Mélanie Béguer
email: melanie.beguer@cemagref.fr

Lionel Feuillassier

Pierre Elie

Philippe Boët

Michel Girardin

Exoskeletal deformities in Palaemonidae: are they a threat to survival?

Keywords: Exoskeletal deformities, Palaemon spp, Gironde estuary, Estuary, Mortality, Disease, Contamination, Rearing, Decapoda

Shrimps of the Palaemon genus have been affected for over fifteen years by a problem of exoskeletal deformities, particularly in the Gironde estuary (France). Given the large numbers of individuals affected, this study focus on a better description of the phenomenon to estimate its impact on shrimps. This study, on P. longirostris and P. macrodactylus, is based on samples collected from 1992 to 2007 in the Gironde estuary with particular focus on the year 2007, and on a 4-month rearing of adult shrimps in experimental system. The different approaches reveal the relative persistence of the phenomenon since 1992 (with on average more than 58% of ovigerous females affected) and its persistence during shrimp life cycle, even after several moults. Important consequences associated with deformities at the individual level were demonstrated: a higher mortality, a lighter egg mass and a lower weight for a given size. The presence of exoskeletal deformities is now a major phenomenon, with profound effects on individual shrimps, and which may in turn have an important impact on the survival of the Palaemon longirostris population in particular.

Introduction

Exoskeletal deformity is a major phenomenon that has affected some shrimp of the Palaemon genus (P. longirostris, P. macrodactylus and P. serratus) for over 15 years [START_REF] Béguer | First description of main skeletal deformations in shrimps populations of the Gironde estuary (France)[END_REF]. This phenomenon has recently been observed and briefly described for Palaemon genus shrimp populations in the Gironde estuary, but it also affects, although to a lesser degree, shrimp in other French and European estuaries, especially the Scheldt (Belgium) and the Rhine (Rotterdam, Netherlands) [START_REF] Béguer | First description of main skeletal deformations in shrimps populations of the Gironde estuary (France)[END_REF]. In 2006, on average, over 40% of shrimp observed in the Gironde population were suffering from deformities [START_REF] Béguer | First description of main skeletal deformations in shrimps populations of the Gironde estuary (France)[END_REF]. Such a phenomenon, affecting a very large proportion of individuals from wild populations, has never been described before. Indeed, although morphological anomalies remain a very well-known and a common occurrence among crustaceans [START_REF] Aguirre | Abnormal rostrum and telson in two species of penaeid shrimp (Decapoda, Dendrobranchiata, Penaeidae) from the Pacific Coast of Mexico[END_REF]De Grave and Mentlak, 2008;[START_REF] Delphy | Pinces anormales de crabes[END_REF][START_REF] Dutt | Notes on abnormal shrimps and prawns[END_REF], they are usually observed in isolated individuals or sometimes in farm rearing.

In the Gironde estuary shrimps, and particularly in P. longirostris and P. macrodactylus, these deformities are mainly in the cephalothorax, which appears either wrinkled or bent instead of being smooth and straight. The rostrum is also often partly affected: either bent to one side, or pointing downwards. Some often spectacular dissymmetries, caused by scaphocerite or uropod hypertrophy, have also been described for both species [START_REF] Béguer | First description of main skeletal deformations in shrimps populations of the Gironde estuary (France)[END_REF].

P. longirostris is the most common and abundant shrimp in the Gironde estuary [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF][START_REF] Girardin | Surveillance halieutique de l'estuaire de la Gironde -suivi des captures 2006-2007 -étude de la faune circulante[END_REF][START_REF] Sorbe | Les Décapodes Natantia de l'estuaire de la Gironde (France). Contribution à l'étude morphologique et biologique de Palaemon longirostris H. Milne Edwards[END_REF]. It is a typical estuary species, completing its entire life cycle in this environment [START_REF] Campbell | Osmoregulation of the estuarine prawn Palaemon longirostris (Caridea: Palaemonidae)[END_REF][START_REF] Gonzalez-Ortegon | An illustrated key to species of Palaemon and Palaemonetes (Crustacea: Decapoda: Caridea) from European waters, including the alien species Palaemon macrodactylus[END_REF][START_REF] Gurney | Some notes on Leander longirostris M. Edwards, and others British prawns[END_REF]. It is fished commercially in the Gironde estuary in large quantities using traditional methods and every year, for over a century, between 27 and 82 tonnes have been fished and consumed [START_REF] Girardin | Surveillance halieutique de l'estuaire de la Gironde -suivi des captures 2006-2007 -étude de la faune circulante[END_REF][START_REF] Holthuis | FAO Species Catalogue. Vol 1: Shrimps and Prawns of the World -An annoted Catalogue of Species of Interest to Fisheries, FAO Fisheries Synopsis[END_REF]. Shrimps are also an important link in the food web in this estuary, since they are prey for many species of exploited fish, like the bass or the meagre [START_REF] Pasquaud | Les relations trophiques : éléments de structuration des peuplements ichtyologiques en milieu estuarien -Application à l'estuaire de la Gironde[END_REF][START_REF] Pasquaud | A preliminary investigation of the fish food web in the Gironde estuary, France, using dietary and stable isotope analyses[END_REF]. P. longirostris has been monitored via monthly faunal surveys in the Gironde since 1978 [START_REF] Girardin | Surveillance halieutique de l'estuaire de la Gironde -suivi des captures 2006-2007 -étude de la faune circulante[END_REF] and today its numbers seem to be in decline compared with the abundant quantities observed at the beginning of the monitoring period.

Deformities first appeared in P. longirostris before 1992 (oldest preserved samples), but after the beginning of the 1980s, since neither [START_REF] Sorbe | Les Décapodes Natantia de l'estuaire de la Gironde (France). Contribution à l'étude morphologique et biologique de Palaemon longirostris H. Milne Edwards[END_REF] nor [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF] observed this phenomenon during their studies. Deformities have also been observed in P. macrodactylus, an exotic species that has colonised the Gironde estuary for about a decade [START_REF] Béguer | First record of the invasive oriental shrimp Palaemon macrodactylus Rathbun, 1902 in France (Gironde estuary)[END_REF] and which is now found here very frequently. This invasive species is very similar to the native species, P. longirostris, both in its morphology and its ecology, especially its feeding habits [START_REF] Sitts | Predation by the estuarine shrimps Crangon franciscorum Stimpson and Palaemon macrodactylus Rathburn[END_REF][START_REF] Sorbe | Les Décapodes Natantia de l'estuaire de la Gironde (France). Contribution à l'étude morphologique et biologique de Palaemon longirostris H. Milne Edwards[END_REF].

The causes of the deformities so far remain unknown, although recent studies seem to dismiss microorganisms, viruses or bacteria as a possible origin, and also parasites (Béguer, unpublished data). Some serious hypotheses regarding the action of organic or other types of micropollutant have since been suggested. Given the scale of this phenomenon, its impact both at the level of the individual and of the population could be considerable, and it needs to be assessed.

The aim of the present study is two-fold: (i) to provide a better description of the phenomenon of exoskeletal deformity on the basis of recent samples from the Gironde estuary (2007) and others collected since 1992, and (ii) to assess its consequences by observing individuals kept in captivity for several months, in a controlled environment.

Materials and methods

The part of the study based on preserved samples deals with P. longirostris and P. macrodactylus, the two species most frequently found. However, as P. longirostris is present in greater numbers and has similar deformities as P. macrodactylus, detailed results are presented for this species only. The experimental part (rearing) deals exclusively with P. longirostris, a species that is easier to harvest in sufficient numbers and for which there was already a methodological basis for rearing them [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF].

Strategy to demonstrate the progress and scale of the phenomenon

Since 1979, a large part of the estuary had been sampled monthly for fauna survey (mainly fishes and crustaceans, Girardin et al., 2008) (Fig 1). The protocol for these campaigns is described in [START_REF] Béguer | First record of the invasive oriental shrimp Palaemon macrodactylus Rathbun, 1902 in France (Gironde estuary)[END_REF]. Temperature and salinity were recorded at each station.

Shrimp samples from each of these campaigns have been preserved since 1992. To assess the scale of the deformity phenomenon and its evolution over the years, sub-samples were observed. For every year until 2007, about thirty ovigerous P. longirostris shrimps collected from the upstream part of the estuary during May or June, depending on abundance, were measured and examined. Carapace length (CL, to the nearest 0.1 mm) was used as standard size for all specimens. It was decided to observe and measure shrimp of the same sex, females carrying eggs, as they are easily recognisable, and give a good representation of the general state of health of the population. The period and the place where sampling took place were similar every year. In all, 539 female shrimps were examined under a binocular microscope to detect the presence of deformities.

