The influence of salinization on seed germination and plant growth under mono and polyculture

Environmental Pollution 260 (2020) 113993

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The influence of salinization on seed germination and plant growth under mono and polyculture*
tia Vena ^ncio a, *, Ruth Pereira b, Isabel Lopes c Ca a

Centre for Functional Ecology (CFE), Department of Life Sciences, University of Coimbra, Coimbra, Portugal GreenUPorto - Sustainable Agrifood Production Research Centre & Department of Biology, Faculty of Sciences of the University of Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal c Department of Biology & CESAM, Campus de Santiago, University of Aveiro, 3810-193, Aveiro, Portugal b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 September 2019 Received in revised form 19 December 2019 Accepted 13 January 2020 Available online 14 January 2020

Sea level rise induced-salinization is lowering coastal soils productivity. In order to assess the effects that increased salinity may provoke in terrestrial plants, using as model species: Trifolium pratense, Lolium perenne, Festuca arundinacea and Vicia sativa, two specific objectives were targeted: i) to determine the sensitivity of the selected plant species to increased salinity (induced by seawater-SW or by NaCl, proposed as a surrogate of SW) and, ii) to assess the influence of salinization in total biomass under different agricultural practices (mono- or polycultures). The four plant species exhibited a higher sensitivity to NaCl than to SW. Festuca arundinacea was the most tolerant species to NaCl (EC50,seed germination and EC50,growth of 18.6 and 10.5 mScm
1, respectively). The other three species presented effective conductivities in the same order of magnitude and, in general, with 95% confidence limits overlapping. Soil moistened with SW caused no significant adverse effects on seed germination and growth of L. perenne. Similar to NaCl, the other three species, in general, presented a similar sensitivity to SW exposure with EC50,seed germination and EC50,growth within the same order of magnitude and with confidence limits overlapping. The agricultural practice (mono-vs polyculture) showed some influence on the biomass of each plant species. When considering total productivity, for aerial and root biomass, it was higher in control comparatively to salinization conditions. Under salinization stress, the practice of polyculture was associated with a higher aerial and root total biomass than monocultures (for instance with combinations with T. pratense and F. arundinacea).Results suggest that the effects of salinity stress on total productivity may be minimized under agricultural practices of polyculture. Thus, this type of cultures should be encouraged in low-lying coastal ecosystems that are predicted to suffer from salinization caused by seawater intrusions. © 2020 Elsevier Ltd. All rights reserved.

Keywords: Sea level rise Seawater intrusion Terrestrial plants Productivity loss Agricultural practices Ecotoxicity

1. Introduction Salinization of soils may arise from primary salinization processes (such as weathering of the parental rock or seawater invasion of low-lying systems near coastal areas) or secondary salinization events (e.g., use of poor-quality ground water for irrigation/consumption purposes) (European Commission et al., 2012;

* €rg Rinklebe. This paper has been recommended for acceptance by Jo * Corresponding author. Centre for Functional Ecology e Science for People & the Planet, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal. E-mail address: [email protected] (C. Ven^ ancio).

https://doi.org/10.1016/j.envpol.2020.113993 0269-7491/© 2020 Elsevier Ltd. All rights reserved.

Intergovernmental Panel on Climate Change, 2018). This may have major implications in soils quality and fertility (FAO, 2008), since many crops, forage and livestock feeding plants are glycophytes. Salt stress exerts immediate effects by changing osmotic pressure, decreasing the amount of water that plant roots can uptake from the surrounding environment (e.g., Osakabe et al., 2014). These alterations trigger signalling mechanisms that promote the production of reactive oxygen species (ROS) which in turn can disrupt normal cellular metabolism by causing lipid peroxidation (Hu et al., 2012), protein denaturation or even targeting nucleic acids (Imlay, 2003). Furthermore, with the increase of salt contents in soils, plants are more likely to absorb ions like sodium (Naþ). The extrusion of Kþ to balance the entrance of Naþ ions impairs many cellular functions that depend on Kþ, namely, protein synthesis

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(e.g., Giri et al., 2007) or stomatal movements (Brugnoli and Bjorkman, 1992), which when restricted, decrease the availability of CO2, impairing photosynthetic rates and, consequently plant’s growth rates (Parida et al., 2004). Nevertheless, plants evolved mechanisms that counteract the effects caused by salt stress (Munns and Tester, 2008). For instance, antioxidant mechanisms may be activated to minimize reactive oxygen species (ROS) effects (e.g. Hu et al., 2012) or polyamines (to deal with salinity increases and consequent potassium deficiency) may be accumulated to hamper or retard cellular growth helping to maintain the Kþ gradient (Ahmad et al., 2013). In the same line of evidence, other solutes that do not interfere with cellular natural processes (like proline e an antioxidant agent and manitol) help the cells to maintain their primary functions as cell turgor, providing the correct gradient for water uptake by the plant (Ahmad and Prasad, 2011) or can act as scavengers of free ions (Diamant et al., 2001). In addition, plants have natural substances (like phytohormones) that help seed germination and growth under adverse conditions (e.g., Jamil and Rha, 2007; Jayakannan et al., 2015). The adverse effects caused by exposure to salinization in terrestrial plants may lead to a decrease in productivity of crops. A scenario that may be aggravated considering the proliferation of monocultures to respond to human food demands (e.g., FAO, 1999). Thus, it is also important to understand if effects of soils salinization on terrestrial plants might be mitigated by the agricultural practice (mono- or polyculture). The re-design of agrosystems to include polyculture, fallow systems or crop rotation, has gained increased attention recently (e.g., Stevens, 2017; Altieri et al., 2017; Crews et al., 2018) and might be a cost-effective and sustainable practice to prevent further soil loss and degradation in face of climate change-derived effects. In the same line, the Common Agricultural Policy (CAP) states that permanent and diverse grassland are fundamental measures to make soil more resilient to those threats (European Commission, 2017). For instance, Picasso et al. (2008) found out that the increasing of species diversity could lead to an increase of total biomass in over 70% when compared to monoculture practices. Following the information mentioned above, this work aimed at assessing the effects that increased soil salinity may pose to seed’s germination and growth of four terrestrial species of plants: Trifolium pratense, Festuca arundinacea, Lolium perenne and Vicia sativa. Two specific objectives were tackled: i) to assess seeds emergence and growth of each species, under salinity stress caused by natural seawater (SW) and by sodium chloride (NaCl), to evaluate the use of the later as a possible surrogate of SW at early stages of ecological risk assessment, and, ii) to assess the effects of salinization in total biomass under different agricultural practices (mono- and polyculture).

