Awantha Dissanayake
email: awantha@nagasaki-u.ac.jp

Shaw D Bamber

Monitoring PAH contamination in the field (South west Iberian Peninsula): biomonitoring using fluorescence spectrophotometry and physiological assessments in the shore crab Carcinus maenas (L.) (Crustacea: Decapoda)

Keywords: Polycyclic aromatic hydrocarbon (PAH), Carcinus maenas, fluorescence spectrophotometry, Gibraltar

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the marine environment, arising predominantly from petrochemical contamination and pyrogenic sources. A biomarker of PAH exposure was employed in a field study (South West, Spain) in both captured (indigenous) and deployed (caged) shore crabs (Carcinus maenas) in the chronic PAH-exposed Bays of Algeciras and Gibraltar (from associated harbour and boating activity) compared to a relatively 'clean' site (Cadiz). Metabolite fluorescence was attributed to the following key priority PAH groups; naphthalenes (NAPs), pyrenes (PYRs) and benzo[a]pyrenes (BAPs). Temporal variability was assessed using deployed populations over an eight week period. Petrogenic and pyrogenic PAH contamination (as an indicator of the PAH type) was demonstrated using a ratio between FF BAP+PYR / FF NAP . Physiological assessments from deployed crabs demonstrated both physiological and cellular alterations as shown by reduced heart rates (at rest) and increased cellular stress in crabs from the PAH contaminated sites.

INTRODUCTION

Coastal marine waters and sediments receiving anthropogenic inputs can contain a large variety of contaminants that are potentially toxic to aquatic organisms [START_REF] Krahn | Determination of metabolites of xenobiotics in the bile of fish from polluted waterways[END_REF] [START_REF] Kennish | Environmental threats and environmental future of estuaries[END_REF]. One group of chemicals of particular concern are the highly lipophilic polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous pollutants of the marine environment. These compounds arrive in coastal waters from a diverse array of sources including petrochemical pollution, incomplete combustion processes [START_REF] Livingstone | Persistent Pollutants in Marine Invertebrates[END_REF]Clarke et al., 2001), metal smelting [START_REF] Msc-E ; Naes | Occurrence of PAH in marine organisms and sediments from smelter discharge in Norway[END_REF][START_REF] Beyer | Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed by caging to polluted sediments in Sorfjorden, Norway[END_REF] and electrolytic production of aluminium using anode technology [START_REF] Beyer | Bioavailability of PAH in effluent water from an aluminium works evaluated by transplant caging and biliary fluorescence measurements of Atlantic cod (Gadus morhua L.)[END_REF]Aas et al., 2000b;[START_REF] Msc-E ; Naes | Occurrence of PAH in marine organisms and sediments from smelter discharge in Norway[END_REF]. Concern over the fate and effect of PAHs in the environment is related to their persistence, bioaccumulation This approach may overlook the risks posed by the potentially much more toxic daughter compounds produced by many organisms as a result of metabolism and biotransformation of the parent chemicals [START_REF] Livingstone | The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish[END_REF]. Metabolites arising from biotransformation processes may be concentrated in body fluids, tissues or

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excreta and the analysis of such biological compartments provides us with an opportunity to detect and measure exposure of organisms to bioavailable contaminants (Fillmann et al., 2002).

The detection of PAH metabolites is made possible due to the fluorescence properties attributed to their delocalised π-electrons. This property was first developed for environmental monitoring purposes as a rapid, inexpensive screening method to detect PAH metabolites in fish bile and used HPLC in combination with a fluorescence detector to process test samples. This technique has subsequently been implemented as a monitoring tool in several environmental monitoring programmes [START_REF] Krahn | Determination of metabolites of xenobiotics in the bile of fish from polluted waterways[END_REF][START_REF] Stagg | The development of an international programme for monitoring the biological effects of contaminants in the OSPAR convention area[END_REF]Aas et al., 2000a[START_REF] Aas | Evidence of uptake, biotransformation and DNA binding of polyaromatic hydrocarbons in Atlantic cod and corkwing wrasse caught in the vicinity of an aluminium works[END_REF] and has been further developed for use in analysing the presence or absence of PAH metabolites in crustacean urine by [START_REF] Watson | A direct fluorimetric technique for the detection of PAH equivalents in crustacean urine and haemolymph: potential for use as an in situ biomarker of PAH exposure[END_REF]. Metabolites in shore crab (Carcinus maenas) urine were analysed at specific fluorescence wavelengths for naphthalene-, pyrene-and benzo [a]pyrene-type compounds. The use of crab urine to monitor environmental contamination has proved to be a useful technique as it provides a measure of contemporary levels of contamination (Watson et al. 2004b

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heart rate measurements and monitoring cellular impact by lysosomal stability measurements) in crabs that in combination could be used to support biomonitoring programmes. Physiological assessments of invertebrates using cardiac activity (Fossi et al. 2000;Camus et al. 2002) and lysosomal stability [START_REF] Lowe | Contaminant induced lysosomal membrane damage in marine mussel digestive cells -an in-vitro study[END_REF]Castro et al. 2004) measurements have been widely used in both laboratory and field scenarios [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF][START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF] as general "health" indicators.

Lysosomal damage arises from the accumulation of PAHs, the resultant formation of oxyradicals leads to increased membrane permeability and subsequent release of acid hydrolases into the cytoplasm, causing cellular damage and/or ultimately, cell death [START_REF] Lowe | Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene[END_REF]. The resulting toxic effect of PAHs is expressed through the hierarchical levels of biological organisation i.e. cellular and physiological levels (Fossi et al. 1994).