Strategy to characterise deformities and relate them to environmental factors

From November 2006 to November 2007, monthly campaigns to collect small mobile fauna (from the bottom and the surface) were carried out over a 60 km length of the Gironde estuary, included the 20 km area around the nuclear power plant (Fig 1). The protocol was the same as that used since the beginning of the 1980s. During these fishing campaigns, standard physico-chemical parameters were taken: temperature, salinity, conductivity and turbidity.

Collected shrimp were preserved in 70% alcohol and sub-samples were prepared in the laboratory: about thirty individuals per station, per month and, where possible, per species were chosen at random. Each shrimp was then measured (CL, to the nearest 0.1mm), sexed, and weighed (Fresh weight, W, to the nearest 0.0001 g). In all, 1,937 individual females, 3,145 males and 111 juveniles of P. longirostris were measured. A total of 2,235 P. macrodactylus were examined in a similar way (776 females, 694 males and 765 juveniles).

The eggs from 247 ovigerous P. longirostris females were detached by immersing whole individuals in a 10% bleach solution; the shrimp were then weighed (Fresh weight, W, to the nearest 0.0001 g). The eggs in each clutch were counted and characterised according to stage of development: visible eye (stage II) or no visible eye (stage I). For P. macrodactylus, 89 clutches were weighed and the eggs counted.

Observation of shrimp in experimental environment

Capturing the shrimps

During February 2008, a batch of P. longirostris was caught using bow nets with mesh size 5 mm, close to the port of Pauillac, about 80 km from the mouth of the Gironde estuary (Fig 1). Captured specimens were observed for damage to their body or appendages, and only undamaged shrimps (360, CL 6.0-16.6 mm) were selected for an experiment of 4 month duration. Half the individuals, which had no external visible morphological deformity, were the control. The other half had at least one deformity.

Rearing conditions

The shrimps were gradually acclimatised to the rearing conditions, which was different from their natural environment in terms of salinity (5 PSU), for about 3 hours. Fresh well water was used, maintained at a dissolved oxygen concentration of 10.3 mg l -1 , pH 8.2, and salted artificially at 15 PSU. Temperature was 15°C at the beginning of the rearing period and 20°C at the end, in accordance with the increase observed in the natural environment. Temperature fluctuations were avoided by the use of a thermoregulated chamber. These parameters were determined from a previous successful rearing of shrimps [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF]. Before the beginning of the experiment, the shrimps were quarantined for 2 weeks, in order to select the least fragile individuals. Individual units were designed and built specially for this experiment (Fig 2). It was essential that all the shrimp were kept separate from each other, (i) to avoid problems of cannibalism that can frequently occur, (ii) to ensure that all individuals were fed identically, and (iii) to be able to monitor and recover the exuvia from each shrimp. The submerged units, consisting of a PVC pipe, 8 cm in diameter and 9 cm high, provided each shrimp with a vital volume of 453 cm 3 , sufficient to allow it to move around and to carry out the moulting processes. The submerged part had a 3 mm mesh which allowed good circulation of the water (oxygenation and waste removal). The units were grouped in batches of 6 and placed in 2 square tanks, 1.98 m x 1.95 m. These two tanks formed a closed system, with the water circulating at a rate of 45 l/min though two sand filters and a 25W UV filter.

Every two days the shrimps were allowed to feed ad libitum on an artificial food (Biomar Ecostart no.1.9, composed of 56% protein, 18% fats, 12.2% brute ash and 0.3% cellulose).

The shrimps were kept in the dark throughout the entire experiment (except when being measured), as these conditions are similar to those found in the natural environment (the Gironde estuary is very turbid) and thus limited any stress related to captivity.

Observations and measurements

Every two days, when the shrimp were feeding, the following physico-chemical parameters were measured: temperature, oxygen, pH, nitrates and nitrites. Each individual shrimp was also checked at this time. When a moult was observed, the shrimps underwent a series of measurements 2 days later, this delay was in order to give the new carapace time to harden [START_REF] Nouvel-Van Rysselberge | Contribution à l'étude de la mue, de la croissance et de la régénération chez les Crustacés Natantia[END_REF]. The shrimps could then be manipulated without serious risk of causing trauma. For each shrimp, CL (to the nearest 0.1 mm), W (to the nearest 0.0001 g) and any deformities (location, size, persistence) were recorded. Photographs were also taken of some individual before and after each moult, to confirm the persistence of external deformities or the appearance of new ones. At the end of the experiment, or when a shrimp died, they were sexed (too difficult to carry out on live individuals without risk of causing trauma, as they are transparent) and dry weight was determined after oven drying (48h, 60°C).

Statistical analyses

All analyses were carried out using the R software (R foundation core team, 2005).

Percentages were compared using the Chi² test. Links between the presence of deformities in the shrimp and environmental factors were explored using General Linear Models (GLM) [START_REF] Mccullagh | Generalized linear models[END_REF]. A binomial error distribution via a logistic link function was applied. To obtain the relative importance of the variables (predictor), we assessed the residual deviance change after excluding each variable in turn from the total model. To evaluate the model, we used its percentage of variance explained (i.e. the difference between the Null model deviance and the residual model deviance, divided by the Null model deviance). ANCOVAs were used to evaluate differences between individuals with and without deformities in respect of size-weight ratio and size-number of eggs ratio in ovigerous females. Differences were considered as significant with a 95% (p<0.05) confidence level.

Results

Scale of the phenomenon since 1992

Deformities in Palaemon species have been observed at least since 1992, the earliest year for which samples are preserved (Fig 3). The proportion of shrimps with at least one deformity is 58.8% on average, but has varied between 30% (1992) and 80% (2000 and 2003). Variations from year to year are significant (Chi² test, p>0.05, N=539) but no linear trend has so far been observed. From the samples, no link could be established between the proportion of deformed females and water temperature recorded during the fishing. However, there was a significant positive correlation with mean annual water temperature: the proportion of deformed females increased with temperature (GLM, binomial family, p>0.05). There was no correlation with salinity.

Characterisation of deformities: analysis carried out on samples harvested during 2006-

Link with size, sex and environmental factors

This analysis concerned a total of 5,193 P. longirostris individuals. Using a GLM (binomial family with a logit link), relationships were established between the presence of deformities and several factors: size of individual, sex, water temperature, salinity and turbidity. The response curve obtained described the probability of the shrimp presenting at least one deformity as a function of the combination of variables (Fig 4, a, b andc).

The model accounts for only 12.5 % of the total variability observed, however some variables were significantly correlated with the presence of deformities. Size and sex accounted for the majority (p<0.05): the probability of finding a shrimp with a deformity increased with size but differently according to sex (Fig 4-a). For example, a female shrimp with a cephalothorax length of about 10 mm has more than a 50% probability of having at least one deformity. The second factor which accounts for the presence of deformity is temperature, independently of other factors. The higher the temperature the greater the probability of finding a deformed shrimp . Salinity is the last factor to be linked significantly with the presence of deformity. The higher the salinity, the lower the probability of finding a deformed shrimp. For example, in fresh water, a female shrimp with a cephalothorax length of about 10.5 mm has a 60% risk of having a deformity compared with 50 % in water at 25 PSU (Fig 4-c).

Turbidity was not linked significantly with the presence of deformity (p>0.1).

The same analysis carried out on the 2,235 P. macrodactylus shrimp gave similar results. The relationship between size, sex and the presence of deformity was therefore identical in both species.

Size-weight relationship

The relationship between shrimp fresh weight, patted dry (abbreviated to W in g) and length of cephalothorax (CL in mm) was examined according to the presence or absence of a deformity, and sex of the shrimp. For P. longirostris, this relationship was weaker in shrimp with a deformity than without a deformity, for both sexes (ANCOVA, p<0.05) (Fig 5). Thus for shrimp of equal size, a deformed shrimp had a smaller weight than a non-deformed one, and this weight difference increased with size. The same difference was observed in P. macrodactylus males; for the females of this species, however, the difference was not significant.

Fecundity

The relationship between the total number of eggs carried by each female and her size was not significantly different between deformed and non-deformed females for both species of shrimp (ANCOVA, p>0.1). The developmental stage of the eggs spawned (I or II) did not affect the results. On the other hand, in the case of P. longirostris a difference was observed for the relationship between total weight of the clutch and size of the female: the deformed shrimp had a lighter clutch than the individuals with no deformity, whatever their size (ANCOVA, p<0.05) (Fig 6). This difference was also seen in P. macrodactylus although it was not statistically significant (ANCOVA, p>0.5).

