macroalgal (fucus vesiculosus) δ 15 n values trace decrease in sewage...

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Ecological Applications, 14(2), 2004, pp. 517–526q 2004 by the Ecological Society of America

MACROALGAL (FUCUS VESICULOSUS) d15N VALUES TRACE DECREASEIN SEWAGE INFLUENCE

CANDIDA SAVAGE1 AND RAGNAR ELMGREN

Department of Systems Ecology, Stockholm University, SE-106 91 Stockholm, Sweden

Abstract. Nutrient discharges from sewage treatment plants can contribute significantlyto coastal and estuarine eutrophication. To counter anthropogenic nitrogen (N) loads in aBaltic Sea coastal embayment, improved (;85%) N removal was implemented in a tertiarysewage-treatment plant. This study used nitrogen stable-isotope ratios (d15N) in attachedbrown macroalgae, Fucus vesiculosus, to map the change in the spatial extent of the influenceof sewage-derived N. Elevated N content and d15N values (d15N 5 8–9‰) suggest thatsewage N was still traceable in algal tissues despite enhanced N removal from the effluent.However, the d15N values decreased significantly with distance and approached backgroundlevels within 10–12 km of the outfall. Compared to a survey conducted in 1989, prior toenhanced N removal in the sewage treatment plant, macroalgae were 2.5–6‰ lower in d15Nvalues within 24 km from the outfall, demonstrating a decline in the importance of sewage-derived nutrients to macroalgae, particularly in the inner reaches of the bay. Beyond 25km from the outfall, d15N values were at background levels (d15N 5 4‰). Two-sourcemixing models estimate that the percentage sewage contribution to total algal N uptake inalgae near the outfall was ;40% in 1989 and ;12% in 1999. Similarly, temporal variationin individual plants estimate that the percentage sewage N contribution to algae within 1km from the outfall declined from .40% in the mid-1990s to ;20% in 2002. Nutrientbudget calculations showed that F. vesiculosus is not an effective sink for N, assimilatingonly ;3% of total annual N loads entering the bay.

Key words: Baltic Sea (Himmerfjarden Bay); coastal eutrophication; d15N; Fucus vesiculosus;macroalgae; N assimilation; nitrogen removal from sewage, measuring effects of; sewage treatment.

INTRODUCTION

Understanding the fate and processing of anthro-pogenic nutrients is crucial for water-quality manage-ment and for assessing the assimilation capacity ofcoastal zones. Increased inputs of wastewater-derivednitrogen (N) to nitrogen-limited (Howarth 1988) es-tuaries and coastal areas has increased primary pro-duction (Bowen and Valiela 2001) and altered pathwaysof N cycling (McClelland and Valiela 1998). Nutrientreduction initiatives have often focused on enhancedN removal in sewage-treatment plants to counter theeffects of coastal eutrophication, as point-source dis-charges of nutrients are easier to manage than diffuseand atmospheric sources. Himmerfjarden Bay, Sweden,is the site of a large-scale experiment to assess theeffects of nutrient discharges from municipal waste-water and the effectiveness of tertiary sewage treatmentto counter coastal eutrophication. Coastal inlets andarchipelagos of the brackish Baltic Sea are particularlyprone to the effects of eutrophication as they are gen-erally shallow, have limited water exchange, and areoften strongly stratified in summer. Nitrogen removalprocedures have been progressively improved in theHimmerfjarden treatment plant and water quality in the

Manuscript received 12 December 2002; revised 20 May2003; accepted 8 June 2003. Corresponding Editor: L. A.Deegan.

1 E-mail: [email protected]

bay has generally improved, as indicated by decreasesin annual average phytoplankton biomass (Elmgren andLarsson 2001). Yet phytoplankton biomass and nutrientconcentrations show strong seasonality and high var-iability in response to nutrient-remediation measures.To better understand the fate and assimilation of sew-age-derived N in the bay and as an aid to assessing theeffectiveness of enhanced nutrient removal over longertime intervals, stable nitrogen isotope ratios in the ses-sile, perennial macroalga, Fucus vesiculosus L., wereused to map the influence of sewage-derived N in thebay.

