REVIEW 7 of 16 treatment. ΦPSII was significantly reduced under aniline stress in a dose-dependent manner. ΦPSII was the lowest under 0 mM NaCl treatment and 8 mg·L −1 aniline stress. The decrease in the ΦPSII value caused by 8 mg·L −1 of aniline was 55.20%, but after the supplementation of 200 mM NaCl, the observed decrease was only approximately 20.73%. These results indicated NaCl alleviated the decrease in ΦPSII due to aniline stress. The photochemical quenching coefficient (qP) measures the proportion of PSII reaction centers capable of photochemistry [33]. There was no significant effect on qP under the 1 mg·L −1 aniline treatment. The qP decreased with the increase in aniline, and 200 mM NaCl significantly alleviated the decrease by 8.90%, 6.40%, 16.80%, and 22.51%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the control ( Figure S4). These results indicated that the application of NaCl increased the quantum yield and protected the photosynthetic system from aniline damage.

Cl -Concentration in S. Salsa Tissues
Both the root and shoot Clconcentrations were increased by the individual effects of NaCl (Figure 5c, Figure 6c). Specifically, 200 mM NaCl increased the root and shoot Clconcentrations by 82.05% and 80.80%, respectively, and 100 mM NaCl increased the root and shoot Clconcentrations by 66.91% and 61.56%, respectively. Furthermore, 4-8 mg·L −1 of aniline significantly decreased the shoot Clconcentrations under 100 mM NaCl, but in the roots, 2, 4, 6, and 8 mg·L −1 of aniline significantly decreased the Clconcentrations under the 100 mM NaCl treatment (p = 0.001). In the treatment with 200 mM NaCl, aniline significantly decreased the shoot Clconcentrations, and decreased the shoot Clconcentrations under 8 mg·L −1 aniline (Figure 5c, Figure 6c). These results indicated that aniline can inhibit the accumulation of Clin S. salsa seedlings. Figure 5. Effect of NaCl on shoot elemental Na + (a), K + (b), Cl -(c), K + / Na + (d), Ca 2+ (e), and Mg 2+ (f) concentrations (mmol·g −1 DW) of S. salsa exposed to aniline. For each parameter, the data are the means ± SE of five replications, with standard errors indicated by the vertical bars. Means denoted by the different letters are significantly different at a p < 0.05 according to the LSD test, the different letters indicate statistically significant differences within different NaCl treatments.

Aniline Inhibition of Na + Absorption and Change in K + /Na + in S. Salsa Seedlings
The NaCl supply significantly increased the root and shoot Na + concentrations (p < 0.01); Na + increased by 34.68% and 195.70% in the shoots and by 59.90% and 86.36% in the roots with a supply Figure 5. Effect of NaCl on shoot elemental Na + (a), K + (b), Cl − (c), K + / Na + (d), Ca 2+ (e), and Mg 2+ (f) concentrations (mmol·g −1 DW) of S. salsa exposed to aniline. For each parameter, the data are the means ± SE of five replications, with standard errors indicated by the vertical bars. Means denoted by the different letters are significantly different at a p < 0.05 according to the LSD test, the different letters indicate statistically significant differences within different NaCl treatments. by 11.17% and 9.05%, under 6 and 8 mg·L −1 , respectively, compared with no NaCl within corresponding aniline concentration. There were interactions between the effects of NaCl and aniline on the shoot and root Na + (p < 0.01), K + (p < 0.01), and K + /Na + ratio (p < 0.01) (Table S1). In addition, the K + /Na + ratio reached homeostasis with NaCl supplementation under aniline stress (Figure 5a, Figure  6a). Figure 6. Effect of NaCl on root elemental Na + (a), K + (b), Cl -(c), K + / Na + (d), Ca 2+ (e) and Mg 2+ (f) concentrations (mmol·g −1 DW) of S. salsa exposed to aniline. For each parameter, the data are the means ± SE of five replications, with standard errors indicated by the vertical bars. Means denoted by the different letters are significantly different at a p < 0.05 according to the LSD test, the different letters indicate statistically significant differences within different NaCl treatments.

