Ben Mounir

Asma Brahim

Imen Hamza

Ahmed-Riadh Hannachi

Othman Rebai

Abderrahmen Jarboui

Lotfi Bouain

Aleya

Mounir Ben Brahim

Asma Hamza

Imen Hannachi

Ahmed Rebai

Othman Jarboui

Abderrahmen Bouain

Lotfi Aleya
email: lotfi.aleya@univ-fcomte.fr

Variability in the structure of epiphytic assemblages of in relation to human interferences in the Gulf of Gabes, Tunisia

Keywords: Macro-epiphytes, Posidonia oceanica, disturbance, spatial variability, Gabès Gulf

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Introduction

Over the past 20 years, the Gulf of Gabes, on the southeast coast of Tunisia, has produced more than 65% of the annual fish yield in Tunisia (Ben [START_REF] Ben Mustapha | The Gulf of Gabes: A case study in the Mediterranean decline in Fishing out the Mediterranean. 21 st session of the G[END_REF][START_REF]Annuaire des statistiques des pêches en Tunisie[END_REF], being favoured by widespread seagrass beds widely recognized as key ecosystems in temperate and tropical infra littoral habitats [START_REF] Hemminga | Seagrass Biology[END_REF][START_REF] Boudouresque | Regression of Mediterranean seagrasses caused by natural processes and anthropogenic disturbances and stress: a critical review[END_REF].

However, fish production is gradually decreasing parallel to the decline of littoral beds of the endemic Mediterranean seagrass Posidonia oceanica (L.) Delile species as the coastal area of the Gulf is now a threatened biotope mainly due to the pressure of anthropogenic expansion and dumping of large quantities of phosphogypsum and other chemical products which severely impacted benthic habitats ( [START_REF] Hamza-Chaffai | Relationship between metallothionein and metals in a natural population of clam Ruditapes decussates from Sfax coast, a non-linear model using Box-Cox transformation[END_REF][START_REF] Bejaoui | Modélisation de la dispersion du phosphogypse dans le Golfe de Gabès[END_REF]. The loss of the native vegetable cover in the Gulf of Gabes is estimated at 90% with the P. oceanica beds being replaced by the opportunistic and caulerpenyne-producing green algae Caulerpa prolifera (Forsskål) Lamouroux in deeper zones [START_REF] Hamza | New mentions of Caulerpa racemosa (Forsskal) J. Agardh in the Gabes Gulf (Tunisia)[END_REF][START_REF] Ramos-Espla | Contribution à la caractérisation des herbiers à Posidonia oceanica (L.) Delile aux Iles Kerkennah (Est de la Tunisie, Mediterranée orientale)[END_REF]. The remaining Posidonia meadows cover the littoral fringes at depths ranging between -3 and -4 m [START_REF] Hattour | L'écosystème du golfe de Gabès; dégradation de son couvert végétal et de sa pêcherie benthique[END_REF]. On the other hand, most biological studies of the Gulf of Gabes have dealt with the distribution of phytoplankton [START_REF] Drira | Dynamics of dinoflagellates and environmental factors during the summer in the Gulf of Gabes (Tunisia, Eastern Mediterranean Sea)[END_REF][START_REF] Bel Hassen | Phytoplankton dynamics related to water mass properties in the Gulf of Gabes: Ecological implications[END_REF] zooplankton [START_REF] Drira | Coupling of phytoplankton community structure to nutrients, ciliates and copepods in the Gulf of Gabes (South Ionian Sea, Tunisia)[END_REF] ciliates [START_REF] Kchaou | Distribution of ciliates in relation to environmental factors along the coastline of the Gulf of Gabes, Tunisia[END_REF][START_REF] Hannachi | Abundance and biomass of the ciliate community during a spring cruise in the Gulf of Gabes (East Mediterranean Sea, Tunisia)[END_REF], demosponges [START_REF] Ben Mustapha | Diversity of Tunisian demosponges[END_REF] while seagrass species and their epiphytes which are known to be highly sensitive to environmental changes [START_REF] Balata | Patterns of spatial variability of seagrass epiphytes in the north-west Mediterranean Sea[END_REF][START_REF] Prado | Marine protection and meadow size alter fish herbivory in seagrass ecosystems[END_REF][START_REF] Montefalcone | Ecosystem health assessment using the Mediterranean seagrass Posidonia oceanica: a review[END_REF] have been given limited attention. Epiphytes of seagrass not only contribute significantly to the primary production of the meadow [START_REF] Buia | Primary production and growth in Posidonia oceanica[END_REF][START_REF] Nelson | Seasonality of eelgrass, epiphyte, and grazer biomass and productivity in subtidal eelgrass meadows subjected to moderate tidal amplitude[END_REF][START_REF] Duarte | Seagrass ecosystems: their global status and prospects[END_REF], but are also more sensitive to environmental changes than the plant host [START_REF] Delgado | Effects of fish farming on seagrass (Posidonia oceanica) in a Mediterranean bay: seagrass decline after organic loading cessation[END_REF][START_REF] Nesti | Variability in the structure of epiphytic assemblages of the Mediterranean seagrass Posidonia oceanica in relation to depth[END_REF][START_REF] Giovannetti | Early warning response of Posidonia oceanica epiphyte community to environmental alterations (Ligurian Sea, NW Mediterranean)[END_REF]. For example, various studies reported increases in epiphyte biomass parallel with nutrient enrichment [START_REF] Armitage | Variable responses within epiphytic and benthic microalgal communities to nutrient enrichment[END_REF]Neckles et al., 1993), eutrophication [START_REF] Balata | Comparison between epiphyte assemblages of leaves and rhizomes of the seagrass Posidonia oceanica subjected to different levels of anthropogenic eutrophication[END_REF][START_REF] Frankovich | Nutrient effects on seagrass epiphyte community structure in Florida Bay[END_REF] and water quality [START_REF] Meric | Toxicity of leather tanning wastewater effluents in sea urchin early development and in marine microalgae[END_REF].

Differences in the spatial heterogeneity of epiphytes [START_REF] Piazzi | Patterns of spatial variability in epiphytes of Posidonia oceanica. Differences between a disturbed and two reference locations[END_REF][START_REF] Martínez-Crego | Composition of epiphytic leaf community of Posidonia oceanica as a tool for environmental biomonitoring[END_REF] and shifts in species composition [START_REF] Nesti | Variability in the structure of epiphytic assemblages of the Mediterranean seagrass Posidonia oceanica in relation to depth[END_REF][START_REF] Fourqurean | Epiphyte loads on seagrasses and microphytobenthos abundance are not reliable indicators of nutrient availability in oligotrophic coastal ecosystems[END_REF] have also been observed. It is widely known that the distribution of epiphytic assemblages of P. oceanica both on leaves ( [START_REF] Van Der Ben | Les épiphytes des feuilles de Posidonia oceanica Delile sur les côtes françaises de la Méditerranée[END_REF][START_REF] Mazzella | Spatio-temporal distribution of algal and animal communities in a Posidonia oceanica (L.) Delile meadow[END_REF] and rhizomes [START_REF] Piazzi | Epiphytic macroalgal assemblages of Posidonia oceanica rhizomes in the western Mediterranean[END_REF][START_REF] Balata | Comparison between epiphyte assemblages of leaves and rhizomes of the seagrass Posidonia oceanica subjected to different levels of anthropogenic eutrophication[END_REF] can change, but these changes have not yet been explored on a horizontal scale in the Gulf of Gabes. Differences in the spatial heterogeneity of epiphytes [START_REF] Piazzi | Patterns of spatial variability in epiphytes of Posidonia oceanica. Differences between a disturbed and two reference locations[END_REF] and shifts in species composition [START_REF] Nesti | Variability in the structure of epiphytic assemblages of the Mediterranean seagrass Posidonia oceanica in relation to depth[END_REF][START_REF] Fourqurean | Epiphyte loads on seagrasses and microphytobenthos abundance are not reliable indicators of nutrient availability in oligotrophic coastal ecosystems[END_REF][START_REF] Martínez-Crego | Composition of epiphytic leaf community of Posidonia oceanica as a tool for environmental biomonitoring[END_REF] have also been observed. Several findings have provided evidence that epiphytes are patchy at 1 to 10 cm [START_REF] Kendrick | Seasonal changes in epiphyte macro-algae assemblages between offshore exposed and inshore protected Posidonia sinuosa Cambridge et Kuo seagrass meadows, Western Australia[END_REF]Jernakoff and Neilsen, 1998) and > kilometer scales, (Vanderklift and[START_REF] Vanderklift | Patchiness in assemblages of epiphytic macroalgae on Posidonia coriacea at a hierarchy of spatial scales[END_REF][START_REF] Lavery | A comparison of spatial and temporal patterns in epiphytic macroalgal assemblages of the seagrasses Amphibolis griffithii and Posidonia coriacea[END_REF], other studies have indicated that epiphytes may be uniform at relatively small spatial scales (e.g., 0.1 m [START_REF] Vanderklift | Patchiness in assemblages of epiphytic macroalgae on Posidonia coriacea at a hierarchy of spatial scales[END_REF]<10 m, Saunders et al., 2003[START_REF] Piazzi | Patterns of spatial variability in epiphytes of Posidonia oceanica. Differences between a disturbed and two reference locations[END_REF]. [START_REF] Moore | Lack of significant change in epiphyte biomass with increasing extent of measurement within seagrass meadows[END_REF] reported that epiphytes biomass is uniform at the scale < 100 m transect. Variability in leaves epiphytic biomass at the scale of meters may be relatable to differences in shoot density [START_REF] Gambi | Distribution and ecology of Polychaetes in the foliar stratum of a Posidonia oceanica bed in the bay of Porto Conte (N.W. Sardinia). International Workshop on Posidonia beds. édit[END_REF], and to impacts of water movements on both settlement and recruitment of propagules [START_REF] Trautman | The distribution of the epiphytic organisms on Posidonia australis and P. sinuosa, two seagrasses with differing leaf morphology[END_REF][START_REF] Vanderklift | Patchiness in assemblages of epiphytic macroalgae on Posidonia coriacea at a hierarchy of spatial scales[END_REF]. It is, therefore, of importance to undertake a study of the effects of urban pressure on P. oceanica dynamics under the impact of human activities versus control conditions. To our knowledge, only the flowering and fructification of P. oceanica along the coastline of the Gulf of Gabes has been the subject of studies in recent years [START_REF] Hattour | L'écosystème du golfe de Gabès; dégradation de son couvert végétal et de sa pêcherie benthique[END_REF]. As part of a comprehensive study intended to understand the dramatic decline of the endemic Mediterranean seagrass P. oceanica in the Gulf of Gabes, we analyzed, for the first time in this ecosystem, the structure and patterns of spatial variability of leaf macro-epiphytic assemblages and tested their relationships with anthropogenic disturbance. We attempted to answer the questions 1) how vulnerable are the leaf macro-epiphytic assemblages to these environmental pressures? 2) Does epiphyte biomass increase parallel with organic and nutrient enrichment and which members of the epiphytic community should be considered as most sensitive species? And 3) Does anthropogenic disturbance modify the spatial scales at which variability of the epiphytic communities is highest/lowest or also influences the partition of total variability among spatial scales? We, thus, compared epiphytic assemblages between locations exposed to urban and industrial effluents and control locations, and examined their variability at spatial scales ranging from cm to km, using a hierarchical sampling design [START_REF] Underwood | On beyond BACI: sampling designs that might reliably detect environmental disturbances[END_REF][START_REF] Chapman | Variability at different spatial scales between a subtidal assemblage exposed to the discharge of sewage and two control assemblages[END_REF].

