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Page 1: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Adv. mar. Biol., Vol. 16, 1978, pp. 381-508.

POLLUTION STUDIES WITH MARINE PLANKTON PART II. HEAVY METALS

ANTHONY G. DAVIES The Laboratory, Marine Biological Association,

Plymouth, England

I. Introduction . . .. .. .. .. .. .. .. ..

The Kinetics and Mechanism of Metal Uptake by Phytoplankton The Effect of the Chemical Form of a Metal upon its Uptake by

The Role of Phytoplankton in the Biogeochemistry of Heavy Metals in the Sea . . .. .. .. .. .. . .

Laboratory Studies of the Toxic Effects of Heavy Metals upon Phyto-

11. The Turnover of Heavy Metals by Phytoplankton . . . . .. . .

Phytoplankton . . .. .. .. .. .. .. A. B.

C.

111. plankton . . .. .. .. .. .. .. .. ..

A. The Effects on the Growth of Phytoplankton . . .. ..

Phytoplankton . . .. .. . * .. .. .. C. The Nature of Metal Toxicity in Phytoplankton . . .. ..

Phytoplankton . . .. .. .. .. .. .. .. A. The Effects on Primary Production Rates . . .. .. .. B. The Effects in Large Volume Sea Water Enclosures . . ..

plankton . . . . .. .. .. .. .. .. .. VI. The Turnover of Heavy Metals by Zooplankton . . .. .. ..

A. Studies of Metal Fluxes through Zooplankton . . .. ..

Zooplankton . . .. .. .. .. .. .. ..

B. Synergism and Antagonism of Mixtures of Heavy Metals towards

IV. Studies of the Toxic Effects of Heavy Metals upon Natural Populations of

V. Heavy Metal Concentrations in Natural Populations of Marine Phyto-

B. C.

D.

Food and Water as Sources of Metals for Uptake by Zooplankton The Effect of the Chemical Form of a Metal upon its Uptake by

The Role of Zooplankton in the Biogeochemistry of Heavy Metals in the sea . . .. .. .. .. .. .. ..

Laboratory Studies of the Toxic Effects of Heavy Metals upon Zooplankton VII. A. The Effects on the Metabolic Activity of Zooplankton . . ..

D. The Effects on the Fecundity of Zooplankton . . .. .. F. The Effects on the Swimming Activity of Zooplankton . . . .

mental Stress upon Zooplankton . . .. .. .. ..

Zooplankton in Large Volume Sea Water Enclosures . . .. ..

B. C.

E.

6.

The Effects on the Feeding and Ingestion Rates of Zooplankton The Effects on the Growth and Development of Zooplankton . .

The Effects on the Phototactic Response of Zooplankton . . . .

The Combined Effects of Heavy Metals and Additional Environ-

VIII. Studies of the Toxic Effects of Heavy Metals upon Natural Populations of

Heavy Metal Concentrations in Natural Populations of Marine Zooplankton IX. 381

382

383 383

391

395

398 398

411 412

416 416 419

426

428 428 434

439

440

446 447 448 460 464 466 456

456

467

460

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382 ANTHONY a. DAVIES

X. Conclusions . . . . . . I . .I .. .. . . . . 463 XI. Acknowledgements . . .. .. .. .. .. . . . . 464

XII. Referenoes . . .. .. .. .. .. .. .. .. 484

Appendix1 .. .. .. .. .. .. .. . . . . 419

Appendix11 .. .. .. .. .. .. .. . . . . 498

Appendix I11 . , .. .. . . . . . . . . .. ,. 601

I. INTRODUCTION

That heavy metals are poisonous towards microscopic marine life has been recognized for most of the present century. As early as the 1920's, paints containing the oxides of toxic metals were being tested for their capacity to prevent the fouling of surfaces immersed in the sea by deterring the settlement of algal spores and the planktonic forms of certain animals, particularly barnacles (Orton, 1929-30), and the continued development of anti-fouling coatings has provided the stimulus for measuring the concentrations of heavy metals necessary to kill both planktonic animal larvae and permanent members of the zooplankton (Corner and Sparrow, 1956, 1957 ; Wisely and Blick, 1967). Attempts have also been made to control the damaging out- bursts of dinoflagellates known as " red tides '' by adding copper sulphate or copper ores to the affected areas, but with little success (Rounsefell and Evans, 1958 ; Marvin, Lansford and Wheeler, 1961).

Thus, until 10 or 15 years ago, interest had centred upon the lethal aspects of metal toxicity. Since then, however, there has been an increasing awareness that the insidious build-up of low-level concen- trations of metals in coastal and estuarine sea areas receiving industrial effluents and sewage could be having a deleterious effect upon the growth and development of the plankton leading to a decrease in the productivity of these regions. Further, it has been realized that the uptake of metals by the plankton provides an entry into marine food chains, the higher trophic levels of which are often used for human consumption. This has given rise to the upsurge in research into the longer term sub-lethal aspects of metal toxicity towards marine plankton and the way in which metals are accumulated at the first and second trophic levels which forms the basis of this review.

The metals considered have been restricted mainly, but not ex- clusively, to the ten which appear to be most poisonous to marine life: mercury, cadmium, silver, nickel, selenium, lead, copper, chromium, arsenic and zinc. They are listed here in the order of decreasing toxicity given by Ketchum, Zitko and Saward (1975) though this particular ranking could obviously be a matter of some debate.

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POLLUTION STUDIES WITH MARINE PLANKTON-11 383

The geochemical aspects of heavy metals in the sea have provided the subjects for several recently published articles and will not, there- fore, be considered again here; information on the inputs of heavy metals to the sea, both due to natural processes and to pollution, may be obtained from reviews by Turekian (1971) and Goldberg (1976), and the chemical behaviour of metals in sea water has been discussed by, amongst others, Dyrssen and Wedborg (1974) and Stumm and Brauner (1975).

In this review, the interactions of metals with phytoplankton and with zooplankton have, for the most part, been dealt with separately though there is inevitably some degree of overlap, especially in the sections dealing with metal uptake by zooplankton from their food supply. The portions concerned with the plants and with the animals progress in a similar manner. They begin with an examination of the rates and mechanisms of uptake and loss of heavy metals, including a discussion of the influence of the plankton upon the biogeochemistry of metals in the sea, then the toxic effects of the metals are considered using data obtained both in the laboratory and with natural populations and, finally, the heavy metal concentrations in natural populations of plankton are discussed. We start with the phytoplankton.

11. THE TURNOVER OF HEAVY METALS BY PHYTOPLANKTON

A. Kinetics and mechanism of metal uptake by phytoplankton

Phytoplankton populations present surprisingly large surface areas to the sea water or culture medium in which they are growing. Cells of the common diatom Skeletonema costatum (Greville) Cleve, for instance, have been found to have areas in the range 235-369 pm2 (Smayda, 1970) so that a million cells would have a total surface exceeding 2 cm2. Under natural conditions, a million diatoms per litre of sea water is by no means unusual (Bainbridge, 1957) and tkis number can be greatly exceeded during the spring bloom (e.g. Butler, Corner and Marshall, 1970), while in laboratory cultures populations of a million cells per ml are commonplace. It is not surprising, therefore, that adsorption onto the outsides of cells represents an important aspect of metal uptake by phytoplankton.

Little information is available on the physico-chemical nature of the surfaces of marine phytoplankton except for the work of Myers, Iverson and Harriss (1975) who showed, using a microelectrophoretic technique, that three estuarine species Nannochloris oczdata Droop, Pavlova (Monochrysis) lutheri (Droop) Green and Cyclotella mene-

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384 ANTHONY 0. DAVIES

ghiniana Kutzing all had negatively charged surfaces due, it was suggested, to the ionization of groups in the polysaccharide-glyco- protein matrices exposed to the sea water. Davies, Haydon and Rideal (1 956) had previously demonstrated that bacterial surfaces are also negatively charged ; by following the variation in surface charge with changing pH in the solution bathing the cells, they established that the acid dissociation constant for the groups on the surface (pK = 2-9) was about the same as that of acidic polysaccharides (pK = 2.95) suggesting that the surface charge was associated with carboxylic rather than phosphatidic groups which have pK values of about 1.8. Davies et al. (1956) also found that the charges on bacterial surfaces could be neutralized and even reversed by heavy metal ions such as copper or lead. Similar experiments have not been carried out with marine phytoplankton, but the decrease in the net charge on their surfaces caused by increasing salinity observed by Myers et al. (1975) was presumably the result of interaction of the cations in sea water with the negative groups on the surfaces.

On this basis and by analogy with more intensely studied cellular systems, e.g. erythrocytes (Bangham, Pethica and Seaman, 1958 ; Passow, 1970), it is possible to arrive at a tentative picture of the physico-chemical nature of the surfaces of phytoplankton as consisting of a mosaic of interspersed cationic and anionic exchange sites, pro- vided by carboxylic, sulphydryl, phosphatidic, amino and other groups, the net charge on the surface being related to the degree to which the sites are occupied by protons and the other ions present in sea water, i.e. being a function of the pH and salinity. The initial uptake of a positively charged heavy metal ion can then be envisaged as occurring by the displacement of the cations already occupying the binding sites, the amount of metal finally bound onto the surface a t equilibrium being determined by the relative affinities of the sites for the metal and the sea water cations and also the concentrations of each remaining in solution, in accord with the principles of ion-exchange. The metal, once bound on to the surface, would be suitably placed for being transported, actively or passively, through the diffusion barrier presented by the cell membrane into the cytoplasm.

From measurements of the amounts of 2osHg taken up by dividing, non-dividing and formalin-killed cultures of Chaetoceros costatum Pavillard in the light and the dark, Glooschenko (1969) concluded that mercury was accumulated passively, and more recent studies of the mechanism by which metals enter marine phytoplankton, namely those on zinc uptake by Pheodactylum tricornutum Bohlin (Davies, 1973), and of mercury uptake by Isochrysis galbana Parke and Duna-

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POLLUTION STUDIES WITH MARINE PLANKTON-11 385

ZieZZa lcrtiolecta Butcher (Davies, 1976), also support the idea of passive uptake. In the latter experiments, cultures were grown in a chelator- free culture medium of enriched natural sea water containing very little of the metals being studied so that the plant cells would initially be virtually devoid of them ; in the case of zinc, this necessitated passing the sea, water used in the medium through a column of a chelating ion-exchange resin in order to remove as much as possible of the metal naturally present.

When the populations were dense enough to make the measurements feasible, the metal, labelled with a radioactive tracer, was added to give a suitable concentration, and its incorporation by the cells followed by filtering samples of the culture a t regular intervals to determine the

t (min) .\/I (mi")+

FIO. I. The uptake of inorganic mercury by A, ~sochryeis galbanu and B, Dunaliella tertiolecta in cultures containing cell populations having approximately the same total surface areas. Initial mercury concentrations (pg/l) : 0, 10 ; A, 20 ; ., 60. During the period ikstrated, the mercury transported across unit area of surface in the Isochryaie galbana cultures was substantially greater than that in the Dunaliella tertiolecta cultures due to differences in the permeabilities of their membranes to the metal ions. In both cases, the weight of mercury taken up was a linear function of the square root of the elapsed time [C, D) indicating that, in the early stages, uptake was diffusion controlled. (From Davies, 1976. Reproduced by kind permission of the Council of the Marine Biological Association of the United Kingdom.)

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386 ANTHONY Q. DAVIES

radioactivity in the cells. I n order that the data could be interpreted in terms of uptake by a fixed number of binding sites, it was necessary that any increase in population during the period of the measurements be as small as possible ; it was, therefore, necessary to limit the analysis of the results to those obtained during the first few hours after the

EDTA added

I 06

I I I I I 2 4

Time (hours)

O b L

FIG. 2. The measurement of the " extracellularly " bound zinc in Phaeodactylum tricornutum. At zero time, EDTA was added to a zinc containing culture to give a concentration of 3 x 10-SM in the medium and the change in the cellular zinc content followed over the period shown. Two typical examples are illustrated. The initial rapid decrease, presumed to be due to desorption of zinc from the extracellular binding sites on the cells, was followed by a slower loss of more firmly bound metal. (After Davies, 1973.)

metal addition. It was found that, both with zinc and mercury, the metal accumulated by the cells increased linearly with the square root of the time elapsed after the addition of the metal to the culture, a relationship characteristic of a diffusion-controlled process (Crank, 1970) ; the results for mercury uptake by the two flagellates-plotted

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POLLUTION STUDIES WITH MARINE F'LANKTON-XI 387

in this instance as the weight of mercury which had been taken up across unit area of cell surface-are shown in Fig. 1.

Zinc uptake by Phueodactylum tricornutum differed from that for mercury uptake by the flagellates in that when the linear plot of cellular zinc content against d(t ime) was extrapolated back to zero time, it had a positive intercept on the ordinate. Davies' (1973) inter- preted this as indicating that, immediately upon addition of the zinc to the medium, rapid equilibration took place between the solution and the exchange sites, some of the metal becoming bound to the exterior surfaces of the cells, and this was followed by the slower movement of the zinc inwards into the cell cytoplasm. This view was supported by the finding that when the chelating agent, disodium ethylenediaminetetraacetate (EDTA) was added at a concentration of 3 x lO-3M to cultures containing zinc-laden cells, there was an almost instantaneous decrease in the metal content of the cells due, it was presumed, to desorption from the extracellular binding sites, and a subsequent much slower loss of intracellular zinc (Fig. 2). The extracellularly bound zinc determined in this way increased hyper- bolically with the concentration of zinc in solution in the medium in accord with the Langmuir adsorption isotherm indicating that there was a fixed number of zinc binding sites available on the cell surfaces. By subtracting the surface bound zinc from the total cellular zinc content, the weight of metal which had been bound intracellularly could be calculated and this too increased linearly with d(tirne).

Similar detcrminations of t,he easily removed mercury in Isochrysis galbana (Davies, 1974) and Dumliella tertiolecta (Davies, 1976) using thioglycollic acid as the chelating agont gave rather different results. In the case of Isochrysis galbana, the amounts of mercury extracted from the cells were greater than those taken up during the period illustrated in Fig. 1 and tended to be larger in older cultures. This suggested that the thioglycollic acid treatment had removed more than just the extracellularly bound metal and Cossa (1976) has since observed much the same effect on adding the sulphur-containing amino acid cysteine to cultures of Phaeodactylum tricornutum containing cadmium. Very little of the cellular mercury could be removed from Dunffiliella tertiolecta, however, probably indicating that most of the metal taken up by this species is precipitated in a refractory form-possibly as the sulphide (Davies, 1976).

As a result, it was not possible to determine the concentrations of surface bound mercury on the two flagellates in this way; but it was obvious from attempts to determine the mercury binding capacities of their surfaces using parachloro-mercuribenzene sulphonate (PCMBS)

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388 ANTHONY 0. DAVIES

which has a very low membrane penetrability and attaches only to exposed sulphydryl groups (VanSteveninck, Weed and Rothstein, 1965) that there were very few mercury binding sites on the exteriors of the cells, as the concentrations of PCMBS bound by the surfaces were too small to be measured accurately (Davies, 1976).

The relationship between the amount of material (Mt) taken up by diffusion across unit surface area of a solid initially free from the material and the elapsed time (t) is, during the early stages of uptake, Mt = 2C (Dt/.rr)* where C is the concentration of the diffusing substance adjacent to the surface of the solid and D is its diffusion coefficient within the solid (Crank, 1970). Davies (1973) showed that the slopes of the plots of the intracellular zinc content of Phaeodactylum tricornutum against z/(time) were in reasonable agreement with the idea that the metal adsorbed on to the exterior cell surfaces provided the driving concentration, C, for inward diffusion. As explained earlier, the values of C in the case of mercury were very low and could not be determined directly. Davies (1976) found that if the driving concentration, C, was assumed to be that of the mercury in the culture medium, the values obtained for the diffusion coefficients for entry of mercury into the cells were too large. As this could only have arisen if the assumed driving concentration was too small, it suggested that a build up in the mercury concentration at the surfaces of the cells, presumably by adsorption, must have taken place. In support of this, it was, in fact, found that the relative slopes of the linear plots of mercury taken up against z/(time) (Fig. lC, D) could be interpreted by assuming that the driving concentrations of mercury on the cell surfaces (C) were related to those in solution [Hg] by the adsorption isotherm

c = Cmax [Hgl/(k + [Hgl),

C,,, being the saturation value for C when [Hg] is large and k, the half saturation constant.

Thus the early stages of uptake of both zinc and mercury by initially metsl-free phytoplankton could be explained as being due to rapid adsorption of the metals on to externally exposed binding sites on the cell surfaces followed by passive, diffusion controlled transport into the cytoplasm at rates proportional to the concentration of surface bound metal.

An interesting feature of the results shown in Fig. lA, B was that the membrane of Dunaliella tertiolecta was markedly less permeable to mercury than that of Isochrysis galbana, which took up almost four times more of the metal across unit area of surface in the period of the experiment than the former species, Sick and Windom (1975) have

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POLLUTION STUDIES WITH MARINE PLA'NKTON-II 389

similarly found that mercury uptake by Dunuliella tertiolecta occurs considerably more slowly than by Carteria sp. and Cylindrotheca closterium (Ehr.) Reimann & Lewin as Nitzschia closterium Ehr. and this is believed to explain, at least in part, why Dunaliella tertiolecta has a greater mercury tolerance than most other species (Davies, 1976).

Cadmium uptake rates were considerably slower than those of mercury for the same species, the rate of uptake by Dunaliella tertiolecta being intermediate between that for Carteria sp. (the slowest) and Cylindrotheca (Nitzschia) closterium (Sick and Windom, 1975) suggesting that the mechanisms of uptake of mercury and cadmium are different.

At first sight, it seems surprising that a metabolically essential metal like zinc should be taken up passively. The work of Davies (1973) showed that metabolic control of zinc uptake is probably exercised indirectly by variations in the protein content of the cyto- plasm. By continuing the measurements of the zinc content of Phaeo- dactylurn tricornutum for several days after the initial period of frequent sampling, Davies (1973) estabhhed that the cellular zinc levels in the growing cultures reached a maximum 12-15 hours after the metal was originally added and then decreased steadily despite the availability of further zinc for uptake in the culture medium. Similar reductions in the zinc content of Phaeodactylum tricornutum cells in growing cultures have been observed by Hayward (1969) and Cossa (1976). Davies ascribed the decrease to a gradual reduction in the intracellular binding capacity of the cells as the population developed and it was suggested that, as most of the zinc taken up by the cells is probably bound to protein, this could have been related to the switch from protein to carbohydrate and lipid production in the cells as the nitrate in the culture medium was used up (Hobson and Pariser, 1971) as has been observed in other species of phytoplankton (Thomas and Dumas, 1970; Berland, Bonin, Daumas, Laborde and Maestrini, 1970).

Recently, Cossa (1976) made the interesting discovery that the cadmium contents of the cells in cultures of Phaeodactylum tricornutum continued to increase during the same period in which the zinc content was decreasing ; that this could have been due to competition between the two metals for the binding sites in the cells, the cadmium being taken up more slowly and displacing the already bound zinc, is sup- ported by the fact that in the stationary phase when the cells would have ceased to manufacture protein, there was a substantial decrease in the cellular cadmium contents. The changes in cadmium concentra- tions in the cultures studied by Motohashi and Tsuchida (1974) indicate that uptake of the metal during the growth phase followed by loss in the stationary phase also occurs in Skeletonem cosjatum.

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390 ANTHONY a. DAVIES

On the basis of the observations described, Davies (1973) proposed that the mechanism of zinc accumulation was along the lines illustrated in Fig. 3 ; i t was suggested that the rate of zinc uptake intracellularly was proportional to the concentration gradient across the cell membrane established by the quantities of zinc, q, and q,, adsorbed on the outside and inside of the diffusion barrier. The value of qo would, in accordance

Z n C a Z n Z n C a Z n

qmco 90 =- k O + G

I I I I I I

Ionic zinc I in intracellular fluid

11 I lntracellular zinc-

protein complexes

FIa. 3. Preliminary model for zinc uptake by Phaeodactylurn trkornu$urn. Initially, the zinc is rapidly adsorbed on to the extracellular binding sites outside the diffusion barrier presented by the cell membrane, displacing other sea water cations already occupying the positions. The quantity of zinc taken up in this way, q,, is related to the concentration in the sea water, C,, by the isotherm shown. Transport across the membrane then takes place a t a rate, AP/dT, which is proportional to the con- centration gradient created by the difference in the quantities of zinc bound to unit surface area of the outside (so) and the inside (q,) of the membrane, that on the inside rapidly attaining equilibrium with the concentration of intracellular, unbound metal ion, CI. Most of the intracellular zinc is bound on to protein, the maximal binding capacity of which (QM) regulates the value of C1 and, in turn, of q, thereby controlling the rate of zinc uptake (or loss when q,, - q, is negative).

with the experimental results, be related to the concentration of zinc in solution in the culture medium (C,) and q,, to the concentration of zinc in solution in the intracellular fluid (GI), in both cases through a Langmuir type adsorption isotherm. Inside tho cells, most of the zinc (Q) was expected to be associated with the binding sites on the protein and this would, therefore, control the value of C, and in turn of q,, equilibration intracellularly being a.ssumed to be rapid relative to the

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POLLUTION STUDIES WITH NAFtINE PLANKTON-I1 391

rate of zinc diffusion through the membrane. The concentration gradient governing the diffusion would thus be an indirect function of the protein content of the cytoplasm; while in the early stages of a culture when the protein in the celIs was high, it would cause an inward movement of zinc, the gradual decrease in the cellular protein as growth continued would eventually release sufficient of the metal into the intracellular fluid to cause a reversal of the concentration gradient (qo-qi) and diffusion of zinc out of the cells giving rise to the maximum observed in their zinc content. Davies (1973) showed that a compu- terized stepwise version of this model which incorporated the idea of the reducing cellular protein content predicted variations in the zinc levels in Phaeodactylum tricornutum which were in very good agreement with those which had been observed experimentally.

B. The effect of the chemical form of a metal upon its uptake by phytoptankton

A metal in sea water may be present in particulate forms andin solution. The particulate forms result from either the precipitation of an insoluble compound of the element, e.g. chromium hydroxide or lead carbonate, or adsorption of the metal ions on to other particulate material such as clay minerals and organic detritus in the water (Stumm and Brauner, 1975). The extent to which metals in these particulate forms are taken up by phytoplankton is difficult to judge, but adhesion of iron hydroxide-or more precisely hydrous ferric oxide-on to their surfaces has been observed (Davies, 1967) and probably represents the initial stage in iron assimilation by marine phytoplankton in chelator- free culture media and under natural conditions (Davies, 1970). Once in the vicinity of the cells, localized decreases in pH and the presence of reducing and chelating agents in the cell surfaces can be envisaged as mechanisms by which the particulate metals might be remobilized for inward transport. As metal hydroxides act as scavengers for other elements in sea water (e.g. Krauskopf, 11956 ; Ishibashi, Fujinaga, Kuwamoto, Sugibayashi, Sawamoto, Ogino and Murai, 1968) this process provides an additional mechanism by which toxic metals could enter phytoplankton.

Due to the lack of thermodynamic data for metals in sea water, the chemical nature of the dissolved forms remains, to a large extent, a matter of conjecture because it usually involves the extrapolation of stability constants for metal complexing obtained in solutions of a different ionic strength and composition. Predictions of the ionic speciation of the metals therefore depend on what are considered to be

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392 ANTHONY Q. DAVIES

the most reliable values for the stability constants and, as a result, often differ from one set of calculations to another (Stumm and Brauner, 1975). However, from an assessment of calculated and experimental data taken from the literature, Stumm and Brauner (1975) have listed the main inorganic species probably present in sea water ; for the metals covered by this review, they are: mercury 11, HgCli-; cadmium 11, CdCli ; silver I, AgC1,; nickel 11, Ni2+, NiCO:(?) ; selenium IV, SeOg-; lead 11, PbCO:, Pb(CO,)$; copper 11, CuCO:, &OH+ ; chromium 111, Cr(OH):, VI, CrOZ-; arsenic V, HASO:-; zinc 11, ZnOH+, Zn2+, ZnCO:.

Many of these elements will also be complexed to some extent by the organic matter dissolved in sea water. Evidence has already been obtained for the existence of organically-bound mercuxy (Fitzgerald and Lyons, 1973), copper (Williams, 1969; Foster and Morris, 1971) and zinc (Fukai, Huynh-Ngoc and Murray, 1973), and as the proportions of the metal complexed in this way have been found to vary on a seasonal basis (e.g. Foster and Morris, 1971; Fukai et al., 1973), it seems likely that the organic matter is released by phytoplankton.

Precisely which chemical species enter the phytoplankton is not known, but there is quite a lot of evidence to suggest that organically- bound metals are not taken up. The extraction of zinc and mercury from phytoplankton by chelating agents was discussed in the previous section (see Fig. 2, p. 386) ; this happens because the formation of the chelates reduces the concentration of the inorganic forms of the metals in solution in the culture medium thereby reversing the concentration gradient across the cell membrane causing the metal to diffuse out from the cells. If the metal chelate itself was taken up, there would have been either no removal of metal if the chelate entered the cells rapidly, or a gradual rise in the cellular metal content after the initial decrease if it entered slowly; as is evident from Fig. 2, neither of these possibilities took place. Other indirect evidence that organic metal complexes are not assimilated by phytoplankton has been obtained by Myers et al. (1975) who found that the presence of humic acids decreased the amount of mercury taken up by Cyclotella menenghiniana, and by Cossa (1976) who showed that Phaeodactylum tricornutum accumulated less cadmium in the presence of 35 pM EDTA than in its absence. The most direct evidence that organically bound metals are not taken up by phytoplankton has been provided by Sunda and Guillard (1976) who grew Thalassiosira pseudonana (Hustedt) Hasle & Heimdal in a culture medium containing copper and a. range of concentrations of the chelating agent TRIS (trishydroxymethylamino methane). The copper content of the cells in 3-4 day old cultures was

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POLLUTION STUDIES WITH MARINE PLANKTON-II 393

shown to be hyperbolically related, not to the total copper concentration present in the medium, but to the activity ( = concentration x activity coefficient) of the unchelated cupric ion (Fig. 4) ; as Sunda and Guillard used the same culture medium for each total copper con- centration, the activity coefficient of the ions can be regarded as having remained constant, so that, in effect, the cellular copper levels were controlled by the free cupric ion concentrations in the culture media. It should however be remembered that although, in principle, the

-log (Icu

FIG. 4. The cellular copper content in 3-4 day old cultures of Thalassk&rapseudonuna as a function of acu, the activity of the uncomplexed cupric ion in the medium. The activity was varied both by changing the total amount of copper present and by adding t,he following concentrations of TRIS (a11 in mM) : A, 1 ; A, 2 ; 0 , 3 ; 0, 6. The curve was calculated from the isotherm: copper/cell = 4-8 X a,./(a,, +

(After Sunda and Guillard, 1976.)

relationship obtained experimentally-copper (fg at)/csll r= 4.8 x acu/(acu + 10-9'2)-depends only upon the ionic strength of the culture medium, variations in the biochemical composition of the phytoplankton at different nutrient concentrations or salinities could cause changes in the values of the two constants outweighing any activity coefficient corrections necessary to allow for differences in ionic strengths.

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304 ANTHONY 0. DAVIES

Thus only the inorganic ionic species and possibly only the free ions in the case of the electropositive elements are taken up by phyto- plankton. As the equilibration between the free hydrated metal ions and the complex ions formed with the anions present in sea water generally takes place very rapidly (Wilkins, 1975), the rate of uptake of a metal, in the absence of organic complexing, is unlikely to be limited by the concentration in the medium of the ionic species which enters the plant cells, as redistribution will quickly occur to replace the depleted species; in these circumstances, the rate of uptake will, in fact, be proportional to the total concentration of metal in the medium. However, the rates of dissociation of metal-organic complexes are often quite slow and if a large part of a metal is bound by an organic compound, its rate of uptake would, once the inorganic species have been incorporated, be limited by the rate at which the metal is released from the complex. This effect can be very important in studies of metal uptake using radioactive tracers, for if the label does not quickly exchange with the other chemical forms of metal present in the water due to the slow dissociation of the organically bound fraction, it could be taken up preferentially by remaining in the inorganic form. This situation appears to have occurred in the experiments of Bernhard and Zattera (1969) who were studying zinc uptake by Phaeodactytum tricornutum. They found that the s6Zn tracer added in an inorganic form was accumulated by the plant cells disproportionately to the stable zinc present in the sea water which formed the basis of the culture medium, and this resulted in a higher specific activity of 66Zn in the phytoplankton than in the water. Later work showed that, even after a year, ionic zinc added to sea water had not equilibrated with the naturally present metal (Piro, Bernhard, Branica and Verzi, 1973).

The presence in culture media or the sea of organic compounds which form complexes with heavy metals will thus not only compete for and thereby reduce the amount of the metals which are taken up by phytoplankton but will also decrease their rate of incorporation into the plant cells, both processes providing a degree of protection against the toxic effects of the metals.

In view of this, the amounts of metals taken up by phytoplankton from culture media containing high concentrations of chelating agents and heavy metals (e.g. Hayward, 1969; Riley and Roth, 1971) would be expected to bear little relation to the levels of metals present in natural populations. Surprisingly, many of the data obtained by Riley and Roth (1971) for the metal contents of cultured phytoplankton lie within the ranges found for metal levels in natural phytoplankton

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POLLUTION STUDIES WITH MARINE PLANKTON-II 395

from different sea areas (Appendix 11), though as there appears to be a correlation between the amount of metal taken up by the cells and the concentration initially added to the culture medium (Riley and Roth, 1971), the agreement must, to a large extent, be fortuitous.

C. The role of phytoplankton in the biogeochemistry of heavy metals in the sea

Because phytoplankton cells accumulate heavy metals, it would seem reasonable to expect, that there should be an inverse correlation- possibly on a seasonal basis-between primary production and the concentrations of the metals present in dissolved form in sea water. Further, the eventual sedimentation of dead or senescent plant cells provides a mechanism by which heavy metals introduced to the surface layers of the sea might be transferred to the bottom sediments.

Direct evidence of the influence of phytoplankton on the geo- chemical cycling of heavy metals in the sea has proved difficult to obtain. Although Morris (1971) found that changes in the particulate concentrations of copper and manganese in the Menai Straits, North Wales, reached maxima which coincided with peaks in the flagellate counts during a bloom of Phaeocystis sp., and there was an inverse relationship between the dissolved and particulate levels of manganese, there was little sign that the Phaeocystis had had a.ny effect upon the concentrations of dissolved copper ; this finding was later confirmed by Foster and Morris (1971) who, over a two year period, were unable to detect any changes in the copper concentration in the same area which correlated with the seasonal growth of phytoplankton. Parti- culate copper concentrations in the sea off Florida had previously proved to be unrelated to chlorophyll a levels (Alexander and Corcoran, 1967) and Spencer and Brewer (1969) had failed to fhd any variations in the concentrations of copper, zinc or nickel in the Gulf of Maine or the Sargasso Sea which could be attributed to biological effects even though it was estimated on the basis of the phosphate utilization in the top 50 m and using a value of 6.5 x 10-3 for the copper/phosphorus ratio in phytoplankton, tha t the copper should have been depleted by about 18% if all of the metal removed by the plant cells sedimented out and none was regenerated in the upper layers. Knauer and Martin (1973) used primary production rates and metallcarbon ratios obtained from their own data to estimate the amounts of cadmium, zinc, copper and manganese likely to be taken up by the phytoplankton population in Monterey Bay, California. They found that only in the case of cadmium would the quantity of metal accumulated by the plankton be

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306 ANTHONY U. DAVIES

large enough to produce a detectable change in its concentration, and the observed reduction in the dissolved cadmium was in quite good agreement with the calculated decrease. The validity of such calcula- tions is strengthened by recent evidence on copper uptake by phyto- plankton communities entrapped in large volume water enclosures where the sinking of phytoplankton occurs unhindered by turbulent mixing (Thomas and Seibert, 1977) for Topping and Windom (1977) showed that, up to values of about 10 mg carbon/m3/h, there is an approximately linear relationship between the rates of primary production and the rate of copper sedimentation in sinking plant cells.

It seems likely, in fact, that it will be impossible to demonstrate directly variations in metal concentrations in the sea due to their uptake by phytoplankton until the methods for measuring metals in sea water are improved; for, as Topping (1974) has pointed out, the biologically induced variations in dissolved metal levels in oceanic or coastal areas of low to medium productivity are, in most cases, smaller than the errors associated with the analytical methods and are, there- fore, unlikely to be detected.

Indirect evidence of the influence of phytoplankton upon metal concentrations is now beginning to be provided by the findings that linear correlations exist between the concentrations of certain metals and the major nutrient ions, nitrate and phosphate. Boyle and Edmond (1975) took samples across the circumpolar current south of New Zealand where there were large horizontal gradients in the chemical properties of the water and found that the copper concentrations were directly proportional to the nitrate ion concentrations. Similarly, Martin, Bruland and Broenkow (1976) showed that, in the waters off Baja California, where upwelling causes sharp gradients in the con- centrations of nutrients in the surface layers, cadmium concentrations were linearly related both to nitrate and to phosphate levels. Boyle and Edmond (1975) have rightly stated that such results do not in themselves establish a biological cause for the relationships between heavy metal and nutrient concentrations, but that this is a satisfactory explanation for them is supported by the finding that there was also it significant correlation (coefficient = 0.71) between the amounts of cadmium and phosphorus in the phytoplankton collected off Baja and analysed by Martin et al. (1976) indicating that the two elements were always taken up in a constant ratio (Cd/P = 4.9 x

The interpretation of trace element concentrations and ratios in particulate materials is increasingly yielding useful information about the biogeochemical cycling of heavy metals in the sea. Spencer and Sachs (1970) showed that, while zinc and copper concentrations in

by atoms).

