Bioaccumulation of 110mAg, 60Co, 137Cs and 54Mn by the Freshwater Crustacean Daphnia magna from Dietary sources (Scenedesmus obliquus and Cyclotella meneghiana)

BIOACCUMULATION OF 110MAg, 60Co, 137Cs AND 54Mn BY THEFRESHWATER CRUSTACEAN DAPHNIA MAGNA FROM DIETARY

SOURCES (SCENEDESMUS OBLIQUUS AND CYCLOTELLAMENEGHIANA)

C. ADAM1∗, J. GARNIER-LAPLACE1 and J. P. BAUDIN2

1 Institut de Protection et de Sûreté Nucléaire, Département de Protection de l’Environnement,Laboratoire de Radioécologie Expérimentale, Centre de Cadarache, Bât. 180, 13108

Saint-Paul-Lez-Durance Cedex, France; 2 Centre National de la Recherche Scientifique; sameaddress

(∗ author for correspondence, e-mail: [email protected], fax: +33 4 42 25 40 74)

(Received 25 August 2000; accepted 11 April 2001)

Abstract. The importance of food as radionuclide source for the crustacean Daphnia magna wasinvestigated using a planktonic food chain composed of young pre-adult daphnids and two algalspecies (Scenedesmus obliquus and Cyclotella meneghiana). Daphnids placed in a tank containingnatural 0.45 µm filtered water were fed on algae previously kept during 4 days in natural watercontaminated by 110mAg, 60Co, 137Cs and 54Mn. After about one week of exposure, daphnids wereplaced in non-contaminated water on a diet of non-labelled algae, in order to monitor radionucliderelease. The results suggest that the Trophic Transfer Factor (TTF) of radionuclides in daphnids wasgenerally greater for the transfer via Scenedesmus than via Cyclotella and that it could be linked to theintracellular fraction of accumulated radionuclides and consequently to their biochemical behaviour.For the radionuclide transfer via Cyclotella meneghiana, the biological periods ranged, for the firstcompartment, from 7 to 30 min and for the second, from 10 h to 1.8 d. As regards the transfer viathe green algae Scenedesmus obliquus the biological half-lives were longer, since Tb1 characterizingthe first compartment, ranged from 11 min to 5.2 h, whereas Tb2 ranged from 1.2 to 2.1 d. From anoperational point of view, this paper underlines the importance of considering the food contaminationin the models of radionuclide transfer through trophic chains, in order to widen their applications indifferent seasons or ecosystems.

Keywords: Cyclotella meneghiana, Daphnia magna, kinetics, Radionuclides, Scenedesmus obliquus

1. Introduction

For the past 40 yr, electronuclear facilities have been releasing radionuclides intoaquatic ecosystems where these pollutants represent a source of contamination foraquatic organisms. Radioactive isotopes of cobalt, caesium, manganese and silverare among the major radionuclides discharged under normal operating conditionsby many nuclear power stations. In 1995, they accounted for about 90% of the non-tritium mean composition of the low-level liquid wastes discharged from a French1300 MW Pressurized Water Reactor (Anonymous, 1996). One of the purposesof freshwater radioecology is to explain and to predict the impact of radionuclide

Water, Air, and Soil Pollution 136: 125–146, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

126 C. ADAM ET AL.

release on aquatic biotic components. An experimental approach can be used toestimate the fate and effect of radionuclides in these systems, provided that astandardized procedure is used to ensure that the laboratory results are independentof the experimental conditions and can be more representative of the field. On thebasis of this methodology, trophic chains or webs have shown great potential asexperimental tools to assess the relative importance of food and water in radionuc-lide accumulation by organisms (Garnier-Laplace, 1991; Vray, 1994; Adam, 1997;Véran, 1998).

Although the freshwater invertebrates occupy an important position as a routefor the transfer of pollutants from phytoplankton to fish (Pont, 1995), their radio-active contamination from planktonic algae have been very poorly investigated.Moreover, the few studies undertaken have mainly dealt with transfers from greenplanktonic algae, such as Scenedesmus, Chlorella or Selenastrum (Kwasnik et al.,1978; Amiard-Triquet, 1979; Kearns and Vetter, 1982; Baudin and Nucho, 1992;Gil Corrisco and Vaz Carreiro, 1992). However, it is worth pointing out that thephytoplanktonic population of various water courses is often made up of othergenera such as the centric diatom Cyclotella that may represent, for example, morethan 90% of the phytoplanktonic population during early summer in the Frenchriver Loire (Lair and Reyes-Marchant, 1997). In that context, it is of great in-terest to study the radionuclide accumulation by herbivorous zooplankton, not onlyfrom green algae, but also from diatoms, that present different morphological andphysiological characteristics.

Given their filtration characteristics, daphnids appear to play a key role in phyto-planktonic biomass control (Riemann and Christoffersen, 1993). For this reason,Daphnia magna was chosen to study the potentiality of transfer of 110mAg, 60Co,137Cs and 54Mn from the two ubiquitous phytoplanktonic species, Scenedesmusobliquus and Cyclotella meneghiana. This study is part of a wider research pro-gram that aims to model the dynamics of transfer of radionuclides occurring inliquid effluents from the Civaux nuclear power plant, located on the Vienne river,a tributary of the river Loire (France).

