The release of trace elements by dying marine phytoplankton

Deep-SeaResearch1, Vol 40, No 4, pp 671~o94,1993 Printedm GreatBntam

0967--0637/93$6 00 + 0 00 © 1993PergamonPressLtd

The release of trace e l e m e n t s NICHOLAS S. FISHER*

by dying marine phytoplankton and MARYANNWENTE*

(Recetved 17 January 1992; m revtsed form 27 May 1992; accepted 10 June 1992)

Abstract--The extent to whtch stoking phytoplankton can d~rectly influence the cychng and vertical transport of metals m the oceans depends largely on the loss rates of the metals from dying cells dunng their descent Thts was examined m a series of ra&otracer experiments in which a diatom, a dmoflagellate, and a coccolithophore each accumulated Se, Ag, Sn, Au and Am and then were maintained m the dark for up to 10 days to assess depuraUon kinetics Concurrent measurements were made of cell counts and particulate C, N and dry wetght. Se was taken up acUvely by the cells, Sn and Am passively, and Ag and Au uptake patterns varied with the species The coccolithophores had less reactive surfaces for these metals than did the other species, as reflected m lower metal uptake and greater metal release. Generally, those metals with greatest parUcle affimty dunng uptake (Am, Sn, Ag) were retained for the longest periods All cells decomposed over ume, so that particulate C and N decreased by up to 10.7 and 9.8% day -1, respectively, m dmtom cultures, up to 4 7 and 4.9% day-1 m dmoflagellate cultures, and up to 6.5 and 6.0% day -1 m coccolithophore cultures. In the dmtoms, all elements except Se were released more slowly than C and N from the particulate phase, resulting m increasing metal:C (or N) ratios wath time, while Se was released faster than C or N With the dmoflagellates, elemental release generally followed C and N release, while with the coccohthophores elements were released more rapidly than C and N from the particulate phase Metals (especaally Am, Ag, Sn) were retained sufficiently long, even by decomposing cells, to suggest that phytoplankton stoking as aggregates at rates of 100 m day -1 would effectively transport these metals hundreds of meters out of oceamc surface waters.

INTRODUCTION

THE oceanic flux of particle-reactive elements is strongly influenced by sinking biogenic debris (CHERaV et al., 1978; COLLIERand EDMOND, 1984; FOWLER and KNAUER, 1986; FOWLERet al., 1983, 1987; MURRAYet al., 1989; WnrrrmLD and TURNER, 1987). Both field studies and modeling studies have demonstrated that the flux of these elements may be particularly influenced by zooplankton (BISHOP et al., 1986; FISHER and FOWLER, 1987; COALEand BRULAND,1987) and that vertical flux from the euphotic zone of such particlereactive elements as Th and Pb is coupled to new production (BRULANr)and COALE,1986; FISHERet al., 1988; MOOREand DYMOND, 1988). The studies of BILLETet al. (1983), SMETACEK(1985), TAKAHASHI(1986), ALLDREDGE and GOTSCHALK(1989), PASSOW (1991), and others have shown that sinking phytoplankton aggregates at times may dominate the mass of downward sinking particles in

*Manne Sciences Research Center, State Umvers~ty of New York, Stony Brook, NY 11794-5000, U.S A. 671

672

N S FISHERand M WENTE

many regions. While several studies have focused on the fate of metals bound to zooplankton debris (especially fecal pellets), few have examined the long-term retention of metals by phytoplankton, particularly of dying cells undergoing decomposition. Such studies may provide quantitative assessments of the impact of such debris on the vertical transport of particle-reactive metals in the oceans. Moreover, since most of the particulate organic carbon (POC) sinking out of the euphotic zone typically decomposes above 1000 m (HARGRAVE, 1985), it is important to relate the retention of metals by particles with that of the organic matrix which carries the metals. By analysing the retention of particulate C (PC) and PN together with the metals, it is possible to determine whether the release of metals simply follows that of the particulate organic matrix itself. This report describes a series of radiotracer experiments to determine the rates of metal release from dying phytoplankton cells held in culture. Three types of phytoplankton were compared--diatoms, dinoflagellates, and coccolithophores---each of which were examined for five metals/metalloids--Se, Ag, Sn, Au and Am. Trace elements were chosen to allow comparisons of non-essential and essential elements and class A and class B metals. Of the elements examined, only Se has been shown to be essential to phytoplankton (PRICE et al., 1987). Se associates with proteins and small organic compounds in the cytoplasm of cells (FISHER and REINFELDER, 1991). Se exists in seawater as selenite, selenate, and organic selenides and displays nutrient-type vertical profiles (MEASURESand BURTON, 1980; CUrrER and BRULAND, 1984), suggesting involvement in the biological cycle. Ag and Au are Group IB class B metals (affinity for S > N > O), and might be expected to associate with S-containing compounds (primarily protein) in the cells. Both are accumulated by algae and associate with S-containing molecules, although Au uptake has not been reported in marine phytoplankton (FISHERet al., 1984; GREENEet al., 1986). Ag displays nutrient-type vertical profiles in the ocean (MARaTN et al., 1983); Au concentrations appear to be uniform with depth, although some profiles suggest surface depletion (FALKNERand EDMOND, 1990). By contrast, Sn and Am are particle-reactive, class A metals (affinity for O > N > S) (NIEBOERand RICHARDSON, 1980), associate with inorganic phases (e.g. diatom frustules) on cell surfaces (FISHERet al., 1983b, 1986), and display scavenging-type vertical profiles in the ocean (WHITFIELDand TURNER, 1987).

