Ecological responses to simulated benthic-derived nutrient subsidies mediated by omnivorous fish

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1 Freshwter Biology (25) 5, doi:1.1111/j x Ecologicl responses to simulted enthic-derived nutrient susidies medited y omnivorous fish STEPHEN P. GLAHOLT JR AND MICHAEL J. VANNI Deprtment of Zoology, Mimi University, Oxford, OH, U.S.A. SUMMARY 1. Fish cn ply n importnt role in coupling enthic nd pelgic hitts y consuming enthic prey nd providing essentil nutrients to lge in dissolved form. However, little is known out the fctors ffecting the mgnitude of this nutrient susidy. 2. Using lortory nd mesocosm experiments we evluted how vrying ingestion rtes of luegill sunfish (Lepomis mcrochirus) ffects fish excretion rtes of oth nitrogen (N) nd phosphorus (P). During the 1-week mesocosm experiment, we lso evluted how vrying ingestion rtes my ffect plnkton community dynmics, nd nutrient flux etween pelgic nd enthic hitts. Lstly, ioenergetic/mss lnce models were used to exmine the nutrient stoichiometry of fish ody composition nd excretion products. 3. Under lortory conditions, oth N nd P excretion rtes incresed with incresed ingestion of enthic prey surrogtes (erthworms). This effect ws more pronounced for N thn P. Furthermore, under the more relistic conditions of the mesocosm experiment ingestion rte hd no significnt effect on P excretion rte. 4. Incresed fish ingestion rte in the mesocosm experiment incresed totl lgl iomss nd the flux of nutrients from the wter column to sediments. Effects of vrile ingestion were much stronger on periphyton iomss nd lgl sedimenttion rtes thn on phytoplnkton or zooplnkton iomss or composition. 5. Fish ody nutrient composition ws gretly ffected y ingestion rte. N content incresed nd P content decresed with ingestion rte. As result, the N : P rtio of fish odies lso incresed with ingestion rte. The N : P rtio of nutrients excreted y fish lso incresed with ingestion rte, counter to predictions of stoichiometric theory, which predicts tht excreted N : P rtio is negtively correlted to ody N : P. However, this finding cn e explined y relxing the ssumption of constnt nutrient ssimiltion rtes, nd our mss lnce dt suggest tht ssimiltion rtes vry indeed with ingestion rte. 6. Our study provides experimentl evidence tht trnsloction of enthic-derived nutrients y fish cn ffect the flux of nutrients mong hitts, while lso suggesting tht stoichiometry models need to etter incorporte how vrile ingestion rtes ffect nutrient ssimiltion nd excretion rtes. Keywords: enthic-pelgic coupling, ioenergetics, fish, ingestion, nutrient excretion, stoichiometry Introduction Correspondence: Stephen P. Glholt, Deprtment of Biologicl Sciences, Drtmouth College, Hnover, NH 3755, U.S.A. E-mil: stephen.p.glholt.jr@drtmouth.edu Relese of nutrients y nimls vi excretion cn e importnt in determining lgl iomss, community structure nd species composition in freshwter ecosystems (Sterner & Elser, 22; Vnni, 22). Fish cn 1864 Ó 25 Blckwell Pulishing Ltd

2 Rtion-specific N nd P excretion y fish 1865 lso ply importnt roles in nutrient cycling nd my hve consequences on primry producers tht outweigh those of fish vi ingestive effects on grzer undnce (Schindler, 1992; Persson, 1997; Vnni, Lyne & Arnott, 1997; Schus & Vnni, 2). Becuse fish cn cycle significnt mounts of limiting nutrients, they re le to promote lgl iomss nd productivity (Brrnd, Ffeng & Nilssen, 199; Crpenter et l., 1992; Persson, 1997; Schus et l., 1997; Attyde & Hnsson, 1999; Schus & Vnni, 2; Schindler, Knpp & Levitt, 21), s well s drive chnges in lgl community composition (Drenner et l., 199; Schindler, 1992; Attyde & Hnsson, 1999, 21; Schus & Vnni, 2). The supply rte of phosphorus, the most common limiting nutrient in freshwters, vi excretion y fish cn e comprle to or exceed externl inputs (i.e. from ctchments or the tmosphere) (Brrnd et l., 199; Reinertsen et l., 199; Persson, 1997; Schindler et l., 21). Consequently, determining which fctors medite nutrient cycling y fish is importnt to our understnding of food-we dynmics in qutic ecosystems. Nutrients re cycled y consumers to primry producers in two sptilly distinct wys, either vi nutrient recycling or nutrient trnsport (Vnni, 22): nutrient recycling occurs when n orgnism consumes, trnsforms, nd excretes nutrients in the sme hitt, such s zooplnkton or fish feeding nd excreting in the pelgic zone. The process y which orgnisms feed in one hitt nd excrete in second hitt is commonly referred to s nutrient trnsport or trnsloction (Kitchell et l., 1979; Crpenter & Kitchell, 1987; Vnni, 1996; Schus et l., 1997). Fish tht feed on enthic orgnic mtter nd relese enthic-derived nutrients into the wter column re good exmples of nutrient-trnsporting orgnisms. Nutrient trnsport is likely to e prevlent in nture ecuse most fish, including most plnktivorous fish, supplement their diet y feeding on enthic orgnic mtter (Hecky & Hesslein, 1995). This trnsfer of nutrients y fish from the enthos to the pelgic zone my ply lrge role in driving food-we dynmics y directly ffecting the community structure nd stility of pelgic food-wes (Schindler & Scheuerell, 22). Bsed on stoichiometric principles, the vriility in fish diets nd ingestion rtes (Kest & Welsh, 1968; Bumnn & Kitchell, 1974; Srker, 1977; Mittelch, Osenerg & Winwright, 1992) could ffect nutrient flux etween pelgic nd enthic hitts, nd the communities receiving these nutrient susidies. Therefore, it is importnt to determine how interctions etween fish nd enthic prey cn ffect the strength with which these two hitts re coupled vi nutrient trnsloction. Nutrient cycling y fish is the result of complex interctions etween fish physiology nd ecology. Physiology sets the upper limits on nutrient excretion rtes, while ecologicl fctors determine ctul rtes (Schindler & Ey, 1997). Fctors such s stress, ody size, temperture, nd diet hve ll een shown to drive excretion rtes (Hopkins & Cech, 1992; Schindler & Ey, 1997; Elser & Ure, 1999; Sterner & Elser, 22; Vnni et l., 22). Effects of vrition in ingestion rtes of fish on nutrient excretion rtes hve een reltively unexplored. The few studies tht hve exmined how vrition in ingestion rte ffects nutrient excretion rtes of fish hve shown tht excretion rtes generlly increse with incresing ingestion rte (e.g. Weiserg & Lotrich, 