Journalof MarineResearch, 56, 713–729, 1998

Excretion oftrace elements bymarine copepods andtheir bioavailability to diatoms

byWen-Xiong Wang 1,2 andNicholas S. Fisher 1,3

ABSTRACT Wemeasuredthe physiological turnover rate ( 5 excretionrate) of Žvetraceelements (Ag, Cd, Co, Se andZn) in the marine copepod Temoralongicornis followingfeeding on radiolabeled diatoms. Theturnover rate constants of trace elements in copepods were high (0.05 to 0.38 d 2 1) and were comparableto published values for N andP excretion.Ag, Cd and Co were excreted at higher rates thanSe andZn. Turnover rate constants of Ag, Cd, and Co also increased with increasing food concentration,whereas excretion of Se andZn was not affected by food concentration. There was littleevidence that copepod grazing regenerated diatom Ag, Co and Zn into the dissolved phase duringthe pre-ingestive phase. Copepod grazing slightly enhanced the release into the dissolved phaseof Cdand Se fromdiatoms, where they were primarily localized in cytoplasm.Excreted Ag, Se andZn were less bioavailable to diatoms than when in inorganic form, but the bioavailability of excretedCd and Co was comparable to inorganic forms. Our study demonstrated that copepod excretionrepresents a signiŽcant route by whichparticulate metals are regenerated to thedissolved phase.Metal excretion by marine zooplankton is analogous to N andP excretionand may thus signiŽcantly affect metal cycling and modify metal speciation in surface waters. Regenerated metals mayre-enter planktonic food chains and be recycledseveral times in surfacewaters before sinking in particulatematter.

1.Introduction Regenerationof dissolvedorganic carbon (DOC) dueto zooplankton excretion, sloppy feedingand fecal pellet decomposition has been well documented (Lampert, 1978; Lehman,1980; Jumars et al.,1989).Biologically mediated regeneration of traceelements insurfacewaters remainsless studied, although there is evidencethat some metals may be recycledmany times before they are exported from surfacewaters (Bruland,1983; Coale andBruland, 1985, 1987). Several essential trace elements (e.g., Fe andZn) have been proposedto limit primary productivity in some oceanic areas (e.g., high nutrient low chlorophyll,or HNLC) dueto theirrestricted supply from externalsources (Martin et al., 1991; Morel et al., 1994).In HNLC areas,primary production can be maintainedby the

1.Marine Sciences Research Center,State Universityof New York,Stony Brook, New York,11794-5000, U.S.A. 2.Present address: Department ofBiology, Hong Kong University of Science andTechnology, Clear Water Bay,Kowloon, Hong Kong. 3.Author to whom reprint requests should be sent. 713 714 Journalof MarineResearch [56, 3 efficientregeneration of metals into the dissolved phase and recycling by phytoplankton (Hutchinsand Bruland, 1994). Several studies have demonstrated that metals can be efficientlyregenerated by nano-,micro- and mesozooplanktonic grazers and that this may playa signiŽcant role in metalcycling in aquatic systems (Hutchins et al., 1993;Hutchins andBruland, 1994, 1995; T wiss andCampbell, 1995; Twiss et al., 1996, Wang et al., 1996).However, it isnot yet clear whether metal is regenerated by excretionof assimilated metals,sloppy feeding of animals (by breaking food particles during the pre-ingestive phase),release from decomposingfecal material, or simply due to re-equilibration of metalsbetween particulate and dissolved phases. Quantitative estimation of the relative importanceof each process in metal regeneration by marine mesozooplankton is also lacking. Freyand Small (1979) argued that iron could be released from zooplanktonin a bioavailableform afteringested algal cells pass through an acidic gut, although direct evidencewas lacking.Recently, we suggestedthat metal excretion by copepodsrepresents asigniŽcant route by which metals associated with phytoplankton cells are regenerated intothe dissolved phase following grazing (i.e., post-ingestive regeneration) (W angand Fisher,1998). Metal release from decomposingfecal pellets, which may also represent a signiŽcant route by whichphytoplankton metals are regenerated into the dissolved phase, hasbeen quantiŽ ed (Fisher et al.,1991a,b;Lee and Fisher, 1992; W ang et al., 1996).In this studywe examinedthe effects of foodconcentration on theturnover of Žvetraceelements (Ag, Cd,Co, Se, and Zn) in a marinecalanoid copepod ( Temoralongicornis ). Therole of copepodgrazing (during the pre-ingestive phase) in metalrelease from diatomswas also quantitativelyassessed. W ealsodetermined the bioavailability of excreted metals to diatoms.W ecomparedbiologically essential and nonessential metals whose distributions insurface waters arewell characterized.

2.Materials and methods a.Excretion of radiolabeledtrace elements in copepods The diatom Thalassiosirapseudonana (clone3H) was radiolabeledwith Ž veradioiso- 110m 109 57 75 topes, Ag(in 0.1 N HNO 3), Cd(in0.5 N HCl), Co(in 0.1 N HCl), Se(indistilled 75 65 water,as selenite, Na 2 SeO3), and Zn(in 0.1 N HCl),as described in Wang et al. (1996). Radioactivityadditions were 37–91 kBq l 2 1 for 109Cd(corresponding to 2.5– 6.2 nM), 57Co(2.8– 7.0 pM), 75Se (0.5–1.1 nM), and 65Zn(4.4– 10.8 nM), and 18– 37 kBq l 2 1 for 110mAg(1.8– 3.7 nM). Immediately prior to isotope additions, microliter amounts of 0.5N SuprapurNaOH were addedso that Ž nalpH was 7.8–8.0. The culture was thengrown underconditions described in Wang et al. (1996)for 3d(5cell divisions), after which the cellswere collectedonto 1 µmpolycarbonatemembranes and resuspended into Ž ltered seawater.This procedure was repeatedtwice to remove radioisotopes weakly bound to algalsurfaces. Surface seawater collected 8 kmoff Southampton,NY was usedfor all experiments.Experiments used trace metal clean techniques throughout to avoid metal contaminationduring the course of theexperiments. Adultcopepods ( Temoralongicornis )were collectedfrom StonyBrook Harbor between 1998] Wang& Fisher:Marine copepod excretion of trace elements 715