Material and methods

Study area

The Gulf of Gabes is located between 35° N and 33° N and extends from "Ras kapoudia" at the 35° N parallel level to the Tunisian-Libyan border (Fig. 1). It shelters various islands (Kerkennah and Djerba) and lagoons (Bougrara and El Bibane). The climate is dry (average precipitation: 210 mm year -1 ) and sunny with strong easterly winds. The tide is semidiurnal, with a maximum range of about 2 m.

Sampling

Because the P. oceanica epiphytic community reaches its optimum seasonal development in summer (Romero, 1988) fieldwork was conducted in July-August 2006. Four locations distant 30 km from each other were chosen for the samplings. The depth of all stations was 5 m and temperature and salinity ranges were 27°C-30°C and 38-40 P.S.U., respectively. Two locations were disturbed by a sewage discharge: Ghannouch (34° 30' 523'' N 10° 54' 743'' E) and Zarrat (33º 43'417'' N 10º 20' 486'' E) labeled herein respectively,D1 and D2. The other two: Hassar (34° 44' 290'' N 11° 08' 590'' E) and Ajim (33° 45' 112'' N 10° 42' 992'' E) were control locations, located north and south of the disturbed area and labeled respectively, C1 and C2. Within each location (D1, D2, C1, C2), three sites were randomly selected, 500-600 m apart, and within each site two sub-sites were also randomly selected. Each sub-site was randomly divided into three plots, each plot then being divided into three quadrats 20 cm apart. We examined variability at tens of centimeter-scale by collecting samples from the same quadrat. This quadrat was 20 cm × 20 cm and divided into a grid of 10 cm × 10 cm subquadrats.

Data collection

The density of P. oceanica shoots was estimated from 216 replicates present within 100 cm² sub-quadrats inside each meadow. Five shoots were randomly collected from each subquadrat during SCUBA diving and preserved in seawater-formalin (5%) solution for species identification in the laboratory. Leaf length corresponds in fact to the average evaluated from each meadow. The samples were examined for leaf surface per shoot and the coverage (expressed as a percentage of leaf surface) of each morphological group which was estimated with a binocular lens, then carefully scraped with a razor blade [START_REF] Libes | Productivity-irradiance relationship of Posidonia oceanica and its epiphytes[END_REF]. Epiphytes and scraped leaves were oven-dried at 60 °C for 48 h before weighing [START_REF] Alcoverro | The influence of herbivores on Posidonia oceanica epiphytes[END_REF][START_REF] Lepoint | Fauna vs flora contribution to leaf epiphytes biomass in a Posidonia oceanica seagrass bed (Revellata Bay Corsica)[END_REF].

Data analysis

Multivariate analysis (MPMANOVA) was used to test the hypothesis that the structure of epiphytic assemblages differed between disturbed and control locations and to evaluate variability at different spatial scales [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. The analysis consisted of a 6-way model with sites (three levels) being nested within locations (two levels), subsites (two levels) being nested within sites, plots (three levels) being nested within subsites, quadrats being nested within plots and sub-quadrats (four levels) being nested within quadrats. Sites, subsites, plots and quadrats were random whereas the contrast of disturbed versus control and the disturbed were fixed. All data were transformed to ln (x + 1) to remove heterogeneity of variances (after Cochran's C-test). SIMPER analysis [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF] was used to identify the percentage contribution of each species to the Bray-Curtis dissimilarity between the average of the disturbed and the control locations. A two-dimensional nMDS (non-metric multidimensional scaling), based on the centroids of the sites of the four locations, was used for a graphical representation of the data for leaf epiphytic assemblages.

Variance components of all assemblages were calculated for each location and for all spatial scales. A discriminant analysis was used for classifying the different meadows into one among possible K classes. These classes were identified by linear combinations of the variables maximizing the homogeneity of each class. The test Wilks' lambda was employed to test whether there are differences between the means of identified groups of subjects among a combination of dependent variables.

Epiphyte biomass was standardized by shoot biomass to be able to compare epiphyte load among locations. This is necessary because a significant positive correlation was found between epiphyte biomass and leaf biomass (Fig. 2), allowing us to express epiphyte biomass as g dw epiphytes /g dw of leaves. Differences in epiphytic biomass and the percentage cover of the main phyla or morphological groups were analyzed through 6-way ANOVA with the same factors and levels used in the multivariate analysis. Student-Newman-Keuls (SNK) test was employed for a posteriori multiple comparisons of means.

Results

A total of 49 taxa were identified in the epiphyte assemblages of control locations: 20 Macroalgae (11 Rhodophyta, 6 Cyanobacteria, 2 Chlorophyta, 1 Heterokontophyta), 11 Bryozoa, 9 Cnidaria (Hydrozoans), 3 Annelida, 2 Tunicata and 3 Porifera. A total of 32 taxa were identified in the epiphyte assemblages of disturbed locations: 13 Macroalgae (7 Rhodophyta, 4 Cyanobacteria, 1 Chlorophyta, 1 Heterokontophyta), 9 Bryozoa, 8 Cnidaria (Hydrozoans), 1 Annelida and 1 Tunicata (Appendix 1). In control locations, leaf assemblages were dominated by filamentous algae belonging to the genera Polysiphonia, Ceramium and Antithamnion; common algal species were also present such as Dasycladus vermicularis, Cladophora sp., Griffithsia opuntoides, Neomonospora sp. and Laurencia obtusa. Among animals, the Bryozoans Alcyonidium and Electra Posidoniae dominated the community. The Tunicata Botryllus schlosseri and Clavelina lepadiformis were numerically dominant. Among the Cnidaria the most abundant species were Dynamina cavolinii, Monotheca sp. and Aglaophenia sp. For encrusting Annelida the genus Spirorbis was the dominant genus, and for the Porifera the genus Ircinia dominated the Porifera assemblage. In disturbed locations, leaf assemblages were dominated by filamentous algae belonging to genera Antithamnion and Polysiphonia; other common algal species were also present such as Laurencia obtusa, dictyota dichotoma and Ceramium gracillimum. Among Bryozoa the genera Alcyonidium, Scrupocellaria and Bowerbankia were the most abundant. The Cnidaria Dynamena and Obelia were the most abundant genera. For the encrusting Annelida, the genus Spirorbis dominated the assemblage.

There was a difference between the two disturbed locations; the location Ghannouch was more affected than the location Zarrat, the number of species and the mean percentage cover were reduced for the majority of taxa. We noted the absence of Aetea truncata, Micropora complanata. Filamentous algae, namely Ceramium tenuissimum, Neomonospora sp. and Dasya sp. were absent in the location of Ghannouch.

The high mean value of shoot density (1022 ± 54.23 shoot m -² ) was recorded in the site 1 of Hassar location (C1), whereas the lowest mean value (119.5 ± 17.67 shoot m -² ) has been detected in site 1 of Ghannouch location (D1) (Fig. 3). The lowest mean value of the leaf surface area (205.28 ± 5.56 cm² shoot -1 ) was recorded in the site 1 of the location Ghannouch (D1), whereas the hightest mean value (467.28 ± 5.82 cm² shoot -1 ) was detected in the site 2 of the location Ghannouch (D1) (Fig. 4).

The hightest mean value of leaf length (59.68 ± 7.87 cm) was recorded in the site b of the control location Hassar (C1), whearas le lowest mean value (29.2 ± 4.87 cm) was observed in the site a of the disturbed location Ghannouch (D1) (Fig. 5).

ANOVA analysis showed that for density, leaf surface area and leaf length there was a significant difference between (i) locations and (ii) between the contrast 'disturbed vs. control' locations. For leaf surface area and leaf length, no significant difference could be detected in the disturbed and the control locations separately. The difference was not significant for shoot density among the control locations (Table 1). The disturbed location D1 has a lower mean epiphyte biomass (g dw epiphytes /g dw of leaves) than the location D2; this value ranging between 0.02 ± 0.07 and 0.1 ± 0.08 g dw epiphytes /g dw of leaves at D1, and between 0.25 ± 0.07 and 0.38 ± 0.09 g dw epiphytes /g dw of leaves in the location D2. In the control location C1, the mean epiphyte biomass varied between 0.46 ± 0.01 and 0.63 ± ± ± ± 0.03 g dw epiphytes /g dw of leaves and between 0.33 ± 0.17 and 0.38 ± 0.19 g dw epiphytes /g dw of leaves in the location C2 (Fig. 6).