Observations monitoring live individuals for 4 months

Ninety per cent of the shrimp monitored in this experiment were females with cephalothorax length of between 7.0 and 16.6 mm CL and fresh weight of between 0.194 and 2.380g. Males measured between 6.7 and 12.0 mm Cl and weighed between 0.268 and 1.234 g.

Retaining deformities over several moults

During the 130 days of monitoring, all individuals with a deformity retained the identical deformity in the course of successive moults (up to 7) (Fig 7). No further deformity was observed in any of these individuals. In shrimps with no deformity at the outset, no deformity appeared during the experimental period.

Moulting frequency

During the observation period, the majority of shrimp moulted between 3 and 4 times (minimum 1, maximum 7) (Table 1). Moulting frequency was examined using a GLM, with size or weight, sex and the presence of deformity being the 4 explanatory factors used. Size or weight accounted best for variations in the number of moults from one individual to another: the larger or heavier the individual, the fewer the moults. During the same period, the males moulted more often than the females (median 3 moults for the females, compared with 4 for the males). Shrimps with deformities moulted more frequently than those without: 25% of shrimps with deformities moulted 5 times or more compared with 16% of those without deformities (Table 1).

The inter-moult period (number of days between each moult) was examined in the same way.

The males had a shorter average inter-moult than the females (median 21 days for the females and 20 days for the males) and this period increased with size. The inter-moult between the first and second moults was significantly shorter for shrimp with deformities, compared with shrimps without deformities (23.3 days on average for the deformed shrimp against 24.9 days for the non-deformed shrimps).

Growth

Growth rate in terms of size between the 1 st and 4 th moults was examined using a GLM, according to initial size, sex and the presence or not of deformities. Average growth rate was 6.5%, varying from 0 to 55% according to the individual. It did not depend on sex, nor on the presence of deformities, but only on size (the larger the individual, the lower the growth rate).

Growth in terms of weight increase was examined in the same way. The growth rate between the 1 st and 4 th moults was 6.5% on average, although some individuals lost weight and others gained considerable weight (up to 100%). This rate did not depend on the presence of deformities, but on initial weight and sex: the females put on more weight than the males and larger individuals gained less weight.

The dry weight measurements were compared and there was no significant difference between individuals with deformities and those without. Dry weight represented about 20% of fresh weight.

Mortality

Mortality was zero until the 7 th week of the experiment (Fig 8). At the end of the observation period, cumulated mortality was 12.2% in the batch of shrimps without deformities (i.e. 22 individuals died out of 180) and 24.4% for those with deformities (i.e. 44 individuals out of 180). This is a significant difference (p<0.01) which does not depend on the size of the shrimp nor on their sex. It was also observed that these deaths usually occurred during the moulting process, or shortly afterwards.

Discussion

Analysis of the preserved samples of P. longirostris ovigerous females collected every year since 1992 revealed the persistence of this phenomenon over the course of several years, on a fairly large scale since, on average, more than 58.8% of the samples observed were affected.

The proportions of individuals affected did vary significantly from year to year, which is explained in part by the positive correlation with temperature.

For both species, P. longirostris and P. macrodactylus, the presence of exoskeletal deformities is related first of all by the size of the shrimp: the larger (i.e. older) the shrimp, the greater the probability of having at least one deformity. According to their growth curve (Béguer, unpublished data;[START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF], the difference between males and females can be explained simply by the difference in their size-age relationship. Two hypotheses can explain that fact: either the factor(s) causing the deformities is(are) accumulated throughout the shrimp's life, or the youngest individuals are less resistant and thus have a higher mortality rate, making way in the environment for the larger, and hence the less fragile individuals. Deformities could also be triggered by the sexual maturity, which is raised at a size of 8 mm CL for females (Béguer et al., in press). Contamination by pollutants [START_REF] Rainbow | Phylogeny of trace metal accumulation in crustaceans[END_REF][START_REF] Ramade | Précis d'écotoxicologie[END_REF] or parasite infestation (Lauckner, 1984) may explain the first hypothesis. However, histological sections have been carried out and didn't show any parasite [START_REF] Larrieu | Déformations exosquelettiques chez les crevettes P. longirostris et P.macrodactylus: les contaminants métalliques sont-ils responsables? Mémoire de Master II Lauckner, G[END_REF]. No differences in heavy metal concentration were also found between deformed and normal shrimps [START_REF] Larrieu | Déformations exosquelettiques chez les crevettes P. longirostris et P.macrodactylus: les contaminants métalliques sont-ils responsables? Mémoire de Master II Lauckner, G[END_REF], but concentrations into the hepatopancreas were fairly high. Further studies are needed in this area, and also on organic pollutants, or even on cocktails of pollutants that can act in synergy.

Independently of the size of the individual, the occurrence of deformities also depends on water temperature or on the season. It is difficult to separate these two factors: the month necessarily incorporates the temperature factor, but other parameters too, such as day length, rainfall, or even the presence of prey and predators. There is a peak in numbers of individuals with deformities in spring and in summer, when temperatures are highest. P. longirostris begins to reproduce at the end of winter and from the end of spring it enters a major growth phase [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF]Béguer et al., in press). After reproduction, the shrimps are weakened and must feed a great deal. One might think, therefore, that their accelerated metabolism during that period would favour the emergence of deformities. Temperature could also be a factor affecting the availability or the toxicity of the factor responsible for the deformities [START_REF] Dimmock | The influence of environment on the morphology of Macrobrachium australiense (Decapoda: Palaemonidae)[END_REF][START_REF] Lemonnier | Summer syndrome" in Litopenaeus stylirostris grow out ponds in New Caledonia: Zootechnical and environmental factors[END_REF]. This seasonality could also be explained by fluctuations in pollutant input: in Gironde, spring and summer are the seasons when pesticides or other micropollutants are discharged and carried into the estuary by rivers and runoff water.

Salinity also proved to be an explanatory factor for the occurrence of deformities: individuals found in saline areas were less likely to have deformities than those in fresh water. Salinity is a factor that can affect the bioavailability of certain substances. Indeed, [START_REF] Pierron | Effects of salinity and hypoxia on cadmium bioaccumulation in the shrimp Palaemon longirostris[END_REF] have shown that the higher the salinity, the lower the accumulation of cadmium by P. longirostris. This accumulation is also favoured by hypoxia. In the context of our study it was not possible to take into account oxygen levels in the environment as insufficient data were available (oxygen levels were not measured at each sampling station and there were difficulties in obtaining a representation of earlier conditions in a single reading).

It has been shown that in shrimp of the same size, individuals affected by at least one deformity have a lower weight than normal individuals, and this is true for both sexes of P. longirostris. This could be attributed to difficulties experienced by deformed shrimp in feeding in the natural environment. There could be mechanical problems, due possibly to a reduced ability to grasp their prey (hypertrophy of scaphocerites), or poor assimilation of food due to a physiological problem, or again due to the energy expenditure linked with trying to combat the factor responsible for these deformities (e.g. in the case of a detoxification mechanism). At the end of the 4-month rearing period, the weight of shrimps with deformities was still significantly lower than the weight of the control shrimp for identical size, even though they were fed in exactly the same way, both in quality and quantity. As it was not possible to check whether both categories of shrimp consumed the same food, the problem can come from food assimilation or extra energy expenditure. Nevertheless, we cannot ignore the fact that in the natural environment it is probably mechanically more difficult for a shrimp with a deformity to catch its prey.

The mass of eggs carried by females of P. longirostris without deformities was heavier than in specimens of equal size with deformities, thus may be indicating a decrease in the reproductive performance of the shrimps. This can have repercussions for the survival of the population. Analysis of the lipid composition of these spawns could be carried out to check whether there is a drop in egg quality and hence in the potentialities of their offspring.

Most of the results for the native P. longirostris are also valid for the exotic P. macrodactylus.

However, in P. macrodactylus, the differences between individuals with deformities and those without (for weight and mass of eggs) were not always statistically significant. The reason may be that fewer data were available for this species, but also because of its different physiology or ecology. Indeed, P. macrodactylus lives further downstream, so in a more saline part of the estuary, and this, according to our results, seems to be less favourable for the emergence of the deformity phenomenon. Moreover, P. macrodactylus is an exotic species which settled here only fairly recently, and so the population is perhaps not yet affected to the same extent as P. longirostris.

Rearing a large number of shrimp in individual units, in a controlled environment for 4 months provided complementary data on the characteristics of deformities in living individuals and in particular helped demonstrate the negative impact of the deformities on the individuals that are affected. Overall mortality during our experiments was very low (zero during the first seven weeks), which shows that the rearing conditions during the 4 months were very good. Indeed, for P. longirostris, [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF] had a mortality of 50% over 70 days, in similar salinity and temperature conditions.

