Hydrobiologia 431: 165–174, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Short-term influence of recolonisation by the polycheate wormNereis succineaon oxygen and nitrogen fluxes anddenitrification: a microcosm simulation

Marco Bartoli, Daniele Nizzoli, David T. Welsh∗ & Pierluigi ViaroliDipartimento di Scienze Ambientali, Universit`a degli Studi di Parma, Parco Area delle Scienze 33/A,I-43100 Parma, ItalyTel: +39 521 905 683. Fax: + 39 521 905 402. E-mail: dave@eagle.dsa.unipr.it

Received 5 March 1999; in revised form 2 March 2000; accepted 21 April 2000

Key words:bioturbation,Nereis succinea, sediment-water column fluxes, denitrification nitrification

Abstract

The short-term effects of sediment recolonisation byNereis succineaon sediment-water column fluxes of oxygenand dissolved inorganic nitrogen, and rates of denitrification, were studied in microcosms of homogenised, sievedsediments. The added worms enhanced oxygen uptake by the sediments, due to the increased surface area providedby the burrow walls and the degree of stimulation was stable with time. Similarly, ammonium fluxes to the watercolumn were stimulated byN. succinea, but declined over the 3 day incubation in all microcosms including thecontrols. Nitrate fluxes were generally greater in the faunated microcosms, but highly variable with time. Deni-trification rates were positively stimulated byN. succineapopulations, denitrification of water column nitrate wasstimulated 10-fold in comparison to denitrification coupled to nitrification in the sediments. Rates of denitrificationof water column nitrate were not significantly different from rates in undisturbed sediment cores with similardensities ofN. succinea, whereas rates of coupled nitrification–denitrification were 3-fold lower in the experimentalset-up. These results may reflect the relative growth rates of nitrifying and denitrifying bacteria, which allow morerapid colonisation of new burrow surfaces by denitrifier compared to nitrifier populations. The data indicate thatrecolonisation by burrowing macrofauna of the highly reduced sediments of the Sacca di Goro, Lagoon, Italy,following the annual dystrophic crisis, may play a significant role in the reoxidation and detoxification of thesediments. The increased rates of denitrification associated with the worm burrows, may promote nitrogen losses,but due to the low capacity of nitrifying bacteria to colonise the new burrow structures, these losses would behighly dependent upon water column nitrate concentrations.

Introduction

Benthic infauna through their feeding, burrowing andsediment irrigation activities can significantly modifythe overall biogeochemistry of marine sediments(Aller, 1988; Kristensen, 1988). Sediment rework-ing due to macrofauna displacement and burrowingmixes fresh organic matter with the bulk sediment,stimulating mineralisation and respiration processes(Kristensen, 1988; Reichardt, 1988; Hansen & Black-

∗ Author for correspondence.

burn, 1992; Hansen et al., 1996) and in particular themineralisation of ‘older’ more refractory material (An-dersen & Kristensen, 1988; Kristensen et al., 1992).Similarly, burrow formation and the sediment irriga-tion activity of certain macrofauna, promotes soluteexchange between the sediment and water column(Aller, 1988, Kristensen, 1988; Kristensen et al.,1991) and can influence the zonation and balancebetween aerobic and anaerobic processes in the sed-iment (Aller, 1988; Kristensen, 1988; Hansen et al.,1996). The sediment surrounding macrofaunal bur-rows is often a zone of high bacterial populations com-

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Table 1. Characteristics of the sampling station

Variable

Sediment composition Silts & fine muds

Organic matter (%) 11

Porosity (ml ml−1) 0.8

Sediment density (g ml−1) 1.2

Temperature (◦C) 18–22

Salinity (‰) 0–25

N. succineadensity (ind m−2) 1200–1600

pared to the bulk sediment (Hylleberg, 1975; Aller &Yingst, 1985; Hansen et al., 1996) and higher bacterialactivities such as sulfate-reduction (Aller & Yingst,1978; Hansen et al., 1996), nitrification, denitrifica-tion and coupled nitrification–denitrification (Sayama& Kurihara, 1983; Pelegrí et al., 1994; Pelegrí &Blackburn, 1995; Gilbert et al., 1998).

