Impact of the brine from a desalination plant on a shallow seagrass (Posidonia oceanica) meadow

Estuarine, Coastal and Shelf Science 72 (2007) 579e590 www.elsevier.com/locate/ecss

Impact of the brine from a desalination plant on a shallow seagrass (Posidonia oceanica) meadow Esperanc¸a Gacia a,*, Olga Invers b, Marta Manzanera b, Enric Ballesteros a, Javier Romero b b

a Centre d’Estudis Avanc¸ats de Blanes, CSIC, Acce´s Cala St. Francesc 14, 17300 Blanes, Spain Departament d’Ecologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain

Received 10 April 2006; accepted 20 November 2006 Available online 16 January 2007

Abstract Although seawater desalination has increased significantly over recent decades, little attention has been paid to the impact of the main byproduct (hypersaline water: brine) on ecosystems. In the Mediterranean, potentially the most affected ecosystems are meadows of the endemic seagrass Posidonia oceanica. We studied the effect of brine on a shallow P. oceanica meadow exposed to reverse osmosis brine discharge for more than 6 years. P. oceanica proved to be very sensitive to both eutrophication and high salinities derived from the brine discharge. Affected plants showed high epiphyte load and nitrogen content in the leaves, high frequencies of necrosis marks, low total non-structural carbohydrates and low glutamine synthetase activity, compared to control plants. However, there was no indication of extensive decline of the affected meadow. This is probably due to its very shallow situation, which results in high incident radiation as well as fast dilution and dispersion of the brine plume. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: desalination; Posidonia oceanica; brine; physiological stress; Mediterranean; Spain; Balearic Islands

1. Introduction Freshwater deficiency is a common and increasing problem in many Mediterranean areas, particularly in SE Spain and the Balearic Islands. It is caused by the imbalance between the limited available resources and the increasing demand, related to changes in land use and development of the tourism industry. Seawater desalination through reverse osmosis (RO) plants has been, within the last 20 years, a very promising alternative to overcome such deficiency. However, while technical improvements of the desalination process has led to increasing efficiency and cost reductions (i.e. Tsiourtis, 2001), very little attention has been paid to the associated environmental issues. Specifically, the effects of the discharge of the by-product of

* Corresponding author. E-mail address: [email protected] (E. Gacia). 0272-7714/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2006.11.021

the freshwater production, i.e., the brine, on marine coastal ecosystems have only rarely been addressed. Brine resulting from marine desalination is essentially seawater concentrated by a factor that depends on the efficiency of the RO-membranes (55e60%, with maximum concentrations reaching close to 90%; Farin˜as, 2001). This hypersaline wastewater originates dense discharge plumes that can potentially affect marine organisms and communities. The effects depend on the ecosystem receiving the discharge, the general hydrodynamic field, the depth of the discharge and the brine flux (Ho¨pner and Wildemberg, 1996; Einav et al., 2002). In addition, a number of other substances (such as anti-scale additives, biocides, surface active agents or solid residues from back flushing of filters) may continuously or sporadically accompany the release (Morton et al., 1996; Einav et al., 2002) and are also likely to have an environmental impact. Seagrass meadows are coastal communities of outstanding ecological importance (see Costanza et al., 1998; Duarte,

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2002). They are likely to receive the impact of desalination plants, which, for technical and logistic reasons, are usually located near sandy shores; the preferred habitat of seagrasses. Yet, the number of studies addressing the effects of brine on seagrasses is surprisingly low. The evidence available suggests some seagrass tolerance to hypersaline conditions, at least for Atlantic and Pacific species (Tyerman et al., 1984; Walker and McComb, 1990). This seems to be confirmed by the report that Thalassia testudinum shows very limited or no effects when it experiences increases of up to 4 units of salinity from RO discharge plumes (Tomasko et al., 2000). Moreover, in the Canary Islands (subtropical eastern Atlantic) patches of Cymodocea nodosa and Caulerpa prolifera were unaffected by nearby brine, although the salinities registered at vegetated points were always below 37.5 (Pe´rez-Talavera and Quesada Ruı´z, 2001), which is within the natural range for both species. However, recent results from experimental work indicate heightened sensitivity in the Mediterranean species Posidonia oceanica (Ferna´ndez-Torquemada and Sa´nchez-Lizaso, 2005). This highlights the need for more in-depth research into the impact of brine, as it seems plausible that desalination activities will increase world-wide in the near future, and particularly in the Mediterranean region, where this endemic seagrass species covers vast areas between the subsurface and 35 m depth. Posidonia oceanica plays a major ecological role with beneficial effects across the whole Mediterranean (see Boudouresque, 2004). Meadows of this species shelter a high biodiversity of associated algae (i.e. Van der Ben, 1971; Panayotidis, 1979), vertebrates (i.e. Harmelin-Vivien, 1982; Francour, 1997) and invertebrates (i.e. Gambi et al., 1997; Katagan et al., 2001), and contribute to improve water quality (De Falco et al., 2000), prevent coastal erosion (Gacia and Duarte, 2001) and regulate biogeochemical fluxes along the coast (Romero et al., 1994; Gacia et al., 2002). Posidonia oceanica is reported to have experienced a widespread decline in recent decades (see, Pere`s, 1984; Sa´nchez-Lizaso et al., 1990, among others). It is classed as a priority habitat type by European Community directive 92/43/CEE and its conservation has become a priority. Therefore, the potential impacts of new activities should be carefully examined especially in the light of plans to build new desalination plants in the near future. This work reports the possible effects on a Posidonia oceanica meadow of RO brine discharge over more than 6 years. Posidonia oceanica; is a perennial long-lived species (shoot age >25 years, Marba` and Duarte, 1997; meadow persistence of the order of millennia, Mateo et al., 1997) that may take considerable time to show signs of stress. Both the inertia associated with the storage capacity of the different tissues (see, for example, Alcoverro et al., 1997a, 2001) and the clonal integration of the shoots (Marba` et al., 2002) may slow down the plant’s response and make it difficult to foresee effects before the system collapses. This study complements laboratory work, since it includes variables that may modify the plant’s response (temperature, light, water motion, etc.) at the same time as showing the response at the ecosystem level.