Every one of the affected shrimp that were monitored retained their deformities during successive moults (up to 7). P. longirostris has a lifespan of 2 years maximum [START_REF] Aurousseau | Eléments d'écologie de la crevette blanche, Palaemon longirostris, dans l'estuaire de la Gironde[END_REF][START_REF] Van Den | Observation on the population dynamics and distribution of the White Prawn Palaemon longirostris H. Milne Edwards, 1837 Crustacea Decapoda Natantia) in the Netherlands, with special reference to its occurrence in the major rivers[END_REF], so although we are not able to assert that shrimp retain these deformities throughout their life, nevertheless we know that this is the case during a significant part of it. The hypothesis of a mutation at the level of the epidermal and/or germinal cells can therefore be envisaged. We also suggest that these deformities can be transmitted to their offspring; this would explain the persistence and the worsening of the phenomenon over several years. If larvae were hatched then monitored in a controlled environment this would show whether individuals were affected from their youngest stage of development.

No deformity appeared spontaneously in the control shrimp. This suggests that either these individuals were the most resistant and will never have the problem, or the conditions in the rearing environment prevented the phenomenon from occurring. However, no reversal was observed too. A rearing environment that incorporated several batches in various controlled environments could be planned to check the influence of physico-chimical parameters on the appearance of the deformities.

We have shown that moulting frequency is significantly higher in shrimp with deformities than in those without, which would indicate a different metabolism in the two categories of shrimp. Although indispensable, the moulting process is nevertheless not without some risk for the life of the shrimp [START_REF] Drach | Mue et cycle d'intermue chez les Crustacés Décapodes[END_REF]. During our experiment we observed that all the shrimp that died did so during the moulting process or soon after. This may therefore explain the significantly higher mortality rate (double) in the shrimp with deformities compared to the controls. During the monitoring period, shrimp with deformities and those without were kept in the same conditions, thus the deformities are the only explanatory factor for the difference observed in mortality. As moulting frequency is accelerated by the increase of temperature [START_REF] Barclay | Development of spawning and mass larva-rearing techniques for the euryhaline shrimp Palaemon macrodactylus[END_REF][START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF][START_REF] Nouvel-Van Rysselberge | Contribution à l'étude de la mue, de la croissance et de la régénération chez les Crustacés Natantia[END_REF], deformed shrimps should have a higher mortality rate than non deformed ones particularly in summer.

We present a diagram summarising the consequences observed and the possible links and action/functioning of the factors responsible for the deformities (FRD) (Fig 9). The FRD could be permanently present in the environment, acting on the shrimp throughout their lifespan, on all developmental stages. This could explain why in the natural environment we found a big proportion of deformed shrimps although a higher mortality rate was demonstrated in our experiment. The FRD could then be accumulated during the shrimp growth and cause a general physical weakening. The shrimps with deformities would then have offspring of inferior quality, a higher frequency of moult and then a higher level of mortality than those without deformities. The first stage could be affected directly by the FRD, including during the egg stage, or though a parental transmission (genetic or metabolic).

The action of the FRD could be influenced directly or indirectly by environmental parameters: increased effects as temperature increases, and on the contrary, inactivity of the effects in very high levels of salinity. Finally, the big proportion of deformed shrimps and the individual effects associated could have serious consequences on population viability. The next step is now to look for the possible causes of these deformities in order to evaluate the possibility of reducing or eliminating it, the possibility of using shrimps as a bio-indicator species, and to detect probable effects on other inhabitants of the estuary.

List of figures

Fig 1 .

 1 Fig 1. The sampling stations in the Gironde estuary.

Fig 2 .

 2 Fig 2. Individual units built specially for the experiment.

Fig 3 .

 3 Fig 3. Proportion of deformed ovigerous shrimps P. longirostris since 1992, in samples from the Gironde estuary collected during the spring (numbers in bars are the number of shrimps observed).

Fig 4 .

 4 Fig 4. Predicted values from the GLMs: mean probability for a shrimp (P. longirostris) to be affected by a deformity according to its size and sex (A), the water temperature (B, example for females of different sizes) and the salinity (C, examples for females for different sizes).

Fig 5 .

 5 Fig 5. Relationship between weight and cephalothorax length (CL) according to the sex and the presence of deformities for the shrimp P. longirostris (Deformed females :W=0,0017Cl2,60 r²=0.86, p<0.001 : Not deformed females W=0.0012 x CL 2.78 , r²=0.94, p<0.001; Deformed males W=0.0019(CL)+2.52 r²=0,78 p<0.001; Not deformed Males W=0.0013 x CL 2.73 , r²=0,90, p<0.001).

Fig 6 .

 6 Fig 6. Relationship between clutch weight (CW) and female size (CL), for P. longirostris deformed and not deformed. (Deformed : CW=(0.0245 x CL) -0.1537 (r²=0.42, p<0.001); Not deformed : CW=(0.00355 x CL ) -0.250 (r²=0.56, p<0.001).)

Fig. 1

 1 Fig. 1

  Fig. 2

Table 1 :

 1 Moulting frequency during the experimental rearing study (4 month) of P. longirostris adults.

Table 1 :

 1 Moulting frequency during the experimental survey (4 month) of P. longirostris adults

	Number of moults per	Proportion of	Proportion of	Total number
	shrimp during	deformed	non-deformed	of individuals
	experimental time (4	individuals	individuals
	month)	(%)	(%)
	1 moult	9	10	33
	2 moults	14	20	61
	3 moults	29	32	109
	4 moults	23	22	80
	5 moults	17	12	52
	6 moults	6	3	15
	7 moults	2	1	5

Acknowledgements

The authors would like to thank Bernard Ballion, Romaric Le Barh, Jean-François Bigot, Gérard Castelnaud, Christine Gazeau, Aymeric Guibert, Philippe Jatteau, Mario Lepage and everyone who took part in the sampling process. Grateful thanks also to all the staff at the St-Seurin station where the shrimp were reared. Thanks also to Aude Lochet and Hilary Koziol for language help. This study was financed by the CNPE du Blayais and the Aquitaine Regional Council.


J M Ruiz
email: jmruiz@mu.ieo.es

C Marco-Méndez

J L Sánchez-Lizaso

Remote influence of off-shore fish farm waste on Mediterranean seagrass (Posidonia oceanica) meadows

Keywords: Fish farms waste, Posidonia oceanica, Epiphytes, Mediterranean Sea, Stable isotopes

The aim of this study was estimating the remote influence of waste dispersed from a large offshore fish farm complex (6,197 ton year -1 ) on the nearshore Posidonia oceanica meadow (26-27 m deep) located at a distance of 3 km. Measurements of isotopic nitrogen content in epiphytes and seagrass leaf tissues, epiphyte biomass, shoot size, herbivory pressure, shoot density and seagrass meadow cover, performed in this meadow (FA area) were compared with those obtained in an undisturbed control meadow (CA area) to evaluate: (1) the remote influence of waste and (2) the impact of such influence on seagrass condition. In adition, δ 15 N measurements in particulate organic matter of natural and anthropogenic origin were used in a single isotope mixing model to elucidate the relative contributions of these sources to the isotopic N signal measured in epiphytes and leaf tissues. Total tissue N content was similar between meadow areas, but δ 15 N signatures were significantly higher in the FA area than in the CA area both in epiphytes and seagrass leaf tissues. Results from the mixing model, together with available information on local currents and previous studies, support the conclusion that the dispersion of farm wastes over large areas (spanning kilometres) are responsible for the elevated δ 15 N signatures found in the FA meadow area. Despite this, no changes in meadow structure were detected and only some changes at the level of seagrass community (epiphytes abundances and herbivores activity) could be interpreted at the light of nutrient-induced effects in the FA area. Results from this study indicate that concentrating aquaculture facilities in offshore areas is a strategy not totally exempt of environmental risk on nearshore sentitive habitats such as seagrass meadows.

Introduction

The increasing use of floating net cages in the Mediterranean Sea is a subject of major concern, due to the impact of farm waste on marine ecosystems (FAO, 2006).