Whilst some effects of bioturbation may simplyreflect the increased sediment surface area providedby the burrow walls for exchanges and/or the irrig-ation activity of the animals, others are dependentupon the development of specific bacterial populationswithin the burrow wall sediments and would be ex-pected to show some evolution with the age of theburrows. Therefore, the formation of new structuresduring recolonisation of areas defaunated by cata-strophic events, such as the anoxic/dystrophic eventscharacteristic of highly eutrophic environments (Jør-gensen, 1980; Caumette, 1986; Rosenberg et al., 1990;Llansó, 1992), may create different effects on sedi-ment biogeogeochemistry than those associated withmature burrows.

In this study, we have investigated the short-termeffects of new burrow structures ofNereis succineaonsediment-water fluxes of oxygen, dissolved inorganicnitrogen and denitrification rates in sediments from theeutrophic Sacco di Gora, Adriatic coast, Italy. This la-goon suffers annual dystrophic crises, with prolongedperiods of anoxia and release of toxic sulfides to thewater column associated with the crash ofUlva rigidablooms (Viaroli et al., 1992; Viaroli et al., 1993). Ourstudies were performed in experimental microcosms,with the aim of elucidating the potential role of mac-rofaunal recolonisation on sediment reoxidation andin promoting N-losses via denitrification following adystrophic event.

Figure 1. Location of the Sacca di Goro on the Adriatic coast ofItaly (circled) and detailed map of the lagoon, showing the positionof the sampling station, near to the mouth of the Po di Volano canalin the eastern part of the lagoon (filled circle).

Materials and methods

Study site

All sediment and water samples were collected inthe Sacca di Goro (44◦ 47–50′ N 12◦ 15–20′ E),a eutrophic lagoon of the Po River Delta, Adriaticcoast, Italy, which suffers annual summer dystrophiccrises associated with the crash and decomposition ofblooms of the macroalgaUlva rigida (Viaroli et al.,1992; Viaroli et al., 1993). The sampling station wassituated close to the mouth of the Po di Volano (Figure1) in an area not normally subject to dystrophic crises,with high abundance of matureN. succinea(Table 1).

Collection ofN. succineaindividuals

Sediment cores (20 cm internal diameter) were im-mediately transported to the laboratory (∼1 h), sievedover a 500µm mesh.N. succineaindividuals retainedon the mesh were transferred to small aquariumscontaining a few cms of sieved sediment and main-tained under a constantly aerated water column untilrequired. Animals were stored maximally for 24 hbefore the experiments.

Microcosm incubations

Sediment collected at the sampling station was sievedthrough a 500µm mesh to remove large infauna. Thesieved sediment was mixed with an equal volume ofwater from the sampling station and dispensed to com-pletely fill 40 × 8 (internal diameter) cm plexiglasscores. During dispensation to the cores, the slurry

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Table 2. Densities and biomasses ofN. succineain the exper-imental treatments at the end of the 3 day incubation period.Figures in parenthesis represent one standard deviation (N =3). NA not applicable, no animals were added.∗ The smallbiomasses present were made up of small juvenileN. succineaindividuals, which had passed through the 500µm mesh used tosieve the sediments used in the microcosms

Treatment Added worm density Biomass(No. animals added core−1) (Individuals m−2) (g dry weight m−2)

0 NA∗ 2.1 (1.3)∗8 1600 21.7 (3.0)16 3200 49.0 (7.5)

was continually mixed in order to maintain homo-geneity. The sediment was allowed to settle for 3 hand magnetic stirrers were fitted inside the cores ap-proximately 5 cm above the sediment surface. Coreswere transferred to a constantly aerated 60 l incubationtank containing a central motor with mounted mag-nets to drive the individual magnetic stirrers withinthe cores. The cores were pre-incubated in the darkfor 3 days to allow the establishment of an oxidisedsurface layer and solute gradients within the sediment.Medium sizedN. succineaindividuals were added totriplicate cores to give densities of 0 (control), 8 or16 individuals per core (see Table 2 for biomassesin the individual treatments), which correspond toapproximately 1× and 2× the naturalin situ popu-lation densities (Table 1). Flux rates of oxygen andinorganic-N species were determined at intervals (seetext) over the following 3 days and denitrification rateswere measured following the final flux incubation.