Anthropogenic sources of N can be explicitly linkedto N in estuarine plants (McClelland et al. 1997), thusmaps of stable N isotopic ratios in macroalgae providea useful tool to delineate the influence of sewage incoastal areas (Hobbie et al.1990, Costanzo et al. 1999,Rogers 1999, Gartner et al. 2002). Stable isotopes re-flect an integrated history of sources, pathways, andfates of organic matter (Robinson 2001). Primary pro-ducers often have distinctive N stable isotopic ratiosthat reflect the d15N of their inorganic-N sources plusa variable amount of fractionation. Secondary and ter-tiary sewage treatment normally enriches the d15N sig-nal of dissolved inorganic nitrogen (DIN) relative toautochthonous marine sources through nitrification anddenitrification processes (Heaton 1986), thereby pro-viding a convenient tracer of sewage N. Isotope anal-

518 CANDIDA SAVAGE AND RAGNAR ELMGREN Ecological ApplicationsVol. 14, No. 2

FIG. 1. Map of Himmerfjarden Bay, Sweden, showing thesampling stations where Fucus vesiculosus were collected in1999. The 17 stations sampled in only 1999 are illustrated assolid circles, and the 19 stations also sampled in 1989 areillustrated as empty circles. Environmental monitoring sta-tions, H2–H6, X1, and B1, where total N was measured, arealso indicated.

yses have shown the uptake and incorporation of sew-age-derived nutrients in macroalgae (Hobbie et al.1990, Costanzo et al. 1999, Rogers 1999, Wayland andHobson 2001, Gartner et al. 2002), soft-bottom benthicorganisms (Van Dover et al. 1992, Waldron et al. 2001),corals (Heikoop et al. 2000), and zooplankton and fish(Spies et al. 1989, Hansson et al. 1997), yet to the bestof our knowledge, no studies have used stable isotopesto quantify reduction of impact following improvedsewage treatment.

The objective of this study was to use stable isotopesin macroalgae to quantify reductions in sewage-derivedN following enhanced sewage treatment. This studyfollows an initial isotopic survey conducted in 1989 inHimmerfjarden Bay that showed that d15N in Fucusvesiculosus was elevated (d15N 5 8–13‰) in the prox-imity of and within 25 km of a sewage outfall, beyondwhich it gradually decreased to background levels(d15N 5 4‰) at the seaward end of the bay (Hobbie etal. 1990). Using the stable N isotope ratio in F. vesi-culosus following enhanced N removal from sewageeffluent, we map a lessened influence of sewage-de-rived nutrients in a coastal embayment, integrated overtime. Moreover, we use estimates of macroalgal densityand records of total Nitrogen (TN) discharge to esti-mate N uptake by macroalgae within the embayment.

MATERIALS AND METHODS

Site description

Himmerfjarden Bay (598009 N, 178459 E) is a 30-km-long inlet of the brackish Baltic Sea, located about60 km south of Stockholm, Sweden (Fig. 1). The 174-km2 bay is oriented mainly along the north–south axisand has a drainage basin of 1286 km2. The bay has amean depth of 17 m, mean surface salinity of 6‰, andexperiences only minor salinity fluctuations. Verticalwater exchange in the estuary is normally driven bywind-induced mixing while water exchange with theopen Baltic Sea is driven by atmospheric pressure gra-dients. Tides are negligible. Temperature stratificationin summer traps sewage-derived nutrients in the ther-mocline below the upper mixed layer where an estu-arine circulation carries discharged effluent in a pre-dominantly northerly direction. In winter, there is nothermocline, resulting in vertical mixing of the sewage-derived nutrients.

Nutrient-loading history

Himmerfjarden Bay receives treated sewage fromabout 250 000 people, discharged from a diffuser lo-cated ;1.5 km offshore at 20 m depth in the northernend of the bay. Almost all phosphate (;96%) is re-moved from the effluent by chemical precipitation. Thesewage treatment plant (STP) has undergone progres-sive improvement in N removal. In 1988, the year pre-ceding the first isotopic macroalgal survey, a total of848 3 103 kg TN was discharged and sewage N con-

tributed ;70% of total N loads to the bay. During 1998-2000, denitrification in the STP removed ;85% of thesewage TN so that sewage contributed 171 3 103 kgTN (only ;30%) of the total N loading entering thebay. Other nutrient sources are land runoff (estimatedas 282 3 103 kg TN in 1998), precipitation (18 3 103

kg TN in 1998) and Lake Malaren through a regulatedsluice at the northwest inlet (78 3 103 kg TN in 1998).About 90% of the effluent N is in the form of DIN(dissolved inorganic N), with the proportion of NH4

1

and NO22/NO3

2 varying temporally. In 1986 and 1987,the effluent TN was composed of 47% NH4

1 and 44%NO2

2/NO32. In 1998 and 1999, following enhanced de-

nitrification, the effluent was composed of ;7% NH41

and 45% NO22/NO3

2.