MATERIALS AND METHODS

Metal accumulation Unialgal, clonal cultures of the small centric diatom Thalassiosira pseudonana (clone 3H), the coccolithophore Emiliania huxleyi (clone MCH No. 1), and the dinoflagellate Prorocentrum minimum (clone EXUV) were maintained in f/2 medium (GuILLARD and RrraES, 1962) prepared with sterile-filtered (passed through sterilized 0.2 Nuclepore polycarbonate membranes) surface seawater (35 ppt) collected 5 miles off Southampton, NY. Cultures were incubated at 18 + I°C under 200/~Ein m -2 s -1 cyclic illumination (14:10, L:D) provided by cool-white fluorescent lamps. Log-phase cells were removed from their media for metal uptake studies by centrifugation (E. huxleyi) or by filtration using a sterilized 1.0 /~m Nuclepore filter (T. pseudonana and P. minimum), and resuspended into 1 1 (500 ml for E. huxleyi) of unenriched sterile-filtered Southampton seawater held in sterilized 1-1 flasks with Teflon-coated screw caps. Metal uptake by T. pseudonana and by P. minimum was measured for both living and dead cells in separate

Release of trace elements by dying phytoplankton

673

flasks. Comparison of living and dead cells enables an assessment of the influence of metabolic activity on the bioaccumulat~on and retention of metals in the cells. Aliquots of both species were heat-killed in a 50°C water bath, 5 min for T. pseudonana and 15 min for P. mmtmum; dead cells of both species remained intact for the duration of the metal uptake experiments. Heat-killed E. huxleyt cells did not stay intact, and therefore only live cells of this species were examined. Equal densities of living and dead cells were tested. The cell densities employed were chosen to produce initial suspended particle loads of approximately 1-20 mg dry wt 1-1; over the course of the uptake period, the maximum load reached 35.6 mg 1-1 (Table 1). Epifluorescence microscopy revealed bacterial contamination of all cultures. Gamma-emitting radioisotopes of Se, Ag, Sn, Au and Am were added in mlcroliter quantities to the flasks, to yield the final molar additions given in Table 1. The isotopes used were: 75Se (/1/2 = 120 days), ll°mAg (tl/2 = 250 days), 1135n (tl/2 = 115 days), 195Au (q/2 = 186 days) and 241Am (tl/2 = 433 years). The 75Se (obtained from Amersham) was added as selenite in 0.5 N Ultrex HCI, the 11°mAg (Amersham) was in 0.1 N Ultrex HNO3, the 113Sn (from New England Nuclear) was added as hexachlorostannic acid in 4 N Ultrex HC1, the 195Au (New England Nuclear) was in 4 N Ultrex HCI, and the 241Am (Amersham) was in 3 N Ultrex HNO3. (Appropriate microliter amounts of dilute Suprapur (Merck) NaOH were added to the cultures immediately prior to radioisotope additions so that, after addition of the acidic solutions, the pH of the water was 8.0.) The cultures received 74 kBq 1-1 of 75Se (for all species), 37 kBq 1-1 of 11°mmg for T. pseudonana and P. minimum and 74 kBq 1-1 for E. huxleyi, 74 kBq 1-1 of ll3Sn for all species, 74 kBq 1-1 of 195Au for T. pseudonana and P. minimum and 148 kBq 1-1 for E. huxleyi, and 74 kBq 1-1 of 241Am for all species. The 755e and 241Am were added in combination, the 1135n and 195Au were added as a separate combination, and the ll°mAg was added by itself. Combinations were chosen on the basis of analytical compatibility using an LKB Compugamma automated gamma counter equipped with a well-type NaI(TI) crystal and LKB's Ultroterm software package, which accounted for spillover effects of one isotope into another's analytical "window." For counting the radioactivity of the samples, the photon emissions of 75Se were detected at 265 keV, of ll°mAg at 658 keV, and of 241Am at 60 keV. llasn decays by electron capture to 113In, which, like ll3Sn, has a gamma emission at 392 keV and which has a half-life of 99 min. Since the 113Sn stock solution obtained from New England Nuclear also contains 113In, it was necessary to wait seven 113In half-lives (i.e. 11.6 h) before counting 1135n samples, to ensure that > 99% of the 113In in the sample remaining was that which was in radioactive equilibrium with the accumulated 113Sn. 195Au decays by electron capture to 195pt, whose Kal X rays were detected at 67 keV. All samples were contained in plastic counting tubes and had the same counting geometries; counts were corrected for decay with appropriate standards and counting times were adjusted to give propagated counting errors <5%. Parallel cultures, treated identically, were exposed to equimolar concentrations of stable metals (analytical grade metal salts, prepared for use as AAS standards, were used); these cultures were later sampled for CHN analyses. Periodically, metal uptake by the cells (living and dead) was determined using an established protocol (FISHER et al., 1983a). Briefly, cultures were swirled vigorously to produce a homogeneous suspension and 10 ml from each culture was gently filtered (<20 kPa) onto a 1 pm Nuclepore filter, rinsed with 10 ml of filtered non-radioactive seawater,

Metal

Ag Sn & Au Se & Am

Ag Sn & Au Se & Am

Ag Sn & Au Se & Am

Ag Sn & Au Se & Am

Ag Sn&Au Se & Am

Clone

3H-hve

3H-dead

EXUV-live

EXUV-dead

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10.5 1.2 & 0 004 0.3 & 2.4

10.5 1.2 & 0.004 0 3&2 4

10 5 1.2 & 0.004 0.3 & 2.4

10.5 1.2 & 0 004 0.3 & 2 4

Metal added (nM)

88 88 88

110 110 110

110 110 110

112 112 112

112 112 112

Duration of uptake (h)

3.2 4.1 35

6.0 4.9 4.4

42 59 3.8

36 5.4 50

36 42 35

Cell denstty (107 cells l -l)

1.6 2 1 1.8

22.2 18.2 16 3

15.6 21.9 14 1

08 12 1.1

08 09 08

Cell dry wt (mg l -I)

Start of uptake

4.6 59 50

64 3 52 5 47.2

45.0 63 3 40 7

22 3.3 3 1

22 2.6 2 1

Cell volume (mm 31-1)

36 2 19 8 43.5

5.2 5.1 4 1

85 7.4 96

3.9 30 44

31 4 32.2 31 4

Cell density (107 cells 1-1)