The water in the incubation tank was changed ona daily basis during the pre-incubation period and fol-lowing each flux incubation during the experimentalincubation using natural unfiltered water collected atthe sampling station.

Determination of sediment–water fluxes

In order to ensure similar initial conditions betweencores at the beginning of incubations, the water inthe cores was exchanged with that in the incubationtank, by repeatedly removing water from within thecores using a 100 ml syringe, whilst they were sub-merged. To initiate flux measurements, the water levelin the tank was lowered below that of the top of thecores, initial time water samples were collected foroxygen and inorganic nutrient concentrations and thecores sealed with floating plexiglass lids (Risgaard-

Petersen & Rysgaard, 1995). Cores were incubatedfor 1 h in the dark, the floating lid removed and finaltime samples for oxygen and nutrients immediatelycollected.

To determine the oxygen consumption rates ofN.succinea, known numbers of individuals were ad-ded to microcosms containing autoclaved fine sand assubstrate. After the worms had completely burrowedinto the substrate oxygen, fluxes were determinedas described for the experimental microcosms andcalculated on a per individual basis.

Flux rates were calculated using the general fluxequation:

F = (C0− CT)V /A∗T ,

whereC0 andCT are the species concentrations at timezero and timeT , respectively;V is the volume of wa-ter in the core;A is the sediment surface area andTthe time of incubation.

Determination of denitrification rates

Denitrification rates of water column nitrate andcoupled nitrification–denitrification were determinedby the isotope pairing technique (Nielsen, 1992). Priorto experiments, the water in the cores was exchangedwith that in the incubation tank to ensure homogeneity.To initiate measurements, the water in the incubationtank was lowered below the level of the cores and azero time sample collected for determination of theinitial nitrate concentration. Sufficient of a stock solu-tion of 15N-nitrate (9915N atom %) was added togive a nominal final concentration of 30µM, a secondwater sample was collected to determine the actual15N-nitrate addition by difference and the cores wereclosed with floating plexiglass lids (Risgaard-Petersen& Rysgaard, 1995).

Cores were incubated in the dark for 1 h. Afterincubation, biological activity was stopped by additionof 7 M ZnCl2 to a final concentration of 10 mM andthe cores were gently slurried using a metal bar in or-der to homogenise the dissolved N2 pools in the watercolumn and porewater. Subsamples of the final slurrywere transferred to completely filled 12 ml Exetainervials (Exetainer, Labco, High Wycombe, U.K.), fixedwith 100 µl of 7M ZnCl2 and stored at 4◦C untilanalysed.

Samples were analysed for14N15N and 15N15N–N2 by GC-mass spectroscopy as previously described(Rysgaard et al., 1995) and the production rates ofthe isotope pairs were calculated from the combined

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volume of the water column and porewater (calculatedby multiplying the sediment volume by the porosity,determined as loss of wet weight after drying at 90◦C for 24 h). Denitrification rates were estimated fromthe production rates using the equations derived byNielsen (1992):

D15=p(14N15N)+ 2p(15N15N),

D14=p(14N15N) /2p(15N15N)∗ D15,

where D15 and D14 are the rates of denitrifica-tion of 15N-nitrate and14N-nitrate, respectively, andp(14N15N) and p(15N15N) are the production rates ofthe two15N labelled N2 species,14N15N and15N15N,respectively. The calculated rate D15 expresses denitri-fication of the added15N-nitrate, whilst, D14 expressesthe totalin situ denitrification rate of14N-nitrate. Theproportion of D14 based on nitrate diffusing from thewater column (DW14) was calculated from D15 and the15N enrichment of the water column nitrate pool asbelow:

DW14=D15[14NO3−]W/ [15NO3

−]W,

where [14NO3−]W and [15NO3

−]W are the concen-trations of unlabelled and15N-labelled nitrate in thewater column.