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POLLUTION STUDIES WITH MARINE PLANKTON--II 397

suspended material collected from deep water (> 150 m) in the Gulf of Maine were both linearly correlated with the aluminium content of the solids, the levels of both heavy metals in the particulates present in the surface waters were considerably higher than would have been expected on the basis of their aluminium content. Spencer and Sachs attributed these elevated zinc and copper contents to biological accumulation and pointed out that the much lower concentrations present in the suspended matter in the deep water implied that a large proportion of the heavy metals taken up by the solids in the surface layers must have been released back into solution as the particulates sank through the water column. The importance of biological accumulation in the recycling of heavy metals seems to depend on the particular element, however, for Chester and Stoner (1975) concluded from a study of trace element concentrations in suspended matter gathered from the surface waters of several oceans that, while lead and zinc are probably associated mainly with plankton, copper is distributed between plankton and other particulate materials.

An interesting suggestion about the way in which phytoplankton might modify the geochemical cycling of heavy metals in certain locations was put forward by Schutz and Turekian (1965). It was found that the waters in upwelling areas tended to have higher than normal concentrations of the metals silver, cobalt and nickel and that, furthermore, the concentrations generally increased with depth. Schutz and Turekian postulated that the localized build-up of metals could be due to the metals being removed from the newly upwelled water by the abundance of phytoplankton resulting from the high nutrient levels and then carried downwards in the sinking plant cells, the metal-depleted water moving away from the area. As the cells sink downwards through the water, it was suggested that regeneration of the metals would elevate their concentrations in the inflowing sub- surface currents which provide the source of the upwelling water ; thus, over a long period of time, there would be a gradual increase in the metal concentrations present in the water coming to the surface until, finally, a steady state situation would be established. The absence of normal mixing processes would, as a result, produce extended residence times for the metals in such situations.

An increase in cadmium levels which might have resulted from such a process has been observed in the upwelling area off Baja California for concentrations of the metal in phytoplankton collected from the area were substantially higher than in oceanic plankton (Appendix 11) (Martin and Broenkow, 1975; Martin et al., 1976). Martin et al. (1976) found that the cadmium concentrations in the water

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398 ANTHONY Q. DAVIES

increased rapidly with depth from about 7ng/l at 25 m to almost 70 ng/l at 80 m ; similar increases occurred in the nitrate and phosphate concentrations as expected in upwelling situations. The observations support the view that heavy metals contained in sinking phytoplankton are returned to the water before t,he decaying cells reach the bottom and, 5 ~ s Schutz and Turekian (1965) have stated, there appears to be no evidence to suggest " that significant quantities of these elements are permanently removed as deep sea sediment by this mechanism ",

111. LABORATORY STUDIES OF THE TOXIC EFFECTS OF HEAVY METALS UPON PHYTOPLANKTON

A. The effects on the growth ofphtJoplankton

Numerous studies have been made of the effects of heavy metals upon the growth and metabolism of laboratory cultures of marine phytoplankton and most of the results currently available are sum- marized in Appendix I. An immediately noticeable feature of the data is the remarkable diversity of techniques and culture conditions used to obtain them; the phytoplankton cells have been grown in batch cultures-the most usual experimental method, continuous cultures (Rice, Leighty and McLeod, 1973 ; Kayser, 1976 ; Bentley- Mowat and Reid, 1977) and dialysis cultures (Jensen, Rystad and Melsom, 1974, 1976) under various conditions of temperature and illumination ; culture media have ranged from unenriched natural sea water (Erickson, 1972; Rice et al., 1973; Jensen et al., 1974, 1976) to artificial sea water containing high concentrations of nutrients and chelating agents (Nuzzi, 197.2)) and the parameters used as indices of the toxicities of the metals have included changes in growth rate (e.g. Davies, 1974; Sunda and Guillard, 1976)) the cell population after a certain period of growth (Erickson, Lackie and Maloney, 1970; Rosko and Rachlin, 1975) and effects on the rate of photosynthesis measured by 14CO, utilization (Tkachenko, Mortina and Lukankina, 1974; Zingmark and Miller, 1975) or oxygen production (Saraiva, 1973; Overnell, 1976).

Each of the culture methods has advantages and disadvantages which should be borne in mind when considering the data obtained with them. Batch cultures are straightforward to set up and allow the simultaneous study of the effect of a wide range of metal concentrations upon cell populations all taken from the same stock culture and, therefore, initially in the same physiological condition ; however, the growth of the phytoplankton in batch cultures causes changes in the

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POLLUTION STUDIES WITH MARINE PLANKTON-= 399

culture medium, and variations in the biochemical composition of the plant cells due to depletion of the nutrients, and the possible modifi- cation of the chemical form of the metal by the extracellular products could both influence the outcome of the experiments. Continuous cultures have the advantage of allowing the establishment of a physio- logically stable population growing in an unchanging environment so that the effects of metals can be studied both in the short term as a single pulse of contamination, or in the longer term, by continued exposure to a chosen concentration of a metal ; to examine the effects of a range of metal concentrations takes a considerable time, however, due to the necessity of allowing the chomostat to reequilibrate when the concentration is changed and a further disadvantage of continuous cultures is that, for many metals, i t is not possible to use chelator-free culture media as adsorption of the metals would contaminate the complex system of pipe work associated with the apparatus. Dialysis cultures are useful as they permit the study, over an extended period, of the growth of a unialgal culture in natural unenriched sea water. The nutrient levels are maintained by inward diffusion from the water surrounding the membrane containing the culture and the build-up of extracellular products avoided as they diffuse out of the culture. Jensen et al. (1974, 1976) have demonstrated that it is possible to maintain a constant metal concentration in such cultures by the continuous feed of a stock solution. The main disadvantage of dialysis cultures is, as in batch cultures, that as the population increases, it eventually outstrips the nutrient supply and this is likely to cause changes in the biochemical status of the cells and hence their susceptibility to metals.

Because of the wide range of techniques and conditions used, analysis of the data in Appendix I poses many problems. It is certainly impossible to extrapolate the results from most of the experiments to predict the effects of metals upon phytoplankton in the sea for, whereas the cultures have usually contained high densities of cells growing in nutrient-rich media under near optimal conditions, natural populations of phytoplankton are normally of low density and their growth, often in nutrient-depleted water, is regulated by a complex pattern of interacting and ever changing chemical, physical and meteorological factors.

The experimental data summarized in Appendix I do, however, point to the factors which influence the toxicity of heavy metals towards phytoplankton; the most important ones appear to be: (1) the phytoplankton species, (2) the composition of the sea water supporting the plankton, (3) the cell population, (4) changes in the

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400 ANTHONY a. DAVIES

metal tolerance of the cells or in the chemical state of the metal during the period of growth and, of course, ( 5 ) the concentration of the metal. Although temperature and the level of illumination are also likely to influence the effects of metals because they regulate the metabolic activity of phytoplankton, they have been insufficiently investigated to warrant discussion a t the present time.

1. Metal toxicity in relation to the species of the phytoplankton Where workers have used the same culturing conditions and

medium to study the effect of one metal upon a range of species of phytoplankton, it is possible to place the species in order of their metal tolerance ; examples for mercury and copper are given in Table I. In comparing the susceptibilities of different species to the metals, it must be remembered that because the measurements have been made in a standardized way, some species may have been growing under sub- optimal conditions and may, therefore, have been less resistant to the effects of the metals than they might have been in a more favourable situation.

The order of metal tolerance in Table I confirms previous experience with anti-fouling paints that green algae are generally more mercury tolerant than brown algae and diatoms, and that the situation, although less clear-cut, is largely reversed with copper (G. T. Boalch, private communication). Dunaliella tertiolecta is the exception to this being extremely resistant both to mercury (Davies, 1976) and copper (Mandelli, 1969). It is significant, in this respect, that this species releases hydrogen sulphide during growth (Craigie, McLachlan, Majek, Ackman and Tocher, 1966) though Davies (1976), who found that the concentrations of sulphide which appeared in cultures of Dunaliella tertiolecta were insufficient to remove the mercury by precipitation until the populations were entering the stationary growth phase, has suggested that the metals are probably detoxicated intracellularly by the formation of their highly insoluble sulphides. It is interesting that while the production of dimethyl-@-propiothetin by Tetraselmis spp. (Craigie et al., 1966) seems to confer mercury but not copper tolerance upon this genus, with Phaeodactylum tricornutum which is believed to produce dimethyl sulphide (Armstrong and Boalch, 1960), the reverse is true. The biochemical basis of heavy metal tolerance is, in general, very poorly understood.

2. Metal toxicity in relation to the composition of the culture medium The addition of soil extracts or synthetic chelating agents to marine

culture media in order to maintain the availability of essential trace

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t K

TABLE I. ORDER OF HEAVY METAL TOLERANCE IN PWTOPLANKTON SPECIES ~

Reference 1 ~ n d e z of tolerance Species in order of increasing metal tolerance - 01

A. MERCURY

w Growth Isochry& galbana < Skeletonema costatum < Phaeodactylum tricornutum < Dunaliella tertiolecta

coatatum = Attheya decora West < Brachiomonaa aubmarina Bohlin < Dunaliella tertiolecta G y m d i n i u m splendens Lebour < Scrippsiella faeroeme (Paulsen) Balech & Soares < Pro-

Davies (1974) Oxygen evolution Pavlova (Monochrysis) lulheri (Droop) Green < Phaeodactylum tricornutum = Skeletonema Overnell (1976) 0

3 rocentrum micana Ehrenberg 0

E I 3

2

3

Growth

Growth

Kayser (1976)

Berland et al. Lauderia borealis Gran < Skeletonema wstatum < Thalmawaira paeudonana = Exuviaella mariae-

Ehrenberg = Phueodactylum triwrnnutum < Pavlova (Monochrysis) lutheri = Monallantw salina Bourelly = Porphyridium marinum Kylin < Prasinockadua marinua (Cienk.) Waern < Cylin-

w lebouriae Parke & Ballantinen < Amphidinium carterae Hulburt < Pavlova pinguia Green = Chaetoceros didymzrs Ehrenberg < Chlamydomonm palla Butcher = Fragillaria phna ta

(1976)

drothecu closterium (Ehrenb.) Reimann & Lewin < Heterothrix sp. = Cryptomonas paeudo- bdtica Butcher < Tetraaelmis atriata Butcher

1 B. COPPER Growth Coccachloris elabena (Breb.) Dr. & D. < Exuviaella ~ p . ~ < alenodinium foliaceum Stein < Cylindro- Mandelli (1969)

them (Nitzachia) doaterium < Skeletonema costaturn < Thulaaaioaira jluviatilia Hustedt < Thalaaaioaira paeudonana (Cyclotella nana) << Dunaliella tertiolecta

!4

T3 8 I

M

ii Growth O&9thdkCU8 luteua N. Carter < Iaochryaia gdbana < Amphidinium carteme < Thalaaaiosira

paeudonanh (Cyclotella nana) < Skeletonema wstatum < Dundiella tertwlecta Oxygen evolution Brachhaonaa submarina < Skeletonema wstutum < Attheya dewra = Dunaliella tertwlecta <

Pavlova (Monochryk) lutheri < Phaeodactylum tricornutum Growth Exuviaella mariae bbouriae" < Porphyridium marinum < Thulaasioaira paeudonana < Amphidi-

nium carterm = Chaetoceros didymua < Lauderia borealis < Chlumydomonas palla < Prasino- cladua marinua < Pavlova (Monochryma) lnutheri < Tetraaelmis atriata = Skeletonema coatatum = Cryptomonaa paeudobaltiea < Cylindrothecu closterium < Monallantua salina < Fragilarh pinnatu < Pavlova pinguis = Phaeodactylum tricornutum = Heterothrix sp.

Eriokson et d.

Overnell (1976)

Berland et al.

(1970)

I3 (1976)

a. Now in Proorcentrum minimum (Pav.) J . Schiller. 0- 0 b. Now in Prorocentrum sp. rp E

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402 ANTHONY a. DAVIES

metals-especially iron-by keeping them in solution is now routine, as is the use of organic pH buffers such as TRIS and glycylglycine which are also metal chelators (McLachlan, 1973). Most of the data given in Appendix I were obtained using culture media containing such compounds.

The calculations of Spencer (1958) indicate that even at EDTA concentrations as low as 10 pM-the order of magnitude of that present, for example in the much-used f/2 culture medium of Guillard and Ryther (1962)-metals like copper and zinc are present largely in chelated form. The data of Sunda and Guillard (1976) similarly show that in a culture medium containing 2 mM TRIS, as used by Berland, Bonin, Kapkov, Maestrini and Arlhac (1976), only about 0.03y0 of the copper added to give a concentration of 1000 pg/l would be present as uncomplexed cupric ion (assuming an activity coefficient of 0.2).

In view of the evidence discussed earlier (Section 11, B) that the quantity of a metal taken up by phytoplankton is related to the concentration of unchelated metal in the culture medium, the earlier observations that the toxicity of, for example, copper is moderated by the presence of chelating agents (Steemann-Nielsen and Wium- Anderson, 1970; Davey, Morgan and Erickson, 1973) can be explained as being due to the lower cellular burdens of the metal accumulated by the phytoplankton in the presence of the chelators. The toxicity of a metal, determined a t a certain to td concentration in a culture medium, will therefore bear little relation to the effects caused by the same concentration of the metal in natural waters having considerably lower levels of chelating materials, and it is significant that the lowest inhibitory metal concentrations have been observed in cultures using either natural sea water or enriched sea water containing no added chelating agents.

Another aspect of the composition of culture media used to study the effects of heavy metals upon marine phytoplankton which is often overlooked is the high concentrations of the nutrient ions, nitrate and phosphate, which are frequently present. Hannan and Patouillet (1972) have, in fact, suggested that the toxicity of metals towards phytoplankton may be inversely related to the available nutrient levels but as, in their experiments, the concentrations were varied by dilution of the culture medium with 3.5% sodium chloride, the concomitant changes in the cation balance may also have influenced their results.

On the other hand, although it is not feasible to carry out extended growth studies at the nutrient levels normally present in sea water, the enrichments made to culture media have often been considerably in excess of those necessary to allow the cultures to develop for the

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POLLUTION STUDLES WITH MARINE PLANETON-II 403

minimal period required-in batch cultures, 1 to 3 weeks depending on the growth rate of the plankton-to determine the effects of heavy metal additions. By keeping the nutrient levels as low as possible, the density of the cell populations is reduced and, as will be seen in the next section, this allows a more realistic appraisal of the toxicity of a metal to be made.

3. Metal toxicity in relation to the cell population Whilst it is possible to determine the effect of a heavy metal upon

the growth of unialgal cultures containing cell populations similar in numbers to thoso which occur naturally by measuring their photo- synthetic rates over short periods using 14C0, (e.g. Zingmark and Miller, 1975), in order to study the effects on the growth and physiology of cultures over extended periods, it is necessary to use cell densities which are considerably higher than those normally present in the sea in order to facilitate measurements of the changes in the population with time. However, as can be seen in Appcndix I, it has not been uncommon for extremely high cell densities to be used and this can give rise to an underestimate of the toxicity of a metal when determined as the concentration which must be added to the culture to cause growth inhibition.

The reason for this lies in the fact that i t is the quantity of metal taken up by the cells which ultimately determines its effect upon their growth and, in dense cultures, more metal is required to produce a given cellular burden. The effect of different populations upon the amount of metal accumulated by individual cells may be illustrated by assuming that metals are bound by the cells in accord with the Langmuir adsorption isotherm

qmc c l =

k + c

where g = weight of metal bound per cell, q, = maximal value of q when c, the concentration of metal in solution in equilibrium with the cells, is very large and k is the half-saturation constant. (Sunda and Guillard (1976) have shown that copper uptake by Thalassiosira pseudonana can be described by an expression of this form.) For the purpose of these calculations, qm is put equal to 100 fg/cell, as Davies (1976) found that the maximal mercury binding capacity of Isochrysis galbana was about 300fg/cell, and the data of Sunda and Guillard (1976) indicate that Thalassiosira pseudonanu can accommodate up to 30 fg copper/cell ; and the half-saturation constant k is given the value of 2 pg/l, the figure for any particular phytoplankton species depending

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404 ANTHONY Q. DAVIES

upon the affinity of its binding sites for the metal. The values of q and c for a total metal concentration, T, may then be calculated by solving the equation

for different values of N, the cell population in numbers/ml. The results for two values of T are given in Table 11. The actual values obtained for q and c depend, of course, upon the constants q, and k but it can be seen that whereas, up to densities of lo4 cells/ml, the metal burdens of the cells would be little affected by changes in population, a t higher

TABLE 11. AN ILLUSTRATION OF THE EFFECT OF PHYTOPLANKTON POPULATION DENSITY UPON THE METAL BURDEN ACCUMULATED BY INDIVIDUAL CELLS

T N 108 108 104 106 106

2-4 2.3 1.6 0.42 0.06 ca. 0.050 0.048 0.033 0.008 ca. 0

71 68 38 4.9 4.93 4-32 1-22 0.10

0*060

5.0

T: Total metal concentration (pg/l). q : Metal burden of each cell (fglcell). c : Concentration of metal in solution at equilibrium (pg/l).

N : Population density (number of cells/ml).

cell numbers, because most of the metal is contained by the cells, the cellular metal content becomes approximately inversely proportional to the population density. Thus, if the response of the cells to the metal is a function of the amount they have taken up, there would be an apparent inverse relationship between the toxicity of the metal and the cell numbers. Although this effect has not been demonstrated using marine phytoplankton, it has been shown to occur in cultures of Chlorella pyrenoidosa Chick containing copper (Steemann-Nielsen, Kamp-Nielsen and Wium-Anderson, 1969) and mercury (Kamp- Nielsen, 197 1 1.

Quite a few of the data in Appendix I were obtained using cultures containing populations of more than 105 cells/ml and in those cases where changes in optical density or turbidity were used as a measure of growth, the cell numbers could have been substantially higher than this. Measurements of the effects of metals upon such dense cultures provide little ecologically useful information.

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POLLUTION STUDIES WITH MARINE P-KTON-If 406

4. Metal toxicity in relation to changes in the metal tolerance of the celle or in the chemical state of the metal during the growth of the culture Stockner and Antia (1976) have suggested that short term investi-

gations lasting only a few hours or days of the effects of pollutants upon marine phytoplankton take insufficient account of the possibility that the cell populations being studied might, given the time, adapt to the hostile conditions created by the presence of the contaminant in the culture. They have given several examples of how, after extended lag phases, cultures containing high levels of pollutants have grown at near normal rates ultimately producing populations comparable with those in uncontaminated controls.

While the existence of strains of other microorganisms and higher plants with acquired metal tolerance is well documented (Ashida, 1965 ; Antonovics, Bradshaw and Turner, 1971), there is little informa- tion on the development of heavy metal resistance in marine phyto- plankton although it is certainly possible to " train " them to withstand levels of metals which would normally be lethal (A. G. Davies, unpublished work).

The resurgence of growth in polluted cultures may not always be of biological origin, however, because the detoxication of the contaminant due to its transformation chemically can cause a similar result. Stockner and Antia (1976) cited several examples of such changes but none were related to metals. There are, though, many cases in the literature where cultures have been found to recover from the toxic effects of mercury added in the inorganic form (e.g. Rice et at., 1973; Davies, 1974; Zingmark and Miller, 1975; Kayser, 1976). The sequence of events leading to a delayed outburst of growth in a mercury-conhining culture of Isochrysis galbana is shown in Fig. 5. The concentration of mercury present in particulate form, i.e. in the plant cells, at first increased as the phytoplankton grew but although, on the fifth day, the cell numbers stopped increasing, the plankton continued to take up more of the mercury. During the same period, the total concentration in the culture (medium plus cells) decreased steadily due to the chemical reduction of the ionic mercury by the extracellular products from the cells, the resulting elemental form being removed by volatilization into the air being bubbled through the culture. By the fifteenth day of the experiment, all of the mercury remaining in the culture was contained by the plant cells and, at this time, the cells began to grow again eventually attaining a population density similar to that in the mercury- free control. It is not clear why the disappearance of mercuric ions from the solution bathing the cells enabled growth to recommence but

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406 ANTHONY 0. DAVIES

it may be because the metal interferes with the uptake of the nutrients required for growth.

The effective concentrations of metals in cultures may also be decreased by precipitation, adsorption on to the culture vessels and chelation by extracellular products. Studies of the effects of metals

lo r

c c o n

Days

FIQ. 6. The changes which took place in a mercury containing culture of Isochysis galbana. 0, total mercury concentration (pg/l) in culture (medium + cells); 0, particulate mercury concentration (pg/1) in cells ; a, mean mercury concentration in cell material (fglpms); X, volume of cell material in culture (pms/ml o f medium). By day 5, the mercury content of the cells W R Y sufficient to prevent growth but it continued to increase after this. Volatilization of metallic mercury from the culture caused the total concentration to decrease steadily until day 15 when the metal remaining was all contained in the phytoplankton. This led to a renewed outburst of exponential growth giving a final cell population, measured as the volume of cellular material present in the culture, approaching that attained in the mercury free control. (From Davies, 1974. Reproduced by kind permission of the Council of tho Marine Biological Association of the United Kingdom.)

upon phytoplankton based merely on observing changes in the cell populations with time obviously take no account of these possibilities.

5. Metal toxicity in relation to Concentration In view of the foregoing discussion, it will be appreciated that the

concentration of metal added to a culture medium a t the start of an experiment gives very little guide to the amount of metal incorporated by the cells and which will, therefore, influence their metabolic activity ; and although it was demonstrated some time ago that quantitative

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POLLUTION STUDIES WITH MARINE PLANKTON-II 407

relationships exist between the growth rates of phytoplankton and the cellular contents of nutrients such as vitamin B,,, iron and silicate ion when they are rate-limiting (Droop, 1968 ; Davies, 1970 ; Paasche, 1973), few attempts have been made to establish similar expressions for growth-inhibiting toxic metals, most authors having been content to

Experiment 1 Culture

' C D E F A U V X

X X

I I\ x-xJx .. . I

M

B. Experiment 2 Symbols as above

0.6

0.4

0.2

X

X

x ' I X-

1 2 3 4 5 6 0

4r (fg H g l w 3 )

FIG. 6. The ratio of the specific growth rate (pH.) of mercury containing cultures of 18ochq& galbana to that in the mercury free control (p,) plotted &B a function of the mercury content of the cell material (qt). The concentrations of mercury initially addedtothecultureswere (inpg/l) :Experiment 1 ;B, 16S;C, 3.30; D, 6.08;E,7.60; F, 10.5. Experiment 2 ; B, 0.77 ; D, 3.30 ; E, 6.08 ; F, 7.60. Most of the data approximate to the line pEs/pm = 1 - q,/K,, K,, the intercept on the abscissa, representing the concentration of mercury in the cell material which just prevented growth. The points having q, values greater than K, correspond to the renewed growth which took place after loss of the mercury from the culture medium (see Fig. 6). (From Davies, 1974. Reproduced by kind permission of the Council of the Marine Biological Association of the United Kingdom.)

present their data simply as the reduction in growth rate or the cell population after a certain time as compared to the values in a metal- free control.

That the growth rate of a culture is related to the heavy metal content of the phytoplankton cells has been shown in two cases-for mercury containing Isochrysis galbana (Fig. 6) (Davies, 1974) and copper containing Thalassiosira pseudonana (Fig. 7) (8unda and

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408 ANTHONY G. DAVIES

Guillard, 1976). However, as most of the metal taken up by cells is likely to be bound on to structural rather than metabolic sites and the number of the former decreases as the nutrients are used up due t,o the switch from protein to carbohydrate and lipid production (Section 11, A), it might be expected that a given cellular burden of a heavy metal would prove to be more inhibitory in older cultures as more of the

Mean cellular copper content (fg at Cu/cell)

FIQ. 7. The specific growth rate (p) of copper containing cultures of Thalussiosira pseudonunu plotted as a function of the mean copper content of the cells. Concentra- tions of TRIS present in culture medium were (mM) : A, 1 ; A, 2 ; 0, 3; 0, 5. The shape of the plot suggests that there are at least two types of copper-sensitive metabolic sites in the cells. (After Sunda and Guillard, 1976.)

metal content of the cells would be free to attach to vulnerable enzyme systems.

Davies (1 974) found no evidence that this was the case for the effect of mercury upon Isochrysis galbana, but preliminary experiments with cadmium (A. G. Davies, unpublished data) have shown that as the cultures develop, the cells in them become less metal resistant. The effect of cadmium upon the growth of Isochrysis galbana is illustrated in Fig. 8. When the specific growth rates of the phytoplankton were plotted against the cadmium content of the cells using data taken from cultures containing differing levels of cadmium but having the same content of cell material and therefore the same remaining nutrient

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POLLUTION STUDIES WITH MARINE PLANKTON-= 409

Fro. 8. The effect of cadmium upon the growth of Isoch.rysis galbana. Culture conditions were as given in Davies (1974), the population being measured, as previously, as the volume of cell material/ml of culture. Analysis of the experimental data in terms of the fractional reduction in specific growth rate caused by the cadmium w8.s carried out for the four cell densities designated A, B, C and D (see Fig. 9).

concentrations (A, B, C and D in Fig. 8 ) , it was found (Fig. 9) that they lay on a series of curves having the general form

! % = 1 - _ - - "qGd Pmax b-qm

where pGa was the specific growth rate in the culture containing cadmium, pmax was that in the control, q,, was the cadmium content of the cell material (in ag cadmium/pm3 in Fig. 9) and a and b were constants for a given nutrient level. The increasing curvature of the plots supports the idea, as outlined earlier, that cells growing in batch

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410 ANTHONY Q. DAVIES

cultures could become more susceptible to the effects of metals as the nutrients are utilized and this would obviously be quite important environmentally.

Further investigations into relationships of this type are required, for once it is established that there is a definite link between the growth rates of phytoplankton and their heavy metal contents, variations in

a b A 1.5 360

2 0 4 0 ao 120 140

a b

FIG. 9. The ratio of the specific growth rate (pea) of cadmium containing cultures of Isoehrysis galbana to that in the cadmium free control (pmax) plotted as a function of the cadmium content of the cell ma.terial (qcd). The four sets of data A, B, C and D correspond to the four cell densities shown in Fig. 8, the curves being calculated from the expression pCd/pmsx = 1 - aqcd/(b-qca) using the values for the constants a and b given in the diagram. The increasing curvature of the plots and their decreas- ing intercepts on the abscissa as the populations in t,he cultures increased both reflect the reduction in cadmium tolerance of the older cells.

the concentrations and availability of metals in culture media will become of secondary importance to the amount of metal taken up by the cells which can easily be determined; and furthermore, as shown in Section V, it would become possible to utilize the data on the heavy metal contents of natural phytoplankton populations to assess the extent to which their growth might be affected by the metals present.

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POLLUTION STUDIES WITH MARINE PWKTON-II 41 1

B. Synergism a d antagonism of mixtures of heavy metals towards phytoplankton

The only studies of the effects of mixtures of metals upon marine phytoplankton seem to be those of Braek, Jensen and Mohus (1976) who measured the growth rates of Amphidinium mrterae Hulburt, Thalassiosira pseudomna, Skeletonem costatum and Phaeodactylum tricornutum in the combined presence of copper and zinc. The two metals were found to interact synergistically towards the first three species but antagonistically to the last. The results obtained with Amphidinium carterae are summarized in Table I11 which clearly illustrates that the reductions in growth rate caused by mixtures of the metals were greater than the sum of the effects due to each separately but at the same concentrations; further work on metal mixtures is required to discover whether other, possibly more serious, cases of synergism exist.

TABLE 111. THE SYNERGISTIC EFFECT OF COPPER AND ZINO UPON TEE GROWTH RATE (IN RELATIVE UNITS) OF AMPEIDIHIUM OARTERAE

Added metal

c u 0On.c. (yg l l )

Zn

0 60

100 200 400 500

1 000 2 000

0 60 75 100 160 200 260

1 .o 0.66 0.47 0.41 0.36 0.18 0 1 s o 0.47 0-36 0.18 0-94 0.47 0-23 0.82 0.35 0 0.66 0.69 0.23 0.12

The data are derived from Braek et al. (1976). Culture medium: 76% Sea water + 0.18 mM NO, + 7.26 pM HaPO; + micro- nutrients + 4.6 @I EDTA. Illumination : 2.6 klux, 14 hours/day. Initial number of cellslml : (2-4) x 104.

Braek et at. considered that the decrease in the toxicity of copper towards Phaeodactylum tricornutum caused by the presence of zinc was due to competition of the two metals for the same binding sites, and they found that the toxicity of zinc towards the same species could similarly be moderated by increasing the levels of magnesium in the culture medium. It will be recalled that Cossa (1976) obtained evidence

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412 ANTHONY Q. DAVIES

that seems to indicate that cadmium and zinc are also bound by the same groups in Phaeodactylum tricornutum supporting the view of Braek et al. that all divalent ions taken up by this species compete for the same binding positions.

C. The nature of metal toxicity in phytoplankton

Some heavy metals, notably mercury, silver and copper, have very high affinities for sulphur and, as a result, it is generally believed that, in living material, they are bound mainly by the sulphydryl groups of proteins and enzymes. In support of this view, Shaw (1954) demon- strated that there is a correlation between the toxicity of metals and their tendency to bind to sulphur expressed in terms of the solubility of their sulphides. The effect of metals upon phytoplankton might therefore be expected to be related to their disruption of the metabolic processes involving sulphur-containing cellular constituents and there are signs that this is, in fact, the case. The available evidence is con- cerned with changes in membrane permeability and the inhibition of cell division brought about by the presence of metals.

1. Membrane permeability Rothstein (1959) has pointed out that the interaction of metals

with the sulphydryl groups in a cell membrane, probably to form -S-metal-S- bridges, would produce considerable stress at the mole- cular level which could cause an increase in the permeability of the membrane leading to the loss of intracellular constituents as observed with several different types of microorganism ; mercury, for instance, has been found to cause potassium leakage from bakers’ yeast (Passow and Rothstein, 1960), human erythrocytes (Weed, Eber and Rothstein, 1962) and phytoplankton (Kamp-Nielsen, 1971 ; Shieh and Barber, 1973; Overnell, 1975).

As it is necessary to use dense cell suspensions to allow the accurate determination of any changes in the levels of the intracellular con- stituents, the concentrations of heavy metals which have had to be added to cause membrane leakage have, in most cases, proved to be very high in order to reach the cellular burdens of metals necessary to produce the effect. However, Kamp-Nielsen (1971) using cultures of the freshwater phytoplankton Ghlorella pyrenoidom showed that the loss of cellular potassium occurred at the same concentrations of mercury or copper as those which caused a reduction in the rates of photosynthesis in the cultures. This suggested to Kamp-Nielsen that the damage to the membrane represented the primary cause for the decrease in growth ;

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POLLUTION STUDIES WITH MARINE PLANKTON--II 413

but the more recent experiments of Overnell (1975) indicate that this may not always be the case for, while the range of copper concentrations which caused potassium leakage from Dunaliella tertiolecta was the same as that which inhibited oxygen evolution, the mercury levels which reduced the rate of oxygen production were an order of magnitude lower than those causing loss of potassium. Davies (1976) similarly found that the mercury concentrations which inhibited the growth of 1sochrysis galbana caused changes in the potassium contents of the cells which were no greater than those which occurred in Dunaliella tertio- lecta, the growth of which WM unaffected by the presence of mercury. Thus, in the case of marine phytoplankton a t least, there need not always be a link between growth inhibition and membrane leakage due to metal uptake, though the fact that metals can cause severe disrup- tion of phytoplankton membranes is shown by the observations of Erickson (1 972) that large aberrant cells with cytoplasm exuding from them were present in copper-containing cultures of Thalassiosira pseudonana.

Other morphological abnormalities in phytoplankton due to the presence of heavy metals have been reported by Nuzzi (1972) who found that phenylmercuric acetate caused the normally biradiate cells of Phaeodwtylum triwrnutum to become vacuolated or ovoid in shape, by Kayser (1 976) who noted that the thecae of Scrippsiella faeroense (Paulsen) Balech & Soares split open in the presence of mercury allowing the escape of a motile, naked form of the dinoflagellate- though this was thought to be the response of the organism to the unfavourable environment created by the metal, and by Bentley- Mowat and Reid (1977) who observed swelling of the cell contents of Ditylum brightwellii (T. West) Grunow ex Van Heurck.

2. Giant cells in metal-containing cultures of phytoplankton An unexpected effect of some metals upon phytoplankton is that

they cause the production of very large cells ; these have been observed both in copper and in mercury containing cultures (Erickson, 1972 ; Davies, 1974, 1976). Davies (1974) found that the size spectrum of cclls of Isochrysie galbana was very dependent upon the mercury concen- tration and that, at the highest sub-lethal concentration studied (initially 10.5 pg mercury/l), there was almost a doubling in the mean cell volume relative to that in the mercury-free control (Fig. 10). Giant cells were produced even in cultures of Dunaliella tertiolecta containing mercury at concentrations which had no effect upon its growth rate (Davies, 1976). It thus appears that metals inhibit the process of cell division independently of any effect they have upon the

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414 ANTHONY a. DAVIES

production of new cell material so that the phytoplankton increase in size.