2. Materials and Methods

2.1. EXPERIMENTAL CONDITIONS

The freshwater crustacean D.magna was cultured at 20 ± 1 ◦C, under 45 µEm−2

s−1 lighting on a 16-h light 8-h dark cycle, on a diet of the green algae Chlorellavulgaris. Crustaceans to be used in the contamination experiment were collectedfrom the rearing aquarium through two plastic sieves, with a mesh size of 560and 800 µm, in order to separate the young pre-adults. The selection of this sizeclass made it possible to limit death, molt or reproduction processes, which maychange the population size. Selected organisms were acclimatized for 2 weeks to

BIOACCUMULATION OF 110MAg, 60Co, 137Cs AND 54Mn 127

experimental conditions, with a density of 250 individuals per litre, usually usedfor similar studies (Boudou and Ribeyre, 1981; Watras et al., 1985).

Scenedesmus obliquus and Cyclotella meneghiana were obtained from Göttin-gen University. Stock cultures were maintained in modified B3N medium (Nicholsand Bold, 1965), and in a f/2 medium (Guillard and Ryther, 1962), for Scenedesmusand Cyclotella respectively. The green algae Scenedesmus were grown in 3-L glassflasks at 20 ± 1 ◦C, under ‘cool white’ fluorescent lights on a 16 : 8 day : nightcycle, whereas Cyclotella was maintained at 15 ± 1 ◦C, under constant light ona 12 : 12 day : night cycle. Air was bubbled continuously into the algal culturesto keep cells in suspension and to ensure an adequate supply of CO2. All cultureswere acclimatized to experimental conditions at least 2 weeks before assays. Inparticular, the levels of stable manganese and cobalt concentrations in the nutritivemediums (in the chemical form of MnCl2, CoCl2 and B12 vitamin) were adjustedto the concentrations observed in the Vienne river, by adding 20 µg L−1 of Mn, andstopping the input of stable cobalt entering into the culture medium composition.Algae, in stationary phase of growth, were then removed from stock cultures byfiltering on 0.45 µm cellulose acetate membranes under very gentle vacuum andwashed with filtered Vienne river water (pH 7.85, 11.2 mg L−1 Ca, 1.1 mg L−1

Mg, 13.1 mg L−1 Na, 2.1 mg L−1 K, 7.6 mg L−1 NO3, 10.3 mg L−1 Cl, 12.8 mgL−1 SO4, 49 mg L−1 HCO3, 3 mg L−1 TOC, conductivity 177 µS cm−1) to removenutritive medium. Algal cells were placed in 1-L glass flasks containing 0.45 µmfiltered Vienne water contaminated at a level of 150 Bq mL−1. The radionuclideswere obtained from the Amersham International Radiochemical Centre (UK) andwere added to the water of the separate flasks in the form of 60CoCl2, 137CsCl,54MnCl2 and 110mAgNO3 respectively. Due to the presence of carrier element incommercialized radionuclide solutions, this radioactive contamination came withthe addition of a stable element. Thus, the concentrations of stable cobalt, caesium,manganese and silver during experimentation were 2.7 × 10−2 µg L−1, 1.3 × 10−1

µg L−1, 4.4 × 10−1 µg L−1 and 2 µg L−1 respectively. Algae were maintained inthis medium during 4 days, under the same light and temperature regime as thestock cultures. Preliminary experiments showed that this period was long enoughto reach the steady state contamination of algal cells. Finally, they were removedby filtering under very gentle vacuum and introduced into the experimental tanks,containing 4 L of filtered Vienne water, softly aerated in order to maintain the algaein suspension. The density was set to 2 × 105 cells mL−1, which is consistent withdensities observed under natural conditions (Lair and Reyes-Marchant, 1997; Lairet al., 1999). One thousand daphnids were placed in this aquarium, the organismdensity being then of 250 individuals L−1. Throughout the accumulation phase,water and contaminated algae were renewed daily. To take into account a possibleloss of radionuclide from algae or daphnids to water that may lead to a directtransfer, samples of 5 mL filtered water were measured twice a day. To monitorthe algae contamination, algae were removed from the medium by gentle filtrationon a 0.45 µm cellulose acetate membrane and weighed. In parallel, 50 to 200

128 C. ADAM ET AL.

daphnids were collected with a sieve (mesh size 560 µm), rinsed to remove anycontaminated particles, blotted dry with absorbent paper and weighed. Each samplewas introduced into a polyethylene tube and their radioactivity was measured bygamma spectrometry in a multichannel analyzer, connected to a sodium iodidewell probe. All results were corrected for the physical decay of the radionuclides,by relating them to the first day of experiment.

At the end of the uptake phase, the remaining daphnids were transferred toanother aquarium. In order to follow the radionuclide elimination kinetics, theywere fed with non-labelled algae, whose characteristics (species and density) wereidentical to those used during the contamination phase. Uncontaminated water andfood were changed after 4 and 8 hr on the first day, and then daily until the end ofthe depuration phase.

