Marine Pollution Bulletin 115 (2017) 252–260

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Effects of high salinity from desalination brine on growth,

photosynthesis, water relations and osmolyte concentrations of seagrass
Posidonia australis
M L Cambridge a,⁎, A Zavala-Perez a, G R Cawthray b, J Mondon c, G A Kendrick a
a
UWA Oceans Institute and School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia
b
School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia
c
School of Life and Environmental Science, Deakin University, PO Box 423, Warrnambool, Victoria 3280, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Highly saline brines from desalination plants expose seagrass communities to salt stress. We examined effects of
Received 12 September 2016 raised salinity (46 and 54 psu) compared with seawater controls (37 psu) over 6 weeks on the seagrass, Posidonia
Received in revised form 19 November 2016 australis, growing in tanks with the aim of separating effects of salinity from other potentially deleterious com-
Accepted 30 November 2016
ponents of brine and determining appropriate bioindicators. Plants survived exposures of 2–4 weeks at 54 psu,
Available online 16 December 2016
the maximum salinity of brine released from a nearby desalination plant. Salinity significantly reduced maximum
Keywords:
quantum yield of PSII (chlorophyll a fluorescence emissions). Leaf water potential (Ψw) and osmotic potential
Bioindicators (Ψπ) were more negative at increased salinity, while turgor pressure (Ψp) was unaffected. Leaf concentrations
Brine of K+ and Ca2+ decreased, whereas concentrations of sugars (mainly sucrose) and amino acids increased. We
Amino acids recommend leaf osmolarity, ion, sugar and amino acid concentrations as bioindicators for salinity effects, associ-
Ions ated with brine released in desalination plant outfalls.
Sugars © 2016 Elsevier Ltd. All rights reserved.

1. Introduction occur in some coastal zones where the desalination industry is

experiencing significant development (Portillo et al., 2014; Roberts et
Desalination plants are increasingly being developed in coastal areas, al., 2010). Some seagrass species appear to be highly vulnerable to hy-
sometimes with discharge of brines into relatively shallow nearshore persaline conditions created by brine plumes, such as the Mediterra-
zones. Brine resulting from marine desalination is essentially seawater nean species Posidonia oceanica (Fernández-Torquemada and
concentrated by a factor that depends on the efficiency of the reverse os- Sánchez-Lizaso, 2005, Gacia et al., 2007; Sanchez-Lizaso et al. 2008;
mosis membranes, with the addition of a number of other substances, Ruiz et al., 2009). Effects on growth were evident at only 1–2 units
such as anti-scale additives, biocides, surface-active agents or solid resi- above ambient (39 psu) and significant mortality was found at 4 units
dues from back-flushing of filters (Morton et al., 1996; Einav et al., above ambient (42 psu), whereas others species, such as Thalassia
2002). If brines are discharged at high salinity, then, depending on diffus- testudinum, showed very limited or no effects when exposed to in-
er port design and hydrodynamic conditions (Portillo et al., 2014), a creases of up to 4 units of salinity from reverse osmosis discharge
plume of denser water may spread out over areas of seafloor posing a po- plumes (Tomasko et al., 2000). The relatively low tolerance of P.
tential threat to benthic marine communities (Fernández-Torquemada et oceanica occurring in open coastal areas with stable salinities contrasts
al., 2009). Following predictions of a drying climate, a large desalination with the higher tolerances reported for P. oceanica growing in a hyper-
plant using reverse osmosis of seawater (ca 100 GL pa) has been built saline lagoon up to 48 psu in Sicily (Tomasello et al., 2009), indicating
on the Western Australian coast 200 km south of Perth. Hypersaline the development of ecotypes. More recently, physiological responses
wastewater up to 54 psu (practical salinity units, equivalent to g kg−1 of P. oceanica to raised salinity have been studied in mesocosms
or parts per thousand, ‰) is discharged into the wave-exposed nearshore (Marín-Guirao et al., 2011; Sandoval-Gil et al., 2012a, 2012b;
environment approx. 10 m deep where the benthos is predominantly a Garrote-Moreno et al., 2014). They showed that after 6 weeks of hyper-
mosaic of seagrasses on sand, rock pavement and sand sheets. saline exposure, organic osmolytes had accumulated in leaf tissues,
Seagrass meadows are coastal communities of outstanding ecologi- thereby avoiding dehydration, but at a metabolic cost (Sandoval-Gil et
cal importance (see Costanza et al., 1997; Orth et al., 2006), but they al., 2012a). The salt-stressed plants had lower photosynthetic rates
and increased respiration rates, which was associated with reduced
⁎ Corresponding author. growth and caused some shoots to die (Marín-Guirao et al., 2011).
E-mail address: [email protected] (M.L. Cambridge). Early laboratory work on the Australian sister species, Posidonia australis

http://dx.doi.org/10.1016/j.marpolbul.2016.11.066
0025-326X/© 2016 Elsevier Ltd. All rights reserved.
 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260 253

