F Forster

S Goodsir

Jennings

M Schratzberger
email: michaela.schratzberger@cefas.co.uk

R M Forster

F Goodsir

S Jennings

Nematode community dynamics over an annual production cycle in the central North Sea

Keywords: Nematodes, Taxonomic and functional community composition, Benthic-pelagic coupling, Food quality, Food quantity, North Sea

Nematode community dynamics over an annual

production cycle in the central North Sea

Introduction

The ecology of marine food webs at higher latitudes is strongly influenced by broadly predictable production cycles, driven by changes in nutrient supply, solar radiation and wind stress [START_REF] Colebrook | The seasonal cycle of the plankton in the North Sea and the north-eastern Atlantic[END_REF]. The production cycle in stratified temperate seas is characterised by one or more distinct phytoplankton blooms. The spring bloom is often dominated by larger-celled groups such as diatoms, followed by proliferation of nano-and picoplankton populations throughout the summer and autumn. The fate of carbon generated by phytoplankton depends on whether energy and nutrients fixed during blooms are recycled in the water column, exported to higher trophic levels or transferred to the seabed [START_REF] Jago | Size and settling velocity of suspended flocs during a Phaeocystis bloom in the tidally stirred Irish Sea, NW European shelf[END_REF]. After the bloom, fresh organic material sinking to the bottom is partly incorporated into the sediment as detrital particles or becomes adsorbed to sediment grains.

Sedimentation of organic matter represents the major input of production to the benthic community on an annual basis and is a major factor influencing benthic community structure, biomass and metabolism [START_REF] Graf | Benthic response to sedimentation of a spring phytoplankton bloom: process and budget[END_REF]. Energy produced during annual production cycles propagates through the food web, with increasing lag and attenuation as it passes to larger animals with slower turnover times [START_REF] Pope | Successful surf-riding on size spectra: the secret of survival in the sea[END_REF]. Smaller benthic animals are likely to track the production cycle most closely, as their high relative rates of consumption and fast turnover times allow them to rapidly assimilate available production. The ecology and life histories of meiofauna (invertebrates that pass through a 500 -1000 m mesh sieve but are retained on a 32 -63 m mesh sieve; [START_REF] Giere | Meiobenthology -the microscopic fauna in aquatic sediments[END_REF] suggest that their population and community dynamics will respond to the seasonality of production.

Meiofauna play an important role as intermediaries between the microbial/detrital compartments (by influencing bacterial density and biomass; [START_REF] Mikola | Effects of microbivore species composition and basal resource enrichment on trophic-level biomasses in an experimental microbial-based soil food web[END_REF], and larger organisms (by serving as major food sources for higher trophic levels; [START_REF] Danovaro | Trophic importance of subtidal metazoan meiofauna: evidence from in situ exclusion experiments on soft and rocky substrates[END_REF] in food webs [START_REF] Chardy | Carbon flows in a subtidal fine sand community from the western English Channel: a simulation analysis[END_REF][START_REF] Schmid-Araya | The importance of meiofauna in food webs: evidence from an acid stream[END_REF]. However, little is known about how food quality and quantity affect natural populations [START_REF] Findlay | Effect of detrital nutritional quality on population dynamics of a marine nematode (Diplolaimella chitwoodi)[END_REF][START_REF] Danovaro | Biodiversity and trophic structure of nematode assemblages in seagrass systems: evidence for a coupling with changes in food availability[END_REF]. Seasonal data on meiofauna abundance, reproduction and species composition are essential for conceptualising and modelling trophic dynamics [START_REF] Fleeger | Meiofaunal densities and copepod species composition in a Louisiana, USA, estuary[END_REF] but to date, relationships between faunal characteristics and phytoplankton sedimentation have proven elusive. This is partly a result of the rarity of surveys which monitor phytoplankton sedimentation and faunal responses adequately. Thus most ecological studies of meiofauna have focused on intertidal or shallow subtidal habitats. When deeper water environments were studied, sampling designs rarely permitted repeated sampling at multiple stations or over more than one season (e.g. Vanaverbeke et al., 2004a and references therein).

Phytoplankton sedimentation is likely to affect meiofauna communities as many species live close to the sediment surface, feeding on diatoms and epigrowth (i.e. algal cells attached to particles). Fresh organic material is expected to reach deeper dwelling, deposit and bacteria feeding species during later stages of phytoplankton decomposition [START_REF] Graf | Benthic response to sedimentation of a spring phytoplankton bloom: process and budget[END_REF][START_REF] Rudnick | The lags between the deposition and meiobenthic assimilation of phytodetritus[END_REF]. Indeed, temperature and food availability are the factors most often invoked to explain seasonal changes in meiofaunal abundance [START_REF] Heip | The ecology of marine nematodes[END_REF][START_REF] Ólafsson | Seasonal dynamics of sublittoral meiobenthos in relation to phytoplankton sedimentation in the Baltic Sea[END_REF].

Taxon-based approaches are commonly used to study temporal dynamics of meiofauna.

Aggregating taxonomically similar species or different developmental stages of the same species, however, does not take account of the diverse biology and autecological requirements of the organisms [START_REF] Schratzberger | Functional diversity of nematode communities in the southwestern North Sea[END_REF]. These issues have been taken into account by adopting functional and size-based approaches. The functional group approach classifies organisms by morphological characteristics that are thought or known to represent an important ecological function [START_REF] Chalcraft | Mapping functional similarity of predators on the basis of trait similarities[END_REF]. However, links between morphology and function can be weak and inconsistent, in some cases leading to an uncertain separation of taxonomic and functional approaches. The size-based approach focuses on the aggregation of organisms based on individual body weight, often independent of their taxonomy. Size is linked to processes such as metabolism and productivity, but size-based approaches may not reflect complex differences in the feeding ecology of different sized organisms.

Since taxonomic, functional and size-based approaches focus on different aspects of assemblage structure, a combination of all three methods is likely to provide better understanding of seasonal changes in assemblage structure, dynamics and responses to changing food supply. Here, we study meiofaunal nematode community dynamics in the central North Sea to assess how taxonomic, trophic and biomass distribution patterns varied during a seasonal production cycle. We relate community changes to changing food quality and quantity in the sediment to investigate two questions:

(1) Does a local increase in food promote consistent temporal changes in the taxonomic, trophic and size structure of nematode communities?

(2) Do selected nematode species, representing the main taxonomic and trophic categories available to the phylum, respond in a differential way to a pulsed food supply?

Material and methods

Study site

We studied nematode communities in the Silver Pit, central North Sea (Fig. 1). The Silver Pit is predominantly 60 -80 m deep with a muddy-sand substratum and the water column stratifies in summer months. Parts of the Silver Pit are fished by beam trawlers but there is limited evidence that trawling disturbance has significant effects on nematode community structure. Thus a previous experimental study in the Southern North Sea, investigating acute effects of trawling disturbance on nematodes, showed that seasonal changes in assemblage structure were greater than those attributed to trawling [START_REF] Schratzberger | Impacts of trawling on the diversity, biomass and structure of meiofauna assemblages[END_REF]. Subsurface temperature at 35 m depth at the sampling stations was low during spring and summer (6 -8 °C February to June, 9 -10 °C in July), peaked in late summer (11 -16 °C August to October) and declined again during autumn and winter months (8 -10 °C November to January) (Paul McCloghrie, unpublished data).

Sample collection

Sediment samples were collected with a NIOZ corer on six cruises in [START_REF] Deprez | NeMys[END_REF]and 2006(21 -26 May, 12 -16 July, 29 September -4 October, 5 -10 November 2005;13 -17 February, 26 April -5 May 2006) from seven randomly chosen locations within 50 m of each of three stations (Fig. 1). The NIOZ corer takes a circular core of 0.1 m 2 to a depth of > 30 cm. The surface of the sediment core is relatively undisturbed, and the corer retains a few cm of the overlying water.

From each deployment, two sediment sub-samples were collected with a perspex corer (2 cm diameter, 3.14 cm 2 surface area) to a depth of 5 cm. On each sampling date, sub-samples were combined by station and frozen at -20 °C prior to the analysis of particle size and organic carbon content. From each of the first three deployments, two further sub-samples were collected, one for the analysis of sediment chlorophyll a, the other for the analysis of sediment porosity. These sub-samples were frozen separately at -20 °C pending analysis. As sediments reflect a time-averaged input of phytodetritus to the benthic system, sediment chlorophyll a concentrations are likely to show a more reliable pattern of annual algal biomass and sedimentation than near-bottom water samples. Boon et al. (1998) and [START_REF] Franco | Effect of phytoplankton bloom deposition on benthic bacterial communities in two contrasting sediments in the southern North Sea[END_REF] showed that the typical algal spring bloom is quantitatively reflected in the chlorophyll a concentration of the majority of North Sea sediments.