2.2. THEORETICAL BASIS FOR THE ESTIMATION OF THE TRANSFER

PARAMETERS

Uptake and depuration were analyzed using a compartmental model characterizedby first order kinetics. According to this approach, each compartment represents agroup of organs or tissues which can be described by the same kinetic parameters.Initial conditions and basic assumptions required for the resolution of the differ-ential equations formalizing this model have been described in detail by Garnier-Laplace et al. (1997). All the mathematical terms used in the following formulationare listed in Table I. The growth of the organism follows an exponential law mD(t)= mD(0)eGt where mD(t) is the mass of a daphnid (g w.w.) at time t, mD(0), themass at the beginning of the experiment and G, the relative mass gain (d−1).∣∣∣∣∣∣∣∣∣∣∣∣

CDFood(t) = FR CA

m∑i=1

ufi

(dfi + G)

(1 − e−(dfi+G)t

)(accumulation phase)

CDFood(t − t0) = CDFood(t0)

m∑i=1

Eie−(dfi+G)(t−t0) (elimination phase)

where CDFood(t) is the daphnid radionuclide concentration (Bq g−1 w.w.) dueto algae ingestion, FR the feeding rate (d−1), CA the algae radionuclide concen-tration (Bq g−1 w.w.), ufi (gFood. gDaphnid

−1 w.w.) and dfi (d−1) the uptake anddepuration parameters characterizing the radionuclide transfers, considered to beinvariable for a ‘radionuclide – contamination vector – organism’ triplet. Duringthe elimination phase, the change in daphnid radionuclide concentration is repres-ented by CDFood(t-t0), with t0, the initial time of the elimination phase and Ei , thecontribution of compartment ‘i’ to the total concentration in the receptor organism.

The first step in the evaluation of the kinetic parameters is to process the exper-imental data resulting from the depuration phase. The dfi values are estimated bylinear (m = 1) or nonlinear regression (m > 1) on the basis of the experimental dataset (t-t0, CD(t – t0)).


TABLE I

Main variables and parameters used in the model describing 110mAg, 60Co, 137Cs and 54Mntransfer from Cyclotella meneghiana and Scenedesmus obliquus to daphnids

Variable or Meaning Units

parameter

mD(t) Mass of a daphnid g w.w.

G Relative mass gain d−1

CDFood(t) Radionuclide concentration in the daphnid due to the Bq g−1 w.w.

transfer from algae ingestion

FR Feeding rate of daphnids ind−1 d−1

CA Radionuclide concentration in the algae Bq g−1 w.w.

ufi Accumulation parameter characterizing the transfer of gFood. gDaphnid−1

radionuclide from food (algae) w.w.

dfi Depuration kinetic parameter characterizing the d−1

physiological turn-over of the radionuclide

accumulated after algae ingestion

Ei Contribution of the compartment ‘i’ to the total dimensionless

radionuclide concentration in daphnid (� Ei=1)

Tj , Tj+1 Time interval on which feeding rate and radionuclide

concentration in algae are assumed as constant functions

DFoodi,j+1 Intermediary variable used to simplify the formulation Bq g−1 w.w.

XFoodi(t) Intermediary variable used to simplify the formulation Bq g−1 w.w.

in order to perform a simple linear regression

d Depuration kinetic parameter characterizing the d−1

physiological turn-over of radionuclide in algae

µ Multiplication rate of the algae d−1

Eff Efficiency of grazing dimensionless

F Individual filtration rate mL d−1 ind−1

nD Number of daphnid per unit of volume mL−1

mA Algae mass per unit of volume g w.w.mL−1

Wj Radionuclide concentration in the water, constant Bq mL−1

function on each time interval

uwi Uptake kinetic parameter describing the radionuclide d−1

transfer from water to the daphnid

dwi Depuration kinetic parameter describing the d−1

physiological turn-over of the radionuclide

accumulated from water

CDWater (t) Radionuclide concentration in the daphnid due to the Bq g−1 w.w.

transfer from water

XWateri (t) Intermediary variable used to simplify the formulation Bq g−1 w.w.

in order to perform a simple linear regression

130 C. ADAM ET AL.

For the accumulation phase, the formulation is only valid if the radionuclideconcentration in algae remains constant, and if the concentration in daphnid at thestart of the experiment is negligible. To take into account the fluctuations of thealgae radionuclide concentration, the observed series were transformed into func-tions which are constant by time interval. For each interval, the model describedabove is used to calculate the concentration accumulated by the daphnids, and isalso corrected of the previously accumulated concentration (Le Fur, 1991) (to makeeasier the reading of the equations, daphnid radionuclide concentration is presentedfor the case of prior correction by the growth of the organism).

By considering n time intervals ]Tj , Tj+1] (j ∈ N∗), on which feeding rate andradionuclide concentration in algae can be described by constant functions, thechange in daphnid radionuclide concentration due to food ingestion CDFood(t) canbe written.

For each time t included in a time interval ]Tj , Tj+1],CA(t) = CAj+1

CDFood(t) =m∑

i=1

ufi

dfi

n∑

j=1

CAjFRj

(e−dfi (t−Tj ) − e−dfi(t−Tj−1)

)

+ CAj+1FRj+1(1 − e−dfi (t−Tj )

)

With the intermediary variable:

DFoodi,j+1 = CAjFRj

dfi

(edfiTj − edfiTj−1

) + DFoodi,jwithDFoodi,1 = 0

Introducing it in CDFood(t):

CDFood(t) =m∑

i=1

ufi

[e−dfi tDFoodi.j+1 + CAj+1FRj+1

dfi

(1 − e−dfi(t−Tj )

)]

Knowing the values CAj, FRj and dfi , the variable XFoodi

(t) can be calculated foreach compartment i and each time step, with the formula:

XFoodi(t) = e−dfi tDFoodi,j+1 + CAj+1FRj+1

dfi

(1 − e−dfi(t−Tj )

)

∀t ∈]Tj , Tj+1] with DFoodi,1 = 0

Thus, the ufi values are estimated by a multiple linear regression without aconstant term, since CDFood(t) may be given as:

CDFood(t) =m∑

i=1

ufi XFoodi(t) (1)


During experimentation, the monitoring of algae radionuclide concentrationrapidly became very difficult because of the presence in the solid phase of aggreg-ates composed of partially digested algae, particles and organic/mineral complexes.The algae radionuclide concentration 24 hr after their addition to the medium wasthus estimated on the basis of the following equation:

CAj+1 = CAje(−d−µ)(Tj+1−Tj ) (2)

with CAj and CAj+1, the algae radionuclide concentration (Bq g−1 w.w.) measuredat the beginning and at the end of the 24 hr time interval, d, the depuration kineticparameter (d−1) characterizing the algae decontamination and µ, its multiplicationrate (d−1). The parameters d and µ were estimated during preliminary experimentsof radionuclide transfers to both algal species, where the contamination mediumused was also filtered Vienne water (Adam, 1997).

For the same reason, there was considerable difficulty associated with the meas-urement of the daphnids’ feeding rate during the 24 hr intervals, between tworenewals of the medium. The theoretical feeding rate was written for each timeinterval ]Tj , Tj+1]:

FRj+1 = EffFj+1nDmAje(µ−EffFj+1nD)(Tj+1−Tj )

nDmD(0)GTj+1(3)

with Eff, the efficiency of grazing, nD, the total number of daphnids per unit ofvolume (mL−1), mD, the daphnid mass (g w.w.) and mAj , the algae mass (g w.w.mL−1) per unit of volume; Fj+1 is the individual filtration rate (mL d−1 ind−1)calculated from Burns (1969):

Fj+1 = 4.99

(mD(0)eG(Tj+1)

1.45 10−4

) 2.802.67

Moreover, the radionuclide loss from algae and daphnid to the water com-partment resulted in a direct transfer of dissolved radionuclides to daphnids. Thisphenomenon, estimated from radionuclide measurements, was integrated into themodel by treating the radionuclide water concentrations with the same method asthat used for the algae. The change in daphnid radionuclide concentration due todirect transfer was calculated by replacing in all previous equations CAj by Wj ,(FR × ufi) by uwi and dfi by dwi . Wj represents the radionuclide concentration inwater (Bq mL−1), uwi (d−1) and dwi (d−1) are the uptake and depuration kineticparameters describing the radionuclide transfer from water.

On the hypothesis of the additivity of the transfer pathways, the observed radi-onuclide concentration in daphnids can be modelled as:

CD(t) = CDFood(t) + CDWater(t) =m∑

i=1

ufi XFoodi(t) +

m∑i=1

uwi XWateri (t) (4)

132 C. ADAM ET AL.

TABLE II

Growth rate (d−1) of Daphnia magna during radionuclide transfers from Cyclotella meneghianaand from Scenedesmus obliquus

110mAg 60Co 137Cs 54Mn

Cyclotella meneghiana

uptake phase a0.27 ± 0.024 0.25 ± 0.0043 0.24 ± 0.020 0.32 ± 0.015

(R2 = 0.93) (R2 = 0.83) (R2 = 0.95) (R2 = 0.99)

depuration phase 0.099 ± 0.0067 0.17 ± 0.021 0.12 ± 0.028 0.076 ± 0.018

(R2 = 0.99) (R2 = 0.95) (R2 = 0.75) (R2 = 0.70)

Scenedesmus obliquus

uptake phase 0.19 ± 0.017 0.046 ± 0.0037 0.069 ± 0.0053 0.28 ± 0.015

(R2 = 0.94) (R2 = 0.91) (R2 = 0.91) (R2 = 0.98)

depuration phase 0.035 ± 0.0046 0.060 ± 0.0093 0.052 ± 0.0031 0.018 ± 0.011

(R2 = 0.90) (R2 = 0.80) (R2 = 0.95) (R2 = 0.65)

a Estimated growth rate ± 1 A.S.E; values determined by non linear regression on the basis of theexponential equation mD(t) = mD (0) eGt .

Uptake and depuration kinetic parameters characterizing the radionuclide trans-fer from water (uwi and dwi) have been estimated during other specific exper-iments, whose aim was to study the direct transfer of 110mAg, 60Co, 137Cs and54Mn from water to the daphnids (Adam et al., 2001). Knowing the radionuclideconcentration accumulated by direct transfer, the estimation of the parameters ufiand dfi , characterizing the transfer from algae, was done by subtracting CDWater(t)

from the observed CD(t). All statistics were performed using the Systat softwarefor Windows, version 5 (Systat Institute, Evanston, Illinois Deltasoft).

3. Results and Discussion

3.1. WEIGHT CHANGE IN DAPHNIDS

During radionuclide transfer from C.meneghiana, the growth rate values (G) ofdaphnids varied from 0.076 to 0.32 d−1 (Table II), whereas they ranged from 0.018to 0.28 d−1 in the case of the ingestion of S.obliquus. During a similar study, wheredaphnids were fed 2 hr a day with a solution of meat extract and glucose, G valuesranged only from 0.027 to 0.089 d−1(Adam et al., 2001). This result indicates thatthe general physiological conditions of the daphnids were better when they werecontinuously fed with algae suspensions.