showed a broad range of tolerance at salinities ranging from 27 to withstand raised salinity. Posidonia australis shoots were collected
N60 psu (Tyerman et al., 1984), reflecting its occurrence in both estua- in the field from a shallow meadow 0.5–1 m deep at Woodman
rine and hypersaline habitats (e.g., Cambridge and Kendrick, 2009; Point near Fremantle. Each seagrass plant unit consisted of two api-
Walker et al., 1988), and suggesting either broad tolerances or develop- cal shoots and a 15–20 cm length of rhizome. Plants were
ment of ecotypes. transported in seawater in an insulated carrier and transplanted
Plant species differ widely in salinity tolerance (Munns, 2002). High into the culture tank system within 3 h of collection. Tanks were
salinity is well-known as a problem for most plant species, due to two placed in indoor laboratories to allow culture with constant back-
effects: osmotic stress and ion toxicity. These affect plant water rela- ground conditions (temperature, light, day-length) with adjust-
tions, ion concentrations in cytoplasm and vacuole, and many metabolic ments to salinity of natural seawater using artificial marine salt
processes, particularly growth and photosynthesis, leading in turn to (Aquavitro, Seachem, Wilmington, USA). Calcareous sand dredged
mortality. Osmotic stress occurs when the salinity of the external medi- from seagrass habitat was placed in nursery trays to provide the
um increases, corresponding to increasingly negative water potentials. growing medium for seagrass plants. Organic matter was added to
Since water flows towards more negative water potentials, plant cells the sediment using 9 g of Posidonia leaves, roots, and fibrous leaf
must have a more negative water potential than the external medium, sheaths sourced from beach wrack, mixed with 600 g of sediment
or osmotic stress will result as the cells lose turgor and the tissues dehy- to yield a total of 1.5% dry weight organic content, following
drate. Ion toxicity occurs because Na+ and Cl− accumulate in leaf tissue methods developed in Statton et al. (2013). All roots on each trans-
or their concentration increases with dehydration. High Na+ and Cl− plant unit were cut off close to the rhizome just before planting.
concentrations disrupt electrochemical potential gradients across mem- The mesocosm system consisted of nine 200 l tanks with 3 tanks per
branes, denature proteins, increase levels of reactive oxygen species treatment. Each treatment consisted of a closed seawater circuit feed
(ROS), or displace cellular K+ - a vital cofactor for many enzymes from a 1000 l reservoir tank. Water was circulated using a 2500 l h−1
(Munns and Tester, 2008). Seagrasses maintain very negative osmotic pump and drained back into the reservoir tank continuously. Artificial
potentials due to very high concentrations of ions and the formation light ensured a consistent light quality simulating natural summer con-
of compatible solutes such as sugars and amino acids (Tyerman, 1989; ditions, provided by one overhead Sylvania BT28 Metal Halide 250 W
Torchette, 2007). Water stress induces the up-regulation of the synthe- lamp (Osram Sylvania Inc., Danvers, MA, USA) per tank, set to 100 ±
sis of specific proteins to counteract the increasing concentration of 20 μmol m−2 s−1, measured with a LI-1400 Data Logger (LI-COR Inc.,
damaging ions such as Na+ and to a lesser extent, Cl−. These proteins Lincoln, NE, USA) spherical quantum sensor in a 12 h light:dark cycle.
include aquaporins (Serra et al., 2013), and enzymes involved in the Water temperatures in the tanks ranged between 21 and 26 °C (mean
synthesis of various compatible solutes, e.g., specific amino acids 23.9 ± 0.8 °C). The plants were held in the tanks for 5 days before com-
(Pulich, 1986; Tyerman, 1989). mencing the experiment in natural seawater with a salinity of 37 psu
Establishing causal relationships between brine discharge and im- (practical salinity units, equivalent to g kg−1 or parts per thousand,
pacts on key species is particularly difficult in environments where ‰) with a range of 36.4 to 36.8 psu, which is the same as ambient salin-
other activities may confound or mask the effects of brine plumes, or ity in the area where plants were collected. Salinity treatments were im-
areas are inaccessible/not appropriate for benthic surveys. In this series posed without a period of gradually increasing salinity, in order to
of experiments, we first separated salinity responses from the effects of simulate sudden increments in salinity associated with a brine dis-
whole brine that includes both salinity and the chemical additives used charge. Natural seawater was used for controls. Salinity was raised in
mostly in various membrane-cleaning processes. These experiments two treatments: to 46 and 54 psu by adding artificial marine salt
form part of a larger study aimed at assessing the effects of desalination (Aquavitro, Seachem, Wilmington, USA) to the reservoir tanks until
brine using biomarkers that could be tested in laboratory mesocosm the desired salinity was reached. These salinity levels were maintained
conditions, rather than in the field. In this study, we examined the ef- for six weeks until the end of the experiment. Salinity and temperature
fects of salinity alone without the additives present in desalination were monitored daily using a multi-parameter field logger (TPS 90-FLT,
brine, exposing mature seagrass shoots of Posidonia australis to three TPS Pty Ltd., Springwood, Brisbane, Australia).
levels of salinity (36, 46, 54 psu) over six weeks in tanks held at constant
conditions, basing the experiment on the maximum salinity of brines 2.2. Survival
produced by a large seawater reverse-osmosis desalination facility re-
cently completed on the south-western Australian coast. The highest sa- Survival over the experimental period was identified by the pres-
linity corresponds to the undiluted brine, and the intermediate level to ence of aboveground healthy shoots for each species under each salinity
50% dilution. Responses to raised salinity were measured for survival, treatment. Plants were photographed when harvested to provide a re-
growth, photosynthesis and aspects of osmoregulation, including devel- cord of salt damage, such as presence of salt scorch and blackened
opment of higher osmotic pressure and increasing concentrations of patches.
sugars and amino acids. We selected P. australis as a test species because
of its distribution around the temperate southern half of Australia 2.3. Growth
(some 5000 km) from Shark Bay on the west coast to north of Sydney
on the east coast, where several of the new desalination developments Leaf and root tissue were measured every two weeks on three ran-
have been sited. It has also been investigated in details in studies on domly-selected harvested plants. Leaf growth rates were measured fol-
physiology (Tyerman et al., 1984, 1989) and recent work on techniques lowing the method by Short et al. (2001). Seagrass leaves were marked
for tank culturing under controlled conditions (Statton et al., 2013, 5 to 7 days prior to harvesting. After harvesting, new tissue was mea-
2014). sured to estimate shoot leaf growth rate (cm2 shoot− 1 day− 1).
Completely new leaves (leaves with no marks) were counted and mea-
2. Methods sured entirely as new tissue. Additionally, root lengths were measured
on harvested plants and growth rates (cm shoot− 1 day− 1) were
2.1. Tank system and experimental design estimated.

