Valentine K Mubiana

Ronny Blust

Effects of Temperature on Scope for Growth and Accumulation of Cd, Co, Cu and Pb by the Marine Bivalve Mytilus edulis

Keywords: Mussels, Heavy metals, Bioaccumulation, Temperature, Speciation, Scope for Growth

In many aquatic organisms including Mytilus edulis, the role of temperature on bioaccumulation of metals is still not clearly understood. In this study, uptake and accumulation of Cu, Co, Cd and Pb in mussels were investigated at different temperatures (6 -26 °C). Results from exposure of isolated gills showed a positive relationship between temperature and metal uptake. But in whole organism experiments, only the accumulations of non-essential metals (Cd, Pb) showed a similar trend while the two essential metals Co and Cu were either independent or inversely related to temperature, respectively. With exception of Cu, elimination process appeared to be independent of temperature. The study also showed that neither changes in scope for growth (SFG) of mussels nor chemical speciation could fully account for the observed temperature-effects. Overall, these results suggest that fundamentally (i.e. at epithelial membranes), temperature-effects on uptake are largely due to changes in solution chemistry and physical kinetics, which favours higher uptake at high temperature. But at whole organism level, complex physiological responses appears to mask the relationship, particularly for biologically essential metals like copper.

Introduction

Bioaccumulation of heavy metals in aquatic organisms such as mussels depends not only on environmental concentrations but also on a variety of biological and environmental factors. The common marine mussel M. edulis is widely used in monitoring metal pollution in coastal and estuarine waters (e.g. [START_REF] Goldberg | US Mussel Watch: 1977-1978 results on trace metals and radionuclides[END_REF][START_REF] De Kock | Active Biomonitoring (ABM) by Translocation of Bivalve Molluscs[END_REF]. Therefore, understanding the possible roles of temperature and other environmental factors on metal uptake and accumulation in these biomonitors is critical in order to correctly relate tissue concentrations to those in the surrounding environment. Temperature affects both metal chemistry in seawater [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF] and physiology of mussels [START_REF] Dame | Ecology of marine bivalves an ecosystem approach[END_REF]. Temperature affects metal chemistry by changing chemical speciation, pH, solubility, reaction rates or physical kinetics [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF]Blust, Van Ginneken, & Decleir, 1994). Theoretical calculations of chemical speciation in seawater indicate that changes in temperature and pH have most effects on strongly hydrolysed and carbonate complexes and less effect on chlorides and free metal ions [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF]. Though it is widely known that uptake is largely controlled by the free metal ion, some studies have also shown significant contribution of other species, particularly metals bound to weak complexes (i.e. [START_REF] Campbell | Interactions between Trace Metals and Aquatic Organisms: A Critique of the Free-ion Activity Model[END_REF][START_REF] Hudson | Which aqueous species control the rates of trace metal uptake by aquatic biota? Observations and predictions of non-equilibrium effects[END_REF][START_REF] Lorenzo | Copper uptake by Mytilus edulis in the presence of humic acids[END_REF]. Chemical speciation indicates that increase in temperature generally results in increase in the concentrations and activities of bioavailable metal forms, therefore, enhance uptake, at least in theory.

Like most aquatic organisms, mussels cannot regulate their body temperature in accordance with surrounding environment, as a result most physiological and biochemical processes are temperature dependent [START_REF] Dame | Ecology of marine bivalves an ecosystem approach[END_REF]. One measure of animal's integrated physiological status is Scope for growth (SFG), which is essentially an energy balance equation of an individual determined from energy absorbed from ingested food items minus the energy lost through respiration and excretion [START_REF] Widdows | Physiological indices of stress in Mytilus edulis[END_REF][START_REF] Widdows | Physiological Energetics of Mytilus edulis: Scope for Growth[END_REF]. During the past 15 years, many studies have applied SFG approach as a quantitative measure of the impact of environmental stress due to i.e. salinity and temperature [START_REF] Widdows | Physiological indices of stress in Mytilus edulis[END_REF][START_REF] Sobral | Effects of elevated temperatures on the scope for growth and resistance to air exposure of the clam Ruditapes decussates (L), from Southern Portugal[END_REF][START_REF] Smaal | The scope for growth of bivalves as an integrated response parameter in biological monitoring[END_REF][START_REF] Vercauteren | Uptake of Cd and Zn by the mussel Mytilus edulis and inhibition by calcium channel and metabolic blockers[END_REF] and chemical pollution [START_REF] Widdows | Physiological Energetics of Mytilus edulis: Scope for Growth[END_REF][START_REF] Widdows | Effects of pollution on the scope for growth of mussels (Mytilus galloprovincialis) from the Venice Lagoon, Italy[END_REF][START_REF] Widdows | Measurement of stress effects (Scope for growth) and contaminant levels in mussels (Mytilus edulis) collected from the Irish Sea[END_REF][START_REF] Smaal | The scope for growth of bivalves as an integrated response parameter in biological monitoring[END_REF]. The role of physiological energetics such as SFG in ecotoxicology is discussed in detail in a review by [START_REF] Widdows | Role of physiological energetics in ecotoxicology[END_REF]. [START_REF] Smaal | The scope for growth of bivalves as an integrated response parameter in biological monitoring[END_REF] outlined the justification for use of SFG as an integrated response in biological monitoring programmes.

The role of temperature on metal uptake and accumulation in marine mussels has not been well studied. This is despite the fact that mussels are important biomonitoring organisms. Furthermore, there seem to be no general consensus among the few available studies on the subject. [START_REF] Carpene | Absorption of cadmium by gills of Mytilus edulis (L.)[END_REF] found no temperature effects on Cd uptake in isolated gills. However, lack of prior temperature acclimation of mussels used in that study may have influenced the results. In whole organism experiments, [START_REF] Phillips | The common mussel Mytilus edulis as an indicator of pollution by Zinc, Cadmium, Lead and Copper: effects of environmental variables on uptake of metals[END_REF] detected no temperature effects on accumulation of Cd, Cu, Pb and Zn except at low salinity (<18 ppt). [START_REF] Fischer | Influence of temperature, salinity, and oxygen on the cadmium balance of mussels Mytilus edulis[END_REF] also observed that Cd accumulation by mussels was only significantly decreased at very low temperatures (<7 °C). In both studies temperature exposures were coupled with different salinity treatments, therefore it is not clear whether salinity or interaction between the two may have influenced the results. Some recent studies have reported uptake that is dependent and increases with increase in temperature [START_REF] Wang | Metal and oxygen uptake in the green mussel Perna viridis under different metabolic conditions[END_REF]. [START_REF] Baines | Influence of temperature on dietary metal uptake in Arctic and temperate mussels[END_REF] also found a positive relationship between assimilation efficiency of dietary metals with temperature. These studies are consistent with results involving many different organisms, which tend to show a positive correlation between temperature and metal uptake, accumulation or toxicity [START_REF] Mclusky | The effects of temperature and salinity on the toxicity of heavy metals to marine and estuarine invertebrates[END_REF][START_REF] Blust | Effect of temperature on the uptake of copper by the brine shrimp, Artemia fransiscana[END_REF][START_REF] Bervoets | Effects of temperature on Cadmium and Zinc uptake by the Midge Larvae Chironomus riparius[END_REF][START_REF] Chowdhury | Effect of temperature on the uptake of waterborne strontium in the common carp, Cyprinus carpio (L)[END_REF].

