Branka Pevalek-Kozlina

Ecotoxicological effects of aluminum and zinc on growth and antioxidants in Lemna minor L.

The present study aimed at investigating effects of zinc and aluminum (0.15 and 0.3mM) in duckweed (Lemna minor L.) over a 15-day period. High bioaccumulation of both metals was accompanied by an increase in dry weight under higher metal treatments. Antioxidant response was observed under both metal stresses, with large increases in superoxide dismutase and peroxidases. Catalase activity declined only in duckweed exposed to Zn while lipid peroxidation as well as H(2)O(2), proline and ascorbate levels increased. The results suggest induction of oxidative stress under both aluminum and zinc toxicity, and also demonstrate duckweeds capacity to upregulate its antioxidative defense. Additionally, Zn was found to be more toxic than Al to duckweed for the concentrations applied. Due to its high bioaccumulation potential and tolerance via increased antioxidant capacity, duckweed has a potential for phytoremediation of water bodies polluted by low levels of zinc and aluminum.

Effects of radiofrequency electromagnetic fields on seed germination and root meristematic cells of Allium cepa L.

The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on seed germination, primary root growth as well as mitotic activity and mitotic aberrations in root meristematic cells were examined in Allium cepa L. cv. Srebrnjak Majski. Seeds were exposed for 2h to EMFs of 400 and 900MHz at field strengths of 10, 23, 41 and 120Vm(-1). The effect of longer exposure time (4h) and field modulation was investigated at 23Vm(-1) as well. Germination rate and root length did not change significantly after exposure to radiofrequency fields under any of the treatment conditions. At 900MHz, exposures to EMFs of higher field strengths (41 and 120Vm(-1)) or to modulated fields showed a significant increase of the mitotic index compared with corresponding controls, while the percentage of mitotic abnormalities increased after all exposure treatments. On the other hand, at 400MHz the mitotic index increased only after exposure to modulated EMF. At this frequency, compared with the control higher numbers of mitotic abnormalities were found after exposure to modulated EMF as well as after exposure to EMFs of higher strengths (41 and 120Vm(-1)). The types of aberration induced by the EMFs of both frequencies were quite similar, mainly consisting of lagging chromosomes, vagrants, disturbed anaphases and chromosome stickiness. Our results show that non-thermal exposure to the radiofrequency fields investigated here can induce mitotic aberrations in root meristematic cells of A. cepa. The observed effects were markedly dependent on the field frequencies applied as well as on field strength and modulation. Our findings also indicate that mitotic effects of RF-EMF could be due to impairment of the mitotic spindle.

The evaluation of surface and wastewater genotoxicity using the Allium cepa test.

Screening for mutagens in complex environmental mixtures, such as surface water or industrial wastewater, is gradually being accepted as a routine method in environmental monitoring programs. In the present work, the simplified Alliumcepa root assay was utilized to evaluate the possible cyto- and genotoxic effects of surface and wastewaters collected near the Sava River (Croatia) over a three-month monitoring period. Physicochemical characterization of the water samples included measurements of conductivity, chemical and biological oxygen demand, levels of suspended matter and salts, nitrate, nitrite, ammonium, total nitrogen and total phosphorus. Morphological modifications of the A. cepa roots, inhibition of root growth, cell division and induction of mitotic and chromosomal aberrations were observed. The most highly polluted water samples (industrial effluents) caused an inhibition of root growth of over 50%, a decrease in the mitotic index of over 40%, and a considerable increase in chromosomal aberrations compared to the control. The measured biological effects of some water samples appeared related to the physicochemical characteristics. Therefore, mutagenicity/genotoxicity assays should be included, along with conventional chemical analysis, in water quality monitoring programs. Their use would allow the quantification of mutagenic hazards in surface and wastewaters.