MATERIALS AND METHODS

Study sites

The study sites were situated in the Southwest Iberian Peninsula in the Bays of Algeciras and Gibraltar and further west in the Bay of Cadiz (Fig 1). Gibraltar is a small peninsula situated at the mouth of the Mediterranean Sea with a 30km coastline. There is a heavy industrial presence from within the Bays of Algeciras and

Gibraltar and an apparent lack of data concerning environmental quality with regard to specific types of contaminants within the region. The area has proximity to chemical plants, refineries, thermal power plants, ironworks, paper mills, shipyards and two major ports which serve the economically important sea route of the Straits of Gibraltar [START_REF] Truver | The United States and the Strait of Gibraltar[END_REF][START_REF] Mavor | Ship traffic through the Gibraltar Strait[END_REF]. The Bay of Algeciras receives input from two major rivers; The Palmones and the Guadarranque, which carry sewage from urban and industrialised activities corresponding to populations of 15000 and 2500 respectively, (Carballo et al., 1996). Polyaromatic hydrocarbons in the Bay of Algeciras indicate chronic contamination in this area (21.4 µg kg -1 sediment) originating from accidental spills, bunkering activities and deliberate discharges from commercial shipping [START_REF] Jimenez-Tenorio | Chronic bioassay in benthic fish for the assessment of the quality of sediments in different areas of the coast of Spain impacted by acute and chronic oil spills[END_REF]. The Bay of Cadiz is characterised by urban and harbour activities with low sediment contamination (Cesar et al., 2007), however, the area outside the Bay of Cadiz (San Fernando) was used as the reference site as it is situated in a highly productive protected natural site where there is no urban activity [START_REF] Perez | Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cadiz bay (SW Spain)[END_REF]. These particular sites were chosen due to the highly industrialised nature of the surrounding area, including the Gibraltar Harbour [inner (36° 07' 55 N, 5° 21 18W)] (high: major boating activity) (Fig. 1).

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Shore crab deployment and collection

Carcinus maenas individuals (green intermoult males; carapace width 50-75 mm) sampled from the reference site [San Fernando,Cadiz,6° 11 50 W)] and were used for the deployment st udy (June -August 2001) using cages at selected field sites within the Bay of Gibraltar, Southwest Iberian Peninsula (Fig. 1). Prior to deployment, adult individuals (> 60 mm) (Crothers, 1967) were to caging experiments, a preliminary test for the effects of caging was performed at the reference site (Cadiz) where shore crab physiological responses (see 2.5) were measured after one week and no significant differences were observed (heart rates:

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F 1, 14 = 0.6, P = 0.45; lysosomal stability (neutral red retention time): F 1, 14 = 1.24, P = 0.28). Local indigenous populations were collected using squid-baited traps or handcollected at the various field sites in the month of June. During transportation (to field sites or the laboratory) all crabs were placed in cool boxes with icepacks and damp absorbent paper.

Urine Extraction and Analysis

Urine from each crab was extracted and analysed using the techniques described by Watson et al. (2004b). In summary, crabs were physically restrained prior to urine extraction by using elastic bands to hold them securely onto to small wooden boards with their ventral surface presented uppermost. The third maxillipeds were moved aside and restrained using further elastic bands to allow access to the epistome and

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antennal gland opercula. The epistome was dried in order to prevent seawater diluting the urine. Lifting of the antennal gland opercula, with the aid of a fine hooked seeker, triggers the release of urine from restrained crabs. Extracted urine (20 -1000µl) was transferred to siliconised micro-centrifuge tubes. All urine samples were stored at -80°C until subsequent analysis.

Spectrophotometric analysis

Fixed fluorescence (FF) spectrometry analysis was performed on diluted urine samples (1:20) whereby there are no significant inner filter effects (Hitachi F-4500 fluorescence spectrophotometer). All FF analyses were carried out in a 3.5 ml quartz fluorescence cuvette and performed with excitation and emission slit widths of 2.5 nm. Fluorescence contributions can be attributed to three PAH groups; 2 ring, 4 ring & 5/6 ringed PAHs typified by naphthalene-type (NAP-type), pyrene-type (PYR-type)

and benzo[a]pyrene-type (BAP-type) metabolites respectively as their behaviour is similar to other compounds of similar ring number [START_REF] Reeves | Partitioning and desorption behavior of polycyclic aromatic hydrocarbons from disparate sources[END_REF]).

Fluorescence analyses were not undertaken to quantify individual PAH compounds, but to discriminate between sites of varying PAH exposure (Dissanayake and Galloway, 2004). The assigned wavelength pairs and peak area measurements were used to quantify the metabolite groups as follows; FF 290/335 (315-375), FF 341/383

(380-390nm) & FF 380/430 (400-490nm) for 2&3-rings (naphthalene-type PAHs), 4-rings (pyrene-type PAHs) and 5-6 ringed PAHs respectively (benzo[a]pyrene-type PAHs).

Pyrene-type metabolites only were quantified by using 1-OH pyrene standards (Sigma, U.K). The ratio between the sum of BAP & PYRs over NAPs (FF BAP+PYR / FF NAP ) was calculated in order to determine PAH contamination origin [i.e petrogenic (NAPs) or pyrogenic (PYR and BAP)]. Cysteine (50 mg ml -1 ) was added as an anti-coagulant [START_REF] Smith | Host defence reactions of the shore crab, Carcinus maenas (L.), in vitro[END_REF].

Shore crab physiological assessment

Haemolymph (40 µl) was transferred onto a glass slide cells were allowed to adhere in a humidity chamber (15 mins) before incubating (15 min) with the neutral red dye.

Cells were observed under a high powered microscope over time (15, 30, 60, 75, 90 120, 150, 190 min) and the retention of the dye within the haemocyte lysosomes was recorded (as employed by [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF].

Statistical analysis

Analysis of variance (ANOVA) tests were performed for the individual PAHs (NAP, PYR and BAP) physiological (heart rates) and cellular (NRR) endpoints (transformed where necessary to meet parametric normality assumptions) (GMAV 5 for [START_REF] Underwood | Experiments in Ecology: Their logical design and interpretation using analysis of variance[END_REF]. Student-Newman-Keuls (SNK) tests were used to isolate differences between sites or time (weeks).