The objective of the current study was to investigate the influence of temperature on uptake and accumulation of two types of metals i.e. Co, Cu as biologically essential metals and Cd and Pb as non-essential metals. Accumulation kinetics of these metals were determined in long-term exposure experiments. Short-term exposures involving isolated gills were also conducted. These experiments involved exposure of gills to either single metal solutions or metal mixtures. The two treatments were intended to check whether there were significant interactions among metals during uptake. Isolated gills experiments minimised the influence of animal physiology and hence provide a better insight into the fundamental mechanisms and processes involved. The main consequences of temperature changes namely, metal speciation, pH and dissolved oxygen were also determined. SFG was determined as an integrated measure of the animal physiological status in response to differences in ambient temperature.

Materials and methods

Test animals

Adult mussels of 40 -50 mm shell length were collected from Eastern Scheldt (The Netherlands), a former estuary turned into a semi-enclosed marine bay after construction of dams across river channels in the early 1980s. This area is largely a marine tidal bay with relatively low levels of metal pollution [START_REF] Mubiana | Temporal and spatial trends in heavy metal concentrations in the marine mussel Mytilus edulis from the Western Scheldt estuary (The Netherlands)[END_REF].

Exposure medium

Artificial seawater was prepared by dissolving 35 g/L sea salt hw Sea Salt professional (Wiegandt GmbH, Germany) in deionised water. Elemental composition of this salt is well described and contains only traces of heavy metals i.e. Cu (200 ng/kg), Pb (19 ng/kg), Co (3 ng/kg) and Cd (0.1 ng/kg) (http://www.hw-wiegandt.de). Metal exposure solutions were then prepared by dissolving high purity salts of CdCl 2 , CoCl 2 , CuCl 2 and PbNO 3 either as a mixture of all four metals or single metal solutions.

Exposure concentrations (see Table 1) were neither too low for measurable uptake nor too high to be environmentally relevant. However, in view of the short exposure times for isolated gills (1-h), relatively high exposure concentrations (0.5 µM) were used in order to achieve measurable uptake in the gills.

Prepared solutions were always left for 48-hrs to equilibrate, analysed for metal concentrations and if necessary additional metals were added. In long-term exposures, water was changed regularly to avoid significant drop in concentrations. Metal concentrations in water samples were analysed with ICP-AES (Liberty series II, Varian Australia) and as shown in Table 1 metal concentrations did not fluctuate by more than 10 % within or among tanks. Metal speciation in prepared seawater as a function of temperature and pH was calculated using chemical speciation model Visual MINTEQ 2.32 [START_REF] Gustafsson | Visual MINTEQ ver[END_REF]. Metal speciation calculations were only performed for the water that was used in isolated gills experiments. Concentration of DOC in the exposure water (in gills experiments) was found to be below method detection limits (0.01 mg/l) and was therefore not included in the speciation model.

Whole organism experiments

After sampling, animals were transported on ice to the laboratory and on arrival 20 and 14 individuals were chosen randomly for metal analysis and SFG measurements, respectively. Remaining mussels were divided into three groups of about 250 mussels and each group was placed in 500 L tanks. Initially, temperature was set at 13 °C (field temperature) then after a week and during two weeks it was slowly adjusted to obtain 6 °C, 16 °C and 26 °C after which acclimation was then continued for another week. Mussels were always fed 10 mg /individual of enriched yeast cells (Lanzy PZ, Inve, Belgium) twice a week. To minimise organic matter loading in the experimental tanks, feeding of mussels was in separate tanks during 3-hrs with conditions identical to experimental tanks. Furthermore, organic carbon was monitored every 3 days using a TOC 5000 Analyzer (Shimadzu) fitted with a 0.8 mm diameter suspended particle sampler. Both DOC and POC varied insignificantly with values consistently below 0.1 mg/L and 0.01 mg/L, respectively.

At time intervals 7 -9 mussels were collected from each tank for metal analysis. On day 28, animals were transferred to clean seawater for elimination during 28 days. In the first 14 days water was replaced every three days and then every six days for the remaining period. After sampling, each animal was partially opened and the inside cavity sprayed with purified water to flush-out trapped seawater. They were then dissected and soft tissues were individually placed in pre-weighed polypropylene vials.

The tissues were freeze-dried at -60 °C for 72-h, after which tissue dry weight was determined.

Five ml high purity concentrated HNO 3 and 0.25 ml of 27% H 2 O 2 were then added and the digest was further destructed by microwave heating according to [START_REF] Blust | Evaluation of microwave-heating digestion and graphite-furnace atomic-absorption spectrometry with continuum source background correction for the determination of Fe, Cu and Cd in brine shrimp[END_REF]. Samples were heated in four steps of five minutes at 80, 160, 240 and 320 watts, and then diluted with purified water to bring acid concentration to about 2%. Metal concentrations in the final solutions were measured with ICP-MS (Ultra Mass 700, Varian Australia) employing yttrium and indium as internal standards. As quality control, three reference samples (mussel tissue CRM 278R, IRMM, Geel Belgium) and three preparation blanks were included in each 40sample rack and were carried along in every step of the analysis. Recoveries for all four metals in the reference samples were consistently within 96 -101 % of the certified values.

Measurements of scope for growth

Scope for growth measurements were conducted according to a procedure adopted from [START_REF] Widdows | Physiological Energetics of Mytilus edulis: Scope for Growth[END_REF] and [START_REF] Widdows | Practical procedures for the Measurement of Scope for growth[END_REF]. Unless stated, all the constants used in the equations were adopted from these references. The first SFG parameter determined was clearance rate (CR). Mussels were placed in clean filtered seawater for 1-hr to flush-out any particles enclosed inside shell valves and then individually placed in 2-L seawater, which was previously filtered (0.45 µm) and then spiked with yeast cells (Lanzy PZ, Inve, Belgium) at 50,000 cells/ml. The vessels were aerated to provide homogenous distribution of cells and then every 15 minutes for 90 minutes, 10 ml water samples were taken for cell counting with a Coulter Counter. By definition, CR is the volume of water cleared of particles per gram mussel per hour and was calculated according to the expression:

CR (L g -1 h -1 ) = volume of water (L) x (log e C 1 -log e C 2 ) / DW x time interval (h)
Where C 1 and C 2 are cell concentrations at the beginning and end of each time increment and DW is soft tissue dry weight (g). CR during each time interval was calculated and for each individual the highest CR or 'maximum clearance rate' (MCR) was converted into energy equivalent according to the formula: Energy absorbed (J/h/g) = MCR (L g -1 h -1 ) x POM x 23 J mg -1 x 0.5

Where, POM is concentration of yeast cells (mg/L) with energy of 23 J mg -1 ash free dry weight (obtained from the manufacturer, Inve, Belgium) and 0.5 is absorption efficiency from yeast cells, which was calculated from ash free dry weight of ingested cells and faeces produced by the mussels.

