Accumulation of cadmium by Dunaliella tertiolecta Batcher

Joanal of Pfamktoa Bemrek Vohnoe 1 Number 1 1979

Accumulation of cadmium by Dunaliella tertiolecta Batcher

J.R. Jennings, P.S. Rainbow

Department of Zoology and Comparative Physiology, Queen Mary College, MileEnd Road, London El 4NS

(Received August 1978; accepted October 1978)

Abstract Cultures of Dunaliella tertiolecta were exposed to five concentration* of cadmium in solution(0.1, 0.5, 1, Sand 10ppm(mg 1-'))• The accumulation of cadmium by the algae was found to have twophases, an initial rapid uptake followed by a stabilisation of the cellular cadmium levels. D. tertiolectaconcentrated cadmium from solution (cone, factor approx. 1350) at exposures up to 1 ppm Cd butexposure to the higher concentrations caused no further increase in the accumulated cadmium concentra-tion of the algae which reached a nmriimim at about 1.5 ng Cd mg~' Dunaliella.

Introduction

Although cadmium levels in the sea are apparently not toxic (<10 ppb (jig 1"') inBritish coastal waters, (Preston et al., 1972a)) the metal is recognised as a hazardousenvironmental pollutant (Hiatt and Huff, 1975) and its uptake by phytoplankton atthe first level of marine food chains may be of ecological significance.Phytoplankton play a major role in the geochemical distribution of cadmium, formuch of the cadmium in the surface layers of the ocean at times of high primary pro-ductivity is bound by the plankton (Knauer and Martin, 1973) and such cadmiummay be transported to higher trophic levels of food chains. As Preston et al. (1972b)have pointed out, it is the initial accumulation of metals by phytoplankton andparticulates which provides much of the momentum for the transfer of metals alongfood chains.

Information is therefore required on the acumulation of particular heavy metalsalong marine food chains if any attempt, however crude, is to be made at predictivemodelling of the effect of heavy metals on marine ecosystems. This paper desribesan investigation into the accumulation of cadmium by the phytoplankton speciesDunaliella tertiolecta Butcher, and is part of a detailed laboratory study of thetransfer of cadmium through a simulated marine food chain consisting also ofArtemia salina (see Jennings and Rainbow, 1979b) and Carcinus maenas (see Jenn-ings and Rainbow, 1979a).

The accumulation of trace metals and their radioisotopes by planktonic algae inculture has been the object of numerous studies. In the case of essential trace metals,Jensen et al. (1976) have investigated the uptake of copper by three diatoms andBemhard and Zattera (1969) and Parry and Hayward (1973) have studied the uptakeof zinc by Phaeodactylum tricomutum and Dunaliella tertiolecta respectively. Someemphasis has also been placed on the uptake of non-essential metals such ascadmium (Cossa, 1976; Gerhards and Weller, 1977; Saraiva and Fraizier, 1975),lead (Schulz-Baldes and Lewin, 1976), mercury (Gerhards and Weller, 1977) andnickel (Gerhards and Weller, 1977; Skaar et al., 1974).

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Materials and Methods

Cultures of Dunaliella tertiolecta Butcher (ChlorophytarChorophyceae) were main-tained axenically in Foyn's Erdschreiber medium under continuous fluorescentillumination at 18°C. Experimental cultures were grown in artificial seawater(Instant Ocean, Aquarium Systems Inc., Ohio) with added NaNo3 (1 g I"1),NaH2PO4 (0.1 g 1 -•), vitamin B, (1 mg 1"') and vitamin B,2 (5 /« 1 -1). The use ofartificial seawater ensured the absence of any chelators of heavy metals that mightbe present in natural seawater (see Johnston, 1964) and affect the chemical nature ofthe metal under test (see Cossa, 1976; Jensen et al., 1974).