NaCl Alleviation of the Decrease in Ca 2+ and Mg 2+ Caused by Aniline in S. Salsa
Ca 2+ and Mg 2+ are essential elements for plant growth. The intracellular Ca 2+ concentration can regulate ion transport across the membrane via its role in cell wall and membrane integrity [34]. Aniline stress significantly influenced the shoot and root Ca 2+ concentrations, which shows that 4-8 Figure 6. Effect of NaCl on root elemental Na + (a), K + (b), Cl − (c), K + / Na + (d), Ca 2+ (e) and Mg 2+ (f) concentrations (mmol·g −1 DW) of S. salsa exposed to aniline. For each parameter, the data are the means ± SE of five replications, with standard errors indicated by the vertical bars. Means denoted by the different letters are significantly different at a p < 0.05 according to the LSD test, the different letters indicate statistically significant differences within different NaCl treatments.

Aniline Inhibition of Na + Absorption and Change in K + /Na + in S. salsa Seedlings
The NaCl supply significantly increased the root and shoot Na + concentrations (p < 0.01); Na + increased by 34.68% and 195.70% in the shoots and by 59.90% and 86.36% in the roots with a supply of 100 mM and 200 mM NaCl, respectively (Figure 5a, Figure 6a). In shoots, 1-2 mg·L −1 of aniline showed no effect on the Na + content, while 6 mg·L −1 and 8 mg·L −1 of aniline clearly decreased the Na + content under the treatments of 100 mM and 200 mM NaCl (Figure 5a). However, aniline had no effect on the content of Na + in the roots (Figure 6a). In addition, the increments/decrements caused by NaCl were less with 8 mg·L −1 of aniline than those with 1-2 mg·L −1 and 4-6 mg·L −1 of aniline ( Figure 6), thereby indicating that 8 mg·L −1 of aniline inhibited NaCl absorption. NaCl significantly (p < 0.01) decreased the root K + /Na + ratio in comparison with that caused by individual aniline stress, especially for the 8 mg·L −1 aniline treatment.
There was no significant effect on shoot K + concentrations under the 1 mg·L −1 , 2 mg·L −1 , and 4 mg·L −1 aniline treatments. With the application of 200 mM NaCl, the K + concentrations decreased by 23.4%, 17.5%, and 15%, under 4, 6, and 8 mg·L −1 , respectively, compared with no NaCl within corresponding aniline concentration There was no significant effect on the root K + concentrations under the 1, 2, and 4 mg·L −1 aniline treatments, and the root K + concentrations decreased with the increase in aniline (Figure 6b). With the application of 200 mM NaCl, the K + concentrations increased by 11.17% and 9.05%, under 6 and 8 mg·L −1 , respectively, compared with no NaCl within corresponding aniline concentration. There were interactions between the effects of NaCl and aniline on the shoot and root Na + (p < 0.01), K + (p < 0.01), and K + /Na + ratio (p < 0.01) (Table S1). In addition, the K + /Na + ratio reached homeostasis with NaCl supplementation under aniline stress (Figure 5a, Figure 6a).