After measuring CR, each mussel was then transferred into a 500-ml respirometer filled with clean filtered (0.45 µm) water and fitted with an oxygen electrode connected to a computer, which recorded dissolved oxygen over time (90 minutes). Measured respiration rates were then converted into energy equivalent according to the formula: Energy respired (J/h/g) = Respiration rate (µmoles O 2 g -1 h -1 ) x 0.456 Where 0.456 J µmole -1 O 2 is the heat equivalent of oxygen uptake.

Energy excreted was calculated from ammonia produced during respiration measurements. Water samples were taken before and after incubation and were filtered (0.45 µm) and then analysed for ammonia concentration according to the phenolhypochlorite method of [START_REF] Solorzano | Determination of ammonia in natural water by the phenolhypochlorite method[END_REF]. Ammonia produced by each mussel was then converted into energy equivalents as follows:

Energy excreted (J/h/g) = Ammonia excretion rate (µmoles NH 3 -N g -1 h -1 ) x 0.349

Where excretion of 1 µmole NH 3 -N h -1 is equivalent to energy loss of 0.349 J h -1 .

Finally SFG was calculated as: SFG = Energy absorbed -(Energy respired + Energy excreted)

Metal uptake in isolated gills

For each temperature treatment, three groups of gills consisting of 14 -15 replicates were obtained from fully temperature acclimated mussels. After extraction, gills from one group were rinsed in clean seawater and individually incubated for 1-h in 300 ml of clean seawater (controls). The second and third groups were incubated under the same conditions and at 0.5 µM single and metal mixture solutions, respectively.

After incubation gills were washed three times in 300 ml clean seawater and then in purified water. This procedure was found to be sufficient to remove loosely adsorbed metals. It is generally accepted that strongly adsorbed metals are associated more with internalised than external metal fraction [START_REF] Endo | Cadmium uptake from apical membrane of LLC-PK1 cells via inorganic anion exchanger[END_REF]. After washing gills were placed in pre-weighed polypropylene tubes and freeze -dried at -60 °C during 72-hrs. Dry samples were then digested in 1 ml HNO 3 and 0.05 ml H 2 O 2 as described earlier and reference material and preparation blanks were also included. Metal concentrations in diluted solutions were then analysed with ICP-MS as earlier described.

Modelling bioaccumulation

Metal accumulation in mussels was best fitted by linear regressions while elimination data was best fitted by single exponential elimination model [START_REF] Newman | Quantitative methods in aquatic ecotoxicology[END_REF]): is elimination rate constant (day -1 ) and t is time (days). The data were fitted in the software GraphPad Prism 4.0.

Results

Scope for growth

Figure 1a shows soft tissue dry weights (mean of 14 -20 mussels) at the time of sampling (field) and at the end of the experiment. Since sample sizes were small data were tested for differences using Kruskal-Wallis ANOVA and results showed no significant differences among the four groups. Figure 1b & c shows MCR and respiration rates of mussels after sampling (field) and after acclimation (crossed bars)

and at end of experiment (grey bars). Both data were subjected to Kruskal-Wallis test with Dunn's post-hoc and results showed higher MCR under field conditions and there were significant differences among temperature treatments (as shown in the Figures).

Respiration rate was only significantly lower at 6 °C. Both respiration rate and MCR showed no differences between measurements performed after acclimation and after metal exposures (results not shown for clarity), similar trend was shown for energy excreted (as ammonia) but there were differences among temperature treatments with highest energy excreted at 26 °C (1.26 +0.2 J h -1 g -1 ) followed by 16 °C (0.23 +0.05 J h -1 g -1 ), then field (0.13 +0.03 J h -1 g -1 ) and the least was at 6 °C (0.05 +0.01 J h -1 g -1 ).

Energy excreted contributed less 1 % towards SFG while clearance rate (or food acquisition) contributed most, consistent with literature [START_REF] Sobral | Effects of elevated temperatures on the scope for growth and resistance to air exposure of the clam Ruditapes decussates (L), from Southern Portugal[END_REF][START_REF] Widdows | Practical procedures for the Measurement of Scope for growth[END_REF]. Finally, as shown in Figure 1d, SFG was highest under field conditions, lowest at 26 °C and there was no difference between 6 °C and 16 °C.

Metal accumulation in whole organisms

Figure 2 shows metal concentrations in mussels at sampling (day 0), during metal exposure (day 28 -56) and during depuration (day 56 -84). In order to determine whether acclimation and temperature had effect on metal concentrations, day 0 and day 28 data in all three temperature treatments were subjected to ANOVA. Results showed that overall there were significant differences (p<0.05) as shown in the figure and posthoc test results for specific differences are summarized in Table 2. Metal accumulation increased linearly with time and in order to evaluate the effect of temperature, slopes of the linear lines were compared using F-statistics. Results showed significant differences except for Co concentration whose pooled slope was calculated as 0.046. Post-hoc test (Table 2) showed that slopes increased with increase in temperature for the nonessential metals Cd and Pb while the slope for Cu decreased with temperature.

Metal elimination data were best fitted using single exponential elimination kinetics [START_REF] Newman | Quantitative methods in aquatic ecotoxicology[END_REF] and results showed no effect of temperature (F-statistics, p>0.05) on the elimination rate constants (k e ) with exception of Cu, which showed lower k e at 6 °C (Table 2). Finally net metal accumulation calculated as a difference between preexposure (day 28) and after depuration (day 84) showed that the two essential metals Co and Cu did not register significant net accumulation (see Figure 2).

Metal uptake in isolated gills

Figure 3 shows metal concentrations in control gills and those exposed to either single or metal mixtures at three test temperatures. The data were analysed for significant differences among the groups using Kruskal-Wallis ANOVA with post-hoc test. For all four metals, uptakes in gills exposed to metals were higher than the controls, except for Cu 6 °C. For the purpose of clarity only post-hoc test results comparing uptake from single metal and metal mixtures are shown (p-values on top of bar graphs).

As shown in Figure 3, uptake from single metal solutions were consistently higher than from metal mixtures, except in a few cases i.e. Co at 6 °C and 16 °C, Cu at 16 °C and 26

°C and Pb at 6 °C.

Inserted graphs in Figure 3 show metal uptakes in gills exposed to single metal solutions. The values were calculated as averages (+SD) of the differences between mean controls and concentration in individual gills. The data were subjected to Kruskal-Wallis ANOVA and results as shown in Table 3 indicated significant differences, confirming that uptake was higher at higher temperature.