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RESULTS

Fluorescence spectrophotometry of diluted crab urine revealed significant PAH exposure characterised by strong fluorescence signals for both petrogenic and pyrogenic PAHs from both indigenous and deployed (caged crabs).

No differences were observed between crabs caught from the different sites with regard to Naphthalene-type metabolites (Fig. 2A), however, significant differences were observed between sites with both PYR and BAP-type metabolites significantly higher in urine from Gibraltar Harbour crabs than Palmones Estuary or Cadiz 2). Over the 8 week monitoring period, PAH contamination shifted from pyrogenic origin (due to high levels of PYR and BAP metabolites) (week 1) to petrogenic as observed in Gibraltar middle harbour site (week 8). The high levels of PYR and BAP metabolites also found in Palmones Estuary crabs indicate PAHs from a pyrogenic source (weeks 4) (Table 2). The observed variations in PAH levels in urine were also associated with significant physiological and cellular alterations.

Physiological assessments revealed a significant acute reduction (approx. 50%) in heart rates in crabs from all sites (at rest) at week 1 compared with controls (week 0) (two-way ANOVA; F 10, 126 = 2.51, P < 0.001) (Fig. 5A). Significant impacts at the cellular level were observed with significant reductions in neutral red retention times with the biggest reduction between week 0 and 1. Although, reductions in neutral red retention times were observed in all sites over the 8 week monitoring period, significant differences were also observed between sites with Palmones Estuary crabs displaying significantly higher NRR times compared to both Gibraltar Harbour sites (weeks 1-4) (two-way ANOVA; F 10, 126 = 6.20, P < 0.001) (Fig. 5B). where there is relatively less boating activity. Cadiz was chosen as a reference site even though a low level of metabolites was detected. Pyrene metabolite concentrations in urine are approximately 30 % higher than that of the parent compound in seawater (when exposed continuously) (Dissanayake et al., 2008a) and reflect short-term exposure (upto 4 days) rather than bioaccumulation (Watson et al., 2004b). Mean concentrations of pyrene found here (Cadiz, 20 µg l -1 ; Palmones, 13 µg l -1 and Gibraltar Harbour 51 µgl -1 ) are consistent with values found in relatively clean coastal sites (17 -20 µg l -1 ) and PAH contaminated sites (40 -90 µg l -1 ) (Watson et al., 2004a;Dissanayake et al., 2010).

DISCUSSION

Results

The mobility of PAHs due to both aqueous and aerial transport makes it difficult also to locate completely 'pristine' locations, although the Bay of Cadiz has been classed as a 'clean' area due to undetectable levels of PAHs in sediments [START_REF] Riba | Sediment quality in littoral regions of the Gulf of Cadiz: a triad approach to address the influence of mining activities[END_REF].

Other studies have also shown low metabolite levels present in various crustacean and fish species taken from areas assumed to be 'clean' [START_REF] Klumpp | Toxic contaminants and their biological effects in coastal waters of Xiamen, China. I. Organic pollutants in mussel and fish tissues[END_REF]Eickhoff et al., 2003b;[START_REF] Ruddock | Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries[END_REF][START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. The 2b). Indigenous sampled crabs, however, demonstrated that Palmones Estuary-caught crabs indicated PAHs of a petrogenic origin (Table 2a), thereby indicating a change in the PAH origin over the study period. It could be argued that the pattern observed here (of increasing and decreasing levels PAH metabolites levels) may occur due to possible downregulation dose-dependent effects (thus reflecting environmental levels) or saturation processes of the biotransformation system. A previous study has shown that in Carcinus, when exposed to a set concentration similar metabolite levels are observed after 7 and 14 days exposure to a sustained dose (200 µg l -1 for 7 and 14 days exposure; see Dissanayake et al. 2008a). However, saturation processes of the biotransformation (mixed function oxidase) system may occur with high doses as observed by bell-shaped dose response curves as shown in vertebrate systems (Bosveld et al. 2002;[START_REF] Lu | The dose-response relationships for EROD and GST induced by polyaromatic hydrocarbons in Carassius auratus[END_REF]). In shore crabs, however, there is conflicting evidence for saturation of the biotransformation system as shown by Fossi et al. control. The objective of rapid screening techniques is to be able to discriminate between sites of varying contamination, thereby providing a rapid assessment of pollution (Galloway et al., 2002). The FF BAP+PYR / FF NAP ratio was used in order to indicate the source of PAH contamination (Aas et al., 2000a). The rationale for using this technique is based on the fact that PAHs occur in combination and PAH profiles (e.g. ratio of high to low molecular weight PAHs) can indicate sources of PAHs [START_REF] Law | Polycyclic Aromatic-Hydrocarbons (PAH) -Problems and progress in sampling, analysis and interpretation[END_REF][START_REF] Wang | Distribution of PAHs in burn residue and soot samples and differentiation of pyrogenic and petrogenic PAHs-the 1994 and 1997 mobile burn study[END_REF]. The FF technique is regarded as a measure currents and/or dredging activities may have an effect on suspended matter levels [START_REF] Zhou | Fluoranthene and pyrene in the suspended particulate matter and surface sediments of the Humber estuary, UK[END_REF][START_REF] Rogers | Assessment of PAH contamination in estuarine sediments using the equilibrium partitioning-toxic unit approach[END_REF][START_REF] King | Dynamic behaviour of polycyclic aromatic hydrocarbons in Brighton marina, UK[END_REF]. The atmosphere can be classed as a secondary dominant source of hydrocarbons in areas where the marine input is the primary dominant source [START_REF] Zhou | Seasonal distribution of dissolved pesticides and polynuclear aromatic hydrocarbons in the humber estuary and humber coastal zone[END_REF]. In the context of environmental monitoring, it is therefore imperative that all modes of PAH inputs are evaluated, not only oil spills. PAH spectrofluorimetric analysis in crustacean urine and associated physiological assessments are therefore an integrated approach which represents a realistic, relatively-inexpensive means of monitoring the bioavailability of PAHs (Dissanayake and Galloway, 2004;[START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]Watson et al., 2004a;2004b). The potential for PAH-induced biological impact, or specifically, cellular stress, as observed here by reduced lysosomal dye retention times corroborates previous evidence of reduced retention times with contaminant exposure [START_REF] Nicholson | Cardiac and lysosomal responses to periodic copper in the mussel Perna viridis (Bivalvia : Mytilidae)[END_REF][START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. Previous evidence of PAH-induced physiological effects includes tachycardia (increased heart rates) [START_REF] Bamber | Responses of shore crabs to physiological challenges following exposure to selected environmental contaminants[END_REF]Fossi et al. 2000;Camus et al. 2002;Dissanayake et al. 2008b) with acute exposure. In this study, however, only bradycardia (depressed heart rates) was observed and is indicative of chronic PAH exposure [START_REF] Hagger | Application of biomarkers to assess the condition of European Marine Sites[END_REF].

