of elements ingested by marine planktonic bivalve larvae

Transcription

1 Limnol. Oceanogr., 39(l), 1994, , by the American Society of Limnology and Oceanography, Inc. The assimilation bivalve larvae of elements ingested by marine planktonic John R. Reinfeldeer and Nicholas S. Fisher Marine Sciences Research Center, State University of New York, Stony Brook Abstract The assimilation efficiencies of nine elements were measured in planktonic bivalve mollusc larvae (oysters, Crassostrea virginica, and hard clams, Mercenaria mercenaria) fed uniformly radiolabeled phytoplankton cells (Zsochrysis galbana) in order to test whether the liquid digestion strategy observed in marine copepods operates in other planktonic hcrbivorcs with gut morphologies different from that of crustacean zooplankton. Of the elcmcnts studied (Ag, Am, C, Cd, Co, P, S, Se, and Zn), americium was assimilated the least by both the larval oysters (7.9%) and clams (2.6Oh), while selenium was assimilated with the highest efficiency by the larvae (oysters, 97%; clams, 1 OOO/o). Assimilation efficiencies were directly related to the fraction of each clement present in the cytoplasm of the ingested algae. Like copepods, bivalve larvae have short gut passage times and assimilate only the easily mobilized, cytoplasmic fraction of ingested phytoplankton cells. The cytoplasmic fraction of some elements (Se, Zn, and Cd) and ofprotein in I. galbana increased inversely with algal growth rate. Larvae feeding on seneseent cells would therefore be expected to assimilate proportionately more ofthcse elements and protein than when feeding on rapidly dividing cells. The trophic transfer of material through marine food webs is of primary interest to marine ecologists because it is the means by which energy and nutrients flow from one organism to another. Trophic transfer processes in marine animals are also of interest with regard to the accurate assessment of trace element toxicity which may depend on how much of a particular toxic substance an organism accumulates from its food. Quantitative resolution of contaminant accumulation from the dissolved phase and from ingested food has proven to be difficult but would greatly enhance predictive models of the accumulation of potentially toxic elements in marine organisms (Thomann 198 1; Luoma et al. 1992). The flow of material along trophic pathways can also influence the geochemical fate of contaminants in marine environments, including potential routes to man, as trophic transfer processes will generally affect the geochemical behavior of all elements and compounds in I Present address: Ralph Parsons Laboratory, Department of Civil and Environmental Engineering, MIT, Cambridge, Massachusetts Acknowledgments This research was supported by National Science Foundation grant OCE and by a grant from the New York Sea Grant Institute. Contribution 914 from the Marine Sciences Research Center. marine systems. The influence of trophic transfer processes on the geochemical cycles of elements is most evident in planktonic food webs where elements that are assimilated by zooplankton will be recycled with organic matter and therefore have longer residence times in the water (Fisher and Reinfelder in press). Elements that are egested with sinking fecal matter will accumulate in deeper water and in the benthos (Cowman et al ) and have shorter residence times in the water (Whitfield and Turner 1987). To assess the importance of trophic transfer to nutrient flow, contaminant bioaccumulation, or biogeochemical cycles in marine environments, it is necessary to know what portion of ingested matter is assimilated by consumer animals. Assimilation efficiency, de- 12 fined as the proportion of ingested material that crosses an animal s gut lining, is a quantitative measure of the trophic transfer of ingested substances in marine animals that can be used to compare the bioaccumulation of different elements. The assimilation of ingest- ed elements in marine herbivores is of interest because it is the first step in the transfer of matter from plants to animals and because the bioaccumulation and trophic transfer of potentially toxic elements among marine animals and terrestrial animals (including humans) that consume seafood will be influenced by trophic transfer processes at the lowest levels of marine food webs.

