Brenda Burd

Robie Macdonald

Albert Van Roodselaar

Cindy Wright

Axinopsida serricata shell encrustation: A potential indicator of organic enrichment conditions in sediments in the southern Strait of Georgia, British Columbia, Canada By

Keywords: bivalves, shell encrustation, iron oxidation, acid volatile sulphides, sediment, outfall

come

Introduction

One of the difficulties in determining the effects of organic enrichment on the biology and chemistry of sediments is relating periodic sediment chemistry measurements to the long-term ambient conditions experienced by the organisms inhabiting the benthos. For environments such as seabeds surrounding municipal outfalls, it would be particularly helpful to develop a simple method for evaluating the intensity of organic loading within sediments, integrated over time.

The bivalve Axinopsida serricata (F. Thyasiridae) proved to be of interest when, during the processing of taxonomic samples from a monitoring survey of the Iona outfall receiving environment in the Strait of Georgia on the west coast of Canada, it was noted that the shells showed considerable rust-coloured encrustations that appeared to be related to distance from the outfall.

A. serricata is a small infaunal bivalve (<5mm), which can be found from southern California to Alaska and is an abundance dominant in a variety of subtidal substrate types and depths [START_REF] Bright | Tissue variability in the infaunal bivalve Axinopsida serricata (Lucinacea:Thyasiridae) exposed to a marine-tailings discharge and associated population effects[END_REF][START_REF] Burd | Qualitative and quantitative studies of benthic infaunal communities in British Columbia coastal waters[END_REF][START_REF] Burd | Evaluation of mine tailings effects on a benthic marine infaunal community over 29 years[END_REF]Burd, , 2003a,b;,b;[START_REF] Llansó | The distribution and structure of soft-bottom macrobenthos in Puget Sound in relation to natural and anthropogenic factors[END_REF]McPherson et al., 2004a,b,c), including the receiving environment of the Iona and Lions Gate outfalls (south-eastern margin Strait of Georgia -McPherson et al. 2007a,b) and the Macaulay Point outfall near Victoria, BC (Burd, 2003b) (see Fig. 1). It appears to be relatively tolerant of conditions near these three major outfalls, but the population declines within the zone of maximum particulate deposition north of the Iona outfall (McPherson et al., 2007a) and near the Macaulay Point outfall (Burd, 2003b).

Distributional patterns for particulates from the Lions Gate outfall in Burrard Inlet have not been clearly defined (McPherson et al., 2007b), however.

Acid-volatile sulphides (AVS) have been used to infer general sediment geochemical conditions and particle loadings in the 3 outfall monitoring programs (McPherson et al., 2007a,b;CRD, 2005), and is highest in areas that receive the greatest organic loading. Because AVS values tend to be spatially heterogeneous and ephemeral in sediments (Morse and Rickard, 2004), confident extrapolations from these geochemical parameters to the long-term sediment conditions experienced by the benthos cannot presently be made.

Organic deposition from a source such as an outfall depletes oxygen in sediments and, depending on the strength of depletion and diffusion of oxygen, other electron acceptors (e.g. nitrate, sulphate and oxides of Mn and Fe) can become important in the oxidation process [START_REF] Bender | Organic carbon oxidation and benthic nitrogen and silica dynamics in San Clemente Basin, a continental borderland site[END_REF][START_REF] Brendel | Development of a gold amalgam voltametric microelectrode for the determination of dissolved Fe, Mn, O 2 and S(-II) in pore-waters of marine and freshwater sediments[END_REF]. During redox processes, iron and manganese shift between dissolved phase components subject to diffusion and solid-phase precipitated minerals which may be manifest as deposition on shells. Based on the rusty colour of the shell encrustations, and the burrowing nature of this bivalve, it is probable that the encrustations derive from oxides of either iron or manganese, as found in bivalves from other habitats (Swinehheart and Smith, 1979;[START_REF] Gillan | Morphology of a ferric iron-encrusted biofilm forming on the shell of a burrowing bivalve (Mollusca)[END_REF][START_REF] Huettel | Advective transport affecting metal and nutrient distributions and interfacial fluxes in permeable sediments[END_REF].

The purpose of this paper is to contribute to the understanding of geochemical conditions under which shell encrustation occurs, or does not occur. Shell encrustation patterns may provide insights into the sediment redox conditions and organic loading experienced by the sediments, and provide a more biologically-relevant monitoring tool than spot-measurements of sediment geochemistry. The geographic and within-sediment distribution patterns of shell encrustations on A. serricata in relation to organic enrichment gradients revealed by sediment AVS, were examined near 3 municipal outfalls in the Strait of Georgia, BC, and a background area elsewhere in the Strait. The composition of the encrustations, and the mechanisms for producing these encrustations were also explored. Finally, we propose a schematic model to show how shell encrustation on A. serricata might occur under different sediment conditions.

