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pH of water, time of contamination, presence or absence of other plants or animals, stagnancy or move of water, water temperature belong among the most important.

The concentration factor (CF) is the quantity, which characterizes the amount of a radionuclide accumulation by water plant under steady state conditions. It is defined as a quotient of the radionuclide concentration in the organism or tissue and of the radionuclide concentration in the ambient water. CF gives the evidence neither about the uptake and elimination kinetics of radionuclide nor the character of uptake processes. Nevertheless, CF is a useful indicator for the biota’s suitability for biomonitoring or bioindication of the surface water radioactive pollution. Mathematical models of the radionuclides transfer from water to plants are developed to provide more precise assessment of water pollution [10, 11, 12].

Vltava River : Cs-137 distribution in hydrobionts

riverbank plants

water mosses

algae

other submerged plants

bivalve - soft tissue bivalve - shells

Fig. 1. Distribution of the 137Cs activity concentration in hydrobionts from the Vltava River (Czech Rep.) before the running of NPP.

Hydrophytes are also used for the assessment of the radionuclides activity in the hydrosphere before and after starting-up the nuclear power plants operation. In the Czech and Slovak countries the NPP Bohunice, Dukovany and Temelin were involved in [13, 14, 15]. The algae and the mosses have proved to be suitable for this biomonitoring.

For example, in the years 1996-2000, before the start of the PWR type nuclear power plant in Temelin (South Bohemia, Czech Rep.), the monitoring of the Vltava River, where the liquid radioactive effluents will be discharged, was accomplished („point zero“ assessment). The biomonitoring of the hydrosphere was made by means of water plants and bivalves. The representation of the 137Cs activity concentration in hydrobionts from the Vltava River is shown in Figure 1. It can be seen that 137Cs is concentrate most of all in micro-algae mixture and in water mosses while least of all in bivalve shells. The results from the year 2001 - the first year after the running of the NPP Temelín - are similar to those of the point zero assessment.

Considering a more precise interpretation of measured values of radionuclides activity concentrations in water plants and for deeper understanding

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of radionuclides uptake mechanisms by individual plant species as well it is suitable to study the chosen phenomena at laboratory conditions.

In this contribution, some results of the laboratory experiments of the radionuclides uptake and elimination by the freshwater and saltwater algae are presented. The radionuclides, 60Co and 137Cs, that are relatively important from the point of view of the nuclear industry environmental impact are chosen.

2. Materials and methods

Algae and cultivation

The experiments were pursued in batch cultures with the freshwater diatom Navicula (KÜTZ.) GRUN, strain KOVÁÈIK 1978/9, the freshwater chlorococal algae Chlorella kessleri FOTT & NOVÁK. strain LARG/1 and on the marine diatom Phaeodactylum tricornutum BOHLIN. Strain COUGH 632. The algae were supplied by the Culture Collection of Algal Laboratory of the Institute of Botany of Trebon (Czech Republic).

The saltwater diatom Phaeodactylum tricornutum is one of the most used algal species in the marine biotest because of its easy cultivation [16]. Navicula atomus is the representative of freshwater diatomae. Chlorella kessleri is the representative of common cocal algae and it is often used in the biotests.

These algae species have a different shape but comparable dimensions. This is important when comparing the capability of the algae to adsorb radionuclides on their surface for individual species.

The freshwater and saltwater algae were cultivated in solution (medium) Zehnder [17] and in the artificial seawater after ISO 10253 [18]. The experiments on the algae were then pursued in volumes of 1 litre with solutions of the same composition, which was used for algae cultivation. The solutions preparation is given in Tables 1 (freshwater) and 2 (saltwater).

Table 1. Freshwater solution preparation

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Table 2. Artificial seawater preparation

For each experimental culture the equations of the optical density conversion to dry weight of the algae biomass (g.mL-1) and to the number of cells (mL-1) were set.

The algal suspension was put into polyethylene bottles with supply of an air and CO2 mixture. The flow rate of CO2 was regulated to maintaining the pH value of suspension at a constant value. The mean pH values of the individual batch cultures during the experiment are given in the table 3.