From each of the last four deployments at each location, a meiofauna sub-sample was collected with a 5.5 cm internal diameter perspex tube (surface area 23.76 cm 2 ) to a depth of 5 cm. All faunal samples were fixed and preserved in 10 % formalin in filtered seawater.

Sample processing

intervals, down to 1 phi (500 µm). The fraction < 500 µm was freeze dried and analysed on a Coulter LS 130 Laser sizer. The laser sizer results were combined with the dry sieve results to give the full particle size distribution. The mean particle size diameter (hereafter referred to as grain size) was calculated from these results [START_REF] Dyer | Coastal and estuarine sediment dynamics[END_REF]. The total organic carbon content of the sediment fraction < 63 µm was determined with a Leeman CE 440 elemental analyser.

Total organic carbon in the sediment consists of a variety of compounds with different decomposition rates based on their structures and susceptibility to biological attack [START_REF] Henrichs | Decomposition of 14 C-labelled organic substances in marine sediments[END_REF]. Organic matter in the majority of marine sediments is complex and generally of less degradable (i.e. refractory) nature [START_REF] Fabiano | Composition of organic matter in sediments facing a river estuary (Tyrrhenian Sea): relationships with bacteria and microphytobenthic biomass[END_REF].

Defrosted sediment chlorophyll a samples were extracted in neutralised 90 % acetone for 24 h at 4 °C in the dark. Measurements of extracted chlorophyll fluorescence were made (before and after acidification with 8 % hydrochloric acid to distinguish phaeopigments) using a Turner Design (Model 10 AU) filter fluorometer [START_REF] Tett | Plankton[END_REF]. Sediment chlorophyll a and phaeopigment concentrations in the interstitial water were corrected for porosity, determined from the dry weights and wet weights of known volumes of sediment. Sediment chlorophyll a concentrations were used as an index of phytodetrital concentration (Boon and Duineveld, 1998). Temporal changes in chlorophyll a breakdown products (phaeopigments) and chlorophyll a:phaeopigment ratios were tracked as indices of phytoplankton bloom degradation.

After washing the meiofauna samples onto a 38 µm sieve, fauna were extracted with Ludox TM 40 [START_REF] Somerfield | Meiofauna in marine pollution monitoring programmes[END_REF]. The extraction was repeated three times. The extracts were sub-sampled following the method described by [START_REF] Somerfield | Meiofauna in marine pollution monitoring programmes[END_REF]. Sub-samples of 5 % of the extracted material (containing 85 -448 nematodes, average = 226 nematodes) were evaporated slowly in anhydrous glycerol and mounted on slides for further analysis. The numbers of individuals identified should give reasonable, and comparable, estimates. Nematodes consistently comprised > 95 % of total meiofauna abundance in all samples. Temporal dynamics of nematode communities in the Silver Pit were investigated by analysing changes in their taxonomic and trophic composition as well as changes in their size distribution.

Taxonomic analysis

All nematodes in each sub-sample were identified to genus or species level, using the taxonomic keys of Platt andWarwick (1983, 1988) and [START_REF] Warwick | Free-living marine nematodes[END_REF] and the online information system NeMys [START_REF] Deprez | NeMys[END_REF]www.nemys.ugent.be). These are based on a phylogenetic classification scheme, where the structure of the taxonomic tree approximates the evolutionary relationships among organisms. Changes in phylogenetic diversity of species assemblages, however, are not explicitly linked to changes in functional diversity and so their ecological significance can be difficult to assess [START_REF] Walker | Plant attribute diversity, resilience, and ecosystem function: the nature and significance of dominant and minor species[END_REF][START_REF] Warwick | Practical measures of marine biodiversity based on relatedness of species[END_REF]

Trophic analysis

Despite the uniform organisation of free-living nematodes, a great diversity in mouth structure exists which reflects the ecological role of the species [START_REF] Heip | The ecology of marine nematodes[END_REF]. Based on these morphological characteristics, [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF] devised a classification of feeding types including selective deposit feeders consuming bacteria and small-sized organic particles, nonselective deposit feeders also feeding on organic deposits but targeting larger-sized particles, epigrowth feeders scraping food off surfaces or feeding on diatoms and microalgae and predators feeding on nematodes and other small invertebrates. The disadvantage of this scheme is that it confines species to a single trophic role when many non-selective deposit feeders are known to be omnivores. For example, while the omnivores may feed on particles of a restricted size, their food can include bacteria, small flagellates and non-living aggregates. Moreover, many selective deposit and epigrowth feeders feed on diatoms but these are only numerous in shallow sediments and little is known about their diet in deeper sediments [START_REF] Heip | The ecology of marine nematodes[END_REF] and some genera classed as predators may scavenge dead organic material as juveniles. Thus various authors have revised and modified Wieser's classification [START_REF] Romeyn | Food selection and consumption by estuarine nematodes[END_REF][START_REF] Jensen | Feeding ecology of free-living aquatic nematodes[END_REF][START_REF] Moens | Observations on the feeding ecology of estuarine nematodes[END_REF][START_REF] Moens | Use of carbon and energy sources by nematodes[END_REF]. However, in the absence of experimental evidence to describe the feeding habits of most species, Wieser's scheme remains the most feasible trophic classification of freeliving marine nematodes from a variety of habitats.

Size-based analysis

Many biological and ecological processes are related to body size [START_REF] Peters | The ecological implications of body size[END_REF][START_REF] Brown | Towards a metabolic theory of ecology[END_REF]. In contrast to taxonomic and functional approaches, size-based analyses assume that all organisms of the same size, independent of their taxonomic or functional identity, will behave in approximately the same way in bioenergetic terms.

Individual body sizes were determined for nematodes in three of the four faunal replicates.

One percent of all nematodes in each replicate (corresponding to a total of 2454 individuals), irrespective of species identity, were randomly selected for the calculation of individual body mass from allometric relationships linking body dimensions to mass. Nematode length (except long, filiform tails where present) and maximum width were measured using image analysis (Image Pro Plus version 4.5). The body mass of individuals was then calculated with the formulae of [START_REF] Andrassy | Die Rauminhalts-und Gewichtsbestimmung der Fadenwürmer (Nematoden)[END_REF]. Although more laborious, this volumetric, as opposed to a gravimetric, method was chosen since it is non-destructive and keeps measured specimens intact for taxonomic identification and the analysis of size distributions [START_REF] Udalov | Depth-related pattern in nematode size: what does the depth itself really mean?[END_REF]. Nematode size distributions were described for each sample by assigning individuals to log 2 body-mass classes and calculating the proportion of all individuals in each class.

The similarities percentages procedure (SIMPER; [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF] was used to identify species that consistently contributed to spatio-temporal differences between assemblages. These included the epigrowth feeder Spilophorella paradoxa, the non-selective deposit feeder Sabatieria punctata and the selective deposit feeder Leptolaimus elegans and individual body mass was therefore also estimated for these species separately. The percentages of organisms measured were 3 % of all Sabatieria punctata (corresponding to a total of 1479 individuals) and 5 % of all Spilophorella paradoxa and Leptolaimus elegans (corresponding to a total of 372 and 1129 individuals respectively).

Coupling of sediment and water column chlorophyll a

An archive of surface chlorophyll a concentration images, derived from MODIS-Aqua satellite ocean colour data using a modified version of the case-II water algorithm 'OC5' [START_REF] Gohin | A five channel chlorophyll concentration algorithm applied to Sea WiFS data processed by SeaDAS in coastal waters[END_REF], was processed and supplied by Ifremer, France. This regional algorithm differs from generic, global satellite chlorophyll products in that additional sensor bands are used to correct for the presence of suspended sediments and the chromophoric (coloured) fraction of dissolved organic matter (cDOM). The surface chlorophyll maps cover the northwest Atlantic shelf at a resolution of 1 km 2 and were made available for the time period June

Data processing 2.5.1. Univariate methods

Spatial and temporal patterns of environmental variables were investigated using Kruskal-Wallis one-way analysis by ranks where measures are transformed to ranks, ignoring group membership. The ranks were used to test the null-hypothesis (H 0 ) that there was no significant shift in the centre of the groups between stations and over time. There was no statistically significant co-variation between the environmental measures included in the analyses.

