**4. Discussion**

In this study the impact of salinity (150 mM NaCl) on plant growth, quality and self-life of *C. maritimum* was analysed. The salinity had no effect on the biomass and

growth traits measured, indicating that sea fennel is a facultative halophyte with moderate tolerance to salinity, which does not require salt for maximal growth [29,30]. Similarly, Jiménez-Becker et al. [14] did not find differences in this species with respect to biomass growth when NaCl concentrations of 100 mM, 200 mM, and 300 mM were used. Nevertheless, the response to the salinity of *C. maritimum* is population-dependent, being this trait often correlated with the growth of the plants in their natural habitat [29]. The leaf area was reduced under 150 mM NaCl, in agreemen<sup>t</sup> with previous results of Hamed et al. [29], provoking a reduction of the specific leaf area and consequently an increase in leaf thickness and succulence (measured as leaf FW:leaf area ratio [31], the latter being one of the major factors involved in plant salt tolerance, the main quality trait for stimulated growth in halophytes [32]. However, Jiménez-Becker et al. [14] detected a decrease in leaves FW:DW ratio (leaves succulence) together with a decrease in plant water content in the plants grown with 300 mM with respect to that of those grown with 100 mM NaCl; therefore, the salt concentration in the nutrient solution is one of the main factors to consider in leaf succulence of halophytes plants. It is important to bear in mind that succulence together with firmness and juiciness procure leaf texture, which is an important sensory attribute for determining the post-harvest quality and consumers' acceptance [33]. Consequently, acquiring leaf succulence in the crop cycle and keeping it during post-harvest through adequate technology could be a useful strategy for guaranteeing the quality and shelf-life of sea fennel.

Sea fennel can be also a good source of daily minerals required in a healthy diet. The increase in Na+ and Cl− as the result of NaCl salinity was a common and expected response that was previously reported in sea fennel [32,34] since these elements are compartmentalized in vacuoles to avoid causing cytotoxicity [35]. However, differences between Na+ and Cl− accumulation in the aerial part were observed. It was postulated that differences in ion charge are responsible for the more expensive energetically sequestration of Na+ compared to the sequestration of Cl<sup>−</sup>, as the potential inside the vacuole is positive relative to the cytoplasm [36]. This would explain that the Cl− content in the aerial part was found to be systematically higher than Na+, under control and saline conditions. In this study, the K+/Na+ ratio dropped dramatically with salinity treatment as it was reported in Tunisian [29,37] and Argelian *C. maritimum* populations [34], when increasing salinity concentrations were applied, although the degree of resilience was population dependent. Maintaining a high K+/Na+ ratio is likely to be important to avoid the effects of ion toxicity under salt stress [38]. In our study, the accumulation of Na+ in the control plant leaves was lower than in other halophytes [39]. In addition, K+ was accumulated 4.68-fold higher than Na+, which could mean that sea fennel grown in a floating system could be suitable to cover part of the amount of K+ required daily. However, due to the high Na+ concentration, it would be better to use it as a meal accompaniment or as a condiment [7,39], instead of as a main fresh vegetable dish. In control plants, Ca2+ concentration was 2-fold than the Ca2+ accumulation found by Sánchez-Faure et al. [40] in sea fennel plants grown in their natural habitat. Despite saline treatment reducing the available Ca2+, its content in sea fennel leaves remained relatively high (532.05 mg kg−<sup>1</sup> FW), with the potential benefit of preventing salt-induced oxidative damages, due to the protecting function of Ca2+ when plants face extreme heat, dry, or saline conditions [41,42]. The above-mentioned Ca2+ reduction with the salinity treatment could be due not only to the Na+ accumulation but also to its reduce mobility and transport to the shoot under salinity stress [43,44]. An adequate Ca2+ intake for adults of 750 mg per day was marked by EFSA. Therefore, 100 g of fresh sea fennel grown in our conditions may represent 15.8% (control plants) and 7.6% (plant grown with 150 mM NaCl) of the daily recommended doses.