Experimental aliquots were taken from a single dilution of stock phytoplanktonculture to give equal initial cell concentrations in each subculture. Duplicate 230 mlexperimental subcultures were exposed to each of the cadmium concentrationstested (0.1, 0.5, 1, 5 and 10 ppm (mg 1 '*)). The algae in each subculture were in thelinear growth phase during the experimental period. Algal growth was followed bydirect cell counts using a Segwick-Rafter cell.

To give the required experimental concentration, cadmium was introduced to thecultures in aliquots from a stock solution of cadmium chloride (1,000 ppm Cd) indistilled water. Since only small amounts of cadmium are taken up by the algae, itwas decided to employ a radioisotope of cadmium as a tracer and carrier-freeCd-109 Cl2 (Radiochemical Centre, Amersham, England) was added to give anactivity of 10 microcuries I"1. The accumulation of cadmium was followed bymonitoring daily 10ml samples of culture which were filtered through pre-weighedmembrane filters (Nuflow Filters) and then rinsed three times with Instant Ocean,after which radioactivity levels (i.e. cadmium as Cd-109) in the filtrate wereinsignificantly different from background radioactivity levels.The filter paper with deposited algal cells was dried at 60°C, weighed and digested

in 5 ml concentrated nitric acid, before the sample was made up to 10 ml with distill-ed water. The concentration of accumulated cadmium in each sample was measuredusing an automatic gamma counter system (Nuclear Chicago 1185). As a control forthe weight of the seawater which remained with the algae on the membrane filter andfor its associated activity, a further 10 ml sample of culture was taken at each sampletime and centrifuged to remove the algae. The supernatant was filtered,rinsed, weighed, digested and counted as above. Cadmium levels quoted are themean for the two experimental cultures.

Measurements of cadmium bound by the algae and that remaining in the mediumdid not account for all the cadmium originally added. A possible explanation forthis apparent loss of cadmium is adsorption onto the walls of the glass containers(the adsorption characteristics of cadmium on borosilicate glass have been describedby Struempler, 1973). Schulz-Baldes and Lewin (1976) similarly could not accountfor 20 to 40% of lead added to media containing the phytoplankton Platymonas andPhaeodactylum, and also concluded that the missing fraction had probably beenadsorbed to the surface of the experimental vessel. Furthermore, any loosely boundcadmium may have been lost when the algae were rinsed in fresh seawater prior todetermination of the cellular cadmium.

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Accnmnbttion of rartmtam by DunaUttta tertbUcta Butcher

Results

The results showing the accumulation of cadmium by Dunaliella tertiolecta atincreasing concentrations of cadmium in solution are shown in Table I and theseresults are plotted graphically in Figure 1. At an exposure concentration of 0.1 ppmcadmium, D. tertiolecta showed a rapid accumulation of cadmium for the first twodays, and then the bound cadmium stabilised at about 0.125 pg Cd mg"1 Dunaliella.

Table I. Dunaliella tertiolecta — Uptake of cadmium by algal cells growing at five concentratons ofdissolved rarimhim

0.1 ppm

0.3 ppm

1 ppm

5 ppm

10 ppm

Time(d«yi)

00.230.51234

00.512345

00.51234

00.512345

00.2512345

No. of cdl*per 10ml(xl0«)

8.08.58.58.59.5

11.513.5

8.08.08.0

17.524.528.534.5

8.08.57.09.0

10.511.5

8.07.57.5

17.023.528.034.5

8.08.67.58.09.25

10.515.0

Atnorbed Cd 0 «per mgdry wtDunaBetla)

0.0240.0340.0860.1230.1250.126

0.3550.5940.6660.7020.6860.608

0.2530.5600.6870.9651.062*

0.530.551.491.631.571.57

0.410.601.141.361.501.30

Absorbed Cdper 10* cefljOigxlO-2)

0.1690.2420.6070.8470.9210.948

2.905.554.174.204.145.10

1.772.144.666.857.39

7.313.310.213.413.914.2

2.95.88.49.6

10.710.3

Total atnorbedCd in 10 mlDunaUttia(W)