18.1 9.9 21 8

19 3 18 9 15.2

31.5 27.5 35.6

0.9 0.7 1.0

7.0 7.2 70

Cell dry wt (mg l- l )

End of uptake

52.1 28 5 62 6

55 7 54 7 44 0

91 1 79 3 102 9

24 18 27

19 2 19 6 19.2

Cell volume (mm 3 1-1)

Table 1 Expertmental condittons durmg uptake o f metals Ag denotes cell grown tn presence o f Ag, Sn & Au denotes cells grown wtth tm and gold, Se & Am denotes cells grown wtth selemum and amertcmm. 3H: Thalassiosira pseudonana, EXUV: Prorocentrum minimum, MCHI: Emdlama huxleyl

Oq

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Release of trace elements by dyingphytoplankton

675

and placed into a separate counting tube for radioactive counting. At the same time, 1 ml was removed from each culture and counted without filtering, to determine the total radioactivity in the water (i.e. dissolved plus particulate) at each sample time. Aliquots (1 ml) were also fixed with 5% Lugol's solution and cell numbers were counted microscopically with a hemacytometer. Thus, at each sample time, the fraction of total "water column" radioactivity for each radioisotope associated with suspended particulate matter was determined, and was normalized on a cell, cell volume or cell dry wt basis.

Metal retention Following the uptake period, cells of all species were transferred to non-radioactive water to determine the cellular retention of the various metals and measure the rates and patterns of metal release. To accomplish this, T. pseudonana and P. minimum cells were removed from each uptake flask by resuspension off sterile 1 /zm Nuclepore filters, whereas E. huxleyi cells, which could not be quantitatively resuspended off filters, were removed by centrifugation (9000 g). Cells of all species were resuspended into unenriched, non-radioactive, sterile-filtered seawater (from the same batch of seawater as the water used for metal uptake) so that the cell density for each culture was not different from that at the end of the uptake phase (Table 1). The contents of each flask (resuspended radioactive cells in non-radioactive seawater) were then divided into four equal batches, all contained in separate sterilized flasks with Teflon-coated screw caps. Half the flasks were incubated in the dark at 18°C, while the other half were in the dark at 8°C (that is, two replicate flasks per temperature). Resuspended non-radioactive cells, from the parallel cultures exposed to stable metals, were incubated in the dark at 18°C only. The coefficient of variation of the data from replicate flasks was -<6% for all isotopes. Periodically, aliquots of the radioactive cells were taken and examined for their radioactivity with the same protocol used for measuring metal uptake; however, with time, larger sample volumes were taken to compensate for loss of radioactivity, to ensure sufficient radioactivity in the filtered samples for accurate measurements. During this depuration period, intact cells also were enumerated as above; cell debris was not counted as an intact cell. Aliquots of the non-radioactive cells were also periodically removed by filtration onto precombusted Whatman GF/C glass fiber filters, which were subsequently analysed for C and N content with a Perkin-Elmer CHN elemental analyser. Dry wt measurements of suspended particulate matter were made using both 0.2 and 1.0/zm Nuclepore filters after washing the filtered cells with isotonic ammonium formate to remove salts and drying at 60°C. RESULTS

Metal accumulation During the metal uptake period, there was appreciable cell growth in all cultures containing living cells, despite the lack of added nutrients to the seawater; cell densities in dead cultures remained essentially constant over time (Table 1). There was no evidence of metal toxicity in any of the cultures. Cells numbers (or equivalent dry wt or cell volume) increased about 10-fold in living T. pseudonana and E. huxleyi cultures, and about twofold in the P. minimum culture. The fraction of total metal associated with cells increased with time as well, although not always with the same pattern for all species (Figs 1-5). In

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Fig. 1. Accumulation over time of 75Se in Thalasslos~ra pseudonana (3H), Prorocentrum mtmmum ( E X U V ) and Emdiama huxley, (MCH-1). Data are presented as percentage of total 75 Se in the water associated with the cells and as amoles (10- is moles) cell- 1 over time. (11) L~ving cells, ([]) dead cells

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Fig. 2 Accumulation over time of 11°mAg m Thalasslos,ra pseudonana (3H), Prorocentrum minimum (EXUV) and Em,hanta huxleyl (MCH-1) Data are presented as percentage of total 11°mAg in the water associated with the cells and as amoles (10 -18 moles) cell-I over time (11) Lwing cells, (D) dead cells

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Accumulation over ume of ll3Sn in Thalasswszra pseudonana (3H), Prorocentrum minzmum ( E X U V ) and Emihania huxleyz (MCH-1). Data are presented as percentage of total Fig. 3.

lI3Sn in the water associated wRh the cells and as amoles (10-18 moles) cell-z over time. ( I ) Living cells; ([:]) dead cells.

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Release of trace elements by dying phytoplankton

681

Table 2 Metal concentranon factors, on volume ( VCF) and dry weight (DCF) bases, computed at the end of uptake period for each algal spectes 3H" Thalassloslra pseudonana, EXUV Prorocentrum mlmmum, MCH1 Emdlania huxley1

Metal

VCF ( × 104)

DCF ( × 104)