NaCl Alleviation of the Decrease in Ca 2+ and Mg 2+ Caused by Aniline in S. salsa
Ca 2+ and Mg 2+ are essential elements for plant growth. The intracellular Ca 2+ concentration can regulate ion transport across the membrane via its role in cell wall and membrane integrity [34]. Aniline stress significantly influenced the shoot and root Ca 2+ concentrations, which shows that 4-8 mg·L −1 aniline affects the shoot and 6-8 mg·L −1 aniline affects the Ca 2+ concentration of S. salsa root (Figure 5e, Figure 6e). With the application of 200 mM NaCl, the shoot Ca 2+ concentration decreased by 4%, compared with that of the non-NaCl treatment (p > 0.05) (Figure 5e). Nevertheless, there was no significant effect on the root Ca 2+ concentration under the 1 mg·L −1 aniline treatment. The root Ca 2+ concentration decreased with the increase in aniline, but 200 mM NaCl increased the root Ca 2+ concentration by 1.60%, 11.10%, 12.03%, and 11.48%, under 2, 4, 6, and 8 mg·L −1 , respectively, compared with that of the control. The results suggested that 200 mM NaCl alleviated the Ca 2+ reduction in the shoots and roots of S. salsa seedlings under high aniline (8 mg·L −1 ) concentration.
Mg in tissues is closely related to the salt tolerance of halophytes. The direct contribution of Mg indicates the succulent characteristics of S. salsa cells, which may be used as an isolation pool for salt ions [35]. Both the shoot and root Mg 2+ concentrations increased with NaCl supply (100 mM and 200 mM) (Figure 5f, Figure 6f). The 8 mg·L −1 aniline treatment decreased the shoot Mg 2+ concentration by 15.24%, 7.86%, and 1.23% under the 0, 100, and 200 mM NaCl treatments, respectively (Figure 5f), and also decreased the root Mg 2+ concentration by 4.72%, 4.45%, and 2.73% under the 0, 100, and 200 mM NaCl treatments, respectively (Figure 6f). The results indicated that aniline stress inhibited Mg 2+ accumulation in S. salsa and the supply of NaCl alleviated the inhibition of the Mg 2+ concentration caused by aniline.

Ordination Analysis
Redundancy analysis (RDA) is used to detect the relationship between environmental factors and samples, and important environmental driving factors affecting sample distribution can be obtained [35][36][37]. Figure 7 indicates the relationship between visual response and explanatory variables (aniline and NaCl) (Figure 7a,b), which is based on a linear model. The Monte Carlo test with forward selection of variables was conducted, and highly significant conditional effects of all explanatory variables (p = 0.002) were indicated, with NaCl showing the strongest effect, followed by salinity on root and shoot (F = 85.45 and 70.85, respectively). The response variables were divided into variables related to plant growth (including root DW and shoot DW) and ion concentrations in shoots and roots (including Cl − , K + , Na + , K + /Na + , Ca 2+ , and Mg 2+ ).
There were several positive and negative trends in the relationships between the ion balance-related variables and explanatory variables. The K + and Na + concentrations in the plants had the strongest response to NaCl (Figure 7a,b). There was a significantly positive correlation between the Na + concentrations and NaCl, while a negative correlation between the K + concentrations and NaCl was also observed, but was not significant. The K + /Na + ratio was also negatively correlated with NaCl. The Cl − concentrations were negatively correlated with aniline, and most correlations were not significant. The Ca 2+ concentrations were negatively correlated with aniline and Mg 2+ was negatively correlated with NaCl and aniline, but most correlations were not significant. Meanwhile, there was a strong correlation between plant growth and NaCl.
The ANOVA rendered similar results to those of the RDA (Table S1). The results indicated that the elemental composition of S. salsa is related to salt-tolerance categories, and this relationship is mainly driven by elements directly related to salinity, such as Na, Cl, Mg, K, and Ca.
with forward selection of variables was conducted, and highly significant conditional effects of all explanatory variables (p = 0.002) were indicated, with NaCl showing the strongest effect, followed by salinity on root and shoot (F = 85.45 and 70.85, respectively). The response variables were divided into variables related to plant growth (including root DW and shoot DW) and ion concentrations in shoots and roots (including Cl − , K + , Na + , K + /Na + , Ca 2+ , and Mg 2+ ). ; Na, Na + concentration; K, K + concentration; K/Na, K + /Na + ratio; Ca, Ca 2+ concentration; Mg, Mg 2+ concentration; environmental variables: A, aniline; S, salinity. Partial effects of all explanatory variables were highly significant; (a) A, and S explained 12.87%, and 85.4% of data variability, respectively. (b) Partial effects of all explanatory variables were highly significant; A and S explained 28.1% and 70.8% of data variability, respectively.
There were several positive and negative trends in the relationships between the ion balancerelated variables and explanatory variables. The K + and Na + concentrations in the plants had the ; Na, Na + concentration; K, K + concentration; K/Na, K + /Na + ratio; Ca, Ca 2+ concentration; Mg, Mg 2+ concentration; environmental variables: A, aniline; S, salinity. Partial effects of all explanatory variables were highly significant; (a) A, and S explained 12.87%, and 85.4% of data variability, respectively. (b) Partial effects of all explanatory variables were highly significant; A and S explained 28.1% and 70.8% of data variability, respectively.