Shrift (1959) found that selenomethionine caused a similar un- coupling of growth from division in cultures of Chlorella vulgaria Beij. and it was shown that the resulting giant cells contained no methionine which reappeared in the cells only when they had recovered from the

75

65

C rrr

i! 35

15

Initial mercury concentration (jrgll)

A B E F 0 1.55 7.5 10.5

0 4 8 12 16 20 24 28 Days

Bra. 10. The effect of mercury upon the mean cell volume in cultures of Isochrysis galbana. Even a t concentrations as low as 1.55 pg/l, mercury caused a temporary increase in mean cell size by inhibiting cell division without proportionally reducing the growth rate and the effect was especially evident at higher concentrations, almost a doubling in the mean cell volume relative to that in the control occurring in the culture initially containing 10.5 pg mercury/l. Here also, however, the cell population eventually reverted to a normal size distribution. (From Davies, 1974. Reproduced by kind permission of the Council of the Marine Biological Association of the United Kingdom.)

effect of the selenomethionine and had returned to a normal rate of division. Davies (1976) has suggested that mercury and copper might also prevent the production of methionine which appears to be neces- sary for celldivision to occur, for iodoacetamide, which like mercury and copper reacts with sulphydryl groups, inhibited the reduction of sulphate ions in Chlorella pyrenoidosa, an important product of the reduction being S-adenosyl methionine (Schiff, 1959). As the cultures eventually recovered from the uncoupling effects of selenomethionine

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POLLUTION STUDIES WITH M I N E PLANKTON-XI 416

and meroury, it seems unlikely, even if the same effect did occur under natural oonditiom, that it would cause a permanent change in cell size distributions.

From the foregoing, it appears that the reduction in the growth rate of phytoplankton caused by mercury is due to the inhibition of intra- oellular metabolic processes which are quite separate from those controlling cell division. The data of Sunda and Guillard (1976) (Fig. 7) are interesting from this point of view, for the two-step curve which describes the decrease in the growth rate of Thalasaioeira pseudonana due to oopper suggests that inhibition results from the binding of the metal a t two independent sites of reaction each with a different affinity for copper ; only the decrease in growth rate associated with the higher levels of cellular copper was accompanied by increases in cell volume indicating the presence of even more highly copper sensitive processes- possibly photosynthesis as suggested by Overnell (1976)-in the cellular metabolic system.

IV. STUDIES OF THE TOXIC EFFECTS OF HEAVY METALS UPON

NATURAL POPULATIONS OF PHYTOPLANKTON

In order to study the effects of heavy metals upon natural phyto- plankton populations, they have to be contained in some way in order to prevent their dispersion and dilution by turbulence and diffusion. Two main approaches to the problem have been adopted. In one, samples of water together with the indigenous population are placed in bottles and the photosynthetic rates of the phytoplankton measured, usually in terms of the rate of 14C uptake, in the presence of known concentrations of an added metal. By suspending the bottles either in eitu at the appropriate depth or m o d i f ~ g their transparency to simulate the light reaching the location from which the water was removed and placing them in a tank through which is flowing the sea water, it can be ensured that the temperature and illumination inside the bottles are approximately the same as those of the water and phytoplankton which the sample represents. The technique is relatively straightforward and allows the simultaneous study of a wide range of pollutant concentrations. The large area of container surface in contact with the sample will, however, usually cause a decrease in the concen- tration of the metal added to the water due to adsorption and may, especially if the experiments are prolonged, give rise to other problems such aa bacterial proliferation. Thus, another shortcoming of this method is that the measurements cannot be made over extended periods so that the results reflect only the immediate impact of the

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416 ANTHONY Q. DAVIES

metals upon the phytoplankton. This weakness, to some extent, can be overcome by incubating the populations with the metals prior to the measurements in order to allow equilibration between the added metal and the plant cells, but there is a limit to the time over which this can be done before the unwanted effects described earlier become too prominent. This type of measurement cannot, as a result, give reliable information about the longer term changes brought about by metal pollution, such as alterations in the species composition or the development of resistant populations.

Most of the difficulties can, at least in principle, be overcome by using large volume enclosures of sea water containing its natural populations of flora (and fauna) and floating in the water used to fill them so that the entrapped communities are subject to the same meteorological conditions as those outside. The quantity of water contained (2 500 m3) allows an extensive sampling programme to be carried out for monitoring a wide range of parameters of significance to the growth of the phytoplankton without serious depletion taking place. Preliminary studies indicated that the development of the enclosed populations in replicate containers was, provided they were filled simultaneously, similar both to each other and to that of the phytoplankton in the water surrounding the enclosures (Takahashi, Thomas, Seibert, Beers, Koeller and Parsons, 1975). More recently, however, it has been indicated that anomalies in the behaviour of the enclosed phytoplankton could arise due to shading and the absence of turbulence allowing the heavier diatoms to settle out (Tekahashi and Whitney, 1977). The chief difficulty associated with the use of these systems is their great expense so that the range of pollutant concen- trations which can be used in a given experiment is limited by the number of operational enclosures.

In this section, the results obtained using both techniques will be examined.

A. The effects on primary production rates

Knauer and Martin (1972) were probably the first to use the 14C uptake technique of Steemann-Nielsen (1952) for measuring the decrease of photosynthesis in natural populations of marine phyto- plankton caused by a heavy metal. They found that up to 1 pg/l, inorganic mercury (added as mercuric chloride) actually caused a slight increase in the rate of primary production of phytoplankton taken from Monterey Bay, California, but at higher levels of the metal, photo- synthesis was markedly inhibited ; methylmercury proved to be even

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POLLUTION STUDIES WITH MARINE PLANKTON-II 417

more toxic than the inorganic form (Fig. 11). Later studies on popu- lations taken from the Caspian Sea (Patin, Tkachenko, Ibragim and Fedotova, 1974), the Sargasso Sea and Gulf Stream. (Zingmark and Miller, 1976) and the Eastern Mediterranean and Red Sea (Ibragim and Patin, 1976) have all demonstrated similar effects with inorganic mercury though, in general, the degree of inhibition resulting from a

Mercury concentration (pg/l)

FIG. 11. The effect of mercury in the inorganic form (0) and as methylmercury (@) upon the photosynthesis of a natural population of phytoplankton collected from Monterey Bay, California. (After Knauer and Martin, 1972.)

given level of contamination ha8 tended to be substantially smaller in the more recent work (Table IV) ; the reason for this is not apparent.

Patin et al. (1974) and Ibragim and Patin (1976) also measured the decreases in primary production rates caused by copper, cadmium, lead and zinc and their results (examples are given in Table IV) were in accord with the recognized decrease in toxicity in the metals in the order given in the Table. Additions of zinc, the least toxic metal studied, of up to at least 100 pg/1 in fact caused increases in the photo- synthetio rates of the Caspian Sea phytoplankton.

Ibragim and Patin (1976) extended their work to investigate the results of longer term exposure of the Mediterranean phytoplankton

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418 ANTHONY Q. DAVIES

TABLE IV. INHIBITION OF PRIMARY PRODUCTIVITY OF PHYTOPLANKTON POPULATION^ TA-N FROM DIFFERENT SEA AREAS BY METALS AT A CONCENR~ATION OF 10 pggh

Productivity Metal Source of phytoplankton (% of control) Reference

~ ~

Mercury Monterey Bay, California W. Caspian Sea Estuary, S. Carolina Sargasso Sea Gulf Stream Red Sea E. Mediterranean Sea

Red Sea E. Mediterranean Sea

Red Sea E. Mediterranean Sea

Lead Red Sea E. Mediterranean Sea

Zinc W. Caspian Sea

Copper W. Caspian Sea

Cadmium W. Caspian Sea

12 28 76 49 55 28 50 46 * 56 54 79 65 82 86 92 ll5*

Knauer and Martin (1972) Patin et al. (1974) Zingmark and Miller (1976) Zingmark and Miller (1976) Zingmark and Miller (1976) Ibragim and Patin (1976) Ibragim and Patin (1976) Patin et al. (1974) Ibragim and Patin (1976) Ibragim and Patin (1976) Patin et al. (1974) Ibragim and Patin (1976) Ibragim and Patin (1976) Ibragim and Patin (1978) Ibragim and Patin (1976) Patin et al. (1974)

* Estimated from values at lower and higher concentrations.

to the metals by adding a range of concentrations to sea water con- taining the natural population and measuring the photosynthetic rates a t each metal concentration over the following five days. In general, the toxic effects of the metals were found to be less in the longer term experiments than in the short term studies where the productivity was measured soon after the metal additions had been made. At a concentration of 1 pg/l of copper, cadmium or lead, for instance, primary production rates were increased over that in the control even on the first day after the metals had been added, and on the second day, mercury added to give 1 pg/l and the other metals at 10 pg/l were either non-toxic or stimulatory. Mercury at 10 pg/l, though, perma- nently inhibited photosynthesis as did copper at 100 pg/l and cadmium and lead at 1000 pg/L Ibragim and Patin ascribed the reduction in toxicity in the longer-term experiments to " biological dilution ", i.e. the decrease in the metal burdens of the plant cells as they increased in number in the presence of a fixed amount of metal ; a decrease in the total metal concentrations due to losses on to the container would have reinforced this effect. Unfortunately, the metal contents of the phyto- plankton were not determined. A further possible explanation of the apparent moderation in the effects of the metals could be that the phytoplankton which grew during the experiment were more metal resistant than those initially present. It is interesting to note that

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POILUTION STUDIES WITH MARINE PLANRTON-II 419

while, at first, the phytoplankton community consisted mainly of Cylindrotheca (Nitzschia) closterium, Coscinodiscus granii Gough and Rhizosolenia alata Brightw., the final population was dominated by the first of these. As will be seen later, further evidence of the copper resistance of pennate diatoms has been obtained from the work with large scale water enclosures discussed in the next section.

B. The effects in large volume sea water enclosures

A series of experiments performed in the summer of 1974 with the CEPEX (Controlled Ecosystem Pollution Experiment) enclosures operated in Saanich Inlet (near Victoria, B.C., Canada) has provided the data to be discussed here on the longer term effects of copper on the growth and metabolism of natural phytoplankton populations and, in a later section, on the behaviour of natural zooplankton communities.

Two separate sets of measurements were carried out. In Experiment I, copper was added to two of the test enclosures on the second day after filling to give initial concentrations of 10 and 50 pg/l and, in Experiment 11, the copper additions of 5 and 10 yg/l were delayed until the ninth day after filling. The copper concentrations in the enclosures to which were initially added 10 pg/l (both experi.ments) and 50 pg/l gradually decreased due to loss by sedimentation and the 10 yg/1 enclosures were replenished several times in Experiment I, and once in Experiment I1 (Topping and Windom, 1977). No further copper was added to the enclosure initially containing 50 pg copper/] and at the end of the experiment after almost four weeks, it contained only about 20 pg/l (Topping and Windom, 1977). Nitrate, phosphate and silicate additions were made periodically to maintain the nutrients at pre- determined concentrations.

In both experiments, regardless of the presence or absence of copper, the phytoplankton populations (measured as chlorophyll a and phytoplankton carbon concentrations in the top 10 m) decreased during the first four or five days after filling (Thomas, Holm-Hansen, Seibert, Azam, Hodson and Takahashi, 1977). It was suggested that this fall-off in the levels of phytoplankton might have been due to the absence of turbulent mixing in the enclosures; however, in a later series of experiments, Takahashi and Whitney (1977) noticed that the centric diatoms4huetoceros spp.-settled out from the top 20 m within 3 weeks, both in the enclosures and in the surrounding water.

The copper additions, especially in Experiment I, caused an even greater decline in the phytoplankton levels in the test enclosures than

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420 ANTHONY Q. DAVl3S

in the controls. This appeared to be due largely to the copper sensitivity of the centric diatoms-mainly of the genus Chaetoceros-which dominated the populations in the water used to fill the enclosures (Thomas and Seibert, 1977; Goering, Boisseau and Hattori, 1977); for, while in the controls, the proportion of the total phytoplankton carbon in the upper lOm represented by Chaetoceros spp. steadily increased during the four week period of Experiment I, that in the copper polluted enclosures decreased gradually for the first two weeks and then, during the third week, dropped almost to zero, simultaneously being replaced-roughly on a reciprocal basis-by the development of microflagellates. Pennate diatoms seemed to be more resistant to copper than the Chaetoceros spp. because, for a short period, there was a temporary increase in the numbers of Nitzschia delicatissima Cleve in the 10 pg/l enclosure and, in the last week of Experiment I, Navicula distans (W.Sm) Cleve became the dominant diatom in the 50pg/1 enclosure.

In Experiment 11, the proportion of the phytoplankton population represented by centric diatoms declined almost to zero over the first nine days after filling even in the copper free enclosure, the balance consisting maiilly of microflagellates (Fig. 12A) (Thomas and Seibert, 1977). From Day 9, however, the centric diatoms in the controls increased in number and, after four weeks, the population consisted mainly of these and dinoflagellates, some pennate diatoms and micro- flagellates also being present (Fig. 12A). (This succession of diatom- microflagellatdiatom was the converse of that observed by Takahashi et al. (1975) where the initial phytoplankton population consisted mainly of microflagellates which were temporarily displaced by Thalossiosira spp.) In the copper polluted enclosures of Experiment 11, the centric diatoms failed to recover and were replaced by pennate diatoms (Fig. 12B, C) (Thomas and Seibert, 1977) providing further evidence of the greater resistance to copper of the pennate than the centric diatoms.

It will be recalled that Ibragim and Patin (1976) had similarly found that the pennate diatom Cylindrotheca (Nitzschia) closterium eventually became dominant in Eastern Mediterranean phytoplankton which had been exposed for some time to heavy metals, in contrast to the earlier work of Maiidelli (1969) with cultures of Thalassiosira pseudonana (as Cyclotella nana) and Cylindrotheca (Nitzschia) closterium which indicated that the former was the more copper tolerant.

At the end of both experiments, the phytoplankton crops in the polluted enclosures, as measured by the particulate nitrogen content or the concentrations of chlorophyll a, phytoplankton carbon and ATP

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CEPEX Copper Experiment II,September 1974

A C

c Opg C u l l added 4 Control 5pg Cu/l added

FIG. 12. The effect of copper on the proportions of phytoplankton carbon in the upper 10 m of the CEPEX enclosures represented by microfiagellates (light stippling), dinoflagellates (cross hatching), pennate diatoms (heavy stippling) and centric diatoms (white). The copper prevented the resurgenoe of the centric diatoms following the decrease which took place after the enclosures were filled and was lethal to the dinoflagellates, the pennate diatoms ultimately dominating the population. (After Thomas and Seibert, 1977.)

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422 ANTHONY 0. DAVIES

in the top 10 m, exceeded those in the controls (Harrison, Eppley and Renger, 1977; Thomas et al., 1977). Due to the almost complete absence of herbivorous zooplankton in the copper-containing enclosures (Gibson and Grice, 1977), it was not, however, possible to attribute this positively either to the stimulatory effect of the copper or to the lower grazing pressure on the phytoplankton. Jt could also have been caused

CEPEX Copper Experiment I.June 1974

Cu' added Days afterfilling

FIG. 13. The effect of copper on the mean productivity in the upper 5 m of the CEPEX enclosures. Control enclosures, J ( 0 ) and K ( 0) ; experimental enclosures, L with 10 pg copper/l (A) and M, initially containing 50 pg copper/l (0). The metal caused a marked reduction in the rates of photosynthesis, though after the fift,h day, due to the growth of microflagellates, it started to increase again eventually reaching values higher than in the controls. (After Thomas et al., 1977.)

by the communities-mainly microflagellates-present in the polluted enclosures having slower sinking rates than the chain-forming diatoms present in t-he controls (Harrison et al., 1977).

The variations in the phytoplankton populations brought about by the copper were paralleled by changes in the rates of photosynthesis in the enclosures. On the day after the copper additions in Experiment I, for instance, primary productivity rates in the presence of copper were

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POLLUTION STUDIES WITH MARINE PLANKTON-11 423

only 15-30% of those in the controls and, two days later, were even lower (Fig. 13) (Thomas et al., 1977). Thereafter the photosynthetic rates increased with the build-up of the copper resistant population of flagellated and diatoms eventually reaching values exceeding those in the controls, though the recovery of the population in the 50 pg copper/l enclosure was much slower than that growing in the presence of 10 pg copper/l.

More detailed investigations of the effect of the copper additions on the metabolism of phytoplankton were carried out by Harrison et al. (1977) who studied nitrogen uptake and utilization, and Goering et al. (1977) who measured silicic acid uptake rates. Harrison et al. (1977) found that while the specific rates of uptake of both nitrate and ammonium ions by the enclosed phytoplankton communities were extremely variable, they were generally in accord with previously determined values for coastal phytoplankton. In both experiments, reductions in the specific uptake rates occurred only during the days immediately following the copper additions, at other times the rates of nitrate and ammonium assimilation being regulated mainly by the concentrations of these ions present in the water. The formation of nitrate reductase in phytoplankton taken from either the control enclosures or the surrounding water was found to be inhibited by copper additions of 10 and 20 pg/l while the resistant population which developed in the enclosure to which 50 pg copper/l had been added was relatively unaffected in its ability to produce the enzyme, even in the presence of an additional 20 pg copper/l. This observation may provide at least a partial explanation of the copper tolerance of some species of phytoplankton.

Goering et al. (1977) found that the rates of silicic acid uptake by the phytoplankton communities present in the enclosures was decreased by concentrations of copper as low as 2.5 pg/l. Lewin (1954) had pre- viously shown that the incorporation of silicic acid into diatoms required the presence of reduced sulphur-possibly sulphydryl groups on the cell membrane or in an enzyme-and that it could be prevented by the presence of sulphydryl inhibitors such as iodoacetamide, sodium arsenite and cadmium chloride; the effeot of copper which also has a high affinity for sulphur is therefore perhaps not surprising. It will also be seen later that Martin and Knauer (1973) found that an un- usually large proportion of the copper content of natural phytoplankton was associated with the silica frustules. Goering et al. suggested that the greater copper tolerance of pennate diatoms as compared to the centric forms might be because the silicic acid uptake mechanism in the former was less susceptible to the effect of copper. At first sight, the results of

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424 ANTHONY Q. DAVIES

Lewin (1954) seem to support this view for total inhibition of silicic acid uptake by Navicula pelliculosa (Brr5b.) Hilse required a cadmium concentration of about 106 pg/l ; part of the need for such high cadmium levels can, though, probably be explained in terms of the lower affinity of cadmium than of copper for sulphur, and by the use in Lewin’s studies of very dense cell suspensions (6-10 x 106 cells/ml) so that considerably greater amounts of the metal would be required to produce cell burdens comparable with those in the CEPEX studies, as the diatom popu- lations in the water used to fill the enclosures were more than a thousand times smaller (Goering et al., 1977). The possibility that copper might accelerate the redissolution of the silica frustules of diatoms was also examined but up to 25 pg/l of the metal appeared to have no effect upon the rate of this process.

An unexpected finding in Experiment I was the high proportion of the carbon taken up by the phytoplankton which reappeared in the water (Thomas et al., 1977). In the control enclosure alone, this reached levels of up to 70% of the fixed carbon and the copper additions caused the release of even greater proportions, though levels in the polluted enclosures became the same as or lower than those in the controls after about two weeks. Thomas et al. have pointed out that, in the controls, some of the organic compounds could have been released by the grazing of zooplankton or from cell rupture during the separation of the phytoplankton from the water by filtration. A possible explanation of the additional release of organic carbon caused by the copper is the disruption of the cell membranes and consequent leakage of intra- cellular constituents as discussed earlier (Section 1II.C).

In order to investigate whether the phytoplankton populations which developed in the copper-polluted enclosures really were copper resistant or whether the copper had in some way been detoxicated- possibly by the organic compounds released by the cells, Harrison et al. (1977) measured the rates of photosynthesis of phytoplankton taken from the control enclosures in the presence of water taken from the polluted enclosures, the copper contaminated population in this having first been removed by filtration. When only short periods (3-4 h) of incubation in the contaminated water preceded the measurements, the copper had little effect upon the rates of photosynthesis, but after a 24 hour incubation, copper levels as low as 5 pg/l caused significant reductions in productivity indicating that the metal was still present in a form which was toxic to the communities in the control enclosures. The relative tolerances to copper of the phytoplankton from each enclosure were assessed by measuring their photosynthetic rates in the presence of a range of added concentrations of the metal. The

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POLLUTION STUDIES WITH MARINE PLANKTON-II 425

results, when plotted on a semilogarithmic basis (Fig. 14), allowed the determination of the highest copper concentration which caused no inhibition and this confirmed the greater copper resistance of the communities in the polluted enclosures.

r 0 A

I I I I 0 I 10 102 lo3 0

Copper concentruttan [&I)

FIQ. 14. Measurement of the susceptibility to copper of the phytoplankton populations which developed in the enclosures used for the CEPEX Copper Experiment I. Samples of the populations were incubated for 24 h with copper at vwious concen- trations and their photosynthetic rates then measured over 4 h. The populations studied were taken from the two control enclosures, (0 and .), and from the experimental enclosures with 10 pg copper/l (A) and with 50 pg oopper/l (0). The intercepts on the 100% line represent the threshold copper concentrations causing inhibition of photosynthesis ; these were higher for the populations from the experi- mental enclosures due to their greater copper tolerance. (Aft,er Harrison et el., 1977.)

Out of all of the laboratory studies of the effect of copper on marine phytoplankton cultures (Appendix I), only in the work of Erickson (1972) with Thlassiosira pseudonana and of Jensen et al. (1976) using Skeletonema costatum was it found that copper concentrations as low as 5 or 10 pg/l affected the growth of the experimental populations. It is significant that, in both cases, unenriched natural sea water was used for the measurements.

v. HEAVY METAL CONCENTRATIONS I N NATURAL POPULATIONS OF

MARINE PHYTOPLANKTON It is extremely difficult to obtain reliable data on the metal content

of natural phytoplankton, for apart from the obvious problem of collecting samples without simultaneously entrapping the small zoo- plankton, detritus and inorganic particulate matter also present in the

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426 ANTHONY 0. DAVIES

water, contamination by rust from the sampling gear or chips of paint from the ship is hard to avoid (Topping, 1972; Martin and Knauer, 1973). Even when great care is taken in obtaining the samples, other difficulties can arise; Martin et al. (1976), for instance, found that plankton collected off Baja California contained shiny black magnetic particles of unidentifiable origin which were certainly not a result of the sampling procedure. A further complication which makes it difficult to compare data for phytoplankton from different locations is caused by the variations in analytical technique used in different laboratories, though the increasing and welcome practice of inter-laboratory cali- bration exercises (see, for example, Fukai, Oregioni, Huynh-Ngoc and Vas, 1976) will eventua(l1y remove this latter problem.

The data available for the metal concentrations in marine phyto- plankton from many different sea areas are listed in Appendix 11; metal levels in “ microplankton ” have also been included because the inseparable mixture of phytoplankton, microzooplankton and detrital matter to which the term applies represents an important source of food for many types of zooplankton.

The data of Vinogradova and Koval’skiy (1962) and Szabo (1968) were originally published as the concentrations of metals in ashed samples and, in converting the figures to a dry weight basis to facilitate comparison with the data of other workers, some error may have been introduced into their results, though any discrepancies in the Szabo (1968) data should be quite small as the correction factor used was obtained from other results provided by the same author (Szabo, 1967).

The data for most metals range over at least an order of magnitude. Whether the very high levels of metals found in some of the earlier work were actually present in the phytoplankton, were due to contamination or were caused by experimental artifacts it is impossible to judge, but it may be significant that many of them were obtained by spectro- graphic analysis.

While no distinct gradients in the metal contents of phytoplankton or microplankton seem to have been found in near-shore to off-shore transects, Martin and Broenkow (1975) and Martin et al. (1976) have demonstrated that higher than normal levels of metals can occur in phytoplankton in upwelling areas as discussed earlier (Section 1I.C). Mercury levels were consistently low, even off Minamata (Hirota, Fujiki and Tajima, 1974) where they were only slightly higher than those found on a California-Hawaii transect of the Eastern Pacific Ocean (Knauer and Martin, 1972). The low mercury contents of phyto- plankton probably result from its volatilization from sea water as has been observed in laboratory cultures (Section 1II.A).

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POLLUTION STUDIES WITH MARINE PLANKTON-If 427

Martin and Knauer (1973) classified their phytoplankton samples into three groups (I, I1 and I11 in Appendix 11) on the basis of the metal content of the organic fractions of the samples; in Group I, titanium was undetectable, Group I1 contained titanium in measurable amounts and in Group 111, the concentration factors for strontium were greater than 2 . The Group I samples consisted of almost pure phytoplankton collected whilst actively growing and dispersion of the limited quantity of metals available through the plankton biomass probably accounted for the generally lower metal levels in these samples. The Group I1 plankton had the highest median levels of chromium, nickel and zinc and Group I11 samples the highest levels of cadmium, copper and lead probably because of the presence of the strontium accumulating organisms, radiolarians and dinoflagellates. By digesting away the organic fractions of the phytoplankton, Martin and Knauer were able to determine the metal contents associated with the silica frustulcs ; they found that while only about 2.5% of the zinc was bound to the silica, the proportion of copper was about 20% though the reason for this was not clear.

Because the concentrations of metals present in the water from which the phytoplankton samples were collected have not usually been determined, and anyway, Knauer and Martin (1973) and Fowler, Oregioni and LaRosa (197617) found little correlation between the levels in the water and in the plankton, there is little point in calculating concentration factors from the analytical data. It is, however, an interesting exercise to convert the concentration in the phytoplankton to a wet weight basis in order to compare the values with the metal burdens of cells in cultures where a reduction in growth rate has been observed. Using a value of 19 for the wet weightldry weight ratio in the phytoplankton in Monterey Bay, California (obtained from Table 2 of Knauer and Martin, 1972), the highest levels of mercury, cadmium and copper observed in this area by Martin and Knauer (1973) may be calculated to be Hg, 0.031 ; Cd, 0.34, and Cu, 2.4, all in parts per million wet weight or ag/pm3 of fresh material. The values for mercury and cadmium are considerably lower than the intracellular concentratioiis of these metals which were found to inhibit the growth of Isochryeis galbana (Figs 6 and 9), and the copper concentration would correspond to a level of 6.6 ag at/cell in Thalassiosira pseudonana (using a cell volume of 176 pm3, Eppley, Holmes and Strickland, 1967) which is again well below the cellular burden found to affect the growth rate of this species (Fig. 7).

It may thus be concluded that provided the phytoplankton analysed by Martin and Knauer (1973) were not more susceptible to the effects

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428 ANTHONY 0. DAVLES

of the metals than the species used in the experiments, their growth was probably unaffected by the levels of heavy metals present. Even the mercury concentrations in the phytoplankton off Minamata (Hirota et al., 1974) appear to have been too low to cause any inhibition of growth, but the cadmium levels observed off Baja California (Martin and Broenkow, 1975; Martin et al., 1976) and the copper contents of Black Sea diatoms (Vinogradova and Koval’skiy, 1962) could, on this basis, be causing slight reductions in primary productivity rates.

VI. THE TURNOVER OF HEAVY METALS BY ZOOPLANKTON

Zooplankt,on accumulate metals in two ways. Adsorption on to their body surfaces and also from the water passing over their gills and through their bodies represents the primary process of uptake from solution, and assimilation of metals from food and detrital particles ingested by the animals provides thc other pathway for uptake. Metals initially accumulated in these ways are eventually translocated, by active and passive transport mechanisms, throughout the body tissues.

Elimination of metals by living zooplankton may occur by ex- cretion, both in particulate form in faecal pellots and in solution, by moulting and by egg-laying. The instantaneous metal content of any particular animal thus depends upon t,he degree of imbalance between the in-going and out-going fluxes of the metal, especially as some of the elimination processes, e.g. moulting and egg-laying, are discontinuous. Hence, individual zooplankton probably never reach a state of equi- librium with their environment and in considcring metal fluxes in zooplankton, it is necessary to discuss rates of uptake and loss averaged over a population of animals encompassing the whole range of possible physiological states.

A. Studies of metal 9uxes th!rough zooplankton

So far, few attempts have been made to quantify rates of metal uptake and loss by the smaller zooplankton. Probably the first measure- ments mere carried out by Kuenzler (1969a, b) using zooplankton from the Pacific Ocean, some of which had become radioactive as a result of a nuclear test explosion and others which were labelled with carrier-free radioisotopes after collection and sorting. Using animals from a wide range of groups, the rates of elimination of radioactivity into non- radioactive sea water were measured and expressed as the fraction of the radioisotope content of the animal lost per hour. Kuenzler separated

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TABLE v. FRACTIONAL ELIMINATION RATES OF Z 1 ~ c - 6 6 AND COBALT-68 OR 60 FROM PACIFIC OCEAN ZOOPLAKKTON (DATA FROM KUENZLER, 1969a, b)

Mean Fractional Elimination R a h (hr-l)

06Zn w o 07 ooco Particdate Catixmict Aniolvict Total Particulate Catwnkt Anionic$ Total

Copepoda Neocalanua gracilis (Dana) Pleuromamma xiphiaa (Giesbrecht) P . abdominalie (Lubbock)* Candacia ethiopica (Dana) Euchirella (splendem?) * E m h t a marina (Prestandrea)

Pyrosoma vertieillatum Neumann Pyrosoma vertieillatum*

Sdpa cy l indrk Cuvier Salpa fu8iforrni.s Cuvier Cycloaalpa pinnata (Forskal)

Sagitta eraflda Grassi S. hexaptera d'Orbigny

Thysanopoda tricwpidata Milne-Edwards Mixed species

Cavolinia injexa (Lesueur) Cavoliniu injlexa* Cuaierina columnella (Rang)*

Carinaria lamarcki

Pyrosomatidae

Salpidae

Chaetognatha

Euphausiidae

Pteropoda

HeteropodR

(Peron & Lcsueur)

0.006 0.0007 0.001

0.003

0.010 0.004

0.002

0.015

0.005

0.001

0 0 0.0071 0.0009 0.0130 0.0004

0.007

0.011 0.026 0-002 0.008

0.012 0.007

0.006 0.012

0,001 0,003

0.001 0.001

0.012 0.009 0.016

0.011

0-047 0.014

0-031

0.041

0.014

0.003

0.007 04012 0.030

0 0.006

0.007

0.006

0.001 1

0.009 0.003 0.004

0.022 0 0.012 0 0.014 0.001

0.017 0.011 0.001

0.042 0

0.064 0.001

0.0043 0.0003

0 0 0.007 0.001 0.001 0

0.057 0.043 0.069

0-017 0.032

0.081

0.107

0.011

0.009 0.018 0.016

* Results from shipboard labelling of animals with carrier-free "Zn or 6Fo.

t Cationic : retained on strong cation exchange resin. $ Anionic : retained on strong anion exchange resin.

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430 ANTHONY G. DAVIES

the radioactivity released into the water into soluble and particulate fractions by membrane filtration, and, using ion-exchange resins, also made a preliminary identification of the chemical nature of the soluble forms. The data obtained for '35Zn and a B W 2 0 are summarized in Table V.

Significant quantities of particulate zinc and cobalt were eliminated by most species, presumably in faecal pellets, excretion in this form accounting for up to 50% of the metal loss in the case of some copepods. In thc soluble fraction, the greater part of the cobalt retained by the ion-exchange resins was cationic but there was no such distinc- tion with zinc. That fraction of the soluble metals not accounted for in Table V was not retained by either of the resins and, though this could have been a result of their being organically bound, Kuenzler felt that the evidence available was insufficient to be sure of this.

On the assumption that the zooplankton populations examined by Kuenzler were in equilibrium with the sea water from which they were collected so that the rates of metal accumulation and loss were the same, the elimination rates in Table V could be equated to the metal Buxes through the animals over short periods of time during which growth would not be significant. On this basis, a species having a fractional elimination rate of 0.042 h-l or more would, each day, turn over an amount of metal a t least equal to its body burden, and such was the case for several of the types of animal examined.

By extending this idea, it is possible to calculate the flux of a metal through a mixed zooplankton population provided the biomass and motal content of each species present is known. Using such data, Kuenzler (1 969b) calculated that the daily cobalt turnover through the zooplankton population present in the Pacific Ocean at the time of sampling was 31 pg/l, equivalent to 0.02y0 of the total cobalt present in the upper 100 m, the greater part of the cobalt flux being caused by the chaetognaths.

In a more detailed study of 65Zn uptake and loss by Euphausia pacifica Hansen, Small (1969) found that when animals which had taken up the isotope solely from solution were transferred to non- radioactive water, the radionuclide was released to some extent with the moults but most of it was lost by exchange with the stable zinc in the sea water; 65Zn accumulated by animals feeding on radioactive phytoplankton was, however, eliminated mostly in the faeces. By assuming that the three modes of elimination-moulting, exchange and faecal pellet production-were independent and additive, Small showed that it was possible to calculate the effect of various times of exposure to BSZn, both in the absence and the presence of food, on the

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POLLUTION STUDIES WITH MARINE PLANKTON-11 431

transfer of radio-zinc out of the surface layers of the sea by vertically migrating euphausiids.

The approaches of Kuenzler (1969a, b) and Small (1969) to the problem of metal uptake and loss by zooplankton have since been developed into a more elaborate form for studying metal fluxes in the euphausiid Neganyctiphanes norvegica (M. Sars). The methods initially devised by Small, Fowler and KeCkeS (1973) for investigating zinc accumulation and loss have also been utilized for similar studies with cadmium (Benayoun, Fowler and Oregioni, 1974), selenium (Fowler and Benayoun, 1976) and mercury (Fowler, Heyraud and LaRosa, 1976a). Related work, 0.g. on polonium-210 (Heyraud, Fowler, Beasley and Cherry, 1976), will not concern us here.

