Prof. Dr Angela Köhler
email: angela.koehler@awi.de

Bourque, B.J M S H Graham

Gonadal lesions of female sea urchin (Psammechinus miliaris) after 1 exposure to the polycyclic aromatic hydrocarbon phenanthrene 2 3

Keywords: histopathology, gonad, sea urchin, oocyte atresia, phenanthrene, Psammechinus miliaris, 36 lipofuscin 37 38

Histopathological alterations in the reproductive tissue of the female sea urchin 22 Psammechinus miliaris after exposure to the polycyclic aromatic hydrocarbon (PAH) 23 phenanthrene were investigated using light microscopy. Sexually mature sea urchins were 24 exposed to 500 µg L -1 phenanthrene for 20 days. A severe disorganisation of the acinal 25 structure of the gonads was observed in phenanthrene-treated animals, mainly as a result of oocytes were inhibited, whereas there were no obvious effects on vitellogenic oocytes and 28 mature ova. The acinal wall showed signs of fibrosis as well as dilation of the two epithelial 29 layers. Phenanthrene severely affected the reproductive function of the sea urchins since a 30 proper oogenesis of the ovarian tissue is very unlikely. Generally, the observed alterations 31 are similar to histopathological changes in reproductive tissue in other invertebrates after 32 exposure to PAHs and oil.

after experimental exposure to phenol and cadmium were investigated by electron 83 microscopy [START_REF] Au | Reproductive impairment of sea urchin upon 487 chronic exposure to cadmium. Part II: Effects on sperm development[END_REF][START_REF] Au | Sublethal effects of phenol on 495 spermatogenesis in sea urchins (Anthocidaris crassispina)[END_REF]. [START_REF] Aarab | Biochemical and histological responses in mussel (Mytilus edulis) 476 exposed to North Sea oil and to a mixture of North Sea oil and alkylphenols[END_REF]Littorina littorea -Cajaraville et al., 1990;mammals -Sakamaki, 92 2003). Degeneration and absorption of oocytes, so called oocyte atresia, is a normal 93 physiologic event which may become pathologic after chemical exposure [START_REF] Blazer | Histopathological assessment of gonadal tissue in wild fishes[END_REF] 94 Dietrich et al., in press). Furthermore, contamination with PAHs has been shown to be 95 associated with an increased accumulation of lipofuscin in marine organisms (Krishnakumar et al., 1994;Krishnakumar et al., 1997;[START_REF] Au | Relationship between ultrastructural 491 changes and EROD activities in liver of fish exposed to Benzo[a]pyrene[END_REF][START_REF] Au | The application of histo-cytopathological biomarkers in marine pollution 480 monitoring: a review[END_REF]. Lipofuscin, also known as 97 age pigment, is widely regarded as end product of protein and lipid peroxidation due to 98 oxidative stress [START_REF] Au | Relationship between ultrastructural 491 changes and EROD activities in liver of fish exposed to Benzo[a]pyrene[END_REF][START_REF] Au | The application of histo-cytopathological biomarkers in marine pollution 480 monitoring: a review[END_REF]Terman and Brunk, 2004). In the sea urchin 99

96
Lythechinus variegatus phenanthrene significantly retarded embryo development at 100 environmentally relevant concentrations (Steevens et al., 1999). In Lythechinus anemensis

205

Further quantifications were performed on sections stained with Alcian Blue/PAS method.

206

Aggregates of degenerating oocytes were quantified by applying an eyepiece graticule with a according to the classification of the reproductive stages by [START_REF] Byrne | Annual reproductive cycles of the commercial sea urchin Paracentrotus 513 lividus from an exposed intertidal and a sheltered subtidal habitat on the west coast of 514 Ireland[END_REF] and Kelly (2000).

252

The animals were partly spawned which was observed by empty spaces which had been

253

occupied by oocytes before. At the end of the experiment (day 20) the sea urchins in the

292

We could not detect pathological changes of mature ova and vitellogenic oocytes while 293 previtellogenic oocytes were often degenerating after maternal exposure to phenanthrene.

294

Degenerating oocytes were characterized by fading and vacuolisation of the cytoplasm as

295

well as cell lysis. Further, infoldings of the nuclear membrane of the GV occurred which was 296 followed by disruption of the nuclear membrane (Figure 2 c). The nucleolus appeared to be

302

Degenerating previtellogenic oocytes identified by fading and vacuolisation of the cytoplasm,

303

cell lysis and / or infoldings and disruption of the nuclear membrane were quantified in the oocytes were classified as degenerating based on the above mentioned criteria, whereas in 306 controls 24 % degenerating previtellogenic oocytes were found. In the solvent control group 307 the frequency of degenerating oocytes was quite variable with a few animals exhibiting high 308 amounts of degenerating oocytes. However, differences were only significant between the 309 control group and phenanthrene-treated animals (one-way ANOVA, p = 0.01, Holm-Sidak).

311

As mentioned above, the meshwork of NPs was disrupted after exposure to phenanthrene.

312

Moreover, in phenanthrene-exposed animals the NPs appeared to be more detached from 326

Histological scoring shows that phenanthrene exposure resulted in a significantly increased 327 dilation of the acinal wall (one-way ANOVA, p < 0.001, Holm-Sidak, Figure 3

365

In the Baltic clam (Macoma balthica) necrosis of primary and secondary oocytes as well as 366 ripe ova was reported after 28 days exposure to sediments highly contaminated with PAHs, 367 polychlorinated biphenyls (PCBs), and heavy metals (Tay et al., 2003). Astonishingly, in 368 clams exposed to sediment with a very high concentration of PAHs no ovarian lesions were 369 detected (Tay et al., 2003).

371

In the present study, previtellogenic oocytes showed a reduction in size and vitellogenic

372

oocytes showed a reduction in numbers in P. miliaris after maternal exposure to 373 phenanthrene. It is unclear whether inhibition of oocyte growth and maturation by 374 phenanthrene exposure is the underlying mechanism of these findings. The low number of 375 vitellogenic oocytes may also be a result of spawning induced by exposure to phenanthrene 376 since spawning is known to be accelerated in mussels (Mytilus galloprovincialis) exposed to 377 high doses of oil [START_REF] Cajaraville | Comparative effects of the water 521 accommodated fraction of three oils on mussels -1. Survival, growth and gonad Walker[END_REF]. However, no increased spawning events in the 378 phenanthrene-treated group were observed during the experiment in comparison to controls.

379

Regarding the previtellogenic oocytes, it could be speculated that their small size may, next

401

(2001) also found lipofuscin deposition in NPs and in the hemal sinuses of the acinal walls.

402

However, in contrast to our findings, the authors further identified lipofuscin accumulation in 403 germ cells of S. intermedius (Vaschenko et al., 2001;Vashchenko et al., 2001). Indeed,

404

preliminary studies with a wild population of P. miliaris indicate that lipofuscin accumulation in

405

the reproductive tissue is dependent on the reproductive stage and the sex of the organism

406

(Schäfer & Köhler, unpublished). It has to be further investigated whether lipopigment 407 deposition in reproductive tissue of sea urchins is a useful biomarker for pollutant stress.