.014

.021

.052

.081

.106

.128

0.230.440.731.031.161.73

0.150.280.420.720.85

0.551.001.733.143.894.89

0.250.440.670.891.131.54

had not stabilised yet

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1-6

pgCd mg--

Dunolwtla

1-2

0-8

0-4

>

J/A

ra '

—0

Sppra

- ^ W p p m

Ippm

0-5 ppm

0-lppm

Doyl

Fig. XDunalldla tertiolecur. The concentration of r*Am\nm bound by the algae at increasing levels ofa in solution (ppm - mg 1 ~ ' ) .

At a concentration of 0.5 ppm cadmium in solution, the accumulation showed arapid increase in two days of exposure and then stabilised at about 0.68 \tg Cd mg"'Dunaliella. In all previous experiments stabilisation of accumulated cadmiumconcentrations had occurred within three days and so the cultures exposed to 1 ppmcadmium were stopped after four days. Accumulated cadmium concentrations hadhowever surprisingly not stabilised by then, probably as a result of a low growth rate(note cell numbers in Table I)- The bound cadmium concentration observed was1.062 fig Cd mg'1 Dunaliella, but this value may be an underestimate of themaxiumum level attainable. Cultures of D. tertiolecta exposed to 5 and 10 ppmcadmium showed similar rates of accumulation of cadmium, stabilising after threedays at bound cadmium concentrations of about 1.S7 and 1.36 ng Cd mg"1

Dunaliella respectively.From the graphical representation of these results (figure 1), it is possible to see

that the concentration of cadmium bound at the different cadmium concentrationsstabilises after a period of rapid uptake. Increasing the concentration of cadmium insolution from 5 to 10 ppm caused no further increase in this stabilised boundcadmium concentration. Table II shows the stabilised bound cadmium concentra-tions of Dunaliella at the different concentrations of cadmium in solution as deter-mined from Figure 1. These concentrations are plotted in Figure 2 against externalcadmium concentration and from this graph it is possible to estimate that a max-imum bound concentration of about 1.5 \tg Cd mg ~' Dunaliella would first be reach-ed at an exposure concentration of about 1.05 ppm cadmium in solution. Table IIalso shows the calculated concentration factors for cadmium by the algae at eachconcentration of dissolved cadmium.

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AttmimulMtlum of •-*'••'••» try DumtitOa tertiokcta Butcher

Table D. Dunalidla tertiolecttr. stabilised bound cadmium levels reached when exposed to increasinglevels of r^inlnm. and the concentration factor that these bound concentrations represent over the

concentration.

External Cd concentration Dunalieila stabilised bound Concentration(ppm •» mg 1 - ' ) Cd concentration factor

(pg Cd m g - ' dry wt) (w/w)

0.1 0.125 1,2820.5 0.686 1,4061.0 1.062 1,0895.0 1.570 308

10.0 1.360 134

Discussion

The accumulation of cadmium by D. tertiolecta at all external cadmium concentra-tions had two phases, an initial rapid uptake followed by a stabilisation of thecellular cadmium levels. The initial uptake phase usually took approximately twodays. The time taken to reach a stabilised cellular cadmium level is probably relatedto the growth of the algae, which is lower at higher levels of cadmium exposure (J.R.Jennings, unpublished Ph.D. thesis 1978, University of London). Following thisphase of rapid uptake, the concentration of accumulated cadmium appears to reacha maximum of about 1.5 fig Cd mg"1 D. tertiolecta. This level of accumulatedcadmium is reached on exposure to a minimum of about 1 ppm cadmium and thepresence of any further cadmium externally has no effect on acumulated cadmiumconcentrations. These results suggest that the algae have a limited number ofbinding sites for cadmium which become saturated at just over 1 ppm cadmium inthe external medium.