Fucus vesiculosus

Fucus vesiculosus is a perennial brown macroalga(Phaeophyta) with peaks in biomass found between 0.5and 3 m in the study area (Wallentinus 1979). It as-

April 2004 519MACROALGAE TRACE DECREASE IN SEWAGE

similates DIN mostly between November and Marchwhen nitrate is abundant and before spring bloom phy-toplankton and filamentous algae deplete the DIN con-centrations (Lehvo et al. 2001). The N stored in winteris then used for rapid growth in spring and summer(Lehvo et al. 2001). The new growth tips thus mirrorthe DIN conditions of the ambient water from the pre-vious winter.

Fucus vesiculosus plants for the spatial survey werecollected on 10 and 11 May 1999 along a gradient ofincreasing distance from the sewage outfall to thecoastal reference station, B1, about 36 km seawards(Fig. 1). In the initial isotopic survey in May 1989(Hobbie et al. 1990), one plant was collected from eachof 19 stations and analyzed for its d15N and d13C value.The same 19 stations were sampled in May 1999 plusan additional 17 stations, totaling 36 stations. If F.vesiculosus plants were absent from stations sampledin 1989, the closest plants at the same distance fromthe outfall were obtained. On average, three plants fromeach station were collected in 1999, within half a meterbelow the water surface. New growth tips (;2 cm) wereremoved from vegetative fronds, cleaned of epiphytesand organisms by hand, and oven-dried at 608C over-night.

Three Fucus vesiculosus plants were also collectedon 16 May 2002 from a station ;1 km from the outfalland three plants near the B1 coastal reference station.The samples were cleaned of epiphytes and subsampledaccording to frond dichotomies so that temporal chang-es in isotopic composition within individual plantscould be determined. Holdfast material was disregard-ed as it grows in thickness as well as height and cannottherefore be reliably subsampled to represent annualgrowth patterns. Although the number of dichotomiescannot be directly linked with age, we could roughlyassign age to the frond tissue using a combination ofdichotomy counts, frond length, and the presence ofbladder pairs, based on the finding that F. vesiculosusgrows ;7–9 cm (Wallentinus 1979) and produces onepair of bladders per year in the study area (Lena Kaut-sky, personal communication). Samples were oven-dried at 608C overnight.

Stable-isotope analyses

Macroalgal samples were ground and homogenizedusing a mortar and pestle. Samples were combustedand analyzed for their nitrogen and carbon isotopicratios and elemental composition in a Finnigan MATDelta Plus mass spectrometer (Thermo Electron, Bre-man, Germany) interfaced with a Carlo Erba NC2500elemental analyzer. Only d15N values are presented inthis paper as we are interested in tracing sewage N.Precision for replicates was better than 0.15‰. Stable-isotope values are given as

d (‰) 5 [(R 2 R )/R ] 3 1000sample standard standard

where R is the ratio 15N:14N. Values are expressed rel-

ative to a standard, namely, atmospheric air. Sampleswith a lower d value are referred to as ‘‘lighter’’ or‘‘depleted,’’ while samples with a higher d value arereferred to as ‘‘heavier’’ or ‘‘enriched’’ relative to 15N.

Water-column variables

Water-column variables, including nutrient profiles,have been measured regularly in Himmerfjarden since1976 (Elmgren and Larsson 1997) using standard pro-cedures at environmental monitoring stations (Fig. 1).Total nitrogen (TN) concentrations in milligrams percubic meter measured in the surface water (0–10 m)during the winter months preceding macroalgal col-lection (November 1989 through April 1999) were cor-related to d15N values in Fucus vesiculosus. On average,each monitoring station was sampled 16 times betweenNovember 1989 and April 1999 and the mean TN valuefor this period was used in the correlation. Macroalgald15N values were compared to the TN values at theclosest environmental monitoring station or the averageTN value for the two nearest equidistant stations.

Effluent sample

Five liters of treated sewage effluent was collectedin October 2000, stored at 68C, and filtered throughpre-combusted Whatman glass-fiber filters (GF/F)within 24 hours. The filtrate was analyzed for its d15N-NO2

2/NO32 value according to Sigman et al. (1997).

This value was then used in a two-source mixing modelas the sewage d15N value in 2000, to assess the con-tribution of sewage N to total algal N uptake near thesewage outfall.