1982; Wiesmnn, Scheid & Pfeffer, 1988; Gershnovich & Pototskij, 1992; Leung, Chu & Wu, 1999; Rodehutscord, Gregus & Pfeffer, 2). However, these studies were ll conducted under quculturl or lortory conditions nd hve focused on single excreted nutrient, usully N. Therefore, we need to evlute the effects of ingestion rte on oth N nd P excretion rtes nd how these chnges in excretion rtes medite susequent effects on nturl recipient communities nd ecosystems. Nutrient flow within nd through n orgnism must conform to the constrints of mss lnce nd stoichiometry; therefore, effects of rtion size on excretion rtes my e understood within the frmework of these principles (Sterner, 199; Sterner & Elser, 22). Ecologicl stoichiometry theory sttes tht the mount (or rtio) of nutrients relesed y consumer is function of the imlnce etween the ody nutrient composition (or rtio) of the consumer nd its prey, s well s the efficiency t which the consumer incorportes nutrients into iomss (Sterner, 199; Sterner, Elser & Hessen, 1992; Ure, 1993; Elser & Ure, 1999). Therefore, it stnds to reson tht incresed ingestion rte should result in incresed excretion rtes (Vnni, 1996; Schindler & Ey, 1997) nd tht ingestion rte could lso ffect the rtios t which fish excrete nutrients. Stoichiometric models often ssume tht oth the nutrient content Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

3 1866 S.P. Glholt nd M.J. Vnni nd ssimiltion efficiency of consumers re reltively constnt in the fce of vrition in food nutrient content nd ingestion rte. However, it is still not known precisely how chnges in ingestion rte ffect oth N nd P excretion rtes of fish, nd how these chnges cn then ffect communities nd ecosystems. The primry ojective of this study ws to evlute how vrition in ingestion rtes of enthic prey ffects the rtes nd rtios y which fish excrete N nd P, nd how this vrition ffects pelgic communities nd ecosystem functioning. We ssessed how vrile ingestion rte ffects nutrient excretion in oth lortory nd mesocosm experiment, in which we mnipulted the ingestion rte of Lepomis mcrochirus Rfinesque (luegill sunfish) y feeding them different mounts of model enthic orgnism Lumricus terrestris L. (erthworm) nd mesuring N nd P excretion rtes. We lso ssessed community nd ecosystem effects of vrile ingestion rte y conducting 1-week mesocosm experiment in which luegill ingestion rtes were mnipulted nd oservtions mde on plnkton iomss nd community composition, nd nutrient dynmics. In experiments in which, we explicitly quntified N nd P relesed y fish, we considered only excretion nd not egestion (feces production) ecuse excreted nutrients re more redily ville to primry producers. Nutrients contined in fecl mtter must first e roken down y microil ctivity nd then trnsported ck up into the wter column efore ecoming ville to phytoplnkton (Brrnd et l., 199). However, we did estimte oth N nd P egestion rtes in the mesocosm experiment using mss lnce/ioenergetics pproch (see Methods section), llowing comprison with excretion rtes, which were directly mesured. Methods Lortory experiment Under lortory conditions, we investigted how vriility in ingestion rtes ffects N nd P excretion rtes of luegill over 24 h period. Bluegills of similr size (15 25 g wet mss) were collected from pond locted t Mimi University s Ecology Reserch Center (ERC), Oxford, OH, U.S.A. Fish were then cclimted to lortory conditions nd fed erthworms for 8 dys. After the initil cclimtion period, ll luegill were strved for 24 h, then rndomly ssigned to tretment. In order to eliminte the high within-tretment vriility tht results from using strved orgnisms, s found in preliminry experiments, fish were given their llotted food (sed on their ssigned tretment) fter the strvtion period, then held for 24 h prior to strting the experiment. The experimentl tretments consisted of fish receiving: (1) no rtion; (2) 3% rtion; (3) 5% rtion; nd (4) 1% rtion. Rtions were provided in the form of the erthworm. Lumricus terrestris, which ws used s enthic food surrogte to simplify the experimentl design. Erthworms hve ody nutrient composition (1.6% N nd.89% P y dry mss) remrkly similr to qutic enthic invertertes (Penczk, 1985; Shoup, 21; Frost et l., 23), fish quickly lern to consume them, nd do so t relistic rtes (see elow), nd they re redily ville. The mount of erthworms fed to the fish (i.e. rtion size) ws determined s per cent of fish wet mss. Rtion sizes of 3 nd 5% were determined sed on literture vlues of luegill ingestion rtes found in nture (Gerking, 1954; Seurg & Moyle, 1964; Kest & Welsh, 1968; Whitledge & Hywood, 2). The 1%- rtion tretment ws set slightly elow the level needed for optiml fish growth s determined in lortory studies (12 15%; Hnson et l., 1997) nd is similr to levels used in prior studies of excretion rtes y fish (Ttri, 1981; Mther et l., 1995). In ddition, we included Fishless tretment to ccount for ny chnge in nutrients unrelted to the presence of fish. Ech tretment hd five replictes except the Fishless tretment which hd three ecuse vriility mong replictes in preliminry studies ws low. To strt the experiment, luegill in the 3, 5 nd 1%- rtion tretments were gin fed the pproprite rtion size. After ll erthworms were consumed, ech luegill ws immeditely moved into n erted 6-L Ziploc Ò g (S. C. Johnson, Rcine, WI) contining filtered pond wter tken from the sme pond in which the luegills were collected. The pond wter ws filtered through.3-lm memrne filter to remove lge nd other orgnisms tht my tke up nutrients relesed y the fish. The luegill were then held without dditionl food for 24 h under 16 : 8 h light : drk cycle. Dissolved nutrients (N nd P) smples were collected t, 2, 6, 18, nd 24 h fter fish were plced in the gs to otin temporl ptterns in excretion rtes. Becuse stress hs een Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