2 Bivalve larvae assimilation 13 It has previously been shown that three species of herbivorous copepods use a digestion strategy in which only the readily mobilized fraction of ingested phytoplankton is assimilated (Reinfelder and Fisher 199 1). The amount of an element assimilated in these copepods can be predicted from examination of their food, regardless of the biological usefulness of that element. Communities of marine herbivorous zooplankton, however, include a diverse collection of animals, many ofwhich may have very different digestive systems than those of crustacean zooplankton. The larvae of many bivalve molluscs are important members of coastal herbivorous zooplankton communities during certain times of the year and, being in an entirely different phylum from copepods, * are expected to have a different digestion strategy from crustacean herbivores. Assimilation efficiencies of nine elements were measured in planktonic bivalve mollusc larvae as a comparison study to that involving copepods (Reinfelder and Fisher 199 1). Materials and methods Planktonic hard clam (Mercerzaria mercenaria Linnaeus) and oyster (Crassostrea virginica Gmelin) larvae were obtained from local commercial shellfish hatcheries (Bluepoints Co. Inc. and Frank M. Flower & Sons Inc.). The bivalve larvae were kept in GF/C-filtered seawater adjusted to 26$& with deionized water at ambient laboratory temperature (22-28 C) and fed the prymnesiophyte Isochrysis galbana (clone ISO). Preliminary experiments were conducted in which the assimilation of Ag, Am, Cd, Co, Se, and Zn was measured in oyster larvae that had ingested uniformly radiolabeled diatoms (Thalassiosira pseudonana, clone 3H). None of the trace elements associated with ingested diatoms was assimilated by the oyster larvae. Since this result was attributed to the inability of the oyster larvae to break open ingested diatoms which have a siliceous cell wall, I. galbana, a phytoplankter lacking a cell wall, was used in all other assimilation efficiency experiments. The assimilation efficiencies of Ag, Am, C, Cd, Co, P, S, Se, and Zn were measured in bivalve larvae that ingested bana. uniformly radiolabeled Z. gal- Radiolabeled phytoplankton -I. galbana cells used for feeding to bivalve larvae were labeled with 1 *OrnAg, 241Am, 14C, 09Cd, 57C~, 32P 35S, 75Sc, and 65Zn. Two groups of isotopes (241Am, 75Se and 65Zn and Cd 57Co and I IonlAg) were used to produce double or tiiplelabeled phytoplankton cells. The peak gamma emissions of the three nuclides in each group can be measured with a minimum of spillover which was corrected for in all multiradiotracer experiments. Cells were grown in seawater which was sterile filtered through 0.2- pm Nuclepore filters (SFSW) and enriched with modified f/2 nutrients (Guillard and Ryther 1962) but without any added Si. Cells exposed to 241Am, 75Se, and 65Zn were grown in SFSW enriched with f/2 N, P, and vitamins and with f/20 (clam larvae experiments) or f/50 (oyster larvae experiments) trace metals, minus Cu, Zn, and EDTA. For llomag, lo9cd, 57Co, 32P, and 35S exposures, I. galbana cells wcrc grown in f/2, minus Si (except for 32P, f/50 PO,), and f/10 trace metals, minus Cu, Zn, and EDTA. 14C-labeled I. galbana cells were grown in SFSW with f/2 nutrient additions, minus Si enrichment. I. galbana cultures were labeled as follows: 59.2 kbq liter -I (16.8 nm) of OrnAg from a solution of 0.1 N HNO,; in different experiments, a range of kbq liter- ( nm) of 241Am in 3 N HNO,; 50 kbq liter- of 14C as NaH14C0, in distilled water; 148 kbq liter- (69.7 PM) of lo9cd in 0.1 N HCl; 148 kbq liter- (9.1 PM) of 57Co in 0.1 N HCl; 148 kbq liter- (4 nm) of 32P as NaH232P04 in distilled water; 3.7 MBq liter-l of 35S as Na235S04 in distilled water; in different experiments, a range of kbq liter- (0.13-o. 14 nm) of 75Se as selenite in 0.5 N HCl; and kbq liter- ( pm) of 05Zn in 0.1 N HCl. Assimilation eficiency