Rates of denitrification coupled to nitrate producedby nitrification activity in the sediment compartment(DN) were calculated by difference:

DN=D14−DW14.

Following determination of denitrification rates, thefinal sediment slurries were sieved through a 500µmmesh and theN. succineaindividuals retained on themesh were fixed in 2% (v/v) formaldehyde for laterdeterminations of abundances and biomass. Biomasswas determined as dry weight following drying at 90◦C for 24 h.

Dissolved oxygen concentrations were determinedby the Winkler titration method as described (A.P.H.A.1975), ammonium concentrations were measured bythe indophenol-blue method (Koroleff, 1970), ni-trite was determined spectrophotometrically followingdiazotation (A.P.H.A. 1975) and nitrates were determ-ined as nitrite by the same method following reductionover cadmium columns and data were corrected for theambient nitrite concentration.

Statistical analysis

Normality of data was assumed and homoscedasticitywas confirmed using the Cochrane test. Data were

Figure 2. Sediment oxygen uptake recorded over the 3 day micro-cosm incubations for control microcosms (Open bars), microcosmswith 8 worms (Grey bars) and microcosms with 16 worms (Blackbars). Error bars indicate one standard deviation (n=3). Negativevalues indicate that the flux was from the water column to thesediment.

analysed by ANOVA analysis with ana posterioricomparison of the means using the T-method (Sokal& Rolf, 1995).

Results

Following addition ofN. succineapopulations to themicrocosms, the animals immediately burrowed intothe sediments and burrows extending in depth togreater than 10 cm depth were visible through the corewalls after a few hours. These burrows developed anorange brown coloured layer of oxidised sediment 1–2mm thick within a few hours, which vividly contras-ted with the surrounding black, reduced sediments.Similarly, there was a 2–3 mm thick oxidised layer ofsurficial sediment. Some smaller burrows penetrating1–3 cm into the sediment of the control microcosmswere formed by small juvenileN. succineaindividualswhich had passed through the 500µm mesh used tosieve the sediments. However, the overallN. succineabiomasses in these microcosms were small comparedto the experimental treatments (Table 2).

The addition ofN. succineato the microcosmscaused an immediate and significant stimulation ofthe sediment oxygen demand (ANOVAP < 0.001),with oxygen fluxes to the sediments in microcosmscontaining 8 or 16 individuals being 1.9 and 3.3-foldgreater than those of the controls (Figure 2). Thisaugmentation of oxygen fluxes in the worm amendedmicrocosms remained stable throughout the 3 day in-cubation period and variations in oxygen fluxes with

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Figure 3. Time evolution of sediment water column fluxes of am-monium in control (Open bars), microcosms with 8 added worms(Grey bars) or 16 added worms (Black bars). Error bars indicateone standard deviation (n=3). Positive values indicate an efflux ofammonium to the water column, whereas, negative values representfluxes directed towards the sediments.

time were not significant for any of the treatments(ANOVA P= 0.876, 0.612 and 0.227, for controls,+ 8 worms and +16 worms, respectively). Similarly,the presence ofN. succineastimulated effluxes of am-monium from the sediment to the water column in theexperimental treatments, with the mean fluxes in theincubations performed over the first 8 h after animaladditions, being 1.4 and 2.2-fold greater than the con-trols for microcosms containing 8 and 16N. succineaindividuals respectively (Figure 3). However, in con-trast to oxygen fluxes, ammonium effluxes were notstable with time and showed a significant decline inboth the control and worm-amended microcosms overthe entire 3 day incubation period (ANOVAP < 0.001for all 3 treatments). Although, at each time interval,the fluxes in the worm amended microcosms remainedgreater than those of the controls (Figure 3). However,whilst the differences in effluxes between treatmentswere highly significant for the early incubations (AN-OVA P < 0.01 for incubations within the first 24h), the level of significance of these differences alsodeclined with time and ammonium fluxes during thefinal 3 incubations showed no significant differencesbetween treatments (ANOVAP > 0.05).