Epiphyte biomass vs. leaf biomass displayed a fairly similar relationship in all locations, suggesting that the leaf biomass pattern and hence the leaf surface availability was an important factor governing the epiphytic biomass pattern as also reported by Borowitzka et al.

(2006) (Fig. 2). The two control locations C1 and C2 had the upper slope whereas the disturbed locations present the lower what strongly suggests that anthropogenic disturbance affects epiphyte load (Test of homogeneity of slope between control and disturbed locations; F (n=3) = 430.07; p = 0.00002). Analyses of variance indicate a wide variability in the epiphytic biomass among locations, sites, subsites and quadrats, whereas variation at the scale plot was insignificant (Table 2). Variation between the two disturbed locations was important for both the largest and the smallest scales (quadrat), whereas significant difference was detected only at the subsite and quadrat scale for the control locations. The structure of epiphytic assemblage of leaves differed between the contrast of disturbed vs. control locations (Table 3), while no significant difference was observed among disturbed and control location separately. Significant difference was observed in particular at the smallest scales plots and quadrats. Epiphytes were generally abundant on leaves of P. oceanica (Fig. 7). Algae, bryozoans and encrusting Annelida were common and abundant at all locations. Also hydrozoans were widespread, but their percentage cover was generally low. The two disturbed meadows were different from each other with an extra variability in percentage covered in hydrozoans. Four morphological groups of organisms were sufficiently abundant to be included in univariate analyses. For example, and concerning the percentage cover of filamentous algae and hydrozoans, we recorded a significant difference between (i) locations when considered separately and between (ii) the contrast 'disturbed vs. control locations'. For encrusting Annelida a significant difference was detected only for the scale locations when considered separately, whereas, no significant difference was found for the contrast 'disturbed vs. control locations'. For bryozoans, there was no significant difference between these two scales (Table 4). On a scale site, a significant variability was detected for encrusting Annelida.

At the scale subsite, a significant variability was detected for algae, whereas at the scale plot, variability was detected for all parameters. For the scale quadrat, a difference was significant for all variables but no difference was found between the percentage covers of disturbed and control locations.

nMDS ordination of leaf assemblages at the four locations showed a clear separation between the centroids based on sites of each control and disturbed locations (Fig. 8). The species responsible for the difference between disturbed and control locations changed. For example, the cover percentages of the algae Antithamnion sp., Polysiphonia elongata, Ceramium tenuissimum and Ceramium codii, the bryozoans such as Micropora complanata and the hydrozoan Dynamena cavolinii increased on leaves in the control locations, though some species showed similar trends in these locations (Table 5).

Biomass, epiphytic leaf assemblages and the four abundant morphological groups of epiphyte displayed different responses to the spatial variability among different scales at the disturbed and the control locations (Table 6). Variance components indicated that most of the variability occurred not only at the plot scale but also between locations and quadrats, with the exception of biomass that displayed larger variation at the quadrat scale in disturbed locations.

Discriminate analysis, for both disturbed and control locations showed that there was a strongly significant variation including all the factors of the P. oceanica meadows. Wilks' lambda showed a high variability between locations for biomass, hydrozoans and density, and high variability within locations especially for bryozoans (Table 7).

The projection on the first factorial design crossing the first two factorial discriminant axes corresponding to the first two discriminating linear functions as in table 8, allows a description and a classification of the characterized variables (Fig. 9). The first axis shows discrimination between the two control and the two disturbed locations. The second axis divides locations in three groups: the control location C1, the crossing of the two disturbed locations, and the control location C2.

Discussion

The multivariate analysis illustrates a decrease in seagrass vitality (i.e. shoot density, leaf surface area and leaf length) in disturbed versus control locations, thus indicating the inability of disturbed meadows to withstand increasing urban interferences. [START_REF] Cancemi | Effects of organic matter input from a fish farming facility on a Posidonia oceanica meadow[END_REF] and [START_REF] Balestri | Variability in patterns of growth and morphology of Posidonia oceanica exposed to urban and industrial wastes: contrasts with two reference locations[END_REF] reported that the decline of the phenologic parameters used to assess the seagrass bed vitality among the disturbed locations they sampled is function of the distance from the disturbance source as compared to control locations. In fact, the mean biomass in the inner Ghannouch location (D1) was lower than that recorded close to sewage outlet at Zarrat location (D2). Similar results were reported by others (Short and Wyllie-Echeverria, 1996; Leriche et al., 2006). We suspect the water column stability and the quality of sewage as probably being the most important factor affecting the growth of P. oceanica in the coastal disturbed area of the Gulf of Gabes. This is supported by at least two observations. First, the disturbed locations D1 and D2, which have a muddy bottom, were mechanically turbulent from the constant input of sewage outlet, conveying uniformity throughout the shallow water column [START_REF] Soussi | Contribution à l'étude de la pollution marine dans le Golfe de Gabès[END_REF][START_REF] Zaouali | Les peuplements benthiques de la petite Syrte, Golfe de Gabès -Tunisie. Résultats de la campagne de prospection du mois de juillet 1990[END_REF], and therefore probably reducing leaf photosynthetic activity [START_REF] Mascaro | Experimental manipulation of sediment organic content and water column aeration reduces Zostera marina (eelgrass) growth and survival[END_REF]. Decreases in shoot density and leaf length related to decreases in light environment have already been reported from inner disturbed locations [START_REF] Ruiz | Effects of disturbances caused by coastal constructions on spatial structure, growth dynamics and photosynthesis of the seagrass Posidonia oceanica[END_REF]. In contrast, [START_REF] Giovannetti | Structural variability of the epiphytic community in a Posidonia oceanica meadow (Ligurian sea, NW mediteranean)[END_REF] found no difference in the shoot density, the Conservation Index and the epiphyte coverage between the control (marine protected area) and the disturbed (urbanized area) locations examined in their study.

Only a shift in the epiphyte species composition was observed in the urban meadows subjected to the increase of the sewage outfalls during summer, which showed a considerable development of the brown algae. Indeed, [START_REF] Terrados | Epiphyte load on the seagrass Posidonia oceanica (L.) Delile does not indicate anthropogenic nutrient loading in Cabrera Archipelago National Park (Balearic Islands, Western Mediterranean)[END_REF] found no difference in shoot density between the control and the disturbed locations that they sampled. However, these authors studied locations that received inputs of nutrients and organic matter from only the resident population ( [START_REF] Giovannetti | Structural variability of the epiphytic community in a Posidonia oceanica meadow (Ligurian sea, NW mediteranean)[END_REF] and park visitors ( [START_REF] Terrados | Epiphyte load on the seagrass Posidonia oceanica (L.) Delile does not indicate anthropogenic nutrient loading in Cabrera Archipelago National Park (Balearic Islands, Western Mediterranean)[END_REF], whereas the Gulf of Gabes is exposed to a plethora of toxic inputs. For example, studies here have reported high phosphogypsum concentration (10 g l -1 ) at the sewage outlet, which spread over more than 60 km² [START_REF] Bejaoui | Modélisation de la dispersion du phosphogypse dans le Golfe de Gabès[END_REF]. Dissolution of phosphogypsum was carried out by releasing cadmium and fluorine in sea water [START_REF] Bejaoui | Modélisation de la dispersion du phosphogypse dans le Golfe de Gabès[END_REF], and cadmium has also been shown to be toxic to clams in the Gulf of Gabes [START_REF] Hamza-Chaffai | Relationship between metallothionein and metals in a natural population of clam Ruditapes decussates from Sfax coast, a non-linear model using Box-Cox transformation[END_REF][START_REF] Smaoui-Damak | Preliminary study of the clam Ruditapes decussatus exposed in situ to metal contamination and originating from the Gulf of Gabes, Tunisia[END_REF], 2006). In the control locations Hassar and Ajim, P. oceanica beds seemed healthy and no death was observed [START_REF] Hattour | L'écosystème du golfe de Gabès; dégradation de son couvert végétal et de sa pêcherie benthique[END_REF][START_REF] Ramos-Espla | Contribution à la caractérisation des herbiers à Posidonia oceanica (L.) Delile aux Iles Kerkennah (Est de la Tunisie, Mediterranée orientale)[END_REF][START_REF] Ben Brahim | Proceedings of the third Mediterranean symposium on marine vegetation[END_REF]. Also, since P. oceanica is very sensitive to low salinity in Tunisian coastal regions such as the Gulf of Tunis (Northern Tunisia) [START_REF] Ben Alaya | Répartition et conditions d'installation de Posidonia oceanica Delile et Cymodocea nodosa Ascherson dans le Golfe de Tunis[END_REF], fresh waters from sewage may also have lowered water salinity, thus affecting meadow growth too, but to a lesser extent.

In terms of epiphytic leaf biomass, we also found the lowest mean biomass at the inner disturbed Ghannouch location (D1). As previously mentioned for the meadow biomass, increases in water turbidity in disturbed locations are also detrimental to leaf epiphytic biomass since light is restricted [START_REF] Cebrian | Epiphyte accrual on Posidonia oceanica (L.) Delile leaves: implications for light absorption[END_REF]. A similar general loss of epiphyte biomass and the regression of P. oceanica meadow according to disturbance level has also been reported [START_REF] Cambrige | The loss of seagrasses in Cockburn Sound. Western Australia. I. the time course and magnitude of seagrass decline in relation to industrial development[END_REF][START_REF] Guidetti | Detecting environmental impacts on the Mediterranean seagrass Posidonia oceanica (L.) Delile: the use of reconstructive methods in combination with "beyond BACI" designs[END_REF][START_REF] Piazzi | Patterns of spatial variability in epiphytes of Posidonia oceanica. Differences between a disturbed and two reference locations[END_REF]. In addition, macrograzers are known to feed preferentially on leaf tips where maximum epiphyte biomass is reached [START_REF] Alcoverro | The influence of herbivores on Posidonia oceanica epiphytes[END_REF][START_REF] Ruiz | Effects of disturbances caused by coastal constructions on spatial structure, growth dynamics and photosynthesis of the seagrass Posidonia oceanica[END_REF][START_REF] Peterson | Response of seagrass epiphyte loading to field manipulations of fertilization. Gastropod grazing and leaf turnover rates[END_REF]. This may have accounted, at least partially, for the natural loss of the epiphyte biomass both in disturbed and control locations in the Gulf of Gabes. This decrease in epiphyte biomass was also associated with a reduction of species sharing the epiphyte total biomass (49 species in disturbed location vs 32 species in control locations, see Appendix). Our findings are also supported by those of Mannino et al. ( 2010) who found a well-structured algal assemblage close to the sea (with low anthropogenic pressure) compared to the locations subjected to human disturbance. We, therefore, propose that in the Gulf of Gabes the spectrum of existing conditions was narrow and thus unfavorable for the coexistence of a high number of species.