Isotopic mixing model

Assuming only two sources of DIN to the bay’s ma-croalgae, sewage-derived and open Baltic Sea DIN, atwo-source mixing model of the form

15 15 15d N 5 X(d N ) 1 (1 2 X)(d N )x effluent y

from Spies et al. (1989) and adapted by Wayland andHobson (2001), was used to calculate the amount ofsewage-derived N in the algal tissues, where X is thepercentage sewage contribution, d15Nx is the mean d15Nvalue of the plants near the outfall (d15Nx 5 12‰ in1989 and 8‰ in 1999), and d15Ny is the backgroundd15N-DIN value. Since d15N in DIN in the Baltic sea-water could not be successfully determined, we as-sumed consistent fractionation effects for the plantsacross all sites and years. Therefore, the backgroundd15N-DIN value (d15Ny), could be estimated as the d15Nvalue in plants at the reference area (d15Ny 5 4‰ in1989 and 1999). This value is also in accordance withthe d15N value for PON (particulate organic nitrogen)in the open Baltic (Voss and Struck 1997) and phy-toplankton isotopic data at the end of the spring bloom(Rolff 2000), which should be approximately equal tothe background DIN conditions. The d15Neffluent was38‰ in 2000, as determined for NO2

2/NO32 by the


method above, and the d15Neffluent for the 1989 surveywas taken as 24‰, the concentration weighted averageof d15N-NH4

1 5 23.6‰ and d15N-NO22 5 29.3‰

(Hansson et al. 1997), using a 47:44 ratio (Himmer-fjarden sewage treatment plant, unpublished data). Sta-ble-isotopic mixing models could therefore be used toassess the decrease in percentage sewage N in algaltissues between the 1989 and 1999 surveys.

To assess temporal changes in the percentage sewageassimilated in individual plants, the isotopic mixingmodel was solved using the mean d15N value for thethree plants collected within 1 km from the outfall in2002, a background d15N value of 4‰, and a d15Neffluent

value of 38‰ for years 2002 to 1998, the year when;85% denitrification commenced in the treatmentplant, and a d15Neffluent value of 24‰ for years 1997 to1994.

RESULTS

Spatial distribution of nitrogenstable isotopes and content

Macroalgal nitrogen was enriched (d15N 5 8–9‰)within 4 km of the sewage treatment plant, both down-stream and upstream of the outfall, and then decreasedsignificantly (n 5 36 samples, F 5 25.8, P , 0.001)with distance from the sewage outfall (Fig. 2a). Beyond4 km, d15N values decreased from ;7‰ to 5–6‰ by14 km, and then decreased further to 3–4‰ at themouth of the bay and at the coastal reference site. Rep-licate plants from each sampling station exhibited verysimilar d15N values, except at some seaward stations.

N content in Fucus vesiculosus tissue decreased sig-nificantly (n 5 36 samples, F 5 10.85, P , 0.001)with distance from the outfall to the mouth of the bay(Fig. 2b). F. vesiculosus in the inner reaches of the bayhad an N content of ;2–2.5 mmol N/mg dry tissuecompared to the regions beyond 16 km from the outfall(;1–1.5 mmol N/mg dry tissue). In contrast, carboncontent in the algal tissue was fairly constant through-out the bay at ;30 mmol C/mg dry tissue.

Compared to plants collected in 1989, F. vesiculosussampled in 1999 were depleted in d15N values withinthe first 25 km of the sewage outfall (Fig. 2c). Samplescollected after onset of N removal in the treatment plantwere between 2.5 and 6‰ lighter in d15N than plantssampled at the same stations in 1989. Beyond 25 kmfrom the outfall, d15N values were very similar or evenslightly enriched (;1‰) compared to 10 years earlier.

The d15N value of macroalgae sampled in 1999 waspositively correlated both with total N (TN) concen-tration in the surface water during the preceding winter(November 1989 through April 1999) (R2 5 0.86, P ,0.01) (Fig. 3) and N content in the F. vesiculosus tissue(R2 5 0.40, P , 0.05).