Fluxes of nitrate were much very more variable,mostly due to the use of natural seawater for the in-cubations which varied between approximately 50 and80µM nitrate and masked any evolution of the fluxeswith time in the experimental microcosms. Fluxeswere predominantly directed towards the sedimentin all the treatments and were stimulated in worm

Figure 4. Example of the effect ofN. succineaadditions on the wa-ter column to sediment fluxes of nitrate for control microcosms andmicrocosms to which 8 or 16 worms had been added. The incub-ation was performed between 7 and 8 h after the worm additionsand the initial water column nitrate concentration was 49.9µM.Error Bars indicate one standard deviation (n=3). The negative fluxrates indicate that the nitrate flux was from the water column to thesediments.

Figure 5. Rates of total denitrification (Open bars), denitrifica-tion of water column nitrates (DW; Grey bars) and denitrificationcoupled to nitrification in the sediment (DN; Black bars), for ex-perimental microcosms and intact cores of undisturbed sedimentscollected at the same sampling site and having natural macrofaunapopulation densities approximately equivalent to the treatment with8 worms (Table 1). Denitrification rates for experimental micro-cosms were determined approximately 80 hours after the wormadditions and approximately 24 h after collection for intact cores.Error bars represent one standard deviation (n=3 for experimentalmicrocosms and 5 for intact sediment cores).

amended compared to control microcosms during in-dividual flux incubations (Figure 4). Although for themajority of incubations, this tendency towards higherfluxes in the worm amended microcosms was notsignificant (ANOVAP > 0.05).

Denitrification rates measured at the end of themicrocosm incubations, were positively influenced bythe presence ofN. succinea(Figure 5) and rates of total

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Figure 6. Relationship between rates of DN (open circles) and Dw(filled circles) andN. succineabiomass. Data points represent thebiomasses and rates determined for the individual experimentalmicrocosms.

denitrification were significantly different between thetreatments (ANOVAP < 0.01). This stimulation wasdue almost entirely to increased rates of denitrifica-tion of nitrate diffusing from the water column (DW),which increased linearly withN. succineabiomass(Figure 6) and was significantly different between thetreatments (ANOVAP < 0.01). In contrast, rates ofdenitrification coupled to nitrification in the sedimentwere similar in both control and worm amended mi-crocosms (ANOVAP=0.093) and showed only a weakrelationship with theN. succineabiomasses (Figure6). Comparison of the regressions for DW and DNwith N. succineabiomass shows that there was a 10-fold greater stimulation of DW compared to DN, withrates increasing by 5.1 and 0.5µmol N m−2 h−1 gdry weight−1 for DW and DN, respectively. Similarly,whilst 63 and 76% of total DW activity was associatedwith the burrow walls in microcosms amended with8 and 16 individuals. Only 23 and 34% of total DNactivity was associated with theN. succineaburrowstructures. Additionally, comparison of the data forcores to which 8 worms were added and undisturbedsediment cores with natural macrofaunal populations(Figure 5), which had similar densities and biomassesof N. succinea(Table 1). Demonstrates that whilstrates of DW were similar (216± 76 and 228± 49µmol N m−2 h−1, respectively), rates of DN were al-most 3-fold greater in the undisturbed cores comparedto the experimental microcosms (202± 53 and 71±5µmol N m−2 h−1, respectively).

Figure 7. Calculated contributions ofN. succinea respiration(White portion), burrow surfaces (Grey portion) and the surficialsediment (Black portion) to the overall oxygen flux. The contri-bution of the surficial sediment was estimated as being equivalentto that of the control microcosms, the contribution of the animalswas determined experimentally to be 0.525µmol ind−1 h−1 andthe contribution of the burrow walls was calculated by difference.The mean values for all incubations were used for the calculationsand the mean total oxygen fluxes to the sediments are shown inparenthesis.