This is most likely due to toxicity from several compounds such as Cd (10-15 ppm, [START_REF] Soussi | Contribution à l'étude de la pollution marine dans le Golfe de Gabès[END_REF] and, as previously mentioned, phosphogypsum with a sedimentary section reaching 50 cm in Ghannouch location. In addition, concentrations in hydrocarbon were relatively high (between 882 µg g -1 and 4087 µg g -1 ) compared to those recorded from other coastal Mediterranean sediments [START_REF] Zaghden | Hydrocarbons in surface sediments from the Sfax coastal zone, (Tunisia) Mediterranean Sea[END_REF]. The spatial distribution of these polluting elements is favored by dominant winds (west-east in summer and north-east and south-west in winter) which permanently induced not only the mixing of sediment but also the remobilization of the surface deposits [START_REF] Soussi | Contribution à l'étude de la pollution marine dans le Golfe de Gabès[END_REF]. Others have also reported a decrease in the vitality of the Posidonia meadow due to trace metal pollution [START_REF] Pergent-Martini | Marine phanerogams as a tool in the evaluation of marine trace-metal contamination: an example from the Mediterranean[END_REF][START_REF] Ben Chikha | L.) Delile, vis-à-vis de la contamination mercurielle dans les côtes nord de la ville de Sfax[END_REF]. Furthermore, animals on leaves exhibited different responses to disturbance. For example, encrusting Annelida and Bryozoa displayed a reduced amount of variation in mean cover percentage at large spatial scales; in fact, the increase of the relative dominance in mean cover near the sewage outlet indicates a functional change in the assemblage related with the increase of the trophic component of micropredators [START_REF] Fraschetti | Structural and functional response of meiofauna rocky assemblages to sewage pollution[END_REF]. On the other hand, the sewage causes a significant decrease in terms of mean cover of Hydrozoans as previously reported by [START_REF] Cifuentes | Effects of temporal variability of disturbance on the succession in marine fouling communities in northern-central Chile[END_REF]. The hydrozoan Gonothyraea gracilis and the two bryozoans Amathia lendigera and Beania hirtissima (Bryozoa) were presumably the most sensitive species to environmental stress as they were absent in the disturbed locations. We infer that this decrease is probably related to the water turbulence close to the discharge which affected the availability of potential preys such as zooplankton and particulate matter [START_REF] Wahl | Marine epibiosis. I. Fouling and antifouling: some basic aspects[END_REF][START_REF] Mckinney | Bryozoan evolution[END_REF]Ben Brahim et al., unpublished). In addition, bryozoans and hydrozoans showed different behavioral patterns related to the degree of disturbance, with bryozoans seeming more adapted than hydrozoans to increased disturbance [START_REF] Mckinney | Bryozoan evolution[END_REF]. Obviously, further studies are needed, for example by comparing the response of different epiphytes taxa, in order to elucidate the resilience of each group of epiphytes against disturbance levels and their strategy facing disturbed conditions. The present study showed that the variation in both the spatial pattern and abundance of macroalgal species, rather than their presence/absence, are indicative of disturbance. For example, the assemblage of epiphytic algae dominated by the Rhodophyta (order Ceramiales) tends to decrease in the two disturbed locations D1 and D2. In this context, filamentous algae, especially the Ceramiales, may be considered the most sensitive to disturbance. In other Mediterranean regions, Dictyotales were the dominant species and grew abundantly in the disturbed sampled areas [START_REF] Balata | Patterns of spatial variability of seagrass epiphytes in the north-west Mediterranean Sea[END_REF]. On the other hand, our results showed that the decrease of Ceramiales algae was paralleled with the decrease of the green algae Dasycladus vermicularis, the cyanobacteria Lyngbia sp., Rivularia sp., Gloeotrichia sp., Phormidium sp. and the red algae Dasya sp., Griffithsia sp. and Pleonosporium sp., a decrease also paralleled with increases of anthropogenic disturbance.

Brown algae such as Myrionema orbiculare and Giraudia sphacelarioides are considered characteristic of epiphyte community of P. oceanica leaves and have been already reported along the northern Tunisian coast [START_REF] Ribera | Checklist of Mediterranean seaweed. I. Fucophyceae (Warming, 1884)[END_REF]. In many Mediterranean regions, M.

orbiculare was abundant [START_REF] Giovannetti | Structural variability of the epiphytic community in a Posidonia oceanica meadow (Ligurian sea, NW mediteranean)[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][START_REF] Sliskovic | The fouling of fish farm cage nets as bioindicator of aquaculture pollution in the Adriatic Sea (Croatia)[END_REF].

However, along the Tunisian coast this taxon was detected only in Bechateur which is located in northern Tunisia [START_REF] Menez | The Marine Algae of Tunisia[END_REF]. In the present study (southern Tunisia) brown algae such as M. orbiculare, G. sphacelarioides were absent and only the species Dictyota dichotoma was recorded. We infer that this absence might be related to the high level of disturbance in the Gulf of Gabes. Our suggestion may be supported by the study of [START_REF] Giovannetti | Structural variability of the epiphytic community in a Posidonia oceanica meadow (Ligurian sea, NW mediteranean)[END_REF] at Prelo cove in the Ligurian Sea (NW Mediterranean Sea) showing that some brown algae such as Myrionema orbiculare, Giraudia sphacelarioides and Fenestrulina johannae did not play a significant role in the epiphytic community structure, whereas some Rhodophyta such as Hydrolithon-Pneophyllum spp. were the main epiphytes that were present throughout the year at all stations. In control locations, leaf epiphytes biomass of P. oceanica were most variable at the small scales investigated (among plots), whereas variation at the intermediate scale (among sites, subsites) was negligible. In this study, hierarchical sampling designs enabled an identification of homogeneity of epiphyte assemblage distribution for the majority of functional groups and biomass among the scale site (500-600 m), while heterogeneity is displayed at large scales among regions and at small scales among plots. Our study supports the results of [START_REF] Pardi | Spatial variability of Posidonia oceanica (L.) Delile epiphytes around the mainland and the islands of Sicily (Mediterranean Sea)[END_REF][START_REF] Piazzi | Patterns of spatial variability in epiphytes of Posidonia oceanica. Differences between a disturbed and two reference locations[END_REF], in terms of variability of epiphyte load on Posidonia oceanica which was greatest at the extremes of the spatial extents they investigated, with patchiness greatest at the amongshoot level (i.e. within quadrats), and at sites separated by a few hundred meters. [START_REF] Moore | Lack of significant change in epiphyte biomass with increasing extent of measurement within seagrass meadows[END_REF] and [START_REF] Balata | Patterns of spatial variability of seagrass epiphytes in the north-west Mediterranean Sea[END_REF] also considered that the epiphytes biomass within the 100 m long transect sampled was homogenous, and more generally, Jernakoff and Neilsen (1997) and [START_REF] Balestri | Variability in patterns of growth and morphology of Posidonia oceanica exposed to urban and industrial wastes: contrasts with two reference locations[END_REF] indicated that patchiness in epiphytes as well as in seagrass morphology is significant over a range of scales (typically cm to km).

Conclusion

Our study illustrated a spatial variability related to anthropogenic disturbance for (i) the phenologic parameters of the plant host P. oceanica, such as shoot density, leaf length and leaf surface area in the disturbed locations, for (ii) the mean percentage cover of targeted organisms and for (iii) the epiphytic biomass on leaves of P. oceanica.

The hierarchical sampling designs used in this study similarly to other investigations dealing with the spatial structuring of epiphytes-seagrass relations, pointed out a homogeneity in the distribution of epiphyte assemblages for the majority of functional groups and biomass among the scale site (500-600 m transect) while a heterogeneity was displayed at large scales among regions and at small scales among plots. Seagrass beds separated by few hundred of meters in both disturbed and control locations will apparently be subjected to unique environmental conditions resulting in equally epiphytic leaves biomass and assemblages. P. oceanica meadow and its associated epiphytes appear to be strongly affected by human pressure in the Gulf of Gabes through sewage outlet (this study), in addition to other disturbances such as trawling, mooring, fish farming and coastal pollution. The regression trend will significantly increase unless there is efficient implementation of legal protection along with a reduction in human interference. While restrictions on trawling have been introduced in Tunisia, this practice being responsible for the loss of about 80% of the surface area of seagrass meadows in the Gulf of Gabes (Zaouali, 1992), the legislation is not respected. In a recent review, many authors regretted the lack of data in some Mediterranean regions, particularly indicating the urgent need for studies on P. oceanica in North Africa (Ruiz et al., 2009). We, thus, believe that our data may be, on a North African scale, not only worthwhile but also useful in initiating other investigations. For example, the construction of "matte" by P. oceanica, a feature unique among Mediterranean seagrasses and which may be useful for assessing the regression of P. oceanica [START_REF] Leriche | A one-century suite of seagrass bed maps: can we trust ancient maps?[END_REF][START_REF] Boudouresque | Regression of Mediterranean seagrasses caused by natural processes and anthropogenic disturbances and stress: a critical review[END_REF], should be further addressed. Finally, it is urgent to propose the best management plans to save the remaining 20% of P. oceanica in the Gulf of Gabes and its associated epiphytes which have been reported to be among the most productive marine and terrestrial ecosystems [START_REF] Duarte | Seagrass biomass and production: a reassessment[END_REF].