409

The NPs showed increased phagocytic activity in the phenanthrene-treated animals. This

412

echinoderms that occurs in both sexes at the end of the spawning season (Kalachev and gamete resorption in the gonads is also reported in the prosobranch Littorina littorea after 416 short-term exposure to high concentrations of the PAH 1-napthol [START_REF] Cajaraville | Short-term toxic effects of 1-naphthol 517 on the digestive gland-gonad complex of the marine prosobranch Littorina littorea (L): A light 518 microscopic study[END_REF].

418

The acinal wall of Psammechinus miliaris was severely affected by phenanthrene exposure

419

showing dilation of the epithelial layers and increased formation of collagen. Further, 420 augmentation of basophilic structures occurred which can not be identified so far. The 421 thickness of the gonadal wall of the sea urchin Arbacia punctulata is reported to change 422 during the year (Palmer Wilson, 1940). Increased deposition of collagen in conjunction with a 423 thickened appearance of connective tissue, however, is generally known as fibrosis (Dietrich et al., in press). In reproductive tissue of invertebrates fibrosis is described from gonadal 425 neoplasms in mussels (Peters et al., 1994). And in the prosobranch Littorina littorea fibrosis 426 was shown after short-term exposure to high concentrations of the PAH 1-napthol 427 [START_REF] Cajaraville | Short-term toxic effects of 1-naphthol 517 on the digestive gland-gonad complex of the marine prosobranch Littorina littorea (L): A light 518 microscopic study[END_REF]. When immune cells are present simultaneously fibrosis is assumed 428 to be a scarring process. In a few studies infiltration with immune cells have been described

424
429 in invertebrates after exposure to oil and PAHs [START_REF] Berthou | The occurrence of 499 hydrocarbons and histopathological abnormalities in oysters for 7 Years Following the Wreck 500 of the Amoco-Cadiz in Brittany (France)[END_REF][START_REF] Cajaraville | Short-term toxic effects of 1-naphthol 517 on the digestive gland-gonad complex of the marine prosobranch Littorina littorea (L): A light 518 microscopic study[END_REF] 430 [START_REF] Cajaraville | Comparative effects of the water 521 accommodated fraction of three oils on mussels -1. Survival, growth and gonad Walker[END_REF]

  (PAH) are lipophilic organic compounds which are 86 ubiquitously found in the marine environment. Phenanthrene is a low molecular weight, 3-87 ring PAH and is classified as a priority pollutant by the United States Environmental 88 Protection Agency (US EPA). It is an important petroleum-source PAH and a major 89 component of the total content of PAH compounds in the marine environment. PAHs are 90 known to induce degeneration of oocytes and ovarian lesions in several species (Mytilus 91 edulis -

101

  phenanthrene disrupted axial development in embryos in a dose-dependent fashion which 102 could be linked to nuclear accumulation of the protein -catenin(Pillai et al., 2003). Pillai et 103 al. (2003) have shown that among five investigated PAHs phenanthrene was most potent in 104 causing exogastrulation in sea urchin embryos. 105 106 The aim of the present study was to give a detailed description of the histopathological 107 alterations of the reproductive tissue of the female sea urchin Psammechinus miliaris after 108 exposure to phenanthrene. The findings are compared with effects of hydrocarbons on the 109 reproductive tissue of other aquatic invertebrates. Changes of the general acinal structure, effects on the different developmental stages of oocytes and the nutritive phagocytes, as well 111 as alterations of the acinal wall are described. Lipofuscin is identified and quantified in the 112 tissue and its usefulness as biomarker of pollutant exposure in the gonads of sea urchins is 113 discussed. Histopathological alterations of the gonads such as aggregates of degenerating 114 oocytes, the percentage of degenerating oocytes, lipofuscin accumulation and dilation of the 115 acinal wall are quantitatively or semi quantitatively assessed. Furthermore, the gonad weight 116 and the gonadosomatic index in the different treatments are compared. collection and experimental set up 120 Adult Psammechinus miliaris were collected with a beam trawl close to the Island of Sylt 121 (Germany) at 55° 02, 40 N and 08° 27, 25 E in May 2006. They were separated by sex and 122 transported to the laboratory in Bremerhaven. In a range finding experiment, sea urchins 123 were exposed to set concentrations of 1, 3, 12, 42, 145 and 500 µg L -1 phenanthrene, to 124 0.01% acetone, as the solvent control, and to sea water only for 10 days. Each treatment 125 consisted of two replicate tanks (glass vessels with a volume of 1.5 L) with 4 sea urchins 126 each. Since no mortality occurred in this preliminary experiment 500 µg L -1 phenanthrene 127 was chosen as sublethal test concentration for the subsequent experiment. Female sea 128 urchins of a minimum size of 20 mm horizontal test diameter were selected and acclimatized 129 to experimental conditions for 4 weeks. Sea urchins were exposed to 500 µg L -1 3.2 Sampling 139 For comparison of growth parameters, 24 samples were taken at the beginning (day 0) and 140 30 replicate samples per treatment were taken at the end of the exposure period (day 20). 141 Animals were weighed and horizontal and vertical test diameters were measured with a 142 calliper. The animals were dissected and the gonads were removed and weighed. The gonad 143 index (GI) was calculated as the wet weight of the gonad divided by the total somatic wet 144 weight of the sea urchin expressed as a percentage.

  ZEISS). Image intensity was measured using grey values from 1 (clear, white) to 255 198 (dark, black). Three black and white images were randomly taken from each duplicate 199 section of gonadal tissue (6 measurements per individual) with a 10x objective. In a special 200 macro individual ovarian acini were marked as 'region of interest'. Lipofuscin granules within 201 the defined acini were identified with the aid of a threshold function which discriminates the 202 darker stained lipofuscin granules from the background. The software computed the areal 203 density of lipofuscin [%] within the marked acinus ('region of interest') and mean values for 204 each individual were calculated.

207220

  100 squares grid (10 x 10) as an overlay. The number of squares lying on the acinus and the 208 number of squares lying on aggregates of degenerating oocytes were counted at 400x 209 magnification. In each duplicate section five areas were randomly chosen (10 measurements 210 per animal). The areal density of oocyte aggregates [%] within the acini was calculated. 211 Dilation of the acinal wall was semi quantitatively assessed. For this purpose, in each sample 212 5 randomly chosen areas were graded according to a scaling scheme from 0 (no change) to 213 2 (severe alteration) at 100 x magnification. Degenerating previtellogenic oocytes identified 214 by fading and vacuolisation of the cytoplasm, cell lysis and disruption of the membrane of the 215 germinal vesicle at 600 x magnification were counted. Oogonia were not considered due to 216 their small size and the resultant difficulty in diagnosis. 100 randomly selected previtellogenic 217 oocytes were counted per sample and the data are presented as a percentage. Residuals were tested for normality and variance homogeneity (Levene's test). Data on 221 gonad weight and gonad index were box cox-transformed and a one-way ANOVA with Tukey At the beginning of the experiment (day 0) test animals (n=10) were in the premature stage 251