Total nematode abundance, species diversity of the entire community and diversity within the four feeding types (H') were calculated from the species abundance data [START_REF] Shannon | The mathematical theory of communication[END_REF]:

) (ln ' 1 i S i i p p H = =
where p i is the number of individuals in the i th species (n i ) divided by the total number of individuals (N).

The Index of Trophic Diversity (ITD), based on the proportion of each of four feeding types was calculated following [START_REF] Heip | The ecology of marine nematodes[END_REF]. ITD values range from 0.25 (highest trophic diversity with the four trophic groups accounting for 25 % each) to 1.0 (lowest trophic diversity when a single feeding type is present):

ITD = θ 2
where θ is the percentage contribution of each of four feeding types according to [START_REF] Wieser | Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF].

Average individual body mass was calculated from the body dimensions across all species present in the total nematode community and for populations of Spilophorella paradoxa, Sabatieria punctata and Leptolaimus elegans.

Bartlett's and Cochran's tests were used to test for homogeneity of variance before spatiotemporal variations in all community attributes were analysed by two-way analysis of variance (ANOVA). Following the detection of significant 'station' and sampling 'month' effects, the Tukey HSD multiple comparisons test was used in pair-wise comparisons of samples. When the interaction term station x month was significant, the two-way ANOVA was broken down to a one-way ANOVA.

All univariate statistical analyses were performed using the software package Statgraphics Plus version 3.3.

Multivariate methods

Non-metric multi-dimensional scaling (MDS) ordination using the Bray-Curtis similarity measure (Clarke et al., 2006) was applied to the relative abundance (i.e. proportion) of species, feeding types and geometric size classes [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF]. The statistical significance of spatial differences (averaged over the 12-month study period) and changes in distribution patterns over time (averaged over the three stations) were assessed by two-way crossed analysis of similarities (ANOSIM; [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF], a non-parametric analogue of the univariate ANOVA. BIO-ENV [START_REF] Clarke | A method of linking multivariate community structure to environmental variables[END_REF] and BVSTEP [START_REF] Clarke | Quantifying structural redundancy in ecological communities[END_REF] were used to investigate relationships between environmental variables and the species, feeding type and size composition of nematode communities. Spearman rank correlations (R) between resemblance matrices derived from faunal data (based on Bray-Curtis similarity) and matrices derived from various sub-sets of environmental data (based on normalised Euclidean Distance) were used to identify suites of variables that best explained observed community patterns. The significance of the correlations was determined using a permutation procedure [START_REF] Stoffels | Spatial scale and benthic community organisation in the littoral zones of large oligotrophic lakes: potential for cross-scale interactions[END_REF].

All multivariate statistical analyses were performed using Primer version 6.1.5 [START_REF] Clarke | PRIMER v6. User manual[END_REF].

Results

Benthic habitat in the Silver Pit

All stations comprised soft bottom, muddy habitat. Stations were comparable with respect to water depth (A: 63 m, B: 64 m, C: 55 m) and sediment granulometry, although grain size at station C was slightly smaller than at station A and B (Table 1). The silt-clay content of the sediment did not differ significantly between stations and seasons, nor did the content of total organic carbon (Table 2). A temporal trend in chlorophyll a and phaeopigment concentrations in the sediment was apparent with a distinct peak in May 2005 and to a lesser extent in September 2005 (Tables 1 and2, Fig. 2).

Coupling of sediment and water chlorophyll a

Pelagic chlorophyll a concentrations were typically higher to the north and to the south of the Silver Pit than over the central Silver Pit (see Fig. 1 for a monthly composite image of April 2005). Time series analysis showed that the spring bloom started in the first week of April in both 2005 and 2006. Water chlorophyll a concentration was low in summer in both years, especially at station C (Fig. 3). It is likely that a deep chlorophyll maximum is present during this period of strongly stratified conditions [START_REF] Olesen | Comparison of the sedimentation of a diatom spring bloom and of a subsurface chlorophyll maximum[END_REF][START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF], but this cannot be detected by the satellite. There was an autumn bloom in both years peaking during late September and early October. The spring bloom of 2006 was more pronounced than in 2005.

The most productive station was station A with a mean water chlorophyll a concentration of 0.75 (± 0.60) mg m -3 over the study period. Station B was intermediate with 0.64 (± 0.44) mg m -3 and station C was lowest with a mean value of 0.55 (± 0.37) mg m -3 . Hence there was a gradient in pelagic productivity through the Silver Pit from west (high) to east (low), with station A being closest to the areas of high primary production to the north and south of the Silver Pit (Fig. 1). Higher phytoplankton abundance at the surface was reflected in higher pigment concentrations in the upper layers of the sediment, particularly during the spring bloom of both years (Fig. 3). However, the relationship between surface production and benthic deposition differed considerably between 2005 and 2006 (Fig. 3). It is unknown to what extent the settling particles are buried in the sediment at the place they arrive on the seafloor and how much is lost to other areas by advection near the sediment surface.

The temporal variability of pulses of primary production reaching the seafloor along the gradient of pelagic productivity allowed us to investigate the relationship between nematode taxonomic, trophic and size structure and changes in food quality and quantity.

Nematode species and feeding types

The analysis of spatio-temporal differences in community-related measures (Table 3) showed that density, species diversity and trophic diversity were higher at station A and B than at station C. Similar trends were recorded for species diversity within the four feeding types 3).

As shown in Table 4, the relative abundance of selective deposit feeders (F = 78.56, p < 0.001) and predators (F = 15.43, p < 0.001) were lowest at station C whereas communities at station A were characterised by relatively low proportions of epigrowth feeders (F = 26.75, p < 0.001). Lowest proportions of non-selective deposit feeders were recorded at station B (F = 6.12, p = 0.004). Temporal patterns of nematode feeding types were inconsistent amongst stations and generally not statistically significant at p > 0.05. There were significant spatial differences in distribution patterns of nematode species (R = 0.583, p = 0.001) and feeding guilds (R = 0.492, p = 0.001) and these generally dominated temporal dissimilarities (species assemblages: R = 0.213, p = 0.001, trophic groups R = 0.065, p = 0.089; Fig. 5).

Distribution patterns of nematode species were correlated with grain size, total organic carbon content in the sediment and sedimentary chlorophyll a:phaeopigment ratios (R = 0.533, p = 0.010). Spatial differences in species distribution patterns were most marked in May 2005 when station-specific differences in chlorophyll a:phaeopigment ratios were highest (Table 1).

A combination of grain size and total organic carbon content in the sediment best explained the trophic structure of assemblages (R = 0.424, p = 0.010). Spatial differences in the trophic structure of nematode assemblages were most pronounced in April 2006 and coincided with low levels of total organic carbon in the sediment (Table 1).

Nematode body size

Spatially significant differences in individual body mass across the entire community included a higher average individual body mass at station C than at station A and B whereas the opposite trend was observed for populations of the epigrowth feeder Spilophorella paradoxa and the non-selective deposit feeder Sabatieria punctata (Table 5). Average individual body mass remained stable over the 12-month study period for the total community and for Sabatieria punctata and Leptolaimus elegans populations. Individual body mass of Spilophorella paradoxa, in contrast, peaked in September 2005 at all stations (Fig. 6).

In contrast to functional analyses, the investigation of the distribution of both absolute (not shown) and relative nematode abundance across body size classes revealed significant temporal differences. Conversely, spatial differences were less pronounced when the total community was taken into account (Table 6). We observed a shift from larger to smaller individuals over the 12-month production cycle but this could not be linked to any of the measured environmental variables (p > 0.05).

At all three sampling stations, populations of the epigrowth feeder Spilophorella paradoxa were dominated by mature adults, as indicated by an abundance peak in size class -1 (Fig. 7).