Discussion

The study showed that with exception of Cu, tissue concentrations decreased during acclimation as was expected due to low background levels of metals in the acclimation water. However, the decreases appeared to depend on temperature of acclimation, with animals in the lowest temperature losing most metals. This effect was in contrast with later results during elimination phase where no effects of temperature were observed with the exception of copper (Figure 2 & Table 2). Therefore, temperature-effects during acclimation appeared to be an initial response, which disappeared once animals were fully acclimated and had been exposed to higher metal concentrations. It is not clear what kinds of processes are responsible for such responses. However, it is reasonable to assume that some physiological and biochemical changes related to temperature acclimation may be somehow responsible for influencing metal loss during the early stages of acclimation. These types of effects may be relevant for inter-tidal organisms such as mussels, which are often exposed to temperature fluctuations during tidal cycle.

The general lack of temperature effects on elimination especially for non-essential metals Cd and Pb is consistent with some studies on marine bivalves [START_REF] Amiard | Contribution to the ecotoxicological study of cadmium, lead, copper and zinc in the mussel Mytilus edulis: I. Field study[END_REF][START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF][START_REF] Hutchins | Effects of arctic temperature on distribution and retention of the nuclear waste radionuclides 241 AM, 57 Co, and 137 Cs in the bioindicator bivalve Macoma balthica[END_REF], echinoderms [START_REF] Hutchins | Temperature effects on uptake and retention of contaminant radionuclides and trace metals by the brittle star Ophiothrix fragilis[END_REF] and Japanese eel [START_REF] Yang | Uptake and elimination of cadmium by Japanese eel, Anguilla japonica, at various temperatures[END_REF]. In field studies seasonal variations in tissue metal concentrations may to some extent imply temperature effects. However, many studies seem to indicate that most of the seasonal variability may well be due to changes in animal body weight, since total body burdens remained largely unchanged [START_REF] Cossa | Sexual maturation as a source of variation in the relationship between cadmium concentration and body weight of Mytilus edulis L[END_REF][START_REF] Amiard | Contribution to the ecotoxicological study of cadmium, lead, copper and zinc in the mussel Mytilus edulis: I. Field study[END_REF][START_REF] Mubiana | Temporal and spatial trends in heavy metal concentrations in the marine mussel Mytilus edulis from the Western Scheldt estuary (The Netherlands)[END_REF]. However, size related factors (i.e. growth rate, age and body size), can still significantly influence not only concentrations but can also affect bioaccumulation of metals in marine mussels (Mubiana, Vercauteren, & Blust (in press). Therefore, changes in body weight during long-term exposure experiments can have an influence on accumulated metals.

However, in this particular study, differences in tissues weights among the treatments before and after exposures were not significantly different (Fig 1a). Therefore, change in tissue weight could not have been a significant factor responsible for the observed metal accumulation patterns, other factors must be evoked.

As demonstrated in this study and evidence from literature, it seems more likely that in the long-term, temperature has little or no influence on elimination of most metals, except perhaps for some essential metals such as Cu. In the case of Co, the lack of temperature-effects on both accumulation and elimination in whole organisms despite clear temperature effects at isolated gills level is further indication of physiological regulation. It is well known that most organisms are capable of controlling internal concentrations of essential metals up to a certain threshold level [START_REF] Rainbow | Ecophysiology of trace metal uptake in crustaceans[END_REF][START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF].

Animals regulate metal concentrations either in response to external metals exposures and /or in response to environmental factors including temperature or due to attainment of critical body levels [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF].

Lack of clear evidence for the effect of temperature on the elimination of metals is surprising since temperature has such a profound effect on physiology and metabolism of marine mussels [START_REF] Hawkins | Physiological interrelations, and the regulation of production[END_REF][START_REF] Dame | Ecology of marine bivalves an ecosystem approach[END_REF]. Furthermore, in almost all aquatic invertebrates many essential metals are known to play a variety of important roles in physiological and metabolic processes [START_REF] Rainbow | Ecophysiology of trace metal uptake in crustaceans[END_REF][START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF]. [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF] described different types of metal accumulation patterns in invertebrates. It is clear that the fate of metabolically available metals in an organism can involve direct excretion or storage in a detoxified form prior to excretion. The role of changes in physiology in all these processes leading to metal excretion is still not well understood in many organisms. Therefore, the general role (or lack of involvement) of physiology and metabolism on metal elimination process deserves further investigation. Unlike elimination, there is a bulk of literature showing evidence for temperature effect on metal uptake, accumulation and toxicity (i.e. [START_REF] Mclusky | The effects of temperature and salinity on the toxicity of heavy metals to marine and estuarine invertebrates[END_REF][START_REF] Blust | Effect of temperature on the uptake of copper by the brine shrimp, Artemia fransiscana[END_REF][START_REF] Hutchins | Temperature effects on uptake and retention of contaminant radionuclides and trace metals by the brittle star Ophiothrix fragilis[END_REF][START_REF] Hutchins | Effects of arctic temperature on distribution and retention of the nuclear waste radionuclides 241 AM, 57 Co, and 137 Cs in the bioindicator bivalve Macoma balthica[END_REF][START_REF] Yang | Uptake and elimination of cadmium by Japanese eel, Anguilla japonica, at various temperatures[END_REF].

In seawater, temperature influences a variety of physiochemical factors and the important ones include changes in pH and chemical speciation of metals [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF]. As shown in Figure 4 and consistent with literature, temperature showed little effect on metal speciation, particularly on the free metal ion activity, which is the most bioavailable form for most metals and organisms [START_REF] Campbell | Interactions between Trace Metals and Aquatic Organisms: A Critique of the Free-ion Activity Model[END_REF][START_REF] Hudson | Which aqueous species control the rates of trace metal uptake by aquatic biota? Observations and predictions of non-equilibrium effects[END_REF].

Generally, increase in temperature results in the increase in free metal ion activity and a few other metal species like carbonates. Some studies have indicated that besides free metal ions, other metal forms such as OH -and CO 3 complexes can somehow be bioavailable to organisms [START_REF] Hudson | Which aqueous species control the rates of trace metal uptake by aquatic biota? Observations and predictions of non-equilibrium effects[END_REF][START_REF] Laporte | Combined effects of water pH and salinity on the bioaccumulation of inorganic mercury and metheylmercury in the shore crab Carcinus maenas[END_REF].