experiments -Labeled I. galbana cells were collected by centrifugation at 8,000 x g (10 min) and resuspended into loo-300-ml batches of SFSW (26%0) to yield a cell density of - 5 x 1 O4 cells ml-. This corresponds to a suspended algal load of 0.8 mg dry wt liter-l, 0.55 mg algal C liter - l, or 2.95 mm3 cell vol. liter- I. After allowing the resuspended phytoplankton to equilibrate for 45 min, several thousand oyster or hard clam larvae (40-90 ml-l) were added to each 1 OO-300-ml batch with a small volume of SFSW. Bivalve larvae were fed for h and allowed to purge their guts while grazing on unlabeled I. galbana for 12 h. In prelimi-

3 14 Reinfelder and Fisher Table 1. Bivalve larvae fed labeled Z.sochrysis gulbana: experimental conditions. Duralion Radio- Larvae Larvae Feeding of fccdtracer age (d) ml I vol. (ml) Cells ml I /.a~ cell ing (W Oyster larvae (Crassostwa virginica) I lnmag x Am x C x lo x lo Cd x Co x p x lo S ~ lo ~ lo Se X Zn x Hard clam larvae (Mercenuria mercenaria) I 10mAg x Am x lo JC x x Cd X Co X p X s ~ lo x lo ?k! x X Zn X nary studies, the clearance of radioactive feces was found to be complete within 1 h. The amount of ingested phytoplankton was measured by detecting changes in cell density using in vivo chlorophyll a fluorescence (Brand et al ); fluorescence was converted to cell density with standard curves for each culture. The experimental conditions under which the oyster and hard clam larvae were exposed to labeled I. galbana are shown in Table 1. Assimilation efficiencies were measured by dividing the amount of each tracer retained in the larvae after gut clearance (12 h) by the amount ingested. Since dissolved 75Se released from I. galbana cells into the dissolved phase in the feeding suspension may have contributed to the amount of 75Se retained by the larvae, the assimilation efficiency of Se in oyster larvae was also calculated according to the ratio method described by Fisher and Reinfelder ( 199 1) for comparison. In their method, the ratios of 75Sc to 241Am, an unassimilated tracer of bulk ingested material, are compared in the food and fecal pellets, and the assimi- Table 2. Percentage distribution of elements in different subcellular fractions of Isochrysis gulbanu fed to oyster (Crusostreu virginicu) and hard clam (Mercenuriu mercenuria) larvae. Cell division rates (p) are given as divisions per day. Oyster larvae Super- Pellet 1 natant Ele- (cell (cytoment P membrane) plasm) Ag Am C Cd co i Se Zn Hard clam larvae Super- Pellet 1 natant (cell (cyto- P membrane) plasm) lation efficiency of Se is calculated by 75Se/241Am (food) - 75Se/241Am (feces) 75Se/241Am (food) I. galbana cells were collected on l-pm polycarbonate filters, and bivalve larvae feces were collected on 20-pm Nitex mesh by gravity filtration, Both were rinsed twice with 5 ml of SFSW before radioanalysis. Phytoplankton cell fractionation - Immediately prior to the feedings, the intracellular compartmentalization of each radiotracer in aliquots of homogenized I. galbana cells was measured. I. galbana cells were Zysed by resuspension in deionized water (ph 7.0) followed by freezing. Cellular components were separated by centrifugation at 10 C yielding pellet (750 g, 5 min) and supernatant fractions (Fisher et al. 1983). The pellet contained plasmalemmae, nuclei, and chloroplasts; the supernatant contained mitochondria, lysosomes, peroxisomes, the cytosol, the endoplasmic reticulum, ribosomes, and Golgi complex (Sheelcr 198 1). The cellular components present in the supernatant are considered here as the cytoplasmic fraction. Both fractions were assayed for radioactivity. Because surface-bound 09Cd and 57Co rapidly desorbed into the distilled water after cell breakage, noncytoplasmic lo9cd and 57Co were quantified as the radioactivity washed off cells with a lo-ml rinse of a sterile-filtered seawater solution of 10-3 M EDTA (for 2 min) in addition to the radio-.