Acknowledgements

This

Figure 1 .

 1 study was conducted by Mounir Ben Brahim in the framework of his PhD and supported by grants from by JICA (Japan International Cooperation Agency) and ESREB project (Étude des Stocks et des Ressources Exploitables des Bivalves). It was conducted as part of a collaborative project between the INSTM (Institut National des Sciences et Techniques de la Pêche), the Faculty of Sciences of Sfax (Tunisia) and the University of Franche-Comté (Chrono-Environnement, UMR CNRS 4269). The authors wish to thank the two reviewers for helpful suggestions which improved our contribution.List of figures: Geographical map focussing on sampling locations in the Gulf of Gabes.

Figure 2 .

 2 Figure 2. Leaf biomass vs. epiphyte biomass (g dw leaf -1 ) for disturbed (D) and control (C)

Figure 3 .

 3 Figure 3. Average ± SD values of the shoot density at the control (C1, C2) and disturbed D1,

Figure 4 .

 4 Figure 4. Average ± SD values of the leaf surface area at the control (C) and disturbed (D)

Figure 5 .

 5 Figure 5. Average ± SD values of leaf length at the control (C) and disturbed (D) locations

Figure 6 .

 6 Figure 6. Average ± SD values of the mean biomass of leaf epiphytes of P. oceanica at the

Figure 7 .

 7 Figure 7. Mean percentage cover of the main epiphyte groups at the control and disturbed

Figure 8 .

 8 Figure 8. nMDS showing the dissimilarity among centroids of sites of epiphytic assemblages

Figure 9 .

 9 Figure 9. Global projection on the first factorial design discriminating of variables and group

  Figure 1.

  Figure 4.

Figure 1 .

 1 Figure 8.

Figure 2 .

 2 Figure 2. Leaf biomass vs. epiphyte biomass (g dw leaf -1 ) for disturbed (D) and control (C)

Figure 3 .

 3 Figure 3. Average ± SD values of the shoot density at the control (C1, C2) and disturbed D1,

Figure 4 .

 4 Figure 4. Average ± SD values of the leaf surface area at the control (C) and disturbed (D)

Figure 5 .

 5 Figure 5. Average ± SD values of leaf length at the control (C) and disturbed (D) locations

Figure 6 .

 6 Figure 6. Average ± SD values of the mean biomass of leaf epiphytes of P. oceanica at the

Figure 7 .

 7 Figure 7. Mean percentage cover of the main epiphyte groups at the control and disturbed

Figure 8 .

 8 Figure 8. nMDS showing the dissimilarity among centroids of sites of epiphytic assemblages

Figure 9 .

 9 Figure 9. Global projection on the first factorial design discriminating of variables and group

Table 1 .

 1 Analysis of variance on shoot density, leaf surface area and leaf length. Bold numbers indicate significant effects. C = control locations; D= disturbed locations.

			Shoot density			Leaf surface area		Leaf length
	Source of Variation	d.f	MS	F	P	MS	F	P	MS	F	P
	Locations = L		2.68 10 7	176.90	0.00	1.03 10 6	11.71	0.00	9.31	8.25	0.00
	Disturbed vs. Controls (DC)		6.88 10 7	503.81	0.00	2.44 10 6	16.55	0.02	20.15	12.78	0.02
	Among Disturbed (=D)		1.17 10 7	63.28	0.00	5.52 10 5	4.579	0.10	0.07	0.06	0.82
	Among Controls (=C)		215.90	0.002	0.97	1.03 10 5	1.868	0.24	7.71	7.46	0.05
	Site (DC)		1.36 10 5	6.19	0.03	147 10 5	9.46	0.01	1.57	0.17	0.94
	Site (D)		1.85 10 5	18.95	0.00	120 10 5	13.3	0.00	1.22	0.24	0.91
	Site (C )		1.18 10 5	5.43	0.03	55474.20	7.71	0.02	1.03	0.22	0.92
	Subsite (Site (DC))		22030.05	1.19	0.34	15550.04	3.879	0.01	9.46	3.66	0.01
	Subsite (Site (D))		9767.25	0.97	0.47	9060.70	4.58	0.00	5.19	4.08	0.00
	Subsite (Site (C))		21758.39	1.30	0.30	7192.68	3.56	0.01	4.68	3.17	0.02
	Plot (subsite (Site (DC)))	24 18454.41	0.82	0.70	4009.17	6.925	0.00	2.58	3.44	0.00
	Plot (subsite (Site (D)))	26 10079.36	0.45	0.99	1992.08	7.32	0.00	1.27	3.04	0.00
	Plot (subsite (Site (C)))	26 16742.5	1.42	0.13	2020.14	6.39	0.00	1.48	3.82	0.00
	Quadrat (plot(subsite( Site (DC))))	72 22433.62	0.82	0.86	578.97	0.339	0.99	0.75	6.76	0.00
	Quadrat (plot(subsite( Site (D))))	72 22550.45	12.38	0.00	271.83	0.802	0.86	0.42	3.78	0.00
	Quadrat (plot(subsite( Site (C))))	72 11816.26	2.69	0.00	316.04	0.95	0.58	0.39	5.59	0.00
	Residual	432 3108.5			334.92			0.09
	Residual D	216 1820.99			339.03			0.11
	Residual C	216 4396.01			330.8			0.07
	Transformation		None				None			Ln(x+1)
	Cochran's C test		C=0.05, p<0.01				C=0.403, p<0.01		C=0.288, not significant

Table 4 .

 4 Analysis of variance on mean percentage cover of epiphytes of Posidonia oceanica.

	Bold numbers indicate significant effects.
			Filamentous algae		Bryozoans			Hydrozoans		Encrusting Annelida
	Source of Variation	d.f	MS	F	P	MS	F	P	MS	F	P	MS	F	P
	Locations = L	3 43.014	10.5	0.004 0.11	0.06	0.986 147.13	12.08	0.002 64.1	4.83	0.033
	Disturbed vs. Controls = DC	1 104.59	19.6	0.001 0.2	0.1	0.755 395.59	29.12	0	44.75	1.78	0.211
	Disturbed =D	1	12.68	1.85	0.245 0.02	0.01	0.929 31.92	1.54	0.281 86.09	6.19	0.068
	Controls = C	1	8.812	7.87	0.049 0.08	0.03	0.864	8.12	2.44	0.194 59.74	4.85	0.092
	Site (L) = SL	8	4.07	0.46	0.861 2.53	1.8	0.173 12.18	1.75	0.183 13.28	2.59	0.067
	Site (DC) = SiDC	10	5.32	0.61	0.781 1.95	1.41	0.282 13.58	1.95	0.136 25.07	4.87	0.006
	Site (D) = SiD	4	6.83	0.46	0.759 2.43	0.95	0.495 20.61	2.25	0.178 13.9	1.94	0.222
	Site (C ) = SiC	4	1.12	0.39	0.813 2.42	11.43 0.006	3.33	0.69	0.623 12.32	3.91	0.067
	Subsite (SL)	12	8.8	2.3	0.02	1.4	1.29	0.25	6.93	1.18	0.322 5.12	0.9	0.551
	Subsite (SiDC)	12	8.76	2.28	0.022 1.38	1.33	0.232	6.96	1.19	0.312 5.15	0.91	0.544
	Subsite (SiD)	6	14.62	2.56	0.046 2.55	1.67	0.17	9.12	1.17	0.351 7.14	0.82	0.567
	Subsite (SiC)	6	2.91	1.47	0.23 0.21	0.38	0.882	4.81	1.24	0.321 3.15	1.221	0.33
	Plot ( Subsite (SL))	48	3.81	0	0	0	0	0	5.87	0	0	5.67	0	0
	Plot (Subsite(SiDC))	48	3.83	1569.1	0	1.04 867.34	0	5.81	1346.47	0	5.65 2220.02	0
	Plot (subsite(S(D))	24	5.69	1386.2	0	1.52 1456.2	0	7.75	1242.7	0	8.73 4416.98	0
	Plot (subsite(SiC))	24	1.97	2528.35	0	0.55 409.47	0	3.87	1616.59	0	2.58	826.63	0
	Quadrat(Plot(Subsite (SL)))	144	0	0	0	0	0	0	0.001	0	0	0	0	0
	Quadrat(Plot(Subsite(SiDC)))	144 0.002	0.92	0.74 0.001	0.92	0.746 0.004	1.01	0.457 0.01	0.98	0.539
	Quadrat(plot(subsite(S(D)))	72 0.004	0.91	0.679 0.001	1	0.484 0.006	1	0.484 0.01	0.95	0.584
	Quadrat(plot(subsite(S(D)))	72 0.001	0.94	0.607 0.001	0.86	0.775 0.002	1.04	0.402 0.01	1.01	0.478
	Residual L	648	0			0			0			0
	Residual DC	648 0.003			0.001			0.004			0.003
	Residual D	324 0.004			0.001			0.006			0.002
	Residual C	324 0.008			0.002			0.002			0.003
	Cochran's C-test		0.309, p < 0.01	0.307, p < 0.01	0.406, p < 0.01	0.206, p < 0.01
	Transformation			Ln (x+1)			Ln (x+1)			Ln (x+1)			Ln (x+1)
	S-N-K test		D2<D1<C2<C1				D2<D1<C2<C1	D2<C1<D1<C2

Table 5 .

 5 Major species, ranked in order of importance, contributing to the average dissimilarities between the disturbed and the control locations as determined by similarity percentages (SIMPER).

	C	D

Table 6 .

 6 Variance components expressed as percentage calculated for the epiphytic biomass and the epiphytic assemblages on leaves in the disturbed and the control locations (D = disturbed locations, C = control locations).

	Encrusting

Table 7 .