Discussion

Addition of N. succineaindividuals to the experi-mental microcosms significantly stimulated sedimentoxygen uptake. This stimulation of exchange is due tothe increased surface area offered by the burrow sur-faces and the activity of the animals which circulatesoxygenated water within the burrows promoting theexchange of solutes between the sediment and watercolumn (Aller, 1988; Kristensen, 1988; Kristensenet al., 1991). In parallel incubations ofN. succineain sterile autoclaved substrate, the respiratory oxygendemand of the worms themselves was determined tobe 0.525µmol O2 ind.−1 h−1. Thus, by calculationonly 19 and 21% of the total oxygen flux in micro-cosms containing 8 and 16 individuals, respectively,could be attributed to the respiration of the worms. Ifwe assume that the oxygen consumption of the sur-ficial sediments was unaffected by worm additions,then 31 and 49% respectively of the total oxygen de-mand in the amended cores was associated with theburrow wall sediments (Figure 7). Several other au-thors have also reported increased sediment oxygendemands associated with the presence of macrofaunain both marine and freshwater sediments (Aller, 1988;Pelegrí & Blackburn, 1995; Svensson & Leonard-son, 1996). Hansen & Kristensen (1997) reported thatoxygen uptake was stimulated 2–3-fold during sim-

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ulations of recolonisation byNereis diversicolorandestimated that 54–68% of the increased oxygen uptakewas by the sediment rather than the worms respira-tion. Similarly, N. virensincreased sediment oxygenuptake by 43%, but only 11% of this was due to theirrespiration the remainder being due microbial andchemical uptake associated with their burrow walls(Kristensen, 1985). The increased oxygen consump-tion associated with the burrow walls ofN. succineainour experiments would result in a shift in the balancebetween aerobic and anaerobic processes in the sedi-ments, due to the increased oxygen supply to aerobicheterotrophs, and for the biotic and abiotic reoxida-tion of reduced compounds (Aller, 1988; Kristensen,1988; Hansen et al., 1996). Increased rates of sulfideand iron oxidation have been shown to occur aroundthe burrows of the bivalveMya arenaria, creatinga more oxidised sediment zone despite the fact thatsulfate reduction rates in the burrow walls were 1.5–2-fold higher than in the bulk sediments (Hansen etal., 1996). This effect would be expected to be greaterin polycheate burrows, as the siphon ofM. arenariarepresents a more effective barrier to oxygen diffusionto the burrow wall sediments compared to the mucuslined burrows ofNereisspecies. Indeed, in a compar-ative study, Pelegrí & Blackburn, (1995) reported thatthe bivalvesM. arenariaandCerastodermasp. causedonly a minor stimulation of water column-sedimentoxygen fluxes compared to aNereis sp. indicatingthat polycheates more effectively stimulate reoxida-tion processes in the burrow wall sediments comparedto bivalves. Thus, in the Sacca di Goro, recolonisa-tion of the sediment by burrow forming macro-faunalspecies such asN. succineafollowing the annualdystrophic crisis, could play a major role in the re-oxidation/detoxification of the highly reduced surficialsediments which are generated during the period ofprolonged anoxia associated with the crash of theUlvarigida blooms (Viaroli et al., 1992; Viaroli et al., 1993;Giordani et al., 1997).

The presence ofN. succineaalso stimulated am-monium effluxes, although in contrast to oxygenfluxes, these changes were not stable and effluxes de-clined with time in all the microcosms (Figure 3).Similar pulses in ammonium effluxes have been ob-served during recolonisation of sediments byN. di-versicolor (Hansen & Kristensen, 1997) and can beattributed to a number of effects. Firstly, sieving ofthe sediments during preparation of the microcosmswill have mixed labile organic matter into the bulksediment, stimulating rates of ammonification (Black-