S. salsa Absorbance and Utilization of Salinity of Wastewater and Removal of Aniline
NaCl inhibited the growth of glycophytes, and the damage mainly included the comprehensive response of osmotic stress, ion toxicity, and mineral nutrient deficiency [34,[38][39][40]. Halophytes can grow normally and are able to complete their seed-to-seed cycle at 200 mM NaCl or higher [19]. For most halophytes, low salinity promotes plant growth, but if the NaCl level reaches higher than 200-400 mM, halophytes will die [24,41]. In this study, optimal growth of S. salsa occurred at 200 mM NaCl. This suggested that the salt tolerance of S. salsa is extremely broad, and S. salsa is more adapted to high salinity conditions. The 200 mM NaCl treatment significantly promoted the growth of S. salsa (Figure 1), the increase in these indicators of the root system affected the growth of S. salsa. This may mainly be attributed to the relationship between Na + and Cl − , which can clearly induce leaf succulence of the species [42].
Aniline is often detected in aquatic environments with higher concentrations in aquatic organisms owing to its strong lipophilic properties and low water solubility [8,43]. The environmental fate of aniline is mostly dependent on the molecular size. Generally, the octanol-water partition coefficients of aniline range from 0.5-3.0 (log Kow = 0.5-3.0). In this study, S. salsa removed aniline and salt improved the RE (Figure 2) because of the lower lipophilicity and lower metabolic ability of the plants [44,45]. This may have happened because the vacuole isolation of halophytes is not only limited to salt ions, but also extended to a kind of toxic ions and organic pollutants [46,47]. The strong subcellular isolation of toxic ions and compounds by S. salsa is an important feature of its salt tolerance and stress of organic pollutants [48,49]. It has been reported that phenanthrene and pyrene are transported via the apoplastic pathway [50]. The transportation of organic pollutants in plant phloem and xylem depends on the size of the molecule. It can be speculated that, because halophytes partition salt ions more efficiently than glycophytes, theoretically they can isolate any other toxic ions or compounds.