Significant spatial and temporal dissimilarities in the body size distribution (Table 6) were clearly related to differences in the total organic carbon content and the progression of chlorophyll a decomposition (i.e. chlorophyll a:phaeopigment ratio) in the sediment throughout the annual production cycle (R = 0.368, p = 0.040). There was a clear temporal succession in the size distribution of individuals in the population of Spilophorella paradoxa (Fig. 8) following deposition of the spring phytoplankton bloom in May 2005 and its decomposition over the following 11 months. Relatively high levels of both fresh (i.e. high chlorophyll a:phaeopigment ratios) and refractory (i.e. total organic carbon content) organic

Discussion

It has been estimated that sedimentation of organic matter accounts for approximately half of net primary production in stratified parts of UK shelf seas [START_REF] Jago | Size and settling velocity of suspended flocs during a Phaeocystis bloom in the tidally stirred Irish Sea, NW European shelf[END_REF]. Here we show from paired observations of pelagic and benthic pigment concentration that the degree of transfer between surface and sediment is not constant, and can differ greatly between locations and years. The approximately two-fold higher concentration of phytoplankton in the water column over the Silver Pit during April 2006 did not result in enhanced benthic chlorophyll or carbon concentrations. This could be due to the combined effects of differences in (1) the rate of pelagic consumption (e.g. grazing), (2) local hydrodynamics, resulting in sedimentation of the phytoplankton at other locations and (3) the timing of benthic sample collection (i.e. time elapsed following the pelagic phytoplankton bloom). In the absence of light measurements and live observation of microalgal cells, in situ benthic primary production in the Silver Pit cannot be ruled out [START_REF] Mcgee | Live benthic diatoms from the upper continental slope: extending the limits of marine primary production[END_REF]. However, investigating sediments from the Dogger Bank in the central North Sea, [START_REF] Reiss | Microphytobenthos of the Dogger Bank: a comparison between shallow and deep areas using phytopigment composition of the sediment[END_REF] showed that stations below 40 m water depth were characterised by a phytopigment composition originating from pelagic phytoplankton settled on the sea floor.

Nematode assemblages were studied at three stations in the Silver Pit area of the central North Sea during a 12-month production cycle to identify the relative importance of different environmental factors (including the quality and quantity of organic material reaching the seafloor) in controlling their taxonomic, trophic and biomass distribution patterns. Our data suggest that spatio-temporal differences in nematode species composition were partly a response to the sedimentation of pelagic production and appeared to reflect species-specific responses to its nutritional quality. These correlative relationships provide valuable pointers to the mechanisms potentially causing the observed variation.

Does a local increase in food promote consistent temporal changes in the taxonomic, trophic and size structure of nematode communities?

The taxonomic and trophic structure of nematode communities differed significantly among stations over relatively short distances, potentially masking some of the temporal dynamics.

Nematode assemblages appeared to respond to spatial gradients of declining grain size and pelagic productivity in the Silver Pit from west to east as evidenced by decreasing density, species diversity and trophic diversity. Taking the entire community into account, average individual body size increased with decreasing grain size and pelagic productivity. This result was counter to the analysis of [START_REF] Udalov | Depth-related pattern in nematode size: what does the depth itself really mean?[END_REF] who investigated the relationship between mean nematode individual weight, sediment granulometry and food availability from 120 worldwide locations in the depth range 0 -8260 m. In their analysis, nematode size increased with particle diameter and the concentration of sediment-bound chloroplastic pigments.

However, the overall relationships masked more complex patterns within habitat types.

Variability of generally more diverse, coarser sediments at shallower depths compared to fluid clays and oozes in the deep sea were mainly responsible for the changes in the nematode size structure from shelf to slope. Thus, the relationships between sediment granulometry and nematode size were strong for intertidal and shallow subtidal habitats but weaker for deeper subtidal areas. Equally, although [START_REF] Udalov | Depth-related pattern in nematode size: what does the depth itself really mean?[END_REF] found evidence for decreases in the size of nematodes with decreasing food availability, the correlation with nematode size was significant for oligotrophic areas only and weakened when organic input was sufficient to cover the higher energy consumption rates of larger species [START_REF] Warwick | Ecological and metabolic studies on free-living nematodes from an estuarine mud-flat[END_REF]. Low pelagic productivity and small grain size at the easternmost station in the Silver Pit resulted in high proportions of large-bodied non-selective deposit and epigrowth feeders in the nematode community whereas the density and diversity of their smaller-sized bacteria feeding congeners was reduced.

Although spatial differences in species composition dominated temporal dynamics, significant temporal changes were recorded over the 12-month production cycle. These were linked to changes in the quality and quantity of organic material reaching the seabed, as indicated by the chlorophyll a:phaeopigment ratio and the total organic carbon content. [START_REF] Davies | Supply of organic matter to the sediment in the northern North Sea during a spring phytoplankton bloom[END_REF] provided evidence of a large settlement of diatoms immediately after the spring bloom in the northern North Sea, based on changes in the biochemical composition of material collected in moored sediment traps. The significant relationship between species distribution patterns and the state of phytoplankton remineralisation might thus point towards a species-specific response to the nutritional quality of sedimenting organic material and the biochemical changes in the sediment associated with its decomposition (e.g. oxygenation, alterations in decomposer communities such as bacteria etc.).

There was no consistent temporal relationship between the trophic composition of nematode communities and spring chlorophyll a or carbon sedimentation, most likely a result of the trophic plasticity of most feeding types. Thus different species belonging to one trophic guild may be able to forage on different food sources and/or exploit their food at different rates [START_REF] De Mesel | Topdown impact of bacterivorous nematodes on the bacterial community structure: a microcosm study[END_REF][START_REF] Postma-Blaauw | Within-trophic group interactions of bacterivorous nematode species and their effects on the bacterial community and nitrogen mineralization[END_REF]. Investigating the uptake of a radio-labelled diatom by meiofauna species in a laboratory experiment, Ólafsson et al. (1999) found that nematodes with similar buccal cavities and of similar size showed surprisingly large differences in uptake, suggesting a strong selection both for and against the diatom among epigrowth and non-selective deposit feeders. Likewise, also in a laboratory study, Moens et al. (1999) demonstrated a highly species-specific response of bacteria feeding nematodes to different types, densities and age structures of bacterial strains.

Do selected nematode species, representing the main taxonomic and trophic categories available to the phylum, respond in a differential way to a pulsed food supply?

Phytoplankton sedimentation in stratified temperate marine waters is distinctly seasonal, but the majority of studies in subtidal environments found no unequivocal influence of sedimentation on the density and biomass of meiofaunal nematodes [START_REF] Heip | The ecology of marine nematodes[END_REF][START_REF] Ólafsson | Species specific uptake of radio-labelled phytodetritus by benthic meiofauna from the Baltic Sea[END_REF]. However, the apparent absence of a community response may be misleading. Phytoplankton during and after bloom conditions may settle quickly to the seafloor and these fresh cells may be largely intact and rich in nutrients [START_REF] Graf | Benthic-pelagic coupling: a benthic view[END_REF][START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF]. Sedimented phytoplankton might be directly ingested or contribute to the microbial loop, ultimately increasing meiofaunal food supply. [START_REF] Fleeger | Meiofaunal responses to sedimentation from an Alaskan spring bloom. I. Major taxa[END_REF] hypothesised that this variation in phytoplankton settlement might affect meiofaunal survivorship and reproductive success. Our results suggest that populations of species, assumed to feed on fresh organic material such as Spilophorella paradoxa, can exhibit temporal patterns in their abundance and size distribution. Given the seasonal cycle of detrital sedimentation, the balance of benthic food input and demand may shift seasonally, with spring surpluses followed by deficits later in the year. Across all three stations, relative abundance of Spilophorella paradoxa declined following the spring phytoplankton bloom in May 2005 and reached the lowest values the following November. This suggests that food deficits may be particularly acute for those species that feed at the sediment-water interface and depend on fresh inputs, although other unmeasured factors, such as competition for food, are also likely to influence population dynamics.

Temporal dynamics of Spilophorella paradoxa populations were clearly linked to the quality and quantity of organic material present in the sediment throughout an annual production cycle, confounded by annual fluctuations in sub-surface temperature. Temperature may trigger or terminate reproductive activities of nematodes and determine the development time of juveniles. Generally, higher ambient temperatures result in shorter development times; however, little is known of the development time of most species [START_REF] Warwick | Ecological and metabolic studies on free-living nematodes from an estuarine mud-flat[END_REF] and references therein; Moens and Vincx, 2000a) Many deeper dwelling, deposit-feeding nematodes, including populations of Sabatieria punctata and Leptolaimus elegans may not be able to use sedimenting phytoplankton as food directly and they may rely on the microbial loop to stimulate bacterial production from sedimented material. Results from experimental studies, investigating the importance of freshly produced phytoplankton detritus in the diet of meiofauna are conflicting. [START_REF] Rudnick | The lags between the deposition and meiobenthic assimilation of phytodetritus[END_REF] hypothesised that, in contrast to surface-dwelling nematodes, the food resources of sub-surface deposit feeders are more stable because they have access to and assimilate a portion of the buried pool of older organic matter. Consequently, one might not expect consistent temporal changes in their density and size distribution. Ólafsson et al. (1999) contradicted this hypothesis by revealing that sub-surface dwelling nematode species can assimilate as much phytoplankton carbon per unit biomass as surface dwellers. Recently, [START_REF] Franco | Density, vertical distribution and trophic responses of metazoan meiobenthos to phytoplankton deposition in contrasting sediment types[END_REF] showed that Sabatieria-species can benefit from sedimenting phytoplankton by moving to the sediment surface.