2. Material and methods 2.1. Tested solutions Natural seawater (SW), from the North Atlantic Ocean, was collected at a reference site (40 380 3300 N 8 440 5500 W, Aveiro, Portugal), classified by the Foundation for Environmental Education from good to excellent in terms of water quality, as it is frequently monitored according to the Directive 2006/7/CE of the European Union and of the Council of February 15th, transposed to national Portuguese law by Decree-Law no. 135/2009 of June 3rd ^ncio et al., 2017; Vena ^ncio et al., 2018a, 2018b). Prior to its use (Vena for phytotoxicity assays, seawater was filtered through cellulose nitrate membranes of 0.20 mm (ALBET-Hannemuehle S.L., Barcelona, Spain). This procedure aimed at removing particles in

suspension and plankton. All tested SW dilutions were made with distilled water (Gesellschaft für Labortechnik mbH Water Still 2012). Eleven major ions contribute to 99.9% of SW composition, namely, Cl-, Naþ, Mg2þ, Ca2þ, Kþ, HCO-3, Br-, H2BO-3, Sr2þ and F(Wiesenburg and Little, 1988). From those ions, sodium (Naþ) and chloride (Cl-) are predominant, making up almost 80% (Wiesenburg and Little, 1988). Considering this,sodium chloride (NaCl; supplied by Merck, St Louis, MO, USA) was tested as a possible surrogate of SW. A stock solution of 15 g/L of NaCl was obtained by adding and dissolving directly the salt into distilled water. To initiate the phytotoxicity assays, the stock solution was always prepared fresh and all the tested NaCl concentrations were prepared from that stock solution. 2.2. Tested species To conduct the present work, four species of terrestrial plants were chosen: two dicotyledonous species (Trifolium pratense and Vicia sativa) and two monocotyledonous species (Lolium perenne and Festuca arundinacea). These species are proposed as test species in terrestrial ecotoxicity standard protocols (ISO, 1995; OECD, 2006) since they are easily found worldwide, being commonly used as cover crops, forage or pasture for livestock. 2.3. Standard monospecific assays on emergence and growth of higher plants Standard monospecific assays on seed emergence and growth of higher plants were carried out by exposing each of the four selected plant species to a gradient of increasing soil salinities generated with SW or with a NaCl stock solution plus to a control. For this, the standard ISO guideline 11269-2 (1995) was followed. For each treatment (SW dilutions, NaCl concentrations or controls) four replicates were carried out, each consisting of a plastic container (11.7 cm diameter, 6.2 cm height) filled with 200 g of artificial OECD soil (2006; adjusted pH to 6.0 ± 0.5 and approximately a volume of 58 cm3/seed), spiked with the respective SW dilution, NaCl concentration or control water (distilled water). The volume of the test solution added to soil was calculated for adjusting the soil moisture to 45% of the maximum Water Holding Capacity (WHCmax) of the soil. To maintain the soil moisture constant over the duration of the assay, each test vessel was introduced in a larger plastic container filled with distilled water. Furthermore, each test vessel was perforated and equipped with a rope that allowed the communication with the inferior plastic container and enabled the ascending of distilled water by capillarity to the test soil. The level of the soil moisture was regularly checked during the assay and water level in inferior plastic containers was kept constant. At each replicate, ten seeds (for V. sativa) or twenty seeds (for the remaining tested species, with very small seeds) were added and carefully covered with a thin layer of the spiked soil. All assays were run under controlled conditions of temperature (20 ± 2  C), photoperiod (16L: 8D) and light intensity (25.000 lux). According to the ISO guideline, every day the number of emerged seeds was counted but only the first five to emerge were left to grow, while the remaining were counted and harvested (ISO 11269-2, 1995). At the end of the assay, after 14 days of exposure (starting to be counted from the emergence of 50% of seeds from the control group), plants were gently removed from soil and then cut at the interface point of below and above soil surface biomass. The biomass above soil (expressed as mg of dry weight) of each sample was assessed after samples were dried in the oven at 60  C for 12 h and, by weighting in a balance (Kern, EW1500-2M, resolution of 0.01 g). To moisture the soil, a total of 12 nominal concentrations/dilutions (corresponding to a range of nominal conductivities) were