The main assumption embodied in this work was that the popu- lations of animals taken from the sea for study were in equilibrium with the water so that the rate of uptake of a metal (k,) by the euphau- siids could be taken to be equal to the rate of elimination (Ae) plus the rate of metal incorporation by the population into newly-formed animal tissue (p,), i.e. k, = A, + pe. (The units of these rates were respectively weight of metal taken up, lost or incorporatedlaverage weight of dry animal/day.) Hence, by determining the values of A, and pe, the flux of metal into the population could be calculated.

As mentioned earlier, metal elimination takes glace in four ways- by moulting, faecal pellet production, soluble excretion and egg production-and each of these can be assigned an individual rate. If, for instance, the rate of moulting isp, (weight of moult produced/weight of dry animal/day) and the metal content of the moult is Q,, then the rate of metal elimination by this route is Q,p,. Similar terms were used for all the particulate forms of elimination, but the rate of metal excretion in soluble form was expressed as the fraction of the animal burden lost each day as in Kuenzler’s work. As the growth term p, was equal to Qgpg where pg was the fractional or specific growth rate of the animals and Qg the concentration of the metal in the animal tissue, the average overall metal flux through the animal population expressed in terms of unit weight of dry animal could then be written as

ke = Qmpm + Qfpf + Qgpe + Q x ~ x + Q g p g -

Elimination of the metals from the animals as a result of egg pro- duction was found to be relatively insignificant (except possibly in the case of selenium where it represented 3 4 % of the total flux) and so this aspect of the studies will not be considered further.

The rates ( p ) of the individual physiological processes were

moults faeces excretion eggs growth

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432 ANTHONY 0. DAVIES

determined by carrying out separate laboratory measurements on populations collected from the Mediterranean (Small et al., 1973). The moulting rate (p,) was obtained by maintaining the euphausiids on an adequate supply of food (algae-fed Artemia nauplii) through an average of over two moults per animal; it was found to be 0.092 g moult/g dry animal/day and independent of the size of the animal.

Days

FIG. 15. Excretion of losCd in dissolved form by Meganyctiphanes norvegica. The data were obtained by placing uniformly labelled euphausiids in clean sea water containing Arlemia nauplii as food and following the decrease in radioactivity of the animh. The results could be interpreted as representing the simultaneous loss from three separate compartments, each with its own characteristic rate, from the sum of which the proportion of lo°Cd excreted daily could be calculated. (After Benayoun el al., 1974.)

The faecal pellet production rate (pf) depended upon the quantity of food available 60 the euphausiids. Of the three significant food levels identified by Small et al., two will be used to illustrate their findings. These relate to a maximal and a near-minimal food supply, the values for pf based on 12 hours of feeding per day being respectively 0.061 and 0.018 g faeceslg dry animal/day.

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POLLUTION STUDIES WITH HAEINE P-ETON-11 4%

Excretion rates were determined by initially labelling the euphau- siids radioactively by allowing them to feed on a mixture of Artemia nauplii and phytoplankton containing an appropriate tracer for the metal being studied until the concentration factor for the tracer in the animals was approaching constancy, i.e. isotopic equilibration had almost been attained. The animals were then transferred to non- radioactive water, which was changed daily, and fed on a non-radio- active diet and the loss of radioactivity from the animals followed over a period of time. In all cases it was found that, within the experimental error, the excretion of the metal in dissolved form corresponded to the sum of the losses from three compartments within the animals each having its own characteristic rate ; a typical example is shown in Fig. 15. The value of pe was taken to be the sum of the daily fractional losses from each compartment.

The fractional growth rates (pg) of the euphausiids when maintained on a food supply of Artemia nauplii and phytoplankton were determined by Fowler, Benayoun and Small (1971) and found to vary with the size of the animal from a minimal value of about 0.007 day-l for animals of dry weight in the range 20-25mg up to 0.03day-' for animals of dry weight around 5 mg.

Data on the metal contents of the moults, faecal pellets and whole animals were obtained by analysing material collected from the Mediterranean and these are given, together with a summary of the various rate constants, in Table VI. In the Table, the near-minimal fluxes were calculated using the near-minimal faecal pellet production rate and the minimal growth rate and the maximal fluxes were obtained using the maximal values of these rates. As the soluble excretion rates were assumed to be independent of the food supply, they always represent larger proportions of the near-minimal fluxes.

From columns 5 and 6 of Table VI, it will be seen that net uptake of the metals into newly-formed tissue was significant only with mercury and selenium. In the series-zinc, cadmium, mercury-an increasing proportion of the elimination of the metals was accounted for by excretion of the metals in dissolved form (column 4). This mechanism was particularly important in the case of mercury, selenium being intermediate between cadmium and mercury in this respect. Zinc and cadmium excretion, on the other hand, occurred largely in the form of faecal pellets (columns 1 and 2) and although the difference in behaviour of mercury from the other two Group IIB metals is in accord with normal experience, it is not clear why it happens here, though it is probably related to the fact that most of the mercury taken up by the euphausiids is also in solution (Fowler et al., 197th).

d.l.B.-lS 17

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434 ANTHONY 0. DAVIES

TABLE VI. FLUXES OF METALS THBOUOI

Faecal pellets Soluble Nearminimum Maximum excretaola

Column number 1 2 3 4

Fractional rates

Zinc

Cadmium

Mercury

Selenium*

Q va,lues Pa Partial fluxes yo of near-min flux yo of max flux Q values Po Partial fluxes yo of near-min flux yo of max flux Q values Pa

Partial fluxes YQ of near-min flux

,yo of max flux Q values Pe Partial fluxes yo of near-min flux

,% of max flux

pm = 0-009

Q,=l60

1.35 2.6 1 a 0

Qm=2.1

0.019 7.2 3.2

Q,=0*17

0*0016 2.9 2.1

Q,=1.71

0.015 3.6 2.7

pf=o.O18 p i =0*051

Qr=2 300

41.4 117.3 80.8

91.1 Q r ~ 9 . 6

0-17 0-49 64.1

81.5 Q 1 ~ 0 . 3 4

0.0061 0.0173 11.9

24-6 Q,=6*66

0.12 0.34 27.9

46.5

0.109 7.96

16.5 6.2

0.102 0.071

26.8 11.8

0.116 0-041

80.2 68.3

0.076 0.27

62.8 36.9

* As selenite ion. t Includes elimination in eggs.

B . Pood and water as sources of metals for uptake by zooplankton

The relative importances, under natural conditions, of the two possible modes of metal uptake by zooplankton-directly from the water, and by assimilation from ingested food and detritus-remains to be clearly resolved, but most of the available evidence favours the latter. Polikarpov (1966) considered that, at the phytoplankton concentrations normally present in the sea, uptake from the water would greatly exceed that from the food, the latter becoming important only when the amount of food available was considerably greater. Lowman, Rice and Richards (1971) pointed out that it was also necessary to take into account the concentration factor for an element in the food being consumed, and that all of the experimental data used by Polikarpov to support his contention related to elements with low concentration factors.

To illustrate their argument, Lowman et al. considered the uptake

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POLLUTION STUDIES WITH MARINE PWKTON-11 435

Mqanyctiphanes norvegica

Growth Lowest rate Highest rate

5 6

p,=0.007 p , =0*030

Q,=73

0.51 2.19 1 -0

1.7 Q,=0.7

0.005 0.021 1.9

3.5 Q , = 0.35

0.0025 0.0105 4.9

14.9 QE = 3.63

0.025 0.106 5.8

14.5

Total Juxes Units Reference8 Near-minimum Max4mum

1+2+4+5 1+3+4+6

51.2 1284

0.265 0.601

0.0511 0.0703

0.4307 0-731t

g/g dry animal/day

pg Zn/g dry material

pg Zn/g dry animal/day Small et al.

Small et al. (1973)

days-'

(1973)

pg Cd/g dry material

pg/Cd/g dry anirnal/day Benayoun days-'

et al.(1974)

pg Hg/g dry material davs -

pg Hg/g dry animal/day Fowleretal. (197th)

p g Se/g dry material

pg Se/g dry animal/day Fowler and Benayoun

(1976)

days-'

of two metals by a medium-sized copepod, e.g. Calanus of volume 1.6 x (31113, one metal-strontium-having a concentration factor of 20 in phytoplankton, the other-zirconium-with a concentration factor of 6 x lo4. On the assumption that one copepod ingests 0.3 times its body weight of food each day and assimilates 85% of the metal contained in the food, it may be calculated that it would be necessary for all of the strontium in a volume of water equal to about five times the animal's body volume to be removed daily by the animal in order for uptake from the food and the water to be identical. In the case of zirconium, however, assuming the same efficiency of extraction of the element from the food, a volume of water equal to 15 300 times the animal body volume, i.e. about 2-4 cm3, would need to be depleted of the element each day to equal uptake from the food consumed.

From the data of Marshall and Orr (1 955), Lowman et al. calculated that, for respiratory purposes, one Calanus would each day need to

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436 ANTHONY Q. DAWES

remove all of the oxygen from a volume of air-saturated water equal to 20-25 times its body volume. As this water would pass in close proximity to the animal, it would also be able to provide enough strontium to equal that taken up from the food but too little zirconium.

Lowman et al. ruled out the possibility that uptake from the water being filtered to obtain the food would bring enough of the elements into contact with the animal to provide amounts equivalent to those available in the food on the grounds that an animal while feeding would " see " a volume of water only about 500 times that of its body volume (0.08 om3) each day. Current evidence, however, indicates that the volume of water swept clear by grazing copepods is inversely related to the level of foodstuff in the water and, in order to satisfy their basic food requirements when phytoplankton concentrations are low, considerable volumes of sea water are processed daily by individual animals. Paffenhafer (1971) found, for instance, that female Calanus helgolandicus (Claus) feeding on Lauderia borealis Gran or Cymnodinium splendens Lebour at levels corresponding to the carbon concentrations observed in the sea water off La Jolla, California, grazed at mean rates in the range 286 to 773 cm3/copepod/day ; Frost (1972), in experiments with Calanus pacificus Brodsky feeding on various diatoms, calculated that the volume swept clear was between 72 and 288 om3 water/copepod/ day depending on the size of cell being grazed, and Corner, Head and Kilvington (1972) showed that the volume swept clear by Calanus helgolandicus grazing on Biddulphia sinensis Grev. reached a maximal value of about 700 cm3/copepod/day, when the cell concentration was just over 100/1. While it is doubtful that all of the water swept clear would be in as intimate contact with the animals as that used to satisfy their oxygen requirements, it can be seen that, even at a relatively modest grazing rate of 240 cm3 (1.5 x 106 body volumes)/animal/day, only one atom of zirconium in each 100 present in the water being grazed would need to be taken up by an animal for it to obtain an amount equal to that accumulated from its food.

Data on the actual efficiency of metal uptake from solution are, however, difticult to obtain. There are indications that, below a certain threshold food concentration, zooplankton cease to graze, possibly because the energy expended in obtaining the food would exceed that provided by it (Corner et al., 1972; Mullin, Stewart and Puglister, 1976). In carrying out measurements of metal uptake in the absence of food, i t is, therefore, not easy to assess the volume of water passing over the animal.

It seems likely, on the other hand, that metal uptake from solution occurs largely by adsorption on to the body surfaces of zooplankton ;

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POLLUTION STUDIES WITH MARINE PLANKTON-II 437

although Osterberg, Small and Hubbard (1963b) had formed the opinion from the analysis of animals taken from the sea that surface uptake played a comparatively minor role in the accumulation of radionuclides by copepods, euphausiids and salps, more recent evidence (Fowler and Small, 1967; Small, 1969; Fowler, Small and Dean, 1969) indicates that metal adsorption on to the body surfaces of zooplankton is quite important, large proportions of the animals’ burden of a metal being shed on moulting. If this is the ca.se, once equilibrium between the metal bound to sites on the body surface and that in solution is established, no further uptake would take place regardless of the volume of water passing over the animal, whereas the internal body tissues would continue to accumulate the metal by assimilation from the food being ingested.

The results of Nassogne (1974) on the uptake of zinc by the harpacti- coid copepod Euterpina acw.tifrons (Dana) are relevant to this argument. In the absence of food, it was found that exchange of 66Zn-labelled zinc between the solution and the animals ceased after four days even though the specific activity of the radionuclide in the animals was lower by two orders of magnitude than in the solution. This indicated that isotopic equilibration was incomplete and that a large part of the zinc contained by the Euterpina was not freely exchangeable with that in solution. When the animals were fed with a ssZn-labelled suspension of Platymonas suecica Kylin, however, over 95% of the zinc in the animals had excha,nged by the end of five days. These observations are consistent with the idea that most of the zinc bound within the animals had been obtained, not from the water but from their food, and that zinc uptake from solution occurs only on to sites which are easily accessible to the dissolved metal, that is, on the body surface.

In earlier experiments (Nassogne, 1970, 1971), a direct comparison was made of 65Zn accumulation from radioactively-labelled food and water and, as can be seen in Fig. 16, it was found that both the rates and maximal levels of uptake from the food supply were substantially higher than those resulting from uptake from the water. When extra- polating such data to natural conditions, there are, however, two important details to be borne in mind, both relating to the cell densities used in the experiments. Firstly, while the phytoplankton populations used in the laboratory (about 1.7 x lo5 cells/ml) may be typical of bloom conditions, for most of the year, cell levels will normally be lower, by perhaps a factor of lo3. Under these conditions, the volume of water swept clear by the animals would be greater-possibly by a factor of 70 (from data of Nassogne, 1971)-than those which occurred in the experiments. Secondly, Small (1969) demonstrated that the rate

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438 ANTHONY 0. DAVIES

of 66Zn uptake from solution by Euphausia pacifwa decreased when the extracellular products from Skeletonema costatum were also present. At a concentration of 5 x lo4 cells/ml, the initial rates of e6Zn uptake from the phytoplankton and from a solution containing the organic exudates released by the same number of cells were equal ; below this food level, uptake rates from solution increased sharply as the cell numbers decreased whereas that from the food suspension fell away towards zero.

4 6 8 10 12 14 16

Days

Fro. 16. The uptake of s6Zn by Euterpina acutifrona in the presence and absence of labelled and unlabelled Platymonaa auecica as food. (a) Algae and sea water both radioactive, (b) algae radioactive but sea water inactive, (0) sea water radioactive but algae inactive, (d) radioactive sea water only. During the first seven days of the measurements, the algal populations remained virtually constant a t about 1.7 x lo6 cells/ml. The curves clearly demonstrate the importance of E6Zn uptake from the food relative to that from the water when the phytoplankton are plentiful. (After Nassogne, 1971.)

Both of these factors obviously favour uptake from solution relative to that from food under natural conditions and it therefore seems likely that an increase in the ambient levcls of a metal in the sea would most quickly be reflected in the amount of metal adsorbed onto the surfaces of the zooplankton and only, some time later, in the metal content of the animal tissue.

Once animals have reached equilibrium both internally and externally with their environment, however, it would be expected that, if most of their metal content is exchangeable only with that assimilated from their food, then the metal flux through an animal would be related to the rate of food ingestion and the metal content of the food supply.

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POLLUTION STUDIES WITH MARINE PLANKTON-11 439

Fowler et al. (1971) measured the specific food ingestion rate of Meya- nyctiphunes norvegica as a function of food availability and showed that, at the food levels corresponding to the near-minimal and maximal fluxes in Table VI, the food ingestion rates would be respectively 113 and 320 mg dry weight of food/g dry animallday. On this basis, it was possible to calculate the metal contents of the food necessary to provide the metal fluxes in Table V I and compare them with the metal levels in the natural food supply-mostly phytoplankton, microcrustaceans and detritus-available to the euphausiids in the water from which they were collected. The data obtained were as follows :

Mdal conc i n food necessary for fluxes

in Table V I Metal conc i n food

present in am water (I*s/s dry weight) (I*s/s d7y w&ht)

Zinc 400-600 570&113 Small et al. (1973) Cadmium 1.9-2.3 2.1 Benayoun et at. (1974) Mercury 0.22-0.45 <0-05 Fowler et al. (1976a) Selenium 2.3-3.8 2.7 Fowler and Benayoun (1976)

Except in the case of mercury, the data were thus consistent with the view that metal turnover rates in zooplankton are largely related to the rates of ingestion of the metals contained in their food ; mercury, however, seems to be accumulated mainly from the water.

C . The effect of the ckmical form of a metal upon its uptake by zooplankton

Much of the discussion about the effect of the chemical form of a metal upon its uptake by phytoplankton (Section 1I.B) will also apply here. Little research appears to have been carried out on this subject in connection with zooplankton. Lowman and Ting (1973) showed that an unidentified species of zooplankton-a pela.gic macruran crustacean collected from the sea off Costa Rica-preferentially accumulated ionic cobalt from sea water containing the metal both in the ionic form and as cobalamin. This happened both in the presence and absence of food. O6Zn uptake by zooplankton is decreased by the presence of EDTA (Nassogne, 1974) or the chelating substances released by phytoplankton (Small, 1969) and Nassogne (1974) con- sidered that data obtained on the accumulation of zinc by Euterpina acutifrons could be interpreted as indicating that only the particulate and ionic forms of the metal in sea water are available for uptake by

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440 ANTHONY a. DAVIES

the animals. These observations suggest that organically-bound metals are not taken up by zooplankton, and the reduction in the toxicity of copper to copepod nauplii caused by the presence of chelating agents (e.g. Lewis, Whitfield and Ramnarine, 1972) is presumably a result of this.

Organo-mercury compounds, which contain covalent metal-carbon bonds rather than the coordinate linkages between the metal and the organic compound present in chelates are taken up much more rapidly by zooplankton than inorganic forms of mercury: when added as n-amylmercuric chloride, mercury entered larvae of the barnacle Eliminius modestus Darwin about 20 times faster than the inorganic form (Corner and Rigler, 1958). Fowler et nl. (1976a) similarly found that, while the conceiitration factor for mercury in the methylated form in the euphausiid Meganyctiphanes norvegica reached a value of about lo* after 28 days and was still increasing, that for inorganic mercury after the same time was levelling off at just under 2.5 x lo3.

Methyl mercury was also retained more firmly by these euphausiids, only about 10% of that taken up from food being released in just over three weeks, whereas over 90% of the inorganic mercury was eliminated in the same period. Biological half-times for the turnover of the two forms of mercury in these animals were calculated to be respectively 450 days for the organic form but only 10 days €or the inorganic form.

D. The role of zooplankton in the biogeochemistry of henu!/ metals in the sea

The possibility that the sinking of detrital material from a popa- lation of zooplankton-faecal pellets, moults and carcasses-might represent an important mechanism for rapidly transferring metals from the upper to the deeper parts of the oceans has been raised on several occasions and it has been pointed out (Goldberg, 1965) that the rain of trace element containing particulates of biological origin could provide an important source of the heavy metals found in bottom sediments.

Osterberg, Carey and Curl (1963a) had noticed that sea-cucumbers collected from a depth of 2 800 m in the north-eastern Pacific Ocean contained short-lived isotopes of zirconium/niobium and cerium which could have been present only if the radionuclides had sedimented out more rapidly than would have been expected on a purely physical basis; a granite particle of 10 pm diameter, for instance, would, according to Stokes’ Law, take about a year to sink 2 800 m and, as the particles of radioactive fallout rarely exceeded 4.5 pm in diameter, they

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POLLUTION STUDIES WITH MARINE PLANKTON-II 441

should have taken rather longer than this. From the isotopic ratios present in the sea-cucumbers, however, it was calculated that the surface to bottom transit time for the radionuclides must have been between only 7 and 12 days, corresponding to sinking rates in the range 400 to 233 m/day.

Osterberg et al. suggested that the small radionuclide particles entering the sea surface were probably being ingested by filter-feeding zooplankton and processed into faecal pellets which would have very much higher rates of sedimentation. On investigation, they found that the pellets voided by Euphausia pacijica feeding on Skeletonema costatzcm sank at 43 mlday. This was, of course, substantially slower than the rates necessary to explain their observations, but. more recently Fowler and Small (1972) have shown that euphausiid-produced faecal pellet sinking rates generally lie in the range 126-862 m/day which encom- passes the settling speeds estimated by Osterberg et al.

Using values for the concentration factors for metals in phyto- plankton and zooplankton largely derived from the data of Vinogradova and Koval’skiy (1962), Lowman et al. (1971) examined the question of the downward transport of metal in some detail. Zooplankton were assumed to ingest daily phytoplankton equivalent to 30% of their body weight, a half of this food intake being utilized for the production of new zooplankton tissue. To illustrate the calculations involved, they considered a population of migrating zooplankton equivalent to 1-64 g animal/m2 in an upper mixed layer of 100 m depth and assumed that the population spent a half of each day in the mixed layer and the other half in deep waters. It was then possible to estimate the contribution of the zooplankton towards vertical transport of the metals both due to diurnal vertical migration of the animals and as a result of the sinking of faecal material. An example of the calculation, as carried out for iron, follows :

The concentration of iron in sea water was taken to be 3 pg/l.

3 yg/l = 3 x lo6 pg/100m3 fi 3 x 1.64 g of zooplankton in upper 100 m ingest 0.246 g phytoplankton/ day. (Another 0.246 g food is consumed in deeper waters.) Concentration factor for iron in phytoplankton = 4.5 x lo4. Hence, weight, of iron ingested daily = 0-246 x 4.5 x lo4 x 3 x

Weight of zooplankton tissue formed daily = 0.5 x 0.246 = 0.123 g.

Concentration factor for iron in zooplankton = 2.5 x lo4.

pg/g sea water.

= 33.2 pg.

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442 ANTHONY a. DAVIES

Hence, weight of iron incorporatt- illto new zooplankton tissue =

:. weight of iron excreted h i zooplankton population as faecal pellets and carcasses 1 33.2 - 9.2 = 24 pg/day. Thus each day, 9.2 pg of iron would be transported downwards due to vertical migration and 24 pg of iron due to the sinking of the detritus.

Using similar calculations for the non-migrating zooplankton (2.32 g animal/m2) and combining the two sets of figures, Lowman et a2. found that below each square metre of sea surface, 105 pg of iron would be transported downwards each day from the upper mixed layer by the sinking of zooplankton-derived solids but only 9-2 pg by vertical migration. From these figures, the fractions of the total iron present in the upper 100 m of water removed annually by the two processes may be calculated to be 0.128 and 0.011. Lowman et al. carried out similar estimations for several other metals and for three types of sea area having different productivities. The fractions of these elements removed annually as derived from their data are given in Table VII where, for the purpose of comparison, the fractions of the same elements lost annually from the upper layers due to geochemical sedimentation, calculated from the residence times estimated by Goldberg and Arrhenius (1958), are also given.

In all cases, the metals listed would be removed from the surface layers of the sea more rapidly by the sinking faecal pellets and carcasses than by vertical migration or geochemical processes. In areas of high productivity, those metals having high concentration factors in the plankton would be completely depleted from the upper layers in less than a year due to the activities of the zooplankton unless replaced by regenerative processes or advection.

The sinking of moults from zooplankton may also contribute towards the downward movement of heavy metals in the sea (Fowler and Small, 1967 ; Martin, 1970 ; Small and Fowler, 1973). On moulting, euphausiids, for instance, lose a substantial proportion of their total metal content. Fowler et al. (1969) found that moults from Euphusia pacijica and Thysanoessa spinifera Holmes carried, on average, about 41% of the animal body burden of 65Zn though it was thought that this figure might have been slightly high due to adsorption of the isotope on to the newly-exposed surfaces of the moults. From the data of Small et al. (1973) and that in Table VI, it may be calculated that the euphausiid Xeganyctiphanes norvegica moulted, on the average, every 8.5 days under the experimental conditions used for the mertsurements

0.123 x 2.5 x 104 x 3 x 10-3 = 9 2 pg.

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TABLE vn. ESTIMATES OF TEE FRACTIONS OF CERTAIN BlETALS REMOVED FROM THE UPPER MIXED LAYER DUE TO

ZOOPLANKTON ACTIVITY IN AREAS OF DIFFERING PRODUCTIVITY AND BY GEOCHEMIOAL PROCESSES (DATA DERIVED FROM LOWMAN ET AL. (1971) AND GOLDBERG AND ARRHENIUS (1968)) m

R 3

0.04 cc Zooplanktonlma 0.44 cc Zooplanktonlm' 1.0 cc Zooplanktonlm8 Geochemical p Fracttion of element eatimatecl to be removed annually from surface layers of the sea

1 Phytoplankton Zooplankton migration detritus migration detritus migration detrittw El

Element Concentration factor e.g. Eastern North Paci,lio e.g. Coastal areaa e.g. Upwelling areas sedimentation Vertical Zooplankton Vertical Zooplankton Vertical Zooplankton

Manganese 4 x loa 1.5 X 10' 6.7 x 1.2 X 7.1 x 10-8 0.14 1.7 x 0.33 7 . 1 ~ 1 0 - ' 1 Iron 4.5 x 104 2.5 X lo4 1.1 x 10-2 0.13 0.12 1 *4 0-28 3.3 7.1 X lo- '

5*6X10-* H Zinc 2.6 x 104 8 X 10' 3.6 X l o - $ 7.7 X 4 x 10-2 0.83 9.1 x 10-2 2.0 Cobalt 1.6 X 10' 7 X l oa 3.1 X lo- ' 4.2 X lo- ' 3.4 X lo- ' 4.5 x 7.7 X 0.10 6.6 X Zirconium 6 x 10' 2.6 X lo4 1.1 X 10-8 0.17 0.12 2.0 0.28 6.0 - Lead 4 x 10' 3x1Oa 4 . 6 ~ l O - ~ 0.14 6 . 0 ~ 1 0 - ' 1.4 1 . 1 x 1 0 - ~ 3.3 6.0 x 10-4 t

5l I fi

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444 ANTHONY Q. DAVIES

(13"C, ample food supply), each time losing moults which represented a mean of 8% of the body weight and which contained the following percentages of the total body burden of metal : zinc, 16% ; cadmium, 23% ; mercury, 3.7% and selenium, 3.7%.

Euphausiid moults, when first shed, sink much faster than faecal pellets though their sinking rates gradually decrease as they disinte- grate; Small and Fowler (1973) found, for instance, that the rates for moults from Meganyctiphanes norvegica decreased from an initial value of 1 700 to 400 "/day 8 days later.

A similar decrease in the sinking rates of euphausiid carcasses was observed by Small and Fowler (1973), those from Meganyetiphanes norvegica reducing from about 4 000 m/day soon after death to about 2 400 m/day after 24 days. The same authors found that the death rate in a euphausiid population corresponded to 7-5 mg carcass/g animal/day during the breeding season (mid-March to May) but was only 1.0 mg carcasslg animallday at other times of the year. Using these death rates and the Q g values in Table VI, the maximal and normal levels at which the metals are immobilized in carcasses may be calculated to be (in pg metal/g dry animallday): zinc, 0.55, 0.07; cadmium, 0.005, 0.0007 ; mercury, 0.003, 0.0003 ; selenium, 0.026, 0,003.

Comparison of these rates with those shown in Table VI shows that, of the fluxes of the metals into the various euphausiid-derived detrital material, that into the faecal pellets, in most cases, greatly outweighs the rest and thus should potentially provide the greatest supply of particulate metals to the sediments. However, Small and Fowler (1973) found that zinc was gradually released from the detritus so that not all of the metals leaving the surface layers in it would reach the bottom. The zinc content of faecal pellets and moults decreased most quickly (by about 90% in 6 days), that of the carcasses falling by only about 45% in the same time. Using their experimentally observed values for the rates of zinc release from the faecal pellets, moults and carcasses and their measured sinking rates, Small and Fowler were able to calculate the flux of zinc through the 500 m and 2 500 m depths of the Ligurian Sea due to the sinking of these solids. Examples of the values obtained are given in Table VIII where, to correspond to the low fertility of the Mediterranean, the data presented relate to the faecal pellet production rate observed when the euphausiids were living on a near-minimal food supply (column 2 of Table VI).

From the data in Table VI and that of Small and Fowler (1973), the fractions of the zinc content of the Ligurian Sea leaving the top 50 m in the sinking detritus and the fractions reaching depths of 500 m and 2 500 m have been calculated, the figures being based on a euphausiid

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TABLE VIII. AN ASSESSMENT OF DOWNW~RD ZINC TWSPORT IN TEE LIGURIAN SEA DUE TO THE SINKING OF DETRITUS FROM

A POPULATION OF ~ E B A N Y O T I P E A N E B IORVEGICA

(BASED ON SMALL AND FOWLER, 1973) Cd

8 Units Total

fraction Faeces Moults Carcasaea

z cn

Fraction of zinc annually leaving top 50 m in sinking 6.5 X lo-' 2.1 X 1.1 X lo-( 6-8 X lo-' ! Depth : 500 m Fi

d E

E

detritus U

Mean time to reach this depth 1.25 0.39 0.16 Days

Flux of zinc reaching this depth* 24.8 1.12 0.07 pg zinc/g dry animal/day Fraction of zinc in top 50 m annually sinking to this

Depth : 2 500 m

Fraction of zinc content lost while sinking 0.40 0.17 0.03 -

3.9 x lo-' 1.8 x 1.1 x 4.1 x lo-' depth?

Q Mean time to reach this depth 6.25 2-50 0.78 Days M Fraction of zinc content lost while sinking 0.91 0.66 0.21 - +d

9

mortality rate. E

Flux of zinc reaching this depth* 3.73 0.46 0.06 pg zinc/g dry animal/day Fraction of zinc in top 50 m annually sinking to this 5.9 x 7.3 x 9.5 x 6.7 x

0 u I

dept,ht

* Based on faecal pellet production rate corresponding to near-minimal food supply (column 2 of Table VI) and non-breeding season

t Based on the average value in the Mediterranean Sea of the euphausiid population equivalent to 0.1 rng dry animal/ms (derived by Small and Fowler (1973) from the dat.a of Franqueville (1970)) and a mean zinc concentration of 2.3 pg/L (Fukai and Huynh-Ngoc, 1976.)

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446 ANTHONY a. DAVIES

population equivalent to 0.1 mg dry animal/m3 and a zinc concentration of 2.3 p.g/l. These fractions are also given in Table VIII where it can be seen that, of the zinc sinking from the surface layers in the solids, whereas 60% reaches 500 m, only 10% arrives at 2 500 m, most of the downward zinc movement being due to the faecal pellets. The residence time for zinc in the oceans calculated from geochemical considerations is 1-8 x 105 years (Goldberg and Arrhenius, 1958) so that the fraction of zinc sedimenting out annually on this basis would be 5-6 x 10-8. Thus, assuming that this figure can also be applied to the Mediterranean, and that the euphausiids provide the major source of faecal pellets, the initial downward movement of zinc from the surface in the detritus produced by the euphausiids would, even in a region of relatively low fertility, exceed by two orders of magnitude that taking place due to geochemical processes. However, it seems likely that, in deep waters, due to the loss of zinc from the sinking solids, the downward movement of zinc in the zooplankton-derived particulates and that due to geo- chemical processes would approach similar values.

VII. LABORATORY STUDIES OF THE TOXIC EFFECTS OF

HEAVY METALS UPON ZOOPLANKTON

The use of LC,, values-the concentrations of pollutants causing a 50% mortality in a given time-as a means of assessing acute toxicities has been commonplace, and compilations of data, including LC,, values for the effect of meta,ls upon zooplankton, continue to be published (e.g. Bernhard and Zattera, 1975; Black, Hinton, Johnston and Sprague, 1976; Taylor, 1977a, b). While data of this type provide a convenient means for comparing the toxicities of a range of pollutants, or the susceptibilities of different species to a given pollutant, the LC,, values available have been obtained under widely differing experimental rdgimes. Furthermore, as only relatively short exposures of the animals to the toxic substances are necessary for their determination, it is increasingly being recognized that LC,, values are inadequate for assessing the environmental impact of pollutants because they give no guide to the " influence of long-term sub-lethal concentrations on behaviour, survival, reproduction and community structure " (Barber, Barrett, Blaxter, Jannasch, McGowan, Quinn, Pomeroy and Provasoli, 1971).

It has of course long been realized, even from acute toxicity determinations, that low concentrations of metals can prove to be lethal after extended periods of time, but it is only comparatively recently, apart from some early work on the response of barnacle larvae to

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POLLUTION STUDIES WITH MARME PLCNKTON-11 447

sub-lethal levels of heavy metals (e.g. Pyefinch and Mott, 1948), that attempts have been made to assess the chronic effects of low con- centrations of metals upon zooplankton by examining sub-lethal physiological responses to the metals. In this section, we will con- centrate largely on these recent results of laboratory experiments and, in the next section, deal with those obtained using large-scale water enclosures ; the discussion includes data obtained for the planktonic larval forms of certain animals and fish.

/

A. The e#ects on the metabolic activity of zooplankton

There have been several investigations into the effects of heavy metals upon the metabolic activity of zooplankton as measured by their rate of oxygen consumption.

Bernard and Lane (1963) found that a copper concentration of 500 pg/l increased the respiration rate of planktonic cyprids of the barnacle Balanus amphitrite (Darwin) and only at concentrations greater than 5 mg/l were the rates lower than in the controls. It should be noted that in this work, in order to keep the copper in solution at the high levels used, it was added to the sea water as the acetate ; complexing by this anion would have reduced the effective concentrations of free cupric ions to levels significantly lower than the total amounts intro- duced into the experiments. An increase of about 30% in the respiration rate of larvae of the surf clam Spisula solidissima (Dillwyn) when exposed to 50 pg silver/l has similarly been noted by Thurberg, Cable, Dawson, MacInnes and Wenzloff (1975).