The radionuclides uptake by algae

The experimental suspensions were contaminated in one-shot way with the radionuclides, 60Co and 137Cs, in the form of chlorides. After the contamination, three parallel samples of the suspension were taken from each experimental bottle. The activities of the unfiltered suspension, the filtered suspension and filters with algae were measured. Polycarbonate membrane filters Sartorius of the pore diameter of 0,45 µm were used for the filtration, which was made through two filters placed one on the other. The filter underneath served for the correction of radionuclides self-adsorption on the filter. After the filtration the individual filters were mineralised directly in the measuring vessel.

Table 3. Mean pH values of the individual batch cultures (experiment duration period in days is indicated in parenthesis).

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At every sampling, the values of pH and the suspension optical density were measured. The pH values were measured by means of pH meter WTW 330i with gel electrolyte electrode Sentix 41-3. The optical density at 750 nm was measured by Titertek Uniscan II for samples in the plate of well volume of 3 mL. During the experiment the samples for bacteriological test were taken and fixed.

The radionuclides elimination from algae

About ten days later when radionuclides were sorbed, the algae were gently filtered on the membrane filter and then washed from the filter into the inactive medium. In this way the medium was periodically changed for a fresh one during the elimination experiment.

At given time intervals, three parallel samples of suspension were taken and filtered for the algae activity measurement. It was found that the self-adsorption on the filters was negligible in that case.

The pH value and the optical density were measured at every sampling.

3. Results and discussion

The radionuclides uptake by algae

The time course of the radionuclides uptake by individual algal species is shown on Figures 2, 4 and 5. The corresponding decrease of radioactivity in the filtered medium is also depicted on these figures. The separation of the volume activity between the medium and the algae relatively to the initial volume activity in the batches is represented on these figures. The part of activity adsorbed on the surface of

experimental vessels was deduced from the time course of suspension (medium with algae) activity. The estimated values are 5-10% for 137Cs and 10 -50% for 60Co (the

upper limit is valid for the batch with Chlorella kessleri).

The values of concentration factor, CF, were estimated in steady state, about 7 days after the time of contamination of the algal suspension in batch. They are shown in Table 4. The concentration factor is calculated according to the following formula:

Table 4. The estimation of the concentration factors CF for the radionuclides, 60Co and 137Cs in three algal species.

The volume activity in algae relatively to the unitary optical density of the algal suspension is shown in figure 6. It can be seen that the uptake varies significantly both for the type of radionuclide and for the algal species. For the freshwater diatom Navicula atomus the uptake of cobalt is substantially higher than the uptake of caesium. In the case of cocal algae Chlorella kessleri the difference in the uptake of these two radionuclides is much less evident. For the saltwater diatom

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located in the ISO 10253 medium, the difference of the uptake for two radionuclides were not observed and the quantity of radionuclides concentrated by this algae species was very small.

The cobalt uptake of Navicula atomus is much higher than the one of Chlorella kessleri. The uptake of Phaeodactylum tricornutum in ISO 10253 medium is one to two order and two to three orders of magnitude less for caesium and cobalt, respectively, than the uptake of two freshwater algal species.

The radionuclides elimination from algae

The elimination kinetics of radionuclides, 60Co and 137Cs, is shown in Figure 3 for the algal species Navicula atomus and Chlorella kessleri. Because of the very weak radionuclide uptake by Phaeodactylum tricornutum, the elimination kinetics was not measured in frame of this experiment. For Chlorella kessleri, the activity of both radionuclides decreased in one hour to 30% of the initial value and than remained

practically constant. At Navicula atomus, the decrease is slower than at the Chlorella kessleri. The activity decreased to 50% in about 1 and 4 days for 137Cs and 60Co, resp.

After about 12 days the initial activity of Navicula atomus fell to 15% and 40% for 137Cs and 60Co, respectively.

Conversion curves

The conversion curves were calculated for each culture. The summary of curves is shown in Table 5. The regression coefficients R2 > 0.99.

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Actual values -- o -- Cs-137 , -- Ŷ -- Co-60

Fig. 5. a) The time course of accumulation of the radionuclides, 137Cs and 60Co, in algae Phaeodactylum tricornutum. b) The relative decrease of activity in the filtered medium. A0 is the initial radionuclide volume activity in the batch.

Fig. 6. The uptake of radionuclides, Co-60 (--Ŷ--) and Cs-137 ( o ), by two algae species. OD = optical density of algal suspension measured at 750 nm.

4. Acknowledgement

This project ME 419 is supported by the Ministry of Education, Youth and Sports of Czech Republic, and K 60 051 14 of Acad. Sci. Czech Republic. The authors thank Mr. K. Svadlenka for cooperation with the data processing.