burn & Blackburn, 1992; Sloth et al., 1995) and theresultant high porewater ammonium concentrationswould favour diffusive ammonium fluxes to the watercolumn. These, fluxes would be further enhanced bytheN. succineaindividuals initially burrowing into theammonium rich sediments, the increased surface areaprovided for diffusive effluxes by the burrow walls,burrow irrigation and the excretion of ammonium bythe animals themselves, which can contribute signific-antly to overall ammonium fluxes in bioturbated sed-iments (Kristensen, 1984; Kristensen, 1985; Gardneret al., 1993; Hansen & Kristensen, 1997). However,with time this porewater ammonium reservoir wouldbe depleted especially in the presence of bioturbatinginfauna (Kristensen, 1988; Reichardt, 1988; Hansen& Blackburn, 1992; Hansen et al., 1996) and this ef-fect may account for the decline in the significanceof treatment effects on overall ammonium fluxes weobserved. The presence of macrofauna has previouslybeen demonstrated to reduce the overall porewater am-monium concentration, compared to equivalent non-bioturbated sediments (Hylleberg & Henriksen, 1980;Henriksen et al., 1980; Kristensen, 1984), therebyreducing the concentration gradient between the pore-water and water column and hence the diffusive fluxrate. Secondly, the development of an oxidised layer ofsediment around the burrow walls and at the sedimentsurface and depletion of the ammonium pools withinthis layer (Hylleberg & Henriksen, 1980; Henriksenet al., 1980; Kristensen, 1984) would increase thediffusional path-length between the ammonium rich,anaerobic sediments and the water column, whichwould further limit diffusional effluxes. Finally, thegrowth of bacterial populations within these oxic sed-iment layers would create an assimilatory sink forammonium and the development of ammonia oxid-ising, nitrifier populations would further increase thisammonium sink. Thus, it is probable that a combin-ation of these factors was responsible for the overalldecline in ammonium fluxes recorded in all the mi-crocosms and the reduced significance of treatmenteffects with time, that occurred in our experiments.

Nitrate fluxes showed a high degree of variabil-ity between the individual incubations and no cleartrends were evident, due to the large variation (50–80µM) in the nitrate concentration in the natural seawa-ter used for the incubations. In bioturbated sediments,the water column nitrate concentration is a major de-terminant of overall nitrate fluxes, influencing both itsmagnitude and direction (Kristensen, 1984), since theconcentration difference between the porewater and

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water column is the driving force for diffusive fluxes.Thus, it is probable that any tendencies of the nitratefluxes with time resulting from the relative changesin the sedimentary sinks (assimilation, denitrificationand dissimilatory nitrate reduction to ammonium) andsources (nitrification) in the surface and burrow wallsediments, were obscured by the variability in watercolumn nitrate concentrations. However, the generalstimulation of nitrate fluxes byN. succineaalthoughnot significant for most incubations is consistent withother studies of the effect of benthic infauna (Hen-riksen et al., 1980; Kristensen, 1984; Hansen &Kristensen, 1997).

Rates of total denitrification were significantlystimulated by the presence ofN. succinea(Figures 5and 6), as has been reported for other burrow formingpolycheate worms (Henriksen et al., 1980; Sayama &Kurihara, 1983; Kristensen, 1985; Kristensen, 1988;Pelegrí & Blackburn, 1995; Gilbert et al., 1998). Thestimulation of denitrification in our experiments wasdue almost exclusively to increased rates of denitri-fication of nitrate from the overlying water column(Figure 5), which increased linearly with increasingN. succineabiomass (Figure 6) and an estimated 63and 79% of this activity was associated with the bur-row structures for microcosms amended with 8 and16 worms, respectively. Indeed, rates of DW for mi-crocosms amended with 8N. succineaindividualswere highly similar to those recorded for intact coreswith natural macrofaunal populations at similar bio-mass densities (Figure 5; Tables 1 and 2). These dataindicate that within the 3 day incubation period, de-nitrifying bacteria had rapidly colonised the burrowsurfaces and exhibited similar activities to thein situpopulations of natural mature burrow structures. Incontrast, rates of coupled nitrification–denitrificationin the sediments were weakly stimulated byN. suc-cinea additions (Figures 5 and 6) and were approx-imately 3-fold lower than rates in intact cores withsimilar N. succineapopulation densities (Figure 6).Additionally, only 23 and 34% of the total couplednitrification–denitrification was associated with theburrow structures of microcosms amended with 8 and16 worms, respectively. These results indicate that the3 day incubation period was insufficient to allow nitri-fier populations to re-establish to levels and activitiesequivalent to those in natural mature burrow struc-tures and reflects the slow growth rates of this bacterialgroup (Henriksen & Kemp, 1988). These relative dif-ferences in the rates of recolonisation of new burrowsby nitrifying and denitrifying bacteria could have a