DeCoursey and Vernberg (1972) found that oxygen consumption by larvae of the fiddler crab Uca pugilator (Bosc.) maintained under optimal conditions of salinity (30%,) and temperature (25%) was reduced by a 6 hour exposure to 180 yg/1 of mercury, the Stage I11 zoeae being the least susceptible to the metal. As this concentration proved to be lethal to all zoeal stages after 24 hours, the observed reduction in metabolic rate was not surprising. In a similar experiment with cadmium at a concentration of only 1 yg/l (Vernberg, DeCoursey and O’Hara, 1974), the rate of oxygen consumption by Stage V larvae was reduced by about 50%, but that of the earlier stages was increased.

The experiments of DeCoursey and Vernberg (1972) on the effect of mercury were extended by Vernberg, DeCoursey and Padgett (1973), to include studies of the effect of varying salinity and temperature. In the absence of mercury, metabolic rates were decreased under non- optimal conditions of salinity and temperature and the general effect of mercury a t a concentration of 1.8 yg/l was to stimulate respiration

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448 ANTHONY 0. DAVIES

relative to the mercury-free controls at low temperatures (20°C) and to decrease it at higher temperatures (25" and 30").

On the basis of their results, Vernberg et al. (1974) suggested that the reduction in metabolic activity caused by the mercury under non- optimal conditions could adversely affect the survival of the fiddler crab larvae due to their decreased ability to feed or escape predators. The possibility thus arises that the zooplankton inhabiting inshore and estuarine locations with widely and rapidly changing salinity and temperature regimes might be at greater risk when subjected to the additional stresses imposed by the presence of heavy metals than off-shore or oceanic zooplankton.

Reeve, Grice, Gibson, Walter, Darcy and Ikeda (1976), however, found by determining 24 hour LC,, values, that the susceptibilities to copper of a wide range of species of zooplankton gathered from all types of sea areas-estuarine, off-shore and oceanic-were a function mainly of their size rather than of their habitat. Further, in short-term experiments with samples of sub-tropical Sagitta hispida Conant and Undinula vulgaris (Dana) and temperate Calanus plumchrus Marukawa and Metridia paci$ca Brodskii exposed to copper and mercury, Reeve, Walter, Darcy and Ikeda (1977b) found that concentrations up to, and in the case of Undinula vulgaris considerably exceeding the 24 hour LC,, vaIues for the metals, had little effect upon the respiration rates. Even in longer term measurements (up to 30 days) with Calanus plumchrus (Reeve et al., 1976, 1977b) there was no indication that exposure to 5 or 10 pg/l of copper had any effect upon the oxygen consumption of the animals which appeared to be reduced only when their death was imminent. It was also found that excretion of ammonia and phosphate by Calanus plumchrus was similarly unaffected by the same copper concentrations.

While Reeve et al. have concluded, as a result of their work, that respiration and excretion do not provide suitable criteria for assessing the sub-lethal toxicity of metals, more information about the syner- gistic effects of salinity, temperature and heavy metal pollution on the metabolic activities of planktonic animals would be useful.

B. The eSfects on the feeding and ingestion rates of zooplankton

Reeve et al. (1976) examined the effect of copper at concentrations up to 50 pgll on the feeding of Calanus plumchrus and Metridia pacijca ; it was assumed that if the animals were provided with a standardized food supply, then the rates of feeding would be reflected by the more easily determined faecal pellet production rates. At 5 pg copper/l,

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POLLUTION STUDIES WITH MARINB PLANKTON-II 449

a reduction of about 40% in the rate of faecal pellet production by the Calanus occurred in 14 days but with the Metridia, the same reduction took place in only four days (Fig. 17). These differences in the sensi- tivity of the two species to copper were borne out by the separately determined 24 hour LC,, values of respectively 2 800 and 180 pg/l.

Similar experiments with natural populations containing a mixture of species taken from a sub-tropical (21°C) and two temperate (9°C) regions have also been carried out (Reeve et al., 1977b). Even on the

0

P Days after copper addition

FIG. 17. The effect of copper on faecal pellet production by A, Calanwr plumchrwr and B, Metridia pacijca feeding on diatoms in sea water containing the following con- centrations of the metal (pg/l): 0, 5; ., 10; 0, 20; X, 50. (After Reeve et al., 1976.)

first day of exposure of the sub-tropical zooplankton (mainly Acartia tonsa Dana) to the metal, copper, added at a concentration of 20 pg/l, caused a decrease in faecal pellet production of about 50% its compared to a control with no added copper and by the fourth day, the addition of 10 pg/l was having a similar effect. The faecal pellet production rates of the zooplankton taken from the temperate sea areas-mainly Temora, Paracalanus and Acartia in one case and Paracalanus spp., Pseudocalanus spp. and Acartia toma in the other-were less dependent upon the copper concentration but there were signs, after five days of exposure, that concentrations of 50 pg/l and upwards were beginning to cause a decrease in these rates.

With the sub-tropical zooplankton, mercury, predictably, proved to be more toxic on its own than copper (Reeve et al., 1977b), only 2 pg/l

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450 ANTHONY a. DAVIES

causing a 30% reduction in the faecal pellet production rate on the first and second days of exposure. Subsequently, however, the effect of this concentration was less marked, possibly due to its disappearance from the experiments by volatilization, though the addition of 10 pg mercury/l continued to repress faecal pellet production even on the tenth day of exposure. As with copper, the zooplankton from the colder sea areas were less sensitive to the effects of mercury and it was found that the two metals individually at concentrations of 5 pg/l and as a mixture containing 2-5pg/l of each metal had much the same effect upon faecal pellet production rates.

A factor related to feeding which appears to have been overlooked in studying the responses of zooplankton to heavy metals is that the toxic effects are ultimately related to the amounts of the metals actually taken up by the zooplankton rather than to the concentrations in the water bathing the animals. The effect of feeding upon the accumulation of metals by zooplankton has already been described in a previous section and it might be expected that, as internal accumulation of metals takes place mainly through the food suppIy, then, in short term experiments at least, the toxicity of a metal might be influenced by the availability of food. When Shealy and Sandifer (1975) com- pared the effect of mercury upon fed and unfed larvae of the grass shrimp Palaemonetes vulgaris (Say), it was found that, during the early stages of exposure to concentrations of mercury of 18 pg/l and upwards, survival of the unfed larvae was indeed greater than that of the fed animals. At lower concentrations, the mercury had little effect and starvation led to a greater mortality of the unfed larvae. Whether the differences in the sensitivity to metals of the sub-tropical and temperate zooplankton populations studied by Reeve et al. (1977b) arose from differing levels of food supplies or from other factors such as the temperature difference is not clear. The problem is further complicated in the case of mercury, by the finding that this metal seems to be accumulated, at least in the case of euphausiids, not through the ingestion of food but directly from the water (Fowler et al., 1976a).

C . The effects on the growth and development of zooplankton There have been surprisingly few studies of the effects of sub-lethal

heavy metal concentrations upon the growth and development of zooplankton and planktonic larvae; Table IX contains a list of the concentrations of the metals which caused a reduction in growth rate, and also some results on the effects of metal mixtures.

Only two sets of data are available from studies with permanent

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POLLUTION STUDIES WITH MARINE PLANKTON-I1 461

members of the zooplankton. Lewis et al. (1972) found that the hatching of the eggs and subsequent development of the first two naupliar stages of the copepod Euchaeta japonica Marukawa were very sensitive to the presence of copper added to sea water to give total concentrations as low as 6 or 7 pg/l. It was also shown that, at any given concentration, the toxic effect of the metal expressed in terms of the percentage survival through the first two naupliar stages, varied with the time of year at which the water was collected. As the addition of both natural and artificial chelating agents and partioulate material increased the survival of the nauplii in copper-enriched water by complexing or adsorbing some of the metal and thus lowering its biological availability (Lewis et al., 1972, 1973), it has been suggested that variations in the degree of complexing of naturally occurring copper and other metals by the organic compounds present in the sea water could give rise to the seasonal differences observed in the " quality '' of sea water for the survival of copepod nauplii (Lewis, Ramnarine and Evans, 1971 ; Whitfield and Lewis, 1976). With the increasing indications of the relationship between metal availability/ toxicity and the presence of natural chelators in sea water (Johnston, 1964 ; Barber andRyther, 1969 ; Steemann-Nielsen and Wium-Anderson, 1970; Davey et al., 1973), more account needs to be taken of such effects in the design and interpretation of experiments with low concentrations of heavy metals in natural sea water.

In the report of the work with Tigriopus japonicus Mori (D'Agostino and Finney, 1974) there is some confusion as to whether mono- or divalent copper chloride was used in the experiments. Starting with egg-bearing females, development of the F, and the early stages of the F, generations was followed at several concentrations of copper and cadmium and mixtures of the two metals, The criterion used for assessing toxicity was the metal concentration which caused a doubling of the time elapsed before the F, generation was hatched though, in fact, the main effect of the metals at the concentrations given in Table IX was to delay development of ovigerous females of the F, generation. Although the concentrations of the two metals which caused this were similar, it was found that, at higher concentrations, whereas develop- ment from the F, naupliar stages was prevented by 640 pg copper/l, in the presence of 440 pg cadmium/l growth continued up to the first F, adult stage thus demonstrating the greater toxicity of copper. Synergism between copper and cadmium was very marked, the con- centrations of the metal mixtures which caused an equivalent delay in the appearance of the F, generation being a factor of 10 lower than for the individual metals.

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8

TABLE IX. CONCENTRATIONS OF HEAVY METALS AFFECTING GROWTH AND DEVELOPMENT OF ZOOPLANKTON AND

PLANKTONIC LARVAE

Metal Animal Species

Copper Calanoid copepod Emhaeta japonim nauplii

Sea-urchin larvae Paracentrotus lividus

Harpacticoid copepod Tigriopzra japonkus

Zinc Mud-crab larvae Rhithropanopeus harriaii

Oyster larvae Crassostrea gigm

Cadmium Harpacticoid copepod Tigrhpus japonicus

Mud-crab larvae Rhithropccnopeus harrisii

Experinaen.ta2 temperature

("C)

8.6, 10

20

20-22

23.5

20-22

20-22

20, 25, 30, 36

salinity Concen- tration Effect Reference

k - 6-7 Reduction in survival Lewisetal. 8

beyond second naupliar (1972) 3 stage P

- 11 Retardat.ion of develop- Bougis (1959) ment

- 64 Time to reach F, gener- D'Agostino and ationmore thandoubled Finney (1974)

20 25 Hatch to megalopa de- Benijts-Claus velopment time in- and Benijts creased (1976)

structural abnormali- (1973) ties

ation more than doubled Finney (1974) 10,20,30 50 Hatoh to megalopa de- Rosenberg and

velopment time in- Costlow (1976) creased especially under non-opt,imal conditions

5

29 100 Growth retardation; Brereton et aZ.

- 44 Time to reach F, gener- D'Agostino and

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Mussel larvae Mytilm gallopr0ci~- 206 I 37-38 80 Growth retardation PaviEi6 and cial is Lam. J&rvenp&&

(1974)

of larvae hatched in con- (1975) taminated water then Fd transferred to clean

d water Mercury Grass shrimp larvae Palaemonetea vulgaris 28 33-34 10 Development time and Shealy and San- 1

0 2

period between moults difer (1975) increased after 48 h ex-

Garpike larvae Belone belone 23 32 2 000 No effect upon growth Dethlefsen et al.

E

posure and then trans- 3 fer to clean water 3

creased (1976) 3 Lead Mud-crab larvae Rhithropampeuir 23-5 20 50 Hatch to megalopa de- Benijts-Claw

hUT&i velopment time in- and Benijts Go

Copper + Harpacticoid copepod Tigriopue japonkus 20-22 - 4.4 Cd+ Time to reach F, gen- D’Agostino and $ Cadmium 6.4 Cu eration more than Finney (1974)

Lead + Mud-crab larvae Rhithropnopeus 23-5 20 28 Zn+ Hatch to megalopa de- Benijts-Claus E Zinc hUWiaii 24 Pb velopment time de- and Benijts

doubled

3 C T e a S e d (1975)

E z 2 I U U

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454 ANTHONY a. DAVIES

There are some indications that even brief contact with metal- containing water may cause effects on the development of planktonic larvae, for those of the grass-shrimp Palaemonetes vulgaris, after spending only two days in water containing 10 pg mercury/l followed by transfer to unpolluted water, had a higher mortality rate, increased development time and longer periods between moults than in the controls, these effects still being apparent up to three weeks after exposure to the metal (Shealy and Sandifer, 1975). Similarly, larvae of the oyster Crassostrea gigas (Thurberg) grown for five days in sea waters containing zinc concentrations between 50 and 200 pg/l and then transferred to unpolluted sea water (14 pg zinc/l) for a further five days showed no signs of recovery from the retardation of growth caused by the metal at 100 pg/1 and above (Brereton, Lord, Thornton and Webb, 1973). On the other hand, larvae of the Baltic garpike, Belone belone L., hatched in water containing 2 000 pg cadmium/l, although a t first exhibiting abnormal body curvature, were reared for 30 days in unpolluted sea water into apparently normal young fish (Dethlefsen, von Westernhagen and Rosenthal, 1975).

The work of Rosenberg and Costlow (1976) was chiefly concerned with demonstrating synergistic effects between cadmium, salinity and temperature. Under optimal conditions of salinity (25%,) and tem- perature (25"C), more than 80% of the larvae of the mud-crab Rhithro- panopeus harissii Gould exposed to 100 pg cadmium/l survived to become crabs though their development was delayed relatively to the control animals. Survival of the zoeae was, however, significantly reduced under all the salinity/temperature regimes by 160 pg cadmium/l, but less so by 50 pg/l. When the animals did survive, hatch to megalopa development times at the lower concentration were increased only at low temperatures (20-25°C) and low salinity (lo%,), conditions under which the animals, even in the absence of cadmium, were probably subjected to great physiological stress.

The finding that some mixtures of lead and zinc can be antagonistic in their effects upon the development rate of Rhithropanopeus harissii larvae (Benijts-Claus and Benijts, 1975) were based on rather few experimental data and requires further investigation.

D. The effects on the fecundity of zooplankton

The effect of heavy metals upon egg-production by zooplankton has been studied by Reeve et al. (1976, 1977b). Sub-tropical Acartia tonsa released more eggs after four days exposure to copper added to give 5 pg/l than did copper-free animals, but higher concentrations

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POLLUTION STUDIES WITH MARINE PLANKTON-II 465

caused a reduction in egg production which virtually ceased at 50 pg/l (Reeve et al., 1976). In later experiments with mixed sub-tropical zooplankton populations comprised mainly of Acartia, more eggs were produced during the early days of exposure to 5 and 10 pg copper/l than in the controls, but after a week, fecundity was lower at all concentrations and particularly a t 10 pg/l and upwards (Reeve et al., 197713).

Mercury, too, caused big reductions in egg production by the same populations but, as observed in the faecal pellet production studies, whereas 2 pg/l caused a decrease during the first two days but had no effect thereafter, 10 pg/1 continued to influence the fecundity of the animals after 10 days of exposure (Reeve et al., 1977b).

Egg production by zooplankton from temperate water areas was similarly reduced by copper and mercury, the effect of copper at a given concentration increasing with time.

E. The effects on the phototactic response of zooplankton

The response of zooplankton and planktonic larvae to light represents an important aspect of their behavioural pattern in connec- tion with their feeding requirements (Longhurst, 1976) and, in the case of larvae, seeking out a suitable habitat (Thorson, 1964), or avoiding predators (Forward, 1974). It is of interest, therefore, that the photo- tactic response of some larvae has been found to be highly sensitive to the presence of metals.

Vernberg et al. (1973) measured the changes in the photopositive behaviour of first and third stage zoeae of the crab Uca pugilator reared at 20°C in the absence and presence of 1-8 pg mercury/l and in sea waters of salinity 20%, and 30%, at various temperatures. The results obtained with the Stage I larvae were rather variable and in- conclusive, but it was found, in the case of the Stage I11 zoeae, that while there was a marked reduction in the phototactic response of both the controls and the mercury-treated larvae at salinities of 20%, and 30%, as compared with those kept under the optimal condition of 25%,, the mercury containing zoeae always behaved more photo- positively than the mercury-free animals.

A similar increase in the phototactic response of Stage I larvae of the crab Carcinus maenas (L.) was produced after they had been exposed for one day to less than 1 pg/l of either cobalt or silver (Amiard, 1976) but prolonged exposure caused a reduction in their photopositive behaviour as did higher concentrations of these metals within the first day.

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466 A?STHONY 0. DAVIES

The reason for the remarkable sensitivity of the phototactic response of these larvae towards the metals is not clear-Amiard (1976) has speculated that it could be due to the effect of the metals upon their nervous systems or their swimming mechanism-but it clearly provides a relatively simple but very sensitive means for assessing the sub-lethal toxicity of metals.

Vernberg et al. (1973) have pointed out that if the reduction in the photopositive response observed in the experiments carried out on larvae maintained under sub-optimal conditions represented an adaptation to the environment, then the failure of the mercury-treated larvae to adapt in the same way would place them a t a disadvantage. Should zooplankton be similarly affected, it is conceivable that con- centrations of toxic metals as low as 1 pg/l could influence their diurnal feeding behaviour.

F. The effects on the swimming activity of zooplankton

The effect of mercury and cadmium upon the swimming rates of the larvae of Uca pugilator have been examined by DeCoursey and Vernberg (1972) and Vernberg et al. (1974). In the absence of the metals, swimming activity was greatest in the Stage I11 and least in the Stage I larvae. After a 6 hour exposure to mercury a t a concentration of 180 pg/l (lethal to all zoeae after 24 hours) reductions in the swimming rates of all stages were observed, the greatest effect (a decrease of 67%) taking place with the Stage V zoem. Cadmium, at the much lower concentration of 1 pg/l, caused reductions in swimming rates which were significant only for the Stage I larvae ; however, this sensitivity to such a low concentration of cadmium supports the idea that the reduction in phototactic response caused by other metals at similar concentrations might be related to their effect upon the swimming mechanism of the larvae as suggested by Amiard (1976). Larval herring though appear to be very much less sensitive to the effects of cadmium in this respect as their swimming rates were reduced only at concentrations of 2 000 pg/1 and upwards (Dethlefsen et al., 1976).

G . The combined effects of heauy metals and additional environmental stress upon zooplankton

The possibility that heavy metals a t sub-lethal concentrations might reduce the ability of animals to withstand additional environ- mental stress was, as we have noted, one aspect of the work of Rosenberg and Costlow (1976) on the synergistic effects of cadmium, salinity and temperature upon the survival and development of mud-crab larvae.

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POLLUTION STUDIES WITH MARINE PLmKTON-11 467

Middaugh, Davis and Yoakum (1976) have carried out a related examination of the vulnerability of larvae of the fish Leiostornus xanthurus Lacepkde to two other types of stress-rising temperature and oxygen deficiency-after the larvae had been exposed for four days to a range of concentrations of cadmium. Up to 90 pg/l, the tolerance of the larvae to either form of stress was little affected, though it was considerably reduced at 500 pg/l. This work and that of Dethlefsen et at. (1976) have demonstrated the remarkable indifference of the larvae of two species of fish to short periods of exposure to cadmium. How typical this is of fish larvae in general remains to be seen as both of the species studied-Leiostomus xanthurus and Belone belone- inhabit low salinity areas so that their cadmium resistance might be related to their ability to overcome variation in membrane permeability caused by the metal. An alternative possibility is that the larval fish contain metallothioneins which bind and detoxify the cadmium as observed in other marine vertebrates (Olafson and Thompson, 1974).

VIII. STUDIES OF THE TOXIC EFFECTS OF HEAVY METALS UPON NATURAL POPULATIONS OF ZOOPLANKTON IN

LARGE VOLUME SEA WATER ENCLOSURES

Attempts to study the effect of sub-lethal levels of copper upon the development of natural zooplankton populations by containing them in large-scale water enclosures have, so far, been beset with unforeseen difficulties due to heavy predation of the herbivorous species ; both in Scotland and in Canada, the feeding of ctenophores and medusae gradually reduced the numbers of copepods in the experimental and the control enclosures (Gamble, Davies and Steele, 1977 ; Gibson and Grice, 1977 ; Reeve et al., 1976 ; Reeve, Gamble and Walter, 1977a) an average ctenophore being able to consume more than 100 copepods of the size of Aeartia or Ternora. each day (Gamble et al., 1977).

The fall off in zooplankton numbers in the enclosures to which copper had been added were, however, much more rapid (Fig. 18A, B) and Reeve et al. (1976), by estimating the extent of the decrease due to predation on the basis of the food requirement of the carnivores, showed that, in their absence, there would in fact have been a slight increase in the total numbers of zooplankton in the enclosures to which no copper was added but a decrease when the metal was present, the decrease being more marked at 50 pg copper/l than at 10 pg/l (Fig. 1SC). In the corrections for predation made by Reeve et al., it was assumed that the feeding rates of the carnivorous zooplankton would be unaffected by the presence of added copper, but later experiments

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r n

CEPEX Copper Experiment I , June 1974

rc

I-

\ t " t

"0 I,,,,, 5 10 15 20 25 ' 0 111111 5 10 15 20 25 102 0 - 5 10 15 20 25

Doys ofter copper addition Doys ofter copper addition

FIG. 18. The effect of the copper additions to the CEPEX enclosures up011 their zooplankton populations. A : total numbers of zooplankton/ms (excluding the main carnivores) ; B : numbers of medusae and ctenophores/ms; C : computed numbers of zoo- plankton/mS in the absence of predation. Control enclosures, 0 and 13 ; experimental enclosures containing 10 pg copper/l, ., end initially 50 pg copperll, A. By correcting the numbers of herbivorous zooplankton for the decrease caused by the feeding of the medusae and ctenophores, it was found (C) that whereas in the absence of copper, they would have increased slightly. the presence of the metal caused them to decrease. (After Reeve et al., 1976.)

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POLLUTION STUDIES WITH MARINE PLANKTON-II 469

(Reeve et al., 1977s) have shown that they are, in fact, reduced by the metal. Thus the decrease in numbers of the herbivorous zooplankton caused by the copper would have been even greater than those shown in Fig. 18.

The 24 hour LC,, values for copper toxicity on the two ctenophores Mnemiopsis mccradyi Mayer (29 pg/l) and Pleurobrachia bachei Agassiz (misidentified as P. pileus) (33 pg/l) determined by Reeve et al. (1976) indicated that, for their size, these carnivores were much more sensitive to the metal than chaetognaths and copepods and, in agreement with this, it was found that the numbers of predators in the polluted enclosures decreased even more rapidly than those of the other zooplankton (Fig. 18B) (Reeve et al., 1976).

Gibson and Grice (1977) monitored the changes in the numbers of individual species and groups in the decreasing zooplankton popu- lations and found marked differences in their compositions. In one experiment in early summer, larvaceans and later also the copepods Pseudocalanus sp. and Acartia longiremis (Lilljeborg) dominated the control populations whereas in the 10 pg/l and 50 pg/l copper-polluted enclosures, the larvaceans were quickly replaced by Pseudocalanus sp. and these, in turn, by polychaete larvae. In a second experiment carried out in late summer, on the other hand, the small copepods Pseudocalanus sp. and Paracalanus parvus (Claus) remained dominant throughout. The toxic action of increasing levels of copper in the enclosures was reflected in the length of time taken for the Pseudo- calanus sp. populations to be reduced to 50% of their starting levels- 2.5 days a t 50 pg/l and 7 days at 10 pg/l compared with 8 days in the unpolluted controls in the first experiment, and 4.5 days a t 10 pg/l and 7.5 days at 5 pg/l with 11 days in the controls in the second.

Reeve et al. (1977a) found that both 5 and 10 pg copper/l reduced the rates of food ingestion by Pseudocalanus sp. and Calanus sp. when feeding either on food growing in similarly contaminated water or on a standardized unpolluted food source. Faecal pellet production was also affected in the same way as had been observed in the laboratory studies (Reeve et al., 1977b) described previously. Further, when Calanus plumchrus which had virtually ceased to produce faecal pellets after 4 days exposure to 4 pg copper/l were transferred to unpolluted water, the faecal pellet production rate rapidly returned to a value only slightly below that observed in a control group of animals. This led Reeve et al. (1977a) to reflect that the reduction in the feeding and the related faecal pellet production rates might have been due, not to the toxic action of the copper, but to the response of the animals to the polluted water.

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460 ANTHONY a. DAVIES

Highly copper sensitive species such as Euphccusia paciJica reacted even more quickly to exposure to the metal, for after 24 hours in 5 pg copper/l, overnight faecal pellet production was reduced from 37 per animal in the controls to one per animal in the contaminated popu- lation. A reduction in the feeding rates of ctenophores by between 45 and 70% was similarly caused by exposure to copper at 5 or 10 pg/1.

Reeve et al. (1977a) observed a very great difference between egg production by the zooplankton in the controls and those in the copper- polluted enclosures, the numbers of eggs released a t 5 pg/l a t one point being lower by a factor of 6 than in the controls even though the zooplankton numbers were similar. It was pointed out, though, that there was insufficient evidence to relate the decrease in fecundity directly to the copper as it could equally have been caused by other factors such as a delay in the maturation of the females. At 10 pg/l for example, very few eggs appeared throughout the 21 day period of the experiment and no adult females were discovered in this container after initial sampling. It will be recalled that D’Agostino and Finney (1974) observed delays in the development of ovigerous females of Tigriopus japonicus exposed to copper and cadmium.

IX. HEAVY METAL CONCENTRATIONS IN NATURAL POPULATIONS OF MARINE ZOOPLANKTON

Appendix I11 contains most of the currently available values for heavy metal levels in marine zooplankton. The problems associated with obtaining and comparing such data are much the same as those discussed in connection with the comparable figures for phytoplankton (Section V). In view of this and the many factors which affect the uptake and loss of metals by the animals-temperature, food avail- ability, concentration of metal in the water-it is not surprising that the data, even for a single species obtained from different areas, often show considerable variability and it is almost impossible to decide why, in certain cases, metal contents were found to be unusually high. As already noted with the phytoplankton data, most of the high values- especially for silver and copper-were obtained spectrographically by Nicholls, Curl and Bowen (1959) and Vinogradova and Koval’skiy (1962).

Even data for zooplankton from a relatively restricted area can spread over a wide range but this is usually associated with gradients in the heavy metal concentrations due to pollution. Windom (1972) found that the levels of mercury, copper, lead or zinc in plankton collected from the Atlantic Ocean within 100 km of the United States

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POLLUTION STUDIES WITH MAJXINE PLANKTON-II 461

coast were often very much higher than those obtained well away from terrestrial influence though cadmium and arsenic contents were relatively uniform on a near-shore to off-shore transect. Windom, Taylor and Stickney (1 973) similarly found that while mercury levels in zooplankton collected a t distances greater than 200 km from the North American seaboard were in the range 0.1-0-3 p.p.m. dry weight, nearer to land they increased substantially and a value of 5.3 p.p.m. was recorded in the New York Bight. No such gradient was obvious in the metal levels of microplankton collected during a north-south transect of the Mediterranean off the south-east coast of France (Fowler et al., 1976b) and the Mississippi River plume seems, surprisingly, to have little effect upon metal levels in zooplankton in the north-west Gulf of Mexico (Trefry and Presley, 1976).

Although mercury levels were in general quite low compared to the other heavy metals, some very high concentrations have been reported for samples of mixed zooplankton obtained from certain areas, e.g. the Adriatic Sea during 196P69 (VuEeti6, Vernberg and Anderson, 1974) and the Ria de Arosa, Spain (Corral and Mass6, 1975) though in both cases levels were mostly below 3 p.p.m. dry weight. Zooplankton in Sdrfjord, Norway seem to be heavily contaminarted with mercury containing levels up to 25 p.p.m. dry weight (Skei, Saunders and Price, 1976) due, no doubt, to the industrial activity in that area. These values may be compared with the range 0.14-2.6 p.p.m. mercury found in zooplankton collected off Minamata, Japan (Hirota et al., 1974). Levels of methylmercury in plankton from various locations off Japan were in the range equivalent to 0-15-0-45 p.p.m. of the metal (Hirota et al., 1974) and were thus not very different from the concentration found in Oplophorus sp. collected from the eastern Atlantic Ocean (Leatherland, Burton, Culkin, McCartney and Morris, 1973).

Cadmium concentrations were generally below 5 p.p.m. dry weight though some of the samples of mixed zooplankton collected off Puerto Rico (Martin, 1970) and off the eastern United States coast (Windom, 1972) were substantially higher than this. Certain species, too, appeared to have accumulated unusually large quantities of cadmium ; the copepod Labidocera acutifrons Dana (10 p.p.m.) and the decapod Systellaspis debilis (Milne Edwards) (13 p.p.ni.) both obtained from the eastern Atlantic Ocean by Leatherland et al. (1973) are examples of this. Fowler et al. (197613) similarly found that the amphipod Phrosina senzilunata Risso contained between 5 and 11 times more cadmium than other species of zooplankton gathered from the Mediterranean, and it would be interesting to know why this happens in certain animals.

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462 ANTHONY Gi. DAVIES

Nickel, which came near the top of the list of heavy metals ranked in order of the relative hazards they present to marine life (Ketchum et al., 1976), reached surprisingly high concentrations in some of the samples of zooplankton collected off Puerto Rico by Martin (1970) but it is impossible to know whether the levels found were deleterious to the animals containing them. Lead concentrations varied widely, possibly due to contamination of some samples by paint chips, for Topping (1 972) found that such particles contained 42 000 p.p.m. of the metal.

Both Szabo (1968) and Martin (1970) have observed that zoo- plankton collected from deep waters (> 100 m) tend to contain greater quantities of metals than those gathered from the surface. Martin has sought to explain this on the basis that the lower food supply available at depth would result in slower growth of the animals and, therefore, less frequent moulting; the extended period of contact between the body surfaces of these animals and the water would then allow a greater build-up of metals giving higher body burdens. Such an explanation, of course, takes no account of vertical migration for feeding purposes.

There is still considerable speculation about whether or not metal concentrations in organisms increase at each trophic level of the food chain. Using the data of Vinogradova and Koval’skiy (1962), Lowman et al. (1971) calculated the concentration factors for metals in Black Sea diatoms and zooplankton and in all cases they turned out to be higher in the plants than in the animals. The data of Martin and

TABLE X. MEAN CONCENTRATIONS (P.P.M. WET WEIGHT) OF SOME HEAVY METALS IN PHYTOPLANKTON AND COPEPODS COLLECTED FROM MONTJCREY

BAY, CALIFORNIA DURING 1971

Concentration in Concentration in Ratio (:) phytoplankton ( A ) copepods ( B )

Mercury 0.01 1 0.010 0.9 Cadmium 0.11 0.40 3.6 Silver 0.016 0.003 0.2 Nickel 0.22 0.34 1.5 Lead 0-36 0.33 0.9

Zinc 4-7 10.8 2.3 Copper 0.61 1.18 1 *9

The values are derived from Martin and Knauer (1973) using average wet weight/% weight ratios of 10.8 for t,he copepods as measured by the same authors, and 19.0 for the phytoplankton (calculated from the data of Knauer and Martin (1972) which were obtained during the mme sctmpling pmgramme).

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POLLUTION STUDIES WITH MARINE PLANKTON-11 463

Knauer (1973) may be used for a similar purpose. In Table X, the mean concentrations of some metals in the organic (non-siliceous) fractions of the phytoplankton and also in the copepods in Monterey Bay, California, during 1971 have been calculated on a wet weight basis. The ratios of these concentrations indicate that while mercury and lead were little different be,tween the two t ropl~c levels, silver decreased and the other metals-nickel, copper, zinc and cadmium-- were concentrated more by the copepods than by the phytoplankton.

The absence of amplification in mercury levels at this link in the food chain is probably related to the observation that euphausiids tend to accumulate inorganic mercury more from solution than from their food (Fowler et al., 1976a) so that levels in animals would not be related to those in the diet. Interestingly, Williams and Weiss (1973) found little evidence of a build-up of mercury even in animals at higher trophic levels. On the other hand, Cocoros, Cahn and Siler (1973) showed that mercury concentrations in phytoplankton in estuaries on the south-east coast of the United States were about four times higher than in zooplankton when expressed on a dry weight basis. Even after the appropriate corrections to a wet weight basis, the plant contents would still be higher than the animals indicating that here, at least, mercury contents decreased between the first and second trophic levels.

Prom the pollution point of view, one of the most useful applications of the data in Appendix I11 would be as a means for assessing the extent to which zooplankton in the sea are already at risk due to their heavy metal burdens. Unfortunately i t is not possible to carry out such an exercise a t the present time due to the absence of any inform- ation relating the effects of heavy metals upon zooplankton to the amounts taken up by the animals. It is to be hoped that this gap in our knowledge will be filled in the near future.

X. CONCLUSIONS

This review of our current understanding of the effects of heavy metals upon marine plankton has highlighted the fact that although, at present, the data obtained from laboratory experiments cannot usually be used to assess the degree to which natural populations are at risk due to metal pollution, it might now be possible to begin to rectify the situation.