Marchand May, 1997, acclimated at 15°C inthelaboratory and fed T.pseudonana for 1 d beforethe experiments. Radiolabeled cells were thenfed to 700copepods held in 2Lof Žlteredseawater at afooddensity of 7 3 104 cells ml2 1 (or 1.54mg dry wt l 2 1,equivalent to 560 µg C l2 1)inthe dark. W aterand food were renewedevery 12 h. Copepods were continuouslyfed under these conditions for sixdays, after which they were collectedand depuratedin three replicate beakers (60 individuals per beaker) containing 200 ml of Žlteredseawater and nonradioactive diatoms ( T.pseudonana )atthree food densities: 4 3 103 cells ml2 1 (or 32 µg C l2 1) , 2 3 104 cells ml2 1 (160 µg C l2 1), and 105 cells ml2 1 (800 µg C l2 1).Theradioactivity retained by 25livecopepods was measuredevery 12– 24 h for 5d.Waterand food were replacedevery 12 h.The radioactivity retained in all copepods was notmeasured because there was considerablemortality at the lowest food concentra- tion (4 3 103 cells ml2 1)duringthe depuration period. After 6dradioactivefeeding, 50 copepods were alsofractionated to determine the distributionof radioisotope in exoskeleton, polar (containing proteins, polysaccharide, nucleicacid, small molecular compounds) and nonpolar (containing lipids) components, usinga methodmodiŽ ed from Reinfelderand Fisher(1994). Copepods were collectedonto 10-µm polycarbonatemembranes and rinsed with 10 ml Ž lteredseawater. After afurther rinsewith 10 ml 0.1mM EDT A(dissolvedin Žlteredseawater) for 2min(this fraction was collectedand the radioactivity measured), copepods were extractedwith 3 mlof 0.2 N NaOH at65° C for 3h(necessaryfor completeextraction of soft tissues). Exoskeletons were removedby Ž lteringcopepods onto 10-µ m polycarbonatemembranes and rinsing themwith 0.2 N NaOH. Chloroform(2 ml) was thenadded twice to the Ž ltrateand shaken vigorouslyto separate the polar and nonpolar fractions (10 to 15 min).Radioactivity in the exoskeleton,polar and nonpolar fractions was thencounted. Radioactivity of the Ž ltrate from theEDT Awashwas addedto theexoskeleton fraction to calculate the proportion of radioisotopein the exoskeleton, polar and nonpolar fractions of copepods. b.Bioavailability of excretedtrace elements to marine diatoms Diatoms (T.pseudonana )were radiolabeledwith 110mAg 1 109Cd 1 57Co 1 75Se 1 65Zn, asdescribedabove. Radioactivity additions (in 200 ml of 0.2µ mŽlteredwater) were 370 kBq l2 1 for 109Cd(corresponding to 25 nM), 57Co (28 pM), 75Se (4.7nM), and 65Zn (44nM), and 92 kBql 2 1 for 110mAg(9.2 nM). Cells were collectedafter 3 dgrowthand fed to1000 copepods ( T.longicornis )maintainedin 1.5 L ofŽ lteredseawater at a food concentrationof 5 3 104 cells ml2 1 (400 µg C l2 1).Thewater and radiolabeled food were replacedevery 12 h. Copepods were fedunder these conditions for 2d,afterwhich they were collectedand transferred to 1 Lof0.2 µ mnonradioactiveŽ lteredseawater for 1d withoutfeeding. During this period copepods regenerated trace elements into the dissolved phase.The waterwas dividedinto two 500 mlbatchesand was thenŽ lteredthrough 0.2 µm polycarbonatemembranes and enriched with nutrients as describedabove. A controlgroup containingradioisotopes ( 110mAg 1 109Cd 1 57Co 1 75Se 1 65Zn)in the nutrient medium ( f/2additionsof nitrate,phosphate, silicate and vitamins, f/20additions of Mn,Mo, Co, andFe, and no Cu, Zn, or EDT A;Guillardand Ryther, 1962) but without copepod 716 Journalof MarineResearch [56, 3 excretorymatter was alsoprepared. Radioisotope additions in thecontrol group were 5.2 kBq l2 1 for 110mAg(0.5 nM), 7.4 kBq l 2 1 for 109Cd(0.5nM), 3.7 kBq l 2 1 for 57Co (0.3 pM) and 75Se (47pM), and 10.4 kBq l 2 1 for 65Zn(1.2 nM). Log phase T.pseudonana cells were resuspendedinto 20 mlŽ lteredseawater, and added to the medium at a concentrationof 10 5 cells ml2 1.Therewere 2replicatebeakers for eachtreatment (excreted metal and control). Cellgrowth and metal partitioning in diatomcells were monitoredevery 12 hfor 3d,as describedin Wang et al. (1997).The dry weight concentration factor (DCF) ofmetalsin the cellswas calculatedas:

DCF 5 Cf /Cw (1)