3H-hve

Se Ag Sn Au Am

06 62 2 1 03 94

16 16 9 5.7 08 25 6

3H-dead

Se Ag Sn Au Am

0 1 08 14 0 08 20 9

03 22 38 1 22 56 9

EXUV-live

Se Ag Sn Au Am

0.4 3.9 71 18 81

12 11 3 20 5 52 23 4

EXUV-dead

Se Ag Sn Au Am

0 04 22 30 01 99

0 1 64 87 03 28 6

MCHl-hve

Se Ag Sn Au Am

0 05 0.5 0.4 02 0.4

0 1 1.4 1.2 06 1.2

Clone

part, the increase in total particulate metal reflected cell growth (in living cultures), which resulted in increased particulate surface area to which the metals could bind. Moles of metal accumulated per cell at each sample time were calculated using the specific activity of the radioisotopes, the particulate fraction of radioisotope, and the cell density (Figs 1-5). For those elements which were passively accumulated (see below), the molar concentration of metal cell-1 generally leveled off or peaked sooner than did the total particulate fraction of metal in the cultures (Fig. 2, for example), reflecting greater rates of radioisotope equilibration between dissolved and particulate phases than rates of cell division. Table 2 presents concentration factors computed on volume (VCF) and dry wt (DCF) bases at the end of each uptake period calculated for all metals and species, where VCF = moles metal/~m -3 cell divided by moles metal/~m -3 dissolved in the water, and DCF = moles metal g-1 cell divided by moles metal g-1 dissolved. Am was concentrated to the greatest extent by all algal species, but no other generalizations regarding the order of concentration factors were apparent for all species (Table 2). Emiliama huxleyi

682

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Table 3 Maxzrnumradlotsotopeuptake (percentage on parncles pg- 1dry wt h- 1) for Thalassloslrapseudonana (3H), Prorocentrumminimum(EXUV), and Emlhama huxleyl(MCH1) Also shown are correlanon coeffictents (r) for maximum uptake rates wtth DCF values

Element

3H-hve

3H-dead

EXUV-hve

EXUV-dead

MCH1

Se Ag Sn Au Am

0.017 0 31 3.5 1.8 85

0.0004 0.11 2.5 2.6 70

0 013 0 15 0 12 0 038 018

0 0008 0 038 0 11 0.003 021

0 004 0 11 0 16 0 08 040

r

0 679

0.867

0 876

0 969

0 605

generally had concentration factors an order of magnitude lower than those of living T. pseudonana or living P. minimum (with the exception of Au in T. pseudonana) (Table 2). The maximum metal uptake rates by the cells (calculated on a dry wt basis, for the portion of the uptake curve where the slope was greatest) were generally correlated with metal concentration factors (Table 3); this relationship held more strongly for dead cells than for living cells. For all species, Am showed the highest values, typically followed by Sn, and Se the lowest. Se accumulation was an active process. Se concentration factors were 6-10 times greater in living cells than in dead cells and the maximum uptake rates were about 40 times greater with living than with dead T. pseudonana and 16 times greater with living than with dead P. minimum cells. Over 99% of the total Se in the water remained in the dissolved phase in cultures with dead T. pseudonana or P. minimum cells throughout the uptake period (Fig. 1). Moreover, the uptake of Se by living cells increased steadily with time (Fig. 1). Ag uptake was greater in living than in dead cells (especially for T. pseudonana); total cellular uptake of Ag appeared to level off toward the end of the uptake period for all species (Fig. 2). Bioaccumulation of Sn and A m appeared to proceed by passive sorption, since dead and living cells concentrated these metals appreciably; in general, accumulation of these metals was characterized by a rapid uptake within the first day, gradually levelling off with time (Figs 3 and 5). Au was concentrated to a greater extent by living P. minimum cells than by dead ones, although this was not observed with the diatom (Fig. 4). Uptake of Au proceeded steadily with time for P. minimum and E. huxleyi cells, but levelled off rapidly for T. pseudonana (Fig. 4). Metal retention

Upon transfer of the cells to unlabeled seawater held in the dark, some cells began to decompose, particularly at 18°C. This was evident from the direct cell counts declining over time (Figs 6-8), most particularly for T. pseudonana and E. huxleyi cells, and from the decline in PC measurements (Table 4). The radioactivity of all species also declined with time, although at different rates for each radioisotope (Figs 6-8). The release of all radioisotopes from the cells was characterized by a rapid initial loss, followed by a more gradual loss (e.g. Am loss from E. huxleyi at 18°C), or by no loss at all (e.g. A m loss from P. minimum) (Figs 6--8). For T. pseudonana, A m was lost equally by living and dead cells, and retention of this element was unaffected by temperature (Fig. 6).

Release of trace elements by dying phytoplankton

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Fig. 7. Retention Over t~me of 75Se, 11°mAg, ll3Sn, 195Au and 241Am in Prorocentrum m+n~mum m unlabeled seawater. Data presented are cell density (intact cells m1-1) and the fraction of each radioisotope retained by suspended particles (cells and cellular debris) 75Se and 241Am-labeled cells were m one set of cultures, 113Sn and 195Au-labeled cells were In another set, and ll°mAg-labeled cells were in a third set. (B) Living cells Incubated at 18°C; (&) hvmg cells incubated at 8°C; (D) dead cells incubated at 18°C, (X) dead cells incubated at 8°C

250

685

Release of trace elements by dying phytoplankton

10~

700

600

-6

500

b

n

E O

E

3O0 20O

100

16O 1so ~o =so Hours

16o 1~o ~o ~o Hours

0

50

100

150

200

250

Hours

100J

100

~I~ c

100

sb

10

16o 1~o ~o ~o

0

50

100

150

200

250

Hours

Hours

Ko

16o 1so ~o 2so Hours

100

f

u m~

<

0

50

100

150

Hours

200

250

lC

o

Ko

16o 1so ~ o Hour s

25o

Fig 8 Retention over time of 75Se, n°mAg, ll3Sn, 195Au and 241Am m E m t h a m a huxley, m unlabeled seawater Data presented are cell density (intact cells m1-1) and the fraction of each ra&olsotope retained by suspended particles (cells and cellular debris). 75Se and 241Am-labeled cells were m one set of cultures, H3Sn and 19SAu-labeled cells were m another set, and ]]°mAg-labeled cells were m a third set (11) Living cells incubated at 18°C; ( A ) hvmg cells incubated at 8°C.

