EXPERIMENTAL STUDY REGARDING THE MAGNETIC LIQUID INFLUENCE ON THE SUNFLOWER SEEDLINGS

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1 SCIENTIFIC ANNALS OF ALEXANDRU IOAN CUZA DIN IAŞI UNIVERSITY Tomul II, s. Biomaterials in Biophysics, Medical Physics and Ecology 29 EXPERIMENTAL STUDY REGARDING THE MAGNETIC LIQUID INFLUENCE ON THE SUNFLOWER SEEDLINGS E. L. Foca-Nici 1, M. Ursache 1, G. Capraru 2, D. E. Creanga 1 KEYWORDS: magnetic nanoparticles, photosynthetic pigments, chromosomal aberrations In this study the authors present their results regarding the influence of Zn ferrite colloidal suspensions on plant growth during their very early ontogenetic stages. The magnetic liquid based on Zn ferrite was obtained by stabilization with sodium oleate of magnetic nanoparticles prepared by Massart s modified method. Aliquots of diluted magnetic liquid were supplied to sunflower seeds with controlled genetic origin that were let to germinate and growth on porous paper support in Petri dishes. The young plant response to laboratory simulated pollution with magnetic nanoparticles was investigated by measuring the concentrations of chlorophyll A, chlorophyll B and carotene like pigments in samples harvested from their green tissue. Nikon microscopy device was used to identify and count the cells frozen during various mitosis phases in order to calculate the mitotic index and the chromosomal aberration percentage. 1. INTRODUCTION The development of nanotechnologies has introduced important amounts of manufactured nanoparticles into the environment, including those in the ambient air and water. Very few studies have been conducted to assess the toxicity of nanomaterials to ecological terrestrial species, particularly plants. In order to develop a comprehensive toxicity profile for manufactured nanoparticles, their phytotoxicity the ability to cause injury to plants has to be investigated. Previous researches showed that plant metabolism could be influenced by magnetic nanoparticles addition in the culture medium, since those seem to be able to induce phenotypic and genotypic effects resulting in possible stimulation of the productivity. Magnetic fluids may be taken as useful tools of manipulating uniform dispersions of magnetic nanoparticles for biological applications. In the last time the interest for the study of the biological effects induced by the magnetic nanoparticles administrated in culture medium of microorganisms [4] and vegetal organisms [5] as well as upon animals [3] has increased. 1 Al.I.Cuza University, Faculty of Physics, Carol I Blvd., No.11, 756, Iasi 2 Institute of Biological Research, Iasi

2 2 E.L.Foca-Nici, M. Ursache, G.Capraru, D.E.Creanga Some studies are dedicated to the influence of ferrofluids on the photosynthesis [1] revealing the stimulatory effect of the magnetic nanoparticles on the chlorophylls content, which has induced some stimulatory effects on the plant growth. The iron oxides from ferrofluid composition can be a source of iron for the plant development on a ferrofluid supplemented medium. The experimental study carried out in our laboratory was intended as a simulator phenomenological model of plant pollution with magnetic nanoparticles delivered from natural and artificial sources at all the levels of Earth biosphere. ZnO nanoparticles are being used in personal care products, and coating and paints, on account of their UV absorption and transparency to visible light. Thus, their potential harm to human health has attracted attention. 2. MATERIALS AND METHODS 2.1. Magnetic liquid preparation The basic procedure for magnetic liquid preparation was consistent with the coprecipitation of Zn and ferrous oxides in alkali medium (NaOH 25%) and further stabilization in deionized water in the form of aqueous magnetic fluid was carried out as shown in [2] Biological material Biological samples were constituted by equal number of sunflower seeds, harvested from an experimental population with ensured uniform genophond. The seeds were let to germinate in controlled environmental conditions into a laboratory room, on porous paper support impregnate with the same volume of magnetic fluid suspension in five different volume fractions (2; 4; 6; 8; and 1 microliter magnetic fluid per liter of deionized water (microl/l)) until their roots reached about maximum 3 mm length. The following treatment was applied: the little embryonic roots were kept, for three hours, in distilled water, at room temperature; seeds were further washed for two hours in distilled water, at room temperature; the fixation was carried out, for 1 2 hours, in absolute ethylic alcohol / glacial acetic acid mixture (3:1), at room temperature; the tissue samples were transferred in ethylic alcohol 7%, in refrigerator; the hydrolyze was accomplished in 5% HCl for 2 minutes; Carr solution was used for selective chromosomes staining. The microscope slides were prepared in accordance with squash method in order to get single cell layer for optimal visualization and counting. Photographs were taken using Nikon type microscope, with 1x objective, with immersion, using a camera Nikon Eclipse 6. Five slides were analyzed for each magnetic liquid concentration added to the sunflower seeds. On each slide 1 microscopic areas were analyzed to identify all dividing cells in all mitosis phases: prophase, metaphase, anaphase and telophase.