Floating net cages lead to considerable amounts of dissolved and particulate carbon, nitrogen and phosphorus contained in uneaten feed components, excreta and faeces being released into the water column [START_REF] Hall | Chemical fluxes and mass balances in a marine fish cage farm. I. Carbon[END_REF][START_REF] Hall | Chemical fluxes and mass balances in a marine fish cage farm: IV. Nitrogen[END_REF][START_REF] Holby | Chemical fluxes and mass balances in a marine fish cage farm[END_REF][START_REF] Dosdat | Marine aquaculture effluent monitoringmethodological approach to the evaluation of nitrogen and phosphorus excretion by fish[END_REF]. By altering water quality and sediment characteristics (Holmer et al., 2007;[START_REF] Holmer | Impact of marine fish cage farming on sediment metabolism and sulphate reduction of underlying sediments[END_REF][START_REF] Iwama | Interactions between aquaculture and the environment[END_REF][START_REF] Karakassis | Diel variation of nutrients and chlorophyll in the sea bream and sea bass cages in the Mediterranean[END_REF][START_REF] Wu | Impact of marine fish farming on water quality and bottom sediment: a case study of the sub-tropical environment[END_REF], these aquaculture inputs have been shown to affect both the functioning and structure of microbial [START_REF] Vezzuli | Bacterial community structure and activity in fish farm sediments of the Ligurian Sea (Western Mediterranean)[END_REF], planktonic [START_REF] Pitta | Natural vs. Mariculture induced variability in nutrients and plankton in the Eastern Mediterranean[END_REF], infaunal [START_REF] Karakassis | Impact of cage farming of fish on seabed in three Mediterranean coastal areas[END_REF][START_REF] Mirto | Nematode community response to fish-farm impact in the western Mediterranean[END_REF]) and seagrass [START_REF] Apostolaki | Fish farming enhances biomass and nutrient loss in Posidonia oceanica (L.) Delile[END_REF][START_REF] Cancemi | Effects of organic matter input from a fish farming facility on a Posidonia oceanica meadow[END_REF][START_REF] Delgado | Seagrass regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean)[END_REF]1999;[START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF] communities in the Mediterranean coastal ecosystem.

Seagrass meadows are dominant habitats of recogized ecological relevance in temperate and tropical coastal areas [START_REF] Green | World atlas of seagrasses[END_REF], but they are experiencing a worldwide decline mainly associated to human activity [START_REF] Orth | A global crisis for seagrass ecosystems[END_REF]. Net cage farming developed throughout the 1990s on the Spanish Mediterranean coast, with small fish farms (i.e. with an annual fish production of several hundred tons) installed in shallow, sheltered and semi-exposed environments where the endemic seagrass Posidonia oceanica forms dense and continuous meadows. In these low dispersive environments, high concentrations of dissolved nutrients and particulate (POM) and sedimentary (SOM) organic matter have led to the loss and deterioration of Posidonia oceanica meadows [START_REF] Apostolaki | Fish farming enhances biomass and nutrient loss in Posidonia oceanica (L.) Delile[END_REF][START_REF] Cancemi | Effects of organic matter input from a fish farming facility on a Posidonia oceanica meadow[END_REF][START_REF] Delgado | Seagrass regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean)[END_REF][START_REF] Pergent | Preliminary data on the impact of fish farming facilities on Posidonia oceanica meadows in the Mediterranean[END_REF][START_REF] Pergent-Martini | Impact of fish farming facilities on Posidonia oceanica meadows: a review[END_REF][START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF]. This seagrass decline has been related to the effects of nutrient excess on plant physiology (Pérez et al., 2008), the toxic effects of sediment anoxia and high sulphide concentrations on plant tissues [START_REF] Frederiksen | Effect of increased sediment sulphide concentrations on the composition of stable sulphur isotopes (δ 34 S) and sulphur accumulation in the seagrasses Zostera marina and Posidonia oceanica[END_REF][START_REF] Pérez | Physiological responses of the seagrass Posidonia oceanica to elevated organic matter content in sediments: an experimental assessment[END_REF], and with nutrient-induced interactions between plants and other components of the seagrass community, such as macroalgae, epiphytes and herbivores [START_REF] Delgado | Seagrass regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean)[END_REF][START_REF] Leoni | Morphological responses of Posidonia oceanica to experimental nutrient enrichment of the canopy water[END_REF][START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF]Ruiz et al., in press).

As a result of the limited availability of un-vegetated sheltered sites in nearshore environments and the continuous expansion of this aquaculture industry, it has been necessary to place farms in deeper (>30 m) offshore waters with mainly un-vegetated soft beds [START_REF] Basurco | Marine fish farming in Spain[END_REF] in order to avoid further degradation of P. oceanica meadows. In these offshore environments, higher current speeds and greater depths should lead to a greater dispersion rate of farm waste and a reduction of SOM, with benthic communities underneath fish cages being affected less as a result [START_REF] Aguado-Giménez | Comparisons between some procederes for monitoring off-shore cage culture in Western Mediterranean Sea: sampling methods and impact indicators in soft substrata[END_REF][START_REF] Maldonado | The environmental impact of Mediterranean cages fish farms at semi-exposed locations: does it need a re-assessment?[END_REF]. However, this contention ignores the possible effects of dispersed farm waste on nearby seagrass communities. The influence of aquaculture waste over a large area has tended to be underestimated by previous studies based on traditional approaches involving only a physicochemical and biological characterization of seawater and sediments, which determine that the extent of the impact of fish farms is very local (no more than a few hundred metres), or even nonexistent [START_REF] Aguado-Giménez | Comparisons between some procederes for monitoring off-shore cage culture in Western Mediterranean Sea: sampling methods and impact indicators in soft substrata[END_REF][START_REF] Cromey | DEPOMOD-modelling the deposition and biological effects of waste solids from marine cage farms[END_REF][START_REF] Karakassis | Diel variation of nutrients and chlorophyll in the sea bream and sea bass cages in the Mediterranean[END_REF][START_REF] Pearson | The environmental impacts of marine fish cage culture[END_REF][START_REF] Pitta | Natural vs. Mariculture induced variability in nutrients and plankton in the Eastern Mediterranean[END_REF][START_REF] Pitta | Fish farming effects on chemical and microbial variables of the water column: a spatiotemporal study along the Mediterranean Sea[END_REF][START_REF] Telfer | Monitoring environmental effects of marine fish aquaculture[END_REF].

However, stable isotope techniques applied recently to trace the spatial extent of farm waste in water, sediment, benthic organisms and bioassays revealed that aquaculture products disperse to distances ranging between hundreds of metres and several kilometres [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF][START_REF] Jones | Assessing ecological impacts of shrimp and sewage effluent: biological indicators with standard water quality analyses[END_REF][START_REF] Lin | Macroalgal bioindicators (growth, tissue N, δ 15 N) detect nutrient enrichment from shrimp farm effluent entering Opunohu Bay, French Polynesia[END_REF]Sará et al., 2004;Sanz et al., in press;[START_REF] Vizzini | Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean[END_REF][START_REF] Ye | Tracing the influence on sediments of organic waste from a salmon farm using stable isotope analysis[END_REF]. Therfore, the suitability of the "offshore strategy" to preserve nearshore Posidonia oceanica meadows against aquaculture waste has yet to be properly evaluated.

In this study, we combined the use of seagrass bioindicators, stable isotope techniques and spatial analysis to evaluate: (1) the remote influence on the Posidonia oceanica meadow of waste originated by an offshore fish farm complex, and (2) the impact of such an influence on the condition of the seagrass. To this end, we selected a set of Posidonia oceanica variables that have previously shown to effectively reflect both the influence and impact of farm waste at different levels of seagrass organization [START_REF] Pérez | Physiological responses of the seagrass Posidonia oceanica to elevated organic matter content in sediments: an experimental assessment[END_REF][START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF]: total and isotopic nitrogen content in epiphytes and seagrass leaf tissues, epiphyte biomass, shoot size, herbivory pressure, shoot density and meadow cover. We examined the null hypothesis that there are no differences in these variables between a meadow area in front of the fish farm facilities and an, unpolluted control area of meadow. A mixing model approach was also applied, using isotopic nitrogen signals obtained in epiphytes and seagrass leaves, to determine the specific contribution of farm waste with regard to other natural and anthropogenic POM sources.