Moderate Salinity Enhancement of Protection of Photosystem II under High Aniline Stress in S. salsa
Chl-a is a critical component for light-harvesting and electron transfer in photosynthesis, which can regulate light absorption, transition, and distribution. A previous study indicated that NaCl increases the content of Chl and the Chl a/b ratio, which is the positive response of halophytes to the NaCl environment [51]. A higher Chl content resulted in higher photochemical efficiency of PSII and then higher production (Figure 3, Figure 4). Chl a/b reflects the stacking extent of the thylakoid membrane, and the increase is often interpreted as a sign of less stacking [52]. In this study, aniline significantly reduced the content of Chl-a, and with the lower Chl a/b ratio, the extent of thylakoid stacking and the content of the light harvesting complex were greater. However, with the supply of 200 mM NaCl, the harvested light energy can be used adequately and it is more difficult for photoinhibition to occur.
PSII is believed to play a key role in leaf photosynthesis in responses to environmental perturbations [53]. In order to investigate if NaCl can counteract the aniline-induced inhibition of the photochemical activity of PSII, we detected the maximum PSII photochemical efficiency (F v /F m ). Aniline decreased F v /F m by 47.1%, but the 200 mM NaCl treatment alleviated this decrease by 19.9% (Figure 4a). There were differences in F v /F m with different levels of aniline stress, and non-stress further indicated that aniline stress induced the increase in susceptibility of PSII to photoinhibition. However, no changes in F v /F m were observed between the 200 mM NaCl-treated and non-stressed plants measured at predawn. For S. salsa, the unsaturated fatty acids in membrane lipids protect the photosynthesis system against stress by alleviating the photoinhibition of PSII and PSI; meanwhile, high salinity can increase the unsaturated fatty acids content of membrane lipids [51,54].
We observed a significant decrease in ΦPSII at the 8 mg·L −1 aniline level (Figure 4b), which indicated the impairment of the light-harvesting complex of ΦPSII [55], thereby resulting in the decrease in heat dissipation depending on the xanthophyll cycle [56] and significant damage to the photosynthetic system of PSII in plants. We also noticed that the treatment of NaCl as high as 200 mM increased ΦPSII by only approximately 14.76%, thereby suggesting that PSII is very tolerant to NaCl. S. salsa has effective mechanisms to protect the photosystem under NaCl stress [57]. A possible explanation for this may be that phosphatidylglycerol (PG) is an integral component of photosynthetic membranes. It is important for both development and functioning of the photosynthesis apparatus [58,59]. For S. salsa, PG increased at higher salinity. Maintenance of PSII function and photosynthetic composition under salinity stress should be viewed as an important strategy for the halophyte S. salsa to grow in saline environments [22].
Zhang et al. [60] used transcriptome analysis to assess the salt stress responses in S. salsa leaves, identified different types of candidate genes associated with photosynthesis (PSB33 and ABA4). Although the effect of aniline on Chl fluorescence in S. salsa leaves has not been reported, it has been confirmed that changes in Chl fluorescence can reflect stress [61]. The decrease in F v /F m , ΦPSII, and qP indicated that the photoactivation of PSII was inhibited by aniline, which resulted in the restriction of electron transport from PSII to PSI and the destruction of the structural integrity of the thylakoid membrane [51].

Enhancement of Excretion of Toxic Molecules in S. salsa by Moderate Salinity
Na is not an essential element for most plant species, but it is necessary for osmotic regulation and the optimum growth of halophytes [62]. Our results indicated that aniline inhibited the absorption of Na by S. salsa, which was counteracted by the supplementation of 200 mM NaCl (Figure 5a, Figure 6a). S. salsa with 200 mM NaCl grew well under aniline stress and had high Na concentrations in their leaves (Figure 1, Figure 5a). Halophytes have the ability to retain high Na concentrations in their shoots via Na compartmentalization in the leaf cell vacuoles or by withdrawing it from cells by glands and bladders [63]. It was reported that Na (in the vacuoles) associated with compatible solutes, such as proline and mannitol, can conduce to the osmoregulation in halophytes. The physiological role of Na in succulent halophytes may be associated with the Na + /H + antiporter activity, which is able to participate in Na compartmentalization in vacuoles for osmoregulation [64]. In order to maintain the normal growth of S. salsa, the contents of Na + and K + and the ratio of Na + /K + in cells are important indexes of plant salt tolerance [65].
NaCl has a certain regulatory effect on the adverse environment of halophytes [66,67]. We also observed that the K + contents in the shoots and roots of S. salsa decreased with the increase in NaCl concentration, even when exposed to 8 mg·L −1 of aniline stress (Figure 5b, Figure 6b). This indicated that the addition of NaCl decreased the absorption of K in S. salsa. Na was found to play an important role in osmotic regulation by replacing K under high salinity. It has been reported that halophytes prefer to keep low cation balances (([K + ] + [Ca 2+ ] + [Mg 2+ ])/[Na + ]), while this ratio may be higher in glycophytes [64,68]. However, the salt glands of S. salsa control salinity accumulation in plant tissues [62]. Thus, salt excretion is a phenomenon by which halophytes discharge excess salt reaching their leaves [69] and the high selectivity contributes to maintaining a suitable K + /Na + ratio. However, excess ions such as K + , Mg 2+ , and Ca 2+ can be excreted [70]. It has been reported that salt glands and trichomes on the leaf surfaces of some halophytes can drain excess metals as a possible detoxification mechanism [27]. Zhang et al. [60] reported that potassium transporters (such as KEA3) could regulate balance excess Na + and Cl − ions in the vacuole.