Patterns of food availability for deposit feeding organisms can be spatially and temporally complex and total organic carbon measurements might be inadequate to elucidate the mechanisms by which food abundance and variety regulate the organisation of depositfeeding assemblages (Whitlach, 1981). Whilst such measures provide information on the total standing stock of organic carbon, they generally overestimate its labile, readily utilisable fraction. At the same time, all living components (e.g. bacteria, microphytobenthos, protozoans) represent a high-quality food source but generally account for a minor fraction of the sediment organic carbon pools [START_REF] Fabiano | Composition of organic matter in sediments facing a river estuary (Tyrrhenian Sea): relationships with bacteria and microphytobenthic biomass[END_REF][START_REF] Danovaro | Biodiversity and trophic structure of nematode assemblages in seagrass systems: evidence for a coupling with changes in food availability[END_REF].

Conclusions

Biomass-size distributions showed the most consistent temporal patterns in nematode assemblages. When applied to the whole community, the size-based approach has significant advantages over taxon-based and functional approaches through its simplicity, since it avoids distinguishing between taxonomic groups or defining distinct trophic guilds [START_REF] Soetaert | Size and shape of ocean margin nematodes: morphological diversity and depth-related patterns[END_REF]. Our results from the central North Sea show that the relationship between phytoplankton sedimentation and meiofaunal response is complex and that community responses are weaker than those of some of the component species. Hence, size-based analyses provided the most insightful results when performed on selected species.

The differential responses within communities reflect different life histories and feeding modes and the changes in the resources available during the seasonal production cycle. The rate of response indicates that the life histories of meiofauna allow their populations to track the availability of suitable food resources. Since the feeding habits of most meiofauna species are not well known, the majority of previous studies on the structure and connectedness of aquatic food webs have ignored meiofaunal taxa or aggregated them into broad functional groups. Existing food web data are therefore strongly biased towards trophically higher organisms. Our findings suggest that future investigations of the relationship between sizestructure of meiofauna populations (Vanaverbeke et al., 2004b) and potential food sources [START_REF] Dauwe | Amino acids and hexosamines as indicators of organic matter degradation state in North Sea sediments[END_REF], factors regulating meiofauna fecundity (e.g. food, temperature; Moens and Vincx, 2000b) as well as novel information on species-specific feeding habits and preferences (Moens and Vincx, 1999) are crucial to the understanding of their role in food webs.

  2002 to present by the EU/ESA project MarCoast (Marine and Coastal Environmental Information Services; www.gmes-marcoast.com). Maps in the archive were queried with the 'Zonal Statistics' function of ArcMap version 9.2 in order to extract discrete chlorophyll a values for the surface waters of the sampling stations A, B and C (Fig. 1). This approach provided a time-series of surface chlorophyll a estimates for each station. Valid, cloud-free estimates of chlorophyll a were obtained on approximately 23 % of all days during the time period of the study. The pelagic chlorophyll a values were then assembled, converted to monthly means and compared with chlorophyll a concentrations in the sediment.

(

  data not shown). Spatial differences in univariate community attributes were generally more pronounced than temporal trends. With the exception of trophic diversity (ITD), the magnitude and direction of spatial differences were maintained throughout the 12-month study period. ITD did not change significantly over time at station A and B and decreased within the first 9 months at station C (p = 0.059). Communities at station C were most trophically diverse in February 2006. Across all three stations, proportions of the epigrowth feeder Spilophorella paradoxa were consistently lower in winter (November 2005) than in spring (May 2005 and April 2006, Table 3, Fig. 4). The non-selective deposit feeder Sabatieria punctata dominated nematode communities at station B. Spatial distributions of Leptolaimus elegans were not consistent over time nor were temporal dynamics of this species consistent among stations. While relative abundance remained temporally stable at station B and C, values at station A were significantly increased in November 2005 following the autumn bloom (Table

  material in the sediment in May 2005 coincided with approximately equal proportions of juveniles and adults in the population, indicating continuous asynchronous breeding. Adults clearly dominated in July and September 2005 when sub-surface temperatures peaked and food availability was still high. The abundance of small individuals increased and that of larger individuals decreased over the winter months (November 2005 to February 2006) as both sub-surface temperatures and carbon resources in the sediment declined. The distribution of individuals among size classes was most homogeneous in April 2006, prior to the deposition of material from the next spring bloom.

  . High concentrations of fresh and refractory organic material in the sediment and rising sub-surface temperatures in the Silver Pit in May 2005 resulted in approximately equal proportions of juveniles and adults in the population. Developmental time decreased as ambient temperatures increased, leading to adult-dominated populations in July and September 2005 when food availability was still sufficient to cover their metabolic demands. Declining food availability and temperatures slowed growth and, potentially, reproduction in subsequent months, leading to low numbers of individuals across the entire size spectrum in November 2005. The abundance peak of adults in summer 2005 was followed by an increase of juveniles between February and April 2006 when temperatures in the sediment began to increase prior to the deposition of the spring phytoplankton bloom.

Figure captions Fig. 1 .

 captions1 Figure captions

Fig. 3 .

 3 Fig. 3. Comparison of mean pelagic and benthic chlorophyll a concentrations in each of the sampling months during 2005 and 2006 coded by sampling station. Shaded areas identify the spring bloom period showing the gradient in pelagic primary production throughout the Silver Pit from west (station A) to east (station C).

Fig 4 .

 4 Fig 4. Temporal changes in the mean (± SE) relative abundance of Spilophorella paradoxa across the three stations in the Silver Pit.

Fig. 5 .

 5 Fig. 5. Non-metric multi-dimensional scaling (MDS) ordination of mean relative abundance of nematode species and feeding types (sensu Wieser 1953) at the three stations in the Silver Pit over a 12-month production cycle. Day of year in parentheses.

Fig. 6 .

 6 Fig. 6. Temporal changes in the mean (± SE) individual body mass of Spilophorella paradoxa across the three stations in the Silver Pit.

Fig. 7 .

 7 Fig. 7. Spatial differences in the mean (± SE) absolute abundance of Spilophorella paradoxa over a 12-month production cycle plotted against geometric size class.

Fig. 8 .

 8 Fig. 8. Temporal changes in the mean (± SE) absolute abundance of Spilophorella paradoxa

Figure

  Figure

Figure

  Figure

Figure

  Figure

Table 2

 2 Results from Kruskal-Wallis one-way analysis by ranks: Spatio-temporal differences in

	sediment properties
	Source	Test statistic	p-value
	Grain size
	Station	11.94	0.003
	Month	1.09	0.950
	Silt-clay
	Station	0.92	0.630
	Month	4.13	0.530
	Total organic carbon
	Station	3.82	0.148
	Month	5.88	0.318
	Chlorophyll a
	Station	3.43	0.180
	Month	27.41	< 0.001
	Phaeopigment
	Station	4.95	0.084
	Month	25.80	< 0.001
	Chlorophyll a:Phaeopigment
	Station	2.53	0.282
	Month	8.72	0.121

Table 5

 5 Results from two-way analysis of variance (ANOVA): Spatio-temporal differences in average individual body mass in the total nematode community and for populations of discriminating Results from two-way crossed analysis of similarities (ANOSIM): Spatio-temporal differences in the size distribution of the total nematode community and populations of

	species
		Sum of
	Source	squares	Df	Mean square	F-Ratio	p-value
	Total community
	Station	0.280737	2	0.140369	3.45	0.043
	Month	0.320326	5	0.0640652	1.57	0.193
	Station x Month	0.490307	10	0.0490307	1.2	0.321
	Spilophorella paradoxa
	Station	0.322737	2	0.161369	4.15	0.024
	Month	0.481504	5	0.0963007	2.48	0.050
	Station x Month	0.339041	10	0.0339041	0.87	0.566
	Sabatieria punctata
	Station	0.271078	2	0.135539	4.23	0.022
	Month	0.105911	5	0.0211822	0.66	0.656
	Station x Month	0.316011	10	0.0316011	0.99	0.473
	Leptolaimus elegans
	Station	0.00149259	2	0.000746296	0.41	0.667
	Month	0.0101259	5	0.00202519	1.11	0.372
	Station x Month	0.0280407	10	0.00280407	1.54	0.166

Acknowledgements

We thank the scientists, officers and crew of RV ' Cefas Endeavour' for practical assistance in the collection of samples. Claire Mason conducted the particle size analyses and Olga Andres carried out phytopigment analyses. We are grateful to Paul Whomersley and Tracy Maxwell for processing the meiofauna samples. The work was supported by the Department for Environment, Food and Rural Affairs (research projects MF10 and ME4118).