Therefore, although temperature-effects on metal speciation were generally modest at least within environmentally relevant temperature ranges, nevertheless, it was expected that increase in temperature would generally favour higher uptake. Besides speciation effects, changes of most physiochemical factors such as chemical reaction and diffusion rates would favour higher uptake with increase in temperature [START_REF] Mclusky | The effects of temperature and salinity on the toxicity of heavy metals to marine and estuarine invertebrates[END_REF][START_REF] Blust | Effect of temperature on the uptake of copper by the brine shrimp, Artemia fransiscana[END_REF][START_REF] Bervoets | Effects of temperature on Cadmium and Zinc uptake by the Midge Larvae Chironomus riparius[END_REF][START_REF] Chowdhury | Effect of temperature on the uptake of waterborne strontium in the common carp, Cyprinus carpio (L)[END_REF]). In the case of pH, since it is usually positively correlated with temperature [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF], increase in temperature was expected to decrease metal uptake. However, since seawater has high buffering capacity, only moderate changes in pH between 6 -26 °C were observed (Table 1). On this basis pH was not expected to be a major factor determining metal uptake or accumulation in this study.

The range of SFG determined in this study is comparable with literature concerning M. edulis. As shown in Figure 1, clearance rate of about 7 L g -1 h -1 corresponding to SFG of nearly 14 J g -1 h -1 measured under field conditions, which are within a typical range for mussels from clean environment (i.e. [START_REF] Widdows | Practical procedures for the Measurement of Scope for growth[END_REF][START_REF] Smaal | The scope for growth of bivalves as an integrated response parameter in biological monitoring[END_REF]. SFG measurements representing field conditions (day 0) are consistent with the fact that Eastern Scheldt is well known for its favourable conditions for shellfish farming in The Netherlands. However, after laboratory acclimation SFG decreased almost by half at 6 °C and 16 °C. This decrease was mainly due to low clearance rate, which is not surprising considering the obvious differences between field and laboratory conditions in terms of food quality and quality. Negative SFG at 26 °C implied that animals were utilizing energy reserves, mainly because high temperature seriously inhibited clearance rate. As demonstrated in other studies (i.e. [START_REF] Sobral | Effects of elevated temperatures on the scope for growth and resistance to air exposure of the clam Ruditapes decussates (L), from Southern Portugal[END_REF][START_REF] Smaal | The scope for growth of bivalves as an integrated response parameter in biological monitoring[END_REF] clearance rate (or food acquisition) is the most sensitive parameter for SFG. The study showed that apart from the initial drop in SFG during acclimation, temperature only influenced SFG at the highest temperature while there was no significant difference between 6 -16 °C. This is consistent with expectations, temperate Mytilus spp. are well known for their ability to tolerate very low temperatures (<10 °C) whereas very high temperatures (generally >22 °C) can have very serious detrimental effects on mussels [START_REF] Seed | Population and community ecology of Mytilus[END_REF][START_REF] Dame | Ecology of marine bivalves an ecosystem approach[END_REF]. In this respect, the study has shown that in terms of energy budgets, mussels are physiologically more tolerant to very low temperatures. The study has also shown that SFG can be a useful tool to determine stress from high temperature exposures such as occasionally experienced in the summer periods.

SFG is essentially an integration of many complex physiological processes some of which can be directly or indirectly linked to metal uptake or accumulation in mussels.

For example, change in clearance rate has the potential to affect exposure to dissolved metals, but the nature of the relationship is not yet clear. Theoretically, increased clearance rate implies increased contact with waterborne metals and thus enhanced uptake and accumulation. However, [START_REF] Blackmore | Inter-population differences in Cd, Cr, Se, and Zn accumulation by the green mussel Perna viridis acclimated at different salinities[END_REF] observed low uptake among mussels with high filtration rates, though those results may have been influenced by other factors. Respiration rate also has the potential to affect metal content of mussels, [START_REF] Wang | Metal and oxygen uptake in the green mussel Perna viridis under different metabolic conditions[END_REF] found uptake of Cd and Zn to decrease with decrease in respiration rate. In this study, none of the SFG parameters could be directly correlated with observed metal uptake or accumulation among the temperature treatments. This does not in anyway exclude effects of SFG on metal accumulation, what the results highlight is the fact that temperature related physiological responses are complex and many of them can directly or indirectly influence metal uptake and accumulation.

Further research is required especially including wide differences in terms of SFG, in order to better assess the impact or lack of SFG on metal accumulation in mussels.

The study has shown that when considered at the solution-organism interface such as isolated gills, temperature effects on both essential and non-essential metals is largely controlled by solution chemistry and physical kinetics, which tend to favour higher uptake with increase in temperature [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF][START_REF] Blust | Effect of temperature on the uptake of copper by the brine shrimp, Artemia fransiscana[END_REF][START_REF] Simkiss | Transport of metals across membranes[END_REF]. However, at whole organism level, complex physiological responses tend to mask the relationship, particularly for biologically essential metals like copper, which are subject to regulation over a certain range and conditions [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF][START_REF] Amiard | Contribution to the ecotoxicological study of cadmium, lead, copper and zinc in the mussel Mytilus edulis: I. Field study[END_REF][START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF].

Concentrations of metals (µg/g dry weight) in mussels as a function of time and fitted regressions (see text for details). Also shown are ANOVA results for effect of acclimation on metal concentration and results of F-statistic for effects of temperature on uptake slopes and elimination rate constants (k e values).

Figure 3

Metal uptake in isolated gills and the effect of temperature on gills exposed to single metal (shaded bars) and to metal mixture (crossed bars). Indicated p-values are Kruskal-Wallis results comparing uptake in gills exposed to single and metal mixtures. Inserted graphs show net uptake in gills exposed to single metals (see Table 3 for statistical analysis).

Figure 4

The effect of temperature on metal speciation (percent activities) in chemically defined seawater with pH of 7.8 (at 6 °C), 7.9 (at 16 °C) and 8.0 (at 26 °C). Chemical speciation modelling was performed using the software Visual MINTEQ 2.32.

  metal concentration (µg/g) in soft tissues, a and b are model estimates, k e

  Figure 1

Table 1

 1

	Experimental	Average dissolved metals in µg/l (+ SD)	[O 2 ]	pH
	temperature	Cd	Co	Cu	Pb	(mg/l)
	6 °C	3.3 (0.2)	2.9 (0.2)	4.0 (0.3)	2.2 (0.1) 10.4 (0.7) 7.8 (0.3)
	16 °C	3.1 (0.1)	3.0 (0.2)	4.1 (0.3)	2.3 (0.2)	9.3 (0.8) 7.9 (0.2)
	26 °C	3.2 (0.3)	2.9 (0.1)	4.2 (0.2)	2.3 (0.1)	7.4 (1.0) 8.0 (0.3)

Acknowledgements

We are very thankful to the Belgian Federal Office for Science, Technical and Cultural Affairs (OSTC) who partially financed this work through the CALMARS project contract DWTC-EV/05/04C. We are also thankful to our former post-graduate student Mr. Richard Juma for his contribution in the preliminary experiments.

List of Tables

 andFigures Table 1Mean (+SD) concentrations of dissolved metals (µg/l) measured during 28 days of exposure. The table also shows average dissolved oxygen and pH over the same period.

Table 2

Summary of ANOVA for the effect of temperature on tissue metal concentrations before and after acclimation. The second and third columns show uptake slopes and elimination rate constants (k e values), respectively. Marked * denotes p<0.05, ns p> 0.05 and superscripts a, b & c refers to 6 °C, 16 °C and 26 °C, respectively.