Methods

Composition and inner/outer structure of shell encrustation

Rust-coloured encrustations from the upper valves of a few shells from the 2003 Iona monitoring samples (station 15 -see Fig. 1) were scraped and combined to undergo analyses for crystalline metal oxides and trace metals, using X-ray diffraction. The combined scrapings were ground into fine powder in a corundum mortar and smeared onto a zero-diffraction quartz plate with ethanol.

Step-scan X-ray powder-diffraction data were collected over a range 3-70°2θ with CuKα radiation on a standard Siemens (Bruker) D5000 Bragg-Brentano diffractometer equipped with a diffracted-beam graphite monochromator crystal, 2 mm (1°) divergence and anti-scatter slits, 0.6 mm receiving slit and incident-beam Soller slit. The long sample holder used (43 mm) ensured that the area irradiated by the X-ray beam under these conditions was completely contained within the sample. The long fine-focus Cu X-ray tube was operated at 40 kV and 40 mA, using a take-off angle of 6°. The X-ray diffractogram was analyzed using the International Centre for Diffraction Database PDF-4 and Search-Match software by Siemens (Bruker AXS).

An intact upper valve of Axinopsida serricata from the 2003 Iona monitoring station 15 (see Fig. 1) and from the ER67 location (Satellite Channel) were examined using scanning electron microscopy (SEM) with an energy dispersive spectrometer (EDS) attached. Specimens from these two areas were chosen to determine whether or not the shell encrustations were formed in the same way regardless of sediment conditions or location. This analysis determines relative bulk elemental composition of the shell encrustation. Scans were done a) on the external coating of the encrustations for each location; b) on the shell itself from the Iona location (no encrustations) and; c) within the cross section of broken edges of the encrusted shell material for each location.

Axinopsida serricata size classes, abundance distribution and shell encrustation related to sediment geochemistry

Axinopsida serricata were extracted from archived grab samples collected during various monitoring programs over 8 years in the Strait of Georgia (Table 1). Only shells from live organisms (at the time of sampling) were examined. The general sampling areas, ranging from 32-136 m in water depth, are shown in Fig. 1. Consistent field (sampling) and laboratory protocols for monitoring benthic infauna were utilized by Biologica Environmental Services Ltd.

(Victoria, BC. Canada), and are described in the original monitoring reports; for example, benthic samples were collected using a 0.1 m 2 Van Veen grab and gently washed through a 1 mm mesh screen. All specimens were preserved in 70% ethanol. Sediment AVS samples were available from all studies in Table 1 except the 2000 ER67 Ecological Reserve monitoring program conducted in Satellite Channel (Fig. 1). Sediment Acid volatile sulphide (AVS) samples were prepared by decanting the overlying water in the grab sample, removing and discarding the surface layer of potentially oxidized sediment, and then homogenizing the material from the remaining top 2 cm of the grab prior to chemical analysis in accordance with the method described by USEPA (1991). Hydrochloric acid (6.0 N HCl) was added to each sediment sample within a purge and trap system. To measure AVS, the resulting hydrogen sulphide (H2S) was carried into a basic zinc acetate solution by argon gas and the AVS was determined colourimetrically (using a method in which sulphide reacts with N-N-dimethylphenylenediamine and the resulting methylene blue is then measured). Concentrations were reported on a mol/g dry weight basis (see also McPherson et al. 2007a).

The lengths of A. serricata specimen shells from the preserved samples were measured by passing each specimen over an optical grid with 0.5 mm gradations. The specimens divided naturally into shell length groups as follows: >2.70 mm (size class A: SC-A), and 2.70 mm to 1.0 mm. (size class B: SC-B) (see also McPherson et al. 2004a;2005a;2006a;2007a). All specimens from each sample were counted and assigned into four categories based on the level of shell encrustations: no encrustations (0), light encrustations (1), moderate encrustations (2) and heavy encrustations (3) (Fig. 2). An example of a series of shells with moderate-heavy encrustation on the external surfaces is also shown in Fig. 2. In this paper, encrustations were classified as "light" (categories 0 and 1) or "heavy" (categories 2 and 3); data are presented as proportions of shells with light encrustations.

Data from the 2004 Iona monitoring program were used to show an example of a distribution pattern for abundance and shell encrustation of the two size classes of Axinopsida serricata relative to the organic enrichment gradient around the outfall (as evidenced by sediment AVSsee McPherson et al., 2007a). The proportion of bivalves from all monitoring location (Table 1) samples with light shell encrustation, was plotted against sediment AVS to show trends for the two size classes.

Axinopsida serricata vertical distribution, shell encrustation and AVS within sediment depth layers

In addition to the archived samples from monitoring programs, duplicate 0.1 m 2 Smith-McIntyre grab samples were obtained on October 22/23, 2004 at the Iona monitoring stations 8 and 15 (Fig. 1). Station 8 is located less than 0.5 km north of the Iona outfall, where particulate deposition from the outfall is known to be at a maximum [START_REF] Hodgins | A re-evaluation of Iona effluent solids deposition based on sediment grain size characteristics[END_REF], while Station 15 is located 8 km south of the outfall and outside the influence of the sewage particulate deposition.