4 Bivalve larvae assimilation 15 Zn -A Am Algal growth rate (divisions d-l) Fig. 1. Comparison of the percent of total Ag, Am, C, Cd, Co, P, Se, Zn, and protein in the cytoplasmic fraction of Isochrysis galbana and the ccl1 division rate. activity measured in the pelletized fraction. Protein concentrations were determined in the subcellular fractions of I. galbana cells by the BCA method (Smith et al. 1985). The gamma-emitting isotopes were measured with a Pharmacia-Wallac LKB gamma counter equipped with a well-type NaI(T1) crystal. The gamma emissions of l OrnAg were detected at 658 kev, of 241Am at 60 kev, of 57Co at 122 kev, of 75Se at 264 kev, and of 5Zn at 1,115 kev. The amount of *O Cd in samples was quantified by detecting X-ray emissions at 22 kev. The beta emitters ( C, 32P, and 35S) were measured with an LKB Rack Beta liquid scintillation counter. Quenching of beta-emitting samples was corrected with the external standards ratio method. Counting times were adjusted so that propagated counting errors were (5%. Results The fractionation of different elements in the cytoplasm of uniformly radiolabeled I. galbana cells ranged from 11% for Am to 94% for Se (Table 2). With the exceptions of S, Cd, and Zn, nonmetals were more enriched in the cell s cytoplasm than were the metals (Table 2). The proportion of total Zn in I. galbana cells associated with the cytoplasmic fraction ranged from 39 to 89%, with the percentage of cytoplasmic Zn higher in slower growing cells (Fig. 1). Similar inverse relationships between the proportions of elements in the cytoplasmic fraction of I. galbana cells and log Percent of total protein in each cellular fraction Fig. 2. Relationship between the percent of total cellular 9 and the pcrccnt of total cellular protein in subcellular fractions of Zsochrysis galbana from replicate cultures. Pl (Cl), pellet after centrifugation at 750 x g; P2 (A), pellet after centrifugation at 2,000 x g; S2 Q, supcrnatant after centrifugation at 2,000 x g. linear cell division rates were observed for C, Cd, P, and Se (Fig. 1). The percent of total cellular protein in the cytoplasmic fraction of I. galbana cells was 83% in cells growing at 0.23 div. d- I, 70% in cells growing at 0.50 div. d-- l, and 440/ o in cells growing at 1.12 div. d-l. The cellular fractionation of S was directly relatcd to that of protein (Fig. 2), so the amount of total cellular S in the cytoplasmic fraction of I. galbana cells probably also varies with culture age. Ag, Am, and Co were predominantly associated with the cell membrane fraction of I. galbana (Table 2), and the cellular distribution of these metals did not change markedly with algal growth rate (Fig. 1). Assimilation efficiencies of the nine elemcnts examined ranged from 7.9 and 4.6% for Am to and 100.1% for Se in the larvae of oysters and hard clams, respectively (Tables 3 and 4). Although the nonmetals C, P, and Se were assimilated by both oyster and clam larvae with efficiencies >50%, S was assimilated in both bivalve larvae with an efficiency of only N 35% (Tables 3 and 4). Se assimilation efficiencies in oyster larvae based on the mass balance method (105.4*5.8%, Table 3) were in good agreement with that determined by the ratio method ( %) using 75Sc : 241Am ratios in I. galbana cells and larval egesta of 2.72 and , respectively. The metals Ag,

5 16 Reinfelder and Fisher Table 3. Oyster larvae (Crassostrea virginica) fed labeled Zsochrysis galbana: number of ingested phytoplankton cells, radioactivity ingested and retained, and assimiliation effxiencies (AE). Radioactivity (Bq liter ) Radio- Cells ingcstcd AE* tracer (cells liter ) ingested rctaincd PM Otl Ag 5.77 x 10 1, Arn 1.20x IOx 1, kO.5 14C 1.58 x IO )Cd 1.56x f 5.77 x k3.4 5 Co 3 -P x 10 3,500 2, k x lk2.1 5s 2.23 x x &6.0-f?Se 1.20x 10 2,530 2, k5.8?Zn 1.20x 10X 2,020 1, k4.8 * Plus or mmus propagated error. j Mean AE calculated in replicate cxpcrimcnts. f 1 SD. Am, and Co were assimilated by the bivalve larvae with efficiencies ~33% (Tables 3 and 4), but significantly higher assimilation efficiencies of the group IIb metals Cd (6 1.3%) and Zn (79.3%) were measured (Tables 3 and 4). The assimilation efficiencies of all elements in the bivalve larvae were directly related (r* = 0.926) to the proportion of each in the cytoplasmic fraction of ingested phytoplankton