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set for NaCl or SW (plus a control), for each of the tested species. For NaCl, tested nominal conductivities ranged from 2.6 to 23.1 mScm
1 (with a dilution factor of 1.2x) while for SW the nominal dilutions ranged from 6.5 to 48.0 mScm
1 (with a dilution factor of 1.2x). In both exposure scenarios (NaCl or SW), after addition of the solutions/dilutions to the soil, soil conductivities were recorded. The mean % of variation between the conductivities of the solutions added to the soil at the beginning of the assays and the conductivities of the soil samples measured at the end of the assays were of 14 and 21% for NaCl and SW, respectively (Tables 1S and 2S). Therefore, from this point onwards the values of conductivity measured in the soil samples were applied to compute effective conductivities. At the end of the assays, the physico-chemical parameters were measured in three soil samples randomly selected from each treatment. Soil-deionized water suspensions (in a 1:5 w:v proportion) were used to measure soil pH and conductivity. For pH, soils samples were magnetically stirred for 15 min and left to rest for other 60 min after; pH was measured in the suspensions with a WTW pH 330i meter (ISO 17512-1, 2008). For conductivity measurements, 10 g of soil were mechanically shaken with 50 mL of deionized water (deionized water pH > 5.6 and conductivity<0.2 mScm
1) for 15 min. Samples were left to rest overnight for bulk sediment to settle (FAOUN, 1984). Conductivity was measured the day after with a LF 330/SET meter. 2.4. Mono- vs polyculture assays To assess the effects of increased salinity in terrestrial plants under the contrasting agricultural practices (mono- versus polyculture), only exposure to SW was carried out. This decision was based on the results previously obtained with monospecific seed germination and growth assays, which showed that some of the studied plant species (e.g., L. perenne) could tolerate soil moistened with water whose conductivity matched the conductivity of natural SW (z52 mScm
1). Therefore, it was important to understand how species deal with each other, under long-term exposure to SW. For this purposes each plant species was exposed solely (monoculture) or seeded with any of the other three species (polyculture) to a control, consisting of artificial soil moistened with distilled water, and to a salinized treatment, consisting of artificial soil moistened with SW diluted to a nominal conductivity of 4.0 mScm
1. The conductivity level was chosen taking into consideration that salinity levels above 3.0 mScm
1 may already be considered unsafe (most sensitive plants may start to present stress symptoms and soil use might be restricted to some crops plantation; e.g., James, 1995; Natural Resources Conservation Service, 2012). After the addition of the SW solution to the soil, the conductivity level measured during the mono- and polyculture assays, was on average 10.2 and 11% (respectively) lower (approximately 3.6 mScm
1; Supplementary Table 3S) than the value of 4 mScm
1 of the initial SW solution. The conductivity value of 3.6 mScm
1 will be used from this point onwards. A full cross design was carried out to perform these polyculture exposures, i.e., each species was tested against the other three (a total of six plant pairs were tested): (i) T. pratense versus V. sativa, (ii) T. pratense versus L. perenne, (iii) T. pratense versus F. arundinacea, (iv) V. sativa versus L. perenne, (v) V. sativa versus F. arundinacea, and (vi) L. perenne versus F. arundinacea. The methodology used to run the assays was similar to the one used to carry out the monospecific emergence and growth assays (please see section 2.3 of Materials and Methods). For each treatment six replicates were carried out, which consisted of plastic trays (34.5  23.5 cm) filled with 1.5 kg of artificial OECD soil spiked with distilled water (Control) or with SW solution (4.0 mScm
1). This

3

volume was chosen so that the volume per seed was similar to that previously employed in standard monospecific assays (approximately a volume of 54 cm3/seed). As reported for the monospecific emergence and growth assays, here each tray was perforated at the bottom, where a rope was introduced, and placed in larger plastic containers (43.5  28.5 cm) filled with distilled water to maintain soil moisture constant during the assay. For each treatment six replicates were performed. At each replicate 40 seeds were introduced at the beginning of the assay. For monoculture exposure, the 40 seeds belong to the same species; for the agricultural practice of polyculture, 20 seeds were from one species and 20 seeds from another plant species. Every day the number of emerged seeds was counted and only the first 30 emerged seeds were left to grow. The test began when all 30 seeds in the control vessels emerged. Similarly, in polyculture practice, only the first 15 seeds of each species were left to grow, and the beginning of the test started immediately after the germination of 50% of the seedsin the control vessels. All assays took place at controlled conditions of temperature (20 ± 2  C), photoperiod (16L: 8D) and light intensity (25.000 lux). Two periods of harvesting were performed: at day 14 and day 28 after germination of all 30 seeds. Three of the six replicates were harvested at day 14, while the remaining replicates were harvested at day 28 (end of the assay; to assess prolonged effects of exposure to SW). At the end of each period (14 or 28 days of exposure), aerial biomass (above soil) and root biomass (below soil surface) were assessed for each species. For that, plants were gently removed from the soils and cut at the stem at the point that separated the plant biomass above soil surface from the part below soil surface. Roots were carefully washed to remove soil particles that could influence the final weight and dried with absorbent paper to remove excessive water. Both biomasses (above and below soil, were expressed as mg of dry weight) of each sample were assessed after drying in the oven at 60  C for 12 h. Weighting of biomass was performed in a balance (Kern, EW1500-2M, resolution of 0.01 g). At the end of the assays, five soil samples were taken from each tray (replicate) to assess final physico-chemical parameters. This was chosen because, trays were larger, and this procedure allowed to diminish the variability that could exist due to soil’s high spatial variability. The pH and conductivity were measured as described previously in section 2.3 of Materials and Methods. 3. Data analysis The calculations of effect conductivities provoking X% of reduction on seed’s emergence and growth of higher plants (ECx) were performed using the values of conductivity measured in the soils at the end of the assays. The ECx were estimated by fitting the logistic model to the obtained data, using the program Statistica for Windows 4.3 (StatSoft, Aurora, CO, USA). To infer if NaCl had a similar toxicity as SW, a comparison of ECx and respective confidence intervals was made for each species. Regarding mono- and polyculture assays, data analysis was performed, separately for aerial and root dry mass, to identify possible differences between: i) monoculture control vs monoculture in SW-spiked soil, to assess the effects of the salinity stress under practices of monoculture; ii) polyculture control vs polyculture SW-spiked soil, to assess the influence of salinity under practice of polyculture; iii) monoculture control vs polyculture control, to understand the effects of the interaction between species solely, and iv) monoculture SW-spiked soil vs polyculture SWspiked soil, to assess the influence of both salinity and of polyculture on plants response. For i) and ii) a two-way Anova, followed by Holm-Sidak all pairwise comparison (p < 0.05) was performed to test the effects of the factors plant part (root and aerial) and