4 Rtion-specific N nd P excretion y fish 1867 shown to effect excretion rtes of fish (e.g. Brrnd et l., 199; Mther et l., 1995; Shoup, 21), the experiment ws conducted in climte controlled room nd the gs were well erted nd in coolers to minimise the mount of visul contct with humns nd therefore minimise stress. Furthermore, hndling of the fish prior to strting the excretion experiments is elieved not to hve influenced excretion rtes sed on results of previous studies which used similr methods s descried here ut lso mesured effects of hndling nd showed no effect of hndling on fish excretion rtes (Mther et l., 1995; Shoup, 21). Design of mesocosm experiment Mesocosms were used oth to tke dvntge of potentilly strong pelgic-enthic coupling occurring in the littorl zone of lkes, nd to provide controlled environment in which to investigte how different nutrient excretion rtes nd rtios (resulting from vrile ingestion rte) ffect plnkton communities nd nutrient dynmics. A totl of 16 cylindricl mesocosms 1.8 m in dimeter, 2.2 m high, nd holding c. 5 L were used t the Ecologicl Reserch Center. These high-density polypropylene mesocosms were wrpped in lck plstic to lock light from entering through the sides in n effort to reduce periphyton growth. All 16 mesocosms were filled simultneously with wter from nery fishless pond contining nturl plnkton communities. The wter ws grvity fed through two screens (154- nd 11-lm mesh) to exclude lrge qutic invertertes (such s insects nd snils) nd tdpoles. Screens of 154-lm mesh were plced over the tops of ll mesocosms for the durtion of the experiment, except during smpling, to minimise llochthonous inputs. Sediment trps mde of four 5.4-cm dimeter nd 25.4-cm high PVC pipes strpped together, were plced on the ottom of ech mesocosm just fter the mesocosms were filled. Temperture fluctutions were monitored in one mesocosm per tretment using Hoo Ò temperture loggers (Bourne, MA, U.S.A.) set to smple t 1-min intervls over the course of the experiment. The communities in the mesocosms were left to equilirte for 5 dys efore the tretments were pplied. Conditions in the mesocosms resemled those in the littorl zone of lkes, with multiple lgl groups (phytoplnkton nd periphyton) competing for the sme resources, smll (non-dphnid) zooplnkton dominting the grzer community nd luegill, common omnivorous species found in littorl res of most lkes in the Estern nd Midwestern U.S.A. The mesocosms were oriented north to south in two rows of eight. Therefore, mesocosms were rndomly ssigned to tretment using lock design with the two rows s locks nd hlf of the replictes of ech tretment ssigned to ech row. The tretments were s follows: (1) fishless; (2) no rtion (fish present ut not fed erthworms); (3) low rtion; nd (4) high rtion. Fish in the low- nd high-rtion tretments were fed c. 5 nd 1% of totl fish wet mss ech dy. Ech tretment ws replicted four times. Bluegills of g wet mss were collected from the sme pond from which fish used in the lortory experiment were otined. Ech luegill ws strved for 48 h to empty its gut nd then weighed efore eing plced into the mesocosm. Ech mesocosm (except for the Fishless tretment) received two luegills, which were plced into cge mde of Vexr (DuPont, Wilmington, DE, U.S.A.; 1.2 m in dimeter, 1.2 m tll, with 2.5-cm mesh) tht ws suspended in the mesocosm. The density of fish used in this experiment (c. 1.6 g m )2 wet mss) ws similr to nturl luegill densities found in eutrophic Midwestern lkes (Gerking, 1962). The cges were used to llow esy ccess to the luegill in order to feed them nd to determine ech fish s stte of helth, while still llowing the luegill to feed on pelgic prey. Other experiments hve housed luegill in similr cges with success (e.g. Leiold, 199; González & Tessier, 1997). Cges were lso plced in the mesocosms of the Fishless tretment s control. Bluegills were hnd-fed dily (round 8.m.) in wy tht llowed the investigtors to wtch the luegills fully consume the erthworms nd ensure tht oth fish in ech mesocosm ws fed. The method y which we fed fish consisted of ttching ech erthworm to closed sfety pin nd lowering the erthworm into the mesocosm (just under the surfce of the wter) using fishing line. This llowed us to wtch the luegill pull the erthworm off the sfety pin nd consume the erthworm in its entirety. The luegill styed ner the surfce of the wter throughout the feeding process nd we never witnessed luegill spit out n erthworm fter pulling it from the sfety pin. To initite feeding, erthworms needed to e sumerged in the wter for period of time efore the fish would come up to feed. Therefore, s control for potentil nutrients relesed y erthworms prior Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

5 1868 S.P. Glholt nd M.J. Vnni to fish consuming them, erthworms were floted in the fishless nd no-rtion mesocosms (outside the cges) for 5 min, which ws the verge length of time it took luegills to consume the erthworms. Mesocosms were checked dily for ded or dying fish. If moriund fish ws encountered, it ws removed nd weighed, then replced with luegill of similr size. Smpling The experiment rn for 71 dys from 5 July to 13 Septemer 22. Mesocosms were smpled weekly for temperture, dissolved oxygen, ph, light intensity, zooplnkton composition nd iomss, phytoplnkton iomss (chlorophyll ) nd dissolved, prticulte nd totl N nd P. Integrted zooplnkton, phytoplnkton nd nutrient smples were collected with 2.5-m long PVC tue with ruer ll tied to rope s stopper. Phytoplnkton primry productivity ws mesured in the lortory under vrile light conditions over 2 dys period (hlf the mesocosms ech dy) on 17 nd 18 July 22 using methods descried in Knoll, Vnni & Renwick (23). To evlute how rtion size ffects nutrient cycling y luegill under mesocosm conditions, excretion rtes of luegill in ech mesocosm were mesured twice (24 July nd 25 August 22) over the course of the experiment. To mesure excretion rtes, oth fish were removed from ech mesocosm 4 h fter feeding nd plced for 1 h in plstic lined cooler filled with filtered (.3 lm) pond wter. Fish were removed 4 h fter feeding in order to cpture reltively high excretion rtes for oth N nd P, sed on results of the lortory experiment. An incution time of 1 h ws used to minimise the mount of time the luegill were out of the mesocosms (nd hence not feeding), while llowing time for ccumulting enough N nd P to ccurtely mesure excretion rtes (e.g. Mther et l., 1995; Schus et l., 1997). Dissolved nutrient smples were tken efore, immeditely fter, nd 1 h fter the fish were plced in the cooler. Temperture nd susequent dissolved oxygen levels were mintined t levels similr to those found in the mesocosms. Smpling ws conducted in the sme mnner s the lortory excretion experiment. The temporl dynmics of excretion rtes oserved in the lortory experiment were used