0 140 236

0 110 211

EXUV-dead

MCHl-hve

0 48 188

0 140 236

0 140 236

0 110 211

3H-hve

EXUV-hve

EXUV-dead

MCHl-hve

Clone

0 1~ 236

EXUV-hve

31 26 20

24 22 16

14 2 119 86

20 16 10

21 15 12

22 15 12

lost 120 95

32 14 11

25 18 15

23 19 15

14 3 113 88

32 15 05

76 71 40

400 689 957

1093 1343 688 8

54 45 46

Ag S n & A u S e & A m

121 71 58

830 969 888

lost 1612 164 2

76 33 25

104 63 40

403 591 940

1860 1887 254 9

72 34 12

Ag S n & A u

pg Carbon cell-I Sn

218 15 344 25 473 66

Ag 9 91 14 92 38 109

12 12 17

0.126 91 2 6 1 112 8 1 0 0 3 2 44 1 73 40 2 3 0 0 2 3 39 2 2 1 36 2 2

0003 130036102 0001 168037023 0001208043025 270 290 160

230 230 160

1000 1030 800

85 148 89

190 200 90

230 200 120

lost 1060 890

126 109 84

240 230 120

210 170 130

1030 1020 820

126 232 29

Ag Sn&Au

ng Nitrogen ml-1

Au A r o S e & A m

0 0 0 7 lost 0 0 7 0 1 7 0 2 00050570.09015 02 0 002 0 71 0 11 0 20 0 2

1 69 1 12 085

Se

/~mol metal per g carbon Ag

89 9 11 0 23 2

07 0 2 0 2

29 19 0 5

935 ND 1251

62 9 10 8 I1 1

0 5 0 2 0 3

2 2 06 02

835 ND 206

30700 ND 21800

248 291 216

238 195 587

lost ND 139075

26500 ND 16900

129 231 252

206 195 273

143361 ND 199640

2100 53700 11200

118 64 95

31 30 49

11862 ND 12117

1440 1420 4430

35 74 108

25 43 44

11979 ND 8278

90400 58500 181000

336 40 54

69 53 86

6823 ND 6875

62200 32600 71400

35 74 108

55 76 77

6891 ND 4697

5780 796 2240

322 238 306

83 81 307

50474 ND 160955

Sn

Au

1 4 6 980 0 2 9 315 0 2 8 516

003 124 001 123 001 199

28 14 27

40 42 49

0 1 0 lost 1 0 006 64 09 0 02 7 6 1 2

4050 781 1070

212 249 430

63 25 66

45084 ND 26565

1180 320 439

112 26 28

23 17 2 1

41 5572 385 221 12 4332 162 89 20 5879 1155 655

Se

94 21 27

125 117 168

29 25 3 2

2185 1008 1236

Am

/zmol metal per g mtrogen

nmol metal per g dry wt Ttme (h) S e > 1 #m S e > 0 2 u m A g > l , u m A g > 0 2/~m S n > l ,urn S n > 0 2 u r n A u > 1 g m A u > 0 2,um A m > l , u m A m > 0 . 2 g m

0 48 188

3H-hve

Clone

Time (h) S e & A m

u g Carbon m l - l

Table 4. Carbon, mtrogen, and metal loss from the dtatom T h a l a s s l o s l r a p s e u d o n a n a ( 3 H ) , the dmoflagellate P r o r o c e n t r u m m i n i m u m ( EXUV), and the coccohthophore E m d m n l a h u x l e y l (MCH1). Se & A m denotes cells grown m presence of selemum and amertctum, A g denotes cells grown wtth sdver, Sn & A u denotes cells grown wtth tm and gold Metals are normalized on parttculate C, N, or dry weight bases. ND" not determined

r~

rn

~r

Z

Release of trace elementsby dyingphytoplankton

687

Temperature had relatwely small effects on retention of the other elements in these cells, although living cells retained these other elements to a significantly greater extent than did dead cells (except Sn at 18°C) (Fig. 6). Thus, in T. pseudonana and P. minimum cultures, radioisotopes which were accumulated to a significantly lower extent by dead than by living cells were also lost at greater rates from dead cells (Figs 6 and 7). For P. mimmum, Am, Sn and Ag were retained to the greatest extent and were lost equally by living and dead cells; Au and Se were lost more rapidly from these cells than from living cells and somewhat more rapidly at 18°C than at 8°C (Fig. 7). Loss rates of all radioisotopes were comparable from E. huxleyt cells, generally reflecting the decline in cell numbers (Fig. 8). Overall, metals were retained to a greater extent by the diatom and the dinoflagellate cells than by the coccolithophores. PC declined at 18°C by up to 84% in the T. pseudonana cultures, up to 39 and 45% in hving and dead P. mimmum cultures, respectively, and up to 57% in E. huxleyi cultures (Table 4). Thus the range of PC losses was 6.4-10.7% day -1 for T. pseudonana 2.2--4.0% day -1 for hving P. minimum and 3.4--4,6% day -1 for dead P. minimum, and 4.0-6.5% for E. huxleyi. Increases in C per cell (Table 4) simply reflect a more rapid decomposition of cells (to the extent that the resulting debris was no longer recognizable as an intact cell) than of PC retained by filtration. In the same cultures, PN declined by comparable amounts, with observed loss rates of 0--9.8% day -1 for T. pseudonana, 2.0% day -1 for living P. minimum and 3.1--4.9% day -a for dead P. minimum, and 4.7-6.0% day -1 for E. huxleyi. In all cultures, Se was lost more rapidly than was PC, so Se:C ratios decreased over time (Fig. 9). For E. huxleyi cultures, the ratios of all other metals to PC also declined with time, whereas these ratios all increased m T. pseudonana cultures and did not change substantially in living P. minimum cultures (Fig. 9). The Ag:C and A m : C ratios increased and Au:C decreased in dead P. mmimum cultures (Fig. 9). The ratios of metal:PN in cultures of all speoes over time generally paralleled the metal:PC ratios (Table 4), although Ag and Am in T. pseudonana cultures were notable exceptions. For Ag, the ratio to PC more than doubled while the ratio to PN stayed essentially constant; for Am, the ratio to PC increased slightly and the ratio to PN decreased 43% (Table 4). When normalized to dry weights, loss of metals from the cells reflected a downward shift in the size spectrum of the particulate material over time, That is, even though intact cells of all these species are quantitatively filterable onto 1.0/~m Nuclepore filters, as cells started to decompose with time, the suspended particles decreased in size so that some material passed through a 1.0 ktm filter but was retained by a 0.2 ktm filter. This shift in size spectra resulted in shifts over time in the metal:dry wt ratios and greater differences between the 1.0#m and 0.2/~m filter data at the final samples times (Table 4). Further, this particle size shift sometimes resulted in an increase over time in metal:dry wt ratios using the 1.0#m filter data and a decrease in metal:dry wt ratios using the 0.2/~m filter data from the same cultures (e.g. Se and Am in T. pseudonana cultures). Increases in ratios of metal:dry wt over time (e.g. Sn in E. huxleyt cultures) reflect a greater loss rate for dry weight than for metals from the suspended particles. DISCUSSION The metal uptake and retention data suggest that the diatom Thalassiosira pseudonana and the dinoflagellate Prorocentrum mimmum have more reactive cell surfaces for metals