2. Methods The approach consisted on a comparative field study, in which different environmental variables (ranges of salinity, dissolved nutrients, pH and dissolved inorganic carbon, and interstitial water salinity), plant traits (i.e. N and P content in tissues, morphometrics) and meadow descriptors (i.e. shoot density, cover) were determined within an area receiving the hypersaline waste and compared to those in undisturbed, reference zones. To take into account the strong seasonality of Posidonia oceanica meadows (see Alcoverro et al., 1995), sampling was performed twice (winter and summer). 2.1. Study site The study was carried out in a shallow Posidonia oceanica meadow located at Platja de Mitjorn on the island of Formentera (Balearic Islands, NW Mediterranean, Spain), in February and June 2001. This meadow has received (more or less continuously) the brine discharge form a seawater desalination plant since 1985, the water used by the plant being pumped from the water table. The discharge rate was 2000 m3 day1 during the summer and about half this during the winter. The brine is discharged 22.3 m from the coastline at a depth of 0.9 m. Samples were taken from three transects, perpendicular to the coastline. One transect (named ‘‘I’’, for ‘‘impacted’’ area, see Fig. 1) started at the outfall. The other two were situated 250 m to the west and to the east of I, in supposedly unaffected areas; reference areas R1, and R2 respectively (see Fig. 1). Some additional measurements were also taken in a fourth transect (R3) situated between I and R1 (Fig. 1). The general layout moving out from the shoreline was the same for all the transects: a small fringe between 0 and 1.2 m deep of bare sand followed by a patchy meadow 1.2 to 1.6 m deep and then a continuous dense meadow from 1.6 m deep out (see Fig. 2). To take into account possible differences in meadow and plant traits caused by meadow structure and/or distance from the coastline, sampling was performed at two

Fig. 1. Aerial photograph of the study site at Platja Mitjorn showing the location of the transects. I: impacted transect (outfall); R1, R2 and (R3): reference transects.

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Fig. 2. Bathymetric profiles of the three transects studied at Platja Mitjorn in which the type of vegetation is detailed.

locations within each transect: one in the patchy and one in the continuous meadow. 2.2. Water features Salinity, temperature, pH, dissolved inorganic carbon (DIC) and dissolved nutrients were determined in water samples collected by divers using 100 ml syringes and transferred to sampling vials for analysis. Pore water samples were obtained using 100 ml syringes connected to a finely perforated tube that was introduced 15 cm into the sediment. Salinity and temperature were measured with a WTW-LF 197 conductimeter connected to a submersible TA 197-LF probe (resolution 0.1) and the pH was measured with a CRISON 507 pH meter (resolution 0.1 pH unit). The salinity probe was calibrated using a precision Oceanographic Salinometer Guildline and the results of our measurements re-calculated after a quadratic regression adjustment between the readings ( y ¼ 0.089 þ 1.007x  0.00024x2). Salinity was measured using the practical salinity scale.

To measure the concentration of dissolved inorganic carbon (DIC) samples were kept in Winkler glass bottles (30 ml) 5  C until they were processed at the laboratory, using the Gramm method (Stumm and Morgan, 1981). Dissolved nutrients (phosphates and nitrates þ nitrites) were measured with an Autoanalyzer (Traacs 2000, from Bran Luebbe) using a method based on Grasshoff et al. (1983). 2.3. Meadow and plant traits Shoot density was estimated by counting shoots into a 20  20 cm square randomly situated in the sampling area (n ¼ 20 quadrates for each sampling area). The projected area covered by the leaves in relation to the whole area of the substrate, ‘‘cover’’, was estimated visually in the patchy meadow, and by counting contacts of Posidonia oceanica along 50 m auxiliary transects (perpendicular to the main transect) in the continuous meadow. Between 10 and 30 shoots were collected at random from each sampling area. The length and the width of the