in conjunction with the mesocosm-mesured rtes to estimte dily mesocosm excretion rtes of luegill. This ws done y first otining multiplier tht ws equl to the 4 h rte in the lortory experiment divided y the men dily rte in the lortory experiment. Then, y multiplying the mesocosm excretion rte (tken t hour 4) y this multiplier we otined dily excretion rtes of the luegill in the mesocosms. Before the morning feeding on the finl dy of the experiment, one fish ws tken from ech mesocosm, while the second fish ws given the pproprite rtion nd tken 12 h lter. In oth cses, fish were weighed, scrificed nd put on ice to e rought ck to the lortory where their stomchs were removed, weighed nd dissected for content nlysis, while the rest of the fish ws ground up for ody N nd P content. Removing the fish t different times of the dy llowed us to ccount for dily vrition in feeding known to occur in nturl luegill popultions (e.g. Kest & Welsh, 1968). Gut content nlysis ws crried out y identifying nd counting ll contents identifile in the stomch of ech luegill. Counts were then converted into iomss using estimtes of men species iomss otined from previous studies (Downing & Rigler, 1984; Mther et l., 1995). In ddition to the regulr weekly smpling, sediment, phytoplnkton composition nd periphyton smples were lso collected on the lst dy. Sediment trps were collected nd their contents rought ck to the lortory where they were nlysed for chlorophyll (chl ), prticulte cron (C) nd nitrogen (N), nd prticulte phosphorus (PP). Before emptying the mesocosms, loosely ttched periphyton ws smpled y scrping 9-cm swth from the sides of the tnks from the ottom of the mesocosm to the wter surfce in ll four crdinl directions. Immeditely fter the wter ws removed from ech mesocosm, second periphyton smple ws tken within the swths lredy smpled using rzorlde. This second smple ws crried out to ccount for periphyton ttched to the wlls tht ws not removed y the previous smpling. The two types of periphyton smples were nlysed seprtely for chl, C nd N, nd PP nd then used together to estimte totl periphyton in ech mesocosm. To determine the effect of our tretments on lgl nd nutrient dynmic we constructed udgets for lge (using chl s mesure of lgl iomss) nd N nd P, t the end of the experiment. The udgets included ll the mjor comprtments (seston, including zooplnkton, phytoplnkton, periphyton, sediment, nd fish) nd were Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

6 Rtion-specific N nd P excretion y fish 1869 clculted y simply sutrcting the iomss mesured on the finl dy of smpling (dy 71) from those tken on the dy the tretments were strted. All dissolved nd totl nutrient smples were cidified nd stored in cold room efore eing nlysed. Smples for prticulte nutrient nlysis were filtered onto Gelmn A/E glss fire filters (Est Hills, NY, U.S.A.) nd either frozen (C nd N) or stored t room temperture (PP) until nlysed. Chlorophyll smples were filtered onto Gelmn 45 mm A/E glss fire filter nd frozen. Phytoplnkton composition smples were preserved in Lugol s solution nd zooplnkton smples were preserved using sugr formlin (2 g of sugr dissolved in 1-L of 37% formldehyde) in the mount of 1% of smple volume. Smple nlysis Zooplnkton community composition ws determined under compound microscope. Ech smple ws concentrted to 15 2 ml efore two su-smples were tken using 1-mL Henson Stempel pipette nd counted using Sedgewick Rfter counting cell. Zooplnkton were identified to genus except copepods, which were ctegorised s cyclopoids, clnoids or nuplii. Zooplnkton iomss ws determined using length weight regressions for rotifers estlished y M.J. González (pers. comm.) nd for ll other zooplnkton using Downing & Rigler (1984). Phytoplnkton composition ws determined using n inverted microscope. Biovolumes were clculted using the eqution for geometric volume tht most closely resemles the species shpe. Algl iomss ws estimted y mesuring chlorophyll concentrtions using Turner TD-7 Fluorometer (Mountin View, CA, U.S.A.) fter extrcting smples in cetone for t lest 2 h ut less thn 12 h prior to reding. Solule rective phosphorus (SRP), mmoni, nitrite + nitrte (herefter referred to s nitrte or NO 3 ), ecuse nitrite levels were negligile) nd totl phosphorus (TP) nlyses were run on Lcht 8 series utonlyzer (Hch, Lovelnd, CO, U.S.A.). Totl nitrogen (TN) ws quntified using second-derivtive spectrophotometry (Crumpton, Isenhrt & Mitchell, 1992). TP nd TN smples were digested with potssium persulphte prior to nlysis. Prticulte phosphorus smples were nlysed using HCl digestion nd the molydte method (Stinton, Cpel & Armstrong, 1977; Vnni et l., 22). C nd N smples were run on Perkin Elmer Series 24 CHN nlyzer (Boston, MA, U.S.A.). Bioenergetics nlysis To gin etter understnding of how rtion size (ingestion rte) ffected nutrient dynmics, we used ioenergetic models to quntify N nd P flux through luegill in ech mesocosm tretment. This ws crried out y clculting totl luegill ingestion rtes using the Bjkov (1935) model, in which the weight of stomch contents ws multiplied y the gut pssge time (24 h divided y the digestive rte). To determine the digestive rte of luegill, we needed to djust the literture vlues for luegill digestive rtes (Seurg & Moyle, 1964; Windell, 1966) to ccount for differences in experimentl conditions. The two most importnt differences etween our study nd those of Seurg & Moyle (1964) nd Windell (1966) ws the higher wter temperture nd continuous feeding y our fish, oth likely to increse the digestive rte of fish. The men wter temperture in our study ws c. 4 C higher thn tht reported y Seurg & Moyle (1964) nd Windell (1966). Furthermore, ecuse of the continuous feeding nture of luegill (Srker, 1977) nd the low occurrence of luegill with empty stomch oth in our study nd in previous studies (Seurg & Moyle, 1964; Kest & Welsh, 1968), we determined tht luegill in our experiment were more likely to mintin digestion rte required to evcute 5 75% of their gut contents (tking 5 12 h) thn 1% (tking 18 2 h), s depicted y Seurg & Moyle (1964) nd Windell (1966). Therefore, to ccount for the higher tempertures in our study nd the continuous feeding, we estimted gut pssge time to e 7.5 h. Using this estimte we then clculted ingestion rte (I) s descried ove, which ws then used in comintion with field-mesured growth (G) nd excretion (E) to estimte egestion (F), sed on the mss lnce principle: I ¼ G + E + F. Sttisticl nlysis Tretment differences in N nd P excretion rtes nd the molr N : P rtio t which nutrients were excreted (herefter excretion N : P rtio) in the lortory experiments were determined using the 24 h men excretion rte of ech tretment in one-wy ANOVA, followed y Tukey test. Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