688

N S FISHERand M WENTE Se

Ag

15

25 0

0 "0 E

10~

q) -~ E

os

O0

5O

IO0

150

2OO

20

oo

25O

50

100

Hours

150

200

250

200

250

Hours

Au

Srl 50" (.~

40" 30' 2.0'

21 1(

0C

50

100

150

200

O(

250

50

Hours

100 150 Hours

Am 15 ¢..) O E

--

0 E

10

og

00

50

100

150

200

25O

Hours

Fig 9 Molar ratios over Ume of Se, Ag, Sn, Au and Am to C in particulate phases in Thalasstostrapseudonana (11), Prorocentrum mmtrnurn (mitmlly living. ~ ; initially dead: &----A),andEmdiamahuxleyt(X)culturesheldatl8°C Values are normalizedto initial values at start of depuratlon period; values unavadable for Ag m hvlng P. mtmmurn culture (see Table 4). than does the coccolithophore Emiliania huxleyi. Differences between species can not be attributed to differences in surface:volume ratios of the cells, since E. huxleyi cells have intermediate values between those of the other two species. Since other species of these taxonomic groups were not examined, it is impossible at present to determine whether these relative differences are representative of other diatoms, dinoflagellates, and coccolithophores. Emiliania huxleyi similarly displays less reactive surfaces than diverse diatom and dinoflagellate species for Ba, another non-essential metal (FISHER et al., 1991a). Attempts were made to work with suspended particle loads that approach those in natural surface waters (generally --<1 mg 1-1 in the open ocean). It should be noted that these particle loads are much lower, and therefore less susceptible to experimental artifact, than those used in many studies designed to determine Ka values for metals (HoNEYMANet al., 1988). SCHOONEN et al. (1992), examining A u sorption to pyrite and goethite, found that Ka values also stayed constant for particle loads of 1-20 mg 1-1. Although the use of radioisotopes greatly facilitates the experimental determination of

Release of trace elements by dyingphytoplankton

689

sorptmn kinetics in abiotm and living particles in seawater (e.g. FISHER et al., 1983a; JANNASCH et al., 1988; NYFFELER et al., 1984), the introduction of easily detectable concentrations of radioisotopes (even from carrier-free stocks) into seawater can sometimes overwhelm background levels of their stable analogues. The metal concentrations added via isotope addition for the uptake experiments exceeded typical concentrations in surface seawater, with the exception of Se. The greatest discrepancy was for Am, whose current concentrations in surface waters, primarily from radioactive fallout, are about 10-21 M (COCHRANet al., 1987). Nevertheless, concentration factors for Am in marine phytoplankton derived from laboratory experiments using Am concentrations greatly exceeding these ultra-low background levels are comparable with those calculated from field measurements (FISHER et al., 1983a). The concentration of Sn in surface waters is around 10-11 M (BYRD and ANDREAE, 1986), Ag is about 10-12 M in surface waters (MARTINet al., 1983), Au is about 10 -13 M (KoIDE et al., 1988; FALKNERand EDMOND, 1990), and total Se is about 10 -9 M (MEASURESand BURTON, 1980; CUTrER, 1982). It is assumed that the isotopes added speciated in the water similarly to the stable metals, primarily inorganically, although this was not checked. In surface seawater Se may be primarily in the form of organic selenldes (CuttER and BRULAND, 1984). Much of the variation in metal release data, including differences between living and dead cells, can be explained in terms of equilibrium partitioning of the radioisotopes between dissolved and particulate phases. Correlating the fraction of radioisotopes on particles at the end of the uptake phase with the fraction at the end of the depuration phase produces an r value of 0.833 for all data. Living cells can transport some of these elements (especially Se) into their cytoplasm and make them less susceptible to equilibrium partitioning; if only the dead cell data are considered (i.e. dead T. pseudonana and P. minimum), the r value increases to 0.940. Thus, much of the initial decline in cell radioactivity immediately following resuspension into unlabeled seawater is likely due to a rapid re-equilibration of the radioisotopes between cell surfaces and the ambient water. Isotope dilution (by stable metal exchanging with radioactive metal) also may account for some of this initial loss, although not for 241Am, for which there is no stable isotope. By assuming: (1) that partition coefficients for a radioisotope are constant for varying particle loads (MCKINLEYand JENNE, 1991), (2) all the particle-bound isotope is in equilibrium with that in the ambient water; and (3) no isotope dilution (e.g. for 241Am), it is possible to predict the release via desorption for any combination of particle loads during uptake and during loss. Thus, if X = fraction of isotope on particles after uptake at particle load C1, Y = fraction of isotope on particles after depuration at load C2, K -- constant, and CF = concentration factor or partition coefficient (assumed constant for particle load range C 1. . C 2 ) ;

(X/C1)

CF-(I_X)/K

_

(Y/C2)

(1-Y)/K

(1)

which can be converted to: y_

(XI1 - X ) (C1/C2) + (X/1 - X )