2 1 where Cf isthe radioactivity in diatom cells (dpm g drywt cells),and Cw is the radioactivityin the dissolved phase (dpm ml 2 1 water). Thedry wt ofeach T.pseudonana cellis 22.4pg, and the volume is 61µm 3; thus for T.pseudonana , the DCF 5 2.7 3 the volume-volumeconcentration factor (VCF). c.Partitioning of traceelements in diatoms due to copepod grazing Diatomswere radiolabeledas described above, resuspended twice into unlabeled seawater,and then added to 1Lof0.2µ mŽlteredseawater at aconcentrationof 1.5 3 105 cells ml2 1 (correspondingto 3.3 mg dry wt l 2 1 or 1.2 mg C l2 1).After 10minequilibration, thepartitioning of radioisotopes between the diatom cells and the dissolved phase was determined(Fisher et al.,1983).Copepods ( T.longicornis, 50–60 individuals)were then addedinto the beaker and incubated in the dark for 48h. Acontrolwithout copepods but containingthe same number of diatomcells was usedto follow the loss (desorption) of radioisotopesfrom diatoms.There were tworeplicates for eachtreatment. The fraction of radioisotopesretained by the diatoms and the cell density were determinedperiodically overa 48h incubationperiod. The DCFs ofthe trace elements in the diatoms were calculatedaccording to Eq. (1). d.Radioactivity measurements Radioactivityof live copepods was measurednoninvasively with a largedeep well NaI(Tl) gammadetector; radioactivity of water,labeled phytoplankton, and the biochemi- calfractions was determinedwith a Pharmacia-WallacLKB NaI(Tl) gammadetector. All measurementswere relatedto appropriate standards and calibrated for radioisotope spilloverfrom higherenergy to lower energy windows. The gamma emission of 110mAg was determinedat 658 keV ,of 109Cdat88 keV ,of 57Coat122keV ,of 75Se at264keV ,and of 65Znat 1115 keV .Countingtimes in all samples were adjustedto give propagated countingerrors , 5%.

3. Results a.Efflux rates of traceelements in copepods Theretention of radioisotopes( 110mAg, 109Cd, 57Co, 75Se and 65Zn) in T.longicornis over a5ddepurationperiod are shown in Figure 1. High mortality ( . 50%)was foundfor 1998] Wang& Fisher:Marine copepod excretion of trace elements 717

Figure1. The retention of metalsin the copepod Temoralongicornis feedingat different food levels duringthe depuration period. ( d ):lowfood (32 µ gCL 2 1); (j ):mediumfood (160 µ gCL 2 1); (O): highfood (800 µ gCL 2 1).Valuesare means 6 1 SD, n 5 3. copepodsmaintained at the lowest food concentration (4000 cells ml 2 1), thusmetal depurationin this treatment was followedfor only3 d.Following transfer of copepodsto unlabeledwater, there was aninitialrapid loss of radioactivitywithin the Ž rst dayfor some elements(especially Co), and then a slowerloss between 1 and5 d.Effluxrate constants (theslope of the linear relationship between ln % retainedin copepods and time) were thereforecalculated from thedata between 1 and5 d.Efflux rates determined after 1 d 718 Journalof MarineResearch [56, 3

Table1. Efflux rate constants ( ke)oftraceelements in thecopepod Temoralongicornis atdifferent foodconcentrations following 6 dfeedingon radiolabeleddiatoms. The biological half-life ( t1/2) andtheproportion( a)oftraceelement in theslower exchanging compartment (between 1 and5d) arealso shown. Means 6 SD (n 5 3).A orBindicatesthat there is a signiŽcant difference in efflux rateconstant between two food treatments for each trace element ( P , 0.05).

Food conc. ke t1/2 a Metal (µg C L2 1) (d2 1) (d) (%) r2 Ag 32 0.158 6 0.051A 5.05 6 2.07 81.6 6 8.2 0.882 160 0.221 6 0.065 3.52 6 1.32 93.6 6 9.6 0.938 800 0.289 6 0.024A 2.42 6 0.20 93.6 6 5.2 0.970 Cd 32 0.157 6 0.050A 5.06 6 2.03 100 0.966 160 0.176 6 0.034B 4.06 6 0.71 100 0.947 800 0.375 6 0.093A,B 1.96 6 0.46 100 0.958 Co 32 0.304 6 0.040 2.32 6 0.33 67.2 6 2.3 0.880 160 0.216 6 0.027A 3.25 6 0.37 52.9 6 1.6 0.941 800 0.312 6 0.014A 2.22 6 0.10 48.3 6 1.1 0.916 Se 32 0.194 6 0.041 3.73 6 0.80 100 0.915 160 0.155 6 0.014 4.50 6 0.42 91.5 6 7.5 0.950 800 0.158 6 0.008 4.40 6 0.21 86.7 6 4.3 0.941 Zn 32 0.078 6 0.018 9.44 6 2.18 95.1 6 5.1 0.907 160 0.047 6 0.008 15.1 6 2.47 90.3 6 3.5 0.923 800 0.093 6 0.034 8.48 6 2.81 91.7 6 10.4 0.963 depurationcan be considered equivalent to metal excretion rates because there was negligibleradioactivity in the fecal pellets produced after 1 dofdepuration (data not shown).Most of theradioisotopes ( . 82%for Ag,Cd, Se andZn, and . 48%for Co)were inthe slower exchanging compartment following 6 dfeedingon radiolabeled food and theseproportions remained relatively independent (for Ag,Cd and Zn) or decreased slightlywith increasing food concentration (for CoandSe) (Table1). Thecalculated efflux rate constants were highestfor Ag,Cd, and Co (0.16to 0.38 d 2 1), andlowest for Zn(0.05 to 0.09 d 2 1)(Table1). The efflux rates of Ag,Cd, and Co (butnot Se andZn) increased with the food concentration. For example,efflux rate constants of Ag andCd were 1.8and 2.4 times higher at the highest food ration than at the lowest food ration.For allmetals except Zn, the calculated biological half-lives were , 5d(Table1). Tissuefractionation indicated that most radioactivity was foundin the polar components containingproteins, polysaccharide, nucleic acid, and small molecular compounds. Exo- skeletonscontained , 16%of thetotal radioactivity for Cd,Co and Se and , 35% for Ag andZn (Table 2). The nonpolar component (containing lipids) represented , 6% of total radioactivityin copepods for alltrace elements. b.Bioavailability of excretedtrace elements to marinediatoms After 1dofdepuration in nonradioactive Ž lteredseawater (without food), copepods whichwere previouslyfed with radiolabeled diatoms for 2dexcretedabout 33% of their Ag,27% of their Cd, 34% of their Co, 24% of their Se, and 15% of their Zn into the 1998] Wang& Fisher:Marine copepod excretion of trace elements 719