Ángel

Carmen Morales-Caselles

Inmaculada Riba

Carmen Sarasquete

T Ángel Delvalls

Carmen Morales Caselles

A weight of evidence approach for quality assessment of sediments impacted by an oil spill: The role of a set of biomarkers as a line of evidence

Keywords: PAHs, contamination, toxicity, sublethal, WOE, bioassay, clam, crab

come

A weight of evidence approach for quality assessment of sediments impacted by an oil spill: The role of a set of biomarkers as a line of evidence

Carmen Morales-Caselles, Inmaculada Riba, Carmen Sarasquete, T.

INTRODUCTION

Chemical analyses normally provide the main tool in sediment quality assessment, even though chemical concentrations alone are inadequate for the prediction of biological consequences. Biological effects can be established using laboratory tests that determine toxic responses and field surveys of the communities living within impacted sediments, i.e. observing pollution-induced degradation of the biota [START_REF] Chapman | Evaluation of effects associated with an oil platform, using the sediment quality triad[END_REF].

Weight of evidence (WOE) investigations determine the possible ecological impacts of chemicals or other stressors based on multiple lines of evidence (LOE) [START_REF] Chapman | Determining when contamination is pollution -Weight of evidence determinations for sediments and effluents[END_REF]. They have been widely used in recent years to assess sediment quality around the world [START_REF] Chapman | The Sediment Quality Triad: then, now and tomorrow[END_REF][START_REF] Borgmann | Identifying cause in sediment assessments: bioavailability and the Sediment Quality Triad[END_REF], including different areas within the Iberian Peninsula (DelValls and [START_REF] Delvalls | Site-specific sediment quality values for the golf of Cádiz (Spain) and San Francisco Bay (USA), using the sediment quality triad and multivariate analysis[END_REF][START_REF] Caeiro | Environmental data management in the Sado estuary: weight of evidence to assess sediment quality[END_REF][START_REF] Riba | Sediment quality in littoral regions of the Gulf of Cádiz: a triad approach to address the influence of mining activities[END_REF][START_REF] Martín-Díaz | Determinación de la calidad ambiental de sistemas litorales y de estuario de la Península Ibérica utilizando ensayos de campo y laboratorio[END_REF]Morales-Caselles, 2008 a).

The sinking of the tanker Prestige (2002) spilt about 63,000 tonnes of heavy fuel oil (a mixture of saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes, with most of the PAHs being of medium to high molecular weight) mainly affecting the Galician Coast, NW Spain. Several investigations have focused on determining the biological and environmental effects of this dramatic episode by following single lines of evidence, such as chemical analyses (CSIC, 2003a;[START_REF] Franco | Spatial distribution and ecotoxicity of petroleum hydrocarbons in sediments from the Galicia continental shelf (NW Spain) after the Prestige oil spill[END_REF]González et al., 2006), toxicity [START_REF] Mariño-Balsa | Assessment of the toxicity of sediment and seawater polluted by the Prestige fuel spill using bioassays with clams (Venerupis pollastra, Tappes decussates and Venerupis rhomboideus) and the microalga Skeletonema costatum[END_REF][START_REF] Martínez-Gómez | Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill[END_REF][START_REF] Marigómez | Cell and tissue biomarkers in mussel, and histopathology in hake and anchovy from Bay of Biscay after the Prestige oil spill (Monitoring Campaign 2003)[END_REF][START_REF] Morales-Caselles | Ecotoxicity of sediments contaminated by the oil spill associated with the tanker "Prestige" using juveniles of the fish Sparus aurata[END_REF] or benthic alteration [START_REF] Junoy | The macroinfauna of the Galician sandy beaches (NW Spain) affected by the Prestige oilspill[END_REF][START_REF] Serrano | Spatial and temporal changes in benthic communities of the Galician continental shelf after the Prestige oil spill[END_REF]. Recently, authors presented a report (Morales-Caselles et al., 2008 a) where a classical WOE approach, the Sediment Quality Triad (SQT), based on three lines of evidence (physicochemical characterization of the sediments, determination of acute toxicity and benthic alteration) was carried out in the Galician Coast. This study suggested a general recovery of the environmental health within the area. However, there were signs (Morales-Caselles et al., 2008 a) that other sources of contaminants apart from the Prestige oil spill could be producing some environmental stress. The aim of the present study is to use a biomarker line of evidence (LOE) with the weight of evidence (WOE) approach to further improve the sediment quality assessment of the Galician Coast. [START_REF] Chapman | Should the sediment quality triad become a tetrad, a pentad, or possibly even a hexad?[END_REF] suggested that future applications of the SQT should consider specific LOE in terms of risk assessment, ensuring that both exposure and assessment of biological effects are adequately addressed, as both are of causation and ecological relevance. Thus, the LOE selected in this study constitutes an improvement of the classical WOE laboratory and field studies based on biomarker determinations. Biomarkers can act as an important early warning system by indicating whether environmental pollutants are present at sufficiently high concentrations to cause an effect (Walker et al., 2006). In addition, biomarker responses are specific to certain groups of contaminants, providing information related to possible causation [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]). Chemicals such as the Polycyclic Aromatic Hydrocarbons (PAHs) have a very short biological half-life in most species, yet nevertheless, may have long-term effects (Walker et al., 2006). In this sense, some compounds may not produce acute toxic effects, however sublethal effects are likely to be observed; carefully selected biomarkers can highlight these sublethal effects, thus providing increased sensitivity and robustness to WOE assessments. Thus, the aim of this research was to examine the feasibility and viability of incorporating a biomarker LOE within the classical WOE approach as applied to the assessment of oil contaminated sediments. This new approach is demonstrated by examining the extent of biological impact and identity of contaminants within sediments influenced by the accidental oil spill of the Prestige (2002) on the Galician coast.

METHODOLOGY

Approach

The study was performed in two areas of the Galician coast (NW Spain) affected by the Prestige oil spill in 2002 (Figure 1

Data integration

The data obtained from the different LOE were integrated through a multivariate analysis approach based on linking all the variables obtained [START_REF] Riba | Sediment quality in littoral regions of the Gulf of Cádiz: a triad approach to address the influence of mining activities[END_REF]) and a pie chart representation of comparisons between sites of multivariate factors [START_REF] Riba | Sediment quality in littoral regions of the Gulf of Cádiz: a triad approach to address the influence of mining activities[END_REF]Morales-Caselles et al., 2008 a). The multivariate analysis was performed using principal component analysis (PCA) as the extraction procedure, which is a multivariate statistical technique to explore variable distributions [START_REF] Riba | Deriving sediment quality guidelines in the Guadalquivir estuary associated with the Aznalcóllar mining spill, a comparison of different approaches[END_REF]

RESULTS AND DISCUSSION

Table 1 shows the summarized results of the different parameters analyzed. Sediment Hg and Pb content was significantly higher in all Corme-Laxe sites; The highest metal contents were observed at stations A and F. There was no observed general pattern in toxicity and benthic parameters between the sampling sites.

Factor analysis revealed that the original variables could be grouped into three principle components (factors) that explained 79 % of the total variance (Table 2). The multivariate analysis is a tool that allows us to interpret large groups of different variables by grouping them using correlations; in addition it indicates the importance of each factor in every single study site.