Sediment was extracted from the grabs at 1 cm depth intervals, to within 1 cm of the grab sides.

This was done to a maximum sediment depth of 10 cm, to provide equal volume samples. Below 10 cm, the wall curvature of the grab begins to reduce the sample volume. From each 1 cm depth layer, a 30 mL sub-sample was removed by plastic spatula and then frozen for chemical analysis of AVS (see methods above), silver and elemental sulfur (for analytical methods see McPherson et al., 2004a). In this study, Ag was measured as an indicator of exposure to municipal outfall particulates (e.g., see [START_REF] Gordon | Sedimentary Tracers of Sewage Inputs to the Southern Strait of Georgia[END_REF][START_REF] Gobeil | Silver in sediments from the St. Lawrence River and estuary and the Saguenay Fjord[END_REF], whereas elemental sulphur indicates redox condition of sediments. Live speciments of A. serricata were preserved in 70% ethanol and later sized and classified according to the degree of shell encrustation.

Results

Shell encrustation occurred primarily in the vicinity of the inhalant aperture and secondarily in the region of the exhalant aperture, on the outside of the upper shell. Figure 2 shows a close-up photograph of encrustations on the external surface of the upper valve. The density and extent of encrustation was highly variable, and thick layers of encrustation sometimes flaked off during mechanical handling. Despite this, some reddish accretions were typically evident which appeared to be more persistent, even after the loose material was sloughed off.

Composition of Shell encrustation

The X-ray diffraction showed that the metallic crystalline phases of the composite sample of shell scrapings contained rock-forming minerals commonly found in sediment (magnesium, potassium, aluminum), along with a large proportion of aragonite, likely indicative of bivalve shell. No crystalline Fe-oxides or hydroxides were detected. The only phases found that may contain significant Fe are chlorite and dolomite (if solid solution to ankerite is present).

The results of the SEM/EDS analysis of shell encrustations for the Iona and ER67 shells are summarized as relative proportions of elements in Table 2. Included are data from the most reliable scans showing a) un-encrusted shell composition; b) outer surface of encrustations, and; c) inner surface (broken edges) of encrustations. All analyses revealed that oxygen was the dominant element in the encrustations, and the only notable element evident in the scan of bare shell material. The interior part of the encrustation of the Iona shells had much higher iron and calcium levels, and higher phosphorous levels, than the exterior of the encrustation, whereas the reverse was true for aluminum and magnesium. There was no notable difference in relative composition between Iona and ER67 samples. There was a clear but variable predominance of shells with light encrustations (i.e. categories 0 or 1) within the maximum deposition zone for the outfall (Fig. 4). By extrapolation, specimens with heavy shell encrustation predominated outside the Iona outfall's deposition zone. However, Fig. 4 also shows that outside the maximum deposition zone for the outfall, the proportion of the small size class of bivalves (1.00 to 2.7 mm) with light shell encrustation (categories 0 or 1) was sometimes higher and more variable relative to the enrichment gradient, than for larger bivalves (>2.7 mm).

Distribution of

The proportion of the two size classes of Axinopsida serricata with light shell encrustation was plotted against sediment AVS levels (Fig. 5), for all monitoring samples (see Table 1). Figure 5 demonstrates that shell encrustations were not linearly related to sediment AVS; rather there was a variable response at sediment AVS levels <7 µmol/g for the larger size class animals, followed by a more consistent increase in proportion at AVS levels higher than this (power regression line fit for all large size class data: r 2 =0.54, p<0.001). At sediment AVS levels >7 µmol/g, an increasing proportion of the larger size class Axinopsida serricata had light encrustations. A similar pattern was found for the smaller size class, but with a higher variability (power regression line fit for all small size class data: r2=0.36, p<0.01).

The A. serricata samples from ER67 (Satellite Channel) did not have concurrent sediment AVS measurements, but sediments in this location are generally considered to be well oxygenated and flushed. The area is also subject to low natural sedimentation and has low silt/clay content (i.e. 10-25% silt/clay, Ellis 1968) compared with the other sampling areas in this study (45-98% silt/clay, McPherson et al., 2007a,b). For simplicity, the ER67 samples were plotted with a presumed AVS = 1.0 µmol/g, although experience with similar habitats suggests that it was probably even lower for these samples (Macdonald et al., in press). The proportion of shells from ER67 samples with light encrustation tended to be higher than those found at low AVS levels for the other 4 sampling locations listed in Table 1.

Shell encrustation and AVS in sediment depth layers

More than 90% of A. serricata of both size classes were found within the top 4 cm of sediments (Fig. 6a,b) at station 8 and station 15 (reference area). A. serricata in the smaller size class were rare at station 8 (near the outfall), with only 8 individuals found in both grabs. AVS levels in sediments at station 15 were consistently lower than at station 8 (Figs. 6,e,f). The proportion of large size class Axinopsida serricata with light shell encrustation was higher in all sediment depth samples regardless of AVS levels at station 8 than at station 15. The small size class bivalves found at station 15 showed more variable shell encrustations relative to AVS levels than for the large size class.