Table2. Distribution of trace elements in different components of Temoralongicornis after 6 d feedingon radiolabeleddiatoms. Mean 6 SD (n 5 2). Traceelement Exoskeleton Polar Non-polar Ag 35.0 6 3.5 58.8 6 5.0 6.2 6 1.5 Cd 15.8 6 0.9 79.4 6 0.2 4.8 6 1.1 Co 13.9 6 1.1 82.3 6 0.2 3.8 6 0.9 Se 13.4 6 0.4 83.9 6 6.3 2.6 6 0.5 Zn 33.6 6 5.0 60.0 6 6.3 6.4 6 1.3 ambientwater. For theseexcreted metals, about 70% of the Ag, 95% of theCd, 87% of the Co,99% of theSe and80% of the Zn were foundin thedissolved phase ( , 0.2 µm). Duringthe 3 dexposureperiod to Ž lteredand nutrient-enriched Ž lteredseawater collectedfrom agrazingtreatment, diatoms accumulated less excreted Ag, Se andZn than inorganicforms (i.e.,without excretory matter) of these elements added to seawater (Fig.2). This difference decreased for Se andZn after 2 d,whereasfor Agit increasedwith time.The calculated DCF (whichcan be considered as an index of metalbioavailability to diatoms)of excreted metals decreased by 70% 6 8%for Ag,61% 6 15%for Se,and 53% 6 7%for Zncompared to the controls, indicating that bioavailability of these elementswas considerablylower within the 3dexposureperiod (Fig. 3). For CdandCono signiŽcant effects of excretory matter were consistentlynoted (Figs. 2, 3). During the exposureperiod, the growth rate of diatomswith excreted metals (0.86 6 0.01 d2 1) was slightlylower than that of diatomsin thecontrol cultures (0.91 6 0.02 d2 1). c.Partitioning of trace elements in diatomsdue to copepodgrazing Whenradiolabeled diatoms were resuspendedinto unlabeled seawater, about 24% of the Ag,16% of theCd, 26% of theCo, 6% oftheSe and49% of theZn were releasedinto the dissolvedphase within 10 min.Ag and Co were continuouslyreleased into the dissolved phaseover the Ž rst 10h,butfor theother metals there was relativelylittle further release intothe dissolvedphase. During the 48 hincubationperiod, copepod grazing removed 35% ofthe diatom cells. The fraction of totalmetal associated with diatom cells was slightly lowerfor Cd,Co, Se andZn after24 h ofgrazingthan in the controls (without copepods), whereasthe proportion of Agin food was notaffected by copepod grazing (Fig. 4). The calculatedDCFs howeverremained comparable to the controls for Ag,Co and Zn, indicatingthat copepod grazing did not contribute to metal regeneration into the dissolved phasefrom diatoms(Fig. 5). For CdandSe, the DCFs ofdiatomsincubated with copepods were lowerthan the controls after 2 to8 hofincubation, suggesting that grazing contributedslightly to Cd andSe regenerationinto the dissolved phase from preyparticles.

4.Discussion Metalefflux ( 5 excretion)rate constants measured for Temoralongicornis (0.08 and 0.3 d2 1)areprobably the highest recorded among marine invertebrates (Fowler, 1982). Sucha highturnover rate is presumablyrelated to ahighweight speciŽ c metabolicrate and 720 Journalof MarineResearch [56, 3

Figure2. The uptake of metals by the diatom Thalassiosirapseudonana incubatedwith excreted metals(by copepods) and metals added in inorganicform. V aluesshown are the percentages of totalmetal bound to diatom cells. ( d ):controls(metals added in inorganic form); ( j ): excreted (metalsadded in excretedform). V aluesare means 6 1 SD, n 5 2. 1998] Wang& Fisher:Marine copepod excretion of trace elements 721

Figure3. The calculated dry weight concentration factors (DCF) ofmetalsin thediatom Thalassio- sirapseudonana incubatedwith excreted metals (by copepods) and metals added in inorganic form. (d ):controls(metals added in inorganic form); ( j ):excreted(metals added in excreted form).V aluesare means 6 1 SD, n 5 2. excretionrate associated with small body size. Among the 5 traceelements, Se andZn, bothof whichare biologically essential, are excreted at a muchlower rate than Ag, Cd and Co,consistent with their higher assimilation efficiencies (AEs) from ingestedfood particles(W angand Fisher, 1998). 722 Journalof MarineResearch [56, 3

Figure4. The fraction of totalmetal associated with the diatom Thalassiosirapseudonana incubated withand without copepods. V aluesshown are the percentages of totalmetal bound to diatomcells. (d ):withoutcopepods; ( j ):withcopepods. V aluesare means 6 1 SD, n 5 2.