Table 3

Kruskal-Wallis test results for differences among temperature treatments for the net uptake of Cd, Co, Cu and Pb in isolated gills of mussels.


Ceccherelli Giulia°

Campo Davide^

Milazzo Marco

^i C R A M Struttura

SHORT-TERM RESPONSE OF THE SLOW GROWING SEAGRASS POSIDONIA OCEANICA TO SIMULATED ANCHOR IMPACT

Keywords: anchoring, clonal integration, disturbance, Posidonia oceanica, seagrass

Experimental evaluations about the impact of anchors of small vessels have previously shown that each anchoring can on average damage up to 6 shoots of Posidonia oceanica, removing small amount of biomass and, at the same time, interrupting continuity among shoots. The aim of the paper was to investigate the response of P. oceanica to different damage intensity at two levels of substrata compactness. Three treatments were considered: Control (no damage); Low damage (simulated anchor damage by 3 strokes of a hoe); High damage (6 strokes). Disturbance was higher where the substratum was highly penetrable and after one year significant variation was observed among treatments for both the number of leaves per shoot and shoot density.

Conversely, the number of leaves per shoot by the end of the study was similar among all treatment combinations, suggesting that this was the only phenological feature that recovered, and probably the only result that through clonal integration could be achieved. These data strongly highlighted the role that anchoring might have on the slow growing seagrass P. oceanica meadow.

INTRODUCTION

World-wide seagrass loss has been reported from a variety of both direct and indirect human-induced influences [START_REF] Hemminga | Seagrass Ecology[END_REF]. Among all the causes, some are responsible for large scale seagrass decline, such as eutrophication, industrial runoff, oil spillage, dredging (e.g. [START_REF] Short | Comparison of a current disease to the wasting disease in the 1930's[END_REF][START_REF] Preen | Flood and cyclone related loss, and partial recovery, of more than 1,000 km 2 of seagrass in Hervey Bay, Queensland, Australia[END_REF]. Conversely, propellers and anchors only cause small gaps within the seagrass meadow, by removal of plant biomass depending on anchor size and type [START_REF] Porcher | Impact de mouillages forains sur le herbiers à Posidonia oceanica[END_REF][START_REF] Walker | Effect of boat moorings on sea beds near Perth, Western Australia[END_REF][START_REF] Creed | Disturbance and recovery of the macroflora of a seagrass (Halodule wrightii Ascherson) meadow in the Abrolhos Marine National Park, Brazil: an experimental evaluation of anchor damage[END_REF][START_REF] Rasheed | Recovery of experimentally created gaps within a tropical Zostera capricorni (Aschers.) seagrass meadow, Queensland Australia[END_REF][START_REF] Francour | Effects of boat anchoring in Posidonia oceanica seagrass beds in the Port-Cros National Park (north-western Mediterranean Sea)[END_REF][START_REF] Milazzo | Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, Western Mediterranean): effect of anchor types in different anchoring stages[END_REF].

Because loss of seagrasses within meadows has been quite well documented, information about the recovery capacity of seagrasses to all forms of disturbance have become particularly important, so that proper evaluations on the effective impact could be done. For example, seagrass recovery to physical damage was quite unknown until recently. Overall, recovery seems to be mostly due to vegetative propagation and, therefore, recovery time seems highly variable, being dependent upon species growth rate.

For example, [START_REF] Creed | Disturbance and recovery of the macroflora of a seagrass (Halodule wrightii Ascherson) meadow in the Abrolhos Marine National Park, Brazil: an experimental evaluation of anchor damage[END_REF] found that Halodule wrightii (Ascherson) recovered from 0.25 m 2 gaps in 9 months, while [START_REF] Rasheed | Recovery of experimentally created gaps within a tropical Zostera capricorni (Aschers.) seagrass meadow, Queensland Australia[END_REF] found Zostera capricorni (Ascherson) recovered after 12 months from the same clearing plot size.

However, data for seagrass recovery are particularly needed for slow growing species [START_REF] Meehan | Recovery times for a damaged Posidonia australis bed in south eastern Australia[END_REF]. [START_REF] Zieman | The ecological effects of physical damage from motor boats on turtle grass beds in southern Florida[END_REF] and [START_REF] Williams | Thalassia testudinum productivity and grazing by green turtles in a highly disturbed seagrass bed[END_REF] provided data for recovery of Thalassia testudinum (Banks ex Konig) which seems a much slower recovery species with only minimal colonisation of anchor scars occurring after 7 months. A recent paper by Gonzàlez-Correa, Bayle, Sànchez-Lizaso, Valle, Sàanchez-Jerez & Ruiz (2005) has highlighted the recovery capacity of Posidonia oceanica (L., Delile) to illegal otter trawling, although the performance of this seagrass seems much faster at control sites where meadows are much denser and resuspension of sediments was lower. Other predictions on the recovery rate to mechanical disturbances could only be done indirectly on the basis of data on population dynamics, seasonal and annual growth, and productivity of all seagrasses (e.g. Marbà, Cebriàn, Enriquez & Duarte, 1996;[START_REF] Marbà | Growth and population dynamics of Posidonia oceanica on the Spanish Mediterranean coast: elucidating seagrass decline[END_REF][START_REF] Vermaat | Meadow maintenance, growth and productivity of a mixed Philippine seagrass bed[END_REF][START_REF] Pergent-Martini | Primary production of Posidonia oceanica in the Mediterranean Basin[END_REF][START_REF] Alcoverro | Annual growth dynamics of Posidonia oceanica: contribution of large-scale versus local factors to seasonality[END_REF]Marbà & Duarte, 1997).

Due to the spatial extent of Posidonia oceanica meadows and its high productivity, it is a major component of the Mediterranean coastal system [START_REF] Buia | Primary production and growth dynamics in Posidonia oceanica[END_REF][START_REF] Pergent | Fate and production in Posidonia oceanica meadows of the Mediterranean[END_REF], where it plays a very important role sheltering understoried and speciose assemblages (e.g. [START_REF] Macpherson | Mortality of juvenile fishes of the genus Diplodus in protected and unprotected areas in the western Mediterranean Sea[END_REF][START_REF] Guidetti | Differences among fish assemblages associated with nearshore Posidonia oceanica seagrass beds, rocky-algal reefs and unvegetated sand habitats in Adriatic Sea[END_REF]. P. oceanica ranks among the longest-lived seagrasses [START_REF] Duarte | Variance and the description of nature[END_REF] exhibiting very slow growth rate [START_REF] Cebrian | Magnitude and fate of the production of four co-occurring Western Mediterranean seagrass species[END_REF].