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conductivity level (control or SW) and for iii) and iv) was applied a three-way Anova, followed by Holm-Sidak all pairwise comparison (p < 0.05) to test the effects of the factors plant part, conductivity level and agricultural practice (mono- or polyculture).

4. Results 4.1. NaCl as a surrogate for SW For both exposures to NaCl or SW, effective conductivities were computed based on the median value of real conductivities measured in three soil samples at the end of the assays (please see Table 1S for NaCl and Table 2S for SW). The soil pH ranged between 4.10 and 4.97 in NaCl (Table 1S) and between 4.16 and 5.12 in SW treatments (Table 2S). Conductivity range between 2.25 and 18.2 mScm
1 in NaCl treatments (Table 1S) and between 5.64 and 32.8 mScm
1 in SW treatments (Table 2S). The four plant species showed a similar sensitivity to NaCl as supported by the values of ECx, which were in the same order of magnitude and, in general, the corresponding 95% confidence limits overlapped (Table 1). The only exception was observed for F. arundinacea, which exhibited the highest EC50,NaCl for seedling germination: 18.6 (16.3e21.0) mScm
1. Festuca arundinacea was also the species presenting the lowest difference in sensitivity to NaCl and SW for both measured endpoints: EC50 for NaCl was always 1.6-fold lower than that for SW (EC50,seedling of 18.6 and 25.8 mScm
1, respectively; and EC50,growth of 10.5 and 17.4, respectively) (Table 1). Results obtained for germination and growth with increased salinity levels of SW showed that, among the four-tested species, L. perenne was the most tolerant species, as no adverse effects were observed for seed germination and growth even when soil was moistened with a solution whose conductivity corresponded to natural SW conductivity (z52 mScm
1) (Table 1). Additionally, L. perenne registered an increment in growth of about 30% at the highest concentration comparatively to the control. Regarding germination, T. pratense and V. sativa were the most sensitive species, with computed EC50,SW (CL at 95%) of 14.5 (12.0e16.9) mScm
1 and 15.1 (10.5e19.7) mScm
1, respectively. The two species (T. pratense and V. sativa) were also the ones where significant adverse effects on seed germination started to be observed at lower conductivities (EC20,SW of 10.4 and 10.0 mScm
1, respectively) (Table 1). Regarding growth, with the exception of L. perenne, all the

other three species (T. pratense, F. arundinacea and V. sativa) showed a similar sensitivity to SW: the EC50 values were in the same order of magnitude and, generally, the 95% confidence limits overlapped (Table 1). Overall, NaCl caused higher toxicity than SW to the tested species, both for seeds germination and for growth (except for F. arundinacea where NaCl and SW induced similar toxicity regarding growth). The highest differences between NaCl and SW EC50s were detected for L. perenne (both germination and growth were more than 3-fold higher for SW) followed by T. pratense (more than 3-fold for germination). For the other two species differences between NaCl and SW EC50s were below 2.4-fold. Table 1: Conductivities causing 10, 20 and 50% inhibition (EC10, EC20, and EC50, respectively) of germination and growth of four species of terrestrial plants exposed to sodium chloride (NaCl) and natural seawater (SW). The 95% confidence limits are depicted within brackets. The ECx values were estimated based on real conductivities measured in soil samples at the end of the assays. n.d. e could not be determined as no inhibition on germination or growth could be detected at the water treatment corresponding to 100% SW. 4.2. Influence of SW and agricultural practices in plant biomass The pH values ranged between 4.61 and 4.76 in control soil, 4.58e4.98 under monoculture agricultural practice and between 4.78 and 5.02 for polyculture practice (Supplementary Table 3S). The conductivity ranged between 0.12 and 0.18 mScm
1 in control soil, 3.36e3.81 mScm
1 for monoculture agricultural practice and between 3.36 and 3.95 mScm
1 for polyculture (Supplementary Table 3S). 4.2.1. Effects in aerial biomass 4.2.1.1. Influence of SW in the aerial biomass of tested plants. Overall, exposure to SW under monocultures caused a significant reduction in aerial biomass of V. sativa, after 14 days (p < 0.05; Fig. 1b) and 28 days of exposure (p < 0.001; Fig. 1b and c). For L. perenne, a significant change in aerial biomass was only observed after 28 of exposure to SW (p < 0.001; Fig. 1b and f), though there was not a clear increase/decrease pattern in the observed effects (Fig. 1b and f). For T. pratense, significant changes were only observed after 14 days of exposure (p < 0.05; Fig. 1a). For F. arundinacea, in general, exposure to SW induced no significant