On the other hand, nitrate, bromide, and sulphate were reduced in the leaves of plants treated with NaCl, confirming a reduction in the absorption capacity of nutrients by the roots under salt stress [45]. The difference in nitrate accumulation in response to salinity is generally linked with the inhibition of NO3 − uptake by Cl− [46], which could happen by the interaction between these ions at the site of entry and for ion transport [47,48]. The

nitrate content in the plants studied was generally quite low, and lower than the maximum legislated in the EU (Commission Regulation (EC) No 1258/2011) for other leafy vegetables such as spinach, lettuce, or rocket plant (2000–7000 mg kg−1).

Salinity increased the content of total flavonoids but decreased phenolic content, while total antioxidant capacity was unaffected. Plants vary widely in their phenolic composition and content also accordingly to genetics and environmental conditions [49]. Our results agree with those of Labiad et al. [50], who demonstrated an increase in flavonoids content in NaCl treated sea fennel plants. Similarly, Yuan et al. [51] demonstrated on radish sprouts that moderate concentration of NaCl (100–150 mM) reduced total phenolic content while total antioxidant capacity remained unchanged. More recently, Emami Bistgani et al. [52] observed an increase in total phenolic content by around 20% after saline irrigation (60 mM NaCl) was applied to *Thymus vulgaris* and *Thymus daenensis*, compared with control plants. Additionally, an increase in leaf flavonoid content by 38.6% and 36.6% was observed in plants grown under salt stress conditions after the application of 60 and 90 mM NaCl. Plants cope with salinity-induced stress by altering metabolic processes and stimulating antioxidant activity to scavenge free radicals and ions chelators. Therefore, salt tolerance seems to be favoured by increased antioxidative compounds against oxidative stress induced by a toxic ion action [53]. Flavonoids are frequently induced by abiotic stress and promote roles in plant protection [54] as happened in our study. Hence, based on previous evidence and current data, it is possible to affirm that salt stress (150 mM NaCl) could be a feasible approach to keep, or even increase, the content of health-promoting compounds in *C*. *maritimum*.

Few studies have examined the storage conditions for edible halophytes leaves, with a clear lack of knowledge on *C. maritimum* shelf-life. The storage period of halophytes is usually limited to around a week, so high-tech storage and shipping conditions are required for longer periods [55]. The results presented here show that crop cultivation in controlled soilless conditions, even when salty, can yield production of high quality and good storability. The leaves kept their marketability until 12 days at 5 ◦C. Concerning the colour, NaCl produced clearer leaves, probably due to the presence of salt crystals. To corroborate this, a detailed microscopy study would be needed. These results are in agreemen<sup>t</sup> with D'Imperio et al. [56] who found similar colour parameters on wild sea fennel collected along sea shoreline, which is the natural habitat of this species. Colour is among the first quality parameters catching the attention of consumers with a strong influence on consumers' choice and opinion about the food quality [1,7]. Changes of colour observed in our study were subtle and undetected by the trained sensory panel.

Modified atmosphere packaging is commonly used for fresh produce quality maintenance, prolonging shelf-life, and decreasing the microbial growth on perishable commodities [57]. The atmosphere reached in our experiments seems to be adequate, since no off-odours related to anaerobic metabolism were detected. The high relative humidity inside the packages made the weight loss almost negligible, indicating that the modified atmosphere is convenient for retaining succulence and firmness. The relatively lower firmness of leaves obtained from salinity did not affect the shelf-life.

The reduced microbial load at harvest for leaves grown with 150 mM NaCl would be related to the fact that they had less aerial biomass, so microorganisms appeared later and/or in fewer number than in the control samples. However, at the end of storage, that difference was negligible. Abadias et al. [58] obtained similar values (10<sup>6</sup> to 10<sup>7</sup> CFU g<sup>−</sup>1) of yeas<sup>t</sup> and moulds in fresh-cut lettuce grown under salty conditions. Enterobacteriaceae, a common species in raw vegetables, even when reduced with NaCl, was still present, being an indicator of contamination, that should be carefully avoided in a floating system. The absence of Enterobacteriaceae is an ideal starting point prior to storage and commercialisation.