Duckweed Lemna minor as a tool for testing toxicity and genotoxicity of surface waters

In this investigation growth parameters and certain endpoints (pigment content, peroxidase activity, lipid peroxidation and alkaline comet assay) were used to detect the toxic and genotoxic effects of surface water samples on duckweed plants. The surface waters of different origin and pollutant burdens were collected monthly over a 3-month monitoring period at three sampling sites along the river Sava and its confluents (Croatia). Physicochemical characterization of the water samples included measurements of conductivity, chemical and biological oxygen demand, levels of total suspended solids, nitrate, nitrite, ammonium, Kjeldahl nitrogen and orthophosphate. Surface water samples collected from three stations caused reduction of duckweed growth rates, chlorophylls and carotenoid contents and peroxidase activity. In contrast, damage to membrane lipids (estimated by malondialdehyde content) and especially to DNA (estimated by tail extent moment) markedly increased in duckweed exposed to industrial wastewater samples. The results from the study indicate the ability of selected biomarkers to predict the phyto- and genotoxic effects of complex water mixtures on living organisms as well as the relevance of duckweed as a sensitive indicator of water quality.

Ecotoxicological assessment of industrial effluent using duckweed (Lemna minor L.) as a test organism

This study aimed at assessing the toxic effects of industrial effluents using duckweed (Lemna minor L.) plants as a test system. Growth inhibition test according to standardized protocol (ISO 20079) was performed. The suitability of the Comet assay (indicates DNA damage) and certain parameters such as peroxidase activity and lipid peroxidation level, as biomarkers for environmental monitoring was evaluated. The water samples were collected monthly over a 3-month period from the stream near the industrial estate of Savski Marof, Croatia. All samples caused inhibition of growth rates based on frond number and biomass as well as decrease of chlorophylls content. In contrast, peroxidase activity, malondialdehyde content and tail extent moment (measure of DNA strand breaks) markedly increased. Obtained data demonstrate the relevance of duckweed as sensitive indicators of water quality as well as the significance of selected biological parameters in the reliable assessment of phyto- and genotoxic potential of complex wastewaters.

The rate of elimination of chromosomal aberrations after accidental exposure to microwave radiation

Abstract Analysis of structural chromosome aberrations was performed in a group of radar station personnel who were engaged in repairing radar devices a couple of days earlier. Test results showed a major decline from the values recorded by regular mutagenic monitoring in terms of a significantly increased number of chromosome breaks, acentric fragments, dicentric and polycentric chromosomes with accompanying fragments, ring chromosomes and chromatid interchange. Multiply repeated mutagenic testing demonstrated for all subjects a fall in the total number of chromosome aberrations as a function of time. During a 30-week-long follow-up study a decrease in the total number of chromosome aberrations was observed. In the same period the presence of unstable aberrations such as dicentrics and ring chromosomes persisted, together with a relatively unchanged incidence of stable aberrations.

OXIDATIVE STRESS AND DNA DAMAGE IN BROAD BEAN (VICIA FABA L.) SEEDLINGS INDUCED BY THALLIUM

Thallium (Tl) is a metal of great toxicological concern because it is highly toxic to all living organisms through mechanisms that are yet poorly understood. Since Tl is accumulated by important crops, the present study aimed to analyze the biological effects induced by bioaccumulation of Tl in broad bean (Vicia faba L.) as well as the plants antioxidative defense mechanisms usually activated by heavy metals. Thallium toxicity was related to production of reactive oxygen species in leaves and roots of broad bean seedlings following short-term (72 h) exposure to thallium (I) acetate (0, 0.5, 1, 5, and 10 mg/L) by evaluating DNA damage and oxidative stress parameters as well as antioxidative response. The possible antagonistic effect of potassium (K) was tested by combined treatment with 5 mg/L of Tl (Tl+) and 10 mg/L of potassium (K+) acetate. Accumulation of Tl+ in roots was 50 to 250 times higher than in broad bean shoots and was accompanied by increase in dry weight and proline. Despite responsive antioxidative defense (increased activities of superoxide dismutase, ascorbate peroxidase, and pyrogallol peroxidase), Tl+ caused oxidative damage to lipids and proteins as evaluated by malondialdehyde and carbonyl group levels, and induced DNA strand breaks. Combined treatment caused no oxidative alternations to lipids and proteins though it induced DNA damage. The difference in Tl-induced genotoxicity following both acellular and cellular exposure implies indirect DNA damage. Results obtained indicate that oxidative stress is involved in the mechanism of Tl toxicity and that the tolerance of broad bean to Tl is achieved, at least in part, through the increased activity of antioxidant enzymes.