Seagrass plants in culture were used for experiments on raised 2.4. Photosynthesis: chlorophyll fluorescence
salinity in large aquaria at aquaculture facilities (Western Australian
Department of Fisheries) to study the natural osmoregulatory ca- Chlorophyll fluorescence measurements were conducted using a
pacities, as well as the capacity of basic metabolic functions to pulse-amplitude modulated fluorometer (Diving-PAM Fluorometer,
254 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260

Table 1
Summary of univariate PERMANOVA assessing the effects of raised salinity (Treatment) among different Time exposures, with Tank nested as a random factor within Treatment.
PERMANOVA tests were performed on transformed data (log(x + 1)) for root growth rate, osmolalities, water content and photosynthesis data (ETRmax and Ek). Significant differences
shown in bold.

Source of variation Root growth Osmotic pot Water pot Turgor Water content ETRmax Ek

MS p MS p MS p MS p MS p MS p MS p

Time 0.002 0.660 0.860 0.001 1.008 b0.001 4.1126 0.043 0.008 0.021 1.258 b0.001 1.172 b0.001
Treatment 0.175 0.003 0.112 0.001 0.223 0.001 5.006 0.012 0.019 0.022 0.557 0.029 0.628 0.0428
Tank (treat) 0.009 0.780 0.002 0.278 0.002 0.164 0.39815 0.645 0.001 0.707 0.081 0.200 0.080 0.2178
Ti × treat 0.002 0.815 0.055 0.001 0.098 b0.001 3.135 0.047 0.005 0.049 0.051 0.431 0.052 0.3628
Ti × tank (treat) 0.009 0.773 0.001 0.636 0.004 0.002 0.96999 0.093 0.002 0.260 0.048 0.616 0.044 0.7117
Residual 0.017 0.002 0.001 0.56526 0.001 0.056 0.057

Walz GmbH, Effeltrich, Germany). The potential quantum yield of pho- to avoid leakage of ions from the leaf tissue, wrapped in Al-foil and fro-
tochemistry (Fv/Fm) was determined by subjecting dark-adapted leaves zen immediately in liquid nitrogen, and stored at −80o C until freeze-
to a saturating pulse of light. Measurements were carried out in the dried and ground to a powder in a ball mill grinder.
morning before lights were on to ensure complete dark adaptation; Subsamples of the ground material were dissolved in 5 ml of 0.5 M
four randomly selected leaf replicates were measured per tank (in HCl for Na+/K+ analysis and 20 ml of boiling DI water for Cl− analysis,
total 12 replicates per treatment). In order to minimise the associated and shaken for two days. Leaf tissue cation concentrations (Na+, K+
leaf age variation, leaf clips (Heinz Walz GmbH, Effeltrich, Germany) and Ca++) were determined using an ion chromatograph by flame pho-
were placed on each leaf at the same distance from the base (approx. tometry (Corning, Model 410, Halstead, Essex, UK). Anion concentra-
3 cm). Chlorophyll fluorescence measurements consisted of a saturation tions (Cl−) were determined with a boiling water extraction of
pulse that induced maximal fluorescence yield (Fm) and maximal vari- ground tissue, followed by chlorometric impulse titration with a chlo-
able fluorescence (Fv = Fm − Fo). Fv/Fm indicates the quantum yield of rinity meter (Model 4-2000, Buchler Instruments, New Jersey, USA).
PS II and is typically used as a measure of stress in plants (especially
an increase in Fo). Measurements were performed at the beginning, 2.7. Sugars and amino acids
after one month and at the end of the six weeks experiment.
Rapid Light Curves (RLC) were performed on the middle leaf of four 2.7.1. Extraction
randomly-selected plants per tank (in total 12 replicates per treatment) The non-structural carbohydrates (NSC) of glucose, fructose and su-
at the beginning, after one month and at the end of the experiment. crose and free amino acids (FAA) were determined in freeze dried and
Leaves were clipped at the same distance from the base (approx. finely ground leaf and rhizome tissue obtained from plants collected
3 cm) as in the chlorophyll fluorescence measurements. Parameters es- in the experimental units, as described above for ion analysis, and stored
timated were: photosynthetic efficiency (α), Maximum Electron Trans- at −80 °C. The method of extraction for NSC and FAA was adapted from
port Rate (ETRmax) and saturating irradiance (Ek) (Ralph and Marín-Guirao et al. (2013) and Warren and Adams (2000) in that 20 mg
Gademann, 2005; Schwarz et al., 2000). of ground dried sample was extracted with 1.0 ml of 95% (v/v) ethanol
at 80°C for 15 min, and then centrifuged. After removal of the superna-
2.5. Water relations tant, the pellet was re-suspended and the extraction repeated. The su-
pernatants were combined to provide the extracted sample for NSC
Osmotic and water potentials in leaf tissue were measured at the be- and FAA analysis as detailed below.
ginning, after one month and at the end of the experiment from three For FAA analysis only, aliquots of 700 μl for leaf eand 500 μl for rhi-
randomly-selected harvested plants using a Dewpoint Potential Psy- zome were taken to dryness under vacuum with a SpeedVac (Savant,
chrometer WP4 (Decagon Devices Inc., Pullman, Washington, USA) fol- Farmingdale, NY, USA) and subsequently re-dissolved in 150 μl Milli-Q
lowing methods developed by Tyerman (1982), Murphy et al., 2003, water containing 56.6 μM of the internal standard, α-amino-n-butyric
and Sandoval-Gil et al., 2012a. From each replicate plant, approximately acid.
4 cm of leaf was cut 3 cm above the leaf base. Fresh tissue was kept sub-
merged during handling to minimise evaporation; then quickly blotted 2.7.2. Soluble sugars — analysis
dry before placing into the psychrometer chamber. Water potentials Analysis of NSC was adapted from Slimestad and Vagen (2006) and
were measured in fresh tissue and osmotic potentials in frozen tissue. undertaken using a high performance liquid chromatograph (HPLC)
After water potentials were measured, sample tissues were frozen im-
mediately in liquid nitrogen at -70 °C. The frozen sample tissues were
then placed into the chamber cup to measure osmotic potentials at
zero turgor. Leaf turgor pressure was calculated as the absolute differ-
ence between water and osmotic potentials (Kramer and Boyer, 1995).
Leaf water content was measured from three randomly-selected
harvested plants as the difference in fresh and dry weight at 0, 4,
6 weeks exposure to raised salinities.