Temporal trends of d15N in individual plants

Within individual plants, there was temporal vari-ability in d15N values (Fig. 4a) but no significant var-

iation in percentage N in the algal tissues. Three plantscollected ;1 km from the outfall in 2002 exhibitedmean d15N 5 14‰ in the oldest frond tissue, repre-senting growth that probably occurred about eight yearspreviously (ca. 1994). The d15N value decreased withtime to ;12‰ in the late 1990s, with the most recentgrowth tips having an average d15N value as low as10.5‰. In contrast, plants collected in the referencearea showed no consistent decrease in d15N with time.The oldest tissue exhibited a d15N of 3.8‰ and the tipd15N was 3.4‰, while the intermediate tissue repre-senting the late 1990s had a d15N value of about 3.5‰.There was no significant difference in percentage N inalgal tissues between sites and for the different-agefronds. Percentage N ranged from 1.7% to 2.5% withno directional change temporally and no significant dif-ference between the two sites.

A two-source mixing model using sewage effluent(d15N 5 38‰ for years 1998–2002, and d15N 5 24‰for years 1994–1997), background DIN (dissolved in-organic N) values (d15N 5 4‰) and the mean d15N valuefor algal tissue representing each year’s growth inplants ;1 km from the outfall indicated that the per-centage sewage N in the algae decreased from .40%in the oldest tissues from about 1994 to ;19% in thegrowth tip from 2002 (Fig. 4b).

DISCUSSION

Assimilation of wastewater nitrogen

The influence of nitrogen from the sewage was de-tectable up to 24 km from the effluent outfall in ma-croalgae, but was most pronounced within 10 km ofthe discharge point. The elevated d15N values (.7‰)(Fig. 2a) and high N content (Fig. 2b) in Fucus vesi-culosus tissue in the inner reaches of the bay suggestthat plants close to the outfall utilize sewage-deriveddissolved inorganic N (DIN), both in downstream andupstream directions. The N isotopic ratio of sewageupstream of the diffuser is in agreement with water-circulation models that predict predominant northernmovements of deepwater nutrients including sewageeffluent (Engqvist and Omstedt 1992). A significantisotopic gradient was recorded with increasing distancefrom the sewage outfall, and macroalgal d15N valuesdecreased from 8–9‰ within 4 km to 5–7‰ by 14 kmand to background levels (d15N 5 ;4‰) at 24 km fromthe outfall. Similar findings have been reported for oth-er macroalgae and seagrasses in conjunction with anutrient gradient at Moreton Bay, (Brisbane, Queens-land, Australia) (Costanzo et al. 1999), Tomales Bay,(California, USA) (Fourqurean et al. 1997), and theWapiti River (Alberta, Canada) (Wayland and Hobson2001), with the highest d15N values closest to sewagesources.

Patterns of elevated tissue-N content proximate tothe discharge point (Fig. 2b) showed that Fucus vesi-culosus assimilate wastewater N and that N tissue con-


FIG. 2. Spatial distribution of stable N isotopes and N content in the macroalgae Fucus vesiculosus in HimmerfjardenBay (Baltic Sea). (a) d15N values in F. vesiculosus sampled in 1999 along a gradient from the sewage outfall (km). Samplescollected north of the outfall are indicated as negative distances. Data are means 6 1 SE. (b) F. vesiculosus N content (drymass macroalgal tissue) as a function of distance from the sewage outfall. Data are means 6 1 SE. (c) Difference in d15Nvalues of macroalgae between 1989 and 1999 with increasing distance from the sewage outfall. The 1989 d15N values arefrom a single plant, while the 1999 values are means for three plants sampled at the same station.


FIG. 3. Relationship between the mean d15N value of Fu-cus vesiculosus at each station and mean winter (November1989 through April 1999) surface water (0–10 m) total ni-trogen (TN) concentration, measured at the nearest monitor-ing stations. TN concentrations are the mean values for about16 sampling occasions.

tent reflects N availability. This hypothesis is consistentwith results from Waquoit Bay, (Massachusetts, USA)where the greatest N content and increased nitrate re-ductase activities were recorded in F. vesiculosus fromestuaries with the highest nitrate loads (Thompson andValiela 1999). A positive correlation was also foundbetween wastewater-enriched N and d15N values in eel-grass and the red macroalga Gracilaria tikvakiae acrossestuaries in Waquoit Bay (McClelland et al. 1997).Studies of Ulva lactuca also revealed that N contentwas highest near sewage outfall regions (Rogers 1999).Moreover, experimental addition of fertilizer N to sea-grasses (Udy and Dennison 1997) and sewage N tomacroalgae (Gartner et al. 2002) is known to increasetissue N content and alter d15N values, suggesting thatboth parameters reflect changes in N availability. To-gether these patterns demonstrate that sewage-derivedN is rapidly assimilated by macroalgae and recordedin isotopic and elemental shifts in growth tissues.