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leaves were measured to obtain shoot size (cm2 shoot1). Leaf epiphytes were gently removed with a razor blade, dried to constant weight (70  C, 24 h), and referred to shoot size following Alcoverro et al. (1997b). The herbivory pressure corresponded to Sarpa salpa and Paracentrotus lividus attacks, and was estimated as the percentage of leaves bearing marks (bites) of these herbivores (Alcoverro et al., 1997b). Densities of sea urchins (Paracentrotus lividus) and holothurians were estimated in June by counting individuals in randomly situated (n ¼ 20; 40  40 cm) quadrates within each sampling area. 2.4. Physiology Leaf growth, necrosis marks on the leaves, total nitrogen and phosphorous, non-structural carbohydrates (TNC), glutamine synthetase activity (GS) and d13C were measured in 10 to 30 shoots collected at random from each sampling area. Leaf growth was determined in June in I and R1, in both the patchy and the continuous meadows, following a modification of the Zieman (1974) method (see Romero, 1989). Nutrient content in tissues was measured for different plant organs (young leaves, rhizomes and roots) dried (70  C) to constant weight. Total N (% dry weight) was determined using a Carlo-Erba CNH elemental autoanalyzer and total P (% dry weight) was determined by ICP following wet acid digestion in a microwave oven (Sommers and Nelson, 1972; modified by Mateo and Sabate´, 1993). The non-structural carbohydrate content was measured by following the method described in Alcoverro et al. (1999), based on Yemn and Willis (1954), and the glutamine synthetase activity by following Kraemer and Mazella (1996). The d13C and total C content were determined in a continuous flow isotope radio mass spectrometer Delta C coupled to a Flash 1112 elemental analyser via a conflo III interface (Termo Finnigan). Samples of reference material (internal standards) were used to calibrate the system and compensate for drift with time. Experimental precision, based on the standard deviation of replicates of the internal standard, was 0.05%. The isotope ratios were expressed relative to the Pee Dee Belemnite (PDB) standard and calculated in the usual manner. ICP analysis, and total N and C contents and C

isotopic ratios were determined at the Serveis Cientı´fico-Te`cnics (University of Barcelona). 2.5. Statistical analysis The statistical significance of the variability found among transects (factor ‘transect’ n ¼ 3) and among types of meadow (patchy or continuous, factor ‘zone’ n ¼ 2), was assessed using a two-way ANOVA, with ‘transect’ and ‘zone’ as between-subject fixed factors. In summer, sampling was repeated for some of the variables (shoot size, epiphyte biomass, herbivory marks, necrosis marks and nutrient content in tissues) but only in the impacted transect and in one reference (R-1). The time effect was then tested using a three-way, repeated measures ANOVA, with one within-subject factor (time) and two between-subject fixed factors (‘transect’ n ¼ 2 and ‘zone’ n ¼ 2). In the case of TNC, GS, N, P and d13C, a fourth within-subject factor was added (leaves and rhizomes, factor ‘tissue’ n ¼ 2). Whenever necessary, the significance of differences between given experimental conditions was assessed using Tukey’s post hoc test. 3. Results 3.1. Water features Water samples collected at the outfall showed characteristics differing considerably from those of seawater (Table 1). The discharge had a high salinity (around 50, on average), low pH (7.5), and high concentrations of dissolved inorganic carbon (DIC), orthophosphate and, especially, nitrates þ nitrites (approximately 100 times higher than in normal seawater). The influence of the brine was detected throughout transect I (impacted) both in February and June (Fig. 3a and b) although it was greater in February. This was probably due to a more intense hydrodynamism during the summer sampling. The maximum salinity recorded in an area with living Posidonia oceanica was 41.8. This value was detected at the inner edge of the patchy meadow in I, where dead roots and rhizomes were abundant. Pore water salinity was higher along the impacted transect than in R1, whose values corresponded to those of normal seawater (37.4e37.6; see Table 1). No

Table 1 Range of the pore water and water column salinity, and average dissolved inorganic carbon (DIC), pH and nutrient (nitrates þ nitrites and ortophosphate) concentration in the water column at the outfall and the different sampling points: impacted, reference 1 and 2 transects, in P (the patchy meadow) and C (the continuous meadow). Values in brackets correspond to the standard error of the mean Site

Point

Salinity Pore water

Impacted Reference 1 Reference 2

Outfall P C P C P C

39.74e40.14 38.04e39.24 37.44e37.54 37.54e37.64

Water column 41.44e60.14 38.44e39.84 37.84e39.34 37.44e37.64 37.44e37.64 37.44 37.44

DIC (mM)

pH

 NO 2 þ NO3 (mM)

Pi (mM)

3.27 2.25 2.14 2.28 2.20

7.5 (0.025) 8.13 8.25 8.04 8.05 8.12 8.12

20.8 0.42 0.18 0.13 0.32 0.07 0.26

0.57 (0.040) 0.027 0.015 (0.003) 0.009 (0.020) 0.044 (0.003) 0.091 (0.075) 0.041 (0.004)

(0.15) (0.15) (0.03) (0.02) (0.05)

(1.5) (0.12) (0.06) (0.02) (0.05) (0.09)

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(a)

583

Depth (m)

0 44 42 40 39 46

-1

38 40

38

40

42

-2

40

5

0

38 39

39

10

15

20

25

30

Distance to the outfall (m) Outfall

(b)