2.6. Ion concentrations

Inorganic cations (Na+, K+ and Ca++) and Cl− anions were deter-
mined in leaf tissue from the remaining nine plants in each tank at the
end of the experiment. Fully expanded young leaf material free of at-
tached algal epiphyte growth was selected for analysis. The leaves
were excised, carefully cleaned by wiping with tissue moistened with
seawater, then briefly wiped with tissue soaked in de-ionised water to Fig. 1. Root growth of Posidonia australis in seawater controls (37 psu) compared with
remove salty water on the leaf surface. They were then quickly blotted raised salinities (46, 54 psu).
 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260 255

Fig. 2. Photosynthetic parameters of seagrass Posidonia australis derived from P vs E curves (a) maximum electron transport rate (ETRmax), (b) saturation irradiance (Ek)at 0, 4 & 6 weeks
exposure to seawater controls (37 psu) and raised salinities (46 and 54 psu). Values are means ± SE, n = 12.

consisting of 600E pump, 717 plus autoinjector, (Waters, Milford, MA, kit, reservoir B was acetonitrile and reservoir C was Milli-Q water. All
USA) and an Evaporative Light Scattering Detector (Alltech 500 ELSD, solvents were vacuum filtered to 0.22 μm prior to use and were contin-
Grace Materials Technologies, Deerfield, IL, USA). Separation was ually degassed with helium sparging. The gradient profile was as per the
achieved at 30 ± 0.5 °C on a Prevail ES Carbohydrate column instructions with the kit.
(250 × 4.6 mm i.d. with 5 μm packing; Grace Materials Technologies) The mixed amino acid standard contained aspartic acid, glutamic
using an isocratic mobile phase consisting of 25% Milli-Q water and acid, glycine, arginine, threonine, alanine, proline, γ-aminobutyric
75% acetonitrile at 1 ml min− 1. All solvents were vacuum filtered to acid, cysteine, tyrosine, valine, methionine, lysine, iso-leucine, leucine,
0.22 μm prior to use and were continually degassed with helium sparg- phenylalanine, tryptophan, as well as amino acid pairs, serine + aspar-
ing Samples in the autoinjector were held at 10 °C and the ELSD drift agine and histidine + glutamine, which could not be separated with the
tube was held at 80 °C with high purity nitrogen flow rate of 2.5 litres HPLC method used.
per minute for nebulisation.
Calibration curves for each sugar were generated from ELSD peak
area (log10) versus the mass of standard sugar injected (log 10), and a 2.8. Data analyses
standard analysed every 10 samples to check for any instrument/detec-
tor drift. Data acquisition and processing was with Empower™ 2 (Wa- All the studied parameters tested the null hypothesis that there
ters Corp., Milford, Massachusetts, USA) software. Retention times of were not significant differences among salinity treatments or sampling
sugar standards were used to identify sugars in the sample extracts. times. Analyses consisted of nested analysis of variance (ANOVA) design
Typical sample injections were 10 μl and runtime was 15 min per with Treatment and Time as fixed factors, while Tank was treated as
sample. random factor nested within Treatment. Data were transformed
(log(x + 1)) when necessary and ANOVA assumptions were tested. If
2.7.3. Free amino acids — analysis data presented significant deviations from normality and homogeneity
Analysis of FAA was undertaken using the Waters AccQ.Tag chemical of variances, then a permutational analysis of variance (PERMANOVA)
package for HPLC, with a Waters system consisting of a 600E dual head test was run using the same model. PERMANOVA analyses were based
pump, 717 plus autosampler, a 470 scanning fluorescence detector and on a Euclidean dissimilarity matrix using a minimum of 9999 permuta-
a 996 photodiode array (PDA) detector. Separation was performed on a tions of residuals under a reduced model. PRIMER-E software package
Waters Nova-Pak C18 column (150 mm × 3.9 mm I.D.) with 4 μm par- (Clark and Gorley, 2006) and the PERMANOVA + add on for PRIMER
ticle size, held at 35 °C. All data were acquired and processed with Em- (Anderson et al., 2008) were used for PERMANOVA analyses while R
power® chromatography software (Waters Corp., Milford, (version 3.0.2; R Development Core Team, 2013) was used for all