  254 three treatments were still, except of one control animal, in the premature stage and partially 255 spawned (Figure 1 a). The one control animal was in the recovery period. 256 257 In phenanthrene-exposed animals, the acinal structure was severely affected: Early 258 developmental stages of oocytes were no longer found close to the acinal wall but spread in 259 the acinal lumen, the meshwork of NPs appeared disrupted and aggregates of degenerating 260 previtellogenic oocytes were found in the acini (Figure 2 a, b). Control animals hardly showed 261 any aggregates of degenerating oocytes while in phenanthrene-exposed animals these 262 aggregates filled large areas of most of the acini. The areal density of these aggregates was 263 significantly increased in phenanthrene-treated animals in comparison to the control and the 264 solvent control groups (one-way ANOVA, p = 0.006, Figure 3 a). Remarkably, less 265 vitellogenic oocytes were found in the gonads of animals exposed to phenanthrene in 266 comparison to controls. 267 268 Lipofuscin identified as irregularly shaped and heterogeneously textured blue granules in 269 sections stained with the Schmorl's technique was found in the acini (Figure 4 a, b). High 270 amounts of lipofuscin were localized in extracellular spaces in the acini and inside of NPs 271 (Figure 4 a, b). In addition, lipofuscin was found in epithelial cells of the acinal wall and in 272 conjunction with immune cells, the so called coelomocytes, outside of the acini (Figure 4 a).273No lipofuscin was observed in oocytes. Indeed, early developmental stages of oocytes can 274 hardly be identified in sections stained with the Schmorl's technique due to their small size 275 and the lack of a counterstain. Lipofuscin deposition in oogonia and previtellogenic oocytes 276 can, therefore, not be excluded. Overall, lipofuscin content was increased but not significantly different in phenanthrene-treated animals (one-way ANOVA, p = 0.068, Figure3on germ and somatic cells 281 In all treatments, oogonia, previtellogenic and vitellogenic oocytes as well as mature ova 282 were found in the gonads. Previtellogenic oocytes are usually localized close to the acinal 283 wall. Morphologically, they are characterized by a large germinal vesicle, a high nucleus to 284 cytoplasmic ratio and their small size in comparison to later developmental stages. Their 285 cytoplasm is stained blue in H&E and Alcian Blue/PAS (Figure 1 b). Vitellogenic oocytes, on 286 the other hand, accumulate PAS+ material in the cytoplasm with ongoing maturation visible 287 as pink granules in the Alcian Blue/PAS stained sections. Vitellogenic oocytes are larger and 288 their nucleus to cytoplasmic ratio is reduced in comparison to previtellogenic oocytes (Figure 289 1 c). They also possess a large germinal vesicle which breaks down to form ripe ova (Figure 290 1 d) accumulating in the acinal lumen of mature animals.

297normal.

  No pyknosis or karyomegaly of the GV was identified. In comparison to control 298 samples, cell size of previtellogenic oocytes seemed to be reduced. Oogonia were not 299 studied in detail due to their small size and the resultant difficulty in diagnosis of cellular 300 alterations.

313

  each other and engaged in phagocytotic activity. No karyomegaly, nuclear polymorphism, or 314 nuclear inclusions were observed in NPs. Yolk was identified as globules of various shades 315 of pink and blue in nutritive phagocytes in the Alcian Blue/PAS stain. is usually composed of an inner and an outer layer of epithelial cells which 319 are characterized by their basophilic nuclei. Depending on the plane of the tissue section at 320 least one additional layer is visible being stained pink with H&E as well as with the Picrosirius 321 method indicating the presence of collagen fibres (Figure 4 c).322 323In phenanthrene-exposed animals the two epithelial layers were often separate from each 324 other (Figure4 d, e). Often this dilation was concomitant with an increased formation of 325 collagen between the epithelial layers and augmentation of basophilic material (Figure4 d).

346

  was mainly due to aggregates of degenerating oocytes. This disorganisation makes a proper 347 oogenesis unlikely since the migration of maturing oocytes towards the acinal lumen is 348 hampered. Generally, degeneration of ovarian follicles is described as follicular atresia and 349 stress such as exposure to xenobiotics appears to increase the incidence of atretic follicles 350(Dietrich et al., in press). Degenerating follicles were also observed in Mytilus edulis exposed 351 to a mixture of North Sea oil, alkylphenols and PAHs. Exposure to 0.5 ppm North Sea oil 352 alone, however, increased the size and number of ovarian follicles in the mussels(Aarab et 353 al., 2004). In the present study, a few degenerating acini were also found in control animals 354 which may resorb malfunctional oocytes to preserve normal gonadal function as known from 355 atretic follicles in female fish(Dietrich et al., in press).356 357Furthermore, phenanthrene exposure significantly increased degeneration of previtellogenic found a higher volume density of atretic oocytes in mussels (Mytilus edulis) exposed to 0.5 361 ppm North Sea oil and to a mixture of North Sea oil (0.5 ppm), alkylphenols (0.1 ppm) and 362 extra polycyclic aromatic hydrocarbons (0.1 ppm) for three weeks. In M. edulis 363 experimentally exposed to low (28 ppb) and high (128 ppb) doses of diesel oil for three 364 months the volume of atretic oocytes was also significantly increased(Lowe and Pipe, 1986).

410

  may indicate early phagocytosis and resorption of gonadal and oocyte material as a result of 411 improper ovarian and gamete development. Gamete resorption is a natural phenomenon in

  Further, it has to be considered that release of affected gametes may result in 391 underestimating the adverse effects of phenanthrene on oocytes and eggs in P. miliaris.

	392
	393
	394	After exposure to phenanthrene we observed elevated but not statistically significant
	395	increased deposition of lipofuscin in the ovaries of P. miliaris. In several studies, lipofuscin
	396	accumulation in fish liver and mussel digestive gland is associated with oxidative stress
		caused by pollution with oil and PAHs (Krishnakumar et al., 1994, 1997; Au et al. 1999; Au,
	380
	381	to an inhibition of growth, result from degeneration and their resorption by NPs. Studying the
	382	effects of phenanthrene exposure at different times during oogenesis may clarify whether
	383	phenanthrene exposure inhibits growth and maturation of oocytes. Interestingly, in contrast
	384	to the severe alterations of previtellogenic oocytes, we found no adverse effects of
	385	phenanthrene on vitellogenic oocytes and mature eggs. It has to be considered that later

developmental stages of oocytes may be better protected against pollutant stress: Eggs of 386 sea urchins and sea stars are known to express low levels of efflux transporter genes 397 2004). In the reproductive tissue of the sea urchin Strongylocentrotus intermedius inhabiting 398 polluted areas in the Sea of Japan, lipofuscin accumulation was related to contamination with 399 heavy metals

(Vashchenko and Zhadan, 1993; Vaschchenko et al., 2001; Vashchenko et al., 400 2001)

. As demonstrated in the present study,

Vaschenko et al. (2001)

and Vashchenko et al.