Factor #1

Factor 1 explained 35.2 % of the variance and was influenced by the relationship between different variables related with chemicals, sublethal responses, bioaccumulation and benthic alteration. Sediment PAHs, Pb and Hg contents were positively correlated to the bioaccumulation of PAHs in Arenicola marina (under laboratory conditions), but negatively correlated to their mortality in the acute assays. A set of antioxidant and detoxification biomarkers analyzed in crabs (activity of GPX, GR, GST, EROD and FRAP) and the digestive gland of clams (GR and GST activity) under laboratory conditions were also correlated with Factor #1. EROD and FRAP of organisms exposed to sediments in situ were not significantly correlated with other variables (Table 2). The aforementioned contaminants and the toxicity variables are slightly connected to the benthic alteration explained by alteration of the specific richness and an increase of the polychaete population, while a positive development of crustaceans was detected. Thus, Factor #1 may be interpreted as representing contamination by PAHs, Hg and Pb, which does not produce lethal effects; although PAH bioaccumulation and sub-lethal responses in organisms (under laboratory and field conditions) are associated with a slight alteration of the in situ benthic community.

Environmental alterations due to these contaminants have been reported by other authors (DelValls et al., 1998). Factor 1 scores for stations E (1.0) and F (1.4) located in Corme-Laxe (Figure 2) were rather high, suggesting an accumulation of contaminants including fuel oil from the Prestige [START_REF] Labandeira | La Ría de Corme y Lage: Revisión de su conocimiento[END_REF][START_REF] Prego | Trace elements in the Prestige fuel-oil spill: Levels and influence on Laxe Ria sediments (NW Iberian Peninsula)[END_REF].

Although initial studies did not detect the presence of Pb and Hg in emulsified samples of the Prestige fuel (with 54-59% water) (CSIC, 2003b), the presence of Pb was later corroborated by other authors [START_REF] Andrade | Effect of the Prestige oil spill on salt marsh soils on the coast of Galicia (Northwestern Spain)[END_REF]. Thus, the contamination by PAHs, Although, a good correlation was observed between the biomarkers induced in both invertebrate species, the crab Carcinus maenas was more sensitive i.e., showed stronger responses, than the clam Ruditappes philippinarum to this kind of pollution. On the other hand some of the variables related to the benthic alteration present in Factor #1 corroborate the effects observed in the sub-lethal experiments.

Factor #2

The second factor, Factor #2 (24.5 % of the variance) connects the set of biomarkers 2). The good correlation observed between the biomarkers in both crabs and clams incubated under field conditions suggests that these locations suffer the stress of non-measured variables which, in the case of site D, could be related to the proximity of aquaculture infrastructures (mussel culture). Other authors [START_REF] Otero | Sulphur partitioning in sediments and biodeposits below mussel rafts in the R a de Arousa (Galicia, NW Spain)[END_REF], have described the negative impacts of these mussel aquaculture, including: the discharge of a large volume of bio-deposits containing high concentrations of nutrients; the release of drugs and pesticides into the environment; an increase in sedimentation and accumulation of organic matter and an increase in the concentration of nutrients in sediments and waters (mainly N and P). Negative effects on wild populations of animals have also been observed, ranging from genetic interaction and disease transmission, to changes in the composition of the structure of benthic fauna due to a change from oxic to anoxic conditions [START_REF] Otero | Sulphur partitioning in sediments and biodeposits below mussel rafts in the R a de Arousa (Galicia, NW Spain)[END_REF].

Factor #3

The third factor, Factor #3 explained 19.2 % of the variance and was correlated with sediments contaminated by the metals Zn, Cu and Ni, and PAHs; these contaminants were correlated with the antioxidant responses under laboratory assays with crabs and clams (GR) and the acute toxicity as determined by the amphipods assay, which was not significant (the toxicity detected was not enough to consider sediment samples as toxic according to this acute assay). Samples where the mortality rate of the amphipods was 20 % higher than the mortality recorded in the reference and showed significantly different (*p < 0.05) results compared to those obtained in the reference are considered as toxic (Morales-Caselles et al., 2008 b) The effects on the benthic community were manifested as an alteration in the diversity and percentage of molluscs. This suggests the presence of a stress related to the source of metals (Zn, Cu and Ni) and organic compounds (PAHs), which is different than the source explained by Factor #1. Sites A (1.2) in the AINP and F (1.3) in the Bay of Corme-Laxe had positive Factor #3 values (Figure 2). Previous studies in Bay of Corme-Laxe detected severe contamination of the sediments by Cu [START_REF] Prego | Trace elements in the Prestige fuel-oil spill: Levels and influence on Laxe Ria sediments (NW Iberian Peninsula)[END_REF], however a fuel oil origin was unlikely, with the major source of Cu being antifouling paints from the hulls of fishing vessels [START_REF] Cobelo-García | Land inputs, behaviour and contamination levels of copper in a ria estuary (NW Spain)[END_REF]. Although contamination by Cu and Zn was observed in the uppermost sediment layers of the Prestige shipwreck area [START_REF] Prego | Cadmium, copper and lead contamination of the seawater column on the Prestige shipwreck (NE Atlantic Ocean)[END_REF][START_REF] Cobelo-García | Two opposite cases of metal accumulation in ria sediments: Ferrol and Corme-Laxe (Galicia, NW Iberian Peninsula)[END_REF], this contamination is unlikely not be related to the shipwreck, because levels of Cu in the fuel oil carried by the Prestige were relatively low (3.39 mg kg -1 ) and previous studies have shown that inputs from terrestrial sources are probably higher than from the spilled fuel oil [START_REF] Carballeira | Estado de conservación de la costa de Galicia[END_REF][START_REF] Villares | Use of an environmental specimen bank for evaluating the impact of the Prestige oil spill on the levels of trace elements in two species of Fucus on the coast of Galicia (NW Spain)[END_REF]. Some of the studied variables demonstrate the stress of these contaminants and the effects on the benthic community; however, the results mostly point to chronic contamination with low bioavailability, and potential, but unconfirmed biological risk. Thus, despite the high trace metal content of sediments from the Galician coast, their reactivity and bioavailability were very low [START_REF] Otero | Trace elements in biodeposits and sediments from mussel culture in the Ría de Arousa (Galicia, NW Spain)[END_REF], which maybe related to the high degree of pyritization found for some of the most toxic trace metals, favouring their release by oxidation of the sulphides that they form, thus making them bioavailable to benthic fauna.

Significant differences among stations

Station C, with an absence of PAH contamination and the lowest biological effects, was considered as the most suitable site to use as a reference station. Factor loadings obtained in the MAA were submitted to ANOVA and post-hoc examination using the Tukey test (Figure 3).

Factor # 1 scores of sediments from Cies Island in the AINP were not significantly different, suggesting that the effects of the Prestige oil spill were no longer detectable within the area. However, Factors # 2 and # 3 scores for station A were significantly different (p < 0.01) from the reference station (C), suggesting the influence of nonmeasured stressors from sources other than the Prestige. The factor #3 score of station B was also significantly different (p < 0.05) from the reference station, suggesting the presence of some contaminants considered a potential risk within the area, although in general, the environmental quality of the sediments was relatively good. On the other hand, Factor #1scores of sediments from Corme-Laxe D, E and F were significantly different (p < 0.01) from the reference station, suggesting the remaining contaminants from the Prestige oil spill are still producing sub-lethal effects to the biota of the bay. In addition, the significant difference (p < 0.01) in Factor #2 score observed at station D suggests other unknown sources of contaminants responsible for biological stress in the study site. In the case of station F a mixture of metals and PAHs from different sources could be considered a potential risk in the area as shown by Factor #3 (p < 0.01).

Biomarkers are useful "early warning" indicators of the health status of animals from impacted areas [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]Montserrat et al., in press), including systems impacted by oil (Anderson and Lee 2006), where complex mixtures of pollutants are usually present. The use of a set of biomarkers, as part of a WOE approach designed to assess contaminated sediments, contributed added value to the classical LOE concept and allows for the characterization of the environmental status of the studied area in a more precise and accurate way. In addition, the inclusion of chronic bioassays with two invertebrate species, not only under laboratory conditions but also in field deployments, elucidate different sources of contaminants apart from the sediments permitting a more realistic approach to the original situation of the ecosystem, and the potential ecological risks.

CONCLUSIONS

Multiple lines of evidence suggest that 4 years after the impact of the Prestige oil spill, the fuel is not producing acute toxic effects within the environment (Morales-Caselles et al., 2008 a); however, the use of a biomarker LOE within the WOE approach demonstrated that sub-lethal responses may occur in the area of Corme-Laxe, related to PAHs, Pb and Hg. No effects of oil spill were observed in the study sites located in AINP although contamination by metals, especially Zn, Cu and Ni was observed in some sites on Cíes Island. The inclusion of biomarkers in the SQT indicates that coastal anthropogenic influences (a mixture of pollutants that act as environmental stressors) are evident in both areas and should be considered a potential risk. In the case of Corme-Laxe, a possible impact related to the aquaculture of mussels was detected.