7 187 S.P. Glholt nd M.J. Vnni Anlysis of time series dt in the mesocosm experiment ws crried out y clculting the timeintegrted response using trpezoidl pproximtion to remove the effect of time (Stewrt, 1999) nd the mens were compred vi series of two-smple t-tests (Wlpole et l., 1998). For the t-tests, Stterthwite s pproximtion for unequl vrince nd Bonferroni correction to the overll significnce level were oth utilised. Comprisons mong tretments for ll non-time series dt were nlysed using onewy ANOVAs fter log-trnsformtion (JMP, SAS Institute Inc., 1996). All significnt differences were then tested using Tukey test to determine which tretment mens differed. The locking vrile ws never found to e significnt vrile in ny of the nlyses. The two estimtes of dily excretion rtes mesured in July nd August were nlysed for tretment differences seprtely due to differences in environmentl condition (i.e. chnges in wter temperture), while zooplnkton nd phytoplnkton iomss dt were verged over the course of the experiment efore eing nlysed. Anlysis of overll tretment differences in zooplnkton nd phytoplnkton community composition, lgl nd nutrient concentrtions, s well s luegill ody nutrient composition were ll conducted using dt collected on the finl dy of the experiment. Results Bluegill nutrient excretions in lortory experiment Both N nd P excretion rtes were high shortly fter feeding, then declined over time (Fig. 1). However, potentilly importnt differences were oserved in the dynmics of N nd P excretion rtes. N excretion rtes peked t different times depending on tretment nd did not lwys return to low, constnt rtes over the 24 h period (Fig. 1). In contrst, P excretion rtes peked within 2 h of feeding then rpidly declined to low, constnt level in ll tretments (Fig. 1). N nd P excretion rtes of fed luegill incresed with incresing rtion size. Sttisticl differences were found for oth N nd P excretion rtes mong ll tretments in which the diet of the fish were supplemented with the simulted-enthic prey (Fig. 1). Furthermore, N excretion rtes differed mong ll tretments except the no-rtion nd 3%-rtion tretments (Fig. 1). However, luegill P excretion rtes () Excretion rte (mg N g 1 h 1 ) () Excretion rte (µg P g 1 h 1 ) (c) Molr N : P rtio of excretions No rtion 3% rtion 5% rtion 1% rtion % rtion No rtion 5% rtion 1% rtion No rtion 5% rtion 1% rtion 3% rtion Time fter feeding (h) Fig. 1 () Nitrogen nd () phosphorus excretion rtes nd (c) molr N : P rtios of excretions y luegill fter eing fed one of four rtion sizes under lortory conditions. Brs in ll grphs indicte 1 SD. Letters ove error rs indicte significnt differences etween tretments t the P <.5 level, unless indicted y n sterisk (*), in which cse significnce ws found t P <.1. vried gretly mong replictes in the no-rtion tretment, nd thus the 1%-rtion tretment ws the only tretment tht differed sttisticlly from the no-rtion tretment (Fig. 1). In generl, tretment differences in P excretion rtes were less pronounced thn N excretion rte differences (Fig. 1). Although Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

8 Rtion-specific N nd P excretion y fish 1871 the tretments did not differ in excreted N : P rtios t ny individul time intervl, ll tretments showed the similr trends of low N : P rtios excreted shortly fter feeding, followed y pek excreted N : P rtios 8 h fter feeding nd finlly returning to low molr N : P excretion levels y hour 24. Furthermore, while the excretion N : P rtio verged over the 24 h period did not differ mong tretments in which fish were fed, unfed luegill in the no-rtion tretment tended to excrete t lower N : P rtios thn luegill in the 3%-rtion tretment (P ¼.6). Bluegill diet in mesocosms Anlysis of gut contents tken on the finl dy of the mesocosm experiment showed tht luegill diet (excluding erthworms fed to fish) consisted lmost entirely of emerging chironomid lrve nd zooplnkton (cldocerns, copepods, Choorus nd ostrcods) in ll three tretments with fish. However, no sttisticl differences were detected in the mount of nonerthworm iomss found in the stomch of luegill mong the three tretments with fish (P ¼.91). Gut content nlysis nd estimted ingestion rtes using Bjkov (1935) clcultions suggests tht high-rtion luegill hd twice the ingestion rte of low-rtion luegill nd erthworms mde up sustntil mount of the gut iomss of fish in oth the Low (38% of gut mss) nd high-rtion (52%) tretment (Tle 1). Therefore, sed on the finding tht luegill in oth low- nd high-rtion tretments consumed similr mounts of non-erthworm prey nd tht erthworms fed to the fish represented high percentge of their dily food needs, we feel confident tht we were le to mnipulte totl ingestion rtes s desired. Bluegill excretion rtes in mesocosms The effects of rtion size on luegill excretion rtes of P under mesocosm conditions differed from those found in the lortory experiment, while effects on N excretion rtes were similr to lortory experiments. N excretion rtes incresed with rtion size oth in July (P <.1) nd August (P <.1), s found in the lortory experiment (Fig. 2). In contrst, P excretion rtes did not increse with rtion size on either smpling dte (July P ¼.15, August P ¼.47; Fig. 2). The molr N : P rtios significntly differed Tle 1 Bioenergetic model descriing nutrient flux through luegill using Bjkov (1935) ingestion rte estimtes* (sed on dry mss), mesured prmeters nd the mss lnce eqution to estimte unmesured prmeters P-ssimiltion efficiency (%) P-egestion rte (g dy )1 ) P-excretion rte (g dy )1 ) P-llocted to growth (g dy )1 ) Totl P-ingestion rte* (g dy )1 ) N-ssimiltion efficiency (%) N-egestion rte (g dy )1 ) N-excretion rte (g dy )1 ) N-llocted to growth (g dy )1 ) Totl N-ingestion rte* (g dy )1 ) Worm ingestion rte* (g dy )1 ) Non-worm ingestion rte* (g dy )1 ) Men growth rte (g dy )1 ) Tretment No rtion Low rtion High rtion Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