  . Indeed, a clear diagnosis of immune reactions in the present study Prince William Sound in the Northern Gulf of Alaska in 1989 where around 37 000 metric 441 tons of crude oil were released an average of 0.4 µg L -1 total petroleum hydrocarbon were the West coast of Korea in July 2007 up to 17 mg L -1 and an average of 626 µg L -1 total 444 petroleum hydrocarbons were measured (Shim, 2008). However, direct comparison of 445 concentrations used in the present study and levels of PAHs or phenanthrene found in the This study was financed by the Alfred Wegener Institute for Polar and Marine Research 468 within the Helmholtz Association. It is part of a PhD study conducted at the Jacobs University 603 hydrocarbons (PAH) in seawater around England and Wales. Marine Pollution Bulletin 34(5), Lendrum, A.C., Slidders, W., Fraser, D.S., 1972. Renal hyalin -Study of amyloidosis and 607 diabetic fibrinous vasculosis with new staining methods. Journal of Clinical Pathology 25, Lowe, D.M., Pipe, R.K., 1986. Hydrocarbon exposure in mussels -a quantitative study of the 611 responses in the reproductive and nutrient storage cell systems. Aquatic Toxicology 8, 265-Ortiz-Zarragoitia, M., Cajaraville, M., 2006. Biomarkers of exposure and reproduction-related 615 effects in mussels exposed to endocrine disruptors. Archives of Environmental Palmer Wilson, L., 1940. Histology of the gonad wall of Arbacia punctulata. Journal of Pearse, A.G.E. 1985. Histochemistry: Theoretical and Applied, Vol. 2, Analytical Technology, 622 4th edition, Churchill Livingstone, Edinburgh, London, Melbourne and New York, p. 748. Growth parameters of Psammechinus miliaris in the different treatments. Standard deviations 701 are in parentheses. Different letters in the same row indicate statistical significant differences

	446 695 696 697 699 698	8. Tables & Figures 8.1 Table Table 1 parameter / treatment	0 control	control	days control solvent	20	phenanthrene
	700	mean vertical test	17.5 a (1.7)	17.4 a (1.9)	17.6 a (2.5)	17.4 a (2.8)
	447	marine environment in general or at point sources are difficult since combinations of PAHs diameter [mm]
	448 702	may have different effects than single chemicals (Hylland, 2006). Contamination of sea diameter [mm] between the treatments (control at day 0: n = 24, all other treatments: n = 30, one-way mean horizontal test 30.9 a (3.2) 30.4 a (3.3) 30.6 a (3.9) 31.8 a (3.5)
	449	urchin habitats with PAHs e.g. by oil spills or release of oil from oil platforms may have [g] mean somatic weight 13.0 a (3.6) 12.4 a (2.9) 12.7 a (3.9) 13.5 a (3.8)
	450	severe impacts on sea urchin populations. Daan & Mulder (1996), for example, have shown [mg] mean gonad weight 723 a (445) 712 a (384) 582 a (493) 390 b (257)
	451	that the abundance of the echinoidea Echinocardium cordatum was reduced close to a gonad index [%] 5.4 a (2.2) 5.8 a (2.2) 4.5 a (2.2) 2.9 b (1.9)
	452	drilling station in the North Sea where oil-based drilling muds had been used. Following the
	453	Torrey Canyon oil spill in 1967 large numbers of dead Psammechinus miliaris where found in
	454	the vincinity which may be attributed to the hydrocarbon exposure and the heavy spraying of
	455	hydrocarbon based dispersants in that area (Smith, 1968).
	456
	604
	457 605	In summary, 20 days exposure towards sublethal concentrations of phenanthrene resulted in 306-322.
	606
	458	severe ovarian lesions of Psammechinus miliaris which make a proper oogenesis unlikely.
	608
	431 459 609	Strikingly, the acinal structure was disorganized which may be analogous to follicular atresia 373.
	610
	432 460	was difficult since, to our knowledge, there is no detailed histological description of immune in other invertebrates and fish. Previtellogenic oocytes were severely affected while there
	612
	433 461 613	cells of sea urchins, the coelomocytes, in situ. Furthermore, coelomocytes had a similar were no obvious effects on vitellogenic oocytes and mature ova. For future studies, the 272.
	614
	434 462	appearance as epithelial cells of the acinal wall in Psammechinus miliaris. usefulness of lipofuscin accumulation in gonads of sea urchins as a biomarker needs to be
	616
	435 463 617	further investigated. Moreover, a detailed description of coelomocytes in situ is necessary to Contamination and Toxicology 50, 361-369.
	618
	436 464 619	The concentration of phenanthrene used in the present study is, in general, higher than clearly identify immune reactions in sea urchin tissues and to compare histopathological
	620	Morphology 66, 463-479.
	437 465 621	levels of PAHs found in the marine environment: In seawater around England and Wales effects of pollutants with studies on other invertebrate species.
	438 466 623	background concentrations of PAHs range from none detected to 10.7 µg L -1 total PAHs
	624
	439 467 625	(Law et al., 1997). 'Safe' levels of phenanthrene for aquatic organisms set by several 6. Acknowledgements Peters, E.C., Yevich, P.P., Harshbarger, J.C., Zaroogian, G.E., 1994. Comparative
	626 440 627	histopathology of gonadal neoplasms in marine bivalve mollusks. Diseases of Aquatic countries are equal or below 4.6 µg L -1 (Law et al., 1997). After the Exxon Valdez oil spill in Organisms 20, 59-76.
	628
	631
	632

Pillai, M.C., Vines, C.A., Wikramanayake, A.H., Cherr, G.N., 2003. Polycyclic aromatic 629 hydrocarbons disrupt axial development in sea urchin embryos through a [beta]-catenin 630 dependent pathway. Toxicology 186, 93-108.

aggregates of degenerating previtellogenic oocytes. Growth and maturation of previtellogenic

organism in fertilisation and embryo development assays(Klöckner et al., 1985; Caldwell et

reported byVashchenko and Zhadan (1993), Vaschenko et al. (2001) and Vashchenko et al.

(2001) in areas of Peter the Great Bay (Sea of Japan) polluted with heavy metals.

Histopathological alterations of spermatogenesis in the sea urchin Anthocidaris crassipina

For the Alcian Blue/PAS staining procedure sections were first stained with Alcian Blue

Schiff's reagent for 20 min., bleached in sulfurous acid for 2 min., washed again in distilled

water for 5 min. and stained with Gill's hematoxylin for 15 min. The sections were blued in

running tap water for 10 min., rinsed in distilled water for 2 min., air dried and mounted with

Euparal. The procedure results in blue staining of acidic mucopolysaccharides, magenta

staining of neutral mucopolysaccharides and in a blue/purple staining of a mixture of acidic

4.6.3.0, ZEISS).

lipofuscin content were square-root transformed. For the data on histopathology a one-way

gonadal tissue (Figure3 c). In phenanthrene-treated animals 78 % of the previtellogenic

oocytes in the ovary of P. miliaris. PAHs and oil are known to increase the prevalence of

oocyte degeneration or oocyte atresia in molluscs: Ortiz-Zarragoitia andCajaraville (2006)

(Hamdoun et al., 2004; Roepke et al., 2006). In sea stars efflux transporter activities are

known to increase during oocyte maturation(Roepke et al., 2006). Moreover, younger

Reunov, 2005) but it is also described in the sea urchin Strongylocentrotus intermedius from

polluted areas in the Sea of Japan(Vashchenko and Zhadan, 1993). The PAH-induced

measured at polluted sites(Boehm et al., 1990). However, after the Hebei Spirit incident at

Bremen. The authors thank B. Hussel and the staff of the FS Mya for collecting the sea

(Echinodermata: Echinoidea) in a Scottish sea loch. Journal of the Marine Biological

Association of the UK 80, 909-919.