Previousstudies using the same experimental approach have demonstrated that metal effluxis dominatedby excretioninto the dissolved phase (Hutchins et al., 1995; Wang et al., 1996;W angandFisher, 1998). In these studies, most depurated metals were detectedin thedissolved phase with little association with fecal pellets, suggesting that excretion is the 1998] Wang& Fisher:Marine copepod excretion of trace elements 723

Figure5. The calculated dry weight concentration factors (DCF) ofmetalsin thediatom Thalassio- sirapseudonana incubatedwith and without copepods. ( d ):withoutcopepods; ( j ): with copepods.V aluesare means 6 1 SD, n 5 2. majorroute by which a metalis lostfrom copepodsfollowing assimilation. Egestion via fecalpellet production dominates metal loss during the initial digestive period (W angand Fisher,1998). Sick and Baptist (1979) reported that Cd isregenerated by marinecopepods (Pseudodiaptomuscoronatus )witha rateconstant of 0.13to 0.24 d 2 1,comparableto the 724 Journalof MarineResearch [56, 3 rates(0.16 to 0.38d 2 1)inour study. Recently, we reportedturnover rates of 0.29d 2 1 for Ag, 0.30 d2 1 for Cd,0.28 d 2 1 for Co,0.16 d 2 1 for Se and0.08 d 2 1 for Zn in T.longicornis following2 dradioactivefeeding on diatomsat ahighfood concentration (800 µ gCl 2 1) (Wangand Fisher, 1998). These values are consistent with our presentmeasurements at this foodlevel, but following 6 dradioactivefeeding on diatoms. The fractions of metals distributedin theslowerexchanging compartment are also comparable between 2 dand6 d radioactivefeeding (W angand Fisher, 1998; this study). There is no evidence of an additionalcompartment of metal loss after 2 or6dfeedingon radiolabeled food. With a muchshorter labeling period (1 to 2 h),however, metal regeneration rates increase considerably,probably because most radioisotopes are in the faster exchanging compart- mentdominated by metal defecation (W ang et al.,1996).Thus, metal efflux rates measured usinga short(1– 2 h)exposure period may be confounded by metal loss from thefaster exchangingcompartment. Excretionrates of trace elements measured in our study are directly comparable to turnoverrates of N andP inzooplankton. For P,adailyturnover rate of 10%in marine copepods(CalanusŽ nmarchicus )(Conover,1961), 41% in Calanushelgolandicus (Corner et al.,1972),and 35% to 60% in the freshwater zooplankter Daphniapulex (Lehman, 1980)have been reported, although P turnoverefficiency may depend greatly on the physiologicalstate of algal cells (Olsen and Ø stgaard,1985; Sterner and Smith, 1993). Turnoverrates of N indiversecopepods are typically within the range of 0.05to 0.30 d 2 1 (Checkley et al.,1992).For example,Checkley et al.(1992)found a Nturnoverrate of 0.12 d2 1 (0.05to 0.21d 2 1) in Acartia spp.from theInland Sea of Japanand 0.15 d 2 1 (0.09 to 0.22 d2 1) in Centropagesfurcatus from theGulf of Mexico,values similar to 0.15 d 2 1 (0.06to 0.25 d 2 1) in Acartiatonsa (Kiørboe et al.,1985).Excretion rates of Ninoceanic copepods(from theSargasso Sea) aresomewhat higher (0.2 to 0.3 d 2 1)andincrease inverselywith body size (V erity,1985, W enandPeters, 1994). The efficientregeneration of NandP byzooplanktoncan signiŽ cantly impact primary production in thesea (Johannes, 1968;Smith and Whitledge, 1977; Alcaraz et al., 1994). Theeffects of foodconcentration on metal excretion are metal-speciŽ c. Since all Ž ve traceelements are measured simultaneously in thecopepods,any difference among them is dueto different biological and chemical behavior of themetals; biological variability can beignoredas an explanationfor differentexcretion rates in experimentsusing multilabeled organisms.Because our results are similar to previous Ž ndingswith same copepod species, itappears that inter-experimental variation is low as well.Differences in metalexcretion ratesat different food concentrations may be related to variationsin metabolic rates of the animals.It is likely that excretion of Se andZn, which are essential and have the slowest excretionrates, can be metabolicallyregulated to maintain desired internal concentrations. As withthe metals, con icting results on theeffects of foodconcentration on N excretion incopepods have been reported. Millar and Landry (1984) found no effectof foodlevel on Nexcretionin CalanuspaciŽ cus ,whereasHead et al.(1988)reported that N excretionin copepodsis positively correlated with ambient chlorophyll concentration. Head et al. 1998] Wang& Fisher:Marine copepod excretion of trace elements 725

