Excretion of Trace Elements by Marine Copepods and Their Bioavailability
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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 vetraceelements (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 signicant route by whichparticulate metals are regenerated to thedissolved phase.Metal excretion by marine zooplankton is analogous to N andP excretionand may thus signicantly 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 signicant 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 asignicant 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 signicant route by whichphytoplankton metals are regenerated into the dissolved phase, hasbeen quanti 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 vetraceelements (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 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 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 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 lteredseawater 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 lteredseawater 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 methodmodi ed from Reinfelderand Fisher(1994). Copepods were collectedonto 10-µm polycarbonatemembranes and rinsed with 10 ml lteredseawater. After afurther rinsewith 10 ml 0.1mM EDT A(dissolvedin lteredseawater) 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 lteringcopepods onto 10-µ m polycarbonatemembranes and rinsing themwith 0.2 N NaOH. Chloroform(2 ml) was thenadded twice to the 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 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 mlteredwater) 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 of 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 mnonradioactive lteredseawater for 1d withoutfeeding. During this period copepods regenerated trace elements into the dissolved phase.The waterwas dividedinto two 500 mlbatchesand was then 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