9 1872 S.P. Glholt nd M.J. Vnni ().4 Excretion rte (mg N g 1 h 1 ) () Excretion rte (µg P g 1 h 1 ) (c) Molr N : P rtio of excretions No rtion Low rtion High rtion July only etween the no-rtion nd high-rtion tretments (July P ¼.7, August P ¼.3); however, trend of incresing excretion N : P with rtion size ws found on oth smpling dtes (Fig. 2c). Totl nd dissolved nutrients July July The mount of TP found in the wter column declined stedily over the course of the mesocosm experiment, August August August Fig. 2 () Nitrogen nd () phosphorus excretion rtes nd (c) molr N : P rtios of excretions y luegill for ech fish tretment from mesocosm experiment. Excretion rtes were mesured on 24 July nd gin on 25 August 22. c from c. 4lg PL )1 t the eginning of the experiment to c. 2 lg PL )1 y the experiment s end, in ll four tretments. Mrginlly significnt differences were detected for the mount of TP in the wter column using the time-integrted response nlysis (P ¼.6), with no-rtion tretment hving less TP in the wter column thn oth low- nd high-rtion tretments, respectively. However, these differences my not hve iologicl significnce ecuse the difference in TP mong these tretments ws less thn 2 lg PL )1. TN lso showed decline over the course of the experiment in ll tretments with vlues strting round 1.8 mg N L )1 nd dropping elow 1.3 mg N L )1. No differences were detected mong fish tretments, ut the fishless tretment hd significntly lower mounts of TN compred with ll of the tretments with fish (P <.1). SRP (P ¼.81) nd NO ) 3 (P ¼.85) showed no sttisticl differences mong tretments, nd differences in mmoni were only mrginlly significnt (P ¼.6). In contrst to totl N nd P, concentrtions of dissolved inorgnic nutrients in the wter column remined reltively constnt for the durtion of the experiment. Vlues rnged from 3.4 to 5.5 lg SRP L )1,.2 to.18 mg NH 4 N L )1, nd.2 to.2 mg NO 3 N L )1. Zooplnkton To determine if the fish cges used in the mesocosm experiment creted refuge for zooplnkton outside the cges, we nlysed zooplnkton smples tken within nd outside cges. No sttisticl differences were oserved in rotifer (P ¼.99), copepod (P ¼.28), cldocern (P ¼.17), or totl zooplnkton iomss (P ¼.58), suggesting tht the cges did not crete significnt refuge for zooplnkton from fish predtion. Totl zooplnkton iomss did not sttisticlly differ mong tretments over the course of the experiment (Fig. 3). Similrly, the men iomss of the three mjor zooplnkton groups (rotifers P ¼.35, cldocerns P ¼.38, copepods P ¼.13) lso did not differ sttisticlly mong tretments (Fig. 3). By the end of the experiment the zooplnkton community ws dominted y cyclopoid copepods, followed y the cldocern Simocephlus, with clnoid copepods nd rotifers contriuting only minor prt of overll zooplnkton iomss. No lrge dphnids were present in this experiment. As with iomss verged Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

10 Zooplnkton iomss (mg L 1 ) Rotifer Cldocern Copepod Fishless No rtion Low rtion High rtion Fig. 3 Men iomss, rotifers plnktonic cldocerns nd copepods over the durtion of the experiment. over the entire experiment, no sttisticl differences in zooplnkton totl iomss were detected mong tretments on the finl dy of the experiment (P ¼.46). In summry, neither the ddition of fish nor the rtion size fed to fish hd ny sttisticlly mesurle effect on zooplnkton iomss or community composition. () Chlorophyll (µg L 1 ) () Biovolume (%) Rtion-specific N nd P excretion y fish 1873 Fishless No rtion Low rtion High rtion Unknown Dinophyt Cynocteri Cryptophyt Chlorophyt Bcillriophyt Phytoplnkton The phytoplnkton response to the pplied tretments verged over the course of the mesocosm experiment lso reveled surprisingly few differences. The presence of fish resulted in significnt differences mong tretments, with phytoplnkton iomss (chlorophyll ) eing significntly lower in the fishless tretment thn in ll the tretments with fish (P <.1; Fig. 4), while rtion size hd no sttisticlly significnt effect on phytoplnkton iomss (Fig. 4). Light-sturted primry productivity rtes per unit chlorophyll mesured mid-wy through the experiment lso reveled no significnt differences mong tretments (P ¼.85). Phytoplnkton community composition ws lso very similr mong tretments t the end of the experiment. Among phytoplnkton groups, only Chlorophyt (per cent of totl iovolume) showed significnt differences mong tretments, eing significntly lower in the fishless tretment thn oth the no-rtion nd high-rtion tretments (Fig. 4). However, mong the tretments in which fish were fed, Cynocteri, Dinophyt nd Bcillriophyt ll showed similr decresing trends s rtion size fed to fish incresed in terms of per cent of totl (c) Biovolume (%) Fishless No rtion Low rtion High rtion Fishless No rtion Low rtion High rtion Fig. 4 Phytoplnkton response to tretments, including () men phytoplnkton iomss (chlorophyll ) over the durtion of the experiment; () phytoplnkton composition (per cent of totl iovolume) t the end of the experiment; nd (c) inedile phytoplnkton (per cent of totl iovolume) t the end of the experiment; s determined using time-integrted response curves to clculte mens in series of two-smple t-tests. iovolume, while Cryptophyt incresed s rtion size incresed (Fig. 4c). The reltive contriution of inedile phytoplnkton, sed on size (>3 lm, including Aphnizomenon, Cosmrium nd Certium), lso incresed with rtion size, ut this trend ws only mrginlly significnt (P ¼.7; Fig. 4c). * Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