Roepke, T.A., Hamdoun, A.M., Cherr, G.N., 2006. Increase in multidrug transport activity is

condition of the sea urchin Strongylocentrotus intermedius from Alekseev Bight (Amurskii

Bay) under polluted conditions. Russian Journal of Marine Biology 27, 49-53.

ANOVA, Tukey HSD, p < 0.05).


José M González-Correa

Yolanda Fernández-Torquemada

José Luis Sánchez- Lizaso

José Luis Sánchez-Lizaso

Short-term effect of beach replenishment on a shallow Posidonia oceanica meadow

Keywords: beach replenishment, short-term impact, earlier impact, Posidonia oceanica Introduction

Putative perturbations on a P. oceanica meadow produced by recent artificial beach nourishment were evaluated in relation to four undisturbed meadows. Temporal variations of putative impacted location versus control locations of environmental (light availability and sediment features), plant (associated epiphytes, silt-clay fraction attached to epiphytes, herbivore attack, non-structural carbohydrate reserves) and structural parameters (cover and density) of meadows were tested by asymmetrical analysis of variance beyond BACI (Before/After, Control/Impact). Additionally, two asymmetrical analyses of variance were used to test for differences in vegetative growth of horizontal rhizomes (leaf production, horizontal rhizome growth, biomass production and net secondary rhizome recruitment) before and after beach replenishment.

Environmental effects induced by dumping works were only detected in connection with a higher silt-clay deposition rate. This increase was consistent with the increase of silt-clay cover attached to epiphytes. As a consequence of silt-clay smothering, a decrease of filter feeding epiphytes, starch reserves, shoot surface and shoot biomass was observed. The sensitivity of plants to sediment inputs, leads us to recommend avoidance of dumping or sediment movement in the vicinity of P. oceanica meadows.

 [START_REF] Hamm | A summary of European experience with shore nourishment[END_REF][START_REF] Hanson | Beach nourishment projects, practices, and objectives an European overview[END_REF]. However, despite the fact that from an engineering viewpoint it is considered as an "environmental-friendly" option for coastal protection [START_REF] Miller | Detrimental effects of sedimentation on marine benthos: what can be learned from natural processes and rates?[END_REF], sizeable impacts on several beach ecosystem components (microphytobenthos, vascular plants, terrestrial arthropods, marine zoobenthos, and avifauna) are described in the literature [START_REF] Peterson | Shot-term consequences of nourishment and bulldozing on the dominant large invertebrates of a sandy beach[END_REF][START_REF] Rumbold | Estimating the effect of beach nourishment on Caretta caretta (Loggershead sea turtle) nesting[END_REF][START_REF] Peterson | Exploiting beach filling as an unaffordable experiment: Benthic intertidal impacts propagating upwards to shorebirds[END_REF][START_REF] Speybroeck | Beach nourishment: an ecologically sound coastal defence alternative? A review[END_REF][START_REF] Colosio | Effects of beach nourishment on sediments and benthic assemblages[END_REF].

In the Mediterranean Sea, the endemic seagrass Posidonia oceanica constitutes a key component, which provides valuable ecological goods and services for shallow water ecosystems (Terrados and Borum, 2004). P. oceanica beds cover large areas, spreading from locations near the shoreline to depths of 50-60 meters. When they are established close to sandy beaches they play an important role in the sand budget and shoreline protection. This occurs through: a) enhancement of sedimentation and decantation of suspended particles into the water column and its subsequent retention [START_REF] Terrados | Experimental evidence of reduced particle resuspension within a seagrass (Posidonia oceanica L.) meadow[END_REF][START_REF] Gacía | Sediment retention by a Mediterranean Posidonia oceanica meadow: the balance between deposition and resuspension[END_REF], b) reduction of the erosive energy of waves [START_REF] Gambi | Estimates of water movement in Posidonia oceanica beds: a first approach[END_REF][START_REF] Gacía | Sediment retention by a Mediterranean Posidonia oceanica meadow: the balance between deposition and resuspension[END_REF], c) nourishment of beaches with calcareous parts of organisms that live in them [START_REF] Falco | Relationship between sediment distribution and Posidonia oceanica seagrass[END_REF], and finally d) protection of beaches against erosion through the accumulation on the shoreline of leaves lost annually (Boudouresque and Jeudy de Grissac,1983). P. oceanica is adapted to the natural sediment mobility of sandy bottoms by vertical growth of rhizomes to avoid burial [START_REF] Boudouresque | Posidonia oceanica in the Mediterranean: interactions between the plant and the sediment[END_REF]).

Nevertheless, beach replenishments involve large inputs of sediments in the short-term causing potentially dramatic environmental changes that can overcome response mechanisms. Burial is the most obvious detrimental effect on the plant. [START_REF] Manzanera | Seagrass mortality due to oversedimentation: an experimental approach[END_REF] demonstrated in a manipulative experiment that 100% of rhizomes died after burial to a depth of 15 cm for 200-300 days. In addition, siltation leads to an increase in the turbidity and organic matter load [START_REF] Malmer | Converting tropical rainforest to forest plantation in Sabah, Malaysia. Part II. Effects on nutrient dynamics and net losses in stream water[END_REF], both of which negatively affect seagrass growth [START_REF] Giesen | Eelgrass condition and turbidity in the Dutch Wadden Sea[END_REF][START_REF] Sand Jensen | Interactions among phytoplankton, periphyton and macrophytes in temperate freshwaters and stuaries[END_REF].

Sediments with a high silt-clay fraction can limit seagrass photosynthesis by reflecting and absorbing available light [START_REF] Onuf | Seagrass dredging and light in Laguna Madre, Texas[END_REF][START_REF] Abal | Seagrass depth range and water quality in southern Moreton Bay, Queesland, Australia[END_REF][START_REF] Longstaff | Seagrass survival during pulsed turbidity events: the effects of light deprivation on the seagrasses Halodule pinifolia and Halophila ovalis[END_REF][START_REF] Ruíz | Effects of disturbances caused by coastal constructions on spatial structure, growth dynamics and photosynthesis of the seagrass Posidonia oceanica[END_REF] or by enhancing epiphyte growth on leaves promoted by nutrient input associated with exogenous sediment [START_REF] Neverauskas | Monitoring seagrass beds around a sewage outfall in South Australia[END_REF][START_REF] Cebrian | Epiphyte accrual on Posidonia oceanica (L.) Delile leaves: implications for light absorption[END_REF].

As a consequence of these effects, a wide range of P. oceanica meadow responses to sediment input are described in the literature, including: 1) no effect, as a result of a well planned and managed project [START_REF] Borg | Environmental Monitoring at St George's Bay (Malta) in connection with Beach Replenishment Works[END_REF][START_REF] Ebejer | Creating a sandy beach in St George's Bay-a new experience for Malta[END_REF]; 2) a pulse disturbance [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]; and 3) the regression of meadows [START_REF] Astier | Impacts des aménagements littoraux de la rade de Toulon, liès aux techniques d´endigage, sur ler herbiers à Posidonia oceanica[END_REF] Birzebbuga´s case reported in [START_REF] Borg | Environmental Monitoring at St George's Bay (Malta) in connection with Beach Replenishment Works[END_REF] where surviving plants are continuously submitted to a long-term press disturbance [START_REF] González-Correa | Longterm effect of beach replenishment on natural recovery of shallow Posidonia oceanica meadows[END_REF]. P. oceanica is a resilient plant, and as such it is expected to show first a slight responses in the short-term following the impact, which will lead to more serious symptoms (including death) if the cause of the impact persists. Long-term effects of beach replenishment on P. oceanica have been identified previously [START_REF] González-Correa | Longterm effect of beach replenishment on natural recovery of shallow Posidonia oceanica meadows[END_REF], but early symptoms linked to short-term effects are unknown.