food (Fig. 3). The slope of the curve describing this relationship (1.08 f0.076) was not significantly different from 1, and the intercept (-5.8k8.3) w as not significantly different from zero. Discussion The extent to which different elements were compartmentalized in the cytoplasmic fraction of I. galbana and assimilated in bivalve larvae varied over almost the entire range of possible values (Fig. 3; Tables 2-4). Generally, the nonmetals examined (C, P, S, Se) had a greater proportion of their total cellular content in the cytoplasmic fraction of phytoplankton cells than did the metals (Table 2). Similarly, assimilation efficiencies in zooplankton were higher for nonmetals than for metals (Tables 3 and 4). The different behavior of metals and nonmetals reflects the different modes of accumulation and metabolism characteristic of the two groups of elements. C, P, and S (major components of proteins, lipids, carbohydrates, and nucleic acids) are actively in- Table 4. As Table 3, but for hard clam (Mercenaria mercenaria) larvae. Radioactivity (Bq liter ) Radio- Cells ingcstcd AE* tracer (cells liter ) ingcstcd retained w I IOmA 241/j; 3.74 x 10 4, x 10 3, C 3.41 x Cd 3.57 x k2.7.f 3.74 x co 3.74 x k P x 106 2,450 1, YS 3.37 x x t- 1.7-t 5Se 1.96x x k2.4 5Zn 1.09 x 108 4,430 1, k2.3 * Plus or minus propagated error. f Mean AE calculated in replicate cxpcrimcnts, + 1 SD. corporated into nascent biomolecules by phytoplankton. The fractionation of these three elements in phytoplankton should therefore reflect that of the biochemicals with which they are associated. This pattern of fractionation was evident for S, whose subcellular distribution in I. galbana closely follows that of protein (Fig. 2), as was also shown for T. gseudonana (Fisher and Reinfelder 199 1). The metalloid Se, an essential micronutrient for diverse species of marine (Price et al. 1987; Harrison et al. 1988) and freshwater (Lind- Strom 1983) phytoplankton, is expected to behave analogously to S in biota (Wrench 1978; Wrench and Campbell 198 1). The subcellular fractionation of Se, however, did not match that of protein or S in diatoms (Fisher and Reinfelder 199 1; Reinfelder and Fisher 199 1) or I. galbana (Fig. 1; Table 2). More than 90% of the cellular Se was detected in the cytoplasmic fraction of these phytoplankton cells, possibly as hydrogen selenide, small soluble peptides, or free seleno-amino acids (Wrench 1978). High levels of Se have also been observed in the soluble fractions of the chlorophytes Dunaliella tertiolecta (73% soluble) and ChZorella sp. (98% soluble) (Bottino et al. 1984). The incorporation of Se into small, soluble organic compounds seems, therefore, to be a common feature of many phytoplankton spe- cies. Such small molecular weight compounds may be readily leached from phytoplankton and sinking particulate matter (Lee and Fisher 1992, 1993). Because the reoxidation of selenides in seawater to selenite or selenate is a

6 Bivalve larvae assimilation 17 slow process (Cutter and Bruland 1984) compared with phytoplankton production, dissolved organic selenides should accumulate in the water column, which is consistent with the chemical speciation of Se in coastal (Cutter 1989) and oceanic (Cutter and Bruland 1984) waters. The compartmentalization of trace metals in phytoplankton cells may be controlled by the mode of accumulation. The accumulation of trace metals by phytoplankton can be mediated by carrier compounds, membrane transport channels, and intracellular ligands (Williams 198 1; Gekeler et al ) or may occur through passive processes such as adsorption onto cell surfaces (Fisher 1986). Essential metals are actively accumulated by phytoplankton and are generally expected to exhibit greater penetration into a cell s cytoplasm than would nonessential metals that adsorb to cell surfaces. Thus, a greater proportion of total cellular Zn, an essential enzyme cofactor, was found in the cytoplasm of I. galbana than the nonessential metals Ag and Am. Not all nonessential metals, however, are restricted to accumulation on phytoplankton cell surfaces. The percent of total cellular Cd found in the cytoplasmic fraction ranged from 44 to 58% (Table 2) and, like that of Zn, varied inversely with algal growth rate (Fig. 1). The accumulation of Cd in phytoplankton cells, though incidental, may