A key facet of the urinary PAH metabolite technique is its ability to identify PAH 'hotspots' within relatively large coastal areas impacted by PAH contamination which promotes its use in environmental management and regulation procedures. The PAH metabolite technique has been used successfully in various decapod crustaceans (Watson et al. 2004a;Eickhoff 2003a;[START_REF] Koenig | Non-destructive assessment of polycyclic aromatic hydrocarbon (PAH) exposure by fluorimetric analysis of crab urine[END_REF].

Implementation of the PAH metabolite technique, when used especially in deployed (caged) crabs, and in conjunction with other biological techniques, allows for contaminant-induced physiological impact to be monitored temporally and therefore can be used as important tools in environmental monitoring schemes (Galloway et al. 2002[START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. In summary, this study highlights the use of fluorescence

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spectrophotometry as a rapid, cost-effective biomonitoring tool of environmental contamination. Rapid screening techniques should therefore be incorporated with other physiological techniques (as shown here) as a means of primary modes of investigation within a framework of detailed investigative monitoring procedures, prior to mitigatory action being undertaken (Galloway et al., 2002;2004).

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Bosveld, A.T.C., de Bie, P.A.F., van den Brink, N.W., Jongepier, H. and Klomp, A.V., 2002. In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in The Netherlands. Chemosphere, 49(1), 75-83. Brown, R.J., Galloway, T.S., [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. Differential sensitivity of three marine invertebrates to copper assessed using multiple biomarkers. Aquatic Toxicology, 66(3), 267-278. Camus, L., Jones, M.B., Borseth, J.F., Regoli, F. and Depledge, M.H., 2002. Heart rate, respiration and total oxyradical scavenging capacity of the Arctic spider crab, Hyas araneus, following exposure to polycyclic aromatic compounds via sediment and injection. Aquatic Toxicology, 61(1-2), 1-13. Carballo, J.L., Naranjo, S.A. and García-Gómez, J.C., 1996. Use of marine sponges as stress indicators in marine ecosystems at Algeciras Bay (southern Iberian Peninsula Boca Raton,Florida.,M.C.,Casini,S.,Savelli,C.,Corbelli,C.,Franchi,E.,Mattei,N.,J.C.,Corsi,I.,Bamber,S. and

  potential and acute & chronic toxicity to marine organisms. Three PAHs, naphthalenes, pyrenes & benzo[a]pyrenes, are included in the USEPA priority pollutant list (MSC-E, 2001). Current monitoring techniques employed to determine environmental quality include the chemical analyses of sediment and water samples. PAHs are sparingly soluble in water and are difficult to detect and although they show a much greater association with sediments, chemical analysis of this material is laborious and costly due to the lengthy extraction methodology needed. Furthermore, sediment/waterbased measurements provide little information about contaminant bioavailability and toxic potential. Routine chemical monitoring usually involves determination of the concentration of parent compounds present in the environmental phase of interest.

  physiological (heart rate measurements) and cellular endpoints were used to determine potential physiological impact. Following urine extraction, heart rate measurements were performed on shore crabs by use of the non-invasive CAPMON system (Depledge andAndersen, 1990). Heart rate measurements were performed on individuals in 2 L aquaria (using water collected from each site); sensors were glued to the carapace above the heart and, after 30 min acclimation, cardiac activity (at rest) was recorded for 30 min as (as employed previously byDissanayake et al., 2008b). Lysosomal stability (using neutral red dye) assesses the lysosomal compartment of cells where toxins (e.g. PAHs) are accumulated prior to detoxication. If the lysosomes are overloaded, damage may lead to cell injury, tissue dysfunction, and reduction in animal "health status"[START_REF] Moore | Biological effects of contaminants: Measurement of lysosomal membrane stability ICES Techniques in Marine Environmental Sciences[END_REF]. Haemolymph was extracted by inserting a needle through the arthodial membrane at the base of the third walking leg and extracted into a syringe with 0.5 ml of crustacean physiological saline (0.5 M NaCl, 11 mM KCl, 12 mM CaCl 2 •6H 2 O, 26 mM MgCl 2 •6H 2 O, 45 mM Na 2 HPO 4 •12H 2 O, 45 mM Trisma-Base, 1M HCl; pH 7.4).