Silver and elemental sulfur were much higher in all depth layers in sediments at station 8 than at station 15 (Fig. 7).

Discussion

Encrustations such as those found on A. serricata shells have been documented for sedimentdwelling bivalves from various locations globally (Swinehart and Smith, 1979;[START_REF] Dando | Sulphide mining by lucinid bivalve mollusks: demonstrated by stable sulphur isotope measurements and experimental models[END_REF]. [START_REF] Gillan | Morphology of a ferric iron-encrusted biofilm forming on the shell of a burrowing bivalve (Mollusca)[END_REF] describe a rust colored biofilm and iron oxide deposit on shells of the bivalve Montacuta ferruginosa, which is commensal in burrows of sand urchins (spatangoids). [START_REF] Gillan | Morphology of a ferric iron-encrusted biofilm forming on the shell of a burrowing bivalve (Mollusca)[END_REF] found that biofilms are laid down over bivalve shells by a complex microbial community with filamentous bacteria similar to Thiothrix and Beggiatoa, both of which can oxidize sulphide. Similarly, active pumping of water through the burrow (since this species lack siphons) by A. serricata to support their metabolism creates two zones (inflow and outflow) of reliable O 2 supply in direct contact with the shell, which would become favoured habitat for microbes that produce biofilms and aid in preserving oxidized iron precipitates. [START_REF] Gillan | Iron oxidation and deposition in the biofilm covering Montacuta ferruginosa (Mollusca, Bivalva)[END_REF] and [START_REF] Gillan | Iron encrustation of the bivalve Montacuta ferruginosa[END_REF] proposed possible ontogenetic scenarios relating to the development of rust-coloured bivalve shell encrustations.

In the current study, the lack of calcium or other elements in the clean shell surface scan, and the differences in outer and inner scans for both locations could be due to the presence of a biological film or periostracum. In bivalves, shell colour is mostly concentrated in the periostracum, a horny layer covering the shell. The encrustations found herein do occur on the outside of the periostracum, but their persistence upon mechanical abrasion of the accretions suggests that the material may also become "cemented" to the shells, possibly within biological films, or the periostracum itself. Swinehart and Smith (1979) found considerable concentrations of Fe and Mn within the periostracum of freshwater molluscs, with reduced amounts found in the periostracum of the marine bivalve Mytilus californianus. The authors describe the minerals as being concentrated in amorphous regions in a proteinaceous matrix (see also [START_REF] Gillan | Morphology of a ferric iron-encrusted biofilm forming on the shell of a burrowing bivalve (Mollusca)[END_REF]. How the shell encrustation patterns vary on live versus dead shells has not been documented.

The depth distribution of the bivalves primarily in the top 4 cm of sediments, appears to be related to their size and ability to maintain a viable burrow for bringing overlying oxygenated water to the inhalant aperture. Thyasirids lack siphons, and build an inhalant tube in sediments using their elongated foot [START_REF] Allen | On the basic form and adaptations to habitat in the Lucinacea (Eulamellibranchia)[END_REF][START_REF] Slack-Smith | Axinopsida serricata (Carpenter, 1864), its burrowing behavior and the functional anatomy of its pallial organs (Mollusca: Thyasiridae)[END_REF]. This limits burial depth (see [START_REF] Dufour | Sulphide mining by the superextensile foot of symbiotic thyasirid bivalves[END_REF], particularly in the non-chemosynthetic species [START_REF] Dufour | Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae[END_REF] and is an unusual feature of endobenthic bivalves, making this taxon uniquely situated to reflect geochemical conditions in near-surface sediments. This species is considered by [START_REF] Word | The Infaunal Trophic Index, a Functional Approach to benthic community analysis[END_REF] to be an interface detrital (suspension) feeder (see also [START_REF] Zhukova | Fatty acids as markers of bacterial symbionts of marine bivalve mollusks[END_REF][START_REF] Dufour | Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae[END_REF]. [START_REF] Dufour | Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae[END_REF] did not find any bacterial symbionts in the gills of A. serricata, which suggests that it is unlikely to have any nutritional augmentation from chemoautotrophic symbiosis, as some thyasirid bivalves do.