be an actively regulated process that is influenced by cell physiology. The observation that Cd can support the growth of the marine diatom Thalassiosira weissflogii, apparently by substituting for Zn in a midmolecular weight compound in the cell s cytosol (Price and Morel 1990), is consistent with the accumulation of Cd in the cytoplasm of I. galbana. The incidental incorporation of certain metals like Cd and PO (Fisher et al. 1983), but not others, into algal cytoplasm suggests that essential metal accumulation pathways in phytoplankton operate with limited specificity. Bivalve larvae appear to use a digestion strategy similar to that proposed for copepods (Reinfelder and Fisher 199 1) in which only the easily mobilized, cytoplasmic fraction of ingested phytoplankton is assimilated. Assimilation of only the cytoplasmic fraction of ingested phytoplankton is consistent with the digestive physiology of bivalve larvae. Once Percent of each element in the cytoplasm of Isochrpis galbana or Thalassiosira pseudonana Fig. 3. Assimilation efficiencies (%) of ingested clcmcnts in oyster (m and hard clam (A) larvae fed Zsochrysis gulbana compared with the percentages of those elements in the cytoplasmic fraction of the phytoplankton food 0, = 1.08x , r2 = 0.926). For comparison, data for copepods (0) fed the diatom Thalassiosira pseudonana (from Rcinfelder and Fisher 199 1) are also shown 01 = 1.13x , I.2 = 0.977). in the larval gut, food is exchanged between two digestive diverticula which are the sites of assimilation (Millar 195 5). Regular muscular contractions (5-22 min- ) control the flow of material in and out of the digestive diverticula (Millar 195 5), making the passage of food through bivalve larval guts rapid. Short gut transit times minimize the time available for assimilation of more than the easily mobilized portion of ingested phytoplankton (i.e. the cytoplasm). The striking difference in the larval assimilation of trace elements (particularly Cd, Se, and Zn) from T. pseudonana and I, galbana is consistent with observations that monocultures of diatoms (including T. pseudonana) are good diets for juvenile animals (after larval settlement), but larvae typically favor small flagellates such as I. galbana (Walne 1970; Urban and Langdon 1984; Enright et al. 1986). For example, the diatom Skeletonema costatum is an excellent food for oyster juveniles (Walne 1970) and a poor food for larvae of the same species (Ostrea edulis) which favor flagellates, including I. galbana (Ferreiro et al. 1990). Consequently, hatcheries commonly use diets of small flagellates for bivalve larvae, either alone or sometimes in combination with diatom species. Significant amounts of Cd, Se,

7 18 Reinfelder and Fisher and Zn are associated with the readily assimilated cytoplasmic fraction of T. pseudonana (Reinfelder and Fisher 199 l), so the extremely low assimilation of these elements from T. pseudonana noted in the preliminary experiments would indicate a low assimilation of organic matter, which may account for the poor nutritional value of this diatom for larvae. If copepods and bivalve larvae assimilate only the cytoplasmic fraction of their food (Fig. 3), then elements that are actively transported to the interior of phytoplankton cells will be accumulated by zooplankton while elements on phytoplankton cell surfaces will not. C, Cd, P, Se, and Zn, which were enriched in the cytoplasmic fraction of phytoplankton cells, were assimilated with higher efhciencies in copepods (Reinfelder and Fisher 199 1) and bivalve larvae (Tables 3 and 4) than were Ag, Am, and Co, which were concentrated on cell surfaces. Higher trophic transfer efficiencies of elements enriched in the cytoplasmic fraction of phytoplankton food may result in accumulation by herbivorous zooplankton of trace metals such as Cd, which can be toxic at elevated concentrations. This mechanism of assimilation may also explain why 210Po, which can penetrate into algal cell cytoplasm (Fisher et al. 1983), is highly enriched in marine invertebrates, including herbivores (Cherry et al. 1983). The percentages of cellular C, Cd, P, Se, Zn, and protein in the cytoplasm of 1. galbana were higher in slower growing phytoplankton than in rapidly dividing, log-phase cells (Fig. l), so bivalve larvae should assimilate more of these elements