Other authors investigating metal uptake by phytoplankton, such as Saraiva andFraizier (1975) on the uptake of cadmium by Dunaliella bioculata and Schulz-Baldesand Lewin (1976) on the accumulation of lead by Phaeodactylum tricomutum havealso described an initial phase of rapid uptake. Following this phase of rapid uptake,the results of Saraiva and Fraizier (1975) indicate a phase of low increase ofaccumulated cadmium concentration, in contrast to our results and also to theresults of Schulz-Baldes and Lewin (1976) who found that Phaeodactylum cellsbecome saturated with lead after the initial uptake phase.

At low external cadmium exposures (<1 ppm) the D. tertiolecta cells concentratedcadmium by a factor of about 1350, this value becoming progressively smaller atincreased external cadmium concentration. The concentration factor of 1,350 issimilar to that (1,210) found by Petrocelli et al. (1975) for dieldrin absorption byDunaliella peircei but surprisingly greater than that of 300 obtained by Saraiva andFraizier (1975) for Cd-109 uptake by D. bioculata.

The nature of the cadmium binding sites on or within the cells is as yet unknown.The uptake of heavy metals by aquatic plants seems to be a passive process,although one which can be affected by metabolism (Bryan, 1976). Cossa (1976)concluded that cadmium is adsorbed onto the cell wall of Phaeodactylum tricor-nutum. Similarly Schulz-Baldes and Lewin (1976) interpreted the pattern of lead

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J.R. Jemdngi, P.S.R*inbow

lOGDunohflotfobSMdCdconc".

1 0

0 1

0-1 1-0 nIOG coAmm cone".

Fig. 2. Dunaliella tertlolectcr. Final stabilised concentrations of bound cadmium in the algae afterexposure to different concentrations of cadmium.a — this result may be an underestimate as the cadmium accumulated by D. tertiolecta may not have

stabilised by the end of the experimental period.

accumulation by P. tricornutum in terms of intitial adsorption to the cell surfacefollowed by transport of the metal to other sites either actively or by passivediffusion. Davies (1973) showed that the kinetics of zinc uptake by P. tricornutumcould be explained by the rapid adsorption of zinc onto the cell membrane followedby slower diffusion into the cell. The rate of this diffusion controls the rate of up-take of the zinc which is then bound to proteins within the cell. Binding to proteinmay control the concentration in the cell since, during the growthcycle, the concentration of zinc reaches a maximum and then decreases as theamount of protein in each cell declines (Davies, 1973). A similar pattern has beenobserved for the uptake of nickel by the same species (Skaar et al., 1974). Parry andHayward (1973) concluded that "Zn was bound to protein in Dunaliella terti-olecta and Gerhards and Weller (1977) showed that both mercury and cadmiumbecome bound to high molecular weight substances in Chlorella pyrenoidosa.Unpublished observations show that accumulated cadmium in D. tertiolecta is alsobound to protein.

In conclusion, therefore, it has been shown that D. tertiolecta has a high ability toconcentrate cadmium from seawater. The concentration of accumulatedcadmium appears to be proportional to the concentration of the metal in solution upto about 1 ppm and above this concentration there is a limit, possibly of

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Affnmnlrtloo of fHmhun bj DunalleUa tertiolecta Butcher

further binding sites, when no further accumulation can occur. If a high rate ofaccumulation of heavy metals by phytoplankton provides the impetus for thetransfer of heavy metals along marine food chains (Preston et al., 1972b) then anymechanism which limits this accumulation by the phytoplankton will affect theaccumulation of cadmium by organisms higher up the food chain. Results fromexperiments on the second stage of the food chain {Artemia salina) (see Jennings andRainbow, 1979b) show that the accumulated cadmium concentration of Artemia isincreased by a factor of between 6 and 8 when cadmium is accumulated from D.tertiolecta as a food supply in addition to uptake from solution at cadmium concen-trations of 1 ppm and less. At higher cadmium concentrations however, the lack offurther uptake of cadmium by D. tertiolecta limits the amount of cadmiumaccumulated from the food so that at 10 ppm cadmium, the level of cadmiumaccumulated by Artemia from food and solution is only a factor of about 2 greaterthan the cadmium level accumulated only from solution (Jennings and Rainbow,1979b).