The decrease in abundance of A. serricata in the maximum deposition zone around Iona (e.g. Fig. 3), and the low abundance of this species in the maximum deposition zone around the Macaulay Point outfall (Burd, 2003b) suggest that sediment geochemical conditions in organically enriched sediments near outfalls can be detrimental. Contaminants associated with outfalls might also play a role in near field bivalve declines, although Ag [START_REF] Gordon | Sedimentary Tracers of Sewage Inputs to the Southern Strait of Georgia[END_REF], nonylphenol ethoxylates [START_REF] Shang | Persistence of nonylphenol and ethyoxylate surfactants and their principle primary degradation products in sediments from near a municipal outfall in the Strait of Georgia, British Columbia, Canada[END_REF] and PBDEs (Johannessen et al., in press) all show enrichments that can be assigned to the Iona outfall, these contaminants are rarely found at levels exceeding provincial and national guidelines at any of the three outfalls (Payne, 2003, 2004, McPherson et al. 2007a,b), and are thus not expected to have adverse effects at this level of the food chain. Some sediment contaminants (e.g., PCBs; Johannessen et al., in press) are unusually low near the Iona outfall, possibly due to dilution from the continuous supply of sediment from the Fraser River. In contrast to the uncertainty related to contaminant effects on biota, Burd et al. (2003a,b) describe biotic changes at both the Iona and Macaulay Point outfalls typical of organic enrichment of sediments. This organic enrichment has been inferred from consistently elevated AVS levels (CRD, 2005;McPherson et al. 2007a), from the elevated elemental sulfur and silver in sediments near the Iona outfall, and from the redox profile in a recent sediment core at station 8 (Macdonald et al., in press).

The small size class A. serricata were less tolerant of conditions near the Iona outfall than the large size class (Fig. 3). This may be related to lack of maturity of physiological coping mechanisms or more limited metabolic requirements for smaller specimens, which would prevent successful recruitment in the organically enriched sediments. Other possible reasons include reduced predation on large specimens due to changes in juvenile fish and megafauna composition in the high outfall deposition area (Metro Vancouver, unpublished Iona monitoring data), or to other unknown reasons related to the life history and variable survival of colonizing individuals. In addition, the population distribution of this species may be complicated in the Iona region by the documented transport of sediment down-slope (see [START_REF] Hodgins | A re-evaluation of Iona effluent solids deposition based on sediment grain size characteristics[END_REF]. The potential for sediment transport to shift recently settled bivalves in laboratory experiments has been shown by [START_REF] Roegner | Transport of recently settled soft-shell clams (Mya arenaria L.) in laboratory flume flow[END_REF]. Along with potential patchiness in sediment conditions, this could explain why there are some bivalves with heavy encrustations in the maximum deposition zone around the outfall (Fig. 4). Conversely, [START_REF] Gillan | Iron encrustation of the bivalve Montacuta ferruginosa[END_REF] noted that the thick shell encrustation in larger bivalves commensal in echinoid burrows can sometimes get scraped off during burrowing under laboratory conditions, which may help to explain why, in the present study, there was often a small proportion of larger Axinopsida serricata with light shell encrustation in areas where most of the bivalves had heavy shell encrustation. To further complicate this picture, the reduced encrustations overall in the smaller size class A. serricata compared with the large size class suggests that encrustations accumulate progressively over the life of these animals. [START_REF] Gillan | Iron encrustation of the bivalve Montacuta ferruginosa[END_REF] also noted that the smallest size class of the commensal bivalve Montacuta ferruginosa had a much higher incidence of uncoated or weakly coated iron shell encrustation than the larger size class, and that the encrustations were thicker in larger specimens, suggesting that they build up over time. The above factors could result in a mosaic of shell encrustation patterns, explaining the variable results in the current study.

The pattern and intensity of shell encrustations are presumably related to characteristics of the water pumped through the shell over time. Figure 5 suggests that there are geochemical thresholds (AVS >7umol/g) above which shell encrustation frequencies decrease. However, between about 3 and 7 umol/g AVS, results were highly variable, which means that periodic measurements of AVS might not provide the most reliable measure of sediment geochemical conditions due to spatial and temporal patchiness in sediments (Morse and Rickard, 2004). For example, in outer Burrard Inlet (Lions Gate sampling area -Fig. 1) samples from 2005, the proportion of larger size class A. serricata with light encrustations was unusually high at one of the stations. Although AVS levels were not very high at this station in 2005, they have historically been much higher and more variable than at any other station in the survey area. The proportion of shells with light encrustations in this location remained relatively high throughout the sampling period (2002)(2003)(2004)(2005)(2006) regardless of variability in sediment AVS conditions. This may partially explain why there is considerable scatter in the relationship between shell encrustation and sediment AVS (Fig. 5).

Results suggest that the pattern of shell encrustation is not ephemeral, but rather a cumulative product of substrate conditions. If there is a progressive increase in shell encrustation over the life of the animals, the degree of encrustation in these animals provides a life-long picture of sediment geochemical conditions, which could be verified and clarified by more in-depth studies of the ontogeny of shell encrustation under varying geochemical conditions over the life of the animals.

The biogeochemical mechanism of encrustation

The formation of shell encrustations in A. serricata clearly occurs throughout the southern Strait of Georgia and likely in other coastal seas. However, the degree of encrustation and its distribution in the various sampling areas suggests a relationship with sediment composition and organic loading. Although the organic and geochemical composition of the shell encrustations and the mechanisms of microbiological precipitation of iron on shells have been discussed by [START_REF] Gillan | Iron oxidation and deposition in the biofilm covering Montacuta ferruginosa (Mollusca, Bivalva)[END_REF], there has been no work done on the geochemical conditions required within the sediments to facilitate the shell encrustation.