Increase in Resistance to Aniline by Moderate Salinity Regulating Nutrient Homeostasis in S. salsa
Nutrient homeostasis is one of the major factors of the survival of halophytes under stress [71]. In this study, S. salsa was exposed to a range of aniline concentrations and was more able to maintain its nutrient homeostasis with NaCl (NaCl 200 mM + aniline) than with aniline treatment alone. The effect of NaCl was shown to alleviate the inhibition of aniline toxicity on accumulation of Ca 2+ and Mg 2+ (Figure 5e,f, Figure 6e,f), and NaCl was negatively correlated with Ca 2+ and Mg 2+ accumulation ( Figure 7). Our findings are in agreement with those of several previous reports on the incompatibility of Ca and Na in plant cells under salinity [72]. The uptake competition of Na + with Ca 2+ and Mg 2+ is induced by NaCl [73]. This is probably due to its higher Ca 2+ and K + selectivity [64]. Meanwhile, the K + selectivity in the roots (Figure 6b) indicated that the roots have a regulation mechanism for efficient absorption of K + [74]. The transport of the polycyclic aromatic hydrocarbon phenanthrene into the root cells is coupled with K + influx and H + efflux [75]. This is a new perspective on the role of nutrient homeostasis in plant tolerance to organic pollutants, particularly in halophytes. Mg in the leaves of halophytes plays a major role in osmotic regulation and water uptake in saline soils. The Mg concentration of some halophytes has been shown to achieve similar levels to those of Na [76].
There is a strong relationship between the ion content of plants growing with both salinity and aniline and cross-tolerance. In this study, the halophyte S. salsa, which has succulent organs or salt glands, was characterized by high Na and Cl and low K concentrations in the leaves. Supplementation of 200 mM NaCl can not only assist S. salsa growth, but also alleviate the accumulation strategy of ions under aniline stress.

Conclusions
Halophytes tolerate abiotic stresses through cross-tolerance mechanisms. Moderate salinity alleviated aniline toxicity in the plants by protection of PSII, maintenance of ionic homeostasis, and increase in biomass accumulation. Salinity also enhanced the Na + and Cl − uptake in plants under aniline stress and the nutrient uptake efficiency. Therefore, the present study is valuable for understanding the cross-tolerance of halophytes to salinity and organic pollutants, and is a new attempt at the phytoremediation of aniline wastewater by halophytes. This study provides important information about the toxic effects of aniline on halophytes in saline environments.
Supplementary Materials: The following are available online at http://www.mdpi.com/2071-1050/12/18/7457/s1, Figure S1: Determination of aniline a: 0 mg·L −1 , b: 1 mg·L −1 , c: 2 mg·L −1 , d: 4 mg·L −1 , e: 6 mg·L −1 , f: 8 mg·L −1 by HPLC. Figure S2: Determination of aniline a: standard curve of aniline (Y = 45020.502X − 3488.433, R 2 = 0.999). b: Measurement concentration and configuration concentration of aniline (mean ± SE of three replications). Figure S3: Effect of NaCl on aniline removal efficiency (a) 0 mM NaCl, (b) 100 mM NaCl, (c) 200 mM NaCl. Figure S4: Effect of NaCl on chlorophyll fluorescence parameters qP of S. salsa under aniline stress. For each parameter, the data are the means ± SE of five replications, with standard errors indicated by the vertical bars. Means denoted by the different letters are significantly different at a p < 0.05 according to LSD test. Table S1: Results of ANOVA of aniline, salt on growth parameters