3 EXPERIMENTAL STUDY REGARDING THE MAGNETIC LIQUID Spectral assays Shimadzu spectrophotometer UV type 17 Pharmaspec provided with quartz 1cm cells was used for chlorophylls and carotenes assay. Chlorophyll A, chlorophyll B and carotene extracts in 9% acetone have been assayed on the basis of light extinctions at the wavelengths of 663nm, 645nm and 472nm accordingly to Meyer- Berthenrath s modified method [6]. The calculation formulae for photosynthetic pigments content are: 12.3E( 663).86E( 645) chla = v. (1) 1dw where: chlb 19.3E( 645) 3.6E( 663) = v. (2) 1dw 1E( 472). c. = v. (3) t 2485dw chla/b = chlorophyll A/chlorophyll B content in mg pigment/g fresh tissue t.c.-total carotene content (mg/g) E(λ)-light extinction to the wavelength λ ; d-cell width (=1cm) v-extract volume of acetone 9% (ml);w-tissue weight (g) Statistical analysis. Three repetitions of experimental investigations upon assimilatory pigments were carried out for all experimental variant samples. Average values, standard deviations have been calculated for graphical plots. The statistic significance of the differences between exposed samples and control ones was assessed by means of the Student t-test. 3. RESULTS AND DISCUSSIONS The microscopic investigation upon the cells frozen in different phases of the mitosis as well as in mitotic rest (interphase) revealed several noticeable results. First, the number of cells frozen in mitosis is one order of magnitude lower than the total cell number investigated for every sample. Actually, for other plant tissue, the number of dividing cells is always considerably smaller (interphase cell percentage is still higher) in comparison to the meristemes. Special attention was paid to the total cell number in ana-telophases. From Fig. 1 one can see that the addition of the magnetic liquid in the seedling culture medium has reduced the number of cells in ana-telophase - mainly the number of the normal dividing cells while the percentage of aberrant divisions was significantly increased from.38% in the control sample to more than 4% in the magnetic liquid treated seedlings. The aberrant dividing cells have presented several types of chromosomal aberrations such as: chromosomal bridges, expulsed chromosomes, micronuclei and retard chromosomes.

4 22 E.L.Foca-Nici, M. Ursache, G.Capraru, D.E.Creanga cell number total cells normal mitoses aberrant mitoses aberrant mitoses (%) cell number, (%) magnetic liquid (microl/l) Fig. 1 Situation of normal and aberrant cell divisions in ana-telophase. Table 1. Magnetic liquid influence on the cell division phenomenon. Magnetic liquid (microl/l) Cells in Prophase s (%) Cells in Metaphases (%) Cells in Anaphases (%) Cells in Telophases (%) cells in mitosis aberrant divisions (%) Magnetic liquid (microl/l) Fig. 2 Percentage of cells in all mitosis phases and percentage of aberrant dividing cells.

5 EXPERIMENTAL STUDY REGARDING THE MAGNETIC LIQUID 23 The magnetic liquid influence is better revealed from Table 1 where the inhibitory effect (on the cell division phenomenon) is obvious higher when the magnetic liquid concentration is higher. This is similar for metaphases, telophases and anaphases. Peculiar behaviour was observed for the cells in prophase where an increase of the dividing cells was obtained for the concentrations of 2 and 4microl/l magnetic liquid (38% and respectively 17% increase) in comparison to the control sample (with no magnetic liquid treatment). The inhibitory effect (Fig. 2) upon the cell division rate is given by the diminution of the mitosis cell percentage from about 8 (in the control sample) to less than 5 in the case of the sample corresponding to 8microl/l magnetic liquid. The increasing of the aberrant divisions is given by the enhancing percentage of vegetal cells presenting chromosomal aberrations from less than.4 in the lack of the magnetic liquid to more than 4% for the two highest concentrations tested by us: 8 and respectively 1microl/l. Chlorophyll A content.3 ChlA (mg/g) Magnetic liquid (microl/l)) Fig. 3 Chlorophyll A content for control sample and for different concentrations of magnetic liquid. The contents of photosynthesis pigments (chlorophyll A, B and total carotenoids) in the green tissue of sunflower seedlings (aged of 14 days) belonging to sunflower seedlings in Fig. 3-5 are presented. Accordingly to Fig. 3, the chlorophyll A content the main photosynthesis pigment is generally increased in the magnetic liquid treated plants in comparison to the control ones - except for the lowest magnetic liquid volume fraction tested in the frame of this study (2microl/l) where a non-significant diminution (p>.5) in comparison to the control was noticed. The higher stimulatory effect was obtained for 4microl/l magnetic suspension level where the chlorophyll A content was increased with about 7%; for the samples corresponding to 6 and 8microl/l the chlorophyll A content is increased with only 3% in comparison to the control while for the highest concentration (1microl/l) no difference could be further observed.