62 60 58 56 54 52 50 48 46 44 42 40 38 37

Depth (m)

0 38 40 42

38

-1

38

40 42

40

-2 0

5

10

15

20

25

58 56 54 52 50 48 46 44 42 40 39 38

30

Distance from the outfall (m) Outfall

Fig. 3. Bathymetric profile of salinity measured in February (a) and June (b) in transect I.

data on pore water was obtained from R2, but R3 (the closest to the outfall) showed salinities in the interstitial water higher than those of normal seawater, thus indicating that the brine eventually reached this point. The salinity was always observed to be slightly higher in the sediment pore water than above the leaves in the impacted area (Table 2). Fig. 3 shows that the brine stays close to the sediment. In the area studied, sea-floor irregularities may cause accumulation of water pockets with higher densities and salinities, facilitating retention of the interstitial water. 3.2. Meadow structure Shoot density (Table 3) and cover (Fig. 4) were significantly lower in the shallow patchy meadow in I than in the reference site patchy meadows (Table 4). In contrast, shoot density and cover were similar in all the sampling areas within continuous meadows (see Table 3 and Fig. 4). Shoot size (leaf surface; Fig. 5a) and leaf epiphyte load (Fig. 5b) increased significantly from February to June at

most of the stations, as expected from the well-known seasonal trend in this community (see Alcoverro et al., 2004). Shoot size also varied between the plants in the different sampling areas (Fig. 5a), but these variations were not consistent in winter and summer (Table 5). By contrast, the epiphyte load was considerably higher for the shoots collected in the patchy meadow in I (in both February and June) than elsewhere (Fig. 5b; Table 5). The herbivore pressure was low (less than 40% of leaves had marks) at all the stations in February (Fig. 6a) while in June it was significantly higher (85e60% of leaves with marks) in both the patchy and continuous meadows in I (Table 5; Fig. 6a). The herbivory was mainly due to Sarpa salpa (L.) in I (Fig. 6b), in agreement with the very low densities of sea urchins (Table 3). 3.3. Physiology Leaf elongation was similar between the shoots of the two patchy meadows (reference and impacted) where it was

Table 2 Salinity and temperature ( C, in parentheses) values recorded at different points of the meadows studied in June. The data were collected at the surface, above the plant canopy, within the plant canopy and close to the rhizomes in areas with Posidonia oceanica shoots, or the equivalent depths in the absence of plants. P, patchy meadow; C, continuous meadow Site

Impacted Reference 1 Reference 2

Point

Salinity Surface

Above canopy

Inside canopy

Base rhizome

Pore water

Outfall IP IC R1P R1C R2P R2C

59.54 37.54 37.54 37.64 37.64 37.44 37.44

60.14 38.24 38.04 37.64 37.64 37.44 37.44

38.44 38.44 37.64 37.64 37.44 37.44

38.44 38.04 37.64 37.64 37.44 37.44

39.74e40.14 38.64e39.24 37.44e37.54 37.54e37.64 e e

(23.1) (23.9) (23.8) (23.6) (23.5) (23.9) (23.9)

(23.1) (23.8) (23.7) (23.5) (23.9) (23.9)

(23.8) (23.8) (23.7) (23.5) (23.9) (23.9)

(23.8) (23.8) (23.7) (23.5) (23.9) (23.9)

(23.7) (22.4e23.2) (23.7e23.6) (23.2e22.6)

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Table 3 Shoot densities and presence of echinoderms at the different sampling points. Values in parentheses correspond to the standard error of the mean

Impacted Reference 1 Reference 2

Meadow

P C P C P C

Density (shoots m2)

962 1470 1320 1482

(106.4) (49.3) (57.4) (62.9)

100

Density of echinoderms (ind. m2)

80

Paracentrotus lividus

Holothuria spp.

1 (0.31)

0 (0)

5 (0.89)

5 (0.89)

% cover

Site

120

60

40 I R1 R2

20

1632 (87.2)

10 (3.13)

5 (1.56) 0 5-9

measured (Table 6). However, leaf elongation varied between the two continuous meadows, being significantly lower in I than in R1 (IC and R1C; see Tables 4 and 6). Necrosis at the base of the leaves was significantly more prevalent in plants from I (both the patchy and the continuous meadow) than those from R1 and R2 (Fig. 6c). Necrosis incidence was particularly high in I patchy meadow, where shoots showing necrosis averaged 17%, a value that is dramatically higher than the 2% average reference incidence. In fact, in February, only the shoots from the patchy meadow in I showed signs of necrosis in leaf tissues (Fig. 6c). The total N content in young Posidonia oceanica leaves was significantly higher in February than in June at almost

10-14

15-19

20-24

25-29

30-34

distance from the origin (m) Fig. 4. Posidonia oceanica percentage cover of the substrate (averaged over 5 m intervals) in the patchy meadow between the start of the transects (22.3 m from the shoreline) and the beginning of the continuous meadow. Vertical bars correspond to the standard error of the mean.

all the stations considered (Fig. 7), in agreement with the seasonal pattern of nutrient concentration in tissues described for this species (Alcoverro et al., 1995; Invers et al., 2002). The plants closest to the outfall (in I patchy meadow) showed the highest N concentration, both in young leaves and in rhizomes (Fig. 7; Table 7). Total P content was very similar in February and June at all stations for leaves and rhizomes,