 7 Test of equality of mean variables studied for P. oceanica meadows. F:, df1, df2, Sig.:

		Wilks' Lambda	F	df1 df2 Sig.
	Biomass	0.081	30.85	3	8	0
	Filamentous algae	0.285	6.673	3	8	0.014
	Bryozoans	0.967	0.092	3	8	0.962
	Hydrozoans	0.131	17.61	3	8	0
	Encrusting Annelida	0.363	4.66	3	8	0.036
	Leaf length	0.373	4.476	3	8	0.04
	Shoot density	0.015	175.4	3	8	0

Table 8 .

 8 Standardized canonical discriminant function coefficients of the studied variables forPosidonia oceanica.

			Function
		1	2	3
	Biomass	1.143	1.52	0.07
	Hydrozoans	2.523	1.287	0.866
	Encrusting Annelida	0.608	-0.79	0.984
	Shoot density	-2.992	-0.898	-0.287

Tunicata

Botryllus schlosseri Pallas 0 Clavelina lepadiformis (light-bulb sea squirt) 1

Total number of taxa 32

Appendix. Presence/absence of species and/or genus found growing on Posidonia oceanica leaves in the disturbed (D) and control locations (C) at Gabes gulf.


Ann-Marie Roos-Jansåker

Christel ; Lindahl

Rutger G Persson

Stefan Renvert
email: stefan.renvert@hkr.se

G Rutger Pesson

Long-term stability of surgical bone regenerative procedures of peri-implantitis lesions in a prospective case-control study over three years

Keywords: Peri-implantitis, Surgery, Bone graft, Membrane, Defect fill Peri-implantitis, surgery, bone graft, membrane, defect fill, plaque index Surgical treatment of peri-implantitis

Long-term stability of surgical bone regenerative procedures of peri-implantitis lesions in a prospective

case-control study over three years

Introduction

It is well established that peri-implantitis has an infectious etiology. A complex biofilm around implants with peri-implantitis has been identified (Renvert et al. 2007, 2008[START_REF] Shibli | Composition of supra-and subgingival biofilm of subjects with healthy and diseased implants[END_REF]. The infection results in a pathology that includes soft tissue inflammation, and loss of implant surrounding alveolar bone. In the progression of peri-implantitis, severe bone lesions with crater formed defect characteristics can be found [START_REF] Schwarz | Comparison of naturally occurring and ligature-induced peri-implantitis bone defects in humans and dogs[END_REF]. Data suggest that the prevalence of peri-implantitis is high (Roos-Jansåker et al. 2006a[START_REF] Fransson | Extent of periimplantitis-associated bone loss[END_REF], Koldsland et al. 2010). Data also suggest that within the same subject with periimplantitis the characteristics of the bony lesions are similar and that the rate of untreated lesions appear to increase over time [START_REF] Fransson | Severity and pattern of peri-implantitis-associated bone loss[END_REF]. There is no evidence that the implant surface characteristics can have an influence on the initiation of peri-implantitis [START_REF] Renvert | How do implant characteristics influence peri-implant disease[END_REF]. Surgical treatment of peri-implantitis lesions with an intra bony component has a favorable treatment outcome [START_REF] Schwarz | Impact of defect configuration on the clinical outcome following surgical regenerative therapy of periimplantitis[END_REF].

Non-surgical therapy of peri-implantitis does not seem to be efficacious (Schwarz et al. 2006b[START_REF] Renvert | Mechanical nonsurgical treatment of peri-implantitis: a double-blind randomized longitudinal clinical study. I: clinical results[END_REF][START_REF] Persson | Mechanical nonsurgical treatment of peri-implantitis: a single-blinded randomized longitudinal clinical study. II. Microbiological results[END_REF]. Surgical intervention and cleaning of implants with hydrogen peroxide and adjunct systemic antibiotics have demonstrated limited success over a five year period [START_REF] Leonhardt | Five-year clinical, microbiological,and radiographical outcome following treatment of peri-implantitis in man[END_REF].

Resective surgery and implantoplasty of cases with peri-implantitis seems to be effective over a three year period [START_REF] Romeo | Therapy of peri-implantitis with resective surgery. A 3-year clinical trial on rough screw-shaped oral implants. Part I: clinical outcome[END_REF].

Different bone regenerative therapies have been used attempting to accomplish healing of peri-implantitis defects in humans. A useful surgical treatment modality including bone grafts covered by ePTFE membranes in animal with experimentally induced peri-implantitis is effective in the management of peri-implantitis [START_REF] Schou | Autogenous bone graft and ePTFE membrane in the treatment of peri-implantitis. II. Stereologic and histologic observations in cynomolgus monkeys[END_REF][START_REF] Fiorellini | Osseointegration of titanium implants following guided bone regeneration using expanded polytetrafluoroethylene membrane and various bone fillers[END_REF]. Surgical treatment of circumferential defects around implants in humans using bone grafting and collagen membranes have been shown to be useful [START_REF] Schwarz | Surgical regenerative treatment of peri-implantitis lesions using a nanocrystalline hydroxyapatite or a natural bone mineral in combination with a collagen membrane: a four-year clinical follow-up report[END_REF][START_REF] Schwarz | Impact of defect configuration on the clinical outcome following surgical regenerative therapy of periimplantitis[END_REF]. Available data also suggest that surgical treatment of non-submerged implants with bony dehiscence can successfully be treated with a stiff non-resorbable membrane combined with a xenograft [START_REF] Boever | Guided bone regeneration around nonsubmerged implants in narrow alveolar ridges: a prospective long-term clinical study[END_REF].

Other studies in humans have shown that surgical treatment of implants with bone lesions that were > 4 mm, and with circumferential crater defects can effectively be treated with a mixture of autogenous and xenogenous bone [START_REF] Wiltfang | Regenerative Treatment of Peri-Implantitis Bone Defects with a Combination of Autologous Bone and a Demineralized Xenogenic Bone Graft: A Series of 36 Defects[END_REF]).

Almost complete bone fill in subjects with peri-implantitis treated with surgical intervention using laser debridement and application of bone grafts with collagen membrane coverage has been demonstrated [START_REF] Romanos | Regenerative therapy of deep peri-implant infrabony defects after CO2 laser implant surface decontamination[END_REF]. These findings are supported by other studies demonstrating that surgical treatment of periimplant defects with bone graft substitutes combined with resorbable membrane methods and submerged healing or regenerative treatment with a bone substitute alone or in combination with a resorbable membrane in a non-submerged model results in defect fill (Roos-Jansåker et al. 2007a, b). The aim of the present study was to evaluate three-year stability of defect fill after surgical regenerative therapy of periimplantitis including the use of a bone graft material with, or with a resorbable membrane in an non-submerged mode of wound healing.

Material and Methods

Study design

The study design was a human prospective case-control, single blinded, longitudinal study comparing the radiographic outcome after three years.

Study population

The Institutional Review Board at the University of Lund, Sweden approved the study. All participating individuals signed an informed consent. The CONSORT guidelines for clinical trials were followed (Figure 1). Study subjects were recruited from (I) individuals examined in a survey evaluating the prevalence of peri-implantitis 9-14 years following placements of Brånemark implants (Brånemark system ® , Nobel Pharma, Göteborg, Sweden) and (II) subjects who had been referred for the Thirty-eight consecutive consenting subjects scheduled by the surgeon for surgical treatment of peri-implantitis were enrolled. In order to be included the subject had a minimum of one osseo-integrated implant with peri-implantitis. In order to be included the subject must have an implant demonstrating a progressive bone loss of ≥ 3 threads (≥ 1.8 mm) following the first year of healing [START_REF] Renvert | Infection at titanium implants with or without a clinical diagnosis of inflammation[END_REF]). In addition, bleeding on probing with or without suppuration must be present at enrollment. This is in agreement with the consensus report of the 7 th European Workshop on Periodontology [START_REF] Berglundh | Peri-implant diseases[END_REF]. Furthermore, efforts must first have been to treat the condition using a non-surgical protocol, and that such All subjects had surgical regenerative treatment using a bone graft substitute (Algipore  , Friadent, Malmö, Sweden). The first 19 subjects were treated with the graft substitute that was covered with a resorbable membrane (Osseoquest  , W.L.

Gore & Associates, Inc, Flagstaff, AZ). The next 19 subjects were treated with the bone graft substitute alone.

Clinical and Radiographic examination

At baseline and year-three, the same examiner (A-M.R.J) performed the radiographic examinations. At the time of assessment of the radiographs, the examiner had only access to coded radiographs. Radiographs were obtained of implants using individually made bite-blocks on an Eggen holder (Renvert et al. 1981). The bite-blocks were made of Provil  Novo, Putty Soft (Heraeus Kulzer, GmbH I, Hanau, Germany). The X-ray films (Kodak Insight, EKC, Rochester, NY) were supported by the bite-block to avoid displacement and curving of the films.

Attempts were made to place the film parallel to the long axis of the implant examined. During the exposure the extension arm of the film holder was inserted into an acrylic track mounted on the long cone of the X-ray apparatus. Threads not supported by bone at the mesial and distal site of the implants were assessed.

Plaque index was defined as visible or not visible at the implant. Probing pocket depth measurements were deferred at year three as the super-structures remained in place. Hence the values would not be comparable to pre-surgical baseline.

Surgical treatment

The study population and the surgical techniques have been described in detail previously (Roos-Jansåker et al. 2007a). All subjects received antibiotic coverage (amoxicillin 375 mg x 3 per day, and metronidazole 400 mg x 2 per day) during the first 10 days following surgery. Briefly, following removal of the supra-structure granulomatous tissue was removed. The exposed implant threads were carefully debrided, treated with hydrogen peroxide (3%), and rinsed with copious amounts of a saline solution. The osseous defects were filled with a bone graft substitute (Algipore  , Friadent, Malmö, Sweden). Before insertion of the Algipore  bone fill material, the bone graft material was mixed with blood from the subject. The first 19 consecutive subjects had a resorbable membrane (Osseoquest  , W.L. Gore & Associates, INC, Flagstaff, AZ), placed over the filled defect. The following 19 consecutive subjects where treated with the bone graft substitute without a resorbable membrane. The abutments were then reconnected and the flaps were sutured with non-resorbable sutures (Gore 5-0, W.L. Gore & Associates, INC, Flagstaff, AZ) and the supra-structures were remounted. The wound healing was performed in a non-submerged mode.