The results of this study suggest that increasing enrichment of labile organic carbon related to municipal outfall particulate deposition (as evidenced by sediment silver distributions; Gordon 1997 and Fig. 7), reduces the concentration of oxygen in the sediments (as evidenced by elemental sulfur distributions -Fig. 7) and leads to reduced encrustations on the bivalve shells.

The process of advecting oxygenated water in through the anterior, inhalent siphon and out through the exhalant siphon affects the redox environment surrounding the bivalve's burrow. In particular, given the location, coloration and composition of the encrustations documented here (Fig. 2), it appears that bivalve ventilation produces encrustations by bringing together dissolved oxygen and dissolved iron (Fe II), a process known to lead to ferric iron precipitation in permeable sediments [START_REF] Brendel | Development of a gold amalgam voltametric microelectrode for the determination of dissolved Fe, Mn, O 2 and S(-II) in pore-waters of marine and freshwater sediments[END_REF][START_REF] Huettel | Advective transport affecting metal and nutrient distributions and interfacial fluxes in permeable sediments[END_REF]. This process may be mediated on the bivalve shells in part by microbes (e.g. [START_REF] Gillan | Iron encrustation of the bivalve Montacuta ferruginosa[END_REF]) and the coincident formation of a biofilm, which would help to protect precipitated Fe from re-dissolving during subsequent episodes of hypoxia in surrounding sediments.

The distribution of shell encrustations as a function of distance from an extraordinary labile carbon source (Fig. 4) together with a notable relationship between AVS and shell encrustation (Fig. 5) suggest that the complexity in the shell encrustation process derives from the manner in which the redox cycle affects iron phases (dissolved/particulate) in sediments.

Before proposing a relationship between the oxygen/iron rich encrustations observed on A. serricata shells, and sediment geochemistry, it is necessary to examine how redox chemistry might produce dissolved iron (Fe 2+ ) and precipitated Fe phases (Fe 3+ oxy-hydroxide phases).

Metabolism of organic matter involving the consumption of oxygen creates a gradient in the porewater which then supports diffusion of O 2 from bottom water into the sediments. At a depth in sediments where O 2 has been completely consumed, other electron acceptors may be employed in metabolism, but with decreasing energetic return [START_REF] Bender | Organic carbon oxidation and benthic nitrogen and silica dynamics in San Clemente Basin, a continental borderland site[END_REF].

Accordingly MnO 2 may be reduced producing dissolved Mn 2+ which can diffuse upward to reprecipitate when it reaches dissolved O 2 , and NO 3 -can likewise be reduced during the process of denitrification. With considerably less free-energy return, iron oxy-hydroxides associated with solid phases can be reduced to produce soluble Fe 2+ [START_REF] Brendel | Development of a gold amalgam voltametric microelectrode for the determination of dissolved Fe, Mn, O 2 and S(-II) in pore-waters of marine and freshwater sediments[END_REF], at a depth below the availability of dissolved O 2 . When other electron acceptors have been exhausted, SO 4 2-may be used but this provides a very poor energetic return to microbes [START_REF] Emerson | Sediment diagenesis and benthic flux[END_REF] and also releases S 2-(sulphide) as an important byproduct. Therefore, shell encrustation could occur because Fe 2+ may precipitate when it comes in contact with dissolved O 2 and also when it comes in contact with S 2 -pyritization [START_REF] Berner | Burial or organic carbon and pyrite sulfur in the modern ocean: its geochemical and environmental significance[END_REF].

The processes outlined above are organized vertically in sediments because O 2 is supplied from the sediment surface and because microbes have a preference for O 2 as an electron acceptor due to the available free-energy return. Dissolved Fe 2+ , which may be produced below the oxic zone within the sediments during metabolism, can then diffuse upward to precipitate in oxyhydroxides, but it may also be sequestered by precipitation as sulphides which occur at depth in sediments provided organic metabolism is sufficiently robust to produce S 2-. AVS in sediments is clear evidence of S 2-production and precipitation (Morse and Rickard, 2004). Thus the depth at which dissolved Fe 2+ may buildup in sediment porewater is bounded above by oxic conditions where Fe 2+ is oxidized to Fe 3+ and precipitated as oxyhydroxide, and below by sulphidic conditions where Fe 2+ is precipitated as sulphide.

Figure 8 is a proposed schematic diagram which shows how Fe 2+ buildup in sediments may be affected by labile organic carbon loading and O 2 penetration. A. serratica live predominantly in the top 4-cm of the sediments, and pump water to supply themselves with O 2 . Therefore, it is the supply of dissolved Fe 2+ in porewater, and not O 2 , that controls whether or not strong encrustation occurs. To construct this figure we have used measured AVS profiles from dated cores for the Iona monitoring stations 8 (near the outfall) and station 15 (reference area), (Macdonald et al., in press) and a hypothetical AVS profile at Satellite Channel. Reasonable porewater Fe 2+ profiles are proposed based on literature [START_REF] Reimers | Porewater pH and authigenic phases formed in the uppermost sediments of the Santa Barbara Basin[END_REF][START_REF] Huettel | Advective transport affecting metal and nutrient distributions and interfacial fluxes in permeable sediments[END_REF]. Note that annually measured AVS levels have been relatively consistent at the 2 Iona sampling stations over time (McPherson et al., 2007a).