Biomarkers demonstrated higher sensitivity when compared to acute toxicity approaches providing a more comprehensive and integrated weight-of-evidence approach (Morales-Caselles et al., 2008 a), that as demonstrated, can be easily incorporated as a fourth line of evidence into the classical SQT. Furthermore, this study confirms the suitability of biomarkers as a tool to assess the sources and potential ecological risk of metallic and petrogenic contaminants within sediments. Table 1. Summarized results and SD of chemical analysis (mg kg -1 for metals, µg kg -1

ACKNOWLEDGEMENTS

for PAHs) the acute toxicity tests (Corophium volutator and Arenicola marina: % mortality; Microtox: IC50; bioaccumulation of PAHs: µg kg -1 ), biomarker responses under field and laboratory conditions (glutathione peroxidase activity GPX: nmol min -1 mg prot -1 , glutathione transferase GST activity nmol min -1 mg prot -1 , glutathione reductase GR activity nmol min -1 mg prot -1 , ferric reducing ability of plasma FRAP activity M mg -1 min -1 and EROD activity pmol mg EROD-crab-field 0.13±0.00 2.96±0.00 0.02±0.00 8.51±0.00 0.39±0.00 0.48±0.00 EROD-clam-field 0.15±0.00 0.07±0.00 0.09±0.00 0.63±0.00 0.07±0.00 0.13±0.00 FRAP-crab-field

Biomarkers (field)

FRAP-clam-field 10.39±1.4 3.09±0.53 2.63±0.91 23.58±2.5 2.01±0.65 Table 2. Sorted rotated factor loadings of 36 variables for the three principal factors resulting from the multivariate analysis of the single results obtained from the chemical analysis, the acute toxicity tests, the suite of biomarkers and the alteration parameters for the study of the sediments quality in the Galician Coast. Chemicals: loadings are related to the concentration of contaminants in sediments; Acute effects: loadings explain the toxicity detected by the acute assays and the bioaccumulation of PAHs in Arenicola marina. Sublethal effects: loadings are related to the induction of biomarkers.

Benthic alteration: loadings are related to alteration of the biota (decrease of number of species, specific richness, diversity, diminuition in the percentage of molluscs and crustacea and increase in the polychaete population. The group of variables selected for the interpretation represented a loading of 0.40 or higher for a good association between an original variable and a factor.

  ): Cies Island (A, B, C) within the Atlantic Island National Park (AINP) and the Bay of Corme-Laxe (D, E, F). Cies Island acted as a natural barrier protecting the rias from entry by the fuel. The Bay of Corme-Laxe is also considered a place with high ecological relevance, with a low anthropogenic and industrial influence (fishing and farming being the main economic activities). The 4 LOE employed within the WOE approach were: (a) Sediment contamination; physicochemical characterization of sediments by analyzing PAHs (acenaphtalene, acenaphtylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i) perylene, benzo(k)fluoranthene, chrysene, dibenzo(a,h) anthracene, fenanthrene, fluoranthene, fluorene, indene (1,2,3,cd )pyrene, naphthalene, and pyrene) using GC-MS with selected ion monitoring and trace metals (Zn, Pb, Cu, Ni and Hg) with anodic stripping voltammetry (Morales-Caselles et al., 2006); (b) Acute toxicity and bioaccumulation; by performing sediment bioassays such as the commercial assay Microtox ® (Morales-Caselles et al., 2007), the amphipod mortality test with Corophium volutator (Morales-Caselles et al., 2007), the polychaetes mortality assay (Casado-Martínez et al., in press) and bioaccumulation experiments with Arenicola marina (Morales-Caselles et al., 2008 b); (c) 'In situ alteration'; benthic alteration was selected and determined by measuring parameters in situ based on taxonomic identification and community descriptive statistics (abundance-biomass analysis, species richness, diversity, dominance and proportions of the major taxonomic groups) (DelValls and Chapman, 1998); (d) Laboratory and field studies based on biomarkers; using two invertebrate species the crab Carcinus maenas and the clam Ruditappes philippinarum, and a set of biomarkers (Morales-Caselles et al., 2008 c): mixed function oxygenase activity, which is the first mode of detoxification in many organic pollutants, was measured using the adapted Ethoxyresorufin-O-deethylase (EROD) assay; the Ferric Reducing Ability of Plasma (FRAP) assay allows a measure of the antioxidant capacity; antioxidant Glutathione-Stransferase (GST) activity was determined by monitoring the rate of conjugation of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm; oxidation of 1 mM NADPH by Glutathione Reductase activity (GR) in the presence of 10 mM oxidized glutathione was also monitored at 340 nm; phase II metabolizing enzyme Glutathione Peroxidase activity (GPX); all the biomarker responses were normalized with the total protein content (n=5).

  . The original data set used in the analysis included the variables obtained from the 4-LOE and its objective was to derive a reduced number of new variables as linear combinations of the original variables. This provides a description of correlations between the set of the original variables and their meaning in each study site with a minimum loss of information. For the representation of the pie charts the new factors obtained from the PCA were submitted to ANOVA and Tukey tests which identified significant differences in sensitivity among stations and controls for each factor (Morales-Caselles et al., 2008 a). Every study site has a pie chart divided into the obtained factors which use different colours depending on the level of significant differences in relation to the reference.

  Hg and Pb observed at Corme-Laxe appears to be caused by the remnants of the Prestige (Morales-Caselles et al., 2008 b).Although no lethal effects were observed, the bioaccumulation of PAHs by A. marina demonstrates the high bioavailability of these compounds. The induction of different biomarkers in the hepatopancreas of crabs and in the digestive glands of clams was also correlated with the presence of these contaminants (PAHs, Hg and Pb), suggesting that the deployed organisms suffered stress due to the presence of these substances in the sediments(Morales-Caselles et al., 2008c). The correlation observed among the biomarkers and the different variables defined by Factor #1 was stronger under laboratory conditions, implying that field deployments result in less sensitivity[START_REF] Astley | Multi-variate Analysis of Biomarker Responses in Mytilus edulis and Carcinus maenas from the Tees Estuary (UK)[END_REF]. The flushing action of the open water environment may act to reduce the impacts of sediment contaminants when compared with laboratory incubations.

  measured under field conditions (EROD and FRAP activity in crabs and clams, and GPX and GST in clams), the mortality of Arenicola in the acute experiment, no toxicity for amphipods, the alteration in the number of species and the decrease of the Polychaete population. Positive and negative correlations for a few biomarkers were also detected under laboratory conditions and no concordance with the amphipod toxicity test was observed. The relationships between the biological responses identified in Factor #2 are not correlated with any of the chemicals analyzed suggesting that a contaminant or group of compounds that was not analysed, is the cause of the biomarker responses. Taking into account that the acute toxicity observed in Arenicola and the rest of the bioassays was relatively low (less than 30% mortality) (Morales-Caselles et al., 2008 b), a source of contaminant not related to the sediment is the most likely cause of these effects. Station A (0.1) located on Cies Island and site D (2.0) in Bay of Corme-Laxe had the highest positive correlation with Factor #2 (Figure

  This work was supported by the projects VEM2003-20563/INTER, and CTM2005-07282-C03-01/TECNO financed by the Spanish Education and Science Ministry and by Centro Investigaciones Subacuáticas (CIS) funded by the Ministry of Environment. C Morales-Caselles thanks the Ministry of Education and Science for funding her research fellowship (FPU). The authors would like to thank the members of the CIS for their support and help in the chemical analysis and the benthic community information; special thanks are given to L Martín, N Fernández, A César, P Vidal and A Moreno.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Estimated factor scores for the three factors in each of the 6 cases. The factor

Figure 3 .Figure 1 .

 31 Figure 3. Pie charts which represent the significant differences of the factors score in

Figure 2 .

 2 Figure 2. Estimated factor scores for the three factors in each of the 6 cases. The factor

Figure 3 .

 3 Figure 3. Pie charts which represent the significant differences in the factors' score in

Walker, C.H., Hopkin, S.P., Sibly, R.M.,Peakall, D.B. 2006. Biomarkers. In: Principles

of Ecotoxicology. CRC Press Boca Raton, Florida. 315 pp