(1988)proposed that differences in theinitial gut content of copepodsmay be responsible for thevariation of food concentration effect on N excretionbecause there is a strong couplingbetween defecation and excretion processes. At thelowest food level (32 µ gCl 2 1),individualcopepods appeared to be food-limited duringthe depuration periods and had a highmortality ( . 50%within the 3 dperiod). Similarly,Dam (1986)observed that at a chlorophyllconcentration of 1.7 µ gl 2 1 (T. pseudonana,correspondingto 88 µ gCl 2 1),gutfullness of T.longicornis doesnot differ from thestarvation level, indicating that the animals are not feeding at thisfood level. Thedistribution of metalsin copepodsfollowing 6 dradiolabeledfeeding was compa- rableto thatin apreviousstudy employing a 2dradiolabeledfeeding, and this distribution remainsessentially constant over a 7ddepurationperiod (W angand Fisher, 1998). The highfraction of Agand Zn intheexoskeleton may have resulted in part from uptakefrom thedissolved phase following metal desorption from diatomsduring the feeding period. Thesemetals have a highaffinity for proteinand it is likelythat they combine with chitin andchitosan in theexoskeleton by bindingwith amino groups (Muzzarelli, 1977). Severalstudies have stressed the importance of biologically mediated regeneration in metalcycling in marine and freshwater systems(Frey andSmall, 1979; Hutchins et al., 1993;Hutchins and Bruland, 1994; T wiss andCampbell, 1995; Twiss et al., 1996). Our resultsunequivocally demonstrate that mesozooplankton excretion is animportantroute by whichdiatom metals are regenerated into the dissolved phase. Efficient regeneration of metalsinto the dissolved phase can increase metal residence time in surface waters ina manneranalogous to N andP .Ametalcan thus be recycled several times before being transportedout of surfacewaters withsinking particles. However, metal residence time in surfacewaters canalso be affected by metal AE inanimals,metal release from biogenic particles(e.g., fecal pellets and marine snow), and transport by migrating animals. Zooplanktonhave been shown to play a dominantrole in inuencing metal transport out of surfacewaters (Coaleand Bruland, 1985; Fowler and Knauer, 1986). Thereis littleevidence to indicate that copepod grazing directly causes metal desorption orrelease from phytoplanktoncells. In our experiments, which employed relatively low copepoddensities (50– 60 individualsl 2 1),onlyCd and Se partitioning(DCF) indiatoms was slightlylowered by copepod grazing, and the other elements were unaffected.It is unlikelythat release of Cd and Se intothe dissolved phase is due to release from fecal pellets,since their release rates from fecalpellets are comparable to those of othermetals, includingCo andZn, which show no evidenceof regenerationby copepod grazing (W ang et al., 1996).Lee and Fisher (1994) also concluded that zooplankton grazing would have onlya smalleffect on the release of Ag, Cd, Co, Pb and Po from phytoplanktoninto the dissolvedphase. Therehave been considerably more studies on theproduction of DOC duringzooplank- tongrazing. Generally, a substantialrelease of DOC dueto zooplankton grazing has been attributedto animalexcretion, leakage of DOC from feces(Lampert, 1978), or release from brokencells during ‘ ‘sloppyfeeding’ ’ (Conover,1966). Lampert (1978) suggested that up 726 Journalof MarineResearch [56, 3

Figure6. The calculated percentage of total metal bound to particlesas a functionof particleload (range0 to10 mg l 2 1)andmetal concentration factor (range 5 3 103 to 5 3 106).Seetext for furtherexplanation. to17% of algal carbon ingested by the freshwater zooplankton Daphniapulex is lost as DOC from algaedamaged during feeding. Hygum et al.(1997)however concluded that leakagefrom fecalpellets is more signiŽ cant than sloppy feeding or excretion for mesozooplankton-mediatedDOC production.The release rates of DOC andtrace metals from phytoplanktonand copepod fecal pellets have been shown to becomparable(Fisher andW ente,1993; Lee and Fisher, 1993, 1994). Theimpactof zooplanktongrazing on metalregeneration depends on such conditions as foodconcentration, zooplankton density and the duration of feeding.An additional factor thatmight control the release of metalsfrom algalcells is the decrease in algalabundance duringfeeding and the consequent re-equilibration of metals between particulate and dissolvedphases (Fisher and W ente,1993). Thus, if theDCF remainsconstant for agiven metal,lowering the particle abundance by feeding would lead to a desorptionof surface- boundmetal from theremaining algal cells into the dissolved phase. Figure 6 presentsa simplecalculation showing that the fraction of metal associated with particles must decreasewith decreasing particle load to maintaina constantDCF ,andthat the effects of particleload on metal partitioning depend greatly on metal DCF .For metalswith DCFs , 104,particleload has only a slighteffect on metal partitioning, thus any observed increaseof metal partitioning in the dissolved phase may result from animalregeneration, notre-equilibration of metals between particulate and dissolved phase. For metalswith DCFs . 106,adecreaseof particle load below 4 mgl 2 1 cangreatly decrease metal partitioningin the particulate phase, whereas for particleloads . 4 mg l2 1, metal 1998] Wang& Fisher:Marine copepod excretion of trace elements 727 partitioningremains essentially independent of particle load. Intermediate effects of particleloads on metal partitioning are found for metalswith DCFs between5 3 104 and 5 3 105.Wethereforesuggest that changes in particle load should be considered in experimentsexamining metal regeneration by zooplankton grazing. In our grazing experi- ments,the DCFs ofalltrace elements were between2 3 105 to 3 3 106.Withan initial particleconcentration of 3.3mg l 2 1,adecreasein particle load by copepodgrazing should decreasethe partitioning of all trace elements in diatoms. Consequently, the DCF is probablya morerealistic parameter for quantifyingmetal regeneration due to copepod grazing. ExcretedSe, Zn and especiallyAg were muchless bioavailable to diatoms than metals in inorganicform, suggestingthat zooplankton excretion may signiŽ cantly in uence their speciationin surface waters. This would of course be dependent on the prey and zooplanktonbiomass, since the metal complexing capacity of theexcreted matter would increasewith prey and animal abundance. W estressthat in ourstudy of metal bioavailabil- ity,as in most of the earlier studies cited, the zooplankton abundance was signiŽcantly greaterthan in mostnatural waters. Twiss andCampbell (1995) similarly demonstrated that nanoagellate grazers can regenerate some metals (Gd, Zn,Cd, Cs) from prey Synechococ- cusleopoliensis intothe dissolved phase. They showed that the bioavailability of regeneratedGd, Zn and Cd islower than inorganic species. Insummary, our study demonstrates that copepod excretion represents a signiŽcant routeby whichparticulate metals are regenerated into the dissolved phase. Food concentra- tionappears to affect the excretion of Ag, Cd, and Co, whereas for Se andZn food concentrationdoes not have an appreciable effect, presumably because these two elements areessential and can be metabolicallyregulated by copepods.There is very little evidence thatdiatom metals are regenerated into the dissolved phase by copepods during the pre-ingestivephase. Lower bioavailabilityof excretedmetals to diatoms are found for Ag, Se andZn, whereas for Cdand Co their bioavailability remains comparable to metals addedas inorganic species. Regeneration by zooplankton grazers may therefore affect trace metalspeciation in surface waters. Metals regenerated by copepod excretion should re-enterthe food chain and get recycled several times before sinking as particulate metals.