As we lack sediment geochemical data for Satellite Channel, it is only possible to propose the sediment variables that must exist there in support of the comparatively lighter shell encrustation.

compared with the other three locations. This difference may be related to availability and concentration of Fe 2+ through porewater advection [START_REF] Huettel | Advective transport affecting metal and nutrient distributions and interfacial fluxes in permeable sediments[END_REF]. It is likely that predominantly sandy sediments in Satellite Channel (75-90% -Ellis, 1968) with low organic loading would have low potential to produce iron oxide shell encrustation because O 2 penetration into the surface sediment would be facilitated, thus precipitating out Fe 2+ in the region the bivalves tend to inhabit (upper 4 cm of sediments), and making pore-water Fe 2+ less available for microbial precipitation onto bivalve shells. Therefore, Fe 2+ can build up in porewater only to small concentrations in a restricted zone somewhat below the main habitat of A. serratica. At Iona station 15, sediments are less porous, and have more mud (low O 2 diffusivity) with higher carbon flux supported by terrestrial (Fraser River particulates) and marine carbon. Here, the oxygen penetrates shallowly, and AVS accumulates weakly (due to a moderate supply of carbon) below about 3 cm. As a result, Fe 2+ can build up strongly in A. serratica's prime habitat.

Finally, in the right-hand panel, Station 8 (near the Iona outfall) exhibits low oxygen permeability (or more rapid biological use of oxygen than can be balanced by diffusion into sediment -see Macdonald et al., in press) and high organic loading, supported partly by outfall particulates [START_REF] Hodgins | A re-evaluation of Iona effluent solids deposition based on sediment grain size characteristics[END_REF] and partly by marine and terrestrial organic carbon [START_REF] Macdonald | Recent climate change in the Arctic and its impact on contaminant pathways and interpretation of temporal trend data[END_REF]. In this case, the zone of Fe 2+ build up is again constrained narrowly near the sediment surface because of strong AVS production supported by high organic carbon loading. Redox boundaries shown in Fig. 8 would vary seasonally or interannually with carbon loading [START_REF] Gobeil | Recent changes in organic carbon flux to Arctic Ocean deep basins: Evidence from acid volatile sulfide, manganese and rhenium discord in sediments[END_REF]. Furthermore, sedimentation tends to bury Fe sequestered in solidphase sulphides, whereas when oxyhydroxides near the surface become buried, the iron can be reduced and remobilized into pore water.

Conclusions

The shell encrustations on the Axinopsida serricata from both locations were primarily iron oxides or hydroxy-oxides possibly biologically mediated; the oxides appear to be mostly embedded within an "inner" iron and phosphorus-rich matrix with a different external composition, possibly suggesting a biofilm coating the encrustations. The encrustations appear to be persistent regardless of short-term or small scale changes in sediment geochemisty. These facts suggest that the rust-coloured encrustations on A. serricata shells are similar to those described by [START_REF] Gillan | Morphology of a ferric iron-encrusted biofilm forming on the shell of a burrowing bivalve (Mollusca)[END_REF].

How can Axinopsida serricata shell encrustation be used to diagnose sediment geochemical conditions of interest? Here, we show that the frequency and intensity of A. serricata shell encrustation, particularly in the larger animals, has the potential to provide a map of cumulative labile organic loading and potential oxygen penetration of sediments around a municipal outfall, where sediment AVS levels are >7 umol/g. However, given the many variables that can potentially affect the shells of these animals, it is likely that this approach would only be feasible in areas where abundance of the bivalves is naturally high, and sufficient samples are collected to see a pattern in spite of the variability. In addition, interpretation of results must take into account oxygen permeability of sediments (typically substrate type), as well as possible confounding due to sediment transport, which may move animals in and out of the organic enrichment zone.

Given a pre-survey of shell encrustation within a relatively homogenous and low current substrate regime, a more thorough biogeochemical/contaminant sampling program can be planned in projected areas of organic enrichment impact for sediment geochemistry and benthic ecology. With a better understanding of the development of the encrustations over the life of the bivalves, they may ultimately provide a useful tool to assess integrated organic input in locations where loading is patchy in time or space, for example around fish farms.