and protein from each senescent cell than from each rapidly dividing cell. This finding is consistent with the observation that oyster larvae had increased growth when they were fed flagellates (Tetraselmis maculata) whose growth was slowed by vitamin deficiency (Ukeles and Wikfors 1988) and that juvenile oysters fed stationary-phase diatoms (T. pseudonana) grew somewhat faster than animals fed diatoms in exponential growth (Flaak and Epifanio 1978). To obtain the same amount of nutrient elements and protein, bivalve larvae feeding on younger, rapidly dividing phytoplankton need to eat more cells than larvae feeding on older senescent algae. Since bivalve larvae exhibit the same pattern of assimilation observed in copepods feeding on a very different species of phytoplankton (Fig. 3) a functional group of small suspension-feeding marine animals may exist that maximizes clearance rates at the expense of having to egest the noncytoplasmic portion of ingested phytoplankton which can contain significant amounts of essential elements and protein (Fig. 1; Table 2). Such animals would, like bivalve larvae and many herbivorous copepods, be expected to have simple digestive tracts and very short gut residence times. By contrast, adult oysters and hard clams, which have morphologically and physiologically more complex digestive systems than bivalve larvae, have been found to assimilate more or less than the easily mobilized, cytoplasmic fraction of some trace metals from ingested phytoplankton cells (Fisher and Reinfelder in press). Mature oysters and hard clams have much longer gut residence times (2 12 h) than bivalve larvae and consequently appear to have greater control over the process of assimilation. Enzymatic and physical breakdown of the more refractory portions of ingested phytoplankton cells (e.g. cell membranes, large organelles) may be readily achieved by adult bivalves in 12 h. Metals that tend to accumulate on phytoplankton cell surfaces typically include highly particle-reactive nonessential metals such as Pb (Fisher et al. 1983; Michaels and Flegal 1990), Am (Table 2; Fisher et al., 1983; Reinfelder and Fisher 199 l), and Ag (Table 2). The cytoplasmic fractions of Ag and Am in I. galbana were relatively low (Ag, - 20%; Am, 11 O/o) and did not vary appreciably with culture age (Fig. l), suggesting that the association of these nonessential metals with phytoplankton is largely the result of abiotic processes. The cellular fractionation of the essential trace metal Co was also largely unaffected by phytoplankton culture age in I. galbana (Fig. l), possibly indicating an abiotic uptake pathway. Phytoplankton may not be able to use Co ion that is not associated with cobalamin, as shown for diatoms (Nolan et al. 1992). The accumulation of Ag and Am by marine plankton is comparable to that of Pb which is largely associated with the surfaces of both phyto- and zooplankton (Michaels and Flegal 1990). Michaels and Flegal(l990) showed that in marine organisms with ratios of surface area to volume >O.O I pm2 : pm3, which include most of the plankton, Pb concentrations are overwhelmingly a func-

8 Bivalve larvae assimilation 19 tion of an organism s surface area. Given this model, Pb and other highly particle-reactive metals are expected to associate with zooplankton exoskeletons and the surfaces of phytoplankton cells that are not assimilated in herbivorous zooplankton. The accumulation of Pb and other nonessential particle-reactive metals in marine herbivorous zooplankton from ingested food would therefore appear to contribute only a small portion of the total amounts of these elements to these animals. References BOTTINO, N. R., AND OTHERS Selenium containing amino acids and proteins in marine algae. Phytochemistry 23: BRAND, L. E., R. R. L. GUILLARD, AND L. S. MURPHY A method for the rapid and precise determination of acclimated phytoplankton reproduction rates. J. Plankton Res. 3: CHERRY, R. D., M. HEYRAUD, AND J. J. W. HIGGO Polonium-2 10: Its rclativc cnrichmcnt in the hepatopancreas of marinc invertebrates. Mar. Ecol. Prog. Ser. 13: CUTTER, G. A The estuarine behavior of selenium in San Francisco Bay. Estuarine Coastal Shelf Sci. 28: , AND K. W. BRULAND The marine biogeochemistry of selenium: A re-evaluation. Limnol. Oceanogr. 