Comparisons of spatial patterns in N content andd15N values show that isotopic signals arise from chang-es in N sources, rather than variation in fractionationprocesses associated with enhanced uptake and pro-cessing of nutrients. Fractionation theory supposes thatd15N of a substrate will increase as the reaction pro-ceeds due to the preferential conversion of the substrate14N to product (Owens 1987). The d15N value of Fucusvesiculosus was positively correlated with N contentin the algae (R2 5 0.40) and with the total N (TN) (R2

5 0.86) concentration in the water column (Fig. 3). Ina situation where nitrate is available in excess, onewould therefore expect the algae to have lower d15Nthan the substrate, whereas if the nitrate is all used up,the algae would have the same ratio as the substrate.Fractionation would in this case tend to give propor-tionally higher d15N-DIN values with distance from thesource. Instead, the opposite spatial pattern was ob-served (Fig. 2a), showing that isotopic enrichment was

predominantly indicating changes in N source and wasnot due to fractionation processes. Moreover, N uptakewas mostly in winter when nitrate is in excess at allstudied stations. We therefore conclude that the posi-tive relationship between macroalgal d15N and TN con-centration in the water column is due to a greater con-tribution of sewage-derived N to the inner basin DINpool, which is assimilated by the algae.

Lessening sewage impact

Spatial and temporal variation in macroalgal isotopicratios demonstrates that the proportion of sewage-de-rived N assimilated by the algae has declined and thatthe areal influence of sewage has been reduced sincethe initiation of tertiary N treatment. Comparisons ofisotopic surveys from 1989 and 1999 (Fig. 2c) showthat enhanced N removal measurably decreased the im-pact of sewage effluent up to 24 km. Sewage N stillcontributed to the DIN pool and was assimilated bymacroalgae in the inner reaches of Himmerfjarden Baydespite ;85% N removal from the wastewater. How-ever, compared to the initial survey conducted a decadebefore, d15N values in macroalgae were depleted by2.5–6‰ despite the sewage effluent d15N value beingmore enriched than in 1989. The survey in 1989showed that Fucus vesiculosus d15N was uniformly high(d15N 5 11–13‰) up to about 15 km, but measurablyelevated (d15N . 8‰) within 24 km of the outfall andonly declined to background levels of ;4‰ beyond 25km (Hobbie et al. 1990). In contrast, the d15N gradientin 1999 (Fig. 2a) distinguished between samples col-lected within 10 km of the outfall and those from be-yond 12 km. This distance coincides with a bathymetricsill at Oaxen Island (Fig. 1) that delineates the innerand central basins of the bay and suggests that whileeffluent treatment reduced N throughout the bay, thereductions were most significant within 10 km becauseof the morphometric features of this embayment. Thisis in accordance with changes in macrobenthic com-munities in the bay, which showed that morphometriccharacteristics such as sill depth strengthened the im-pact of sewage N (Savage et al. 2002).

Fucus vesiculosus plants collected ;1 km from theoutfall and subsampled to reflect temporal changes alsoreflected a decrease in d15N values from 14‰ in themid-1990s to 10.5‰ in 2002 (Fig. 4a), consistent witha decline in the percentage contribution from sewageN (Fig. 4b). The stable-isotopic ratios of macroalgaemay vary seasonally, owing to seasonality in sources,irradiance, and temperature (Stephenson et al. 1984).Nevertheless, no decrease in d15N over time was ob-served in three F. vesiculosus plants collected from thereference area. While we cannot exclude the possibilityof some isotopic fractionation associated with nutrienttranslocation or other physiological processes that af-fect the d15N value in different tissues, this is unlikelyto differ between sites and years. We suggest that thedifference between impacted and reference sites is not


FIG. 4. Temporal trends of d15N in Fucus vesiculosus plants. (a) Temporal variance in d15N values of three individual F.vesiculosus plants collected ;1 km from the outfall and three plants at the reference area. Macroalgae were subsampledaccording to frond dichotomies to represent approximately annual growth from years before present (b.p.), i.e., from before2002. Data are means 6 1 SE. (b) Percentage sewage N assimilated by the plants collected within ;1 km of the outfallthrough time, approximated from a two-source mixing model using the mean d15N value for plants within 1 km from theoutfall, sewage d15N, and a background d15N value. Macroalgae were subsampled according to frond dichotomies to representannual growth patterns, indicated as years before present (0–5) and the corresponding approximate year for estimatingpercentage sewage N assimilation.

due to physiological processes but is most likely dueto source value differences. This demonstrates that ifinterannual differences in d15N input exist, analyzingN isotopes in perennial macroalgae can also capturemultiyear trends in N sources.