Materials and Methods

Study area

The study was conducted in a town on the southeast coast of Spain (San Pedro del Pinatar; Murcia; 37º 50´12.88´´N, 0º47´34.26´´W. Fig. 1). A fish farm complex has operated in the waters of this town since 1998, with a current total annual production of 6.197 tons of blue fin tuna (Thunnus thynnus), sea bream (Sparus aurata), meagre (Argyrosomus regius) and sea bass (Dicentrarchus labrax). Available data on current speed and direction in the farming site taken over a six-month period (Sánchez-Lizaso, unpubl. data) indicate that surface currents (7 m depth) most frequently run parallel to the coastline in NW (34%) and SW (25%) directions, with an average speed of 10 cm s - 1 and a maximum speed of 68 cm s -1 . Deeper currents (25 m depth) run mainly in SE (35%) and NW (25%) directions, with an average speed of 7 cm s -1 , and a maximum measured speed of 27 cm s -1 . In this coastal area, the Posidonia oceanica meadow forms a large and continuous band 25 km long (a total surface area of ca. 5.000 ha), between 1 m and a mean depth limit of 27 m (Ruiz 2007;Fig. 1). Hydrodynamic exposure and substrate type are similar throughout the whole meadow area. Oceanographic characteristics (salinity, nutrients, light attenuation coefficient and Chl a) correspond to typical high oligotrophic waters of this geographical area [START_REF] Vargas-Yáñez | Cambio climático en el Mediterráneo español[END_REF], except in the central area, due to the influence of hypersaline waters of the Mar Menor coastal lagoon. A database of 90 sampling points available for this meadow (Ruiz, unpubl. data) shows that the meadow structure is highly homogeneous throughout this area, and corresponds to a healthy meadow in a good state of conservation.

Sampling design and seagrass variables

Seagrass sampling was carried out in August 2005 at the deepest point of the Posidonia oceanica meadow (26-27 m) in front of the fish farming area (hereinafter 'FA', Fig. 1). The nearest distance between the edge of the meadow and the fish cages was 2.8 km. At the time of the sampling (summer), Posidonia oceanica shoot and epiphyte biomasses [START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF][START_REF] Sánchez-Lizaso | Estudio de la pradera de Posidonia oceanica (L.) Delile de la Reserva Marina de Tabarca (Alicante): Fenología y producción primaria[END_REF] were at their maximum, as was fish production, with annual maximums of light and typical temperatures for the time of year in the Mediterranean [START_REF] Klein | Oceanografía y régimen hidrológico[END_REF]. The FA meadow was compared with a control meadow area (CA) 12.2 km in a southerly direction at the same depth, very close to the Marine Reserve of Cabo Palos-Islas Hormigas (Fig. 1). This control meadow area was selected due to its similarity with the FA meadow, and to rule out any possible influence of fish farm effluents, hypersaline waters from the coastal lagoon or any other anthropogenic influence. No alternative control sites could be found either northward, due to the influence of continental inputs, or to the south, due to the intense alteration of deep Posidonia oceanica meadows caused by illegal trawling over recent decades [START_REF] Martín | Cuantificación del impacto de las artes de arrastre sobre la pradera de Posidonia oceanica (L.)[END_REF].

A nested sampling design [START_REF] Quinn | Experimental design and data analysis for biologists[END_REF] was used to incorporate and partition spatial variability of the selected seagrass variables at the different spatial scales (from metres to kilometres) included in each meadow area. Two replicated locations, separated by approximately 1.5 kilometres, were selected in each meadow area (L1-L2 in FA and L3-L4 in CA, Fig. 1). Two sites separated by 100 metres were selected within each location. Meadow structure showed high patchiness in all sites, so three patches separated by a few metres were selected at random. For seagrass variables measured at the level of individual shoots (i.e. shoot size, epiphyte load, total N content and isotopic N signatures), six vertical shoots of Posidonia oceanica were randomly collected within each patch and transported to the laboratory for further measurement and analysis. At the patch level, the selected variables were shoot density and herbivore pressure. Shoot density (number of shoots per m -2 ) was measured by counting shoot numbers in 400 cm 2 quadrants placed in six randomly selected patches within each site.

In each site, herbivory pressure was determined in three randomly selected patches as the frequency (percentage) of leaves with recognizable marks of herbivores bites [START_REF] Boudouresque | Découverte de l'herbier de Posidonies[END_REF], relative to the total number of leaves contained in 40 shoots collected within each patch. At the meadow level (i.e. sites), meadow cover (as a percentage) was measured by visually estimating the percentage of substrate occupied by shoot patches within twelve 1600 cm 2 quadrants placed at regular intervals along a 25 m line transect.

In order to determine the specific contributions of external, natural and anthropogenic organic matter sources to the isotopic N signature measured in plant and epiphytes tissues (see mixing model analysis below), POM was collected in six replicated sediment traps deployed close to the net cages and in one site of the control area (CA) over the seagrass canopy. POM samples at the first site were mainly composed of fish faeces and hence we assume that they represent the external anthropogenic source to be mainly farm waste (FFW); at the second site we assume that POM samples represent the external natural source (NAT) that is composed of a mixture of both autochthonous and allochthonous materials of diverse origin (Dolenec 2006[START_REF] Sarà | Use of stable isotopes to investigate dispersal of waste from fish farms as a function of hydrodynamics[END_REF]. Sediment traps consisted of PVC cylinders of 11 cm diameter and 55 cm height, as recommended by many authors in similar studies [START_REF] Cromey | DEPOMOD-modelling the deposition and biological effects of waste solids from marine cage farms[END_REF].

Trapped particles were concentrated in a small glass tube placed at the bottom end of the cylinder, which was recovered after 24 hours and immediately transported to the laboratory for analysis of stable isotopes (see below).

Laboratory analysis

In the laboratory, epiphytes were scraped and separated from the leaves of each shoot using a razor blade, and the epiphytes were dried to a constant weight (60º C, 24 h) to estimate their biomass (mg DW shoot -1 ). The length and width of each leaf on a shoot were measured to calculate shoot size as leaf surface area per shoot (cm 2 shoot -1 ); epiphyte biomass was normalized to shoot size to estimate epiphyte load (mg cm -2 ).

Dried epiphyte and leaf tissues (young leaves, 0-50 days old) were finely grounded in a mortar and, for each replicate, approximately 2 mg of dry weight was encapsulated to determine total (%DW) and isotopic (δ 15 N, ‰) nitrogen. POM samples obtained in sediment traps were filtered in laboratory through pre-combusted fibreglass filters (Whatman GF/F), and the filters were dried and ground for analysis of total and isotopic nitrogen. Total N content was measured using a Carlo-Erba CNH elemental autoanalyzer. The nitrogen stable isotope composition was measured using an EA-IRMS (Thermo Finnigan) analyzer in continuous flow configuration, and was compared with a worldwide standard (atmospheric N 2 ) to estimate the relative amount of 15 N, or δ 15 N in plant tissues and POM, as described by the following equation [START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]:

δ 15 N (‰) = (R sample / R standard -1) x 10 3
where R is defined as the atomic 15 N/ 14 N ratio. Acetanilide was used as the internal laboratory standard.

Statistical analysis

A nested ANOVA model was used to test the null hypothesis that seagrass variables were similar across meadow areas, locations, sites and seagrass patches. For seagrass variables measured at the individual shoot level, a four-factor model was considered: (1) "areas" (fixed factor with 2 levels: FA and CA), (2) "locations" (random factor nested in "areas" with 2 levels: L1 and L2 in area FA and L3 and L4 in area CA),

(3) "sites" (random factor nested within each "location" with 2 levels) and ( 4) "patches" (random factor nested within each "site" with 3 levels). For seagrass variables replicated at site level (meadow cover, shoot density and herbivory pressure), a model with the first three factors considered was used. Prior to the analysis, Cochran's C-test was used to test the homogeneity of variances [START_REF] Underwood | Experiments in ecology. Their Logical and Interpretation Using Analysis of Variance[END_REF], and data were properly transformed when necessary. When no homogeneous variances were rendered with any type of transformation, the significance level was set at 0.01 instead of 0.05, as ANOVA can withstand variance heterogeneity, particularly for large balanced experiments, reducing the possibility of a Type I error [START_REF] Underwood | Experiments in ecology. Their Logical and Interpretation Using Analysis of Variance[END_REF].

A single-isotope three-source mixing model [START_REF] Phillips | Source partitioning using stable isotopes: coping with too many sources[END_REF] was used to determine the relative contribution of farm sources in the isotopic N signature of epiphytes in relation to other potential external sources present in the study area.

Epiphyte N signatures measured in FA and CA meadow areas were designated as the targets in the model. Seagrass leaf N signature was not included in this analysis, as it is governed not only by external water sources but also by internal physiological mechanisms and sediment pools [START_REF] Romero | Nutrient dynamics in seagrass ecosystems[END_REF][START_REF] Touchette | Review of nitrogen and phosphorus metabolism in seagrasses[END_REF] that were not measured in this study. The isotopic signal of three potential sources were considered as the end-members of the model: fish farm waste (FFW), external natural sources (NAT), both of which were measured in this study (see previous section), and urban waste (UW), reported from literature (δ 15 N = 9.7‰, [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF]. This last external anthropogenic source was introduced into the model due to the presence of an urban sewage outfall 6 km north of the FA area.