major influence on nitrogen losses via denitrificationfollowing the annual dystrophic crisis in the Sacca diGoro. As water column nitrate concentrations are gen-erally low during this period, and ammonium is thedominant N-source in both the water column and sed-iments due to the rapid mineralisation of theUlva bio-masses (Viaroli et al., 1992). Thus, potentially couplednitrification–denitrification would represent the mostsignificant pathway for N-loss. Indeed, the slow re-establishment of nitrifier populations may be evenmore severely limitedin situ, as the environmentalconditions during the dystrophic crisis, particularly theprolonged anoxia and presence of toxic free sulfidesin the water column are not amenable to the growthor survival of obligatory aerobic nitrifiers (Henriksen& Kemp, 1988). Thus, following the dystrophic crisisthe potential inoculum of nitrifying bacteria for newlyformed burrows may be extremely low and this wouldcompound the problem of their slow growth rates. Thiscombination of low water column nitrate concentra-tions, probable low initial nitrifier populations and theslow growth rates of nitrifying bacteria, may limit N-losses via denitrification and favour N-recycling andretention within the lagoon, which could fuel furthermacro-algal blooms and act as a positive feedbackin the spiralling eutrophication status of the Sacca diGoro.

Conclusions

Data presented in this paper demonstrate that recolon-isation of sediments byN. succineacould potentiallyplay a major role in the re-oxidation and detoxificationof the highly reduced sediments generated during theannual dystrophic crisis in the Sacca di Goro, Italy,due to the stimulation of oxygen fluxes to the sedi-ments. Similarly, the recolonisation process may alsostimulate N-losses from the system via denitrification,although these losses may be somewhat limited by thegenerally low water column nitrate concentration (Vi-aroli et al., 1992) and a combination of the probablesmall inoculum of nitrifying bacteria in the immediateenvironment and their low capacity to colonise newburrow structures, due to their inherently slow growthrates.

However, these processes would be dependentupon the capacity of migrating macrofaunal popula-tions, from areas unaffected or not greatly affectedby the dystrophic crisis to recolonise the more heav-ily impacted areas. Many adult macrofauna, including

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polycheate worms, are known to be able to recolonisedefaunated areas (Dauer & Simon, 1976; Santos & Si-mon, 1980; Thrush et al., 1992). However, consideringthe large area of the Sacca di Goro affected by the algalblooms (up to 10 km2) and subsequent anoxic events(Viaroli et al., in press), migration of adult individualsto new areas may be limited due to the large distancesinvolved, unless refuge populations persist within theaffected area. Thus, further research on the survivaland spatial distribution of large macrofaunal speciesin the Sacca di Goro following the dystrophic crisis,is required before a true estimation of the potentialimpacts of recolonisation of defaunated areas can beassessed.

Acknowledgements

This study is a contribution to the ELOISE programme(ELOISE No. 0156) within the frameworks of theprojects NICE (Grant No. MAS3-CT96-0048) andROBUST (Grant No. ENV4-CT96-0218). The authorsgratefully acknowledge the Cooperative Pescatori diGoro, for the provision of laboratory space and facil-ities and the crew of the Hydra (Provincia di Ferrara),Tony Zappata and Giancarlo Grigatti, for assistancewith sample collection.

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