Table 4 Summary of the factorial two-way ANOVA to assess significant differences in Posidonia oceanica variables between transects (I, R1 and R2) and Zones (patchy and continuous meadow). p-Values correspond to those provided by a univariate F-test. df, degrees of freedom; MS, mean squares; n.s., not significant Variable

Effect

df

MS

Shoot densities

Transect Zone Tr*Z Error Transect Zone Tr*Z Error Transect Zone Tr*Z Error Transect Zone Tr*Z Error Transect Zone Tr*Z Error Transect Zone Tr*Z Error Transect Zone Tr*Z Error

1 1 1 37 3 3 9 93 1 1 1 103 2 1 2 133 2 1 2 131 2 1 2 133 2 1 2 131

351070 1146791 305285 52869 2925 7737 883 637 0.1444 0.3542 0.8115 0.0306 81068 140593 28671 3897 37104 183 9359 354 1067 460 558 121 37104 183 9359

% Cover

Leaf growth

Shoot size

Epiphyte biomass

Necrosis marks

% Herbivory

F

p-Value

6.64 21.69 5.77

0.0141 0.0000 0.0214

I < R1 P
4.59 12.14 1.39

0.0048 0.0000 n.s.

I < R1, R2 P
4.72 11.59 26.54

0.0321 0.0009 0.0000

I < R1 P the rest

20.81 36.08 7.36

0.0000 0.0000 0.0009

I < R1 < R2 P
0.0000 n.s. 0.0000

I > the rest

8.81 3.80 4.61

0.0003 n.s. 0.0116

I > R1,R2

104.72 0.52 26.42

0.0000 n.s. 0.0000

I > the rest

105 1 26

IP > the rest

IP > others

IP > IC > the rest

E. Gacia et al. / Estuarine, Coastal and Shelf Science 72 (2007) 579e590

(a)

Table 5 Leaf growth (cm2 shoot1 day1) from May to June in shoots from I and R1. Values in brackets correspond to the standard error of the mean

300 February June

250

200

cm2 shoot-1

585

150

Site

Meadow

Growth (cm2 shoot1 day1)

Impacted

P C P C

0.53 0.43 0.47 0.72

Reference 1

(0.025) (0.030) (0.025) (0.045)

100

resolution characterization of the changes induced in seawater, since salinity probably fluctuates according to hydrodynamic forcing, variability in brine production, etc. However (and since

50

(b)

0

(a)

1.8

100 February June 80

mg cm-2

1.5 1.2

60

0.9 40 0.6 20

0.3

I

R1

R2

Patchy meadow

I

R1

R2

(b)

0

Continous meadow

Fig. 5. (a) Posidonia oceanica shoot size (cm2 leaf shoot1) and (b) epiphyte load in leaves (mg cm2 leaf surface) for plants collected in February (empty bars) and June (grey bars). Vertical bars correspond to the standard error of the mean.

except in the continuous meadow of the impacted zone (IC) were we recorded very high values, particularly in the rhizomes (Fig. 7, Table 7). The glutamine synthetase activity (GS) showed high variability between the different stations, particularly in the leaves (Fig. 8), and was higher in leaves than in rhizomes (Fig. 8, Table 5). The leaves in I tended to show lower activity than the reference leaves. Total non-structural carbohydrates (TNC) content was higher in the rhizomes than in the leaves, and generally higher in June than in February (Fig. 9) as expected from previous work (Alcoverro et al., 2001). TNC tended to be lower in I, especially in the patchy meadow. The carbon isotopic signature (d13C) for young Posidonia oceanica leaves from I showed significantly higher proportions of the heaviest isotope than those from the reference sites (Fig. 10; Table 7) both, in the patchy and continuous meadows. No significant seasonal differences were found in this parameter.

% leaf with marks shoot-1

0.0

(c)

80

60

40

20

0 25 20 15 10 5 0

4. Discussion The Posidonia oceanica meadow near the brine discharge showed characteristics significantly different from those of the reference areas investigated, supporting the notion of a measurable impact from these hypersaline waters. The salinity values obtained during the two periods are not sufficient for a high-

I

R1

R2

Patchy meadow

I

R1

R2

Continous meadow

Fig. 6. Total herbivore pressure as (a) the percentage of leaves with bite marks in a shoot (b) herbivory marks due to Salpa salpa and (c) percentage of leaves in a shoot with necrosis marks (dark spots) at the base of the leaves, for plants collected in February and in June. In February we only found necrosis marks in the patchy meadow of the impacted transect. Vertical bars represent the standard error of the mean.