If the growth rates observed during the transfer via S.obliquus were lower thanwith C.meneghiana (F test, α = 0.05), it must be underlined that the diatom is


significantly bigger than S.obliquus, which means that, for an identical number ofalgal cells provided, the resulting weight in suspension was not the same. Thus,the C.meneghiana mass (w.w.) distributed daily to daphnids was of the order of3 × 10−4 g mL−1, whereas for S.obliquus, it corresponded roughly to 2 × 10−5 gmL−1. The digestibility of the cellular wall and the mean size of the algal cells haveusually been adopted as criteria to explain the growth rates of daphnid populations(Hutchinson, 1967; Gawler et al., 1986). Recently, the biochemical compositionof algal species was chosen as the best criteria to characterize the nutritive qualityof phytoplankton. Ahlgren et al. (1990) demonstrated that the algae content inPoly Unsaturated Fatty Acids (PUFA) significantly increased their nutritive qual-ity. Thus, the authors showed that the ingestion of Cryptomonadines, very rich inPUFA, led to higher growth rates of Daphnia longispina, Eubosmina longispinaand Chydorus sp. Likewise diatoms and flagellates are considered as good-qualityfoods because of their high EPA (EicosaPentaenoic Acid) content (Gulati andDemott, 1997).

3.2. CONTAMINATION VECTOR

During the radionuclide transfer via C.meneghiana, concentrations of dissolvedradionuclides in the water ranged from 2 to 8 Bq mL−1 (Figure 1). In the case ofthe transfer via S.obliquus, water radionuclide concentrations did not significantlydiffer from the background levels. This discrepancy can be explained by the mor-phology of the two algal species: C.meneghiana was characterized by a surface of450 µm2 and a volume of 700 µm3, instead of 77 µm2 and 70 µm3 for S.obliquus.Thus, for an equal number of cells, the exchange surface of C.meneghiana was 5to 6 times greater than for S.obliquus.

Algal radionuclide concentrations corresponding to a 24 hr time interval werecalculated according to Equation 2. For C.meneghiana, the depuration kinetic para-meter values were 0.111 ± 0.024, 0.116 ± 0.021, 1.81 ± 0.54 and 0.533 ± 0.091d−1, for 110mAg, 60Co, 137Cs and 54Mn respectively and the multiplication rate was0.102 ± 0.0179 d−1. For S.obliquus, the depuration kinetic parameter values were0.0619 ± 0.0412, 1.30 ± 0.685, 2.72 ± 1.56 and 0.0509 ± 0.0798 d−1, for 110mAg,60Co, 137Cs and 54Mn respectively, whereas the algae multiplication rate was 0.356± 0.0338 d−1. The algae radionuclide concentration taken into account in themodel was the average value on the time interval ]Tj , Tj+1]. The daily renewalof water and contaminated algae made it possible to preserve a quasi-stable algaeradionuclide concentration and thus to simulate a chronic input of contaminatedphytoplankton.

3.3. ACCUMULATION PHASE

Equation 3 was applied to the data obtained during the exposure phase to estimatethe mean feeding rate between two renewals of the medium, with a value of 250individuals per litre for nD and of 0.5 for Eff. mAj was set to 3 × 10−4 and 2 ×

134 C. ADAM ET AL.

Figure 1. Evolution of 110mAg, 60Co, 137Cs and 54Mn concentrations in contaminated medium.


10−5 g w.w. mL−1 for C.meneghiana and S.obliquus, respectively. No significantdifference could be evidenced for a given algae, between the FR corresponding tothe first and the last days of the exposure phase (F test, α = 0.05). For that reason,a constant feeding rate corresponding to a type of algae and a radionuclide wasused to model the radionuclide uptake. For Cyclotella, the feeding rates estimatedranged from 2.91 to 3.37 d−1 (Table III), while for Scenedesmus the values variedfrom 0.14 to 0.29 d−1 (Table IV).

For 60Co and 137Cs transfers from C.meneghiana, the contamination due towater was less than 5% of the total concentration of the daphnids (Figure 2 A),while for 54Mn, the contribution of the direct pathway reached about 25% of thetotal contamination. For 110mAg, it was not pertinent to estimate this contribu-tion. Radionuclide concentration in daphnids, calculated on the assumption of theircontamination from water, was much higher than the total 110mAg contaminationmeasured during the experiment. Thus, unlike 137Cs, 60Co and 54Mn, the chemicalform of 110mAg released by C.meneghiana was probably not bioavailable to daph-nids. Twiss and Campbell (1995) demonstrated that Gd, Zn and Cd, consumed asradioactive preys and regenerated into the dissolved phase, were less available forresorption by plankton. Likewise, several authors have showed the effect of compl-exation by phytoplankton exudates on the accumulation of heavy metals. Dissolvedorganic compounds such as polypeptides (Fogg, 1966), polysaccharides (Wangand Tisher, 1973), citrate or glycolate (Watt and Fogg, 1966 ; Hunstman, 1972;Tolbert et al., 1985) may form anionic or neutral stable metal complexes. Thesecompounds may be unavailable for algae since adsorption of metals is mainlygoverned by electrostatic attraction between algal cell walls, which are negativelycharged, and cationic species. For those reasons, it was assumed that the accumu-lated 110mAg in daphnids was only due to the trophic transfer of radionuclide. Theconcentration of 110mAg, 60Co and 54Mn in daphnids increased very fast within thefirst 4 hr, whereas for 137Cs, the increase lasted a few days.