Acknowledgements

The authors thank the many people involved in the various monitoring programs that contributed data to this project. In particular, Metro Vancouver (formerly Greater Vancouver Regional District) provided most of the funding for the work. A great deal of help and cooperation were also gratefully obtained from Biologica Environmental Services, Victoria, BC., the Capital Regional District (Victoria, BC), the British Columbia Ministry of Environment, field personnel from Fisheries and Oceans Canada, and several incredibly constructive and professional reviewers. Depth layer in sediments (cm)

Figure Captions

Burd, Macdonald, Wright, van Roodselaar, Fig. 8 Figure Table 2

  Figure 3 shows mean abundance for three replicates (number per 0.1 m 2 ) of the two size classes

Fig

  Fig. 6c,d shows the proportion of A. serricata with light shell encrustation through all sediment

Figure 1 .

 1 Figure 1. (Right) Locations from which Axinopsida serricata specimens, collected as part of

Figure 2 .

 2 Figure 2. Illustration of the range of shell encrustation for A. serricata corresponding to the

Figure 3 .

 3 Figure 3. Abundance of Axinopsida serricata (number per 0.1m 2 ) at the 2004 Iona outfall

Figure 4 .

 4 Figure 4. Proportion of Axinopsida serricata shells with either no encrustations (Category 0) or

Figure 5 .

 5 Figure 5. Empirical relationship between the proportion of Axinopsida serricata shells with light

Figure 7 .

 7 Figure 7. Differences in mean sediment elemental sulfur and silver concentrations with depth

Figure 8 .

 8 Figure 8. A schematic diagram showing how the flux of labile organic carbon to the sediment

  Figure 6a-f Burd, Macdonald, Wright, van Roodselaar, Fig. 1

  Figure1

  precipitates as Fe oxyhydroxides maintaining low[Fe 2+ ] in A. serratica's prime habitat. In the middle panel, moderate organic loading together with less permeable sediments (such as found near Iona station 15 off the south arm of the Fraser River), creates a zone favourable to Fe 2+ in the pore water sandwiched between the surface oxic layer where Fe oxy-hydroxides are precipitated and a deeper, mildly sulphidic layer (AVS buildup) where Fe sulphides are precipitated. This narrow, Fe 2+ enriched zone lies in A. serratica's habitat. In the right hand panel, high organic loading, supported partly by outfall particulates (such as that found at Iona station 8 near the outfall), rapidly consumes oxygen and supports strong SO 4 2-reduction in near-surface sediments as evidenced by high AVS buildup. Because all living A. serratica bivalves pump O 2 containing water through their siphons, we propose that the shell encrustation process is controlled by the diffusion of dissolved Fe 2+ in porewater for which the conditions in the middle panel are most favorable.

	49.5°N	Spanish Bank	Burrard Inlet
					Burrard Inlet
	N o r t h A r m J e t t y			Iona
	49°N	Io na Je tty
			Vancouver
			Airport
		Sturgeon Bank			Satellite Channel
			Lulu Island
	48.5°N S t e v e s t o n J e t t y			Macaulay Point Parry Bay
		124.5°W Roberts Bank	124°W	123.5°W	123°W	122.5°W

8

  Table1. Summary of specimen and data sources used to examine Axinopsida serricata shell deposits on the BC coast. Key: SO = sewer outfall; *includes nearby reference area (Parry Bay).

	Project	Study Area	Year	Depth	A. serricata	Sediment
				Range (m)	Abundance range (# per 0.1m 2 )	Geochemical Data
	Lion's Gate SO	Outer Burrard Inlet	2004	32 -87	42-445	Yes	McPherson et al., 2005b
	Monitoring Program
	Lion's Gate SO	Outer Burrard Inlet	2005	32 -87	34-355	Yes	McPherson et al., 2006b
	Monitoring Program
	Lion's Gate SO	Outer Burrard Inlet	2006	32 -87	40-501	Yes	McPherson et al., 2007b
	Monitoring Program

Table 1

 1

Table 2 .

 2 Comparison of the relative elemental composition, determined using SEM/EDS, of Axinopsida serricata shell deposits. Atomic % is the relative percent composition of noted elements. * indicates < 2 sigma or below detectable range.

		shell	Outer	Inner	Outer
		only (no	Coating	Coating	Coating	Inner
	Elements	deposits)	Iona	Iona	ER67	ER67
			Atomic			Atomic
		Atomic %	% Atomic %	Atomic %	%
	O	97.51	83.1	78.1	80.3	74.63
	Mg	.52*	1.47	1.39	0.85	1.41
	Al	1.09	4.03	1.33	2.49	1.48
	Si	.84*	7.59	3.33	6.72	5.95
	P	-.34*	1.33	4.52	0.73	3.66
	S	.37*	0.13*	.37	1.02	0.53
	K	.01*	0.67	.33	0.32	0.49
	Ca	0.00*	0.9	2.36	5.14	2.73
	Fe	0.00*	0.78	8.26	2.42	9.11
	Total	100	100	100	100	100

Monitoring Program: Sediment Effects Survey, Prepared for the Greater Vancouver Regional District by EVS Consultants Ltd., Burnaby, BC, 262 pp. + appendices. McPherson, C.A., Chapman, M.K., McKinnon, S., Burd, B.J., Fanning, M.L., Olson, J., Hamilton, M.C., Chen, F., 2007a