For if, as might be expected, the damage caused to plankton by metals is more closely linked to the amounts taken up by the organisms than to the concentrations in the water, problems associated with the

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464 ANTHONY Q. DAVIES

differences between laboratory experiments and natural conditions, such as the concentration and chemical form of metals in the water and the periods of exposure of the plankton to them, largely disappear; and once a quantitative relationship between the burden of metal obtained by an organism and an important physiological effectgrowth inhi- bition, for example-has been established, it is then only necessary to measure the metal content of the same organism collected from the sea to discover the extent to which it might be being affected by the metal.

Other problems will also need to be investigated, however, particu- larly the effect of the nutritional status of plankton upon their suscepti- bility to metals. More importantly, the question of adaptation deserves special attention for not only could it give rise to resistant strains in the laboratory and in the field making the extrapolation of experimental data less valid, but the survival of the metal tolerant plankton populations would also increase the likelihood of greater quantities of metals getting into marine food chains and, ultimately, reaching man.

XI. ACKNOWLEDGEMENTS

The author wishes to thank all of the many people who have helped with the preparation of this review, particularly the Library staff of the Plymouth Laboratory and Drs G. T. Boalch, J. C. Green, R. P. Harris and J. H. Wickstead.

He is especially grateful to Jill Sleep who organized the data for the Tables and Appendices into their appropriate orders and compiled the list of references, to G. A. W. Battin for redrawing many of the diagrams and to Marsha Rapson and Sylvia Marriott for typing the manuscript.

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Skei, J. M., Saunders, M. and Price, N. B. (1976). Mercury in plankton from a polluted Norwegian Fjord. Marine Pollution Bulletin, 7, 34-35.

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Takahashi, M., Thomas, W. H., Seibert, D. L. R., Beers, J., Koeller, P. and Parsons, T. R. (1976). The replication of biological events in enclosed water columns. Archiv fiir Hydrobiologie, 76, 5-23.

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Turekian, K. K. (1971). Rivers, tributaries and estuaries. I n “Impingement of Man on the Oceans,” (D. W. Hood, ed.), pp. 9-73. Wiley-Interscience, New York and London.

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478 ANTHONY 0. DAVIES

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Zingmark, R. G. and Miller, T. G. (1975). The effects of mercury on the photo- synthesis and growth of estuarine and oceanic phytoplankton. In “Physiolo- gical Ecology of Estuarine Organisms.” (F. J. Vernberg, ed.), pp. 45-57. University of South Carolina Press, Columbia, South Carolina.

RECENT REFERENCES NOT DISCUSSED IN TEXT Berland, B. R., Bonin, D. J., Gu6rin-Ancey, 0. J., Kapkov, V. I. and Arlhac, D. P.

(1977). Action de m6taux Iourds 8. des doses subl6tales sur les caract6ris- tiques de la croissance chez la diatom& Skeletonemu coetatum. Marine Biology,

Copper sorption and release by Cyclotella meneghiniana (Bacillariophyceae) and Chlamydomonas reinhrdtii (Chlorophyceae). Journal of Phycology, 13, 198-202.

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Saboski, E. M. (1977). Effects of mercury and tin on frustular ultrastructure of the marine diatom, Nitzsohia liebethrutti. Water, Air and Soil Pollution,

Sonntag, N. C. and Greve, W. (1977). Investigation of the impact of mercury on enclosed water columns using a zooplankton simulation model. Journal of the Fisherim Reseamh Board of Canada, 34, 2295-2307.

Weigel, H. P. (1977). On the distribution of particulate metals, chlorophyll and seston in the Baltic Sea. Marine Biology, 44, 217-222.

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18, 3-8.

8, 461-466.

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APPENDIX I Snararaay OF DATA OBTAINED FROM E X P E ~ N T E ON E ~ O T S

OF HEAVY METALS ON CULTURED PHYTOPLANKTON

Notea : a. C d ~ w Au&#i (Lohm.) Kampt. h now in Bmlicmio A M (Lohm.) H8y & Mohler b. Emviaella mamuWou riaS Parka & BslLntlm is now in Prormwnlrum minimum (Pav.) J. &hiller

Medium Effect

MERCURY Ehodophyta PwphVMium marinurn SW +0.2 d d NO,-+U) phi H,PO,-+ 20 102klux 3.0~10' 5 Bangiophyceae Kylin micronutrienta+2 mM TRIS 14 h/day

z t E k $ & e Butcher

25 Clyplmnocuu pseudobaltica 6.0 x 10' 10

Dinophyta Amphidbium curlcrae Dlnophyceae Hulburt

E m w a marmeMwu due Parke & Ballantine

25 SW + 0.88 mM NO,-+ 36 pM HrPO1-+ 23 f 1 6.5 klux 50-70 1-10 micronutrients + 23 @d EDTA 24 h/day

1-10

10-6.0 x 10'

10-5.0 x 10'

? 1-10

Lowest concentration causing growth inhibition (1976) Lowest lethal concentration Loweat concentration causing growth inhibition Lowest lethal eoncentration After 4 h exposure of culture to Zingmark and mebl, ' T O , photosynthetic rate Miller (1976) little affected After 24 h exposure of culture to metal, "CO, photosynthetic rate reduced to about R0"b of that in control After 4 h exposure of culture to metal, "CO, photosynthetic rate reduced to 100-200/,, of that in

Berland el d.

,- control After 24 h exposure of culture to metal, "CO, photosynthetic rate reduced to 8 0 4 % of that in control ___ __ -. Increasing reduction in growth rate and ha1 population

10'-5.0 x 10% No mowth for 3-5 weeks then ? cultbcs recovered; only 3% of mercurv left in culture after 18 days -

sw + 0.2 mM NO*- + 20 &PO1- + 20 10.2 klux 4 0 x loa <5 Lowest concentration causing Berland et d. micronutrients + 2 mM TRIS (1976) growth inhibition 14 h/day

20 Lowest lethal concentration

3.0 x 10' 5 Lowest concentration causing growth inhibition

15 Lowest lethal concentration

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MERCURY (continued) Dinophpta Dinophyceae Lebour Hg added a8 acetate 14 h/day

(mRtmusd) Ehrenberg

@mdiniUm apteduno SW + 1 m31 NO,- + 130 1139 H,PO,- + Prorocenlrum micam

18 f 1 6 klux

Sdflsiella faeroenne (Paulsen) Balech & Soarm

H$&p€~g~~~~ lsoehryms gdbana Parke SW + 0.12mJi SOa- + 1 2 ~ A l € l I ' O ~ - + (Haptophyceae)

14.5 f 4.6 klus rnicronutr ion ts 0.5 24 hiday

- 2 10

6.0 x 109 1-10

- 1-50

ti.0 x 10' 210 - 10'

1.5 x lff 1

1 - 1.5 x 10' 10

10

1.5Xlff 50

-

- 5@-10'

- 108

1.5 X l f f 210' ca. 6.0 x loD 1.5-75

1 0 5

18.6

46

Gradual wash-out of cells in continuous culture Little effect upon growth

Little effect upon cell population in continuous culture Lethal concentration In continuous culture, initial decrease in cell population followed by recovery almost to original level Little effect upon growth rate and final population In continuous culture, cell population lower than in control after 7 days Growth rate reduced but cell population after 16 days eame as in control

g a p e r (1976)

In continuous rulture, growth inhibited after 7 days Initial decrease in cell population followed by recovery arid growth to cell population similar t0 that in control In continuous culture, cell population decreased

In continuous culture, lethal concentration Lethal concentration Increasing reduction in growth rate, measured in terms of biomass due to effect of metal upon cell volume. Growth rate related to cellular content of mercury Growth a t 6mt severely inhibited but cultures later

Davies (1074)

recovered and grew exponentially to 6nal population similar to that in control .- .~...~ .. Initial growth followed by death of culture Lethal concentration

Page 101: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

P a u k a (Monoehryeis) luthen (Droop) Green

SW + 0 2 d NO.- + 20 pM H,PO,- + 20 10.2 k l u 1.1 x 10' 5 Lowest concentration causing Berlandetd. micronutrients + 2 mM TRIS

ArtificialSW + 1mMNO.- + 57pMHP0,'- - P 2.0 x 10' Rate of 0, production in + micronutrients + 0.15 mM EDTA

14 h/day growth inhibition (1976) 25 Lowest lethal concentration

Overnell(1978) saturating red light reduced to 50% of that in control after 15 mini dark incubation with metal

SW + 0.2 mM NO,- + 20 pM H,PO,- + 1 0 2 klUX 1.3 x 10' 5 Loweet conceutration causing Berland el d. micronutrients + 2 mM TRIS

SW + 0.2, mN NO,- + 20 p N H,PO,- + 20 10.2 klux 8.0 x 10' 10 Lowest concentration causing Berland & d. rmcronutrients + 2 mM TRIB

14 h/day growth inhibition (1976)

14 h/day growth inhibition (1976)

2,5 x 10' 5 Lowest concentration causing

9 ? ca. 10' 3 Little effect upon growth Aubert e l al.

20 Lowest lethal concentration

25 Lowest lethal concentration

growth inhibition 25 Lowest lethal concentration

SW + 046 mM NO,- + 25 glycerophosphate + micronutnent; (1972) + 14 mM EDTA

Na

Pavlcma pinguW Green

Eeterolhriz sp.

Aaterimzella glaciolis Csatr. ( A . japmica Cleve & M6ller)

Atlheya deeora West

9 ? 11 Growth severely inhibited P ? 22 Lethal concentration

lo" P 2.0 x lo*- Rateof0,productionin OverneU(1978)

saturating red light reduced to 9CH3% of that in control after 15 mins dark incubation with metal

Arti5cial SW + 1 mM NO - + 57 a 20 - HPO 2- + micronutrients'+ 0.15 E D T ~

SW + 0.2 mM NO,- + 20 pM H,PO,- + 10.2 klnx 3.3 x lo" 5 Lowest concentration causing Berland et d. micronutrients + 2 mM TRIS

SW + 0.18 mM NO,- + 7 H,PO,- + 15 5.4 k l w 1 10' Lethal concentration micronutrients + 5 a EDTA

14 h/day growth inhibition (1876)

24 h/day Patouillet (1972)

20 Lowest lethal concentration Hannsn and

Chastoceroa didymus Ehrenberg

ChaEtOoeros gdvrntonensis Collier & Murphy Cylilurrdhcw doslen'um (Ehr.) Reiman & Lewin

Cylindrolheea cloaterium (Nileichia clostsrium Ehrenb.). Fraqihna pinna4a Ehrenberg

Lauderia borealis Gran

SW + 0 .2 mM NO,- + 20 pal H,PO,- + micronutrients f 2 mM TRIS

SW + 1.1 mM NOa- + 66 p M &PO,-

20 10.2 klux 14 h/day

? ?

6.5 x 1V

?

3.0 x 104

10'

P

P

P

P

10

20 002-0.35

Lowest concentration causing growth inhibition Lowest lethal concentration After 5 days growth, cell population lOCral% of that in Control Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing m w t h inhibition

Berland el d. (1976)

Sick and Windom (1975)

Berland el d. (1976)

SW + 0.2 mM NO,- + 20 a H,PO,- + micronutrients + 2 mY TRIS

<5

25 < 5

10 10'

10'

7-22

Lowest lethal concentration Growth rate reduced relative to that in control Lethal concentration

SW + 0.22 mM NO - + 9 pBi H,PO,- + micronutrients + 6 'a EDTA BW + 0.18 mM NO.- + 7 uM H.PO.- +

P+mdad@bm tnconzutum Bohlin Hannan and

Patonillet (1972) micronutrients + 5 - a EDTA - - Artificial SW + 5.8 mM NO.- + 18 mN 1 8 * 2 2klur

14 h/day After 16 days growth cell population only 7 5 4 % of that in control Rate of 0, production in saturating red light reduced to loo-O% of that in control, a f k 15 mins dark Incubation with the metal

Nuzzi (1972)

OvemeU (1976)

E, glycerophosphata + mic;onutrien&+ 41 mM TRIS + 80 phl EDTA Artificial SW + 1 mM NO,- + 57 a HP0,'- + micronutrients + 0.15 mM EDTA

2.0 x 10'- 1-4 x 10.

Page 102: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

MERCURY (oontinued) Chryaophyta P+codWtulum SW + 0 2 mM NO.- + 20 pM H,PO,- + 10.2 k l u ~ 6.0 X 10' <5 BaciUpriophyceae tracornutua micronutrienta + 2 mM TEIS 14 h/day

Arti5cialSW+lmMNO,- + 57aHP0,'- + micronutrients + 0.15 mM EDTA

SEdstoMcno coataturn SW only 5.4klux 108 06-60 (Qrev.) Cleve 14 h/day

10-25

5 -

SW + 088 mM NO,- + 36 pM H,PO,- + micronutrients + 23 fl EDTA

S W + 0 2 mM NO,- + 20 pM H,PO,- + rmcmnutrients + 2 mM TEIS

Artiflccisl sw + 1 DlM NO,- + 57 HP0,'- + micronutrients + 015 mM EDTA

Thdadtiosira pwUaonan0 (Hut.) Hasle & Hehdal

S W + 0.2.- NO.- + 20 pM HaPo,- + rmcronutrienta + 2 mM TEIS

T ~ E ~ ~ ~ ~ ~ M ~ J o u & ? I M M SW + 018mM NO,- f 7 d H,PO,- + (ovdolsUo MM Haste&) miomnutdents + 6 ILM EDTA

23 f

20

20

15

1 6.5 k h ~ ~ 24 h/day

10.2 khlx 14 h/day

-

10.2 klux 14 h/day

1 5

50-70 1-10'

5 x 10'

1.3 x 10' <5

15 9 5 x 10'

5.0 x 10' 5

15 ? 10'

Loweat concentration causing growth inhibition (1976) Zowest lethal concentration Rate of 0, production in ssturating red light reduced to 50% of that in control after 15 mins dark incubation with metal Increasing reduction of growth rate and llnal population (1973) Decrease in cell nos. a t h t but later recovery to exponential growth In spin-fllter continuous culture, cell population decreased by almost 5% by single metal addition but later recovered

Berland e4 d.

Overnell (1976)

Rice st d.

ibly due to loss of mercury r m system Cultures recovered after lag phase

After 4 h exposure of culture to Zingmark and metal W O , photosyntbetic rate Miller (1975) reduck to 80-10% of that in control After 4 h exposure of culture to metal, W O , photosynthetic rate reduced to zero Lowest concentration causing growth inhibition (1976) Lowent lethd concentration Rate of 0, production in saturating red light reduced to 50% of that in control after 15 minn dark incubation h t h metal Lowest concentration causing growth inhlbition (1976)

Berland e4 d.

Overnell(l976)

Berland el d.

Lowest lethal concentration No growth after 3 days Hannan and

PatouiUet (1072)

? U

Page 103: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Chlomphyta Prssinophyceae (Cienk) Waern mlcronutrients + 2 mM TRIS

Praainododus murinus

TetMsdmi.8 atrhta Butcher

chlomphyta Brodriomonar aubmarina Artiflcial SW + 1 mM NO,- + 57 p M Chlorophyceae Bohlin HP0.'- + micronutrients + 0.15 mM

SW + 0.2 mM NO,- + 20 pM H,PO4- +

Cartsria ap.

CItlamydmnonas sp.

CMam&nmnm pdla Butcher

Dundidla bioculala Butcher

Dundiclla tertidecta Butcher

EDTA

SW + 1.1 m?d NO,- + 56 pM H,PO4-

Artitlcial SW + 5.8 mM NO,- + 18 m?d Kb glyeerophospbate + micronutrients + 41 mM TRIS + 80 EDTA SW + 0.2 m?d NO,- + 20 pM H,PO,- + micronutrients + 2 mM TRIS

Artificial SW + ? NO,- + ? PO,'- + vitamins

ArtiflcialSW + lmMN0,- + 57pMHP0.c- -k micronutrients + 0.15 mM EDTA

SW + 1.1 mM NO,- + 56 pM H,P04-

8W + 0.12 mM NO,- + 12 pM HPO,*- + mmonutrlenta

ArtiEcial SW + 1 mM NO,- + 57 pM HPO,e- + micronutrients + 0.15 mMEDTA

20 10.2kl~r 4.0 X 10. 5

50 10' 15

50

14 h/day

9 8.0 x 10' -

Y ? ? 0.02-0.35

18 f 2 2klux ? 7-15 14 h/day

20

'!

14 5 f 0.6

20

10.2 klux 2.5 X 10' < 5 14 h/day

24 107 4.0 X-ioc - 4.0 X lo5

? 2.0 x lo*- - - 2.0 x 104

9 Y 0.02-0.35

4.6 klux m. 5.0 X 10' 28-4.1 X 24 h/day 10'

10'

2.0 x l W

- ? 2.0 x 10' -

Lowest concentration causing growth inhibition (1070) Lowest lethal concentration Lowed concentration causing growth inhibition Lowest lethal concentration Rate of 0, production in mturating red light reduced to 50% of that in control after 15 mins dark incubation with metal After 5 days growth, cell population 100-0% of that in

Berland el d.

Overnell(1070)

Sick and Windom (1975)

control After 16 days growth, cell population only 5 0 4 % of tbat in control Lowest concentration causing growth inhibition Lowest lethal concentration Oxygen production 834% of that in control when culture intensely illuminated (16 k l u ) Rate of 0, production in saturating red light reduced to OO-lO% of that in control after 15 mins dark incubation with metal After 5 days growth, cell population 100-5% of that in control

Nuzzi (1072)

Berland et d. (1076)

Saraiva (1073)

Overnell(lO75)

Sick and Windom (1975)

Nosigniflcant effect upon growth Davies (1070) rate No effect upon growth rate a t first but later reduced to 60% pf that in control. Final population aho reduced Growth rate 16O/ of that in control after i n i h rapid burst of growth. Final population 87% of tbat in control Rate of 0, production in saturating red light reduced to 50% of that in control after 15 mins dark ineubation with metal

Overnell (1976)

Page 104: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Group Reference + ORGANIC MERCURY COMPOUNDS

Haptophyts Pawlova (Illonochrysis) SW + 1.8 mM NO,- + 0.93 mY NH,+ + 20.5 f 1 5.4 klux Prymnesiophyceae 1uthei-i 24 hiday (Haptophyceae)

EE%$yceae gdoestonensis

0.15 rnM H,PO,- + 27 JIM EDTA

Chaeloceros SW + 0 2 2 mM NOa- + 9 pM H,PO,- + micronutrients + 6 )lM EDTA

SW + 1.8 mM NO.- + 0.93 mM NH,+ + 0.15 mM H.PO,- + 27 JIM EDTA

15

Phaeodactylur/l lriemnulum

20.5 f I

SW + 022 mM NO,- + 9 H,PO,- 15 + micronutrients + 6 pbf EDTA

Artificial SW + 5.8 mM NO,- + 18 mM K, glycerophosphate + micronutrients + 41 mM TRIS + 80 JIM EDTA

18 f 2 2 klux 14 h/day

Artificial SW + 1 mM NO,- + 57 pM HP0,'- 20 - + micronutrients + 0.15 mM EDTA

Thalmaiosira pSt?udOnana SW + 0 2 mM NO,- + 9 pM H,PO,- + (Cydotella mm) Chlumgdomonas sp.

Dunaliella euehlora Lerche 0.15 mM H,PO.- + 27 EDTA

micronutrients + 6 pM EDTA Arti5cial SW + 5.8 mM NO,- + 18 mM K,

+ 41 mM TRIS + 80 pM EDTA SW + 1.8 mM NO,- + 043 mM NH,+ +

Chlorophyta Chloroph yceae glycerophosphate + micronutrients

D u d i e l l a tertioleeta Artificial SW + 1 mM NOa- + 57 pM HP0,'- f micronutrients + 0.15 mM EDTA

Protwoecus sp. SW + 1.8 IIIH NO.- + 093 mhI NIX,+ + 0.16 mM H,PO,- + 27 pM EDTA

15 5.4klux 24 hiday

14 hiday 18 f 2 2kllra

20.5* 1 5.4 klux 24 h/day

20.5&1 5.4klux 24 h/day

1.5 x lo6 0.6 Ethyl mercury phosphate; no Ukeles (1962) effect on growth

?

2.5 x

?

?

?

?

?

?

1.5 X

?

106

a6.0 Ethyl mercury phosphate; no growth after 10-14 days

reduction in growth rate, especially at higher concentrations

06-6.0 Ethyl mercury phosphate ; Ukeles (1962) growth after 10-14 days 5547% of that in control

6.0 Ethyl mercury phosphate: no growth after 10-14 days

on growth rate Patouillet (1972) 5.0 x 10% Dimethyl mercury; marked

reduction in growth rate 0.06-9.0 Phenyl mercuric acetate; after Nuzzi (1972)

16 days growth, cell population only 80-10% of tlmt in control

9.0-15 Phenyl mercuric acetate; after 16 days growth, cell population only 10% of that in control

10-40 x lo* Methyl mercuric chloride; rate Overnell (1975) of 0. production in saturating red light reduced to 9625% of that in control after 15 mins dark

lo', 5.0 x 10' Dimethyl mercury; marked Hannan and Patonillet (1972)

l o p Dimethyl mercury; little effect Hannan and

10',50 x 10'

0.06-3.0

10' 0.6-6.0

60

20-4.0 x 10'

inmihation with metal . Dimethyl mercury; slight Hannan and reduction in growth rate Patonillet (1972) Phenyl mercuric acetate; after Nuzsi (1972) 16 days growth, cell population only O W % of that in control Ethyl mercury phosphate; Ukeles (1962) growth after 10-14 days 64-31 % of that in control Ethyl mercury phosphate; no growth after 10-14 days Methyl mercuric chloride; rate Overnell(1975) of 0 roduction in saturating red l&%t reduced to WlOY of that in control after 15 mini dark incubation with metal

1.5 X 10' 0.6 Ethyl mercury phos hate. Ukeleg (1062) growth after 10-14 &ps 86% of that in control

a6.0 growth Ethyl mercury after 10-14 phosphate; days no

Page 105: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Medium Effect Reference Group

CADMIUM

Porpliyridiuin inarinum SW + 0.2 NO,- + 20 UM H,PO,- + micronutrients + 2 mM TRIS

20 102klux 14 h/day

Rhodophyta Bangiophyceae

Cryptophyta Cryptophyceae

Dinophyta Dinophyceae

3.0 x 10' 25

>5.0 x 10' 6.0 X 10' 25

>5.0 X lo* 4.0 X lo* 25

2.5 x lop 3.0 X loa 50

3 x 10' 1-10 >2.5 x 10'

>10

Lowest Concentration causing growth inhibition Lowent lethal concentration Lowest concentration causing growth inhibit ion Lowest lethal concentration Lowest concentratiun causing growth inhibition Lowest lethnl concentration Lowest coocentrution cawing mowtlr inhibition Lowest Ivtllal concentration Little effect on "CO. photoaynthetic rate Photosyntliesis inhibited especially after prolonged exposure to metal Final cell populatioir 7507: of that in control nt end of exponential yhnse No eflect upou rate of 0, production in saturating red liyl after 15 mins dark incubntion with metal ].owest concentration causing growth inhilJiti(Jn Lowcst lethal concentration Ro l.ncct upon rate of 0, production in saturntiug rod light after 15 mi118 dark incubation with metal Lowest concentration causing growth inhibition

Lowest lethal concent rat ion Lowest concentration causing growth inhihition Lowest lethal conceotration Lowest roncrntration causing growth inhibit ion Lowest lethal roncentration

Berland el d. (1976)

C r y p t m o m pseudobdtica

Amphidhiurn curterae

Exuviaella mariwlebour4ue

Tkachenko el ul. (1974)

%rodinium fcesuin (Levander) Kof. & Swezy + trace elements + 0.1 M citrate

SW + 2 mM NO.- + 0.35 mM HP0,'- 20 2 68 klux P

Arti5cial SW (16% salinity) + 1 mM NO.- + 57 HN HPO.1- + trace elements + 0.13 M EDTA Artificial SW + 1 mM NO*- +

18 4.6 klux 12 h/day

18-20 - -

CU. 3.0 x 103 -3.0 X 10'

1 5.6 x 106- 1.1 x 106

Bentley-Mowat and Reid (1977)

Overnell (1976) I t

Haptophyta l'rymnesioph yceac ( IIaptophgceaa)

H y m e n o m o m (Crdcosphueru) elongata (Droop) Parke & Green ISOeh9ySiS gd6anU

57 a HP0,'- + micronutrients + 015 mM EDTA

SW + 0.2 m X NO,- + 20 9M H,POI- + micronutrients + 2 mM TRIS

Artificial SW + 1 nil1 NOa- + 57 @d HP04'- + micronutrients t 0.15 mM EDTA

SW + 02.m NO,- + 20 micronutrrenh + 2 mIv1 TRIS

H2P0,- +

Pavlova (Monochrllsis) lutkri

20 104klux 14 h/day

13-20 - -

1.1 x 104 50

5.0 x 10' ? 1.1 x 106

Berland et d. (1976)

Overnell(l976)

Pavlowa pinguis 20 10.2 klux 14 h/day

1.3 x 10' 25 Berland e l d. (1976)

> 102 8.0 X10* 2.5 X lo*

> 10'

2.5 X 10'

2.5 x lo4 50

Chrysophyta Xanthophyceae

Heterolhrir sp.

Monullanlus salinu

Page 106: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

SpscieS Medium Initial no. Initial

Temp. IUumina- oj cellal mctal c m . ('C) liMI rnl added (&I)

Effecl

Chaetocero8 didymue

Chaetmros gdveetonenais Cylindrdheea closteriun

Ditylum b r i g h t d i i (West) GrUn.

Frogilaria pinnda

Lauderia boredis

Artiflcial SW + 1 mM NO - + 57 UM HP0,'- + micronnhents + 0.15 mM EDTA

CADMIUM (aontinued)

SW + 0.2 mM NO.- + 20 pM H,PO.- + micronutrients + 2 mY TRIS

SW + 0.18 mnl NO,- + 7 UM H;PO,- + micronutrients + 5 p N EDTA SW f 0.2,mM NO,- + 20 pM H,PO,- +- micronutrients + 2 mM TRIS

SW + 2 mM NO.- + 0.35 d v 1 HP0,'- + trace elements + 0.1 M citrate

SW + 0.2 mM NO.- + 20 pM H.PO,- + micronutrients + 2 mM TRIS

Phaeodaclylum tricmulum

SW + 0.2 mM NO.- + 20 pM H,POI1- + micronutrients + 2 mM TRIS

18-20 - P 5.6 x 104- - 1.1 x 10'

20 102 klux 3.3 x 10' 25

z 50 15 5.4 klux 9 10'

20 102klux 0 5 X 10' 5.0

14 hiday

24 hlday

14 h/day

9

50

10'-10*

20 f 2 68kluX 10 1-10;

Skeletonemu m l a t u m

Artiflcial SW (16% sslinity) + 1 mM NO.- + 57 WM HPO '- + trace elements + 013 mM EdTA SW + 0.2,mM NO,- + 20 pN H,PO,- + micronutrients + 2 mi TRIS

Artiflcial SW + 1 mM NO - + 18-20 57 PM HPO,~- + micronutri&ts + 0-15 DIM EDTA

SW f 0.2 mM NO.- + 20 pM H,PO; + micronutrienta + 2 mM TRIS

SW -4 0.18 mM NO - + 7 pId H,PO,- +

18

20

20

15 micronutrients + 5 ;M EDTA

102klux 3.0 x 10' 50 14 h/day

22.5 x 10' 10% 50

>2.5 x 10' 5.4 k l u ? 10' 24 hiday - P 1.1 x IW

10,2 k l u 1.3 X 10' 2.5 x 10'

- 9 1.1 x 10'

14 h/day >5.0 x 10'

10.2klux 5.0 X 10' 10 14 hiday

5.4 klux P 1 0' 24 h/day

>5.0 x 10'

Overneil(l976) No effect upon rate of 0, production in saturating red light Bfter 15 mins dark incubation with metal Lowest concentration causing growth inhibition Lowest lethal concentration Little effect upon growth

Lowest concentration causing growth inhibition Lowest lethal concentration "CO,-Photosynthetic rate increased relative to control Photosynthesis decreased but still greater than in control Loweat concentration causing growth inhibition Lowest lethal concentration Lowest conceutration causing growth inhibition Lowest lethal concentration Little cEwt upon growth

No effect UDOU rate of 0,

Berland e l al. (1976)

Hanuan and Patouillet (1972) Berland et ad. (1976)

Tkachenko el al. (1974)

Berland et d. (1976)

Berland el d. (1976)

productionin mturatingred light after 15 mins dark incubation with metal

10.2 klux 6.0 X 10' 10' Lowest concentration causing 14 h/day growth inhibition

4.6 klux 12 hlday

> 108 Lowest lethal concentration ca. 3.0 x 10'- Final cell population 100-5% of Bentley-Mowat

and Reid (1977) 3.0 x 10' that in control a t end of

Berland a6 d.

Overnell (1976)

P

exponential phase Lowest concentration musing growth inhibition (1976) Lowest lethal concentration No effect upon rate of 0, production in saturating red light after 15 mins dark incubation with metal Lowest concentration causing growth inhibition (1976) Lowest lethal concentration Little effect upon growth

Berland el d.

Hannan and Patouillet (1972)

Page 107: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Prasinocladus marinue

Tetradelmis strialu

Tetradelmis spp.

Brachiomonaa SUbmaritUZ

Chlamgdmnwm palla

Dundiedla bioculata

Dundiella primdecta Butcher

Dundiclla lerlidecla

SW + 0.2 mM NOs- + 20 pM H,PO,- + 20 micronutrients + 2 mM TRIS

Artificial SW (16% salinity) + 1 mM NO - 4- 57 MM HP0,'- + h c e elements 0.13 mM EDTA

57 pId H P O p + micronutrients + 0.15 mId EDTA

SW + 0.2 mM NO.- + 20 pM H,PO,- + micronutrlents + 2 mM TRIS

Artificial SW + ? NO.- + ? PO4'- + vitamins

18

Artificial SW + 1 mM NOs-. + 18-20

20

? ?

9 9

Artincia1 SW (16% ealiniiy) + 1 mM KO,- + 57 pM HP0,'- + trace elements + 0.13 mM EDTA

57 uId H P O P + micronutrients +

li

Artificial SW + 1 mM NO.-, + 20

0.16 m~ EDTA

SILVER SW + 0.18 mM NO,- + 7 pM H,PO.- + micronutrients + 5 UM EDTA

15

10.2 klux 14 h/day

4.6 klux 12 h/day

10.2 klux 14 h/day

-

?

7 4.6 klux 12 h/day

-

4.0 x lo" 25 Lowest concentration causing growth inhibition

25.0 x 10' Lowest lethal concentration 10' 5.0 x 10' Lowest concentration causing

growth inhibition > 10' Lowest lethal concentration ca. 3.0 x lo*- Final cell population 1055% 3.0 x 10' of that in control a t end of

9

7 1-1 x lo6 Noeffectuponrateof 0, exponential phase

production in eaturating red light after 15 mina incubation

2 .5 x 10'

10'

7

7 9

?

25

5 . 0 ~ 10' 243 x lo6- 2.1 x 10'

39-8 x 10.

>8 x 10s M. 8.0 x 10' -3.0 x lo* 1.1 x 106

with metal Lowest concentration causing growth inhibition hwes t lethal concentration Oxygen production l W 6 % of that in control when culture intensely illuminated (16 klux) Growth inhibited

No growth Final cell population ln&az. 0% of that in control a t end of ex$nential phase Rate of 0, production in saturating red light reduced to 70% of that in control after

Berland et d, (1976)

Bentley-Mowat and Reid (1077)

Overnell(1976)

Berland el d. (1976)

Saraiva (1973)

Peneda- Saraiva (1976)

Beutley-Mowat and Reid (1977)

Overnell(l975)

15 mi& dark incubation with metal

5.4 klux Y 10' Lethal concentratlon Hannan and 24 h/day Patouillet (1972)

? 10' Little effect upon growth

9 10' No growth

Ip 00 4

Page 108: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

le 00 00

Med;um Egmt

LEAD

Rhodophyta Pmphyridturn Bangiophyoeae marinurn

C r y p t O P h ~ ~ Ctllptom-

Dinophyta Amphirlinium carterm Dinophyceae

CrpptophyCCeae p8ei6dObaJtiCa

Emmadla mariadebouriae

Prorocmtrum m k n a

Scrippsiella faeraense

Haptophyta H y m n n m o l ~ a Prymnesiophyceae (Cricosphmra) elonrJaa (Haptoph yceae)

Pavloua (Mmehryg i s ) lutheri

P a v h a pinguis

Heteothrix sp.

.M~ndantua aalina

%%:tthg&%e

Asterimlla glucialia %a!$&eae (A. japonica)

Chaetoceros didymus

SW + 0.2 mM NO,- + 20 pJI H,PO,- + micronutrients + 2 mM TRIS

s7f f 1.2 mM NO,- f 013 mN H,PO,- + soil extract

Artiflcial SW (16% ealinity) + 1 mM NO.- + 57 pM HP0,'- + trace elements + 0.13 mM EDTA YW + 0.2 mM NO.- + 20 ~ d l H,PO,- + micronutrients + 2 mM TRIS

SW + 0.66 mM NO,- f 25 pM Na, glycerophosphate + micronutrients + 14 mM EDTA

SW + 0.2 mM NO.- + 20 micronutrienta + 2 mM TRIS

H,PO,- +

20 102 klux 3.0 x 10' 2.5 x 10' 1 4 h/day

>2.0 x 10. 6.0 x 10. 1.0 x 10.