Results from the F test (significance level 5%) prompted the use of two com-partments to describe the transfer of 110mAg, 60Co and 54Mn, whereas a singlecompartment was sufficient for 137Cs. For the first compartment (Table III), cor-responding to the fast process, the greatest uptake parameter was estimated in thecase of 60Co (4.33 ± 1.00), whereas for the second compartment it was estimatedfor 137Cs (0.0448 ± 0.0167).

The Trophic Transfer Factor TTF (w.w.), defined as the ratio between the con-centration of the radionuclide in the organism and in the algae, may be expressed,for a zero growth rate and without taking into account the physical decay of theradionuclide, as follows:

T T F(t) = FR

m∑i=1

ufi

dfi

(1 − e−dfi t

)(5)

Knowing the corresponding parameters, this equation can be used to estimatethe trophic transfer factor at steady state, as well as the time needed to reach 99% of

136 C. ADAM ET AL.

TAB

LE

III

Est

imat

edpa

ram

eter

sto

quan

tify

the

trop

hic

tran

sfer

of11

0mA

g,60

Co,

137 C

san

d54

Mn

from

Cyc

lote

lla

toth

eda

phni

ds(s

eeTa

ble

Ifo

run

its

and

mea

ning

)

Upt

ake

phas

eD

epur

atio

nph

ase

uf1

auf

2F

R(d

−1)

R2

df1(d

−1)

df2(d

−1)

E1

E2

R2

110m

Agb

1.22

±0.

0828

0.00

103

±0.

0010

12.

910.

7859

.4±1

7.9

1.75

±1.

440.

740.

260.

9860

Co

4.33

±1.

000.

0168

±0.

0159

3.37

0.68

146

±11

.51.

47±

0.13

50.

360.

640.

9913

7 Cs

0.04

48±

0.01

672.

940.

880.

266

±0.

0159

10.

7654

Mn

1.43

±0.

210

0.00

806

±0.

0079

33.

020.

6532

.4±

28.4

0.61

0±

0.18

40.

390.

610.

98

aPa

ram

eter

sar

eex

pres

sed

asm

ean

valu

e±

1A

.S.E

.b

Para

met

ers

are

calc

ulat

edon

the

assu

mpt

ion

ofno

upta

keof

110m

Ag

from

wat

er.


TAB

LE

IV

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138 C. ADAM ET AL.

Figure 2. Uptake and depuration of 110mAg, 60Co, 137Cs and 54Mn from Cyclotella to daphnids.


this maximum value (t99%s−s). The steady state values were of the order of 0.0616,0.138, 0.495 and 0.173 (w.w.), for 110mAg, 60Co, 137Cs and 54Mn respectively, withcorresponding t99%s−s values of 2.5 h, 2.9 d, 23 d and 6.3 d.

Regarding S.obliquus, since no significant radionuclide concentration was de-tectable in water, the transfer of the four radionuclides via this species was mod-elled as a simple trophic transfer. Radionuclide uptake was rapid in D.magna,since significant concentrations were detected at the first sampling time of 15min (Figure 3A). Daphnid concentrations continued to rise until the 2nd , 7th and5th days of experiment for 110mAg, 137Cs and 54Mn respectively, and then rapidlydecreased. For 60Co, the increase was more regular throughout the exposure phase,but concentration in daphnids started to level off by the 4th day.

During this exposure phase, the uptake of radionuclides was characterized by atwo compartment model for 110mAg and 60Co (F test significant at 5% level) and bya mono-compartmental model for 137Cs and 54Mn (Table IV). With regard to thefirst compartment, the highest uptake parameter was estimated for 60Co (uf1 = 9.71± 1.27) whereas for the second compartment the highest value was estimated for137Cs (uf2 = 0.205 ± 0.0114). Knowing the values of uptake parameters, feedingrates and depuration kinetic parameters, the trophic transfer factors were calculatedaccording to Equation 5. The steady state TTF values were 0.0140, 0.0373, 0.0808and 0.0717 (w.w.), for 110mAg, 60Co, 137Cs and 54Mn respectively, with t99%s−s

corresponding to values of 2.9, 6.4, 8.8 and 7.7 days.To our knowledge, radionuclide uptake by daphnids fed on contaminated di-