Massachusetts, USA) with fluorescence detector settings of 250 nm Ex- other statistical analysis and graphs were produced using the ‘ggplot2’
citation; 395 nm Emission, 0.5 filter, 100 gain and the PDA set to add-on package (Wickham, 2009).
250 nm. Positive identification of amino acids was accomplished by
comparing standard retention time for fluorescence and PDA as well
as PDA peak spectral analyses with the samples.
For sample derivatisation, a 10 μl aliquot of the re-dissolved extract
70 μl of the kit-supplied borate buffer was added and samples vortex
Table 2
mixed. Then 20 μl of the AccQ.Fluor reagent was added and samples im-
Survival (%) of Posidonia australis after 6 weeks exposure to raised salinities
mediately vortex mixed. Samples were then incubated at 55 °C for (46, 54 psu) compared to controls (37 psu).
10 min prior to HPLC analysis. Standards and blanks were carried
Salinity treatment (psu) Survival (% ± se)
through the same procedure.
A gradient mobile phase system was employed with reservoir A 37 97.9 ± 3.61
consisting of the Waters AccQ.Tag aqueous buffer, prepared from the 46 66.7 ± 9.55
54 31.3 ± 10.82
concentrate following manufacturers' instructions supplied with the
256 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260

3. Results 3.3. Water relations

3.1. Survival and growth After exposure to increased salinity treatments, P. australis
responded to the more negative external water potentials with more
Survival was strongly reduced at the highest salinity (Table 2). At negative leaf water potentials, achieved by lowering osmotic potentials
54 psu, only 31% of the plants were surviving by 6 weeks, and all while maintaining slightly positive turgor pressures. As the salinity in-
showed signs of salt damage (blackened patches on leaves and dead creased, the mean water potential of the external medium was reduced
roots). At the intermediate salinity (46 psu), most plants (67%) were from approx. −2.4 MPa in ambient seawater (37 psu) to −3.2 MPa in
alive, but also showing signs of morbidity. the 46 psu treatment and −3.8 MPa in 54 psu.
Root growth was significantly inhibited by Treatment (p b 0.003, Water potentials were significantly more negative (p = 0.001) after
Table 1), with almost no growth at the highest salinity. At the interme- 6 weeks exposure in both salinity treatments compared with controls
diate salinity, growth rates at 6 weeks were b50% of controls (Fig. 1). (significant Time × Treatment interaction, p b 0.001, Table 2). There
Leaf growth showed no significant responses to salinity treatments. was a significant pair-wise decrease in water potential after 6 weeks
from 37 to 46 psu, and from 46 to 54 psu (Fig. 3a). There were no signif-
icant changes in osmotic pressure between controls and the intermedi-
3.2. Photosynthesis ate salinity. Osmotic potentials were significantly more negative only
after 6 weeks at the highest salinity treatment compared with controls
Rapid Light Curve parameters showed significant reductions for (Fig. 3b).
Treatment and Time (p b 0.01). Maximum electron transport rates Turgor pressures, derived from the balance of water and osmotic po-
(ETRmax, p b 0.001) and saturating irradiance (Ek, (p b 0.0001) were sig- tentials, showed only minor changes from 0.2–0.8 MPa in controls,
nificantly reduced at the highest salinity (54 psu) by 4 weeks. At the in- whereas mean values remained similar in salinity treatments (0.2–
termediate salinity 46 psu, ETRmax and Ek were maintained at similar 0.3 MPa) (Fig. 3c, Table 1).
values to controls for 4 weeks but were significantly lower than controls Water contents showed significant time (p = 0.02) and treatment
by 6 weeks (Fig. 2 a, b, Table 2 with post-hoc tests). There were no (p = 0.02) responses after six weeks at intermediate and highest salin-
changes in maximum quantum yield (Fv/F) or photosynthetic efficiency ities (Fig. 3d, Table 1). Pair-wise comparisons showed significant de-
(slopes of the rapid light curves in the light limited region, α) at raised creases in water content between 37 psu and 46 psu, and between 46
salinities compared with controls (data not shown). and 54 psu.