Table 1 Conductivities causing 10, 20 and 50% inhibition (EC10, EC20, and EC50, respectively) of germination and growth of four species of terrestrial plants exposed to sodium chloride (NaCl) and natural seawater (SW). The 95% confidence limits are depicted within brackets. The ECx values were estimated based on real conductivities measured in soil samples at the end of the assays. n.d. e could not be determined as no inhibition on germination or growth could be detected at the water treatment corresponding to 100% SW. Conductivity (mScm
1)

Species

Growtha

Seed Germination

Lolium perenne NaCl SW Festuca arundinacea NaCl SW Trifolium pratense NaCl SW Vicia sativa NaCl SW

EC10

EC20

EC50

EC20

EC50

3.04 (1.90e6.10) n.d.

5.44 (3.34e7.55) n.d.

9.21 (7.38e11.0) n.d.

4.96 (1.92e8.01) n.d.

9.61 (6.68e12.6) 29.9%*

1.16 (0.86e1.48) 16.1 (12.2e20.0)

1.38 (0.03e7.26) 19.1 (15.9e22.4)

18.6 (16.3e21.0) 25.8 (23.3e28.4)

5.14 (
) 10.3 (1.69e18.9)

10.5 (2.94e18.0) 17.4 (9.41e25.4)

1.12 (
) 8.59 (5.30e11.9)

1.87 (0.01e3.73) 10.4 (7.39e13.4)

4.43 (1.90e6.95) 14.5 (12.0e16.9)

1.96 (0.91e3.22) 5.83 (2.84e8.81)

4.07 (2.74e5.39) 8.11 (5.60e10.6)

2.92 (1.53e4.31) 7.61 (2.46e12.7)

3.94 (2.51e5.37) 10.0 (4.72e14.9)

6.56 (5.23e7.90) 15.1 (10.5e19.7)

3.76 (1.94e5.57) 5.97 (
)

6.28 (4.56e7.99) 15.1 (5.89e24.1)

*A growth stimulation of 29.9% was recorded at the highest tested concentration. a EC10 could not be estimated for growth since these values were below the range of the conductivities tested.

Fig. 1. Average of total aerial biomass (mg) for each of the six plants pairs exposed for a period of 14 days and 28 days (left and right bars, respectively, in each treatment) to a conductivity level of 3.6 mScm
1, either in agricultural practices of monoculture or polyculture. CTR corresponds to the control treatment and SW to seawater treatment (3.6 mScm
1). Error bars represent standard deviation. Letters (a to d) represent significant differences (Holm-Sidak, p < 0.05) between paired comparisons of control and SW treatments, of the different exposure periods, within the agricultural practice of monoculture and letters (A to B) represent significant differences (Holm-Sidak, p < 0.05) between paired comparisons of control and SW treatments, at the different exposure periods, within the polyculture practice for total biomass.

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effects in the aerial biomass (p > 0.05; Fig. 1; Fig. 1S). This finding is inline with the responses recorded and with effective conductivities obtained for SW after 14 days of exposure in the previous section. Regarding the effects of SW under polyculture agricultural practice, a significant change in aerial biomass was only observed for V. sativa, which decreased after 28 days of exposure when cultured with L. perenne or with T. pratense (p < 0.05; Fig. 1b and c; Fig. 1S). For all the other tested pair of plants SW induced no effects in the aerial biomass (p > 0.05; Fig. 1; Fig. 1S). 4.2.1.2. Influence of the agricultural practice in aerial biomass. Looking at the aerial biomass of each species individually under controlled conditions, by comparing mono- with the polyculture practice, overall after 28 days of exposure, V. sativa biomass decreased when co-cultured with L. perenne and T. pratense (p < 0.05; Fig. 1b and c; Fig. 1S; Table 4S). Regarding individual biomasses under salinity conditions, comparing mono- with polyculture, a significant decrease was observed for L. perenne, after 28 of exposure, when cultured jointly with V. sativa or with F. arundinacea (p < 0.05; Fig. 1b and f; Fig. 1S; Table 6S). The production of total aerial biomass was compared between agricultural practices under control and SW conditions. The total aerial biomass obtained with polycultures (calculated by summing the biomass of both species) of V. sativa with any of the three other species, was always lower than the total aerial biomass obtained when this species was in monoculture, both under controlled or SW conditions (p < 0.05; Fig. 1b, c, 1d; Tables 4S and 6S). Contrarily, the total aerial biomass obtained in polycultures of L. perenne, T. pratense, or F. arundinacea with V. sativa was always higher than the total aerial biomass obtained in monocultures of these three species (p < 0.05; Fig. 1b, c, 1d; Tables 4S and 6S). 4.2.2. Effects in radicular biomass 4.2.2.1. Influence of SW in the radicular biomass of tested plants. Overall, increased salinity, under monoculture practices, lead to significant changes in the root biomass of L. perenne (p < 0.05; Fig. 2a and b), after 14 days of exposure. After 28 days of exposure, significant changes in root biomass, compared to the control, were found for L. perenne (Fig. 2a, b, 2f), for V. sativa (Fig. 2b, c, 2d), T. pratense (Fig. 2C) and F. arundinacea (Fig. 2f). Exposure to salinity under polyculture scenarios influenced V. sativa root biomass depending on the species with which was cocultured: it increased after 14 and 28 of exposure when co-cultured with L. perenne (p < 0.05; Fig. 2b; Fig. 2S) and decreased after 28 of exposure with T. pratense (p < 0.05; Fig. 1c; Fig. 2S). Root biomass of F. arundinacea tended to decrease after 14 and 28 of exposure when co-cultured with V. sativa (p < 0.05; Fig. 2d; Fig. 2S). For L. perenne significant differences were found only when it was co-cultured with V. sativa, but without a clear pattern of increase or decrease (p < 0.05; Fig. 2b; Fig. 2S), while for T. pratense a significant decrease was found in root biomass also when co-cultured with V. sativa, after 28-days of exposure (p < 0.05; Fig. 2c; Fig. 2S). 4.2.2.2. Influence of the agricultural practice in root biomass. Analysing the individual root biomasses, under control conditions, and comparing monoculture with polyculture practices, changes in root biomass were detected for L. perenne, V. sativa and F. arundinacea after 14 and 28 days of exposure (p < 0.05; Fig. 2b, c, 2d; Fig. 2S) and for T. pratense after 28 days of exposure (p < 0.05; Fig. 2c and d; Fig. 2S). Under exposure to salinity, and comparing monoculture with polyculture, for no clear pattern was observed for L. perenne as its root biomass varied depending on the polyculture combination after 14 days of exposure (Fig. 2a, b, 2f; Table 7S); though, after 28 of