(2)

Equation (2) gives the predicted fractionation of radioisotope on particles after equilibrium has been attained during the depuration phase. Deviations from predicted values

690

N S FISHERand M WENTE

could result from any of the above assumptions being wrong. Assumption (2) would not hold for those cases (e.g. 75Se) in which cells transport the radioisotope into the cytoplasm, particularly if it becomes covalently bound to organic molecules within the cell. Assumption (1) is presumed valid, at least for the particle loads (1-20 mg 1-1) employed in these experiments; results of experiments with particle loads in excess of hundreds ofmg 1-1 may be more suspect. The positive correlations between maximum radioisotope uptake rates by the different species (especially for dead cells) and DCFs are consistent with JANNASCHet al.'s (1988) findings that metal uptake rates onto suspended particles in Puget Sound correlate well with their partition coefficients for these particles. The general order of reactivity of metals for phytoplankton and abiotic particles has been shown to conform with the hydrolytic scavenging potential of metal ions (CLEGGand SARMIENTO,1989). Retention of metals by phytoplankton is very variable among metals and species (FISHER et al., 1983a, 1991b; LEE and FISHER, 1992b). While recognizing that experimental studies such as this employ conditions (lack of grazers, different bacterial assemblages, lack of turbulence, etc.) which make extrapolations of absolute rates to the open-ocean environment tenuous, certain generalizations can be made. Generally, the most particle-reactive metals in seawater (e.g. some of the actinides) are retained by planktonic debris for the longest periods. The small effect of temperature on metal retention suggests that biological activity, such as by microorganisms, may not directly release metals from the cells. This is consistent with oceanographic observations that biogenic particle decomposition can occur abiotically by fragmentation and dissolution (KARL et al., 1988) and that > 95% of the bacteria in the ocean are free-living (Cuo and AZAM, 1988). LEE and FISHER (1992b) also report small effects of temperature on release rates of Am, Ce, Co, and Ag from T. pseudonana cells, while Cd and Se retention are significantly greater at 4°C than at 18°C. The retention of metals in crustacean zooplankton fecal pellets produced on monospecific algal diets is typically much longer than in the algal cells themselves (FISHER and FOWLER, 1987), due to lower surface: volume ratios of the fecal material and to the peritrophic membrane barrier of the fecal pellets. Of the elements studied here, all but Se were released from T. pseudonana cells at rates slower than POC. By contrast, all radioisotopes are released from dying E. huxleyt cells more rapidly than C, and at about the same rate as C from dying P. minimum cells which had been living during the radioisotope exposure period. The slower loss of Se and Au from living P. minimum cells than from dead cells suggests that the living cells transported these elements into the cytoplasm, from which they were lost more slowly than from the cell surface. None of the radioisotopes studied systematically followed N during the depuration periods for all algal species, indicating that these elements were not primarily bound to protein in these cells. It would appear, therefore, that in marine snow composed of sinking diatom aggregates (ALLDREDGEand SILVER,1988), some metals are likely to be released more slowly than C. The results suggest that a substantial fraction of a cell's Am, Sn and Ag will likely be retained by sinking cellular debris for days to weeks. HEBEL et al. (1986) have, in fact, reported elevated metal concentrations in marine snow in Monterey Bay comprised of phytoplankton aggregates. Some diatom species aggregate to form rapidly sinking flocs (PASSOW, 1991). Given that newly formed aggregates have sinking rates of approximately 100 m day -I (ALLDREDGE and GOTSCHALK, 1989), sinking phytodetritus is probably an effective vector for these

Release of trace elements by dyingphytoplankton

691

particle-reactive metals out of surface waters. Because of their shorter metal retention times, sinking coccolithophores might be expected to be less effective than diatoms in transporting these metals to deep waters, although the high sinking rates (hundreds of meters per day) of palmelloid formations of certain coccolithophores (SMAVDA, 1971) may compensate for the shorter retention times in the vertical transport of metals. Sediment traps deployed at depths ranging from 378 to 1464 m in open ocean regions contain biogenic debris enriched in particle-reactive metals such as Th, Pb, Pu, and Am (BREWER et al., 1980; LIVINGSTONand ANDERSON, 1983; FISHERet al., 1988). These metals may have been retained by the particles from the beginning of their descent and/or may have been scavenged during the particles' descent in the water column. NORIKIetal. (1985) reported that the flux of Cd, Pb, Ni, Cu, Co, Mn and Fe was carried by settling debris following phytoplankton blooms in Funka Bay, Japan, although they did not examine any of the metals studied in the present report. Ag flux out of surface waters in the northeast Pacific closely follows organic C flux and may be tightly bound to the organic matter of settling biogenic debris (MARTINe t al., 1983), consistent with the loss patterns from the dinoflagellate, Prorocentrum minimum. Inorganic Sn that is accumulated from the dissolved phase by green macroalgae can be released by the decaying algae in organic (primarily methylated) as well as inorganic form (DONARDet al., 1987); the extent to which methylation occurs in marine phytoplankton assemblages remains to be studied. Studies of Au and Sn flux by sinking biogenic particles in the ocean are unavailable. While the biologically mediated flux of Se has not been studied extensively in the oceans, the evidence that Se is essential to phytoplankton (PRICEet al., 1987), is highly assimilated in zooplankton (FISHER and REINFELDER, 1991), is present as organic selenides in surface waters (CUTTERand BRULAND,1984), and is released at a rate ->POC from phytoplankton (this report) suggests that this element enters into the organic cycle and behaves like a "recycled" element (WHITFIELDand TURNER,1987). The flux of Am and Pu (FOWLERet al., 1983) and other particle-reactive radionuclides emanating from the Chernobyl accident (FOWLERet al., 1987) on sinking debris, including phytodetritus, have been described in the Pacific and the Mediterranean. The release rate of C from the particulate phase in the T. pseudonana cultures (6.410.7% day-I), determined with CHN analysis, was comparable to the estimate--9.7% day -1 also at 18°C--for the same species (over the first 7 days of incubation) using a 14C radiolabel method (LEE and FISHER, 1992b). Even without grazing, a phytodetritus decomposition rate of 6--10% day- a would result in a pronounced decrease in the POC flux at 1000 m, even for aggregates sinking at 100 m day -1. This is consistent with oceanographic observations, where most of the sinking POC decomposes during descerit in the upper water column (GARDNERet al., 1985; HARGRAVE,1985; MARTINet al., 1987; CLEGG and WHITFIELD,1990). As cell aggregates sink into colder waters, C remineralization rates should decline. ITURRIAGA(1979) reported a two-fold greater remineralization rate of sedlmenting debris at 20°C than at 5°C, and LEE and FISHER (1992b) found a four-fold difference between T. pseudonana C retention at 18°C and at 4°C. As with the metals, C release rates determined for fecal pellets, which are enriched in refractory organic C, are slower than those for phytoplankton cells. JACOBSEN and AZAM (1984), for example, measured C remineralization rates from zooplankton fecal pellets at 1% day -1 at 18°C, and LEE and FISHER (1992a) found C remineralization rates from copepod fecal pellets (produced on a T. pseudonana diet) of 2.2 and 0.5% day -1 at 18 and 2°C, respectively. Other organisms such as phagotrophic zooflagellates can also mediate the decomposition