During the first 5 weeks all subjects rinsed with 0.1 % chlorhexidine. During the first 3 days all subjects also received an anti-inflammatory and analgesic drug (Ibuprofen 400 mg x 3 days). Six weeks after surgery the first supportive therapy was given, and the subjects were enrolled in a maintenance program with visits every third month. At the visits, full mouth plaque scores were obtained. Plaque was disclosed using an erythrosine dye (Top Dent Lifco Dental AB, Enköping, Sweden). Re-instruction in oral hygiene procedures was performed as necessary. Teeth and implants were cleaned

Statistical methods

Parametric tests (independent t-tests, equal variance not assumed), and nonparametric tests (Mann-Whitney U test, Wilcoxon test, and Pearson χ2 were performed to assess differences over time and between groups. Statistical significance was declared at p < 0.05. The Kolmogorov-Smirnov test was used to define if the data presented with normal distribution or not. The SPSS PASW 18.0 statistical software (SPSS Inc. Chicago IL) for PC was used in the analysis.

Results

Subject characteristics at study baseline are presented (Table 1). At baseline, a total of 38 subjects were enrolled. Two subjects died before the 1-year control, leaving 17 subjects, with 29 treated implants in the group receiving bone graft and membrane treatment and 19 subjects with 36 treated implants in the group treated with bone graft alone. Four subjects in the group treated with bone graft alone were lost to follow-up during the 3-year period, leaving 15 subjects with 27 implants in this group after three years. In the present analysis only the 32 subjects who returned to the follow-up examination and had radiographs taken could be assessed. During the present study period, no clinical complications as a result of the interventions were found in any of the subjects.

One subject in each group had received Astra implants (Astra Tech system  , Astra Tech, Mölndal, Sweden). These implant had the same thread distance between threads as for the Brånemark implants (0.6 mm)other subjects had received machined surfaced implants (Brånemark system ® , Nobelpharma, Göteborg, Sweden).. Subject characteristics are presented (Table 1). Analysis by Mann-Whitney U test confirmed the lack of difference by study method.

The radiographic evidence of defect mean improvement with defect fill between baseline and year three at implants treated with bone graft and membrane was a mean gain of 1.6 mm (S.D. ± 1.2 mm, range: -1 to +7). The corresponding improvement with defect fill at implants treated with bone graft alone was a mean gain of 1.3 mm (S.D. ± 1.3, range -2 to + 6 threads) (NS). Statistical analysis failed to demonstrate differences in the extent of defect fill (mean difference: 0.3 mm, S.E. diff: 0.3 mm, 95% CI: -1.0 to + 0.4 mm, P = 0.34). In the group treated with bone graft material and membrane, a total of 15 implants (51.7 %) had a defect fill > 1.8 mm. In the group treated with bone graft alone, a total of 12 implants (44.4 %) gained more than 1.8 mm in defect fill.

When the data were dichotomized in regards to change in the number of exposed threads as a gain if bone or bone fill could be identified at one or more previously exposed threads, or if no change had occurred or if one or more additional threads had been exposed three years after intervention statistical analysis (Pearson´s χ2) failed to demonstrate differences in gain /loss of thread exposure by surgical treatment modality (p = 0.89). The distribution of bone level changes in the two study groups between baseline and year three after surgery are presented (Table 2).

Statistical analysis failed to demonstrate differences in the extent of defect fill both in regards to number of threads filled and defect fill in mm by study group assignment. The distribution of bone level changes in the two study groups between baseline and year three after surgery are presented (Table 3).

Analysis by Mann-Whitney U test failed to demonstrate a difference in bone fill between year one and year three between the two study groups (P= 0.36). Within each study group related samples Wilcoxon signed rank test failed to demonstrate a difference in bone fill between year one and year three in the group receiving bone graft alone (P=0.18), or in the group treated with bone fill and membrane (P= 0.42).

Bone fill mean values, SD, and range of fill are presented (Table 4).

Non-parametric Wilcoxon test identified that plaque index continued to improve between year one and year three after surgery (p = 0.02). Statistical analysis (Mann-Whitney U test) failed to demonstrate differences in this change of plaque scores by intervention (p = 0.43). The distributions of changes in the mean % plaque scores at various time points up to year three are presented (Figure 4).

Examples of treatment outcome at year three

Background clinical information and radiographs from before treatment of periimplantitis and at year three are presented for one selected case defined as a nonresponding case, and from one case defined as successfully treated (Table 5). Intraoral radiographs taken immediately before and at three years after surgical treatment of peri-implantitis representing a non responding treatment outcome (Figure 5A

Discussion

In a previous report, one-year data following surgical treatment of peri-implantitis using a bone graft substitute demonstrated that the clinical conditions and radiographic conditions had improved (Roos-Jansåker et al.2007a). These results had occurred in spite of the fact that a majority of the subjects included were smokers, and with a past history of periodontitis (Roos-Jansåker et al. 2007a). It has previously been discussed that a smoking history and having a history of periodontitis can negatively influence the outcome of surgical treatment and reconstruction of defects around dental implants (Roos-Jansåker et al. 2006b[START_REF] Karoussis | Association beween periodontal and peri-implant conditions: a 10-year prospective study[END_REF][START_REF] Máximo | Peri-implant diseases may be associated with increased time loading and generalized periodontal bone loss: preliminary results[END_REF]). Smoking has also been considered as a risk factor during wound healing following implant placement [START_REF] Sadig | Risk factors and management of dehiscent wounds in implant dentistry[END_REF]. The fact that smoking may have a negative influence on regenerative procedures in the management of periodontitis is well established [START_REF] Mayfield | Guided tissue regeneration for treatment of intraosseous defects using a bioabsorbale membrane. A controlled clinical study[END_REF], Trombelli et al. 1997, Rosén et al. 1996, Tonetti et al.1995, Kornman & Roberson 2000). It is therefore of interest that also in smokers, the use of a bone graft material placed in bone defects around implants can remain stable also at three years after intervention.

Our findings are consistent with results reported elsewhere using bone substitutes in a non smoking study population (Schwarz et al.2008).

Subjects with a history of periodontitis and specifically in men with dental implants appear to be at risk for peri-implantitis. A history of smoking may be of less significance a s a risk factor for peri-implantitis (Koldsland et al. in press). In spite of the accrued risk factors in the present study population it should be emphasized that both surgical study modalities resulted in a successful three year outcome of surgical The importance of plaque control after periodontal therapy and supportive therapy has been described extensively (i.e. [START_REF] Nyman | Effect of professional tooth cleaning on healing after periodontal surgery[END_REF][START_REF] Nyman | Periodontal surgery in plaque-infected dentitions[END_REF][START_REF] Rosling | Periodontally treated dentitions: their maintenance and prognosis[END_REF][START_REF] Rosling | Longitudinal periodontal tissue alterations during supportive therapy. Findings from subjects with normal and high susceptibility to periodontal disease[END_REF]). In the present study the post-surgical management was focused on a strict oral hygiene program that continued during the study. Thus, the overall average plaque score at year three was very low. This may explain the good results reported.

In a previous study using non-surgical management of peri-implantitis it was not possible to reach a similar oral hygiene level which in part may explain the less favorable clinical outcome [START_REF] Renvert | Mechanical nonsurgical treatment of peri-implantitis: a double-blind randomized longitudinal clinical study. I: clinical results[END_REF][START_REF] Persson | Mechanical nonsurgical treatment of peri-implantitis: a single-blinded randomized longitudinal clinical study. II. Microbiological results[END_REF]. It is possible that the surgical intervention in this study resulted in an improved soft tissue architecture after wound healing facilitating oral hygiene measures. This may, in part, explain the differences in plaque control efficacy in the present surgical intervention study compared to the non-surgical intervention reported in previous papers. Nevertheless, other studies employing surgical intervention in non-smokers with peri-implantitis reported higher plaque scores. However, they also reported a gradual detoriation of results primary obtained (Schwarz et al. 2006a). Higher plaque scores than reported in the present study have also been reported in a long term retrospective study [START_REF] Matarasso | 10-year retrospective analysis of radiographic bone-level changes of implants supporting single-unit crowns in periodontally compromised vs. periodontally healthy patients[END_REF]. Thus, the surgical procedure and the wound healing may not be effective without good plaque control during the follow up period.

The present follow-up, three years after surgical intervention comparing a bone substitute with or without the adjunct treatment with a resorbable membrane demonstrated that the additional effort to obtain stability did no seem to result in enhanced three year results as assessed by radiographs. Our results are consistent with those reported from studies of experimental healing in dogs [START_REF] Nociti | Evaluation of guided bone regeneration and/or bone grafts in the treatment of ligature-induced peri-implantitis defects: a morphometric study in dogs[END_REF], or in a non-human primate model [START_REF] Schou | Autogenous bone graft and ePTFE membrane in the treatment of peri-implantitis. II. Stereologic and histologic observations in cynomolgus monkeys[END_REF]. It is possible that the use of a bone graft with the additional placement of a membrane may be more efficient in specific bone lesions around implants [START_REF] Schwarz | Impact of defect configuration on the clinical outcome following surgical regenerative therapy of periimplantitis[END_REF]. Although somewhat better results have been presented in a 10-year follow clinical trial using bone grafts with the adjunct use of resorbable membranes in comparison to not using such membranes, the clinical relevance appeared to be negligible [START_REF] Nygaard-Østby | Periodontal healing following reconstructive surgery: effect of guided tissue regeneration using a bioresorbable barrier device when combined with autogenous bone grafting. A randomized-controlled trial 10-year follow-up[END_REF]). The study by [START_REF] Nygaard-Østby | Periodontal healing following reconstructive surgery: effect of guided tissue regeneration using a bioresorbable barrier device when combined with autogenous bone grafting. A randomized-controlled trial 10-year follow-up[END_REF] also identified that the resolution of deep intra-bony periodontal defects requires a structured maintenance program emphasizing high oral hygiene standards.