Acknowledgements

The authors wish to thank Mr. A.G. Scott for technical advice. J.R.J. was in tenureof an S.R.C. studentship during this work.

References

Bernhard, M., and Zattera, A.: 1969, 'A comparison between the uptake of radioactive and stable zinc bya marine unicellular alga'. Symposium on radioecology. Proceedings of the 2nd National Symposium,Ann Arbor, Michigan (1967), pp. 389-398.

Bryan, G.W.: 1976, 'Some aspects of heavy metal tolerance in aquatic organisms', In: Effects ofpollutants on aquatic organisms. Ed. A.P.M. Lockwood, pp. 7-34. Cambridge University Press,Cambridge.

Cossa, D.: 1976, 'Sorption du cadmium par une population de la diatomee Phaeodactylum tricornutumen culture', Mar. Biol. 34, 163-167.

Davies, A.O.: 1973, "The kinetics of and a preliminary model for the uptake of radioactive zinc byPhaeodactytum tricornutum in culture', In: Radioactive contamination of the marine environment.Proceedings of a symposium on the interaction of radioactive contaminants with the constituents of themarine environment held by the International Atomic Energy Agency, Seattle, U.S.A. 10-14th July,1972, pp. 403-420. Vienna I.A.E.A.

Oerhards, U. and Weller, H.: 1977, 'The uptake of mercury, cadmium and nickel by Chlordlapyrenoidosa', Z. Pflanzenphysiol. Bd. 82, 292-300.

Hiatt, V. and Huff, J.E.: 1975, 'The environmental impact of cadmium: An overview'. Int. J. Environ.Stud. 7, 277-285.

Jennings, J.R. and Rainbow, P.S.: 1979a, 'Studies on the uptake of cadmium by the crab Carcinusmaenas in the laboratory. I. Accumulation from seawater and a food source', Mar. Biol. 50, 131-139.

Jennings, J.R. and Rainbow, P.S.: 1979b, 'The accumulation of cadmium by Artemia salina ', Mar.Biol. 51, 47-53.

Jensen, A., Rystad, B. and Melsom, S.: 1974, 'Heavy metal tolerance of marine phytoplankton. I. Thetolerance of three algal species to zinc in coastal seawater', J. exp. mar. Biol. Ecol.15, 145-157.

Jensen, A., Rystad, B. and Melsom, S.: 1976, 'Heavy metal tolerance of marine phytoplankton—II.Copper tolerance of three species in dialysis and batch culture', / . exp. mar. Biol. Ecol. 22, 249-256.

Johnston, R.: 1964, 'Seawater, the natural medium of phytoplankton. II. Trace metals and chelation andgeneral discussion', / . mar. biol. Ass. U.K. 44, 87-109.

Knauer, O.A. and Martin, J.H.: 1973, 'Seasonal variations of cadmium, copper, manganese, lead andzinc in water and phytoplankton in Monterey Bay, California', Limnol. Octanogr. 18, 597-604.

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Petrocefli, S.R., Anderson, J.W. and Hanks, A.R.: 1975, 'Controlled food chain transfer of dleldrinresidues from phytoplankters to dams', Afar. blot. 31, 215-218.

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Preston, A., Jefferies, D.F. and Pentreath, R.J.: 1972b, 'The possible contributions of radioecology tomarine productivity studies', Symp. Zool. Soc. London 29, 271-284.

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Schulz-Baldes, M. and Lewin, R.A.: 1976, 'Lead uptake in two marine phytoplankton organisms', Biol.Bull. 150, 118-127.

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Accumulation of cadmium by Dunaliella tertiolecta Batcher