692

N S FISHERand M WENTE

of biogenic debris in the ocean at depths to 2000 m (TAYLOR et al., 1986), and presumably their activity increases with temperature as well. Acknowledgements--This research was supported by NSF OCE8810657 Thls 18 contribution no. 839 from the Marine Sciences Research Center We thank Dr S Fowler for helpful comments on the manuscript

REFERENCES ALLDREDGEA. L and C C GOTSCHALK(1989) Direct observations of the mass flocculatlon of diatom blooms characteristics, settling velocities and formation of diatom aggregates. Deep-Sea Research, 36, 159-171 ALLDREDGEA L and M W. SILVER(1988) Characteristics, dynamics and significance of marine snow Progress m Oceanography, 20, 41--82. BILLET D S. M , R S. LAbIPITT, A L RICE and R F. C MANTOURA(1983) Seasonal sedimentation of phytoplankton to the deep sea benthos Nature, 302, 520-522. BISHOPJ. K. B , J. C STEPIENand P. H. WIEEE (1986) Particulate matter distributions, chemistry and flux in the Panama Basin. Progress in Oceanography, 17, 1-59. BREWERP. G., Y NOZAKI,D W SPENCERand A P FLEER(1980) Se&ment trap experiments in the deep North Atlantic' ISOtOpicand elemental fluxes Journal of Manne Research, 38,703-728. BRULANDK. W and K. H COALE(1986) Surface water 234Th/238U &sequllibria spatial and temporal variations ofscavenglngrateswithinthePaclficOcean.In: Dynamlc processes m the chem~stry of the upper ocean, J D BURTON, P. G. BREWERand R CHESSELET,editors, Plenum, New York, pp 159-172 BYRD J. T and M O. ANDREAE (1986) Dissolved and particulate tin in North Atlantic seawater. Marine Chemistry, 19, 193-200. CHERRYR. D., J J. W HIGGOand S W FOWLER(1978) Zooplankton fecal pellets and element residence times in the ocean Nature, 274, 246-248 CHO B. C and F. AZAM(1988) Major role of bacteria In blogeochemical fluxes in the ocean's interior Nature, 332,441-443. CLEGG S. L. and J. L SARMIENTO(1989) The hydrolytic scavenging of metal Ions by manne particulate matter Progress in Oceanography, 23, 1-21 CLEGO S. L. and M. WHITFIELD (1990) A generalized model for the scavenging of trace metals in the open ocean--I Particle cycling Deep-Sea Research, 37,809-832 COALEK and K. W. BRULAND(1987) Oceamc stratified euphotlc zone as elucidated by 234Th:238 U disequlhbria Ltmnology and Oceanography, 32, 189-200 COCHRANJ K , H. D. LIVINOSrON, D. J. HIRSCnRER6 and L D SUPRENA~rr(1987) Natural and anthropogenic radlonuchde distributions in the northwest Atlantic Ocean Earth and Planetary Science Letters, 84, 135152 COLLIER R and J. EDMOND (1984) The trace element geochemistry of manne blogemc particulate matter. Progress In Oceanography, 13, 113-199. CUTrER G. A. (1982) Selenium in reducing waters. Scwnce, 217, 829--831. Cu'rrEa G A and K. W. BRULAND(1984) The manne blogeochemistry of selenium: a re-evaluation L~mnology and Oceanography, 29, 1179-1192 DONARDO F X , F. T. SHORTand J. H. WEBER(1987) Regulation of tin and methyltm compounds by the green alga Enteromorpha under simulated estuanne conditions Canadian Journal of Ftsherles and Aquattc Sciences, 44, 140-145 FALKNERK K and J. M EDMOND(1990) Gold in seawater. Earth and Planetary Science Letters, 98,208-221 FISHERN S andS W. FoWLER(1987)Theroleofblogenicdebnsinthevertlcaltransportoftransuramcwastesof the sea. In Oceamc processes tn marme pollutlon. 2" Physico-chemlcal processes and wastes in the ocean, T P. O'CONNOR, W V BURr and I W. DUEDALL,editors, Kneger, Malabar, pp 197-207. FISHER N S. and J. R REINFELDER(1991) The assimilation of selenium In the manne copepod Acartla tonsa studied with a radiotracer ratio method. Marme Ecology Progress Series, 70, 157-164 FISHER N S , F AZAMand J -L TEYSSII~(1986) Accumulation of ll3Sn by a marine diatom In. Speclauon of fisston and activation products in the envtronment, R A BULMANand J. R COOPER, editors. Elsevier, London, pp 361-367

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