586

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Table 6 Summary of repeated ANOVA measurements performed at the two sampling times (February and June) to assess differences in the selected Posidonia oceanica variables in I and R1 for the different meadows (patchy and continuous). p-Values correspond to those provided by a univariate F-test. df, degrees of freedom; MS, mean squares; n.s., not significant. For the variable glutamine synthetase (GS) the within-subject factor corresponds to the different tissues instead of time Variable

Effect

Shoot size

Between subjects Transect Zone Within subjects Time Tr*Z Tr*Ti

Epiphyte biomass

Herbivory

Necrosis marks

d13C

Glutamine synthetase

Between subjects Transect Zone Within subjects Time Tr*Z Tr*Ti Between subjects Transect Zone Within subjects Time Tr*Z Tr*Ti Between subjects Transect Zone Within subjects Time Tr*Z Tr*Ti Between subjects Transect Zone Within subjects Time Tr*Z Tr*Ti Z*Ti Tr*Z*Ti Between subjects Transect Zone Within subjects Tissue Tr*Z Tr*Tissue Z*Tissue Tr*Z*Tissue

df

MS

F

p-Value

1 1

23561 250177

12.40 131.63

0.0012 0.0000

1 1 1

57501 395 23281

20.58 0.21 8.33

0.0001 n.s. 0.0065

1 1

2.83 2.20

56.18 43.77

0.0000 0.0000

1 1 1

1.10 1.97 0.19

18.83 39.14 3.16

0.0004 0.0000 n.s.

1 1

15865 533

89.72 3.02

0.0000 n.s.

1 1 1

21887 2839 2346

70.76 16.06 7.59

0.0000 0.0003 0.0092

1 1

1039 1348

13.88 17.99

0.00067 0.00015

1 1 1

6 1348 43

0.04 17.99 0.28

n.s. 0.00015 n.s.

29.48 0.02

56.14 0.05

0.0001 n.s.

1.59 0.74 0.87 0.33 1.39

2.52 1.40 1.37 0.52 2.20

n.s. n.s. n.s. n.s. n.s.

1 1 1 1 1 1 1 1 1

2 1

190 161

4.47 3.79

0.0355 n.s.

1 2 2 1 2

2929 161 283 167 212

68.69 3.79 6.64 3.92 4.98

0.0000 n.s. 0.0115 n.s. 0.0266

the measurements were obtained in relatively calm weather conditions), our values probably reflect a situation of low dilution. Yet in these calm conditions, the water reached the shoots rather diluted, 39e40 in the patchy area and 38e39 at the edge of the continuous meadow, compared to a background salinity of 37, and 41e60 for the brine. Some dispersed shoots surrounded by abundant dead rhizomes were also found at salinities of 41.8 in June. The pore water showed a greater increase in salinity

than did the water column, and even in some areas where the water column salinity was unaltered at the time of measurement. Given the fact that turnover in pore water is lower than in the water column (see Huettel et al., 1996), interstitial water salinity can be considered a tracer of brine. Consequently, we conclude that brine influence can extend beyond what we considered the impacted transect, up to 50 m west from the outfall (R3), where we registered salinities of 38 in the pore-water indicating that the

E. Gacia et al. / Estuarine, Coastal and Shelf Science 72 (2007) 579e590

% N (DW)

(a)

% P (Dw)

(b)

(a)

Young leaf 4

Rhizome 4

February

February

June

June

3

3

2

2

1

1

0

(b)

0

0.35

0.35

0.30

0.30

0.25

0.25

0.20

0.20

0.15

0.15

0.10

0.10

0.05

0.05

0.00

I

R1

R2

Patchy meadow

I

R1

587

R2

Continous meadow

0.00

I

R1

R2

Patchy meadow

I

R1

R2

Continous meadow

Fig. 7. (a) Total N content and (b) total P content in young leaves (empty bars) and rhizomes (dashed bars) of shoots collected in February and June. Vertical bars correspond to the standard error of the mean.

brine eventually reached that point. This view is supported by the data on the carbon isotopic ratio (d13C) in leaf tissues, which increases in plants from the impacted zone. This increase in the heaviest isotope is probably due to a different isotopic composition of the water used in the treatment plant, which, as mentioned, is groundwater. Metabolic fractionation of DIC in ground water is expected since methanogenic bacteria strongly discriminate against d13C (Lajtha and Michener, 1994). In R3, the plants also showed a slightly heavier isotopic signal in June, confirming the occasional influence by the discharge plume indicated by the interstitial water data. The discharge seemed to cause significant changes in plant traits such as an increase in nitrogen content in tissues and an associated decrease in glutamine synthetase activity, together with deterioration in plant health, especially in the area closest to the discharge (patchy meadow). This deterioration was reflected in the high incidence of necrosis, and the decrease in the total nonstructural carbohydrates content. Carbohydrate storage is a crucial feature of the Posidonia oceanica carbon balance, allowing survival in winter (Alcoverro et al., 2001). Carbohydrate decrease can thus compromise growth and vitality of the affected plants. This, in addition to the higher epiphytic load and the increased frequency of herbivore attacks, determined lower