(

  reference) crabs (F 2, 21 = 49.77, P< 0.001 and log-transformed F 2, 21 = 32.89, P < 0.001, for PYR-and BAP-type metabolites, respectively) (Fig.2B and C). No differences in levels in any of the three PAH metabolite groups were observed between Palmones Estuary or Cadiz collected crabs. Physiological assessment of shore crabs collected from each site revealed that both Palmones Estuary and Gibraltar Harbour crabs were impacted as demonstrated by lower basal heart rates (log-transformed; F 2, 21 = 35.73, P< 0.001) (Fig.3A) and lower neutral red retention times (F 2, 21 = 450.19, P< 0.001) (Fig.3B) than Cadiz (reference) crabs.The investigation of the Bay of Algeciras (Palmones Estuary) and Gibraltar Harbour sites using caged crabs demonstrated the presence of PAHs (NAPs and BAPs) as observed in shore crab urine (Table1). High levels were recorded for all PAH metabolite types, especially PYR as significantly high concentrations (approx 125 -160 µg l -1 ) were observed in Palmones Estuary crabs (Fig.4A) over the 8 week period (F 5, 42 = 3.70, P < 0.01). Significantly high PYR concentrations (153-221 µg l -1 ) continuously in Gibraltar Harbour crabs (Coaling Island) (F 5, 42 = 4.01, P < 0.01). (Fig.4B), however, in the inner harbour site high levels were observed initially with a subsequent return to background levels (Fig.4C) (logtransformed; F 5, 42 = 4.41, P < 0.01). A ratio between 4 & 5 ring PAHs & 2-3 ring PAHs (FF BAP+PYR / FF NAP ) was calculated in order to determine PAH contamination source (Table

  from fluorescence spectroscopy indicates the presence of PAH metabolites at the sample sites characterised by strong PAH-associated fluorescence responses at specified wavelengths. Shore crabs caught from each site revealed inter-site variation with significant differences occurring between both Cadiz and Palmones Gibraltar harbour crabs, where high levels of urinary PAH were observed. The observed high levels of PAH metabolites verify the fact that Gibraltar Harbour has a greater amount of boating and harbour activity due in large part the presence of the Naval Base and dockyard when compared to San Fernando, Cadiz,

  similarity of metabolite levels found in indigenous populations of crabs from the reference site (Cadiz) and the intermediate Palmones Estuary may only reflect the bio-available water-borne PAH levels, however, previous studies have shown that the Palmones Estuary has high PAHs levels in sediments (641-725 mg kg -1 (Morales-[START_REF] Morales-Caselles | Sublethal responses in caged organisms exposed to sediments affected by oil spills[END_REF][START_REF] Ramos-Gómez | Acute toxicity measured in the amphipod Ampelisca brevicornis after exposure to contaminated sediments from Spanish littoral[END_REF]. High levels of all PAH metabolites were observed in the Palmones Estuary over the 8 week monitoring period, which was unexpected and may be largely attributed to a reported minor oil spill in the area over the background of a low level boating activity (inferred by the levels of NAP calculated pyrene concentrations in seawater). The presence of 4-ringed PAHs can also be inferred due to the elevated pyrene concentrations. The high FF BAP+PYR / FF NAP ratios, especially at week 4 (13.02) compared to control levels (4.87), indicates that the source of the PAHs may have been pyrogenic through the dominance of 4-and 5-ringed PAHs (Table

(

  2000) evaluated by measuring both benzo(a)pyrene monooxygenase activity and ethoxyresoru®n-O-deethylase activity (EROD) in both hepatopancreas and gills and remains to be tested. In this study, however, the pattern observed of increasing pyrene metabolite levels (weeks 1 -8) as seen in the Palmones Estuary deployed crabs (Fig.4a) does not suggest saturation of the biotransformation system and thus may reflect exposure levels.The results from this study indicate the effectiveness of using fluorescence spectrophotometry as a rapid, cost-effective technique for the detection of PAH exposure in field situations in tandem with physiological assessments to assess especially in large coastal bays. By extrapolating the pyrene concentrations from the metabolite concentrations, the use of the FF method when used on urine represents a very useful tool indeed as it facilitates PAH temporal monitoring. External control sites have therefore limited importance benchmarking environmental concentrations as each site or location can be used as an 'internal'

  of groups of PAH metabolites due to the incorporation of fluorescence contributions of other PAH compounds, such as chrysene, which fluoresces at both naphthalene & pyrene wavelength pairs(Aas et al., 2000a). It is recognised that FF 290/335 comprises mainly 2 & 3 ringed PAHs and 4 & 5 ring PAHs for wavelength pairs FF 341/383 & FF 380/430 respectively. Any monitoring programme which includes FF as a rapid screening technique for PAH exposure will, therefore, highlight the most frequent and prioritised PAH compounds associated with environmental monitoring(Aas et al., 2000a;[START_REF] Msc-E ; Naes | Occurrence of PAH in marine organisms and sediments from smelter discharge in Norway[END_REF]). The differences observed in PAH sources (pyrogenic and petrogenic) among the three deployment sites over time may also occur either by minor oil spills or from bunkering activities, characterised by high molecular weight PAHs (i.e. 4-5 ringed), or light engine fuels (low molecular weight PAHs; 2-3 ringed) and /or the atmospheric emissions of an oil refinery in the vicinity, which remains to be tested. PAH emissions are distributed via the atmosphere over a large area and its fate is dictated by and dependant on wind direction(Eickhoff et al., 2003a). Inputs from non-point sources such as atmospheric deposition and from roads would be diffuse and pollutants would be quickly dispersed in the water column and local

Fig. 4 .

 4 Fig. 4. Pyrene-type metabolite concentrations (µg l -1 ) (mean ± SE) from urine of deployed (caged) crabs over an 8 week monitoring period from A; Palmones Estuary, B; Gibraltar Harbour middle and C; Gibraltar Harbour inner sites (n = 8) (different letters denote statistical difference between weeks; same letters denote no statistical difference).

Fig. 5 .

 5 Fig.5. Shore crab physiological assessment (mean mean ± SE): A; heart rate (at rest) and B; neutral red retention time of deployed (caged) crabs over an 8 week monitoring period from Palmones Estuary and Gibraltar Harbour (middle and inner sites) (n = 8) (different letters denote statistical difference between sites within each week; same letters denote no statistical difference).

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M.H., 2002.

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Table 1 .

 1 Naphthalene-and benzo[a]pyrene-type metabolite fluorescence contributions (peak area mean ± SE) from urine of deployed crabs (caged) over an 8 week monitoring period from Palmones Estuary and Gibraltar Harbour (middle and inner sites) (n = 8) (different letters denote statistical difference between weeks; same letters (e.g. ab) denote no statistical difference to either groups a or b).