>2.0 x 10' 4.0 X loa 2.5 x 10'

2.0 x 10' 3.0 x 10'1.0 x 10s

>2.0 x 10' 15 6klux 1.2 x 10'25-1W

1.3 or 7.2 25-10' x 10'

1 4 h/day

>lo* 18 4.6 klux ? 2.1 X 10'-

12 h!day 1.2 x 105

50 10.2kln~ 1.1 X 10' 1.0 X loa 1 4 h/day

>2.0 x 10' 1.3 x 10' 5.0 x 10'

>e.o x 108 8.0 X 10' 2.0 x 10.

>2,0 x 10'

>2.0 x 10.

20 10.2 WUX 2.5 X 10' 2.0 X 10.

2 ? ea. 10' 5.6 x 10'

1 4 hiday

3.2 Y

Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Little effect on growth rate

Little effect on exponential growth rate Final population less than in control Cell population 85-5OA of that in controi at end of exponential

t%&t concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration No effect upon growth

Growth severely inhibited Lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration

Berland st al. (1976)

rc P

Bentley-Idowat u and Reid (1977)

Berland st al. 8 (1976) m

Aub& 6t d . (1972)

Berland ct al. (1976)

Page 109: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Coscinodiseus granii Gough

Cylindrotlreea closlen'um

Ditylum brightrodlii

Friragilaria pinnnta

I,nuden'a borealis

Thdaamouira peeudonnrta

SW + 2 mM NO,- + 0.35 mJI HPO.a- + trace elements + 0.1 If citrate

SW + 0.2 mM NO.- + 20 pM H,PO,- + micronutrienta + 2 mM TRIS 9 W + 2 mM NO.- + 0.35 mM HP0,'- + trace elements + 0.1 M citrate

SW + 0.2 mM NOS- + 20 pM H,PO.- + micronutrients + 2 mM TRIS

SW + 0.22 mM NO,- + 9 pM H,PO.- + micronutrients + 6 pM EDTA YW + 9 mM Na glutamate + 160 pR.1 Na? glycerophosphate + trace elements

Artificial SW + 1 mN HN0,- + 57 pM HP0,'- + micronutrients + 0.15 mM EDTA

S W C 0.2 mY NOJ- + 20 pM II,PO.- + micronutrients + 2 mM TRIS

SW + 10 mM NO,- + 320 pM Na glycerophosphate + trace element:

20 f 2

20

20 f 2

20

15

22

20

20

25* 30 35

Artificial SWz<16% salinity) + 1 mY NO,- + 57 pM HPO, 0.13 mM EDTA SW + 0.2 mM NO.- + 20 pM H,PO,- + micronutrients + 2 mM TRIS

18

20

+ trace elements +

68 klux 5 1-10

kina ?

10.2 6.5 x i o G i O a 14 h/day 68!$ug 10 1-10

1, ~ - _ .. _ _

10' 2.0 x 102

W O , Photosynthetic rate increased (1974) Photosvuthesis inhibited

Tkachenko et d.

Lowesccnncentration causing I3erland el al. growth inhibit ion (1976) "VO. Photiiwnthetic rstc Tkachenko ef al. incrgased (1974) Photosynthesis inhibited Lowest concentration causing Berland et al. q growth inhibition (1976) Lowest lethal concentration

z Lowest Concentration causing growth inhibition

>2.0 x 10s Lawest lethal concentration u)

5.4 k l w ? 10' Little effect on growth rate Hannan and 4 24 h/day Patonillet (1972) 3

ca 24 h/day

4

2 klux 3.0 X 10' 10' Lag phase increased from 37 to Dayton and (1.8 x 108 51 days. Mean generation time Lewin (1975) in solution) 1.3 times that in control.

Maximum yield 40% of that in

- ? 2.1 x 10' - -4.2 x 104

1 0 . 2 l d U X 6.0 x 10' 105 14 h/day

v

>2.0 x 102 4.3 klux 4 10*-104

4.6 k l w ? 6.5 x 10' 12 h/day

10.2 k l u 1.3 X l W 10' 14 h/day

22.0 x 10' 2.5 X 10' 5.0 X 10'

2.0 x lo"

control Rate of 0, production in saturating red light redured to 95-40% of thst in control after 15 mine dark incubation with

Overnell(1975)

metal Lowest concentration causing Berland 6f d. growth inhibition (1976) Lowest lethal concentration Photosynthetic rate decreased Woolery and with rising concentration to Lewin (1076) only 2 5 4 % of control a t highest level. also decrPAsd with lOnwx _ _ tinii'ofisposure to metal

Cell population little different from that in control a t end of

Bentley-Nowat 4 and Reid (1977)

exponential phase Lowest concentration causing Berland c1 al. 7 growth inhibition Lowest lethal concentration Lowest concentration causing rowth inhibitlon e oweat lethal concentration

(1976) E

Page 110: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Inilial no. Initial M e d i U n , Temp. Illumina- of eeUn/ metal e m . Eflect Refwema

("C) /ion mJ aJiM(WI0

LEAD (continued) SW + 9 mM Xa glutamate + 320 pM Na, 21 2 klux ? 10' Gradual decrease in numbers of Hessler (1974)

(2.5 x 10' mobile cells, and increase in ceUs in solution) in arrested division and colony

formation, espccially with older cultures

(1.8 x 10' i n 31 days due to non-adhesion of Lewin (1975) solution) cells to culture vessel. Mean

generation time 1.9 times that in control. Miximum yield 30% of that in control

Chlorophyh P'alfnymu PMlnophyoeae subcordtfmis glycerophosphate f trace elements 24 h/day

(Wille) Hazen

SW + 9 mM Na glutamate + 160 pM Na, 22 1.1 x 10' 105 Lag phase decreased from 43 to Dayton and glycerophosphate + trace elements

SW + 9 mM Na glutamate + 320 pM 21 9 10'-106 No increase in cell mutation Kessler (1975) Na, glycerophosphate 't trace elements (2.5 x 1P-

6 x 10'in

Chlorophyta Chlorophyceae

Prasidadu.8 marinus SW + 0.2 mM NO.- + 20 pM H,PO,- + micronutrients + 2 mM TRIS

Telroselmia slriala

Tetraudmir spp.

Chlamydomonae palla

Artiflcial SW (10% salinity) + 1 mY NO.- + 57 pM HP0,'- + trace elements + 0.13 DIN EDTA SW + 0.2 mM NO,- -?- 20 gM H,PO.- + micronutrients + 2 mM TRIS

Dudidla biaculata Artificial SW + ? F0,- + ? PO,'- + vitamins

DudicUa primdacla Artiflcial SW (16% salinity) + 1 mM NO,- -t 57 pM HP0.'- + trace elements + 0.13 mM EDTA

Dulaalislla lertioleda Artificial SW + 1 mM NO.- + 57 pM HP0,'- + micronutrients + 0.15 DIN EDTA

20

20

Id

20

20

18

20

10.2 klux 4.0 X 10' 14 h / b y

1 0'

4.6 klux ? 12 h/day

14 h/day 10.2 klux 2.5 X 10'

- 10'

4 4 klux ? 12 h / b y

solution) 5.0 x 10'

2 2 . 0 x 10' 10'

>2.0 x 10'

1.2 x 101

5.0 x 10'

2.8 x 104-

12.0 x 10' 2.5 x lo* -10'

6.5 X 10" 6.5 x 104

2.1 x 104

Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Cell population 12&5% of that in control at end of exponential nhnur! I.owmt ronrenlration causing growth inhibition Lowrut lethal voncentration Oxygen production reduced to 80-55% of that in control when culturea intensely illuminated (16 klux) Cell population 110-15% of that in control at end of exponential phase No effect upon rate of 0, production in saturating red light aft!er 15 mins dark incubaton with metal

Berland el d. (1970)

Bentley-Mowat and Reid (1977)

Berland st al. (1976)

Saraiva (1973)

Beutley-Mowat and Reid (1977)

Overnell(l975)

14

?

Page 111: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Cyanophyta Cpanophyceae Ehodophyta Bangiophyceae

Cocwchlori8 dabens (BrBb.) Dr. & D. Porphyridium marinuin

Cryplomonan pneudobaltim

A mphidiniocm mrt.roe

COPPER S W + 1 8 , M NO,- + 726 pM H,PO,- + micronutrients 4- 9.3 pM EDTA

micronutrients 4- 2 mM TRIS

40

20 SW + 0 . 2 . d NO.- + 20 pM H,PO,- +

SW + 1.2 mM NO,- + 57 pM HP0,'- + micronutrients

90 f 2

Artificial S W + 1.2 mM NO,- + 57 pM HP0,'- i- micronutrients SW + 0.2 mi NO.- + 20 uM H.PO.- + 20

4.0 kiux CQ. 10"

10.2 klux 3.0 X 10' 24 h/day

14 h/day

6.0 x 1P

2.7 klux 1.5 X 10' 14 h/day

102klux 4.0 x los 14 h/day micronutrients + 2 mM TRIS

S W + 1.5 mi\I NO - + 72.5 pM H,PO,- + micronutrients + 6.3 p~ EDTA S W + 0.2 mM NO,- + 20 pM H,PO,- + micronutrients + 2 mM TRIS

S W + 1.8 mM NO - + 72.5 p M H,PO,- + micronutrients + 6.3 p~ EDTA

20-30

20

20-30 4.0 klux 9.0 x 10'

6.0 x 10' 24 h/day

4.0 klm 2.0 X 10' 24 h/day 10.2 UUX 3.0 X 10' 14 h/day

30

10

50 50

25.0 X 10' 2.5 X 10'

l o p

25

1 0' 25-45

10

20 30-55

30-55

Lowest concentration preventing growth Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Cell population 93% of control after 14 days, smaller still at high metal concentrations Cell population 7% of control after 14 days Lowest concentration causing growth inhibition Lowest lethal concentration Lowest concentration preventing growth Lowest concentration causing growth inhibition Lowest lethal concentration Lowest Concentration preventing growth Lowest concentration

Mandeili (196Q)

Berland et at. (1976)

Erickson el d. (1970)

Berland et d. (1976)

Mandelli (1969)

Berland el d. (1976)

Mandelli (1969)

preventing growth SW + 1.1 mM NO,- + 56 pM HPO.1- 18 ? P 10-50 No effect upon growth Bernhardand k

12 h/day Zattera (1970)

? (1-2) x 10' Growth inhibited esWcially a t d ..

higher concentrations 3 1 2 x 10' No growth. cells viable

Artificial SW1('6% salinity) + 1 mM NO,- + 18 4.6 klnx 1 1.1 x loa- Cell popuiaition slightly higher Bentley-Mowat 67 12 h/day 2.0 x lo4 than in control a t end of and Reid (1977) HPOI + trace elements + 013 mM EDTA exponential phase H

? 2.0 x 10'- Cell population 1054% of that 0 1.1 x 10' in control at end of exponential u

phase 22 5.2 klm - 6.3 x 10'- Single dose in continuous culture

24 hldav 6.3 x 10' bad little effect on Cell . . opulation ingle dose in continuous culture

caused big decrease in cell population

6.3 x 104 8 I El

Page 112: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

arocap Specie8 fiJedium Effect Referencs

COPPER (continued) SW + 1.2 mM NO,- + 57 pJI HPO,P- + micronutrients

Haptophyta Isochryais galbana Prymnesiophyceae (Haptophyceae (oonlitsued)

20&2 2.7 klux 3.5 x lo3 14 h/day

1.5 x l o * Cell population 90% of that in control after 14 davs: smaller

Erickaon et d. (1970)

at higher metal conc&trations Cell population 89% of that in control after 14 day8 ' smaller a t higher metal concenthions Rate of 0, production in saturating red light reduced to 9570% of that in control after 15 mins dark incubation with metal

Artificial SW + 1.2 inM NO,- HPO,a- + micronutrients

+ 57 p M 50

Artiflcial SW + 1 mM NO.- + 57 HPO,*- + micronutrients + 0.15 mY EDTA

18-20

20

18-20

20

1.3 x 10'- 1.3 x lo*

Ovcrnell(l976)

Pauloiia (fiJonoch7yain) lntheri

SW + 0 2 mM KO8- + 20 p M H,PO,- + micronutrients + 2 mN TRIS

Artificial SW + 1 mM X0.- + 57 pM HPO,%- + micronutrients + 0.15 mM EDTA

SW + 0.2 m31 NOl- + 20 p?vi H,PO,- + micronutrients + 2 mM TRTS

SW + 1.2 mM NO,- + 57 pll HPO,a- + micronutrients

10.2 klux 1.1 x 10' 14 h/day

Lowest concentration cansing growth inhibition

5.0 x 10' Lowest lethal concentration ? ea. 1.3 x 10' Rate of 0. Droduction iu

50 Berland et al. (1976)

Overnell(1976) saturatingied light reduced to SO,% of that in control, after 15 mins dark incubation with metal Lowest Concentration causing Berland el d. rowth inhibition (1976) f owest lethal concentration

Cell population 73% of that in Erickson el at. control after 14 days, smaller a t (1970) higher metal concentrations Lethal concentration Cell population 57% of that in control after 14 days, smaller a t higher metal concentrations Lethal concentration Lowest concentration causing growth inhibition (1976) Lowest lethal concentration Lowest concentration causing growth inhibition Lowest lethal concentration Growth rate increased slightly

Growth severely inhibited Lethal concentration Rate of 0, production in saturating red light reduced to W 2 5 % of that in control after 15 mins dark incubation with metal

Beriand t6 al.

Aubert et d. (1972)

Overnell (1976)

10.2 klux 1 9 x lo' 50 14 h/day

> 108 20 2 2.7 klux 2.0 X loa 50

14 h/day

3 x 10' 50 Artiflcial SW + 1.2 mM NOt- +

57 pM HP0,'- + micronutrients

3 x 10' 10.2 klux 8.0 X 10' 5.0 x 10' 14 h/day

SW -t 0.2 ma1 NO,- + 20 pM H,PO,- + micronutrient,s + 2 mM TRIS

20

?

? ?

18-20

>lo" 7 (25)xlO' 60 SW + 0.66 mM NO,- + 25 fl! Na,

glycerophosphate + micronutrients + 14 mM EDTA

? 5.0 x 10' ? lo" - ? 6.4 x 10'. - 1.3 x 10'

Artillcia1 9W - 1 mJ1 NO,- - 57 p.11 HP0,'- 7 inirronutrients + 0.13 m31 EUTA

Page 113: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Chaetmroe didymus

Cylindrotheca closlm'um

Cylindrothma (Nilzsdria) clostariurn

Phaeodactylum tdcmnutum

SW + 0.2 mM NO.- + 20 pM H-PO,- + micronutrients + 2 mM TRIS

SW + 1.8 mM NO,- + 72.5 pM H,PO,- -i micronutrients + 9.3 pM EDTA Arti0cial SW + 0.84 DIM NO,- + 0.11 mhl H PO - 8W + 0.2 $M h0 - + 20 pM H,PO,- + micronutrients + d mM TRIS

SW + 1.1 mM NO,- + 56 pM HP0,'-

SW + 0.22 mM NO,- + 9 pM H,PO,- + micronutrients Artiflcial SW + 1 mM NO,- + 57 pM HP0,'- + micronutrients + 0.15 mM EDTA

SW + 0.2 mM NO.- + 20 pM &PO,- + micronutrients + 2 mM TRIS

Natural SW (dialysis cnlture)

75% SW + 0.18 mM,NO.- 7.2 fl H,PO,- + micronutrients + 4.6 pM EDTA

20 10.2 klux 3.3 x 10' 25 Lowest concentration causing Berland ct d. 14 hiday growth inhibition (1976)

6.5 x loa 10' Lowest concentration causinrc >lo* Lowest lethal concentration

- growth inhibition

>5.0 x 10' Lowest lethal concentration 20-30 4.0 klux

15.5 f 5 klux (1.4-2.0) x 18-12 Cell population after 90 hours 0.5 16 hlday loo only 77-38% of that in eontrol

20 1 0 4 klux 3.0 x 10' 2.5 x 10' Lowest concentration causinc

1.6 x lo4 (1.3-1.9) x 10'Lowest concentration preventing Mandelli (1969) 24 h/day growth

1s

15

20

20

6 -8

13

Artiflcial SW (16% salinity) + 1 mM NO,- + 57 pM RPO I- + trace elements + 0.13 mM E I ~ T A

18

2.2

SW + 1.8 mM NO,- + 72.5 &PO,- + 18 micronutrients + 9.3 pM EDTA

20-30

SW + 1.2 mM NO.- + 57 pM HP0,'- + micronutrients Artiflcial SW + 1.2 mM NO,- + 57 pM HP0.I- + micronutrients

20 f 2

14 h/day

? ?

? 12 h/day

5.4 klux ? 24 h/day

? -

10s

growth inhibition > 10' Lowest lethal Concentration

25 Lowest eonrentration causing growth inhibition

2.5 x 10' Lowest lcthal concentration 10-50 No effect upon growth

(1-4) x 10% Growth inhibited especially a t higher concentrationq

8.0 x 10' Lethal concentration 10' Little effect on growth

(1.3-6.4) x 10' Rate of 0, production in saturating red light reduced to 9(rlO% of that in control after 15 mins dark inrubation with metal

10.2 klux 8.0 X 10' 5.0 X 10' 14 h/day

Daylight 1.6 x 10' 10 > 103

103 >2.5 x 10%

3 klux ? >2.5 x 10' 16 h/day

4.6 klux ? 1.1 x 10'- 12 h/day 1.1 x 106

542kIux - 6.4 x loa - 1.4 x loa

? 108

24 h/day

4.0 klux 3.0 X 10' 50 15 h/day

(1.6-2.5) X

2.7 klug 9.0 x 10'

. Lowest concentration causing growth inhibition Lowest lethal concentration Stimulating in dialysis culture, 1.1 times growth rate in control Qroatli rate decreased Lethal concentration Growth rate decreawd

Lethal Concentration Cell population 100-0:i, of that in control a t end of exponential nhasn

Rosko and Rachlin (1975) Berland ct el. (1976)

Bernhard and Zattera (1970)

Ilannan and Patouillet (1972) Overnell(l975)

Berland el d. (1970)

Jensen el al. (1970)

Jensen et al. (1976)

Bentley-Mowat and Reid (1977)

gingie dose in continuous culture had little effect Single dose in continuous culture caused increase in cell population Lowest concentration Mandelli (1969) Lowest preventing concentration growth

preventing growth Cell population 80 rt 4% of that in control after 14 davs (.ell~opulation inrreised over that in control after 14 days Cell population 97% of that in

I Erickaoo et al (1970)

controiafter 14 days

Page 114: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Medium

COPPER (continued) pW;T- Skel&onenso coslatum SW + 0.2 mM NO,- + 20 pM H,P04- + 20 10.2 klm 1.8 x 10' 50

micronutrients + 2 mM TRIS

Natural SW (dialysis culture) 6-8 Daylight 1.6 x lo6 10

14hlday >5*0 x 10' (00,rrnWB)

75% sw + 0.18 mM,NO,- + 7.2 pM H PO - + micronutr~euts + 4 8 pM E b T i

25 13 3kIux P 10

16 h/day

25

18-20 - P 3.2 X lo* - Artificial SW + 1 mM NO,-, +

57 pM HP0,'- + micronutrients + 0.15 mllI EDTA

Thdaesiorira plloialilia Host.

pseudonana

SW + 1.8.mM NO,- + 72.5 pM &PO.- + micronutrients + 9.3 pM EDTA

20-30

Tholasrwsim SW, unenriched 20*1

SW + 0.2,mJI NO.- + 20 fl &PO,- + mcronutrients + 2 mM TRIS

20

Natural SW (dialysis culture) 8-8

75,% SW + 0.18mMNO.- + 7.2pMHyP0.- + mcronutrients + 4 8 fl EDTA SW + 0.88 mM NO.- + 36 fl H,PO,: + micronutrlents + 1.0 pM EDTA + variable levels of TRIS bufFer

13

20 f 1

Thala8&8ita SW + 1.8 mM NO - + 72.5 pX H,PO,- + 20 micronutrients + 8.3 p~ EDTA

mcronutnents SW + 1.2.mM NO;. + 57 pM HP04'- + 20 f 2

4.0 klux

2.7 klm 24 h/day

14 hiday

10.2 klm 14 h/day

Daylight

3 klux 16h/day 7.2 klux 14 h/day

4.0 klux 24 h/day

2.7 klux 14 h/day

3.0 X 10' (1.8-2.6) X 10.

5.0 X lo" 25.0

5.0 x lo1 210 5.0 x 10' 10

2.5 X 10' 1.6 x 10' 10

50 4 lo*

W. 104 -

2.0 X 106 2.3 X 10'

7.0 X 10' 50-3.5 X 10' 4 0 x lo*

Lowest concentration causing growth inhibition (1876) Lowest lethal concentration Growth rate 19% of control value (1876) Lethal concentration Growth rate 83% of control value

Growth rate 50% of control value Rate of 0, production in saturating red light reduced to 50% of that in control after 15 mins dark incubation with

Berland et d.

Jensen el d.

k 8

OverneU(l976)

metal 0

rc Mandelli (1969) Lowest concentration preventing growth Growth and W O , flxation rate Ericknon (1972) Q decressed especially on extended Lxposure to metal Mean cell volume increased by extended exposure to metal Lowest concentration causing

Growth rate 77% of control value (1976) Lethal concentration Growthrate 73% ofcontrolvniue

Berland et d.

Jensen et d.

growth Lowest inhibition lethal concentration (1878) v1

Growth inhibition was related to calculated concentration of free. uncomplened Cult ions:

Sunda and Guillard (1976)

values > 1.6 ng/l reduced erowth rate which became zero 'st c0. 0.3 pg/l Lowest concentration Mandelli (1969) preventing growth

Cell population increased over that in control after 14 days Cell population 87% of control after 14 days, smaller still at higher metal concentrations

Ericbon el d. (1970)

Page 115: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Artiflcial SW + 1.2 mM NOs- + 57 p M HPO.'- + micronutrients

SW + 0.2.1nM NO.- + 20 pM H,PO,- t mfcronutrients + 2 mM TRIS

1.5 x 10' Cell population 89% of control after 14 days; smaller a t higher metal concentrations

4.0 x loa 50 Lowest concentration rsusing Berland ct al. growth inhibition (1976) Lowest lethal concentration Lowest concentration cansing growth inhibition Lowest lethal concentration

9 1.1 x 10'- Cell population 80-100% of that Bentley-Mowat in control at end of exponential and Reid (1977)

>2.5 x lo3

>5.0 x 10'

2.0 x 10'

104 50

Ohlorophyta Prsainophyceae

Chlorophyta Chlorophyoeae

20 1 0 2 k l m 14 hiday

TelraeclmiS spp. Artificial S y J l 6 % salinity) + 1 mM NO,- + 57 pM HPO, 0.13 mM EDTA

+ trace elements + 18 46klux 12 h/day

Phase ? 2.0 x 104- Cell population 1000% of that

1.1 x 108 in control at end of exponential phase

saturating red light reduced to 50% of that in control after 15

1 (1.3-3.2) x Rate of 0, production in Overnell (1976) 8 L4

mins dark incubation with %

16-20 - - Arti5cial SW + 1 m?I NO.-. + 57 pM HPO.'- + micronutrients + 0.15 mM EDTA

metal 2.5 X 10' 50 Lowest concentratlon causing Berland el al. a

growth inhibition (1976) !2 2.5 x 10' Lowest let,hal concentration

? 1.1 x 10'- Cell population little different Bentley-Mowat 2.0 x 10' from control a t end of

exponential phase ? 2.0 x 10'- Cell population loO-Oo/, of that

1.1 x 10' in control at end of exponential

and Reid (1977)

w

Chlamydoinonas p a l k

Dundiclla primdccta

SW + 0.2 mM NO.- + 20 micronutrients + 2 mM TRIS

H,PO,- + 20 10.2 klm 14 h/day

Artiflcial SW (16% salinity) + 1 mbl NO; + 57 pM HP0,'- + trace elements + 0.13 mM EDTA

18 46klux 12 h/day

SW + 1.8 mM NO.- + 72.5 pM H,P04- + micronutrients + 9.3 pM EDTA S W + 1.2 mM NO,- 3.57 pM HP0,'- + micronutrients Artillcial SW + 1.2 mM NO,- + 57 pM HPO,' - + micronutrients Artiflcial SW + 1 mM NO.- + 57 pM HP0,'- + micronutrients + 0.15 mY EDTA

35

20*2

20

1e-20

20 f 1

4.0 k l U

2.7 khm 24 h/day

14 h/day

5.0 x 104

8.0 x 10'

>6.0

4.5

4.6

1.3 2.5

1.3 2.5 4.4

-

x 103

x 103

x 10'

x 10'- x 10'

x 10'- x 10' x 10'

L%st concentration preventing growth Cell population 80% of control after 7 days Cell population 84% of control after 7 days Rate of 0, production in saturating red light reduced to 9 5 4 % of that in control, after 15 mins dark incubation with metal Potassium content of celb 8&10% of thnt in control Rate of 0. production in saturating red light reduced to 50% of that in control after 15 mins dark incubation with

Mandelli (1969)

Erickson et a[. (1970)

Overnell(1975) ?

Overnell(l976)

metal Growth inhlhition was related to Sunda and calculated Concentration of free, Quillard (1976) nnromplexed Cu' + ions; growth rate reduced by values > 2.5 ug/l and became zero at M. 0.3 pg/l

Nannoelrlatw atomus Butcher

SW + 0.88 mM NO,- + 36 pJI H.PO.- + micronutrlenta + 1.0 pM EDTA + variable levels of TRIS buffer

7.2 k l u 14 hjday

cu. 6.0 x 104

Page 116: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Medium Form and effect R e f e m

chlorophyta Chlorophyceae

PAaeodoetyl UNl

lrieonzutum

CHROMIUM sw + 1.1 mar NO,- + 56 or ~ ~ 0 , s - 18

SW + 0.60 mM NO,- + 25 p M Na, 9

?

glycerophosphata + micronutrient9 + 14 mM EDTA

? 18 SW + 1.1 mN NO,- + 56 uM HP0;-

Artificial SW + 7 XO.,- + ? PO,’- + 20 vitamins

? 12 h/day

1

?

?

P ?

12 h/day ?

- -

?

f

?

? (1-8) x lo* Cr 111; No effect upon growth Bernhard and Zattera (1970) rate

lW

ca. 10‘ ?

107

?

?

?

P

2.0 x 2.8 x los Cr M; Growth rate reduced at Anbert er al.

(1-4) x 10. Cr I11 ; Growth inhibited by up to 50% at higher concentrations

first but later recovered; final population lower than in control

5.6 x lo8 Cr VI. Initial decrease In cell popudtion but culture recovered and grew giving flnal DoDulation onlv about loo/, of

(1972)

.- that in control-

1.1 x 10‘ Cr VI; Lethal concentration 10*-2.0 x 10’ Cr I11 ; No effect upon growth Bernhard and

rate Zattera (1970) 4.0 x lo* Cr 111. Growth rate

slight& inhibited 2.0 x 106- Cr VI; Oxygen production Saraiva (1973) 2.0 x 10’ lOO-QZ% of that in control when

cultures intensely illuminated (10 klux)

4 4 x loa Cr VI ’ Growth stimulated Pepeda-Saraiva 4 4 x los Cr VI Growth inhibited a t b t (1976)

but cultures later recovered giving 0nal populations similar to that in control Cr VI ; Qrowth increasingly

Cr V1; No growth

9.2 x 10’- 9.2 x 10‘ inhibited 1.8 x 10’

Page 117: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

c % Haptophyb Cornlithue huZleyi .w Prymnwiophyceae I (Haptophyceae) I 4 cn

ZINC SW + 1.1 mM NO.- + 66 pb1 EP04a- 18 ?

12 hiday 2 2.0 x 10'

? 4.0 X 10'-10'

? (3.3-6.6) x 104

Xo effect upon growth rate Bernhard and Zattera (19iO)

Growth inhibited especially a t higher concentrations Rate of 0, production in ( Artificial SW + 1 rnM NO - + 57 pbl 18-20 -

HP0,'- + micronutrients'+ 0.15 mil1 - EDTA

hernell(1976) saturatingfed light reduced to 90-85% of that in control after 15 mins dark incubiLtion with mf+A

? (1.3-2.0) x 105

~. .-. Rnte of 0, production in saturating red light reduced to SOo& of that in control after 15 mini dark incubation with metal Aubert et al. Little effect upon growth

(1972) Chrysophyta Bacillariophyceae

Asterionella glacialis (A. japonica)

SW + 0.66 mM NO,- + 25 pM Sa, ? ?

? ?

glycerophosphate + micronutrients + 14 mM EDTA

10' 1.7 x 103 5.4 x 103

? (9.3-6.6) x 10'

No growth. cell numbem constant Lethal conEentration Rate of0,productionin Overnell(1976) saturating red light reduced to 80-700/ of that in control after 15 m i d dark incubation with mpt,nl

Artiflcial SW f 1 mM NO,- f 67 WN HP0,'- + micronutrients + 0.15 m31 EDTA

18-20 1 - Attheya dceora

CylindrotWa (Nttz8chia) closterium Pllaeodactylum rricornl4tuTR

Artiflcial SW + 084 ml\I NO,- f 15.5f 5 klux 0.11 mM HaPO4- 0..5 16 hjday SW + 1.1 mM NO,- + 56 3 1 HPO,*- 16 ?

(144.0) x 16-42 10'

? (2-8) x 10'

103-104

6.7 X 10'- 5.0 X 10a-lO' 44 x 10'

2.2 x 106

4 4 X 10'- 5.0-5 x loe 4.9 x 101

( 2 . 2 4 9 ) x 10' 10' 6.4 x 10'- 50-10' 107 9.0 x 104- 2.5 x 108-108

6.7 x i o c 2.5 x 104

105 25

4.0 X 10"

Cell populationafter 96 hours only 70-80% of that in control No effect uDon growth rate

llosko and Rachlin (1975) Bernhard and

1 2 h/day

Natural SW (dialysis culture) 1 Daylight

~- Zattera (1070)

Growth inhibited. esoeciallv at higher concentrations . Average growth rate greater than in control, little effect

Jensen et al. (1974)

upon h a 1 population Average growth rate only about 77OL of that in control

Skeletonema costalum Grokth rate greater than in rnntrol . . - .. . . Average growth rate only "-45% of that in control; flnal population also decreased kkhal concentration

Average growth rate greater t,han in cnntrol ~

Average growth rate only 89-33O' of that in control. final &ulation also decrhased

? ArtiEf!l SW + 1 mM NO - + 67 @I

EDTA

18-20 - HPO, + rnicronutrients'+ 0.15 m u -

4.0 X 110' 104 . 6.6 X 10' Lethal concentration Rate of 0, production in saturating red light reduced to 80 7 of that in control after 15 gins dark incubation with metal

Overnell(1 Chlorophy ta Chlorophyceae

Y

DunalisUa terfidcda ,976)

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APPENDIX II CONCENTRATIONB (P.P.M. DRY WEIQHT) OF H?EAVY METALS

IN PHYTOPLANKTON AND MICROPWETON COLLECTED FROM VARIOUS SEA AREAS

Mjcroplankton oonsiat of a mixture of phytoplankton microzooplankton and detritus. The data of Vinogradovs and Koval'skiy (1962) were'converted from an ash weight to a dry weight basis using a factor obtained from Fujita (1972); the data of Szabo (1968) were similarly converted using a factor obtained from Szabo (1967). The data of Thomnson el al. (1967) were obtained from Suencer and Sachs (1970). Concentrations in brackets are m d a n values. ND = Not detectable. SA = Spectrographic analysis. AA =Atomic absorption analysis.

Drying Concentration Reference Location Method temp. YC) Range Mean Maah

size (wm)

Mixed phytoplankton

Mixed phytoplankton I Mied phytoplankton Miged phytoplankton I1 Mixed phytoplankton 111 Mixed phytoplankton

Microplankton

Mixed phytoplankton

MIxed phytoplankton I11 U e d phytoplankton Mixed phytoplankton I1 Mixed phytoplankton Mixed phytoplankton I Mixed phytoplankton

Microplankton

?

i6 76 153 76 76

76 132 60

7

?

?

!

i G 64 76 64 76 37 64 37 64 64 64 64 1$2

60 76

Yatsushiro-kai, Japan Ariake-kai, Japan E . Pnriflr nj

Yatsushiro-kai, Japan Off Minamata Japan Atiake-kai, Jipan

Monterey Bay, Californie

Paoiflc, off Hawaii Monterey Bay Caliornia Korthwest d f of Mexico

E. Paci5c Off Los Angela, California Nediterranean Sea

E. Paci0c

MERCURY ? 60

? 60 ? 60 AA $5 AA AA i

METHYL MERCURY ? ? ?