Results

Table 1 shows the results of the nested ANOVA test on seagrass variables.

Meadow cover varied between 14.5 ± 3.4% and 27.0 ± 2.4% (Fig. 2A), but no significant differences were detected between areas or at any other spatial scale within them. Shoot density was also similar (116-200 shoots m -2 ) between areas and sites, with the exception of the significant higher mean values founded in location L4 in the CA meadow (295.2 ± 12.5 shoots m -2 ) (Fig. 2B; Table 1). Herbivore pressure on seagrass leaves was generally very low (0-7%), but showed highly significant variation between locations due to the extremely high values measured in location L2 of the FA meadow area (8.3-28%; Fig. 2C andTable 1). This herbivore pressure was accounted for by the herbivorous sparid fish Sarpa salpa in L2, but by other unidentified herbivore species (possibly isopods) in the CA meadow locations.

Shoot size was slightly (but not significant) lower in the FA meadow (421.4 ± 17.8 cm 2 shoot -1 ) than in the CA meadow (472.2 ± 22.0 cm 2 shoot -1 ); spatial variability of this variable was only significant at the smaller scale i.e. between patches (Fig. 3A, Table 1). Epiphyte load showed lower mean values in the FA area (1.11 ± 0.23 mg DW cm -2 ) than in the CA area (1.77 ± 0.14 mg DW cm -2 ) (Fig. 3B), but differences were not significant at this and any other spatial scale (Table 1). Subsequent calculations of the observed power of the test for 'Area' effects (13.8%) indicated a low capability to detect differences observed in epiphyte load (i.e. high probability of Type II error); the non-significant term 'Location' (Table 1) was then pooled to increase the power of the test after considering a conservative significance level of α = 0.25 for the pooled term [START_REF] Sokal | Biometry: The Principles and Practice of Statistics in Biological Research[END_REF][START_REF] Quinn | Experimental design and data analysis for biologists[END_REF]. After pooling, the power of the test for the 'Area' effect on epiphyte load increased to 70%, and significant differences in epiphyte load between areas were then detected (df = 1,6; F = 10.29; P = 0.0184).

Mean epiphyte N content varied between 0.7 and 1.0% throughout the area studied with maximum values concentrated in the FA meadow, but no significant differences were detected at any spatial scale considered in the analysis (Fig. 3C, Table 1). Mean leaf N content varied between 1.4 and 1.9% and was also similar between areas, but very significant variability was detected between locations and seagrass patches (Fig. 3E, Table 1). The isotopic signal of nitrogen (δ 15 N) in epiphyte tissues was consistently and significantly higher in the FA meadow (5.36 ± 0.013 ‰) than the CA meadow (3.75 ± 0.013 ‰) (Fig. 3D, Table 1). Variability between localities within each meadow area was also significant for this variable (SNK, Table 1). Isotopic nitrogen measured in seagrass leaves also showed significantly higher mean values in the FA meadow (3.69 ± 0.02 ‰) than at CA (2.98 ± 0.08 ‰) (Fig. 3F), although the variability between seagrass patches within sites was also significant (Table 1).

Mean values of isotopic N measured in POM captured by sediment traps showed significant differences between FFW and NAT sources, with higher values for the former (6.67 ± 0.32 ‰) than for the latter ( 3.69 ± 0.17 ‰) (n = 6; t-test, p < 0.05).

Table 2 shows the results of the mixing model applied to determine the relative contributions of distinct isotopic N signatures measured in various sources to the mixed N signature measured in epiphytes. For each meadow area, the table indicates the range (1-99th percentile) and the mean and standard deviation of the distribution of all feasible contributions from each source to the epiphyte N signature. In the FA area, fish farm waste (FFW = 12-56%) and natural sources (NAT = 44-66%) both provided the most significant contributions to the epiphytes N signature. The contribution of urban waste was very low and ambiguous, since it included zero contributions (UW = 0-22%).

In the CA area, epiphyte N signal was unambiguously accounted by external natural sources (NAT = 98-99%) while external anthropogenic contributions (FFW and UW)

were almost negligible (0-2%).

Discussion

Both epiphytes and seagrass leaf tissues were particularly and significantly 15 Nenriched in Posidonia oceanica plants located 3 km from the fish farm complex (i.e. the FA meadow area) in comparison with the control meadow, which seems to reflect the nature of external N sources influencing each meadow area. In the control meadow area, POM (NAT source) had an isotopic N signal similar to that measured in water samples from unpolluted natural sites in near-shore Mediterranean environments (2-4‰, e.g. [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF]Sará et al., 2004). It may therefore represent a natural or "background" signal composed by a mixture of detritus material of marine and terrestrial origin, phyto-and zooplankton [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF], and, in this case, seagrass detritus. By contrast, POM collected close to the fish cages (FFW) consisted mostly of uneaten food pellets and fish faeces, and exhibited higher positive δ 15 N values as those typically measured in these anthropogenic sources (6-11‰, e.g. [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF]Sará et al., 2004). In the water column, rapid mineralization of this initially N-enriched POM results in an impoverishment of its isotopic N signature and an enrichment in the dissolved N pool, due to isotopic fractionation by physical and biological process [START_REF] Macko | Pollution Studies Using Stable Isotopes[END_REF]Sará et al., 2004). Excreta from farmed fish [START_REF] Dosdat | Marine aquaculture effluent monitoringmethodological approach to the evaluation of nitrogen and phosphorus excretion by fish[END_REF] and wild fish around the cages [START_REF] Fernández-Jover | Seasonal patterns and diets of wild fish assemblages with Mediterranean coastal fish farms[END_REF], as well as re-suspension of enriched SOM (Sará et al., 2006), contribute significantly to this dissolved 15 N-enriched pool, which currents immediately disperse toward surrounding areas [START_REF] Sarà | Effects of fish farming waste to sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Castellamare, Sicily): a multiple stable isotope study (δ 13 C and δ 15 N)[END_REF][START_REF] Sarà | Use of stable isotopes to investigate dispersal of waste from fish farms as a function of hydrodynamics[END_REF].

The isotopic N composition of benthic primary producers reflected that of external N sources [START_REF] Fry | Stable Isotope Ecology[END_REF]. High δ 15 N values, such as those observed in epiphytes and seagrass tissues in the FA meadow, are associated with the influence of external anthropogenic sources such as aquaculture loads and land-derived wastewater [START_REF] Cole | Assessment of a δ 15 N isotopic method to indicate anthropogenic eutrophication in aquatic ecosystems[END_REF][START_REF] Cole | Macrophytes as indicators of land-derived wastewater: Application of a δ 15 N method in aquatic systems[END_REF]Constanzo et al., 2001;Constanzo et al., 2004;[START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF][START_REF] Jones | Assessing ecological impacts of shrimp and sewage effluent: biological indicators with standard water quality analyses[END_REF][START_REF] Lin | Macroalgal bioindicators (growth, tissue N, δ 15 N) detect nutrient enrichment from shrimp farm effluent entering Opunohu Bay, French Polynesia[END_REF][START_REF] Pérez | Physiological responses of the seagrass Posidonia oceanica as indicators of fish farm impact[END_REF][START_REF] Vizzini | Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean[END_REF]. Several findings support this hypothesis in our case study: (1) fish farm production in our study was approximately 4.2 times higher than a case study that recorded elevated δ 15 N signatures in Posidonia oceanica leaves over an area located up to 3 km from the fish farm [START_REF] Dolenec | Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in the central Adriatic[END_REF]); (2) the dominant current direction was NW (34%), but the frequency of currents directed toward the FA meadow area (i.e. SW) was also important (25%); (3) the mean velocity of SW currents was 10 cm s -1 and reached maximum values of 58 cm s -1 , which has shown to be enough to disperse farm waste over a distance covering several kilometres (Sará et al., 2006); (4) a spatial gradient of δ 15 N between the fish farm and the FA meadow area was obtained in a previous study by measuring isotopic N in benthic macrophytes incubated in pelagic bioassays deployed at increasing distances from fish cages (García-Sanz et al., in press); and finally, (5) the mixing model used in this study provided consistent evidence of the significant contribution of farm waste (i.e. FFW source) to the isotopic N signature of epiphyte tissues in the FA meadow area. Therefore we can consider that farm waste is responsible for the high δ 15 N signatures measured in the seagrass and epiphyte tissues of the FA meadow located 2.8 km from the aquaculture facilities.