11 1874 S.P. Glholt nd M.J. Vnni Algl iomss No significnt mong-tretment differences in phytoplnkton iomss (chlorophyll ) were oserved on the finl dy of the mesocosm experiment (P ¼.33; Fig. 5). Furthermore, phytoplnkton iomss mde up reltively smll frction of the totl lgl iomss in the mesocosms (Fig. 5). Periphyton iomss comprised out hlf the totl lgl iomss found in the mesocosms, nd ws significntly lower in the low-rtion tretment thn in the high-rtion tretment (P ¼.4; Fig. 5). The mount of sedimented lge ws comprle with tht of periphyton nd ws significntly higher in the high-rtion tretment thn in the fishless tretment (P <.5). In ddition, there ws trend towrd incresed lgl sedimenttion with incresing rtion size lthough it ws not significnt (Fig. 5). Lstly, totl lgl iomss (sum of chlorophyll in phytoplnkton, periphyton nd sedimented orgnic mtter) ws significntly higher in the high-rtion tretment thn ll other tretments (P <.1; Fig. 5). Nutrient udgets The N nd P udgets constructed on the finl dy of the mesocosm experiment show consistent trends oth within nd mong tretments for ech of the four mjor nutrient pools mesured (Fig. 6,). Overll, sediment sequestered the most nutrients (oth N nd P), followed y periphyton nd fish. However, the mss of N nd P in seston showed net loss (Fig. 6). Concentrtions in ll mjor nutrient pools generlly incresed with the ddition of fish or with rtion size fed to fish, except P in the seston pool, which remined constnt mong tretments (Fig. 6). The mount of N lost from the seston pool differed mong the fishless nd high-rtion tretment, with other tretments intermedite (P <.5; Fig. 6), while the loss of P from the wter column did not differ mong tretments (P ¼.84; Fig. 6). The sequestrtion of N y periphyton over the course of the experiment did not differ sttisticlly mong tretments (P ¼.25; Fig. 6); however, the mount of P sequestered y periphyton ws significntly lower in the Fishless tretment thn in ll tretments with fish (P <.1; Fig. 6). The mounts of oth sedimented N nd P were significntly higher in the high-rtion tretment thn in the fishless tretment, with other tretments intermedite (N: P ¼.2; P: P <.5; Fig. 6,). The proportion of P stored in fish compred with the totl P pool ws sustntilly greter thn the mount of N stored in fish compred with the totl N pool (Fig. 6,). Lstly, oth N nd P sequestrtion y fish incresed s rtion size incresed, however, only P sequestrtion y fish resulted in tretment differences. Furthermore, luegill in the no-rtion tretment sequestered significntly less N nd P thn fish in the low- nd highrtion tretments (N: P ¼.2; P: P ¼.4; Fig. 6,). Nutrient composition of luegill N nd P content of luegill ody mss mesured t the end of the mesocosm experiment vried significntly mong tretments. In generl, N content of Chlorophyll (mg mesocsom 1 ) Fishless No rtion Low rtion High rtion Phytoplnkton Periphyton Sediment Totl Fig. 5 Distriution of lgl iomss (chlorophyll) in mesocosms on the lst dy of the mesocosm experiment. Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

12 Rtion-specific N nd P excretion y fish 1875 () Chnge in N mss (g) Fishless No rtion Low rtion High rtion Seston Periphyton Sediment Bluegill 1 ().9 Chnge in P mss (g).6.3. Seston Periphyton Sediment Bluegill.3 Fig. 6 Nitrogen () nd phosphorus () udgets, expressed s the chnge in mss from eginning to end of the mesocosm experiment. luegill incresed while P content decresed s rtion size incresed (Fig. 7). However, luegill P content differed mong ll tretments (P <.1), while N content differed only mong the no-rtion nd highrtion tretments (P <.1; Fig. 7). The opposing trends in luegill N nd P content resulted in significntly higher ody N : P rtio of fish s rtion size incresed (P <.1; Fig. 7). Fish ioenergetics/mss lnce As predicted, ioenergetics nlyses using the Bjkov (1935) model showed tht the high-rtion tretment in the mesocosm experiment hd the highest estimted ingestion rte, followed y the low-rtion tretment, nd then the no-rtion tretment (Tle 1). Both P nd N ssimiltion efficiencies differed >2.5 mong Percent N, P or Molr N : P rtio No rtion Low rtion High rtion %N %P Molr N : P Fig. 7 Bluegill nutrient content nd molr N : P rtio in response to tretments. tretments (Tle 1). Bluegill in oth the no-rtion nd high-rtion tretments hd lower ssimiltion efficiencies for P compred with luegill in the c c Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

13 1876 S.P. Glholt nd M.J. Vnni low-rtion tretment (Tle 1). However, the lloction of P to growth ws lmost 1.5 times higher for luegill in the high-rtion tretment compred with luegill in the low rtion, while luegill in the nortion tretment hd very low lloction of P to growth over the course of the experiment (Tle 1). Alloction of N to growth followed the sme generl trend s ssimiltions efficiencies ut gin the highrtion luegills tended to show surprisingly high N lloction to growth in comprison with its ssimiltion efficiency (Tle 1). Finlly, s for P, the mount of N sequestered in luegill of the no-rtion tretment ws not significntly different from zero (Tle 1). Discussion Nutrient excretion rtes nd rtios Our lortory excretion results suggest tht N : P rtios excreted y fish cn vry, ecuse of temporl differences in the relese rte of N versus P, over 24- h period. Mther et l. (1995) found similr fluctutions in excreted N : P rtios for luegill feeding on chironomids over 24 h period. The fluctution in N : P excretion rtes over 24 h period could hve potentil consequences for communities in qutic ecosystems. While it is unrelistic to think tht luegills feed only once dy, temporlly vrile luegill N : P excretion rtios could lter lgl community composition, if pek dily feeding times of luegill coincide with dily migrtions etween different hitts. Bsed on the findings y Kest & Welsh (1968) tht luegill hve two pek feeding times per dy shortly fter they migrte to nd from the littorl zone fter sunrise nd efore sunset, our dt suggests tht pek excretion rtes y luegill my occur c. 4 h fter oth sunrise when the luegill re still feeding in the littorl zone nd sunset when the luegill hve moved into the pelgic zone to feed (Kest & Welsh, 1968; lso shown to occur in L. giosus L. Collins & Hinch, 1993). These two peks in excretion rtes, occurring in either the littorl or pelgic zone, could e very different from one nother ecuse of potentil differences in the type of prey cptured (e.g. enthic invertertes in the littorl zone nd zooplnkton in the pelgic zone) nd wter temperture. Becuse oth diet nd temperture ffect excretion rte (Sterner & Elser, 22; Vnni et l., 22), it is likely tht the pek excretion rtes occurring in the littorl nd pelgic zone re different nd could ffect lgl community structure s well s nutrient dynmics etween these two hitts. The significnt increses in N nd P excretion rtes with incresing rtion sizes, found under lortory conditions suggests tht the dily excretion rtes of oth N nd P cn e ltered y chnges in ingestion rtes of fish. Although differences mong tretments were much more pronounced for N excretion thn for P excretion, differences in the N : P excretion rtio mong tretments were miniml, nd temporl vrition in