atoms has never been studied by freshwater ecologists. Some experiments havebeen carried out on the green algae. Thus, Gil Corrisco and Vaz Carreiro (1992)reported a TTF (wet weight) of 1.69 for D.magna contaminated by 134Cs labelledSelenastrum capricornutum cells. This value is 20 times higher than the TTF cal-culated in our experiment for 137Cs (0.0808). It must be underlined that duringthe experiment conducted by these authors over a period of 4 weeks, water andcontaminated algae were renewed only five times a week. Moreover, as the authorsdo not give any information on the observed feeding rate or on the biomass ofboth algae and daphnids, the steady-state TTF value cannot be accurately com-pared. Amiard-Triquet (1979) measured, after 6 weeks, a TTF of 0.0019 (w.w.) forD.magna fed with 60Co contaminated Chlorella cells. The experimental feedingrate was close to the values applied to our study and varied from 0.08 to 0.3 d−1,but the TTF was 20 times lower than the value of 0.0373 obtained in our study. Thisdiscrepancy may be explained, on the hypothesis of the homeostasis of total cobaltin the daphnids, by the lower specific activity of 60Co used by Amiard-Triquet (1× 106 instead of 5.6 × 106 Bq µg−1 corresponding to our experiments). Garnier-Laplace (1991) determined, after 30 days, a trophic transfer factor of 0.0702 (w.w.)for 110mAg accumulated by D.magna via Scenedesmus, which is 5 times greaterthan the value calculated in our study. This higher value may be attributed to thedifferent experimental protocol (lower density of daphnids, different chemical formand specific activity).

140 C. ADAM ET AL.

Figure 3. Uptake and depuration of 110mAg, 60Co, 137Cs and 54Mn from Scenedesmus to daphnids.


To explain these dissimilarities between the trophic transfer factor values, it isimportant to know the way the radionuclide associated with algal cells is takenup by daphnids. In vertebrates, the low pH of the stomach helps to solubilizemetals, which are then absorbed in the intestine. In contrast, the digestive tractof invertebrates generally lacks highly acidic extracellular conditions (Roesijadiand Robinson, 1994). Zooplankton that have short gut residence time (< 30 min)most likely employ a ‘liquid’ digestion strategy in which only the easily mobilizedfractions of ingested phytoplankton, i.e. cytoplasmic material, are absorbed (Fisherand Reinfelder, 1995). Thus, the distribution of radionuclide between the differentcomponents of algal cells is a major parameter influencing their trophic transfersthrough the aquatic food web.

The links between chemical and biological characteristics of the consideredradionuclides determine their localization in the phytoplanktonic cells. Nieboerand Richardson (1980) proposed a classification of the metal elements on the basisof the stability of the compounds they may form with biological ligands. Class Ametals, such as caesium, firstly bind to ligands with oxygen as the donor atom.Potassium, that is a chemical analog of caesium, belongs to the same class. It playsan important role in the control of the osmotic pressure in cells and is crucial inmembrane electrophysiology. Consequently, in the same way as this macronu-trient, 137Cs should easily be uptaken by phytoplankton. Sombré et al. (1987)who studied the contamination of Scenedesmus obliquus by 137Cs in a turbidostat,demonstrated that the intracellular fraction of this radionuclide represented 64% ofthe contamination.

Metals that exhibits Class B characteristics, such as silver, have the strongestaffinities for sulfur functional groups that are ubiquitous and crucial to the integrityof proteins or the functioning of various enzymes. Garnier and Baudin (1989), whocontaminated Scenedesmus obliquus with 110mAg in a batch culture, concluded thatthe accumulation was due to adsorption to the cell surface, rather than to activeuptake into the cells. In a similar study, neither mechanical disruption of the cells,low pH, nor enzymatic degradation (usually by digestion) was able to remove silverfrom the cell walls (Ratte, 1999). The binding of heavy metals, such as cadmiumand copper, has been described by a two-site Langmuir isotherm, where they bindfirst to the surface groups with highest affinity and subsequently to groups withlower affinity (Xue et al., 1988). Nevertheless, silver is not expected to migrateinto cells in general, but to be strongly bound on the exterior ligands and later tobe sloughed off or converted into Ag2S (Bell and Kramer, 1999).

Finally, most of the transition metals which act as essential cofactors for en-zymes and components of cytochrome systems used in electron transport are Bor-derline metals with relatively strong affinity for a large quantity of biological lig-ands. These metals may initially bind to cell surfaces, after which they can diffuseor be actively transported into the interior of cells. Nucho (1989) contaminatedScenedesmus obliquus with 60Co in a medium constituted of natural freshwater.She observed that the intracellular fraction of radionuclide reached about 50 %

142 C. ADAM ET AL.

of the total contamination. Reinfelder and Fisher (1991, 1994) showed that thecytoplasmic fraction of cobalt in marine phytoplankton (Isochrysis galbana andThalassiosira pseudonana) varied from 30 to 40%.

Our results tend to confirm that a clear relationship exists between the as-sumed intracellular fraction of radionuclides and their corresponding trophic trans-fer factor. Thus, for both freshwater phytoplankton species considered, 110mAgexhibits the lowest values of TTF. This radionuclide may only pass through thegut of the daphnids and then be released into fecal pellets with algal cell walls. Incontrast, 137Cs is characterized by the highest trophic transfer factor values, whichis in agreement with its great intracellular fraction in phytoplankton.

On the other hand, it is worth noting that the TTF calculated were generallyhigher for the transfer via the green algae than for the transfer via the diatom.A similar phenomenon was highlighted for 241Am in marine mussels (Fisher andTeyssié, 1986) and for silver, cadmium and cobalt in the hard clam Mercenariamercenaria (Fisher and Reinfelder, 1995). The role of food quality in metal accu-mulation by herbivorous invertebrates has never been evidenced, but it could beimportant. In the case of our study, the mean growth efficiency (dimensionless)calculated as the ratio between growth rate and feeding rate, is greater for allexperiments carried out with Scenedesmus (2700) than with Cyclotella (340). Thesiliceous diatom frustule may be less digestible than the green algae cell wall, andconsequently may have shorter residence time in the daphnid gut, which reducesthe possibility of radionuclide accumulation through the digestive tract.