exposure its root biomass significantly decreased when cultured with T. pratense and V. sativa (p < 0.05; Fig. 2a and b; Table 7S). When in combination with L. perenne, the root biomass of F. arundinacea decrease after 14 days (p < 0.05; Fig. 2f; Table 7S) while the root biomass of V. sativa significantly decreased after 28 days (p < 0.05; Fig. 2b; Table 7S). Comparing the two agricultural practices, under controlled conditions, the root biomass was lower in the polyculture practice for V. sativa when cultured with L. perenne and T. pratense after 14 and 28 days exposure (p < 0.05; Fig. 2b and d; Table 5S) and for F. arundinacea when cultured with L. perenne after 14 or 28 days of exposure (p < 0.05; Fig. 2b; Table 5S). Furthermore, a higher root biomass was observed when any of the other three species were in polyculture with V. sativa after 14 or 28 of exposure, comparatively to the monoculture practice: L. perenne (p < 0.001; Fig. 2b; Table 5S), T. pratense (p < 0.05; Fig. 2c; Table 5S) and F. arundinacea (p < 0.001; Fig. 2d; Table 5S).

5. Discussion 5.1. NaCl as a surrogate for SW The results obtained after exposing the four plant species to increased salinities revealed that, overall, NaCl exerted higher adverse effects in seed germination and biomass than SW. In some cases, L. perenne (germination and biomass) and T. pratense (germination), the difference in the toxicity exerted by NaCl and SW exceeded 3-fold. Based on the results of previous studies for soil ^ncio et al., invertebrates and fungi (e.g., Pereira et al., 2015; Vena 2017), NaCl was already expected to be of similar or higher toxicity than SW. The complex mixture of ions in SW might explain its lower toxicity (Wiesenburg and Little, 1988), since the presence of ions, other than Naþ and Cl
, in small dosages, may present an advantage for the organisms (e.g., Shaul, 2002; Tuteja and Mahajan, 2007). As an example, the presence of calcium (Ca2þ) in SW, may constitute an advantage as this ion intervenes in the abscisic acid cycle (ABA), which plays a major role in membrane integrity and ionic regulation/transport (e.g., Parida et al., 2004). Furthermore, through regulation of ABA levels, Ca2þ may also buffer plants against the effects of Cl
, by decreasing the accumulation of these
mez-Cadenas et al., 2002) and, on its ions (Cl
) in the leaves (Go turn the ABA signalling cascade is also involved in stomatal closure/ aperture, preventing further water loss when the plant is submitted to salt stress (Shinozaki and Yamaguchi-Shinozaki, 2007; Mittler and Blumwald, 2015). Plants respond to saline stress in two phases, but the time of response may vary from species to species (Munns and Tester, 2008). The first phase involves response to osmotic stress, which is rapidly induced; the second phase, commonly denominated ionspecific, involves accumulation of ions inside the cells. In the first phase, due to osmotic stress the ability of roots to extract water from the external environment decreases with increasing salt concentration (i.e. osmotic pressure), which leads to a reduction in shoot growth (Munns and Tester, 2008). This osmotic stress may also be determinant during germination, since the growth of the embryo is stimulated through imbibition, i.e., water uptake by the dehydrated seed (Bewley, 1997). If the embryo growth is affected at an early stage, the posterior growth of the plant will also be affected. Zapata et al. (2003) reported 100% germination rates at a salinity level of 150 mM of NaCl (approximately 17.5 mScm
1) in seven out of nine species of lettuce cultivars. Yet, these authors also found that, all cultivars, despite the high germination rate, presented higher respiration rates, ethylene production and, later, reduction in fresh weights, suggesting that seeds and embryos