In the present study, the extent of bone fill obtained at one year after surgical treatment using either a bone graft alone or bone graft and a resorbable membrane could be maintained up to three years after treatment. Thus, provided oral hygiene is excellent both procedures resulted in a clinically relevant defect fill. A pre-requisite for a successful treatment outcome might also be the anatomy of bone defect. A well defined crater-like defect might be the most reliable factor for stable conditions. The limitation of using the radiographic assessment method used in the present study is obvious. The radiographic evaluation does not allow distinguishing between osseo-integration of the graft in direct contact to the implant, or if the bone graft simply remains as a defect fill. However in comparison to the one year results (Roos-Jansåker et al. 2007a) the present findings at three years demonstrated stable results without progression of bone loss. Our results are consistent with histological results obtained from animal studies demonstrating that hard tissue fill and osseo- integration can occur in experimentally created defects around implants [START_REF] Abushahba | Effect of grafting materials on osseointegration of dental implants surrounded by circumferential bone defects. An experimental study in the dog[END_REF]. This would also be the result at implants previously exposed to an infectious biofilm, and animal studies it has also been demonstrated that it is possible to obtain re-osseointegration on previously infected implant surfaces [START_REF] Alhag | Re-osseointegration on rough implant surfaces previously coated with bacterial biofilm: an experimental study in the dog[END_REF].

The two cases identified with the similar age and clinical conditions, albeit with opposite gender represent examples of a successful and a non-successful treatment outcome of circumferential advanced lesions at implants placed in the maxilla. Both subjects had had a previous history of peri-implantitis at the same or other implants, and both of them were former smokers. At the time of surgical intervention in the study the clinical conditions were similar in both cases. The presences of circumferential defects with a significant vertical component were in both cases suitable for the assigned therapy. The study treatment was the same in both cases using a bone graft material without membrane coverage. Both subjects complied with the post-surgical instructions and maintained excellent oral hygiene. The differences identified between the two cases were that the subject with a non-successful outcome had a medical diagnosis of diabetes mellitus and clinical lack of keratinized and attached mucosa at the buccal aspect of the implant in treatment. Furthermore, the infection at the implant appeared to harbor pathogens that are commonly therapy resistant. The present observations from these two cases and the observations made should only be used to suggest further studies on the role of keratinized attached tissues, systemic health, and local infection in relation to therapy in cases with periimplantitis. In conclusion, defect fill obtained at one year after surgical treatment with a bone graft and membrane or with bone graft alone remain stable between one and three years in cases on maintenance care.

CONSORT flowchart

Figure 2

Examples of radiographs taken from one subject at baseline, year one, and year 3 after follow-up.

Figure 3

Example of radiographs taken from another subject at baseline, year one, and year 3 after follow-up.

Figure 4

Mean plaque index from baseline to 3 years in Group 1 (bone graft substitute + membrane) and Group 2 (bone graft substitute).

Figure 5A

Baseline radiograph illustrating the alveolar bone conditions at the implant (replacing tooth 24) at a non-responding defect. Notice the number of implant threads not supported by bone and the crater defect. Clinical details are presented in table 5. Radiograph from year three after surgical intervention and placement of bone graft material illustrating the alveolar conditions at the implant (replacing tooth 24) at a non-responding defect, and similar extent of bone defect and implant threads exposed as at baseline. Notice the number of implant threads not supported by bone and the crater defect. Clinical details are presented in table 5. Radiograph from year three after surgical intervention and placement of bone graft material illustrating the alveolar conditions at the implant (replacing tooth 24) at a non-responding defect, and similar extent of bone defect and implant threads exposed as at baseline. Notice the number of implant threads not supported by bone and the crater defect. Clinical details are presented in table 5. Radiograph from year three after surgical intervention and placement of bone graft material illustrating the alveolar conditions at the implant (replacing tooth 14), and in principle complete fill of crater defect with bone fill material used. Notice that no threads are exposed. Clinical details are presented in table 5. The image suggests that the lesion has been filled with radiopaque material and this should not be interpreted as bone regeneration or "osseo-integration".

  treatment of peri-implantitis to the Clinic of Periodontology, Public Dental health Services and University of Kristianstad, Sweden. The present paper is a follow-up report based on the publication by Roos-Jansåker et al. 2007.

  . Subjects with radiographic evidence of horizontal bone loss only and without evidence of a vertical crater-like defect were not included.

  cup and a low-abrasive paste. During the follow-up period, none of the subjects was prescribed antibiotics.

  and 5B) and a successfully treated case (Figure6Aand 6B) are shown. In these cases, the immediate postoperative wound healing was uneventful. The maintenance effective as illustrated by the low plaque scores during the study period.

  fill around implants with a previous history of periimplantitis (2006).

  Figure 5B

Figure 6 A

 6 Figure 6 A

Figure 6B Radiograph

 6B Figure 6B

  radiograph from baseline illustrating the alveolar bone conditions at the implant (replacing tooth 14). Notice the number of implant threads not supported by bone and the crater defect. Clinical details are presented in table 4. The opaque area visible on the radiograph is part of the stent used to standardize the positioning before image exposure.

Table 2 .

 2 Defect changes between baseline and year three after surgery (implant level).

	Bone level change	Bone graft and	Bone graft alone
	(mm)	membrane including 29	including 27 implants
		implants
		Number	%	Number	%
	-1.2	0	0	1	4
	-0.6	2	7	2	7
	0.0	3	10	4	15
	0.6	2	7	3	11
	1.2	4	14	5	18
	1.8	6	21	4	15
	2.4	5	17	2	7
	3.0	5	17	4	15
	3.6	0	0	2	7
	4.2	2	7	0	0

Table 3 .

 3 Defect changes between year one and year three of follow -up after surgery (implant level).

	Bone level change	Bone graft and	Bone graft alone
	(mm)	membrane including 29	including 27 implants
		implants
		Number	%	Number	%
	-1.8	1	3	0	0
	-1.2	0	0	0	0
	-0.6	1	3	1	4
	0.0	21	73	24	89
	0.6	3	10	0	0
	1.2	2	7	1	4
	1.8	1	3	0	0
	2.4	0	0	1	4

Table 4

 4 Results of treatment at the implant level observed after three years compared to one year for implants treated with bone graft and membrane, or bone graft alone.

	Variable	Bone graft and membrane Bone graft alone
		Mean S.D. Range	Mean S.D. Range
	Defect fill (number of	0.2	± 0.9	2.5:-3.0	0.2 ± 0.8 3.5:-1.0
	threads)
	Defect fill (mm)	0.1	± 0.5	1.5:-1.8	0.1 ± 0.5 2.1:-0.6

Table 5 .

 5 Characteristic findings for two cases illustrating either a non-responding or a responding outcome of surgical treatment of peri-implantitis, and with an observation period of three years.

	Variables	Non responding case	Responding case
	Gender and age	Male, 68 years	Female, 69 years
	Medical /smoking	Diabetes mellitus (type II),	No systemic diseases reported
		former smoker	former smoker
	Periodontal therapy	No case history	Periodontal therapy in the past
	Peri-implantitis	Previous treatment at other	Previous treatment for peri-
		implants	implantitis at this location
	Implant(location and years in function	14,	10 years	24;	5 years
	Probing pocket depth	mes: 4 mm, buc: 4mm	mes: 6 mm, buc: 5mm
		dist:4 mm, pal:4 mm	dist:5 mm, pal:6 mm
	Bone sounding	mes: 7 mm, buc: 8 mm	mes: 10 mm, buc: 12 mm
		dist: 8 mm, pal: 6 mm	dist: 10 mm, pal: 11 mm
	Surgical procedure	Open flap debridement with	Open flap debridement with
		Algipore ® treatment without	Algipore ® treatment without
		membrane technique, antibiotic	membrane technique, antibiotic
		coverage	coverage
	Baseline thread exposure (xray)	mes: 6 threads and dist: 6	mes: 4 threads and dist: 5
		threads		threads
	Threads exposed at surgery	mes:6, buc:7 dist:7, pal:7	mes: 3, buc:6, dist:6, pal:7
	Horizontal destruction at surgery	mes 3 mm, buc: 5 mm	mes:0 mm, buc:5 mm,
		dist:2 mm, pal:5 mm	dist: 0 mm, pal: 3mm
	Vertical destruction assessed at	mes:2 mm, buc: 0 mm dist:3	mes: 3 mm buc;0 mm, dist: 1
	surgery (depth)	mm, pal: 0 mm	mm, pal: 2 mm
		circumferential	circumferential
	Year 3 mesial thread exposure (xray)	6		0
	Year 3 distal thread exposure (xray)	6		0
	Buccal mucosa baseline	non keratinized, no change to	keratinized 3 mm, no change to
		year 3		year 3
	Mucosal recession baseline	Buccal: 4 mm, Pal:2 mm
	Bacterial findings at the time of	Enterococci positive	S.epidermis, F.nucleatum,
	surgery	Antibiotic resistance consistent	S.sanguinis
		with Pseudomonas infection
	Bacterial findings at year 3	negative	negative
	Oral hygiene	Excellent, plaque scores on	Excellent plaque scores on
		average < 10%	average < 10 %

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Acknowledgements

This project was supported by research foundations from the Public Dental Health Service, County of Skåne, Sweden and Kristianstad University, Sweden. We thank Mr. Nuray Güner, (MA, Medical Statistics, MSc, Engineering Physics), for the help with the statistics.

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Conflict of interest and sources of funding statement:

None of the authors declare a conflict of interest. The present study was supported by research foundations from the Public Dental Health Service, County of Skåne,

Sweden and Kristianstad University