With a high availability of nutrients, seagrass and its epiphytes can assimilate and store excess nutrients in their tissues, increasing their total N content [START_REF] Lepoint | Field measurements of inorganic nitrogen uptake by epiflora components of the seagrass Posidonia oceanica (Monocotyledons, Posidoniaceae)[END_REF][START_REF] Invers | Effects of nitrogen addition on nitrogen metabolism and carbon reserves in the temperate seagrass Posidonia oceanica[END_REF][START_REF] Touchette | Review of nitrogen and phosphorus metabolism in seagrasses[END_REF]. However, no significant increase in nitrogen was observed in either Posidonia oceanica leaves or epiphytes in the FA meadow, despite the influence of farm waste. A possible explanation is that the nutrients supplied are too low to merit long-term N storage, and are probably used to sustain growth, as suggested for macroalgae species exposed to moderate nutrient increase in fertilization experiments [START_REF] Mcglathery | Nutrient and grazing influences on a tropical seagrass community[END_REF] and from shrimp farm loads [START_REF] Lin | Macroalgal bioindicators (growth, tissue N, δ 15 N) detect nutrient enrichment from shrimp farm effluent entering Opunohu Bay, French Polynesia[END_REF]. This argument can be particularly valid for epiphytic algae, which quickly exploit any increase in water column nutrient availability [START_REF] Lepoint | Field measurements of inorganic nitrogen uptake by epiflora components of the seagrass Posidonia oceanica (Monocotyledons, Posidoniaceae)[END_REF][START_REF] Short | Mesocosm experiments quantify the effects of eutrophication on eelgrass, Zostera marina[END_REF]. Furthermore, nutrients could favour opportunistic epiphytic algae over seagrass leaves [START_REF] Duarte | Submerged aquatic vegetation in relation to different nutrient regimes[END_REF][START_REF] Mcglathery | Nutrient and grazing influences on a tropical seagrass community[END_REF][START_REF] Prado | Seasonal response of Posidonia oceanica epiphyte assemblages to nutrient increase[END_REF][START_REF] Wear | Effects of water column enrichment on the production dynamics of three seagrass species and their epiphytic algae[END_REF], the rapid growth of which prevents N accumulation in their tissue.

Nonetheless, epiphytes showed greater variability of N content between seagrass patches within some of the FA meadow sites, as a consequence of the maximum values obtained only in this meadow area (Fig. 3C), which can be interpreted as a heterogeneous influence of farm waste on epiphyte N content at a small spatial scale. In seagrass leaves, the lack of N response to water-dissolved nutrients has a more complex interpretation, as it can also be governed by other limiting factors (e.g. light), competition for nutrients by epiphytes, internal N and C metabolism and sediment nutrient pools [START_REF] Invers | Effects of nitrogen addition on nitrogen metabolism and carbon reserves in the temperate seagrass Posidonia oceanica[END_REF][START_REF] Romero | Production ecology of Posidonia oceanica (L.) Delile meadows in Nueva Tabarca Marine Reserve: Growth biomass and nutrient stocks along a bathymetric gradient[END_REF][START_REF] Romero | Nutrient dynamics in seagrass ecosystems[END_REF][START_REF] Touchette | Review of nitrogen and phosphorus metabolism in seagrasses[END_REF].

In response to fish farm load, epiphyte abundance in Posidonia oceanica leaves can either rise [START_REF] Cancemi | Effects of organic matter input from a fish farming facility on a Posidonia oceanica meadow[END_REF][START_REF] Delgado | Seagrass regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean)[END_REF][START_REF] Delgado | Effects of fish farming on seagrass (Posidonia oceanica) in a Mediterranean bay: seagrass decline after organic loading cessation[END_REF] or decrease due to enhanced consumption of epiphyted leaf tips by seagrass macro-herbivores [START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF][START_REF] Ruiz | Respuesta de la fanerógama marina Posidonia oceanica (L.) Delile a perturbaciones antrópicas[END_REF]Ruiz et al., in press). Previous experimental studies support the existence of this nutrient-macrograzers interaction in P. oceanica meadows [START_REF] Prado | Magnitude of herbivory in Posidonia oceanica and factors responsible for spatial variation[END_REF][START_REF] Ruiz | Respuesta de la fanerógama marina Posidonia oceanica (L.) Delile a perturbaciones antrópicas[END_REF] and in tropical seagrass communities [START_REF] Mcglathery | Nutrient and grazing influences on a tropical seagrass community[END_REF][START_REF] Goecker | Effects of nitrogen content in turtlegrass, Thalassia testudinum, on consumption by the bucktooth parrotfish, Sparisoma radians[END_REF]. However, our data did not support the existence of such interaction in the FA meadow since the spatial pattern showed by the herbivorous fish S. salpa activity was not coupled with that of epiphyte abundance (Figs. 2C and 3B,respectively).

Alternatively, we suggest that nutrient induced changes in the composition of the epiphytic assemblage and/or enhanced mesoherbivores (mainly gastropods) activity [START_REF] Lin | Responses of epiphytes on eelgrass, Zostera marina L., to separate and combined nitrogen and phosphorus enrichment[END_REF], see Borowitzka et al., 2006[START_REF] Valentine | The central role of grazing in seagrass ecology[END_REF][START_REF] Burkholder | Seagrass and eutrophication[END_REF] for a recent review of this topic) might also account for the reduction in epiphyte abundance reported in the FA meadow. Nonetheless, since no measurements of these variables are available this possibility is speculative and other factors must be considered including those responsible for natural spatial patterns of P. oceanica epiphyte assemblages (e.g. [START_REF] Balata | Patterns of spatial variability of seagrass epiphytes in the north-west Mediterranean Sea[END_REF][START_REF] Pardi | Spatial variability of Posidonia oceanica (L.) Delile epiphytes around the mainland and the islands of Sicily (Mediterranean Sea)[END_REF]. Apart from these possible changes in seagrass community, no other evidence of any nutrient-related effect was obtained at the level of meadow structure. Smaller shoot size and altered meadow structure (shoot density and meadow cover) are commonly reported responses for Posidonia oceanica meadows directly exposed to aquaculture loads (e.g. [START_REF] Delgado | Seagrass regression caused by fish cultures in Fornells Bay (Menorca, Western Mediterranean)[END_REF][START_REF] Ruiz | Effects of fish farm loadings on seagrass (Posidonia oceanica) distribution, growth and photosynthesis[END_REF] or experimental nutrient enrichment [START_REF] Leoni | Morphological responses of Posidonia oceanica to experimental nutrient enrichment of the canopy water[END_REF], though this kind of alteration was not detected in our study.

In conclusion, results based on stable isotope analysis, together with available information on hydrodynamics and from previous studies in the study site, provide consistent evidence that waste delivered by offshore farms has a large area of influence (spanning kilometres) that affects Posidonia oceanica meadows located in remote nearshore environments. Results obtained also indicate that such influence was not sufficient to produce significant meadow alterations, unlike those described in nearshore case studies under a more severe influence of nutrients. Only changes in the abundance of epiphytes (and possibly in the activity of grazers) could be interpreted in the light of nutrient-induced effects at the community level, but further evidence would be required to confirm it. Finally, based on this study case, the idea that concentrating aquaculture facilities in offshore areas is a strategy totally exempt of environmental risk cannot be supported and hence site-selection processes for new aquaculture facilities must be careful with regard to the distribution of this and other sensitive habitats, even when these are located at distances greater than one kilometre.

Figure 1 .

 1 Figure 1. Map of the study area indicating the location of the fish farms (empty polygons) and sampled meadow areas (see the Materials and Methods section).

Figure 2 .

 2 Figure 2. Mean ± SE of A the percentage of meadow cover; B the shoot density (shoots m -2 ); and C herbivore pressure (% of leaves eaten) at sites sampled within each location.

Figure 3 .

 3 Figure3. Mean ± SE of A the shoot size (as leaf surface area, cm 2 shoot -1 ); B epiphyte load (mg DW cm -2 ); C epiphyte N content (% DW); D epiphyte isotopic nitrogen (δ 15 N); E leaf N content (% DW); and F leaf isotopic nitrogen composition (δ 15 N).

Figures

  Figures 3:

Acknowledgments

This study is a part of a project funded by the Plan Nacional de Cultivos Marinos of the Junta Nacional Asesora de Cultivos Marinos (JACUMAR) to JMR during the period 2004-2007. We would like to thank IEO technician Rocío García Muñoz for her valuable help in laboratory work and field sampling. JMR would like to thank TAXON Estudios Ambientales S.L. and the students Teresa Castro and Eva Vázquez for field assistance. We acknowledge to the Servizos de Apoio à Investigación of the University da Coruña for their support in the analysis of stable isotopes.