Here, our goal was to identify early warning symptoms of P. oceanica as a consequence of stress of artificial beach nourishment. We hypothesized that short-term environmental changes (light availability and sediment features) related to the input of sediment may have a negative impact on the vitality of meadows. Both the accumulation of sediment on seafloor and the increase of silt-clay fraction into water column are environmental consequences expected from dumping. If burial occurs, with significant changes in the height sediment on the seafloor, then structural changes could be detected in meadows in short-term, such as changes in density and cover. If only siltclay fraction suspended into water column reach the meadow, then a subtle cascade of effects could be trigged. Early effects may begin with the decrease of the light available and the filter feeding epiphytes and increases in the silt-clay deposition rate, the silt-clay fraction attached to epiphytes, the epiphyte load and herbivore attack. These changes could lead to modifications in the non-structural carbohydrate content (decreasing starch and soluble carbohydrates) and in vegetative growth (slowing the growth of leaves and rhizomes). This first symptoms may subsequently be followed by structural changes in meadows, such as changes in density and cover.

2. Material and methods.

Study area and sampling design.

The study was carried on between 2004 and 2006 on the upper depth limit of a P. oceanica meadow spread along nine kilometres of the northern Alicante shoreline (SE Spain, SW Mediterranean Sea). In this area, the shoreline has retreated due to touristrelated beach management practices and building, including the systematic elimination of the protective remainds of P. oceanica leaves from the shoreline and the dune plant communities. In order to counteract the ensuing erosion, a replenishment project was carried out on Deveses beach from July 2005 to June 2006. Sand was trucked from a nearby quarry and dumped along a 1300 m stretch of beach. It was observed that sediments were eroded and the beach outline reached a new equilibrium slope. As a consequence, coarse sediment particles will be transported only short distances from their origin, while small particles (silt-clay) can be suspended for longer periods and move farther, potentially reaching on the nearby P. oceanica meadows. Therefore, we randomly chose a putative impacted location off Deveses beach (the only replenished beach in the area; hereafter impacted location: (I)) and four control locations (hereafter; C1, C2, C3 and C4). We replicated control locations in order to describe the natural variability of P. oceanica meadows in the area. Understanding natural variability is critical to assess how impacted locations vary from controls [START_REF] Kingsford | Procedures for establishing a study[END_REF]. The impacted location was placed 440 m from the dumping location at the upper depth limit of the meadow. We assumed that this was the closest point to the dumping site for beach replenishment and that it would therefore be the most sensitive. Control locations were placed distant enough from the planned dumping to prevent any negative effects. They were spaced between 1000 to 3000 m apart. Locations depths ranged from 2.2 Environmental variables.

Sediments

For sediment characterization, two 4 x 10 cm cores were taken at each location and at the dumping area. The upper 10 cm portion of each core was used to estimate the organic matter and silt-clay concentrations (<0.063 mm). Sediment was dried for 24 h, weighed and sifted through a five sieve column (2, 0.5, 0.25, 0.125 and 0.063 mm; sifter CISA model RP-03). The fraction smaller than 63 m was estimated by subtracting initial dry weight from final dry weight of sediment retained on sieves [START_REF] Buchanan | Sediment analyses[END_REF]. Loss-On-Ignition (LOI) was used to estimate the organic matter content in the sediments following the method of [START_REF] Luczak | Simple standard procedure for the routine determination of organic matter in marine sediment[END_REF]. Water content was determined at 105°C. Thereafter, the oven-dried samples were heated in a furnace at 500 o C for 6 hr.

The samples were cooled in a dessicator before measurement.

In order to measure annual variations in sediment height, we drove twelve labelled sticks (80 cm long) into the sea floor (approximately half of the stick) at each location.

The distance from the top of each labelled stick to the sea floor was measured in July of 2004, 2005 and 2006. Sticks were placed 1 m apart each other.

The silt-clay deposition rate (mg DW m -2 d -1 ) was evaluated by installing two sediment traps (diameter 8.5 cm, height 28 cm) on seafloor at each location for a period of 20 days in June 2006. They were closed before the sediment traps were withdrawn. In the laboratory, they were vigorously shaken and three sub-samples of 200 ml were subsequently taken from each sediment trap. Each sub-sample was filtered first by a 63 m sieve (sifter CISA model RP-03) and later re-filtered through a 0.7 m glass microfiber filter disc (Whatman type GF/F). We obtained the weight of sediment retained by subtracting the total dry weight of the 0.7 m filter and the sediment retained (dried for at least 1 hr at 103-115ºC) from the initial dry weight of the filter.

The methodology applied followed the ESS Method 340.2 (1993).

Light conditions

To assess light conditions at each locality, light intensity (lux) was measured in situ using photometer loggers (ONSET Stow Away LI) with an accuracy of ±0.01 lux.

Photometers were placed at canopy level by attaching them to sticks pushed vertically into sediment in empty spaces in the meadow. Light measurements were continuously recorded each 15 min from 07.00 to 20.00 GMT for a 14 day period each year (in June 2005 and 2006), which we considered short enough to prevent shading effects by fouling. Photometrics units (lux) were converted to quantum units (µmol PAR m -2 s -1 ) applying the experimental algorithm Log Y (µmol PAR m -2 s -1 ) = (0.9828 x log X (lux))-0.038. This algorihtm was calculated through an experimental regression in Gonzalez-Correa (2008).

Plant variables.

P. oceanica meadow structure was surveyed at each location by estimating density and cover of living and dead plant material. Shoot density was estimated by counting living shoots in three random 40 x 40 cm quadrats per location. Living and dead P. oceanica cover were estimated as the proportion of living and dead P. oceanica patches on three replicated 25 m transects at each location.

To estimate leaf, epiphyte variables and non-structural carbohydrate content, 10 vertical shoots were harvested in each location 'before' and 'after' dumping. Shoot surface (cm 2 shoot -1 ), shoot biomass (g DW shoot -1 ) and epiphyte biomass per leaf surface unit (mg DW cm -2 ) were estimated following the method described for these parameters in Ruíz and Romero (2001). Damage by herbivore attack was estimated calculating the percentage of bitten leaves at each shoot (% bitten leaves). Cover of epiphytes and filter feeding epiphytes, and the cover of silt-clay attached to epiphytes were estimated by 15 randomly distributed 5 x 5 mm quadrats. Each of five quadrats were allocated to young, medium and old leaves. We used a binocular microscope and a sampling square printed on a transparent plastic sheet subdivided into smaller squares of 1 mm 2 in order to enhance the accuracy of the method.

