Health Phys. Vol. 37 (September), pp. 315-321 Pergamon Press Ltd., 1979. Printed in the U.S.A.

STUDY OF ”’Cs ABSORPTION BY LEMNA MINOR P. G. BERGAMINI, G. PALMAS, F. PIANTELLI and M. SANI

Istituto di Fisica dell’universita’, Siena, Italy and P. BANDITELLI, M. PREVITERA and F. SOD1

C.A.M.E.N., San Piero a Grado, Pisa, Italy

(Received 9 March 1978; accepted 12 February 1979)

Abstract-The absorption of I3’Cs by the Lemma minor was measured in relation to the concentration of the radioisotope in water; the temperature, pH, and luminosity were kept under control. A method for the analysis of the results is illustrated. When applied to the present case, this method evidences the influence on the absorption of the potassiumcesium change, due to chemical affinity, of the natural growth of the organism’s colony and of the light effect. The concentration factors have been determined for the above mentioned processes. INTRODUCTION

IN REACTOR safety, knowledge of the aquatic environment behaviour assumes particular importance in relation to the discharge of the liquid wastes. There are several mechanisms through which diffusion of the radionuclides in the water occurs. It is, therefore, important to collect as much information as possible on such mechanisms, to assess the capacity of an aquatic environment to receive the radioactive nuclides without injury to the ecosystem. For this purpose we studied the behaviour of the Lemnu minor, an aquatic monocotyledon classified in the order Spathiflorae, which is very abundant in the aquatic environment around the nuclear centre of C.A.M.E.N. at San Piero a Grado (Pisa). In particular we studied the time-dependence of the absorption of the I3’Cs by Lemna minor cultivated in an environment where the concentration of the radioisotope, the pH, the potassium content, the temperature and the average daily natural light intensity had been controlled. The radioisotope I3’Cs was chosen because, together with the 90Srand the I3’I, it

presents the greatest potential hazard among fission products. EXPERIMENTAL DESIGN

The Lemna minor (Hi6la) is a floating plant which lives on the surface or just underneath relatively still, fresh water. The individual green structures of the Lemna minor produce new “fronds” (daughter) from two cavities on each side of the narrowest end of an older frond (mother), very close to the point where the root or roots branch off. Each daughter frond becomes a mother in its turn, usually when it is still joined to its own mother. A group of such fronds, old and new, can be called a “colony”. Each mother bears a considerable number of daughters during its life, the exact number being influenced by several environmental factors. The fruits can float and the seeds can germinate on the surface (occasionally they remain rooted to the genitrix root) or underneath it. Even in the second case the aerial part develops and, immediately after, the young plant floats.

315

316

STUDY OF I3’Cs ABSORPTION BY LEMNA MINOR

In the literature (Hi6la) we find that the Lemna minor tolerates an upper limit of salinity of 2.5% and a pH ranging between 3.5 and 8.5; its growth is influenced by light intensity and by environmental temperature (Hi6lb; Ik58). When an extremely low temperature or a persistent drought create adverse conditions for life, the Lemna minor is capable of surviving until growth is again possible. The peculiar aquatic environment, in which the Lemma minor lives, consists of a canal with slightly moving water. Chemical analysis of the water has shown a potassium content of 17 mg/l, a pH of 8.1 and a salinity of 220 mg/l. The average temperature and the average relative humidity in the spring are: t = 16”C, U = 72%. The experiment was performed during the spring. To simulate life conditions in a natural environment, the Lemna minor was cultivated into Perspex containers with a wide open surface, each one containing 401 of filtered water pumped from a canal. The containers were placed in a greenhouse next to the canal, where temperature and relative humidity were kept to the above mentioned values and light intensity was the same as in the external environment. The values of light intensity were measured by the local weather station. The characteristics of the growth and of the vital cycle of the Lemna minor, previously mentioned, make impossible the separation of organisms at the same stage of growth from the totality of the population. Therefore, it is necessary to study the global behaviour of the whole population and to take samples that contain organisms at different stages of growth. We thought it right to begin from a condition in which the presence of dead organisms could be considered negligible. Hence we put a sufficient amount of Lemna minor in a container in which we bubbled pressured oxygen. The bubble action caused the separation of the living organisms from the dead ones, which deposited themselves at the bottom. Because the mass of every single organism is extremely small, the number of organisms in every sampling must be sufficiently large to be representative of the average condition of