5. References

1.TIMOFEYEVA-RESOVSKAYA,E.A. (1963) Radioisotopes distribution in the main components of freshwater reservoirs. Trudy Uralskogo filiala AN SSSR, vyp. 30., 77 pp. (in Russian)

2.VYMAZAL, J. (1990a): Uptake of lead, chromium, cadmium and cobalt by Cladophora glomerata. – Bull.Environ. Contam.Toxicol. 44: 468-472.

3.ŽÁKOVÁ, Z. & KOÈKOVÁ, E. (1998): Biomonitoring and assessment of heavy metal contamination of streams and reservoirs in the Dyje/Thaya river basin, Czech Republic – Water Sci. Technol. 39: 225-232.

4.FOULQUIER, L. & BAUDIN-JAULENT, Y. (1992) Impact Radioécologique de l´Accident de Tchernobyl sur les Ecosystèmes Aquatiques. CEC - Radiation protection 58, UIR, No. 90-ET-014.

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5.VANDERPLOEG, H.A. et al. (1975) Bioaccumulation Factors for Radionuclides in Freshwater Biota. Oak Ridge Nat. Lab., Environ. Sci. Div. Public. No. 783, ORNL-5002, 222 pp.

6.BARINOV, G.B. (1970) Dynamic characteristics of the radioecological concentration process. In: Marine radioecology. Kiev, Naukova Dumka, p. 69. (In Russian)

7.GLOYNA, E.F. et al. (1966) Transport of radionuclides in model water. In: Disposal of Radioactive Water into Seas, Oceans and Surface Waters, Proc. Symp. Vienna, IAEA, p. 11-32.

8.SUTCLIFFE, J.F. (1964) Mineral Salts Absorption in Plants. (ruský pøeklad) Moskva, Mir, 221 pp.

9.THORNLEY, J.H.M. (1976) Mathematical Models in Plant Physiology. London, Academic Press, 318 pp.

10.KULIKOV, N.V. & TCHEBOTINA, M.Ya. Radioecology of Freshwater Biosystems. AN SSSR, Ural´s Dept., Sverdlovsk 1988 (in Russian)

11.RICE, D.L. (1984) A simple mass transport model for metal uptake by marine macroalgae growing at different rates. J. Exper. Marine Biol. Ecol. 82: 175-182.

12.SVADLENKOVA, M. -DVORAK, Z. -SLAVIK, O. (1989) A mathematical model for 137Cs uptake and release by filamentous algae. Internat. Revue der Gesamten Hydrobiologie, 74 (4): 461-469.

13.SLÁVIK, O. - OBDRŽÁLEK, M. - JANEÈKA, S. (1983) Occurrence and behaviour of the radionuclides in hydrosphere of the NPP Bohunice environment (in Slovak). Research Report VÚJE Jaslovské Bohunice 1983. 42 pp. (in Slovak)

14.STANÌK, Z. (1988) Impact of the Nuclear Power Plant Dukovany on the River Jihlava. Vater Menagement 8: 214-217. (in Czech)

15.ŠVADLENKOVÁ, M. & HANSLIK, E.: Biomonitoring of the Vltava River Radioactivity. In: Proceedings of the int. conf. ECORAD 2001: Radioécologie - ecotoxicologie des milieux continentaux et estuariens. France, Aix-en-Provence, 3-7 Sept 2001 (in press)

16.LUKAVSKÝ, J. & SIMMER, J. (2001): Microprocedure for the standard marine algal bioassay (ISO 10253). Algological Studies 101: 137-147.

17.STAUB, R.(1961): Untersuchungen an der Blaualge Oscillatoria rubescens DC. - Schweiz. Z. Hydrol. 23: 83-198.

18.ISO 10253 (1995): Water quality – Marine algal growth inhibition test with Skeletonema costatum and

Phaeodactylum tricornutum. 8 pp.