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went through osmotic stress. In the present work, it was possible to observe that for NaCl, the EC50’s computed for germination were, in general, very close to those computed for biomass. Therefore, in the case of NaCl, it is suggested that effects observed in biomass could result from the direct action of the salt in the plant at the embryo phase. In the case of SW, the EC50’s computed for germination were higher than those computed for biomass, suggesting that in response to SW, the second phase of reaction to saline stress (ionspecific) is probably the main response. The second phase of response to saline stress occurs mainly through the accumulation of Naþ ions in the leaves, which has a direct effect on chlorophylls activity. Old leaves are known to be especially sensitive to the accumulation of this ion and rapidly died, resulting in the reduction of total photosynthetic activity and subsequently in growth (Hu et al., 2014; Qados, 2011). The sequester of ions by the plant’s leaves may be partially responsible for the reduction of the conductivity values on soil samples at the end of the assays by 14e21%, helping to explain also the fact that some plant species were able to cope with water with a salinity level corresponding approximately to one third of SW (washing of the ions from the soil was discarded as a possibility since water was provided through the base and not at the top layer of the soil). Plants response to osmotic stress may involve the activation of ion pumps that either restrict Naþ influx into the plant (called SOS1) or compartmentalize the excess of Naþ into tissues or vacuoles (HKT1 and NHX1 ion channels, respectively) (Munns and Tester, 2008) and/or may further involve activated selective transmembrane channels, the aquaporins, that are known to also intervene in salt tolerance response by regulating the transport of water into and out of the cell (Glenn et al., 1999; Hill et al., 2004). The results of monospecific assays also revealed that the two monocotyledonous (F. arundinacea and L. perenne) species were, in general, more salt-tolerant (considering the EC50) than the dicotyledonous species (T. pratense and V. sativa). Data already available in the literature support these results. Glenn et al. (1999) stated that, the Naþ uptake rate is much lower in monocotyledonous species than in dicotyledonous, probably due to its lower cation exchange capacity, which underlies a low cellular Naþ:Kþ ratio and different ion accumulation strategies, during saline stress, between this two groups (Conn and Gilliham, 2010). Finally, grounded on the monospecific assay results, it seems that NaCl may be used as a protective surrogate for early stages of risk assessment of SW intrusion in low-lying coastal terrestrial ecosystems. Pereira et al. (2015) also made this recommendation after running toxicity assays with soil invertebrates and registering that NaCl seemed to exert a similar or higher toxicity than SW. However, care should be taken in the use of NaCl for these assessments since risk may be highly overestimated, as shown here for L. perenne and T. pratense. 5.2. Influence of SW and agricultural practices in plant biomass In this work the effect of agricultural practices (mono- or polyculture) on the biomass of different plants species, while dealing

Fig. 2. Average of total root biomass (mg) for each of the six plant pairs exposed for a period of 14 days and 28 days (in every 2 bar set corresponding to the left and right bar, respectively) to a salinity level of 3.6 mScm
1, either in the absence (Monoculture) or presence of a different species (Polyculture). CTR corresponds to the control treatment and SW to the seawater treatment (3.6 mScm
1). Error bars represent standard deviation. Letters (a to g) represent significant differences (Holm-Sidak, p < 0.05) between paired comparisons of control and SW treatments, at the different exposure periods, within the monoculture practice; and letters (A to B) represent significant differences (Holm-Sidak, p < 0.05) between paired comparisons of control and SW treatments, at the different exposure periods, within the polyculture practice for total biomass.

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with exposure to saline stress induced by natural SW was assessed. The natural SW had a conductivity of 4.0 mScm
1, though when mixed with the soil, the soil conductivity decreased (to approximately 3.6 mScm
1) probably due to the presence of organic matter. Still, salinization of soil is a cumulative process so the difference between 3.6 and 4 mScm
1 might be easily exceeded, thus not invalidating the conclusions drawn along this section and furthermore, conductivity (measured as a surrogate of salinity) levels above 3.0 mScm
1 may be already considered unsafe (e.g., James, 1995; Natural Resources Conservation Service, 2012). Moreover, if considering the forecasted scenarios of climate change (Intergovernmental Panel on Climate Change, 2018), it is expected that these conductivity levels may be easily achieved or surpassed in coastal terrestrial ecosystems, possibly inducing even more severe effects. Other areas suffering from seawater intrusion, like south-eastern Sardinia in Italy (considered a representative area for many Mediterranean zones at risk of salinization), estimated an average conductivity level for soils (from 198 samples) of 10.78 mScm
1; while for Baixo Vouga Lagunar (a highly agricultural area), the irrigation water used by farmers for agricultural fields may reach almost 15 mScm
1 (e.g., IDAD, 2008). Regarding the effects of salinity itself (control versus SW within monoculture agricultural practice), overall, it did not change the root and aerial biomass of the four plants species. This was quite an expected result since the EC20,SW after 14 d of exposure to a gradient of salinity were higher than 5.83 mScm
1, i.e., above the median conductivity level measured at the end of the assays in the agricultural practice of monoculture and polyculture (approximately 3.6 mScm
1). The lack of significant effects at this salinity level may indicate that the mechanisms associated with salt tolerance, mentioned in the previous section, may reveal efficient to cope with this salinity level. However, it must be highlighted that, at this conductivity level an exception on this no-effect pattern was observed for V. sativa (one of the most sensitive species to SW in the monospecific assays): after 28 d of exposure to soil moistened with a conductivity level of z3.6 mScm
1 SW, this species revealed a significant decrease in root and aerial biomass comparatively to the control. This higher sensitivity of V. sativa may be related with the fact that this was the species exhibiting, under controlled conditions, the highest weights for root and aerial biomass. It would be expected that a bigger plant needs more water for the maintenance of cell turgor, consequently under osmotic stress could undergo a greater desiccation stress ending up in a reduced growth (Schwinning and Weiner, 1998). Furthermore, plants’ response to salinity stress may also vary according to their life cycle. All species here tested are considered perennial, except for V. sativa. Munns (2002) referred that perennial species respond to salinity stress within months or years, while for annual species the timescale is reduced to days or weeks. Thus, ionic toxicity may be exerted in a faster and shorter period for annual species. Furthermore, annual species (like V. sativa) are programmed to invest earlier in reproduction, maybe investing less on mechanisms to counteract saline stress. This finding highlights the importance of long-term studies (e.g., employment of 28 days assays) as a complement to standard guidelines (e.g., 14 days), as the later ones may not be long enough for a more realistic evaluation of the effects on terrestrial plants. Summarising, the results obtained with V. sativa alert for two important concerns that must be consider when carrying out risk assessment of SW intrusion on terrestrial ecosystems: (i) the need to assess long-term effects, since a 14 d exposure did not identified significant effects (considered as a no effect concentration; EC10,SW always above 9.88 mScm
1) at salinity levels that induce significant effects after a period of 28 d of exposure (3.6 mScm
1); and (ii) the thresholds currently accepted to consider a soil saline should be revised since it may induce sublethal effects, at least in some plant