the population and to allow the weighting and measuring of the residues after treatment; at the same time, the sampling must be small compared to the total mass of the population, so as not to alter the ratio between the mass of the Lemna minor and that of the water, even after numerous samplings. From preliminary experiments we established that the optimum mass of the Lemna minor for every sample is 0.6g. To study the absorption of 137Csby the Lemna minor, we contaminated the water of three containers to the following concentrations: C1= 2.4

pCi/cm3

C2= 2.4 lo-* pCi/cm3 C3= 2.4 lo-’

pCi/cm3

These concentrations are such that the total content of 137Csis negligible compared to the potassium content. A fourth container with Lemna minor in uncontaminated water served as a reference cultivation. To make sure that all organisms of each batch started the absorption of 137Cssimultaneously, we proceeded as follows: a rigid and fine meshed nylon net was placed on the bottom of a container with uncontaminated water. On the open surface of the container a mass of 300 g of Lemna minor was uniformly distributed. By lifting the net, we extracted the Lemna minor without changing the superficial distribution. The net with the Lemna minor was subsequently immersed in the container with contaminated water. This procedure was repeated for the three containers with contaminated water. Then from each container we extracted the samples in spaced succession. Every sample was washed with 50cm3 of water taken from the canal and filtered. We used that water instead of distilled water so that we would not alter the I3’Cs concentration in the samples. At the same time, reference background samples were taken from the uncontaminated container. Each sample was dried at 110°C to a constant weight and then calcified at 400°C. Because of the hygroscopical character of the

P. G. BERGAMINI et al.

3 17

samples, all the weighings were carried out in a sample at 1lOOC) by the ratio of the total mass of Lemna minor and the mass of conhumidity-controlled room ( U = 30%). We measured the total beta radioactivity of taminated solution present in each container each ash residue with a low-background G.M. at the beginning of the experiment. B ( t ) , counter (window 0.9 mg/cm2) for the low then, expresses the concentration of 137Csin activity samples, and with a scintillation the Lemna minor per gram of water, and the counter for the others. The time variation of sum A ( t )+ B ( t ) is the fixed concentration of the 137Csconcentration in the contaminated 137Csin the water container at the beginning water was determined by total gamma of the experiment. In this first stage the effect of the growth of activity measurement on 2 cm3 samples. the Lemna minor is negligible. We can then ANALYSIS OF RESULTS AND CONCLUSIONS assume that the content of potassium in the A hypothesis for the analysis and an inter- water and in the Lemna minor remains conpretation of the results must be formulated. stant (stationary state) (Br57; G141; Ha60; First of all we assume that the absorption of Re61). This requires that the rate of the total the 137Csby the Lemna minor occurred by exchange of potassium be zero. With such a two main processes: (i) exchange of potas- hypothesis, the boundary conditions for the sium-cesium by chemical affinity, and (ii) concentration of 137Csin the water are accumulation due to the development of the colony. We also consider two stages. In the initial stage, the first process is preponderant until a relative equilibrium is reached; in the = -D(O), second stage, the first process becomes neglt=O igible compared to the second. We consider in the initial stage a closed where Q represents the fixed initial concensystem of two compartments: water and tration of 137Csdue to the contamination and Lemna minor. D ( t ) the slope of the tangent to the curve of We call A ( t ) the concentration of 137Csin the Concentration of 137Csin the water as a the various water samples and B ( t ) the function of time. product of the concentration of 137Csin the Figure 1 shows such a curve for the conLemna minor samples (in pCi per gram of centration in one of the containers (the one I

[y]

!

2 a r = 260

200 220

I I

I

I

FIG. 1. Variation with time of the concentration of 13’Cs in fresh water for an initial concentration of 2.4 x pCi/cm3. The closed circles represent the experimental data. The solid curves were calculated from equations (2) and (9) using: Q = 246.71 pCi/cm3; d-*; K2 = 17.68 K l = 79.85 d - ’ ; D(0)= 9.46 pCi/(dg); Bp = 8.58 pCi/g; to = 9.05 d. The non-linear correlation coefficient r was 0.99.