Fig. 3. (a) Leaf water content, (b) water potential, (c) potential, (d) turgor pressure, measured for Posidonia australis leaves at 0, 4 and 6 weeks exposure to seawater controls (37 psu) and
raised salinities (46 and 54 psu).
 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260 257

3.4. Osmolyte concentrations 3.4.3. Amino acids

In leaf tissue, several amino acids showed significant increases after
3.4.1. Ions 6 weeks exposure to salinity treatments (ARG, HIS/GLN, ILE, LEU, LYS
Leaf potassium (K+) and calcium (Ca++) concentrations significant- ALA, PHE, PRO, SER/ASN, THR, VAL, abbreviations listed in figure legend,
ly decreased as salinity increased. At the highest salinity, there were sig- Fig. 5a, b, Table 3).
nificant decreases in leaf concentrations of K+ (p b 0.0002) from In rhizome tissue, concentrations of several amino acids showed sig-
32.47 ± 0.81 mg K g−1 at 37 psu, 31.59 ± 0.78 mg K g−1 at 46 psu to nificant increases after 6 weeks exposure to salinity treatments (ALA,
13.18 ± 2.58 mg K g−1 at 54 psu, and Ca++ (p b 0.03) from 8.92 ± GABA, GLY, HIS/GLN, PHE, PRO, abbreviations list in figure legend, Fig.
0.01 mg Ca g− 1 at 37 psu, 8.10 ± 0.40 mg g− 1at 46 psu to 5.03 ± 5c, Table 3).
0.60 mg Ca g−1 at 54 psu. Sodium and chloride (Cl−) did not change
with increased salinity (Fig. 4a, Table 3). The decreases in K and Ca re-
sulted in significant decreases in the molar ratios of Ca:Na and K:Na 4. Discussion
(Fig. 4b).
In the rhizomes, only Na+ showed a significant increase at the Desalination plants discharge brines with raised salinities and addi-
highest salinity from 2.17 ± 0.07 mg Na g− 1 at 37 psu, 2.32 ± tives derived from the reverse osmosis process. Separating the effects of
0.17 mg Na g−1at 46 psu and 2.87 ± 0.15 mg Na g−1 at 54 psu. In con- elevated salinity is the first step in considering the likely impacts of
trast to leaves, Ca++ and K+ remained constant with increased salinity. brine discharge into nearshore environments. In this first series of ex-
Mean values for ion concentrations in rhizomes were similar to those in periments on an important temperate Australian species of seagrass,
leaves. Posidonia australis, we observed significant negative responses to raised
salinities with increased plant morbidity and mortality, reduced growth
of roots, highly negative water potentials, increases in known
3.4.2. Compatible solutes: sugars osmoregulators, including sucrose and amino acids, and reduced con-
The concentration of sucrose in leaf tissue significantly increased centrations of K and Ca ions.
with salinity (p b 0.001), but there were no significant changes for glu- Elevated salinities presented a major obstacle to survival and growth
cose or fructose (Fig. 4c, Table 3). in Posidonia australis through lowering the water potential, and making
The concentration of fructose, and sucrose in rhizomes was not sig- it increasingly difficult to sustain growth. We found leaf water and os-
nificantly different between treatments and there was no glucose motic potentials in P. australis became more negative as a response to
detected. higher salinities in both salinity treatments (46 and 54 psu), but not

Fig. 4. (a) Concentrations of ions: potassium (K) and calcium (Ca), sodium (Na) and chloride (Cl) in leaf tissue of seagrass Posidonia australis after 6 weeks exposure to seawater controls
(37 psu) and raised salinities (46 and 54 psu). (b) Molar ratios of ions K:Na, K:Cl, Ca:Na, Ca:Cl, (c) Concentrations of sugars (sucrose, glucose, fructose) after 6 weeks.
258 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260

Table 3 Table 3 (continued)