		Site		Palmones Estuary		Gibraltar Harbour (middle)			Gibraltar Harbour (inner)
	Metabolite	Week	Peak area	Week	Peak area		Week	Peak area
		type
			0	2784 ± 362 a	0	2784 ± 362 a		0	2784 ± 362 a
	Naphthalene-type	metabolites (NAP)	1 2 4 6 8	4101 ± 576 c 4817 ± 716 c 1742 ± 86b b 4016 ± 202 c 4488 ± 322 c	1 2 4 6 8	5659 ± 1175 b 4480 ± 286 b 4020 ± 287 b 2109 ± 345 a 4625 ± 528 b		1 2 4 6 8	4635 ± 500 b 4627 ± 337 b 2897 ± 182 a 3027 ± 185 a 3469 ± 367 ab
		Statistical		F5, 42 = 13.82, P < 0.001	log-transformed; F5, 42 = 4.82, P < 0.01		log-transformed; F5, 42 = 6.25, P < 0.001
		difference
	Benzo[a]pyrene -type	metabolites (PYR)	0 1 2 4 6	4781 ± 955 a 10236 ± 1739 12110 ± 1651 b b 3668 ± 761 a 11821 ± 1310 b	0 1 2 4 6	4781 ± 955 a 11570.19 ± 2015 12309 ± 2614 c 7679 ± 1426 b 3183 ± 977 a	c	0 1 2 4 6	4781 ± 955 a 9863 ± 2065 b 14766 ± 2816 bc 6401 ± 837 a 3760 ± 839 a
			8	14093 ± 2904 b	8	10153 ± 1454 bc		8	8919 ± 1713 b
		Statistical	log-transformed; F5, 42 = 10.64, P < 0.001	log-transformed; F5, 42 = 7.22, P < 0.001		log-transformed; F5, 42 = 45.47, P < 0.001
		difference

Table 2 .

 2 FF BAP+PYR / FF NAP ratio as an indicator of PAH contamination in a) indigenous sampled populations and in b) deployed shore crabs (n = 8). PETRO (predominance of 2-3 ringed PAHs) indicates petrogenic contamination and PYRO (predominance of 4-and 5-ringed PAHs) indicates pyrogenic contamination (i.e. ratio values of < 4.87 and > 4.87, respectively, relative to that of the control site). Only the lowest (i.e. petrogenic) and highest (pyrogenic) ratios are indicated here. Map showing the location of sites (see insets) within the Southwest Iberian Peninsula. Insets (1.) denote Cadiz (C) (relatively 'clean') (2) within the Bay of Algeciras (Palmones Estuary; P) and within Bay of Gibraltar (2a); Gibraltar Harbour inner (I) and middle (m). Fig. 2. Relative fluorescence contributions (peak area mean ± SE) of each of the PAH groups (A; NAP; naphthalene-type, B; PYR; pyrene-type and C; BAP; benzo[a]pyrene type in crabs caught from Cadiz, Palmones Estuary and Gibraltar Harbour (n = 8) (different letters denote statistical difference between sites; same letters denote no statistical difference).Fig. 3. Shore crab physiological assessment (mean ± SE): A; heart rate (at rest) and B; neutral red retention time of crabs caught from Cadiz, Palmones Estuary and Gibraltar Harbour (n = 8) (different letters denote statistical difference between sites; same letters denote no statistical difference).

	2a
			Site
		Cadiz (control)	Palmones Estuary	Gibraltar Harbour
		4.87	3.95 PETRO	36.14 PYRO
	2b
				Site
	Week	Palmones	Gibraltar Harbour	Gibraltar Harbour
		Estuary		(middle)	(inner)
	0	4.87		4.87	4.87
	1	5.87		7.42	5.90
	2	6.32		6.75	7.94
	4	13.02 PYRO	7.54	7.99
	6	8.36		9.32	5.10
	8	7.62		8.81	6.05

Acknowledgements

We thank the Royal Navy Dive Team for shore crab collection (HMS Naval Base) and the Queen's Harbour Master for sampling permission. We thank Darren Torres and Neil Perera for logistical assistance. We thank also the University of Cadiz (T.A del Valls), University of Sevilla (J. C. Garcia-Gomez) and Gibraltar Museum (Darren Fa) for use of laboratory facilities. Project financial support by grants from Gibraltar Government Ministry of Education (educational grant) and University of Plymouth (Erasmus exchange grant) awarded to AD.


Rafik Absi
email: r.absi@ebi-edu.com

Hitoshi Tanaka
email: tanaka@tsunami2.civil.tohoku.ac.jp

TOWARD IMPROVING PREDICTION OF SEDIMENT TRANSPORT OVER WAVE-INDUCED RIPPLES

 for sinusoidal and asymmetric waves;

(3) The vertical eddy viscosity profile depends on the wave non-linearity parameter and requires therefore a specific calibration.

INTRODUCTION

Coastal zones are of high vulnerability to natural hazards/disasters. Waves, currents and tides make coastal zones areas changing due to erosion and deposition of sediments. Thus, an understanding of sediment transport process in coastal zones is of crucial importance for accurate predictions of coast line evolution and sea-bed changes. However, the modeling of coastal sediment transport needs a compromise between two types of models: detailed mathematical models and engineering approaches. This compromise is imposed by on the one hand the accuracy of predictions and on the other hand the usability in practical applications [START_REF] Absi | Engineering modeling of wave-related suspended sediment transport over ripples[END_REF]. In coastal engineering practical accurate engineering models which take into account the more important involved physics, are needed.