Experimental evaluations of the impact of anchors have suggested that Posidonia oceanica is differently affected by anchors depending on the anchor model used (4 kg in weight) [START_REF] Milazzo | Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, Western Mediterranean): effect of anchor types in different anchoring stages[END_REF]. In particular, the use of the Hall type anchor has a lower impact than Danforth and Folding grapnel anchors. Each complete anchoring cycle (e.g. anchor fall, dragging/lock-in and weighing) can uproot or break on average up to 6 shoots and create discontinuity among shoots within the meadows.

In this paper we examine the short-term response of Posidonia oceanica to simulated anchoring. By experimentally scarring the meadow we reproduced the damage provoked by a Folding grapnel anchor, which was found to be the highest impacting anchor [START_REF] Milazzo | Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, Western Mediterranean): effect of anchor types in different anchoring stages[END_REF]. The aim of the paper was to estimate P. oceanica changes in above ground biomass after being damaged: the response to different damage intensity at two levels of substrata compactness was evaluated.

Substratum compactness, and thus penetration, was evaluated as a possible important source of variation in damage intensity. In particular, the lower the compactness the higher is the probability expected for rhizomes to be uprooted. The higher the compactness the lower should be the number of shoots uprooted.

MATERIALS AND METHODS

The study was carried out in a shallow (2 m depth) Posidonia oceanica meadow located at Stagnone, (37° 52' 32"N and 12° 26' 17"E) along the western coast of Sicily, which is a particularly sheltered and little frequented site. Within this meadow 12 areas (about 3 × 3 m in size) were chosen so that 6 were on substratum highly penetrable and the other 6 on substratum which was less penetrable. Penetration of the substratum was quantified by using a penetrometer such as that used by [START_REF] Francour | Effects of boat anchoring in Posidonia oceanica seagrass beds in the Port-Cros National Park (north-western Mediterranean Sea)[END_REF]. A 2 m long, 8 mm diameter rod was placed perpendicularly to the bottom and a 5 kg weight was dropped 50 cm on to a stop in the middle of the rod. The impact of the weight hitting the stop provided a constant force to drive the rod into the rhizome mat. This measurement was repeated randomly at the site 20 times to define areas of different compactness. Weak substratum compactness refers to a mean penetration depth of 26.6 cm (±2.1 SD), while high compactness refers to a mean penetration of 9.2 cm (±1.9 SD). At the high penetration areas a higher shoot density and proportion of orthotropic shoots was found, while at low penetration areas a higher sediment accumulation was observed.

In each of these areas 3 replicated experimental units (40 × 40 cm in size) were randomly selected and marked at the corners with plastic stakes (35 cm in length). Within these areas anchor damage was experimentally simulated by using a garden hoe. It was impractical to use a real anchor because it was not possible to throw an anchor several times in the same experimental area. The three treatments considered are: Control (no simulated anchor damage); Low damage (3 strokes); High damage (6 strokes). Strokes were given as simulation of the damage caused by folding grapnel anchor [START_REF] Milazzo | Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, Western Mediterranean): effect of anchor types in different anchoring stages[END_REF]; the hoe was drawn through the sediment face on so that it ploughed like an anchor. The three treatments were randomly assigned to the six areas within each compactness group, so that two areas belonged to the same compactness × damage combination of treatments. All the 36 experimental units established occurred within a 120 m 2 area.

Damage treatments were carried out on December 2000 and recovery after damage was evaluated by sampling every month until November 2001, as we believed that it was a feasible time period to detect a response of the plant. The response variables considered were plant leaf number per shoot, leaf length and shoot density, as they are the common phenological variables to evaluating the aboveground biomass performance of this seagrass. They were all evaluated in the field: shoot density was estimated by counting the number of shoots in each experimental unit, while leaf length (the length of the longest leaf in the shoot) and leaf number (adult, intermediate and juvenile leaves in the shoot) were estimated by choosing two shoots at random within each unit.

A multifactorial analysis of variance was performed on data taken at the last sampling time for each response variable. 'Penetration' (2 levels) and 'damage' (3 levels) were treated as fixed and orthogonal, while 'area' (2 levels) as random nested in their interaction. For leaf length and number 'experimental unit' (3 levels) was also considered and treated as random nested in area. Cochran's test was performed for all analyses to check for homogeneity of variances. Post-hoc multiple comparisons were done using SNK test [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF].

RESULTS AND DISCUSSION

Estimation in the field of the biomass of Posidonia oceanica removed or broken due to strokes of the hoe, because it was not possible to collect fragments of leaves in the water column. However, the impact on shoot density was estimated by counting the shoots in each unit before and right after damaging: at the low and high damage treatment 11.16 (±1.44) and 13.80 (±2.24), 7.00 (±0.68) and 11.3 (±1.33) were the mean number of shoots (± SE) removed per plot, at weak and high compactness, respectively (Fig. 1). The mean number of shoots removed in the experiment at low damage and weak compactness was equivalent to about the effect of one anchoring cycle, while in the other three combinations of treatment the mean number of shoots removed was equivalent to about two anchoring cycles.

Each response variable analysed in this study has given a specific result to the experimental damage of the plants. For example, after one year, leaf number per shoot was not affected by damaging (Fig 2a andb, Table 1). Although differences among treatments were evident after a few months, especially at high penetration of substratum (Fig 2b), analysis performed using November data did not detect significant differences. The patterns found suggest that plant recovery in leaf number per shoot occurred during the study period.

Conversely, leaf length was still affected by experimental damage even after one year (Table 1 and Fig 2c andd). In fact, analysis of data has detected that the shortest size was found at a high level of damage, while leaf length in control and medium damaged plots were similar (SNK test, Table 1). Although no analysis has been performed on previous sampling times, differences among treatments could have changed through time being more evident in the latter period at low penetration condition (Fig. 2c). Evident temporal variations were found through the study period at both types of substratum, according to patterns already described extensively [START_REF] Pergent-Martini | Primary production of Posidonia oceanica in the Mediterranean Basin[END_REF][START_REF] Alcoverro | Annual growth dynamics of Posidonia oceanica: contribution of large-scale versus local factors to seasonality[END_REF]Marbà, Cebriàn, Enriquez & Duarte, 1996;Cebriàn, Duarte, Marbà & Enrìquez, 1997).

Overall, among the three response variables, P. oceanica shoot density was the most affected by experimental damaging especially where the substratum was highly penetrable (Table 1 and Fig 2e andf). A significant interaction between substratum penetration and damage intensity was found. In fact, at low penetration of substratum, high and medium levels of damage had a similar effect on shoot density but one year after the damage were similar to the controls. Conversely, at high penetration three very different shoot densities occurred among levels of damage intensity. Possibly, where substratum is highly compact (low penetration) P. oceanica was better able to resist the simulated anchor strokes and a lower number of shoots were uprooted, compared to the low compactness (high penetration) areas. This could be corroborated by the similarity in mean shoot density among levels of damage at low penetration, rather that at high penetration, in December 2000. In fact, data taken before and right after damaging estimated mean values of 11.16 and 7.0 shoots removed where damage was low, while 13.83 and 11.33 where damage was high, at high and low compactness, respectively (Fig. 1). However, although at high compactness the initial shoot density was apparently very similar among the three treatments, it was very evident that through time differences among them increased.