vitality, probably resulting in shorter leaves and a reduction in abundance (as expressed by both shoot density and cover). However, the impact detected seems to have been caused not only by the high salinity, but also by the high nutrient concentration, especially of nitrates, which reached 100 times the base level. The origin of the water obtained for desalination (groundwater with possible agricultural influence) accounts for such a high concentration of dissolved inorganic nitrogen. In effect, some of the symptoms detected should have been caused by a nutrient increase, as indicated by the high nitrogen content in the seagrass tissues (Duarte, 1990; Alcoverro et al., 1997a). Increased nitrogen availability is known to increase epiphyte load (Gacia et al., 1999; Moore and Wetzel, 2000), and it stimulates herbivore activity (Ruiz, 2001). Moreover, a high nitrogen supply can cause a high demand for energy and carbon skeletons for protein synthesis, reducing the carbohydrate bulk (Burkholder et al., 1992; Invers et al., 2004). All these plant responses to eutrophication are well documented, and can cause seagrass deterioration and/or mortality (e.g. Ruiz et al., 2001). Surprisingly, however, the activity of the enzyme glutamine synthetase is significantly reduced in the leaves affected by the brine and this may indicate salinity stress (Lin Kao and Kao, 1996). Hypersaline conditions have

E. Gacia et al. / Estuarine, Coastal and Shelf Science 72 (2007) 579e590

588

Table 7 Summary of the four-way ANOVA performed for the two between factors; Transect (I, R1) and Zone (P and C) and two within factors; time (February and June) and tissue (young leaves and rhizomes). Only statistically significant interactions ( p < 0.005) are shown F

MS Effect

1 1 1 1 1 1 1 1 1

4.96 1.21 0.64 6.92 0.45 0.58 1.55 0.22 0.27

Young leaf Rhizome 40

p-Level

131.78 32.20 12.54 183.97 14.35 11.31 49.79 5.78 6.96

µmol g-1Fw h-1

df Effect Total N Transect Zone Tissue Tr*Z Tr*Tm Tr*Ts Tr*Z*Tm Tr*Tm*Ts Z*Tm*Ts

50

0.0000 0.0005 0.0076 0.0000 0.0053 0.0099 0.0001 0.0429 0.0298

30

20

10

0

I

R1

R2

I

Patchy meadow

Total P Transect Zone Tr*Z Tr*Tm Z*Tm Tr*Ts Tm*Ts Tr*Z*Tm Tr*Z*Ts Tr*Tm*Ts

1 1 1 1 1 1 1 1 1 1

0.02 0.04 0.01 0.00 0.01 0.01 0.00 0.00 0.01 0.00

23.93 61.48 9.71 19.24 72.53 9.82 6.88 13.51 10.44 6.49

0.0012 0.0001 0.0143 0.0023 0.0000 0.0139 0.0305 0.0063 0.0120 0.0343

TNC Transect Time Tissue Tr*Z Tr*Tm Z*Tm Z*Ts Tm*Ts Tr*Z*Tm Tr*Z*Ts

1 1 1 1 1 1 1 1 1 1

24.99 43.94 20.08 4.11 5.68 5.83 0.98 14.99 3.07 1.07

101.16 184.46 198.87 16.63 23.83 24.46 9.75 31.83 12.88 10.62

0.0000 0.0000 0.0000 0.0035 0.0012 0.0011 0.0142 0.0005 0.0071 0.0115

% TNC (DW)

R2

Fig. 8. Activity of the enzyme glutamine synthetase in young leaves (white bars) and rhizomes (grey bars) of Posidonia oceanica collected in June. Vertical bars correspond to the standard error of the mean.

been shown to decrease the assimilation of inorganic N via a reduction of GS activity in rice roots (Lin Kao and Kao, 1996) and result in an accumulation of intracellular ammonia in seagrasses (Pulich, 1986) causing cellular death due to ammonia toxicity (Mehrer and Mohr, 1989). This last effect agrees with the high frequency of necrotic marks encountered, as necrosis is one of the first symptoms identified in plants subjected to hypersaline stress in the aquarium (Ferna´ndezTorquemada and Sa´nchez-Lizaso, 2005). Overall, it seams that two major factors have an impact: increased nitrogen and hypersaline conditions. From our present data we cannot establish the relative contribution of each of them on the observed meadow deterioration. The influence of the brine also reached the continuous meadow, where the plants showed some evidence of salinity stress (lower leaf growth rates, increased leaf necrosis, lower GS activity) but no evidence of stress caused by an excess

Young leaf

Rhizome

4

40 February

February

June

June

3

30

2

20

1

10

0

R1

Continous meadow

I

R1

R2

Patchy meadow

I

R1

R2

Continous meadow

0

I

R1

R2

Patchy meadow

I

R1

R2

Continous meadow

Fig. 9. Total non-structural carbohydrates content in young leaves (empty bars) and rhizomes (dashed bars) of shoots collected in February and June. Notice the different scales of the plots. Vertical bars correspond to the standard error of the mean.

E. Gacia et al. / Estuarine, Coastal and Shelf Science 72 (2007) 579e590

589

Ferna´ndez-Torquemada and Sa´nchez-Lizaso (2005); salinity has an impact above 39.1. Therefore, care should be taken to dilute brine considerably, before it reaches seagrasses in order to preserve these ecosystems.