In the engineering approach, the net (averaged over the wave period) total sediment transport is obtained as the sum of the net bed load and net suspended load transport rates [START_REF] Fredsoe | Mechanics of coastal sediment transport[END_REF]. For suspended load, the net sand transport is defined as the sum of the net current-related and the net waverelated transport components. The wave-related suspended transport component requires computation of the time-averaged suspended sediment concentration (SSC) profile and its integration in the vertical direction [START_REF] Van Rijn | United view of sediment transport by currents and waves II: Suspended transport[END_REF]. Computation of SSC needs the sediment diffusivity s  which is related to the eddy viscosity t  by the parameter β (i.e., the inverse of the turbulent Schmidt number). For moderate wave conditions and/or deep water, wave ripples can be formed on the sea bottom. If the ripples are relatively steep (η r /λ r ≥ 0.12, where η r is the ripple height and λ r is the ripple wavelength), the mixing close to the bed is dominated by coherent, periodic vortex structures. Above rippled beds, the mixing in the near bed layer is dominated by the mechanism of vortex shedding which entrains sediments. The aim of our study is to improve the prediction of SSC over ripples by using simple analytical tools which take into account the more important involved physics, for practical use in coastal engineering.

TIME-AVERAGED CONCENTRATIONS OVER WAVE-INDUCED RIPPLES

Sediment diffusivity ε s describes the disorganized ""diffusive" process. The process of vortex formation and shedding at flow reversal above ripples is a relatively coherent phenomenon. The associated convective sediment entrainment process may also be characterized as coherent, instead of a pure disorganized ""diffusive" process represented in the classical gradient diffusion model [START_REF] Thorne | Suspended sediments under waves measured in a largescale flume facility[END_REF]. [START_REF] Nielsen | Coastal bottom boundary layers and sediment transport[END_REF] indicated that both convective and diffusive mechanisms are involved in the entrainment processes. In the combined convection-diffusion formulation, the steady state advection-diffusion equation is given by 0

   conv s s F dz dc c w  (1)
The respective terms in (1) represent downward settling, upward diffusion (given by gradient diffusion , where F(z) is a function describing the probability of a particle reaching height z above the bed [START_REF] Nielsen | Coastal bottom boundary layers and sediment transport[END_REF]. [START_REF] Thorne | Observations and analysis of sediment diffusivity profiles over sandy rippled beds under waves[END_REF] wrote



(2)

where

s s     *
and  is a parameter related to convective sediment entrainment process associated to the process of vortex shedding above ripples

    c w F s conv / 1 / 1   
. With the upward convection

) ( 0 z F c w F s conv   ,  becomes equal to     ) ( / 1 / 1 0 z F c c  , while with w w conv c w F   (Thorne et al., 2009)     c w c w s w w / 1 / 1   
. The condition of [START_REF] Sheng | Sediment eddy diffusivities in the nearshore zone, from multifrequency acoustic backscatter[END_REF]  

2 . 0 /  c w c w s w w
shows therefore that when the convective transfer is very small (above low steepness ripples), [START_REF] Absi | Engineering modeling of wave-related suspended sediment transport over ripples[END_REF]. From equations ( 1) and ( 2), it is possible to write

1   and therefore s s    *
s diff conv s F F             1 * (3) and therefore   diff conv F F / 1  
. This equation shows that  depends on the relative importance of coherent vortex shedding (related to In order to allow adequate predictions of suspended sediment transport, it is important to understand interaction between sediment particles and turbulence of fluid flow. The turbulent diffusion of suspended sediments s  is given by

t s     (5)
where  = inverse of the turbulent Schmidt number, describes the difference between diffusivity of momentum (diffusion of a fluid "particle") and diffusivity of sediment particles. It should depend on the particle Stokes number [START_REF] Absi | Turbulent diffusion of suspended particles: analysis of the turbulent Schmidt number, Defect and Diffusion Forum[END_REF]). However, for simplicity and in order to allow analytical analysis, we suggested a simple equation

      / exp z C b 
; where b  = the value of  close to the bed and  C = coefficient [START_REF] Absi | Concentration profiles for fine and coarse sediments suspended by waves over ripples: An analytical study with the 1-DV gradient diffusion model[END_REF]. This  (y) profile increases with z for  C > 0 and decreases for  C < 0. We used an analytical eddy viscosity given by 1.

Table 1. Flow parameters 2) [START_REF] Absi | Concentration profiles for fine and coarse sediments suspended by waves over ripples: An analytical study with the 1-DV gradient diffusion model[END_REF].

Figure 1 shows time-averaged concentrations of fine (○) and coarse (×) sediments suspended by waves over ripples. The present method allows a good description of concentration profiles for both fine (dashed line) and coarse sand (solid line). The parameters were chosen to give a good fit.

For an eddy viscosity given by: However in order to allow practical use for predictive purpose, the method needs calibration. Before calibrating parameters of α and β, we need to assess and validate the eddy viscosity profile given by Eq. ( 6).

ANALYTICAL EDDY VISCOSITY FORMULATION

Eq. ( 6) was used as an empirical equation. However in order to allow more general use, we need a deeper theoretical analysis.

Steady plan channel flows: analysis by DNS data

In the equilibrium region z+>50, the turbulent kinetic energy (TKE) is given by [START_REF] Nezu | Turbulence in Open-Channel Flows[END_REF]. Since in the inner region the streamwise velocity profile is given by the loglaw, it is possible to write a mixing length as

) / exp( *  z C u k k   (Nezu
) / exp(   z C z l k m  
and therefore Eq. ( 6) for eddy viscosity. Figure 2 shows TKE profiles given by two analytical solutions [START_REF] Nezu | Turbulence in Open-Channel Flows[END_REF]Nakagawa 1993, Absi 2008) and eddy viscosity profiles (white dashed lines) given by Eq. ( 6). In figure 2, variables with the superscript of + are those nondimensionalized by the friction velocity and the kinematic viscosity as

 / * u z z   ; * / u k k   ;    / t t   .
Comparisons with DNS data (data of [START_REF] Iwamoto | Database of fully developed channel flow[END_REF], Iwamoto et al. 2002[START_REF] Hoyas | Scaling of velocity fluctuations in turbulent channels up to Re τ = 2003[END_REF] show that Eq. ( 6) provides accurate description of DNS in the equilibrium region [START_REF] Absi | Turbulent diffusion of suspended particles: analysis of the turbulent Schmidt number, Defect and Diffusion Forum[END_REF]. [START_REF] Absi | Analytical solutions for the modeled k equation[END_REF][START_REF] Absi | Turbulent diffusion of suspended particles: analysis of the turbulent Schmidt number, Defect and Diffusion Forum[END_REF].