species (James, 1995; Natural Resources Conservation Service, 2012). When analysing the effects of polyculture solely on the biomass of the plants (control conditions: agricultural practice of monoversus polyculture), it was observed that, though some effects were observed after a 14-d exposure, major alterations in aerial and root biomass were observed after 28 d of exposure, here again denoting the importance of long-term studies. Both for aerial and root biomass, an overall tendency for a decrease in biomass under interspecies polyculture practice was observed. These results may be explained, for instance, by the root capacity, of the four plants, to produce exudates, composed of allellopaths, that inhibit the growth of the competitors’ roots and consequently of their aerial biomass (Schwinning and Weiner, 1998). The comparison of aerial and root total biomass between control and SW, under polyculture practice, revealed significant decreases for V. sativa. This species exhibited a significant decrease in aerial biomass when exposed to SW for 28 d with L. perenne and T. pratense. These results are most probably directly linked with the highest sensitivity that this species demonstrated to salt stress, which is likely even more compromised by the presence of other species, that could be better water and nutrient competitors, as they are less affected by salinity. When analysing the results obtained for root biomass, significant effects were observed just for a few cases. Vicia sativa exhibited lower root biomass when competing with F. arundinacea (14 d) and with T. pratense (28 d), which is inline with the results observed for aerial biomass. Furthermore, L. perenne and F. arundinacea had their root biomass diminished when exposed for 28 d to SW with V. sativa. Finally, the comparison of biomass of plants exposed to SW, revealed a general increase in root and aerial biomass for each species individually under practice of polyculture (except for V. sativa) when compared to the practice of monoculture, suggesting that the use of polyculture practices might be a better alternative than monocultures ones in salinized soils. This finding of higher total biomass in polyculture agricultural practices is inline with the extensive review performed by Weißhuhn et al. (2017) as well as CAP policies (European Commission, 2017). The several positive factors to which polycultures are associated (e.g., prevention of soil erosion, contributing to soil fertility and protection, biomass production) are the answer to the global challenges that we face: demand for food, and quality of the same, water availability and regulation of climate. Considering this, polyculture agricultural practices may be an appropriate choice for coastal areas with salinized soils or at risk of salinization.

6. Conclusions The obtained results showed that the use of NaCl as a surrogate of SW for risk assessment of soil salinization may overestimate the risk and, thus, its use should be cautious. Standard monospecific assays with plants seem to underestimate the risk of soil salinization since they could not detect sub-lethal effects observed after prolonged exposures (28 d) at low levels of salinity, under the practices of monoculture. Furthermore, results suggest that the threshold value above which a soil is considered saline should be revised since at conductivity values very close to the existing threshold, adverse effects were observed both for aerial and root biomass in the studied plant species, under such scenario, and because these conductivity values may be easily surpassed. The reduced root biomass in some species, under monoculture practices, observed at the salinity treatment of 3.6 mScm
1, foresees the worsening of costal soil erosion processes namely under the framework of climate changes associated with sea level rise, which

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may subsequently result in the disruption of the soil structure and communities. However, when assessing effects of salinization under agricultural practice of polyculture, overall the productivity, for aerial and root biomass, and consequently, total biomass, increased. The practice of polyculture should also be encouraged in coastal terrestrial ecosystems that are predicted to suffer from salinization, as has already been done to mitigate erosion and increase soil fertility. Declaration of competing interest The authors declare no conflict of interests. CRediT authorship contribution statement
tia Vena ^ncio: Methodology, Investigation, Formal analysis, Ca Writing - original draft, Writing - review & editing. Ruth Pereira: Conceptualization, Methodology, Writing - review & editing, Funding acquisition. Isabel Lopes: Conceptualization, Methodology, Writing - review & editing, Funding acquisition. Acknowledgements This research was supported by national funds (OE) through FCT/MEC and co-funded by FEDER, through COMPETE (POFC), by FSE and POPH and the research projects SALTFREE (PTDC/AAC-CLI/ 111706/2009) and SALTFREE II (POCI-01-0145-FEDER-031022). This research was also partially supported by Strategic Funding to CFE (UID/BIA/04004/2019) and CESAM (UID/AMB/50017/2019) through national funds provided by FCT d Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2020.113993. References IDAD e Instituto do Ambiente e Desenvolvimento, 2008. Projeto de Desenvolvi~o da Fauna mento Agrícola do Baixo Vouga Lagunar: Programas de Monitorizaça
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