Summary of ANOVA and PERMANOVA tests assessing the effect of salinity treatments after
six weeks exposure on concentration of ions, free amino acids in leaf and rhizome tissue of Variable Source of variance df MS p
Posidonia australis. ANOVA tests were performed on transformed data on ions (K, Ca, Na, Na+ Treatment 2 0.355 0.987
Cl), carbohydrates (fructose and sucrose) and amino acids concentrations. PERMANOVA Residual 6 26.345
tests were performed on concentrations of ions (Ca), carbohydrates (glucose) and amino Ca++ Treatment 2 0.342 0.032
acids. Residual 6 0.016
Cl− Treatment 2 191.460 0.654
Variable Source of variance df MS p
Residual 6 419.250
Free amino acids in leaf
ALA Treatment 2 0.267 b0.001
Residual 11 0.010
ARG Treatment 2 0.000 0.014
Residual 11 0.000
THR Treatment 2 0.038 b0.001 turgor pressure, which remained similar across treatments and controls.
Residual 11 0.002 The osmotic adjustment was associated with increased sucrose concen-
ASP Treatment 2 0.000 0.728 tration in the leaves, minor increases in fructose and increases in some
Residual 11 0.001
amino acids, mostly after 6 weeks. Water and osmotic potentials in P.
GABA Treatment 2 0.021 0.004
Residual 11 0.007
australis were much more negative than those observed in Posidonia
GLU Treatment 2 0.001 0.602 oceanica, a sister species from the Mediterranean, considered to be
Residual 11 0.001 very sensitive to only small increases in salinity
GLY Treatment 2 0.004 0.262 (Fernández-Torquemada and Sánchez-Lizaso, 2005; Sandoval-Gil et
Residual 11 0.003
al., 2012a), indicating that P. australis can withstand greater changes
HIS.GLN Treatment 2 0.021 0.026
Residual 11 0.005 to salinity by maintaining these potentials. For example, when P.
ILE Treatment 2 0.052 b0.001 oceanica was exposed to salinities from 37 to 43 psu over 47 days,
Residual 11 0.001 water potentials decreased from −3.04 to −3.74 MPa, and osmotic po-
LEU Treatment 2 0.019 b0.001 tentials decreased from −3.61 to −4.14 MPa, with a significant loss of
Residual 11 0.000
LYS Treatment 2 0.000 0.036
turgor at 41 psu and 43 psu salinities, 35 and 44%, respectively
Residual 11 0.000 (Sandoval-Gil et al., 2012a). Posidonia australis, in contrast, had a
NH3 Treatment 2 2.375 0.001 mean water potential at 4 weeks of −4.5 MPa for both the intermediate
Residual 11 0.044 and highest salinity. After 6 weeks, water potentials decreased even fur-
SER.ASN Treatment 2 0.348 0.001
ther at the highest salinity (54 psu) to almost −6 MPa, compared with
Residual 11 0.033
PHET Treatment 2 0.041 b0.001 the water potential of the medium, less than −4 MPa.
Residual 11 0.000 This study showed that the photosynthetic capacity of P. australis
TYR Treatment 2 0.003 0.258 was maintained with little change for several weeks when subjected
Residual 11 0.002 to a considerable increase in salinity, in contrast to the sensitivity of P.
VAL Treatment 2 0.126 b0.001
Residual 11 0.002
oceanica to increased salinity demonstrated by Marín-Guirao et al.
(2011). Salinity-induced inhibition of photosynthesis in plants mea-
Soluble sugars in leaf sured at 4 weeks was evident only at the highest salinity (54 psu). Spe-
Fructose Treatment 2 9.706 0.663
Residual 11 22.761
cifically, there were no changes in maximum quantum yield (Fv/F) or
Glucose Treatment 2 0.001 0.602 photosynthetic efficiency (alpha); while ETRmax and saturated irradi-
Residual 11 0.001 ance (Ek) were maintained at the same level at intermediate salinity
Sucrose Treatment 2 0.144 b0.001 (46 psu) as for controls (37 psu) for 4 weeks. Only after 6 weeks, were
Residual 11 0.005
these variables reduced in both high salinity treatments. Information
Free amino acids in rhizome available for a few seagrass species indicates that high salinity can re-
ALA Treatment 2 349.320 b0.001 duce photosynthetic performance by altering chloroplast number and
Residual 12 20.190
ultrastructure (Iyer and Barnabas, 1993), capacity of the photosynthetic
ARG Treatment 2 0.336 0.026
Residual 12 0.066 apparatus to harvest light (Ralph, 1998), PSII photochemical efficiency
ASP Treatment 2 0.007 1.000 (Ralph, 1998, 1999; Koch et al., 2007), and inhibition of enzymes for
Residual 12 0.007 photosynthetic carbon assimilation and reduction (e.g., Beer et al.,
GABA Treatment 2 123.047 b0.001 1980). Decline in photosynthetic parameters has been attributed to
Residual 12 2.229
GLUT Treatment 2 0.021 0.519
ion imbalances (with Na+ and Cl− influx and K+ deficiencies) and
Residual 12 0.030 membrane destabilization as well as indicated possible damage to PSII
GLY Treatment 2 0.088 0.001 reaction centres (Ralph, 1998).
Residual 12 0.007 When salinity increases, a low water potential in the surrounding
HIS.GLN Treatment 2 5.531 0.008
medium results in water being drawn away from the plant's tissues,
Residual 12 0.735
ILE Treatment 2 0.121 0.717 and while both raised salinity and desiccation lead to an increase in cel-
Residual 12 0.352 lular ion concentrations, salinity stress also results in ion imbalances
LEU Treatment 2 0.112 0.053 due to changes in the concentrations and ratios of specific ions. In our
Residual 12 0.030 experiment, there was a decrease in the ion concentrations of Ca2 +
LYS Treatment 2 0.011 0.296
Residual 12 0.008
and K+ suggesting ion exclusion, but no change in concentration of
NH3 Treatment 2 1.189 b0.001 Na+ or Cl was observed in whole leaves. Tyerman et al. (1984) showed
Residual 12 0.059 that P. australis leaf cells have a large elastic modulus, ε, so that shrink-
PHE Treatment 2 0.122 0.001 ing or swelling did not contribute significantly whereas ionic concentra-
Residual 12 0.008
tions played a greater role in adjusting turgor. Garrote-Moreno et al.
Ions in leaf (2014) found osmotic potentials, Ψπ in P. oceanica, became more nega-
K+ Treatment 2 355.680 b0.001 tive with increasing salinities, but the contribution by ions was reduced,
Residual 6 7.920
suggesting the activation of ion-exclusion mechanisms at high salinities.
 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260 259

Fig. 5. Concentrations of free amino acids in seagrass Posidonia australis showing significant increases after 6 weeks exposure to raised salinities (46 and 54 psu) compared to seawater
controls (37 psu). (a) Amino acids with concentrations b 0.3 μmol g−1 DW in leaf tissue: argentine ARG, histidine/glutamine HIS/GLN, isoleucine ILE, leucine LEU, lysine LYS (b) amino
acids with concentrations b 1.0 μmol g−1 DW in leaf tissue: alanine ALA, phenylalanine PHE, proline PRO, serine/asparagine SER/ASN, threonine THR, valine VAL. (c) rhizomes: alanine
ALA, gamma-amino butyric acid GABA, glycine GLY, histidine/glutamine HIS/GLN, PHE phenylalanine, proline PRO.