PRINCIPAL COMPONENT ANALYSIS OF CHRONIC INFLUENCE OF LOWDOSES OF IONIZING RADIATION AND CADMIUM ON ORGANISMS

V. VOITSITSKY, S. HIZHNYAK, O. KYSIL N. KUCHERENKO

National Taras Shevchenko University of Kyiv, Biological Faculty, 64 Volodymyrska street, 01033 Kyiv, UKRAINE

E-mail: vmv@biocc.univ.kiev.ua

A. KURASHOV, National Taras Shevchenko University of Kyiv, Radiophysical Faculty, 64 Volodymyrska street, 01033 Kyiv, UKRAINE E-mail: vnk@mail.univ.kiev.ua

L. BEZDROBNAYA, Institute for Nuclear Research, National Academy of Sciences of Ukraine, 47 Nauki Ave., 03680 Kyiv, UKRAINE

1. Introduction

Exceptional variety of different physical and chemical actions on biota outlines the topical environmental problems resulting from combined biological influence of external agents on organism [1, 2]. The reaction of a biological object on ionised radiation and chemical agents over the area of low dose effects (LDE) becomes highly versatile and ambiguous. The study of organism status by conventional sequential statistical analysis of individual indices as compared with their control levels by variation model does not provide a way of estimating its manifold reactions. Adequate valuation of organism reaction on such complex influence may be done only by manifold correlation of various biological indices. The most perfect method to solve a problem is provided by the principal component analysis (PCA) [3, 4]. With this method, it becomes possible:

ξ to evaluate latent but objective regularity in the control mechanism of the processes in the biological object under consideration;

ξ to obtain essential information by process description with minimum number (much less than the number of initial indices) of resulting appreciable variables;

ξ to reveal and study statistical dependence of biological status on external influences from whence one can draw a valid conclusion about the effectiveness of agent action and predict the process evolution.

The objective of this paper consists in the investigation of LDE under combined chronic action of low-dose ionizing radiation and cadmium ions on the basis of morphologic and biochemical indices of various rats’ organs by PCA method.

2. Materials and methods

The white underbred rats (180-200g) were divided into 10 groups (6-8 animals in each group) according to the experimental conditions: control (1),

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© 2005 Springer. Printed in the Netherlands.

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received 0,01mg/l of CdCl2 solution during 42 days (2), 84 days (5), 145 days (8); subjected to ϑ-irradiation during 42 days (3), 84 days (6), 145 days (9); subjected to combined influence of irradiation and CdCl2 during 42 days (4), 84 days (7), 145 days (10). Animals were chronically irradiated by ϑ-quanta from 60Co (dose rate 0,72 cGy/per twenty-four hours) at installation "ETALON" (Scientific centre of INP, NAS of Ukraine) and decapitated on the 42, 84 and 145 days after gaining the total absorbed doze of 0,3; 0,6 and 1,0 Gy, respectively. The objects of the research were blood, liver and intestinal parts (duodenum and jejunum) according to their high sensitivity to cadmium effect along with the differences in their radiosensitivity. Cytophotometric researches were carried out by light microscopy [5]. Enzyme activities were determined by routine procedures [6, 7]. Lipid peroxidation products were detected as described in [8]. The lipids content was assayed by enzyme methods according to [9]. The measured variables are grouped in Table 1. Hereinafter only the parameter 43 numbers are used for short.

3. Main features of PCA model in LDE estimation

The general condition of a biological object is qualitatively characterized by a great set of indices, which account for the dose-dependent action of external agents on the organism. The conventional approach to the problem based on the sequential analysis of the finding (a correlation between every individual index and its value in control group) does not ensure an unambiguous interpretation of experimental results. The adequate estimation of the combined action of small dose of radiation and xenobiotics must be based on radically different integral vital signs, which correctly determine the effect of the action. It should be mentioned that the procedures presently used (see, for example, [2, 10]) cannot be tolerated just for this reason. Consequently, the problem of the mathematical analysis of LDE under the combined action of several external agents is the development of proper multi-factor and informative estimation, which is valid far from lethal doses. We propose for this purpose the multi-factor PCAbased model, which was effectively used when studying the acute damaging action of ionizing radiation and cadmium ions on the organism of rats [11].

The PCA model is used in the analysis of a great many interdependent characteristics of the objects under consideration with regard to their structure and associations [3]. According to this model, the set of observed variables

x x1 ,x2 ,...,xN ` is represented as a linear combination of some hypothetic

variables, principal components, which most closely match their correlation. The ultimate aim of the analysis consists in the determination of such transformed data

a a1 ,a2 ,...,aM `, M N , which maximize statistical compatibility of observed correlation with number M of used variables ai as few as possible.