Although the hoeing of low penetration substrate could not uproot but only initially damage shoots, the detachment of shoots occurred later over a period of three months.

Nevertheless, this finding might indicate a different impact of simulated anchors on

Posidonia oceanica shoot density depending on the substratum penetration. Unfortunately, the effect of Time×Damage×Substratum penetration interaction could not be estimated because the assumption of independence of data by time would have not been met. However, graphical inspection could easily bring further evidence to the model that differences found at the end of the study are basically due to the different impact and not to a different recovery pattern. We believe that different features that are likely to occur at the two substratum types, such as sediment mobility and nutrient availability for roots, did not affect recovery but are merely affecting natural spatial variability of P. oceanica shoot density independently of the impact (e.g. [START_REF] Balestri | Spatial variation in Posidonia oceanica structural, morphological and dynamic features in a northwestern Mediterranean coastal area: a multi-scale analysis[END_REF].

In summary, damage caused by folding grapnel anchors, although considered small scale damage, can create an impact on Posidonia oceanica, particularly to shoot density, from which it is very difficult to recover in the short-term. This result was not unexpected given the slow pattern of growth demonstrated in several studies for this species [START_REF] Pergent-Martini | Primary production of Posidonia oceanica in the Mediterranean Basin[END_REF][START_REF] Alcoverro | Annual growth dynamics of Posidonia oceanica: contribution of large-scale versus local factors to seasonality[END_REF][START_REF] Vermaat | Meadow maintenance, growth and productivity of a mixed Philippine seagrass bed[END_REF]Marbà, Cebriàn, Enriquez & Duarte, 1996;[START_REF] Marbà | Growth and population dynamics of Posidonia oceanica on the Spanish Mediterranean coast: elucidating seagrass decline[END_REF][START_REF] Marbà | Rhizome elongation and seagrass clonal growth[END_REF].

Moreover, as the holes created by the anchors and other impacts can take a long time to be re-colonised, the possible increase in sediment mobility due to holes could be a further indirect cause of disturbance affecting recovery through time. Conversely, the most interesting result could be that the number of leaves per shoot was the only response variable recovered within one year, probably due to resource reallocation through modules.

Overall, this gives good estimates on the quantitative and qualitative short-term response of P. oceanica suggesting that biomass allocation after a stress is addressed in terms of the number of leaves per shoot.

Consideration should be given to the possible influence of clonal integration, among shoots of Posidonia oceanica. Indeed, anchor damage may be responsible for module discontinuity, through breakage of rhizomes, besides biomass removal, through both uprooting shoots and breakage of leaves (apex up to portions of several centimetres in length). For the findings about the importance of nutrient mobility through modules collected for P. oceanica and other seagrasses (e.g. [START_REF] Libes | Uptake and long-distance transport of carbon in the marine phanerogam Posidonia oceanica[END_REF][START_REF] Nielsen | Growth, photosynthesis and nutrient content of seedlings and mature plants of Cymodocea nodosa-the importance of clonal integration[END_REF][START_REF] Marbà | Carbon and nitrogen translocation between seagrass ramets[END_REF] and stressed by [START_REF] Kendrick | Clonality in seagrasses, emergent properties and seagrass landscape[END_REF], module interruption could deplete efforts from neighbouring shoots of the same clone. So, whether interruption among shoots occurs, leaf production rate could be decreased and not be able to cope with the leaves' removal within 1 year. This could be the reason for the low leaf length of shoots at highly impacted plots, even at the end of the study.

Unfortunately, in this experiment, treatments have been attributed to several areas within a P. oceanica meadow regardless of clonality, and whether shoots occurring in the same plot belong to the same clone remains unknown. Also, identification of clone dimension at the study site could help to interpret the very high recruitment rate of shoots (e.g. [START_REF] Vermaat | Meadow maintenance, growth and productivity of a mixed Philippine seagrass bed[END_REF]Marbà, Cebriàn, Enriquez & Duarte, 1996) observed at controls. This finding could lead to the possibility that biomass removal of a clonal individual could off set by an increase growth in other 'communicating' portions. However, only properly designed experiments could investigate the influence of clonality on recovery from damage, estimating the distances to which integration among shoots could occur in the field.

Overall, the results obtained within this study strongly highlighted the role that anchor activity, even by small vessels, might have on P. oceanica meadow representing a small-scale but affective cause of degradation. The frequency of anchoring at some visited sites, in terms of number of boats and extension of the visitation period, could make this chronic disturbance very stressful to the growth of the seagrass over several years.

ACKNOWLEDGEMENT

This work was in part supported by PADI AWARE.

Legends

Fig. 1 Posidonia oceanica. Mean (± SE) shoot density (n=6) before and after hoeing at low and high substratum penetrability for control, medium and high damage.

Fig. 2

 2 Fig. 2 Posidonia oceanica. Temporal changes in mean (± SE) number per shoot (n=12),

  Fig 1:

Fig 2:

  Fig 2:

Table 1

 1 Analysis of variance on P. oceanica shoot density, longest leaf length and leaf number per shoot with two different types of substratum penetration (high vs. low) one year after performing damage intensity (control, medium and high) at the two experimental areas. Shoot density was estimated in each experimental units (n=3), while leaf length and number were estimated in duplicate in each experimental unit (n=2). on the interaction term P × D (Table1) for Posidonia oceanica shoot density:(i) low (Lp) and high (Hp) substratum penetration; (ii) damage intensity, control, medium and high. SE=3.04 df=24.

					shoot density		leaf length			leaf number
	Source of variation		df	MS	F	P	df	MS	F	P	MS	F	P
	Penetration	=P	1	342.2			1	2.07	1.86	0.2213	0.34	0.41	0.5457
	Damage	=D	2 1667.6			2	12.68	11.43	0.0090	0.59	0.70	0.5309
	P × D		2	299.2	5.32	0.0166* 2	0.31	0.28	0.7674	0.26	0.31	0.7435
	Area (P × D)	=A	6	68.1	1.23	0.3270 6	1.11	0.16	0.9838	0.84	2.18	0.0809
	Ex.unit.(A(P × D))		24				24	6.73	1.45	0.1511	0.39	0.34	0.9966
	Residual		36	55.5			36	4.63			1.15
	*on Residual
	Cochran's test				C=0.3383 ns			C=0.2430 ns			C=0.1084 ns
	Table 1A
	SNK test (i)	Penetration			(ii)		Damage
	Damage					Penetration
	Control		Hp>Lp			Hp	Control>Medium>High
	Medium		Hp>Lp			Lp	Control>Medium=High
	High		Hp=Lp
	SNK test on damage factor for Posidonia oceanica leaf length. SE=0.21 df=6
	Control(31.16)=Medium(30.63)>High(29.72)