STUDY OF 137CsABSORPTION BY LEMNA MINOR

318

with concentration C,). The scheme of the system is of the type

The analytical scheme is

ly

=

representation

+K,A(t) - K , B ( t ) ,

of

the

(1)

where K1and K , are the respective constants of exchange. By solving the system (1) we have A(t) = Q - B,( 1 - e-(Ki+K2)' I (2)

the same sample dried at 110°C. Such a ratio is shown as a function of time in Fig. 2. The agreement between the calculations and the experimental data shows that the ratio of the weights expresses correctly the population growth of equation (5). Values for the parameter s can be easily inferred from the experimental data. It must also be remembered that the variation of the population of the Lemna minor is determined not only by the positive factor of growth, but also by the negative factor of death at the end of the vital cycle. Indicating with s1 the constant of death and with x , ( t ) the entity of the present population, we have: d x l = sxl(t) d t - s l x l ( t ) d t ; 047

(6)

-

043 039

In the second stage, the growth of the Lemna minor cannot be overlooked. It is necessary to remember that Lemna minor and its habitat constitute a natural system, which, lacking external perturbations, tends towards a situation of equilibrium in which the entity of the population of the Lemna minor remains constant. Indicating with X , such an entity at equilibrium, with x(t) the entity at a generic instant t and with to the initial instant for this s'econd stage, we have

where s is the growth constant of the population. Let x ( t o ) = p . By integrating, we have

Considering that with the aging of the colony the ratio between the more solid roots and the fronds increases, we took, as an index of the aging itself, the ratio between the weight of the calcified sample at 400°C and that of

~

043 -

039 -

042 -

038 -

Days

FIG.2. Ratio between the weight of the calcified sample at 400°C and that of the same sample dried at 110°C. The closed circles represent the experimental data. The solid curves were calculated from equation ( 5 ) using: CI: X , =0.4299; p = 0.2352; s = 40.36 x 10-3d-1; for C2: X , = 0.4522; p = 0.2460; s = 42.08 X 10-3d-'; for C3: X , = 0.5286; p = 0.2302; s = 31.37 x lO-'d-'.

P. G. BERGAMINI et al.

taking into consideration equation ( 5 ) and its boundary conditions, and integrating, we obtain

319

B ( t )= s B m - B, fe-s(t-t,,)

-

-s,(t-rd]

SI - s

+ B, e-s,('-'d

(8)

where B, and B, are the concentrations of I3'Cs corresponding to p and X,. The In the second stage, in which the first parameters of equation (8) are determined by process is negligible with respect to the fitting the experimental points with the second, we assume that the concentration of minimization method of x2 (Be77). Figure 3 137Cs in the Lemna minor is directly related to shows the values of the 137Csconcentration in what we call entity of population and as such the Lemna minor, measured in pCilg of obeys an equation analogous to equation (7). organism dried at 110°C. An analysis of the We can write for the concentration of 137Cs curves in Fig. 3 shows clearly the presence of the two processes previously considered. The in the Lemna minor and in the water:

* *

*

9

1.8 -

*

.

e -

16

*

t

1.6 -

c

* ( € & * €

-

1.4

L 1.21

0

16

c

t

1.7

0

3

9

15

21

27

33

39

' ' 1'2 ' 1'8

6

I t

1

1

24

1

'

30

1

* ** * : * **

* *

*

1

36

*

'

1

42

'

1

48

* * *

*

45

Days

. t

... .

FIG.3. Variation of the concentration of I3'Cs in the Lemna minor. The closed circles represent the t experimental data. The solid curves were calculated from equations (3) and (8) using: K I = 79.85 x 10-4d-1; K2 = 17.68 x 10-*d-'; to = 9.OSd and for CI: B, = 10.61 pCi/g; B, = 9.46 pCi/g; B, = 8.58 pCi/g; s = 4 . 3 6 x d-'; s1 = 44.98 Days x 10-3d-1; for C2: B , = 763.27 pCi/g; B, = 723.10 pCi/g; B, = 617.23 pCi/g; s = 42.08 FIG. 4. Ratios p(t)/p(O) and B(t)/B(O) for the x 10-3d-'; sI= 47.08 x for C3: B , = second stage. Closed circles: concentration CI; 6238.65 pCi/g; B, = 5703.78 pCi/g; B, = 5044.19 asterisks: concentration C2; stars: concentration pCi/g; s = 31.37 x 10-3d-'; s, = 36.38 x 10-3d-'. c3.

320

STUDY OF I3’Cs ABSORPTION BY LEMNA MINOR

process related to the stationary stage, equations (2) and (3), has occurred in the first nine days. Starting from the ninth day, the second process, relative to the influence of growth and death, equation (8), has overcome the former, making it negligible. Figures 1 and 3 show a fine agreement between the experimental data and the theoretical values obtained from equations ( 2 ) , (3), (8) and (91, the parameters for which have been obtained by utilizing the curves of Figs. 2 and 3. In Fig. 4 the ratio B ( t ) / B p= p ( t ) is plotted as an index of the increase of 137Csconcentration in the Lemna minor, relative to the second stage, and the ratio p ( t ) / p ( O ) is plotted as index of the population growth.