Acknowledgments. Wethanktwo reviewers for many thoughtful comments. This study was supportedby grants from ONR (No.N00014– 95-1– 1229), NAPM, andNSF (OCE-9617675)to N. Fisher.This is MSRC contributionno. 1072.

REFERENCES Alcaraz,M., E. Saizand M. Estrada.1994. Excretion of ammoniaby zooplanktonand its potential contributionto nitrogenrequirements for primary production in the Catalan Sea (NW Mediterra- nean).Mar. Biol., 119, 69–76. Bruland,K. W.1983.Trace elements in sea-water, in ChemicalOceanography 8,J.P.Rileyand R. Chester,eds., Academic Press, London, 157– 220. Checkley,D. M.Jr.,M. J.Daggand S.-I. Uye. 1992. Feeding, excretion and egg production byindividualsand populationsof themarine, planktonic copepods, Acartia spp. and Centropages- furcartus.J.PlanktonRes., 14, 71–96. 728 Journalof MarineResearch [56, 3

Coale,K. H.andK. W.Bruland.1985. 234Th:238Udisequilibriawithin the California Current. Limnol. Oceanogr., 30, 22–33. ——1987.Oceanic stratiŽ ed euphotic zone as elucidated by 234Th:238Udisequilibria.Limnol. Oceanogr., 32, 189–200. Conover,R. J.1961.The turnover of phosphorusby CalanusŽ nmarchicus .J.Mar.Biol. Assoc. U.K., 41, 484–488. ——1966.Feeding on large particles by Calanushyperboreus (Kroyer), in SomeContemporary Studiesin MarineScience, H. Barnes,ed., Allen & Unwin,London, 187– 194. Corner,E. D.S.,R. N.Headand C. C.Kilvington.1972. On the nutrition and metabolism of zooplankton.8. Thegrazing of Biddulphia cells by Calanushelogolandicus .J.Mar.Biol. Assoc. U.K., 52, 847–861. Dam,H. 1986.Short-term feeding of Temoralongicornis Mu¨llerin thelaboratory and the Ž eld.J. Exp.Mar. Biol. Ecol., 99, 149–161. Fisher,N.S., P .Bjerregaardand S.W.Fowler.1983. Interactions of marineplankton with transuranic elements.1. Biokineticsof neptunium, plutonium, americium, and californium in phytoplankton. Limnol.Oceanogr., 28, 432–447. Fisher,N.S., C.V .Nolanand S.W .Fowler.1991a. Scavenging and retention of metalsby zooplankton fecalpellets and marine snow. Deep-Sea Res., 38,1261–1275. ——1991b.Assimilation of metalsin marinecopepods and its biogeochemical implications. Mar. Ecol.Prog. Ser., 71, 37–43. Fisher,N. S. andM.Wente.1993. Release of traceelements by dyingmarine phytoplanton. Deep-Sea Res., 40, 671–694. Fowler,S. W.1982.Biological transfer and transport processes, in PollutantTransfer and Transport intheSea, G. Kullenberg,ed., CRC Press,Boca Raton, 1– 65. Fowler,S. W.andG. A.Knauer.1986. Role of largeparticles in the transport of elements and organic compoundsthrough the oceanic water column. Prog. Oceanogr., 16, 147–194. Frey,B. E.andL. F.Small.1979. Recycling of metabolized iron by the marine dino agellate Amphidiniumcarterae .J.Phycol., 15, 405–409. Guillard,R. R.L.andJ. H.Ryther.1962. Studies on marineplanktonic diatoms. I. Cyclotellanana Hustedt and Detonulaconfervacea (Cleve)Gran. Can. J. Microbiol.,8, 229–239. Head,E. J.,A.Bedoand L.R. Harris.1988. Grazing, defecation and excretion rates of copepodsfrom inter-islandchannels of theCanadian Arctic archipelago. Mar. Biol., 99, 333–340. Hutchins,D. A.andK .W.Bruland.1994. Grazer-mediated regeneration and assimilation of Fe, Zn andMnfrom planktonic prey. Mar. Ecol. Prog. Ser., 110, 259–269. ——1995.Fe, Zn, Mn and N transferbetween size classes in acoastalphytoplankton community; tracemetal and majornutrient recycling. J. Mar.Res., 53, 297–313. Hutchins,D. A.,G.R.DiTullioand K. W.Bruland.1993. Iron and regenerated production: evidence forbiological iron recycling in two marine environments. Limnol. Oceanogr., 38,1242–1255. Hutchins,D. A.,W .-X.W angandN. S.Fisher.1995. Copepod grazing and the biogeochemical fate of diatomiron. Limnol. Oceanogr., 40, 989–994. Hygum,B. H.,J. W.Petersenand M. Sondergaard.1997. Dissolved organic carbon released by zooplanktongrazing activity: a high-qualitysubstrate pool for bacteria. J. PlanktonRes., 19, 97–111. Johannes,R. E.1968.Nutrient regeneration in lakes and oceans, in Advancesin Microbiology of the Sea,M. R.Droopand E. J.F.Wood,eds., Academic Press, NY ,203–213. Jumars,P .A.,D. L.Penry,J. A.Baross,M. J.Perryand B. W.Frost.1989. Closing the microbial loop: dissolvedcarbon pathway to heterotrophic bacteria from incomplete ingestion, digestion and absorptionin animals. Deep-Sea Res., 36, 483–495. 1998] Wang& Fisher:Marine copepod excretion of trace elements 729