Non-structural carbohydrates were estimated by cutting the first 3 cm from the apical part of the rhizomes to obtain samples. Samples were dried (100ºC for 24 h) and converted into powder. Rhizome powder was heated with ethanol (80ºC, 15 min in the Heating Block), centrifuged (3000 rpm, 10 min) and finally the supernatant was separated from insoluble residues in different tubes. Soluble carbohydrates were extracted from the supernatant by evaporating ethanol and dissolving the residues in deionised water for further analysis. Starch was extracted from insoluble residues by keeping it for 24 h in NaOH (0.1N). Soluble carbohydrates and starch content of extracts were determined spectrophotometrically using anthrone assay, with sucrose as a standard [START_REF] Yemm | The estimation of carbohydrates in plant extracts by anthrone[END_REF].

To evaluate vegetative growth, 10 principal horizontal rhizomes separated by 1-2 m, were labelled at the apex at each location before dumping. They were identified and pulled out one year later, removing the longest portion of horizontal rhizome possible.

Since the distance from the label to the apex can be considered to represent growth of 1 yr, we directly obtained annual vegetative growth estimates by measurement. The total age of each horizontal rhizome was estimated by the lepidochronology technique [START_REF] Pergent | Lepidochronological analyses of the seagrass Posidonia oceanica (L.) Delile: a standardized approach[END_REF]. This technique is based on the thickness cycle of sheaths attached to rhizomes; identifying an annual period which equates to the length of rhizome as measured between two sheaths of minimum thickness. Minimum thickness sheaths are obvious in vertical but not in horizontal rhizomes. Hence, the age of a horizontal piece of rhizome was calculated as the age expressed by subtracting the youngest from the oldest vertical rhizome to which it was connected. This dating method was not sensitive to horizontal rhizomes younger than 1 yr; therefore, only rhizomes older than 3 yr were selected in order to minimize such imprecision (González-Correa et al 2005).

Both methods (direct and indirect) allowed us to obtain annual estimates of the following seagrass variables: leaf production of horizontal rhizomes (leaves y -1 ), estimated by counting sheaths; horizontal rhizome growth, which was obtained by measuring its length following rhizome contour; biomass production of horizontal rhizomes (g DW y -1 ), estimated after drying rhizomes for 24 h at 80 o C; and net production of secondary rhizomes (new secondary rhizomes y -1 ), calculated as the difference between branches which formed and those that died per year. While it is possible to distinguish between secondary, tertiary and quaternary shoots [START_REF] Molenaar | Modelling and growth patterns of Posidonia oceanica[END_REF], we focused on secondary rhizomes because they are the only ones that are likely to branch on the one-year-old horizontal rhizome fraction.

Data analyses.

To test differences in environmental parameters (sediment features and light availability), epiphyte features, phenological parameters, carbohydrate reserves, and structural parameters (cover and density) and examine temporal differences between the potentially impacted location and the randomly-selected control locations, we used a "beyond BACI" (Before/After, Control/Impact) asymmetrical analyses of variance. The "beyond BACI" design employs various controls in order to avoid problems of spatial confounding (or "pseudoreplication"; [START_REF] Hurlbert | Pseudoreplication and the design of ecological field experiments[END_REF]. A new level of spatial replication was not considered necessary because of the relatively short stretch of shoreline replenished (approximately 1300 m) during the study period versus the longer stretch of impacted location (approximately 40-50 m, similar to controls). The mechanics and the logical structure of these analyses are fully explained by [START_REF] Underwood | Beyond BACI: the detection of environmental impacts on populations in the real, but variable, world[END_REF][START_REF] Underwood | The mechanics of spatially replicated sampling programmes to detect environment impacts in a variable world[END_REF][START_REF] Underwood | On beyond BACI: sampling designs that might reliably detect environmental disturbances[END_REF].

To test vegetative growth variables of horizontal rhizomes, two separate asymmetrical analyses of variance for each time (before/after) were used. The change in analysis with respect to the above variables was necessary because vegetative growth variables emanate from different methods. The 'before' dumping values were obtained by an indirect method and represent the average of various previous years, while 'after' dumping values were obtained by a direct method and only represent the value of a year (the impact period). We tested if vegetative growth of horizontal rhizomes at the impacted location was significantly different with respect to those from control locations, before and after sediment dumping, but without temporal interaction. The mechanics and the logical structure of asymmetrical analyses of variance are fully explained in [START_REF] Glasby | Analysing data from post-impact studies using asymmetrical analyses of variance: A case study of epibiota on marinas[END_REF]. Finally, to test for differences in silt-clay deposition in the last year, we used an asymmetrical analysis of variance. Before analysis, Cochran's test was used to test for homogeneity of variance for all variables. Where significant differences were found, the data were ln(x+1) transformed.

Results

Environmental parameters.

Grain size analyses of dumped sediment on the shoreline revealed a low content of both silt-clay (< 2.5 %) and organic matter fraction (<1%) at each of the three sampling times (Fig. 2). Consequently, dumped sediment volume was not sufficient to provoke a significant variation of sediment height at the impacted location. Sediment height ranged approximately ±2 cm for each annual period measured, which was similar to the values recorded at the control locations (Fig. 3.1). The silt-clay fraction and organic matter at the impacted location did not differ significantly from the control locations either before or after dumping (Fig 3.2,3.3). We detected a large silt-clay and organic matter fraction in C1, which came from the nearby Racons creek (see Fig. 1). However, the silt-clay deposition rate was significantly higher at the impacted location after dumping in comparison to the controls, including C1 (181 mg DW m -2 d -1 at impacted location vs 75 mg DW m -2 d -1 average of controls; Fig 3 .4). Finally, light availability at the impacted location did not differ significantly either before or after dumping compared to the control locations (Fig 4).

Plant variables.

Shoot surface and biomass at the impacted location decreased from before to after dumping (shoot surface: 199.7±12.8 cm 2 shoot -1 to 109.8±7.6 cm 2 shoot -1 ; shoot biomass: 1.005±0.007 to 0.735±0.008 g DW shoot -1 ; Fig 5). Whereas, shoot surface and shoot biomass at control locations did not show significant interactions in relation to dumping time. Herbivorous attack did not differ before and after dumping at control locations (p>0.05) and was always highly variable (28.3 to 54% of bitten leaves). In contrast, herbivory was always high at the impacted location (59.5 to 59.6 % of bitten leaves), therefore no significant changes in herbivory in impacted location were detected (p>0.05). Dumping provoked increased epiphyte biomass, an increased load of silt-clay attached to epiphytes and decreased the cover of filter feeding epiphytes at the impacted location, while in controls, no significant differences were detected (Fig. 6).

Epiphyte cover was not affected (p>0.05), which indicated that although weight increased, as a consequence of silt-clay attached to epiphytes, the area of settled surface did not.

After dumping, starch reserves of rhizomes at the impacted location dropped to half of the levels measured in 2005 (from 29.7±4.9 to 13.0±1.2 mg starch g DW rhizome -1 ), while values at control locations remained within a similar range (Fig. 7.1). Significant variability in soluble carbohydrate concentration occurred at control locations in relation to time (p<0.05), which prevented the decreasing trend at the impacted location from being significantly detected (Fig. 7.2).

Vegetative growth of horizontal rhizomes at the impacted location was similar for leaf production, net production of secondary rhizomes and biomass production compared to the controls before and after dumping. Horizontal rhizome growth at impacted location was significantly lower the year after dumping. However this difference may be better explained by an increase of C3 location than by a significant decrease in impacted location (Fig 8)

Finally, no significant changes were detected in structural parameters (shoot density and cover of alive and dead P.oceanica) in impacted location as a consequence of dumping (p>0.05).