3.4. ELIMINATION PHASE

During depuration phase, radionuclide concentration in daphnids reached the back-ground levels rapidly (Figure 2A and 3A). In the case of contamination via Cyclo-tella, the results of the F test (significance level 5%) showed that two compartmentswere statistically necessary to describe 110mAg, 60Co and 54Mn concentrations indaphnids during elimination phase. On the other hand, the use of a single compart-ment was statistically significant for 137Cs. The corresponding biological half-livesranged from 7 to 30 min for the first compartment and from 2 hr to 1.8 d for thesecond (Figure 2A). 110mAg was eliminated very quickly, since only 4% of the ini-tial concentration remained on the second day after the beginning of the depurationphase, while for 137Cs, 50% of the initial concentration was still measured. For thetransfer via Scenedesmus, the results of the F test (5%) prompted the use of twocompartments in the case of 110mAg and 60Co, and of a single compartment for137Cs and 54Mn. The biological half-lives (Tb) corresponding to the first compart-ment were 5 hr and 11 min, for 110mAg and 60Co respectively (Figure 3A). For thesecond, they ranged from 1.2 to 2.1 d. The remaining percentage observed on thesecond day of the depuration phase was only 3% in the case of 110mAg but close to37% for 137Cs.


The longer biological half-lives estimated after the transfers via Scenedesmus,particularly for the second compartment, tend to confirm the better accumulationof radionuclides from ingestion of this green algae compared to the diatom Cyc-lotella. Nevertheless, the elimination of radionuclides by herbivorous zooplanktonis generally considered as a rapid phenomenon, closely linked to the ejection oflabelled algae. Thus, Garnier-Laplace (1991) reported a Tb value of 22 min, for110mAgNO3 transfer from Scenedesmus to D.magna. After the transfer of 134Csfrom Selenastrum to D.magna, Gil Corrisco and Vaz Carreiro (1992) calculateda depuration kinetic parameter of 0.245 d−1, which corresponds to a biologicalhalf-life of 2.8 days.

The relative importance of water and food as radionuclide or metal sources forfreshwater organisms is still widely discussed. Thus, Vray et al. (1993) reporteda contribution of the trophic pathway of 70 and 15%, for the transfer of 106Ruto daphnids from Scenedesmus and Chlorella respectively. 60Co accumulation bydaphnids was much greater from water than from algae sources (Amiard-Triquet,1979). A similar result was reported for heavy metals by Hatakeyama and Yasuno(1981) and Watras et al. (1985), who found that crustaceans accumulated Cd andNi directly from water rather than from labelled algae. In contrast, Munger andHare (1997) studied the accumulation of Cd within a food chain constituted ofSelenastrum, Ceriodaphnia and Chaoborus and concluded that the food was themajor source of contamination. From in situ studies, Hare and Tessier (1996) haveshown that Cd concentration in Chaoborus could be predicted accurately fromCd concentrations in the water column. Nevertheless, this interrelation does notrule out the hypothesis that Cd concentrations in Chaoborus were linked to ac-cumulation from water at a lower level in the food web. Under that assumption,it is of major importance to take into account the influence of food concentrationin biomonitoring models, in order to increase their accuracy and to widen theirapplications in different environments or seasons. In this regard, the parameterscharacterizing the trophic transfers studied in the current paper were used to calib-rate the TRANSAQUA model, that aims to simulate the contamination of aquaticfood-chains (Garnier-Laplace et al., 1997). The results have shown that secondorder consumers, such as cyprinids, were 3 to 8 times more contaminated duringsummer, characterized by the development of green algae in the phytoplanktonicpopulation, than during autumn, corresponding to the preponderance of diatoms(Adam, 1997).

4. Conclusion

The transfer of 137Cs from labelled algae to the daphnids, was described by aTrophic Transfer Factor (wet weight basis) ranging from 0.495 for C. meneghianato 0.808 for S. obliquus. For 60Co and 54Mn, the steady state TTF value was muchlower and varied from 0.0373 to 0.173. Finally, the lowest values were observed in

144 C. ADAM ET AL.

the case of 110mAg (TTFS.obliquus = 0.0140 and TTFC.meneghiana = 0.0616). It mightbe hypothesized that the distribution of radionuclide between algal cell wall andcytoplasm influence the trophic transfer, since the radionuclides characterized by ahigher intracellular localisation are more accumulated than those that adsorb to thecell surface.

The fraction of radionuclide accumulated in the daphnids’ tissue strongly affectsits recycling in the food web and in the environment. Thus, 110mAg that showsalmost no absorption, is defecated after ingestion. Fecal pellets are released inthe water column or sink to the sediment, representing contamination sources forfiltering invertebrates of higher biological organisation level such as bivalves or forbenthic organisms. On the contrary, radionuclides that are transported into algaecytoplasm may be a contamination source for the upper levels of the pelagic foodchain.

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Documents

Bioaccumulation of 110mAg, 60Co, 137Cs and 54Mn by the Freshwater Crustacean Daphnia magna from Dietary sources (Scenedesmus obliquus and Cyclotella meneghiana)