Oscillatory flows: analysis by a two-equation model

Eq. ( 6) for eddy viscosity was validated for the case of steady plane channel flow. However for use in wave boundary layers, we need to assess this equation for the case of oscillatory flows. Eq. ( 6) is therefore analyzed by the baseline (BSL) k-ω model. This model allows accurate prediction of velocity profiles in oscillatory boundary layers [START_REF] Suntoyo | Effect of bed roughness on turbulent boundary layer and net sediment transport under asymmetric waves[END_REF]. Figure (3.b) shows comparison between period-averaged eddy viscosity obtained by BSL k-ω model (symbols) and analytical profile of Eq. (6) (dashed line). Even if the eddy viscosity is highly time-dependent (figure 3.a), the period-averaged dimensionless eddy viscosity (Figure 3.b) has a shape which is well described by the analytical profile given by Eq. ( 6) for z/z h < 0.6 (figure3.b) where z h is the water depth or the distance from the wall to the axis of symmetry or free surface. Figure (3.c) presents temporal and spatial variation of dimensionless eddy viscosity for asymmetric waves. Figure (3.d) shows comparison between period-averaged eddy viscosity obtained by BSL k-ω model (symbols) and analytical profile of Eq. (6) (dashed line). Even for the case of asymmetric wave, the periodaveraged dimensionless eddy viscosity has a shape which is well described by Eq. ( 6) for z/z h < 0.5 (figure3.d).

Figures (3.b) and (3.d) shows that the period-averaged eddy viscosity profile for sinusoidal wave is different from the profile for asymmetric wave. This indicates that the period-averaged eddy viscosity profile should depend on the wave non-linearity parameter given by N i =U c /û, where U c is the velocity at wave crest and û is the total velocity amplitude. We need therefore a specific calibration for parameters of Eq. ( 6) using full-range equations of friction coefficient [START_REF] Tanaka | Full-range equation of friction coefficient and phase difference in a wavecurrent boundary layer[END_REF] and wave boundary layer thickness [START_REF] Sana | Full-range equation for wave boundary layer thickness[END_REF].

CONCLUSIONS

The main conclusions of the present study are:

-A modified advection-diffusion equation with an additional parameter α related to the process of vortex shedding above ripples allows a good description of suspended sediment concentration profiles -For practical applications the period-averaged eddy viscosity could be described by a simple analytical formulation -The shape of the analytical period-averaged eddy viscosity formulation was validated by BSL k-ω model for sinusoidal and asymmetric waves -Period-averaged eddy viscosity profile depends on the wave non-linearity parameter and requires therefore a specific calibration.

  of concentrations and vertical velocity and the overbar denotes time averaging. It is possible to write (1) in the form of a diffusion equation. The time-averaged (over the wave period) advection-diffusion equation is given therefore by[START_REF] Absi | Concentration profiles for fine and coarse sediments suspended by waves over ripples: An analytical study with the 1-DV gradient diffusion model[END_REF]

  4) provides a link between upward concavity/convexity of concentration profiles (in semi-log plots) and increasing/decreasing of * s  . Increasing * s  allows upward concave concentration profile, while decreasing * s  allows an upward convex concentration profile.

  = the friction velocity (m/s), δ = the boundary layer thickness (m), κ = the Karman constant (=0.41) and C a parameter =1.12[START_REF] Hsu | Calibration of Businger-Arya type of eddy viscosity models parameters[END_REF][START_REF] Absi | Discussion of Calibration of Businger-Arya type of eddy viscosity models parameters[END_REF][START_REF] Absi | Concentration profiles for fine and coarse sediments suspended by waves over ripples: An analytical study with the 1-DV gradient diffusion model[END_REF]). Using the  -function and eddy viscosity (6), the sediment diffusivity is given therefore by and h s are two parameters.Test case: Fine and coarse sediments over rippled beds in the same flow (McFetridge and Nielsen, 1985)Maximum value of the free stream velocity, wave period, mean depth of the flow, orbital amplitude or near-bed flow semi-excursion, mean ripple height, mean ripple wavelength, equivalent roughness

Figure 1 :

 1 Figure 1: Time-averaged concentration profiles over wave-induced ripples. Symbols: measurements (McFetridge and Nielsen, 1985), (○) fine; (×) coarse; Curves: solutions of Eq. (2) (Absi 2010).

  could be related to an inaccurate estimation of the boundary layer thickness. For coarse sediments, the profile of * s  (Absi 2010, solid line in figure7) shows the effect of parameter  which indicates that vortex shedding occurs at z <0.015m.

Figure 2 :

 2 Figure 2: Turbulent kinetic energy (left) and eddy viscosity (right) profiles in plan channel flows for different friction Reynolds numbers. Symbols: DNS data; Lines: analytical[START_REF] Absi | Analytical solutions for the modeled k equation[END_REF][START_REF] Absi | Turbulent diffusion of suspended particles: analysis of the turbulent Schmidt number, Defect and Diffusion Forum[END_REF]

  Figure (3.a) presents temporal and spatial variation of dimensionless eddy viscosity for a sinusoidal wave.

Figure 3 :

 3 Dimensionless eddy viscosity; Left: Temporal and Spatial Variation; Right: Period-averaged dimensionless eddy viscosity; Top: sinusoidal wave; Bottom: asymmetric wave.

ACKNOWLEDGMENTS

The first author is grateful for the financial support provided by Japan Society for the Promotion of Science (JSPS), within the FY2010 JSPS Invitation Fellowship Program for Research in Japan (No. S-10168).