Macroalgal dependence on sewage-derived nutrientsdeclined from 1989 to 1999. Sewage-derived nutrientsdecreased from ;70% of total N loads entering the bayfrom land in 1989 to ;30% in 1999. Simultaneously,the enhanced denitrification procedure enriched the ef-fluent d15N value from ;24‰ to ;38‰. Two-sourcemixing models estimate the sewage N percentage con-tribution to the algal N uptake as ;40% in 1989 and;12% in 1999. Similarly, for plants within 1 km ofthe outfall, the percentage N from sewage declined

from .40% in the oldest tissues, representing the Nconditions in the mid-1990s, to ;19% in 2002. Therewas a marked decrease in percentage sewage N in ma-croalgae from ;40% to ;24% between 1997 and 1998(Fig. 4b), which is partly because the d15N effluentvalue used in the mixing model was 24‰ until 1997and 38‰ from 1998 onwards, when the improved de-nitrification step was implemented. These estimatesshould be interpreted with caution for two reasons.First, they are based on only two sources (sewage d15Nand background d15N) and exclude diffuse inputs. Sec-ond, the effluent d15N value is based on single samplesfrom 1988 and 2000, and thus do not account for sea-sonal and annual variation in the d15N value of effluent.Despite these limitations, the calculations clearly show


that there has been a substantial decline in the pro-portional contribution of sewage N to macroalgal Nuptake in recent years.

Macroalgae integrate nutrient conditions

Attached macroalgae are good integrators of ambientnutrient conditions over longer timescales than phy-toplankton and other conventional monitoring indica-tors. Fucus vesiculosus is a perennial alga with a rel-atively long tissue-turnover time and is therefore agood indicator of ambient water nutrient conditionsover timescales of several years. Furthermore, usingmacroalgae to trace anthropogenic N sources has theadvantage that they assimilate DIN, which is more bi-ologically available than particulate organic nitrogenand is therefore an ecologically relevant measure ofresponses in the ecosystem to elevated nutrient levels.Consequently, stable-isotope values in macroalgae canbe used to delineate the influence of sewage-derivednutrients in estuarine and coastal areas (Hobbie et al.1990, Costanzo et al. 1999, Rogers 1999, Wayland andHobson 2001) and to map sewage dispersal over dif-ferent timescales using different functional forms(Gartner et al. 2002). Here we show that macroalgaecan also be a useful complement to monitoring studiesto trace a decrease in sewage impact over integratedtimescales following enhanced sewage treatment.

Limitations of approach

This study was possible as sewage N and backgroundDIN were isotopically distinct, there was limited nat-ural denitrification in the water column in the estuary,and the other possible N sources and in situ denitrifi-cation remained largely unchanged between the twosurveys. The larger the isotopic distinction of the con-tributing sources, the easier it is to interpret isotopicresults. Sensitivity analyses have shown that the pre-dictive power of a two-source isotopic mixing modelis directly related to the degree of isotopic differencebetween sources and the number of replicates (Phillipsand Greg 2001). We were therefore able to exploit theclear distinction between sewage N and background Nto partition sources. Moreover, the cold temperaturesin Himmerfjarden Bay in early May (;78C on averagein May) limit natural denitrification that could frac-tionate background d15N-DIN values in the water col-umn. Natural nitrification and denitrification processesin the sea have resulted in d15N fractionation greaterthan 10‰ for d15N values of suspended organic matter(Mariotti et al. 1984, Owens 1987) and surface waterinorganic N (Lindau et al. 1989), which can maskchanges in d15N values of sources. While there is ev-idence of in situ denitrification in the bay, especiallyin the sediments (Larsson and Engqvist 1997), we haveno indication that the rate of denitrification has changedbetween the two surveys. Finally, the other N sourceshave not changed noticeably. If the other contributingsources or denitrification had changed markedly at the

same time as the sewage-load N, we would not havebeen able to interpret the changes as due to the sewage-reduction initiatives. We can therefore assume thatchanges in d15N values in macroalgae must mainly bedue to nitrogen removal in the wastewater treatmentplant.