They found osmotic acclimation was more dependent on the accumula- 5. Management implications
tion of organic osmolytes (i.e. soluble sugars), which would involve
higher metabolic costs. We observed a range of responses to changes in salinity in the tem-
Most halophytes exhibit a strategy of partially excluding NaCl from perate seagrass, Posidonia australis that may be suitable as bioindicators.
the cytoplasm and equalising vacuolar and cytoplasmic osmotic pres- Bioindicators are used to monitor biotic responses to environmental
sure with organic solutes more compatible with metabolism. Solutes pressures (e.g., McMahon et al., 2013). Good bioindicators should reflect
that fill this role include amino acids, such as proline (Tyerman et al., a cause-and-effect pathway, where the state of the organism relates to a
1984; Sandoval-Gil et al., 2012a). We found the highest concentrations predictable response to some pressure. They should also be cost-effec-
of alanine, proline and serine/asparagine developed in leaves after tive, easy to measure and interpret (Niemi and McDonald, 2004).
6 weeks at the highest salinity. Tyerman et al. (1984) also found total Posidonia australis is tolerant of high salinity over several weeks: at
amino acids in P. australis varied in parallel with internal osmotic pres- the highest salinity we tested, up to 2 weeks (54 psu), and N6 weeks
sure, but did not identify the specific amino acids responsible. Proline at the intermediate salinity (46 psu). The high salinity tolerance is facil-
increased with salinity for Halodule wrightii, Thalassia testudinum and itated by very negative osmotic and water potentials (osmolarity) that
Ruppia maritime and the concentration of alanine increased in Halophila are developed in the leaves, which counteract the desiccating effects
engelmanii under elevated salinities (Pulich, 1986). Concentrations of of high salinity in the external medium. The physiological adjustments
sucrose in Zostera capricorni also increased with salinity (Tyerman, included modification of osmotic potential (ion sugar and amino acid
1989). Sandoval-Gil et al. (2012a) found an increase in concentrations concentrations) to avoid desiccation and ion toxicity. These sub-lethal
of free amino acids with an increase in salinity from 37 to 43 psu, mostly effects on physiology place extra metabolic demands on the plant that
asparagine (25–45 μmol g−1 FW), with most other amino acids present will be reflected in reduced growth and greater shoot mortality.
in much lower concentrations, b1 μmol g− 1 FW including proline The experimental study has shown that sub-lethal responses to sa-
(0.9–0.16 μmol g− 1 FW). We analysed both soluble sugars and linity are dependent on both concentration and time of exposure. Po-
amino acids and found that the concentration of sucrose was much tential bioindicators for salinity effects associated with brine released
higher (4 orders of magnitude) than any increases in amino acids. in desalination plant outfalls included leaf osmolarity, ion, sugar and
Production of any organic compound is energetically costly, depend- amino acid concentrations. We suggest that although these
ing on the amount produced, so accumulation of sucrose to counter- bioindicators were effective in elucidating the cause-effect pathway as-
act increased salinity, combined with loss of photosynthetic perfor- sociated with increased salinity, they are not field-based and would re-
mance, will tend to reduce photosynthates available to other plant quire time -consuming specialised laboratory analysis. Once
functions, particularly growth. physiological thresholds have been established, we suggest a practical
260 M.L. Cambridge et al. / Marine Pollution Bulletin 115 (2017) 252–260

alternative in the management of impacts of desalination brine dis- Orth, R.J., Carruthers, T.J.B., Dennison, W.C., Duarte, C.M., Fourqurean, J.W., Heck, K.L.,
Randall Hughes, A., Kendrick, G.A., Kenworthy, W.J., Olyarnik, S., Short, F.T.,
charge would be to set an environmental threshold for increases in sa- Waycott, M., Williams, S.L., 2006. A global crisis for seagrass ecosystems. Bioscience
linity around desalination outfalls, based on experimental findings. 56, 987–996.
Then by continuously monitoring salinity utilizing a spatial array of re- Portillo, E., de la Rosa, R., Louzara, M.G., Ruiz, J.M., Marín-Guirao, L., Quesada, J., González,
J.C., Roque, F., González, N., Mendoza, H., 2014. Assessment of the abiotic and biotic
motely sensed salinity meters any breach of the designated threshold effects of sodium metabisulphite pulses discharged from desalination plant chemical
would then result in monitoring, using the suggested bioindicators to treatments on seagrass (Cymodocea nodosa) habitats in the Canary Islands. Mar.
assess loss of seagrass health and ecosystem function. Pollut. Bull. 80, 222–233.
Pulich, W.M., 1986. Variations in leaf soluble amino acids and ammonium content in sub-
G_ tropical seagrasses related to salinity stress. Plant Physiol. 80, 283–286.
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