N : P excretion rtio were greter thn differences mong tretments (Fig. 1). The reltionship etween ingestion rte nd excretion rte ppered liner for oth N nd P, sed on the finding tht excretion rte tended to increse proportionlly to rtion size. This trend grees with results of erlier findings on how rtion size ffects N relese in lortory experiments (Ttri, 1981; Cui & Wootton, 1988). Furthermore, this liner reltionship etween ingestion rte nd excretion rte lso suggests tht fish mintin reltively homeosttic levels of ody nutrients nd ssimiltion efficiency under vrying ingestion rtes (over the short-time scle of such experiments) resulting in vrile excretion rtes, s predicted y ecologicl stoichiometry theory (Sterner et l., 1992; Sterner & Elser, 22) nd ssumed in mny fish ioenergetic models (e.g. Hnson et l., 1997). However, the sme tretments pplied under mesocosm conditions did not show liner reltionship etween ingestion rte nd excretion rte, suggesting tht consumer s ody nutrient content nd ssimiltion efficiency my not e s constnt s previously thought (see Bioenergetics nd mss lnce section elow). We evluted the reltive importnce of luegill in trnsporting nutrients into the pelgic zone using dt on excretion rtes, diets nd nutrient pools in the different mesocosm tretments. Although we were not le to directly mesure oth the mount of recycled nutrients (i.e. nutrients tken nd relesed in the sme hitt) nd trnsported nutrient (i.e. nutrients tken from one hitt nd relesed into nother), we could estimte nutrient trnsport rtes y sutrcting the mount of nutrients excreted y luegill in the no-rtion tretment (ll recycled nutrients) from the mount excreted y luegill in oth the low- nd high-rtion tretments (oth recycled nd trnsported nutrients). The difference is roughly equl Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,

14 Rtion-specific N nd P excretion y fish 1877 to the mount of nutrients trnsported into the pelgic zone y luegill nd derived from simulted enthic prey (i.e. erthworms). This pproch is only vlid ecuse no sttisticl differences were found mong tretments in the mount of non-erthworm prey consumed y luegill. We estimted tht etween 2 nd 4% of the totl mount of SRP found in the wter column ws dded to the pelgic zone dily in the low- nd high-rtion tretments, respectively. This is not trivil mount. For exmple, y the end of the experiment luegill in the low-rtion tretment would hve dded nerly 3 the mount of SRP found in the pelgic zone. Plnkton Fish cn ply importnt roles in driving pelgic foodwe dynmics (e.g. Crpenter et l., 1992; Schus & Vnni, 2). However, it is still uncler whether the reduction of grzing pressure on phytoplnkton y fish feeding on herivorous zooplnkton (i.e. topdown process) or nutrients supplied y fish excretions (i.e. ottom-up process) is the more importnt mechnism driving food-we dynmics (Vnni et l., 1997; Vnni & Lyne, 1997; Attyde & Hnsson, 1999). The enhncement of phytoplnkton iomss (chlorophyll) in the presence of fish occurred despite the fct tht there were no detectle effects of fish on zooplnkton iomss, species composition nd thus presumly grzing pressure on phytoplnkton. This suggests tht the role of nutrient cycling y fish is more importnt thn the effects of fish on zooplnkton grzing rtes, in terms of driving phytoplnkton iomss, in greement with some other experimentl studies (Schindler, 1992; Persson, 1997; Vnni et l., 1997). However, the wek grzing effect oserved in this experiment my e ecuse of the sence of lrge grzers such s dphnids, which generlly drive grzing-induced chnges in phytoplnkton, s well s the reltively low consumption of zooplnkton y luegill. We predicted tht differences in the mount of nutrients excreted y fish (ecuse of vrile ingestion rtes) mong tretments would result in differences in pelgic community composition. However, the lck of differences in zooplnkton communities mong tretments suggest tht zooplnkton were unle to tke dvntge of the incresed nutrient supply to the system vi incresed excretion y luegill fed supplementry enthic prey in the form of erthworms. Similrly, ecuse our fish gut nlysis suggests tht the mounts nd types of pelgic prey consumed y luegill were consistent mong tretments, t lest t the end of the experiment, we cn eliminte the hypothesis tht consumption of zooplnkton y fish compensted for lck of enthic prey, nd tht totl ingestion rtes were similr mong tretments. An lterntive hypothesis to explin why tretments given dditionl nutrients (vi erthworms) did not hve higher zooplnkton iomss is tht inedile lge dominte systems with higher nutrient supply rtes (Evns, Rorts & Arts, 1995), therey constrining zooplnkton growth. If inedile species dominte the phytoplnkton community, much of the nutrients could e locked up in the lge nd not trnsferred up the food-we (Leiold, 1989; Hnsson et l., 1998). The phytoplnkton compositionl dt give some support for this ide, in tht the reltive undnce of inedile phytoplnkton (per cent of totl iomss) tended to increse with incresed rtion size fed to fish (Fig. 4c). However, inedile lge mde up less thn 25% of the phytoplnkton community nd it is uncler whether or not this mount of inedile lge could cuse the lck of response y zooplnkton to tretment differences found in our experiment. Although the N : P excretion rtio incresed significntly with rtion size in the mesocosm experiment, we oserved few mong-tretment differences in phytoplnkton community composition. The pprently wek tretment effect on phytoplnkton communities could e explined y ny or ll of severl resons. One possile reson for the lck of response of phytoplnkton to tretments is tht N : P excretion rtios were generlly well ove the Redfield rtio (Fig. 2c), which should promote P limittion in ll tretments. Alterntively, ny shift in community composition towrd lrge filmentous or colonil lge could hve een missed ecuse of the likelihood of these lge to settle out of the wter column nd ecome included in the sedimented lgl pool. Although different mounts of nutrients were eing supplied to the different tretments s desired, we did not oserve the lrge community response we predicted. This suggests tht nutrient flow, even through reltively simple food-we, is complex. Consequently, we compred lgl iomss nd constructed nutrient udget in n ttempt to Ó 25 Blckwell Pulishing Ltd, Freshwter Biology, 5,