29: 1179-l 192. ENRIGHT, C. T., G. F. NEWKIRK, J. S. CRAIGIE, AND J. D. CASTELL Evaluation of phytoplankton as diets for juvenile Ostreu edulis L. J. Exp. Mar. Biol. Ecol. 96: FERREIRO, M. J., AND OTHERS Changes in the biochemical composition of Ostrea edulis larvae fed on different food regimes. Mar. Biol. 106: FISHER, N. S On the reactivity of metals for marinc phytoplankton. Limnol. Oceanogr. 31: , K. A. BURNS, R. D. CHERRY, AND M. HEYRAUD Accumulation and cellular distribution of 24 Am, 2 0Po, and 210Pb in two marine algae. Mar. Ecol. Prog. Ser. 11: , AND J. R. REINFELDER. 1991, Assimilation of selenium in the marine copepod Acartia tonsa studied with a radiotracer ratio method. Mar. Ecol. Prog. Ser. 70: 157-l 64. AND J. R. REINFELDER. In press. The trophic transfer of metals in marine systems. In D. R. Turner and A. Tessier [eds.], Metal speciation and bioavailability. Lewis. FLAAK, A. R., AND C. E. EPIFANIO Dietary protein levels and growth of the oyster Crussostrea virginica. Mar. Biol. 45: GEKELER, W., E. GRILL, E.-L. WINNACKER, AND M. H. ZENK Algae scqucster heavy metals via synthesis of phytochelatin complexes. Arch. Microbial. 150: GUILLARD, R. R. L., AND J. H. RYTHER Studies of marine planktonic diatoms 1. Cyclotelfa nana Hus- tcdt, and Detonula conjkvuceu (Clcve) Gran. Can. J. Microbial. 8: HARRISON, P. J., P. W. Yu, P. A. THOMPSON, N. M. PRICE, AND D. J. PHILLIPS Survey of selenium requircments in marine phytoplankton. Mar. Ecol. Prog. Ser. 47: s LEE, B.-G., AND N. S. FISHER Degradation and elemental release rates from phytoplankton debris and their geochemical implications. Limnol. Oceanogr. 37: 1345-l , AND Rclcase rates of tract elcments and protein from decomposing planktonic debris. 1. Phytoplankton debris. J. Mar. Res. 51: LINDSTR~M, K Selenium as a growth factor for plankton algae in laboratory experiments and in some Swedish lakes. Hydrobiologia 101: LOWMAN, F. G., T. R. RICE, AND F. A. RICHARDS Accumulation and redistribution of radionuclidcs by marine organisms, p In Radioactivity in the marinc environment. Natl. Acad. Sci. LUOMA, S. N., AND OTHERS Determination of selenium bioavailability to a benthic bivalve from particulatc and solute pathways. Environ. Sci. Tcchnol. 26: MJCHAELS, A. F., AND A. R. FLEGAL Lead in marine planktonic organisms and pelagic food webs. Limnol. Oceanogr. 35: MILLAR, R. H Notes on the mechanism of food movement in the gut of the larval oyster, Ostrea edulis. Q. J. Microsc. Sci. 96: NOLAN, C., S. W. FOWLER, AND J.-L. TEYSSI~ Cobalt speciation and bioavailability in marine organisms. Mar. Ecol. Prog. Ser. 88: 105-l 16. PRICE, N. M., AND F. M. M. MOREL Cadmium and cobalt substitution for zinc in a marine diatom. Nature 344: , P. A. THOMPSON, AND P. J. HARRISON Selenium: An essential element for growth of the coastal marine diatom Thalassiosiru pseudonana (Bacillariophyceae). J. Phycol. 23: 1-9. REINFELDER, J. R., AND N. S. FISHER The assimilation of elements ingested by marine copepods. Science 251: SHEELER, P Centrifugation in biology and medical science. Wiley. SMITH, P. K., AND OTHERS Measurement of protcin using bicinchoninic acid. Anal. Biochcm. 150: THOMANN, R. V Equilibrium model of fate of microcontaminants in diverse aquatic food chains. Can. J. Fish. Aquat. Sci. 38: UKELES, R., AND G. H. WIKFORS Nutritional value of microalgae cultured in the absence of vitamins for growth of juvenile oysters, Crassostrea virginica. J. Shellfish Res. 7: 38 l-387. URBAN, E. R., AND C. J. LANGDON Reduction in costs of diets for the American oyster Crassostrea virginica (Gmelin), by the use of non-algal supplements. Aquaculture 38: WALNE, P. R Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria, and Mytilus. Fish. Invest. (Ser. 2) 26: l-62.

9 20 Reinfelder and Fisher WHITFIELD, M., AND D. R. TURNER The role of phytoplankters Tetmselmis tetrathele and Dunaliella particles in regulating the composition of seawater, p. minuta. Mar. Biol. 49: Zn W. Stumm [ed.], Aquatic surface chem- -, AND N.C. CAMPBELL Protein bound seistry: Chemical processes at the particle-water inter- lenium in some marine organisms. Chemosphere 10: face. Wiley WILLIAMS, R. J. P Physico-chemical aspects of inorganic element transfer through membranes. Phil. Submitted: 15 March 1993 Trans. R. Sot. Lond. Ser. B 294: Accepted: 1 July 1993 WRENCH, J. J Selenium metabolism in the marine Amended: 23 August 1993