CADMIUM

0.09-0.79 0.11-0.70 0.10-0.27 - 0.12-048 0.15-0.59 0.01-0,52

L

0.11-0$3 0,058-0.26 0.028-0.26

0.46 Hirota et d. (1974) 0.41 Knauer and Mnrtin (1972) (0.19) Martin and Knauer (1973) 0.19 Cocofos et al. (1973) (016) Martin and Knaner (1973) (0.16) 0.15 Hiota et al. (1974) . , 0.10

0.132 Fowler et d. (1976b) 0.099

Martin and Knauer (1973) -

GO 0 , 0 5 4 5 7 0.21 Hirota et al. (1974) 60 004-040 FO 005-0.17

0.19 -

AA 65 AA ? AA 05 AA ? AA 65 AA ? AA AA

AA AA A.4 ? AA ? AA ? AA ? AA 65

9, AA i - -

2.2-65 04-695 1.1-35 1.0-2,o 0.4-4.8

<0.0543 07-14 - 5.620.9 24-24.7 2.244.1 14-15.0

- 0.7-34 1.0-2.2

Nartin and Kmuer (1973) Martin and Broenkow (1975) Martin and Knauer (1973) Martin and Broenkow (1975) Martin and Enauer (1973) Sims (1975) Mnrtin Sims (1975) and Broenkow (1975)

W t i n and Broenkow (1975) Hartin et d. (1976)

Martin and Broenkow (1975) Fowler eL d. (1976b) Benayonu et d. (1974) Fowler et d. (1976b) Martin and KnRner (1873)

+ W

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SILVER

Diatoms s Black Sea W e d phytoplankton P Irish Sea Mixed phytoplankton I1 76 Monterey Bay, California Mixed phytoplankton I 76 AUxfid phytoplankton 111 76 Microplankton 64 E. Paciflc

64 Off Baja California 76 E. Paciflc

Diatom Mixed phytoplankton

&ed phytoplankton I1 Mixed phytoplankton 111 Mixed uhytoulankton I ?viicwpiad&n

Mcroplankton

DiatoIIlS Mixed phytoplankton

Mixed phytoplankton 111 Mixed phytoplankton I1 Mixed phytoplankton I Microplankton

? ? 37 37 76 76 76

200 76 64 64

Black Sea Irish Sea Northwest Gulf of Mexico Corpna Christi Bay, Texaa Mmterey Bay, California

Off Bahamaa E. Paciflo

Off Baja California

? Mediterranean Sea

SA SA AA AA M M AA AA

NICKEL

SA SA AA Ad M AA M SA AA AA AA

?

65 65 65

-

- - 65

?

? ? 65 65 65

65

-

go

- -

SELENIUM

? ?

? ? 37 37 76 76 76 76 64 64

Black Sea SA Irinh sea SA Northwest Gulf of Mexico AA Corpus Christi Bay, Texas AA Monterey Bay, California AA

AA AA

E. Paciflc AA AA

Off Baja California AA

LEAD

? - ; 65 65 65 65 - -

1.1-17 - Vinogradova and Koval'skiy (1962) - 3.3 Riley and Roth (1971)

0%0.9 (0.6) Martin and Knauer (1973)

ND-O.1 0.05-0.24 0.13 Marti et al. (1976) 0.03-0.51 0.10 ND-O.4 - Martin and KnaUer (1973)

ND-O.6 iy";,

23-86

0.9-11.0

2.3-12.8 13-65 0.6-5%3 6-67

- -

1152-1 728

2.6-29.0 - -

ND-26.4 2%46.6 ND-13.0 164-38.8 43-134 08-8.8

Vinogradova and Koval'skiy (1962) Rdey and Roth (1971) Sims (1975)

Martin and Knauer (1973)

Szabo (1968) Martin and Knauer (1973) Martin el al. (1976)

2.7 Fowler and Benayoun (1976)

Vinogradova and Koval'skiy (1962) Riley and Roth (1971) Sims (1975)

Martin and Knauer (1973)

Nartin el a/. (1976)

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Comentrdion 8 Range Mean Reference Meuh m w

size (pm) LocaliOn Method t a p . (“C)

Diatoms Msed phytoplankton

Mixed phytoplankton III IKlxed phytoplankton

hUxd phytoplankton I1 Mixed phytoplankton Mixed phytoplankton I Mcroplankton

nirtnma Wxed phytoplankton Hixed phytoplankton II med DhYtODlankton 111 Mixed bh,toiiankton I Microplankton

Diatoms =xed phytoplankton

hUxed phytoplankton I1 Mixed phytoplankton

Mixed phytopIankton III Mixed phytoplankton I Microplankton

? 7 9 76 37 76 76 37 76 76 60

132 200

64 64

9

i 6 76 76

200 76

? 100

i 6 37 76

i 6 78 76 ?

132 60

200 64 64

‘P

f 7

Black Sea Atlantic Wish Sea Monterey Bay California Northweat Gnif of Mexico Off Nova Scotia Monterey Bay, California Corpus Chriiti Bay, Texas Monterey Bay, California E. Paciflc Mediterranean Sea

Off Bahamas E. Pacific Off Baja California

Black Sea Irish Sea Monterey Bay, California

Off Bahamas E. Paciflc

Black Sea Ise Bay, Japan W. Paciflc Irish Sea Off Nova Scotia Northwest Gulf of Mexico Monterey Bay, California Corpus Christi Bay, Texas Atlantic Monterey Bay, California

E. Paciflc Mediterranean Sea

Off Bahamas E. PacUlc Off Baje California

COPPER ? - SA

? SA - AA 65 AA 9 AA 105 AA 65 AA P AA 65 AA 65 AA ? AA 9 SA AA

90 -

AA 65 ~~~

AA 9 AA 105 AA 65 AA P AA 65 AA 65 AA ? AA 9 SA AA

90 - - AA

CHROMIUM

AA 65 SA 90 AA 65

ZINC SA 9 AA 105 AA 105 SA - AA 105 AA ? AA 65 AA P - ? AA 65 AA 65 AA 65 9 P AA ? AA 4 SA AA AA

90 - -

- ? AA 65 ~ ~~

AA 65 AA 65 9 P AA ? AA 4 SA 90 - - AA AA

115-575 - - 16-45.4 1.2-25.4 107-13.5 1.7-42.0

1.3-19.0 40-104 12-161 13-172 13-48 4.4-17.7 2.9-8.9

-

1.1-46 - 1.0-21.4 ND-1.6 ND-1.3

<l9-9*5 ND3.7

578-8 640 140-1 757 228-1 061 - 149-157 13-129 16-445 - - 11-703 8-64 285-4 190 443-753 224-769 79-1 276 114-764 11-87 3-72

- 33 36

(1 4.8) -

7,5 (39)

- - - 282 153

(40) 26 (24) (19)

570 446 385 305 62 31

(122)

-

Vinogradova and Koval’skiy (1062) Thompson et d. (1967) Riley and Roth (1971) Martin and Enauer (1973) Sims (1975) Mayzaud and Martin (1975) Martin and Knaner (1973) S im (1975) Martin and Knauer (1973)

Fowler et al. (1976b)

Szabo (1968) Martin el al. (1976)

Vinogradova and Eoval’skiy (1962) Riley and Roth (1971) Martin and Knaner (1973)

Szabo (1968) Martin and Knauer (1973)

Vinogradova and Koval’skiy (1962) E’njita (1972)

Riley and Roth (1971) Mayzaud and Martin (1975) Sims (1975) Martin and Knauer (1973) Sims (1975) Thompson et d. (1967) Martin and Knaner (1973)

Small et el. (1973) Fowler el al. (1976b)

Szabo (1968) Martinet al. (1976)

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a b

c

APPENDLX I11 CONCENTRATIONS (P.P.M. DRY WEIGHT) OF HEAVY METALS

IN ZOOPLANKTON COLLECTED FROM VARIOUS SEA AREAS D = Dried in uaeuo over silica gel. The data of Viogradova and Eoval’skiy (1962) were converted from ash weight to dry weight using factors obtained for the appropriate genera by Nicholls e t d . (1959). ND = Not detectable.

Group Loeation Reference

Protozoa Radiolaria Coeienterata crnataoea copepoda

Cruatacea Mysidacea Cmtaoee Euphausiacea

CrustaceaDecapoda

Urochordata Tbaliaees Mixed zooplankton

PdmD SO.

MERCURY Monterey Bay, California E. Atlantic

Am&a h u e Giesbrecht Elcfsis-Bay, Greece Labi&cera acutifrm Dana E. Atlantic Copepods Monterey Bay, California Euc@u snrlplicauda Faxon E. Atlantic Mwantrctiphanee nwoeoica Claus Mediterranean Sea . . -

E. Atlantic Monterey Bay, California Mainly Euphausia paci&a

H a B n E u p h a d p&&u or Thysanoeeea N.E. Paciflc spinifera Holmes usually domlnsnt AContheph#ra ezivnia Smith E. Atlantic SysWlaspiS debiliS (Bfilne Edwards)

(mainly copepods and S@la sp.) E. and N.E. United States gI2z;p

rnnat,

65 ?

6 s ?

P,

; 65

?

? ?

?

;

- 0 . 0 5 4 1 5

O.l(M.50

0 1 6 Martin and Knauer (1973) 0.07 Leatherland et al. (1973) 0.29 Zafiropoulosand Qnma1iis(l977) 0.12 Leatherland el al. (1973) 0.11 Martin and Knauer (1973) 0.20 Leatherland el al. (1973) 0 3 5 Fowler et d. (19768) 0.26 Leatherland el al. (1973) 0.09 Martin and Knauer (1973)

- Cutshall and Holton (1972)

0.38 Leatherland at d. (1973) 0.22

0 3 6 0.06 0 5 8 Wiudom (1972)

F.W; m i c a n coast ? 0.28-0&l 0 4 8 0.06-0.93 0 3 2 N.W. Atlantic 9 - Windom el d. (1973) 80 0.1-0.41

? 0.079-0.132 - Williams and Weias (1973)

mainly euphausiids and copepods) E. Paci5c 65 0.039-0.448 - Knauer and Martin (1972)

(mainly copepods) E. Atlantic Mixed zooplankton (450/ mpepod~ and 45% chaetognaths) ? 0.1234388 -

!60{ copepods and 30% chaetognaths) ? 0.055-0.189 - 0.78 Corral and -6 (1975) Mixed zooplankton Riade Arona Spain 60 0.5-164

Central and &orth Adriatic ? 2-16 VuCetiO et d. (1974) 1.10 Hlrota el d. (1974) OffBfinamata Japan 60 0.14-2.59

Hardangerfjoh, Norway 80 0.5%0,66 0 5 4 Skei et d. (1976) N.W. Atlantic ? 0.09-0.51 0 3 0 Fitrgerald et d. (1972)

0.14 Martin and Knauer (1973) E. Paciflc 65 0.04-0.45 W. Atlantic estuarim 80-100 0.11-0.13 Yatsushiro-kai, Japan 60 0 ~ 0 1 3 4 1 6 8 0.083 Hirota et d. (1974) Ariake-kai, Japan 60 0.01-0.036 0.023

Cocoros el d. (1973) -

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Qroup Ladion ReJemwe

METHYL MERCURY

Wx0d zooplankton 60 60 60

0.18-0.92 0.0.1-0.53 0.10-0.23

045 0.22 0.15

6.11 5.3 041 9.8

4.27

-

- 6.6 7

2.0 1.3 0.7 0% 2.98 -

C.6 3.0 0.8 13 13 0.9

0.44 5.2 3.1

-

1 .a 5.0

4.47 4.4 3.15

2.4 0.93

-

- -

Hirota el al. (1874)

CADMIUM Protozoa Radiolaria Cwlenterata Crustacea Copepoda

Monterev Bav. California 65 Msrtio sud Knauer (1973) Ifitherlandel aL(1973) Zallropoulos and Grimanis(l977) Leathwland et d. (1973)

P&in an. E. Atla& " ' Elefsis Bay, Greece E. Atlantic Firth of Clyde

?

! i

65 105

? 105

?

60 9

- -.-~ .- .~ . Bmrlia daua' Labidocera acutifrom Mainly Celanua ~ D D . and Steele et al. (1973)

Martin and Knaner (1973) Bohn and McElroy (1976)

?CU RIonterey Bay, California Strathcona Sound, N. Baffin Island Mediterranean Sea Strathcona Sound, N. BatRn Inland E. Atlantic Mediterranean Sea

Crustacea Amphipoda

Cmtacea Myaidncea Cruetseea Euphausiacen

Fowler el al. (1976b) Bohn and McElroy (1976)

Leatherland el ul. (1973) Fowler et al. (1976h~

B. Atlantic ? U h l y Euphausia pacijtea Monterey Bay, California 65 Euphauaia pacifica or N.E. Pacitlc 90 Thu8anosesa avinifera usually dominant Euphauda spp. Xediterranean Sea Acanthephyra ezimia E. Atlantic l3ennudua fiegay.(Smith) Mediterranean Sea ? Systeuospas debalzs E. Atlantic ? Oplophorus sp. ? Sergestes epp. Mediterranean Sea ?

;

Sagittu sp. Strathcona Sound, 105 N. Baffin Island

04-1.4 - 07-1.1 - - 0 4 .la5 1.2 -1.3

1443% 04-24

-

Fowler et al. I1 976hl Crustacea Decapoda

Fowler el al. (1978b) Bohn and McElroy (1976)

Leatherland el ul. (1973) Windom (1972)

Chaetognatha

Urochordata Thaljacea Mixed zooplankton : PVTOSOma Sp. E. Atlantic

(mainly copepods and SagiCla sp.) N.W. African coast E. and N.E. United States I coast N.W. Atlantic Off Pucrto Rico

? 80

0.6-3.7 2-12 Mixed zooplankton

Mixed zooplankton

(75% copepods) Xartin (1970)

Topping (1972) Martin and Broenkow (1975)

North Sen E. Pacitlc

10;

N.W. Gulf of Mesico 4

lY i

E. Paciflc 65 Firth of Ciyde

Sims (1975) Martin and Knauer (1973) Topping (1872) Steele et al. (1973)

04-4.4 1.8-3.5 0.31-2.34 O.QZ1.09 0437-087

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Protozoa Radiolaria Ctenophora

Crustacea Copepoda

Cmtacea Euphausiacea Chaetognatha

Mmed zooplankton

Protozoa Radiolaria Coelenterate Ctenophora

Crustacea Copepoda

Crustacea Euphauaiacea

Pteropoda Gymnosomata Pteropoda Themsomata

NOllUsCa Cephalopoda Chaetopatha

Uroohordata Thsliacea Mixed zooplankton

Nixed zooplankton

Crastacea copepods

Crustacea Euphausiacea

Pleurobraehia pileus (0. F . Miiller) Calanus Mgolandicua Claw. Anmaloma sp. and Pontella sp. Small coueoods Copepodi Xainly Euphnusia pacifia Sagitta setosa J. Miiller and S. euxim Moltschanoff

Cyanaa capillala (L.) Beriie cucumis FabriciaR Pleurobraehia pileus

Cdanua fznrmzrchicus (Runncrus)

Cdanus Mgolandicus

Cgnlropagea typieue (Lilljeborg) and C . hantalu6 Kroyer Anonedocera sp. and P ~ n l d l a sp.

Small conenods

SILVER

Monterey Bay, California Black Sea

Monterey Bay, California

Black Sea

I.:. Pacific

NICKEL Monterey Bay, California W. Atlantic

Black Sea

W. Atlantic

Black Sea

W. Atlantic

Black Sea

Monterey Bay, California W. Atlantic Monterey Bay, California W. Atlantic

Mixed c&p& Euphuuia krohnii (Brandt) Mainly Eup?aull"a pacijim Clione limaczna (Phipps) Spiralella (Limacim) retroversa (Fleming) Ommaslrephea iUicebrosa (ki ieur) Sositla degam Verrill Sagi6ta aelosa and S. euxina

Sdpa fusifonnis Cuvier W. Atlantic (75% copepods) Off Puerto Rico

Black Sea

E. Pacific N.W. Gulf of Mexico

SELENIUM

65 >

>

; 65 65

1

ti5

ti5 D D ?

D ? ?

D

?

? 65 D

65 D u 1)

: D 80

65 1

Acarfia &u8i Elefsis Bay, Greece P

Meganyeliphw noroegica Mediterranean Sea 4

ND4.4 2.1-2.8

- - 0.549

ND-02 NDd-5 6.6-13.2

0,144

ND-7-1 - - - - - - - -

10-30 ND-114

ND-6.6 - - - - -

44-66

- 17-113

5-13 <05-7.8

141-308

-

0.1

1 6

0.03 0.13

0.26

-

-

-

3.63 < 0.63 < 0.70

35

29 0.12 40

1254

30

20 3.7 1.12 3.44 1.33 1.28

<0.08 104

42.3 42

8.2

-

-

1.86

3.53

Nartin and Knauer (1973) Vinogadova and Koval'skiy (1962)

Martin and Knauer (1973)

Hartin and Knauer (1973) Nicholls et al. (1959)

Vinogradova and Koval'ekiy (1962) NichoUs el al. (1959) Vinogradov (1953) Vinogadova and Koval'skiy

Nicholls et nl. (1959) (isez)

Vinogadova and Koval'skiy (1962)

Martin and Knauer (1973) Nichob el al. (1969) Martin and Knauer (1973) Nicholls el d. (1959)

Vinomadova and Koval'skiy (1962) Nicholls sf al. (i959) Martin (1970)

Ifartin and Knaiier (1973) S im (1975)

Zaflropoulos and Grimanis (1977) Fowler and Benayoun (1976)

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8 k P

Refersnes ConemUration Rauge Meun

Drsring temp. “(C) Qroup species LoeGclion

Protozoa Radiolaria Coelenterata Ctenophora

Crustacea Copepoda

Crustacea Euphansiacea

Pteropoda Cynmosomata Pteropoda Thocosomata Mollusca Cephalopoda Chae t ognat ha

Urochordata Thaliacea Mixed zooplankton

Mixed zooplankton

Mixed zooplankton

Mixed zooplankton Mxed zooplankton Mixed large zooplankton Mixed zooplankton

LEAD

Monterey Bay, California Cyanea capillata W. Atlantic Ber& codcumi8 Pleurobrachia pileu8 Black Sea

Calanus finmarchicus N. Atlantic Cdanue helgdandicus Black Sea

Centrqpages typicns and W. Atlant,ic

Amalocera sp. and Pontellfl sp. Black Sea

Mainly Calanus spp. and Firth of Clyde Pssudocalanus elongatus Small copepods Black Sea

Copepods Monterey Bay, California Euphaueia krohnii W. Atlantic Mainly Euphausia pm‘jica Yonterey Bay, California Euphauda pmjica or N.E. Paci0c Thysamessa spinifera nanaily dominant Clbm l i d n a W. Atlantic SpirateUa ( L i d n n ) retroversa Ommastrephe8 illkebrosa S@ta *am Sagitta eetoea and S. euxina Black Sea

Salpa fusiformi8 W. Atlantic (mainly copepods and Sagifta sp.) N.W. African coast

E. United Statcs coast N.W. Atlantic North Sea N.W. Gulf of Mexico

C. hamatu8

(75% copepods) Off Pnerto Rico

Firth of Clyde

E. Pacific N.W. Atlantic

05 D D ?

: D

?

?

?

05 D 05 90

1) D D D 1

li’ !,

100 ?

so . ?

100

05 ?

XU-12.2 I -

17-5-26

- 50-60

- I

2.4-14.3

12-30

ND-70

ND-10.9 4.34377

-

- - - -

176-330

- 22-340 2-250 2-97

< 3.1-1 000 05-62.5 8-107

20-47.5 1.2-103 10.2-11.6 2.2-144 1.8-16.0

3 4 1 3.78 4.22 -

101 -

290

60

- - 3432 3.72 3.09 -

21.7 12s 0.39 65 -

2.31 124 94 1 5 143

49

15

6.6 5.01

-

- -

Martin and Knauer (1973) Nicholls et al. (1959)

Vinogradova and Koval’skiy (1962) Nicholls el al. (1959) Vinogradova and Koval’skiy (1962) Nicholls et d. (1959)

Vinogradova and Koval’skiy (1962) Steele el al. (1973)

Vinogradova and Koval’skiy (1973) Martin and Knauer (1973) Nicholla ef al. (1959)- Martin and Knauer (1973) Cutshall and Rolton (1972)

Nicholls et al. (1959)

Vinogradova and Koval’skiy (1962) Nicholls et d. (1959) Windom (1972)

Topping (1972) Sims (1975) Martin (1970)

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COPPER

Nonterey Bay, California 65 W. Atlantic D

D Black Sea ?

Elcfsis Bay, Greece ?

W. Atlantic D Off Nova Scotia 105 Black Sea ?

W. Atlantic D

Black Sea ?

Firth of Clyde ?

Black Sea ?

Protozoa Radiolaria Coelenterata Ctenophora

Cyawu mpiUola Bet2 ~1curni.3 Pleurobrachia pilcus

Acartia clawri

CaEanus finmarchicus

Calunue he3.golandieu.s

4.4-12.4 - -

210-350

34-107

- 17-18 -

7.7 8.19 492

Hartin and Knauer (1973) Nicholls et d. (1959)

Vinogradova and Koval’skiy (1962) Zaflropoulos and Grimanis (1977) Nicholls el al. (1959) Mayzaud and Martin (1975) Vinogradova and Koval’skiy (1959) Nicholls el d. (1859)

Crustacea Copepoda 65

238 17.5 400

Ceniropages typicua and c. hamatus Anmaloeera sp. and Pontella sp.

137

300-400

8.5-17.3

30-60

9-22.6 3.4-3.7

214-26.7 -

Viuogradova and Koval’skiy (1962) Steele el al. (1973) M%inly Calunurr spp. and

Pueudoealanus &mgatus Small copepods

-4

Vinogradova and Koval’skiy m

!! 4 (1962)

Martin and Knauer (1973) Bohn and McElroy (1976)

-4

Vinogradova and Koval’skiy m

!! 4 (1962)

Martin and Knauer (1973) Bohn and McElroy (1976)

Monterey Bay, California 65 Strathcona Sound, N. BaffIn 105

12.7 - Island Mediterranean Sea ? Strathcona Sound, N. 105 BafBn Island W. At,lant,ic T)

Crnstacea Amphipoda

Crnstacea Euphausiacea

Phrosinu semilunnta Amphipods

Eupplrausia krohnii Meganyetiphanur norvegiea Mainly Euphausia paclfca Euphausia p&&a or T h y e a w m spinifera

Gknnadaa @am

C l i m lirnaana Spimteuo (Limacinu) rctroacren Omma8trepha Uicebrma Sagilta W a n 8

Sagilta 8sCma and S . euxina

Euphausia spp.

sergcstcs spp:

sagata sp.

24.3 26

Fowler et al. (1976b) Bohn and McElroy (1976) *

112 65.6 15.1 -

2 Nicholls et d. (1959)

Martin and Knauer (1973) Cutshall and Holton (1072) Fowler et al. (1978b) w

Fowler el al. (1976b) E . .. .. . -

Mediterranean Sea ? Monterey Bay, California 65 N.E. Paciflc 90

58.671.1 7.5-21.3 8 4 4 2 . 2

lkditerranean Sea 1 ?

25-41.4 24.5497 202-314

32.9 38.7 27,7

Cmtacea Decapoda

Pteropoda Gymumomata Pteropodn Thecosomata Mollwa Cephalopoda Chaetognatha

Nicholls et d. (1959) W. Atlantic u I) D D

Off Nova Scotia 105 Black Sea ?

23.8 ~~ . 19.3 208 238 3.0 660

Mayzaud and Nartin (1975) Vinogradova and Koval’skiy (1982)

Nicholls et al. (1959) Windom (1972)

Martin (1970)

Sims (1975) Steele et d. (1973)

! Bohn and McElroy (1976) #

1 n Strathcona Sound, N.

laland Baffin 105

D 9 a 80

Salpa funiformi8 (mainly copepods

(75% copepods)

Urochordata Thaliaoea Mixed zooplankton

Mixed zooplankton

Mired xonnlankton

______ W. Atlantic N.W. African coast E. United States coast N.W. Atlantic Off Puerto Rieo

386 54 33 11 41

and Sagitla 3&98 1476 1-19 10-207

N.W. Gulf of Mexico ? Firth of Clyde 9

34-71 13-27.2 141-10.8 2.3-72.6 3.2-44.5 84-32.5 64-58.4

North Sea 106 N.W. Atlantic 9 Firth of Clyde 100 E. Paciilc 65

10.4 18.2 16.4 15.4

8 Topping (1972) Fitzgerald st d. (1972) Topping (1972) ,Ifartin and Knauer (1973)

o(

?lankton

Page 126: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Refer- Drying Conmmllation &P. (“0 Range Mean &WP Species Loeation

or a

Coelenterata Ctenophora

Crustacea Copepoda

Cntstacea Euphausiacea Pteropoda Thecosomata Mollusca Cephalopoda Chaetognatha

Urochordata Thaliacea

Coelenterata Cmstacea Copepoda

Crustacea Srnphipodn Crustacea Mysidaces Crustacea Euphausiacen Crustacea necapoda

Cbaetoguat ha

Urochordata Thaliacea Mixed zooplankton

Mixed zooplankton

Mixed zooplankton Mixed zooplankton

Mixed zooplankton

Cyanea rapillata Be?& cueumis Pleurobraehia pileus

Acurtia elausi

Cdanus hclgdandicus

Centropages typiezrs and C . hamatus Anomaloeera sp. and Pontella sp-

Clauswalanus sp. and Paracalunus spp. Small copepods

Euphausia krohnii Spiratdla (Limaeina) retrovevsa Ommastrepha illieebrosa Sagitta elegans Sagitta setosa and S. eum‘na

Salpa fusiformin

Pelagia sp. Acartia clausi

Calanus finmarehieus Labidmra acutifronr Mixed copepods

Xixed amphipods Emopia nculptieauda Meganyctiphanes notvegica Acanthqhyta ezimia Oplophorua sp. Syetellaspie debilis Chaetognaths

P y m m sp. Mainly Acartia centropages and Braehionus sp.) Mainly Calaniveda SP. and Acarlia

CHROMIUM W. Atlantic D

TI - Black Sea 9

Elefsis Bay, Greece ? N. Mediterranean Sea 1M)-110 Black Sea ?

W. Atlantic D

Black Sea ?

N. Mediterranean Sea 100-110

Black Sea ?

W. Atlantic D D n

Black Yea

W. Atlantic

ARSENIC E. Atlantic Elefsis Bay, Greece

Barents Sea E. Atlantic Strathcona Sound, N. Baffln Island

E. Atlantic

Strathcona Sound, N. Baffin Island E. Atlantic Sea of Azov

centrosages) (Mainly copepods) Off W. Greenland (Mainly ropepods and Sagitla sp.) N.W. Atlantic

E. and N.E. United Ststas coast N.W. African coast N.W. Gulf of Mexico

- D P

D

? ?

i 1b5

1$5

9

i i ?

105

? ?

?

? ? 1

? ?

- < 1.0-8.4 <1-14

1G-2.5 1.0-20.5

< 0.63 < 0.70

7

3.26 1.5 8

59.3

10

2.1

-

<0.19 <0.64 < 0.08 < 0.22

< 0.77

-

11 2.9

10 145 -

7.9 30 42 17 235 22 -

1.5 49

18

6.0 <3

2.2

t l . 4 -

eJ Nicholls et J. (1959)

Vinogradova and Kovai’skiy (1962) Za5ropoulos and Grimanis (1977) Fukai and Broquet (1965) Vinogradova and Koval’skiy (1962) Nicholls et d. (1959)

Vinogradova and Koval’skiy (1962) Fukai and Broquet (1965)

Vinogradova and Koval’skiy (1962) Nicholls et al. (1950)

ti E

Vinogradova and Koval’skiy 0 (1962) 2 Nicholls et al. (1959) *

Leatherland et al. (1973) ZaBroponlos and Grimanis

Vinoaradov (1953) (1977) 3 Leat6erlandkt d.’(1973) Rohn and McElroy (1976)

Leatherland et al. (1973)

Bohn and 3fcELroy (1976)

Leatherland et d. (1973) Vinogradov (1953)

Bohn (1975) Windom (1972)

Sims (1075)

Page 127: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

ZINC

Mouterey Bay, California 65 A g h d h a digitale Milller Japan Sea 1!5 Pekgia sp. E. Atlantic Pleurobrachia pdeua Black Sea ?

Evadne tergeulina Claus Ise Bay, Japan 105 Penilia schmackeri Richard Sagami Bay, Japan 105 Auzrtia dausi Elefsis Bay, Greece ?

Protozoa Radiolaria Coelenterata Hydrozoa Coelenterata Ctenophora

Crustacea Cladocera

Cmtacea Copepoda

63-279 - -

560-630

I

135 235 28

Nartin and Knaucr (1973) Fujita (1972) Leatherland et al. (1973) Vinomadova and Koval'skiy (1968 Fujita (1972) 242

253 1 270

1069

282

99

- _ _ - -

- 80&2 500

Ise Bay, Japan N. Pacitlc Ocean Japan Sea

N. Pnci0c Ocean OtTNova Scotia

C&nus crislatw Krtiyer

Calanwr finmarchicue

Calanue helgolandiczts Black Sea

105 105 105 105 105 105

?

8OG1 600 75-248 79-205 - 38-149 94-103 2 000-3 000

224-230 - -

20-106

82-62

2 00&3 000

163-234

30&2 000

62-170 6&75

- -

- - 1OM92

- - - - -

66-112 - -

164-200 64-83

50-131

Maymaud and Martin (1975) Vinogradova and Koval'skiy (1962) Fujita (1972) Sagami Bay Japan

Suruga Bay,'Japan Japan Sea N. Paciflc Ocean Suruga Bay, Japan h e Bay, Japan Sagami Bay, Japan Black Sea

105 105 105 105 105 105 1!5

140 114 ('donus plumchrua Marukawa

Eitculanus bungii Oiesbrecht Bfichat'la marina Giesbrecht O i l h nana Gieabrerht Pareuchaeta sp. Anomdocera sp. and PunleUa sp.

Mainly Calanus spp. and Pseudoealanur dongalua

Mixed copepods Mixed copepods

Paralhembtu obZibitria (Kxayer)

Phroeina semilunata Themblo japonica Bovallius ViMia g i h a a Bovalliua Amphipods

Neolnyds sp. Btcphaueia rimiliu 0. 0. Sam Meqanycliphanes nomegica

Thysa?uxaaa rauchii (Sam.)

Mixed euphausids mainly Buphauaia paeifica Buphawia pacifrca or Tht/sanoursa npinifera usually dominant

Thysanoesaa sp.

Small copepods

Euphausia sp.

- 206

Vinogradova and Koval'skiy (1962) Steele et al. (1973) Firth of Clyde ?

Black Sea 1

Monterey Bay California 65 Strathcona Sobud, N. Ba5n 105 Island Japan Sea 105 N. Pacltlc Ocean I t 5 Mediterranean Sea Japan Sea 105 Japan Sea 105 Strathcona Sound, N. Baffln 105 Island Japan Sea 105 Suruga Bay, Japan I f 5 E. Atlantic

W. Psci0c Ocean 105 Japan Sea 105 Monterey Bay, California 65

N.E. Paci5c 90

Sagami Bay, Japan 1f5 Mediterranean Sea Japan Sea 105

Mediterranean Sea i 9

Vinomadova and Koval'akiv E L! Y

(196b Martin and KnaUer (1973) Bohn and McElroy (1976)

Cmtaeea Amphipoda 166 114 150 456 95 43

Fujita (1972)

Fowler et al. (1976b) Fujita (1972)

Bohn and McElroy (1976)

Fujita (1972)

Leatherland et d. (1873) Fowler el al. (1976b) Small et al. (1973) Fujita (1972)

Martin and Knauer (1973)

Cutshall and Holton (1972)

Fujita (1972) Fowler et al. (1976b) Fujita (1972)

CNshcea Mgsidacea Cmtacea Euphausiacea

385 214 104 85 73 463 - 71

01 s 211 108 128

Page 128: [Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton

Drying ConcelUrntion temp. ("C) Range Mean croup Species LooalimL

Crustacea Decapoda

Pteropoda Oymnosomata Pteropoda Thecosomata

Chaetognatha

Urochordata Thaliaeea

Mixed zooplankton

bfixed zooplankton

Mixed zoophnkton Mixed )r zooplankton Mixed zooplankton

Aeanthephyra exi,ma'a Qennadau dqana Lucifer reynaudii H. Milne Edwards Swt&a.mW debilis O>lqphoh48 Sp. Serpeates sp. C l i m sp. SpirrUella (Limocina) t i&iJm& (D'Orbigny) Sagitca bipumlala Quoy Sagitta -and

Sagitta minima Orassi Sagitla paci~%% Tokolka SW.tta r o h t a Doncaster Sagitta setosa and S. ew'na

ZINC (continued)

E. Atlantic Mediterranean Sea Sagami Bay, Japan

E. Atlantic

Neditenanean Sea N. Pacific Ocean

Sagami Bay, Japan Japan Sea Off Nova Scotia Sagami Bay, Japan

Black Sea

Sdpa Jusifomi.9 Japan Sea

%rotoma sp. E. Atlantic (malnly copepoda and S-Ia sp.) N.W. African coast

N. Pacific Ocean

E. and N.E. United Stater coast N.W. Atlantic Off Puerto Rim

North Sea FUh of Clyde

E. Pacinc N.W. Gulf of Mexico Firth of Clyde

9

i 105

? ? ? 105 105

105 105 105 105 105 105

?

105 105

9 ? 1

86

100 P 100 65 4

9

- 60-79

- 156-170 - - -

3 300-4 400

- - -

90-2 700 40-1 200

92-591 120-1 200

54-1 220 207-252 Ql-892 50-385 41-200 110-139

65 69 745

50 98 68 160 113

75 224 162 394 134 86

633 121 105 1 468 237

236 428

301

228 199

-

- - -

Refe?ena

Leatherland el al. (1973) Fowler et d. (1976b) Fujita (1972)

Leatherland et d. (1973)

Fowler Fujih (1972) et d. (1976b) b 8 2 +4

Nayaaud and Martin (1975) Fujita (1972) ?

!i Vinogradova and Koval'skiy (1962) Fujita (1972)

Leatherland el al. (1973) Windom (1972)

Nartin (1970)