6 24 E.L.Foca-Nici, M. Ursache, G.Capraru, D.E.Creanga Chlorophyll B content ChlB (mg/g) Magnetic liquid (microl/l) Fig. 4 Chlorophyll B content for control sample and for different concentrations of magnetic liquid. So, one might say that the presence of magnetic liquid in the seedling culture medium was able to stimulate chlorophyll. A biosynthesis in the samples corresponding to the concentrations of 4-6-8microl/l while non-significant effect for 2 and 1microl/l was recorded. In Fig. 5 the situation of the carotene like pigments is given. Apparently similar response as in the case of chlorophyll B was obtained; however the quantitative insight revealed only 1% increased level in the samples corresponding to microl/l in comparison to the control with no statistical significance while 16% and respectively 28% diminution (with statistical significance) in the samples corresponding to the concentrations of 2microl/l and 1microl/l were measured. Carotenes content.1 Carotenes (mg/g) Magnetic liquid (microl/l) Fig. 5 The carotenoid pigment content for control sample and for different concentrations of magnetic liquid.

7 EXPERIMENTAL STUDY REGARDING THE MAGNETIC LIQUID CONCLUSIONS Magnetic liquid addition to the young seedlings of sunflower during germination is able to induce cytogenetic changes evident in the microscope slides analyzed by us. So, the diminution of the dividing cell percentage reached about 4% in comparison to the control while the presence of the chromosomal aberrations was revealed by the increase with about one order of magnitude of the total aberrant dividing cells. Atypical effect of the two lowest magnetic liquid concentrations (2 and respectively 4microl/l) was noticed regarding the cells in prophase where an increase in comparison to the control was observed (38% and respectively 17% increase). The administration of magnetic liquid to the sunflower seedlings up to the age of 14 days seems to have a discernable influence at the level of pigment biosynthesis. In the case of 4, 6 and 8microl/l the increase with 7% of chlorophyll. A was evidenced as well as the increase with 75% of chlorophyll B. All pigment sum was increased with only 4% while the ratio of chlorophylls sum to all pigment sum was related to the lowest stimulatory effect 7.5% increased. Negative effect was revealed at the level of carotene synthesis - 16% and respectively 28% diminution in the samples corresponding to the concentrations of 2microl/l and 1microl/l magnetic liquid were measured. The positive influence could be related to the generally benefic effect of low levels of iron oxide while the negative influence to the toxic effect of zinc oxide could be related. REFERENCES 1. C. Cotae, D. Creangă, LHC II System Sensitivity to Magnetic Fluids, Journal of Magnetism and Magnetic Materials, 289, p , Foca-Nici, E.L., Capraru, G., Creanga, D.E., Experimental study of some cytogenetic Zn ferrite colloidal suspension, The Annals of Dunarea de jos University of Galati, p , Goovaerts M.J., Dhaene J., Lacava Z.G.M., Azevedo R.B., Martins E.V., Lacava L.M., Biological effects of magnetic fluids: toxicity studies, J. Magn. Magn. Mater., 21(1), p (4), Manoliu Al, Antohe L., Creanga D.E., Cotae C., The influence of the petroleum ferrofluids upon the cellulosolytic fungi Chaetomium globosum Kunze, Fr. J. Magn. Mater., 21(1); p (3), Răcuciu M., Creangă D.E., Influence of water based ferrofluid upon chlorophylls in cereals, J. Magn. Magn. Mater., 311(1), p , Stirban, M., Procese primare in fotosinteza, Ed. Didactica si Pedagogica, Bucuresti, 229, 1985.

TMA-OH COATED MAGNETIC NANOPARTICLES INTERNALIZED IN VEGETAL TISSUE

TMA-OH COATED MAGNETIC NANOPARTICLES INTERNALIZED IN VEGETAL TISSUE MIHAELA RÃCUCIU 1, DORINA-EMILIA CREANGÃ 2 1 Lucian Blaga University, Faculty of Science, 10 Blvd Victoriei, 550012, Sibiu, Romania,