Cytogenetic stability of Centaurea ragusina long-term culture

Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings.

Growth and photosynthetic responses of Lemna minor L. exposed to cadmium in combination with zinc or copper.

Abstract Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F’m) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms. Izloženost metalima može izazvati različite štetne učinke u biljaka. Vodene leće izložili smo solima teških metala CdCl2 (5 μmol L-1), ZnCl2 (25 μmol L-1 ili 50 μmol L-1) i CuCl2 (2,5 μmol L-1 ili 5 μmol L-1) te kombinaciji CdCl2 sa svakom od navedenih koncentracija ZnCl2 i CuCl2. Rast biljaka, količina fotosintetskih pigmenata i učinkovitost fotosistema II (PSII) mjereni su nakon četiri i sedam dana tretmana. Utvrđeno je da su svi tretmani uzrokovali značajnu inhibiciju rasta te akumulaciju metala u biljci. U biljaka koje su bile izložene kombinacijama teških metala količina pojedinog metala u tkivu bila je niža u odnosu na količinu istog metala u biljaka izloženih samo tom metalu. Nakon četiri dana tretmana sva su tri metala, neovisno o tome jesu li bila primijenjena zasebno ili u kombinacijama, uzrokovala smanjenje količine klorofila a i pad vrijednosti maksimalnog (Fv/Fm) i efektivnog (ΔF/F’m) prinosa PSII. Međutim, u biljaka koje su bile istovremeno izlagane kadmiju i cinku, vrijednosti količine pigmenata i učinkovitost PSII vratile su se nakon sedam dana na kontrolnu razinu, a bakar u koncentraciji 5 μmol L-1 te kombinacija kadmija i bakra i dalje su imali inhibitorni učinak. Budući da smanjeno primanje pojedinog metala uočeno u biljaka izloženih kombiniranim tretmanima nije ublažilo inhibitorni učinak na rast, možemo zaključiti da je inhibicija rasta uzrokovana apsolutnom količinom metala primljenog u tkivo. Povećanje količine fotosintetskih pigmenata i učinkovitosti PSII nakon sedam dana tretmana kadmijem i cinkom upućuje na oporavak biljaka, što se može objasniti ublažavajućim djelovanjem cinka na učinak kadmija uslijed smanjenog primanja kadmija u biljku. Suprotno tome, dugotrajni inhibitorni učinak istovremenog tretmana biljaka kadmijem i bakrom te samim bakrom u koncentraciji 5 μmol L-1 može se objasniti oksidacijskim stresom uzrokovanim bakrom. Rezultati ovoga istraživanja pridonose saznanjima o štetnim učincima antropogenih i okolišnih onečišćivača, koji narušavanjem mehanizama fotosinteze ugrožavaju ne samo biljke i njihove zajednice nego i osjetljivu ravnotežu ekosustava.

Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra

Symbiotic associations are of a wide significance in evolution and biodiversity. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells, it harbors individuals of unicellular green algae. Morphological characteristics of isolated algae determined by light and electron microscopy are presented. Cytological morphometric parameters (cell area, cell radius, chloroplast area) of isolated algae from green hydra (Cx), as well as from reference species Chlorella kessleri (Ck) and Chlorella vulgaris (Cv), revealed similarity between the isolated endosymbiont and C. kessleri. Isoenzyme patterns of esterase (EST), peroxidase (POX), and catalase (CAT) were used for the investigation of genetic variability in endosymbiotic algae isolated from green hydra. Out of 14 EST isoenzymes observed in Cx species, 9 were expressed in the Cx sample. Results of the EST isoenzyme analysis indicated a higher degree of similarity between Cx and Cv than between Cx and Ck. Due to much higher heterogeneity, EST isoenzymes seem to be more suitable genetic markers for identification of different Chlorella species than CAT and POX isoenzymes. Results obtained suggest that symbiogenesis in green hydra has probably not been terminated yet.