0

-5

-10

δ13C

Acknowledgements

-15

-20

February June

-25 I

R1

R3

Patchy meadow

I

R1

R3

Continous meadow

Fig. 10. d13C isotopic signature measured in young leaves of shoots collected in February and June. Vertical bars correspond to the standard error of the mean.

of nutrients. In any case, the meadow showed no decline, as cover and shoot density were similar to those found in the reference zones. The decrease in leaf growth found in the continuous meadow (but not in the patchy one) together with the fact that necrosis incidence was similar in the two areas, seems contradictory. However, it could be explained by a compensatory effects on growth of increased nutrients (see Alcoverro, 1997a) and higher light availability. Specific experimentation is required to confirm this. The only measured change in ecosystem integrity in the continuous meadow was the absence of echinoderms, holothurians and sea urchins. Laboratory tests have shown that invertebrates, particularly echinoderms (Lloret-Oltra and Sa´nchez-Lizaso, 2001) and crustaceans (Barbera` et al., 2001), are very sensitive to brine. Our observations confirm this finding, and suggest possible changes in trophic fluxes, as both sea urchins and holothurians are important elements in the seagrass food-web (Zupi and Fresi, 1984). 5. Conclusions This study shows that under field conditions Posidonia oceanica is very sensitive to brine discharges from desalination plants. This is due in part to the hypersaline conditions, and in part to the associated eutrophication. This eutrophication, which has already been described as an indirect consequence of desalination (Chestner, 1975; Tomasko et al., 2000) seems particularly relevant when the water for desalination is obtained from ground wells, potentially enriched in nutrients from agricultural drainage, as is the case in this study. The scarcity of salinity measurements in our study prevents us from extracting robust conclusions about critical salinity thresholds. However, based on the fact that there is a significant impact on the patchy meadow, a slight impact on the continuous meadow and only a trace impact on transect 3, we suggest as the critical threshold a value of 39.3. This value is in extremely good agreement with the experimental results of

This project was financially supported by a contract with CEDEX-Ministerio de Fomento, within the framework of a joint project with ACSEGURA. We wish to thank J. Ruı´z and B. Hereu for their valuable help in the field, in the cold, February waters of Formentera and J. Buceta for the good time during the fieldwork in June. We also wish to thank J. Camp for allowing us to use the oceanographic salinometer to calibrate our probe. Finally, we would like to thank our colleagues from the UA and IEO, ‘the salt team’, for their stimulating discussions during the course of the project. Comments from two anonymous referees significantly contributed to improving the final version of this paper. References Alcoverro, T., Duarte, C.M., Romero, J., 1995. Annual growth dynamics of Posidonia oceanica: contribution of large-scale versus local factors to seasonality. Marine Ecology Progress Series 120, 203e210. Alcoverro, T., Romero, J., Duarte, C.M., Lo´pez, N.I., 1997a. Spatial and temporal variations in nutrient limitation of seagrass Posidonia oceanica growth in the NW Mediterranean. Marine Ecology Progress Series 146, 155e161. Alcoverro, T., Duarte, C.M., Romero, J., 1997b. The influence of herbivores on Posidonia oceanica epiphytes. Aquatic Botany 56, 93e104. Alcoverro, T., Zimmerman, R.C., Kohrs, D.G., Alberte, R.S., 1999. Resource allocation and sucrose mobilization in light-limited eelgrass Zostera marina. Marine Ecology Progress Series 187, 121e131. Alcoverro, T., Manzanera, M., Romero, J., 2001. Annual metabolic carbon balance of the seagrass Posidonia oceanica: the importance of carbohydrate reserves. Marine Ecology Progress Series 211, 15e116. Alcoverro, T., Perez, M., Romero, J., 2004. Importance of within-shoot epiphyte distribution for the carbon budget of seagrasses: the example of Posidonia oceanica. Botanica Marina 47, 307e312. Barbera`, C., Sa´nchez, P., Sa´nchez-Lizaso, J.L., 2001. Test agudo de ecotoxicidad para estimar el efecto combinado de la salinidad y temperatura en la mortalidad del misida´ceo Leptomysis posidoniae Wittmann, 1986 (Crustacea). In: AEDYR (Ed.), Actas II Congreso Nacional AEDYR, Madrid, pp. 1e14. Boudouresque, C.F., 2004. Marine biodiversity in the Mediterranean: status of species, populations and communities. Traveaux Scientifiques Parc National de Port-Cross 20, 97e146. Burkholder, J.M., Mason, K.M., Glasgow, H.B., 1992. Water-column nitrate enrichment promotes decline of eelgrass Zostera marina: evidence from seasonal mesocosm experiments. Marine Ecology Progress Series 81, 163e178. Chestner, R.H., 1975. Biological impact of a large-scale desalination plant at Key West, Florida. R.H. Office of Research and Monitoring, Environmental Protection Agency, Washington, 150 pp. Costanza, R., Andrade, F., Antunes, P., van der Belt, M., Boersma, D., Boesch, D.F., Catarino, F., Hanna, S., Limburg, K., Low, B., Molitor, M., Pereira, J.G., Rayner, S., Santos, R., et al., 1998. Principles for sustainable governance of the oceans. Science 281, 198e199. De Falco, G., Ferrari, S., Cancemi, G., Baroli, M., 2000. Relationship between sediment distribution and Posidonia oceanica Seagrass. Geo-Marine Letters 20, 50e57.

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