Ecosystem and management implications

Eutrophication often leads to a decline of perennialmacrophyte species and an increase in opportunisticfilamentous algae (McClelland and Valiela 1998, Dee-gan et al. 2002). Fucus vesiculosus has decreased indensity and vertical distribution in some areas of theBaltic Sea, which is normally attributed to worseninglight conditions with increasing eutrophication (Kaut-sky et al. 1986, Kautsky 1991). Yet, these macrophytesassimilate N from sewage and could be potentially im-portant sinks and sites of biogeochemical transforma-tions among N pools. We were therefore interested inquantifying the potential biological uptake of anthro-pogenic N by the macroalgae. Specifically, using Him-merfjarden Bay as a model and given the known andestimated N loads, the amount of N assimilated by F.vesiculosus averaged over the bay can be calculatedusing quantitative estimates of algal cover, algal growthestimates, and percentage N in the algal tissue.

Assuming that ;4 3 106 m2 (;10% of the total areaof Himmerfjarden Bay) has suitable substrate for Fucusvesiculosus plants (Bjorklund and Larsson 1979), andthat the plants exhibit a biomass peak of 210 g/m2 drymass at 1–2 m depth in the reference area (Jansson andKautsky 1977), we estimated a total standing stock of840 3 103 kg dry mass F. vesiculosus in the bay. Ifthe mean P:B (production-to-biomass ratio) for F. ves-iculosus in the reference area is 0.975 (Kautsky andKautsky 1995), we can assume annual production ofnew tissue is ;840 3 103 kg in the bay. If the algaecontained ;2% N on average in their tissues (Fig. 2band Wallentinus 1984, Schramm et al. 1988), thenabout 16.8 3 103 kg N was contained in the F. vesi-culosus produced in the bay. In 1998, the bay had atotal load of 549 3 103 kg TN, of which 171 3 103 kgwere of sewage origin. Therefore, the F. vesiculosusbelt could potentially assimilate ;3% of the total Nload in the bay in 1998. Since the algae primarily as-similate and store N during the winter months (Lehvoet al. 2001) when DIN concentration peaks in Him-merfjarden, and since there is no long-term biomassaccumulation of F. vesiculosus, it cannot act as a sig-nificant long-term sink of N, but will accumulate someN in winter when uptake by phytoplankton is minimal.

CONCLUSION

In conclusion, stable isotopes in attached macroalgaereflected a decrease in the percentage contribution ofsewage N in a coastal Baltic embayment with limitedwater exchange. Both spatial and temporal patterns ofd15N values in Fucus vesiculosus suggested a lessening


of sewage impacts within 24 km from the outfall, withmore pronounced responses within 10 km from the out-fall, because of morphometric basin features. Stable-isotopic values in macroalgae can therefore provide anintegrated picture of ambient nutrient conditions andcan trace declines in sewage influence following im-proved wastewater treatment. Sewage-derived N ac-counted for ;12% of algal N demand in 1999 near theoutfall, but removed only about 3% of total annual Nloads entering the bay on average. Calculations indicatetherefore that F. vesiculosus is not an effective sink forretaining the sewage-derived nutrients within the coast-al zone and reducing export to the open Baltic Sea, atmost assimilating a small percentage of the winter Ndischarge.

ACKNOWLEDGMENTS

This study was financed by SUCOZOMA (SUstainableCOastal ZOne MAnagement), a program of the SwedishFoundation for Strategic Environmental Research (MISTRA),and The Southwestern Stockholm Sewerage Company. Ad-ditional funding for stable-isotope analyses was provided bythe Stockholm Marine Research Centre, Sweden. John Hob-bie, Brian Fry, Sture Hansson, and Ulf Larsson kindly allowedus to use the macroalgal d15N values from 1989. The effluentsample from 2000 was collected by Anna Axelsson and an-alyzed by Maren Voss and co-workers at the Baltic Sea Re-search Institute, Rostock, Germany. Heike Sigmund at theDepartment of Geology and Geochemistry, Stockholm Uni-versity, Sweden, and Dunling Wang and Nicole Knezacek atthe Environmental Quality Analysis Laboratory at ReginaUniversity, Canada, are thanked for performing the stable-isotope and elemental analyses of the macroalgae for the spa-tial and temporal surveys, respectively. Susanne Ericsson isthanked for field assistance during sample collection. We alsothank Peter Leavitt, Sture Hansson, and Lena Kautsky forhelpful discussions and valuable comments on the manu-script. Linda Deegan and two anonymous reviewers arethanked for insightful comments that improved the manu-script.

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