cp=

mental values are situated more to the right. This can be explained by the fact that the container was placed in a location with a slightly different light exposition. We have calculated three values for the concentration factor @ of the ‘37Cs in the Lemna minor because there are three different processes that influence the absorption of the radioisotope. The first value is calculated in correspondence with the asymptote of the curve in equation (3) (Ba75). The second value has been calculated at the maximum of the curve in equation (8). The third value is calculated at the maximum of the perturbations caused by the influence of the light. Table 1 shows these values obtained from the expression

concentration in pCi/(g of L e m m a minor dried at 110°C) initial concentration in pCi/(cm3 of environmental solution)’

For the concentration C3 the ratio p ( t ) / p ( O ) shows a gradient higher than the one relative to the other two concentrations, revealing a bigger growth factor and consequently a smaller constant s. Correspondingly, the ratio B ( t ) / B pfor the concentration C3 presents a slower decrease in accordance with a more intensive development of the colony, while the shape of the curves B ( t ) / B pfor the concentrations C , and C2 agrees with the curves of p ( t ) / p ( O ) for the same concentrations. This agreement confirms the choice of the ratio p(t)/p(O) as an index for colony development. In our analysis we do not account for the additional effect from sunlight. The meteorological data have indicated a greater general luminosity of the environment from the 20th day onward, with an interval of overcast sky from the 31st to the 35th day. In this period of higher luminosity, the experimental data of Fig. 3 have, undoubtedly, higher values than the corresponding ones in the calculated curves, while for the concentration of 137Csin the water (Fig. l), the experimental values are smaller than the calculated ones. Such concomitance has induced us to associate the increase of the absorption intensity with the effect of light. The curve in Fig. 3 that is relative to the concentration C3 shows that higher experi-

Table I . The concentration factor of the I3’Cs for the three processes influencing the absorption and for the concentrations C,, C, and C,

Concentration

1st Process

2nd Process

3rd Process

Cl

86.26 84.20 83.68

92.44 123.44 122.82

136.75 147.71 183.71

-

c2 C,

It is obvious that, by definition, the concentration factor depends upon the conditions of the experiment; therefore, the values found in the literature (Ti63; W166; P066) cannot, in general, be compared either among themselves or with those found here, although each one maintains its own validity. However, our mathematical treatment has general validity because the potassiumcesium change and the natural growth of the organism colony are always present, whatever the experimental conditions may be. REFERENCES

Ba75 Banditelli P., Bergamini P. G., Palmas G., Previtera M., Sani M. and Sodi F., 1975, “Determinazione dei Fattori di Concentrazione Negli Ecosistemi”, CAMEN document 1079. Be77 Bergamini P. G., Palmas G., Piantelli F., Rigato M. and Sani M., 1977, “La Valutazione Statistica pei Fotopicchi Gamma in Esperimenti con Traccianti Radioattivi”, SOC. Italiana di Biofisica Pura ed Applicata, General Assembly, Proc. 3rd Session, Siena.

P. G. BERGAMINI et al. Br57 Bray G. N. and White K., 1957, Kinetics and Thermodynamics in Biochemistry (London: Churchill) . GI41 Glasstone S., Laidler K. J. and Eyring H., 1941, The Theory of Rate Processes (New York: McGraw-Hill). Ha60 Hart H. E., 1%0, “The Kinetics of General n-Compartment System”, Bull. Math. Biophys. 22, 41. Hi6la Hilmann W. S., 1%1, “The Lemnacee or Duckweeds: A Review”, Botanical. Rev. 27, 221. Hi6lb Hilmann W. S., 1961, Light and Life (Baltimore: John Hopkins University Press). Ik58 Ikusima I. and Kira T., 1958, “Effects of Light Intensity and Concentration of Culture

321

Solution on the Multiplication of Lemna Minor”, Physiol. Ecol. 8, 50. Po66 Polikarpov G. G., 1%6, Radioecology of Aquatic Organisms (Amsterdam: North Holland). Re61 Rescigno A. and Segre G., 1961, La Cinetica dei Farmaci e dei Traccianti Radioattivi (Torino: Boringhieri). Ti63 Timofeyeva Resovskaya Y. A., 1%3, “Distribution of Radioisotopes in the Main Components of Fresh Water Bodies”, USAEC Rep. TID-63. Wl66 Wlodek S., 1%6, “The Behaviour of Cesium-137 in Fresh Water Reservoir”, Radioecological Concentration Processes (Edited by B. Aberg) (Oxford: Pergamon Press).