Kiørboe, T .,F.Møhlenberg and K. Hamburger.1985. Bioenergetics of the planktonic copepod Acartiatonsa :relationbetween feeding, egg production and respiration, and composition of speciŽc dynamicaction. Mar. Ecol. Prog. Ser., 26, 85–97. Lampert,W .1978.Release of dissolved organic carbon by grazing zooplankton. Limnol. Oceanogr., 23, 831–834. Lehman,J. T.1980.Release and cycling of nutrients between planktonic algae and herbivores. Limnol.Oceanogr., 25, 620–632. Lee,B.-G. and N. S.Fisher.1992. Decomposition and elemental release from zooplankton debris. Mar.Ecol. Prog. Ser., 88, 117–128. ——1993.Release rates of trace elements and protein from decomposing planktonic debris. 1. Phytoplanktondebris. J. Mar.Res., 51, 391–421. ——1994.Effects of sinking and zooplankton grazing on the release of elements from plankton debris.Mar. Ecol. Prog. Ser., 110, 71–281. Martin,J. H.,R. M.Gordonand S.E. Fitzwater.1991. The case for iron. Limnol. Oceanogr., 36, 1793–1802. Miller,C. A.andM. R.Landry.1984. Ingestion-independ entrates of ammonium excretion by the copepod CalanuspaciŽ cus .Mar.Biol., 78, 265–270. Morel,F .M.M.,J.R.Reinfelder,S. B.Roberts,C. P.Chamberlain,J. G.Leeand D. Yee.1994. Zinc andcarbon co-limitation of marinephytoplankton. Nature, 369, 740–742. Muzzarelli,R. A.A.1977.Chitin. Pergamon Press, Oxford, 309 pp. Olsen,Y .andK. Østgaard.1985. Estimating release rates of phosphorusfrom zooplankton: models andexperimental veriŽ cation. Limnol. Oceanogr., 30, 844–852. Reinfelder,J. R.andN. S. Fisher.1994. Retention of elementsabsorbed by juvenileŽ sh (Menidia menidia, Menidiaberyllina )fromzooplankton prey. Limnol. Oceanogr., 39,1783–1789. Sick,L. V.andG. J.Baptist.1979. Cadmium incorporation by the marine copepod Pseudodiaptomus coronatus.Limnol.Oceanogr., 24, 453–462. Smith,S. L.andT .E.Whitledge.1977. The role of zooplanktonin the regeneration of nitrogenin a coastalupwelling system off Northwest Africa. Deep-Sea Res., 24, 49–56. Sterner,R. W.andR. F.Smith.1993. Clearance, ingestion and release of N andP by Daphiaobtusa feeding on Scenedesmusacutus ofvaryingquality. Bull. Mar. Sci., 53, 228–239. Twiss,M. R.andP .G.C.Campbell.1995. Regeneration of trace metals from picoplankton by nanoagellate grazing. Limnol. Oceanogr., 40,1418–1429. Twiss,M. R.,P.G.C.Campbelland J.-C.Auclair. 1996. Regeneration, recycling, and trophic transfer oftrace metals by microbial food-web organisms in the pelagic surface waters of Lake Erie. Limnol.Oceanogr., 41,1425–1437. Verity,P .G.1985.Ammonia excretion rates of oceaniccopepods and implications for estimates of primaryproduction in the Sargasso Sea. Biol. Oceanogr., 3, 249–283. Wang,W .-X.and N. S.Fisher.1998. Accumulation of traceelements in a marinecopepod. Limnol. Oceanogr., 43, 273–283 Wang,W .-X.,S. B.Griscomand N. S.Fisher.1997. Bioavailability of Cr(III)and Cr(VI) to marine musselsfrom solute and particulate pathways. Environ. Sci. T echnol., 31, 603–611. Wang,W .-X.,J. R.Reinfelder,B.-G. Lee and N. S.Fisher.1996. Assimilation and regeneration of traceelements by marinecopepods. Limnol. Oceanogr., 41, 70–81. Wen,Y .H.andR. H.Peters.1994. Empirical models of phosphorusand nitrogen excretion rates by zooplankton.Limnol. Oceanogr., 39,1669–1679.

Received: 11August, 1997; revised: 17March, 1998.