Discussion

This beach replenishment was characterised by the small amount of sediments dumped with a very low silt-clay fraction (< 2.5%). Sediments eroded from the dumping area reached the meadows increasing silt-clay deposition rate and the silt-clay cover attached to epiphytes, but it was not enough to provoke any measurable changes to sediment features on seafloor (sediment level, silt-clay fraction and organic matter) or to light availability at the impacted location.. The increase in silt-clay deposition rate was consistent with the diminishing of cover of filter feeding epiphytes. It should be noted that differences observed are due to the high proportion of filter feeding epiphytes before the dumping in comparison with controls. Filtration mechanisms of epiphytes become clogged if the silt-clay fraction in the water column is high, and this is known to interfere with ingestion and respiration [START_REF] Mcfarland | Lethality of a suspended clay to a diverse selection of marine and estuarine macrofauna[END_REF][START_REF] Waters | Sediment in streams: sources, biological effects and control[END_REF][START_REF] Ellis | Determining effects of suspended sediment on condition of a suspension feeding bivalve (Atrina zelandica): results of a survey, a laboratory experiment and a field transplant experiment[END_REF], which subsequently leads to changes in the distribution of epibenthic species [START_REF] Cohen | Epibenthic community structure in port Philip Bay, Victoria, Australia[END_REF][START_REF] Lohrer | Assessing far-field effects of terrigenous sediment loading in the coastal marine environment[END_REF]. Due to the high temporal and spatial variability of epiphytes, further studies should be performed in order to confirm this trend.

P. oceanica starch reserves at the impacted site is higher than at control sites before dumping and decreased by half after the beach replenishment. This fall is consistent with the reduction in shoot surface and biomass of leaves. Before dumping, leaves were longer in impacted location than controls locations. Since sampling was simultaneous at all locations differences in shoot surface, shoot biomass and starch reserves before the dumping are not related with seasonality and may indicate a higher vitality of the plants of the impacted locality before the beach replenishment. After dumping sampling (same week one year later), similar features on leaves and starch level would indicate that vitality of plants was similar in all locations. The reduction of vitality of the plants after the dumping in the impacted location is consistent with the increase of epiphyte biomass and the increase of silt-clay attached to epiphytes. It could involve a lengthy smothering effect, which may affect the carbon budget of the plant, although no decrease in light availability was detected at the time of sampling. Reserves depletion could occur in two possible ways: a) clogging the pass of nutrients and bicarbonates necessary for plant development or b) preventing the normal transfer of light through the leaf epidermis.

In any case these results should be interpreted with caution since it should be better to have sampled several times before the impact to be sure that this meadow had a higher vitality in relation to the meadows used as controls.

Soluble carbohydrates showed a highly variable behaviour at controls both before and after dumping, preventing the decrease at the impacted location from being statistically significant. The variable behaviour of soluble carbohydrates matches the results of a previous study [START_REF] González-Correa | Longterm effect of beach replenishment on natural recovery of shallow Posidonia oceanica meadows[END_REF] and could be explained by the fast response of soluble carbohydrates to growth status [START_REF] Touchette | Overview of the physiological ecology of carbon metabolism in seagrasses[END_REF] and photosynthethic problems (Ruíz et al., 2001).

No effect was observed on the vegetative growth of rhizomes. This result contrasts with the 45 % reduction of net total rhizome recruitment determined by a long-term effect of beach replenishment study on shallow P. oceanica meadows [START_REF] González-Correa | Longterm effect of beach replenishment on natural recovery of shallow Posidonia oceanica meadows[END_REF]. Discrepancies between the results can be explained by the different sets of environmental conditions that affected the health of plants in the two studies. In the long-term effect study, beach replenishment was undertaken using marine sediments and affected two sheltered meadows which retained a high silt-clay and organic matter fraction in the sediment 18 yr after dumping took place. In contrast, the more exposed impacted meadow in the present study was characterized by a low silt-clay and organic fraction in the sediment. In addition, different volumes of sediment were dumped on the beaches used in the two studies (personal observation).

Natural processes may increase sediment inputs on the meadows. It is well known that P. oceanica is not present in front of important rivers that produce a continuous input of sediments. In our study one of the controls is placed in front of a gully that is usually dry except after periods of heavy rainfall which in the Mediterranean climate may be separated by decades. The high amount of silt-clay in the C1 location may be related to historical sediment inputs, although this kind of event was not observed during our study period. This increase in the silt-clay fraction in the meadow sediment seems not to affect the plant features. This result indicates that the organic matter load of the sediments is more relevant than the silt-clay content to explain the low recruitment after the beach replenishment with marine sediments [START_REF] González-Correa | Longterm effect of beach replenishment on natural recovery of shallow Posidonia oceanica meadows[END_REF]. While in that study the amount of organic matter of the impacted locations ranged between 15 and 20%, in this study it was lower than 4% at all locations.

.

Conclusions

In conclusion, the small inputs of sediments into the water column due to sediment dumping for beach replenishment provoked the increase of the sedimentation rate and the sediments attached to the seagrass leaves but it was not sufficient to produce measurable changes in light availability or sediment level. Early warning symptoms of response of P. oceanica, such as starch depletion, shoot size reduction, and changes in epiphytes may be related to the smothering effects on leaves. Further studies on the smothering effects on leaves are necessary to determine if the impacts observed in the short-term produce a negative carbon budget that in turn results in regression of the meadow in the long-term. The sensitivity of plants to small sediment inputs, leads us to recommend avoiding undertaking work that involves dumping or sediment movement in the vicinity of P. oceanica meadows.

Aknowledgements

Thanks are due Tim Dempster for improving an earlier version of the manuscript and two anonymous referees by their constructive criticisms.

5

  to 6 m (Fig 1). Light, sediment and plant parameters were sampled before beach replenishment (June 2005) and one year after. Sediments dumped on the shoreline were sampled on three occasions during dumping work (in July 2005, November 2005 and June 2006).

Figure 1 .

 1 Figure 1. Study area. Location of beach replenishment area, impacted (I) and control sites (C1,

Figure 2 .

 2 Figure 2. Average of 1) silt-clay fraction (±SE, n=2) and 2) organic matter fraction (±SE, n=2)

Figure 3 .

 3 Figure 3. Average of 1) sediment height (±SE, n=12), 2) silt-clay fraction (±SE, n=2), 3) and

Figure 4 .

 4 Figure 4. Average of light availability in the study period (±SE, n=14) before and after beach

Figure 5 .

 5 Figure 5. Average of 1) shoot surface and 2) shoot biomass before and after dumping at

Figure 6 .

 6 Figure 6. Average of 1) epiphyte biomass (± SE, n=10), 2) cover of silt-clay attached to

Figure 7 . 1 )

 71 Figure 7. 1) Starch reserves (± SE, n=10) and soluble carbohydrates (± SE, n=10), before and

Figure 8 .

 8 Figure 8. Average of horizontal rhizomes growth (± SE, n=10) before and after dumping at

  Fig.2

Terrados, J., Borum, J., 2004. Why are seagrasses important? Goods and services

Fig.1